CYTOTOXIC AGENTS

FIELD OF THE INVENTION

The invention relates to DNA-alkylating units comprising fused rings. In particular it relates to compounds comprising a naked C-ring and C-ring substituted (e.g., aryl) pyrridinobenzodiazepine group linked via the A-ring to other aromatic alkylating units (e.g., PBD), and to pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents. BACKGROUND

The pyrrolobenzodiazepines (PBDs) are a group of compounds some of which have been shown to be sequence-selective DNA minor-groove binding agents. The PBDs were originally discovered in Streptomyces species ^{2 6} They are tricyclic in nature, and are comprised of of fused 6-7-5-membered rings and can be identified as an anthranilate (A ring), a diazepine (B ring) and a pyrrolidine (C ring) 4 They are characterized by an electrophilic Nio=Cn imine group (as shown below) or the hydrated equivalent, a carbinolamine [NH-CH(OH)], or a carbinolamine alkyl ether ([NH-CH(OR, where R = alkyl)] which can form a covalent bond to a C2-amino group of guanine in DNA to form a DNA adduct ⁷ . The natural products interact in the minor groove of the DNA helix with excellent fit (i.e., good "isohelicity") due to a right-handed longitudinal twist induced by a chiral Cua-position which has the (^-configuration ⁸ .

Carbinolamine Imine Carbinolamine alkyl ether

The DNA adduct has been reported to inhibit a number of biological processes including the binding of transcription factors ^{9 10} ' ¹¹ and the function of enzymes such as endonucleases ¹² ' ¹³ and RNA polymerase ¹⁴ . PBD monomers (e.g., anthramycin) have been shown by footprinting ⁸ , NMR ¾ ^ molecular modeling and X-ray

crystallography ¹⁸ to span three base pairs and to have a thermodynamic preference for the sequence s'-Pu-G-Pu-3' (where Pu = purine, and G is the reacting guanine) ^ and a kinetic preference for the sequence 5'-Py-G-Py-3'. PBDs are thought to interact with DNA by first locating at a low-energy binding sequence (i.e., a s'-Pu-G-Pu-3' triplet) through Van der Waals, hydrogen bonding and electrostatic interactions 9. Then, once in place, a nucleophilic attack by the exocyclic C2-amino group of the central guanine

SUBSTITUTE SHEET RULE 26 occurs to form the covalent adduct 9 (Fig 2). Once bound, the PBD remains anchored in the DNA minor groove, avoiding DNA repair by causing negligible distortion of the DNA helix ¹⁸ . The ability of PBDs to form an adduct in the minor groove and crosslink DNA enables them to interfere with DNA processing and, hence, their potential for use as antiproliferative agents.

WO 2004/087711, WO 2011/117882 and WO 2013/164593 disclose PBD (6-7-5) dimers linked via their A-rings, and more recently WO2012128868 and WO2016115191 disclose G-alkylating agents containing a D-ring (i.e., 6-7-5-6 and 6-7-6-6 respectively), all of which have been shown to act as cytotoxic agents in vitro and as anti-tumour agents in vivo in animal tumour models. Furthermore, the C8/C8'-linked PBD dimer SJG-136 ²⁰ has completed Phase I clinical trials for leukaemia and ovarian cancer ²¹ and has shown sufficient therapeutic benefit to progress to Phase II studies, and two antibody-drug conjugates (ADCs) containing PBD dimers have now entered Phase III clinical trials.

SJG-136

The present application reports pyrridinobenzodiazepines (PDDs) and aryl-PDDs, which are related to PBDs but contain an expanded 6-membered C-ring as compared to the 5-membered C-ring of PBDs, and/ or contain an extra bulky group on the C-ring. The PDD is coupled to various G-alkylating moieties through its A-ring. The inventors have discovered that symmetrical and unsymmetrical dimers based on the PDD or C- ring substituted PDD provide properties, such as cytoxicity and DNA binding, that results in effective compounds. In addition, compounds according to the present invention are less hydrophobic than currently available dimer compounds.

Hydrophobicity is a key problem associated with the conjugation of PBD dimers to antibodies and other scaffolds and it can have a considerable impact on the successful use of such compounds as therapeutics. The disclosed PBD dimers also show sequence selectivity.

The present invention seeks to overcome problem(s) associated with the prior art. SUMMARY

In a first aspect, the present invention provides a compound of formula (I) or formula (Π):

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SUBSTITUTE SHEET RULE 26

wherein;

each double line — independently represents a single bond or a double bond;

m is o or l;

Ri, R ₂ , R ₃ and R4 are independently selected from H and R _ig ;

or one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3

independently selected optional R ₂₀ groups;

R ₅ and R ₆ are selected such that either (i) R ₅ is selected from H, OH and OCi- ₆ alkyl; and R ₆ is selected from H, S0 ₃ H, nitrogen protecting groups, -L ₂ -R ₂ s and RA; (ii) R ₅ is oxo or H, and R ₆ is H or Ci- ₆ alkyl; or (iii) R ₅ and R ₆ together form a double bond;

R ₇ , R ₉ , R16 and R ₁ 8 are independently selected from H and R ₂₀ ;

Rs and R _i7 are independently selected from H, SR ₂₂ , SCH ₂ Ph and R ₂₀ ;

Rio, R11, Ri ₂ and R _i3 , are independently selected from H and R _ig ;

or one of R _i0 and Rn, Rn and R _i2 , R _i2 and R _i3 , or R _i3 and R _i4 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6- membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups;

R14 and R1 ₅ are selected such that either (iv) R _i4 is selected from H, OH and OCi-6 alkyl; and R1 ₅ is selected from H, S0 ₃ H, nitrogen protecting groups, -L ₂ -R ₂ s and RB; (v) R14 is oxo or H, and R _i5 is H or Ci- ₆ alkyl; or (vi) R _i4 and R _i5 together form a double bond; with the proviso that if R ₅ and R ₆ are selected from (ii) then R _i4 and R1 ₅ are selected from (iv) and (vi); and if R _i4 and R _i5 are selected from (v) then R ₅ and R6 are selected from (i) and (iii);

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SUBSTITUTE SHEET RULE 26 each R _A and R _B is independently selected from (CH ₂ )j-0H, (CH ₂ )j-C0 ₂ R26, C(=0)-0- (CH ₂ ) _k -NR ₂₆ R ₂₇ , (CH ₂ )j-NR ₂₆ R ₂₇ , and C(=0)-NH- (CH ₂ ) _k -C(=NH)NR ₂₆ R ₂₇ ;

Xi is O, S, NR ₂₄ , CR ₂₄ R ₂₅ , CR ₂₄ R ₂₅ 0, C(=0), C(=0)NR ₂₄ , NR ₂₄ C(=0), O-C(O), C(0)-0 or is absent;

Li is selected from an amino acid, a peptide chain having from 2 to 12 amino acids, a paraformaldehyde chain -(0CH ₂ )i- ₂₄ -, a polyethylene glycol chain

-(OCFLCFQ^- and -(CH ₂ ) _n -Yi-(CH ₂ ) _p - wherein

n is selected from o, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

p is selected from o, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and

Yi is selected from -(CH ₂ )i- ₅ -, -C(0)-NH-, -NH-, -S(0) ₀ - ₂ -, -C[(CH ₂ ) ₀ - ₅ Y ₂ ]-, -ATI-C(O)- NH-(Ar ₂ ) ₀ -i-Ar ₃ -, -Ar ₃ -(Ar ₂ )o-i-NH-C(0)-Ari-, optionally substituted 3- to 7- membered cycloalkylene or heterocycloalkene, optionally substituted 6- membered arylene, and optionally substituted 5- to 9-membered heteroarylene; Y ₂ is H or R ₂₀ ;

ΑΓΙ is an optionally substituted 5-membered heteroarylene;

Ar ₂ is an optionally substituted 6-membered arylene or heteroarylene;

Ar ₃ is an optionally substituted 5- to 9-membered heteroaryl ring;

wherein the optionally substituted groups of Yi, Ar _i; Ar ₂ and Ar ₃ are optionally

substituted with 1, 2 or 3 independently selected optional R ₂₀ groups;

X ₂ is O, S, NR ₂₄ , CR ₂₄ R ₂₅ , CR ₂₄ R ₂₅ 0, C(=0), C(=0)NR ₂₄ , NR ₂₄ C(=0), O-C(O), C(0)-0 or is absent;

each R ₁₉ is independently selected from R ₂₀ , R ₂ i, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , =CH-(CH ₂ ) _S - R ₂₁ , =0, (CH ₂ )s-0R ₂₁ , (CH ₂ )s-C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , 0-(CH ₂ ) _t -NR ₂₁ R ₂₃ , NH- C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _s -S0 ₂ R ₂₁ , O-

S0 ₂ R ₂₁ , (CH ₂ )s-C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ ;

each R ₂₀ is independently selected from F, CI, Br, (CH ₂ )j-0H, Ci- ₆ alkyl, OCi-6 alkyl, 0CH ₂ Ph, (CH ₂ )j-C0 ₂ R ₂₆ , 0-(CH ₂ ) _k -NR ₂₆ R ₂₆ , C(=0)-0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , C(=0 NR ₂₆ R ₂₇ , (CH ₂ )j-NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ ,

(CH ₂ )j-R ₂₆ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NR ₂₆ R ₂₇ , -L ₂ -R ₂₈ , 8(0) ₂ -( ₆ alkyl), O-

(CH. -O-Cd-e alkyl), (CH ₂ )j-S(0) ₂ -NR ₂₆ R ₂₇ , alkyl), (CFQk-O-Cd-e alkyl), CN, NCO, Cy, C(0)-NH-(CH ₂ )j-Cy, C(0)-Cy, NH-C(0)-NR ₂₆ R ₂₇ and

each j and s is independently selected from o, 1, 2, 3, 4, 5 or 6;

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SUBSTITUTE SHEET RULE 26 each k and t is independently selected from 1, 2, 3, 4, 5 or 6;

each R ₂ i is independently selected from H, d-i ₂ alkyl, C ₅ -6 heterocyclyl, C ₅ - _g heteroaryl, C6- ₁ 5 heteroarylalkyl, phenyl and C _7- i ₂ aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups;

each R ₂₂ , R2 ₃ , R24, R2 ₅ , R26 and R ₂₇ is independently selected from H and Ci-12 alkyl; each Cy is independently selected from a C ₅ -6 heterocyclyl or C ₅ -6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R ₂₀ groups;

L2 is a bond or a linker moiety having 1-200 non-hydrogen atoms selected from C, N, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and

R ₂ 8 is an azide, alkyne, bisulfone, carbohydrazide, hydrazine, hydroxylamine,

iodoacetamide, isothiocyanate, maleimide, phosphine, pyrridopyridazine, semihydrazide, succinimidyl ester, sulfodichlorophenol ester, sulfonyl halide, sulfosuccinimidyl ester, 4-sulfotetrafluorophenyl ester, tetrafluorophenyl ester, thiazole, RA, 0-(CH ₂ )k-NR ₂ 6R26, NHNH ₂ , or is a targeting agent wherein the targeting agent is selected from a protein, a portion of a protein, a peptide, a nucleic acid, or an antibody;

or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof;

with the proviso that either the compound is:

(a) a compound of formula (I), wherein at least one of R ₃ or R _i2 is an optionally

substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - ₉ heteroaryl or an optionally substituted phenyl;

(b) a compound of formula (I), wherein both R ₂ and R ₃ , and Rn and R _i2 , together with the carbon atoms to which they are attached form an optionally substituted 5- membered cyclic, heterocyclic, or heteroaryl ring;

(c) a compound of formula (II), wherein m is 1; R _i2 is an optionally substituted C ₅ _6 heterocyclyl, an optionally substituted C ₅ _ _g heteroaryl or an optionally substituted phenyl; and Ri, R ₂ , R ₃ , R4, Rio, Rn and R _i3 are independently selected from H and

(d) a compound of formula (II), wherein m is 1; Rn and R _i2 , or R _i2 and R _i3 , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring; and Ri, R ₂ , R ₃ , R4, are independently selected from H and R _ig ;

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SUBSTITUTE SHEET RULE 26 (e) a compound of formula (II), wherein m is o; R _i2 is an optionally substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - _g heteroaryl or an optionally substituted phenyl; and R ₂ and R ₃ , or R ₃ and R ₄ , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or a 5- or 6- membered cyclic, heterocyclic, or heteroaryl ring; or

(f) a compound of formula (II), wherein m is 1; R _i2 is =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ or =CH-

(CH ₂ )s-R ₂ i; and Ri, R ₂ , R ₃ , R4, R _i0 , Ru, Ri ₂ and R _i3 are independently selected from

In another aspect, the present invention provides a compound of formula (I) or formula (II), or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, for use as a medicament.

In a further aspect, there is provided a compound of formula (I) or formula (II), or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, for use in a method of therapy.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or formula (II), or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition of the present invention may further comprise one or more (e.g. two, three or four) further active agents.

In another aspect, the present invention provides a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, or a pharmaceutical composition comprising a compound of formula (I) or formula (II), for use in the treatment of a proliferative disease.

In a further aspect, the present invention provides a method of treatment of a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, or a pharmaceutical composition comprising a compound of formula (I) or formula (II).

In a further aspect, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be

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SUBSTITUTE SHEET RULE 26 administered alone or in combination with other treatments, either simultaneously or sequentially depending upon the condition to be treated.

In a further aspect, the present invention provides a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, for use as a drug in an antibody-drug conjugate.

In certain aspects, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be used as a payload on a tumour-targeting agent (e.g., antibody, antibody fragment, hormone, etc.).

In a further aspect, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be linked, either directly or indirectly, to a targeting agent (e.g., antibody, antibody fragment, hormone, etc.) to provide a targeted conjugate. The target conjugates of the present disclosure may contain one or multiple compounds of formula (I) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof. A variety of target conjugates are known in the art and may be used with a compound of formula (I) and salts or solvates thereof. For example, in a particular aspect the target conjugate is an antibody-drug conjugate, wherein one or more compounds of formula (I) are linked, directly or indirectly, to the antibody. Therefore, the compound of formula (I) and salts or solvates thereof, may be used as a payload on a targeted conjugate. Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims. DEFINITIONS

The following abbreviations are used throughout the specification: Ac acetyl; Alloc allyloxycarbonyl; BAIB bis(acetoxy)iodobenzene/ (diacetoxyiodo)benzene; Boc tert- butoxycarbonyl; DCM dichloromethane; DMAP 4-dimethylaminopyridine; DMF dimethylformamide; DMSO dimethylsulfoxide; Et ethyl; Et ₂ 0 diethyl ether; EtOAc ethyl acetate; EtOH ethanol; HATU (i-[Bis(dimethylamino)methylene]-iH-i,2,3- triazolo[4,5-b]-pyridinium 3-oxid hexafluorophosphate); Me methyl; MeOH methanol; PBDs pyrrolo[2,i-c][i,4]benzo-diazepines; PDDs pyrridinobenzodiazepines; Ph phenyl;

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SUBSTITUTE SHEET RULE 26 Pyr pyridine; TBAF tetrabutylammonium fluoride; TEMPO (2,2,6,6-tetramethyl- piperidin-i-yl)oxyl; and TBS-C1/TBDMSC1 tert- butyldimethylsilyl chloride.

"Substituted", when used in connection with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and that a chemically stable compound results from the substitution. "Optionally substituted" refers to a parent group which may be unsubstituted or which may be substituted with one or more substituents. Suitably, unless otherwise specified, when optional substituents are present the optional substituted parent group comprises from one to three optional substituents. Where a group maybe "optionally substituted with 1, 2 or 3 optional groups", this means that the group may be substituted with o, 1, 2 or 3 of the optional substituents. Suitably, the group is substituted with 1, 2 or 3 of the optional substituents. Where a group may be "optionally substituted with one or two optional substituents", this means that the group may be substituted with o, 1 or 2 of the optional substituents. Suitably groups may be optionally substituted with o or 1 optional substituents. In some aspects, suitably the group is not optionally substituted. In other aspects, suitably the group is substituted with 1 of the optional substituents.

Optional substituents may be selected from Ci _-7 alkyl, C ₂ - ₇ alkenyl, C ₂ - ₇ alkynyl, C ₅ - ₂₀ aryl, C _3- i ₀ cycloalkyl, C _3- i ₀ cycloalkenyl, C _3- i ₀ cycloalkynyl, C ₃ - ₂₀ heterocyclyl, C ₃ - ₂₀ heteroaryl, acetal, acyl, acylamido, acyloxy, amidino, amido, amino, aminocarbonyloxy, azido, carboxy, cyano, ether, formyl, guanidino, halo, hemiacetal, hemiketal, hydroxamic acid, hydroxyl, imidic acid, imino, ketal, nitro, nitroso, oxo, oxycarbonyl, oxycarboyloxy, sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, uredio groups.

"Independently selected" is used in the context of statement that, for example, "each R _2i and R ₂₂ are independently selected from H and Ci _-6 alkyl,..." and means that each instance of the functional group, e.g. R _2i , is selected from the listed options

independently of any other instance of R ₂ i or R ₂₂ in the compound. Hence, for example, H may be selected for the first instance of R _2i in the compound; methyl may be selected for the next instance of R _2i in the compound; and ethyl may be selected for the first instance of R ₂₂ in the compound.

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SUBSTITUTE SHEET RULE 26 Ci-12 alkyl: refers to straight chain and branched saturated hydrocarbon groups, generally having from l to 12 carbon atoms; more suitably Ci _-7 alkyl; more suitably Ci _-6 alkyl; more suitably Ci _-3 alkyl. Examples of alkyl groups include methyl, ethyl, n- propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-i-yl, pent-2-yl, pent-3-yl, 3- methylbut-i-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-i-yl, n-hexyl, n- heptyl, and the like.

"Alkylene" refers to a divalent radical derived from an alkane which may be a straight chain or branched, as exemplified by -CH2CH2CH2CH2-.

"Monocyclic cycloalkylene" refers to a divalent radical derived from a saturated monocyclic hydrocarbon group (or cycloalkane). The cycloalkylene group maybe attached to the rest of the compound at any ring atom unless such attachment would violate valence requirements. Suitably, the monocylic cycloalkylene group is a C _3- i ₀ cycloalkylene group that is a cycloalkyl group having from 3 to 10 carbon atoms that comprise the ring. Suitably the monocylic cycloalkylene group is a C ₃ - ₇ cycloalkylene group, more suitably a C ₆ cycloalkylene group (i.e. a cyclohexylene group). The term "amino acid" refers to both the twenty "canonical" or "natural" amino acids, as well "non-canonical" amino acids, also referred to as "unnatural" amino acids, such as modified or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, i.e. they are amino acids selected from alanine, argenine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Modified amino acids include, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

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SUBSTITUTE SHEET RULE 26 "Aryl": refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring and having a specified number of carbon atoms that comprise their ring members (e.g., 6-membered aryl refers to an aryl group having 6 carbon atoms as ring members and C ₆ -i4 aryl refers to an aryl group having 6 to 14 carbon atoms as ring members). The aryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non- hydrogen substituents unless such attachment or substitution would violate valence requirements. Suitably, a C6-14 aryl is selected from a Ce-iz aryl, more suitably, a C6-10 aryl. Examples of aryl groups include phenyl.

"Arylene" refers to a divalent radical derived from an aryl group, e.g. -ΰ ₆ Η ₄ - which is the arylene derived from phenyl.

"C7-12 aralkyl" refers to an arylalkyl group having 7 to 12 carbon atoms and comprising an alkyl group substituted with an aryl group. Suitably the alkyl group is a Ci _-6 alkyl group and the aryl group is phenyl. Examples of C _7- i ₂ aralkyl include benzyl and phenethyl. In some cases the C ₇ -i ₂ aralkyl group may be optionally substituted and an example of an optionally substituted C _7- i ₂ aralkyl group is 4-methoxylbenzyl. "C3-C8 carbocyclyl" by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7- or 8- membered monovalent, substituted or unsubstituted, saturated or unsaturated non- aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative C ₃ -Cs carbocyclyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1 ,3-cyclohexadienyl, 1 ,4-cyclohexadienyl, cycloheptyl, 1 ,3-cycloheptadienyl, 1 ,3,5-cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo(i.i .1 pentane, and bicyclo(2.2.2.)octane. A C ₃ -Cs carbocyclyl group can be optionally substituted. Halogen: refers to a group selected from F, CI, Br, and I. Suitably, the halogen is CI.

"heteroalkyl," refers to a stable straight or branched chain hydrocarbon, or

combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S maybe placed at any interior position of

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SUBSTITUTE SHEET RULE 26 the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive. Heteroalkyl groups typically comprise from 1 to 1 5 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 8 carbon atoms, and most preferably from 1 to 4 carbon atoms. Heteroalkyl groups may be optionally substituted.

"heteroalkylene" refers to a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Heteroalkylene groups may be optionally substituted.

"C ₅ - ₉ heteroaryl": refers to unsaturated monocyclic or bicyclic aromatic groups comprising from 5 to 9 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms. Suitably, any monocyclic heteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ring heteroatoms. Suitably each ring heteroatom is

independently selected from nitrogen, oxygen, and sulfur. The bicyclic rings include fused ring systems and, in particular, include bicyclic groups in which a monocyclic heterocycle comprising 5 ring atoms is fused to a benzene ring. The heteroaryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

Ni: pyrrole, pyridine;

Oi: furan;

Si: thiophene;

N1O1: oxazole, isoxazole, isoxazine;

N ₂ 0i: oxadiazole (e.g. i-oxa-2,3-diazolyl, i-oxa-2,4-diazolyl, i-oxa-2,5-diazolyl, l-oxa- 3,4-diazolyl);

N ₃ O1: oxatriazole;

N1S1: thiazole, isothiazole;

N ₂ : imidazole, pyrazole, pyridazine, pyrimidine, pyrazine;

N ₃ : triazole, triazine; and,

N ₄ : tetrazole.

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SUBSTITUTE SHEET RULE 26 Examples of heteroaryl which comprise fused rings, include, but are not limited to, those derived from:

Oi. benzofuran, isobenzofuran;

Ni: indole, isoindole, indolizine, isoindoline;

Si: benzothiofuran;

NiOi: benzoxazole, benzisoxazole;

NiSi: benzothiazole;

N ₂ : benzimidazole, indazole;

0 ₂ : benzodioxole;

N ₂ 0i: benzofurazan;

N ₂ Si: benzothiadiazole;

N ₃ : benzotriazole; and

N ₄ : purine (e.g., adenine, guanine), pteridine; "5- or 6-membered heteroaryl": refers to unsaturated monocyclic aromatic groups comprising from 5 or 6 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms. Suitably, any monocyclic heteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ring heteroatoms. Suitably each ring heteroatom is

independently selected from nitrogen, oxygen, and sulfur. The heteroaryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from the list given above in relation to the definition for C ₅ - ₉ heteroaryl.

"heteroarylene" refers to a divalent radical derived from a heteroaryl group (such as those described above) and preferably contain 5-14, 6-14, or 6-20 carbon atoms in addition to one, two or three heteroatoms. Heteroarylenes may be monocyclic, bicyclic, or tricyclic ring systems. Representative heteroarylenes, are not limited to, but may be selected from triazolylene, tetrazolylene, oxadiazolylene, pyridylene, furylene, benzofuranylene, thiophenylene, benzothiophenylene, quinolinylene, pyrrolylene, indolylene, oxazolylene, benzoxazolylene, imidazolylene, benzimidazolylene, thiazolylene, benzothiazolylene, isoxazolylene, pyrazolylene, isothiazolylene, pyridazinylene, pyrimidinylene, pyrazinylene, triazinylene, cinnolinylene,

phthalazinylene, quinazolinylene, pyrimidylene, azepinylene, oxepinylene, and quinoxalinylene. Heteroarylenes are optionally substituted.

12

SUBSTITUTE SHEET RULE 26 "Monocyclic heteroarylene" refers to a divalent radical derived from a monocyclic heteroaryl group (in particular those derived from this list of monocyclic heteroaryl groups provided above). "C0-15 heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. Suitably the alkyl is a Ci _-6 alkyl group and the heteroaryl group is C ₅ - ₉ heteroaryl as defined above. Examples of C ₆ -i ₅ heteroarylalkyl groups include pyrrol-2-ylmethyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, pyrrol-3-ylethyl, pyrrol-4-ylethyl, imidazol-2- ylmethyl, imidazol-4-ylmethyl, imidazol-4-ylethyl, thiophen-3-ylmethyl, furan-3- ylmethyl, pyridin-2-ylmethyl, pyridin-2-ylethyl, thiazol-2-ylmethyl, thiazol-4-ylmethyl, thiazol-2-ylethyl, pyrimidin-2-ylpropyl, and the like.

"C3-20 heterocyclyl", "heterocyclic" or "heterocyclo": refers to saturated or partially unsaturated monocyclic, bicyclic or polycyclic groups having ring atoms composed of 3 to 20 ring atoms, whether carbon atoms or heteroatoms, of which from 1 to 10 are ring heteroatoms. Suitably, each ring has from 3 to 7 ring atoms and from 1 to 4 ring heteroatoms (e.g., suitably C ₃ - ₅ heterocyclyl refers to a heterocyclyl group having 3 to 5 ring atoms and 1 to 4 heteroatoms as ring members). The ring heteroatoms are independently selected from nitrogen, oxygen, and sulphur.

As with bicyclic cycloalkyl groups, bicyclic heterocyclyl groups may include isolated rings, spiro rings, fused rings, and bridged rings. The heterocyclyl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

Ni : aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine;

Oi: oxirane, oxetane, tetrahydrofuran, dihydrofuran, tetrahydropyran, dihydropyran, pyran, oxepin;

Si: thiirane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane;

0 ₂ : dioxolane, dioxane, and dioxepane;

0 ₃ : trioxane;

N ₂ : imidazoiidine, pyrazolidine, imidazoline, pyrazoline, piperazine, uracil;

13

SUBSTITUTE SHEET RULE 26 NiOi: tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine;

NiSi: thiazoline, thiazolidine, thiomorpholine;

N ₂ 0i: oxadiazine;

OiSi: oxathiole and oxathiane (thioxane); and

NiOiSi: oxathiazine.

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses, such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses, such as aliopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

"5- or 6-membered heterocyclic" refers to saturated or partially unsaturated monocyclic examples of "C ₃ - ₂₀ heterocyclyl" groups. 5- or 6-membered heterocyclic having ring atoms composed of 5 to 6 ring atoms, whether carbon atoms or heteroatoms, of which from 1 to 4 are ring heteroatoms. More suitably, each ring has from 5 to 6 ring atoms and from 1 to 2 ring heteroatoms. The ring heteroatoms are independently selected from nitrogen, oxygen, and sulphur.

"Monocyclic heterocyclylene" refers to a divalent radical derived from a monocyclic heterocyclyl group (in particular those derived from this list of monocyclic heterocyclyl groups provided above). "Monocyclic cycloalkenylene" refers to a divalent radical derived from a cycloalkyl that contains at least one double bond. Suitably, the cycloalkenylene group comprises one or two double bonds. The cycloalkenylene group may be attached to the rest of the compound at any ring atom unless such attachment would violate valence

requirements. Suitably the monocylic cycloalkenylene group is a C ₃ _ ₇ cycloalkenylene group, more suitably a C ₆ cycloalkenylene group (i.e. a cyclohexenylene group).

"Nucleic acid", refers to a linear polymer of nucleosides (including deoxyribo- nucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Nucleic acid may encompass the term "polynucleotide" as well as "oligonucleotide". The linear polymer may be represented by a sequence of letters, such as "ATGCCTG," where it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes

14

SUBSTITUTE SHEET RULE 26 deoxyguanosine, and "T" denotes deoxythymidine, unless otherwise noted. Another natural nucleotide is "U", denoting uridine. The letters A, C, G, T and U can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art. In naturally occurring nucleic acids, the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as "nucleotides." Nucleic acids may also include other inter-nucleoside linkages, such as phosphoro- thioate linkages, and the like. Such analogs of nucleotides that do not include a phosphate group are considered to fall within the scope of the term "nucleotid"" as used herein, and nucleic acids comprising one or more inter-nucleoside linkages that are not phosphodiester linkages are still referred to as "polynucleotides", "oligonucleotides", etc.

Nitrogen protecting groups

Nitrogen protecting groups are well known in the art and are groups that block or protect the nitrogen groups from further reaction. Nitrogen protecting groups are exemplified by carbamates, such as methyl or ethyl carbamate, 9-fluorenylmethyloxy- carbonyl (Fmoc), substituted ethyl carbamates, carbamates cleaved by 1,6-beta- elimination, ureas, amides, peptides, alkyl and aryl derivatives. Carbamate protecting groups have the general formula:

In this specification a zig-zag line indicates the point of attachment of the shown group (e.g. the protecting group above) to the rest of the compound of formula (I) or formula (II). Suitable nitrogen protecting groups may be selected from acetyl, trifluoroacetyl, t- butyloxy-carbonyl (BOC), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxy-carbonyl (Fmoc).

A large number of possible carbamate nitrogen protecting groups are listed on pages 706 to 771 of Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4 ^th Edition, Wiley-lnterscience, 2007, and in P. Kocienski, Protective Groups, 3rd Edition (2005) which are incorporated herein by reference.

Particularly preferred protecting groups include Alloc (allyloxycarbonyl), Troc (2,2,2- Trichloroethyl carbonate), Teoc [2-(Trimethylsilyl)ethoxycarbony], BOC (tert- butyloxycarbonyl), Doc (2,4-dimethylpent-3-yloxycarbonyl), Hoc (cyclohexyloxy- carbonyl), TcBOC (2,2,2-trichloro-tert-butyloxycarbonyl), Fmoc (9-

SUBSTITUTE SHEET RULE 26 fluorenylmethyloxycarbonyl), l-Adoc (i-Adamantyloxycarbonyl) and 2-Adoc (2- adamantyloxycarbonyl).

Hydroxyl protecting groups

Hydroxyl protecting groups are well known in the art, a large number of suitable groups are described on pages 16 to 366 of Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4 ^th Edition, Wiley-lnterscience, 2007, and in P. Kocienski, Protective Groups, 3rd Edition (2005) which are incorporated herein by reference. Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates. Particularly preferred protecting groups include THP (tetrahydropyranyl ether).

An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does

16

SUBSTITUTE SHEET RULE 26 not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/ or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At ²¹¹ , l ¹ ^, I ¹² s, Y9o _; R _e ⁱ 86 _; ^R e ⁱ ss, Sm^, Bi ²¹² , P3 ² , Pb ²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other

intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

17

SUBSTITUTE SHEET RULE 26 By "co-administering" is meant intravenously administering two (or more) drugs during the same administration, rather than sequential infusions of the two or more drugs. Generally, this will involve combining the two (or more) drugs into the same IV bag prior to co-administration thereof.

A drug that is administered "concurrently" with one or more other drugs is

administered during the same treatment cycle, on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs. For instance, for cancer therapies given every 3 weeks, the concurrently administered drugs are each administered on day-i of a 3-week cycle.

A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol;

colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin;

podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and

ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammail and calicheamicin omegali (see, e.g., Nicolaou et al., Angew. Chem lntl. Ed. Engl, 33 : 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as

neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin,

18

SUBSTITUTE SHEET RULE 26 daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2- pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxonibicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;

aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;

triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,

FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;

methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE® ) ; etoposide (VP- 16); ifosfamide;

mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin;

ibandronate; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®);

bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),

SUBSTITUTE SHEET RULE 26 etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC- alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF- R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU- 11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus,

RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,

SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include "anti-hormonal agents" or

"endocrine therapeutics" which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/ antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene

(FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti- estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARFMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RrvTSOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5 imidazoles;

lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including

SUBSTITUTE SHEET RULE 26 progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as

fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti- progesterones; estrogen receptor down- regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);

phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. The term "epitope" refers to the particular site on an antigen molecule to which an antibody binds.

The "epitope 4D5" or "4D5 epitope" or "4D5" is the region in the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. This epitope is close to the transmembrane domain of HER2, and within domain IV of

HER2. To screen for antibodies which bind to the 4D5 epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of HER2 (e.g. any one or more residues in the region from about residue 550 to about residue 610, inclusive, of HER2 (SEQ ID NO: 39).

The "epitope 2C4" or "2C4 epitope" is the region in the extracellular domain of HER2 to which the antibody 2C4 binds. In order to screen for antibodies which bind to the 2C4 epitope, a routine cross-blocking assay such as that described in Antibodies, A

Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2. The 2C4 antibody and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328 (2004)). Anti-HER2 murine antibody 7C2 binds to an epitope in domain I of HER2. See, e.g., PCT Publication No. WO 98/ 17797.

21

SUBSTITUTE SHEET RULE 26 This epitope is distinct from the epitope bound by trastuzumab, which binds to domain IV of HER2, and the epitope bound by pertuzumab, which binds to domain II of HER2. By binding domain IV, trastuzumab disrupts ligand- independent HER2-HER3 complexes, thereby inhibiting downstream signaling (e.g. PI3K/AKT). In contrast, pertuzumab binding to domain II prevents ligand-driven HER2 interaction with other HER family members (e.g. HER3, HERl or HER4), thus also preventing downstream signal transduction. Binding of MAb 7C2 to domain I does not result in interference of trastuzumab or pertuzumab binding to domains IV and II, respectively, thereby offering the potential of combining a MAb 7C2 ADC with trastuzumab, trastuzumab emtansine (T-DM-i), and/or pertuzumab. Murine antibody 7C2, 7C2.B9, is described in PCT Publication No. WO 98/ 17797. An anti-HER2 7C2 humanized antibody is disclosed in WO2016/040723 Al.

The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of

Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FRi, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FRi-Hi(Li)-FR2-H2(L2)-FR3-H3(L3 FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the

22

SUBSTITUTE SHEET RULE 26 progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non- human source that utilizes human antibody repertoires or other human antibody- encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hi, H2, H3), and three in the VL (Li, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops

23

SUBSTITUTE SHEET RULE 26 and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (Li), 50-52 (L2), 91-96 (L3), 26-32 (Hi), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-Li, CDR-L2, CDR-L3, CDR-Hi, CDR-H2, and CDR- H3) occur at amino acid residues 24-34 of Li, 50-56 of L2, 89-97 of L3, 31-35B of Hi, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDRi in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity

determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a- CDRs. Exemplary a-CDRs (a-CDR-Li, a-CDR-L2, a-CDR-L3, a-CDR-Hi, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13 : 1619- 1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

The term "immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens.

Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as Cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5- lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol;

dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments;

cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL®

methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as

24

SUBSTITUTE SHEET RULE 26 chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta, or -gamma antibodies, antitumor necrosis factor(TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti- interleukin-6 (IL-6) receptor antibodies and antagonists (such as ACTEMRA™

(tocilizumab)); anti-LFA-i antibodies, including anti-CDna and anti-CDi8 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187); streptokinase; transforming growth factor-beta (TGF-beta); streptodornase; RNA or DNA from the host; FK506; RS-61443;

chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et al, Science, 251 : 430-432 (1991); WO 90/11294; Ianeway, Nature, 341 : 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies and ZTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol, 23 : 113-5 (2002) and see also definition below); biologic agents that interfere with T cell helper signals, such as anti- CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al, Science, 261 : 1328-30 (1993); Mohan et al, J. Immunol, 154: 1470- 80 (1995)) and CTLA4-Ig (Finck et al, Science, 265: 1225-7 (i994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Some preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate. An "isolated antibody" is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

25

SUBSTITUTE SHEET RULE 26 "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term "HER2," as used herein, refers to any native, mature HER2 which results from processing of a HER2 precursor protein in a cell. The term includes HER2 from any vertebrate source, including mammals such as primates (e.g. humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of HER2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human HER2 precursor protein, with signal sequence (with signal sequence, amino acids 1-22) is shown in SEQ ID NO: 64. The amino acid sequence of an exemplary mature human HER2 is amino acids 23-1255 of SEQ ID NO: 64.

The term "HER2 -positive cell" refers to a cell that expresses HER2 on its surface. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants

(epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

26

SUBSTITUTE SHEET RULE 26 "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric

glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative

substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid

27

SUBSTITUTE SHEET RULE 26 sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program

The term "PD-i axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-i axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-i signaling axis - with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, a PD-i axis binding antagonist includes a PD-i binding antagonist, a PD-Li binding antagonist and a PD-L2 binding antagonist.

The term "PD-i binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- 1 with one or more of its binding partners, such as PD-Li, PD-L2. In some embodiments, the PD-i binding antagonist is a molecule that inhibits the binding of PD-i to one or more of its binding partners. In a specific aspect, the PD-i binding antagonist inhibits the binding of PD-i to PD-Li and/or PD-L2. For example, PD-i binding antagonists include anti-PD-i antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-i with PD-Li and/or PD-L2. In one embodiment, a PD-i binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-i so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-i binding antagonist is an anti-PD- 1 antibody. In a specific aspect, a PD-i binding antagonist is MDX-1106 (nivolumab) described herein. In another specific aspect, a PD-i binding antagonist is MK- 3475 (lambrolizumab) described herein. In another specific aspect, a PD-i binding

28

SUBSTITUTE SHEET RULE 26 antagonist is CT-oi 1 (pidilizumab) described herein. In another specific aspect, a PD-i binding antagonist is AMP-224 described herein.

The term "PD-Li binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- Li with either one or more of its binding partners, such as PD-i, B7-1. In some embodiments, a PD-Li binding antagonist is a molecule that inhibits the binding of PD- Li to its binding partners. In a specific aspect, the PD-Li binding antagonist inhibits binding of PD-Li to PD-i and/or B7-1. In some embodiments, the PD-Li binding antagonists include anti-PD-Li antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-Li with one or more of its binding partners, such as PD-i, B7-1. In one embodiment, a PD-Li binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signalling through PD-Li so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-Li binding antagonist is an anti-PD-Li antibody. In a specific aspect, an anti-PD-Li antibody is YW243.55. S70 described herein. In another specific aspect, an anti- PD-Li antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD- Li antibody is MPDL3280A described herein. In still another specific aspect, an anti-PD-Li antibody is MEDI4736 described herein.

The term "PD-L2 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- L2 with either one or more of its binding partners, such as PD-i. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-i. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-i. In one embodiment, a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less

29

SUBSTITUTE SHEET RULE 26 dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.

A "fixed" or "flat" dose of a therapeutic agent herein refers to a dose that is

administered to a human patient without regard for the weight (WT) or body surface area (BSA) of the patient. The fixed or flat dose is therefore not provided as a mg/kg dose or a mg/m ² dose, but rather as an absolute amount of the therapeutic agent.

A "loading" dose herein generally comprises an initial dose of a therapeutic agent administered to a patient, and is followed by one or more maintenance dose(s) thereof. Generally, a single loading dose is administered, but multiple loading doses are contemplated herein. Usually, the amount of loading dose(s) administered exceeds the amount of the maintenance dose(s) administered and/or the loading dose(s) are administered more frequently than the maintenance dose(s), so as to achieve the desired steady-state concentration of the therapeutic agent earlier than can be achieved with the maintenance dose(s).

A "maintenance" dose herein refers to one or more doses of a therapeutic agent administered to the patient over a treatment period. Usually, the maintenance doses are administered at spaced treatment intervals, such as approximately every week, approximately every 2 weeks, approximately every 3 weeks, or approximately every 4 weeks, preferably every 3 weeks.

"Infusion" or "infusing" refers to the introduction of a drug-containing solution into the body through a vein for therapeutic purposes. Generally, this is achieved via an intravenous (IV) bag.

An "intravenous bag" or "IV bag" is a bag that can hold a solution which can be administered via the vein of a patient. In one embodiment, the solution is a saline solution (e.g. about 0.9% or about 0.45% NaCl). Optionally, the IV bag is formed from polyolefin or polyvinal chloride.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g.,

30

SUBSTITUTE SHEET RULE 26 Kindt et al. Kuby Immunology, 6 ^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.

Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of

complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." A "free cysteine amino acid" refers to a cysteine amino acid residue which has been engineered into a parent antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge.

"Drug", "drug substance", "active pharmaceutical ingredient", and the like, refer to a compound (e.g., compounds of Formula (I) and compounds specifically named above) that may be used for treating a subject in need of treatment.

"Excipient" refers to any substance that may influence the bioavailability of a drug, but is otherwise pharmacologically inactive.

The term "or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof means that pharmaceutically acceptable salt, solvate, tautomeric, stereoisomeric forms of the shown structure are also included. Mixtures thereof means that mixture of these forms may be present, for example, the compounds of the invention may include both a tautomeric form and a pharmaceutically acceptable salt.

"Pharmaceutically acceptable" substances refers to those substances which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like,

commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

31

SUBSTITUTE SHEET RULE 26 "Pharmaceutical composition" refers to the combination of one or more drug substances and one or more excipients.

The term "subject" as used herein refers to a human or non-human mammal.

Examples of non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.

"Therapeutically effective amount" of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect. The therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things. "Treating" refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition. "Treatment" refers to the act of "treating", as defined immediately above.

As used herein the term "comprising" means "including at least in part of and is meant to be inclusive or open ended. When interpreting each statement in this specification that includes the term "comprising", features, elements and/or steps other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

The term "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. When the phrase "consisting essentially of appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause.

The term "consisting of excludes any element, step, or ingredient not specified in the claim; "consisting of defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consists of appears in a clause of the body of a claim, rather than immediately

32

SUBSTITUTE SHEET RULE 26 following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. It should be understood that while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment is also described using "consisting essentially of or "consisting of language.

Provisos

The compound of formula (I) or formula (II) is a compound according to proviso (a); (b); (c); (d); (e) or (f). Suitably, the compound of formula (I) or formula (II) is a compound according to proviso (a); (b); (c); (d) or (e). Suitably, the compound of formula (I) or formula (II) is a compound according to proviso (a); (b); (c) or (d).

Suitably, the compound of formula (I) or formula (II) is a compound according to proviso (a); (b) or (c). More suitably, the compound of formula (I) or formula (II) is a compound according to proviso (a) or (b). Most suitably, the compound of formula (I) or formula (II) is a compound according to proviso (a).

Proviso (a)

Suitably, the compound of formula (I) or formula (II) is (a) a compound of formula (I), wherein at least one of R ₃ or R _i2 is an optionally substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - ₉ heteroaryl or an optionally substituted phenyl. Suitably, such a compound may be represented as a compound having the structure of formula (Ai) or (A

wherein Ar ₄ is an optionally substituted an optionally substituted C ₅ -6 heterocyclyl, G heteroaryl or an optionally substituted phenyl, optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

33

SUBSTITUTE SHEET RULE 26 Suitably, (Ai) or (A2) has a single double bond in the C-ring between R ₄ and Ar ₄ . More suitably, (Ai) or (A2) has a single double bond in the C-ring between Ar ₄ and R ₂ .

In an aspect, suitably, both R ₃ and R _i2 are selected from an optionally substituted C ₅ _6 heterocyclyl, an optionally substituted C ₅ - ₉ heteroaryl or an optionally substituted phenyl. Suitably, such a compound may be represented as a compound having the structure of formula (A3):

wherein Ar ₄ and Ar ₅ are independently selected from an optionally substituted C ₅ -6 heterocyclyl, optionally substituted C ₅ _ _g heteroaryl or optionally substituted phenyl, optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, (A3) contains a single double bond in each C-ring and those double bonds are between R,, and Ar ₄ ; and between R _i3 and Ar ₅ .

Suitably, the compound of formula (I) or formula (II) has the structure of formula (A4):

More suitably, the compound of formula (I) or formula (II), or formula (A4) has the structure of formula (A5):

More suitably, the compound of formula (I) or formula (II) has the structure of formula (A6):

34

SUBSTITUTE SHEET RULE 26

wherein R _2g , R x ₃ g ₀ o,j R ^χ ₃ 3i ₁ ,; R ⁱ 332 ₂ ,j R ^x ₃ 3 ₃ 3 a ^l n ^{1 1} d ^l R ^x ₃ 3 ₄ 4 are each independently selected from H and R _;

Hence :, R ₂₉ , R ₃ o, R ₃ i, R ₃ 2, R ₃₃ and R ₃₄ represent the optional substituent groups

The above structures are drawn showing the bonds for groups R ₂₉ and R ₃₀ going into the middle of the phenyl ring on the left-hand side of the molecule (A6). This indicates that these groups are attached to this phenyl ring without specifying the exact positions of the R29 and R ₃₀ groups. Hence, these groups may be present on any of the four available position of phenyl ring on the left-hand side that would meet the valence requirements (i.e. the four positons for which no bound is shown).

More suitably, the compound of formula (I), formula (II), or formula (A6) has the structure of formula (A7) or formula (A8):

wherein R _2Q , R ₃₀ and R ₃ i are each independently selected from H and R ₂₀ .

More suitably, the compound of formula (I), formula (II), formula (A6) or formula (A7) has the structure of formula (A9) or formula (A10):

35

SUBSTITUTE SHEET RULE 26

(Aio).

In another aspect, suitably, either (i) R ₃ is an optionally substituted an optionally substituted C ₅ -6 heterocyclyl, C ₅ - _g heteroaryl or an optionally substituted phenyl, and Rn and Ri ₂ , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or a 5- or 6-membered heteroaryl ring; or (ii) R _i2 is an optionally substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - ₉ heteroaryl or an optionally substituted phenyl, and R ₂ and R ₃ , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or a 5- or 6- membered heteroaryl ring. The optionally substituted 6-membered aryl, or a 5- or 6- membered heteroaryl rings are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably, such a compound may be represented as a compound having the structure of formula (An), (A12), (A13) or (A14):

36

SUBSTITUTE SHEET RULE 26

wherein one of Z _A , Z _B , Zc and Z _D is CH, one of Z _A , Z _B , Zc and Z _D is C-R ₃₅ , one of Z _A , Z _B , Zc and Z _D is N or C-R ₃ 6, and the remaining one of Z _A , Z _B , Zc and Z _D is N or C-R ₃₇ ; R ₃₅ , R ₃ 6 and R ₃₇ are independently selected from H and R ₂₀ ; and

Zi is selected from NH, N(Ci- ₆ alkyl), S and O; and Z ₂ and Z ₃ are independently selected from N, CH, C-R ₂₀ ; or Zi and Z ₂ are independently selected from N, CH, C-R ₂₀ ; and Z ₃ is selected from NH, N(Ci- ₆ alkyl), S and O.

In this aspect, where there is more than one R ₂₀ group present, each R ₂₀ group is independently selected. Again, Ar ₄ and Ar ₅ are independently selected from an optionally substituted C ₅ -6 heterocyclyl, optionally substituted C ₅ - ₉ heteroaryl or optionally substituted phenyl, wherein these groups are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Thus, for (An) and (A12), the 6-membered D-ring containing groups Z _i; Za and Zg is either an optionally substituted heterocyclic ring (i.e. is selected from an optionally substituted pyridine, pyridazine, pyrimidine or a pyrazine ring) or is an optionally substituted phenyl ring. For (A13) and (A14), the 5-membered D-ring containing groups Zi, Z ₂ and Z ₃ is a heterocyclic ring.

More suitably, the compound of formula (I), (II), (Αι), (A2), (An) or (A12) has the structure of formula (A15) or (A16):

37

SUBSTITUTE SHEET RULE 26

wherein R _2g , R ₃₀ , R31, R ₃₅ , R ₃ 6 and R ₃₇ are each independently selected from H and R ₂

More suitably, the compound of formula (I), (II), (Ai), (A2), (An), (A12) (A15) has the structure of formula (A17) or (A18):

In another aspect, suitably the compound of formula (I) or formula (II), has the structure of formula (Ai), or formula (A2), wherein Ri, R ₂ , R ₃ , R4, Ri ₀ , Rn, R12 and R _ig are independently selected from H and R ₂₀ ; wherein Ar ₄ is an optionally substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - _g heteroaryl or an optionally substituted phenyl, optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, the compound of formula (I), formula (II), formula (Ai) or formula (A2), the structure of formula (A19), or formula (A20):

38

SUBSTITUTE SHEET RULE 26

wherein Ri, R ₂ , R ₃ , R4, Ri ₀ , Rn, R12 and R _i3 are independently selected from H and R ₂₀ .

Suitably, the compound of formula (I), (II), (Ai), (A2), (A3) or (A6) is

(A21).

In some aspects, suitably, the compound of formula (I) or formula (II) is a compound of formula (Ai), (A2), (A3), (An), (A12), (A13) or (A14); suitably, a compound of formula (Ai), (A2), (A3), (An) or (A12). More suitably, the compound of formula (I) or formula (II) is a compound of formula (Ai), (A2) or (A3).

Proviso (b)

Suitably, the compound of formula (I) or formula (II) is (b) a compound of formula (I) wherein both R ₂ and R ₃ , and Rn and R _i2 , together with the carbon atoms to which they are attached form an optionally substituted 5-membered cyclic, heterocyclic, or heteroaryl ring. Suitably, the optionally substituted 5-membered cyclic, heterocyclic, or heteroaryl rings are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably, such a compound may be represented as a compound having th

39

SUBSTITUTE SHEET RULE 26

wherein Z ₄ is selected from NH, N(Ci _-6 alkyl), S and O; and Z ₅ and Z ₆ are independently selected from N, CH, C-R ₂₀ ; or Z ₄ and Z ₅ are independently selected from N, CH, C-R ₂₀ , and Z ₆ is selected from NH, N(Ci _-6 alkyl), S and O; and

Z' ₄ is selected from NH, N(Ci _-6 alkyl), S and O; and Z' ₅ and Z' ₆ are independently

selected from N, CH, C-R ₂₀ ; or Z' ₄ and Z' ₅ are independently selected from N, CH, C-R20, and Z' ₆ is selected from NH, N(Ci- ₆ alkyl), S and O.

Zy, Zs, Ζ'ό, Z' ₇ , Z's and Z' _g are each independently selected from NH, N(Ci _-6 alkyl), S, O and CHR ₃ 8 wherein each R ₃ s is independently selected from H and R ₂₀ .

Suitably, the compound of formula (I), formula (II) is a compound having the structure of formula (Bi).

Suitably, the compound of formula (I), or formula (II) is a compound having the structure of formula (Bi) or formula (B2), wherein Z ₄ = Z' ₄ , Z ₅ = Z' ₅ and Z ₆ = Z' ₆ ; and Z ₇ = Z' ₇ , Zs = Z's and Z ₉ = Z' ₉ . Hence, suitably, the D-rings are the same.

More suitably, both R ₂ and R ₃ , and Rn and R _i2 , together with the carbon atoms to which they are attached form an optionally substituted 5-membered heteroaryl ring.

More suitably, the compound of formula (I), formula (II) or formula (Bi) is a compound having the structure of formula (B3) or formula (B4):

SUBSTITUTE SHEET RULE 26 Proviso fc)

Suitably, the compound of formula (I) or formula (II) is (c) a compound of formula (II), wherein R _i2 is an optionally substituted C ₅ _6 heterocyclyl, an optionally substituted C ₅ _ _g heteroaryl or an optionally substituted phenyl and Ri, R ₂ , R ₃ , R4, Rio, Ru and R _i3 are independently selected from H and R _ig . Suitably, such a compound may be represented as a compound having the structure of formula (Ci):

wherein Ar ₄ is selected from optionally substituted C ₅ -6 heterocyclyl, optionally substituted C ₅ _ _g heteroaryl or optionally substituted phenyl, optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, the compound of formula (I), formula (II), or formula (Ci) is a compound having

(C2) wherein R ₂₂ , R ₃₃ and R ₃₄ are each independently selected from H and R ₂₀ .

Suitably, the compound of formula (I), formula (II), formula (Ci) or formula (C2) is a compound having the structure of formula (C3):

(C ₃ ).

Proviso (d)

41

SUBSTITUTE SHEET RULE 26 Suitably, the compound of formula (I) or formula (II) is (d) a compound of formula (II), wherein Rn and R _i2 , or R _i2 and R _i3 , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring, and Ri, R ₂ , R ₃ , R4, are independently selected from H and R _ig . Suitably, the 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl rings are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably, such a compound may be represented as a compound having the structure of formula (Di), (D2), (D3) or (D4):

wherein w is o or 1, and

when w is 1, then Di, D ₂ , D ₃ and D ₄ are independently selected from N and C-R _3g ;with the proviso that no more than two of Di, D ₂ , D ₃ and D ₄ are N;

when w is o, then D ₂ is selected from NH, N(Ci- ₆ alkyl), S and O; and D ₃ and D ₄ are independently selected from N, C-R ₃₉ ; or D ₂ and D ₃ are independently selected from N, C-R ₃₉ , and D ₄ is selected from NH, N(Ci- ₆ alkyl), S and O; and

42

SUBSTITUTE SHEET RULE 26 when w is l, then D\, D' ₂ , D' ₃ and D' ₄ are independently selected from S, O, N-R _3g and CH-R ₃₉ ;with the proviso that l, 2, 3, or 4 of D\, D' ₂ , D' ₃ and D' ₄ are CH-R ₃₉ ;

D'i, D' ₂ , D' ₃ and D' ₄ are independently selected from S, O, NH, N(d- ₆ alkyl) and CH-R ₃₉ ; and

each R _3g is independently selected from H and R ₂₀ .

