POLYCYCLIC EPOXIDES AND COMPOSITIONS THEREOF WITH

ANTI-CANCER ACTIVITIES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 62/159826, filed May 11, 2015, incorporated herein by reference.

FIELD

[0002] The present technology relates generally to polycyclic expoxides and compositions thereof useful for inhibiting cancer cell proliferation and tumor angiogenesis, inducing apoptosis of cancer cells, as well as for treating leukemia, ovarian, breast, prostate, liver, pancreatic and colon cancers.

BACKGROUND

[0003] The following description is provided to assist the understanding of the reader.

None of the information provided or references cited is admitted to be prior art to the present technology.

[0004] Triptolide is the main active ingredient of the Chinese herbal medicine

Tripterygium willfordii Hook f (TWHF). Low-dose herbal preparations of triptolide have been used in China for the treatment of rheumatoid arthritis, systemic lupus erythematosus, purpura, psoriasis, Behcet's syndrome and glomerulonephritis. Triptolide has also shown some potential as an anti -cancer and anti -fertility agent since its isolation in 1972. In certain model systems, triptolide can function as an inhibitor of tumor angiogenesis. The combination of triptolide and tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance apoptosis under some circumstances. In certain animal models triptolide has been shown to be synergistic with chemotherapeutic agents such as 5- fluorouracil ("5-FU") and irinotecan ("CPT-11") as well as with radiation. [0005] Despite its use in herbal remedies, the clinical development of triptolide as a pharmaceutical agent has been limited due to the low separation between its therapeutic and toxic doses. In particular, triptolide displays efficacies at a dose of approximately 0.5 mg/kg, yet the LD ₅₀ of triptolide is approximately only 1.9 mg/kg. Furthermore, triptolide is extensively metabolized and has a very short half-life. It is unlikely that any low dose extract preparation of triptolide will overcome its narrow therapeutic window.

[0006] Efforts to develop commercially viable analogs and prodrugs of triptolide have been as challenging as triptolide itself. Such analogs and prodrugs often suffer from narrow therapeutic windows, high toxicities, rapid metabolization, and short half-lifes. Moreover, the isopropyl side chain of triptolide is exceedingly difficult to modify. Preparation of structurally diverse libraries from triptolide itself is not practical. Only a handful of hydroxyl-substituted isopropyl derivatives of triptolide are known, primarily isolated as natural products.

SUMMARY

[0007] The present technology provides polycyclic epoxide compounds, methods of using them, as well as intermediates for the synthesis of such compounds. The polycyclic epoxides share certain features with triptolide but are not prepared from triptolide. As a result, the present compounds possess chemical structures and biological profiles that differ significantly from triptolide. The present polycyclic expoxides, compositions thereof and medicaments containing the same are useful for for treating certain cancers, including leukemia, ovarian, breast, prostate, liver, pancreatic, and colon cancers, as well as inhibiting cancer cell proliferation and tumor angiogenesis and promote apoptosis. Unlike triptolide, certain of the present compounds are significantly less toxic and have a greater therapeutic index.

[0008] In one aspect, the present technology provides compounds represented by

Formula I, as well as stereoisomers thereof, tautomers thereof, prodrugs thereof, and pharmaceutically acceptable salts of any of the foregoing,

Formula I wherein

Ri is -H or a substituted or unsubstituted alkyl group;

R ₂ and R3 are each independently -H or a substituted or unsubstituted alkyl group; Rt is -H or a C ₁ -60 substituted or unsubstituted alkyl or heteroalkyl group which optionally comprises a substituted or unsubstituted cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl or aryl group;

R ₅ is -OR';

Re is -H or -OR', or R ₅ and Re together are =0;

R7 is -H or an unsubstituted alkyl group;

each R' is independently -H, a hydroxyl protecting group, -CO ₂ R", -C(0)R", or a substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or heterocyclylalkyl group; and each R" is independently a substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or heterocyclylalkyl group.

[0009] In some embodiments of the compounds of Formula I, when R ₅ is OH and Re is -H, R ₄ is not an isopropyl group and/or R ₄ is not a hydroxy substituted isopropyl group, e.g., 1-hydroxyisopropyl.

[0010] In another aspect, the present technology provides pharmaceutical compositions for medical treatment. Each pharmaceutical composition includes a compound of Formula I, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing, and pharmaceutically acceptable carrier.

[0011] In another aspect, the present technology provides a method of treatment comprising administering to a subject having leukemia, ovarian, breast, prostate, liver, or colon cancer a therapeutically effective amount of a compound of Formula I or a monoclonal antibody conjugate thereof, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing, or a composition comprising any of the foregoing and pharmaceutically acceptable carrier.

[0012] In another aspect, the present technology provides a method of inhibiting cancer cell proliferation and tumor angiogenesis, promoting apoptosis, or treating leukemia, ovarian, breast, prostate, liver, or colon cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I or a monoclonal antibody conjugate thereof, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing, or a composition comprising any of the foregoing and pharmaceutically acceptable carrier.

[0013] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features will become apparent by reference to the following drawings and the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the identification of triptolide (TL)-binding proteins in different cell lines, based on silver-stained gels of pull down products of biotin-TL or biotin. Cell lysates were incubated with 5 μΜ biotin or Biotin-TL. The bottom arrows indicate bands confirmed as Peroxiredoxin I. The top arrows indicate bands confirmed as HSP90.

DETAILED DESCRIPTION

[0015] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0016] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0017] The following terms are used throughout as defined below.

[0018] As used herein, singular articles such as "a", "an", and "one" are intended to refer to singular or plural.

[0019] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C ¹⁴ , P ³² and S ³⁵ are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0020] In general, "substituted" refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include: halogens (i.e., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; imines; nitro groups (i.e., NO2); nitriles (i.e., CN); phosphates, and the like.

[0021] Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0022] Alkyl groups include straight chain and branched chain saturated hydrocarbon groups having (unless otherwise specified) from 1 to 12 carbon atoms, and in some embodiments from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like. [0023] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, 6, or 7 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1. l]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with, non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but are not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with substituents such as those listed above.

[0024] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups have from 4 to 16 carbon atoms, or in some embodiments, 4 to 12 carbon atoms, or 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above.

[0025] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, and in some embodiments from 2 to 10 carbons, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carbon-carbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH ₃ ), -CH=C(CH ₃ ) ₂ , -C(CH ₃ )=CH ₂ , -C(CH ₃ )=CH(CH ₃ ), -C(CH ₂ CH ₃ )=CH ₂ , among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above. [0026] Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, or cyclohexadienyl.

[0027] Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups have from 5 to 14 carbon atoms, or, in some embodiments, 5 to 12 carbon atoms, 5 to 10 carbon atoms, or even 6, 7, or 8 carbon atoms. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted

cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0028] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, or in some embodiments from 2 to 10 carbons or from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to - C≡CH, -C≡CCH ₃ , -CH ₂ C≡CCH ₃ , -C≡CCH ₂ CH(CH ₂ CH ₃ ) ₂ , among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above.

[0029] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms.

Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Aryl groups have 6 to 14 carbons, and in some embodiments 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, the aryl groups are phenyl or naphthyl. Although the phrase "aryl groups" includes groups containing fused rings, such as fused aromatic- aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like), it does not include aryl groups that have other groups, such as alkyl or halo groups, bonded to one of the ring members. Rather, groups such as tolyl are referred to as substituted aryl groups.

Representative substituted aryl groups may be mono-substituted or substituted more than once, such as di-, tri-, tetra- or penta-substituted. In some embodiments, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0030] Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl groups have 7 to 18 carbaon atoms, or, in some embodiments 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7, 8, 9 or 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group.

Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above.

[0031] Heteroalkyl groups are alkyl groups, as described above, in which one or more carbon atoms are replaced with heteroatoms such N, O, S or combinations thereof, or the carbon chain is interrupted by such heteroatoms. Where valence allows, heteroalkyl groups may be further substituted with substituents as described herein. The term heteroalkyl also includes alkyl groups in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atoms are oxygen, nitrogen, sulfur, or combinations thereof, in a stable configuration. Examples of heteroalkyl groups include, but are not limited to -

CH ₂ NH ₂ , -CH ₂ CH2N(CH3)CH ₂ SCH3, -CH2OCH2CH2OCH2CH2OCH3, -CH ₂ S(0)CH ₂ , -CH ₂ CH2SO2CH3, and the like, as well as, e.g., polyether and polyamino alkyl groups, including but not limited to poly(oxyalkylene) groups.

[0032] Heterocyclyl groups are non-aromatic rings containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. Heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass partially unsaturated and saturated ring systems, such as, for example, imidazolinyl and imidazolidinyl groups. While heterocyclyl groups include fused rings, at least one of which contains one or more heteroatoms, they do not include fused rings that include a heteroaryl or aryl ring. Heterocyclyl groups also includes bridged poly cyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. However, the phrase does not include heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members. Rather, these are referred to as "substituted heterocyclyl groups". Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, thianyl, thiepanyl, pyrrolinyl, thiazolinyl, imidazolinyl, piperazinyl, pyranyl, morpholinyl, dioxolanyl, oxaziridinyl, quinuclidinyl, pyrrolinyl, dithianyl, 2-oxabicyclo[3.3.0]octane, thiazolidinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl,

tetrahydrothiopyranyl, and indolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, e.g., di- or tri-substituted with various substituents such as those listed above.

[0033] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. In some embodiments the heteroaryl group includes 1, 2, 3, or 4 heteratom ring members. Heteroaryl groups have from 5 to 14 ring members, or 5 to 12, 5 to 10 or 5 or 6 ring members, and may be mono-, bi- or tricyclic. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and also include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Although the phrase "heteroaryl groups" includes fused ring compounds, the phrase does not include heteroaryl groups that have other groups bonded to one of the ring members, such as alkyl groups. Rather, heteroaryl groups with such substitution are referred to as "substituted heteroaryl groups." Examples of heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl

(azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

[0034] Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl,

tetrahydrofuran-2-yl -methyl, tetrahydrofuran-2-yl -ethyl, and piperidin-2-yl-propyl.

Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0035] Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Representative heteraralkyl groups include but are not limited to imidazol-2-yl- methyl, indol-2-yl-ethyl. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.

[0036] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, "ene." For example, divalent alkyl groups are alkylene groups, divalent heteroalkyl groups are heteroalkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Alkylene groups may be straight or branched and may be substituted with substituents indicated and/or may be substituted with substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl groups.

[0037] Poly(oxyalkylene) is an alkylene group interrupted by two or more oxygen atoms so long as the oxygen atoms are not attached to each other. In some embodiments the poly(oxyalkylene) is poly(C2-C4 oxyalkylene). In certain embodiments the poly(oxyalkylene) is selected from poly(ethylene glycol), poly(propylene glycol) and poly(tetramethylene glycol). In certain embodiments, the poly(oxyalkylene) has 4 to 100, 4 to 50, 4 to 24 or 4 to 12 carbon atoms, or has 10 to 100, 10 to 50, or 10 to 24 carbon atoms.

[0038] Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0039] The terms "alkanoyl" and "alkanoyloxy" as used herein can refer,

respectively, to -C(0)-alkyl groups and -0-C(0)-alkyl groups.

[0040] The term "heteroalkanoyl" as used herein refers to a -C(0)-heteroalkyl group.

[0041] The terms "aryloxy" and "arylalkoxy" refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include, but are not limited to, phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.

[0042] The term "carboxylate" as used herein refers to a -COOH group.

_R 30

[0043] The term "ester" as used herein refers to -COOR ³⁰ groups. is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.

[0044] The term "amide" (or "amido") includes C- and N-amide groups,

i.e., -C(0)NR ³¹ R ³² , and -NR ³¹ C(0)R ³² groups, respectively. R ³¹ and R ³² are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include, but are not limited to, carbamoyl groups (-C(0)NH ₂ ) and formamide groups (-NHC(O)H). In some embodiments, the amide is -NR ³¹ C(0)-(Ci_5 alkyl) and the group is termed

"carbonylamino," and in others the amide is -NHC(0)-alkyl and the group is termed "alkanoylamino . "

[0045] The term "nitrile" or "cyano" as used herein refers to the -CN group.

[0046] Urethane groups include N- and O-urethane groups, i.e., -NR ³³ C(0)OR ³⁴

33 34 33 34

and -OC(0)NR R groups, respectively. R and R are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R ³³ may also be H.

[0047] The term "amine" (or "amino") as used herein refers to -NR ³⁵ R ³⁶ groups, wherein R ³⁵ and R ³⁶ are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH ₂ , methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0048] The term "sulfonamido" includes S- and N-sulfonamide groups,

i.e., -S0 ₂ NR ³⁸ R ³⁹ and -NR ³⁸ S0 ₂ R ³⁹ groups, respectively. R ³⁸ and R ³⁹ are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include, but are not limited to, sulfamoyl groups (-S0 ₂ NH ₂ ). In some embodiments herein, the sulfonamido is -NHS0 ₂ -alkyl and is referred to as the "alkylsulfonylamino" group.

[0049] The term "thiol" or "mercapto" refers to -SH groups, while sulfides include

-SR ⁴⁰ groups, sulfoxides include -S(0)R ⁴¹ groups, sulfones include -S0 ₂ R ⁴² groups, and sulfonyls include -S0 ₂ OR ⁴³ , R ⁴⁰ , R ⁴¹ , R ⁴² , and R ⁴³ are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or

heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl. [0050] The term "urea" refers to -NR ⁴⁴ -C(0)-NR ⁴³ R ^4b groups. R ⁴⁴ , R , and R ^4b groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0051] The term "amidine" refers to -C(NR ⁴⁷ )NR ⁴⁸ R ⁴⁹ and -NR ⁴⁷ C(NR ⁴⁸ )R ⁴⁹ , wherein R ⁴⁷ , R ⁴⁸ , and R ⁴⁹ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0052] The term "guanidine" refers to -NR ⁵⁰ C(NR ⁵¹ )NR ⁵² R ⁵³ , wherein R ⁵⁰ , R ⁵¹ , R ⁵² and R ⁵³ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0053] The term "enamine" refers to -C(R ⁵⁴ )=C(R ⁵⁵ )NR ⁵⁶ R ⁵⁷

and -NR ⁵⁴ C(R ⁵⁵ )=C(R ⁵⁶ )R ⁵⁷ , wherein R ⁵⁴ , R ⁵⁵ , R ⁵⁶ and R ⁵⁷ are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0054] The term "halogen" or "halo" as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0055] The term "hydroxy' as used herein can refer to -OH or its ionized form, -O ^" .

[0056] The term "imide" refers to -C(0)NR ⁵⁸ C(0)R ⁵⁹ , wherein R ⁵⁸ and R ⁵⁹ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0057] The term "imine" refers to -CR ⁶⁰ (NR ⁶¹ ) and -N(CR ⁶⁰ R ⁶¹ ) groups, wherein R ⁶⁰ and R ⁶¹ are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R ⁶⁰ and R ⁶¹ are not both simultaneously hydrogen.

[0058] The term "phosphate" as used herein refers to -OP(O)(OR ⁶⁰ )(OR ⁶¹ ) groups, wherein R ⁶⁰ and R ⁶¹ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0059] The term "azide" (or "azido") as used herein refers to

[0060] The term "biotin" when referenced as a substituent group herein is a monovalent radical of biotin or a derivative thereof. Biotin derivatives are compounds which include the tetrahydrothienoimidazole ring system of biotin and still bind the biotin receptor with a sub-micromolar binding constant. Such compounds may be, e.g., homologs with a shorter or longer alkyl carboxylic acid chain, and/or may include one or two additional functional groups or heteroatoms such as O, N or S, and/or may lack one or more atoms, especially heteroatoms. Thus biotin derivatives include, but are not limited to, norbiotin, oxybiotin, iminobiotin, desthiobiotin, diaminobiotin, biotin sulfoxide, and biotin sulfone. In some embodiments, the monovalent radical of biotin is that obtained by removal of the H atom from the carboxylic acid group of biotin. In other embodiments, the monovalent radical of biotin is that obtained by removal of the OH radical from the carboxylic acid group of biotin.

[0061] The term "folic acid" when referenced as a substituent group herein is a monovalent radical of folic acid or a derivative thereof. Folic acid derivatives are compounds which include the pteroyl ring system or a reduced analog thereof and still bind a folate receptor with a sub-micromolar binding constant. Such compounds may be, e.g., homologs with a shorter or longer alkyl carboxylic acid chain, and/or may include one or two additional functional groups or heteroatoms such as O, N or S, and/or may lack one or more atoms, especially heteroatoms. In some embodiments, the monovalent radical of biotin is that obtained by removal of the H atom from the carboxylic acid group of biotin. In other embodiments, the monovalent radical of biotin is that obtained by removal of the OH radical from the carboxylic acid group of biotin.

[0062] The term "dye" as used herein refers to any reporter group whose presence can be detected by its light absorbing or light emitting properties. The term includes, but is not limited to, chromaphores and fluorophores. A dye may be synthesized with reactive groups so that it can be chemically linked to a compound of the present technology. Labeling of the present compounds with dyes may be done for visualization and quantification purposes. Examples of suitable dyes include, but are not limited to, fluorophores such as fluorosceins (e.g., fluorescein isothiocyanate ("FITC"), carboxyfluorosceins, etc.), cyanines (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5 Cy7, Cy7.5, etc.), rhodamines (e.g., rhodamine B, rhodamine 6G, rhodamine 123, sulforhodamine 101, etc.), xanthenes, azo dyes, coumarins, fluorones, fluorenes, boron-dyprromethenes (e.g., BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, etc.) and the like, as well as derivatives thereof.

[0063] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and are not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group,

pharmaceutically acceptable salts can be formed with inorganic acids (e.g., hydrochloric acid, organic acids (e.g., acetic acid benzoic acid, or acidic amino acids (e.g., aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as, for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals, ammonia or organic amines (e.g., dicyclohexylamine, or basic amino acids (e.g., arginine and lysine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0064] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and/or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and/or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms. [0065] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, imidazoles may exhibit the following isomeric forms, which are referred to as tautomers of each other:

[0066] As readily understood by one skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of compounds as described herein are within the scope of the present technology.

[0067] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds of the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0068] A "compound" or "derivative" as used herein refers to a chemical compound, either in partially purified or substantially pure form, which has been produced by chemical synthesis from any desired starting materials. A compound or derivative according to present technology can be used either as a racemic mixture, a non-racemic mixture, or a pure or essentially pure stereoisomer. Preferred are pure or essentially pure stereoisomers which have activity in the treatment of cancers such as leukemia, ovarian, breast, prostate, liver, pancreatic, and colon cancers, or which have activity in inhibiting cancer cell proliferation and tumor angiogenesis, or in enhancing apoptosis. [0069] A "partially purified" (or "crude") compound or derivative as used herein refers to a compound or derivative thereof which is present in a chemical mixture that has been subjected to at least one separation or purification step resulting in the removal of at least one other chemical substance originally present in the initial extract or synthetic mixture containing the compound or derivative. A "substantially pure" compound or derivative is one which has been separated or purified to render the compound or derivative as the major chemical component of the substantially pure compound or derivative, i.e., comprising at least 50%, or in some embodiments at least 70%, at least 90%, or at least 95% or 99% on a molar basis.

[0070] "Treating" or "treatment" within the context of the present technology, means an alleviation, in whole or in part, of symptoms associated with a disorder or disease, or slowing, or halting of further progression or worsening of those symptoms. As a non-limiting example of treatment, a subject can be successfully treated for colon cancer if, after receiving through administration an effective or therapeutically effective amount of one or more compounds or compositions described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of colon cancer such as, but not limited to, reduced tumor size (e.g., reduced tumor volume), reduced morbidity and mortality, or improvement in quality of life relating thereto.

[0071] "Effective amount" refers to the amount of a compound or composition required to produce a desired effect. Hence, a "therapeutically effective amount" of a compound or composition of the present technology in the context of treatment refers to an amount of the compound or composition that alleviates, in whole or in part, symptoms associated with a disorder or disease, or slows or halts further progression or worsening of those symptoms. In the context of prevention, a therapeutically effective amount prevents or provides prophylaxis for the disease or disorder in a subject at risk for developing the disease or disorder. Determining a therapeutically effective amount of a compound described herein for treating a particular disorder or disease is well within the skill in the art in view of the present disclosure. In the case of colon or pancreatic cancer, the effective amount of the compound or composition may reduce the number of cancer cells; reduce the tumor size; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis. For the treatment of tumor dormancy or micrometastases, the therapeutically effective amount of the compound or composition may reduce the number or proliferation of micrometastases; reduce or prevent the growth of a dormant tumor; or reduce or prevent the recurrence of a tumor after treatment or removal (e.g., using an anti -cancer therapy such as surgery, radiation therapy, or chemotherapy). To the extent the compound or composition may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, disease free survival (DFS), time to disease progression (TTP), duration of progression free survival (PFS), the response rates (RR), duration of response, time in remission, and/or quality of life. The effective amount may improve disease free survival (DFS), improve overall survival (OS), decrease likelihood of recurrence, extend time to recurrence, extend time to distant recurrence (i.e., recurrence outside of the primary site), cure cancer, improve symptoms of cancer (e.g., as gauged using a cancer specific survey), reduce appearance of second primary cancer, etc.

[0072] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or

micrometastatses. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0073] The term "metastasis" refers to the spread of cancer from its primary site to other places in the body. Cancer cells can metastasize by breaking away from a primary tumor, penetrating into lymphatic and blood vessels, circulating through the lymphatic system or bloodstream, exiting the lymph or blood vessels and attaching and growing in normal tissues elsewhere in the body. Metastasis can be local or distant. At the new site, the cells reproduce, may establish a blood supply and can grow to form a secondary tumor, i.e., a metastatic tumor. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

[0074] The term "micrometastasis" refers to a small number of cells that have spread from the primary tumor to other parts of the body. Micrometastasis may or may not be detected in a screening or diagnostic test.

[0075] The term "apoptosis" refers to programmed cell death as signaled by the nuclei in normally functioning human and animal cells when age or state of cell health and condition dictates. "Apoptosis" is an active process requiring metabolic activity by the dying cell, often characterized by cleavage of the DNA into fragments that give a so called laddering pattern on gels. Cells that die by apoptosis do not usually elicit the inflammatory responses that are associated with necrosis, though the reasons are not clear. Cancerous cells, however, are unable to experience, or have a reduction in, the normal cell transduction or apoptosis-driven natural cell death process. Morphologically, apoptosis is characterized by loss of contact with neighboring cells, concentration of cytoplasm, endonuclease activity- associated chromatin condensation and pyknosis, and segmentation of the nucleus, among others. [0076] The term "enhancing apoptosis" refers to increasing the number of cells that undergo apoptosis, or the rate by which cells undergo apoptosis, in a given cell population. Preferably the increase is at least 1.25, 1.5, 2, 5, 10, 50, 100, 500 or 1000 fold increase as compared to normal, untreated or negative control cells.

[0077] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0078] The term "tumor angiogenesis" refers to the proliferation of a network of blood vessels that penetrate into tumors, including cancerous and pre-cancerous tumors, in order to supply nutrients and oxygen and remove waste products, thus leading to tumor growth. Tumor angiogenesis involves hormonal stimulation and activation of oncogenes, expression of angiogenic growth factors, extravasation of plasma protein, deposition of a protein extracellular matrix (ECM), degradation of ECM, and migration, proliferation, and elongation of endothelial capillaries.

[0079] The term "inhibiting tumor angiogenesis" includes inhibiting tumor vasculogenesis, and refers to causing a decrease in the extent, amount, or rate of

neovascularization of a tumor, for example by decreasing the extent, amount, or rate of endothelial cell proliferation or migration in/by a tumor.

[0080] The term "subject" or "patient" refers to a mammal, such as a cat, dog, rodent or primate. Typically the subject is a human, and, preferably, a human having or suspected of having a cancer such as pancreatic and/or colon cancer. The term "subject" and "patient" can be used interchangeably.

[0081] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0082] As shown below, triptolide is a diterpernoid possessing three epoxides and an unsaturated butyrolactone (butenolide) ring. The numbering system shown below will be used herein for triptolide as well as compounds related thereto.

Triptolide

[0083] Epoxide-containing compounds are generally recognized to be toxic to a variety of living organisms, including animals and bacteria. The toxicity of epoxide- containing compounds may be due to a number of factors, including the ability for epoxides to irreversibly alkylate proteins, DNA, and/or RNA. Generally, molecules which possess multiple epoxide groups are more toxic than corresponding molecules with a single epoxide group. In the case of triptolide, cDNA microarray analysis of Jurkat T cells incubated with 10 mg/L of triptolide for 2 hours indicated that 171 genes were suppressed by 5- to > 100-fold (30% genes without known function, 13% transcription factors, 9% signal transduction pathway regulators, and 9% DNA binding proteins. (Wang et al, Acta Pharmacol. Sin. 2003, 24, 864). The multiple epoxide functionality present on triptolide is thought to contribute to both to its therapeutic and toxic effects.

[0084] In various aspects, the present technology provides novel polycyclic epoxides, pharmaceutical compositions containing such compounds, and methods of using the compounds and compositions in anti-cancer treatments. In particular the present compounds are useful in treating leukemia, ovarian, breast, prostate, liver, pancreatic, and colon cancers, especially for colon cancer, as well as for inhibiting cancer cell proliferation and tumor angiogenesis. Advantageously, certain poly cyclic epoxides disclosed herein are less cytotoxic than triptolide and exhibit an improved therapeutic index compared to triptolide. Certain of the present compounds also exhibit more potent activity than triptolide against some types of cancer.