The proviso that no more than two of Di, D ₂ , D ₃ and D ₄ are N means that o, 1 or 2 of Di, D ₂ , D ₃ and D ₄ are N.

Suitably, the compound of formula (I) or formula (II) is a compound of formula (Di) or (D2).

Suitably, the compound of formula (I), (II), (Di) or (D2) is a compound of formula (D5)

Proviso (e)

Suitably, the compound of formula (I) or formula (II) is (e) a compound of formula (II), wherein m is o; R _i2 is an optionally substituted C ₅ _6 heterocyclyl, an optionally substituted C ₅ _ _g heteroaryl or an optionally substituted phenyl; and R ₂ and R ₃ , or R ₃ and R ₄ , together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or an optionally substituteds- or 6-membered cyclic, heterocyclic, or an optionally substituted heteroaryl ring. Suitably, each optionally substituted ring maybe optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably, such a compound may be represented as a compound having the structure of formula (El), (E2), (E3) or (E4):

43

SUBSTITUTE SHEET RULE 26

wherein w is o or l, and

when w is l, then Di, D ₂ , D ₃ and D ₄ are independently selected from N and C-R ₃₉ ;with the proviso that no more than two of Di, D ₂ , D ₃ and D ₄ are N;

when w is o, then D ₂ is selected from NH, N(Ci- ₆ alkyl), S and O; and D ₃ and D ₄ are independently selected from N, C-R ₃₉ ; or D ₂ and D ₃ are independently selected from N, C-R ₃₉ , and D ₄ is selected from NH, N(Ci- ₆ alkyl), S and O; and

when w is l, then D\, D' ₂ , D' ₃ and D' ₄ are independently selected from S, O, N-R _3g and

CH-R ₃₉ ;with the proviso that l, 2, 3, or 4 of D\, D' ₂ , D' ₃ and D' ₄ are CH-R ₃₉ ;

D'i, D' ₂ , D' ₃ and D' ₄ are independently selected from S, O, NH, N(Ci- ₆ alkyl) and CH-R ₃₉ ; each R ₃₉ is independently selected from H and R ₂₀ ; and

Ar ₄ is selected from optionally substituted C ₅ _6 heterocyclyl, optionally substituted C ₅ _ ₉ heteroaryl or optionally substituted phenyl, optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

44

SUBSTITUTE SHEET RULE 26 The proviso that no more than two of Di, D ₂ , D ₃ and D ₄ are N means that o, l or 2 of D D ₂ , D ₃ and D ₄ are N.

Suitably, the compound of formula (I) or formula (II) is a compound of formula (El), (E2), (E3) or (E4), wherein Ar ₄ is an optionally substituted phenyl.

Suitably, the compound of formula (I), (II), (E2) or (E4) is a compound having the formula (

More suitably, the compound of formula (I), (II), (E2), (E4), (E5) or (E6) is a compound having the formula (E7) or (E8):

wherein R ₃₂ , R ₃₃ and R ₃₄ are each independently selected from H and R ₂₀ .

More suitably, the compound according to proviso (e) is a compound of formula (II), wherein m is o; R _i2 is an optionally substituted C ₅ -6 heterocyclyl, an optionally

45

SUBSTITUTE SHEET RULE 26 substituted C ₅ - _g heteroaryl or an optionally substituted phenyl; and R ₃ and R4, together with the carbon atoms to which they are attached form an optionally substituted 6- membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring. Alternatively, more suitably, the compound according to proviso (e) is a compound of formula (II), wherein m is o; R _i2 is an optionally substituted C ₅ -6 heterocyclyl, an optionally substituted C ₅ - ₉ heteroaryl or an optionally substituted phenyl; and R ₂ and R ₃ , or R ₃ and R ₄ , together with the carbon atoms to which they are attached form an optionally substituted 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring.

Alternatively, more suitably, where the compound is a compound according to proviso (e) Yi is selected from -(CH ₂ ) _1-5 -, -C(0)-NH-, -NH- or -S(0) ₀ - ₂ -.

Proviso (f)

Suitably, the compound of formula (I) or formula (II) is (f) a compound of formula (II), wherein m is 1; R ₁₂ is =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ or and R _1} R ₂ , R ₃ , R4, R ₁₀ R11, Ri ₂ and R _i3 are independently selected from H and R _ig . Suitably, such a compound may be represented as a compound having the structure of formula (Fi):

wherein R is H, -(CH ₂ ) _S -CH ₃ or (CH ₂ ) _S -R ₂ i.

Suitably, the compound of formula (I), (II) or (Fi) is a compound having the structure

Other Structures

In some aspects, suitably, the compound of formula (I) or formula (II) is a compound having the structure of formula (Oi):

46

SUBSTITUTE SHEET RULE 26

In some aspects, suitably, the compound of formula (I) or formula (II) is a compound having the structure of formula (O3):

In some aspects, the compound of formula (I) or formula (II) is a compound according to proviso (c) or (e) having the structure of formula (O4):

wherein R ₃₂ , R ₃₃ and R ₃₄ are each independently selected from H and R ₂₀ -

Optional double bonds in the C-ring of the PBD or PDD moieties

The compounds of formula (I) or formula (II) comprise PBD or PDD moieties with C- rings containing dotted lines — that indicate the optional presence of one or more double bonds. Thus, for example, for compounds of formula (I) or for compounds of formula (II) when m is 1, and an optional double bond is present in the C-ring, the left hand side moiety may have one or more double bonds and may be selected from:

47

SUBSTITUTE SHEET RULE 26

Suitably, the compound has a double bond and the left hand side moiety is selected from (DB2) or (DB3).

Suitably, where the compounds have an optional double bond present, the left hand side moiety is (DB3).

More suitably, where the compounds have an optional double bond present, the left hand side moiety is (DB2).

Similarly, for compounds of formula (I) when an optional double bond is present in the -ring, the right hand side moiety may have one or more double bonds and may be

Suitably, the compound has a double bond and the left hand side moiety is selected from (DB6) or (DB7).

48

SUBSTITUTE SHEET RULE 26 Suitably, where the compounds have an optional double bond present, the left hand side moiety is (DB7).

More suitably, where the compounds of formula (I) have an optional double bond present, the right hand side moiety is (DB6).

For compounds of formula (II) when an optional double bond is present in the C-ring, the right hand side moiety may have a double bond as shown below:

Suitably, where the compounds of formula (II) have an optional double bond present, the right hand side moiety is (DB10).

More suitably, where the compounds of formula (II) have an optional double bond present, the right hand side moiety is (DB9).

In some aspects, optional double bonds are absent from the compounds of formula (I) or formula (II).

For the options where any of Ri, R ₂ , R ₃ and R ₄ are each independently selected from

=CH ₂ , =CH-(CH ₂ )s-CH ₃ , and =0, the carbon of the C-ring to which it is attached cannot have an optional double bond in order for the valence requirements of the molecule to be met. For example, if R4 is =CH ₂ then there must be a single bond between the carbons to which R4 and R ₃ are attached, this may be represented as follows (LHMi):

In the aspects where one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered

49

SUBSTITUTE SHEET RULE 26 cyclic, heterocyclic, or heteroaryl ring optionally substituted with up to three optional substituent groups, groups then the left hand moiety contains a further fused ring [not drawn in formula (I) or (II)]. In these aspects, then the remaining groups (from R _l5 R ₂ , R ₃ and R4) that do not form the further fused ring are each independently selected from the normal specified list of groups, i.e. from H and R _ig . For example, where Ri and R ₂ together with the carbon atoms to which they are attached form a 6-membered aryl ring the structure of the left hand moiety can be shown as follows (LMH2):

(LMH2).

In LMH2 groups R ₃ and R4 do not form the further fused ring and so are each independently selected from the normal specified list of groups for R ₃ and R4, i.e. from H and Ri ₉ . In addition, the H groups shown on the further fused ring of (LMH2) may be substituted with up to three independently selected optional R ₂₀ groups.

Suitably, Ri, R ₂ , R ₃ and R ₄ are each independently selected from H, R ₂₀ , R ₂ i, =CH ₂ , =CH-(CH ₂ )s-CH ₃ , (CH ₂ ) _s -0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , O- (CH ₂ ) _t -NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _S - C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ ;

or one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3

independently selected optional R ₂₀ groups.

Suitably, Ri, R ₂ , R ₃ and R ₄ are each independently selected from H, R ₂₀ , R ₂ i, (CH ₂ ) _S - 0R ₂₁ , (CH ₂ )s-C0 ₂ R ₂₁ , (CH ₂ )s-NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , (CH ₂ ) _s -C(0)R ₂₁ and (CH ₂ ) _S - C(0)NR ₂₁ R ₂₃ ;

or one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3

independently selected optional R ₂₀ groups.

50

SUBSTITUTE SHEET RULE 26 Suitably, where one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4 together with the carbon atoms to which they are attached form an optionally substituted 5- or 6-membered heterocyclic or heteroaryl ring the heterocyclic or heteroaryl ring comprises one nitrogen atom. Suitably, the optionally substituted 5- or 6-membered heterocyclic or heteroaryl ring is a pyrrolyl, N-methylpyrrolyl or a pyridyl ring. An N-methylpyrrolyl is an example of where the 5- or 6-membered heterocyclic or heteroaryl ring is optionally substituted with a methyl group.

Suitably, where one of Ri and R ₂ , R ₂ and R ₃ , or R ₃ and R4 together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or an optionally substituted 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring, the ring is selected from optionally substituted phenyl, pyrrolyl, N-methylpyrrolyl and pyridyl rings. In some aspects, suitably Ri, R ₂ , R ₃ and R ₄ are each independently selected from H and R ₂ o

In another aspect, suitably R ₂ and R ₃ together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably R ₂ and R ₃ together with the carbon atoms to which they are attached form optionally substituted phenyl, pyrrolyl, N-methylpyrrolyl and pyridyl rings.

Suitably 1, 2, 3 or 4 of Ri, R ₂ , R ₃ and R ₄ are H.

More suitably, Ri is H.

More suitably, R ₂ is H. More suitably, R ₃ is selected from H, optionally substituted C ₅ _ _g heteroaryl and optionally substituted phenyl; wherein the heteroaryl and phenyl groups are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, R ₃ is H or optionally substituted phenyl.

In some aspects, suitably, R4 is H.

51

SUBSTITUTE SHEET RULE 26 and Re

Suitably, R ₅ and R ₆ are (i), wherein R ₅ is selected from H, OH and OCH ₃ or OCH ₂ CH ₃ ; and R6 is selected from H, S0 ₃ H, nitrogen protecting groups, OH, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ )j-C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , (CH ₂ )j-C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k - NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH- CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ and -L ₂ -R ₂₈ ; (ii) R ₅ is oxo or H, and R6 is H or Ci-6 alkyl; or (iii) R ₅ and R6 together form a double bond. More suitably, R ₅ and R ₆ are (i), wherein R ₅ is selected from H, OH and OCH ₃ or OCH ₂ CH ₃ ; and R ₆ is selected from H, S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH ₂ , (CH ₂ ) _r NH ₂ and -L ₂ -R ₂₈ ; (ii) R ₅ is oxo or H, and R6 is H or Ci _-6 alkyl; or (iii) R ₅ and R ₆ together form a double bond. Suitably, R ₅ and R ₆ are (i), wherein R ₅ is selected from H, OH and OCH ₃ or OCH ₂ CH ₃ ; and R ₆ is selected from H, S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ C(=0)-0-(CH ₂ ) _k -NH ₂ and -L ₂ -R ₂₈ ; (ii) R ₅ is oxo or H, and R ₆ is H or d- ₆ alkyl; or (iii) R ₅ and R ₆ together form a double bond. More suitably, either:

(1) R ₅ is H or OH, and R ₆ is S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ or -L ₂ -R ₂₈ ;

(2) R ₅ is OH, OCH ₃ or OCH ₂ CH ₃ , and Re is H;

(3) R ₅ is oxo or H, and R ₆ is H or Ci _-6 alkyl; or

(4) R ₅ and R ₆ together form a double bond.

In some aspects, R ₅ and R ₆ are (ii) wherein R ₅ is oxo or H, and R ₆ is H or Ci _-6 alkyl in such aspects then the left hand moiety of the compound of formula (I) will not alkylate DNA. In such aspects, the options for R _i4 and R _i5 are limited to options (iv) and (vi) which are those that ensure that the right hand moiety of the compound of formula (I) does alkylate with DNA. Examples of compounds such of formula (I) are shown below:

52

SUBSTITUTE SHEET RULE 26

Most suitably, R ₅ and R ₆ are (iii), wherein R ₅ and R ₆ together form a double bond.

In some aspects, R ₆ is S0 ₃ H and the compound of formula (I) is a salt thereof. Suitably, in this aspect, R ₆ is S0 ₃ H and the compound of formula (I) is an alkali metal salt thereof (AM) ⁺ ; hence, in this aspect, R ₆ maybe written as S0 ₃ ^~ (AM) ⁺ . Suitably, R ₆ is S0 ₃ H and the compound of formula (I) is an alkali metal salt thereof chosen from Li ⁺ , Na ⁺ and K ⁺ . More suitably, R ₆ is S0 ₃ H and the compound of formula (I) is a Na ⁺ salt thereof; hence, in this aspect, R ₆ may be written as S0 ₃ ^~ Na ⁺ .

Suitably, R ₇ , R ₉ , R16 and R ₁ 8 are independently selected from H, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , O- (CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ and -L ₂ -R ₂₈ .

Suitably, R ₇ , R ₉ , R16 and R ₁ 8 are independently selected from H, OH, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , (CH ₂ ) _r NH ₂ and -L ₂ -R ₂₈ .

More suitably, R ₇ , R ₉ , R _i6 and Ris are independently selected from H, OH, methyl, ethyl, OCH ₃ and OCH ₂ CH ₃ .

Most suitably, R ₇ is H.

Most suitably, R ₉ is H.

Most suitably, R16 is H. Most suitably, R18 is H. Rs and

Suitably, Rs and R _i7 are independently selected from H, SH, SCH ₃ , SCH ₂ Ph, (CH ₂ ) 0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ and - -R^.

53

SUBSTITUTE SHEET RULE 26 Suitably, Re and R _i7 are independently selected from H, OH, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , (CH ₂ )j-NH ₂ and - - R ₂₈ .

More suitably, Re and R _i7 are independently selected from OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph.

More suitably, Re and R _i7 are OCH ₃ .

Suitably, R _i0 , Rn, Ri ₂ and R _i3 are each independently selected from H, R ₂₀ , R _2i , =CH ₂ , =CH-(CH ₂ ) _s -CH ₃ , =CH-(CH ₂ ) _s -R ₂₁ , (CH ₂ ) _s -0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , O- (CH ₂ ) _t -NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _S - C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ ;

or one of R _i0 and Rn, Rn and R _i2 , or R _i2 and R _i3 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, R _i0 , Rn, Ri ₂ and R _i3 are each independently selected from H, R ₂₀ , R _2i , =CH ₂ , =CH-(CH ₂ ) _s -CH ₃ , =CH-(CH ₂ ) _s -R ₂₁ , (CH ₂ ) _s -0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , NH- C(0)-R ₂₁ , (CH ₂ )s-C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ ;

or one of R _i0 and Rn, Rn and R _i2 , or R _i2 and R _i3 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

In some aspects, suitably R _i0 , Rn, Ri ₂ and R _i3 are each independently selected from H and R ₂₀ .

Suitably, where one of R _i0 and Rn, Rn and R _i2 , or R _i2 and R _i3 together with the carbon atoms to which they are attached form an optionally substituted 5- or 6-membered heterocyclic or heteroaryl ring the heterocyclic or heteroaryl ring comprises one nitrogen atom. Suitably, the optionally substituted 5- or 6-membered heterocyclic or heteroaryl ring is a pyrrolyl, N-methylpyrrolyl or a pyridyl ring. An N-methylpyrrolyl is

54

SUBSTITUTE SHEET RULE 26 an example of where the 5- or 6-membered heterocyclic or heteroaryl ring is optionally substituted with a methyl group.

Suitably, where one of Ri ₀ and Rn, Rn and R _i2 , or R _i2 and R _i3 together with the carbon atoms to which they are attached form an optionally substituted 6-membered aryl, or an optionally substituted 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring, the ring is an optionally substituted phenyl, pyrrolyl, N-methylpyrrolyl or pyridyl ring.

In another aspect, suitably Rn and R _i2 together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. Suitably Rn and R _i2 together with the carbon atoms to which they are attached form an optionally substituted phenyl, pyrrolyl, N-methylpyrrolyl or pyridyl ring. Suitably 1, 2, 3 or 4 of R _i0 , Rn, Ri ₂ and R _i3 are H.

More suitably, Ri ₀ is H.

Suitably, Rn is H.

More suitably, R ₁₂ is selected from H, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , =CH-(CH ₂ ) _S -R ₂₁ , optionally substituted C ₅ _ _g heteroaryl and optionally substituted phenyl; wherein the heteroaryl and phenyl groups are optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, R _i2 is selected from H, , =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , and optionally substituted phenyl.

More suitably, R _i3 is H.

Suitably, R _i4 and R _i5 are (iv), wherein R _i4 is selected from H, OH and OCH ₃ or

OCH ₂ CH ₃ ; and R _i5 is selected from H, S0 ₃ H, nitrogen protecting groups, OH, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ ) C0 ₂ H, (CH ₂ ) C0 ₂ CH ₃ , (CH ₂ ) C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ ,

(CH ₂ )j-NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ - (CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ and -

55

SUBSTITUTE SHEET RULE 26 L ₂ -R ₂ 8; (v) Ri ₄ is oxo or H, and R _i5 is H or Ci _-6 alkyl; or (vi) R _i4 and R _i5 together form a double bond.

More suitably, R _i4 and R _i5 are (iv), wherein R _i4 is selected from H, OH and OCH ₃ or OCH ₂ CH ₃ ; and R ₁₅ is selected from H, S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH ₂ , (CH ₂ ) NH ₂ and -L ₂ -R ₂₈ ; (v) R ₁₄ is oxo or H, and R _i5 is H or Ci _-6 alkyl; or (vi)R _i4 and R _i5 together form a double bond.

Suitably, Ri ₄ and R _i5 are (iv), wherein R _i4 is selected from H, OH and OCH ₃ or

OCH ₂ CH ₃ ; and R ₁₅ is selected from H, S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ C(=0)-0-(CH ₂ ) _k -NH ₂ and -L ₂ -R ₂₈ ; (v) R ₁₄ is oxo or H, and R15 is H or Ci-6 alkyl; or (vi) R _i4 and R6 together form a double bond.

More suitably, either:

(1) R ₁₄ is H or OH, and R ₁₅ is S0 ₃ H, OH, OCH ₃ , OCH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ or -La-Rae;

(2) R ₁₄ is OH, OCH ₃ or OCH ₂ CH ₃ , and R ₁₅ is H;

(3) R14 IS oxo or H, and R _i5 is H or Ci _-6 alkyl; or

(4) Ri ₄ and R _i5 together form a double bond. In some aspects, R _i4 and R _i5 are (v) wherein R _i4 is oxo or H, and R _i5 is H or Ci _-6 alkyl; in such aspects then the right hand moiety of the compound of formula (I) will not alkylate DNA. In such aspects, the options for R ₅ and R ₆ are limited to options (i) and (iii) which are those that ensure that the left hand moiety of the compound of formula (I) does alkylate with DNA. Examples of compounds such of formula (I) are shown below:

56

SUBSTITUTE SHEET RULE 26 Most suitably, R _i4 and R _i5 are (vi), wherein R _i4 and R _i5 together form a double bond.

In some aspects, R _i5 is S0 ₃ H and the compound of formula (I) is a salt thereof.

Suitably, in this aspect, R _i5 is S0 ₃ H and the compound of formula (I) is an alkali metal salt thereof (AM) ⁺ ; hence, in this aspect, R _i5 may be written as S0 ₃ ^~ (AM) ⁺ . Suitably, Ri is S0 ₃ H and the compound of formula (I) is an alkali metal salt thereof chosen from Li ⁺ , Na ⁺ and K ⁺ . More suitably, R _i5 is S0 ₃ H and the compound of formula (I) is a Na ⁺ salt thereof; hence, in this aspect, Ri ₅ may be written as S0 ₃ Na ⁺ .

Suitably, the group is substituted with 1, 2 or 3 of the optional substituents. Where a group may be "optionally substituted with one or two optional substituents", this means that the group may be substituted with o, 1 or 2 of the optional substituents.

Eoa

Suitably, each R _ig is independently selected from R ₂₀ , R21, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , (CH ₂ )s-0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , 0-(CH ₂ ) _t -NR ₂₁ R ₂₃ , NH- C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _s -C(0)R ₂₁ and (CH ₂ ) _S - C(0)NR ₂₁ R ₂₃ .

Suitably, each R _ig is independently selected from R ₂₀ , R ₂ i, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , (CH ₂ )s-0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , (CH ₂ ) _S - C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ .

In one aspect, suitably, one R _ig is R _2i ; and each of the remaining R _ig groups are independently selected from R ₂₀ , =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , (CH ₂ ) _S - 0R ₂₁ , (CH ₂ )s-C0 ₂ R ₂₁ , (CH ₂ )s-NR ₂₁ R ₂₃ , 0-(CH ₂ ) _t -NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH- C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _s -C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ .

In another aspect, suitably, two R _ig groups are independently selected R _2i groups; and each of the remaining R _ig groups are independently selected from R ₂₀ , =CH ₂ , =CH- (CH ₂ )s-CH ₃ , (CH ₂ ) _s -0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , 0-(CH ₂ ) _t - NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -NH-C(0)-R ₂₁ , 0-(CH ₂ ) _t -C(0)-NH-R ₂₁ , (CH ₂ ) _s -C(0)R ₂ and (CH ₂ )s-C(0)NR ₂₁ R ₂₃ .

Suitably, each R _ig is independently selected from R ₂₀ , R ₂ i, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , (CH ₂ )s-0R ₂₁ , (CH ₂ ) _s -C0 ₂ R ₂₁ , (CH ₂ ) _S -NR ₂₁ R ₂₃ , NH-C(0)-R ₂₁ , (CH ₂ ) _S - C(0)R ₂₁ and (CH ₂ ) _s -C(0)NR ₂₁ R ₂₃ .

57

SUBSTITUTE SHEET RULE 26 More suitably, each R _ig is independently selected from R ₂₀ , =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , and CH ₂ -CH ₂ -C(0)-N(CH ₃ ) ₂ .

In some aspects, each R _ig is independently selected from H, d- ₁₂ alkyl, F, CI, Br, (CH ₂ )j- OH, Od-6 alkyl, 0CH ₂ Ph, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , and an optionally substituted C ₅ -6 heterocyclyl, optionally substituted C _5-g heteroaryl or an optionally substituted phenyl; optionally substituted with l, 2, 3 optional groups, independently selected from F, CI, Br, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , (CH ₂ )j-NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j-R ₂ 6, C(=0)-NH-(CH ₂ ) _k - C(=NH)NR ₂₆ R ₂₇ , - -R^, C ₅ -6 heterocyclyl, -S(0) ₂ -(d- ₆ alkyl), 0-(CH ₂ ) _k -0-(C ₁ - ₆ alkyl), (CH ₂ )j-S(0) ₂ -NR ₂₆ R ₂₇ , C(=NH)-0-(d- ₆ alkyl), (CH ₂ ) _k -0-(d- ₆ alkyl), CN, NCO, C(O)- NH-(CH ₂ )j-Cy, C(0)-Cy, NH-C(0)-NR ₂₆ R ₂₇ and

In some aspects, each R _ig is independently selected from H, Ci alkyl, F, CI, Br, (CH ₂ )j- OH, Od-6 alkyl, 0CH ₂ Ph, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , =CH-(CH ₂ ) _S -R ₂₁ and an optionally substituted C ₅ _6 heterocyclyl, an optionally substituted C ₅ _ _g heteroaryl or an optionally substituted phenyl; optionally substituted with 1, 2, 3 independently selected optional R ₂₀ groups; wherein each R ₂₀ group is independently selected from F, CI, Br, methyl, ethyl, OCH ₃ and OCH ₂ CH ₃ . Suitably, in this aspect R _ig is independently selected from H, Ci alkyl, F, CI, Br, (CH ₂ )j-0H, Od- ₆ alkyl, 0CH ₂ Ph, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ and

In this aspect, suitably, each R _ig is independently selected from H, methyl, ethyl, F, CI, Br, OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph.

Suitably, where an R _ig group may be optionally substituted it is substituted with 1, 2 or 3 of the optional substituents; suitably, an R _ig group is substituted with 1 or 2 of the optional substituents. More suitably, an R _ig group is substituted with 1 of the optional substituents. Alternatively, suitably an R _ig group is not substituted.

Suitably, each R ₂₀ is independently selected from F, CI, Br, (CH ₂ )j-0H, methyl, ethyl,

OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ )j-C0 ₂ R ₂₆ , 0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , C(=0)-0-(CH ₂ ) _k -

58

SUBSTITUTE SHEET RULE 26 NR ₂₆ R ₂₇ ,

NH-C ₆ H ₄ -(CH ₂ )j-R ₂₆ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NR ₂₆ R ₂₇ ,-L ₂ -R ₂₈ , S(0) ₂ -(C ₁ _ ₆ alkyl), O- (CH. -O-Cd-e alkyl), (CH ₂ ) _r S(0) ₂ -NR ₂₆ R ₂₇ ,

alkyl), CN, NCO, Cy, C(0)-NH-(CH ₂ ) _r Cy, C(0)-Cy, NH-C(0)-NR ₂₆ R ₂₇ and

Suitably, each R ₂₀ is independently selected from (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ ) _r C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , (CH ₂ )j-C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ , C(=0)-NH ₂ , N(CH ₃ )-NH ₂ , NHNH ₂ , C(=0)-NH-NH ₂ , C(=0)-NH-CH ₂ - NH ₂ , C(=0)-NH-(CH ₂ )j-NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ , -L ₂ -R ₂₈ , S(0) ₂ - CH ₃ , S(0) ₂ -CH ₂ CH ₃ , S(0) ₂ -CH(CH ₃ ) ₂ , 0-CH ₂ -0-CH ₃ , 0-CH ₂ -0-CH ₂ CH ₃ , S(0) ₂ -NH ₂ , S(0) ₂ -NHCH ₃ , S(0) ₂ -N(CH ₃ ) ₂ , C(=NH)-0-CH ₃ , C(=NH)-0-CH ₂ CH ₃ , CH ₂ -0-CH ₃ , CH ₂ - 0-CH ₂ CH ₃ , CN, NCO, Cy, C(0)-NH-Cy, C(0)-NH-CH ₂ -Cy, C(0)-Cy, NH-C(0)-NH ₂ , NH-C(0)-NH ₂ and

More suitably, each R ₂₀ is independently selected from (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH ₂ , (CH ₂ )j- NH ₂ , NH-CH ₃ , -L ₂ -R ₂₈ , S(0) ₂ -CH ₃ , S(0) ₂ -CH ₂ CH ₃ , S(0) ₂ -CH(CH ₃ ) ₂ , 0-CH ₂ -0-CH ₃ , O- CH ₂ -0-CH ₂ CH ₃ , S(0) ₂ -NHCH ₃ , S(0) ₂ -N(CH ₃ ) ₂ , C(=NH)-0-CH ₃ , C(=NH)-0-CH ₂ CH ₃ , CH ₂ -0-CH ₃ , CH ₂ -0-CH ₂ CH ₃ , Cy, C(0)-NH-Cy, C(0)-NH-CH ₂ -Cy, C(0)-Cy, NH-C(O)- NH ₂ and NH-C(0)-NH ₂ .

In some aspects, suitably, one R ₂₀ group is selected from (CH ₂ )j-C0 ₂ R ₂ 6, 0-(CH ₂ ) _k - NR ₂₆ R ₂₇ , C(=0)-0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , (CH ₂ ) _r NR ₂₆ R ₂₇ , NHNH ₂ , C(=0)-NH-(CH ₂ ) _r NR ₂₆ R ₂₇ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j-R ₂₆ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NR ₂₆ R ₂₇ and -L ₂ -R ₂₈ ; and the remaining R ₂₀ groups are each independently selected from F, CI, Br, (CH ₂ )j-

59

SUBSTITUTE SHEET RULE 26 OH, d-6 alkyl, Od- ₆ alkyl, 0CH ₂ Ph, S(0) ₂ -CH ₃ , S(0) ₂ -CH(CH ₃ ) ₂ , S(0) ₂ -NHCH ₃ , S(0) ₂ - N(CH ₃ ) ₂ , 0-CH ₂ -0-CH ₂ CH ₃ , Cy, C(0)-NH-CH ₂ -Cy, NH-C(0)-NH ₂ and NH-C(0)-NH ₂ .

In one aspect, more suitably, one R ₂₀ group is-I^-R^; and the remaining R ₂₀ groups are each independently selected from F, CI, Br, (CH ₂ )j-0H, Ci- ₆ alkyl, OCi-6 alkyl, 0CH ₂ Ph, S(0) ₂ -CH ₃ , S(0) ₂ -CH(CH ₃ ) ₂ , S(0) ₂ -NHCH ₃ , S(0) ₂ -N(CH ₃ ) ₂ , 0-CH ₂ -0- CH ₂ CH ₃ , Cy, C(0)-NH-CH ₂ -Cy, NH-C(0)-NH ₂ and NH-C(0)-NH ₂ .

More suitably, one R ₂₀ group is selected from (CH ₂ ) C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , 0-(CH ₂ ) _k - NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ - (CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ and C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ ; and the remaining R ₂₀ groups are each independently selected from F, CI, Br, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, S(0) ₂ -NHCH ₃ and S(0) ₂ -N(CH ₃ ) ₂ . More suitably, the remaining R ₂₀ groups are each independently selected from F, CI, Br, methyl, ethyl, OCH ₃ and OCH ₂ CH ₃ .

More suitably, one R ₂₀ group is selected from (CH ₂ ) C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , 0-(CH ₂ ) _k - NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ - (CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ and C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ ; and the remaining R ₂₀ groups are each independently selected from F, CI, Br, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph.

More suitably, one R ₂₀ group is selected from 0-(CH ₂ ) _k -NH ₂ and (CH ₂ )j-NH ₂ ; and the remaining R ₂₀ groups are each independently selected from F, CI, Br, methyl, ethyl, OCH ₃ and OCH ₂ CH ₃ .

In some aspects, suitably R _2i is selected from C ₅ _6 heterocyclyl, C ₅ _ _g heteroaryl, C6-15 heteroarylalkyl, phenyl, benzyl and phenethyl; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 optional groups independently selected optional R ₂₀ groups. In another aspect, suitably R _2i is selected from H, Ci-12 alkyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, uracilyl, tetrahydropyridinyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl,

60

SUBSTITUTE SHEET RULE 26 benzimidazolyl, N-methylbenzo-imidazolyl, benzooxazolyl, benzothiazolyl, pyrrol-3- ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3- ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1 2 or 3 independently selected optional R ₂₀ groups.

Suitably R21 is selected from H, Ci _-6 alkyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, uracilyl, tetrahydropyridinyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl, pyrrol-3- ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3- ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1 2 or 3 independently selected optional R ₂₀ groups.

Suitably R21 is selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i- butyl, t-butyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, uracilyl, tetrahydropyridinyl, indolyl, N- methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4- ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl, furan-3- ylmethyl, phenyl, benzyl and phenethyl optionally substituted with 1, 2 or 3

independently selected optional R ₂₀ groups.

Suitably R _2i is selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i- butyl, t-butyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, uracilyl, tetrahydropyridinyl, indolyl, N- methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl, phenyl, benzyl and phenethyl optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups .

Suitably, the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 optional groups independently selected from F, CI, Br, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , (CH ₂ ) NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ , C(=0)-NH-(CH ₂ )

heterocyclyl, -S(0) ₂ -(d-6 alkyl), 0-( Η ₂ ) _¾ -0-(α- ₆ alkyl), (CH ₂ )j-S(0) ₂ -NR ₂₆ R ₂₇ ,

61

SUBSTITUTE SHEET RULE 26 C(=NH)-0-(d- ₆ alkyl), (CH ₂ ) _k -0-(d- ₆ alkyl), CN, NCO, C(0)-NH-(CH ₂ ) Cy, C(0)-Cy, NH-C(0)-NR ₂₆ R ₂₇ and

In some aspects, suitably, each R _2i is independently selected from:

wherein X' is N, CH or CR'";

X" is O, NH, N-CCi-6 alkyl) or S; and

each R" and R'" are independently selected from H, S(0) ₂ -(d- ₆ alkyl), 0-(CH ₂ ) _k -0-(d- alkyl), (CH ₂ )j-NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ , -L ₂ -R ₂₈ , (CH ₂ )j-S(0) ₂ -NR ₂₆ R ₂₇ , C(=NH)-0-(d- ₆ alkyl), (CH ₂ ) _k -0-(d- ₆ alkyl), CN, NCO, Cy, C(0)-NH-(CH ₂ )j-Cy, C(0)-Cy, NH-C(O)- NR ₂₆ R ₂₇ , R ₂₀ groups and

More suitably, each R" and R'" are independently selected from S(0) ₂ -(Ci-6 alkyl), O- (CH ₂ ) _k -0-(d-6 alkyl), (CH ₂ )j-NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ , (CH ₂ ) _r S(0) ₂ -NR ₂₆ R ₂₇ , C(=NH)-0-(d- ₆ alkyl), (CH ₂ ) _k -0-(d- ₆ alkyl), CN, NCO, Cy, C(0)-NH-(CH ₂ ) _r Cy, C(0)-Cy, NH-C(O)- NR ₂₆ R ₂₇ , R ₂₀ groups and

62

SUBSTITUTE SHEET RULE 26 embodiments, R ₂ i is selected from:

embodiments, more suitably R ₂ i is selected from:

63

SUBSTITUTE SHEET RULE 26

In some embodiments, R ₂ i is selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl.

Suitably, where an R _2i group may be optionally substituted it is substituted with 1, 2 or 3 of the optional substituents; suitably, an R _2i group is substituted with 1 or 2 of the optional substituents. More suitably, an R _2i group is substituted with 1 of the optional substituents. Alternatively, suitably an R _2i group is not substituted.

Cy

Suitably, each Cy is independently selected from a C ₅ heterocyclyl or C ₅ heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R ₂₀ groups.

Suitably, each Cy is independently selected from pyrrolyl, tetrazolyl, triazolyl, furanyl, thiazolidinyl and pyrrolidinyl, wherein these groups are optionally substituted with 1 or 2 R ₂₀ groups.

More suitably, each Cy is independently selected from:

64

SUBSTITUTE SHEET RULE 26

Suitably each R ₂₂ , R ₂₃ , R ₂₄ , R ₂₅ , R ₂₆ and R ₂₇ is independently H, Ci alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C _g alkyl, C ₁₀ alkyl or C _n alkyl.

Suitably, each R ₂₂ , R ₂₃ , R ₂₄ , R ₂₅ , R ₂₆ and R ₂₇ is independently selected from H and Ci- ₆ alkyl.

Suitably each R ₂₂ , R ₂₃ , R ₂₄ , R ₂₅ , ₂ 6 and R ₂₇ is independently selected from H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl and t-butyl.

More suitably each R ₂₂ , R ₂₃ , R ₂₄ , R ₂₅ , R ₂₆ and R ₂₇ is independently selected from H, methyl, and ethyl. More suitably each R ₂₂ , R ₂₃ , R ₂₄ , R ₂₅ , R ₂₆ and R ₂₇ is independently selected from H and methyl.

Suitably, Xi is O, S, NR ₂₄ , CR ₂₄ R ₂₅ , C(=0), C(=0)NR ₂₄ , NR ₂₄ C(=0) or is absent.

The amide is drawn in both directions, i.e. C(=0)NR ₂₄ and NR ₂₄ C(=0). Hence, it may be attached in either direction. Thus, for example, Xi may be C(=0)NR ₂ 8 that links the left hand moiety to group Li as follows: -C(=0)NR ₂ s-Li-X ₂ -, whereas when Xi is

X ₂ -.

In some aspects, suitably, Xi is O, S, NH, N-CH ₃ , CH ₂ , CH ₂ 0, C(=0), C(=0)NH, C(=0)NCH ₃ , NHC(=0), NCH ₃ C(=0), O-C(O), C(0)-0 or is absent.

More suitably, Xi is O, NH, CH ₂ , C(=0), C(=0)NH, NHC(=0) or is absent.

In one aspect, suitably, Xi is O.

In one aspect, suitably, Li is an amino acid or a peptide chain having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids.

In another aspect, suitably, Li is a paraformaldehyde chain -(0CH ₂ )i_ ₂₄ - having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 repeating units; or

65

SUBSTITUTE SHEET RULE 26 a polyethylene glycol chain -(0CH ₂ CH ₂ )i-i ₂ - having l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 repeating units.

In another aspect, more suitably, Li is -(CH ₂ )n-Yi-(CH ₂ )p-.

More suitably, is selected from -(CH ₂ ) _m -(CH ₂ ) _q -(CH ₂ ) _n -, -(CH ₂ ) _m -Ar ₁ -C(0)-NH- (Ar ₂ )o-i-Ar ₃ -(CH ₂ ) _n -, -(CH.im-Arg-CAr.io-i-NH-CCOi-Ari-CCH.),,-,

wherein q is 1, 2, 3, 4 or 5;

Y ₃ .is C-H or N;

R4 ₀ , R41 and R42 are independently selected from H and R ₂₀ ; and

R43 is H or methyl.

The above structures are drawn without specifying the positions of any of the groups, i.e. groups R4 ₀ , R41, R42, and the two groups (shown by bonds that end in a zig-zag line) where the ring is attached to the rest of the molecule. Hence, these groups may be present on any position of the ring except for Y ₃ or Y ₄ (as positioning a group, such as R ₂₀ at Y ₃ or Y ₄ would not meet the valence requirements). The linker groups -(CH ₂ ) _m - Ar ₁ -C(0)-NH-(Ar ₂ ) ₀ -i-Ar ₃ -(CH ₂ )n- and -(CH ₂ ) _m -Ar ₃ -(Ar ₂ ) ₀ -i-NH-C(0)-Ar ₁ -(CH ₂ ) _n - are drawn in both directions and can be attached to Xi and X ₂ in either direction.

More suitably, L is sel

66

SUBSTITUTE SHEET RULE 26

More suitably, L is selected from -(CH ₂ )o-io-(CH2)i-5-(CH ₂ )o-io- and

suitably, L is selected from -CH ₂ -, -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -

12-, -CH ' ₂ 2-CH ₂ -CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -CH ₂ -

CH ₂ -CH ₂ -CH ₂ - and

In another aspect, suitably one of R ₄₀ , 41 and Rz, ₂ is R ₂₀ ; and the remaining of R ₄ o _> R 1 and Rz, ₂ are H. More suitably, one of R ₄₀ , R41 and Rz, ₂ is selected from (CH ₂ )j-C0 ₂ R ₂ 6, O- (CH ₂ ) _k -NR ₂₆ R ₂₇ , C(=0)-0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , C(=0)-NR ₂₆ R ₂₇ , (CH ₂ )j-NR ₂₆ R ₂₇ , NR ₂₆ NH ₂ ,

C(=NH)NR ₂₆ R ₂₇ , - -R ₂ 8, (CH ₂ )j-S(0) ₂ -NR ₂₆ R ₂₇ and NH-C(0)-NR ₂₆ R ₂₇ ; and remaining of R40, R41 and R4 ₂ are H. More suitably, one of R ₄₀ , R ₄ i and R ₄₂ is (CH ₂ )j-NR ₂₆ R ₂₇ or - L2-R ₂ 8; and remaining of R ₄₀ , R ₄ i and R ₄₂ are H.

Suitably, X ₂ is O, S, NR ₂₄ , CR ₂₄ R ₂₅ , C(=0), C(=0)NR ₂₄ , NR ₂₄ C(=0) or is absent.

67

SUBSTITUTE SHEET RULE 26 In some aspects, suitably, X ₂ is O, S, NH, N-CH ₃ , CH ₂ , CH ₂ 0, C(=0), C(=0)NH, C(=0)NCH ₃ , NHC(=0), NCH ₃ C(=0), O-C(O), C(0)-0 or is absent. More suitably, X ₂ is O, NH, CH ₂ , C(=0), C(=0)NH, NHC(=0) or is absent.

In one aspect, suitably, X ₂ is O or is absent.

J

Suitably, each j is independently selected from o, l, 2 or 3.

In some aspects, j is 1.

In other aspects, j is o. k

Suitably, each k is independently selected from 1, 2 or 3.

More suitably, k is 1 or 2.

In some aspects, k is 1.

IB

Suitably m is o.

More suitably, m is 1. n

Suitably, n is selected from o, 1, 2, 3, 4, 5 or 6. Suitably, n is selected from o, 1, 2 or 3. In one aspect, suitably n is 1, 2, 3, 4, 5 or 6; suitably, n is 1, 2 or 3; suitably, n is 1.

P.

Suitably, p is selected from o, 1, 2, 3, 4, 5 or 6. Suitably, p is selected from o, 1, 2 or 3. In one aspect, suitably p is 1, 2, 3, 4, 5 or 6; suitably, p is 1, 2 or 3; suitably, p is 1. a

Suitably, q is selected from 1, 2 or 3.

More suitably, q is 1. s

Suitably, each s is independently selected from o, 1, 2 or 3.

68

SUBSTITUTE SHEET RULE 26 In some aspects, s is 1.

In other aspects, s is o. t

Suitably, each t is independently selected from 1, 2 or 3.

More suitably, t is 1 or 2.

In some aspects, t is 1. w

Suitably w is o.

More suitably, w is 1.

Suitably, Ari is selected from pyrrolylene, N-methylpyrrolylene, furanylene, thiophenylene, imidazolylene, N-methylimidazolylene, oxazolylene or thiazolylene, wherein these groups maybe optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, Ar is:

wherein one of Y ₆ and Y ₇ is independently selected from N-R ₄₂ , S and O; and the other of Y and Y ₇ is CH; and Y ₅ is independently selected from C-R ₄₂ , N, S and COH; and R ₄ is selected from H or R ₂₀ .

Suitably, Ari is selected from pyrrolylene, N-methylpyrrolylene, imidazolylene or N- methylimidazolylene, wherein these groups may be optionally substituted with 1 or 2 independently selected optional R ₂₀ groups.

Suitably, Ar is:

wherein R ₄₄ is H or methyl.

SUBSTITUTE SHEET RULE 26 Suitably, Ar ₂ is an optionally substituted phenylene or pyridylene.

More suitably, Ar ₂ is:

wherein Ys is N or CH; Y _g is N or CH; and wherein at least one of Ys and Y _g is CH; and

More suitably, Ar ₂ is an optionally substituted phenylene. Ara

Suitably, Ar ₃ is selected from pyrrolylene, N-methylpyrrolylene, furanylene, thiophenylene, imidazolylene, N-methylimidazolylene, oxazolylene, thiazolylene, pyridylene, indolylene, N-methylindolylene, benzofuranylene, benzothiophenylene, benzimidazolylene, N-methylbenzoimidazolylene, benzooxazolylene or

benzothiazolylene wherein these groups may be optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups. pyrrolylene, N-methylpyrrolylene, thiophenylene, imidazolylene, N- methylimidazolylene, oxazolylene, thiazolylene, indolylene, N-methylindolylene, benzofuranylene, benzothiophenylene, benzimidazolylene, N- methylbenzoimidazolylene,

Suitably, Ar ₃ is selected from pyrrolyl, N-methylpyrrolyl, thiophenyl, N-methyl- imidazolyl, oxazolyl, thiazolyl, benzothiophenyl, N-methylbenzoimidazolyl and benzothiazolyl wherein these groups may be optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, Ar ₃ is

wherein Z ₁₀ is selected from NH, N-CH ₃ , S and O;

Z11 is selected from CH and N;

70

SUBSTITUTE SHEET RULE 26 Z ₁₂ is selected from NH, N-CH ₃ , S and O;

Z13 is selected from CH and N; and

R46 is selected from H and R ₂₀ .

Ar and Ar _¾

Suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, pyridazinyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl or phenyl wherein these groups maybe optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl or phenyl wherein these groups may be optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

Suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzooxazolyl, benzothiazolyl or phenyl wherein these groups may be optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl or phenyl wherein these groups maybe optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl or phenyl wherein these groups maybe optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

More suitably, Ar ₄ and Ar ₅ are independently selected from pyrrolyl, N-methylpyrrolyl, pyridyl or phenyl wherein these groups may be optionally substituted with 1, 2 or 3 independently selected optional R ₂₀ groups.

71

SUBSTITUTE SHEET RULE 26 Suitably, Ar ₄ is a group that is substituted with 1, 2 or 3 independently selected R ₂₀ groups. More suitably, Ar ₄ is a group that is substituted with one R ₂₀ group.

Suitably, Ar ₅ is a group that is substituted with 1, 2 or 3 independently selected R ₂₀ groups. More suitably, Ar ₅ is a group that is substituted with one R ₂₀ group.

More suitably, Ar ₄ and Ar ₅ are identical groups.

Suitably, R ₂₉ , R ₃₀ , R ₃ i, R325 R335 R345 R355 R365 R375 R385 R39, R405 R415 R425 R45 ^an d R46 are each independently selected from H, F, CI, Br, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ ) _r C0 ₂ R ₂₆ , 0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , C(=0)-0-(CH ₂ ) _k -NR ₂₆ R ₂₇ , (CH ₂ )j-NR ₂₆ R ₂₇ , NHNH ₂ ,

C(=0)-NH-(CH ₂ ) _k -C(=NH)NR ₂₆ R ₂₇ and -L ₂ -R ₂₈ .

Suitably, R _2g , R ₃₀ , R325 R335 R345 R355 R375 R385 R39, R425 R45 ^an d R46 are each independently selected from H, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , 0CH ₂ Ph, (CH ₂ )j-C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , (CH ₂ )j-C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k - NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH- CH ₃ , NHNH ₂ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ - (CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ and - L^-R^.

More suitably, R ₂₉ , R ₃₀ , R ₃ i, R32, R ₃₃ , R ₃ 4> R355 R365 R375 R385 R39, R405 R415 R425 R45 ^an d R46 are each independently selected from H, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH ₂ , (CH ₂ )j-NH ₂ and -

In one aspect, suitably, one of R ₂₉ , R ₃₀ , R ₃ i, R ₃₂ , R ₃₃ , R ₃₄ , R ₃₅ , R36, R37, R38, R39, R40, R41, R^, and R ₄₆ is selected from (CH ₂ ) _r C0 ₂ R ₂₇ , 0-(CH ₂ ) _k -NR ₂₇ R ₂₈ , C(=0)-0-(CH ₂ ) _k -

NR ₂₆ R ₂₇ , (CH ₂ )j-NR ₂₇ R ₂₈ , NHNH ₂ ,

R ₂₇ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NR ₂₇ R ₂₈ and -L ₂ -R ₂₈ ; and the remaining of R ₂₉ , R ₃₀ , R31, R ₃₂ , R ₃₃ , R ₃₄ , R ₃₅ , R ₃ 6, R ₃₇ , R ₃₈ , R ₃₉ , R ₄₀ , R41, R42, R45 and R4 ₆ are each independently selected from H, F, CI, Br, (CH ₂ ) 0H, d- ₆ alkyl, Od-6 alkyl and 0CH ₂ Ph.

In this aspect, suitably, one of R ₂₉ , R ₃₀ , R ₃ i, R ₃₂ , R ₃₃ , R ₃₄ , R ₃₅ , R ₃ 6, R ₃₇ , R38, R39, R40, R41, R ₄₂ , R4 ₅ and R ₄₆ is selected from (CH ₂ ) C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , 0-(CH ₂ ) _k -NH ₂ , O-

72

SUBSTITUTE SHEET RULE 26 (CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j- NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j- H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ and C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ ; and the remaining of R _2G , R ₃₀ , R ₃ i, R ₃₂ , R ₃₃ , R ₃₄ , R ₃₅ , R ₃ 6, R375 R38J R39, R40 _J R^ R42J R 5 ^an d R46 are each independently selected from F, CI, Br, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph.

In this aspect, more suitably, one of R ₂₉ , R ₃₀ , R31, R ₃₂ , R ₃₃ , R ₃₄ , R35, R36, R37, R38, R39, R ₄ o, R4 ₁ , R4 ₂ , R45 and R46 is selected from 0-(CH ₂ )k-NH ₂ and (CH ₂ )j-NH ₂ ; and the remaining R _2G , R ₃ o, R31, R3 ₂ , R33, R3 ₄ , R35, R36, R37 _J R38 _J R39, R ₄ oj R ₄ ij R ₄ 2, R ₄₅ and R46 groups are each independently selected from (CH ₂ )j-0H, methyl, ethyl, OCH ₃ and OCH ₂ CH ₃ .

In one embodiment, R43 is H.

More suitably, R ₄₃ is methyl.

R

In one embodiment, R44 is H.

More suitably, R44 is methyl.

Suitably, each R _A and R _B is independently selected from (CH ₂ )j-0H, (CH ₂ )j-C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , (CH ₂ )j-C0 ₂ CH ₂ CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ and C(=0)-NH-(CH ₂ ) _k -C(=NH)NH-CH ₃ .

More suitably, each R _A and RB is independently selected from (CH ₂ )j-0H, C0 ₂ H, C0 ₂ CH ₃ , C0 ₂ CH ₂ CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ and (CH ₂ )j-NH ₂ .

Linker L ₂ is a bond or is a moiety having 1-200 nonhydrogen atoms selected from C, N, O, S, or halogen, and optionally incorporates alkyl, ether, oxo, carboxyl, carboxamide, carboxamidyl, urethanyl, branched, cyclic, unsaturated, amino acid, heterocyclyl, aryl or heteroaryl moieties. Linker L ₂ may be unbranched or branched, flexible or rigid, short or long and may incorporate any combination of moieties as deemed useful. In some embodiments, at least a portion of the linker L ₂ may have a polyalkylene oxide polymeric region, which may enhance solubility of the compound of formula (I) or (II).

73

SUBSTITUTE SHEET RULE 26 In some embodiments, the linker L ₂ may have a repeating unit of ethylene glycol, and may have a number of repeating ethylene glycol units of about 1 to about 25, or any number therebetween. In some embodiments, L ₂ may include about 3 to about 20, about 4 to about 15, about 5 to about 12 or about 6 to about 10 ethylene glycol units. In some embodiments, at least a portion of Linker L ₂ may include one or more amino acid moieties which may provide enhanced solubility for the compound of formula (I) or (II) or may provide amino acid sequences to enhance target binding, enhance compatibility with a targeting agent, or enhance target binding recognition. In other embodiments, the linker L ₂ may include one or more amino acid moieties that provide a suitable substrate motif for a protease. When a set of amino acid moieties are incorporated into the linker L ₂ that provide a substrate motif specific for a selected protease, the cytotoxic drug compound of formula (I) or (II) may be released from a target bound conjugate to provide localized cytotoxic effects. In other embodiments, the linker L ₂ may include an alkylene chain. Suitably, the alkylene chain is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 carbons in length; and suitably the alkylene chain comprises -CH ₂ - groups. Such substrate motifs are known in the art and may be incorporated into the linker L ₂ as desired to provide selective release from the target bound conjugate. This selectivity can be based on known presence of a desired protease within the localized delivery region of the conjugate drug. Other polymeric types of moieties may be incorporated in the linker L _¾ such as polyacids, polysaccharides, or polyamines. Other moieties such as substituted aromatic or heteroaromatic moieties may be used to enhance rigidity or provide synthetically accessible sites on substituents therein for linking to reactive moieties or to the compound of formula (I) or (II). For example, the linker L ₂ can include ethylene glycol repeating units, and/or an amino acid sequence. In some embodiments, linker L ₂ includes the formula: wherein X _AA is an amino acid sequence.

Any suitable number of ethylene glycol units can be used in the linker L of the present invention. For example, the linker L ₂ can include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, 16, 19, 20, 23, 24, 35, 36, 37, 48, 49, or more ethylene glycol units. In some

embodiments, the linker L ₂ can include 8 ethylene glycol units. Several commercially available ethylene glycol groups (polyethylene glycol, PEG) are suitable in the linker L ₂ , such as H ₂ N-dPEG®8-C(0)OH, having a discrete ("d") polyethylene glycol having 8

74

SUBSTITUTE SHEET RULE 26 ethylene glycol repeating units. Other discrete PEG units are commercially available and known to one of skill in the art, such as by Advanced ChemTech, In some embodiments, the linker L ₂ includes the formula: wherein PEG has 1-50 ethylene glycol units, and XAA is an amino acid sequence.

In another example, the linker L ₂ can include an alkylene chain, and/or an amino acid sequence. In some embodiments, linker L ₂ includes the formula: wherein XAA is an amino acid sequence; and the linker L ₂ can include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more -CH ₂ - moieties.

The amino acid portion of the linker L ₂ can include any suitable number of amino acid moieties, as described above. For example, the amino acid sequence XAA can include from 1 to 100 amino acid moieties, or from 1 to 10 amino acid moieties, or from 1 to 5 amino acid moieties. The linker I^ can include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid moieties. In some embodiments, the linker L ₂ includes 2 amino acid moieties. In some embodiments, the linker L ₂ includes the amino acid sequence Val- Ala. In some embodiments, the linker L ₂ includes the formula:

-HN-PEG ₈ -C(0)-Val-Ala- wherein PEGs has 8 ethylene glycol units.

The linker L ₂ can also include a variety of other connecting groups that connect the ethylene glycol portion to the amino acid sequence, or connect the ethylene glycol or amino acid sequence to R ₂ 8, or the compound of formula (I) or (II). For example, the amino acid sequence can be connected to the compound of formula (I) or (II) via a 4- amino benzyl carboxylate group. In some embodiments, the ethylene glycol portion ca be directly linked to R ₂ 8. In some embodiments, the linker L ₂ has the formula:

75

SUBSTITUTE SHEET RULE 26

R ₂ 8 is an azide, alkyne, bisulfone, carbohydrazide, hydrazine, hydroxylamine, iodoacetamide, isothiocyanate, maleimide, phosphine, pyrridopyridazine,

semihydrazide, succinimidyl ester, sulfodichlorophenol ester, sulfonyl halide, sulfosuccinimidyl ester, 4-sulfotetrafluorophenyl ester, tetrafluorophenyl ester, thiazole, R _A , 0-(CH ₂ )k-NR ₂₆ R ₂ 6, NHNH ₂ , or is a targeting agent wherein the targeting agent is selected from a protein, a portion of a protein, a peptide, a nucleic acid, or an antibody.

Hence, R ₂ 8 is a reactive moiety capable of reacting with a targeting agent, or is a targeting agent. Where R ₂ 8 is a reactive moiety it can react with functional groups such as aldehdes, amines, disulfides, ketones thiols in the targeting agent, or in Staudinger reactions, Pictet-Spengler reactions and/or Click-type chemistry with the targeting agent. For some reactive moieties suitable coupling reagents are used to react the reactive moiety with a targeting agent, e.g. where R ₂ s is a carboxylic acid [when R _A is(CH ₂ )j-C0 ₂ R ₂ 6] carbodiimide coupling reagents maybe used. Suitably, R ₂ 8 is an azide, alkynes, bisulfone, carbohydrazide, hydroxylamine, iodoacetamide, isothiocyanate, maleimide, phosphine, semihydrazide, succinimidyl ester and sulfonyl halide, RA or is a targeting agent wherein the targeting agent is selected from a protein, a portion of a protein, a peptide, a nucleic acid, or an antibody. In one aspect, suitably, R ₂₈ is an azide, alkynes, bisulfone, carbohydrazide,

hydroxylamine, iodoacetamide, isothiocyanate, maleimide, phosphine, semihydrazide, succinimidyl ester and sulfonyl halide or RA.

A number of other chemistries are known for attachment of compounds to antibodies. US 7,595,292 (Brocchini et al.) refers to linkers that form thioesters with the sulfurs in a disulfide bond of an antibody. US 7,985,783 (Carico et al.) refers to the introduction of aldehyde residues into antibodies, which are used to couple compounds to the antibody.

76

SUBSTITUTE SHEET RULE 26 In another aspect, R ₂ 8 is a targeting agent wherein the targeting agent is selected from a protein, a portion of a protein, a peptide, a nucleic acid, or an antibody. The targeting agent may bind to a tumor- associated antigen, a cancer-stem-cell associated antigen or a viral antigen.

In various embodiments, the targeting agent may bind to a target selected from an acute myeloid leukemia (AML M4) cell, an acute promyelocyte leukemia cell, an acute lymphoblastic leukemia cell, an acute lymphocytic leukemia cell, a chronic lymphocytic leukemia cell, a chronic myeloid leukemia cell, a chronic T-cell lymphocytic leukemia, a myelodysplasia syndromic cell, a multiple myeloma cell, a prostate carcinoma cell, a renal cell adenocarcinoma cell, a pancreatic adenocarcinoma cell, a lung carcinoma cell or a gastric adenocarcinoma cell, a gastric adenocarcinoma cell, a breast cancer cell, a colon cancer cell, a melanoma cell, a thyroid cancer cell, an ovarian cancer cell, a bladder cancer cell, a liver cancer cell, a head and neck cancer cell, an esophageal cancer cell, a hodgkin lymphoma cell, a non- hodgkin lymphoma cell, a mesothelioma cell, a neuroblastoma cell, a neuroendocrine tumor cell, a neurofibromatosis type 1 (NFi) cell, a neurofibromatosis type 2 (NF2) or an osteosarcoma cell.

Other features

Suitably, the compound of formula (I) or formula (II) is selected with the proviso that when -L ₂ -R ₂ 8 is present in the compound of formula (I) or (II), there is only one-L ₂ - R ₂ 8 group present.

In some embodiments, -L ₂ -R ₂ 8 is absent from the compound of formula (I) or (II).

Suitably, the compound of formula (I) or formula (II) contains only one primary or secondary amine.

Suitably, the compound of formula (I) or formula (II) contains only one primary amine, secondary amine or -L ₂ -R ₂ 8 group.

Suitably, the compound of formula (I) or formula (II) contains only one primary amine, secondary amine, RA, RB or -I^-R^ group. In another aspect, each R _IG is independently selected from H, Ci-i ₂ alkyl, F, CI, Br, OCi-6 alkyl, 0CH ₂ Ph, =CH ₂ , =CH-(CH ₂ ) _S -CH ₃ , and an optionally

77

SUBSTITUTE SHEET RULE 26 substituted C ₅ - _g heteroaryl or an optionally substituted phenyl; optionally substituted with 1, 2, 3 independently selected optional R ₂₀ groups; and each R ₂₀ group is independently selected from F, CI, Br, methyl, ethyl, OCH ₃ and

OCH ₂ CH ₃ ; and

either:

(i) R ₆ is RA or -L ₂ -R ₂ 8; and R _i5 is selected from H, S0 ₃ H, and nitrogen protecting

groups or together with R _i4 forms a double bond; or

(ii) Ri ₅ is RB or -L ₂ -R ₂ 8; and R6 is selected from H, S0 ₃ H, and nitrogen protecting

groups or together with R ₅ forms a double bond.

In another aspect, one R ₂₀ group is selected from (CH ₂ )j-C0 ₂ H, (CH ₂ )j-C0 ₂ CH ₃ , O-

(CH ₂ ) _k -NH ₂ , 0-(CH ₂ ) _k -NH-CH ₃ , C(=0)-0-(CH ₂ ) _k -NH ₂ , C(=0)-0-(CH ₂ ) _k -NH-CH ₃ , (CH ₂ )j-NH ₂ , (CH ₂ )j-NH-CH ₃ , C(=0)-NH-(CH ₂ ) _k -NH ₂ , C(=0)-NH-(CH ₂ ) _k -NH- CH ₃ , C(=0)-NH-C ₆ H ₄ -(CH ₂ )j-H, C(=0)-NH-(CH ₂ ) _k -C(=NH)NH ₂ and C(=0)-NH- (CH ₂ ) _k -C(=NH)NH-CH ₃ ;

the remaining R ₂₀ groups are each independently selected from F, CI, Br, (CH ₂ )j-0H, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph;

each Ri ₉ is independently selected from H, methyl, ethyl, F, CI, Br, OCH ₃ , OCH ₂ CH ₃ and 0CH ₂ Ph;

R ₅ is selected from H, OH and OCi-6 alkyl; and R ₆ is selected from H, S0 ₃ H and nitrogen protecting groups; or R ₅ and R ₆ together form a double bond; and

R ₁ 4 is selected from H, OH and OCi- ₆ alkyl; and R _i5 is selected from H, S0 ₃ H and

nitrogen protecting groups; or R _i4 and R _i5 together form a double bond. Applications

The invention finds application in the treatment of disease, more specifically of a proliferative disease.

In certain aspects a method of treating a proliferative disease is provided, the method comprising administering to a subject a therapeutically effective amount of a compound of the formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, or a composition comprising a compound of formula (I) formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof.

In certain aspects a method of treating a proliferative disease is provided, the method comprising administering to a subject a therapeutically effective amount of a targeted

78

SUBSTITUTE SHEET RULE 26 conjugate comprising a compound of the formula (I) or formula (II) or

pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof. In certain aspects a method of treating a proliferative disease is provided, the method comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate comprising a compound of the formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof.

The term "proliferative disease" refers to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breast cancer, glioblastoma, cervical cancer, ovarian cancer, oesophageal [or esophageal] cancer, oral cancer, prostate cancer, testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, uterine cancer, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Suitably the proliferative disease is selected from bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer. Suitably the proliferative disease is selected from breast cancer and cervical cancer.

Any type of cell may be treated, including but not limited to, bone, eye, head and neck, lung, gastrointestinal (including, e.g. mouth, oesophagus, bowel, colon), breast

(mammary), cervix, ovarian, uterus, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

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SUBSTITUTE SHEET RULE 26 A skilled person is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type. Suitably subjects are human, livestock animals and companion animals.

In a further aspect, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be linked, either directly or indirectly, to a targeting agent (e.g., antibody, antibody fragment, hormone, etc.) to provide a targeted conjugate. The target conjugates of the present disclosure may contain one or multiple compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof. A variety of target conjugates are known in the art and may be used with a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof. For example, in a particular aspect the target conjugate is an antibody-drug conjugate, wherein one or more compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, are linked, directly or indirectly, to the antibody. Therefore, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be used as a payload on a targeted conjugate.

Suitably, a compound of formula (I) and salts and solvates thereof, for use as a drug in targeted conjugate is prepared by attaching a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, to a targeting agent, either directly or via an optional linker group. Suitably, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, is attached to a targeting agent via a linker group. Suitably, the targeted conjugate is for use in the treatment of a disease, more specifically of a proliferative disease. Suitably, the drug may be attached by any suitable functional group that it contains to the targeting agent either directly or via a linker group. Typically, the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the targeting agent either directly or via a linker group. In some aspects, one or more atoms or groups of the compound of formula (I) or (II) may be eliminated during the attachment of the drug to the antibody. In some aspects, the targeting agent binds to a cell surface receptor or a tumor-associated antigen. In some aspects, the

80

SUBSTITUTE SHEET RULE 26 targeting agent is an antibody. In some aspects, the targeting agent is a hormone. In some aspects, the targeting agent is a protein. In some aspects, the targeting agent is a polypeptide. In some aspects, the targeting agent is a small molecule (for example, folic acid).

In some aspects, the present invention relates to a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, for use as a drug in an antibody-drug conjugate. Suitably, a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, for use as a drug in an antibody-drug conjugate is prepared by attaching to an antibody or an antibody fragment either directly or via an optional linker group. Suitably, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, is attached to an antibody or an antibody fragment via a linker group. Suitably, the antibody-drug conjugate is for use in the treatment of a disease, more specifically of a proliferative disease.

In some aspects, the present invention relates to the use of a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, as a drug in an antibody-drug conjugate. Suitably, the use of a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, as a drug in an antibody-drug conjugate is accomplished by attaching to a compound of formula (I) and salts and solvates thereof to an antibody or an antibody fragment either directly or via an optional linker group. Suitably, the compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, is attached to an antibody or an antibody fragment either directly or via a linker group. Suitably, the antibody-drug conjugate is for use in the treatment of a disease, more specifically of a proliferative disease.

In such aspects, suitably, the drug may be attached by any suitable functional group that it contains to the antibody or antibody fragment either directly or via a linker group. Typically, the drug contains, or can be modified to contain, one or more functional groups such as amine, hydroxyl or carboxylic acid groups for attaching the drug to the antibody or antibody fragment either directly or via a linker group.

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SUBSTITUTE SHEET RULE 26 The compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, find application as payloads for antibodies or antibody fragments or other targeting moieties (e.g. hormones, proteins and small molecule targeting agents such as folic acid). The compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, readily allow conjugation to antibodies or antibody fragments or other targeting moieties.

Suitably, a compound of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be used in preparing a targeting conjugate by being modified to contain one or more linker groups, wherein the targeting agent (such as an antibody) is attached to the drug through one or more linker groups. Therefore, the present invention provides for a compound of formula (I) or formula (II) further comprising one or more linker groups or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof. Suitably, a compound of formula (I) or formula (II) further comprises 1, 2 or 3 linker groups.

Suitably, a compound of formula (I) or formula (II) further comprises 1 or 2 linker groups. Suitably, a compound of formula (I) or formula (II) further complies 1 linker group. In some aspects, one or more atoms or groups (such as H atoms or hydroxyl groups) of the compound of formula (I) or formula (II) may be eliminated during the attachment of the drug to the targeting agent (such as an antibody) or the attachment of the linker to the drug or the modification of the drug to contain one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the antibody either directly or via a linker group. In some aspects, where the compound of formula (I) or formula (II) further comprises a linker group that is attached to the rest of the compound of formula (I) or formula (II) by eliminating one or more atoms or groups, such as H atom or atoms or hydroxyl groups.

Linkers can either be cleavable or non-cleavable, with cleavable linkers normally represented by combinations of amino acids. The list of cleavable linkers includes, but is not limited to, valine-citruline, valine-alanine and any combination of two to eight amino acids. A self-immolative unit (e.g., a PAB spacer) can be included to assist with clean cleavage, and optionally hydrophilic groups (e.g., PEG) can be added to increase hydrophilicity of the construct. In some aspects, more suitably, the linker group comprises a self-immolative unit. A range of self immolative units are known in the art, e.g. A. Blencowe, A. T. Russell, F. Greco, W. Hayes, D.W. Thornthwaite Polymer

82

SUBSTITUTE SHEET RULE 26 Chemistry (2011) 2, 773-790; and have been described in, for example, US Patent No. 7 _> 754 _> 68i, European Patent Publication No. 0624377.

A variety of suitable linker groups are known in the art and may be used as described herein. For example, the maleimide methodology is routinely used as a method to attach antibodies to drug compounds by providing a linker attached to the drug with a terminal maleimide group. In addition, methodologies using diarylcyclooctyne moieties (such as, but not limited to, DBCO, dibenzylcyclooctyne) are known in the art. Diarylcyclooctynes react with stable azides to provide attachment via the formation of stable triazoles. Diarylcyclooctynes are thermostable with very narrow and specific reactivity toward azides, resulting in almost quantitative yields of stable triazoles.

Furthermore, the reaction does not require a cytotoxic Cu(I) catalyst (that is toxic to most organisms) and thus, prevents its use in many biological systems. Still further, alkoxyamine methodologies are also alternatives in the art. For site-specific

conjugation of the drug to the antibody, the antibodies may comprise a "tag" (which may be proprietary) that will react with a diarylcyclooctyne (for example DBCO), an alkyoxyamine and/or maleimide group to attach the antibody to the drug. The tag in some instances may be a mutated amino acid. Suitably linker groups incorporating the various groups described above are available in the art.

The substituent groups of the compounds of formula (I) or formula (II) or

pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may interact with DNA sequences and may be selected so as to target specific sequences.

Antibody and antibody fragments

The term "antibody" specifically covers monoclonal antibodies, polyclonal antibodies, dimers, mul timers, multispecific antibodies (e.g., bispecific antibodies), intact antibodies and antibody fragments, so long as they exhibit the desired biological activity, for example, the ability to bind CD19 (Miller et al (2003) Journal, of

Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen.

(Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may

83

SUBSTITUTE SHEET RULE 26 have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgGi , lgG2, lgG3, lgG4, IgAi and lgA2) or subclass, or allotype (e.g. human Gi mi , Gi m2, Gi m3, non-Gi mi [that, is any allotype other than Gi mi], Gi mi7, G21T123, G31TL21 , G31TL28, G31TLI 1 , G31TL5, G3mi3, G31TL14, G3mio, G31TLI5, G3mi6, G3m6, G31TL24, G31TL26, G31TL27, A2mi , A2m2, Kmi , Km2 and Km3) of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

As used herein, "binds an epitope" is used to mean the antibody binds an epitope with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA,

Genbank accession no. CAA76847, version no. CAA76847.1 Gl:3336842, record update date: Jan 7, 201 1 02:30 PM). In some embodiments the antibody binds an epitope with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, 105 or io ⁶ -fold higher than the antibody's association constant for BSA, when measured at physiological conditions.

"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR

(complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be

84

SUBSTITUTE SHEET RULE 26 synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991 )

Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459)·

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to

corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :68si -6855). Chimeric antibodies include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non- human primate (e.g. Old World Monkey or Ape) and human constant region sequences. An "intact antibody" herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHi , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Ci q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes." vThere are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further

85

SUBSTITUTE SHEET RULE 26 divided into "subclasses" (isotypes), e.g., lgGi , lgG2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The antibodies disclosed herein may be modified. For example, to make them less immunogenic to a human subject. This may be achieved using any of a number of techniques familiar to the person skilled in the art, such as humanisation. Antibody-drug conjugates

Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders (Carter, P. (2006) Nature Reviews Immunology 6:343-357). The use of antibody-drug conjugates (ADC), i.e.

immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer, targets delivery of the drug moiety to tumors, and intracellular accumulation therein, whereas systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3)1281 -291 ; Kovtun ef a/ (2006) Cancer Res. 66(6):3214-3121 ; Law et al (2006) CancerRes. 66(4)12328-2337; Wu et al (2005) Nature Biotech. 23(9): 1 137-1 145; Lambert J. (2005) Current Opin. in

Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9): 1087-1 103; Payne, G. (2003) Cancer Cell 3:207-212; Trail ef a/ (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605- 614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug-linking, drug/antibody ratio (loading), and drug-releasing properties (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Doman ef a/ (2009) Blood Ii4(i3):272i -2729; US 7521541 ; US 7723485; WO2009/052249;

McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina ef a/ (2006) Bioconj. Chem. 17:114-124; Erickson ef a/ (2006) CancerRes. 66(8): 1-8; Sanderson et a/ (2005) Clin. CancerRes. 1 1 :843-852; Jeffrey et al (2005) J. Med. Chem. 48:1344- 1358; Hamblett et al (2004) Clin. Cancer Res. 10:7063- 7070). Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition. Some cytotoxic drugs tend

86

SUBSTITUTE SHEET RULE 26 to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

TUMOR- ASSOCIATED ANTIGENS:

(1) BMPRiB (bone morphogenetic protein receptor-type IB, Genbank accession no.

NM_001203)

ten Dijke,P., et al Science 264 (5155): 101-104 (1994), Oncogene 14 (11): 1377- 1382

(1997); WO2004063362 (Claim 2); WO2003042661 (Claim 12); US2003134790-A1 (Page 38-39); WO2002102235 (Claim 13; Page 296); WO2003055443 (Page 91-92);

WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim 6);

WO2003024392 (Claim 2; Fig 112); WO200298358 (Claim 1; Page 183);

WO200254940 (Page 100-101); W0200259377(Page 349- 350); WO200230268

(Claim 27; Page 376); WO200148204 (Example; Fig 4) NP_ooii94 bone

morphogenetic protein receptor, type IB /pid=NP_ooii94.i - Cross-references:

MIM:603248; ΝΡ_οοιΐ94.ΐ; AY065994

(2) E16 (LATi, SLC7A5, Genbank accession no. NM_003486)

Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699): 288- 291 (1998), Gaugitsch, H.W., et al (1992) J. Biol. Chem. 267 (16): 11267-11273);

WO2004048938 (Example 2); WO2004032842 (Example TV); WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example 2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages 222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33; Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (Fig 3); WO2003025138 (Claim 12; Page 150);

NP_003477 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 /pid=NP_003477.3 - Homo sapiens; Cross-references: MIM:6ooi82;

ΝΡ_003477·3; NM_oi5923; NM_003486_i (3) STEAPi (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_oi2449)

Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R.S., et al (1999) Proc. Natl. Acad. Sci. U.SA. 96 (25): 14523-14528); WO2004065577 (Claim 6); WO2004027049 (Fig lL); EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Fig 2); WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; Fig 13A,

87

SUBSTITUTE SHEET RULE 26 Example 53; Page 173, Example 2; Fig 2A); NP_036s8i six transmembrane epithelial antigen of the prostate; Cross-references: ΜΙΜ:6θ44ΐ5; NP_036s8i.i; NM_oi2449_i

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486)

J. Biol. Chem. 276 (29)127371-27375 (2001)); WO2004045553 (Claim 14);

WO200292836 (Claim 6; Fig 12); WO200283866 (Claim 15; Page 116-121);

US2003124140 (Example 16); US 798959; Cross-references: GL34501467;

AAK74120.3; AF36i486_i (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_oos823) Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.SA. 96 (20): 11531-11536 (1999), Proc. Natl. Acad. Sci. U.SA. 93 (1): 136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14); (WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page 308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57); Cross- references: MIM:6oi05i; NP_oos8i4.2; NM_oos823_i

(6) Napi2b (Napi3b, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34

(sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_oo6424) J. Biol. Chem. 277 (22): 19665-19672 (2002), Genomics 62 (2):28i-284 (1999), Feild, J.A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (Claim 2); EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19); WO200157188 (Claim 20; Page 329); WO2004032842 (Example TV); WO200175177 (Claim 24; Page 139-140); Cross-references: ΜΙΜ:6θ42ΐ7; NP_oo64i5.i; NM_oo6424_i

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type l-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al (2000) DNA Res. 7 (2): 143-150); WO2004000997 (Claim 1); WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20);

WO2003101400 (Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC: 10737;

(8) PSCA hlg (2700050Ci2Rik, C5300o80i6Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002) Cancer

88

SUBSTITUTE SHEET RULE 26 Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim 12);

US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056 (Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5); EP1347046 (Claim 1);

WO2003025148 (Claim 20); Cross-references: GL37182378; AAQ88991.1;

AY358628_i

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);

Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al

Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al Jpn. Circ. J. 56, 1303- 1307, 1992; Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A.,

Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663, 1991;

Elshourbagy N. A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J.

Cardiovasc. Pharmacol. 20, S1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;

Strausberg R.L., et al Proc. Natl. Acad. Sci. U.SA. 99, 16899-16903, 2002; Bourgeois C, et al J. Clin. Endocrinol. Metab. 82, 3116- 3123, 1997; Okamoto Y., et al Biol. Chem.

272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002;

Hofstra R.M.W., et al Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E.G., et al

Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;

Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R.M.W., et al Nat. Genet. 12, 445-447, 1996; Svensson PJ., et al Hum. Genet. 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault

V., et al (2002) Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938

(Example 2); WO2004040000 (Claim 151); WO2003087768 (Claim 1);

WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087 (Fig 1);

WO2003016494 (Fig 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim

1; Page 124-125); EP522868 (Claim 8; Fig 2); WO200177172 (Claim 1; Page 297-299);

US2003109676; US6518404 (Fig 3); US5773223 (Claim la; Col 31-34);

WO2004001004; (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no.

NM_oi7703);

WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1); WO2003024392 (Claim 2; Fig 93); WO200166689 (Example 6); Cross-references: LocusID: 54894; NP_o6o233.2; NM_oi7763_i

89

SUBSTITUTE SHEET RULE 26 (11) STEAP2 (HGNC_8639, IPCA-i, PCANAPi, STAMPi, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138)

Lab. Invest. 82 (11): 1573-1582 (2002); WO2003087306; US2003064397 (Claim 1; Fig 1); WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; Fig 4B);

WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612 (Claim 12; Fig 10); WO200226822 (Claim 23; Fig 2); WO200216429 (Claim 12; Fig 10); Cross-references: GL22655488;

AAN04080.1; AF455i38_i

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_oi7036)

Xu, X.Z., et al Proc. Natl. Acad. Sci. U.SA. 98 (19): 10692-10697 (2001), Cell 109 (3):397- 407 (2002), J. Biol. Chem. 278 (33)130813-30820 (2003); US2003143557

(Claim 4); WO200040614 (Claim 14; Page 100-103); WO200210382 (Claim 1; Fig 9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14; Fig lA-D); Cross-references: MIM:6o6936;

NP_o6oio6.2; NM_oi7036_i

(13) CRIPTO (CR, CRi, CRGF, CRIPTO, TDGFi, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212)

Ciccodicola, A., et al EMBO J. 8 (7): 1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555- 565 (1991); US2003224411 (Claim 1); WO2003083041 (Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53); WO2003024392 (Claim 2; Fig 58); WO200216413 (Claim 1; Page 94-95, 105); WO200222808 (Claim 2; Fig 1);

US5854399 (Example 2; Col 17-18); US5792616 (Fig 2); Cross-references: MIM:

187395; NP_003203.i; NM_0032i2_i (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004)

Fujisaku et al (1989) J. Biol. Chem. 264 (4):2ii8-2i25); Weis J. J., et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. U.SA. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.SA. 83, 5639-5643, 1986; Sinha S.K., et al (1993) J. Immunol. 150, 5311- 5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401

90

SUBSTITUTE SHEET RULE 26 (Claim 9); WO2004045520 (Example 4); WO9102536 (Fig 9.1-9.9); WO2004020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79D (CD79B, CD79 , IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_ooo626 or 11038674)

Proc. Natl. Acad. Sci. U.SA. (2003) 100 (7):4126-4131, Blood (2002) 100 (9)13068- 3076, Muller et al (1992) Eur. J. Immunol. 22 (6): 1621-1625); WO2004016225 (claim 2, Fig 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, US6534482 (claim 13, Fig 17A/B); WO200055351 (claim 11, pages 1145-1146); Cross-references: MIM:

147245; NP_ooo6i7.i; NM_ooo626_i

(16) FcRH2 (IFGP4, IRTA4, SPAPiA (SH2 domain containing phosphatase anchor protein la), SPAPiB, SPAPiC, Genbank accession no. NM_030704, AY358130)

Genome Res. 13 (io):226s-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.SA. 98 (i7):9772-9777 (2001), Xu, M.J., et al (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (Claim 2); WO2003077836; WO200138490 (Claim 5; Fig 18D-1-18D-2);

WO2003097803 (Claim 12); WO2003089624 (Claim 25); Cross-references:

MIM: 606509; NP_ii039i.2; NM_030704_i

(17) HEPv2 (ErbB2, Genbank accession no. M11730)

Coussens L., et al Science (1985) 230(4730): 1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.SA. 82, 6497- 6501, 1985; Swiercz J.M., et al J. Cell Biol. 165, 869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A, et al (1993) Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (Fig ll);

WO2004009622; WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1); US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; Fig 1 A-B); WO2003025228 (Claim 37; Fig 5C); WO200222636 (Example 13; Page 95- 107); WO200212341 (Claim 68; Fig 7); WO200213847 (Page 71-74); WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46); WO200141787 (Page 15);

WO200044899 (Claim 52; Fig 7); WO200020579 (Claim 3; Fig 2); US5869445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7);

WO2004043361 (Claim 7); WO2004022709; WO200100244 (Example 3; Fig 4);

Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

91

SUBSTITUTE SHEET RULE 26 (18) NCA (CEACAM6, Genbank accession no. M18728);

Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R.L., et al Proc. Natl. Acad. Sci. U.SA. 99: 16899- 16903, 2002; WO2004063709; EP 1439393 (Claim 7); WO2004044178

(Example 4); WO2004031238; WO2003042661 (Claim 12); WO200278524 (Example 2); WO200286443 (Claim 27; Page 427); WO200260317 (Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728; (19) MDP (DPEPi, Genbank accession no. BC017023)

Proc. Natl. Acad. Sci. U.SA. 99 (26): 16899-16903 (2002); WO2003016475 (Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (Fig 6-8); W09946284 (Fig 9); Cross-references: MIM: 179780; AAH17023.1; BCoi7023_i (20) IL20Ra (IL20Ra, ZCYTOR7, Genbank accession no. AF 184971);

Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Mungall A.J., et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; Dumoutier L., et al J.

Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517- 47523, 2002; Pletnev S., et al (2003) Biochemistry 42: 12617-12624; Sheikh F., et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63);

WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57- 59); WO200146232 (Page 63-65); W09837193 (Claim 1; Page 55-59); Accession:

Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF 184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)

Gary S.C., et al Gene 256, 139-147, 2000; Clark H.F., et al Genome Res. 13, 2265- 2270, 2003; Strausberg R.L., et al Proc. Natl. Acad. Sci. U.SA. 99, 16899-16903, 2002;

US2003186372 (Claim 11); US2003186373 (Claim 11); US2003119131 (Claim 1; Fig 52); US2003119122 (Claim 1; Fig 52); US2003119126 (Claim 1); US2003119121 (Claim 1; Fig 52); US2003119129 (Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; Fig 52); US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1);

(22) EphB2R (DRT, ERK, Heks, EPHT3, Tyros, Genbank accession no. NM_004442) Chan,J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905

(1995), Annu. Rev. Neurosci. 21 :309-345 (1998^ Int. Rev. Cytol. 196: 177-244 (2000); WO2003042661 (Claim 12); WO200053216 (Claim 1; Page 41); WO2004065576

SUBSTITUTE SHEET RULE 26 (Claim l); WO2004020583 (Claim 9); WO2003004529 (Page 128-132);

WO200053216 (Claim 1; Page 42); Cross-references: MIM: 600997; NP_004433.2; NM_004442_i (23) ASLG659 (B7I1, Genbank accession no. AX092328)

US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (Fig 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Fig 60); WO2002102235 (Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6; Fig 10); WO200194641 (Claim 12; Fig 7b); WO200202624 (Claim 13; Fig lA- lB); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2; Page 320- 321, Claim 34; Page 321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; Fig 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12); WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;

(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436) Reiter R.E., et al Proc. Natl. Acad. Sci. U.SA. 95, 1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3)1783- 788; WO2004022709; EP1394274 (Example 11); US2004018553 (Claim 17);

WO2003008537 (Claim 1); WO200281646 (Claim 1; Page 164); WO2003003906

(Claim 10; Page 288); WO200140309 (Example 1; Fig 17); US2001055751 (Example 1; Fig lb); WO200032752 (Claim 18; Fig 1); WO9851805 (Claim 17; Page 97);

W09851824 (Claim 10; Page 94); WO9840403 (Claim 2; Fig lB); Accession: 043653; EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763);

AAP14954 lipoma HMGIC fusion-partner-like protein /pid=AAPi4954.i - Homo sapiens Species: Homo sapiens (human)

WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45); Cross-references: GL30102449; AAP14954.1; ΑΥ26θ703_ι

(26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456); BAFF receptor /pid=NP_443i77.i - Homo sapiens

Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309;

WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (Claim 35; Fig 6B); WO2003035846 (Claim 70; Page 615-616); WO200294852 (Col 136-137);

SUBSTITUTE SHEET RULE 26 WO200238766 (Claim 3; Page 133); WO200224909 (Example 3; Fig 3); Cross- references: MIM:6o6209; NP_443l77.l; NM_052945_l; AF 132600

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2,

FLJ22814, Genbank accession No. AK026467);

Wilson et al (1991) J. Exp. Med. 173 : 137-146; WO2003072036 (Claim 1; Fig 1); Cross- references: MIM: 107266; NP_00l702.l; NM_00l77l_l

(28) CD79a (CD79A, CD790, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pi: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: I9qi3.2, Genbank accession No.

NP_ooi774.io)

WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); W09958658 (claim 13, Fig 16); WO9207574 (Fig 1); US5644033; Ha et al (1992) J. Immunol. 148(5): 1526-1531; Mueller et al (1992) Eur. J. Biochem. 22: 1621-1625; Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp. Immunol. 90(1): 141-146; Yu et al (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al (1988) EMBOJ. 7(n):3457-3464;

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HrV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pi: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 1 iq23.3, Genbank accession No. NP_ooi707.i)

WO2004040000; WO2004015426; US2003105292 (Example 2); US6555339

(Example 2); WO200261087 (Fig 1); WO200157188 (Claim 20, page 269);

WO200172830 (pages 12- 13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); W09928468 (claim 1, page 38); US5440021 (Example 2, col 49-52); W09428931 (pages 56-58); W09217497 (claim 7, Fig 5); Dobner et al (1992) Eur. J. Immunol. 22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779;

(30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pi: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p2i.3, Genbank accession No. NP_002in.i)

Tonnelle et al (1985) EMBO J. 4(n):2839-2847; Jonsson et al (1989) Immunogenetics 29(6):4ii-4i3; Beck et al (1992) J. Mol. Biol. 228:433-441; Strausberg et al (2002)

94

SUBSTITUTE SHEET RULE 26 Proc. Natl. Acad. Sci USA 99: 16899-16903; Servenius et al (1987) J. Biol. Chem.

262:8759-8766; Beck et al (1996) J. Mol. Biol. 255: 1-13; Naruse et al (2002) Tissue Antigens 59:512-519; W09958658 (claim 13, Fig 15); US6153408 (Col 35-38);

US5976551 (col 168-170); US6011146 (col 145-146); Kasahara et al (1989)

Immunogenetics 3θ(ι):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26): 14111- 14119;

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pi: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17P13.3, Genbank accession No. NP_002552.2)

Le et al (1997) FEBSLett. 418(1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al (2000) Genome Res. 10: 165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full maeaity...tafrfpd (1..359; 359 aa), pi: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9Ρ13·3 _> Genbank accession No. NP_ooi773.i)

WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655

(pages 105-106); Von Hoegen et al (1990) J. Immunol. i44(i2):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903;

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus

erythematosis); 661 aa, pi: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5qi2, Genbank accession No. NP_005573.i)

US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996) Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822; WO2003083047; W09744452 (claim 8, pages 57-61); WO200012130 (pages 24-26);

(34) FcRHi (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B- lymphocyte differentiation); 429 aa, pi: 5.28, MW: 46925 TM: 1 [P] Gene

Chromosome: iq2i-iq22, Genbank accession No. NP_443i70.i)

95

SUBSTITUTE SHEET RULE 26 WO2003077836; WO200138490 (claim 6, Fig 18E-1-18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17)19772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7); (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and

lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pi: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: iq2i, Genbank accession No. Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse:

AK089756, AY158090, AY506558; NP_ii257i.i WO2003024392 (claim 2, Fig 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1): 124-127;

WO2003077836; WO200138490 (claim 3, Fig 18B-1-18B-2); (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPPi, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin);

374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276;

NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No.

AF179274; AY358907, CAF85723, CQ782436

WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8);

WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944

(pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706);

US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;

US2004005563; US2003124579; Horie et al (2000) Genomics 67: 146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al (2000) Cancer

Res. 60:4907-12; Glynne- Jones et al (2001) Int J Cancer. Oct 15594(2): 178-84;

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gpioo) BC001414; BT007202; M32295; M77348; NM_oo6928; McGlinchey, R.P. et al (2009) Proc. Natl. Acad. Sci. U.SA. 106 (33), 13731-13736; Kummer, M.P. et al (2009) J. Biol. Chem. 284 (4), 2296-2306;

(38) TMEFFi (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-i); H7365; C9orf2; C9ORF2; U19878; X83961; NM_o8o655;

NM_003092; Harms, P.W. (2003) Genes Dev. 17 (21), 2624-2629; Gery, S. et al (2003) Oncogene 22 (i8):2723-2727;

96

SUBSTITUTE SHEET RULE 26 (39) GDNF-Rai (GDNF family receptor alpha l; GFRAi; GDNFR; GDNFRA; RETLi; TRNRi; RETiL; GDNFR-alphai; GFR- ALPHA- 1) ; U95847; BC014962; NM_145793 NM_005204; Kim, M.H. et al (2009) Mol. Cell. Biol. 29 (8), 2264-2277; Treanor, J.J. et al (1996) Nature 382 (6586)180-83;

(40) Ly6E (lymphocyte antigen 6 complex, locus E, Ly67,RIG-E,SCA-2,TSA-l);

NP_002337.i; NM_002346.2; de Nooij-van Dalen, A G. et al (2003) Int. J. Cancer 103 (6), 768-774; Zammit, D.J. et al (2002) Mol. Cell. Biol. 22 (3):946-952; WO

(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_00i007539.i;

NM_00i007538.i; Furushima, K. et al (2007) Dev. Biol. 306 (2), 480-492; Clark, H.F. et al (2003) Genome Res. 13 (10)12265-2270; (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGTl);

NP_o67079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80 (1): 113-123;

Ribas, G. et al (1999) J. Immunol. 163 (1)1278-287;

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); NP_003058.i; NM_003667.2; Salanti, G. et al (2009) Am. J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al (2003) Hematology 37 (3):528-533;

(44) RET (ret proto-oncogene; MEN2A; HSCRi; MEN2B; MTCi; PTC; CDHF12;

Hs.168114; RET51; RET-ELEi); NP_o66i24.i; ΝΜ_020975·4; Tsukamoto, H. et al (2009) Cancer Sci. 100 (10): 1895-1901; Narita, N. et al (2009) Oncogene 28

(34):3058-3o68;

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); ΝΡ_059997·3; NM_o 17527.3; Ishikawa, N. et al (2007) Cancer Res. 67 (24): 11601- 11611; de Nooij-van Dalen, A G. et al (2003) Int. J. Cancer 103 (6)1768-774;

(46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_oo6i34.i; NM_oo6i43.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105 (1-2): 162-164; O'Dowd, B.F. et al (1996) FEBS Lett. 394 (3):325"329;

97

SUBSTITUTE SHEET RULE 26 (47) GPR54 (KISSi receptor; KISSiR; GPR54; HOT7T175; AXOR12); NP_ii5940.2; NM 032551.4; Navenot, J.M. et al (2009) Mol. Pharmacol. 75 (6): 1300-1306; Hata, K. et al (2009) Anticancer Res. 29 (2):6i7-623; (48) ASPHDi (aspartate beta-hydroxylase domain containing 1; LOC253982);

NP_859069.2; NM_i8i7i8.3; Gerhard, D.S. et al (2004) Genome Res. 14 (ioB):2i2i- 2127;

(49) Tyrosinase (TYR; OCAIA; OCAiA; tyrosinase; SHEP3); NP_000303.i;

NM_000372.4; Bishop, D.T. et al (2009) Nat. Genet. 41 (8)1920-925; Nan, H. et al (2009) Int. J. Cancer 125 (4): 909-917;

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ 14627);

NP_ooii03373.i; NM 001109903.1; Clark, H.F. et al (2003) Genome Res. 13

(10)12265- 2270; Scherer, S.E. et al (2006) Nature 440 (7082)1346-351

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; DisErtd747e); NP_078807.i; NM_024531.3; Ericsson, T.A. et al (2003) Proc. Natl. Acad. Sci. U.SA. 100 (ii):6759-6704; Takeda, S. et al (2002) FEBS Lett. 520 (i-3):97-ioi.

(52) CD33, a member of the sialic acid binding, immunoglobulin-like lectin family, is a 67- kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells in addition to committed myelomonocytic and erythroid progenitor cells. It is not seen on the earliest pluripotent stem cells, mature

granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et al, (1985) J. Clin. Invest. 75:756-56; Andrews et al, (1986) Blood 68: 1030-5). CD33 contains two tyrosine residues on its cytoplasmic tail, each of which is followed by hydrophobic residues similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.

(53) CLL-i (CLEC12A, MICL, and DCAL2), encodes a member of the C-type lectin/C- type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signalling, glycoprotein turnover, and roles in inflammation and immune response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.

98

SUBSTITUTE SHEET RULE 26 This gene is closely linked to other CTL/ CTLD superfamily members in the natural killer gene complex region on chromosome I2pi3 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9 (5)1585-90; van Rhenen A, et al, (2007) Blood 110 (7):2059-66; Chen CH, et al. (2006) Blood 107 (4): 1459-67; Marshall AS, et al. (2006) Eur. J. Immunol. 36 (8)12159-69; Bakker AB, et al (2005) Cancer Res. 64 (22)18443-50; Marshall AS, et al (2004) J. Biol. Chem. 279 (15): 14792-802). CLL-i has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain and a short cytoplasmic tail containing an ITIM motif.

Anti-CD22 Antibodies

In certain embodiments, the anti-CD22 antibodies of an ADC comprises three light chain hypervariable regions (HVR-Li, HVR-L2 and HVR-L3) and three heavy chain hypervariable regions (HVR-Hi, HVR-H2 and HVR-H3), according to US 8226945:

HVR-Ll RSSQSIVHSVGNTFLE (SEQ ID NO: 1)

HVR-L2 KVSNRFS (SEQ ID NO: 2)

HVR-L3 FQGSQFPYT (SEQ ID NO: 3)

HVR-Hl GYEFSRSWMN (SEQ ID NO: 4)

HVR-H2 GR1YPGDGDTNYSGKFKG (SEQ ID NO: 5)

HVR-H3 DGSSWDWYFDV (SEQ ID NO: 6)

Anti-Ly6E Antibodies

In certain embodiments, an ADC comprises anti-Ly6E antibodies. Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoic acid induced gene E (RIG-E) and stem cell antigen 2 (SCA-2). It is a GPI linked, 131 amino acid length, ~8.4kDa protein of unknown function with no known binding partners. It was initially identified as a transcript expressed in immature thymocyte, thymic medullary epithelial cells in mice (Mao, et al. (1996) Proc. Natl. Acad. Sci. U.SA. 93 :59io-59i4). In some embodiments, the invention provides an immunoconjugate comprising an anti-Ly6E antibody described in PCT Publication No. WO 2013/177055.

In some embodiments, the invention provides an antibody-drug conjugate comprising an anti-Ly6E antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) HVR-Li comprising the

99

SUBSTITUTE SHEET RULE 26 amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 14.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 14; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising the amino acid

100

SUBSTITUTE SHEET RULE 26 sequence of SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In any of the above embodiments, an anti-Ly6E antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-Ly6E antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework. In another aspect, an anti-Ly6E antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Ly6E antibody comprising that sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 8. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/ or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibody comprises the VH sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

In another aspect, an anti-Ly6E antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:7 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Ly6E antibody comprising that sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7. In certain embodiments, a total of 1 to 5 amino acids have been substituted,

101

SUBSTITUTE SHEET RULE 26 inserted and/or deleted in SEQ ID NO: 7. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibody comprises the VL sequence of SEQ ID NO: 7, including post- translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an antibody-drug conjugate comprising an anti-Ly6E antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, an antibody-drug conjugate is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 8 and SEQ ID NO: 7, respectively, including post-translational modifications of those sequences.

In a further aspect, provided herein are antibody-drug conjugate comprising antibodies that bind to the same epitope as an anti-Ly6E antibody provided herein. For example, in certain embodiments, an immunoconjugate is provided comprising an antibody that binds to the same epitope as an anti-Ly6E antibody comprising a VH sequence of SEQ ID NO: 8 and a VL sequence of SEQ ID NO: 7, respectively.

In a further aspect of the invention, an anti-Ly6E antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-Ly6E antibody of an antibody-drug conjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein. In some embodiments, an immunconjugate (ADC) comprises an anti- Ly6E antibody comprising a heavy chain and a light chain comprising the amino acid sequences of SEQ ID NO: 16 and 15, respectively.

Table of Ly6E AntibodySequences

102

SUBSTITUTE SHEET RULE 26 I1U9B12 vi2 GKTVKLLIYYTSNLHSGVPS RFSGSGSGTD YTLTISSLQP light chain

variable region EDFATYYCQQ YSELPWTFGQ GTKVEIK

anti-Ly6E EVQLVESGPA LVKPTQTLTL TCTVSGFSLT

antibody GYSVNWIRQPPGKAL EWLGMIWGDG STDYNSALKS hu9Bi2 vi2 RLTISKDTSK NQWLTMTNM DPVDTATYYC

heavy chain ARDYYFNYAS WFAYWGQGTL VTVSS

variable region

anti-Ly6E SASQGISNYLN

antibody

hu9Bi2 vi2

HVR-Li

anti-Ly6E YTSNLHS

antibody

hu9Bi2 vi2

HVR-L2

anti-Ly6E QQYSELPWT

antibody

hu9Bi2 vi2

HVR-L3

anti-Ly6E GFSLTGYSVN

antibody

hu9Bi2 vi2

HVR-Hi

anti-Ly6E MIWGDGSTDY NSALKS

antibody

hu9Bi2 vi2

HVR-H2

anti-Ly6E DYYVNYASWFAY

antibody

hu9Bi2 vi2

HVR-H3

anti-Ly6E DIQMTQSPSS LSASVGDRVT ITCSASQGIS NYLNWYQQKP antibody GKTVKLLIYYTSNLHSGVPS RFSGSGSGTD YTLTISSLQP hu9Bi2 vi2 EDFATYYCQQ YSELPWTFGQ GTKVEIK RWAAPSVFIF K149C kappa PPSDEQLKSG TASWCLLNN FYPREAKVQW

light chain CVDNALQSGN

103

SUBSTITUTE SHEET RULE 26 SQESVTEQDS KDSTYSLSST LTLSKADYEK

HKVYACEVTH

QGLSSPVTKS FNRGEC

16 anti-Ly6E EVQL VESGPA LVKPTQTLTL TCTVSGFSLT GYSVNWIRQP antibody PGKALEWLGM IWGDGSTDYN SALKSRLTIS

I1U9B12 V12 KDTSKNQWL

IgGi heavy TMTNMDPVDT ATYYCARDYY FNYASWFAYW

chain GQGTLVTVSS

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG

PSVFLFPPKP KDTLMISRTP EV CVWDVS HEDPEVKFNW YVD GVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK

EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV

MHEALHNHYT

QKSLSLSPGK

Anti-HER2 Antibodies

In certain embodiments, an ADC comprises anti-HER2 antibodies. In one embodiment of the invention, an anti-HER2 antibody of an ADC of the invention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-i, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5- 8, as described in Table 3 of US 5821337, which is specifically incorporated by reference herein. Those antibodies contain human framework regions with the complementarity- determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also referred to as trastuzumab, commercially available under the tradename HERCEPTIN®. In another embodiment of the invention, an anti- HER2 antibody of an ADC of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US7862817. An exemplary humanized 2C4 antibody is pertuzumab, commercially available under the tradename PERJETA®.

104

SUBSTITUTE SHEET RULE 26 In another embodiment of the invention, an anti-HER2 antibody of an ADC of the invention comprises a humanized 7C2 anti-HER2 antibody. A humanized 7C2 antibody is an anti-HER2 antibody. In some embodiments, the invention provides an antibody-drug conjugate comprising an anti-HER2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the invention provides an antibody-drug conjugate comprising an anti-HER2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29. In one aspect, the invention provides an immunoconjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

105

SUBSTITUTE SHEET RULE 26 In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO : 21. In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 24 or 29; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 24; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of

106

SUBSTITUTE SHEET RULE 26 SEQ ID NO: 23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

In any of the above embodiments, an anti-HER2 antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-HER2 antibody of an antibody- drug conjugate comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-HER2 antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 18 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-HER2 antibody comprising that sequence retains the ability to bind to HER2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 18. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 18. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- HER2 antibody comprises the VH sequence of SEQ ID NO: 18, including post- translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In another aspect, an anti-HER2 antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 17 contains substitutions (e.g., conservative

substitutions), insertions, or deletions relative to the reference sequence, but an anti-

107

SUBSTITUTE SHEET RULE 26 HER2 antibody comprising that sequence retains the ability to bind to HER2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 17. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 17. In certain

embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprises the VL sequence of SEQ ID NO: 17, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

In another aspect, an antibody-drug conjugate comprising an anti-HER2 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, an antibody-drug conjugate comprising an antibody is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 18 and SEQ ID NO: 17, respectively, including post-translational modifications of those sequences.

In one embodiment, an antibody-drug conjugate comprising an antibody is provided, wherein the antibody comprises the humanized 7C2.V2.2.LA (hu7C2) K149C kappa light chain sequence of SEQ ID NO: 30 In one embodiment, an antibody-drug conjugate comprising an antibody is provided, wherein the antibody comprises the HU7C2 A118C IgGi heavy chain sequence of SEQ ID NO: 31

In a further aspect, provided herein are antibody-drug conjugates comprising antibodies that bind to the same epitope as an anti-HER2 antibody provided herein.

For example, in certain embodiments, an immunoconjugate is provided, comprising an antibody that binds to the same epitope as an anti-HER2 antibody comprising a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 17, respectively. In a further aspect of the invention, an anti-HER2 antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-HER2 antibody of an

108

SUBSTITUTE SHEET RULE 26 immunoconjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, an immunoconjugate comprises an antibody that is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.

Table of humanized 7C2 anti-HER2 antibody sequences

109

SUBSTITUTE SHEET RULE 26 TKSFNRGEC

Humanized EVQLVQSGAE VKKPGASVKV SCKASGYSFT 7C2.V2.2.LA GYWMNWVRQA PGQGLEWIGM IHPLDAEIRA (hu7C2) IgGi NQKFRDRVTI TVDTSTSTAY LELSSLRSED heavy chain TAVYYCARGT YDGGFEYWGQ GTLVTVSSAS

TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

HU7C2. MIHPMDSEIRANQKFRD

V2.1.S53M

HVR-H2

HU7C2. MIHPLDSEIRANQKFRD

V2.1.S53L

HVR-H2

HU7C2. GTYDGGFKY

V2.1.E101K

HVR-H3

Humanized DIVMTQSPDS LAVSLGERAT INCRASQSVS 7C2.V2.2.LA GSRFTYMHWY QQKPGQPPKL LIKYASILES (hu7C2) K149C GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY kappa light YCQHSWEIPP WTFGQGTKVE IKRTVAAPSV chain FIFPPSDEQL KSGTASWCL LNNFYPREAK

VQWCVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Humanized EVQLVQSGAE VKKPGASVKV SCKASGYSFT 7C2.V2.2.LA GYWMNWVRQA PGQGLEWIGM IHPLDAEIRA (hu7C2) A118C NQKFRDRVTI TVDTSTSTAY LELSSLRSED IgGi heavy TAVYYCARGT YDGGFEYWGQ GTLVTVSSCS

110

SUBSTITUTE SHEET RULE 26 chain TKGPSVFPLA PSSKSTSGGT AALGCLVKDY

FPEPVTVSWN SGALTSGVHT FPAVLQSSGL

YSLSSWTVP SSSLGTQTYI CNVNHKPSNT

KVDKKVEPKS CDKTHTCPPC PAPELLGGPS

VFLFPPKPKD TLMISRTPEV TCVWDVSHE

DPEVKFNWYV DGVEVHNAKT KPREEQYNST

YRWSVLTVL HQDWLNGKEY KCKVSNKALP

APIEKTISKA KGQPREPQVY TLPPSREEMT

KNQVSLTCLV KGFYPSDIAV EWESNGQPEN

NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ

GNVFSCSVMH EALHNHYTQK SLSLSPGK

Anti-MUCi6 Antibodies

In certain embodiments, an ADC comprises anti-MUCi6 antibodies.

In some embodiments, the invention provides an antibody-drug conjugate comprising an anti-MUCi6 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34· In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32; (b)

111

SUBSTITUTE SHEET RULE 26 HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (c) HVR- L3 comprising the amino acid sequence of SEQ ID NO: 34.

In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 37; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35 (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34· In any of the above embodiments, an anti-MUCi6 antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-MUCi6 antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-MUCi6 antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 39 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MUCi6 antibody comprising

112

SUBSTITUTE SHEET RULE 26 that sequence retains the ability to bind to MUC16. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 39. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 39. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- MUC16 antibody comprises the VH sequence of SEQ ID NO: 39, including post- translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37·

In another aspect, an anti-MUCi6 antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:38 contains substitutions (e.g., conservative

substitutions), insertions, or deletions relative to the reference sequence, but an anti- MUC16 antibody comprising that sequence retains the ability to bind to MUC16. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 38. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 38. In certain

embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-MUCi6 antibody comprises the VL sequence of SEQ ID NO: 38, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR- L2 comprising the amino acid sequence of SEQ ID NO: 33; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

In another aspect, an antibody-drug conjugate comprising an anti-MUCi6 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

113

SUBSTITUTE SHEET RULE 26 In one embodiment, an antibody-drug conjugate is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 39 and SEQ ID NO: 38, respectively, including post-translational modifications of those sequences. In a further aspect, provided herein are antibody-drug conjugate comprising antibodies that bind to the same epitope as an anti-MUCi6 antibody provided herein. For example, in certain embodiments, an immunoconjugate is provided comprising an antibody that binds to the same epitope as an anti-MUCi6 antibody comprising a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 38, respectively.

In a further aspect of the invention, an anti-MUCi6 antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-MUCi6 antibody of an antibody-drug conjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGi antibody, IgG2a antibody or other antibody class or isotype as defined herein.