[0085] In one aspect, the present technology provides compounds represented by

Formula I, as well as stereoisomers thereof, tautomers thereof, prodrugs thereof, and pharmaceutically acceptable salts of any of the foregoing,

I

wherein

Ri is -H or a substituted or unsubstituted alkyl group;

R ₂ and R3 are each independently -H or a substituted or unsubstituted alkyl group; R _t is -H or a C ₁ -60 substituted or unsubstituted alkyl or heteroalkyl group which optionally comprises a substituted or unsubstituted cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl or aryl group;

R ₅ is -OR';

R5 is -H or -OR', or R5 and R5 together are =0;

R7 is -H or an unsubstituted alkyl group;

each R' is independently -H, a hydroxyl protecting group, -CO ₂ R", -C(0)R", or a substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or heterocyclylalkyl group; and each R" is independently a substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, or heterocyclylalkyl group.

[0086] In some embodiments of the compounds of Formula I, when R ₅ is OH and Re is -H, Rt is not an isopropyl group or Rt is not an unsubstituted or mono-hydroxyl-substituted isopropyl group. For example, in some embodiments, Rt is not 1-hydroxyisopropyl.

[0087] In some embodiments of compounds of Formula I, Ri is methyl. In certain embodiments of Formula I, R2 is -H. In some embodiments of Formula I, R ₃ is -H. In certain embodiments of Formula I, R7 is -H.

[0088] In some embodiments of compounds of Formula I, R5 and Re, together are =0.

In certain embodiments, R5 is -OH or -OR' and Re is -H. In some embodiments of compounds of Formula I, each R' is independently -H, -CO2R", -C(0)R", or a substituted or unsubstituted alkyl group.

[0089] In some embodiments of compounds of Formula I, Ri, R2, R ₃ , and R ₇ are each

-H; R ₅ and Re are together are =0, or Re is -H and R5 is -OH or -OR20; and R4 is a C1-60 substituted or unsubstituted alkyl or heteroalkyl group which optionally comprises a substituted or unsubstituted cycloalkyl, cycloalkylalkyl, or heterocyclyl group, wherein R20 is a hydroxyl protecting group or substituted or unsubstituted alkyl group.

[0090] In some embodiments of compounds of Formula I, R4 is a substituted or unsubstituted Ci-26 alkyl or heteroalkyl group, a substituted or unsubstituted Ci _-7 alkyl or hydroxyalkyl, or a substituted C4-26 heteroalkyl group. In some embodiments, R ₄ is a C4-26 heteroalkyl group substituted with 1, 2 or 3 groups selected from heterocyclyl,

heterocyclylalkyl, or heteroaryl groups. In other embodiments, R ₄ is -H, an unsubstituted Ci_ 7 alkyl or C4-8 cycloalkylalkyl group, or a Ci _-7 alkyl group substitututed with a substituent selected from a hydroxy, carboylic acid, ester, amide, urethane, azide, epoxy, or dialkylamino group. In certain embodiments, R ₄ is -H, or an isopropyl, hydroxyethyl, hydoxypropyl, hydroxybutyl, 1,3-dihydroxyisopropyl, epoxyethyl, or cyclopropylmethyl group. [0091] In some embodiments of compounds of Formula I, R4 has the Formula, -L-Q, wherein L is a linker selected from a substituted or unsubstituted alkylene, poly(oxyalkylene), or piperazinyl group, or a combination of any two more more thereof; and Q is selected from a substituted or unsubstituted alkyl, alkynyl, heterocycloalkyl group, an azide, alkylazide, araylazide, biotin, or dye. In some embodiments Q is a dye, and the dye is selected from the group consisting of Cy3-cyanine, rhodamine, fluorescein, and boron-dipyrromethene (BODIPY).

[0092] In some embodiments of compounds of Formula I, wherein L is selected from an alkylene, poly (oxy alkylene), -alkylene-OC(0)NR'-poly(oxyalkylene)-CH ₂ CH ₂ NR'- , -alkylene-OC(0)NR'-poly(oxyalkylene)-CH2CH ₂ NR'-alkylene-, -alkylene-OC(O)- , -alkylene-NHC(O)-, -alkylene-C(0)NH-, -alkylene-OC(0)-piperazinyl- group, an amino acid, or combination of any two or more thereof.

[0093] In some embodiments of compounds of Formula I, Q is selected from the group consisting of a carboxylic acid, ester, azide, alkylazide, araylazide, dye, biotin, folic acid and amine group.

[0094] In some embodiments, L is an amino acid selected from the group consisting of lysine, aspartic acid, and glutamic acid. In other embodiments of compounds of Formula I, L is -Ci-5-alkylene-0(CO)NH-. In still other embodiments, L is -Ci-5-alkylene-OC(0)- piperazin-yl-.

[0095] In some embodiments, the compounds represented by Formula I are compounds

Formula IA Formula IB Formula IC [0096] In one aspect, the present technology provides conjugates of the present compounds of Formula I to target and deliver the compounds to cancer cells. For example, folic acid may be conjugated to compounds of Formula I, including compounds of formula IA, IB, and IC, as part of variable R4. The folic acid may be attached to the compound through one or more of the available carboxylic acid groups. In other embodiments, monoclonal antibodies designed to target specific types of cancer cells, may be conjugated to a suitable functional group in R4 such as a carboxylic acid, amine, hydroxyl or thiol group using methods well known to those skilled in the art.

[0097] In another aspect, the present technology provides pharmaceutical compositions for medical treatment. Each pharmaceutical composition includes a compound of Formula I, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a

pharmaceutically acceptable salt of any of the foregoing, and pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions may include an effective amount of a compound of Formula I, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing, for treating leukemia, ovarian, breast, prostate, liver, and/or colon cancers, and/or inhibiting cancer cell proliferation and tumor angiogenesis, as well as promoting apoptosis. In some embodiments, the pharmaceutical composition includes a conjugate of a compound of Formula I (a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing) with folic acid or a monoclonal antibod that binds to the cancer cells.

[0098] The pharmaceutical compositions may be prepared by mixing one or more compounds of the present technology, stereoisomers thereof, tautomers thereof, prodrugs thereof, or pharmaceutically acceptable salts of any of the foregoing, with pharmaceutically acceptable carriers, excipients, binders, diluents or the like to prevent and treat disorders associated with the effects of cancers such as breast, ovarian, prostate, liver, or colon cancer, or the effects associated with tumor growth and/or angiogenesis. The compounds and compositions described herein may be used to prepare formulations and medicaments that prevent or treat a variety of disorders associated with pancreatic cancer and colon cancer. Such compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The instant compositions can be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or via implanted reservoir.

Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injections. The following dosage forms are given by way of example and should not be construed as limiting the present technology.

[0099] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the present technology, or stereoisomers, tautomers, prodrugs and/or pharmaceutically acceptable salts thereof, with at least one additive such as a starch or other additive. Suitable additives include, but are not limited to, sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0100] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. Pharmaceutical formulations and medicaments may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, alcohol, and combinations of these. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or parenteral administration.

[0101] As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.

[0102] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. Typically, the oil or fatty acid is nonvolatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

[0103] For injection, the pharmaceutical formulation and/or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

[0104] Compounds of the present technology may be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation include solutions, sprays, dry powders, or aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant) aerosols are typically used for delivery of the present compounds by inhalation.

[0105] Dosage forms for the topical (including buccal and sublingual) or transdermal administration of compounds of the present technology include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier or excipient, and with any preservatives, or buffers, which may be required. Powders and sprays can be prepared, for example, with excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Absorption enhancers can also be used to increase the flux of the inventive compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane (e.g., as part of a transdermal patch) or dispersing the compound in a polymer matrix or gel.

[0106] Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present technology. Such excipients and carriers are described, for example, in "Remingtons Pharmaceutical Sciences" Mack Pub. Co., New Jersey (1991), which is hereby incorporated by reference in its entirety for all purposes as if fully set forth herein.

[0107] The formulations of the present technology may be designed to be short- acting, fast-releasing, long-acting, and sustained-releasing as described below. Thus, the pharmaceutical formulations may also be formulated for controlled release or for slow release.

[0108] The instant compositions may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect. Therefore, the pharmaceutical formulations and medicaments may be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may employ known inert materials such as silicones and biodegradable polymers.

[0109] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the present technology.

[0110] Those skilled in the art are readily able to determine an effective amount by simply administering a compound of the present technology to a patient in increasing amounts until the progression of the disease state is decreased or stopped. The progression of the disease state can be assessed using in vivo imaging, as described, or by taking a tissue sample from a patient and observing the target of interest therein. The compounds of the present technology can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kg of body weight per day is sufficient. The specific dosage used, however, can vary or may be adjusted as considered appropriate by those of ordinary skill in the art. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to those skilled in the art.

[0111] In another aspect, the present technology provides a method of treatment comprising administering to a subject having leukemia, ovarian, breast, prostate, liver, or colon cancer a therapeutically effective amount of a compound of Formula I (or a conjugate thereof that binds to the cancer cells), a stereoisomer thereof, a tautomer thereof, a prodrug thereof, a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition comprising a compound of Formula I or a conjugate thereof that binds cancer cells, a stereoisomer thereof, a tautomer thereof, a prodrug thereof, a pharmaceutically acceptable salt of any of the foregoing, and a pharmaceutically acceptable carrier.

[0112] In another aspect, the present technology provides a method of inhibiting cancer cell proliferation, tumor angiogenesis, or promoting apoptosis, as well as for treating leukemia, ovarian, breast, prostate, liver, or colon cancer in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a compound of Formula I (or a conjugate thereof that binds to the cancer cells), a stereoisomer thereof, a tautomer thereof, a prodrug thereof, a pharmaceutically acceptable salt of any of the foregoing, or a pharmaceutical composition comprising a compound of Formula I (or a conjugate thereof that binds to the cancer cells), a stereoisomer thereof, a tautomer thereof, a prodrug thereof, a pharmaceutically acceptable salt of any of the foregoing, and a pharmaceutically acceptable carrier.

[0113] The compounds of the present technology can also be administered to a patient along with other conventional therapeutic agents that may be useful in the treatment or cancers such as leukemia, ovarian, breast, prostate, liver, or colon cancer, or that may be useful in inhibiting tumor angiogenesis or enhancing apoptosis in such cancers. In one aspect, a method is provided for administering a therapeutically effective amount of one or more compounds of the present technology or a monoclonal antibody conjugate thereof (that binds to the cancer cells), a stereoisomer thereof, a tautomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt of any of the foregoing, to a subject suffering from cancer, wherein the cancer is leukemia, ovarian, breast, prostate, liver, or colon cancer. Moreover, the present technology relates to inhibiting cancer cell proliferation and tumor angiogenesis, orenhancing apoptosis in such cancers. Thus, in another aspect, a method is provided for administering a therapeutically effective amount of one or more compounds of the present technology or a conjugate thereof, or stereoisomers, tautomers, prodrugs, and/or

pharmaceutically acceptable salts thereof to a subject in need thereof. The methods of the present technology can include administering, either sequentially or in combination with one or more compounds of the present technology, a conventional therapeutic agent in an amount that can potentially or synergistically be effective for the treatment of cancers, such as leukemia, ovarian, breast, prostate, liver, or colon cancer. The methods of the present technology can also include administering, either sequentially or in combination with one or more compounds of the present technology or a monoclonal antibody conjugate thereof (that binde to the cancer cells), a conventional therapeutic agent in an amount that can potentially or synergistically be effective for inhibiting tumor angiogenesis or enhancing apoptosis. Exemplary therapeutic agents for use in combination therapies with one or more compounds of the present technology include, but are not limited to, chemotherapeutic agents such as 5- fluorouracil and Irinotecan (CPT-11).

[0114] Additionally or alternatively, the compounds or conjugates of the present technology (or stereoisomers, tautomers, prodrugs, or pharmaceutically acceptable salts thereof) or compositions of the present technology, may also be administered to a subject receiving radiation therapy. By "radiation therapy" is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.

[0115] In one aspect, a compound of the present technology or a conjugate thereof (or stereoisomers, tautomers, prodrugs, or pharmaceutically acceptable salts thereof) is administered to a patient in an amount or dosage suitable for therapeutic use. Generally, a unit dosage comprising a compound of the present technology will vary depending on patient considerations. Such considerations include, for example, age, protocol, condition, sex, extent of disease, contraindications, concomitant therapies and the like. An exemplary unit dosage based on these considerations can also be adjusted or modified by a physician skilled in the art. For example, a unit dosage for a patient comprising a compound of the present technology can vary from 0.1 mg/kg to lg/kg, preferably, 1 mg/kg to 1 g/kg. Dosage of a compound of the present technology can also vary from 0.01 mg/kg to 100 mg/kg or, preferably, from 0.1 mg/kg to 10 mg/kg.

[0116] Useful adjunctive therapeutic agents in combinatorial formulations and coordinate treatment methods include, for example, one or more chemotherapeutic agents. As used herein, the term "chemotherapeutic agent" or "chemotherapy agent" or

"chemotherapeutic drug" refer to an agent that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically-effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease. Chemotherapeutic agents include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and

methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); 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, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related

chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate 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; podophyllinic acid; 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;

dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;

GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid;

capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (<?.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0117] Also included in the definition of "chemotherapeutic agent" are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti -estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen

(including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON'toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0118] A compound of the present technology can bind to one or more targets of interest with a dissociation constant (for example, an equilibrium dissociation constant, ¾) from, for example, about 0.0001 to 10 μΜ (or from 0.0001 to 7 μΜ, 0.0001 to 5 μΜ, 0.0001 to 1 μΜ, 0.001 to 5 μΜ, 0.01 to 5μΜ and/or 0.1 to 5 μΜ) as measured by any suitable techniques routine to those of ordinary skill in the art. The present technology contemplates measurement of a dissociation constant (for example, ¾ and Κ;) or performing competition, saturation and kinetics experiments by conventional techniques routine to one of ordinary skill in the art. Moreover, a compound of the present technology can compete with a reference compound for binding to and/or with targets of interest with a dissociation constant of inhibition (for example, Κ;) from, for example, about 0.01 nM to > 10,000 nM (or from 0.001 to 7,000 nM, 0.001 to 5,000 nM, 0.001 to 1,000 nM, 0.01 to 5,000 nM, 0.01 to 2,000 nM and/or 0.1 to 5,000 nM).

[0119] A compound or probe of the present technology can bind to one or more targets of interest with a dissociation constant (for example, an equilibrium dissociation constant, ¾) from, for example, about 0.0001 to 10 μΜ as measured by binding to a synthetic peptide or tissue associated with a target of interest. The present technology contemplates measurement of a dissociation constant (for example, ¾ and Κ;) or performing competition, saturation and kinetics experiments by conventional techniques routine to one of ordinary skill in the art. Moreover, a compound or probe of the present technology can compete with a reference compound for binding to a target of interest with a dissociation constant of inhibition (for example, Κ;) from, for example, about 0.01 nM to >10,000 nM.

[0120] A compound of the present technology can interact with cancer or other cells with a IC50 constant (the concentration at which 50% cells were viable) from, for example, about 0.0001 to 10 μΜ (or from 0.0001 to 7 μΜ, 0.0001 to 5 μΜ, 0.0001 to 1 μΜ, 0.001 to 5 μΜ, 0.01 to 5μΜ and/or 0.1 to 5 μΜ) as measured by any suitable techniques routine to those of ordinary skill in the art.

[0121] In one aspect, binding, interaction or association with can mean the contact between a compound (stereoisomers thereof, tautomers thereof, prodrug, and

pharmaceutically acceptable salts of any of the foregoing) or conjugate thereof and a target of interest with a binding affinity of at least lO ^-6 M, preferably, at least about 10 ^~7 M, and more preferably 10 ^~8 M to 10 ^~9 M, 10 ^~10 M, 10 ^"11 M, or 10 ^~12 M. In one aspect, binding affinities include those with a dissociation constant or ¾ less than, but not limited to, 5 ^χ 10 ^~6 M, 10 ^~6 M, 5 10 ^~7 M, 10 ^~7 M, 5 χ 10 ^~8 M, 10 ^~8 M, 5 χ 10 ^~9 M, 10 ^~9 M, 5 χ 10 ^~10 M, 10 ^~10 M, 5 χ 10 ^{" 11} M, 10 ^_11 M, 5 x 10 ^~12 M, 10 ^~12 M, 5 χ 10 ^~13 M, 10 ^~13 M, 5 χ 10 ^~14 M, 10 ^~14 M, 5 χ 10 ^~15 M, and 10 ^~15 M.

[0122] A compound of the present technology can also be modified, for example, by the covalent attachment of an organic moiety or conjugate to improve pharmacokinetic properties, toxicity or bioavailability (e.g., increased in vivo half-life). The conjugate can be a linear or branched hydrophilic polymeric group, fatty acid group or fatty acid ester group. A polymeric group can comprise a molecular weight that can be adjusted by one of ordinary skill in the art to improve, for example, pharmacokinetic properties, toxicity or

bioavailability. Exemplary conjugates can include a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrrolidone and a fatty acid or fatty acid ester group, each of which can independently comprise from about eight to about seventy carbon atoms. Conjugates for use with a compound of the present technology can also serve as linkers to, for example, any suitable substituents or groups, radiolabels (marker or tags), halogens, proteins, enzymes, polypeptides, other therapeutic agents (for example, a pharmaceutical or drug), nucleosides, dyes, oligonucleotides, lipids, phospholipids and/or liposomes. In one aspect, conjugates can include polyethylene amine (PEI), polyglycine, hybrids of PEI and polyglycine, polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). A conjugate can also link a compound of the present technology, for example, a label (fluorescent or luminescent) or marker (radionuclide, radioisotope and/or isotope) to comprise a probe of the technology. Conjugates for use with a compound of the present technology can, in one aspect, improve in vivo half-life. Other exemplary conjugates for use with a compound of the present technology as well as applications thereof and related techniques include those generally described by U.S. Patent No. 5,672,662, which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0123] Similarly, a compound of the present technology may be a prodrug. The term

"prodrug" or "prodrugs" is also meant to include any covalently bonded carriers that release the compound of the present technology in vivo when such prodrug is administered to a subject. A prodrug includes a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the present technology. Thus, the term "prodrug" includes a metabolic precursor of a compound of the present technology that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the present technology. Prodrugs are typically transformed in vivo to yield the parent compound of the present technology, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). Prodrugs of a compound of the present technology may be prepared by modifying functional groups present in the compound of the present technology in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the present technology. Prodrugs include compounds of the present technology wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the present technology is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present technology and the like.

[0124] In another aspect, the present technology provides methods of identifying a target of interest including contacting the target of interest with a detectable or imaging effective quantity of a labeled compound of the present technology. A detectable or imaging effective quantity is a quantity of a labeled compound of the present technology necessary to be detected by the detection method chosen. For example, a detectable quantity can be an administered amount sufficient to enable detection of binding of the labeled compound to a target of interest including, but not limited to, one or more cellular proteins. Suitable labels are known by those skilled in the art and can include, for example, radioisotopes, radionuclides, isotopes, fluorescent groups, biotin (in conjunction with streptavidin complexation), and chemoluminescent groups. Upon binding of the labeled compound to the target of interest, the target may be isolated, purified and further characterized such as by determining the amino acid sequence.

[0125] The terms "associated" and/or "binding" can mean a chemical or physical interaction, for example, between a compound of the present technology and a target of interest. Examples of associations or interactions include covalent bonds, ionic bonds, hydrophilic-hydrophilic interactions, hydrophobic-hydrophobic interactions and complexes. Associated can also refer generally to "binding" or "affinity" as each can be used to describe various chemical or physical interactions. Measuring binding or affinity is also routine to those skilled in the art. For example, compounds of the present technology can bind to or interact with a target of interest or precursors, portions, fragments and peptides thereof and/or their deposits.

[0126] The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compounds of the present technology or salts, pharmaceutical compositions, derivatives, metabolites, prodrugs, racemic or other stereoisomeric mixtures, or tautomeric forms thereof. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or aspects of the present technology as described above. The variations, aspects or aspects described above may also further each include or incorporate the variations of any or all other variations, aspects or aspects of the present technology.

EXAMPLES Example 1: Preparation of Compounds Scheme 1 below summarizes a general synthetic route to Biotin-TL and Cy3-

[0128] Reagents and conditions: a) cerium(VI) ammonium nitrate, MeCN, 0 °C, 99

%; b) IBX, acetone, reflux, 98 %; c) A1C1 ₃ , MeCN, reflux, 100 %; d) Sc(OTf) ₃ , NIS, AcOH, rt, 82 %; e) THPOCH ₂ C≡CH, PdCl ₂ (PPh ₃ ) ₂ , Cul, Et ₃ N. THF, 35 °C, 97 %; f) H ₂ . Pd/C, EtOH, 40-50 °C, 100 %; g) TsOH H ₂ 0, MeOH, rt, 95 %; h) NaBH ₄ , EtOH, CH ₂ C1 ₂ , 0 °C, 99 %; i) NaI0 ₄ , Me OH, H20, rt, 80 %; j) CF ₃ COCH ₃ , OXONE, NaHC0 ₃ , CH ₃ CN, Na ₂ (EDTA), 0 °C; k) /?-nitrophenylchloroformate, pyridine, CH ₂ CI ₂ , rt, the yield of 14 was 13% from 10; 1) DMAP, Et ₃ N, CH ₂ Cl ₂ /MeOH, rt, 24 %; m) K-Selectride, THF, 40 °C, 39 %. n) DMAP, ET ₃ N, CH ₂ Cl ₂ /MeOH, rt, 85%; o) K-Selectride, anhydrous THF, -78 °C; p) 30% TFA in CH ₂ C1 ₂ (v/v), rt; q) Cy3-NHS, CH ₂ C1 ₂ , DMAP, rt, 42%.

Preparation of compound 2:

[0129] Compound 1 was synthesized in accordance with or by slight modification of the method set forth in Gao, Q.; Xue, J.; Zheng, B.; Liu, R.; (Shanghai Haoyuan Chemexpress Co., Ltd., PR. China). Application: CN101638426 (2010) p. 13. To a suspension of 1 (10.0 g, 35.2 mmol) in CH ₃ CN (250 mL) was added eerie ammonium nitrate (CAN) solution (40.5 g in 25 mL water) dropwise at 0 °C over a period of 30 min. The resulting mixture turned transparent red, and was stirred for 30 min and monitored by TLC (EtOAc: «-hexane =3:7). When the starting material was consumed and the mixture turned yellow, Na ₂ S0 ₃ solution (102 g Na ₂ S0 ₃ in 300 mL H ₂ 0) was added. The mixture was stirred vigorously for 10 min and filtered through sand funnel. The filtrate was extracted with CH ₂ C1 ₂ (300 mL ^χ 3), and the solid was washed with CH ₂ C1 ₂ (200 mL ^χ 3). The combined organic layer was dried over anhydrous Na ₂ SC>4 and was filtered via short pad silica gel. After the solvent was removed by evaporation, (10.5 g, 99%) of yellow powder 2 was obtained, which was used directly in the next step without further purification.

[0130] Compound 2, analytical TLC (silica gel 60), 40% EtOAc in «-hexane, Rf =

0.16; i NMR ^OO MHz) 7.30 (t, / = 8.0 Hz, 1H), 7.04 (d, / = 8.2 Hz, 1H), 6.83 (d, / = 8.1 Hz, 1H), 5.13 (t, / = 2.3 Hz, 1H), 4.80 (AB system, 2H), 3.88 (s, 3H), 3.17 - 3.12 (m, 1H), 2.54 - 2.40 (m, 3H), 2.11 - 2.05 (m, 2H), 1.74 - 1.72 (m, 1H), 1.00 (s, 3H); ¹³ C NMR (100 MHz) δ 174.0, 163.1, 157.9, 146.3, 129.0, 125.2, 125.1, 117.1, 108.3, 70.5, 62.2, 55.5, 36.8, 36.7, 32.6, 27.9, 21.9, 18.2; IR (CH ₂ C1 ₂ ) 1757, 1682 cm ^"1 . LRMS (EI, 20 eV) m/z 300.2 (M ⁺ , 4), 267.1 (100); HRMS (EI) for Ci ₈ H ₂₀ O ₄ (M ⁺ ): calcd 300.1356, found 300.1353. Preparation of compound 3:

[0131] Compound 3 was prepared in accordance with or by slight modification of the method set forth in Gao, Q.; Xue, J.; Zheng, B.; Liu, R.; (Shanghai Haoyuan Chemexpress Co., Ltd., PR. China), CN101638426 (2010) p 13. Compound 2 prepared in the previous step (10.5 g, 35.0 mmol, crude), 2-iodoxybenzoic acid (IBX, 14.8 g, 52.8 mmol) and acetone (120 mL) was heated under reflux. The reaction was monitored by TLC (EtOAc: «-hexane =3 :7 twice). After 6 h, the mixture was cooled and the solid was removed by filtration and washed with CH ₂ CI ₂ (100 mL). The combined organic layer was dried over anhydrous NaiSOzj. After solvent removal, crude compound 3 ( 10.2 g, 98%) was obtained and was directly used for next step without further purification. The crude compound was a grey solid.