Table of MUC16 Antibody Sequences

114

SUBSTITUTE SHEET RULE 26 37 Anti-Mucl6 ARWASGLDY

antibody

HVR-H3

38 Anti-Mucl6 DIQMTQSPSS LSASVGDRVT ITCKASDLIH

antibody light NWLAWYQQKP GKAPKLLIYG ATSLETGVPS chain variable RFSGSGSGTD FTLTISSLQP EDFATYYCQQ

region YWTTPFTFGQ GTKVEIKR

39 Anti-Mucl6 EVQLVESGGG LVQPGGSLRL SCAASGYSIT

antibody heavy NDYAWNWVRQ APGKGLEWVG YISYSGYTTY chain variable NPSLKSRFTI SRDTSKNTLY LQMNSLRAED

region TAVYYCARWA SGLDYWGQGT LVTVSS

Anti-STEAP-i Antibodies

In certain embodiments, an ADC comprises anti-STEAP-i antibodies.

In some embodiments, the invention provides an antibody-drug conjugate comprising an anti-STEAP-i antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45·

In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43; (b)

115

SUBSTITUTE SHEET RULE 26 HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR- L3 comprising the amino acid sequence of SEQ ID NO: 45.

In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 42; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40 (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45· In any of the above embodiments, an anti-STEAP-i antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-STEAP-i antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-STEAP-i antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94% _> 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 46 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-STEAP-i antibody comprising

116

SUBSTITUTE SHEET RULE 26 that sequence retains the ability to bind to STEAP-i. In certain embodiments, a total of l to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 46. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 46. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- STEAP-i antibody comprises the VH sequence of SEQ ID NO: 46, including post- translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

In another aspect, an anti-STEAP-i antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 47 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- STEAP-i antibody comprising that sequence retains the ability to bind to STEAP-i. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 47 In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-STEAP-i antibody comprises the VL sequence of SEQ ID NO: 47, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 43; (b) HVR- L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

In another aspect, an antibody-drug conjugate comprising an anti-STEAP-i antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

117

SUBSTITUTE SHEET RULE 26 In one embodiment, an antibody-drug conjugate is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 46 and SEQ ID NO: 47, respectively, including post-translational modifications of those sequences. In a further aspect, provided herein are antibody-drug conjugate comprising antibodies that bind to the same epitope as an anti-STEAP-i antibody provided herein. For example, in certain embodiments, an immunoconjugate is provided comprising an antibody that binds to the same epitope as an anti-STEAP-i antibody comprising a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47, respectively.

In a further aspect of the invention, an anti-STEAP-i antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-STEAP-i antibody of an antibody-drug conjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.

Table of STEAP Antibody Sequences

118

SUBSTITUTE SHEET RULE 26 TAVYYCARER NYDYDDYYYA MDYWGQGTLV TVSS

47 Anti-STEAP-i DIQMTQSPSS LSASVGDRVT ITCKSSQSLL

light chain YRSNQKNYLA WYQQKPGKAP KLLIYWASTR

variable region ESGVPSRFSG SGSGTDFTLT ISSLQPEDFA

TYYCQQYYNY PRTFGQGTKV EIK

Anti-NaPi2b Antibodies

In certain embodiments, an ADC comprises anti-NaPi2b antibodies. In some embodiments, the invention provides an antibody-drug conjugate comprising an anti- NaPi2b antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52 and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR

119

SUBSTITUTE SHEET RULE 26 sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 48, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 50; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 48 (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53·

In any of the above embodiments, an anti-NaPi2b antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-NaPi2b antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-NaPi2b antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 54. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 54 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-NaPi2b antibody comprising that sequence retains the ability to bind to NaPi2b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 54. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 54. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- NaPi2b antibody comprises the VH sequence of SEQ ID NO: 54, including post- translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino

120

SUBSTITUTE SHEET RULE 26 acid sequence of SEQ ID NO: 48, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50. In another aspect, an anti-NaPi2b antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 55. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 55 contains substitutions (e.g., conservative

substitutions), insertions, or deletions relative to the reference sequence, but an anti- NaPi2b antibody comprising that sequence retains the ability to bind to anti-NaPi2b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 55. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 55. In certain

embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2b antibody comprises the VL sequence of SEQ ID NO: 55, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 51; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

In another aspect, an antibody-drug conjugate comprising an anti-NaPi2b antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, an antibody-drug conjugate is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 54 and SEQ ID NO: 55,

respectively, including post-translational modifications of those sequences.

In a further aspect, provided herein are antibody-drug conjugate comprising antibodies that bind to the same epitope as an anti-NaPi2b antibody provided herein. For example, in certain embodiments, an immunoconjugate is provided comprising an antibody that binds to the same epitope as an anti-NaPi2b antibody comprising a VH sequence of SEQ ID NO: 54 and a VL sequence of SEQ ID NO: 55, respectively.

121

SUBSTITUTE SHEET RULE 26 In a further aspect of the invention, an anti-NaPi2b antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-NaPi2b antibody of an antibody-drug conjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, F(ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.

Table of NaPi2b Antibody Sequences

Anti-CD79b Antibodies

In certain embodiments, an ADC comprises anti-CD79b antibodies. In some embodiments, the invention provides an antibody-drug conjugate comprising an anti- CD79b antibody comprising at least one, two, three, four, five, or six HVRs selected

122

SUBSTITUTE SHEET RULE 26 from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In one aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60. In a further embodiment, the antibody comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; and (c) HVR- H3 comprising the amino acid sequence of SEQ ID NO: 60.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In another aspect, an antibody-drug conjugate of the invention comprises an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 60; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In another aspect, the invention provides an antibody-drug conjugate comprising an antibody that comprises (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; (c) HVR-

123

SUBSTITUTE SHEET RULE 26 H3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In any of the above embodiments, an anti-CD79b antibody of an antibody-drug conjugate is humanized. In one embodiment, an anti-CD79b antibody comprises HVRs as in any of the above embodiments, and further comprises a human acceptor framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-CD79b antibody of an antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 56. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 56 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CD79b antibody comprising that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 56. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 56. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- CD79b antibody comprises the VH sequence of SEQ ID NO: 8, including post- translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hi comprising the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60.

In another aspect, an anti-CD79b antibody of an antibody-drug conjugate is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 57. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 57 contains substitutions (e.g., conservative

substitutions), insertions, or deletions relative to the reference sequence, but an anti-

124

SUBSTITUTE SHEET RULE 26 CD79I) antibody comprising that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 57. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 57. In certain

embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprises the VL sequence of SEQ ID NO: 57, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-Li comprising the amino acid sequence of SEQ ID NO: 61; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

In another aspect, an antibody-drug conjugate comprising an anti-CD79b antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

In one embodiment, an antibody-drug conjugate is provided, wherein the antibody comprises the VH and VL sequences in SEQ ID NO: 56 and SEQ ID NO: 57, respectively, including post-translational modifications of those sequences.

In a further aspect, provided herein are antibody-drug conjugate comprising antibodies that bind to the same epitope as an anti-CD79b antibody provided herein. For example, in certain embodiments, an immunoconjugate is provided comprising an antibody that binds to the same epitope as an anti-CD79b antibody comprising a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ ID NO: 57, respectively.

In a further aspect of the invention, an anti-CD79b antibody of an antibody-drug conjugate according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti-CD79b antibody of an antibody-drug conjugate is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein. Table of CD79b Antibody Sequences

SEQ ID Description Sequence

125

SUBSTITUTE SHEET RULE 26 NO

56 anti-CD79b EVQLVESGGG LVQPGGSLRL SCAASGYTFS

huMA79bv28 SYWIEWVRQA PGKGLEWIGE ILPGGGDTNY heavy chain NEIFKGRATF SADTSKNTAY LQMNSLRAED variable region TAVYYCTRRV PIRLDYWGQG TLVTVSS

57 anti-CD79b DIQLTQSPSS LSASVGDRVT ITCKASQSVD

huMA79bv28 YEGDSFLNWY QQKPGKAPKL LIYAASNLES light chain GVPSRFSGSG SGTDFTLTIS SLQPEDFATY variable region YCQQSNEDPL TFGQGTKVEI KR

58 anti-CD79b GYTFSSYWIE

huMA79bv28

HVR-Hl

59 anti-CD79b GEILPGGGDTNYNEIFKG

huMA79bv28

HVR-H2

60 anti-CD79b TRRVPIRLDY

huMA79bv28

HVR-H3

61 anti-CD79b KASQSVDYEGDSFLN

huMA79bv28

HVR-Ll

62 anti-CD79b AASNLES

huMA79bv28

HVR-L2

63 anti-CD79b QQSNEDPLT

huMA79bv28

HVR-L3

Human HER2 Precursor Protein

Details of an exemplary human HER2 precursor protein with signal sequences is provided below

SEQ ID Description Sequence

NO

64 Exemplary MELAALCRWG LLLALLPPGA ASTQVCTGTD

human HER2 MKLRLPASPE THLDMLRHLY QGCQWQGNL precursor ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ

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SUBSTITUTE SHEET RULE 26 protein, with VRQVPLQRLR IVRGTQLFED NYALAVLDNG signal DPLNNTTPVT GASPGGLREL QLRSLTEILK sequence GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA

LTLIDTNRSR ACHPCSPMCK GSRCWGESSE

DCQSLTRTVC AGGCARCKGP LPTDCCHEQC

AAGCTGPKHS DCLACLHFNH SGICELHCPA

LVTYNTDTFE SMPNPEGRYT FGASCVTACP

YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR

CEKCSKPCAR VCY GLGMEHL REVRAVTSAN

IQEFAGCKKI FGSLAFLPES FDGDPASNTA

PLQPEQLQ ETLEEITGYL YISAWPDSLP

DLSVFQNLQV IRGRILHNGA YSLTLQGLGI

SWLGLRSLRE LGSGLALIHH NTHLCFVHTV

PWDQLFRNPH QALLHTANRP EDECVGEGLA

CHQLCARGHC WGPGPTQCVN CSQFLRGQEC

VEECRVLQGL PREYVNARHC LPCHPECQPQ

NGSVTCFGPE ADQCVACAHY KDPPFCVARC

PSGVKPDLSY MPIWKFPDEE GACQPCPINC

THSCVDLDDK GCPAEQRASP LTSIISAWG

ILLVWLGW FGILIKRRQQ KIRKYTMRRL

LQETELVEPL TPSGAMPNQA QMRILKETEL

RKVKVLGSGA FGTVYKGIWI PDGENVKIPV

AIKVLRENTS PKANKEILDE AYVMAGVGSP

YVSRLLGICL TSTVQLVTQL MPYGCLLDHV

RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR

LVHRDLAARN VLVKSPNHVK ITDFGLARLL

DIDETEYHAD GGKVPIKWMA LESILRRRFT

HQSDVWSYGV TVWELMTFGA KPYDGIPARE

IPDLLEKGER LPQPPICTID VYMIMFVKCWM

IDSECRPRFR ELVSEFSRMA RDPQRFWIQ

NEDLGPASPL DSTFYRSLLE DDDMGDLVDA

EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS

STRSGGGDLT LGLEPSEEEA PRSPLAPSEG

AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ

RYSEDPTVPL PSETDGYVAP LTCSPQPEYV

NQPDVRPQPP SPREGPLPAA RPAGATLERP

KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ

127

SUBSTITUTE SHEET RULE 26 GGAAPQPHPP PAFSPAFDNL YYWDQDPPER

GAPPSTFKGT PTAENPEYLG LDVPV

Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of≤ ΐμΜ,≤ loo nM,≤ 50 nM,≤ 10 nM,≤ 5 nM,≤ 1 nM,≤ 0.1 nM,≤ 0.01 nM, or≤ 0.001 nM, and optionally is > lo ^ M. (e.g. 10 ⁸ M or less, e.g. from 10 ⁸ M to lo ^ M, e.g., from 10^ M to lo ^ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti- Fab antibody-coated plate (see, e.g., Chen et ah, J. Mol. Biol. 293 :865-88i(i999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo

Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature

(approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et ah, Cancer Res. 57:4593- 4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μΐ/well of scintillant (MICROSCF T-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmon resonance assays using a BIAC0RE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~io response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with

128

SUBSTITUTE SHEET RULE 26 N- ethyl-^'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μΐ/min. Association rates (k _on ) and dissociation rates (k ₀ ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k ₀ ff/k _on , See, e.g., Chen et al., J. Mol. Biol. 293 :865-88i (1999). If the on-rate exceeds 10 ⁶ M ¹ s ¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instalments) or a 8000-series SLM-AMINCO spectrophotometer (ThermoSpectronic) with a stirred cuvette.

Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment.

Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448

129

SUBSTITUTE SHEET RULE 26 (1993)· Triabodies and tetrabodies are also described in Hudson et ah, Nat. Med. 9: 129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

Chimeric and Humanized Antibodies In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et ah, Proc. Natl. Acad. Sci. USA, 81 :685i-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13 : 1619-1633 (2008), and are further described, e.g., in Riechmann et ah, Nature 332:323-329 (1988); Queen et ah, Proc. Nat'lAcad. Sci. USA

130

SUBSTITUTE SHEET RULE 26 86: 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and

7,087,409; Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing");

Dall'Acqua et al, Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68 (2005) and Klimka et al, Br. J. Cancer, 83 :252-26o (2000) (describing the "guided selection" approach to FR shuffling).

Human framework regions that maybe used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 12296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151 12623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13 : 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al, J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al, J. Biol. Chem. 271 :226n-226i8 (1996)).

Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin.

Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23 : 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing

HuMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE®

technology, and U.S. Patent Application Publication No. US 2007/0061900, describing

131

SUBSTITUTE SHEET RULE 26 VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133 : 3001 (1984); Brodeur et ah, Monoclonal Antibody Production Techniques and Applications, pp. 51- 63 (Marcel Dekker, Inc., New York, 1987); and Boerner et ah, J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et ah, Proc. Natl. Acad. Sci. USA, 103 :3557-3562 (2θθ6). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4)1265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3)1927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et ah, ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et ah, Nature 348:552-554; Clackson et ah, Nature 352: 624-628 (1991); Marks et ah, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et ah, J. Mol. Biol. 338(2): 299-310 (2004); Lee et ah, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol.

Methods 284(1-2): 119-132(2004).

132

SUBSTITUTE SHEET RULE 26 In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et ah, Ann. Rev. Immunol, 12: 433-455 (1994). Phage typically display antibody fragments, either as single- chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et ah, EMBO J 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

Multispecific Antibodies In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, bispecific antibodies may bind to two different epitopes of the same target. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express the target. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et ah, EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). The term "knob-into-hole" or "KnH" technology as used herein refers to the technology directing the pairing of two

133

SUBSTITUTE SHEET RULE 26 polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact. For example, KnHs have been introduced in the Fc:Fc binding interfaces, CL:CHi interfaces or VH/VL interfaces of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, Zhu et ah, 1997, Protein Science 6:781-788, and WO2012/106587). In some embodiments, KnHs drive the pairing of two different heavy chains together during the manufacture of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can be also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions).

The term "knob mutation" as used herein refers to a mutation that introduces a protuberance (knob) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

The term "hole mutation" as used herein refers to a mutation that introduces a cavity (hole) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation. A brief nonlimiting discussion is provided below.

A "protuberance" refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the heteromultimer, and thereby favor heteromultimer formation over homomultimer formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). In some embodiments, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one "import" amino acid residue which has a larger side chain volume than the original amino acid residue. It will be appreciated that there can

134

SUBSTITUTE SHEET RULE 26 be more than one original and corresponding import residue. The side chain volumes of the various amino residues are shown, for example, in Table 1 of US2011/0287009. A mutation to introduce a "protuberance" maybe referred to as a "knob mutation." In some embodiments, import residues for the formation of a protuberance are naturally occurring amino acid residues selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments, an import residue is tryptophan or tyrosine. In some embodiment, the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

A "cavity" refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). In some embodiments, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. In some embodiments, import residues for the formation of a cavity are naturally occurring amino acid residues selected from alanine (A), serine (S), threonine (T) and valine (V). In some embodiments, an import residue is serine, alanine or threonine. In some embodiments, the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. A mutation to introduce a "cavity" may be referred to as a "hole mutation."

The protuberance is "positionable" in the cavity which means that the spatial location of the protuberance and cavity on the interface of a first polypeptide and second polypeptide respectively and the sizes of the protuberance and cavity are such that the protuberance can be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of a protuberance with a corresponding cavity may, in some instances, rely on modeling the protuberance/cavity pair based upon a three-dimensional structure such as that obtained by X-ray

135

SUBSTITUTE SHEET RULE 26 crystallography or nuclear magnetic resonance (NMR). This can be achieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgGl constant region is T366W (EU numbering). In some embodiments, a hole mutation in an IgGl constant region comprises one or more mutations selected from T366S, L368A and Y407V (EU numbering). In some embodiments, a hole mutation in an IgGl constant region comprises T366S, L368A and Y407V (EU numbering).

In some embodiments, a knob mutation in an IgG4 constant region is T366W (EU numbering). In some embodiments, a hole mutation in an IgG4 constant region comprises one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, a hole mutation in an IgG4 constant region comprises T366S, L368A, and Y407V (EU numbering).

Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et ah, Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et ah, J. Immunol, 148(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et ah, Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber et ah, J. Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et ah J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to the target as well as another, different antigen (see, US 2008/0069820, for example).

Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/ or other biological properties of the antibody. Amino acid sequence

136

SUBSTITUTE SHEET RULE 26 variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid

substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown below in a Table of conservative substitutions under the heading of "preferred substitutions." More substantial changes are provided in the Table under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased

immunogenicity, or improved ADCC or CDC.

Table of conservative substitutions

137

SUBSTITUTE SHEET RULE 26 Pro (P) Ala Ala

Ser (S) Thr Thr

Thr (T) Val; Ser Ser

Trp (W) Tyr; Phe Tyr

Tyr (Y) Trp; Phe; Thr; Ser Phe

Val (V) He; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(l) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/ or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a- CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been

138

SUBSTITUTE SHEET RULE 26 described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid

substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants maybe screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/ or carboxyl -terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl

139

SUBSTITUTE SHEET RULE 26 residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Glycosylation variants

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl

glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some

embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%). The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108; US 2004/0093621. Examples of publications related to "defucosylated" or "fucose- deficient" antibody variants include: US

2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US

2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US

140

SUBSTITUTE SHEET RULE 26 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; wo

2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha-1,6- fucosyl transferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94(4)1680-688 (2006); and

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/ or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean- Mairet et al); US Patent No. 6,602,684 (Umana et al); and US 2005/0123546 (Umana et al). Antibody variants with at least one galactose residue in the

oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

Fc region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions. In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK

141

SUBSTITUTE SHEET RULE 26 cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non- limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'lAcad. Sci. USA 83 17059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'lAcad. Sci. USA 82: 1499-1502 (1985);

5,821,337 (see Bruggemann, M. et al, J. Exp. Med. 166: 1351-1361 (1987)).

Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood

mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'lAcad. Sci. USA 95:652-656 (1998). Ciq binding assays may also be carried out to confirm that the antibody is unable to bind Ciq and hence lacks CDC activity. See, e.g., Ciq and C3C binding ELISA in WO 2006/029879 and WO 2005/ 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al, J. Immunol.

Methods 202: 163 (1996); Cragg, M.S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103 : 2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int Ί. Immunol. 18(12): 1759-1769 (2006)). In some embodiments, one or more amino acid modifications may be introduced into the Fc portion of the antibody provided herein in order to increase IgG binding to the neonatal Fc receptor. In certain embodiments, the antibody comprises the following three mutations according to EU numbering: M252Y, S254T, and T256E (the "YTE mutation") (US Patent No. 8,697,650; see also Dall'Acqua et al, Journal of Biological Chemistry 28i(33):235i4-23524 (2006). In certain embodiments, the YTE mutation does not affect the ability of the antibody to bind to its cognate antigen. In certain embodiments, the YTE mutation increases the antibody's serum half-life compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 3-fold compared to the native (i.e., non- YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 2-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the

142

SUBSTITUTE SHEET RULE 26 antibody by 4-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by at least 5-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by at least 10-fold compared to the native (i.e., non-YTE mutant) antibody. See, e.g., US Patent No. 8,697,650; see also Dall'Acqua et ah, Journal of Biological Chemistry 28i(33):235i4-23524 (2006).

In certain embodiments, the YTE mutant provides a means to modulate antibody- dependent cell-mediated cytotoxicity (ADCC) activity of the antibody. In certain embodiments, the YTEO mutant provides a means to modulate ADCC activity of a humanized IgG antibody directed against a human antigen. See, e.g., US Patent No. 8,697,650; see also Dall'Acqua et ah, Journal of Biological Chemistry 281(33)123514- 23524 (2006).

In certain embodiments, the YTE mutant allows the simultaneous modulation of serum half-life, tissue distribution, and antibody activity (e.g., the ADCC activity of an IgG antibody). See, e.g., US Patent No. 8,697,650; see also Dall'Acqua et ah, Journal of Biological Chemistry 281(33)123514-23524 (2006).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No.

6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

In certain embodiments, the proline at position 329 (EU numbering ) (P329) of a wild- type human Fc region is substituted with glycine or arginine or an amino acid residue large enough to destroy the proline sandwich within the Fc/Fc gamma receptor interface, that is formed between the P329 of the Fc and tryptophane residues W87 and WHO of FcgRIII (Sondermann et ah, Nature 406, 267-273 (20 July 2000)). In a further embodiment, at least one further amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S and still in another embodiment said at least one further amino acid substitution is L234A and L235A of the human IgGl Fc region or S228P and L235E of the human IgG4 Fc region, all according to EU numbering (U.S. Patent No. 8,969,526 which is incorporated by reference in its entirety).

143

SUBSTITUTE SHEET RULE 26 In certain embodiments, a polypeptide comprises the Fc variant of a wild-type human IgG Fc region wherein the polypeptide has P329 of the human IgG Fc region substituted with glycine and wherein the Fc variant comprises at least two further amino acid substitutions at L234A and L235A of the human IgGl Fc region or S228P and L235E of the human IgG4 Fc region, and wherein the residues are numbered according to the EU numbering (U.S. Patent No. 8,969,526 which is incorporated by reference in its entirety). In certain embodiments, the polypeptide comprising the P329G, L234A and L235A (EU numbering) substitutions exhibit a reduced affinity to the human FCYRIIIA and FCYRIIA, for down-modulation of ADCC to at least 20% of the ADCC induced by the polypeptide comprising the wildtype human IgG Fc region, and/or for down-modulation of ADCP (U.S. Patent No. 8,969,526 which is

incorporated by reference in its entirety). In a specific embodiment the polypeptide comprising an Fc variant of a wildtype human Fc polypeptide comprises a triple mutation: an amino acid substitution at position Pro329, a L234A and a L235A mutation according to EU numbering (P329 / LALA) (U.S. Patent No. 8,969,526 which is incorporated by reference in its entirety). In specific embodiments, the polypeptide comprises the following amino acid

substitutions: P329G, L234A, and L235A according to EU numbering.

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Ciq binding and/ or Complement Dependent

Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)), are described

144

SUBSTITUTE SHEET RULE 26 in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No.

7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants. Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., a "THIOMAB™" or TDC, in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at sites of the antibody that are available for conjugation. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: K149 (Kabat numbering) of the light chain; V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; A140 (EU numbering) of the heavy chain; L174 (EU numbering) of the heavy chain; Y373 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. In specific embodiments, the antibodies described herein comprise the HC- A140C (EU numbering) cysteine substitution. In specific embodiments, the antibodies described herein comprise the LC-K149C (Kabat numbering) cysteine substitution. In specific embodiments, the antibodies described herein comprise the HC-A118C (EU numbering) cysteine substitution. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541.

In certain embodiments, the antibody comprises one of the following heavy chain cysteine substitutions:

145

SUBSTITUTE SHEET RULE 26 HC L 179 175

HC T 187 183

HC T 209 205

HC V 262 258

HC G 371 367

HC Y 373 369

HC E 382 378

HC S 424 420

HC N 434 430

HC Q 438 434

In certain embodiments, the antibody comprises one of the following light chain cysteine substitutions:

A nonlimiting exemplary hu7C2.v2.2.LA light chain (LC) R149C THIOMAB™ has the heavy chain and light chain amino acid sequences of SEQ ID NOs: 26 and 30, respectively. A nonlimiting exemplary hu7C2.v2.2.LA heavy chain (HC) A118C

THIOMAB™ has the heavy chain and light chain amino acid sequences of SEQ ID NOs: 31 and 25, respectively.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for denvatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-i, 3- dioxolane, poly-i, 3, 6-trioxane, ethylene/maleic anhydride

146

SUBSTITUTE SHEET RULE 26 copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propylene glycol

homopolymers, polypropylene oxide/ethyl ene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unb ranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et ah, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody- nonproteinaceous moiety are killed.

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/ or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or

147

SUBSTITUTE SHEET RULE 26 lymphoid cell (e.g., Yo, NSo Sp20 cell). In one embodiment, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789, 199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NT, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et ah, Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

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SUBSTITUTE SHEET RULE 26 Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CVi line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham etal., J. Gen Virol. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23 : 243-251 (1980); monkey kidney cells

(CVi); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et ah, Annals N.Y. Acad. Sci. 383 :44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et ah, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Yo, NSo and Sp2/o. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Administration & Dose

Compounds of formula (I) or formula (II) or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, may be administered alone or in combination with one or another or with one or more pharmacologically active compounds which are different from the compounds of formula (I) or formula (II).

Compounds of the invention may suitably be combined with various components to produce compositions of the invention. Suitably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical

composition (which maybe for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Useful pharmaceutical compositions and methods for their preparation may be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA) and Remington: The Science and Practice of

149

SUBSTITUTE SHEET RULE 26 Pharmacy, 21st Edition (ed. D. B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA) which are incorporated herein by reference.

The compounds of the invention may be administered by any suitable route. Suitably the compounds of the invention will normally be administered orally or by any parenteral route, in the form of pharmaceutical preparations comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. The compounds of the invention, their pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of either entity can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the compounds of the invention or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof, can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, controlled-release or pulsatile delivery applications. The compounds of the invention may also be administered via fast dispersing or fast dissolving dosages forms.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate,

croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents

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SUBSTITUTE SHEET RULE 26 and with diluents such as water, ethanol, propylene glycol and glycerin, and

combinations thereof.

Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device. Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof. Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients. Release rate modifying excipients maybe present both within the dosage form i.e. within the matrix, and/ or on the dosage form i.e. upon the surface or coating.

Fast dispersing or dissolving dosage formulations (FDDFs) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin,

hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, or they may be administered by infusion techniques. For such parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Suitably formulation of the invention is optimised for the route of administration e.g. oral, intravenously, etc.

Administration may be in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) during the course of treatment. Methods of determining

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SUBSTITUTE SHEET RULE 26 the most effective means and dosage are well known to a skilled person and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and the dose regimen being selected by the treating physician, veterinarian, or clinician.

Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. For example, a typical dosage for an adult human may be 100 ng to 25 mg (suitably about 1 micro g to about 10 mg) per kg body weight of the subject per day.

Suitably guidance may be taken from studies in test animals when estimating an initial dose for human subjects. For example when a particular dose is identified for mice, suitably an initial test dose for humans may be approx. 0.5X to 2x the mg/Kg value given to mice.

Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N ⁺ HR ⁱ R ² ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0 ), a salt or solvate thereof, as well as conventional protected forms.

Isomers, Salts and Solvates

Certain compounds may exist in one or more particular geometric, optical,

enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1- forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; alpha- and beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and

combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

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SUBSTITUTE SHEET RULE 26 Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e.

isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH ₃ , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, - CH ₂ 0H.

A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. Ci _-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not apply to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,

thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro. In some cases, the compounds of formula (I) can exist as tautomers. Suitably, the compounds of formula (I) include the keto-enol tautomers of the drawn structures.

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including Ή, Ή (D), and 3H (T); C may be in any isotopic form, including ¹² C, ^C, and ^C; O may be in any isotopic form, including ^l6 0 and ^l8 0; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.

Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below. Compounds formula (I) or formula (II), which include compounds specifically named above, may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include nontoxic acid addition salts (including di-acids) and base salts.

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SUBSTITUTE SHEET RULE 26 If the compound is cationic, or has a functional group which maybe cationic (e.g. -NH ₂ may be -NH ₃ ⁺ ), then an acid addition salt may be formed with a suitable anion.

Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,

hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfonate, naphthylate, 2- napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO ^" , or -S0 ₂ H maybe -S0 ₂ ^~ ), then a base salt maybe formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations, such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, as well as amines. Examples of suitable metal cations include sodium (Na ⁺ ) potassium (K ⁺ ), magnesium (Mg ²⁺ ), calcium (Ca ²⁺ ), zinc (Zn ²⁺ ), and aluminum (Al3 ⁺ ). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4 ⁺ ) and substituted ammonium ions (e.g. NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR^). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine,

154

SUBSTITUTE SHEET RULE 26 as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ . Examples of suitable amines include arginine, Ν,Ν'-dibenzylethylene-diamine, chloroprocaine, choline, diethylamine,

diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N- methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane-i,3-diol, and procaine. For a discussion of useful acid addition and base salts, see S. M. Berge et al., J. Pharm. Sci. (1977) 66:1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2011) Pharmaceutically acceptable salts may be prepared using various methods. For example, one may react a compound of formula (I) of formula (II) with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of formula (I) of formula (II) with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of formula (I) of formula (II) to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.

It maybe convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" describes a molecular complex comprising the compound and one or more pharmaceutically acceptable solvent molecules (e.g., EtOH). The term "hydrate" is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include those in which the solvent may be isotopically substituted (e.g., D ₂ 0, acetone-d6, DMSO-d6).

A currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between isolated site, channel, and metal-ion coordinated solvates and hydrates. See, e.g., K. R. Morris (H. G. Brittain ed.)

Polymorphism in Pharmaceutical Solids (1995). Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound. In channel solvates, the solvent molecules lie in lattice channels where they are next to other solvent molecules. In metal-ion coordinated solvates, the solvent molecules are bonded to the metal ion.

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SUBSTITUTE SHEET RULE 26 When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions.v In such cases, non- stoichiometry will typically be observed.

Compounds of formula (I) or formula (II), include imine, carbinolamine and carbinolamine ether forms of the PBD or PDD. The carbinolamine or the

carbinolamine ether is formed when a nucleophilic solvent (H ₂ 0, ROH) adds across the imine bond of the PBD or PDD moiety. The balance of these equilibria between these forms depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.

These compounds may be isolated in solid form, for example, by lyophilisation.

Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

SYNTHETIC STRATEGIES

The compounds of Formula (I) may be prepared using the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation, chromatography, filtration, trituration, crystallization, and the like), and analytical procedures, which are known to persons of ordinary skill in the art of organic chemistry. The details of such reactions and techniques can be found in a number of treatises, including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 2nd Ed (2010), and the multi-volume series edited by Michael B. Smith and others, Compendium of Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be obtained from commercial sources or may be prepared using literature methods. Some of the reaction schemes may omit minor products resulting from chemical transformations (e.g., an alcohol from the hydrolysis of an ester, C0 ₂ from the decarboxylation of a diacid, etc.). In addition, in some instances, reaction intermediates may be used in subsequent steps without isolation or purification (i.e., in situ).

156

SUBSTITUTE SHEET RULE 26 In some of the reaction schemes and examples below, certain compounds can be prepared using protecting groups, which prevent undesirable chemical reaction at otherwise reactive sites. Protecting groups may also be used to enhance solubility or otherwise modify physical properties of a compound. For a discussion of protecting group strategies, a description of materials and methods for installing and removing protecting groups, and a compilation of useful protecting groups for common functional groups, including amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chemistry, 4th Edition, (2006) and P. Kocienski, Protective Groups, 3rd Edition (2005).

Generally, the chemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants.

Additionally, many of the reactions disclosed throughout the specification may be carried out at about room temperature (RT) and ambient pressure, but depending on reaction kinetics, yields, and so on, some reactions may be run at elevated pressures or employ higher temperatures (e.g., reflux conditions) or lower temperatures (e.g., -78°C. to o°C). Any reference in the disclosure to a stoichiometric range, a temperature range, a pH range, etc., whether or not expressly using the word "range," also includes the indicated endpoints.

Many of the chemical transformations may also employ one or more compatible solvents, which may influence the reaction rate and yield. Depending on the nature of the reactants, the one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination. Representative solvents include saturated aliphatic hydrocarbons (e.g., n-pentane, n-hexane, n- heptane, n-octane); aromatic hydrocarbons (e.g., benzene, toluene, xylenes);

halogenated hydrocarbons (e.g., methylene chloride, chloroform, carbon tetrachloride); aliphatic alcohols (e.g., methanol, ethanol, propan-i-ol, propan-2-ol, butan-i-ol, 2- methyl-propan-i-ol, butan-2-ol, 2-methyl-propan-2-ol, pentan-i-ol, 3-methyl-butan-i- ol, hexan-i-ol, 2-methoxy-ethanol, 2-ethoxy-ethanol, 2-butoxy-ethanol, 2-(2-methoxy- ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol, 2-(2-butoxy-ethoxy)-ethanol); ethers (e.g., diethyl ether, di-isopropyl ether, dibutyl ether, 1,2-dimethoxy-ethane, 1,2- diethoxy-ethane, i-methoxy-2-(2-methoxy-ethoxy)-ethane, i-ethoxy-2-(2-ethoxy- ethoxy)-ethane, tetrahydrofuran, 1,4-dioxane); ketones (e.g., acetone, methyl ethyl ketone); esters (methyl acetate, ethyl acetate); nitrogen-containing solvents (e.g.,

157

SUBSTITUTE SHEET RULE 26 formamide, Ν,Ν-dimethylformamide, acetonitrile, N-methyl-pyrrolidone, pyridine, quinoline, nitrobenzene); sulfur-containing solvents (e.g., carbon disulfide, dimethyl sulfoxide, tetrahydro-thiophene-i,i,-dioxide); and phosphorus-containing solvents (e.g., hexamethylphosphoric triamide).

Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

Figure l shows a snapshot of molecular dynamics simulation of a PDD-Aryl Dimer (Structure 1, see Molecular Modelling Section) covalently bound to G4 and G14 of 5'- GCAGATC (GJTGC-3 ' .

Figure 2 shows a snapshot of molecular dynamics simulation of SGD-1882 (Seattle Genetics PBD dimer) covalently bound to G3 and G15 of 5'-GCGATC(G)TCGC-3', illustrating a similar lack of DNA base-pair distortion (yellow).

Figure 3 shows a snapshot of molecular dynamics simulation of a THIQBD-PDD-Aryl Dimer (Structure 2, see Molecular Modelling Section) covalently bound to G4 and G14 of 5'-GCAGATC(G)TGC-3'.

Figure 4 shows a snapshot of molecular dynamics simulation of a 6-7-6-5 Dimer (Structure 3, see Molecular Modelling Section) covalently bound to G4 and G14 of 5'- GCAGATC (GJTGC-3 ' .

Figure 5 shows a sequence of the labelled strands of HexA, TyrT and MSi DNA fragments used in the biophysical characterisation study.

Figure 6 shows an autoradiograph of a denaturing polyacrylamide gel showing DNA interstrand cross-linking by 34 in linear 3 ² P-end-labelled HexA DNA following overnight incubation at 37 °C at various concentrations.

Figure 7 shows an autoradiograph of a denaturing polyacrylamide gel showing DNA interstrand cross-linking by the PBD dimer Talirine in linear ³² P-end-labelled TyrT DNA following overnight incubation at 37 °C at various concentrations.

Figure 8 shows DNA footprinting gel showing the interaction of 26 (bottom panel) and 34 (top panel) with HexA and MSi DNA fragments. Ligand concentrations are shown at the top of the gel. Tracks labelled "GA" are markers for specific purines, and solid black lines represent strong footprints.

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SUBSTITUTE SHEET RULE 26 Figure 9 shows a sequence of the HEXA and MSi DNA fragments showing the possible adducts formed relating to both 26 and 34.

Figure 10 shows fluorescently labelled DNA duplexes used in the FRET melting study to study the formation of interstrand (top) and intrastrand (bottom) cross-links. The labels were fluorescein (F) and dabcyl (Q).

Figure 11 shows FRET Denaturation data for 5'-AAAAAAGAGAAGAAAAAA-3' (Seq 21) and 5'-AAAAAAGAGAGAAAAAA-3' (Seq 20), 5'-AAAAAAGAGAACAAAAAA-3' (Seq 23) and 5'-AAAAAAGATCAAAAAA-3' (Seq 12) shown in Figure 6. EXAMPLES

General remarks

Unless otherwise stated, all synthetic building blocks and reagents were purchased from standard commercial suppliers including Maybridge Chemicals (UK), Fluorochem (USA), and Sigma-Aldrich (UK) and used as purchased. Solvents were purchased from Sigma-Aldrich (UK), VWR and Fisher Scientific (UK). Anhydrous reactions were carried out in pre-oven-dried glassware under an inert atmosphere of nitrogen.

Anhydrous solvents were used as purchased without further drying. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F ₂₅₄ ), and flash column chromatography was carried out either manually, using silica gel (Merck 9385, 230-400 mesh ASTM, 40-63 μΜ) (whilst monitoring by thin layer chromatography: UV (254 nm) and an aqueous alkaline solution of potassium permanganate as stain), or using a Biotage Isolera 1® automated Flash

Chromatography System. All NMR spectra were obtained at room temperature using a Bruker DPX400 spectrometer or a Varian Mercury Vx, Agilent 400 MHz spectrometer, for which chemical shifts are expressed in ppm relative to the solvent and coupling constants are expressed in Hz. Microwave reactions were carried out on an Biotage Initiator® microwave synthesis reactor. Yields refer to isolated material (homogeneous by TLC or NMR) unless otherwise stated and names are assigned according to IUPAC nomenclature. All Liquid Chromatography Mass Spectroscopy (LCMS) analysis was performed on a Waters Alliance 2695 with water (A) and acetonitrile (B) comprising the mobile phases. Formic acid (0.1%) was added to both acetonitrile and water to ensure acidic conditions throughout the analysis. Function type: Diode array (535 scans). Column type: Monolithic C18 50 X 4.60 mm. Mass spectrometry data were collected using a Waters Micro mass ZQ instrument cou led to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V)j 35; Extractor (V), 3.0; Source temperature (°C), 100; De-solvation

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SUBSTITUTE SHEET RULE 26 Temperature (°C), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. LCMS gradient conditions are described as follows. Hydrophobicity can be determined through computational methods (e.g., calculation of LogP), experimental

(measurement of LogP) or through the use of chromatography-based techniques ²² ^.

Method A (10 min): from 95% A/5% B to 50% B over 3 min. Then from 50% B to 80% B over 2 min. Then from 80% B to 95% B over 1.5 min and held constant for 1.5 min. This was then reduced to 5% B over 0.2 min and maintained to 5% B for 1.8 min. The flow rate was 0.5 mL/ min, 200 was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220- 400 nm.

Method B (5 min): from 95% A/5% B to 90% B over 3 min. Then from 90% B to 95% B over 0.5 min and held constant for 1 min. This was then reduced to 5% B over 0.5 min. The flow rate was 1.0 mL/ min, 100 was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm.

Example 1- Molecular Modelling

Molecular dynamics simulations were undertaken on a series of our novel PDD dimers and D-ring containing G-alkylating analogues.

Simulations (see Figure 1) suggest that the C2-aryl substituted PDD (Structure 1 shown below) sits in the minor groove in a similar manner to the PBD dimer SGD-1882 (see Figure 2). Extensive van der Waals interactions are formed between the central methylene linker and minor groove floor (cyan) and both aryl components enhance binding to DNA As can be seen from Figure 1 the PDD-Aryl Dimer (Structure 1) is covalently bound to G4 and G14 of 5'-GCAGATC(G)TGC-3'. Little base-pair distortion (cyan) is observed in the central binding region, and both imines sit in the minor groove in a similar manner to the PBD molecules

Structure 1 - PDD-Aryl Dimer

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SUBSTITUTE SHEET RULE 26

SGD-1882 (Seattle Genetics PBD dimer)

Similarly, the tetrahydroisoquinoline benzodiazepine fused ring system THIQBD-aryl PDD dimer (see Structure 2 below) shows excellent binding in the DNA with both imines sitting in the minor groove in a similar manner to the PBD dimers (see Figure 3). Limited DNA distortion (orange) occurs in the central base-pairing, suggesting favourable isohelicity with the minor groove floor. Extensive van der Waals interactions are also formed between the trimethylene linker and DNA bases, which assist in restrainin the molecule in the minor groove.

Structure 2 - THIQBD-aryl PDD dimer

The 6-7-6-5 dimer (Structure 3, shown below) also performs similar interactions with the minor groove of DNA, with excellent isohelicity observed (see Figure4). The molecular dynamics simulation of this 6-7-6-5 Dimer show that it is covalently bound to G4 and G14 of 5'-GCAGATC(G)TGC-3'. Little base-pair distortion (yellow) is observed in the central binding region, and both imines sit in the minor groove in a similar manner to the PBD molecules.

Structure 3 6-7-6-5 dimer.

General synthetic scheme 1

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SUBSTITUTE SHEET RULE 26

General synthetic scheme 2

General synthetic scheme 3

SUBSTITUTE SHEET RULE 26

General synthetic scheme 4

General synthetic scheme 5

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General synthetic scheme 9

Alloc

General synthetic scheme 10

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Greneral synthetic scheme 11

Greneral synthetic scheme 12

167

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Greneral synthetic scheme 14

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General synthetic scheme 15

Alloc O—

General synthetic scheme 16

SUBSTITUTE SHEET RULE 26

General synthetic scheme 17

General synthetic scheme 18

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General synthetic scheme 20

General synthetic scheme 21

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General synthetic scheme 22

General synthetic scheme 23

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SUBSTITUTE SHEET RULE 26

4-(Benz loxy)-3-methoxybenzaldehyde (l)

A mixture of compound vanillin (50.0 g, 328.6 mmol), benzyl bromide (59.0 g, 345.1 mmol) and potassium carbonate (136.3 g, 985.9 mmol) in methanol (300 mL) was refluxed for 4 h. The reaction mixture was filtered, and the filtrate evaporated under reduced pressure to afford the title compound (75.0 g, 94%) as a pale yellow solid. Ή NMR (400 MHz, CDCI ₃ ) 59-83 (s, lH), 7·47"7·35 (m, 6H), ₇ -33 (d, J=7-2 Hz, iH), 6.98 (d, J=8.2 Hz, iH), 5.24 (s, 2H), 3-94 (s, 3H). izyloxy)-5-methoxy-2-nitrobenzaldehyde (2)

174

SUBSTITUTE SHEET RULE 26

A solution of 4-(benzyloxy)-3-methoxybenzaldehyde (l) (65.00 g, 268.3 mmol) in trifluoroacetic acid (300.0 mL) was charged with a solution of potassium nitrate (32.55 g, 322.0 mmol), in trifluoroacetic acid (300 mL) dropwise at o°C. The reaction mixture was stirred for 1 h and then diluted with water (1.2 L). The resulting precipitate was filtered and washed with cold water (50 mL ^χ 2) to afford the title compound (64.0 g, 83%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 10.43 (s, lH), 7.67 (s, lH), 7.46- 7.40 (m, 5H), 5.27 (s, 2H), 4.02 (s, 3H).

4-(Benzyloxy)-5-methoxy-2-nitrobenzoic acid (3)

A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (2) (36.00 g, 125.3 mmol) in acetone (500 mL) was charged with a hot solution of potassium permanganate (10% w/v in water) (19.80 g, 125.3 mmol) and then stirred at 70 °C for 2 h, before cooling to room temperature. The mixture was acidified to pH = 4 using hydrochloric acid (2 N), and then extracted with ethyl acetate. The combined organic extracts were washed with cold water and dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica) and eluted with dichloromethane/methanol (20:1) to afford the title compound (24.0 g, 63%) as a yellow solid. Ή NMR (400 MHz, DMSO-d6) δ 7.64 (s, lH), 7.47-7.37 (m, 5H), 7.26 (s, lH), 5.22 (s, 2H), 3.90 (s, 3H).

Methyl (2iS,4iS')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydr oxy- piperidine-2-carboxylate (4)

A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (3) (20.00 g, 66.0 mmol) in A V-dimethylformamide (100 mL) was charged with HATU (37.61 g, 98.9 mmol) and A V-diisopropylethylamine (21.31 g, 164.9 mmol) and stirred for 30 min. The reaction

175

SUBSTITUTE SHEET RULE 26 mixture was then cooled to o °C and methyl (2S,4S)-4-hydroxypiperidine-2-carboxylate hydrochloride (12.90 g, 66.0 mmol) was added. After the reaction was judged to have completed by TLC, it was diluted with water (300 mL) and extracted with ethyl acetate (500 mL x 3). The combined organic extracts were washed with water and dried over solid anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with acetone/dichloro- methane, from 5% to 40% acetone, to afford the title compound (22.00 g, 75%) as a pale yellow solid. Ή NMR (400 MHz, DMSO-d6) mixture of rotamers, δ 7.77 and 7.72 (2 x s, lH), 7-40-7-33 (m, 2H), 7-31-7-20 (m, 3H), 6.94 and 6.73 (2 x s, lH), 5.14 and 5.12 (2 x s, 2H), 4-05-3-99 (m, lH), 3.91 and 3.89 (2 x s, 3H), 3.67 and 3.63 (2 x s, 3H) 3.60-3.58 (m, lH), 3.40-3.30 (m, lH) 2.52 and 2.24 (2 x d, lH, J=144 and 14.1 Hz), 1.99-1.90 (m, lH) 1.89-1.80 (m, lH) 1.73-1.60 (m, lH), 1.57-1.48 (m, lH); MS (ES+): m/z = 445 (M+H) ⁺ ; LCMS (Method B): t _R = 3.25 min. (4-(Benzyloxy)-5-methoxy-2-nitrophenyl)((2iS',4iS')-4-hydrox y-2-(hydroxyl- methyl)piperidin-i-yl)methanone (5)

A stirred solution of methyl (2S ^, ,4S)-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- hydroxypiperidine-2-carboxylate (4) (4.70 g, 10.5 mmol) in anhydrous tetrahydrofuran (100 mL) was charged with lithium borohydride (2 M in tetrahydrofuran, 7.9 mL, 15.8 mmol) dropwise at o°C. The reaction was then stirred at room temperature. After 1 h, the reaction was judged to be complete by TLC and was quenched using water (50 mL) and hydrochloric acid solution (1 N, 10 mL). The mixture was then extracted with ethyl acetate (100 mL ^χ 3) and the combined organic extracts were washed with sodium hydrogen carbonate (50 mL x 2) and brine (50 mL x 2), dried over solid anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the title compound (4.0 g, 91%) as an off-white solid. Ή NMR (400 MHz, CD ₃ OD) mixture of rotamers, δ 7.76, 7-74 and 7.71 (3 x s, lH), 7.39-7.22 (m, 5H), 7.01 and 6.91 (2 x s, lH), 5.12 (2 x s, 2H), 4.39 (d, J=i3.5 Hz, lH), 4.20 and 4.10 (2 x dd, lH, J=9.8, 12.0 and 8.8, 12.0 Hz) , 3.88 and 3.83 (2 x s, 3H), 3.65 (dd, J=4-4, 12.0 Hz, 0.38H), 3-57-3-52 (m, 0.8H), 3-50-3-43 (m, 0.6H), 3.34 (dd, J=4-0, 12.0 Hz, 0.6H), 3.26 (dd, J=2.7, 13.3 Hz, 0.5H), 3.12-3.00 (m, 0.5H), 1.90-1.85 (m, 0.5H), 1.85-1.77 (m, lH), 1.75-1-70 (m, lH), 1.70-1.55 (m, 2H), 1.52-1.42 (m, 2H); MS (ES+): m/z = 417 (M+H) ⁺ ; LCMS (Method B): f _R = 3.08 min.

176

SUBSTITUTE SHEET RULE 26 (4-(Benzyloxy)-5-methoxy-2-nitrophenyl)((2iS',4iS')-2-(((ter t-butyldimethyl- ilyl)oxy)methyl)-4-hydroxypiperidin-i-yl)methanone (6)

A solution of (4-(benzyloxy)-5-methoxy-2-nitrophenyl)((2S,4.S)-4-hydroxy-2 - (hydroxylmethyl)piperidin-i-yl)methanone (5) (7.50 g, 18.0 mmol) in dry N,N- dimethylformamide (15 mL) was charged with imidazole (3.68 g, 54.0 mmol) and tert- butyldimethylsilyl chloride (2.99 g, 19.8 mmol) and then stirred at room temperature. After 2 h, the reaction was found to have partially completed. Additional ieri-butyl- dimethylsilyl chloride (0.54 g, 3.6 mmol) was then added. After stirring for another 30 min, the reaction was judged to have completed by TLC and was diluted with water (100 mL). The mixture was then extracted with ethyl acetate (300 mL x 2) and the combined organic extracts washed with citric acid (1 M) (150 mL) and brine (150 mL) and dried over solid anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (3:1 to 1:1) to afford the title compound (6.17 g, 65%) as an orange oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers, δ 7.61, 7.60 and 7.57 (3 x s, lH), 7-30-7-15 (m, 5H), 6.61, 6.56 and 6.50 (3 x s, lH), 5.04 and 5.03 (2 x s, 2H), 4.86-4.45 (m, 2H), 4.17 (dd, J=3-2, 10.6 Hz, 0.5H), 4.01-3.84 (m, 2H), 3.69 (dt, J=5-9, 3.9 Hz, lH), 3.60 (dd, J=io.6, 2.9 Hz, lH), 3-47-3-34 (m, lH), 3.11 and 2.96 (2 x t, J=12.2 and 14.5 Hz, lH), 2.15-1.97 (m, lH), 1.89 and 1.80 (2 x d, J=i4-4, 14.9 Hz, lH), 1.63-1.30 (m, 3H), 0.80-0.67 (m, 9H), 0.15-0.05 (m, 6H); MS (ES+): m/z = 531 (M+H) ⁺ ; LCMS (Method B): t _R = 4.38 min.

(iS)-i-(4-(Benz loxy)-5-methoxy-2-nitrobenzoyl)-2-(((tert-butyldimethyl- silyl)oxy)methyl)piperidin-4-one (7)

A solution of (4-(benzyloxy)-5-methoxy-2-nitrophenyl)((2S,4S ^, )-2-(((ieri- butyldimethylsilyl)oxy)methyl)-4-hydroxypiperidin-i-yl)metha none (6) (14.0 g, 26.4 mmol) in dichloromethane (200 mL) was charged with TEMPO (0.41 g, 2.60 mmol) and (diacetoxyiodo)benzene (11.05 g, 34-3 mmol) and stirred at room temperature. After 24 h, the reaction was judged to have completed by TLC. The mixture was then

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SUBSTITUTE SHEET RULE 26 extracted with ethyl acetate (300 mL ^χ 2) and the combined organic extracts were washed with a saturated aqueous solution of sodium metabisulfite (150 mL) and brine (150 mL) and dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (3:1 to 1:1) to afford the title compound (13.00 g, 90%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers, δ 7.69, 7.68 and 7.67 (3 x s, lH), 7.40-7.20 (m, 5H), 6.71, 6.70 and 6.65 (3 x s, lH), 5.14 and 5.13 (2 x s, 2H), 4.12 and 3.91 (2 x dd, J=io.6, 2.6 and 10.4, 2.7 Hz, lH), 3.89, 3.88 and 3.87 (3 x s, 3H), 3-80-3.35 (m, 3H), 2.77-2.15 (m, 4H), 0.85-0.72 (m, 9H), 0.02- -0.10 (m, 6H); ^!3 C NMR (100 MHz, CDC1 ₃ ), mixture of rotamers, δ 205.7, 167.5, 154-9, 148-3, 148.2, 137.6, 137.3, 135-2, 130.0, 128.9, 128.7, 128.6, 127.6, 127.3, 127.1, 127-0, 109.9, 109-2, 108.6, 71-4, 65-6, 65.1, 64-6, 56.8, 56.8, 56.7, 56-5, 51-3, 5ΐ·2, 43-3, 4ΐ·9, 4ΐ·5, 41-3, 39-9, 39-8, 39-6, 37-5, 25.8, 25.8, 25.8, 20.3, 18.3, ι8.2, ι8.2, ι8.ι, -5-7, "5-7; MS (ES+): τη/ζ = 529 (Μ+Η) ⁺ ; LCMS (Method A): t _R = 8.28 min.