[0132] Compound 3, analytical TLC (silica gel 60), «-Hexane:CH ₂ Cl ₂ :EtOAc = 3: 1 : 1,

Rf = 0.1; ¾ NMR (300 MHz) S T.51 (t, / = 8.2 Hz, 1H), 7.05 (d, / = 7.7 Hz, 1H), 6.95 (d, / = 8.4 Hz, 1H), 4.70 (br s, 2H), 3.93 (s, 3H), 3.15 - 3.09 (m, 1H), 2.81 (dd, / = 6.0, 18.2 Hz, 1H), 2.63 (dd, / = 18.2, 13.7 Hz, 1H), 2.64 - 2.55 (m, 1H), 2.54 - 2.50 (m, 1H), 2.50 - 2.28 (m, 1H), 1.84 (td, / = 12.5, 7.3 Hz, 1H), 1.13 (s, 3H); ¹³ C NMR (75 MHz) δ 194.6, 173.4, 160.5, 160.1, 153.5, 134.8, 125.8, 121.1, 1 15.2, 110.9, 70.1, 56.2, 39.7, 37.8, 36.9, 32.0, 21.4, 17.8; IR (CH ₂ CI ₂ ) 1757, 1682 cm ^"1 ; LRMS (EI, 20 eV) m/z 298 (M ⁺ , 100), 265 (27), 175 (60); HRMS (EI) calcd. for Ci ₈ H ₁₈ 0 ₄ (M ⁺ ) m/z 298.1205, found 298.1215.

[0133] Preparation of compound 4:

[0134] Compound 4 was synthesized in accordance with or by slight modification of the method set forth in Gao, Q.; Xue, J.; Zheng, B.; Liu, R.; (Shanghai Haoyuan Chemexpress Co., Ltd., PR. China), CN101638426 (2010) p 13. To a solution of compound 3 (10.2 g, 34.2 mmol, crude) in redistilled CH ₃ CN (60 mL) was added anhydrous A1C1 ₃ (14.8 g, 105.6 mmol). The mixture was heated to reflux, and the reaction was monitored by TLC (EtOAc:«-hexane = 4:6). After 12 h, 1 M HC1 (50 mL) solution was added to the reaction mixture while stirring. The mixture was extracted with CH ₂ CI ₂ (300 mL ^χ 3), and the organic layer was filtered via short pad of silica gel. After the solvent was removed by evaporation, yellow solid 4 (8.9 g, 100%) was obtained, which was used in the next step without further purification. [0135] Compound 4, analytical TLC (silica gel 60), «-Hexane: EtOAc :0¾θ2 =

1: 1 : 1, R _f = 0.5; ^ NMR ^OO MHz) 12.61 (s, 1H), 7.49 (t, / = 8.1 Hz, 1H), 6.92 (t, / = 7.6 Hz, 2H), 4.85 - 4.71 (m, 2H), 3.25 - 3.21 (m, 1H), 2.81 (d, / = 2.7 Hz, 1H), 2.79 (s, 1H), 2.75 - 2.52 (m, 3H), 1.84 (td, / = 12.5, 7.3 Hz, 1H), 1.16 (s, 3H); ¹³ C NMR (75 MHz) δ 201.9, 173.2, 164.1, 159.5, 152.1, 137.4, 126.2, 117.1, 115.4, 114.2, 69.9, 40.3, 36.7, 36.3, 31.7, 21.8, 17.8; IR (CH ₂ C1 ₂ ) 1763, 1265 cm ^"1 ; LRMS (EI, 20 eV) m/z 284 (M ⁺ , 100), 269 (17), 187 (35); HRMS (EI) calcd. for Ci ₇ H ₁₆ 0 ₄ (M ⁺ ) m/z 284.1049, found 284.1034.

Preparation of compound 5:

[0136] To a solution of 4 (8.9 g, 31.1 mmol) in AcOH (100 mL) were added N- iodosuccinimide (7.5 g, 34.4 mmol) and scandium (III) triflate (1.5 g, 3.1 mmol). The resulting suspension was stirred in the absence of light at room temperature, and the reaction was monitored by TLC («-hexane: EtOAc: : l : l).After stirring overnight the reaction mixture was diluted with solution (10 g in 1000 mL water) and mixed at room temperature for 1 h. The product was collected by filtration, washed with water (200 mL ^χ 5), and EtOH/H ₂ 0 (1 : 1 v/v, 200 mL ^χ 5), dried under vacuum to give product (10.8 g, 82%) as alight yellow solid.

[0137] Compound 5, analytical TLC (silica gel 60), EtOAc: «-hexane: CH ₂ CI ₂ =1 : 1 : 1,

Rf= 0.50; ^: H NMR (300 MHz, CDC1 ₃ ) S 13.5 (s, 1H), 7.98 (d, / = 8.3 Hz, 1H), 7.01 (d, / = 8.4 Hz, 1H), 4.77 (AB system, 2H), 3.14 (d, / = 13.6 Hz, 1H), 2.83 (dd, / = 6.0, 18.3 Hz, 1H), 2.72 - 2.58 (m, 2H), 2.57 - 2.40 (m,2H), 1.88 - 1.78 (m, 1H), 1.14 (s, 3H); ¹³ C NMR (75 MHz, CDCI ₃ ) S 193.5, 173.1, 160.8, 159.5, 153.1, 144.1, 126.4, 125.9, 120.9, 93.3, 69.9, 62.4, 39.5, 37.4, 36.9, 31.9, 21.4, 17.7; LRMS (EI) m/z 410.0 (M ⁺ ); HRMS (EI) calcd. for CnH ₁₅ I0 ₄ (M ⁺ ) m/z 410.0015; found 410.0007.

Preparation of compound 6:

[0138] Pd(PPh ₃ ) ₂ C1 ₂ (685 mg, 0.97 mmol), Cul (372 mg, 1.95 mmol) and alkyne

(1.02 g, 7.31 mmol) were added to a 250 mL round-bottom flask, evacuated by vacuum and filled with argon. 100 mL of dry THF saturated with argon was injected to the round-bottom flask in an argon atmosphere. Compound 9 (2.0 g, 4.88 mmol) was added to the above solution, and the distilled triethylamine (3.5 mL) was finally added. The resulting mixture was stirred at 35°C until compound 9 was consumed as determined by TLC (EtOAc: n- hexane = 2:8). Upon completion, the mixture was allowed to cool to room temperature, filtered through silica gel to desalt, and washed with 250 mL EtOAc/CH ₂ Cl ₂ (1 : 1, v/v). Removal of solvewerent under reduced pressure afforded a residue, which was purified by flash chromatography to afford arylated alkyne 6 (2.0 g, 97%) as white solid.

[0139] Compound 6, analytical TLC (silica gel 60), EtOAc: «-hexane = 1 :4, R/ =

0.25; ¾ NMR (300 MHz, CDC1 ₃ ) ^ 13.15 (s, 1H), 7.64 (d, / = 8.0 Hz, 1H), 6.92 (d, / = 8.0 Hz, 1H), 4.94 (t, / = 3.4 Hz, 1H), 4.84-4.70 (m, 2H), 4.55 (t, / = 5.3 Hz, 2H), 3.89-3.60 (m, 2H), 3.25 - 3.21 (m, 1H), 2.83 (d, / = 9.1 Hz, 2H), 2.75 - 2.52 (m, 3H), 1.90 - 1.45 (m, 7H), 1.15 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) «5 201.8, 173.0, 164.5, 159.1, 152.4, 140.6, 126.2, 115.1, 114.0, 111.8, 96.9, 90.8, 80.1, 69.9, 62.0, 54.9, 40.1, 36.9, 36.3, 31.5, 30.3, 25.4, 21.6, 19.0, 17.7; LRMS (EI) m/z 422.2 (M ⁺ ); HRMS (EI) calcd. for C ₂₅ H ₂₆ O ₆ (M ⁺ ) 422.1729; found 422.1729.

Preparation of compound 7:

[0140] Compound 6 (400 mg, 0.95 mmol) in ethanol (50 mL) was hydrogenolyzed in the presence of triethylamine (0.6 mL, 4.29 mmol) and 5% Pd/C (400 mg) using a balloon filled with H ₂ gas at room temperature. After 12 h, the mixture was heated to 45-50 °C for 2 h. The mixture was filtered through silica gel, washed with EtOAc: CH ₂ CI ₂ (1 : 1, v/v, 100 mL x 2), and the filtrate was concentrated to give product as white powder without further purification (400 mg, 100%).

[0141] Compound 7, analytical TLC (silica gel 60), EtOAc: «-hexane = 1 :4, R/ =

0.30; ¾ NMR (300 MHz, CDC1 ₃ ) δ 12.93 (s, 1H), 7.38 (d, / = 7.8 Hz, 1H), 6.92 (d, / = 7.8 Hz, 1H), 4.84-4.70 (m, 2H), 4.59 (t, / = 4.2 Hz, 1H), 3.89 - 3.72 (m, 2H), 3.56 - 3.38 (m, 2H), 3.15 (m, 1H), 2.82 - 2.72 (m, 4H), 2.60 - 2.30 (m, 3H), 1.95 - 1.62 (m, 6H), 1.59 - 1.52 (m, 3H), 1.45 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 202.1, 162.2, 159.6, 149.5, 137.2, 129.6, 126.1, 115.2, 113.4, 112.0, 98.9, 69.9, 67.0, 62.3, 40.3, 36.4, 36.3, 31.6, 30.7, 29.0, 26.0, 25.4, 21.7, 19.6, 17.7; LRMS (EI) m/z 426.2 (M ⁺ ); HRMS (EI) calcd. for C ₂ 5H30O6 (M ⁺ ) m/z 426.2042; found 426.2042. Preparation of compound 8:

[0142] To a solution of compound 7 (400 mg, 0.95 mmol) in methanol (50 mL) was added TsOH H ₂ 0 (72 mg, 0.38 mmol). The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc :«-hexane = 2:8). After 2 h, the solvent was evaporated under vacuum. The residue was dissolved in 100 mL of dichloromethane, washed with brine (30 mL x 3), and dried over anhydrous NaiSOzt. The solvent was removed by evaporation, and product 8 (308 mg, 95%) was obtained as a yellow powder, which was purified by flash column chromatography.

[0143] Compound 8, analytical TLC (silica gel 60), EtOAc: «-hexane = 1 :4, R/ =

0.15; ¾ NMR (300 MHz, CDC1 ₃ ) δ 15.05 (s, 1H), 7.39 (d, / = 7.8 Hz, 1H), 6.89 (d, / = 7.8 Hz, 1H), 4.80 - 4.76 (m, 2H), 3.66 (t, / = 6.0 Hz, 2H), 3.18 (m, 1H), 2.82 - 2.73 (m, 4H), 2.60 - 2.40 (m, 3H), 1.92 - 1.83 (m, 4H), 1.15 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 202.2, 162.1, 159.5, 149.8, 137.6, 129.3, 126.1, 118.2, 114.8, 113.8, 69.9, 61.7, 40.4, 36.5, 36.3, 32.4, 31.6, 25.1, 21.7, 17.7; LRMS (EI) m/z 342.1 (M^; HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₅ (M ⁺ ) m/z 342.1467; found 342.1462.

Preparation of compound 9:

[0144] Compound 8 (500 mg, 1.46 mmol) was stirred in the mixture of ethanol (20 mL) and CH ₂ C1 ₂ (5 mL), and treated with NaBH ₄ (110 mg, 2.92 mmol). After 2 h, the reaction mixture was concentrated, dissolved in 1 M HC1 (5 mL) and extracted with CH ₂ CI ₂ (30 mL x 3), then the organic layer was washed with saturated NaCl solution (30 mL ^χ 1), dried over NaSO/t, filtered and concentrated to give colorless oil crude product (500 mg, 99%), which has high purity for next step.

[0145] Compound 9, analytical TLC (silica gel 60), EtOAc: ra-hexane= 4: 1, R/= 0.40;

¾ NMR (300 MHz, DMSO-<4) δ 9.50 (br s, 1H), 6.98 (d, / = 7.9 Hz, 1H), 6.81 (d, / = 8.0 Hz, 1H), 6.50 (br, s, 1H), 5.00 (t, / = 7.9 Hz, 1H), 4.89 (br, s, 2H), 4.40 (br, s, 1H), 3.41 (t, / = 6.6 Hz, 2H), 2.81 (d, / = 13.1 Hz, 1H), 2.60 - 2.40 (m, 3H), 2.38 - 2.12 (m, 3H), 1.95 - 1.80 (m, 1H), 1.71 - 1.59 (m, 2H), 1.54 (td, / = 11.4, 6.8 Hz, 1H), 1.02 (s, 3H); ¹³ C NMR (75 MHz, DMSO-ifc) δ 173.2, 163.4, 154.6, 143.7, 128.5, 126.3, 123.0, 122.2, 114.7, 70.4, 67.9, 60.5, 36.0 (2C), 32.5, 32.1, 29.3, 25.7, 22.8, 17.7; LRMS (EI) m/z 344.2 (M ⁺ ); HRMS (EI) calcd. for C ₂ 0H ₂₂ O ₄ (M ⁺ -H ₂ 0) m/z 326.1518; found 326.1522.

Preparation of compound 10:

[0146] To a solution of compound 9 (100 mg, 0.29 mmol) in methanol (5 mL) at 0 °C was added a solution of NaIC>4 (93 mg, 0.43 mmol, in 1.0 mL distilled water). The reaction flask was covered with aluminum foil and the reaction was stirred at room temperature and monitored by TLC (EtOAc: «-hexane=8:2). A yellow solution with white precipitate was obtained. After 2 h, the precipitate was filtered by a short silica gel column and rinsed with EtOAc:CH2Cl2 (1 : 1, v/v, 200 mL). The combined filtrates were concentrated, and the residue was separated with flash chromatography to give compound 10 (80 mg, 80%) as white solid.

[0147] Compound 10, analytical TLC (silica gel 60), EtOAc: «-hexane= 4: 1, R =

0.30; ¾ NMR (300 MHz, CDC1 ₃ ) S 7.06 (d, / = 6.6 Hz, 1H), 6.40 (d, / = 6.6 Hz, 1H), 4.70 (br, s, 2H), 4.08 (d, / = 5.3 Hz, 1H), 3.61 (t, / = 6.3 Hz, 2H), 2.62 - 2.57 (m, 1H), 2.50 - 2.40 (m, 3H), 2.40 - 2.24 (m, 2H), 2.24 - 2.06 (m, 2H), 1.90 - 1.72 (m, 4H), 1.16 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) S 194.9, 172.9, 159.6, 151.0, 138.5, 135.8, 125.3, 120.7, 69.8, 66.9, 61.5, 57.0, 43.7, 38.3, 32.8, 31.6, 29.6, 24.8, 24.2, 17.5; LRMS (EI) m/z 342.2 (M ⁺ ); HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₅ (M ⁺ ) m/z 342.1467; found 342.1462.

Preparation of Compounds 11 and 12

[0148] To a solution of crude compound 10 (240 mg, 0.701 mmol) in acetonitrile (15 mL) was added an aqueous Na ₂ (EDTA) solution (4 ^χ 10-4 mol/L, 10 mL). The resulting homogeneous solution was cooled to 0-l°C, followed by the addition of 1, 1,1- trifluoroacetone (4.0 mL) in a pre -cooled syringe. To this homogeneous solution was added in portions a mixture of sodium bicarbonate (765 mg, 7.22 mmol) and OXONE (1.73g, 5.63 mmol) over the period of 1 h (pH 7.0-7.4). The reaction was monitored by TLC (EtOAc: Hexane = 1 : 1, v/v). When the reactant was consumed, the mixture was poured into water (30 mL), and extracted with dichloromethane (60 mL ^χ 3). The organic extracts were dried with anhydrous Na2SC>4, filtered, and concentrated to give a yellow oil for the following step without further purification. Due to the similar polarity of two products 11 and 12 on TLC (80% EtOAc in Hexane as developing solution, R = 0.2) and the instability of product 12 under basic conditions, products 11 and 12 could be directly used for next step without further purification.

Preparation of Compounds 13 and 14

[0149] To a solution of the mixture of crude compounds 11 and 12 obtained in the previous procedure in dry THF ( 10 inL) were added 4-nitrophenyl chloroformate (283 mg, 1.402 mmol) and 1 13 of pyridine. The reaction mixture was stirred under argon at room temperature and monitored by TLC (EtOAc:Hexane = 1 : 1, v/v). When the reactant was consumed, the solvent was evaporated under vacuum and the residue was dissolved in 100 mL dichloromethane, washed with saturated NaHCC solution (30 mL ^χ 3) and brine (30 mL x 3). The organic layer was dried over anhydrous NaiS0 ₄ and the solvent was then removed. The crude product was purified by flash chromatography. Evaporation of solvents yielded compounds 13 (140 mg, 39% yield for 2 steps) and 14 (50 mg, 13% yield for 2 steps) as a colorless solid.

[0150] Compound 13: analytical TLC (silica gel 60), EtOAc: Hexane=5 :5, R = 0.125;

¾ NMR (300 MHz, CDC1 ₃ ) δ 8.28 (d, / = 9.1 Hz, 2H), 7.39 (d, / = 9.1 Hz, 2H), 7.08 (d, / = 4.6 Hz, 1H), 4.69 (AB system, 2H), 4.28 (t, / = 6.4 Hz, 2H), 3.83 (d, / = 4.7 Hz, 1H), 3.62 (d, / = 5.7 Hz, 1H), 2.75 (d, / = 13.5 Hz, 1H), 2.42-2.40 (m, 1H), 2.39-2.25 (m, 2H), 2.22-2.10 (m, 2H), 2.08-1.84 (m, 3H), 1.68 (dd, / = 4.5, 12.5 Hz, 1H), 1.27 (dt, / = 6.0, 12.1Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 190.8, 173.1, 159.5, 155.4, 152.3, 145.4, 142.9, 140.0, 125.7 (2C), 125.3, 121.7 (2C), 69.9, 68.4, 64.2, 62.3, 60.9, 51.4, 40.8, 35.0, 30.0, 26.1, 25.9, 23.4, 17.0, 13.7; HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₆ (M ⁺ -N0 ₂ C ₆ H ₅ OCO+H) m/z 358.141 1, found 358.1416.

Compound 14: analytical TLC (silica gel 60), EtOAc: Hexane=5 :5, = 0.25; i NMR ^OO MHz, CDCI ₃ ) δ 8.28 (d, / = 9.2 Hz, 2H), 7.39 (d, / = 9.2 Hz, 2H), 4.71 (AB system, 2H), 4.30 (t, / = 6.3 Hz, 2H), 4.08 (d, / = 2.7 Ηζ, ΙΗ), 3.91 (d, / = 2.7 Hz, 1H), 3.45 (d, / = 5.4 Hz, 1H), 2.82 (d, / = 13.2 Hz, 1H), 2.40-2.30 (m, 1H), 2.21-2.01 (m, 3H), 1.99-1.92 (m, 2H), 1.90- 1.85 (m, 2H), 1.62 (dd, / = 4.5, 12.5 Hz, 1H), 1.27 (dt, / = 6.0, 12.1Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) «Π97.1, 173.0, 159.3, 155.4, 152.4, 145.5, 125.8, 125.3 (2C), 121.8 (2C), 69.9, 68.5, 65.3, 63.0, 61.4, 60.7, 55.9, 40.6, 35.4, 30.6, 29.7, 25.3, 23.6, 23.2, 17.1, 13.8; LRMS (ESI) m/z 562.1 (M ⁺ +Na).

Preparation of Compound 15

[0151] To a solution of biotin-amine (30 mg, 0.0802 mmol) in CH2CI2 (4

mL)/CH ₃ OH (1 mL) was added compound 14 (20 mg, 0.037 mmol), followed by Et3N (11 0.0802 mmol) and DMAP (1 mg, 0.00802 mmol). The mixture was stirred at rt overnight. After that, the mixture was concentrated and purified by column chromatography to give compound 15 as a white solid (10 mg, 24 %).

[0152] ¾ NMR (500 MHz, CDCI ₃ ) δ 6.41 (br s, 1H), 5.81 (br s, 1H), 5.54 (br s, 1H),

5.17 (br s, 1H), 4.98-4.69 (m, 2H), 4.49 (t, / = 4.9 Hz, 1H), 4.33 (t, / = 4.7 Hz, 1H), 4.10- 4.08 (m, 2H), 3.92 (d, / = 2.6 Hz, 1H), 3.62-3.55 (m, 8H), 3.49-3.43 (m, 3H), 3.42-3.36 (m, 2H), 3.19-3.14 (m, 1H), 2.92 (dd, / = 4.9, 12.9 Hz, 1H), 2.85-2.81 (m, 1H), 2.73 (d, / = 12.9 Hz, 1H), 2.39-2.35 (m, 1H), 2.27-2.08 (m, 4H), 2.04-1.95 (m, 2H), 1.77-1.51 (m, 8H), 1.49- 1.39 (m, 3H), 1.37-1.29 (m, 1H), 1.07 (s, 3H); ¹³ C NMR (125 MHz) δ 197.3, 173.1, 173.0, 163.3, 159.4, 156.6, 125.8, 70.2, 70.0(4), 69.9(8), 69.9(1), 65.4, 64.1, 63.3, 61.8, 61.1, 60.8, 60.1, 56.2, 55.3, 55.3, 40.8, 40.6, 40.5, 39.2, 35.9, 35.4, 30.6, 29.7, 28.5, 28.1, 25.7, 25.5, 24.1, 23.2, 17.1, 13.9; LRMS (ESI) m/z 797.1 (M ⁺ +Na).

Preparation of Biotin-triptolide (Biotin-TL)

[0153] To a solution of compound 15 (10 mg, 0.0129 mmol) in THF (2.5 mL) was added K-Selectride solution (1 M in THF, 50 0.05 mmol) at -40 °C. The reaction was stirred for 3 h at -40 °C. After completion of reaction, the mixture was concentrated and purified by column chromatography to give biotintriptolide as a white solid (4 mg, 39 %).

[0154] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH = 9: 1, R/= 0.15; ¾ NMR

(500 MHz, CDCI ₃ +CD ₃ OD) δ 4.75 (AB system, 2H), 4.51 (dd, / = 4.9, 7.8 Hz, 1H), 4.32 (dd, / = 4.6, 8.3 Hz, 1H), 4.14-4.08 (m, 1H), 4.07-4.02 (m, 1H), 3.65-3.62 (m, 5H), 3.61-3.52 (m, 4H), 3.44-3.38 (m, 3H), 3.37-3.34 (m, 3H), 3.20-3.15 (m, 1H), 2.93 (dd, / = 5.0, 12.9 Hz, 1H), 2.74 (d, / = 12.8 Hz, 2H), 2.33-2.29 (m, 1H), 2.26-2.20 (m, 3H), 2.19-2.13 (m, 1H), 2.00-1.90 (m, 1H), 1.76-1.53 (m, 9H), 1.48-1.33 (m, 3H), 1.30-1.23 (m, 1H), 1.11 (s, 3H); ^lj C NMR (125 MHz, CDCI ₃ +CD ₃ OD) δ 1743, 164.3, 161.7, 161.2, 157.4, 125.4, 73.5, 70.5, 70.3, 70.0, 69.0, 66.5, 65.7, 64.8, 63.5, 62.4, 62.1, 60.6, 60.3, 56.5, 55.8, 55.3, 40.6, 40.5, 39.7, 39.2, 36.0, 35.9, 30.3, 29.8, 28.6, 28.3, 25.6, 24.3, 23.7, 17.2, 13.7; HRMS (ESI) calcd. for C ₃ 7H ₅ 3N ₄ Oi ₂ S (M ⁺ +H) m/z 777.3375, found 777.3380.

[0155] Chemical synthesis of cyanine 3-triptolide (Cy3-TL)

[0156] Reagents and conditions: n) DMAP, Et ₃ N, CH ₂ C1 ₂ , rt, 85 %; o) K-Selectride, anhydrous THF, -78 °C; p) 30 % TFA in CH ₂ C1 ₂ (v/v), rt; q) Cy3-NHS, CH ₂ C1 ₂ , DMAP, rt, 42 %. Compound 19 was synthesized according to procedure described in C. Gnaccarini et al. Bioorg. Med. Chem. 2009, 17, 6354-6359.

Preparation of compound 16

[0157] To a mixture of 14 (50 mg, 0.093 mmol) and compound 19 (28 mg, 0.111 mmol) in CH ₂ C1 ₂ (5 mL) was added one small crystal of DMAP. The reaction mixture was stirred at room temperature overnight under argon, after which time TLC analysis

(CH ₂ C1 ₂ :CH ₃ 0H = 15: 1) indicated the reaction was complete. CH ₂ C1 ₂ (45 mL) was added to the crude mixture, and the mixture was washed with NaHCC>3 solution until the water layer was colorless. The organic layer was dried over anhydrous Na ₂ SC>4 and the solvent was then removed to give a crude product, which was purified by flash column chromatography to afford product 16 as a white solid (51 mg, 85% yield).