(iS)-i-(4-(Benz loxy)-5-methoxy-2-nitrobenzoyl)-2-(((tert-butyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethane- sulfonate and (iS)-i-(4-(benz loxy)-5-methoxy-2-nitrobenzoyl)-6-(((tert- butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l trifluoro- methanesulfonate (8) (1:1)

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-bu tyldimethyl- silyl)oxy)methyl)piperidin-4-one (7) (13.00 g, 24.6 mmol) in anhydrous tetrahydro- furan (100 mL) was cooled to -78 °C, and charged with sodium bis(trimethylsilyl)amide (2 M in tetrahydrofuran, 18.4 mL, 36.9 mmol) dropwise. The reaction mixture was stirred for 1 h, before a solution of iV-phenyl-bis(trifluoromethanesulfonimide) (11.42 g, 32.0 mmol) in anhydrous tetrahydrofuran (100 mL) was added dropwise. The resulting mixture was then allowed to warm to room temperature and stirred for 4 h. At this point, the reaction was judged to have completed by TLC. The mixture was then concentrated in vacuo and the residue purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (10:1) then dichloro- methane/acetone (100:1) to afford the title compound (12.8 g, 79%) as a cream solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 7.70-7.60 (m, lH),

SUBSTITUTE SHEET RULE 26 7-39-7-25 (m, 5H), 6.77-6.60 (m, iH), 5-91-5-45 (m, iH), 5-15-5-00 (m, 2H), 3-95-3-77 (m, iH), 3-90-3-85 (m, 3H), 3.75-3.20 (m, 3H), 2.90-2.08 (m, 2H), 0.85-0.6 (m, 9H), 0.07-0.22 (m, 6H); MS (ES+): m/z = 661 (M+H) ⁺ ; LCMS (Method A): t _R = 9.27 min. (iS')-(4-(Benz loxy)-5-methoxy-2-nitrophenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2 Ji)-yl)- methanone and (<S)-(4-(benz loxy)-5-methoxy-2-nitrophenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihyd ropyridin- -yl)methanone (9) (1:1)

9

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (S)-i- (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (3.00 g, 4.53 mmol) in acetonitrile (5 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (0.369 g, 0.45 mmol), 2-(4-methoxy- phenyl)-4,4,5,5-tetramethyl-i,3,2-dioxaborolane (1.87 g, 5.90 mmol) and an aqueous solution of potassium carbonate (2 M, 4.5 mL, 9.06 mmol) and irradiated with microwaves at 50 °C for 10 min. The resulting mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residual oil was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1) to afford the title compound (2.11 g, 75%) as an off-white solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 7.75-7.60 (m, iH), 7.40-7.00 (m, 7H), 6.80-6.70 (m, 2H), 6.69-6.62 (m, iH), 6.11-5.56 (m, iH), 5.15-4.76 (m, 2H), 4.04-3.91 (m, iH), 3.85 and 3.84 (2 x s, 3H), 3.82-3.73 (m, iH), 3-70-3-69 (2 x s, 3H), 3-65-3-46 (m, iH), 3-45-3-24 (m, iH), 3.06-2.55 (m, iH), 2.55-2.10 (m, iH), 0.90-0.57 (m, 9H), 0.10-0.37 (m, 6H); MS (ES+): m/z = 619 (M+H) ⁺ ; LCMS (Method A): t _R = 9.38 min.

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SUBSTITUTE SHEET RULE 26 (iS)-(2-Amino-4-(benz loxy)-5-methoxyphenyl)(2-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2 Ji)-yl)- methanone and (iS)-(2-amino-4-(benz loxy)-5-methoxyphenyl)(6-(((tert- butyldimethylsilyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihyd ropyridin- -yl)methanone (10) (1:1)

10

A solution of (S')-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2 H)-yl)methanone and (S3-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((ieri-butyld imethylsilyl)oxy)methyl)- 4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2H)-yl)methanone (9) (0.50 g, 0.80 mmol) in formic acid (5% v/v in absolute ethanol, 10 mL) was charged with zinc powder (1.94 g, 29.6 mmol) and stirred for 10 min, whilst monitoring by TLC and LCMS. Upon completion, the reaction was quenched by addition of a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and concentrated in vacuo. The residue was then partitioned between ethyl acetate (100 mL) and brine (50 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo to give the title compound (0.428 g, 90%) as a yellow oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.42-7.27 (m, 7H), 7.26-7.21 (m, iH), 6.89-6.85 (m, 2H), 6.80-6.74 (m, iH), 6.28-5.98 (m, iH), 5.12 (s, 2H), 4.07-3.82 (m, 2H) 3.82, 3.82 and 3.81 (3 x s, 3H), 3-79, 3-79 and 3.78 (3 s, 3H), 3·75"3·43 (m, 2H), 2.84-2.50 (m, iH), 2.49-2.35 (m, iH), 0.95-0.75 (m, 9H), 0.15-0.10 (m, 6H); MS (ES+): m/z = 589

(M+H) ⁺ ; LCMS (Method A): t _R = 8.93 min. Allyl (S')-(5-(benz loxy)-2-(2-(((terf-butyldimethylsilyl)oxy)methyl)-4-(4- methoxyphenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-meth oxy- phenyl)carbamate and allyl (<S)-(5-(benz loxy)-2-(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-i,2,3,6-tetrahydropyri dine-i- carbonyl)-4-methoxyphenyl)carbamate (11) (1:1)

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SUBSTITUTE SHEET RULE 26

A solution of (S')-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2 H)-yl)methanone and (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(6-(((ieri-butyld imethylsilyl)oxy)- methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2H)-yl)meth anone (10) (0.40 g, 0.67 mmol) in dichloromethane (30 mL) was charged with pyridine (0.124 mL, 1.54 mmol) and allyl chloroformate (0.074 ^m L _> 0.70 mmol). After 1 h, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (2 x 20 mL) and brine (20 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with dichloro- methane/acetone (95:5) to afford the title compound (389 mg, 85%) as a brown oil. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.52-7.69 (m, iH), 7-44-7-35 (m, 2H), 7-34-7-17 (m, 5H), 6.87-6.60 (m, 3H), 6.04-5-90 (br, iH), 5.85 (ddd, J=i6.i, 10.9, 5-7 Hz, iH), 5.28-5.20 (m, iH), 5-15-5-05 (m, 3H), 4.59-4.46 (m, 2H), 3.98-3-80 (m, iH), 3.76 (s, 3H), 3-72 (s, 3H), 3-66-3-33 (m, 2H), 2.77-2.30 (m, 2H), 0.87-0.68 (m, 9H), 0.15-0.07 (m, 6H); MS (ES+): m/z = 673 (M+H) ⁺ ; LCMS (Method A): t _R = 9.50 min.

Allyl (iS)-(5-(benz loxy)-2-(2-(hydroxymethyl)-4-(4-methoxyphenyl)-i,2, 3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S')-(5-(benz loxy)-2-(6-(hydroxymethyl)-4-(4-methoxyphenyl)-i,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate (12) (1: 1)

181

SUBSTITUTE SHEET RULE 26

A solution of allyl (S')-(5-(benzyloxy)-2-(2-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(4- methoxyphenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-meth oxyphenyl)carbamate and allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(4-methoxy- phenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphen yl)carbamate (11) (0.35 g, 0.52 mmol) in tetrahydrofuran (30 mL) was cooled to o °C and charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 0.65 mL, 0.65 mmol). The reaction was allowed to warm to room temperature and after 1 h, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (100 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (50 mL) and brine (50 mL) and dried over magnesium sulfate. After concentrating in vacuo, the title compound (276 mg, 95%) was afforded as a brown oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 8.21- 7-77 (m, iH), 7.48 (d, J=8.o Hz, 2H), 7.42-7.28 (m, 5H), 6.88 (d, J=8.7 Hz, 2H), 6.83 (br, iH), 6.00-5.83 (m, 2H), 5·37"5·26 (m, iH), 5·24"5·19 (m, iH) 5.18 (s, 2H), 4.54- 4.44 (t, J=5.5 Hz, 2H), 3-97-3-78 (m, iH), 3.77 and 3.74 (2 x s, 3H), 3.73 and 3.70 (2 x s, 3H), 3-69-3-52 (m, 2H), 3-41-3-23 (m, iH), 2.74-2.17 (m, 3H); MS (ES+): m/z = 559 (M+H) ⁺ ; LCMS (Method B): t _R = 3.77 min.

Allyl (6aS')-3-(benz loxy)-6-hydroxy-2-methoxy-8-(4-methoxyphenyl)-i2- oxo-6, 6a, 7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)- carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2 -a][i,4]- diazepine-5(i2ii)-carboxylate (13) (1:1)

182

SUBSTITUTE SHEET RULE 26

A solution of allyl (S')-(5-(benzyloxy)-2-(2-(hydroxymethyl)-4-(4-methoxyphenyl) - i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carba mate and allyl (S)-(5- (benzyloxy)-2-(6-(hydroxymethyl)-4-(4-methoxyphenyl)-i,2,3,6 -tetrahydropyridine-i- carbonyl)-4-methoxyphenyl)carbamate (12) (0.25 g, 0.45 mmol) in dichloromethane (15 mL) was charged with TEMPO (0.017 g, 0.11 mmol) and (diacetoxyiodo)benzene (0.363 g, 1.13 mmol) and stirred for 16 h. Dichloromethane (15 mL) was then added to the reaction mixture, which was washed sequentially with a saturated aqueous solution of sodium metabisulflte (30 mL) and brine (30 mL). The organic extract was then dried over magnesium sulfate and concentrated under reduced pressure. Purification by flash column chromatography (silica), eluting with dichloro methane/acetone (85:15) gave the title compound (137 mg, 55%) as a red oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of regioisomers, δ 7.35-7.21 (m, 5H) 7.19-7.11 (m, lH), 7.10-7.04 (m, 2H), 6.84-6.76 (m, 2H), 6.65-6.57 (m, lH), 6.15-6.01 (m, lH), 5·73"5·85 and 5.60-5.44 (m, lH), 5-H-4-96 (m, 3H), 4.73-4.66 and 4.42-4.28 (m, 2H), 4.23 (dd, J=3.6 Hz, lH), 4.09-3.90 (m, lH), 3.84 and 3.82 (2 x s, 3H), 3.74 and 3.72 (2 x s, 3H) 3.69-3.61 (m, lH), 3·35"3·26 (m, lH), 2.99 (d, J=i4-9 Hz, lH), 2.64-2.46 (m, 2H); MS (ES+): m/z = 557 (M+H) ⁺ ; LCMS (Method B): t _R = 3.50 min.

Allyl (6aS)-3,6-dihydroxy-2-methoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)- carboxylate and allyl (6aS')-3,6-dihydroxy-2-methoxy-8-(4-methoxyphenyl)- 12-0x0-6, 6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H) - carboxylate (14) (1:1)

183

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS ^, )-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4-methoxyphenyl )-i2- oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4-methoxyphenyl) -i2-oxo- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate (13) (0.125 g, 0.23 mmol) in anhydrous dichloromethane (10 mL) was charged with boron trichloride (1 M in dichloromethane, 0.67 mL, 0.67 mmol) under an inert atmosphere of nitrogen. After 15 min, the reaction was deemed to have completed by TLC and LCMS and was subsequently cooled to -78 °C and added to a cold saturated aqueous solution of sodium hydrogen carbonate (20 mL). Dichloromethane (20 mL) was then added and after separating layers, the organic extract was washed with brine (20 mL), dried over magnesium sulfate, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (94:6) gave the title compound (44 mg, 42%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of regioisomers, δ g.8o and 9.78 (2 x s, lH), 7.40-7.36 (m, 2H), 7.04 and 7.02 (2 x s, lH), 6.94-6.91 (m, 2H), 6.65 and 6.62 (2 x s, lH), 6.30 and 5.99 (2 x s, lH), 5.86-5.70 (m, lH), 5-67-5-36 (m, lH), 5-15-4-95 (m, lH), 4.65-4.30 (m, 2H), 4-12-3-91 (m, lH), 3.80 and 3.79 (2 x s, 3H), 3.75 and 3.73 (2 x s, 3H), 3.68-3.43 (m, lH), 3.11-2.90 (m, lH), 2.69-2.35 (m, 2H); MS (ES+): m/z = 467 (M+H) ⁺ ; LCMS (Method B): t _R = 3.18 min.

Allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 ii)- carboxylate and allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)- 12-0x0-6, 6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii )- carboxylate (15) (1:1)

184

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS)-3,6-dihydroxy-2-methoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate and allyl (6aS)-3,6-dihydroxy-2-methoxy-8-(4-methoxyphenyl)-i2-oxo-6,6 a,9,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (14) (44 mg, 0.094 mmol) in methanol was concentrated in vacuo to afford the title compound (35 mg, 77%) as a yellow solid, which was used in the subsequent step without further purification. MS (ES+): m/z = 481 (M+H) ⁺ ; LCMS (Method B): t _R = 3-57 min.

Allyl (6aS)-3-(3-bromopropoxy)-2,6-dimethoxy-8-(4-methoxyphenyl)-i 2- oxo-6, 6a, 7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2Ji)- carboxylate and allyl (6aS')-3-(3-bromopropoxy)-2,6-dimethoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2 - «][i _? 4]diazepine-5(i2H)-carboxylate (16) (1:1)

A solution of allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate and allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6 a,9,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (15) (0.035 g, 0.073 mmol) in A V-dimethylformamide (5 mL) was charged with potassium carbonate

185

SUBSTITUTE SHEET RULE 26 (o.oio g, 0.073 rnmol) and 1,3-dibromopropane (0.015 mL, 0.15 mmol) and stirred at room temperature for 16 h. Water (50 mL) was then added and the resulting suspension extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were then washed with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and brine (3 x 50 mL), dried over magnesium sulfate and concentrated in vacuo. Flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1) gave the title compound (20 mg, impure) as a brown oil. MS (ES+): m/z = 603 (M+H) ⁺ ; LCMS (Method A): t _R = 8.75 min. Diallyl 3,3 ^l -(propane-i,3-diylbis(oxy))(6aS',6a ^l iS')-bis(2,6-dimethoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2 -a][i,4]- diazepine-5(i2Ji)-carboxylate) and diallyl 3,3'-(propane-i,3-diylbis(oxy))- (6aS ^, ,6a'iS')-bis(2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)-carbox ylate) and allyl (6aS')-3-(3-(((6aS')-5-((allyloxy)carbonyl)-2,6-dimethoxy-8- (4- methoxyphenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo[e]pyrido [i,2-a]- [i,4]diazepin-3-yl)oxy)propoxy)-2,6-dimethoxy-8-(4-methoxyph enyl)-i2- oxo-6, 6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii )- carboxylate (17)

186

SUBSTITUTE SHEET RULE 26

17

A solution of allyl (6aS')-3-(3-bromopropoxy)-2,6-dimethoxy-8-(4-methoxyphenyl)- i2- oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6aS')-3-(3-bromopropoxy)-2,6-dimethoxy-8-(4-methoxyphenyl)- i2-oxo- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate (16) (0.020 g, 0.033 mmol) in A V-dimethylformamide (5 mL) was charged with potassium carbonate (0.005 g, 0.033 mmol) and allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2 -a][i,4]diazepine-

5(i2H)-carboxylate and allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2- oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate (15) (0.014 mg, 0.030 mmol). The reaction mixture was heated to 60 °C and stirred for 4 h. Water (50 mL) was then added to the solution and the resulting suspension extracted with ethyl acetate (3 x 50 mL). The combined organics were washed a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and brine (3 x 50 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloro methane/acetone (9:1) gave the title compound (17 mg, 51%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of regioisomers, δ 7.48-7.30 (m, 4H), 7.23 and 7.20 (2 x s, 2H), 6.97-6.84 (m, 4H), 6.77- 6.66 (m, 2H), 6.23 and 6.02 (2 x br, 2H), 5.87-5.69 (m, 2H), 5.63 and 5.41 (2 x d, J = 9.1 and 8.6 Hz, 2H), 5·ΐ8-4·99 (m, 3H), 4·77"4·54 (m, 4H), 4-52-4-37 (m, 2H), 4.33- 4.08 (m, 5H), 4.01-3.85 (m, 8H), 3.82 and 3.79 (2 x s, 6H), 3-69-3-52 (m, lH), 3.18- 2.90 (m, 2H), 2.73-2.51 (m, 3H), 2.45-2.33 (m, 2H); MS (ES+): m/z = 1024 (M+Na) ⁺ ; LCMS (Method A): t _R = 9.32 min.

(6aS ^, ,6a'iS')-3,3 ^l -(Propane-i,3-diylbis(oxy))bis(2-methoxy-8-(4-methoxy- phenyl)-9,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diazepin-i2(6a ii)-one) (18)

187

SUBSTITUTE SHEET RULE 26

18

A solution containing diallyl 3,3'-(propane-i,3-diylbis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]pyrido[i,2- <3][i,4]diazepine-5(i2H)-carboxylate) and diallyl 3,3'-(propane-i,3-diylbis(oxy))- (6aS',6a'S ^, )-bis(2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7, io-tetrahydrobenzo- [e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxylate) and allyl (6aS)-3-(3-(((6ai>)-5- ((allyloxy)carbonyl)-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-ox o-5,6,6a,7,io,i2-hexa- hydrobenzo[e]pyrido[i,2-a][i,4]diazepin-3-yl)oxy)propoxy)-2, 6-dimethoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2 -a][i,4]diazepine- 5(i2H)-carboxylate (17) (0.017 g, 0.017 mmol) in dichloromethane (3 mL) was charged with pyrrolidine (0.0013 mL, 0.02 mmol) and tetrakis(triphenylphosphine)- palladium(o) (0.001 g, 0.00085 mmol). This was stirred for 15 min, then concentrated in vacuo. Diethyl ether (10 mL) was charged and the residue concentrated in vacuo once again and this process was repeated twice more. Purification (separation of regioisomers) was carried out by flash column chromatography (silica), eluting with

188

SUBSTITUTE SHEET RULE 26 ethyl acetate (100%) to give the title compound (10 mg, 76%) as a cream solid. Ή NMR (400 MHz, CDCI ₃ ) 87-87 (d, J=5-3 Hz, 2H), ₇ -47 (s, 2H), 7-39 (d, J=7-2 Hz, 4H) , 6.92 (d, J=7.2 Hz, 4H), 6.88 (s, 2H), 6.50 (d, J=3-7 Hz, 2H), 4.56-4.45 (m, 2H), 4.36-4.19 (m, 6H), 3.93 (s, 6H), 3.82-3.76 (m, 6H), 3-48-3-37 (m, 2H), 2.72-2.65 (m, 4H), 2.48- 2.36 (m, 2H); MS (ES+): m/z = 770 (M+H) ⁺ ; LCMS (Method A): t _R = 7.18 min.

Diallyl 3,3 ^l -((i,3-phenylenebis(methylene))bis(oxy))(6aS',6a ^l iS)-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]diazepine-5(i2Ji)-carboxylate) (19)

19

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6, 6a,9, io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carb oxylate (15) (0.035 g, 0.073 mmol) in A V-dimethylformamide (2 mL) was charged with potassium carbonate (0.010 g, 0.073 mmol) and i,3-bis(bromomethyl)benzene (0.011 g, 0.039 mmol) and irradiated with microwaves 45 °C for 45 min. Water (20 mL) was then added, and the mixture extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with brine (3 x 100 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (28 mg, 73%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-54 (s, 2H) 7.41 (br, 4H), 7.33 (d, J=8.i Hz, 4H), 7.22 (s, 2H), 6.88 (d, J=7.8 Hz, 4H), 6.71 (s, 2H), 6.07-6.00 (m, 2H), 5.80-5.66 (m, 2H), 5.66 (d, J=9-7 Hz, 2H), 5-19-5-00 (m, 8H), 4-72-4-35 (m, 6H), 3.92 (s, 6H), 3.81 (s, 6H), 3.51 (s, 6H), 3-16-3-05 (m, 2H), 2.63-2.51 (m, 4H); MS (ES+): m/z = 1064

(M+H) ⁺ ; LCMS (Method A): t _R = 9.43 min.

(6aS ^, ,6a'iS')-3,3 ^l -((i,3-Phenylenebis(methylene))bis(oxy))bis(2-methoxy- 8- (4-methoxyphenyl)-9,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diaz epin-i2- (6aH)-one) (20)

189

SUBSTITUTE SHEET RULE 26

20

A solution of diallyl 3,3'-((i,3-phenylenebis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]pyrido[i,2-a]- [i,4]diazepine-5(i2H)-carboxylate) (19) (0.015 g, 0.014 mmol) in dichloromethane (3 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (0.001 g, 0.00085 mmol) and pyrrolidine (0.0026 mL, 0.04 mmol) and stirred for 5 min, then

concentrated in vacuo. Diethyl ether (10 mL) was charged and the residue concentrated in vacuo once again, and this process was repeated twice more. Purification was carried out by flash column chromatography (silica), eluting with dichloromethane/ acetone (3:1) to give the title compound (7.6 mg, 65%) as a cream solid. Ή NMR (400 MHz, CDCU 8 7-84 (d, J=5-3 Hz, 2H), 7-53-7- (m, ₃ H), 7·44"7·35 (m, 7H), 6.91 (d, J=8.4 Hz, 4H), 6.85 (s, 2H), 6.05 (d, J=3-9 Hz, 2H), 5.21 (q, J=12.4 Hz, 4H), 4·54"4·4ΐ (m, 2H), 4.32-4.26 (m, 2H), 3.96 (s, 6H), 3.83 (s, 6H), 3·49"3·37 (td, J=i3-9, 7-6 Hz, 2H), 2.72-2.65 (m, 4H); MS (ES+): m/z = 831 (M+H) ⁺ ; LCMS (Method A): t _R = 7.33 min.

Diallyl 3,3'-((i,3-phenylenebis(methylene))bis(oxy))(6aS,6a'S)-bis(2 ,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxylate) (21)

190

SUBSTITUTE SHEET RULE 26

21

A solution of allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6 a, 7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-ca rboxylate (15) (0.010 g, 0.021 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (0.0028 g, 0.021 mmol) and i,3-bis(bromomethyl)benzene (0.003 g, 0.010 mmol) and irradiated with microwaves 45 °C for 45 min. Water (10 mL) was then added, and the mixture extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine (3 x 30 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloro- methane/acetone (9:1) gave the title compound (7.7 mg, 70%) as a cream solid. Ή NMR (400 MHz, CDCI ₃ ) δ 7-52 (s, 2H), 7.40 (br, 4H), 7.36 (d, J=8.5 Hz, 4H), 6.90 (d, J=8.5 Hz, 4H), 6.69 (s, 2H), 6.26-6.20 (m, 2H), 5-76-5-64 (m, 2H), 5-45-5-35 (m, 2H), 5.18-4.99 (m, 8H), 4-61-4-36 (m, 4H), 4-31-4-05 (m, 6H), 3.93 (s, 6H), 3.83 (s, 6H), 3-70-3-59 (m, 2H), 3-42-3-38 (s, 6H), 2.99-2.62 (m, 2H); MS (ES+): m/z = 1063 (M+H) ⁺ ; LCMS (Method B): t _R = 4.17 min.

(6aS ^, ,6a'iS')-3,3 ^l -((i,3-Phenylenebis(methylene))bis(oxy))bis(2-methoxy- 8- (4-methoxyphenyl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diaz epin- i2(6aH)-one) (22)

191

SUBSTITUTE SHEET RULE 26

22

A solution of diallyl 3,3'-((i,3-phenylenebis(methylene))bis(oxy))(6aS,6a'S)-bis(2 ,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydroben zo[e]pyrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate) (21) (0.0041 g, 0.0039 mmol) in

dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (1 mg) and pyrrolidine (3 ^L) and stirred for 5 min, then concentrated in vacuo. Diethyl ether (10 mL) was charged and the residue concentrated in vacuo once again, and this process was repeated twice more. Purification was carried out by flash column chromatography (silica), eluting with dichloro methane/acetone (3:1) to give the title compound (1.9 mg, 60%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.59 (d, J=5-4 Hz, 2H), 7.54 (s, 2H), 7.50 (s, lH), 7-43-7-38 (m, 7H), 6.93 (d, J=7-9 Hz, 4H), 6.83 (s, 2H), 6.39-6.35 (m, 2H), 5.20 (q, J=12.8 Hz, 4H), 4.46 (dd, J=i8.o, 6.1 Hz, 2H), 4.11 (d, J=i8.i Hz, 2H), 3-97 (s, 6H), 3-94 (d, J=5-0 Hz, 2H), 3.84 (s, 6H), 2.94 (m, 4H); MS (ES+): m/z = 831 (M+H) ⁺ ; LCMS (Method A): t _R = 7.42 min.

Diallyl 3,3 ^l -((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]d-iazepine-5(i2ii)-carboxylate) (23)

192

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate (15) (0.030 g, 0.062 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (0.0086 g, 0.062 mmol) and i,3-bis(bromomethyl)benzene (0.008 g, 0.031 mmol) and irradiated with microwaves 45 °C for 40 min. Water (20 mL) was then added, and the mixture extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with brine (3 x 100 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (24 mg, 73%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-79 (t, J=7-7 Hz, lH), 7.54 (d, J=7-7 Hz, 2H), 7.33 (d, J=8.7 Hz, 4H), 7.24 (s, 2H), 6.88 (d, J=8.8 Hz, 4H), 6.73 (s, lH), 6.01 (d, J=2.8 Hz, 2H), 5-76-5-34 (m, 2H), 5.59 (d, J=9-i Hz, 2H), 5.27 (s, 4H), 5-13-4-98 (m, 4H), 4.72 (d, J=12.7 Hz, 2H), 4.61-4.38 (m, 4H), 3.95 (s, 6H), 3.92 (d, J=ii.7 Hz, 2H), 3.82 (s, 6H), 3.49 (s, 6H), 3.17-3.07 (m, 2H), 2.66-2.50 (m, 4H); MS (ES+): m/z = 1064 (M+H) ⁺ ; LCMS (Method B): t _R = 4.17 min.

(6aS ^, ,6a'S')-3,3 ^l -((Pyridine-2,6-diylbis(methylene))bis(oxy))bis(2-meth oxy- 8-(4-methoxyphenyl)-9,io-dihydrobenzo[e]pyrido[i,2-a][i,4]di azepin- i2(6aH)-one) (24)

193

SUBSTITUTE SHEET RULE 26

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]pyrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate) (23) (0.025 g, 0.023 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (2 mg, 0.0013 mmol) and pyrrolidine (4.2 μΐ,, 0.065 mmol) and stirred for 5 min, then concentrated in vacuo. Diethyl ether (10 mL) was charged and the residue concentrated in vacuo once again, and this process was repeated twice more. Purification was carried out by flash column chromatography (silica), eluting with dichloromethane/ acetone (3:1) to give the title compound (6.6 mg, 34%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.86 (d, J=5-3 Hz, 2H), ₇ -74 (t, J=7-8 Hz, iH), 7.48 (d, J= ₇ .8 Hz, 2H), 7·43"7·35 (m, 4H), 6.95-6.89 (m, 4H), 6.86 (s, 2H), 6.06 (d, J=4-0 Hz, 2H), ₅ -34 (s, 4H), 4.51 (dt, J=13.2, 4.6 Hz, 2H), 4.30 (t, J=4-7 Hz, 2H), 4.00 (s, 6H), 3.83 (s, 6H), 3.44 (dt, J=i3-3, 6.8 Hz, 2H), 2.76-2.64 (m, 4H); MS (ES+): m/z = 832 (M+H) ⁺ ; LCMS (Method A): t _R = 7-35 min.

Diallyl 3,3 ^l -((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate) (25)

194

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate (15)

(0.030 g, 0.062 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (0.0086 g, 0.062 mmol) and i,3-bis(bromomethyl)benzene (0.008 g, 0.031 mmol) and irradiated with microwaves 45 °C for 40 min. Water (20 mL) was then added, and the mixture extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with brine (3 x 100 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (23 mg, 72%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.78 (t, J=7-8 Hz, iH), 7.53 (d, J=7-7 Hz, 2H), 7.35 (d, J=8.6 Hz, 4H), 7.27 (s, 2H), 6.90 (d, J=8.6 Hz, 4H), 6.69 (s, 2H), 6.26-6.19 (m, 2H), 5-74-5-58 (m, 2H), 5.40 (d, J=9-6 Hz, 2H), 5.25 (s, 4H), 5-H-4-95 (m, 4H), 4-58-4-33 (m, 4H), 4.26 (dd, J=i8.i, 5.6 Hz, 2H), 4.14 (d, J=i8.i Hz, 2H), 3-95 (s, 6H), 3.81 (s, 6H), 3.63 (dd, J=8.o, 6.1 Hz, 2H), 3.38 (s, 6H), 2.93 (d, J=i5-i Hz, 2H), 2.72-2.55 (m, 2H); MS (ES+): m/z = 1064 (M+H) ⁺ ; LCMS (Method B): t _R = 4.02 min.

(6aS ^, ,6a'iS')-3,3 ^l -((Pyridine-2,6-diylbis(methylene))bis(oxy))bis(2-meth oxy- 8-(4-methoxyphenyl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]di azepin- i2(6aH)-one) (26)

195

SUBSTITUTE SHEET RULE 26

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydroben zo[e]pyrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate) (25) (0.025 g, 0.023 mmol) in dichloromethane (1 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (2 mg, 0.0013 mmol) and pyrrolidine (4.2 μΐ,, 0.065 mmol) and stirred for 5 min, then concentrated in vacuo. Diethyl ether (10 mL) was charged and the residue concentrated in vacuo once again, and this process was repeated twice more. Purification was carried out by flash column chromatography (silica), eluting with dichloromethane/ acetone (3:1) to give the title compound (12 mg, 60%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-72 (t, J=7.8 Hz, iH), 7.58 (d, J= ₅ -5 Hz, 2H), ₇ -55 (s, 2H), 746 (d, J= ₇ .8 Hz, 2H), 7.42- 7.36 (m, 4H), 6.95-6.89 (m, 4H), 6.84 (s, 2H), 6.36 (dd, J=3-0, 1.6 Hz, 2H), 5.31 (s, 4H), 445 (dd, J=i8.3, 5-9 Hz, 2H), 4-15-4-07 (m, 2H), 3.99 (s, 6H), 3.83 (s, 6H), 3.96- 3-90 (m, 2H), 2.96-2.91 (m, 4H); MS (ES+): m/z = 832 (M+H) ⁺ ; LCMS (Method A): £ _R = 7.47 min.

(iS')-4-(i-(4-(Benz loxy)-5-methoxy-2-nitrobenzoyl)-6-(((tert-butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-JV -methyl- benzenesulfonamide and (S)-4-(i-(4-(benz loxy)-5-methoxy-2-nitro- benzoyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-i,2,3,6-te trahydro- pyridin-4-yl)-JV-methylbenzenesulfonamide (27) (1:1)

196

SUBSTITUTE SHEET RULE 26

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (S)-i- (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (2.30 g, 3.45 mmol) in acetonitrile (5 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (0.284 g, 0.345 mmol), (4-(iV-methyl- sulfamoyl)phenyl)boronic acid (0.951 g, 4.40 mmol) and an aqueous solution of potassium carbonate (2 M, 3.45 mL, 6.90 mmol) and irradiated with microwaves at 50 °C for 10 min. The resulting mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residual oil was purified by flash column chromatography (silica), eluting with dichloro methane/acetone (9:1) to afford the title compound (2.09 g, 88%) as an orange solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.88-7.71 (m, 3H), 7-55-7-31 (m, 5H), 6.89-6.73 (m, iH), 6.44-5.93 (m, 4H), 5.28-4.90 (m, iH), 5.24 and 5.23 (2 x s, 2H), 4.38-4.28 (m, iH), 4.08-3.41 (m, 3H), 3-97 (2 x s, 3H), 3.21-2.70 (m, iH), 2.70-2.63 (m, 3H), 2.61-2.23 (m, iH), 0.94- 0.71 (m, 9H), 0.17-0.15 (m, 6H); MS (ES+): m/z = 682 (M+H) ⁺ ; LCMS (Method B): t _R = 4.13 min.

(iS')-4-(i-(2-Amino-4-(benz loxy)-5-methoxybenzoyl)-6-(((tert-butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-JV -methyl- benzenesulfonamide and (S 4-(i-(2-amino-4-(benz loxy)-5-methoxy- benzoyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-i,2,3,6-te trahydro- pyridin-4-yl)-JV-methylbenzenesulfonamide (28) (1:1)

197

SUBSTITUTE SHEET RULE 26

A solution of (S')-4-(i-(4-(Benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri -butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-A^ -methylbenzene- sulfonamide and (S')-4-(i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri - butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l)-A^-methylbenzene- sulfonamide (27) (2.40 g, 3.52 mmol) in formic acid (5% v/v in absolute ethanol, 80 mL) was charged with zinc powder (8.45 g, 130.2 mmol) and stirred for 30 min, whilst monitoring by TLC and LCMS. Upon completion, the reaction was quenched by addition of a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and concentrated in vacuo. The residue was then partitioned between ethyl acetate (200 mL) and brine (100 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (2.07 g, 90%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 8.00 (s, lH), 7.82 (dd, J=8.4, 1.7 Hz, 2H), 7.51 (d, J=8.4 Hz, 2H), 7·45"7·27 (m, 5H), 6.81- 6.72 (2 x s, lH), 6.31-6.26 (2 x s, lH), 6.27-6.08 (m, lH), 5.13 (s, 2H), 4.40 (q, J=5.2 Hz, lH), 4-05-3-58 (m, 3H), 3.81 (s, 3H), 2.91-2.80 (m, lH), 2.70-2.62 (m, 3H), 2.53-2.36 (m, 2H), 0.92-O.78 (m, 9H), 0.13-O.04 (m, 6H); MS (ES+): m/z = 652 (M+H) ⁺ ; LCMS (Method B): t _R = 3.97 min.

Allyl (S)-(5-(benzyloxy)-2-(6-(((tert-butyldimethylsilyl)oxy)methy l)-4-(4- (JV-methylsulfamoyl)phenyl)-i,2,3,6-tetrahydropyridine-i-car bonyl)-4- methoxyphenyl)carbamate and allyl (<S)-(5-(benzyloxy)-2-(2-(((tert-butyl- dimethylsilyl)oxy)methyl)-4-(4-(JV-methylsulfamoyl)phenyl)-i ,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate (29) (1: 1)

198

SUBSTITUTE SHEET RULE 26

29

A solution of (S')-4-(i-(2-amino-4-(benzyloxy)-5-methoxybenzoyl)-6-(((ieri -butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-A^ -methylbenzene- sulfonamide and (S')-4-(i-(2-amino-4-(benzyloxy)-5-methoxybenzoyl)-2-(((ieri - butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l)-A^-methylbenzene- sulfonamide (28) (2.20 g, 3.37 mmol) in dichloromethane (200 mL) was charged with pyridine (0.27 mL, 3.38 mmol) and allyl chloroformate (0.429 mL, 4.25 mmol). After 15 min, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (2 x 100 mL) and brine (100 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with dichloromethane/acetone (95:5) to afford the title compound (2.06 g, 83%) as a brown oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and

regioisomers, δ 8.43-7.90 (m, 2H), 7.83 (d, J=8.4 Hz, 2H) 7.56-7.28 (m, 7H), 6.97-6.77 (2 x s, lH), 6.23 (s, lH), 5.92 (ddd, J=22.6, 10.9, 5.7 Hz, lH), 5.40-5.27 (m, lH), 5.24- 5.12 (m, 3H), 4-08-3.35 (m, 3H), 3.81 (s, 3H), 2.88-2.76 (m, 0.5H), 2.69-2.64 (m, 3H), 2.62-2.36 (m, 1.5H), 0.94-0.75 (m, 9H), 0.20-0.06 (m, 6H); MS (ES+): m/z = 736 (M+H) ⁺ ; LCMS (Method B): t _R = 4.22 min.

Allyl (iS')-(5-(benz loxy)-2-(6-(hydroxymethyl)-4-(4-(JV-methylsulfamoyl)- phenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphen yl)- carbamate and allyl (<S)-(5-(benz loxy)-2-(2-(hydroxymethyl)-4-(4-(JV- methylsulfamoyl)phenyl)-i,2,3,6-tetrahydropyridine-i-carbony l)-4- methoxyphenyl)carbamate (30) (1:1)

199

SUBSTITUTE SHEET RULE 26

30

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(4- (A^-methylsulfamoyl)phenyl)-i,2,3,6-tetrahydropyridine-i-car bonyl)-4-methoxy- phenyl)carbamate and allyl (S')-(5-(benzyloxy)-2-(2-(((ieri-butyldimethylsilyl)oxy)- methyl)-4-(4-(A^-methylsulfamoyl)phenyl)-i,2,3,6-tetrahydrop yridine-i-carbonyl)-4- methoxyphenyl)carbamate (29) (1.90 g, 2.58 mmol) in tetrahydrofuran (50 mL) was cooled to o °C and charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 0.32 mL, 3.23 mmol). The reaction was allowed to warm to room temperature and after 1 h, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (100 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (50 mL) and brine (50 mL) and dried over magnesium sulfate. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (4:1) gave the title compound (1.52 g, 95%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 8.iy- 8.06 (br, iH), 7.83-7.70 (m, 3H), 7·53"7·28 (m, 7H), 6.89-6.77 (s, iH), 6.23-6.08 (m, iH), 5.91 (ddd, J=22.8, 10.8, 5.6 Hz, iH), 5.38-5.18 (m, 2H), 5.15 (s, 2H), 4.66-4.57 (m, 2H), 4-54-4-45 (m, iH), 4-14-3-89 (m, iH), 3-85-3-83 (2 x s, 3H), 3-81-3-32 (m, 2H), 2.91-2.78 (m, 0.5H), 2.68-2.60 (2 x d, J=5-4 Hz, 3H), 2.59-2.31 (m, 1.5H); MS (ES+): m/z = 622 (M+H) ⁺ ; LCMS (Method B): t _R = 3.37 min.

Allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4-(JV-methylsulfa moyl)- phenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4 ]diazepine- 5(i2ff)-carboxylate and allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-8- (4-(JV-methylsulfamoyl)phenyl)-i2-oxo-6,6a,7,io-tetrahydrobe nzo[e]- pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate (31) (1:1)

200

SUBSTITUTE SHEET RULE 26

31

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(hydroxymethyl)-4-(4-(A^-methylsulf amoyl)- phenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphen yl)carbamate and allyl (-S)-(5-(benzyloxy)-2-(2-(hydroxymethyl)-4-(4-(A^-methylsulf amoyl)phenyl)-i,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate (30) (1.50 g, 2.41 mmol) in dichloromethane (50 mL) was charged with TEMPO (0.037 g _> 0.24 mmol) and (diacetoxyiodo)benzene (0.853 g, 2.61 mmol) and stirred at room temperature for 16 h. Dichloromethane (15 mL) was then added to the reaction mixture, which was quenched by addition of a saturated aqueous solution of sodium metabisulfite (50 mL). The organic phase was separated, washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (748 mg, 50%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.82 (apparent t, J=7.6 Hz, 2H), 7.54 (apparent t, J=7.8 Hz, 2H), 7.46-7.29 (m, 6H), 7.25-7.22 (2 x s, iH), 6.79-6.68 (m, iH), 6.39-6.29 (m, iH), 5-87-5-57 (m, 2H), 5-24-5-03 (m, 4H), 4-89-4-79 (m, iH), 4.59-4.34 (m, 2H), 4.24-4.01 (m, iH), 3-95-3-93 (2 x s, 3H), 3-81-3-72 (m, iH), 3-14-3-05 (m, iH), 2.76-2.73 (m, 3H); MS (ES+): m/z = 620 (M+H) ⁺ ; LCMS (Method B): t _R = 3.32 min.

Allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-(JV-methylsulfamoyl)pheny l)-i2- oxo-6, 6a, 7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2Ji)- carboxylate (32)

201

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4-(A^-methyl- sulfamoyl)phenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]pyrido[ i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-8-(4-(iV- methylsulfamoyl)phenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]p yrido[i,2-a][i,4]- diazepine-5(i2H)-carboxylate (31) (700 mg, 1.13 mmol) in anhydrous dichloromethane (20 mL) was charged with boron trichloride (1 M in dichloromethane, 3.39 mL, 3.39 mmol) and stirred under an inert atmosphere of nitrogen for 30 min. Methanol (20 mL) was then charged and the resulting mixture irradiated with microwaves at 55 °C for 1 h. After concentrating the resulting mixture in vacuo, purification was carried out by (multiple) flash column chromatography (silica), eluting with petroleum spirit, 40- 60 °C/ethyl acetate (4:1) to afford the (regiopure) title compound (203 mg, 33%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) 87.84 (d, J=8.5 Hz, 2H), 7-57-7-50 (m, 2H), 7.21 (s, iH), 6.74 (s, iH), 6.47-6.39 (m, iH), 5.81-5.68 (m, iH), 5.40 (d, J=9.6 Hz, iH), 5-16-5-01 (m, 2H), 4.56 (dd, J=i4-6, 4-4 Hz, iH), 4.45 (dd, J=io.7, 5-3 Hz, iH), 4.23- 4.15 (m, 2H), 3.91 (s, 3H), 3-73-3-64 (m, iH), 3.42 (s, 3H), 2.96 (d, J=i4-9 Hz, iH), 2.78-2.59 (m, 5H); MS (ES+): m/z = 544 (M+H) ⁺ ; LCMS (Method B): t _R = 2.97 min.

Allyl (6aS)-3-((6-((((6aS)-5-((allyloxy)carbonyl)-2,6-dimethoxy-8- (4-(JV- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]- pyrido[i,2-a][i,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)meth oxy)-2,6- dimethoxy-8-(4-(JV-methylsulfamoyl)phenyl)-i2-oxo-6,6a,7,io- tetrahydro- benzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate (33)

202

SUBSTITUTE SHEET RULE 26

33

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-(A^-methylsulfamoyl)phen yl)- i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepi ne-5(i2H)-carboxylate (32) (0.045 g _> 0.083 mmol) in A V-dimethylformamide (2 mL) was charged with potassium carbonate (0.012 g, 0.083 mmol) and 2,6-bis(bromomethyl)pyridine (0.011 g, 0.042 mmol). The resulting mixture was irradiated with microwaves at 55 °C for 1 h, at which point TLC and LCMS showed completion of the reaction. Water (20 mL) was added and the resulting suspension extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were then washed with brine (5 x 100 mL), dried over magnesium sulfate, and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:4 to 0:1) gave the title compound (22 mg, 44%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-86 (d, J=8.4 Hz, 4H), 7.79 (t, J=7-8 Hz, lH), 7.54 (apparent d, J=8.4 Hz, 6H), 6.71 (s, 2H), 6.49-6.41 (m, 2H), 5-74-5-59 (m, 2H), 5.39 (d, J=2 Hz, 2H), 5.25 (s, 4H), 5.05 (d, J=io.3 Hz, 2H), 4-76-4-57 (m, 2H), 4-56-4-36 (m, 4H), 4-36-4-i6 (m, 4H), 3-96 (s, 6H), 3-74-3-63 (m, 2H), 3.38 (s, 3H), 2.99-2.91 (m, 2H), 2.78-2.70 (m, 2H), 2.67 (d, J=5.i Hz, 6H); MS (ES+): m/z = 1190 (M+H) ⁺ ; LCMS (Method B): t _R = 3.47 min. 4,4'-((6aS ^, ,6a'iS')-((Pyridine-2,6-diylbis(methylene))bis(oxy))bi s(2- methoxy-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2-a][i, 4]diazepine- 3,8-diyl))bis(JV-methylbenzenesulfonamide) (34)

203

SUBSTITUTE SHEET RULE 26

34

A solution of allyl (6aS)-3-((6-((((6aS')-5-((allyloxy)carbonyl)-2,6-dimethoxy-8 -(4-(A^- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido[i,2-a][i,4]- diazepin-3-yl)oxy)methyl)pyridin-2-yl)methoxy)-2,6-dimethoxy -8-(4-(A^-methyl- sulfamoyl)phenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[ i,2-a][i,4]diazepine- 5(i2H)-carboxylate (33) (0.022 g, 0.018 mmol) in dichloromethane (2 niL) was charged with tetrakis(triphenylphosphine)palladium(o) (0.001 g, 0.0009 mmol) and pyrrolidine (0.0036 mL, 0.04 mmol). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with dichloromethane/acetone (4:1) gave the title compound (12 mg, 66%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-91 (d, J=8.6 Hz, 4H), 7.73 (t, J=7-8 Hz, iH), 7.60 (d, J=8.6 Hz, ₄ H), ₇ -57 (d, J=4-7 Hz, 2H), ₇ -45 (d, J=7-8 Hz, 2H), 6.86 (s, 2H), 6.65-6.56 (m, 2H), 5·34"5·3ΐ (m, 4H), 4-65 (q, J=5-3 Hz, 2H), 4.52 (dd, J=i8.7, 5.9 Hz, 2H), 4.22-4.13 (m, 2H), 4.01 (s, 6H), 3.05-2.92 (m, 4H), 2.74-2.64 (m, 6H), 1.61 (m, 2H); MS (ES+): m/z = 958 (M+H) ⁺ ; LCMS (Method A): t _R = 6.37 min. (iS)-5-(i-(4-(Benz loxy)-5-methoxy-2-nitrobenzoyl)-6-(((tert-butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-i, 3-dimethyl- pyrimidine-2,4(iff,3ii)-dione and (iS)-5-(i-(4-(benz loxy)-5-methoxy-2- nitrobenzoyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-i,2,3 ,6-tetrahydro- pyridin-4-yl)-i,3-dimethylpyrimidine-2,4(iJi,3ii)-dione (35) (1:1)

204

SUBSTITUTE SHEET RULE 26

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (2.68 g, 4.06 mmol) in acetonitrile (4 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (0.334 g, 0.41 mmol), (i,3-dimethyl-2,4- dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)boronic acid (0.968 g, 5.26 mmol) and an aqueous solution of potassium carbonate (2 M, 4.05 mL, 8.10 mmol) and irradiated with microwaves at 50 °C for 40 min. The resulting mixture was diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residual oil was purified by flash column chromatography (silica), eluting with dichloro methane/acetone (9:1) to afford the title compound (1.93 g, 73%) as a brown solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.73-7.70 (m, lH), 7.49-7.30 (m, 5H), 7.17-7.06 (m, lH), 6.91-6.72 (m, lH), 6.36-6.04 (m, lH), 5.26-5.18 (m, 2H), 5.17-4.76 (m, lH), 3.96 (s, 3H), 3-93-3-57 (m, 3H), 3-41-3-39 (2 x s, 3H), 3-34 (s, 3H), 2.70-2.19 (m, 2H), 0.97- 0.69 (m, 9H), 0.19 -0.18 (m, 6H); MS (ES+): m/z = 651 (M+H) ⁺ ; LCMS (Method B): £ _R = 4.03 min.

(iS)-5-(i-(2-Amino-4-(benz loxy)-5-methoxybenzoyl)-6-(((tert-butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-i, 3-dimethyl- pyrimidine-2,4(iff,3Ji)-dione and CS)-5-(i-(2-amino-4-(benzyloxy)-5- methoxybenzoyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-i,2 ,3,6-tetra- hydropyridin-4-yl)-i,3-dimethylpyrimidine-2,4(iJi,3Ji)-dione (36) (1:1)

205

SUBSTITUTE SHEET RULE 26

36

A solution of (S')-5-(i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri -butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-i, 3-dimethylpyrimi

2,4(iH,3H)-dione and (S')-5-(i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri - butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l)-i,3-dimethyl- pyrimidine-2,4(iH,3H)-dione (35) (1.90 g, 2.91 mmol) in formic acid (5% v/v in absolute ethanol, 65 mL) was charged with zinc powder (7.00 g, 108 mmol) and stirred for 30 min, whilst monitoring by TLC and LCMS. Upon completion, the reaction was quenched by addition of a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and concentrated in vacuo. The residue was then partitioned between ethyl acetate (200 mL) and brine (100 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with chloroform/acetone (1:0 to 7:3) gave the title compound (1.43 g, 79%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.54-7.24 (m, 5H), 7.13-7.05 (m, lH), 6.83-6.72 (m, lH), 6.32-6.16 (m, 2H), 5.12 (s, 2H), 4.03-3.81 (m, 2H), 3.80 (s, 3H), 3·67"3·57 (m, lH), 3.44-3.37 (m, 3H), 3.35 (s, 3H), 2.66-2.55 (m, lH), 2.44-2.34 (m, lH), 0.92-0.76 (m, 9H), 0.13-0.08 (m, 6H); MS (ES+): m/z = 621 (M+H) ⁺ ; LCMS (Method B): t _R = 3.85 min.

Allyl (S)-(5-(benz loxy)-2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-4-(i,3- dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-i,2,3,6 - tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)- (5-(benzyloxy)-2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4 -(i,3-dimethyl- 2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-i,2,3,6-tetrahyd ropyridine-i- carbonyl)-4-methoxyphenyl)carbamate (37) (1:1)

206

SUBSTITUTE SHEET RULE 26

A solution of (S')-5-(i-(2-Amino-4-(benzyloxy)-5-methoxybenzoyl)-6-(((ieri -butyl- dimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl)-i, 3-dimethylpyrimi

2,4(iH,3H)-dione and (S')-5-(i-(2-amino-4-(benzyloxy)-5-methoxybenzoyl)-2-(((ieri - butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l)-i,3-dimethyl- pyrimidine-2,4(iH,3H)-dione (36) (1.41 g, 2.27 mmol) in dichloromethane (200 mL) was charged with pyridine (0.22 mL, 2.27 mmol) and allyl chloroformate (0.253 mL, 2.38 mmol). After 15 min, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (2 x 100 mL) and brine (100 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1) to afford the title compound (1.34 g, 84%) as a brown oil. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 8.33-7.78 (m, iH), 7.52-7.28 (m, 5H), 7.10 (s, iH), 6.90-6.75 (m, iH), 6.25-6.11 (m, iH), 6.00-5.88 (m, iH), 5.34 (dd, J=i7-2, 1.5 Hz, iH), 5.23 (dd, J=io.4, 1.2 Hz, iH), 5.16 (s, 2H), 4.69-4.54 (m, 2H), 4.09-345 (m, 6H), 3.42 (s, 3H), 3.35 (s, 3H), 2.66-2.33 (m, 2H), 0.98-0.75 (m, 9H), 0.12-0.07 (m, 6H); MS (ES+): m/z = 705 (M+H) ⁺ ; LCMS (Method B): t _R = 4.18 min.

Allyl (iS)-(5-(benz loxy)-2-(4-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydro- pyrimidin-5-yl)-6-(hydroxymethyl)-i,2,3,6-tetrahydropyridine -i-carbonyl)- 4-methoxyphenyl)carbamate and allyl (<S)-(5-(benz loxy)-2-(4-(i,3- dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-2-(hydr oxymethyl)- i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carba mate (38) (1:1)

207

SUBSTITUTE SHEET RULE 26

38

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4- (i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-i, 2,3,6-tetrahydropyridine- i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)-(5-(benzyloxy)-2-(2-(((ieri- butyldimethylsilyl)oxy)methyl)-4-(i,3-dimethyl-2,4-dioxo-i,2 ,3,4-tetrahydro- pyrimidin-5-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-met hoxyphenyl)carbamate (37) (ΐ·35 g _> 1-91 mmol) in tetrahydrofuran (50 mL) was cooled to o °C and charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 2.38 mL, 2.38 mmol). The reaction was allowed to warm to room temperature and after 1 h, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (100 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (50 mL) and brine (50 mL) and dried over magnesium sulfate. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (4:1) gave the title compound (1.04 g, 92%) as a cream solid. Ή NMR (400

MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.28-7.70 (m, iH), 7.51-7.27 (m, 5H), 7-15-7-07 (m, iH), 6.84-6.75 (m, iH), 6.20-5.87 (m, 2H), 5.33 (dd, J=i7-2, 1.5 Hz, iH), 5-25-5-20 (m, iH), 5.16 (s, 2H), 4-64-4-59 (m, 2H), 4-07-3-63 (m, 6H), 3-42-3-41 (2 x s, 3H), 3-35-3-34 (2 x s, 3H), 2.61-2.37 (m, 2H); MS (ES+): m/z = 591 (M+H) ⁺ ; LCMS (Method B): t _R = 3.22 min.

Allyl (6aS)-3-(benzyloxy)-8-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrah ydro- pyrimidin-5-yl)-6-hydroxy-2-methoxy-i2-oxo-6,6a,9,io-tetrahy drobenzo- [e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxylate and allyl (6aS)-3- (benzyloxy)-8-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyri midin-5-yl)-6- hydroxy-2-methoxy-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[ i,2-a] [i,4]diazepine-5(i2ii)-carboxylate (39) (1:1)

208

SUBSTITUTE SHEET RULE 26

A solution of allyl (S')-(5-(benzyloxy)-2-(4-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tet rahydro- pyrimidin-5-yl)-6-(hydroxymethyl)-i,2,3,6-tetrahydropyridine -i-carbonyl)-4-methoxy- phenyl)carbamate and allyl (S)-(5-(benzyloxy)-2-(4-(i,3-dimethyl-2,4-dioxo-i,2,3,4- tetrahydropyrimidin-5-yl)-2-(hydroxymethyl)-i,2,3,6-tetrahyd ropyridine-i-carbonyl)- 4-methoxyphenyl)carbamate (38) (1.05 g, 1.78 mmol) in dichloromethane (50 mL) was charged with TEMPO (0.055 g, 0.35 mmol) and (diacetoxyiodo)benzene (1.25 g, 3.89 mmol) and stirred at room temperature for 16 h, after which TLC showed only partial consumption of starting material. Further TEMPO (0.014 g, 0.090 mmol) and

(diacetoxyiodo)benzene (0.224 g, 0.700 mmol) were added and the mixture stirred for a further 2 h. Dichloromethane (15 mL) was then added to the reaction mixture, which was quenched by addition of a saturated aqueous solution of sodium metabisulfite (50 mL). The organic phase was separated, washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/acetone (9:1) gave the title compound (366 mg, 35%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.45-7.27 (m, 5H), 7.23-7.22 (2 x s, lH), 7.20-7.12 (2 x s, 2H), 6.85-6.12 (m, lH), 5-84-5-77 (m, lH), 5·73"5·59 (m, lH), 5.20-5.00 (m, 4H), 4.78- 4-43 (m, lH), 4.37 (dd, J=i7-8, 6.0 Hz, lH), 3·99"3·88 (m, 3H), 3-71-3-63 (m, lH), 3.47- 3-39 (m, 3H), 3-39-3-31 (m, 3H), 3.12-2.96 (m, lH), 2.58-2.37 (m, lH); MS (ES+): m/z = 571 (M+H) ⁺ ; LCMS (Method B): t _R = 3.12 min.

Allyl (6aS)-8-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin- 5-yl)-3- hydroxy-2,6-dimethoxy-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyr ido[i,2- «][i _? 4]diazepine-5(i2H)-carboxylate (40)

209

SUBSTITUTE SHEET RULE 26

40

A solution of allyl (6aS')-3-(benzyloxy)-8-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetra hydro- pyrimidin-5-yl)-6-hydroxy-2-methoxy-i2-oxo-6,6a,9,io-tetrahy drobenzo[e]pyrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate and allyl (6aS')-3-(benzyloxy)-8-(i,3-dimethyl-2,4- dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-6-hydroxy-2-methoxy- i2-oxo-6,6a,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (39) (350 mg, 0.595 mmol) in anhydrous dichloromethane (20 mL) was charged with boron trichloride (1 M in dichloromethane, 1.78 mL, 1.78 mmol) and stirred under an inert atmosphere of nitrogen for 30 min. Methanol (20 mL) was then charged and the resulting mixture irradiated with microwaves at 55 °C for 1 h. After concentrating the resulting mixture in vacuo, purification was carried out by (multiple) flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1) to afford the (regiopure) title compound (67 mg, 22%) as a white solid. Ή NMR (400 MHz, CDCI ₃ ) δ 7-21 (s, iH), 7.21-7.08 (3 x s, iH), 6.75 (s, iH), 6.30-6.24 (m, iH), 5.95 (s, iH), 5·84-5·7ΐ (m, iH), 5.52 (d, J=9-6 Hz, iH), 5.16-5.04 (m, 2H), 4.62-4.54 (m, iH), 4.51-4.41 (m, iH), 4.23-4.07 (m, 2H), 3.95 (s, 3H), 3.62-3.54 (m, iH), 3.50 (s, 3H), 3-44 (s, 3H), 3-37 (s, 3H), 2.82 (d, J=i5-i Hz, iH), 2.69-2.52 (m, iH); MS (ES+): m/z = 512 (M+H) ⁺ ; LCMS (Method B): t _R = 2.85 min.

Diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(8- (i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl)-2, 6-dimethoxy- 12-0x0-6, 6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii )- carboxylate) (41)

210

SUBSTITUTE SHEET RULE 26

41

A solution of allyl (6aS')-8-(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin -5-yl)- 3-hydroxy-2,6-dimethoxy-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]p yrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate (40) (0.050 g, 0.097 mmol) in N,iV-dimethyl- formamide (2 mL) was charged with potassium carbonate (0.013 g, 0.097 mmol) and 2,6-bis(bromomethyl)pyridine (0.013 g, 0.048 mmol). The resulting mixture was irradiated with microwaves at 55 °C for 1 h, at which point TLC and LCMS showed completion of the reaction. Water (20 mL) was added and the resulting suspension extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were then washed with brine (5 x 100 mL), dried over magnesium sulfate, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with

dichloromethane/methanol (95:5) gave the title compound (10 mg, impure) as a brown solid, which was used in the subsequent step without further purification.

MS (ES+): m/z = 1128 (M+H) ⁺ ; LCMS (Method B): t _R = 3.22 min.

5 _? 5'-((6aS',6a ^l iS')-((Pyridine-2,6-diylbis(methylene))bis(oxy))bis(2- methoxy- i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazep ine-3,8- diyl))bis(i,3-dimethylpyrimidine-2,4(iff,3Ji)-dione) (42)

42

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(8-

(i,3-dimethyl-2,4-dioxo-i,2,3,4-tetrahydropyrimidin-5-yl) -2,6-dimethoxy-i2-oxo-

211

SUBSTITUTE SHEET RULE 26 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate) (41) (0.010 g, 0.009 mmol) in dichloromethane (2 mL) was charged with tetrakis(triphenyl- phosphine)palladium(o) (0.5 mg) and pyrrolidine (0.002 mL, 0.02 mmol). After 15 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with dichloromethane/methanol (9:1) gave the title compound (2.0 mg, 25%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.92 (d, J=5-3 Hz, 2H), 7-74 (t, J=8.o Hz, iH), ₇ -54 (s, 2H), 7.48 (d, J= ₇ -9 Hz, 2H), 6.87 (s, 2H), 6.33-6.27 (m, 2H), 5.32 (s, 4H), 5.30 (s, 2H), 4.41 (dd, J=i8.8, 5.3 Hz, 2H) 4.05 (d, J=i8.i Hz, 2H), 4.00 (s, 6H), 3-92-3-85 (m, iH), 3.69-3.60 (m, iH), 3.46 (s, 6H), 3.38 (s, 6H), 2.99 (d, J=i5-7 Hz, 2H), 2.82-2.71 (m, 2H); MS (ES+): m/z = 896 (M+H) ⁺ ; LCMS (Method A): t _R = 5.78 min.

(iS')-(4-(Benz loxy)-5-methoxy-2-nitrophenyl)(6-(((tert-butyldimethylsilyl) - oxy)methyl)-4-(pyrimidin-5-yl)-3,6-dihydropyridin-i(2H)-yl)m ethanone and (iS)-(4-(benz loxy)-5-methoxy-2-nitrophenyl)(2-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(pyrimidin-5-yl)-3,6-dihydropyridin-i(2J i)- yl)methanone (43) (1:1)

43

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (S)-i- (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (4.00 g, 6.05 mmol) in acetonitrile (2 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (0.497 g, 0.61 mmol), pyrimidin-5- ylboronic acid (0.970 g, 7.86 mmol) and an aqueous solution of potassium carbonate (2 M, 6.05 mL, 12.1 mmol) and irradiated with microwaves at 50 °C for 10 min, at which point TLC showed only partial consumption of starting material. Further pyrimidin-5-

212

SUBSTITUTE SHEET RULE 26 ylboronic acid (0.970 g, 7.86 mmol) was charged and the resulting mixture irradiated with microwaves for a further 10 min at 50 °C. The mixture was subsequently diluted with ethyl acetate (100 mL) and washed with brine (50 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residual oil was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1) to afford the title compound (2.90 g, 81%) as a yellow solid. MS (ES+): m/z = 591 (M+H) ⁺ ; LCMS (Method B): t _R = 4.12 min.

(iS)-(2-Amino-4-(benz loxy)-5-methoxyphenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(pjT*imidin-5-yl)-3,6-dihydropjridin-i(2 Ji)- yl)methanone and (iS)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(pyrimidin-5-yl)-3,6-dihydr opyridin- -yl)methanone (44) (1:1)

44

A solution of (S')-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(pyrimidin-5-yl)-3,6-dihydropyridin-i(2H )-yl)methanone and (S)- (4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (pyrimidin-5-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (43) (2.85 g, 4.82 mmol) in formic acid (5% v/v in absolute ethanol, 61 mL) was charged with zinc powder (11.6 g, 179 mmol) and stirred for 30 min, whilst monitoring by TLC and LCMS. Upon completion, the reaction was quenched by addition of a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and concentrated in vacuo. The residue was then partitioned between ethyl acetate (200 mL) and brine (100 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloro methane/acetone (4:1) gave the title compound (2.22 g, 82%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ g.ii (s, iH), 8.74 (s, 2H), 7.47-7.27 (m, 5H), 6.83-6.72 (m, iH), 6.33-6.22 (m, 2H), 3·96-3·83 (m, 2H), 3.81 (s, 3H), 3-76-3-46 (m, 2H), 2.87-2.57

213

SUBSTITUTE SHEET RULE 26 (m, lH), 2.54-2.35 (m, lH), 0.90-0.80 (m, 9H), 0.11-0.05 (m, 6H); MS (ES+): m/z = 561 (M+H) ⁺ ; LCMS (Method A): t _R = 7.97 min.

Allyl (iS)-(5-(benz loxy)-2-(6-(((terf-butyldimethylsilyl)oxy)methyl)-4- (pyrimidin-5-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-me thoxy- phenyl)carbamate and allyl (<S)-(5-(benz loxy)-2-(2-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(pyrimidin-5-yl)-i,2,3,6-tetrahydropyrid ine-i- carbonyl)-4-methoxyphenyl)carbamate (45) (1: 1)

A solution of (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(6-(((ferf-butyld imethyl- silyl)oxy)methyl)-4-(pyrimidin-5-yl)-3,6-dihydropyridin-i(2H )-yl)methanone and (S)- (2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (pyrimidin-5-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (44) (1.00 g, 1.78 mmol) in dichloromethane (200 mL) was charged with pyridine (0.17 mL, 1.78 mmol) and allyl chloroformate (0.196 mL, 1.86 mmol). After 15 min, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (2 x 100 mL) and brine (100 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with dichloromethane/ acetone (4:1) to afford the title compound (782 mg, 68%) as a brown solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 9.28-7.20 (m, 4H), 7.52- 7.27 (m, 5H), 6.91-6.76 (m, lH), 6.24 (s, lH), 5.98-5-85 (m, lH), 5.32 (dd, J=i7-2, 1.4 Hz, lH), 5.22 (d, J=io.5 Hz, lH), 5.18 (s, 2H), 4.66-4.56 (m, 2H), 4.00-3.86 (m, 2H), 3-84 (s, 3H), 3-81-3-43 (m, 2H), 2.88-2.32 (m, 2H), 0.93-0.73 (m, 9H), 0.12-0.07 (m, 6H); MS (ES+): m/z = 645 (M+H) ⁺ ; LCMS (Method A): t _R = 8.48 min.

Allyl (S)-(5-(benz loxy)-2-(6-(hydroxymethyl)-4-(pyrimidin-5-yl)-i,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)-

214

SUBSTITUTE SHEET RULE 26 (5-(benzyloxy)-2-(2-(hydroxymethyl)-4-(pyrimidin-5-yl)-i,2,3 ,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate (46) (1: 1)

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4- (pyrimidin-5-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-me thoxyphenyl)carbamate and allyl (S')-(5-(benzyloxy)-2-(2-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(pyrimidin- 5-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl )carbamate (45) (0.78 g, 1.21 mmol) in tetrahydrofuran (50 mL) was cooled to o °C and charged with tetrabutyl- ammonium fluoride (1 M in tetrahydrofuran, 1.51 mL, 1.51 mmol). The reaction was allowed to warm to room temperature and after 15 min, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (100 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (50 mL) and brine (50 mL) and dried over magnesium sulfate. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (4:1) gave the title compound (552 mg, 86%) as a cream solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 9.14 (s, lH), 8.77 (s, 2H), 8.09 (s, lH), 7-85-7-69 (m, lH), 7-54-7-30 (m, 5H), 6.87-6.79 (m, lH), 6.26-6.13 (m, lH), 6.01-5.81 (m, lH), 5-37-5-30 (m, lH), 5-24"5-i8 (m, lH), 4.61 (t, J=5.6 Hz, 2H), 4.09- 3.86 (m, 2H), 3.87-3.82 (2 x s, 3H), 3.81-3.38 (m, 2H), 3.00-2.60 (m, lH), 2.50-2.35 (m, lH); MS (ES+): m/z = 531 (M+H) ⁺ ; LCMS (Method B): t _R = 3.18 min.

Allyl (6aS^-3-(benz loxy)-6-hydroxy-2-methoxy-i2-oxo-8-(pyrimidin-5-yl)- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)- carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8- (pyrimidin-5-yl)-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i ,4]diazepine- 5(i2f/)-carboxylate (47) (1:1)

215

SUBSTITUTE SHEET RULE 26

47

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(hydroxymethyl)-4-(pyrimidin-5-yl)- i,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)-(5- (benzyloxy)-2-(2-(hydroxymethyl)-4-(pyrimidin-5-yl)-i,2,3,6- tetrahydropyridine-i- carbonyl)-4-methoxyphenyl)carbamate (46) (0.55 g, 1.03 mmol) in dichloromethane (50 mL) was charged with TEMPO (0.041 g, 0.26 mmol) and (diacetoxyiodo)benzene (0.78 g, 2.42 mmol) and stirred at room temperature for 16 h, at which point TLC and LCMS showed consumption of starting material. Dichloromethane (15 mL) was then added to the reaction mixture, which was quenched by addition of a saturated aqueous solution of sodium metabisulfite (50 mL). The organic phase was separated, washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (9:1) gave the title compound (236 mg, 43%) as a brown solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 9.14 (s, iH), 8.85-8.75 (m, 2H), 7.45-7.29 (m, 5H), 7.26-7.20 (m, iH), 6.72 (s, iH), 6.51-6.33 (m, iH), 5-87-5-60 (m, 2H), 5-21-5-05 (m, 4H), 4.53-4.07 (m, 4H), 3-98-3-90 (m, 3H), 3-87-3-78 (m, iH), 3.09 (d, J=i6.4 Hz, iH), 2.81-2.71 (m, iH); MS (ES+): m/z = 529 (M+H) ⁺ ; LCMS (Method A): t _R = 6.15 min.

Allyl (6aS)-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(pyrimidin-5-yl)-6,6a ,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2Ji)-carbox ylate (48)

216

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(pyrimidin- 5- yl)-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(pyrimidin -5-yl)-6,6a,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (47) (0.235 g, 0.44 mmol) in anhydrous dichloromethane (20 mL) was charged with boron trichloride (1 M in dichloromethane, 1.32 mL, 1.32 mmol) and stirred under an inert atmosphere of nitrogen for 30 min. Methanol (20 mL) was then charged and the resulting mixture irradiated with microwaves at 55 °C for 1 h. After concentrating the resulting mixture in vacuo, purification was carried out by (multiple) flash column chromatography (silica), eluting with dichloromethane/acetone (2:3) to afford the (regiopure) title compound (36 mg, 18%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 9·ΐ6 (S, lH), 8.81 (s, 2H), 7.21 (s, lH), 6.76 (s, lH), 6.48 (s, lH), 5.85-5-68 (m, lH), ₅ -43 (d, J=9-3 Hz, lH), 5.18- 5.03 (m, lH), 4.66-4.41 (m, 2H), 4.30-4.20 (m, 2H), 3.94 (s, 3H), 3·77"3·69 (m, lH), 2.94 (d, J=i6.o Hz, lH), 2.80-2.71 (m, lH); MS (ES+): m/z = 453 (M+H) ⁺ ; LCMS

(Method A): t _R = 5.45 min.

Diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(2,6- dimethoxy-i2-oxo-8-(pyrimidin-5-yl)-6,6a,7,io-tetrahydrobenz o[e]pyrido- [i,2-a][i,4]diazepine-5(i2Ji)-carboxylate) (49)

49

217

SUBSTITUTE SHEET RULE 26 A solution of allyl (6aS)-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(pyrimidin-5-yl)- 6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)-carboxylate (48) (0.035 g _> 0.077 mmol) in A V-dimethylformamide (2 mL) was charged with potassium carbonate (0.011 g, 0.077 mmol), 2,6-bis(bromomethyl)pyridine (0.010 g, 0.039 mmol) and water (0.1 mL). The resulting mixture was irradiated with microwaves at 55 °C for 1 h, at which point TLC and LCMS showed completion of the reaction. Water (20 mL) was added and the resulting suspension extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were then washed with brine (5 x 100 mL), dried over magnesium sulfate, and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with dichloromethane/ methanol (95:5) gave the title compound (22 mg, 57%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 9.16 (s, 2H), 8.80 (s, 4H), 7-8o (t, J=7-8 Hz, lH), 7.54 (d, J=7-8 Hz, 2H), 7.26 (s, 2H), 6.70 (s, 2H), 6.51-6.45 (m, 2H), 5.41 (d, J=9-3 Hz, 2H), 5.25 (s, 4H), 5.07 (d, J=io.5 Hz, 2H), 4.57- 4.36 (m, 4H), 4.33-4.20 (m, 4H), 3.96 (s, 6H), 3·75"3·67 (m, 2H), 3.40 (s, 6H), 2.92 (d, J=15.2 Hz, 2H), 2.81-2.71 (m, 2H); MS (ES+): m/z = 1008 (M+H) ⁺ ; LCMS (Method A): t _R = 6.80 min.

(6aS,6a'S)-3,3'-((Pyridine-2,6-diylbis(methylene))bis(oxy ))bis(2-methoxy- 8-(pyrimidin-5-yl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]dia zepin- -one) (50)

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-i2-oxo-8-(pyrimidin-5-yl)-6,6a,7,io-tetrahydrobenz o[e]pyrido[i,2- a][i,4]diazepine-5(i2H)-carboxylate) (49) (0.024 g, 0.023 mmol) in dichloromethane (2 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (1 mg) and pyrrolidine (5.4 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with

218

SUBSTITUTE SHEET RULE 26 dichloromethane/methanol (95:5) gave the title compound (10 mg, 54%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 9-19 (s, 2H), 8.86 (s, 4H), 7-74 (t, J=7-8 Hz, iH), 7.61 (d, J=5-6 Hz, 2H), 7.55 (s, 2H), 7.47 (d, J=7-5 Hz, 2H), 6.87 (s, 2H), 6.65-6.57 (m, 2H), 5.32 (s, 4H), 4-48 (dd, J=i9-3, 5-2 Hz, 2H), 4-31-4-25 (m, 2H), 4-24-4-17 (m, 2H), 4.00 (s, 6H), 3.03 (d, J=154 Hz, 2H), 2.93 (d, J=i6.4 Hz, 2H); MS (ES+): m/z = 776 (M+H) ⁺ ; LCMS (Method A): t _R = 5.53 min.

(S')-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(6-(((tert-bu tyldimethyl- silyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydropyridin-i(2Ji )- yl)methanone and (iS)-(4-(benz loxy)-5-methoxy-2-nitrophenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydro pyridin- -yl)methanone (51) (1:1)

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (S)-i- (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (5.00 g, 7.56 mmol) in acetonitrile (10 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (0.617 g, 0.756 mmol), 3-thienylboronic acid (1.07 g, 8.35 mmol) and an aqueous solution of potassium carbonate (2 M, 7.56 mL, 15.1 mmol) and irradiated with microwaves at 50 °C for 20 min. The mixture was subsequently diluted with ethyl acetate (50 mL) and sequentially washed with water (25 mL) and brine (25 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (from 1:0 to 0:1) to afford the title compound (3.65 g, 81%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.85-7.73 (m, iH), 7.49-7.34 (m, 5H), 7.33-7.18 (m, 3H), 7.16-7.06 (m, iH), 6.81-6.72 (m, iH), 5.28-5.18 (m, 2H), 5.06 (br. s, iH), 4-01-3-95 (m, 4H), 3.83 (d, J=7-03 Hz, iH), 3.76 (d, J=9-76 Hz, iH), 3-71-3-57 (m, iH), 3-57-3-36

219

SUBSTITUTE SHEET RULE 26 (m, lH), 2.41 (d, J= 19.14 Hz, lH), 0.98-0.82 (m, 9H), 0.18-0.06 (m, 6H); «C NMR (100 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 166.4, 154-7 _? 148.1, 142.0, 135.3, 134.6, 130.6, 128.8, 128.5, 127.6, 127.5, 126.0, 124.6, 122.1, 119.6, 119.2, 109.1, 71.4, 60.4, 56.7, 56.7, 52.5, 52.3, 42.9, 27.4, 25.9, 25.8, 21.0, 18.2, -5.4; MS (ES+): m/z = 595 (M+H) ⁺ .

(iS)-(2-Amino-4-(benz loxy)-5-methoxyphenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydropyridin-i(2ii - yl)methanone and (iS)-(2-amino-4-(benz loxy)-5-methoxyphenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydro pyridin- -yl)methanone (52) (1:1)

A solution of (S')-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydropyridin-i(2H) -yl)methanone and (S)- (4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (thiophen-3-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (51) (3.62 g, 6.08 mmol) in formic acid (5% v/v in absolute ethanol, 60 mL) was charged with zinc powder (15.9 g, 243 mmol) and stirred for 1 h, whilst monitoring by TLC and LCMS. Upon completion, the mixture was filtered over a pad of celite and the resulting cake washed with ethyl acetate. A saturated aqueous solution of sodium hydrogen carbonate (10 mL) was then added to the filtrate and the mixture concentrated in vacuo to remove organics. The residue was then partitioned between ethyl acetate (60 mL) and brine (60 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo.

Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (1:0 to 4:1) gave the title compound (3.24 g, 80%) as a yellow solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 7.46-7.21 (m, 7H), 7.14 (dd, J=2.7, 1.2 Hz, lH), 6.82-6.76 (m, lH), 6.28 (d, J=5-9 Hz, lH), 6.15 (dd, J=3-9, 2.0 Hz, lH), 5.13 (s, 2H), 4.10 (d, J=i6.8 Hz, 2H), 3.96-3.82 (m, lH), 3.81 (s, 3H), 3.79- 3.69 (m, lH), 3-69-3-39 (m, lH), 2.64-2.38 (m, 2H), 0.92-0.85 (m, 9H), 0.07 (s, 3H),

220

SUBSTITUTE SHEET RULE 26 0.02 (br. s, 3H); «C NMR (100 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 150.8, 141-9, 140.1, 136.8, 131.9, 128.6, 127.9, 127.1, 125-9, 124-5, ΐ2ΐ·3, H9-4, H9-3, 118.7, 113-1, 112.3, 103-0, 102.9, 70.7, 64.4, 62.1, 57.1, 30.9, 25.9, 18.3, -5-4; MS (ES+): m/z = 565 (M+H) ⁺ .

Allyl (iS)-(5-(benz loxy)-2-(2-(((terf-butyldimethylsilyl)oxy)methyl)-4- (thiophen-3-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-met hoxy- phenyl)carbamate and allyl (.S)-(5-(benzyloxy)-2-(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(thiophen-3-yl)-i,2,3,6-tetrahydropyridi ne-i- carbonyl)-4-methoxyphenyl)carbamate (53) (1:1)

53

A solution of (S')-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(thiophen-3-yl)-3,6-dihydropyridin-i(2H) -yl)methanone and (S)- (2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (thiophen-3-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (52) (3.00 g, 5.30 mmol) in dichloromethane (20 mL) was cooled to -2°C and charged with pyridine (0.429 mL, 5.30 mmol) and allyl chloroformate (0.564 mL, 5.30 mmol). After 10 min, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (20 mL), water (20 mL) and a saturated aqueous solution of sodium hydrogen carbonate (20 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0:1) to afford the title compound (3.33 g, 97%) as a white solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.57-7.73 (m,

2H), 7.49 (d, J=7.4 Hz, 2H), 7.42-7.22 (m, 6H), 7.15 (br. s, iH), 6.85 (d, J=12.9 Hz, iH), 6.12 (br. s, iH), 5.99-5.82 (m, iH), 5.33 (d, J=i7-2 Hz, iH), 5-24-5-17 (m, 3H), 4-70-4-53 (m, 2H), 3.85 (s, 4H), 3-75 (br. s, iH), 3.58 (br. s, iH), 2.79 (d, J=i4-i Hz, iH), 2.46 (d, J=i6.8 Hz, iH), 0.92-0.81 (m, 10H), 0.12-0.04 (m, 3H), 0.01 (br. s, 2H); NMR (100

221

SUBSTITUTE SHEET RULE 26 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 153.4, 150.2, 144.8, 141.7, 136.4, 132.5, 128.5, 128.0, 127.7, 126.0, 124.5, 1 4-4, U9-4, 118.0, 111.6, 70.7, 65.7, 64.3, 62.1, 56.7, 25.9, 18.3, -5-4; MS (ES+): m/z = 649 (M+H) ^÷ . Allyl (S')-(5-(benz loxy)-2-(2-(hydroxymethyl)-4-(thiophen-3-yl)-i,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)- (5-(benzyloxy)-2-(6-(hydroxymethyl)-4-(thiophen-3-yl)-i,2,3, 6-tetrahydro- pyridine-i-carbonyl)-4-methoxyphenyl)carbamate (54) (1:1)

54

A solution of allyl (S')-(5-(benzyloxy)-2-(2-(((ieri-butyldimethylsilyl)oxy)meth yl)-4- (thiophen-3-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-met hoxyphenyl)carbamate and allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(thiophen-3- yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)c arbamate (53) (3.27 g, 5-03 mmol) in tetrahydrofuran (20 mL) was cooled to o °C and charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 6.03 mL, 6.03 mmol). The reaction was allowed to warm to room temperature and after 20 min, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (50 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (20 mL) and brine (20 mL) and dried over magnesium sulfate. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (1:0 to 0:1) gave the title compound (2.68 g, 99%) as a pale yellow solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.42-8.06 (m, iH), 7.97-7.64 (m, iH), 7.47 (d, J=8.2 Hz, 2H), 7.41-7.27 (m, 4H), 7.24-7.10 (m, 2H), 6.84 (br. s, iH), 6.05 (br. s, iH), 6.00-5.82 (m, iH), 5.38-5.26 (m, iH), 5.24-5.11 (m, 3H),

4.65-4-56 (m, 2H), 3·96-3·85 (m, iH), 3.83-3.81 (m, 3H), 3.72 (br. s, iH), 3.64 (d, J=4-7 Hz, iH), 3.40 (br. s, iH), 3.22 (br. s, iH), 2.52-2.40 (m, 2 H); NMR (100 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 158.5, 153.9, 150.1, 149-8, 145.3, i4i-5, 141.3, 136.4, 136.3, 132.5, 128.5, 127-7, 126. ι, 124.5, 120.0, 119.2, 117.9, o.8, 107.2,

222

SUBSTITUTE SHEET RULE 26 70.8, 65.7, 61.4, 60.4, 56.5, 30.9, 27.9, 25.6, 21.0; MS (ES+): m/z = 535 (M+H) ⁺ ; LCMS (Method B): £ _R = 4.07 min.

Allyl (6S',6aiS')-3-(benz loxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen-3- yl)-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2Ji)- carboxylate and allyl (6S',6aS')-3-(benz loxy)-6-hydroxy-2-methoxy-i2-oxo- 8-(thiophen-3-yl)-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2- «][i _? 4]diazepine-5(i2H)-carboxylate (55) (1:1)

55

A solution of allyl (S')-(5-(benzyloxy)-2-(2-(hydroxymethyl)-4-(thiophen-3-yl)-i ,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)-(5- (benzyloxy)-2-(6-(hydroxymethyl)-4-(thiophen-3-yl)-i,2,3,6-t etrahydropyridine-i- carbonyl)-4-methoxyphenyl)carbamate (54) (2.39 g, 4.48 mmol) in dichloromethane (15 mL) was charged with TEMPO (119 mg, 0.76 mmol) and (diacetoxyiodo)benzene (2.94 g, 9.13 mmol) and stirred at room temperature for 16 h, at which point TLC and LCMS showed consumption of starting material. Dichloromethane (30 mL) was then added to the reaction mixture, which was quenched by addition of a saturated aqueous solution of sodium metabisulfite (20 mL). The organic phase was separated, washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (1:0 to 0:1) gave the title compound (1.70 g, 71%) as a pale yellow solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 7.47-7.17 (m, 10H), 6.76 (d, J=9.o Hz, iH), 6.28 (br. s, iH), 5.66 (dd, J=io.2, 4.3 Hz, 2H), 5.25-5.03 (m, 2H), 4.46 (br. s, 2H), 4-33-4-15 (m, iH), 4.04 (br. s, iH), 3.96-3.88 (m, 3H), 3.80-3.64 (m, iH), 3.13-2.98 (m, iH), 2.69-2.55 (m, 2H); ^C NMR (100 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 168.9, 167.6, 149.1, 141.4, 136.2, 133.3, 131-8, 130.5, 128.6, 128.1, 127.3, 126.1, 125.3, 120.2, 119.0, 118.0, 117.6, 114.3, 110.8, 84.5, 84.1, 71.0, 66.7, 60.4, 56.1, 55-9, 54-6, 41-3, 37-i; MS (ES+): m/z = 533 (M+H) ⁺ ; LCMS (Method A): t _R = 7.62 min.

SUBSTITUTE SHEET RULE 26 Allyl (6aS)-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a, 7,io- tetrahydrobenzo[e]pyrido[i,2-a] [i,4]diazepine-5(i2Ji)-carboxylate (56)

A solution of allyl (6S',6aS ^, )-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen -3- yl)-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6S',6aS ^, )-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen -3-yl)-6,6a,9,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (55) (1.59 g, 2.89 mmol) in anhydrous dichloromethane (10 mL) was charged with boron trichloride (1 M in dichloromethane, 8.94 mL, 8.94 mmol) and stirred at room temperature under an inert atmosphere of nitrogen for 15 min. Methanol (30 mL) was then charged and the resulting mixture irradiated with microwaves at 55 °C for 1 h. After filtering through a cotton pad, washing with dichloromethane and concentrating in vacuo, purification was carried out by (multiple) flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0:1) to afford the (regiopure) title compound (138 mg, 10%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.31 (dd, J=5.i, 2.7 Hz, lH), 7.25-7.23 (m, lH), 7.20-7.17 (m, 2H), 6.78-6.69 (m, 2H), 6.28 (br. s, iH), 5-73 (br. s, lH), 5.41 (d, J=9-4 Hz, lH), 5.07 (d, J=io.9 Hz, 2H), 4.55 (dd, J=i3-i, 5.7 Hz, lH), 4.44 (br. s, lH), 4·23"4·ΐ6 (m, 2H), 3-87-3-83 (m, 3H), 3-44-3-41 (m, 3H), 2.98-2.89 (m, lH), 2.70-2.60 (m, lH); ^¾ »C NMR (100 MHz, CDC1 ₃ ) δ 169.4, 156.0, 148.4, 146.7, 141.2, 131.9, 130.6, 128.8, 126.4, 124-6, ιΐ9·9, H9-2, 117.3, 116.1, 110.3, 91.3, 66.4, 56.1, 54.9, 41.2, 26.9; MS (ES+): m/z = 457 (M+H) ⁺ ; LCMS (Method A): t _R = 7.00 min. Diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a ^l iS')-bis(2,6- dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a,7,io-tetrahydrobenzo [e]-

224

SUBSTITUTE SHEET RULE 26 yrido[i,2-a][i,4]diazepine-5(i2Ji)-carboxylate) (57)

57

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a ,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (56) (69 mg, 0.15 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (21 mg, 0.15 mmol), 2,6-bis(bromomethyl)pyridine (19.9 mg, 0.075 mmol) and water (0.1 mL). The resulting mixture was irradiated with microwaves at 55 °C for 1 h, at which point TLC and LCMS showed completion of the reaction. Ethyl acetate (10 mL) was added and the mixture extracted with water (5 mL) and brine (5 mL). The organic phase was then dried over magnesium sulfate, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0: 1) gave the title compound (35 mg, 46%) as a pale yellow solid.

Ή NMR (400 MHz, CDCI ₃ ) 5 7.84-7-74 (m, iH), ₇ -54 (d, J=7-4 Hz, 2H), ₇ -33 (dd, J= ₅ .i, 2.7 Hz, 2H), 7.31-7.25 (m, 4H), 7.24-7.16 (m, 2H), 6.70 (br. s, 2H), 6.37-6.26 (m, 2H), 5.67 (br. s, iH), 5.40 (d, J=9-4 Hz, iH), 5-31-5-22 (m, 4H), 5-13-4-97 (m, 3H), 4-57-4-45 (m, 2H), 4.42 (br. s, 2H), 4.30-4.18 (m, 3H), 4.00-3.92 (m, 8H), 3-69-3-57 (m, 2H), 3-45-3-36 (m, 6H), 3.00-2.84 (m, 2H), 2.75-2.57 ppm (m, 2H); «C NMR (100 MHz, CDCI ₃ ) δ 169.0, 156.0, 149.4, 141-2, 137-9, 131-9, 130.6, 126.4, 124-6, 120.4, U9-9, U9-3, 114.6, 110.9, 91-3, 73-7, 71-4, 66.4, 60.3, 56.2, 54.8, 41.2, 26.9; MS (ES+): m/z = 1017 (M+H) ⁺ ; LCMS (Method A): t _R = 8.63 min.

(6aS ^, ,6a'iS')-3,3 ^l -((Pyridine-2,6-diylbis(methylene))bis(oxy))bis(2-meth oxy- 8-(thiophen-3-yl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diaz epin- i2(6aH)-one) (58)

225

SUBSTITUTE SHEET RULE 26

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS ^, ,6'S ^, ,6a'S')- bis(2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a,7,io-tetrahy drobenzo[e]pyrido[i,2- <3][i,4]diazepine-5(i2H)-carboxylate) (57) (35 mg, 0.034 mmol) in dichloromethane (2 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (1 mg) and pyrrolidine (5 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with

dichloromethane/methanol (95:5) gave the title compound (15 mg, 55%) as a cream solid. Ή NMR (400 MHz, CDC1 ₃ ) δ ηη^-ηηο (m, lH), 7.60 (d, J=5-9 Hz, 2H), 7.56 (s, 2H), 7.47 (d, J=7.8 Hz, 2H), 7.37 (dd, J=5.i, 2.7 Hz, 2H), 7.31 (dd, J=5.i, 1.2 Hz, 2H), 6.86 (s, 2H), 6.49-6.43 (m, 2H), 5.32 (s, 4H), 4-45 (dd, J=i8.5, 5-7 Hz, 2H), 4.14 (d, J=i8.7 Hz, 2H), 4.01 (s, 6H), 3·97"3·9ΐ (m, 2H), 3-78-3-64 (m, 2H), 2.98-2.91 (m, 4H); MS (ES+): m/z = 784 (M+H) ⁺ ; LCMS (Method A): t _R = 7.23 min.

Diallyl 3,3'-((i,3-phenylenebis(methylene))bis(oxy))(6iS',6aiS',6 ^l iS',6a ^l iS')- bis(2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a,7,io-tetrahy drobenzo[e]- opyrido[i,2-a][i,4]diazepine-5(i2Ji)-carboxylate) (59)

226

SUBSTITUTE SHEET RULE 26

59

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a ,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (56) (63 mg, 0.13 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (18 mg, 0.13 mmol), i,3-bis(bromomethyl)benzene (17.2 mg, 0.065 mmol) and water (0.1 mL). The resulting mixture was irradiated with microwaves at 55 °C for 1 h, at which point TLC and LCMS showed completion of the reaction. Ethyl acetate (10 mL) was added and the mixture extracted with water (5 mL) and brine (5 mL). The organic phase was then dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0:1) gave the title compound (45 mg, 69%) as a pale yellow solid. Ή NMR (400 MHz, CDCI ₃ ) δ 7-55 (s, lH), 748-7.38 (m, 4H), 7-38-7-34 (m, 2H), ₇ -34 (s, lH), 7-31-7-28 (m, 2H), 7.25-7.20 (m, 2H), 6.72 (br. s, 2H), 6.40-6.31 (m, 2H), 5.71 (d, J=3-9 Hz, lH), 5.42 (d, J=9-8 Hz, lH), 5.17 (s, 5H), 5-i4"5-Oi (m, 4H), 4.54 (d, J=12.9 Hz, 2H), 4.45 (br. s, 2H), 4-33-4-17 (m, 4H), 3-95 (s, 6H), 3.67 (t, J=7-0 Hz, 2H), 3.49- 3-39 (m, 7H), 2.78-2.58 (m, 2H), 2.24 (br. s, 2H); ¾»C NMR (100 MHz, CDC1 ₃ ) δ 169.0, 1494, 141-2, 136.6, 131.9, 130.6, 128.9, 126.3, 124-6, ιΐ9·9, H9-2, 117.2, 114.7, no.8, 91-3, 73-7, 71-0, 66.3, 56.1, 41.1, 29.5, 26.7; MS (ES+): m/z = 1016 (M+H) ⁺ ; LCMS

(Method A): t _R = 8.87 min.

(6aS ^, ,6a'iS')-3,3 ^l -((i,3-Phenylenebis(methylene))bis(oxy))bis(2-methoxy- 8- (thiophen-3-yl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diazep in-i2(6aii)- one) (60)

227

SUBSTITUTE SHEET RULE 26

A solution of diallyl 3,3'-((i,3-phenylenebis(methylene))bis(oxy))(6S ^, ,6aS ^, ,6'S ^, ,6a'S')- bis(2,6-dimethoxy-i2-oxo-8-(thiophen-3-yl)-6,6a,7,io-tetrahy drobenzo[e]pyrido[i,2- <3][i,4]diazepine-5(i2H)-carboxylate) (59) in dichloromethane (2 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (1 mg) and pyrrolidine (5 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with dichloromethane/acetone (1:0 to 0:1) gave the title compound (12.7 mg, 74%) as a pale yellow solid. Ή NMR (400 MHz,

CDCI ₃ ) δ 7·6ι (d, J=5-5 Hz, 2H), 7.54 (s, 2H), 7.51 (s, iH), 7-42-7-37 (m, 5H), 7-32 (dd, J=5.i, 1.2 Hz, 2 H), 6.84 (s, 2H), 6.47 (dt, J=4-9, 2.6 Hz, 2H), 5-29-5-13 (m, 5H), 4.45 (dd, J=i8.4, 5-9 Hz, 2H), 4.14 (d, J=i8.4 Hz, 2H), 3-99-3-98 (m, 6H), 3.95 (dt, J=5-4, 2.6 Hz, 2H), 2.95 (d, J=2.7 Hz, 4H); MS (ES+): m/z = 783 (M+H) ⁺ ; LCMS (Method A): t _R = 7.50 min.

(iS')-(4-(Benz loxy)-5-methoxy-2-nitrophenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydropyridin-i(2Ji - yl)methanone and (iS)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydro pyridin- i(2H)-yl)methanone (61) (1:1)

228

SUBSTITUTE SHEET RULE 26

A solution of (S')-i-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-(((ieri-bu tyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate and (S)-i- (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-(((ieri-butyldime thylsilyl)oxy)methyl)- i,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (8) (2.50 g, 3.78 mmol) in acetonitrile (5 mL) was charged with [i,i'-bis(diphenylphosphino)ferrocene]dichloro- palladium(II), complex with dichloromethane (310 mg, 0.38 mmol), 2-thienylboronic acid (534 mg, 4.17 mmol) and an aqueous solution of potassium carbonate (2 M, 3.78 mL, 7.56 mmol) and irradiated with microwaves at 50 °C for 20 min. The mixture was subsequently diluted with ethyl acetate (25 mL) and sequentially washed with water (15 mL) and brine (15 mL), then dried over solid anhydrous magnesium sulfate and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (from 1:0 to 0:1) to afford the title compound (1.78 g, 80%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.87-7.72 (m, iH), 7.51-7.33 (m, 5H), 7.18 (d, J=6.2 Hz, iH), 7.09-6.88 (m, 2H), 6.77 (d, J=5.9 Hz, iH), 6.47-5.78 (m, iH), 5.24 (d, J=3-5 Hz, 2H), 4-01-3-95 (m, 4H), 3-75 (br. s, 2H), 3.65 (br. s, iH), 3.10-2.33 (m, 2H), 1.03-0.81 (m, 9H), 0.78 (s, iH), 0.20-0.02 (m, 6H); ¾»C NMR (100 MHz, CDC1 ₃ ) δ 177-0, 176.6, 137-3, 135-3, 128.8, 128.5, 122.6, 122.3, 119-6, 109.1, 100.1, 96.5, 71-4, 56.7, 52-0, 35-7, 27.6, 25.9, 18.2, 18.0, -5.4; MS (ES+): m/z = 595 (M+H) ⁺ ; LCMS (Method A): t _R = 9.28 min.

(iS)-(2-Amino-4-(benz loxy)-5-methoxyphenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydropja4din-i(2Ji - yl)methanone and (iS)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydro pyridin- i(2H)-yl)methanone (62) (1:1)

229

SUBSTITUTE SHEET RULE 26

A solution of (S')-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydropyridin-i(2H) -yl)methanone and (S)- (4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (thiophen-2-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (61) in formic acid (5% v/v in absolute ethanol, 30 mL) was cooled to o°C and charged with zinc powder (7.71 g, 118 mmol), then stirred for 2 h, whilst monitoring by TLC and LCMS. Upon completion, the mixture was filtered over a pad of celite and the resulting cake washed with ethyl acetate. A saturated aqueous solution of sodium hydrogen carbonate (10 mL) was then added to the filtrate and the mixture concentrated in vacuo to remove organics. The residue was then partitioned between ethyl acetate (30 mL) and brine (30 mL) and the organic phase dried over magnesium sulfate, filtered, and concentrated in vacuo.

Purification by flash column chromatography (silica), eluting with petroleum spirit, 40- 60 °C/ethyl acetate (from 1:0 to 7:3) gave the title compound (1.53 g, 92%) as a yellow solid. Ή NMR (400 MHz, CDC1 ₃ ), mixture of rotamers and regioisomers, δ 7.46-7.29 (m, 5H), 7.20-7.14 (m, lH), 7.01-6.96 (m, 2H), 6.83-6.73 (m, lH), 6.28 (d, J=5-9 Hz, lH), 6.18-6.00 (m, lH), 4.19-4.01 (m, 2H), 3·97"3·85 (m, lH), 3·83"3·79 (m, H), 3-79" 3.69 (m, lH), 3.69-3.60 (m, lH), 2.82 (dq, J=rj.-≥, 3.1 Hz, lH), 2.57-2.41 (m, lH), 0.93- 0.82 (m, 9H), 0.10-0.03 (m, 6H); ¾»C NMR (100 MHz, CDC1 ₃ ) δ 150.8, 144.6, 141.8, 140.1, 136.8, 128.6, 127.9, 127.1, 123.8, 122.2, 120.4, n8-5, 113.1, 112.2, 102.9, 70.8, 64.4, 62.0, 57.0, 25.9, 18.3, -5.4; MS (ES+): m/z = 565 (M+H) ⁺ ; LCMS (Method A): t _R = 9.65 min. Allyl (iS)-(5-(benzyloxy)-2-(6-(((tert-butyldimethylsilyl)oxy)meth yl)-4-

(thiophen-2-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4- methoxyphenyl)- carbamate and allyl (iS)-(5-(benzyloxy)-2-(2-(((tert-butyldimethylsilyl)- oxy)methyl)-4-(thiophen-2-yl)-i,2,3,6-tetrahydropyridine-i-c arbonyl)-4- methoxyphenyl)carbamate (63) (1:1)

230

SUBSTITUTE SHEET RULE 26

63

A solution of (S')-(2-Amino-4-(benzyloxy)-5-methoxyphenyl)(6-(((ieri-butyl dimethyl- silyl)oxy)methyl)-4-(thiophen-2-yl)-3,6-dihydropyridin-i(2H) -yl)methanone and (S)- (2-amino-4-(benzyloxy)-5-methoxyphenyl)(2-(((ieri-butyldimet hylsilyl)oxy)methyl)-4- (thiophen-2-yl)-3,6-dihydropyridin-i(2H)-yl)methanone (62) (1.50 g, 2.65 mmol) in dichloromethane (10 mL) was cooled to -2°C and charged with pyridine (214 μΐ,, 2.65 mmol) and allyl chloroformate (282 ^L, 2.65 mmol). After 10 min, the reaction was judged to be complete by TLC and LCMS and was sequentially washed with a saturated aqueous solution of copper sulfate (10 mL), water (10 mL) and a saturated aqueous solution of sodium hydrogen carbonate (10 mL). The organic extract was then dried over magnesium sulfate and concentrated in vacuo. The resulting oil was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0: 1) to afford the title compound (1.48 g, 87%) as a white solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.14 (br. s, iH), 7.49 (d, J=7.8 Hz, 2H), 7.42-7.30 (m, 3H), 7.21-7.17 (m, iH), 7.02-6.98 (m, 2H), 6.84 (d, 3=\\η Hz, iH), 6.13 (br. s, iH), 5·99"5·85 (m, iH), 5.33 (dt, J=i7-2, 1.6 Hz, iH), 5.26-5.14 (m, 4H), 4-69-4-53 (m, 3H), 3-88-3-79 (m, 4H), 3-76 (d, J=4-7 Hz, iH), 2.51 (d, J=i7-2 Hz, iH), 0.89 (s, 9H), 0.86 (s, 3H), 0.13-0.04 (m, 3 H), 0.01 (br. s, 2H); «C NMR (100 MHz, CDCI ₃ ) δ 153-6, 150.3, 144-3, 136-4, 132-5, 128.5, 127-7, 127-4, 124-0, 122.4, u8.o, 70.7, 65.7, 64.2, 62.0, 56.7, 25.9, 18.3, -5.5; MS (ES+): m/z = 649 (M+H) ⁺ .

Allyl (5)-(5-(Βεηζγ1οχγ)-2-(6-(1ιγάΓθΧ3Ττΐ6ΐ1ι� �1)-4-(ΐ1ιίορ1ΐ6η-2-γ1)-ι, 2,3,6- tetrahydropjTidine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)- (5-(benzyloxy)-2-(2-(hydroxymethyl)-4-(thiophen-2-yl)-i,2,3, 6-tetrahydro- pyridine-i-carbonyl)-4-methoxyphenyl)carbamate (64) (1: 1)

231

SUBSTITUTE SHEET RULE 26

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(((ieri-butyldimethylsilyl)oxy)meth yl)-4- (thiophen-2-yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-met hoxyphenyl)carbamate and allyl (S')-(5-(benzyloxy)-2-(2-(((ieri-butyldimethylsilyl)oxy)meth yl)-4-(thiophen-2- yl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)c arbamate (63) (1.45 g, 2.23 mmol) in tetrahydrofuran (10 mL) was cooled to o °C and charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 2.67 mL, 2.67 mmol). The reaction was allowed to warm to room temperature and after 1 h, TLC and LCMS confirmed consumption of starting material. Ethyl acetate (25 mL) was added to the reaction mixture and the resulting solution washed with a saturated aqueous solution of ammonium chloride (10 mL) and brine (10 mL) and dried over magnesium sulfate. Purification by flash column chromatography (silica), eluting with dichloromethane/ acetone (1:0 to 0:1) gave the title compound (1.19 g, 100%) as a pale yellow solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 8.19 (br. s, iH), 7.73 (br. s, iH), 7.47 (d, J=8.2 Hz, 2H), 7-41-7-29 (m, 3H), 7.19 (ddd, J=9.8, 4.7, 1.6 Hz, iH), 7.02-6.95 (m, 2H), 6.83 (br. s, iH), 6.21-5.75 (m, 2H), 5-39-5-31 (m, iH), 5.24-5.04 (m, 4H), 4·74-4·52 (m, 2H), 3-88-3.79 (m, 4H), 3·79-3·65 (m, 2H), 3.40 (br. s, iH), 3.06- 2.29 (m, 2H); ^¾ »C NMR (100 MHz, CDC1 ₃ ) 5 170.5, 153-9, 153-8, 144-1, 143-8, 136-3, 132.4, 128.5, 127-7, 127-4, 124-4, 122.9, 122.4, H7-8, 110.7, 70.8, 65.8, 56.5, 53-4, 30-9, 27.9; MS (ES+): m/z = 535 (M+H) ⁺ ; LCMS (Method A): t _R = 7.70 min.

Allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen-2 -yl)- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)- carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8- (thiophen-2-yl)-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i, 4]diazepine- 5(i2f/)-carboxylate (65) (1:1)

232

SUBSTITUTE SHEET RULE 26

65

A solution of allyl (S')-(5-(benzyloxy)-2-(6-(hydroxymethyl)-4-(thiophen-2-yl)-i ,2,3,6- tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)carbamate and allyl (S)-(5- (benzyloxy)-2-(2-(hydroxymethyl)-4-(thiophen-2-yl)-i,2,3,6-t etrahydropyridine-i- carbonyl)-4-methoxyphenyl)carbamate (64) (1.16 g, 2.17 mmol) in dichloromethane (10 mL) was charged with TEMPO (33 mg, 0.21 mmol) and (diacetoxyiodo)benzene (837 mg, 2.60 mmol) and stirred at room temperature for 16 h, at which point TLC and LCMS showed consumption of starting material. Dichloromethane (15 mL) was then added to the reaction mixture, which was quenched by addition of a saturated aqueous solution of sodium metabisulfite (10 mL). The organic phase was separated, washed with a saturated aqueous solution of sodium hydrogen carbonate (10 mL), brine (10 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/ethyl acetate (1:0 to 0:1) gave the title compound (574 mg, 50%) as a yellow solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers and regioisomers, δ 7.52-7.29 (m, 6H), 7.25-7.20 (m, iH), 7.26-7.19 (m, iH), 7.08-6.98 (m, 2H), 6.76 (br. s, iH), 6.40-6.13 (m, iH), 5·93"5·58 (m, iH), 5.22-5.06 (m, 3H), 4·87-4·72 (m, iH), 4-56-4-37 (m, iH), 4-33-4-19 (m, 2H), 4.03 (br. s, iH), 3-98-3-91 (m, 3H), 3.80-3.69 (m, iH), 3.12 (d, J=i5-2 Hz, iH), 2.77-2.54 (m, 2H); «C NMR (100 MHz, CDC1 ₃ ) δ 171.2, l68.8, 149.1, 143.7, 136.2, 129.6, 128.6, 127.5, 127.3, 125-3, 124-6, 123.1, 118.7, 117-3, 114-2, 110.8, 84.0, 71.1, 66.7, 60.4, 56.2, 41.3, 37.1, 27.2, 21.0, 14.2; MS (ES+): m/z = 533 (M+H) ⁺ ; LCMS (Method A): t _R = 7.55 min.

Allyl (6aS)-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(thiophen-2-yl)-6,6a, 7,io- tetrahydrobenzo[e]pyrido[i,2-a] [i,4]diazepine-5(i2ii)-carboxylate (66)

233

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen-2 - yl)-6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate and allyl (6aS')-3-(benzyloxy)-6-hydroxy-2-methoxy-i2-oxo-8-(thiophen- 2-yl)-6,6a,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (65) (529 mg, 0.99 mmol) in anhydrous dichloromethane (3.7 mL) was charged with boron trichloride (1 M in dichloromethane, 2.97 mL, 2.97 mmol) and stirred at room temperature under an inert atmosphere of nitrogen for 15 min. Methanol (5.3 mL) was then charged and the resulting mixture irradiated with microwaves at 55 °C for 1 h. After filtering through a cotton pad, washing with dichloromethane and concentrating in vacuo, purification was carried out by (multiple) flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:0 to 0:1) to afford the (regiopure) title compound (16 mg, 3%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.25-7.21 (m, lH), 7.08-7.04 (m, lH), 7.04-7.00 (m, lH), 6.77 (s, lH), 6.35 (dt, J=5-6, 2.9 Hz, lH), 6.18-5.71 (m, lH), 5.45 (d, J=9-4 Hz, lH), 5.16-5.04 (m, 2H), 4·63"4·55 (m, lH), 4.49- 4.41 (m, lH), 4-25-4-19 (m, 2H), 3-97-3-92 (m, 3H), 3.89 (d, J=6.2 Hz, lH), 3.69-3-62 (m, lH), 3-48-3-43 (m, 3H), 3.03-2.97 (m, lH), 2.76-2.65 (m, 1 H); MS (ES+): m/z = 457 (M+H) ⁺ ; LCMS (Method A): t _R = 7.08 min. Diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(2,6- dimethoxy-i2-oxo-8-(thiophen-2-yl)-6,6a,7,io-tetrahydrobenzo [e]pyrido- -a][i,4]diazepine-5(i2ii)-carboxylate) (67)

234

SUBSTITUTE SHEET RULE 26

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-i2-oxo-8-(thiophen-2-yl)-6,6a ,7,io- tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxy late (66) (15 mg, 0.032 mmol) in iV,iV-dimethylformamide (0.5 mL) was charged with potassium carbonate (4.42 mg, 0.032 mmol), 2,6-bis(bromomethyl)pyridine (4.23 mg, 0.016 mmol) and water (0.1 mL). The resulting mixture was irradiated with microwaves at 55 °C for 2 h, at which point TLC and LCMS showed completion of the reaction. Ethyl acetate (10 mL) was added and the mixture extracted with water (5 mL) and brine (5 mL). The organic phase was then dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit, 40- 60 °C/ethyl acetate (1:0 to 0:1) gave the title compound (7 mg, 22%) as a white solid. Ή NMR (400 MHz, CDCI ₃ ) δ 7.85-7.76 (m, lH), 7.59-7.51 (m, 2H), 7.26-7.20 (m, 2H), 7.08-6.97 (m, 5H), 6.71 (br. s, 2H), 6.39-6.31 (m, 2H), 5.68 (br. s, lH), 5.43 (d, J=9.o Hz, lH), 5-34-5-23 (m, 6H), 5-12-4-98 (m, 4H), 4.50 (br. s, 2H), 4.43 (br. s, 2H), 4.27- 4-19 (m, 3H), 4-01-3-93 (m, 8H), 3-68-3-59 (m, 2H), 3.43 (s, 6H), 3.03-2.92 (m, 2H), 2.71 (d, J=io.2 Hz, 2H); MS (ES+): m/z = 1016 (M+H) ⁺ ; LCMS (Method B): t _R = 4.58 min.