[0158] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ = 0.46; ^: H NMR

(300 MHz, CD ₃ OD) δ 4.85 (AB system, 2H), 4.25 (d, / = 2.9 Hz, 1H), 4.09-4.02 (m, 2H), 3.61 (apparent s, 5H), 3.52 (q, / = 6.1 Hz, 5H), 3.43 (d, / = 5.4 Hz, 1H), 3.30-3.26 (m, 1H), 3.23 (t, / = 5.6 Hz, 2H), 2.94-2.90 (m, 1H), 2.38-2.26 (m, 2H), 2.14-2.11 (m, 1H), 1.97-1.89 (m, 2H), 1.82-1.68 (m, 3H), 1.57-1.49 (m, 2H), 1.44 (s, 9H), 1.04 (s, 3H); ¹³ C NMR (100 MHz, CDCI ₃ ) δ 197.1, 173.0,159.4, 156.4, 155.9, 125.6, 79.2, 70.2 (2C), 70.1, 70.0, 69.9 (2C), 65.2, 64.0, 63.2, 61.0, 60.6(2), 60.5(6), 40.7, 40.5, 40.3, 35.3, 30.5, 28.4, 25.5, 24.0, 23.2, 17.0, 13.8 (Boc); LRMS (ESI) m/z 671.2 (M ⁺ +Na).

Preparation of Compound 17 [0159] To a solution of compound 16 (51 mg, 0. 0787 mmol) in THF (2 mL) at 78°C under argon atmosphere was added K-Selectride. The reaction was stirred for 2 h, and then the reaction was quenched by saturated NH ₄ C1 aqueous solution. The mixture was extracted with dichloromethane (50 mL ^χ 3) and washed with brine (20 mL ^χ 3). The organic layer was dried over anhydrous NaiSO/ _t , filtered, concentrated, and separated by flash column chromatography to afford 17 as a white powder (50 mg, 100%), which was used directly in the following step without further purification.

Preparation of Compound 18

[0160] To a solution of compound 17 (50 mg, 0.0768 mmol) in CH2Q2 (1 mL) was added trifluoroacetic acid (0.3 mL). The reaction mixture was stirred at room temperature for 1 h. Saturated NaHCC>3 solution (5 mL) was added to the mixture and stirred for another 30 min. The resulting mixture was extracted with dichloromethane, then the organic layer was dried over anhydrous NaSOzt, filtered, concentrated to give crude product 18 as a yellow oil (50 mg), which was used for the next step without further purification. Preparation of Cyanine 3-TL (Cy3-TL) To a solution of compound 18 (15 mg, 0.027 mmol) in CH2Q2 (1 mL) were added Cy3-NHS (15 mg, 0.023 mmol) and one small crystal of DMAP. The reaction mixture was stirred at room temperature overnight, followed by concentration in vacuo and purification by flash column chromatography to afford Cy3-TL as a red solid (10 mg, 42 %).

[0161] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH = 9: 1, R/= 0.43; ^: H NMR

(400 MHz, CDCI ₃ ) S 8.65 (br s,lH), 8.40 (t, / = 13.5 Hz, 1H), 7.50-7.30 (m, 5H), 7.32-7.20 (m, 2H), 7.18-7.11 (m, 3H), 6.02 (br s, 1H), 4.68 (AB system, 2H), 4.20-4.10 (m, 2H), 3.90- 3.85 (m, 2H), 3.84 (dd, / = 4.8 Hz, 1H), 3.82-3.79 (m, 3H), 3.75-3.71 (m, 1H), 3.64-3.50 (m, 8H), 3.49-3.44 (m, 3H), 3.40-3.33 (m, 4H), 1.69-1.64 (m, 1H), 2.61 (s, 2H), 2.36-2.27 (m, 1H), 2.26-2.05 (m, 2H), 2.00-1.80 (m, 6H), 1.73-1.71 (m, 14H), 1.65-1.58 (m, 1H), 1.53 (dd, / = 4.5, 12.0 Hz, 1H), 1.30-1.24 (m, 1H), 1.10-1.07 (m, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) S 174.3, 173.8, 173.7, 173.3, 160.2, 157.0, 151.0, 142.8, 141.9, 140.6, 140.5, 129.0, 128.9, 125.5, 125.4, 125.3, 122.0, 122.0, 111.2, 110.8, 105.2, 104.6, 73.5, 70.6, 70.3, 70.1(3) (2C), 70.0(8), 70.0, 69.8 (2C), 65.8, 64.0, 62.4, 60.6, 60.2, 56.5, 55.4, 49.0, 48.8, 44.1, 40.8, 40.4, 39.0, 35.8, 35.4, 32.1, 29.8, 28.2, 28.1, 27.1, 23.7, 23.3, 22.6, 17.1, 13.6; HRMS (ESI) calcd. for C ₅ 6H ₇₁ N ₄ 0ii (M ⁺ +C1) m/z 975.5114, found 975.5091.

[0162] Scheme 1A. Synthetic route to ProC3-TN.

[0163] Scheme 1A Reagents and conditions: j) CF ₃ COCH ₃ , OXONE ^® , NaHC0 ₃ ,

CH ₃ CN, Na ₂ (EDTA), 0 °C, 26%.

Preparation of compound ProC3-TN:

[0164] To a solution of compound 10 (419 mg, 1.22 mmol) in CH CN (10.5 mL) and

Na ₂ (EDTA) solution (4.0 ^χ 10 ^"4 M, 7 mL) was added CF ₃ COCH ₃ (2.5 mL) at 0 °C. A mixture of NaHC0 ₃ (1.23 g, 14.64 mmol) and OXONE ^® (3.0 g, 4.87 mmol) were added to the reaction portionwise over 1 h. When TLC indicated completion of reaction after 2.5 h, the mixture was diluted with EtOAc (50 mL) and H ₂ O (10 mL) was added. The aqueous layer was extracted with EtOAc (20 mL ^χ 2). The combined organic layer was dried over NaiSOzt, filtered and concentrated. The crude product was purified via column chromatography using EtOAc to give purified product (112 mg, 26%) and product 11 was obtained and applied to next step directly.

[0165] Compound ProC3-TN, analytical TLC (silica gel 60), «-Hexane: EtOAc = 2:3,

R _f = 0.1; i NMR ^OO MHz, CDC1 ₃ ) 7.04 (d, / = 4.5 Hz, 1H), 4.70 (br. s, 2H), 3.80 (d, / = 4.7 Hz, 1H), 3.62 - 3.60 (m, 3H), 2.76 - 2.71 (m, 1H), 2.45 - 2.36 (m, 1H), 2.36 (t, / = 7.3 Hz, 2H), 2.26 - 2.18 (m, 2H), 2.05 (t, / = 13.8 Hz, 1H), 1.75 - 1.60 (m, 3H), 1.32 (dd, / = 5.8, 12.0 Hz, 1H), 1.14 (s, 3H); ¹³ C NMR (125.8 MHz, CDC1 ₃ ) δ 191.2, 173.1, 159.5, 144.0, 139.8, 125.9, 69.9, 64.1, 62.3, 61.6, 59.2, 51.5, 40.9, 35.0, 31.1, 30.2, 25.7, 23.4, 17.1, 13.6; IR (CH ₂ CI ₂ ) 3686, 2928, 2360, 1756, 1681 cm ^"1 ; LRMS (EI, 20 eV) m/z 340 (M ⁺ -H ₂ 0, 20), 324 (22), 309 (30), 124(100); HRMS (EI) calcd for C ₂ 0H ₂ 0O5 (M -H ₂ 0) m/z 340.1311, found 340.1300 T127.

[0167] Scheme 2 Reagents and conditions: a) 4-nitrophenylchloroformate, pyridine,

CH ₂ CI ₂ , rt, 13% from 10; b) amine, Et3N, CH ₂ Q ₂ , rt, 75-100%. Compound 12 was prepared under reaction conditions similar to compound 14 of Example 1, Scheme 1.

[0168] Compound 12: analytical TLC (silica gel 60), EtOAc: «-hexane= 1 : 1, R =

0.25; ¾ NMR (500 MHz, CDC1 ₃ ) δ 8.28 (d, / = 9.2 Hz, 2H), 7.39 (d, / = 9.2 Hz, 2H), 4.74 - 4.68 (m, 2H), 4.30 (t, / = 6.3 Hz, 2H), 4.08 (d, / = 2.7 Ηζ,ΙΗ), 3.91 (d, / = 2.7 Hz, 1H), 3.45 (d, / = 5.4 Hz, 1H), 2.82 (d, / = 13.2 Hz, 1H), 2.40 - 2.30 (m, 1H), 2.21 - 2.01 (m, 3H), 1.99 - 1.92 (m, 2H), 1.90 - 1.85 (m, 2H), 1.62 (dd, / = 4.5, 12.5 Hz, 1H), 1.27 (dt, / = 6.0, 12.1Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) «5 197.1, 173.0, 159.3, 155.4, 152.4, 145.5, 125.8, 125.3 (2C), 121.8 (2C), 69.9, 68.5,65.3, 63.0, 61.4, 60.7, 55.9, 40.6, 35.4, 30.6, 29.7, 25.3, 23.6, 23.2, 17.1, 13.8; LRMS (ESI) m/z 562.1 (M ⁺ +Na). General procedure for preparation of compound T67, T117, T118, T121, T124, T127:

[0169] To a solution of compound 12 (1 equiv) in CH ₂ CI ₂ (2 mL) were added corresponding amine (1.2 equiv) and Et ₃ N (1.2 equiv). The reaction was stirred at rt for 2 h. The resulting mixture was diluted with CH ₂ CI ₂ ( 10 mL). The organic layer was washed with 1 N HC1 (2 mL ^χ 1), 1 M K ₂ CO3 (3 mL portions until the aqueous layer was colorless) and brine (2 mL ^χ 1). The organic layer was dried over Na ₂ SC>4, filtered and concentrated to give the crude product. The crude product was purified by column chromatography to give the purified product (75 - 100%).

[0170] Compound T67, analytical TLC (silica gel 60), «-Hexane:EtOAc = 1 : 1, R/ =

0.25; ¾ NMR (300 MHz) 4.94 (br. s, 1H), 4.77 - 4.63 (m, 2H), 4.15 - 3.97 (m, 2H), 4.07 (d, / = 2.9 Hz, 1H), 3.89 (d, / = 2.7 Hz, 1H), 3.43 (d, / = 5.4 Hz, 1H), 3.14 (q, / = 6.6 Hz, 2H), 2.84 - 2.80 (m, 1H), 2.39 - 2.34 (m, 1H), 2.26 - 2.1 1 (m, 3H), 1.94 (apparent t, / = 14.6 Hz, 1H), 1.79 - 1.62 (m, 3H), 1.57 - 1.45 (m, 2H), 1.32 (td, / = 12.2, 5.9 Hz, 1H), 1.26 - 1.18 (m, 1H), 1.07 (s, 3H), 0.92 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75.5 MHz) δ 196.6, 172.7, 159.0, 156.2, 125.6, 69.8, 65.2, 63.8, 63.1, 60.8, 60.6, 60.3, 56.1, 51.1, 42.7, 40.5, 35.3, 30.5, 26.2, 24.2, 23.2, 17.1, 13.8, 1 1.2; IR (CH ₂ C1 ₂ ) 3675, 1757, 1726 cm ; LRMS (EI, 20 eV) m/z 459 (M ⁺ , 3), 430 (M ⁺ -Et, 30), 340 (50), 1 13 (100); HRMS (EI) calcd for C ₂₄ H ₂₉ NO ₈ (M ⁺ ) m/z 459.1893, found 459.1895.

[0171] Compound T117, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, Rf =

0.43; i NMR (500 MHz, CDC1 ₃ ) δ 4.70 (td, / = 10.1, 1.6 Hz, 2H), 4.10 - 4.00 (m, 3H), 3.89 (d, / = 2.7 Hz, 1H), 3.89 - 3.48 (m, 4H), 3.43 (d, / = 5.5 Hz, 1H), 2.82 (d, / = 13.2 Hz, 1H), 2.43 - 2.25 (m, 5H), 2.24 - 2.20 (m, 3H), 2.20 - 2.14 (m, 2H), 2.05-1.80 (m, 3H), 1.80 - 1.70 (m, 2H), 1.60 (dd, / = 12.3, 4.8 Hz, 1H), 1.35 - 1.25 (m, 1H), 1.07 (s, 3H); ¹³ C NMR (125 MHz, CDCI ₃ ) δ 191 A, 173.0, 159.3, 155.2, 125.8, 69.9, 65.3, 64.5, 63.2, 61.1, 60.6, 56.0, 54.7 (2C), 46.1, 43.6 (2C), 40.6, 35.4, 30.6, 29.7, 25.3, 24.0, 23.3, 17.1, 13.8; LRMS (ESI) m/z 501.1 (M ⁺ +H).

[0172] Compound T118: ¾ NMR (400 MHz, CDC1 ₃ ) δ 5.35 (s, 1H), 4.79 - 4.63 (m,

2H), 4.05 (d, / = 17.7 Hz, 3H), 3.89 (s, 1H), 3.44 (d, / = 4.2 Hz, 1H), 3.27 (s, 2H), 2.80 (s, 1H), 2.38 (d, / = 16.7 Hz, 1H), 2.19 (t, / = 23.6 Hz, 3H), 1.99 (t, / = 13.9 Hz, 2H), 1.66 (dd, / = 35.0, 15.1 Hz, 7H), 1.32 (s, 2H), 1.08 (s, 3H).

[0173] Compound T121, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, =

0.40; ¾ NMR (500 MHz, CDC1 ₃ ) S 5.37 (br, NH), 4.75 - 4.65 (m, 2H), 4.16 - 4.06 (m, 3H), 3.96 (dd, / = 5.4, 16.8 Hz, 2H), 3.90 (d, / = 2.8 Hz, 1H), 3.76 (s, 3H), 3.44 (d, / = 5.5 Hz, 1H), 2.82 (d, / = 13.3 Hz, 1H), 2.36 (d, / = 13.5 Hz, 1H), 2.25 - 1.94 (m, 4H), 1.81 - 1.66 (m, 3H), 1.60 (dd, / = 4.8, 12.9 Hz, 1H), 1.32 (td, / = 6.0, 12.9 Hz, 1H), 1.07 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) S 197.0, 173.0, 170.6, 159.3, 156.3, 125.9, 69.9, 65.4, 64.7, 63.2, 61.1, 60.7, 56.2, 53.4, 52.3, 42.6, 40.6, 35.4, 30.6, 26.1, 24.1, 23.3, 17.1, 13.8; LRMS (ESI) m/z 512.1 (M ⁺ +Na).

[0174] Compound T127, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, =

0.25; ¾ NMR (500 MHz, CDC1 ₃ ) δ 7.71 - 7.64 (m, 2H), 7.56 - 7.40 (m, 2H), 7.35 - 7.26 (m, 2H), 7.24 - 6.98 (m, 2H), 6.72 (br s, 2H), 4.76 - 4.63 (m, 2H), 4.13 - 4.01 (m, 4H), 3.66 - 3.56 (m, 8H), 3.42 (d, / = 5.5 Hz, 1H), 3.38 - 3.31 (m, 8H), 2.86 - 2.82 (m, 1H), 2.37 - 2.32 (m, 1H), 2.24 (td, / = 5.7, 10.3 Hz, 1H), 2.20 - 2.10 (m, 1H), 1.97 - 1.90 (m, 1H), 1.82 - 1.71 (m, 3H), 1.60 - 1.51 (m, 3H), 1.30 - 1.23 (m, 12H), 1.04 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) <S 173.3, 167.8, 159.8, 157.79, 157.77, 157.79, 157.77, 155.79, 155.71, 155.2, 142.6, 135.2, 132.3, 130.8, 130.24, 130.16, 130.05, 128.6, 128.4, 127.6, 127.3, 127.1, 125.6, 113.9, 96.0, 70.1, 65.2, 64.9, 63.2, 61.3, 60.6, 56.3, 47.4, 46.11, 46.08, 44.0, 43.1, 41.8, 40.5, 35.3,31.9, 30.9, 30.5, 29.7, 29.0, 25.2, 23.8, 23.2, 22.7, 17.1, 14.1, 13.8, 12.6; LRMS (ESI) m/z 911.2 (M^.

Scheme 3. Synthetic route to T115 and T120.

Scheme 3 Reagents and conditions: a) K-Selectride, THF, - 78 °C, 83%; b) K-Selectride, THF, - 78 °C, 30%. Compounds Tl 15 and T120 were prepared under reaction conditions similar to compound 17 of Example 1, Scheme 1 as described above.

[0175] Compound T115, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, =

0.16; ¾ NMR (500 MHz, CDC1 ₃ ) δ 4.72 - 4.64 (m, 2H), 4.40 (s, 1H), 4.15 - 4.05 (m, 2H), 3.76 (d, / = 2.9 Hz, 1H), 3.72 (d, / = 5.7 Hz, 1H), 3.48 (br, 4H), 3.40 (d, / = 2.5 Hz, 1H), 2.73 (d, / = 13.2 Hz, 1H), 2.43 - 2.23 (m, 8H), 2.20 - 2.14 (m, 3H), 1.96 - 1.91 (m, 2H), 1.80 - 1.70 (m, 2H), 1.60 (dd, / = 12.3, 4.8 Hz, 1H), 1.35 - 1.25 (m, 2H), 1.07 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 173.3, 160.2, 155.3, 125.5, 70.0, 67.4, 65.3, 64.8, 62.5, 61.6, 55.9, 55.7, 54.9, 54.7 (2C), 46.2, 43.6 (2C), 40.6, 35.9, 30.9, 30.0, 24.2, 23.6, 17.1, 13.5; LRMS (ESI) m/z 525.0 (M ⁺ +Na).

[0176] Compound T120, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, R/=

0.29; ¾ NMR (500 MHz, CDC1 ₃ ) δ 4.82 (br, 1H, NH), 4.72-4.64 (m, 2H), 4.07-4.04 (m, 2H), 3.90 (d, / = 2.8 Hz, 1H), 3.45 (d, / = 2.7 Hz, 1H), 3.39-3.33 (m, 4H), 3.29-3.20 (m, 2H), 2.74-2.69 (m, 1H, OH), 2.33 (d, / = 16.9 Hz, 1H), 2.20-2.15 (m, 4H), 1.97 (dd, / = 13.6, 7.0 Hz, 1H), 1.80-1.54 (m, 5H), 1.25- 1.18 (m, 1H), 1.12 (s, 3H); ¹³ C NMR (125 MHz, CDCI ₃ ) δ 173.1, 159.8, 156.5, 125.6, 73.9, 69.9, 66.1, 64.3, 62.4, 60.5, 60.2, 56.6, 55.4, 49.1, 40.5, 38.5, 35.9, 29.8, 29.1, 28.1, 23.7, 23.3, 17.1, 13.6; LRMS (ESI) m/z 525.1 (M ⁺ +Na).

[0177] Scheme 4. Synthetic route to T68, T69, T70 and T71.

[0178] Scheme 4 Reagents and conditions: a) NaH, allyl bromide, DMF, rt, 87%; b)

N, N-dimethylaniline, reflux, 87%; c) Os0 ₄ , NaHC0 ₃ , NaI0 ₄ , iBuOH, H ₂ 0, rt, 75%; d) NaBH ₄ , EtOH, 0 °C, 98%; e) NaI0 ₄ , MeOH, H ₂ 0, 0 °C, 90%; f) CF ₃ COCH ₃ , OXONE ^® , NaHC0 ₃ , CH ₃ CN, Na ₂ (EDTA), 0 °C, 40%; g) H ₂ 0 ₂ , NaOH, 0 °C, 45% from 16; h)

Eu(FOD) ₃ NaBH ₄ , MeOH, - 78 °C, 63% for T70 and 23% for T71; i) phenyl chloroformate, DMAP, CH ₂ C1 ₂ , rt, 100%; j) amine, Et ₃ N,CH ₂ Cl ₂ , rt, 85%. Compounds 16-18 were prepared under reaction conditions similar to compounds 9-12 of Example 1, Scheme 1 as described above. Compound 19 was prepared under reaction conditions similar to compound 12 of Example 1, Scheme 2 as described above except the use of phenyl chloroformate and DMAP in step i in place of 4-nitrophenylchloroformate in step a. Compound T69 was prepared under reaction conditions similar to compound sT67, Ti l 7, Ti l 8, T121, T124, and T 127 of Example 1, Scheme 2 as described above wherein the corresponding amine was n- propylamine.

Preparation of compound 13:

[0179] To a suspension of NaH (7.75 mg, 0.194 mmol) in DMF (0.5 mL), was added a solution of 4 (50 mg, 0.176 mmol) in DMF (1 mL) at room tempreature. The resulting brown homogenous solution was stirred for 1 h, and allyl bromide (0.03 mL, 0.352 mmol) was added. The reaction was stirred overnight. The mixture was poured into water, and acidified with 6 N HCl, then extracted with CH ₂ CI ₂ . The organic extracts were dried with anhydrous Na ₂ SC>4, filtered, and concentrated. DMF was removed by vacuum distillation. The residue was purified by flash column chromatography to afford a white solids 13 (50 mg, 87% yield).

[0180] Compound 13, M.p. 141-144 °C; analytical TLC (silica gel 60), n-

Hexane:EtOAc = 3: 1, R _f = 0.4; ¾ NMR (300 MHz) δ 7.48 (t, / = 8.0 Hz, 1H), 7.04 (d, / = 7.8 Hz, 1H), 6.93 (d, / = 8.4 Hz, 1H), 6.14 - 6.02 (m, 1H), 5.58 (dd, / = 1.5, 17.2 Hz, 1H), 5.33 (dd, / = 1.4, 10.6 Hz, 1H), 4.76 (d, / = 1.3 Hz, 2H), 4.74 - 4.56 (m, 2H), 3.16 - 3.11(m, 1H), 2.81 (dd, / = 5.9, 18.2 Hz, 1H), 2.63 (dd, / = 13.7, 18.2 Hz, 1H), 2.58 - 2.49 (m, 2H), 2.47 - 2.34 (m, 1H) 1.83 (td, / = 12.5, 7.3 Hz, 1H), 1.13 (s, 3H); ¹³ C NMR (75.5 MHz) δ 194.3, 173.4, 160.1, 159.4, 153.5, 134.5, 132.4, 125.7, 121.4, 117.8, 115.3, 112.3, 70.1, 69.5, 39.7, 37.8, 36.9, 32.0, 21.4, 17.8; IR (CH ₂ C1 ₂ ) 3700, 2928, 1754, 1681 cm ^"1 ; LRMS (EI, 20 eV) m/z 324 (M ⁺ , 57), 309 (100), 296(27), 280 (36); HRMS (EI) for C ₂₀ H ₂₀ O ₄ (M ⁺ ): calcd 324.1362, found 324.1349.

Preparation of compound 14:

[0181] A solution of 13 (35 mg, 0.11 mmol) in N, N-dimethyl aniline (3 mL) was heated to reflux for 10 h under argon. The solution was poured into ice water and acidified with 6 Ν HCl. The mixture was extracted with CH ₂ CI ₂ . The organic extracts were dried with anhydrous Na ₂ SC>4, filtered, and concentrated. The residue was purified by flash column chromatography to afford a white solid 14 (31 mg, 87% yield). [0182] Compound 14, M.p.200-203 °C; analytical TLC (silica gel 60), ra-Hexane:

EtOAc: CH ₂ C1 ₂ =1:1:1,/?/= 0.65; ^NMR^OO MHz) 12.95 (s, 1H), 7.36 (d, /= 7.8 Hz, 1H), 6.87 (d, /= 7.8 Hz, 1H), 6.06 - 5.92 (m, 1H), 5.14-5.07 (m, 2H), 4.84 - 4.71 (m, 2H), 3.40 (d, /= 6.5 Hz, 2H), 3.23-3.16 (m, 1H), 2.82 (d, /= 3.2 Hz, 1H), 2.78 (s, 1H), 2.75 - 2.55 (m, 2H), 2.48 - 2.35 (m, 1H), 1.81 (td, /= 12.3, 6.6 Hz, 1H), 1.14 (s, 3H); ¹³ CNMR (75.5 MHz) £202.1, 173.3, 162.0, 159.5, 149.9, 137.2, 135.8, 127.9, 126.2, 116.2, 114.9, 113.7, 69.9, 40.4,36.6,36.4,33.2,31.6,21.8, 17.8; IR (CH ₂ C1 ₂ ) 3065, 1755, 1628 cm ; LRMS (EI, 20 eV) m/z 324 (M ⁺ , 46), 309 (24), 71 (100); HRMS (EI) for C ₂₀ H ₂₀ O ₄ (M ⁺ ): calcd 324.1362, found 324.1364.

Preparation of compound 15:

[0183] OsC>4 (100 μΐ, 2% solution in water) was added to a mixture of 14 (100 mg,

0.31 mmol), NaHC0 ₃ (364 mg, 4.3 mmol), and NaI0 ₄ (550 mg, 2.57 mmol) in teri-butyl alcohol (10 mL) and water (2 mL) at room temperature. The reaction was stirred for 4 h, and another portion of OSO4 (100 μΐ) was added. The resulting mixture was stirred overnight and then quenched with 10% aqueous Na ₂ S ₂ C>3. After stirring for 0.5 h, the mixture was extracted with ethyl acetate. The combined organic extracts were washed with water and brine and dried with anhydrous MgSC Removal of the solvent gave a crude residue, which was purified by flash column chromatograph to provide 15 (75 mg, 75% yield) as a white solid.

[0184] Compound 15, M.p.198 - 200 °C; analytical TLC (silica gel 60), n-

Hexane:EtOAc = 3:1, R _f = 0.35; iNMR (300 MHz) δ 12.95 (s, 1H), 9.79 (t, /= 1.5 Hz, 1H), 7.38 ( d, /= 7.9 Hz, 1H), 6.93 (d, /= 7.9 Hz, 1H), 4.85 - 4.72 (m, 2H), 3.74 (apparent d, / = 4.8 Hz, 2H), 3.26 - 3.19 (m, 1H), 2.83 (d, / = 2.6 Hz, 1H), 2.81 (s, 1H), 2.62 - 2.52 (m, 1H), 2.55 (dd,/= 12.2, 5.4 Hz, 1H), 2.49-2.41 (m, 1H) 1.83 (dt,/=5.4, 12.2 Hz, 1H), 1.17 (s, 3H); ¹³ CNMR(75.5 MHz) £202.1, 198.8, 173.1, 162.0, 159.3, 151.5, 138.6, 126.2, 120.6, 115.2, 114.1,69.9, 44.2, 40.3,36.7,36.3,31.6,21.7, 17.8; IR (CH ₂ C1 ₂ ) 1756, 1624 cm ^"1 ; LRMS (EI, 20 eV) m/z 326 (M ⁺ , 1), 298 (100), 283 (36); HRMS (EI) calcd for

Ci ₉ H ₁₈ 0 ₅ (M ⁺ ) m/z 326.1154, found 326.1162. [0185] Compound 16, M.p.230 - 233 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtOAc = 1:1:1, R _f = 0.1; iNMR^OO MHz, DMSO-<¾) £8.46 (br. s, 1H), 7.12(d,/=8.0Hz, 1H), 7.00 (d,/=8.0Hz, 1H), 6.50 (br. s, 1H), 5.16 (dd, /= 6.5, 10.6 Hz, 1H), 4.93 (apparent q, /= 18.8 Hz, 2H), 4.60 (br. s, 1H), 3.62 - 3.50 (m, 3H), 2.96 (d, / = 11.7 Hz, 1H), 2.75 -2.63 (m, 2H), 2.30 - 2.21 (m, 3H), 1.88 (q, /= 12.3 Hz, 1H), 1.74 - 1.55 (m, 1H), 0.96 (s, 3H); ¹³ CNMR(75.5 MHz, DMSO-<¾) £ 173.9, 164.1, 149.8, 143.0, 130.6, 125.0, 123.7, 121.6, 118.6, 70.9, 69.5, 61.2, 37.2, 33.2, 32.3, 30.9, 27.7, 25.6, 18.3; IR (CH ₂ C1 ₂ ) 3650, 2953, 1743, 1675 cm ^"1 ; LRMS (FAB) m/z 329 (M ⁺ , 20), 307 (35), 219 (49), 154 (100); HRMS (EI) calcd for C ₁₉ H ₂₀ O ₄ (M ⁺ - H ₂ 0) m/z 312.1362, found 312.1377.

[0186] Compound 17: M.p.245 - 247 °C; analytical TLC (silica gel 60), ra-Hexane:

THF:EtOAc= 1:1:1, R _f = 0.35; ^: H NMR (300 MHz) 7.12 (d,/=6.6Hz, 1H), 6.41 (d,/ = 6.6 Hz, 1H), 4.70 (br. s, 2H), 4.09 (d, /= 5.3 Hz, 1H), 3.76 (br. s, 2H), 2.64 - 2.56 (m, 1H), 2.58 (q, /= 5.8 Hz, 2H), 2.51 - 2.44 (m, 1H), 2.33 - 2.23 (m, 1H), 2.27 (td, /= 5.7, 14.5 Hz, 1H), 2.16-2.07 (m, 2H), 1.97 (br. s, 1H), 1.65 (td,/= 12.2, 6.3 Hz, 1H), 1.16 (s, 3H); ¹³ C NMR (75.5 MHz) £ 195.6, 173.2, 159.9, 151.8, 140.1, 133.6, 125.5, 121.0, 69.9, 67.4, 61.5, 57.3, 43.8, 38.5, 32.9 (x2), 24.3, 17.5, 16.9; IR (CH ₂ C1 ₂ ) 3625, 2947, 1755, 1645 cm ^"1 ;

LRMS (EI, 20 eV) m/z 328 (M ⁺ , 26), 310 (24), 297 (75), 71 (100); HRMS (EI) calcd for C ₁₉ H ₂₀ O ₅ (M ⁺ ) m/z 328.1311, found 328.1310.

[0187] Compound 18, m.p.263 - 265 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtOAc = 2:3:3, R _f = 0.2; ¾ NMR (300 MHz, DMSO-<4) 7.21 (d,/ = 4.6Hz, 1H), 4.81 (br. s, 2H), 4.58 (t, /= 5.4 Hz, 1H), 3.98 (d, / = 4.7 Hz, 1H), 3.54 (d, /= 5.4 Hz, 1H), 3.42 (dt, /= 5.4, 6.5 Hz, 2H), 2.87 - 2.82 (m, 1H), 2.32 - 2.03 (m, 4H), 2.01-1.98 (m, 1H), 1.92 (t,/= 13.5 Hz, 1H), 1.44- 1.35 (m, 2H), 0.95 (s, 3H); ¹³ C NMR (67.5MHz, DMSO- e) £190.9, 172.8, 161.8, 141.6, 139.8, 123.0, 70.1, 63.8, 62.4, 59.3, 58.9, 51.5, 34.2 (x2), 32.3, 29.4, 22.4, 16.6, 13.7; IR(CH ₂ C1 ₂ ) 3655, 2928, 1756, 1672 cm ^"1 ; LRMS (FAB) m/z 345 (M ⁺ +l, 10), 219 (60), 135 (69), 109 (100).

Preparation of compound T68: [0188] To a solution of compound 18 (3.9 mg, 0.01 13 mmol) in MeOH (1 mL) at 0

°C were added H ₂ 0 ₂ (0.012 mL, 5%, 0.0176 mmol) and NaOH aqueous solution (0.017 mL, 1 M, 0.0176 mmol). The mixture was stirred for 50 min, then diluted with EtOAc, and washed with water. The organic layer was dried with anhydrous Na ₂ SO/t, filtered, and concentrated. The residue was purified by flash column chromatography to afford compound T68 (3.2 mg, 45% total yield from compound 16).

[0189] Compound T68, m.p. 245 - 247 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtOAc = 1 : 1 : 1, R _f = 0.35; i NMR ^OO MHz, CD ₂ C1 ₂ ) 4.71 (br. s, 2H), 4.06 (apparent q, / = 3.0 Hz, 2H), 3.75 (q, / = 5.6 Hz, 2H), 3.45 (d, / = 5.3 Hz, 1H), 2.85 - 2.81 (m, 1H), 2.45 - 2.30 (m, 1H), 2.30 - 1.95 (m, 5H), 1.72 (t, / = 5.2 Hz, 1H), 1.69 - 1.65 (m, 1H), 1.32 (td, / = 1 1.8, 5.6 Hz, 1H), 1.08 (s, 3H); ¹³ C NMR (75.5 MHz, CD ₂ C1 ₂ ) δ 198.4, 173.4, 159.9, 125.9, 70.4, 65.6, 62.6, 61.7, 61.2, 61.1, 58.5, 56.7, 40.9, 35.7, 31.6, 30.9, 23.5, 17.5, 14.0; IR (CH ₂ C1 ₂ ) 3675, 3609, 2949, 1751, 1605 cm ; LRMS (EI, 20 eV) m/z 360 (M ⁺ , 5), 342 (20), 293 (20), 94 (100); HRMS (EI) calcd for Ci ₉ H ₂₀ O ₇ (M ⁺ ) m/z 360.1209, found 360.1214.

Preparation of compound T70 and T71:

[0190] To a solution of compound T68 (9 mg, 0.028 mmol) in MeOH (3 mL) was added Eu(FOD) ₃ (33 mg, 0.03mmol). The resulting homogenous solution was cooled down to - 78 °C. NaBH ₄ ( 1.2 mg, 0.03 mmol) was added. The reaction was stirred for 20 min. The mixture was poured into water, and extracted with EtOAc. The organic extracts were dried with Na ₂ S0 ₄ , filtered, and concentrated. The residue was purified by flash column chromatography to afford white solids T71 (2.1 mg, 23% yield) and T70 (5.7 mg, 63% yield).

[0191] Compound T70, m.p. 275 - 278 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtOAc = 2:3 :3, R _f = 0.35; ¾ NMR (300 MHz, CD ₃ CN) £4.73 - 4.68 (m, 2H), 4.20 (d, / = 5.8 Hz, 1H), 3.84 (d, / = 3.0 Hz, 1H), 3.65 (d, / = 5.7 Hz, 1H) 3.63 - 3.56 (m, 2H), 3.51 (d, / = 3.1 Hz, 1H), 3.47 (d, / = 3.1 Hz, 1H), 2.85 (t, / = 4.5 Hz, 1H), 2.70 - 2.63 (m, 1H), 2.25 - 2.14 (m, 3H), 2.10 - 2.01 (m, 1H), 1.86 (dd, / = 13.4, 15.0 Hz, 1H), 1.57 (ddd, / = 5.8, 7.5, 13.4 Hz, 1H), 1.42 (ddd, / = 1.6, 5.4, 12.5 Hz, 1H), 1.27 (dt, / = 6.2, 1 1.9 Hz, 1H), 1.04 (s, 3H); ^lj C NMR (75.5 MHz, CD ₃ CN) δ 174.4, 162.6, 125.1, 71.2, 67.9, 66.2, 62.7, 62.4, 58.3, 57.3, 57.2, 56.0, 41.2, 36.6, 35.4, 30.1, 23.9, 17.8, 14.1; IR (CH ₂ C1 ₂ ) 3696, 3601, 1755, 1618 cm ^"1 ; LRMS (FAB) m/z 363 (M ⁺ +l, 15), 219 (100).

[0192] Compound T71, m.p. 288-291 °C; analytical TLC (silica gel 60), n-

Hexane : THF : EtO Ac = 2:3:3, R _f = 0.32; ¾ NMR (300 MHz) δ 4.69 (br. s, 2H), 3.93 (d, / = 3.1 Hz, 1H), 3.80-3.75 (m, 2H), 3.56 (d, / = 3.0 Hz, 1H), 3.51-3.41 (m, 2H), 2.96 (d, / = 10.1 Hz, 1H), 2.74-2.69 (m, 1H), 2.36-2.25 (m, 2H), 2.25-2.10 (m, 3H), 2.08-1.93 (m, 2H), 1.65-1.54 (m, 1H), 1.25 (dt, / = 5.6, 11.7 Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (75.5 MHz) δ 173.1, 159.7, 125.6, 74.7, 69.9, 66.0, 61.9, 60.4, 60.3, 58.5, 56.6, 55.4, 40.4, 35.8, 35.3, 29.8, 23.6, 17.0, 13.6; IR (CH ₂ C1 ₂ ) 3683, 3628, 2963, 1755, 1620 cm ^"1 ; LRMS (ESI) m/z 363 (M ⁺ +H).

[0193] Compound 19, m.p. 185 - 188 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtO Ac = 2:3:3, R _f = 0.70; ¾ NMR (300 MHz) 7.42 - 7.36 (m, 2H), 7.29 - 7.23 (m, 1H), 7.20 - 7.17 (m, 2H), 4.71 - 4.68 (m, 2H), 4.45 - 4.31 (m, 2H), 4.07 (d, / = 2.7 Hz, 1H), 3.96 (d, / = 2.7 Hz, 1H), 3.44 (d, / = 5.4 Hz, 1H), 2.84 - 2.79 (m, 1H), 2.39 - 2.34 (m, 1H), 2.31 - 2.26 (m, 2H), 2.19 (dt, / = 14.6, 5.5 Hz, 1H), 2.25 - 2.10 (m, 1H), 2.00 (dd, / = 13.4, 13.2 Hz, 1H), 1.64 - 1.58 (m, 1H), 1.32 (dt, / = 5.9, 11.9 Hz, 1H), 1.08 (s, 3H); ¹³ C NMR (75.5 MHz) δ 196.9, 173.0, 159.3, 153.4, 151.1, 129.5 (x2), 126.2, 125.8, 121.1 (x2), 69.9, 65.3, 63.8, 61.5, 61.3, 60.8, 60.6, 55.9, 40.6, 35.4, 30.6, 28.2, 23.3, 17.1, 13.8; IR (CH ₂ C1 ₂ ) 2969, 1759, 1733, 1605 cm ^"1 ; LRMS (FAB) m/z 481 (M ⁺ +H).

[0194] Compound T69, m.p. 193 - 196 °C; analytical TLC (silica gel 60), n-

Hexane:THF: EtOAc = 2:3:3, R _f = 0.6; ¾ NMR (300 MHz) £4.76 - 4.63 (m, 3H), 4.22 - 4.11 (m, 2H), 4.07 (d, / = 2.8 Hz, 1H), 3.92 (d, / = 2.6 Hz, 1H), 3.45 (d, / = 5.4 Hz, 1H), 3.12 (q, / = 6.7 Hz, 2H), 2.84 - 2.80 (m, 1H), 2.39 - 2.31 (m, 1H), 2.28 - 2.12 (m, 3H), 2.05 (dt, / = 14.8, 6.6 Hz, 1H), 1.99 (apparent t, / = 14.6 Hz, 1H), 1.59 (dd, / = 4.7, 12.8 Hz, 1H), 1.52 (q, / = 7.2 Hz, 2H), 1.32 (td, / = 12.0, 5.9 Hz, 1H), 1.07 (s, 3H), 0.92 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75 MHz) δ 196.9, 173.0, 159.4, 156.1, 125.8, 69.9, 65.2, 61.6, 60.9, 60.6 (x2), 59.7, 56.0, 42.8, 40.6, 35.4, 30.6, 28.4, 23.3, 23.2, 17.1, 13.8, 11.2; IR (CH ₂ C1 ₂ ) 3683, 3447, 2936, 1757, 1727, 1517 cm ^"1 ; LRMS (ESI) m/z 446 (M ⁺ +H); LRMS (FAB) m/z 446 (M ⁺ +H).

[0196] Scheme 5 Reagents and conditions: a) THPOCH ₂ CH ₂ C≡CH, PdCl ₂ (PPh ₃ )2,

Cul, Et ₃ N, THF, 35 °C, 98%; b) H ₂ , Pd/C, EtOH, 40-50 °C, 100%; c) TsOH H ₂ 0, MeOH, rt, 88%; d) NaBH ₄ , EtOH, CH ₂ C1 ₂ , 0 °C; e) NaI0 ₄ , MeOH, H ₂ 0, rt, 70%; f) CF ₃ COCH ₃ , OXONE ^® , NaHC0 ₃ , CH ₃ CN, Na ₂ (EDTA), 0 °C, 34% for 25 and 30% for T125; g) Dess- Martin periodinane, CH ₂ C1 ₂ , rt, 71%. Compounds 20-T126 were prepared under reaction conditions similar to compounds 6-12 of Example 1, Scheme 1 as described above except the use of THPOCH ₂ CH ₂ C≡CH in step a in place of THPOCH ₂ C≡CH in step e.

[0197] Compound 20, analytical TLC (silica gel 60), EtOAc: «-hexane =4:6, R/=

0.17; ¹ H NMR (400 MHz, CDC1 ₃ ) <H3.15 (s, 1H), 7.55 (d, / = 8.0 Hz, 1H), 6.89 (d, / = 8.0 Hz, 1H), 4.85 - 4.69 (m, 3H), 3.92 (dd, / = 8.0, 16.0 Hz, 2H), 3.67 (dd, / =8.0, 16.0 Hz, 1H), 3.55 - 3.50 (m, 1H), 3.40-3.10 (m, 1H), 2.83 - 2.74 (m, 4H), 2.58 - 2.34 (m, 3H), 2.07 - 1.98 (m, 1H), 1.87 - 1.69 (m, 3H), 1.64 - 1.52 (m, 4H), 1.14 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) <S 202.1, 173.1, 164.2, 159.4, 151.8, 140.3, 125.9, 115.0, 114.0, 112.4, 98.7, 92.7, 75.6, 69.9, 65.5, 62.1, 40.0, 36.7, 36.2, 31.4, 30.5, 25.4, 21.5, 21.1, 19.3, 17.7; LRMS (ESI) m/z 437.0 (M ⁺ +H). [0198] Compound 21, analytical TLC (silica gel 60), EtOAc: «-hexane= 4:6, R/=

0.35; ¾ NMR (300 MHz, CDC1 ₃ ) δ 12.9 (s, OH), 7.28 (d, / = 7.3 Hz, 1H), 6.78 (d, / = 7.8 Hz, 1H), 4.80 - 4.60 (m, 2H), 4.47 (s, 1H), 3.90 - 3.60 (m, 2H), 3.50 - 3.25 (m, 2H), 3.20 - 3.10 (m, 2H), 2.73 (d, / = 8.5 Hz, 2H), 2.60 - 2.20 (m, 5H), 1.70 - 1.40 (m, 10H), 1.33 (t, / = 7.3 Hz, 1H), 1.06 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 202.3, 173.2, 162.1, 160.0, 149.6, 137.1, 129.8, 125.8, 114.7, 113.9, 98.8, 70.0, 67.3, 62.3, 40.3, 36.4, 36.3, 32.2, 31.6, 29.4, 28.9, 25.8, 25.4, 21.7, 19.6, 17.7; LRMS (ESI) m/z 441.0 (M ⁺ +H).

[0199] Compound 22, analytical TLC (silica gel 60), EtOAc: «-hexane =4:6, R/=

0.07; ¾ NMR (400 MHz, CDC1 ₃ ) δ 12.9 (s, OH), 7.28 (d, / = 7.3 Hz, 1H), 6.78 (d, / = 7.8 Hz, 1H), 4.80 - 4.60 (m, 2H), 3.67 (s, 2H), 3.19 (m, 1H), 2.80 (d, / = 8.9 Hz, 2H), 2.60 - 2.30 (m, 5H), 2.17 (br s, 1H), 1.90 - 1.50 (m, 5H), 1.14 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) <S 202.3, 173.4, 162.0, 160.0, 149.6, 137.1,129.8, 125.9, 114.7, 113.5, 70.0, 62.5, 40.3, 36.4, 36.2, 32.3, 31.5, 28.8, 25.4, 21.7, 17.7; LRMS (ESI) m/z 379.0 (M ⁺ +Na).

[0200] Compound 24, analytical TLC (silica gel 60), EtOAc: «-hexane=8:2, R =

0.27; ¾ NMR (400 MHz, CDC1 ₃ ) δ 6.98 (d, / = 6.6 Hz, 1H), 6.36 (d, / = 6.7 Hz, 1H), 4.80 - 4.60 (m, 2H), 4.04 (d, / = 5.2 Hz, 1H), 3.63 (t, / = 6.0 Hz, 2H), 2.94 (s, 1H), 2.86 (s, 1H), 2.60 - 2.50 (m, 1H), 2.49 - 2.38 (m, 1H), 2.35 - 2.20 (m, 4H), 2.10 (d, / = 13.8 Hz, 1H), 1.83 (brs, 1H), 1.60-1.50 (m, 6H), 1.13 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 194.7, 173.3, 160.1, 150.8, 137.9, 136.3, 125.4, 121.0, 70.0, 66.9, 62.4, 57.1, 43.7, 38.3, 32.8, 32.2, 28.3, 24.4, 24.2, 17.4, 16.8; LRMS (ESI) m/z 379.0 (M ⁺ +Na).

[0201] Compound 25: analytical TLC (silica gel 60), EtOAc: «-hexane=8:2, R/=

0.28; ¾ NMR (300 MHz, CDC1 ₃ ) δ 7.01 (d, / = 4.6 Hz, 1H), 4.80-4.60 (m, 2H), 3.82 (d, / = 6.2 Hz, 1H), 3.70 - 3.50 (m, 3H), 2.75 (d, / = 17.5 Hz, 1H), 2.40 - 2.10 (m, 6H), 2.02 (t, / = 19.0 Hz, 1H), 1.66 (dd, / = 6.3, 16.5 Hz, 1H), 1.60 - 1.40 (m, 4H), 1.34 (dd, / = 7.9, 15.9 Hz, 1H), 1.10 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 191.0, 173.3, 159.9, 144.1, 139.3, 125.6, 70.1, 64.0, 62.3, 62.1, 59.3, 51.6, 40.8, 35.0, 32.0, 30.0, 29.0, 24.1, 23.1, 17.0, 13.6; LRMS (ESI) m/z 395.0 (M ⁺ +Na).

[0202] Compound T125: analytical TLC (silica gel 60), EtOAc: «-hexane= 8:2, R =

0.15; ¾ NMR (500 MHz, CDC1 ₃ ) δ 4.77 - 4.66 (m, 2H), 4.07 (d, / = 2.5 Hz, 1H), 3.89 (d, / = 2.5 Hz, 1H), 3.63 (t, / = 6.3, 12.6 Hz, 2H), 3.42 (d, / = 5.5 Hz, 1H), 2.83 (d, / = 13.1 Hz, 1H), 2.35 (d, / = 17.2 Hz, 1H), 2.27 - 2.21 (m, 1H), 2.20 - 2.10 (m, 1H), 2.00 - 1.74 (m, 3H), 1.75 - 1.70 (m, 1H), 1.68 - 1.50 (m, 3H), 1.49 - 1.40 (m, 2H), 1.33 (td, / = 5.8, 12.0, 12.0 Hz, 1H), 1.06 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 191 A, 173.1, 159.6, 125.6, 70.0, 65.2, 63.5, 62.2, 61.0, 60.7, 60.6, 56.1, 40.5, 35.3, 32.2, 30.5, 28.1, 23.1, 20.5, 17.0, 13.8. LRMS (ESI) m/z 411.0 (M ⁺ +Na).

[0203] Compound T126, analytical TLC (silica gel 60), EtOAc: «-hexane=8:2, R =

0.15; H NMR (500 MHz, CDC1 ₃ ) δ 4.16 - 4.71 (m, 2H), 4.06 (d, / = 2.7 Hz, 1H), 3.87 (d, / = 2.6 Hz, 1H), 3.43 (d, / = 5.4 Hz, 1H), 2.82 (d, / = 13.2 Hz, 1H), 2.39 - 2.25 (m, 3H), 2.24 - 2.13 (m, 2H), 2.00 - 1.91 (m, 2H), 1.86 - 1.81 (m, 1H), 1.80 - 1.69 (m, 2H), 1.60 (dd, / = 5.2, 12.2 Hz, 1H), 1.31 (td, / = 6.2, 12.0, 12.0 Hz, 1H), 1.07 (s, 3H); ¹³ C NMR (125 MHz, CDCI ₃ ) δ 197.2, 177.7, 173.1, 159.4, 125.8, 70.0, 65.3, 63.3, 61.1, 60.7, 60.6, 56.1, 40.6, 35.4, 33.2, 30.6, 27.8, 23.3, 19.5, 17.1, 13.8; LRMS (ESI) m/z 425.0 (M ⁺ +Na).

[0205] Scheme 6 Reagents and conditions: a) NaOH, Et ₃ BnNCl, 4-benzyloxy-2- butenyl bromide, CH ₂ C1 ₂ , rt, 80%; b) N, N-dimethylaniline, reflux, 90%; c) H ₂ , Pd/C, EtOH, rt; d) NaBH ₄ , MeOH, 0 °C; e) NaI0 ₄ , MeOH, H ₂ 0, 0 °C; f) CF ₃ COCH ₃ , OXONE ^® ,

NaHC0 ₃ , CH ₃ CN, Na ₂ (EDTA), 0 °C, 20% for compound 32 and 16% for compound 33 from 27; g) H ₂ 0 ₂ , NaOH, MeOH, H ₂ 0, 0 °C, 90% for Till and 90% for ΊΊ10. Compounds 28- 33 were prepared under reaction conditions similar to compounds 7-12 of Example 1, Scheme 1 as described above. Compounds Ti l l and Tl 10 were prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above.

Preparation of compound 26:

[0206] To a solution of compound 4 (20 mg, 0.07 mmol) in CH ₂ C1 ₂ (5 mL) was added a solution of NaOH (3 mg, 0.075 mmol) in H ₂ 0 (2 mL) and catalytic amount of Et ₃ BnNCl (3 mg). After stirring for 10 min, 4-benzyloxy-2-butenyl bromide (17 mg, 0.125 mmol) was added slowly and stirring was continued for 5 h. It was diluted with CH ₂ C1 ₂ (30 mL), washed with H ₂ 0, brine, dried over anhydrous Na ₂ SO/ _t . After removing the solvent, the residue was purified by silica gel column chromatography to form compound 26 as white solid in 80% yield.

[0207] Compound 26, ¾ NMR (300 MHz) δ 7.47-6.86 (m, 8H), 5.91 - 5.84 (m, 2H),

4.79 - 4.70 (m, 4H), 4.53 (s, 2H), 4.15 (d, / = 4.8 Hz, 2H), 3.09 - 3.07 (m, 1H), 2.77 (dd, / = 6.0, 18.0 Hz, 1H), 2.66 - 2.48 (m, 4H), 1.83 - 1.72 (m, 1H), 1.11 (s, 3H).

Preparation of compound 27:

[0208] Compound 26 was dissolved in N,N-dimethylaniline (3 mL) and heated to vigorous reflux and kept at this temperature overnight. After cooling to room temperature, it was poured into a mixture of 12M HC1 (5 mL) and ice. And the mixture was extracted with CH ₂ C1 ₂ (15 mLx3), washed with brine, saturated NaHC0 ₃ , brine. The combined organic layers were washed H ₂ 0, brine, dried over anhydrous Na ₂ S04. After removing the solvent, the residue was purified by silica gel column chromatography to get compound 27 as white powder in 90% yield.