(6aS ^, ,6a'iS')-3,3 ^l -((Pyridine-2,6-diylbis(methylene))bis(oxy))bis(2-meth oxy- 8-(thiophen-2-yl)-7,io-dihydrobenzo[e]pyrido[i,2-a][i,4]diaz epin- i2(6aH)-one) (68)

235

SUBSTITUTE SHEET RULE 26

A solution of diallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aS',6a'S ^, )-bis(2,6- dimethoxy-i2-oxo-8-(thiophen-2-yl)-6,6a,7,io-tetrahydrobenzo [e]pyrido[i,2- <3][i,4]diazepine-5(i2H)-carboxylate) (67) (7 mg, 0.0068 mmol) in dichloromethane (0.5 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (0.39 mg) and pyrrolidine (1.5 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (2 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with

dichloromethane/acetone (1:0 to 0:1) gave the title compound (2.0 mg, 38%) as a pale yellow solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.76-7.65 (m, lH), 7.64-7.60 (m, lH), 7.45 (d, J=7-4 Hz, 2H), 7.09 (d, J=3-5 Hz, 2H), 7.06-6.98 (m, 2H), 6.87-6.78 (m, 2H), 6.46 (dt, J=5-5, 2.7 Hz, 2H), 5-35-5-24 (m, 5H), 4.43 (dd, J=i8.7, 5-9 Hz, 2H), 4.11 (d, J=i8.4 Hz, 2H), 4-01-3-95 (m, 7H), 3.93 (dt, J=5-6, 2.9 Hz, 2H), 3-74-3-58 (m, 2H), 3.02-2.86 (m, 4H); MS (ES+): m/z = 784 (M+H) ⁺ ; LCMS (Method A): t _R = 7.17 min.

Methyl (iS)-2-(4-(benz loxy)-5-methoxy-2-nitrobenzoyl)-i,2,3,4-tetrahydro- isoquinoline-3-carboxylate (69)

SUBSTITUTE SHEET RULE 26

A mixture of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (3) (2.0 g, 6.6 mmol), oxalyl chloride (1.70 mL, 19.8 mmol) and anhydrous A V-dimethylformamide (2 drops) in anhydrous dichloromethane (40 mL) was stirred at room temperature for 3 h.

Anhydrous toluene (8 mL) was added to the reaction mixture which was then concentrated in vacuo. A solution of the resulting residue in anhydrous

dichloromethane (10 mL) was added dropwise to a solution of methyl (S)-i,2,3,4- tetrahydroisoquinoline-3-carboxylate (1.65 g, 7.26 mmol) and triethylamine (2.0 mL, 14.5 mmol) in anhydrous dichloromethane (30 mL), at -10 °C. The reaction mixture was stirred at room temperature for 2 h and then washed with hydrochloric acid (1 M, 20 mL) and brine (20 mL), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 30%), to give the title compound (2.5 g, 79%) as a yellow oil. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.49-7.42 (m, 6H), 7.24-7.19 (m, 5H), 5.25 (s, 2H), 4.64-4-60 (m, lH), 4.38-4.26 (m, 2H), 3.93 (s, 3H), 3-58 (s, 3H), 3·33"3·23 (m, 2H); ^!3 C NMR (100 MHz, CDC1 ₃ ) δ 170.8, 170.3, 154-6, 148.4, 135-3, 133-5, 130-5, 130.ι, 128.9, 128.8, 128.6, 128.4, 127-7, 127-4, 126.7, 109-3, 109·ΐ, 71-4, 56.8, 52.6, 31.8, 31.0, 30.5; MS (ES+): m/z = 477 (M+H) ⁺ ; LCMS (Method B): t _R = 4.10 min. (iS)-(4-(Benz loxy)-5-methoxy-2-nitrophenyl)(3-(hydroxymethyl)-3,4- dihydroisoquinolin-2(iii)-yl)methanone (70)

A solution of methyl (S)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-i,2,3,4- tetrahydroisoquinoline-3-carboxylate (69) (2.4 g, 5.0 mmol) in anhydrous

237

SUBSTITUTE SHEET RULE 26 tetrahydrofuran (48 mL) was charged with a solution of lithium boro hydride (2 M in tetrahydrofuran, 3.8 mL, 8.7 mmol) at o °C. The reaction mixture was stirred at room temperature for 3 hours. Water (150 mL) was added dropwise at o °C and the reaction mixture was then extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were then concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloro methane (from 0% to 30%), to give the title compound (2.2 g, 97%) as creamy oil. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.42- 7-39 (m, 4H), 7-36-7-34 (m, 5H), 7.30 (s, lH), 7.29 (s, lH), 5.17 (s, 2H), 4.62 (s, lH), 4.36-4-25 (m, lH), 4-23-4-16 (m, 2H), 3.87 (s, 3H), 3-70-3-63 (m, lH), 3-58-3-50 (m, lH), 3.05-2.97 (m, 2H); «C NMR (100 MHz, CDC1 ₃ ) δ l68.2, 150.2, 148.3, 133.7, 128.9, 128.9, 128.8, 128.6, 127.7, 127.6, 127.5, 127.0, 126.5, 114-4, 110.6, 108.9, 103.9, 91-6, 71.4, 65.4, 54-4, 33-3; MS (ES+): m/z = 449 (M+H) ⁺ ; LCMS (Method B): t _R = 3.78 min. (iS)-(2-Amino-4-(benz loxy)-5-methoxyphenyl)(3-(hydroxymethyl)-3,4- dihydroisoquinolin-2(iii)-yl)methanone (71)

A solution of (S')-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(3-(hydroxymethy l)-3,4- dihydroisoquinolin-2(iH)-yl)methanone (70) (2.20 g, 4.90 mmol) in tetrahydrofuran (50 mL) and methanol (50 mL) was charged with iron (III) chloride hexahydrate (0.80 g, 2.90 mmol), activated charcoal (2.60 g, 221 mmol) and hydrazine (2.90 mL, 58.9 mmol). The reaction mixture was then stirred at reflux (85 °C) for 16 h. The mixture was subsequently allowed to cool to room temperature and filtered through a plug of celite. The filter cake was washed with ethyl acetate and methanol and then

concentrated in vacuo to give the title compound (1.7 g, 83%) as brown solid.

Ή NMR (400 MHz, MeOD) δ 7.48 (s, lH), 7.46 (s, lH), 7.41-7.33 (m, 4H), 7.20-7.18 (m, 3H), 6.84 (s, lH), 6.56 (s, lH), 5.11 (s, 2H), 4.61 (s, lH), 4.54-4.40 (m, lH), 3.77 (s, 3H), 3.62-3.54 (m, 2H), 3.19 (dd, J=i6.2, 5.9 Hz, 2H), 2.92-2.80 (m, 2H); «C NMR (100 MHz, MeOD) δ 169.1, 149.8, 141.0, 135.5, 130-7, 129.0, 128.7, 128.6, 128.5, 128.4, 128.2, 127 , 127-0, 126.7, no.i, 109.1, 71.0, 68.7, 64.8, 56.4, 50.3, 27.9; MS (ES+): m/z = 419 (M+H) ⁺ ; LCMS (Method B): t _R = 3.50 min.

238

SUBSTITUTE SHEET RULE 26 Allyl (iS)-(5-(benz loxy)-2-(3-(hydroxymethyl)-i,2,3,4-tetrahydro- isoquinoline-2-carbonyl)-4-methoxyphenyl)carbamate (72)

A solution of (S')-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(3-(hydroxymethy l)-3,4- dihydroisoquinolin-2(iH)-yl)methanone (71) (1.50 g, 3.6 mmol) and anhydrous pyridine (696 μΐ,, 8.97 mmol) in anhydrous dichloromethane (50 mL) at -10 °C was slowly charged with a solution of allylchloroformate (343 μΐ,, 3.23 mmol) in anhydrous dichloromethane (30 mL). The reaction mixture was stirred at room temperature for 30 min and then sequentially washed with a saturated aqueous solution of copper (II) sulfate (50 mL), water (50 mL) and a saturated aqueous solution of sodium hydrogen carbonate (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 20%), to give the title compound (1.47 g, 81%) as an off-white solid. Ή NMR (400 MHz, MeOD) δ 8.14 (s, iH), 7.81 (s, iH), 7.51 (s, iH), 7.49 (s, iH), 7-42-7-32 (m, 4H), 7-23-7-17 (m, 3H), 6.82 (s, iH), 5-97-5-87 (m, iH), 5.33 (dq, J=i7-2, 1.5 Hz, iH), 5.22 (dq, J=io.6, 1.3 Hz, iH), 5.19 (s, 2H), 4.68-4.64 (m, iH), 4.61 (dd, J=5-5, 1-3 Hz, 2H), 4.44 (br. s, 2H), 3.82 (s, 3H), 3.70-3.64 (m, iH), 3.21-3.15 (m, iH), 2.74 (br. s, iH); ¾»C NMR (100 MHz, CDC1 ₃ ) δ 169-4, 152-9, 148-7, 144-1, 140.1, 135-3, 131-4, 130.5, 129-1, 128.1, 127.5, 127-0, 126.7, 125.9, 125-5, 117-9, 116.8, 109.6, 105.7, 69.7, 674, 66.0, 64.7, 55-3, 53-8, 26.8; MS (ES+): m/z = 503 (M+H) ⁺ ; LCMS (Method B): t _R = 3.95 min.

Allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy- 14-0x0-6, 6a,7,i2-tetra- hydrobenzo[5,6][i,4]diazepino[i,2-b]isoquinoline-5(i4ii)-car boxylate (73)

239

SUBSTITUTE SHEET RULE 26

A solution of allyl (S)-(5-(benzyloxy)-2-(3-(hydroxymethyl)-i,2,3,4-tetrahydro- isoquinoline-2-carbonyl)-4-methoxyphenyl)carbamate (72) (1.4 g, 2.78 mmol) in dichloromethane (80 mL) was charged with 2,2,6,6-tetramethyl-i-piperidinyloxy (44 mg, 0.28 mmol) and (diacetoxyiodo)benzene (1.0 g, 3.33 mmol). The reaction mixture was stirred at room temperature for 16 h and was then sequentially washed with a saturated aqueous solution of sodium metabisulfite (40 mL), a saturated aqueous solution of sodium hydrogen carbonate (40 mL), water (30 mL) and brine (30 mL). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloro methane (from 0% to 20%), to give the title compound (1.2 g, 86%) as an off-white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.44-7.31 (m, 6H), 7.28-7.26 (m, 5H), 6.72 (s, lH), 5.70-5.61 (m, lH), 5.31 (d, J=9.8 Hz, lH), 5.20-5.17 (m, lH), 5-H-5-Q7 (m, 3H), 4.83 (d, J=15.6 Hz, lH), 4.58 (d, J=i ₅ .6 Hz, lH), 4-48-4-34 (m, 2H), ₃ ,94 (s, 3H), 3-74-3-69 (m, lH), 3.17-3.05 (m, 2H); NMR (lOO MHz, CDC1 ₃ ) δ 169.0, 149-0, 136.2, 134-3, 133-7, 131-8, 126.7, 128.2, 127.9, 127-8, 127.3, 126.7, 118.1, 114.0, 111.2, 84.8, 71.0, 66.7, 56.2, 53.5, 50.8, 44-3, 30.2; MS (ES+): m/z = 501 (M+H) ⁺ ; LCMS (Method B): t _R = 3.80 min. Allyl (6aS')-3-hydroxy-2,6-dimethoxy-i4-oxo-6,6a,7,i2-tetrahydrobe nzo- -b]isoquinoline-5(i4H)-carboxylate (74)

74

A solution of allyl (6aS)-3-(benzyloxy)-6-hydroxy-2-methoxy-i4-oxo-6,6a,7,i2- tetrahydrobenzo[5,6][i,4]diazepino[i,2-¾]isoquinoline-5(i4H )-carboxylate (73) (100 mg, 0.199 mmol) in dichloromethane (1 mL) was charged with boron trichloride (1 M solution in dichloromethane, 600 μΐ,, o.6oo mmol) and the resulting suspension was

240

SUBSTITUTE SHEET RULE 26 stirred at room temperature for 10 min, then methanol (2 mL) was added to the reaction mixture which was irradiated with microwaves 60 min at 55 °C. The reaction mixture was subsequently filtered through a cotton pad that was washed with dichloromethane and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with petroleum spirit 40-6o°C/ethyl acetate (1:0 to 0:1) gave the title compound (40 mg, 48%) as a cream powder.

MS (ES+): m/z = 424 (M+H) ⁺ ; LCMS (Method B): t _R = 3.53 min.

Allyl (6aS)-3-((6-((((6aS)-5-((allyloxy)carbonyl)-2,6-dimethoxy-8- (4-(JV- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido- [i,2-a][i,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)methoxy)-2 ,6- dimethoxy-i4-oxo-6,6a,7,i2-tetrahydrobenzo[5,6][i,4]diazepin o[i,2- b] iso quinoline-5 (14H) -carboxylate (75)

75

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-(A^-methylsulfamoyl)phen yl)- i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepi ne-5(i2H)-carboxylate (32) (43 mg, 0.080 mmol) and allyl (6aS)-3-hydroxy-2,6-dimethoxy-i4-oxo-6,6a,7,i2- tetrahydrobenzo[5,6][i,4]diazepino[i,2-¾]isoquinoline-5(i4H )-carboxylate (74) (34 mg, 0.080 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (22 mg, 0.016 mmol), water (0.1 mL) and 2,6-bis(bromomethyl)pyridine (19 mg, 0.072 mmol) and irradiated with microwaves for 1 h at 55 °C, then diluted with ethyl acetate (10 mL), washed with brine (2 x 10 mL), dried over anhydrous magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with dichloromethane/ethyl acetate (from 1:0 to 0:1) gave the title compound (18 mg, 21%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.87 (d, J=8.2

241

SUBSTITUTE SHEET RULE 26 Hz, 2H), 7·83-7·76 (m, lH), 7.58-7.52 (m, 4H), 7.32-7.28 (m, 5H), 7.26-7.22 (m, lH), 6.78-6.65 (m, 2H), 6.47 (d, J=2.7 Hz, lH), 5-79-5-59 (m, lH), 5.41 (d, J=9.8 Hz, lH), 5.26 (d, J=5.i Hz, 4H), 5.19-5.00 (m, 4H), 4.81-4.68 (m, lH), 4.65-4.38 (m, 5H), 4.38- 4.20 (m, 3H), 4-01-3-93 (m, 6H), 3-75-3-65 (m, lH), 3.60 (d, J=3-5 Hz, lH), 3-44-3-37 (m, 6H), 3.12-2.92 (m, 3H), 2.76 (d, J=10.2 Hz, lH), 2.73-2.65 (m, 3H); NMR (100 MHz, CDCI3) 8157-8, 155-9, 153-3, 143-9, 137-9, 127-9, 127-7, 127-5, 127-3, 126.7, 126.5, 125.7, 120.5, 117-4, H4-6, 111.7, 110.9, 108.6, 92.0, 71.4, 70.3, 66.5, 60.4, 56.2, 56.2, 44.1, 41.6, 30.6, 30.4, 29.7, 29.3; MS (ES+): m/z = 1071 (M+H) ⁺ ; LCMS (Method A): £R = 7.78 min.

4-((iS)-2-Methoxy-3-((6-((((iS')-2-methoxy-i4-oxo-6a,7,i2 ,i4-tetrahydro- benzo[5,6][i,4]diazepino[i,2-b]isoquinolin-3-yl)oxy)methyl)p yridin-2- yl)methoxy)-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2-a ][i,4]- diazepin-8-yl)-JV-methylbenzenesulfonamide (76)

A solution of allyl (6aS')-3-((6-((((6aS ^, )-5-((allyloxy)carbonyl)-2,6-dimethoxy-8-(4-(A^- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido[i,2-a][i,4]- diazepin-3-yl)oxy)methyl)pyridin-2-yl)methoxy)-2,6-dimethoxy -i4-oxo-6,6a,7,i2- tetrahydrobenzo[5,6][i,4]diazepino[i,2-¾]isoquinoline-5(i4H )-carboxylate (75) (18 mg, 0.016 mmol) in dichloromethane (0.5 mL) was charged with tetrakis(triphenyl- phosphine)palladium(o) (0.92 mg) and pyrrolidine (3.2 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with diethyl ether (1 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with dichloro methane/acetone (from 1:0 to 0:1) gave the title compound (13 mg, 96%) as a pale yellow solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-92-7-88 (m, 2H), η.ηη-η.η (m, lH), 7.61 (d, J=2.7 Hz, lH), 7-60-7.55 (m, 3H), 7.50-

242

SUBSTITUTE SHEET RULE 26 7-44 (m, 3H), 7-38-7-29 (m, 3H), 6.86 (d, J=9-4 Hz, 2H), 6.64-6.55 (m, iH), 5-34-5-28 (m, 4H), 4.99 (d, J=i5-2 Hz, iH), 4.62-4.44 (m, 3H), 4.20 (dd, J=i8.9, 2.5 Hz, iH), 4.03-3-98 (m, 7H), 3-96-3-91 (m, 2H), 3-77-3-66 (m, iH), 3.27 (dd, J=i5-2, 5-5 Hz, iH), 3.15 (dd, J=i5-6, 4.3 Hz, iH), 3.07-2.92 (m, 2H), 2.73-2.67 (m, 3H); ¾»C NMR (100 MHz, CDCI ₃ ) δ 166.8, 166.4, i6i.8, 156.1, 150.5, 150.3, 148.1, 148.0, 143.7, 140.3, 140.2, 138.2, 137.8, 133.1, 128.0, 127.8, 127.8, 127.5, 126.7, 125-8, 125.7, 120.3, 112.3, 111-9, 110.9, 105 8, 96.5, 71-2, 62.0, 56.2, 49.4, 48.8, 44-0, 41-7, 35-0, 33-7, 31-9, 30-8, 29.3; MS (ES+): m/z = 839 (M+H) ⁺ ; LCMS (Method A): t _R = 6.43 min.

4-Hydroxy-5-methoxy-2-nitrobenzaldehyde (77)

A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (2) (100 g, 348 mmol) in glacial acetic acid (800 mL) was charged with an aqueous solution of hydrobromic acid (48% v/v, 88.0 mL, 522 mmol) and heated to 85 °C, with stirring for 1 h, after which the reaction was judged to be complete by TLC. After allowing the resulting mixture to cool to room temperature, it was then diluted in water (1.60 L), and the resulting precipitate filtered, and washed with cold water (100 mL x 3) to give the title compound (50.0 g, 73%) as a yellow solid. TLC: Rf= 0.2 (eluent: petroleum spirit, 40-60 °C/ethyl acetate = 3:1, v/v)

5-Me hoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (78)

A mixture of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde (77) (50.0 g, 254 mmol), triisopropylsilyl chloride (59.7 mL, 279 mmol) and imidazole (51.8 g, 761 mmol) was heated and stirred at 100 °C for 30 min. The reaction mixture was poured onto ice- water and extracted with ethyl acetate (500 mL ^χ 3). The organic extract was dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (20:1) to give the title compound (57.5 g, 64%) as a yellow solid. TLC: Rf= 0.6 (eluent: petroleum spirit, 40-60 °C/ethyl acetate = 20:1, v/v)

5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (79)

243

SUBSTITUTE SHEET RULE 26

A solution of sodium chlorite (80%, 46.0 g, 407 mmol) and sodium phosphate monobasic dihydrate (35.5 g, 228 mmol) in water (200 mL) was added to a solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (78) (57.5 g, 163 mmol) in tetrahydrofuran (800 mL) at room temperature. Hydrogen peroxide (30% w/w, 235 mL, 2.28 mol) was immediately added to the vigorously stirred biphasic mixture. The starting material dissolved, and the temperature of the reaction mixture rose to 45 °C. After 30 min, the reaction was judged to have completed by TLC. The mixture was subsequently acidified to pH = 3-4 with citric acid and extracted with ethyl acetate (500 mL x 3). The combined organic extracts were washed with water (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (10:1) then dichloromethane/methanol (10:1) to afford the title compound (38.0 g, 63%) as a yellow oil. Ή NMR (400 MHz, CDC1 ₃ ) δ 9·8ι (s, iH), 7.35 (s, iH), 7.25 (s, iH), 3.91 (s, 3H), 1.26 (q, J=7-4 Hz, 3H), 1.09 (d, J=7-4 Hz, 18H); MS (ES-): m/z = 368 (M-l>.

(iS)-(2-(Hydroxymethyl)indolin-i-yl)(5-methoxy-2-nitro-4- ((triisopropyl- silyl)oxy)phenyl)methanone (80)

80

A solution of 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (79) (1.00 g, 2.71 mmol) in dichloromethane (25 mL) was charged with (S)-(+)-2-indolinemethanol (404 mg, 2.71 mmol), HATU (0.54 g, 4.06 mmol) and A V-diisopropylethylamine (875 mg, 6.77 mmol). The reaction mixture was stirred at room temperature for 3 h and then diluted with water (100 mL) and extracted with dichloromethane (100 mL x 2). The combined organic extracts were then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (9:1 to 0:1) to afford the title compound (800 mg, 58%) as a yellow oil. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.22-7.14

244

SUBSTITUTE SHEET RULE 26 (m, iH), 7.08-7.00 (m, iH), 6.95-6.90 (m, iH), 6.80-6.70 (m, iH), 5.69-5.65 (m, iH), 5.23-5.06 (m, iH), 4.00-3.82 (m, 3H), 2.80 (s, 5H), 2.04 (s, iH), 1.34-1.25 (m, 3H), 1.15-1.10 (m, 18H); MS (ES+): m/z = 501 (M+H) ⁺ . (iS)-(2-Amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2- (hydroxyl- methyl)indolin-i-yl)methanone (81)

81

A solution of (S ^, )-(2-(hydroxymethyl)indolin-i-yl)(5-methoxy-2-nitro-4- ((triisopropyl- silyl)oxy)phenyl)methanone (80) (800 mg, 1.60 mmol) in methanol (10 mL) was charged with palladium (10 wt. % loading on carbon, 80 mg). The mixture was stirred at room temperature under an atmosphere of hydrogen for 16 h then filtered through a pad of Celite. The resulting cake was then washed with ethyl acetate (50 mL) and concentrated under reduced pressure. The residue was then purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (4:1 to 1:1) to afford the title compound (500 mg, 66%) as a yellow oil. Ή NMR (400 MHz, DMSO-d6) δ 7-22 (d, J=6.8 Hz, iH), 7.08 (s, iH), 7.00-6.93 (m, 2H), 6.75 (s, iH), 6.37 (d, J=2.8 Hz, iH), 4.98-4.88 (m, 3H), 4·6ι-4·57 (m, iH), 3.58 (s, 3H), 3·47"3·44 (m, iH), 3.32-3.26 (m, iH), 3.01-2.97 (m, iH), 2.69 (s, iH), 1.27-1.21 (m, 3H), 1.08 (d , J=7.2 Hz, 18H); MS (ES+): m/z = 471 (M+H) ⁺ .

Allyl (iS)-(2-(2-(hydroxymethyl)indoline-i-carbonyl)-4-methoxy-5- ((triisopropylsilyl)oxy)phenyl)carbamate (82)

82

A solution of (S ^, )-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl) (2- (hydroxylmethyl)indolin-i-yl)methanone (81) (470 mg, 1.00 mmol) in

dichloromethane (10 mL) at -10 °C was charged with anhydrous pyridine (158

245

SUBSTITUTE SHEET RULE 26 mmol) and allyl chloroformate (127 mg, 1.05 mmol). After 30 min, the reaction was judged to have completed by TLC and was then diluted with dichloromethane (100 mL), then washed with a saturated aqueous solution of copper sulfate (100 mL), water (100 mL) and a saturated aqueous solution of sodium hydrogen carbonate (10 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (5%) to afford the title compound (400 mg, 72%) as a yellow oil. Ή NMR (400 MHz, CDC1 ₃ ) δ 8.36 (s, iH), 7.74 (s, iH), 7.19 (d, J=7.2 Hz, iH), 6.97-6.87 (m, 2H), 6.72 (s, iH), 6.40 (s, iH), 5.98-5.87 (m, iH), 5.31 (d, J=i6.8 Hz, iH), 5.22 (d, J=io.4 Hz, iH), 4.94-4.91 (m, iH), 4.60 (d, J=5.6 Hz, 2H),

3.76 (d, J=6.o Hz, 2H), 3.54 (s, 3H), 3·45-3·38 (m, iH), 2.81-2.76 (m, iH), 1.35-1.28 (m, 3H), 1.12 (d, J=7-6 Hz, 18H); MS (ES+): m/z = 555 (M+H) ⁺ .

Allyl (i2aS')-i2-hydroxy-8-methoxy-6-oxo-9-((triisopropylsilyl)oxy )-i2a,i3- dihydro-6ff-benzo[5,6][i,4]diazepino[i,2-a]indole-ii(i2ii)-c arboxylate (83)

83

A solution of allyl (S)-(2-(2-(hydroxymethyl)indoline-i-carbonyl)-4-methoxy-5- ((triisopropylsilyl)oxy)phenyl)carbamate (82) (391 mg, 0.71 mmol) in dichloromethane (13 mL) was charged with TEMPO (11 mg, 0.07 mmol) and (diacetoxyiodo)benzene (274 mg, 0.85 mmol). The reaction mixture was stirred at room temperature for 18 h and then diluted in dichloromethane (40 mL), washed with a saturated aqueous solution of sodium metabisulfite (10 mL), then a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and lastly, brine (10 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (10%) to afford the title compound (290 mg, 74%) as a yellow oil. Ή NMR (500 MHz, CDC1 ₃ ) δ 8.19 (d, J=8.o Hz, iH), 7.21 (d, J=7-5 Hz, iH), 7.08 (t, J=7.5 Hz, iH), 6.71 (s, iH), 5.78 (s, iH), 5.73 (d, J=io.o Hz, iH), 5.20-5.14 (m, 2H), 4.62-4.58 (m, iH), 4.46 (s, iH), 4·ΐ5-4·θ6 (m, 2H), 3.86-3.84 (m, 3H), 3·49"3·43 (m,

246

SUBSTITUTE SHEET RULE 26 iH), 3.21 (d, J=i7.o Hz, iH), 2.04 (d, J=2.o Hz, iH), 1.28-1.21 (m, 3H), 1.09-1.08 (m, 18H); MS (ES+): m/z = 553 (M+H) ⁺ .

Allyl (i2aiS)-8-methoxy-6-oxo-i2-((tetrahydro-2Ji-pyran-2-yl)oxy)- 9- ((triisopropylsilyl)oxy)-i2a,i3-dihydro-6ff-benzo[5,6][i,4]d iazepino[i,2-

A solution of allyl (i2aS ^, )-i2-hydroxy-8-methoxy-6-oxo-9-((triisopropylsilyl)oxy )- i2a,i3-dihydro-6H-benzo[5,6][i,4]diazepino[i,2-a]indole-ii(i 2H)-carboxylate (83) (280 mg, 0.510 mmol) in tetrahydrofuran (5 mL) was charged with 3,4-dihydro-2H- pyran (429 mg, 5.10 mmol) and para-t oluenesulfonic acid monohydrate (3 mg, 1% w/w). The reaction mixture was stirred at room temperature for 18 h and then diluted with ethyl acetate (30 mL), washed with a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and brine (10 mL), then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (20%) to afford the title compound (300 mg, 92%) as a colourless oil. Ή NMR (500 MHz, CDCU 58.20-8.13 (m, iH), 7.25-7.21 (m, 2H), 7.08-6.61 (m, 2H), ₅ -94 (m, iH), 5.78- 5.66 (m, iH), 5-13-5.04 (m, 2H), 4.96-4.94 (m, 2H), 4.89 (d, J=6.o Hz, iH), 3.86 (d, J=2.o Hz, 3H), 3.65-3.61 (m, iH), 3.47-3.42 (m, iH), 2.07-2.01 (m, iH), 1.98-1.95 (m, iH), 1.79-1.73 (m, 6H), 1.33-1.26 (m, 3H), 1.11-1.08 (m, 18H); MS (ES+): m/z = 637 (M+H) ⁺ . Allyl (i2aS')-9-hydroxy-8-methoxy-6-oxo-i2-((tetrahydro-2ii-pyran- 2- yl)oxy)-i2a,i3-dihydro-6ff-benzo[5,6][i,4]diazepino[i,2-a]in dole-ii(i2ii)- carboxylate (85)

247

SUBSTITUTE SHEET RULE 26 A solution of allyl (i2aS')-8-methoxy-6-oxo-i2-((tetrahydro-2H-pyran-2-yl)oxy)-9 - ((triisopropylsilyl)oxy)-i2a,i3-dihydro-6H-benzo[5,6][i,4]di azepino[i,2-a]indole- ii(i2H)-carboxylate (84) (290 mg, 0.46 mmol) in tetrahydrofuran (5 mL) under an inert atmosphere of nitrogen was charged with tetrabutylammonium fluoride (1 M in tetrahydrofuran, 0.65 mL, 0.65 mmol). The mixture was stirred at room temperature for 1 h and then charged with water (10 mL), extracted with ethyl acetate (30 mL x 2) and the combined organic phases washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (20%) to afford the title compound (100 mg, 45%) as a white solid. Ή NMR (500 MHz, DMSO-d6) δ 8.16 (dd, J=2i.o, 8.0 Hz, iH), 7.28 (d, J=5-0 Hz, iH), 7-25-7-20 (m, iH), 7.10-7.05 (m, iH), 6.77 (m, iH), 6.02 (s, iH), 5·8ι-5·72 (m, iH), 5.20-5.14 (m, iH), 5.13- 4.84 (m, iH), 4.66-4.48 (m, 2H), 4-15-4-07 (m, iH), 3.95 (s, 3H), 3-60-3.42 (m, 2H), 3.30-3-18 (m, iH), 1.88-1.54 (m, 7H), 1.29-1.24 (m, iH); MS (ES+): m/z = 481 (M+H) ⁺ .

Allyl (i2aS)-9-((6-((((6aS)-5-((allyloxy)carbonyl)-2,6-dimethoxy-8 -(4-(JV- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido- [i,2-a][i,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)methoxy)-8 -methoxy-6- oxo-i2-((tetrahydro-2ff-pyran-2-yl)oxy)-i2a,i3-dihydro-6ii-b enzo[5,6]- [i,4]diazepino[i,2-a]indole-ii(i2ff)-carboxylate (86)

86

A solution of allyl (6aS')-3-hydroxy-2,6-dimethoxy-8-(4-(A^-methylsulfamoyl)phen yl)- i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepi ne-5(i2H)-carboxylate (32) (76 mg, 0.141 mmol) and allyl (i2aS)-9-hydroxy-8-methoxy-6-oxo-i2-

((tetrahydro-2H-pyran-2-yl)oxy)-i2a,i3-dihydro-6H-benzo[5 ,6][i,4]diazepino[i,2-

248

SUBSTITUTE SHEET RULE 26 a]indole-ii(i2H)-carboxylate (85) (68 mg, 0.141 mmol) in A V-dimethylformamide (1 mL) was charged with potassium carbonate (39 mg, 0.282 mmol), water (0.1 mL) and 2,6-bis(bromomethyl)pyridine (33 mg, 0.126 mmol) and irradiated with microwaves for 1 h at 55 °C. The resulting mixture was then diluted in ethyl acetate (10 mL), washed with brine (10 mL x 2), dried over magnesium sulphate, filtered and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (from 1:0 to 0:1), followed by acetone (100%) gave the title compound (25 mg, impure) as a white solid. MS (ES+): m/z = 1127 (M+H) ⁺ ; LCMS (Method A): t _R = 8.33 min.

4-((iS)-2-Methoxy-3-((6-((((iS')-8-methoxy-6-oxo-i2a,i3-d ihydro-6Ji- benzo[5,6][i,4]diazepino[i,2-a]indol-9-yl)oxy)methyl)pyridin -2- yl)methoxy)-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2- a] [i,4]diazepin-8-yl)-JV-methylbenzenesulfonamide (87)

87

A solution of allyl (i2aS')-9-((6-((((6aS ^, )-5-((allyloxy)carbonyl)-2,6-dimethoxy-8-(4-( ^- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido[i,2-

<3][i,4]diazepin-3-yl)oxy)methyl)pyridin-2-yl)methoxy) -8-methoxy-6-oxo-i2- ((tetrahydro-2H-pyran-2-yl)oxy)-i2a,i3-dihydro-6H-benzo[5,6] [i,4]diazepino[i,2- a]indole-ii(i2H)-carboxylate (86) (25 mg, 0.022 mmol) in dichloromethane (0.5 mL) was charged with tetrakis(triphenylphosphine)palladium(o) (1.3 mg) and pyrrolidine

(4.4 ^L). After 5 min, the reaction was observed to have completed via TLC. The reaction mixture was concentrated in vacuo to form an oil, which was charged with

249

SUBSTITUTE SHEET RULE 26 diethyl ether (1 mL) and re-concentrated in vacuo (this process was repeated twice more). Purification via flash column chromatography (silica), eluting with

dichloromethane/acetone (from 1:0 to 0:1) gave the title compound (9.5 mg, 53%) as a pale yellow solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 8.27 (d, J=7-8 Hz, iH), 7·94"7·69 (m, 4H), 7-66-7.45 (m, 6 H), 7.12 (d, J=7-4 Hz, iH), 7.01 (s, iH), 6.92-6.85 (m, iH), 6.59 (d, J=3-i Hz, iH), 5-37-5-28 (m, 4H), 4.55 (d, J=5-i Hz, iH), 4.47 (dd, J=6.6, 4-7 Hz, iH), 4.18 (br. s, iH), 4.04-3-97 (m, 6H), 3-95-3-85 (m, 3H), 3-78-3-64 (m, iH), 3.49 (s, iH), 3.05-2.94 (m, 2H), 2.74-2.58 (m, 4H); MS (ES+): m/z = 825 (M+H) ⁺ ; LCMS (Method A): £ _R = 6.58 min.

Allyl ((S')-i-(((iS')-i-((3,5-bis(hydroxymethyl)phenyl)amino)-i-ox opropan-2- yl)amino)-3-methyl-i-oxobutan-2-yl)carbamate (88)

88

A solution of (5-amino-i,3-phenylene)dimethanol (0.50 g, 3.26 mmol) in N,N- dimethylacetamide (20 mL) was charged with which allyl ((S)-i-(((.S)-i-amino-i- oxopropan-2-yl)amino)-3-methyl-i-oxobutan-2-yl)carbamate (0.93 g, 3.42 mmol) and AK3-dimethylaminopropyl)- '-ethylcarbodiimide hydrochloride (1.25 g, 6.52 mmol) and stirred for 4 h at room temperature. Water (100 mL) was then added, and the mixture extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were then washed with brine (5 x 100 mL), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (3:7), gave the title compound (505 mg, 38%) as a white solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-57 (s, iH), 7-49 (s, 2H), 7.10 (s, iH), 6.01-5.82 (m, iH), 5.29 (ddd, J=i7-3, 11.1, 1-5 Hz, iH), 5.20-5.10 (m, iH), 4.57 (s, 4H), 4.56-4.44 (m, 3H), 3-95 (d, J=6.7 Hz, iH), 2.12-1.93 (m, iH), 1.44 (dd, J=7.i, 4.0 Hz, 3H), 1.06-0.86 (m, 6H); MS (ES+): m/z = 408 (M+H) ⁺ ; LCMS (Method A): £ _R = 5.22 min.

(5-((S')-2-((iS')-2-(((Allyloxy)carbonyl)amino)-3-methylb utanamido)- propanamido)-i,3-phenylene)bis(methylene) dimethanesulfonate (89)

250

SUBSTITUTE SHEET RULE 26

A suspension of allyl ((S^-i-CCCSi-i-CCs^-bisChydroxymethy^pheny^amino)-!- oxopropan-2-yl)amino)-3-methyl-i-oxobutan-2-yl)carbamate (88) (0.50 g, 1.22 mmol) in anhydrous dichloromethane (100 mL) and cooled to -5 °C, to which anhydrous triethylamine (0.51 mL, 3.66 mmol) was added, followed by methanesulfonyl chloride (0.09 mL, 1.22 mmol). Immediate consumption of starting material was observed by TLC. The reaction mixture was washed with brine (2 x 50 mL), dried over magnesium sulfate and concentrated under reduced pressure. The resulting foam was purified by flash column chromatography (silica), eluting with petroleum spirit, 40-60 °C/ethyl acetate (1:9), to give the title compound (263 mg, 38%) as a cream solid. Ή NMR (400 MHz, CDCI ₃ ), mixture of rotamers, δ 7.68 (s, 2H), 7.14 (s, iH), 6.98-6.81 (m, iH), 5.99- 5.76 (m, iH), 5.65 and 5.49 (2 x d, J=7.2 Hz, iH), 5.32-5.26 (m, iH), 5.26-5.18 (m, iH), 5.15 (s, 2H), 4.70 (tt, J=i4-2, 7-0 Hz, iH), 4.61-4.49 (m, 2H), 4.03 and 3.92 (2 x t, J=6.7 Hz, iH), 2.99 (s, 6H), 2.21-2.10 (m, iH), 1.50-1.42 (m, 3H), 1.05-0.91 (m, 6H); MS (ES+): m/z = 564 (M+H) ⁺ ; LCMS (Method A): t _R = 6.37 min.

Allyl (6aS)-3-((3-((((6aS)-5-((allyloxy)carbonyl)-2,6-dimethoxy-8- (4-(JV- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]- pyrido[i,2-a][i,4]diazepin-3-yl)oxy)methyl)-5-((2iS')-2-(2-( ((allyloxy)- carbonyl)amino)-3-methylbutanamido)propanamido)benz l)oxy)-2,6- dimethoxy-8-(4-(JV-methylsulfamoyl)phenyl)-i2-oxo-6,6a,7,io- tetra- hydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate (90)

90

251

SUBSTITUTE SHEET RULE 26 A solution of (5-((S ^, )-2-((S ^, )-2-(((Allyloxy)carbonyl)amino)-3-methylbutanamido)- propanamido)-i,3-phenylene)bis(methylene) dimethanesulfonate (89) (60 mg, 0.11 mmol) in anhydrous , -dimethylformamide (5 ml), was charged with allyl (6aS)-3- hydroxy-2,6-dimethoxy-8-(4-(A^-methylsulfamoyl)phenyl)-i2-ox o-6,6a,7,io-tetra- hydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2H)-carboxylate (32) (100 mg, 0.22 mmol) and potassium carbonate (0.06 g, 0.22 mmol) and the resulting mixture stirred at room temperature for 15 h. Water (50 ml) was then added and the resulting solution extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with brine (5 x 50 mL), then dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure. The resulting oil was purified by flash column chromatography (silica), eluting with dichloromethane/acetone (i:i)to give the title compound (30 mg, 19%) as a beige solid. Ή NMR (400 MHz, CDC1 ₃ ) δ 8.8o (m, iH), 7.85 (d, J=8.2 Hz, 4H), 7-61-7-59 (m, 2H), 7.53 (d, J=8.3 Hz, 4H), 7.30 (s, iH), 7.25- 7.20 (m, 2H), 6.84-6.81 (m, iH), 6.70 (s, 2H), 6.45 (s, 2H), 5-91-5-58 (m, 3H), 5.50- 5-33 (m, 2H), 5-25-5-12 (m, 2H), 5-13-4-97 (m, 7H), 4-88 (d, J=4-7 Hz, 2H), 4-71-4-59 (m, iH), 4-59-4-36 (m, 6H), 4-34-4-17 (m, 4H), 3-89 (s, 6H), 3-73-3-63 (m, 2H), 3.40 (s, 6H), 3.00-3.86 (m, 2H), 2.79-2.69 (m, 2H), 2.66 (d, J=5.i Hz, 6H), 2.16-2.14 (m, iH), 1.43 (d, J=6.3 Hz, 3H), 1.05-O.86 (m, 6H); MS (ES+): m/z = 1458 (M+H) ⁺ ; LCMS

(Method A): t _R = 7.08 min.

(S)-2-Amino-JV-((S)-i-((3,5-bis(((( ₁ S)-2-methoxy-8-(4-(JV-methylsulfamoyl)- phenyl)-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2-a][i, 4]diazepin-3- yl)oxy)methyl)phenyl)amino)-i-oxopropan-2-yl)-3-methylbutana mide (91)

252

SUBSTITUTE SHEET RULE 26

91

A solution of allyl (6aS')-3-((3-((((6aS ^, )-5-((allyloxy)carbonyl)-2,6-dimethoxy-8-(4-(A^- methylsulfamoyl)phenyl)-i2-oxo-5,6,6a,7,io,i2-hexahydrobenzo [e]pyrido[i,2-a][i,4]- diazepin-3-yl)oxy)methyl)-5-((2S')-2-(2-(((allyloxy)carbonyl )amino)-3-methylbutan- amido)propanamido)benzyl)oxy)-2,6-dimethoxy-8-(4-(A^-methyls ulfamoyl)phenyl)-i2- oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine- 5(i2H)-carboxylate (90) (0.030 g, 0.020 mmol) in dichloromethane (2 mL) was charged with tetrakis(triphenyl- phosphine)palladium(o) (1 mg) and pyrrolidine (6.5 ^L). After 5 min, the reaction was observed to have only partially completed by LCMS (deprotection of imines). Further tetrakis(triphenylphosphine)palladium(o) (1 mg) and pyrrolidine (6.5 ^L) were added to the reaction mixture, which resulted in immediate completion of the reaction. The reaction mixture was concentrated under reduced pressure to form an oil, which was re-dissolved in diethyl ether (2 mL) and re-concentrated under reduced pressure to remove any residual pyrrolidine (this process was repeated twice more). Purified by flash column chromatography (silica), eluting with dichloromethane/methanol (95:5) gave the title compound (30 mg, 85%) as a yellow solid. MS (ES+): m/z = 1142 (M+H) ⁺ ; LCMS (Method A): t _R = 5.43 min.

253

SUBSTITUTE SHEET RULE 26 JV-((S)-i-(((S)-i-((3,5-bis((((S)-2-Methoxy-8-(4-(JV-methyls ulfamoyl)- phenyl)-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido[i,2-a][i, 4]diazepin-3- yl)oxy)methyl)phenyl)amino)-i-oxopropan-2-yl)amino)-3-methyl -i- oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-iff-pyrrol-i-yl)hexa namide (92)

92

A solution of (S)-2-amino-A^-((S')-i-((3,5-bis((((S ^, )-2-methoxy-8-(4-(A^-methyl- sulfamoyl)phenyl)-i2-oxo-6a,7,io,i2-tetrahydrobenzo[e]pyrido [i,2-a][i,4]diazepin-3- yl)oxy)methyl)phenyl)amino)-i-oxopropan-2-yl)-3-methylbutana mide (91) (20 mg, 0.018 mmol) in ,iV-dimethylacetamide (2 mL) was charged with 6-maleimido- hexanoic acid (4.0 mg, 0.018 mmol) and A^-(3-dimethylaminopropyl)-A ^f '-ethyl- carbodiimide hydrochloride (3.0 mg, 0.018 mmol) and stirred for 15 h, at which point consumption of starting material was observed by LCMS. Water (10 mL) was added to the reaction mixture and the resulting suspension extracted with ethyl acetate (50 mL x 4). The combined organic extracts were washed with brine (40 mL x 5), dried over magnesium sulfate and concentrated in vacuo. Purification by flash column

chromatography (silica), eluting with dichloro methane/acetone (5:95) gave the title compound (4.9 mg, 21%) as a light brown solid. Ή NMR (400 MHz, DMSO-d6) δ 10.08 (br, iH), 9.95 (br, iH), 8.39 (m, iH), 8.15 (d, J=6.3 Hz, 2H), 7.92 (s, iH), 7.70 (d, J=6.8 Hz, 2H), 7-65-7-62 (m, iH), 7.47 (d, J=5-i Hz, 2H), 7.38 (d, J=2.6 Hz, 2H), 7.24 (br, 2H), 7.11 (s, 2H), 6.96 (dd, J=io.2, 9.0 Hz, 2H), 6.92 (d, J=7.2 Hz, 2H), 6.68 (d,

254

SUBSTITUTE SHEET RULE 26 J=i3.6 Hz, 2H), 6.58 (br, 2H), 5.91 (d, J=6.8 Hz, 2H), 5-39 (br, iH), 5.25-5.15 (m, 2H), 5.05 (br, 2H), 4.53 (s, 3H), 4-39 (br, iH), 4.36-4.26 (m, 2H), 4.20-4.13 (m, 2H), 3.94 (br, 3H), 3.86 (s, 2H), 3.71 (s, 2H), 3.51 (s, 6H), 3.11 (d, J=i4-4 Hz, 2H), 3.04-2.86 (m, 6H), 2.79-2.59 (m, 2H), 2.42 (dd, J=io.3, 5-7 Hz, 3H), 1.47-1-30 (m, 2H), 0.88-0.79 (m, 6H); MS (ES+): m/z = 1336 (M+H) ⁺ ; LCMS (Method A): t _R = 6.62 min.

General synthetic scheme 24

255 6

i) HATU, Et ₃ N, 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid, DMF ii) NaBH ₄ ,THF iii) TBDMSC1, DMAP, Imidazole, DMF iv) TEMPO, BAIB, DCM v) Sodium bis

trimethylsilyl amide, N,N-Bis(trifluoromethylsulfonyl)aniline, THF, -60 °C vi) 2-(4- methoxyphenyl)-4,4,5,5-tetramethyl-i,3,2-dioxaborolane, K ₂ C0 ₃ , Pd(dppf)Cl ₂ · CH ₂ C1 ₂ , acetonitrile, 50 °C vii) Zinc, formic acid, EtOH viii) Allyl chloroformate, pyridine, DCM ix) TBAF, THF x) TEMPO, BAIB, DCM xi) BC1 ₃ ,THF xii) 1, 3-dibromopropane, K ₂ C0 ₃ , DMF xiii) 12, K ₂ C0 ₃ DMF, 60 °C xiv) pyrrolidine, Tetrakis(triphenylphosphine)- palladium(o), DCM Methyl (2iS,4iS')-i-(4-(benz loxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxy- piperidine-2-carboxylate (93)

4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (5.0 g, 16.5 mmol), was dissolved in N,N- dimethylformamide (50 mL). To this solution, HATU (9-4g, 24.75 mmol) and trimethylamine (9.67 ml 69.3 mmol) was added. The solution was allowed to stir for 30 minutes. The solution was then cooled to o °C and methyl (2S,4S)-4- hydroxypiperidine-2-carboxylate (2.6 g, 16.5 mmol) was then added to the mixture. After four hours, the reaction was judged to have completed by LC-MS analysis. Water (500 ml) was then added to the solution, causing the formation of a suspension. The crude product was extracted using ethyl acetate (3x 500 ml) and the combined organic extract was washed sequentially with bicarbonate solution (500 ml) and brine (500 ml) and dried with magnesium sulphate. The crude product was purified using flash column chromatography (acetone/ dichloro methane, from 5% to 40% acetone), resolving a white solid in 82% yield. Ή NMR (400 MHz, CD ₃ OD), rotameric mixture, δ 7.77 and 7.72 (2x s, iH), 7-40-7-33 (m, 2H), 7.31-7.20 (m, 3H), 6.94 and 6.73 (2x s, iH), 5.14 and 5.12 (2 x s, 2H), 4-05-3-99 (m, iH), 3.91 and 3.89 (ax s, 3H), 3.67 and 3.63 (2x s, 3H) 3.60-3.58 (m, iH), 3-40-3-30 (m, iH) 2.52 and 2.24 (2x d, iH, J= 14.4, 14.1 Hz), 1.99-1.90 (m, iH) 1.89-1.80 (m, iH) 1.73-1.60 (m, iH), 1.57-1.48 (m, iH); MS (ES+): m/z = 445.0 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.25 min.

The addition of the methyl (2S,4S 4-hydroxypiperidine-2-carboxylate C ring to the A ring precursor generates rotameric intermediates. The addition of the triflate (step v) generated a regioisomeric mix in a 50:50 ratio which was separated the Suzuki step (step vi). This complicated the NMR spectra obtained, causing peak broadening and splitting.

(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)((2iS',4iS')-4-hyd roxy-2- (hydroxymethyl)piperidin-i-yl)methanone (94)

257

SUBSTITUTE SHEET RULE 26

93 94

93 (4-7 g _> 10.5 mmol) was dissolved in tetrahydrofuran (loo ml). The solution was cooled to o °C using ice and lithium borohydride (0.342 g, 15.75 mmol) was added portionwise to the mixture. Some effervescence was observed. After one hour, the reaction was judged to have completed by TLC and LC-MS analysis. The reaction was quenched using water (50 ml) and hydrochloric acid solution (lM, 10 ml). The tetrahydrofuran was removed under reduced pressure and the resulting concentrated solution extracted using ethyl acetate (3 x 100 ml). The combined organic layer was washed sequentially with bicarbonate solution (50 ml) and brine (50 ml), dried with magnesium sulphate and concentrated under reduced pressure to resolve a white solid in 85% yield. No further purification was carried out and the intermediate 94 was carried through to the next step in the synthesis. Ή NMR (400 MHz, CD ₃ OD), rotameric mixture, δ 7.76, 7.74 and 7.71 (3 x s, iH), 7.39-7.22 (m, 5H), 7.01 and 6.91 (2x s, iH), 5.12 (2 x s, 2H), 4.39 (d, iH, J= 13.5Hz), 4.20 and 4.10 (2 x dd, iH, J= 9.8, 12.0 and 8.8, 12.0Hz) , 3.88 and 3.83 (2x s, 3H), 3.65 (dd, 0.38 H, J= 4.4, 12.0Hz), 3·57"3·52 (m, 0.8H), 3-50-3-43 (m, 0.6H), 3.34 (dd, 0.6H, J= 4.0, 12.0Hz), 3.26 (dd, 0.5H, 2.7, i3-3Hz), 3.12-3.00 (m, 0.5H), 1.90-1.85 (m, 0.5H), 1.85-1.77 (m, iH), 1.75-1-70 (m, iH), 1.70-1.55 (m, 2H), 1.52-1.42 (m, 2H); MS (ES+): m/z = 417.0 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.08 min.

(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)((2iS',4iS')-2-((( tert-butyldimethyl- silyl)oxy)methyl)-4-hydroxj^iperidin-i-yl)methanone (95)

94 95

94 (4.0 g, 9.61 mmol) was dissolved in dichloromethane (100 ml). To this solution, imidazole (1.98 g, 28.83 mmol) and tert-Butyldimethylsilyl chloride (1.52 g, 10.09 mmol) was added. After two hours, the reaction was judged to have completed by LC- MS analysis. The reaction mixture was washed sequentially with saturated citric acid solution (100 ml) and brine (100 ml), dried with magnesium sulphate and the

258

SUBSTITUTE SHEET RULE 26 dichloromethane was removed under reduced pressure. The resulting crude oil was purified using flash column chromatography (acetone/DCM, from 5% to 20% acetone), resolving an orange oil 95 in 82% yield. Ή NMR (400 MHz, CDCI3), rotameric mixture, δ 7.61, 7.60 and 7.57 (3x s, iH), 7.30-7.15 (m, 5H), 6.61, 6.56 and 6.50 (3x s, iH), 5.04 and 5.03 (2 x s, 2H), 4.86-4.45 (m, 2H), 4.17 (dd, 0.5H, 3.2, 10.6 Hz), 4.01- 3.84 (m, 2H), 3.69 (dt, iH, 5.9, 3.9 Hz), 3.60 (dd, iH, 10.6, 2.9Hz), 3·47"3·34 (m, iH), 3.11 and 2.96 (2 x t, iH, J= 12.2 and 14.5 Hz), 2.15-1.97 (m, iH), 1.89 and 1.80 (2 x d, iH, J = 14.4, 14.9 Hz), 1.63-1.30 (m, 3H), 0.80-0.67 (m, 9H), 0.05- -0.15 (m, 6H); MS (ES+): m/z = 531.1 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 4.38 min.