[0209] Compound 27, ¾ NMR (400 MHz) δ 13.0 (s, 1H), 7.41 (d, / = 7.9 Hz, 1H),

7.33-7.26 (m, 5H), 6.88 (d, / = 8.0 Hz, 1H), 6.11 - 6.02 (m, 2H), 5.18 - 5.12 (m, 2H), 4.82 - 4.71 (m, 2H), 4.55 - 4.54 (m, 2H), 4.16 (q, / = 6.8 Hz, 1H), 3.79 - 3.68 (m, 2H), 3.20 - 3.15 (m, 1H), 2.81 -2.75 (m, 2H), 2.62 - 2.50 (m, 2H), 2.46 - 2.38 (m, 1H), 1.80 (dt,/=6.4, 12.1 Hz, 1H), 1.14 (s, 3H).

[0210] Compound 28, ¾ NMR (300 MHz) 13.1 (s, 1H), 7.42 (d, /= 7.8 Hz, 1H),

6.92 (d, /= 7.8 Hz, 1H), 4.79 - 4.77 (m, 2H), 3.81 - 3.77 (m, 2H), 3.22 - 3.10 (m, 2H), 2.83

- 2.80 (m, 2H), 2.20 - 2.06 (m, 4H), 1.80 - 1.60 (m, 3H), 1.06 (s, 3H), 0.89 (t, /= 7.5 Hz, 3H).

[0211] Compound 30: H NMR (300 MHz) £7.05 (d, /= 6.7 Hz, 1H), 6.45 (d, / =

6.7 Hz, 1H), 4.70 (d, br, 2H), 4.06 (d, /= 5.3 Hz, 1H), 3.70 - 3.63 (m, 2H), 2.78 - 2.73 (m, 1H), 2.62 - 2.59 (m, 1H), 2.51 - 2.43 (m, 1H), 2.33 - 2.22 (m, 4H), 2.13 (t, /= 13.6 Hz, 1H),

I.70-1.61 (m, 2H), 1.55 (dd,/=5.6, 12.0 Hz, 1H), 1.16 (s, 3H), 0.88 (d,/=7.4Hz, 3H); ¹³ CNMR(75 MHz) δ 195.2, 173.3, 160.0, 151.4, 138.9, 136.6, 125.4, 120.9, 69.9, 67.3, 64.6, 57.3, 43.7, 43.0, 38.4, 32.8, 24.2, 22.6, 17.4, 16.8, 11.9.

[0212] Compound 31: ¹ HNMR(400 MHz) £7.04 (d, /= 6.7 Hz, 1H), 6.44 (d, / =

6.7 Hz, 1H), 4.70 (d, br, 2H), 4.09 (d, /= 5.2 Hz, 1H), 3.70 - 3.66 (m, 2H), 2.79 - 2.78 (m, 1H), 2.65 - 2.55 (m, 1H), 2.52 - 2.45 (m, 1H), 2.32 - 2.26 (m, 2H), 2.21 - 2.08 (m, 2H), 1.69

- 1.62 (m, 3H), 1.52 (dd,/=5.6, 12.0 Hz, 1H), 1.17 (s, 3H), 0.89 (t, /= 7.4 Hz, 3H); ¹³ C NMR (75.5 MHz) δ 195.2, 173.9, 159.9, 151.3, 138.5, 136.9, 125.4, 120.9, 69.9, 67.1, 65.2, 57.3, 43.6, 42.2, 38.4, 32.8, 24.2, 22.6, 17.4, 16.9, 11.9.

[0213] Compound 32, ¾ NMR (300 MHz, CDC1 ₃ ) δ 4.70 (s, br, 2H), 4.08 (d, /= 2.7

Hz, 1H), 3.95 (d, / = 2.7 Hz, 1H), 3.74 - 3.70 (m, 2H), 3.44 (d, /= 5.4 Hz, 1H), 2.84 - 2.80 (m, 1H), 2.35 -2.34 (m, 1H), 2.22 (td, /= 5.8, 14.8 Hz, 1H), 2.21 - 2.10 (m, 1H),1.99 (t, / = 13.9 Hz, 1H), 1.93 - 1.90 (m, 1H), 1.64 - 1.58 (m, 1H), 1.47 - 1.43 (m, 2H), 1.35 (dt, /= 5.6,

II.9 Hz, 1H), 1.08 (s, 3H), 0.95 (t,/= 7.4 Hz, 3H); ¹³ CNMR(75 MHz, CDC1 ₃ )5198.0, 173.4, 159.7, 126.2, 70.4, 65.4, 64.5, 62.4, 61.2, 60.9, 56.6, 56.4, 43.3, 40.9, 35.7, 32.7, 30.9, 19.8, 17.5, 14.2, 12.3.

[0214] Compound 33, ¾ NMR (300 MHz, CDC1 ₃ ) δ 4.76 (s, br, 2H), 4.08 (d, /= 2.8

Hz, 1H), 4.02 (d, / = 2.8 Hz, 1H), 3.65 - 3.56 (m, 2H), 3.44 (d, /= 5.4 Hz, 1H), 2.75 - 2.86 (m, 1H), 2.33 - 2.19 (m, 4H), 2.00 (t, /= 15 Hz, 1H), 1.72 (s, br, 1H), 1.60 (dd, /= 5.4, 12.6 Hz, 1H), 1.46- 1.42 (m, 2H), 1.30 (dt, /= 5.6, 11.9 Hz, 1H), 1.08 (s, 3H), 0.94 (t, /= 7.4 Hz, 3H); C NMR (75 MHz, CDC1 ₃ ) 5 197.1, 173.1, 159.4, 125.7, 70.0, 69.9, 65.1, 64.8, 60.9, 60.5, 60.2, 55.9, 40.8, 40.5, 35.3, 30.6, 23.4, 20.1, 17.1, 13.7, 11.9.

[0215] Compound Till, ¾ NMR (300 MHz, CDC1 ₃ ) 4.70 (s, br, 2H), 4.08 (d, / =

2.7 Hz, 1H), 3.95 (d, / = 2.7 Hz, 1H), 3.74 - 3.70 (m, 2H), 3.44 (d, / = 5.4 Hz, 1H), 2.84 - 2.80 (m, 1H), 2.35 - 2.34 (m, 1H), 2.22 (td, / = 5.8, 14.8 Hz, 1H), 2.21 - 2.10 (m, 1H), 1.99 (t, / = 13.9 Hz, 1H), 1.93 - 1.90 (m, 1H), 1.64 - 1.58 (m, 1H), 1.47 - 1.43 (m, 2H), 1.35 (dt, / = 5.6, 11.9 Hz, 1H), 1.08 (s, 3H), 0.95 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 198.0, 173.4, 159.7, 126.2, 70.4, 65.4, 64.5, 62.4, 61.2, 60.9, 56.6, 56.4, 43.3, 40.9, 35.7, 32.7, 30.9, 19.8, 17.5, 14.2, 12.3; LRMS (EI, 20 eV) m/z 388 (M ⁺ , 2), 373 (10), 357 (26), 341 (74), 151 (100); HRMS (EI) calcd for C ₂₁ H ₂₄ O ₇ (M ⁺ ) m/z 388.1522, found 388.1520.

[0216] Compound T110, ¾ NMR (300 MHz, CDC1 ₃ ) £4.76 (s, br, 2H), 4.08 (d, / =

2.8 Hz, 1H), 4.02 (d, / = 2.8 Hz, 1H), 3.65 - 3.56 (m, 2H), 3.44 (d, / = 5.4 Hz, 1H), 2.75 - 2.86 (m, 1H), 2.33 - 2.19 (m, 4H), 2.00 (t, / = 15 Hz, 1H), 1.72 (s, br, 1H), 1.60 (dd, / = 5.4, 12.6 Hz, 1H), 1.46 - 1.42 (m, 2H), 1.30 (dt, / = 5.6, 11.9 Hz, 1H), 1.08 (s, 3H), 0.94 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 197.1, 173.1, 159.4, 125.7, 70.0, 69.9, 65.1, 64.8, 60.9, 60.5, 60.2, 55.9, 40.8, 40.5, 35.3, 30.6, 23.4, 20.1, 17.1, 13.7, 11.9; LRMS (EI, 20 eV) m/z 388 (M ⁺ , 9), 341 (10), 341 (92), 151 (100); HRMS (EI) calcd for C ₂₁ H ₂₄ O7 (M^ m/z 388.1522, found 388.1524

[0217] Scheme 7. Synthetic route to T109.

[0218] Scheme 7 Reagents and conditions: a) 0 ₃ , MeOH, - 65 °C; PPh ₃ , 0 °C; b)

NaBH ₄ , MeOH, 0 °C, 68% for 2 steps; c) H ₂ , Pd/C, EtOH, rt; d) NaI0 ₄ , MeOH, H ₂ 0, 0 °C, 80% for 2 steps; e) Na ₂ EDTA, acetone, NaHC0 ₃ , MeCN, H ₂ 0, OXONE ^® ; f) H ₂ 0 ₂ , NaOH, H ₂ 0, 0 °C, 90% for 2 steps. Compounds 34 and 36-T109 were prepared under reaction conditions similar to compounds 16-T68 of Example 1, Scheme 4 as described above except CF3COCH3 was omitted in the preparation of compound 38. Compound 35 was prepared under reaction conditions similar to compound 7 of Example 1, Scheme 1 as described above.

[0219] Compound 34, ¾ NMR (400 MHz) δ 13.0 (s, 1H), 9.83 (s, 1H), 7.43 (d, / =

7.9 Hz, 1H), 7.42 - 7.28 (m, 5H), 6.93 (d, / = 8.0 Hz, 1H), 4.79 - 4.76 (m, 2H), 4.56 - 4.54 (m, 2H), 4.22 - 4.19 (m, 1H), 4.14 - 4.10 (m, 1H), 3.93 - 3.89 (m, 1H), 3.20 (br s, 1H), 2.83 - 2.79 (m, 2H), 2.56 - 2.50 (m, 2H), 2.32 - 2.30 (m, 1H), 1.80 (dt, / = 6.4, 12.1 Hz, 1H), 1.16 (s, 3H).

[0220] Compound 37, ¾ NMR (400 MHz) 7.23 (d, / = 6.8 Hz, 1H), 6.43 (d, / =

6.8 Hz, 1H), 4.70 (s, br, 2H), 4.07 (d, / = 5.2 Hz, 1H), 3.85 (m, 4H), 3.05 - 3.02 (m, 1H), 2.66 - 2.60 (m, 1H), 2.52 - 2.49 (m, 1H), 2.32 - 2.25 (m, 2H), 2.15 - 2.08 (m, 2H), 1.69 - 1.64 (m, 1H), 1.16(s, 3H); ¹³ C NMR (100 MHz) δ 195.0, 173.4, 159.9, 152.1, 140.0, 134.5, 125.5, 121.0, 70.0, 67.7, 63.4, 63.3, 57.5, 43.7, 42.5, 38.5, 32.5, 24.2, 17.5, 16.7.

[0221] Compound T109, H NMR (400 MHz) £4.72 - 4.69 (m, 2H), 4.25 (d, / = 2.8

Hz, 1H), 4.08 (d, / = 3.2 Hz, 1H), 3.85 (s, br, 2H), 3.79 (t, / = 5.2 Hz, 2H), 3.44 (d, / = 5.2 Hz, 1H), 2.83 - 2.80 (m, 1H), 2.54 - 2.48 (m, 1H), 2.39 - 2.35 (m, 1H), 2.24 (td, / = 5.6, 14.8 Hz, 1H), 2.21 - 2.18 (m, 1H), 2.04 - 1.96 (m, 1H), 1.61 - 1.59 (m, 1H), 1.31 (dt, / = 6.0, 12.0 Hz, 1H), 1.08 (s, 3H); ¹³ C NMR (125 MHz) δ 197.5, 172.9, 159.1, 125.9, 69.9, 67.7, 65.1, 63.2, 61.3, 60.8, 60.7, 60.5, 52.8, 41.1, 40.6, 35.4, 30.6, 23.3, 17.1, 13.8; LRMS (EI, 20 eV) m/z 379 (M ⁺ +l, 7), 317 (60), 151 (100); HRMS (EI) calcd for C ₂₀ H ₂₆ O ₇ (M ⁺ ) m/z 378.1678, found 378.1663.

[0223] Scheme 8 Reagents and conditions: (a) Methyl crotonate, PdCl ₂ (dppf),

NaHC0 ₃ , TBACl, DMF, 65 °C, 60%; (b) H ₂ , Pd/C, MeOH, rt, 88%; (c) NaBH ₄ , MeOH, 0 °C, 71%; (d) NaI0 ₄ , MeOH/H ₂ 0, 0 °C, 100%; (e) OXONE ^® , CF ₃ COCH ₃ , NaHC0 ₃ , MeCN, Na ₂ (EDTA), 24%; (f) 5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 50%. Compounds 41-T130 were prepared under reaction conditions similar to compounds 16-T68 of Example 1, Scheme 4 as described above. Compound 40 was prepared under reaction conditions similar to compound 7 of Example 1, Scheme 1 as described above.

Preparation of compound 39:

[0224] Compound 5 (206 mg, 0.5 mmol), NaHC0 ₃ (126 mg, 1.5 mmol) and TBACl

(114 mg, 0.5 mmol) were dissolved in DMF (5 mL). The mixture was degassed for at least 1 h using a balloon filled with Ar. Methyl crotonate (213 μί, 1 mmol) and PdCl ₂ (dppf) (37 mg, 0.05 mmol) were added and the reaction mixture was heated to 65 °C for 33 h. When the completion of reaction was indicated by TLC, the mixture was transferred to a separating funnel and diluted with EtOAc (50 mL). The organic layer was washed with 1 M HC1 (2 ^χ 25 mL), H20 (6 ^χ 25 mL) and brine (2 ^χ 25 mL). The organic layer was dried over Na ₂ SO/ _t , filtered via short-pad silica and concentrated. The crude product was purified by flash chromatography (20% EtOAc in «-hexane) to give the purified product as white solid (114 mg, 60% yield). [0225] Analytical TLC (silica gel 60), 40% EtOAc in ra-hexane _, Rf = 0.45; ¾ NMR

(400 MHz, CDC1 ₃ ) δ 13.1 (s, 1H), 7.39 (d, / = 7.9 Hz, 1H), 6.93 (d, / = 7.9 Hz, 1H), 5.99 (t, / = 1.2 Hz, 1H), 4.84-4.73 (m, 2H), 3.75 (s, 3H), 3.22-3.20 (m, 1H), 2.84 (d, / = 3.0 Hz, 1H), 2.82 (s, 1H), 2.65-2.54 (m, 2H), 2.53 (s, 3H), 2.48-2.40 (m, 1H), 1.83 (td, / = 12.0, 6.6 Hz, 1H), 1.18 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 202.2, 173.1, 166.9, 161.3, 159.2, 154.2,

152.2, 136.1, 131.7, 126.2, 119.7, 115.3, 113.9, 69.9, 51.1, 40.2, 36.8, 36.4, 31.6, 21.7, 19.3, 17.8; IR (CH ₂ C1 ₂ ) 2951, 2925, 2853, 1756, 1713, 1628, 1433, 1335 cm ; LRMS (EI) m/z 382.1 (M ⁺ , 18), 351.1 (M ⁺ -OCH ₃ , 13), 350.1 (31); HRMS (EI) calcd for C ₂₂ R ₂₂ 0 ₆ (M ⁺ ) m/z 382.1411, found 382.1414.

Preparation of compound 40:

[0226] Compound 40, analytical TLC (silica gel 60), 40% EtOAc in w-hexane _, R =

0.45; ¾ NMR (400 MHz, CDC1 ₃ ) δ 13.1 (s, 1H), 7.39 (d, / = 7.9 Hz, 1H), 6.93 (d, / = 7.9 Hz, 1H), 4.78 (AB system, 2H), 3.75 (s, 3H), 3.22-3.20 (m, 1H), 2.87-2.72 (m, 3H), 2.57-2.52 (m, 3H), 2.42-2.35 (m, 2H), 1.81 (td, / = 12.0, 6.6 Hz, 1H), 1.31 (d, / = 6.9 Hz, 3H), 1.18 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 202.2, 173.2, 172.9, 161.8, 159.6, 149.9, 134.7, 134.6, 132.7, 126.1, 114.9, 113.7, 69.9, 51.5, 40.3, 40.2, 36.5, 36.4, 31.6, 29.6, 21.7, 19.6, 17.8; IR (CH ₂ CI ₂ ) 2961, 2936, 2867, 1756, 1736, 1435, 1339 cm ; LRMS (EI) 383.1 m/z (M ⁺ -H, 5), 382.1 (18), 351.1 (13), 350.1 (31); HRMS (EI) calcd for C ₂₂ R _u 0 ₆ (M ⁺ ) m/z 384.1567, found 384.1568.

[0227] Compound 41, Analytical TLC (silica gel 60), as a mixture of diastereomers;

40% EtOAc in ra-hexane _, = 0.21; ¾ NMR (400 MHz, CDC1 ₃ ) δ 8.70 (s, 0.8H), 8.62 (s, 0.2H), 7.11-7.08 (m, 1H), 6.88 (d, / = 8.2 Hz, 1H), 5.26-5.22 (m, 1H), 4.78 (AB system, 2H), 4.15-4.14 (d, / = 5.6 Hz, 0.8H), 4.07 (d, / = 5.6 Hz, 0.2H), 3.65 (s, 3H), 3.63-3.56 (m, 1H), 2.77 (d, / = 14.0 Hz, 1H), 2.72-2.67 (m, 1H), 2.59-2.53 (m, 1H), 2.49-2.44 (m, 2H), 2.38- 2.32 (m, 2H), 2.06-1.99 (m, 1H), 1.65 (td, / = 12.0, 6.6 Hz, 1H), 1.28 (t, / = 7.1 Hz, 3H), 1.11-1.10 (m, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 174.5, 174.2, 162.1, 154.1, 144.2, 130.6,

126.3, 125.4, 122.6, 115.9, 70.5, 68.2, 51.8, 41.6, 40.3, 36.6, 32.6, 29.7, 28.8, 22.9, 20.2, 18.0. IR (CH ₂ C1 ₂ ) 3316 (br), 3066, 2956, 2932, 2873, 1753, 1681, 1576, 1441, 1343 cm ; LRMS (EI) m/z 386.0 (M ⁺ , 2), 338.1 (M ⁺ -OCH ₃ -OH, 24), 336.1 (31); HRMS (EI) calcd for

C ₂₂ H ₂₄ O6 (M ⁺ -H ₂ ) m/z 384.1567, found 384.1568. [0228] Compound 42, Analytical TLC (silica gel 60), CH ₂ CI ₂ : EtOAc: «-hexane =

1: 1 : 1 _, R = 0.30; ¾NMR (300 MHz, CDC1 ₃ ) δ 7.01 (d, / = 6.7 Hz, 1H), 6.41 (d, / = 6.7 Hz, 1H), 4.70 (s, 2H), 4.07 (d, / = 5.4 Hz, 1H), 3.65 (s, 3H), 3.21 (sextet, / = 6.9 Hz, 1H), 2.61 (dd, / = 15.3, 6.5 Hz, 2H), 2.50-2.38 (m, 2H), 2.35-2.23 (m, 3H), 2.12 (d, / = 13.8 Hz, 1H), 1.65 (td, / = 12.2, 6.4 Hz, 1H), 1.18-1.16 (m, 6H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 193.8, 173.3, 172.5, 160.0, 151.4, 138.9, 136.9, 125.4, 120.8, 67.0, 66.9, 57.2, 51.5, 43.6, 39.5, 38.3, 32.8, 29.9, 24.2, 19.1, 17.5, 16.8; IR (CH ₂ C1 ₂ ) 3069, 2936, 2861, 1755, 1659, 1441, 1335, 1260 cm ^"1 ; LRMS (EI) m/z 384.1 (M ⁺ , 7), 353.1 (M ⁺ -OCH ₃ , 6), 352.1 (10); HRMS (EI) calcd for C ₂₂ H ₂₄ O ₆ (M ⁺ ) m/z 384.1567, found 384.1566.

[0229] Compound 43, Analytical TLC (silica gel 60), isolated as a mixture of diastereomers (ratio = 3.74: 1), 60% EtOAc in ra-hexane _, R/= 0.19; ¾ NMR (400 MHz, CDCI ₃ ) δ 6.99 (d, / = 4.5 Hz, 0.79 lH), 6.96 (d, / = 4.7 Hz, 0.21 x lH), 4.75-4.65 (m, 2H), 3.82-3.81 (m, 1H), 3.68 (d, / = 11.2 Hz, 0.21H), 3.65 (s, 0.79x3H), 3.63 (s, 0.21 x3H), 3.60 (d, / = 5.2 Hz, 0.21H), 3.19 (sextet, / = 7.0 Hz, 0.21 x lH), 3.12 (sextet, / = 7.1 Hz, 0.79x lH), 2.79-2.70 (m, 1H), 2.60 (dd, / = 15.4, 6.2 Hz, 0.79x ffl), 2.43-2.30 (m, 0.79x2H + 0.21 x3H), 2.24-2.15 (m, 2H), 2.04 (dd, / = 15.5, 11.1 Hz, 1H), 1.70-1.66 (m, 1H), 1.34-1.24 (m, 2H), 1.19-1.07 (m, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 190.2, 173.1, 172.2, 159.5, 147.0, 138.7, 125.9, 69.9, 64.0, 62.1, 59.4, 51.6, 51.4, 40.9, 39.4, 35.0, 30.7, 30.1, 23.4, 18.9, 17.1, 13.7; IR (CH ₂ CI ₂ ) 3058, 2934, 1756, 1682, 1438 cm ^"1 ; LRMS (EI) m/z 400.1 (M ⁺ , 36), 384.1 (M ⁺ -16, 12), 371.1 (11); HRMS (EI) calcd for C ₂₂ H ₂₄ O ₇ (M ⁺ ) m/z 400.1517, found 400.1513.

[0230] Compound T130, Analytical TLC (silica gel 60), isolated as a mixture of diastereomers (ratio = 4: 1), 80% EtOAc in ra-hexane _, = 0.66; ¾ NMR (400 MHz, CDC1 ₃ ) δ 4.71 (AB system, 2H), 4.05 (d, / = 2.7 Hz, 1H), 3.89 (d, / = 2.7 Hz, 0.2* 1H), 3.86 (d, / = 2.7 Hz, 1H), 3.68 (s, 3H), 3.42 (d, / = 5.4 Hz, 1H), 2.83-2.79 (m, 1H), 2.64-2.59 (m, 0.2* 1H), 2.55-2.48 (m, 2H), 2.44-2.32 (m, 1.8H), 2.25-2.13 (m, 2H), 1.99 (dd, / = 13.5, 2.5 Hz, 1H), 1.61-1.58 (m, 1H), 1.35-1.29 (m, 1H), 1.08 (s, 3H), 1.05 (d, / = 6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 196.6, 173.0, 172.2, 69.9, 65.0, 60.8, 60.6, 60.4, 60.3, 55.8, 51.7, 40.6, 36.7, 35.4, 30.9, 30.6, 23.3, 17.1, 14.6, 13.8; IR (CH ₂ C1 ₂ ) 3036, 1651 cm ^"1 ; LRMS (EI) m/z 400.1 (M ⁺ -0, 16), 384.1 (M ⁺ -0 ₂ , 13); HRMS (EI) calcd for C ₂₂ H ₂₄ O ₈ (M ⁺ ) m/z 416.1466, found 416.1426.

[0231] Scheme 9. Synthetic route to T131.

[0232] Scheme 9 Reagents and conditions: (a) EDC, HOBt, Et ₃ N, propylamine,

CH ₂ C1 ₂ , rt, 29%; (b) PdCl ₂ (dppf), TBAC1, NaHC0 ₃ , 44, DMF, 65 °C, 52%; (c) Pd/C, H ₂ , MeOH, 40 °C, 84%; (d) NaBH ₄ , Me OH, 0 °C, 35%; (e) NaI0 ₄ , MeOH, H ₂ 0, rt, 96%; (f) CF ₃ COCH ₃ , OXONE ^® , NaHC0 ₃ , CH ₃ CN, aq Na ₂ (EDTA), 0 °C, 41%; (g) 5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 90%. Compounds 47-T131 were prepared under reaction conditions similar to compounds 16-T68 of Example 1, Scheme 4 as described above. Compound 46 was prepared under reaction conditions similar to compound 7 of Example 1, Scheme 1 as described above.

Preparation of compound 44:

[0233] Compound 44 was prepared in accordance with or by slight modification of the methods set forth in Nitsche, F.; Aumann, R.; Froehlich, R. /. Organomet. Chem. 2007, 692, 2971. To a solution of crotonic acid (4.18 g, 40 mmol) in CH ₂ CI ₂ (100 mL) were added EDC (8.43 g, 44 mmol), HOBt (5.94 g, 44 mmol) and Et ₃ N (11.2 mL, 80 mmol). The mixture was stirred at room temperature for 45 min. Propylamine (6.49 mL, 80 mmol) was then added. The reaction was stirred at room temperature for another 1 h. When the reaction was complete, the content was washed with 1 M HC1 (30 mL ^χ 2), saturated NaHCC (30 mL ^χ 2), H ₂ O (30 mL x 2), then brine (30 mL ^χ 1). The organic layer was dried over Na ₂ S04, filtered and concentrated. The crude mixture was purified by flash chromatography by 10- 20% EtOAc in «-hexane to give purified product as white solid (1.68 g, 29%).

[0234] Analytical TLC (silica gel 60), 60% EtOAc in ra-Hexane _, R/= 0.43; ¾ NMR

(400 MHz, CDCI ₃ ) δ 6.80 (sextet, / = 7.2 Hz, 1H), 6.30 (br s, 1H), 5.88 (dd, / = 14.3, 1.6 Hz, 1H), 3.26 (q, / = 6.7 Hz, 2H), 1.83 (dd, / = 6.9, 1.6 Hz, 3H), 1.54 (sextet, / = 7.3 Hz, 2H), 0.92 (t, / = 7.4 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 166.1, 139.0, 125.3, 41.1, 22.7, 17.5, 11.3; IR (CH ₂ C1 ₂ ) 3450, 3315, 2971, 2875, 1624, 1516, 1443, 1335 cm ^"1 ; LRMS (EI) m/z 128.1 (M ⁺ +H, 36), 127.1 (M ⁺ , 93), 112.1 (95); HRMS (EI) calcd for C ₇ H ₁₃ ON (M ⁺ ) m/z 127.0992, found 127.0989.

Preparation of compound 45:

[0235] Compound 5 (206 mg, 0.5 mmol), NaHC0 ₃ (126 mg, 1.5 mmol) and TBAC1

(114 mg, 0.5 mmol) were dissolved in DMF (5 mL). The mixture was degassed for at least 1 h using a balloon filled with Ar. The amide (127 mg, 1 mmol) and PdCl ₂ (dppf) (37 mg, 0.05 mmol) were added and the reaction mixture was heated to 65 °C for 3 days. When the completion of reaction was indicated by TLC, the mixture was transferred to a separating funnel and diluted with EtOAc (50 mL). The organic layer was washed with 1 M HC1 (25 mL x 2), H ₂ 0 (25 mL ^χ 6) and brine (25 mL ^χ 2). The organic layer was dried over Na ₂ SO/ _t , filtered via short-pad silica and concentrated. The crude product was purified by flash chromatography (5-60% EtOAc in «-hexane) to give the purified product as yellow solid (225 mg, 52%).

[0236] Analytical TLC (silica gel 60), 60% EtOAc in ra-hexane _, = 0.32; ¾ NMR

(300 MHz, CDCI ₃ ) δ 13.1 (s, 1H), 7.40 (d, / = 7.9 Hz, 1H), 6.91 (d, / = 7.9 Hz, 1H), 5.89 (s, 1H), 5.56 (t, / = 4.5 Hz, 1H), 4.48 (AB system, 2H), 3.29 (q, / = 7.0 Hz, 2H), 3.25-3.18 (m, 1H), 2.83 (d, / = 2.2 Hz, 1H), 2.81 (s, 1H), 2.67-2.52 (m, 2H), 2.48 (s, 3H), 2.46-2.42 (m, 1H), 1.82 (dt, / = 12.3, 6.6 Hz, 1H), 1.57 (sextet, / = 7.2 Hz, 2H), 1.17 (s, 3H), 0.96 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 202.3, 173.1, 166.6, 161.2, 159.4, 151.7, 147.7, 136.2, 131.9, 126.0, 123.1, 115.1, 113.8, 69.9, 41.0, 40.1, 36.6, 36.3, 31.5, 22.8, 21.6, 18.6, 17.7, 11.4; IR (CH ₂ C1 ₂ ) 3446, 2927, 1757, 1671, 1634, 1513, 1432, 1337 cm ^"1 ; LRMS (EI) m/z 409.1 (M , 8), 350.1 (23), 323.1 (30); HRMS (EI) calcd for C ₂₄ H ₂₇ O ₅ N (M ⁺ ) m/z

409.1884, found 409.1883.

[0237] Compound 46, analytical TLC (silica gel 60), 60% EtOAc in w-hexane _, R =

0.28; ¾ NMR (300 MHz, CDC1 ₃ ) δ 13.2 (s, 1H), 7.43 (d, / = 7.9 Hz, 1H), 6.90 (d, / = 7.9 Hz, 1H), 5.86 (t, / = 4.5 Hz, 1H), 4.80 (AB system, 2H), 3.58 (q, / = 7.0 Hz, 1H), 3.20-3.11 (m, 3H), 2.82 (d, / = 8.9 Hz, 2H), 2.66-2.51 (m, 3H), 2.46-2.39 (m, 2H), 1.80 (dt, / = 12.3, 6.6 Hz, 1H), 1.46-1.36 (m, 2H), 1.32 (d, / = 6.9 Hz, 3H), 1.15 (s, 3H), 0.80 (t, / = 7.4 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 202.3, 173.2, 171.6, 161.6, 159.6, 135.2, 132.6, 125.9, 114.8, 113.8, 69.9, 43.0, 40.9, 40.2, 36.4, 36.2, 31.5, 30.7, 22.6, 21.6, 19.6, 17.6, 11.1; IR (CH ₂ C1 ₂ ) 3446, 2934, 1756, 1667, 1621, 1521 cm ^"1 ; LRMS (EI) m/z 411.2 (M ⁺ , 6), 386.0 (62); HRMS (EI) calcd for C ₂₄ H ₂₉ O ₅ N (M ⁺ ) m/z 411.2040, found 411.2040.

[0238] Compound 47, analytical TLC (silica gel 60), isolate as a pair of

diastereomers; EtOAc, _, = 0.45; ¾ NMR (400 MHz, CD ₃ OD) δ 7.88 (s, 0.53H, H-bonded phenol), 7.06 (d, / = 7.9 Hz, 1H), 6.87 (d, / = 7.9 Hz, 1H), 5.12 (t, / = 8.7 Hz, 1H), 4.96-4.82 (m, 2H), 3.62-3.57 (m, 1H), 3.13-3.01 (m, 2H), 2.84 (d, / = 13.2 Hz, 1H), 2.60-2.49 (m, 2H), 2.42-2.32 (m, 4H), 1.99 (q, / = 12.1 Hz, 1H), 1.63 (dt, / = 12.3, 6.6 Hz, 1H), 1.41 (sextet, / = 7.2 Hz, 2H), 1.25-1.22 (m, 3H), 1.10 (s, 3H), 0.85-0.80 (m, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 175.1, 173.8, 164.0, 154.6, 144.1, 130.4, 126.2, 124.2, 122.2, 115.2, 70.8, 68.3,

42.7, 40.7, 40.3, 36.3, 32.4, 30.2, 29.4, 22.1, 21.8, 19.1, 17.5, 10.3; IR (CH ₂ C1 ₂ ) 3446, 2934, 1756, 1667, 1621, 1521 cm ^"1 ; LRMS (EI) m/z 395.2 (M ⁺ -H ₂ 0, 10), 386.0 (19); HRMS (EI) calcd for C ₂₄ H ₂ 9O ₄ N (M ⁺ -0) m/z 395.2091, found 395.2091.

[0239] Compound 48, analytical TLC (silica gel 60), isolate as a pair of

diastereomers; EtOAc, _, = 0.33; ¾ NMR (400 MHz, CD ₃ OD) δ 7.07-7.04 (m, 1H), 6.42- 6.40 (m, 1H), 5.69 (t, / = 8.7 Hz, 1H), 4.75-4.65 (m, 2H), 4.07-4.06 (m, 1H), 3.20-3.10 (m, 3H), 2.61-2.58 (m, 1H), 2.46 (dd, / = 14.1, 6.1 Hz, 2H), 2.33-2.21 (m, 3H), 2.15-2.08 (m, 2H), 1.65 (dt, / = 12.3, 6.6 Hz, 1H), 1.51-1.42 (m, 2H), 1.22 (d, / = 9.0 Hz, 3H), 1.15 (s, 3H), 0.85-0.80 (m, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 194.5, 173.2, 171.4, 159.9, 151.4, 139.0, 137.7, 125.5, 121.0, 69.9, 67.0, 57.3, 43.7, 42.4, 41.1, 38.4, 32.8, 31.2, 24.2, 22.8, 18.9, 17.5,

16.8, 11.3; IR (CH ₂ C1 ₂ ) 3449, 3037, 1638 cm ^"1 ; LRMS (EI) m/z 411.1 (M ⁺ , 10), 386.0 (18), 362.1 (11); HRMS (EI) calcd for C ₂₄ H ₂₉ O ₅ N (M ⁺ ) m/z 411.2040, found 411.2040. [0240] Compound 49, analytical TLC (silica gel 60), isolate as a pair of

diastereomers, dr = 4: 1; EtOAc, _, Rf = 0.13; ¾ NMR (400 MHz, CDC1 ₃ ) δ 7.03 (d, / = 4.3 Hz, 0.8H), 6.98 (d, / = 4.3 Hz, 0.2H), 5.69 (t, / = 5.1 Hz, 1H), 4.71 (AB system, 2H), 3.83-3.81 (m, 1H), 3.60-3.57 (m, 1H), 3.19-3.13 (m, 2H), 3.02 (sextet, / = 6.8 Hz, 1H), 2.77-2.74 (m, 1H), 2.44 (dd, / = 15.6, 5.8 Hz, 1H), 2.40-2.30 (m, 1H), 2.27-2.20 (m, 2H), 2.10 (dd, / = 14.1, 9.5 Hz, 1H), 2.11-1.88 (m, 1H), 1.69-1.66 (m, 1H), 1.49 (sextet, / = 7.3 Hz, 2H), 1.32 (dd, / = 12.0, 5.8 Hz, 1H), 1.18-1.14 (m, 3H), 1.12 (s, 3H), 0.90 (t, / = 7.4 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 190.8, 173.1, 171.1, 159.5, 147.1, 139.3, 125.8, 69.9, 63.9, 62.2, 59.5, 51.5, 42.1, 41.2, 40.8, 35.0, 31.9, 30.1, 23.4, 22.8, 18.7, 17.1, 13.6, 11.3; IR (CH ₂ C1 ₂ ) 3032, 2928, 1752, 1644, 1516, 1447 cm ^"1 ; LRMS (EI) 427.1 (M ⁺ , 17), 411.1 (M ⁺ -0, 14), 394.1 (13), 380.1 (17); HRMS (EI) calcd for C ₂₄ H ₂₉ O ₆ N (M ⁺ ) m/z 427.1989, found 427.1986.

[0241] Compound T131, analytical TLC (silica gel 60), isolate as a pair of diastereomers, dr = 4: 1; EtOAc, _, = 0.33; ¾ NMR (300 MHz, CDC1 ₃ ) δ 5.60D5.52 (br m, 1H), 4.70 (AB system, 2H), 4.05 (d, / = 2.8 Hz, 0.63H), 4.00 (d, / = 2.8 Hz, 0.37H), 3.93 (d, / = 2.7 Hz, 0.3H), 3.89 (d, / = 2.7 Hz, 0.7H), 3.48 (d, / = 5.3 Hz, 0.2H), 3.41 (d, / = 5.3 Hz, 0.8H), 3.27-3.14 (m, 2H), 2.87-2.77 (m, 1H), 2.44-2.17 (m, 6H), 2.04-1.93 (m, 1H), 1.58- 1.45 (m, 3H), 1.34-1.28 (m, 1H), 1.12 (d, / = 6.4Hz, 1H), 1.07-1.03 (m, 5H), 0.92 (t, / = 7.4 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 197.3, 173.0, 170.7, 159.3, 125.8, 69.9, 65.1, 62.3, 60.7, 60.3, 55.8, 53.4, 41.3, 40.6, 39.2, 35.4, 31.5, 30.5, 23.3, 22.8, 17.1, 14.3, 13.8, 11.4; IR (CH ₂ CI ₂ ) 3032, 2928, 1752, 1644, 1516, 1447 cm ^"1 ; LRMS (EI) m/z 443.1 (M ⁺ , 12), 428.1 (15), 415.1 (10); HRMS (EI) calcd for C ₂₄ H ₂₉ O ₇ N (M ⁺ ) m/z 443.1939, found 443.1930.

[0242] Scheme 10. Synthetic route to T139.

[0243] Scheme 10 Reagents and conditions: (a) cone. H ₂ S0 ₄ , /-PrOH, 70 °C, 70%;

(b) NaBH ₄ , CH ₃ OH, 0 °C, 99%; (c) NaI0 ₄ , CH ₃ OH, H ₂ 0, rt, 96%; (d) CF3COCH3,

OXONE ^® , NaHC0 ₃ , CH ₃ CN, aq Na ₂ (EDTA), 0 °C, 70%; (e) Pd/C, H ₂ , Et ₃ N, CH ₃ CH ₂ OH, rt, 3 h, 40%. Compounds 51-53 were prepared under reaction conditions similar to compounds 16-18 of Example 1, Scheme 4 as described above.

Preparation of compound 50:

[0244] Compound 50 was synthesized in accordance with or by slight modification of the method set forth in Berchtold, G. A.; Buckanin, R. S.; Chen, S. J.; Frieze, D. M.; Sher, F. T. /. Am. Chem. Soc. 1980, 102, 1200. To a solution of 4 (2.0 g, 7.03 mmol) in concentrated H ₂ SC>4 (4.2 mL) at 0°C was added isopropanol (2.7 mL). The mixture was heated to 70 °C for 3h. The resulting reaction was poured into ice-water and extracted with dichloromethane. The organic layer was dried over anhydrous Na ₂ SC>4, filtered, and concentrated. The residue was purified by flash column chromatography to give the desired product 50 (1.6 g, 70%) as a yellow solid.

[0245] Analytical TLC (silica gel 60), EtOAc: ra-hexane=4:6, R/= 0.57; ^: H NMR

(300 MHz, CDCI ₃ ) S 13.05 (s, 1H), 7.42 (d, / = 7.9 Hz, 1H), 6.88 (d, / = 7.9 Hz, 1H), 4.86- 4.70 (m, 2H), 3.35 (sept, / = 6.9 Hz, 1H), 3.26-3.14 (m, 1H), 2.84-1.70 (m, 6H), 1.25 (d, / = 6.9 Hz, 3H), 1.23 (d, / = 6.9 Hz, 3H), 1.14 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) S 202.2, 173.2, 161.7, 159.6, 149.1, 136.0, 133.7, 126.1, 114.7, 113.6, 69.9, 40.3, 36.4, 31.6, 26.2, 25.3, 22.2, 22.1, 21.7, 17.8. HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₄ (M ⁺ ) 326.1518; found 326.1518. [0246] Compound 51, analytical TLC (silica gel 60), EtOAc: «-hexane=4:6, R =

0.32; ¾ NMR (300 MHz, CDC1 ₃ ) ^ 8.43 (s, 1H), 7.14 (d, / = 8.1 Hz, 1H), 6.85 (d, / = 8.1 Hz, 1H), 5.28 - 5.17 (m, 1H), 4.86-4.70 (m, 2H), 3.78 (d, / = 7.0 Hz, 1H), 3.30 (sept, / = 6.9 Hz, 1H), 2.78 (d, / = 13.3 Hz, 1H), 2.60-1.50 (m, 6H), 1.22 (d, / = 6.9 Hz, 6H), 1.09 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) S 175.0, 162.3, 154.3, 143.2, 133.9, 126.0, 125.5, 121.1, 115.5, 70.6, 69.0, 40.4, 36.5, 32.6, 30.3, 26.5, 23.1, 22.6, 22.5, 17.9; LRMS (EI) m/z 328.2 (M ⁺ ); HRMS (EI) calcd. for C ₂₀ H ₂₄ O ₄ (M ⁺ ) m/z 328.1675; found 328.1659.

[0247] Compound 52, analytical TLC (silica gel 60), EtOAc: «-hexane= 2:3, R/=

0.30; ¾ NMR (300 MHz, CDC1 ₃ ) ^ 6.97 (d, / = 6.7 Hz, 1H), 6.41 (d, / = 6.7 Hz, 1H), 4.86- 4.70 (m, 2H), 4.07 (d, / = 5.0 Hz, 1H), 2.92 (sept, / = 6.9 Hz, 1H), 2.60-1.50 (m, 7H), 1.15 (s, 3H), 1.12 (d, / = 6.9 Hz, 3H), 1.10 (d, / = 6.9 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) S 194.1, 173.3, 160.1, 150.3, 142.3, 135.1, 125.4, 121.0, 70.0, 66.7, 57.1, 43.6, 38.2, 32.8, 26.2, 24.2, 21.7, 21.4, 17.5, 16.9; LRMS (EI) m/z 326.1 (M+); HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₄ (M ⁺ ) m/z 326.1518; found 328.1520.

[0248] Compound 53, analytical TLC (silica gel 60), EtOAc: «-hexane= 1 : 1, R/=

0.18; i NMR (300 MHz, CDC1 ₃ ) S 6.93 (dd, / = 4.7 Hz, 1H), 4.70 (AB system, 2H), 3.82 (d, / = 4.7 Hz, 1H), 3.60 (d, / = 5.3 Hz, 1H), 2.85 (sept, / = 6.9 Hz, 1H), 2.80 (d, / = 13.0 Hz, 1H), 2.40-1.90 (m, 4H), 1.70 (ddd, / = 13.0, 5.4, 1.2 Hz, 1H), 1.36 - 1.25 (m, 1H), 1.13 (s, 3H), 1.08 (d, / = 6.9 Hz, 3H), 1.01 (d, / = 6.9 Hz, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) S 184.0, 159.6, 150.3, 136.6, 134.0, 126.0, 70.0, 62.1, 59.5, 54.7, 51.6, 41.0, 35.1, 30.2, 27.2, 23.5, 21.5, 21.4, 17.2, 13.7; LRMS (EI) m/z 342.1 (M ⁺ ); HRMS (EI) calcd. for C ₂₀ H ₂₂ O ₅ (M ⁺ ) m/z 342.1467, found 342.1464.

Preparation of T139:

[0249] Compound 53 (20 mg, 0.0585 mmol) in ethanol (5 mL) was hydrogenolyzed in the presence of triethylamine (24 μί, 1.72 ^χ 10 ^"4 mmol) and 5% Pd/C (20 mg) using a balloon filled with H ₂ gas at room temperature for 3 h. The mixture was filtered through silica gel, washed with CH ₂ CI ₂ : CH ₃ OH (9: 1, v/v), and the filtrate was concentrated in vacuo. Product T139 (8 mg, 40%) was obtained as a white solid by flash column chromatography.

[0250] Analytical TLC (silica gel 60), EtOAc: ra-hexane= 1 : 1, R/= 0.10; ¾ NMR

(500 MHz, CDCI ₃ ) S 6.73 (d, / = 4.9 Hz, 1H), 4.70-4.67 (m, 2H), 3.54 (d, / = 5.6 Hz, 1H), 2.90-2.87 (m, 1H), 2.78 (dd, / = 6.1, 18.6 Hz, 1H), 2.69 (d, / = 18.3 Hz, 1H), 2.62 (d, / = 12.9 Hz, 1H), 2.39 (d, / = 14.9 Hz, 1H), 2.20-1.90 (m, 5H), 1.53 (td, / = 12.3, 5.2 Hz, 1H), 1.28-0.88 (m, 9H); ¹³ C NMR (125 MHz, CDC1 ₃ ) S 191.8, 173.3, 160.4, 145.2, 139.1, 125.9, 75.3, 70.1, 64.5, 62.9, 40.2, 38.9, 32.9, 28.3, 26.7, 23.0, 21.7, 21.4, 17.2, 13.0; LRMS (EI) m/z 344.1 (M+); HRMS (EI) calcd for C ₂₀ H ₂₄ O ₅ (M ⁺ ) m/z 344.1642; found 344.1615.

[0252] Scheme 11 Reagents and conditions: (a) Et ₂ Zn, CF ₃ C0 ₂ H, -13 °C, CH ₂ I ₂ ,

CH ₂ CI ₂ , 97%; (b) NaBH ₄ , EtOH/CH ₂ Cl ₂ , rt, crude; (c) NaI0 ₄ /H ₂ 0, CH ₃ OH, rt, crude; (d) CF ₃ COCH ₃ , OXONE®, NaHC0 ₃ , CH ₃ CN, aq Na ₂ (EDTA), 0 °C, 29% of T140 for 3 steps and 10% of T141 for 3 steps. Compounds 55-T141 were prepared under reaction conditions similar to compounds 16-18 of Example 1, Scheme 4 as described above.

Preparation of compound 54:

[0253] To a solution of Et ₂ Zn (1 M in «-hexane, 8.3 mL, 6.15 mmol) in CH ₂ CI ₂ (15 mL), CF ₃ CO ₂ H (0.5 mL, 6.15 mmol) in CH ₂ CI ₂ (5 mL) was added dropwise at -13 °C for 15 min, followed by dropwise addition of a solution of CH ₂ I ₂ (0.5 mL, 6.15 mmol) in CH ₂ CI ₂ (5 mL). After stirring for another 30 min at -13 °C, a solution of compound 14 (199 mg, 0.615 mmol) in CH ₂ CI ₂ (2 mL) was added dropwise and the reaction mixture was stirred for 20 min at -13°C. The reaction was then quenched with saturated aqueous NH ₄ CI (5 mL), the organic layer was separated, and the aqueous solution was extracted with CH ₂ CI ₂ (20 mL ^χ 3). The combined organic layer was dried over anhydrous Na ₂ SC>4 and concentrated. Purification by flash column chromatography afforded compound 54 (200 mg, 97%) as a yellow solid. [0254] Analytical TLC (silica gel 60), EtOAc: ra-hexane= 1 :4, R/= 0.16; ¾ NMR

(400 MHz, CDC1 ₃ ) δ 12.67 (s, 1H, OH), 7.51 (d, / = 7.8 Hz, 1H), 6.87 (d, / = 7.8 Hz, 1H), 4.90-4.60 (m, 2H), 3.19 (br s, 1H), 2.85-2.70 (m, 2H), 2.60-2.50 (m, 4H), 2.48-2.30 (m, 1H), 1.81 (td, / = 6.8, 12.8 Hz, 1H), 1.14 (s, 3H), 1.05-0.99 (m, 1H), 0.55-0.50 (m, 2H), 0.22-0.18 (m, 2H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 202.1, 173.3, 162.1, 159.7, 149.5, 136.7, 129.9, 126.0, 1 14.6, 1 13.5, 69.9, 40.4, 36.5, 36.3, 33.3, 31.6, 21.8, 17.8, 10.0, 4.7; LRMS (EI) m/z 338.2 (M ⁺ ) HRMS (EI) calcd. for m/z 338.1518; found 338.1510.

[0255] T140: Analytical TLC (silica gel 60), EtOAc: ra-hexane=l : 1, = 0.20; ¾

NMR (400 MHz, CDC1 ₃ ) δ 7.17 (d, / = 4.6 Hz, 1H), 4.75-4.65 (m, 2H), 3.83 (d, / = 4.6 Hz, 1H), 3.59 (d, / = 5.7 Hz, 1H), 2.75 (d, / = 13.3 Hz, 1H), 2.37 (d, / = 18.3 Hz, 1H), 2.25-2.10 (m, 3H), 2.09-1.97 (m, 2H), 1.72-1.65 (m, 1H), 1.31 (td, / = 12.0, 5.9 Hz, 1H), 1.12 (s, 3H), 0.81-0.70 (m, 1H), 0.55-0.47 (m, 2H), 0.12-0.05 (m, 2H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 191.0, 173.2, 159.7, 144.2, 138.7, 125.7, 70.0, 63.9, 62.2, 59.2, 51.6, 40.8, 35.0, 33.6, 30.1, 29.6, 23.3, 17.1, 13.6, 8.4, 4.6; LRMS (EI) m/z 354.2 (M ⁺ ); HRMS (EI) calcd. for C ₂₁ H ₂₂ O ₅ (M^ m/z 354.1467; found 354.1463.

[0256] T141: Analytical TLC (silica gel 60), EtOAc: ra-hexane=5 :5, = 0.30; ¾

NMR (500 MHz, CDC1 ₃ ) δ 4.75-4.64 (m, 2H), 4.08-4.05 (m, 2H), 3.43 (d, / = 5.5 Hz, 1H), 3.82 (d, / = 13.3 Hz, 1H), 2.38 (d, / = 13.5 Hz, 1H), 2.24-2.12 (m, 2H), 1.98 (t, / = 7.3, 13.5 Hz, 1H), 1.85-1.74 (m, 2H), 1.60 (dd, / = 12.4, 12.4 Hz, 1H), 1.31 (td, / = 6.3, 12.4, 12.4 Hz, 1H), 1.07 (s, 3H), 0.70-0.64 (m, 1H), 0.48-0.45 (m, 2H), 0.12-0.09 (m, 2H); ¹³ C NMR (125 MHz, CDCI ₃ ) δ 197.5, 173.0, 159.4, 125.8, 69.9, 65.2, 63.7, 60.7, 60.6, 60.2, 56.2, 40.6, 35.4, 32.3, 30.6, 23.3, 17.1, 13.8, 5.3, 4.7, 3.8; LRMS (EI) m/z 370.2 (M ⁺ ); HRMS (EI) calcd. for C ₂₁ H ₂₂ O6 m/z 370.1416; found 370.1410.