(iS)-i-(4-(Benz loxy)-5-methoxy-2-nitrobenzoyl)-2-(((tert-butyldimethyl- silyl)oxy)methyl)piperidin-4-one (96)

95 96

95 (3-7 g, 6.98 mmol) was dissolved in dichloromethane (100 ml). To this solution, TEMPO (0.11 g, 0.698 mmol) and bis(acetoxy)iodobenzene (2.92 g, 9.07 mmol) was added, and the solution allowed to stir at room temperature for 16 hours. At this point, the reaction was judged to have completed by LC-MS and TLC analysis. (Note: On some occasions, the reaction was found to have partially completed. In this case, more TEMPO was added until the oxidation was completed). The reaction mixture was washed sequentially with saturated copper sulphate solution (2x 100 ml) and brine (100ml), dried with magnesium sulphate and the dichloromethane was removed under reduced pressure. The crude product was purified using flash column chromatography (acetone/dichloromethane, from 1% to 8% acetone), resolving a white solid 96 in 90% yield. Ή NMR (400 MHz, CDCI3), rotameric mixture, δ 7.69, 7.68 and 7.67 (3x s, iH), 7.40-7.20 (m, 5H), 6.71, 6.70 and 6.65 (3x s, iH), 5.14 and 5.13 (2x s, 2H), 4.12 and 3.91 (2 x dd, iH, J = 10.6, 2.6 and 10.4, 2.7 Hz), 3.89, 3.88 and 3.87 (3x s, 3H), 3.80-3.35 (m, 3H), 2.77-2.15 (m, 4H), 0.85-0.72 (m, 9H), 0.02- -0.10 (m, 6H); «C NMR (400 MHz, CDCI3), rotameric mixture. 205.7, i67-5, 154-9, 148-3, 148.2, 137.6, 137.3, 135-2, 130.0, 128.9, 128.7, 128.6, 127.6, 127.3, 127.1, 127.0, 109.9, 109.2, io8.6, 71.4, 65.6, 65.1, 64.6, 56.8, 56.8, 56.7, 56.5, 51-27, 51-22, 43.3, 41.9, 41.5, 41.3, 39.9, 39.8, 39-6, 37-5, 25.8, 25.8, 25.8, 20.3, 18.3, 18.2, 18.2, 18.1, -5.7, -5-7; MS (ES+): m/z = 529.0 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 8.28 min.

259

SUBSTITUTE SHEET RULE 26 (iS)-i-(4-(benz loxy)-5-methoxy-2-nitrobenzoyl)-2-(((tert-butyldimethyl- silyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-yl trifluoromethane- sulfonate and (iS)-i-(4-(benz loxy)-5-methoxy-2-nitrobenzoyl)-6-(((tert- butyldimethylsilyl)oxy)methyl)-i,2,3,6-tetrahydropyridin-4-y l

trifluoromethanesulfonate (97)

96 (3.3 g, 6.23 mmol) was added to a stirred solution of sodium

bis(trimethylsilyl)amide (1.73 g, 9.34 mmol) in tetrahydrofuran (50 ml) which was cooled to -78 °C. The solution was allowed to stir for one hour. N-Phenyl- bis(trifluoromethanesulfonimide) (2.89 g, 8.09 mmol) was separately dissolved in tetrahydrofuran (50 ml). The N-Phenyl-bis(trifluoromethanesulfonimide) solution was then added to the flask containing 96 and sodium bis(trimethylsilyl)amide. The combined solution was allowed to warm to room temperature and the reaction mixture left to stir for four hours. At this point, the reaction was judged to have completed by TLC and LC-MS analysis. The tetrahydro-furan was removed under reduced pressure and the crude material was immediately purified using flash column chromatography (acetone/dichloromethane, from 1% to 3% acetone) to yield an off-white foam 97 in 75% yield. Ή NMR (400 MHz, CDCI3), rotameric and regiomeric mixture, δ 7.70-7.60 (m, lH), 7-39-7-25 (m, 5H), 6.77-6.60 (m, lH), 5-91-5-45 (m, lH), 5.15-5.00 (m, 2H), 3-95-3-77 (m, lH), 3-90-3-85 (m, 3H), 3.75-3.20 (m, 3H), 2.90-2.08 (m, 2H), 0.85-0.6 (m, 9H), 0.07- -0.22 (m, 6H); MS (ES+): m/z = 661.1 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 9.27 min.

(iS')-(4-(benz loxy)-5-methoxy-2-nitrophenyl)(6-(((tert-butyl-dimethyl- silyl)oxy)methyl)-4-(4-methoxyphenyl)-3,6-dihydropyridin-i(2 ii)- yl)methanone (98a) and (iS)-(4-(benz loxy)-5-methoxy-2-nitrophenyl)(2- (((tert-butyldimethylsilyl)oxy)methyl)-4-(4-methoxyphenyl)-3 ,6- dihydropyridin-i(2H)-yl)methanone (98b)

260

SUBSTITUTE SHEET RULE 26 97 (3 g _> 4-53 mmol) was dissolved in acetonitrile (2 ml). To this solution, 1,1'- bis(diphenylphosphino)ferrocene (0.369 g, 0.45 mmol), methoxyphenyl)-4,4,5,5- tetramethyl-i,3,2-dioxaborolane (1.87 g, 5.9 mmol) and potassium carbonate (i.25g, 9.06 mmol) were added. The solution was heated at 50 °C for 10 minutes under microwave conditions. The solution was diluted in ethyl acetate (100 ml), washed with brine (50 ml), dried under magnesium sulphate and concentrated under reduced pressure. The resulting oil was purified using flash column chromatography (80:20 petroleum ether/ethyl acetate) repeatedly and the two regioisomers resulting from the introduction of the triflate were separated as an orange solid 98a and creamy white solid 98b. The overall yield (both regioisomers combined) was 75%.

98a Characterisation Ή NMR (400 MHz, CDC1 ₃ ), rotameric mixture, δ 7.81-7.72 (m, iH), 7.49-7.27 (m, 7H), 6.92-6.83 (m, 2H), 6.78/6.76 (2x s, iH), 6.20/6.02 (2x s, iH), 5-27-5-17 (m, 2H), 5.06-4.88 (m, iH), 4.07-3.86 (m, 5H), 3.81 (s, 3H), 3-75"3-34 (m, 3H), 2.59-2.21 (m, iH), 0.97-0.69 (m, 9H), 0.17- -0.19 (m, 6H); MS (ES+): m/z = 619.2 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 4.80.

98b Characterisation Ή NMR (400 MHz, CDC1 ₃ ), rotameric mixture, δ 7.84-7.72 (m, iH), 7.49-7.26 (m, 7H), 6.91-6.82 (m, 2H), 6.78-6.72 (m, iH), 6.02/5.73 (2x s, iH), 5.25-4.88 (m, 3H), 3.96 (s, 3H), 3-88-3-67 (m, 6H), 3-76-3-67 (m, 2H), 3-75-3-54 (m, iH), 3.00-2.34 (m, 2H), 1.29-1.19 (m, 9H), 0.96-0.72 (m, 6H); MS (ES+): m/z = 619.2 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 9.38

(iS)-(2-amino-4-(benz loxy)-5-methoxyphenyl)(6-(((tert-butyldimethyl- silyl)oxy)methyl)-4-(4-methoxj^henyl)-3,6-dihydropjridin-i(2 Ji)- yl)methanone (99a) and (iS)-(2-amino-4-(benzyloxy)-5-methoxy-phenyl)(2- (((tert-butyldimethylsilyl)oxy)methyl)-4-(4-methoxyphenyl)-3 ,6- dihydropyridin-i(2H)-yl)methanone (99b)

261

SUBSTITUTE SHEET RULE 26

98a (0.5 g, 0.8 mmol) was dissolved in a 5% solution of formic acid in ethanol (10 ml). To this solution, zinc powder (1.94 g, 29.6 mmol) was added. After approximately 10 minutes, the reaction was observed to have proceeded to completion via TLC and LC- MS analysis. The reaction mixture was quenched through addition of saturated sodium bicarbonate solution (10 ml) and the solution concentrated under reduced pressure. The concentrated aqueous solution was partitioned between brine (50 ml) and ethyl acetate (100 ml). The organic layer was isolated, dried with magnesium sulphate and concentrated under reduced pressure to resolve a yellow oil 99a in 90% yield. The intermediate was carried to the next step without further purification.

A similar reaction procedure was carried out to generate 99b from 98b in 87% yield. 99a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) Rotameric Mixture, δ 7.99 (s, iH), 7-43 (d, 2H, J = 7.8 Hz), 7-37 (m, 2H), 7·34"7·28 (m, 3H), 6.87 (d, 2H, J = 8.0 Hz), 6.78 (s, iH), 6.41/6.00 (2x s, iH), 5.15 (s, 2H), 4.04-3-83 (m, 3H), 3·8ι (s, 3H), 3-72-3-27 (m, 2H), 2.67-2.37 (m, 2H), 0.94-0.77 (m, 9H), 0.16- -0.03 (m, 6H); MS (ES+): m/z = 589.3 LC Retention Time (Method B): f _R = 4.58.

99b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) Rotameric Mixture, δ 7.99 (s, iH), 7-43 (d, 2H, J = 7.8 Hz) 7-39-7-30 (m, ₅ H), 6.86 (d, 2H, J = 8.2 Hz), 6.82 (s, iH), 6.42/5.91 (2x s, iH), 5.12 (s, 2H), 4-97-4-32 (br, 4H), 3.81 (s, 3H), 3-78-3-56 (m, 3H), 2.80 (d, iH, J= 14.7 Hz), 2.44 (d, iH, J = 16.7 Hz), 0.97-0.72 (m, 9H), 0.13- -0.07 (m, 6H); MS (ES+): m/z = 589.3 LC Retention Time (Method B): t _R = 4.57.

Allyl (S)-(5-(benz loxy)-2-(6-(((tert-butyldimethylsilyl)oxy)-methyl)-4-(4- methoxyphenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-

262

SUBSTITUTE SHEET RULE 26 methoxyphenyl)carbamate (100a) and allyl (iS)-(5-(benz loxy)-2-(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-(4-methoxyphenyl)-i,2,3,6-t etrahydro- pyridine-i-carbonyl)-4-methoxyphenyl)carbamate (100b)

99a (0.4 g, 0.67 mmol) was dissolved in DCM (30 ml). To this solution, pyridine (0.124 ml, 1.54 mmol) and allyl chloroformate (0.074 ^m 0.70 mmol) were added

sequentially. After one hour, the reaction was judged to have completed by LC-MS analysis. The reaction solution was then washed with saturated copper sulphate solution (2 x 20 ml) and brine (20 ml). The organic layer was then dried with magnesium sulphate and concentrated under reduced pressure. The resulting oil was purified using flash column chromatography (5:95 acetone/DCM) to resolve a brown oil 100a in 85% yield.

A similar experimental procedure was carried out to produce 100b from 99b in similar yields (90%).

100a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 8.15 (s, iH), 7.86 (s, iH), 7.48 (d, 2H, J = 7.9 Hz), 7.38 (t, 2H, J = 7.2 Hz), 7-34-7-28 (m, 3H), 6.88 (d, 2H, J = 8.4 Hz), 6.83 (s, iH), 6.03-5.85 (m, 2H), 5.31 (d, iH, J = 17.3 Hz), 5-24"5-i5 (m, 3H), 4.65-4.45 (s, 2H), 3-98-3-85 (m, iH), 3-84-3-77 (m, 9H), 3-69-3-31 (m, iH), 2.62-2.36 (m, 2H), 0.93-0.78 (m, 9H), 0.12-0.02 (m, 6H); MS (ES+): m/z = 673.3 (M+H) ⁺ ; LC Retention Time (Method B) t _R = 4.92.

100b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 8.39 (s, iH), 7.96 (s, iH), 7.48

(d, 2H, J = 7.4 Hz), 7.37 (t, 2H, 7.6 Hz), 7-33-7-27 (m, 3H), 6.88 (d, 2H, 8.7 Hz), 6.07-

5.94 (bs, iH), 5.92 (ddd, iH, J = 22.6, 11.0, 5.7 Hz), 5-23-5-10 (m, 2H), 4.67-4.52 (m, 2H), 3.83 (s, 3H), 3-8i (s, 3H), 3-78-3-52 (m, 3H), 2.82-2.69 (m, iH), 2.44 (d, iH, 17.1

263

SUBSTITUTE SHEET RULE 26 Hz), 0.94- 0.66 (m, 9H), 0.14- -0.13 (m, 6H); MS (ES+): m/z = 673.4 (M+H) ⁺ ; LC Retention Time (Method B) t _R = 4.87.

Allyl (iS)-(5-(benz loxy)-2-(6-(hydroxymethyl)-4-(4-methoxy-phenyl)- i,2,3,6-tetrahydropyridine-i-carbonyl)-4-methoxyphenyl)-carb amate (101a) and allyl (iS)-(5-(benz loxy)-2-(2-(hydroxymethyl)-4-(4- methoxyphenyl)-i,2,3,6-tetrahydropyridine-i-carbonyl)-4-meth oxy- phenyl)

100a (0.35 g, 0.52 mmol) was dissolved in THF (30 ml) and the resulting solution cooled to o °C. To this solution, tetra-n-butylammonium fluoride (0.17 g, 0.65 mmol) was then added to the reaction mixture as a lM solution in THF. The reaction was allowed to warm to room temperature and after 1 hour, TLC and LC-MS confirmed complete reaction of the starting material. Ethyl acetate (100 ml) was added to the reaction mixture, with the resulting solution washed with saturated ammonium chloride solution (50 ml), brine (50 ml) and dried with magnesium sulphate. The solution was then concentrated under reduced pressure to yield 101a as a brown oil (95%). No further purification was carried out on the material.

An analogous procedure was carried out to convert 100b to 101b in 90% yield.

101a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 8.i8 (s, iH), 7.80 (s, iH), 7.47 (d, 2H, J = 8.0 Hz), 7.38 (t, 2H, J = 7.9 Hz), 7-34-7-27 (m, 3H), 6.88 (d, 2H, J = 8.5 Hz), 6.83 (s, iH), 6.00-5.84 (m, 2H), 5.33 (d, iH, J = 17.4 Hz), 5.21 (d, iH, 10.3 Hz), 5.17 (s, 2H), 4.62 (d, 2H, J = 5.4 Hz), 4-15-3-88 (m, 2H), 3.84 (s, 3H), 3-82-3.77 (m, 5H), 3.47-

264

SUBSTITUTE SHEET RULE 26 3.31 (br, lH), 2.69-2.54 (m, lH), 2.47 (d, lH, J = 14.4 Hz); MS (ES+): m/z = 559.2 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.72.

101b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 8.14 (s, lH), 7.82 (s, lH), 7.47 (d, 2H, J = 7.8 Hz), 7.37 (t, 2H, J = 7.6 Hz), 7-34-7-27 (m, 3H), 6.92-6.79 (m, 3H), 5.91 (ddd, lH, J = 22.8, 10.8, 5.6 Hz), 5.36-5.28 (m, lH), 5.20 (d, lH, J = 10.4 Hz), 5.16 (s, 2H), 4.60 (d, 2H, J = 5.5 Hz), 4-07-3-84 (m, lH), 3.82 (s, 3H), 3.81-348 (m, 6H), 2.88- 2.73 (m, lH), 2.36 (d, lH, J = 17.9 Hz); MS (ES+): m/z = 559.3 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.70. Allyl (6aS)-3-(benz loxy)-6-hydroxy-2-methoxy-8-(4-methoxy-phenyl)-i2- oxo-6, 6a,9,io-tetr ahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)- carboxylate (102a) and allyl (6aS')-3-(benz loxy)-6-hydroxy-2-methoxy-8-(4- methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]-pyrido[i, 2- «][i _? 4]diazepine-5(i2H)-carboxylate (102b)

101a (0.25 g, 0.45 mmol) was dissolved in DCM (15 ml). To this solution, 2,2,6,6- tetramethyl-i-piperidinyloxy (0.017 g, 0.11 mmol) and (diacetoxyiodo)benzene (0.363 g, 1.13 mmol) were added. The reaction was allowed to stir for 16 hours, at which point TLC showed consumption of the starting material. DCM (15 ml) was added to the reaction mixture and the organic solution washed with saturated sodium

metabisulphite solution (30 ml) and brine (30 ml). The organic layer was dried with magnesium sulphate and concentrated under reduced pressure. Flash column

265

SUBSTITUTE SHEET RULE 26 chromatography (15:85 acetone/DCM) was then used to purify 102a as a red oil in 55% yield. 101b was converted to 102b using the conditions described above in 58% yield. 102a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.46-7.30 (m, 7H), 7.22 (s, iH), 6.87 (d, 2H, J = 8.4 Hz), 6.73 (s, iH), 6.10 (d, iH, J = 3.6 Hz), 5.79 (d, iH, J = 10.1 Hz), 5-68 (br, iH), 5.23-5.06 (m, 4H), 4.78 (d, iH, J = 13.0 Hz), 4·57"4·36 (m, 2H), 4.02 (d, iH, J = 8.9 Hz), 3.92 (s, 3H), 3-8o (s, 3H), 3-77-3-61 (m, iH), 3.10 (dt, iH, J = 14.4, 7.1 Hz), 2.64-2.52 (m, 2H); MS (ES+): m/z = 557.2 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.68.

102b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7-45-7-28 (m, 7H), 6.89 (d, 2H, J = 8.5 Hz), 6.70 (s, iH), 6.19 (s, iH), 5-74-5-57 (m, 2H), 5.20-5.04 (m, 4H), 4-52-4-35 (m, 2H), 4.31 (dd, iH, J = 18.1, 5.8 Hz), 4.14 (d, iH, J = 18.2 Hz), 3-94 (s, 3H), 3-82 (s, 3H), 3-77-3-69 (m, iH), 3.07 (d, iH, J = 16.2 Hz), 2.73-2.63 (m, iH); MS (ES+): m/z = 557.2 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 3.62. Allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo- 6,6a,9,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2 H)- carboxylate (103a) and allyl (6aS)-3-hydroxy-2,6-dimethoxy-8-(4-methoxy- phenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[i,2-a][i,4 ]diazepine- 5(i2f/)-carboxylate (103b)

102a (0.125 g, 0.23 mmol) was dissolved in anhydrous DCM (10 ml) under nitrogen. To this solution, boron trichloride (0.079 g, 0.67 mmol) was added as a 1 M solution in

266

SUBSTITUTE SHEET RULE 26 DCM. After fifteen minutes, it was determined that the starting material had been completely consumed via LC-MS and TLC analysis. An excess of methanol (50 ml) was then added to the reaction mixture and the solution stirred for a further three hours. At this point, LC-MS and TLC analysis showed the formation of 11a. The reaction mixture was then concentrated to yield a yellow oil. Further purification using flash column chromatography (80:20 ethyl acetate/petroleum ether) yielded 103a as an orange solid in 42% yield*.

Using this procedure, 102b was converted to 103b in similar yields (45%, grey solid). * Some interconversion of 103a to 103b (and vice versa) was observed during purification of the reaction mixture. This olefinic interconversion is possibly borane- catalysed. The regiomeric mix obtained in both cases was separated using the flash column chromatography conditions described above.

103a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.35 (d, 2H, J = 8.0 Hz), 7.20 (s, lH), 6.88 (d, 2H, J = 8.2 Hz), 6.78 (s, lH), 6.04-6.01 (m, lH), 5.92 (s, ιΗ), 5·85"5·72 (br, lH), 5.61 (d, lH, J= 9.3 Hz), 5.18-5.06 (m, 2H), 4.71 (d, lH, J = 13.1 Hz), 4.63 (dd, lH, J = 13.5, 5.1 Hz), 4.52-4.49 (m, lH), 3.94 (s, 3H), 3.92-3-89 (m, lH), 3.81 (s, 3H), 3-53 (s, 3H), 3-17-3-06 (m, lH), 2.62-2.55 (m, 2H); MS (ES+): m/z = 481.0 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 3.43.

103b Characterisation MS (ES+): m/z = 481.0 (M+H) ⁺ ; LC Retention Time

(Method A): t _R = 3.32.

Allyl (6aS')-3-(3-bromopropoxy)-2,6-dimethoxy-8-(4-methoxyphenyl)- i2- oxo-6, 6a,9,io-tetr ahydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)- carboxylate (104a)

103a (0.035 g _> 0.073 mmol) was dissolved in DMF (5 ml). To this solution, potassium carbonate (0.010 g, 0.073 mmol) and 1,3-dibromopropane (0.015 ml, 0.15 mmol) were added, and the solution allowed to stir for 16 hours. At this point, LC-MS analysis confirmed the consumption of the starting material. Water (50 ml) was added to the reaction mixture and the resulting suspension was extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were washed with saturated sodium bicarbonate

267

SUBSTITUTE SHEET RULE 26 (50 ml) and brine (3 x 50 ml), dried with magnesium sulphate and concentrated under reduced pressure. The resulting oil was further purified using flash column

chromatography (80:20 petroleum ether/ ethyl acetate to produce 104a as a brown oil in of 75% purity. This impure intermediate was carried through to the next step with LC-MS analysis only; MS (ES+): m/z = 603.1 (M+H) ⁺ ; LC Retention Time (Method A): fe = 8.75·

Diallyl 3,3'-(propane-i,3-diylbis(oxy))(6aS,6a'S)-bis(2,6-dimethoxy- 8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]-pjrido[i, 2- a][i,4]diazepine-5(i2H)-carboxylate) (105a)

104a 105a

104a (0.020 g, 0.033 mmol) was dissolved in DMF (5 ml). To this solution, potassium carbonate (0.005 g, 0.033 mmol) and 103a (0.014 g, 0.030 mmol) was added. The reaction mixture was heated to 60 °C and stirred for four hours. At this point, formation of the dimer was observed via LC-MS analysis of the reaction mixture. Water (50 ml) was added to the solution, with the resulting suspension extracted with ethyl acetate (3 x 50 ml). The organic layer was washed with saturated sodium bicarbonate solution (50 ml) and brine (3 x 50 ml), dried with magnesium sulphate and

concentrated under reduced pressure. Flash column chromatography was carried out on the sample obtained (10:90 acetone/DCM) to resolve 105a as a white solid in 51% yield. Ή NMR (400 MHz, CDC1 ₃ ) δ 7-33 (d, 4H, J = 8.5 Hz), 7.25 (s, 2H), 6.88 (d, 4H, J = 8.2 Hz), 6.71 (s, 2H), 6.05-5.99 (m, 2H), 5-83-5-69 (br, 2H), 5.61 (d, 2H, J = 9.1 Hz), 5-09 (d, 4H, J = 10.7 Hz), 4.71 (d, 2H, J = 12.3 Hz), 4.68-4.57 (m, 2H), 4.50-4.40 (m, 2H), 4-32-4-19 (m, 4H), 3-90 (s, 6H), 3.81 (s, 6H), 3.56 (s, 6H), 3·ΐ7"3·θ6 (m, 2H), 2.64-2.54 (m, 4H), 2.45-2.34 (m, 2H); MS (ES+): m/z = 1023.5 (M+Na) ⁺ ; LC

Retention Time (Method A): t _R = 9.32.

268

SUBSTITUTE SHEET RULE 26 Diallyl 3,3'-(propane-i,3-diylbis(oxy))(6aS,6a'S)-bis(2,6-dimethoxy- 8-(4- methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydrobenzo[e]-pyrido[i, 2- a][i,4]diazepine-5(i2H)-carboxylate) (106a)

105a (0.017 g, 0.017 mmol) was dissolved in DCM (3 ml). To this solution, tetrakis- (triphenylphosphine)palladium(o) (0.001 g, 0.00085 mmol) and pyrrolidine 0.0013 ml, 0.02 mmol) were added. After fifteen minutes, the reaction was observed to have completed via TLC analysis. The reaction mixture was concentrated under reduced pressure to form an oil, which was re-dissolved in diethyl ether (2 ml) and concentrated under reduced pressure (x3) to remove any residual pyrrolidine. The solid obtained was then purified via flash column chromatography (100% ethyl acetate) to resolve 106a in 76% yield. The dimer obtained was estimated 80% regiopure via the NMR reported below. Ή NMR (400 MHz, CDC1 ₃ ) δ 7.87 (d, 2H, J = 5.3 Hz), 7.47 (s, 2H), 7.39 (d, 4H, J = 7.2 Hz) , 6.92 (d, 4H, J = 7.2 Hz), 6.88 (s, 2H), 6.50 (d, 2H, J = 3.7 Hz), 4.56-4.45 (m, 2H), 4-36-4-19 (m, 6H), 3.93 (s, 6H), 3.82 (s, 6H), 3-48-3-37 (m, 2H), 2.72-2.65 (m, 4H), 2.48-2.36 (m, 2H); MS (ES+): m/z = 769.5 (M+H) ⁺ ; LC Retention Time

(Method A): t _R = 7.18 (Method B): t _R = 3.48. Further Dimer Examples

Four further dimer examples are detailed below as two regioisomer pairs.

Pair One: Aryl Linker

Diallyl 3,3 ^, -((i,3-phenylenebis(methylene))bis(oxy))(6aS',6a ^, iS) -bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate) (107a) and diallyl 3,3'- ((i,3-phenylenebis(methylene))bis(oxy))(6aS ^, ,6a'iS')-bis(2,6-dimethoxy-8- (4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydrobenzo[e]pyrido[ i,2-a]- [i,4]-diazepine-5(i2ii)-carboxylate) (107b)

269

SUBSTITUTE SHEET RULE 26

103b 107b

103a (0.035 g _> 0.073 mmol) was dissolved in DMF (2 ml). To this solution, 1,3- bis(bromomethyl)benzene (0.011 g, 0.039 mmol) and potassium carbonate (0.010 g, 0.073 mmol) were added and the solution heated at 45 °C for 45 minutes under microwave conditions. At this point the reaction was judged to have proceeded effectively by LC-MS analysis. Water (20 ml) was then added to the reaction mixture and the resulting suspension extracted with ethyl acetate (3 x 50 ml). The organic extracts were combined and washed with brine (3 x 100 ml), dried using magnesium sulphate and concentrated under reduced pressure to produce a yellow oil. Further purification using flash column chromatography (10:90 acetone/dichloromethane) produced a cream coloured solid 107a in 73% yield.

A similar procedure was carried out using 103b to generate 107b in 70% yield.

107a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.54 (s, 2H) 7.41 (bs, 4H), 7.33 (d, 4H, J= 8.1 Hz), 7.22 (s, 2H), 6.88 (d, 4H, J = 7-8 Hz), 6.71 (s, 2H), 6.07-6.00 (m, 2H), 5.80-5.66 (m, 2H), 5.66 (d, 2H, J= 9.7 Hz), 5.19-5.00 (m, 8H), 4.72-4.35 (m, 6H), 3.92 (s, 6H), 3.81 (s, 6H), 3.51 (s, 6H), 3.16-3.05 (m, 2H), 2.63-2.51 (m, 4H); MS (ES+) m/z = 1063.7 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 9.43.

270

SUBSTITUTE SHEET RULE 26 107b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.52 (s, 2H), 7.40 (bs, 4H), 7.36 (d, 4H, J= 8.5 Hz), 6.90 (d, 4H, J = 8.5 Hz), 6.69 (s, 2H), 6.26-6.20 (m, 2H), 5-76-5-64 (m, 2H), 5-45-5-35 (m, 2H), 5-18-4-99 (m, 8H), 4-6i-4-36 (m, 4H), 4-31-4-05 (m, 6H), 3-93 (s, 6H), 3.83 (s, 6H), 3-70-3-59 (m, 2H), 3-42-3-38 (s, 6H), 2.99-2.86 (m, 2H), 2.75-2.62 (m, 2H); MS (ES+): m/z = 1063.2 (M+H) ⁺ ; LC Retention Time (Method A): ίκ = 4·ΐ7·

(6aS ^, ,6a'iS)-3,3 ^l -((i,3-phenylenebis(methylene))bis(oxy))bis(2-methoxy- 8- (4-methoxyphenyl)-9,io-dihydrobenzo[e]pyrido[i,2-a][i,4]-dia zepin- i2(6aH)-one) (108a) and (eaiSjea^-s^'-iii^- henylenebis- (methylene))bis(oxy))bis(2-methoxy-8-(4-methoxyphenyl)-7,io- dihydro- benzo[e]pyrido[i,2-a][i,4]diazepin-i2(6aii)-one) (108b)

107a (0.015 g, 0.014 mmol) was dissolved in DCM (3 ml). To this solution, tetrakis-

(triphenylphosphine)palladium(o) (0.001 g, 0.00085 mmol) and pyrrolidine 0.0026 ml, 0.04 mmol) were added. After five minutes, the reaction was observed to have completed via TLC analysis. The reaction mixture was concentrated under reduced pressure to form an oil, which was re-dissolved in diethyl ether (2 ml) and concentrated under reduced pressure (x3) to remove any residual pyrrolidine. The solid obtained was

271

SUBSTITUTE SHEET RULE 26 then purified via flash column chromatography (25:75 acetone/dichloro methane) to resolve 108a in 65% yield. The dimer obtained was obtained 84% regiopure based upon the NMR reported below.

A similar procedure was carried out for the regioisomer 108b. The yield was 60% and the regiopurity estimated as 88% based upon integration of the NMR reported below. 108a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ.84 (d, 2H, 5.3 Hz), 7.53-7 '.48 (m, 3H), 7-44-7-35 (m, 7H), 6.91 (d, 4H, J = 8.4 Hz), 6.85 (s, 2H), 6.05 (d, 2H, J = 3.9 Hz), 5.21 (q, 4H, J = 12.4 Hz), 4.54-4.41 (m, 2H), 4.32-4.26 (m, 2H), 3.96 (s, 6H), 3.83 (s, 6H), 3-49-3-37 (td, 2H, J = 13.9, 7.6 Hz), 2.72-2.65 (m, 4H); MS (ES+): m/z = 831.4 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 7.33 (Method B): t _R = 3.58.

108b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7-59 (d, 2H, J = 5.4 Hz), 7.54 (s, 2H), 7.50 (s, lH), 7-43-7-38 (m, 7H), 6.93 (d, 4H, J = 7-9 Hz), 6.83 (s, 2H), 6.39-6.35 (m, 2H), 5.20 (q, 4H, J = 12.8 Hz), 4.46 (dd, 2H, J = 18.0, 6.1 Hz), 4.11 (d, 2H, J = 18.1 Hz), 3-97 (s, 6H), 3-94 (d, 2H, 5.0Hz), 3.84 (s, 6H), 2.94 (m, 4H); MS (ES+): m/z = 831.3 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 7.42 (Method B): t _R = 3.55.

Pair Two: Pyridine Linker

Ddiallyl 3,3'-((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS', 6a\S)- bis(2,6-dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,9,io-tetra - hydrobenzo[e]pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate ) (109a) and diallyl 3,3 ^l -((pyridine-2,6-diylbis(methylene))bis(oxy))(6aiS',6a ^l iS')-bis(2,6- dimethoxy-8-(4-methoxyphenyl)-i2-oxo-6,6a,7,io-tetrahydroben zo[e]- pyrido[i,2-a][i,4]diazepine-5(i2ii)-carboxylate) (109b)

Alloc

272

SUBSTITUTE SHEET RULE 26

103a (0.035 g _> 0.073 mmol) was dissolved in DMF (2 ml). To this solution, 2,6- bis(bromomethyl)pyridine (0.011 g, 0.039 mmol) and potassium carbonate (0.010 g, 0.073 mmol) were added and the solution heated at 45 °C for 45 minutes under microwave conditions. At this point the reaction was judged to have proceeded effectively by LC-MS analysis. Water (20 ml) was then added to the reaction mixture and the resulting suspension extracted with ethyl acetate (3 x 50 ml). The organic extracts were combined and washed with brine (3 x 100 ml), dried using magnesium sulphate and concentrated under reduced pressure to produce a yellow oil. Further purification using flash column chromatography (20:90 acetone/ dichloromethane) produced a cream coloured solid 109a in 68% yield.

A similar procedure was carried out for the regioisomer 103b to produce the compound 109b in 74% yield.

109a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.79 (t, lH, J = 7.7 Hz), 7.54 (d, 2H, J = 7.7 Hz), 7-33 (d, 4H, J = 8.7 Hz), 7.24 (s, 2H), 6.88 (d, 4H, J = 8.8 Hz), 6.73 (s, lH), 6.01 (d, 2H, J = 2.8 Hz), 5-76-5-34 (m, 2H), 5.59 (d, 2H, J = 9.1 Hz), 5.27 (s, 4H), 5.13-4-98 (m, 4H), 4.72 (d, 2H, J = 12.7 Hz), 4-61-4-38 (m, 4H), 3.95 (s, 6H), 3.92 (d, 2H, J = 11.7Hz), 3.82 (s, 6H), 3.49 (s, 6H), 3-17-3-07 (m, 2H), 2.66-2.50 (m, 4H); MS (ES+): m/z = 1064.2 (M+H) ⁺ ; LC Retention Time (Method B): t _R = 4.17.

109b Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.78 (t, lH, J = 7.8 Hz), 7.53 (d, 2H, J = 7.7 Hz), 7.35 (d, 4H, J = 8.6 Hz), 7.27 (s, 2H), 6.90 (d, 4H, J = 8.6 Hz), 6.69 (s, 2H), 6.26-6.19 (m, 2H), 5-74-5-58 (m, 2H), 5.40 (d, 2H, J = 9.6 Hz), 5.25 (s, 4H), 5.11- 4-95 (m, 4H), 4-58-4-33 (m, 4H), 4-26 (dd, 2H, J = 18.1, 5-6 Hz), 4.14 (d, 2H, J = 18.1 Hz), 3-95 (s, 6H), 3.81 (s, 6H), 3.63 (dd, 2H, J = 8.0, 6.1 Hz), 3.38 (s. 6H), 2.93 (d, 2H, J = 15.1 Hz), 2.72-2.55 (m, 2H); MS (ES+): m/z = 1064.3 (M+H) ⁺ ; LC Retention Time (Method B): £ _R = 4.02.

(6aS ^, ,6a'iS')-3,3 ^l -((pjT*idine-2,6-diylbis(methylene))bis(oxy))-bis(2-me thoxy- 8-(4-methoxyphenyl)-9,io-dihydrobenzo[e]pjrido[i,2-a][i,4]di azepin- i2(6aH)-one) (110a) and (6aS ^, ,6a' ₁ S)-3,3 ^l -((pyridine-2,6- diylbis(methylene))bis(oxy))bis(2-methoxy-8-(4-methoxyphenyl )-7,io- dihydrobenzo[e]pyrido[i,2-a][i,4]diazepin-i2(6aH)-one) (110b)

273

SUBSTITUTE SHEET RULE 26

109a (0.025 g, 0.023 mmol) was dissolved in DCM (3 ml). To this solution, tetrakis- (triphenylphosphine)palladium(o) (0.002 g, 0.0013 mmol) and pyrrolidine 0.0042 ml, 0.065 mmol) were added. After five minutes, the reaction was observed to have completed via TLC analysis. The reaction mixture was concentrated under reduced pressure to form an oil, which was re-dissolved in diethyl ether (2 ml) and concentrated under reduced pressure (x3) to remove any residual pyrrolidine. The solid obtained was then purified via flash column chromatography (35:75 acetone/dichloromethane) to resolve 110a in 34% yield. The dimer obtained was obtained was >go% regiopure based upon the NMR reported below.

A similar procedure was carried out for the regioisomer 109b to generate the compound 110b. The yield was 67% and the regiopurity estimated as >go% based upon integration of the NMR reported below.

110a Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7.86 (d, 2H, J = 5.3 Hz), 7.74 (t, lH, J = 7.8 Hz), 7.48 (d, 2H, J = 7.8 Hz), 7.43-7-35 (m, 4H), 6.95-6.89 (m, 4H), 6.86 (s, 2H), 6.06 (d, 2H, J = 4.0 Hz), 5.34 (s, 4H), 4.51 (dt, 2H, J = 13.2, 4.6 Hz), 4.30 (t, 2H, J = 4.7 Hz), 4.00 (s, 6H), 3.83 (s, 6H), 3.44 (dt, 2H, J = 13.3, 6.8 Hz), 2.76-2.64 (m, 4H); MS (ES+): m/z = 832.1 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 7.35 (Method B): t _R = 340.

274

SUBSTITUTE SHEET RULE 26 nob Characterisation Ή NMR (400 MHz, CDC1 ₃ ) δ 7-72 (t, lH, J = 7.8 Hz), 7.58 (d, 2H, J = 5.5 Hz), 7.55 (s, 2H), 7.46 (d, 2H, J = 7.8 Hz), 7-42-7-36 (m, 4H), 6.95-6.89 (m, 4H), 6.84 (s, 2H), 6.36 (dd, 2H, J = 3.0, 1.6 Hz), 5.31 (s, 4H), 4-45 (dd, 2H, 18.3, 5-9 Hz), 4-15-4-07 (m, 2H), 3.99 (s, 6H), 3.83 (s, 6H), 3-96-3-90 (m, 2H), 2.96-2.91 (m, 4H); MS (ES+): m/z = 832.3 (M+H) ⁺ ; LC Retention Time (Method A): t _R = 7.47

(Method B): i _R = 3-43-

Biological and Biophysical Characterisation Write-up for Patent 6

Cytotoxicity in Cell Lines

The cytotoxicity of compounds 26, 34, 50, 58 and 68 were evaluated in a variety of cell lines using the standard MTT assay for a 72 hour incubation period (Table 1).

Biophysical Characterisation

275

SUBSTITUTE SHEET RULE 26 The ability of 26 and 34 to cross-link DNA was determined using an assay involving linear double-stranded HexA. TyrT and MSi DNA fragments (Figure 5). The PBD dimer Talirine (SGD1882) was used as a positive control, as PBD dimers have previously been shown to cross-link DNA ² 4.

Following denaturation conditions (treatment with formamide and heating at 65 °C for 5 min) the DNA strands were completely separated (see control C2, Figure 6). The presence of an interstrand cross-link holds the denatured strands in close proximity, and cross-linked adducts therefore run as double-stranded DNA on polyacrylamide gel. Talirine and 34 were tested at≥ 10 different concentrations, and the assay was repeated twice. The cross-linking ability of 34 is shown in Figure 6. Figure 6 shows an autoradiograph of a denaturing polyacrylamide gel showing DNA interstrand cross- linking by 34 in linear ³² P-end-labelled HexA DNA following overnight incubation at 37 °C at various concentrations. Ci and C2 represent controls where Ci is DNA without formic acid, and C2 is DNA without formic acid, but heated thereby forming single strands. Cross-links are clearly detectable at concentrations as low as ιοηΜ. Using the same assay, the PBD dimer Talirine was also shown to cross-link DNA down to a concentration of 10 nM (Figure 7). These results demonstrate that 34 can produce DNA cross-links at concentrations comparable to the PBD dimer Talirine.

DNA Footprinting

The sequence selectivity of 26 and 34 was investigated using a modification of the previously established DNA footprinting assay ²⁵ . Following an overnight incubation of the ligand-DNA complexes, the mixture was mixed with strand separation buffer containing 10 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue, 80% formamide and incubated at 100 °C for 3 min. The mixture was then immediately cooled on ice and run on an 8% denaturing gel. Examination of the obtained gel (Figure 8) shows distinct footprints produced by 26 and 34. Furthermore, both DNA fragments contain multiple potential binding sites for 26 and 34 (i.e., multiple examples of potential G-G cross- linking sites), but surprisingly only three preferred sites were observed during this experiment. These footprints correspond to the general code XYWWYWYX, where X is any base, Y is G or C and W is A or T. This suggests that the molecule acts in a highly sequence selective manner. The possible adducts formed within both sequences are shown in Figure 9.

FRET DNA Melting

276

SUBSTITUTE SHEET RULE 26 FRET DNA melting studies were undertaken on 26 and 34 using a number of fluorescently labelled sequences. The sequences (Figure 10) were designed to provide additional evidence that 26 and 34 can form inter- and intrastrand cross-links at the site 5'-XYWWYWYX-3'.

The short duplexes used in this FRET study are relatively unstable in the duplex form with a melting temperature below 30°C so that, in the absence of ligand, a large part of the melting occurs below the starting temperature of the experiment. However, the intrastrand cross-links formed by 34 stabilize the duplex form, producing large increases in melting temperature with T _m values of ~η\ °C for 5'-

AAAAAAGAGAAGAAAAAA-3' (Figure 11, 1st panel) suggesting intrastrand cross-link formation in the DNA-footprinting derived binding site and 66 °C for

AAAAAAGAGAGAAAAAA-3' (Figure 11, 3 ^rd panel) suggesting intra-strand cross-link formation and two sites. Interestingly, 5'-AAAAAAGAGAAGAAAAAA-3' directly corresponds to a footprint derived for 34, whereas 5'-AAAAAAGAGAGAAAAAA-3' represents an intra-strand cross-link with one less base-pair, and it is evident from FRET stabilisation that the GAGAAG adduct is preferred. In the case of inter-strand cross-link studies, 34 stabilises 5'-AAAAAAGAGAACAAAAAA-3' with a T _m value of ~7i °C, which suggests strong DNA interactivity. Furthermore, 34 exhibits limited stabilisation of 5'-AAAAAAGATCAAAAAA-3' (which represents a traditional PBD binding site), suggesting the molecules outlined possess an alternative DNA sequence selectivity profile to the prior art.

As can be seen from Figure 11, the melting temperature of each duplex increases significantly in proportion to the concentration of 34 present, providing strong supporting evidence that the compound can produce intra- and inter-strand crosslinks.

Summary of Cross-linking Data

Taken together, the cross-linking data presented above provide strong evidence that 34 (and its analogues - data not shown) produces both intrastrand and interstrand crosslinks which appear to form with a high degree of sequence-specificity {e.g., Figures 8 and 9). It is possible that the compound may also form mono-alkylated adducts with guanines. Together, this population of DNA adduct types may account for the cytotoxicity of this family of compounds in cells.

Biophysical Characterisation Methodology

277

SUBSTITUTE SHEET RULE 26 l. Material

1.1. DNA fragment

The preparation of the DNA fragments (Figure 5) has been previously described M. Briefly, the sequence which had been cloned into the BamHl site of pUCi8 was obtained by cutting with Hindlll and EcoRI. Radiolabelled DNA fragments were prepared by filling in the 3'-end of the Hindlll site with [a- ³² P]dATP using Klenow DNA polymerase (exo-).

The radiolabelled DNA fragment was separated from the remainder of the plasmid DNA on a 6% non-denaturing polyacrylamide gel. The gel (20 cm long, 0.3 mm thick) was run at 400 V in lx TBE running buffer for about i-2h, until the bromophenol blue had run most of the way down the gel. The glass plates were separated and the position of the labelled DNA fragment was established by short (1 min) exposure to an X-ray film. The relevant band was then cut from the gel and the radiolabelled DNA eluted by adding 300 μΐ. ιο mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and gently agitating overnight at room temperature. The eluted DNA was finally precipitated with ethanol and re-suspended in a suitable volume of 10 mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA buffer so as to give at least 10 counts per second/ μΐ, on a hand-held Geiger counter. With fresh plasmid and a-3 ² P-dATP this process typically generated about 150 μΐ, of radiolabelled fragment DNA. The absolute concentration of the DNA is not important, and it is typically lower than 10 nM.

Footprinting reactions were performed as previously described ^[5] using the DNA fragments HexA and HexB, which together contain all 64 symmetrical hexanucleotide sequences ^[6] , and MSi that contains all possible 134 tetranucleotide sequences ^[7] . The DNA fragments were obtained by cutting the parent plasmids with Hindlll and Sacl (for HexA and MSi) or EcoRI and PstI (for HexB), and were labelled at the 3'-end of the Hindlll or EcoRI sites with [a- ³² P]dATP using reverse transcriptase or exo- Klenow fragment. After gel purification, the radiolabelled DNA was dissolved in 10 mM Tris- HC1 pH 7.5 containing 0.1 mM EDTA, at a concentration of about 10 c.p.s per μΐ, as determined on a hand held Geiger counter. 1.5 μΐ, of radiolabelled DNA was mixed with 1.5 μΐ, ligand that had been freshly diluted in 10 mM Tris-HCl pH 7.5, containing 10 mM NaCl. The complexes were left to equilibrate for at least 12 hours before digesting with 2 μΐ, DNase I (final concentration about 0.01 units/mL). The reactions were stopped after 1 minute by adding 4 μΐ, of formamide containing 10 mM EDTA and bromophenol blue (0.1% w/v). The samples were then heated at 100 °C for 3 minutes before loading onto 8% denaturing polyacrylamide gels containing 8 M urea. Gels were

278

SUBSTITUTE SHEET RULE 26 fixed in 10% acetic acid, transferred to 3MM paper, dried and exposed to a phosphor screen overnight, before analysing with a Typhoon phosphorimager.

1.2. Compounds

32 was synthesised as described above and the PBD dimer Talirine was obtained from Aurum Pharmatech LLC. Stock solution was prepared by dissolving the ligands in DMSO to give a concentration of 10 mM. From this stock solution, working solutions of the desired concentration were prepared by diluting with 10 mM Tris-HCl, pH 7.5 containing 10 mM NaCl.

2. Cleavage Assay

2.1. Preparation ofligand-DNA complexes

Radiolabeled DNA (1.5 μί) was mixed with 1.5 μί, ligand solution of various concentrations (10 μΜ-10 nM) and incubated overnight at 37 °C.

2.2. Preparation ofGA marker

Labelled DNA (1.5 μί) was mixed with 20 μΐ, sterile water and 5 μΐ, of denaturing loading solution (80% formamide containing 10 mM EDTA, 10 mM NaOH, 0.01% bromophenol blue). The sample was then incubated at 100 °C for 20 min with the micro- centrifuge tube cap open to allow evaporation.

3. Cross-linking Assay

3.1. Preparation of Ligand-DNA complexes

Radiolabeled DNA (1.5 μΐ,) was mixed with 1.5 μΐ, ligand solution of various concentrations (10 μΜ-io nM) and incubated overnight at 37 °C.

3.2 Cross-linking assay

After overnight incubation, the samples were mixed with 7 μΐ, loading solution (80% formamide containing 10 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue) and incubated at 65 °C for 5 min. Control 1 (Ci) for native double-stranded DNA consisted of 1.5 μΐ. labelled DNA, 1.5 μΐ. ιο mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 μΐ, lx loading dye. Control 2 (C2) for denatured native single-stranded DNA was composed of 1.5 μΐ. labelled DNA, 1.5 μΐ, ιο mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA which was incubated at 65 °C for 5 min. Control 3 (C3) for native double- stranded DNA consisted of 1.5 μΐ. labelled DNA, 1.5 μΐ, ιο mM Tris-HCl, pH 7.5 containing 0.1 mM EDTA and 7 μΐ, SSB. Control 4 (C4) for denatured native single- stranded DNA was composed of 1.5 μΐ. labelled DNA, 1.5 μΐ, ιο mM Tris-HCl, pH 7.5

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SUBSTITUTE SHEET RULE 26 containing o.i mM EDTA and 7 μί, SSB which was incubated at 65 °C for 5 min.

Separation was performed on a 7.5% denaturing polyacrylamide gel (20 cm long, 0.3 mm thick) at 500V for about 4 h until the dye reached the bottom of the gel. The gel plates were then separated, the gels fixed by immersing in 10% (v/v) acetic acid, followed by transfer to Whatmann 3MM paper and drying under vacuum at 80 °C. The dried gel was then exposed to a phosphorimager screen overnight before scanning using a Typhon FLA 7000 instrument.

FRET Studies Methodology

1. General

1.1. Oligonucleotides

Oligonucleotides were obtained from ATDbio (Southampton, UK) in lyophilised form. They were labelled with a fluorophore molecule (F = fluorescein) at the 5'-end and a quencher molecule (Q = dabcyl) at the 3'-end of the complementary strand. Each oligonucleotide was dissolved in distilled H ₂ 0 to form stock solutions of 100 μΜ.

Working solutions of 5 μΜ were prepared by diluting the stock solution with distilled H ₂ 0.

1.2. Buffers

The following buffers were used: 250 mM phosphate buffer pH 7.4 (consisting of sodium dihydrogen phosphate and sodium phosphate diluted in distilled H ₂ 0) and 5 M sodium chloride buffer. All buffers and distilled H ₂ 0 were filtered through a 0.2 μΜ filter prior to use. 1.3. Compound

For the FRET experiments a stock solution of 18 was prepared by dissolving it in DMSO to give a concentration of 10 mM. From this stock solution, working solutions of the desired concentration were prepared by diluting the stock solution with distilled H ₂ 0.

1.4. Preparation ofligand-DNA complexes

The reaction mixture was comprised of 4 ΐ, of 250 mM phosphate buffer (final concentration of 50 mM), 4 μΐ, flourophor and 4 μΐ, quencher molecule of the appropriate oligonucleotide for a final concentration of 0.2 μΜ, 4 μΐ, 5 M sodium chloride (final concentration of 1 M NaCl), and 4 μΐ, of distilled H ₂ 0. This mixture was heated in an Eppendorf tube at 90 °C for 1 min and slowly cooled down to room temperature. This process was carried out to anneal the single strands to double-

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SUBSTITUTE SHEET RULE 26 stranded DNA. Following this, 4 ΐ, of the ligand was added in the desired

concentration and the mixture incubated overnight either at room temperature or 4 °C. A control sample of DNA only was prepared by mixing 4 ΐ, 250 mM phosphate buffer (final concentration of 50 mM) with 4 fluorophore-labelled and 4 ΐ, quencher- labelled oligonucleotides (of the appropriate sequence) to give a final concentration of 0.2 μΜ, 4 μΐ, 5 M sodium chloride (final concentration of 1 M NaCl) and 4 μΐ, distilled H ₂ 0. This mixture was analysed without prior annealing.

1.5. Fluorescence melting

Fluorescence melting profiles were measured using a Roche LightCycler using a total reaction volume of 20 μL. Initially, the samples were denatured by heating to 95 °C at a rate of 1 °C min ¹ . The samples were then maintained at 95 °C for 5 min before annealing by cooling to 25 °C at 1 °C min ¹ . The samples were then held at 25 °C for a further 5 min and finally melted by heating to 95 °C at 1 °C min ¹ . Annealing steps and melting steps were all recorded and changes in fluorescence were measured at 520 nm.

1.6. Data analysis

T _m values were obtained from the first derivates of the melting profiles using the Roche LightCycler software.

MTT Cytotoxicity Methodology

Tumor cell lines were maintained in RPMI1640 medium supplemented with 10% heat- inactivated fetal bovine serum, 2mM L-glutamine and imM sodium pyruvate. 1800 cells per well were seeded in a volume of ιδομΐ in a 96-well flat bottom polystyrene plate. The cells were allowed to adhere overnight at 37°C in a C0 ₂ incubator. Ligands were initially formulated in DMSO, and stocks stored at -8o°C. They were then further formulated at ιοχ concentration in RPMI1640 medium. 20ul of diluted samples were added into each treatment well. On each plate, blank wells with no cells, and untreated wells containing cells, were included. Plates were then cultured at 37°C in a C0 ₂ incubator for 72hrs. Cytotoxicity was evaluated using a tetrazolium salt-based assay, the MTT assay. After 72hours, the supernatant was removed from each well and 200 μΐ of a sterile filtered MTT solution in water added to each well. The plates were then incubated at 37°C in a C0 ₂ incubator for 4hrs. The supernatant was then removed and the formazan crystals formed solubilized by adding Ι50μ1 of DMSO to each well. The plate was then read on a plate reader at 54onm, and percentage cell survival calculated as follows: ((mean absorbance treated wells at concentration x - mean absorbance blank wells) ÷ (mean absorbance untreated wells at concentration x - mean

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SUBSTITUTE SHEET RULE 26 absorbance blank wells)) x 100. Data were plotted as concentration in nM vs. % cell survival in Microsoft Excel, and IC ₅₀ values (concentration where cell survival is reduced by a half) were determined from the graph.

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All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

SUBSTITUTE SHEET RULE 26