[0258] Scheme 12 Reagents and conditions: (a) Boc ₂ 0, CH ₂ C1 ₂ , 0 °C, 10%; (b)

Biotin-NHS, Et ₃ N, DMAP, DMF, 84%; (c)(i) cone. HC1, CH ₂ C1 ₂ , 0 °C (ii) 1M K ₂ C0 ₃ , EtOH extract, crude; (d) 59, DMAP, Et ₃ N, CH ₂ Cl ₂ /MeOH, rt, 24%; (e) K-selectride, THF, -40°C, 39%. Compound T134 was prepared under reaction conditions similar to compound 17 of Example 1, Scheme 1 as described above.

Preparation of compound 57:

[0259] Compound 57 was synthesized in accordance with or by slight modification of the method set forth in Zuckermann, R. N.; Martin, E. J.; Spellmeyer, D. C; Stauber, G. B.; Shoemaker, K. R; Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani, M. A.; Simon, R;

Banville, S. C; Brown, E. G.; Wang, L.; Richter, L. S.; Moos, W. H. /. Med. Chem. 1994, 37, 2678. To a solution of 2,2 '-(ethylene dioxy)bis-(ethylamine) (4.44 g, 30 mmol) in CH ₂ C1 ₂ (100 mL) was added Boc ₂ 0 solution (6.54 g, 30 mmol pre-dissolved in 40 mL CH ₂ C1 ₂ ) at 0 °C. The reaction was slowly warmed to rt. After 2 h, the reaction was stopped and the mixture was concentrated. The crude product was purified by flash chromatography (20% EtOH in CH ₂ C1 ₂ ) to give purified product (771 mg, 10%).

[0260] For characterization data, refer to: Zuckermann, R. N.; Martin, E. J.;

Spellmeyer, D. C; Stauber, G. B.; Shoemaker, K. R.; Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani, M. A.; Simon, R.; Banville, S. C; Brown, E. G.; Wang, L.; Richter, L. S.; Moos, W. H. /. Med. Chem. 1994, 37, 2678. Preparation of compound 58:

[0261] To a solution of compound 57 (316 mg, 1.30 mmol) in DMF (1.3 mL) was added biotinDNHS (435 mg, 1.30 mmol), DMAP (16 mg, 0.13 mmol) and Et ₃ N (0.178 mL, 1.30 mmol). The resulting mixture was stirred overnight. When TLC indicated completion of reaction, DMF was removed. The mixture was redissolved in CH ₂ CI ₂ (30 mL) and was washed with 0.5 M HC1 (2 5 mL), 1 M K ₂ C0 ₃ (2 5 mL) and brine (1 x 5 mL). The organic layer was dried over Na2SC>4, filtered and concentrated. The resulting product had enough purity for next step (521 mg, 84%). The crude product was a light yellow solid.

[0262] Analytical TLC (silica gel 60), 15% MeOH in CH ₂ C1 _2, R/= 0.83; ¾ NMR

(300 MHz, CDCI ₃ ) δ 6.89 (br s, 1H), 6.84 (br s, 1H), 6.16 (br s, 1H), 5.24 (br s, 1H), 4.52- 4.48 (m, 1H), 4.32-4.28 (m, 1H), 3.61 (s, 4H), 3.59-3.53 (m, 4H), 3.43 (q, / = 4.9 Hz, 2H), 3.35-3.28 (m, 2H), 3.14 (q, / = 6.1 Hz, 1H), 2.89 (dd, / = 12.8, 4.7 Hz, 1H), 2.73 (d, / = 12.8 Hz, 1H), 2.23 (t, / = 7.4 Hz, 2H), 1.78-1.67 (m, 4H), 1.50-1.40 (m, 11H); ¹³ C NMR (75 MHz, CDCI ₃ ) δ 173.3, 164.2, 155.9, 79.1, 69.9, 61.7, 60.1, 55.6, 40.3, 40.2, 39.0, 35.8, 28.3, 28.2, 28.0, 25.5. IR (CH ₂ C1 ₂ ) 3035, 2927, 2855, 1799, 1499, 1447 cm ^"1 ; LRMS (ESI) 497.0 (M ⁺ +Na), 475.1 (M ⁺ +H); HRMS (EI) calcd for C21H38O6N4S (M ⁺ ) 474.2507, found

474.2493.

Preparation of compound 59:

[0263] To a solution of compound 58 (30 mg) in CH2CI2 (1 mL) cone. HC1 (0.5 mL) was added dropwisely. The reaction was stirred at room temperature for 20 min. 1 M K2CO3 was added until the pH was basic. The mixture was then concentrated. The product was extracted by washing repeatedly with EtOH (10 mL), the solid was filtered away and the filtrate was concentrated to give product as yellow solid (27 mg, 100%).

[0264] Analytical TLC (silica gel 60), 15% MeOH in CH ₂ C1 _2, R/= 0.08; H NMR

(400 MHz, CD ₃ OD) δ 4.50 (dd, / = 7.8, 4.5 Hz, 1H), 4.31 (dd, / = 7.8, 4.5 Hz, 1H), 3.63 (s, 4H), 3.55 (t, / = 5.6 Hz, 2H), 3.53 (t, / = 5.4 Hz, 2H), 3.37 (t, / = 5.6 Hz, 2H), 3.25-3.19 (m, 1H), 2.93 (dd, / = 12.7, 5.0 Hz, 1H), 2.80 (t, / = 5.3 Hz, 2H), 2.71 (d, / = 12.7 Hz, 1H), 2.22 (t, / = 7.4 Hz, 2H), 1.78-1.57 (m, 4H), 1.48-1.43 (m, 2H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.2, 166.1, 73.4, 71.3, 70.7, 63.3, 61.6, 57.0, 42.0, 41.1, 40.3, 36.7, 29.7, 29.5, 26.8; IR (CH ₂ CI ₂ ) 3035, 2927, 2855, 1799, 1499, 1447 cm ; LRMS (ESI) m/z 375.0 (M ⁺ +H); HRMS (ESI) calcd for C ₁ 6H30N ₄ O ₄ S (M ⁺ +H) m/z 374.1988, found 374.1998.

Preparation of T128:

[0265] To a solution of compound 59 (30 mg, 0.0802 mmol) in CH ₂ CI ₂ (4 mL)/CH ₃ OH (1 mL) was added compound 12 (20 mg, 0.037 mmol), followed by Et ₃ N (11 μί, 0.0802 mmol) and DMAP (1 mg, 0.00802 mmol). The mixture was stirred at rt for an overnight. After that, the mixture was concentrated and purified by column chromatography to give T128 as white solid (10 mg, 24%).

[0266] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, = 0.23; ¾ NMR

(500 MHz, CDC1 ₃ ) ^ 6.41 (br s, 1H), 5.81 (br s, 1H), 5.54 (br s, 1H), 5.17 (br s, 1H), 4.98- 4.69 (m, 2H), 4.49 (t, / = 4.9 Hz, 1H), 4.33 (t, / = 4.7 Hz, 1H), 4.10-4.08 (m, 2H), 3.92 (d, / = 2.6 Hz, 1H), 3.62-3.55 (m, 8H), 3.49-3.43 (m, 3H), 3.42-3.36 (m, 2H), 3.19-3.14 (m, 1H), 2.92 (dd, / = 4.9, 12.9 Hz, 1H), 2.85-2.81 (m, 1H), 2.73 (d, / = 12.9 Hz, 1H), 2.39-2.35 (m, 1H), 2.27-2.08 (m, 4H), 2.04-1.95 (m, 2H), 1.77-1.51 (m, 8H), 1.49-1.39 (m, 3H), 1.37- 1.29 (m, 1H), 1.07 (s, 3H); ¹³ C NMR (125 MHz) δ 197.3, 173.1, 173.0, 163.3, 159.4, 156.6, 125.8, 70.2, 70.04, 69.98, 69.91, 65.4, 64.1, 63.3, 61.8, 61.1, 60.8, 60.1, 56.2, 55.3, 55.3, 40.8, 40.6, 40.5, 39.2, 35.9, 35.4, 30.6, 29.7, 28.5, 28.1, 25.7, 25.5, 24.1, 23.2, 17.1, 13.9; LRMS (ESI) m/z 797.3 (M ⁺ +Na); HRMS (ESI) calcd for C ₃₇ H ₅₁ N ₄ 0 ₁₂ S (M ⁺ +H) m/z 775.3224, found 775.3220.

Preparation of T134:

[0267] Compound T134, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, R/=

0.15; ¾ NMR (500 MHz, CDC1 ₃ +CD ₃ 0D) δ 4.76-4.73 (m, 2H), 4.51 (dd, / = 4.9, 7.8 Hz, 1H), 4.32 (dd, / = 4.6, 8.3 Hz, 1H), 4.14-4.08 (m, 1H), 4.07-4.02 (m, 1H), 3.65-3.62(m, 5H), 3.61-3.52 (m, 4H), 3.44-3.38 (m, 3H), 3.37-3.34 (m, 3H), 3.20-3.15 (m, 1H), 2.93 (dd, / = 5.0, 12.9 Hz, 1H), 2.74 (d, / = 12.8 Hz, 2H), 2.33-2.29 (m, 1H), 2.26-2.20 (m, 3H), 2.19-2.13 (m, 1H), 2.00-1.90 (m, 1H), 1.76-1.53 (m, 9H), 1.48-1.33 (m, 3H), 1.30-1.23 (m, 1H), 1.11 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ +CD ₃ 0D) δ 174.3, 164.3, 161.7, 161.2, 157.4, 125.4, 73.5, 70.5, 70.3, 70.0, 69.0, 66.5, 65.7, 64.8, 63.5, 62.4, 62.1, 60.6, 60.3, 56.5, 55.8, 55.3, 40.6, 40.5, 39.7, 39.2, 36.0, 35.9, 30.3, 29.8, 28.6, 28.3, 25.6, 24.3, 23.7, 17.2, 13.7; LRMS (ESI) m/z 777.3 (M ⁺ +H); HRMS (ESI) calcd. for C ₃₇ H ₅₃ N ₄ 0 ₁₂ S (M ⁺ +H) m/z

111 315, found 777.3380.

[0269] Scheme 13 Reagents and conditions: (a) 57, Et ₃ N, DMAP, CH ₂ C1 ₂ , 89%; (b)

5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 100%; (c)(i) cone. HC1, CH ₂ C1 ₂ , 0 °C (ii) 1M K ₂ C0 ₃ , EtOH extract, crude; (d) Cy3, EDC, HOBt, Et ₃ N, CH ₂ C1 ₂ , 32%. Compound 61 was prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above. Preparation of compound 60:

[0270] To a solution of ProC3-TN (52 mg, 0.0993 mmol) in CH ₂ C1 ₂ (5 mL) amine portion (37 mg, 1.49 mmol), Et ₃ N (21 μί, 1.49 mmol) and DMAP (1.2 mg, 0.00993 mmol) were added. The reaction was stirred at rt overnight. The reaction was diluted with CH ₂ C1 ₂ (20 mL). The organic layer was washed with 0.5 N HC1 (2 x 5 mL), sat. NaHC0 ₃ (1 x 5 mL), brine (1 x 5 mL) and was dried over Na ₂ SC>4. The organic layer was filtered and was concentrated to give product as white solid (56 mg, 89%).

[0271] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, = 0.45; ¾ NMR

(300 MHz, CD ₃ OD) δ 7.17 (t, / = 4.9 Hz, 1H), 4.86-4.83 (m, 2H), 4.01 (t, / = 6.2 Hz, 1H), 3.96 (d, / = 4.7 Hz, 1H), 3.61 (s, 3H), 3.57-3.49 (m, 5H), 3.31-3.26 (m, 2H), 3.22 (t, / = 5.6 Hz, 2H), 2.91-2.87 (m, 1H), 2.38-2.26 (m, 4H), 2.16-2.13 (m, 1H), 2.04-1.95 (m, 1H), 1.76-1.72 (m, 2H), 1.64-1.58 (m, 2H), 1.44 (s, 9H), 1.09 (s, 3H); ¹³ C NMR (75 MHz, CD ₃ OD) δ 193.3, 176.0, 163.6, 159.0, 144.7, 144.1, 142.3, 141.9, 125.7, 80.0, 72.0, 71.3, 71.1, 71.0, 65.2, 65.0, 64.2, 64.1, 62.2, 61.1, 54.8, 53.0, 41.6, 41.2, 36.1, 31.9, 31.1, 28.8, 28.5, 27.0, 24.1, 17.9, 14.0; LRMS (ESI) m/z 655.2 (M ⁺ +Na); HRMS (ESI) calcd for

C ₃₂ H ₄₅ N ₂ O ₁₁ (M ⁺ +H) m/z 633.3023, found 633.3020.

[0272] Compound 61, analytical TLC (silica gel 60), 10% Me OH in CH ₂ C1 ₂ , _, R/ =

0.46; ¾ NMR (300 MHz, CD ₃ OD) δ 4.86-4.83 (m, 2H), 4.25 (d, / = 2.9 Hz, 1H), 4.09-4.02 (m, 2H), 3.61 (s, 5H), 3.52 (q, / = 6.1 Hz, 5H), 3.43 (d, / = 5.4 Hz, 1H), 3.30-3.26 (m, 1H), 3.23 (t, / = 5.6 Hz, 2H), 2.94-2.90 (m, 1H), 2.38-2.26 (m, 2H), 2.14-2.11 (m, 1H), 1.97-1.89 (m, 2H), 1.82-1.68 (m, 3H), 1.57-1.49 (m, 2H), 1.44 (s, 9H), 1.04 (s, 3H); ¹³ C NMR (75 MHz, CD ₃ OD) δ 199.9, 175.9, 163.3, 158.9, 125.6, 80.1, 71.3, 71.1, 71.0, 66.6, 65.2, 64.3, 62.7, 62.5, 62.3, 57.6, 41.5, 41.2, 36.4, 31.5, 28.8, 26.5, 25.2, 23.9, 17.9, 14.3; LRMS (ESI) m/z 671.2 (M ⁺ +Na); HRMS (ESI) calcd for C32H45N2O12 (M ⁺ +H) m/z 649.2972, found 649.2980.

Preparation of compound 62:

[0273] To a solution of compound 61 (23 mg) in CH2CI2 (1 mL) was added cone. HC1

(0.2 mL) slowly at 0 °C. When TLC indicated completion of reaction, the reaction was basicified by 1 M K ₂ CO ₃ solution. The solution was concentrated. The crude slurry of product was extracted by washing with EtOH (-10 mL). The ethanol extract was

concentrated to give product as a yellow sticky solid (14 mg).

[0274] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/= 0.13; H NMR

(400 MHz, CD ₃ OD) δ 4.86-4.83 (m, 2H), 4.24 (d, / = 2.9 Hz, 1H), 4.06-4.01 (m, 2H), 3.63- 3.59 (m, 5H), 3.55-3.50 (m, 5H), 3.43 (d, / = 5.4 Hz, 1H), 3.30-3.23 (m, 2H), 2.94-2.90 (m, 1H), 2.78 (t, / = 5.3 Hz, 2H), 2.36-2.26 (m, 2H), 2.15-2.10 (m, 1H), 2.01-1.92 (m, 2H), 1.78-1.66 (m, 3H), 1.53 (dd, / = 12.6, 5.5 Hz, 1H), 1.44 (td, / = 12.6, 5.9 Hz, 1H), 1.04 (s, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 198.6, 174.6, 161.8, 157.6, 124.3, 72.2, 69.9, 69.6, 65.2, 63.9, 63.0, 61.3, 60.9, 56.2, 40.7, 40.2, 35.0, 30.1, 25.1, 23.9, 22.5, 16.5, 12.8; LRMS (ESI) m/z 549.1 (M ⁺ +H); HRMS (ESI) cacld for C ₂₇ H ₃₇ N ₂ O ₁₀ (M ⁺ +H) m/z 549.2448, found 549.2440.

Preparation of T136: [0275] Compound 62 (13 mg, 0.0237 mmol) and Cy3 (11 mg, 0.0237 mmol) were dissolved in CH ₂ C1 ₂ (1 mL). EDC (6 mg, 0.0308 mmol), HOBt (4 mg, 0.0308 mmol) and Et3N (4 μί, 0.0308 mmol) were added. The reaction was stirred at rt overnight. The reaction mixture was diluted with CH2Q2 ( 10 mL). The organic layer was washed with 0.1 N HQ (1 ^χ 3 mL), sat. NaHCC>3 (1 x 3 mL) and brine (1 x 3 mL). The layer was dried over NaiSO/t, filtered and concentrated. The crude product was purified by column chromatography using 2-5% EtOH in CH2CI2 to give purified product as red solid (7 mg, 32%). The purified product was subjected to io«-exchange resin (amberlite IRA- 120) to perform io«-exchange with CI ^" ion.

[0276] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, R/ = 0.50; ¾ NMR

(400 MHz, CD ₃ OD) δ 8.55 (t, / = 13.5 Hz, 1H), 7.54 (d, / = 7.1 Hz, 2H), 7.45-7.43 (m, 2H), 7.38-7.33 (m, 2H), 7.32-7.29 (m, 2H), 6.50-6.41 (m,2H), 4.61 (br s, 1H), 4.23 (d, / = 2.8 Hz, 1H), 4.18 (t, / = 7.0 Hz, 1H), 4.04 (d, / = 2.7 Hz, 1H), 4.01-3.97 (m, 1H), 3.70-3.62 (m, 5H), 3.57 (s, 3H), 3.40 (d, / = 5.4 Hz, 1H), 3.36 (t, / = 5.4 Hz, 1H), 3.26 (t, / = 5.4 Hz, 2H), 2.92- 2.84 (m, 1H), 2.43 (t, / = 7.0 Hz, 1H), 2.34-2.24 (m, 3H), 2.20-2.02 (m, 1H), 1.98-1.91 (m, 1H), 1.88-1.73 (m, 14H), 1.76-1.66 (m, 3H), 1.55-1.50 (m, 1H), 1.48-1.40 (m, 1H), 1.01 (s, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 201.0, 193.0, 182.1, 176.7, 176.0, 175.5, 163.1, 152.2, 144.1, 143.4, 142.2, 142.1, 130.0 (2C), 126.8, 126.7, 125.7, 123.6, 123.4, 112.5, 112.4, 103.9, 103.7, 71.3, 71.0, 70.6, 65.3, 64.3, 62.7, 62.4, 61.6, 57.6, 50.6, 45.0, 44.9, 41.6, 40.3, 36.4, 36.3, 34.4, 31.9, 31.5, 28.4, 28.3, 28.2 (2C), 27.9, 27.8, 26.8, 25.3, 24.1, 23.9, 23.2, 17.9, 14.3; LRMS (ESI) m/z 974.3 (M ⁺ ); HRMS (ESI) calcd for C56H ₆ 9N ₄ 0ii ⁺ (M ⁺ ) m/z 973.4597, found 973.4602.

[0277] Scheme 14. Synthetic route to T149.

[0278] Scheme 14 Reagents and conditions: (a) DMAP, Et ₃ N, CH ₂ C1 ₂ , rt, 85%; (b)

K-selectride, anhydrous THF, -78°C, crude; (c) 30% TFA in CH ₂ C1 ₂ (v/v), rt, crude; (d) Cy3-NHS, CH ₂ C1 ₂ , DMAP, rt, 42%. Compound 63 was prepared under reaction conditions similar to compound 17 of Example 1, Scheme 1 as described above.

Preparation of compound 61 via compound 12 (Alternative route):

[0279] To a mixture of 12 (50 mg, 0.0928 mmol) and compound 57 (28 mg, 0.1113 mmol) in CH ₂ C1 ₂ (5 mL) was added one small crystal of DMAP. The reaction mixture was stirred at room temperature overnight under argon, after which time TLC analysis

(CH ₂ C1 ₂ :CH ₃ 0H=15: 1) indicated the reaction was complete. CH ₂ C1 ₂ (45 mL) was added to the crude mixture, and the mixture was washed with NaHC0 ₃ solution until the water layer was colorless. The organic layer was dried over anhydrous Na ₂ SC>4 and the solvent was then removed to give crude product, which was purified by flash column chromatography to afford product 61 as a white solid (51 mg, 85%).

[0280] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ Rf = 0.46; ¾ NMR

(300 MHz, CD ₃ OD) δ 4.86-4.83 (m, 2H), 4.25 (d, / = 2.9 Hz, 1H), 4.09-4.02 (m, 2H), 3.61 (s, 5H), 3.52 (q, / = 6.1 Hz, 5H), 3.43 (d, / = 5.4 Hz, 1H), 3.30-3.26 (m, 1H), 3.23 (t, / = 5.6 Hz, 2H), 2.94-2.90 (m, 1H), 2.38-2.26 (m, 2H), 2.14-2.11 (m, 1H), 1.97-1.89 (m, 2H), 1.82-1.68 (m, 3H), 1.57-1.49 (m, 2H), 1.44 (s, 9H), 1.04 (s, 3H); ¹³ C NMR (75 MHz, CD ₃ OD) δ 199.9, 175.9, 163.3, 158.9, 125.6, 80.1, 71.3, 71.1, 71.0, 66.6, 65.2, 64.3, 62.7, 62.5, 62.3, 57.6, 41.5, 41.2, 36.4, 31.5, 28.8, 26.5, 25.2, 23.9, 17.9, 14.3; LRMS (ESI) m/z 671.2 (M ⁺ +Na); HRMS (ESI) calcd for C ₃₂ H ₄₅ N ₂ O ₁₂ (M ⁺ +H) m/z 649.2972, found 649.2980. Preparation of compound 64:

[0281] To a solution of compound 63 (50 mg, 0.0768 mmol) in CH ₂ Q ₂ (1 mL) was added 0.3 mL of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 1 h. Saturated NaHCC>3 solution (5 mL) was added to the mixture and stirred for another 30 minutes. The resulting mixture was extracted with dichloromethane, then the organic layer was dried over anhydrous NaSO/ _t , filtered, concentrated to give crude product 64 as a yellow oil (50 mg), which was used for the next step without further purification.

Preparation of T149:

[0282] To a solution of compound 64 (15 mg, 0.02700 mmol) in CH2Q2 (1 mL) were added Cy3-NHS (15 mg, 0.02250 mmol) and one small crystal of DMAP. The reaction mixture was stirred at room temperature overnight, followed by concentration in vacuo and purification by flash column chromatography to afford T149 as a red solid (10 mg, 42%).

Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, Rf = 0.43; H NMR (400 MHz, CDC1 ₃ ) δ 8.65 (br s, 1H), 8.40 (dd, / = 13.5, 13.5 Hz, 1H), 7.50-7.30 (m, 5H), 7.32-7.20 (m, 2H), 7.18-7.11 (m, 3H), 6.02 (br s, 1H), 4.73-4.63 (m, 2H), 4.20-4.10 (m, 2H), 3.90-3.85 (m, 2H), 3.84 (dd, / = 4.8 Hz, 1H), 3.82-3.79 (m, 3H), 3.75-3.71 (m, 1H), 3.64-3.50 (m, 8H), 3.49-3.44 (m ₅ 3H), 3.40-3.33 (m, 4H), 1.69-1.64 (m, 1H), 2.61 (s, 2H), 2.36-2.27 (m, 1H), 2.26-2.05 (m, 2H), 2.00-1.80 (m, 6H), 1.73-1.71 (m, 14H), 1.65-1.58 (m, 1H), 1.53 (dd, / = 4.5, 12.0 Hz, 1H), 1.30-1.24 (m, 1H), 1.10-1.07 (m, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 174.3, 173.8, 173.7, 173.3, 160.2, 157.0, 151.0, 142.8, 141.9, 140.6, 140.5, 129.0, 128.9, 125.5, 125.4, 125.3, 122.0, 122.0, 111.2, 110.8, 105.2, 104.6, 73.5, 70.6, 70.3, 70.1, 70.08, 70.0, 69.8, 65.8, 64.0, 62.4, 60.6, 60.2, 56.5, 55.4, 49.0, 48.8, 44.1, 40.8, 40.4, 39.0, 35.8, 35.4, 32.1, 29.8, 28.2, 28.1, 27.1, 23.7, 23.3, 22.6, 17.1, 13.6; LRMS (ESI) m/z 975.5 (M ⁺ ); HRMS (ESI) calcd. for m/z 975.5114, found 975.5091.

[0284] Scheme 15 Reagents and conditions: (a) NHS, DCC, DMF, 50 °C, 34%; (b)

H-Lys(Boc)-OH, Et ₃ N, DMF, 60 °C, 67%; (c)(i) EDC, HOBt, Et ₃ N, CH ₂ C1 ₂ (ii) 59, rt, 62%; (d) TFA, CH ₂ C1 ₂ , rt, 100%;(e) Compound 12, Et ₃ N, DMAP, CH ₂ Cl ₂ /MeOH, 60%.

Preparation of compound 65:

[0285] To a solution of 4-benzoylbenzoic acid (1.36 g, 6 mmol) in DMF (15 mL) was added DCC (1.49 g, 7.2 mmol) and NHS (829 mg, 7.2 mmol). The mixture was heated at 50 °C overnight. The solid was filtered and DMF was removed. The crude product was recrystallized by -PrOH and yield purified product as yellow solid (656 mg, 34%).

[0286] Analytical TLC (silica gel 60), 20% EtOAc in ra-hexane _, = 0.55; ¾ NMR

(300 MHz, CDCI ₃ ) δ 8.26 (d, / = 8.2 Hz, 2H), 7.90 (d, / = 6.7 Hz, 2H), 7.80 (d, / = 7.3 Hz, 2H), 7.64 (t, / = 7.3 Hz, 1H), 7.52 (t, / = 7.3 Hz, 2H), 2.94 (s, 4H); ¹³ C NMR (125 MHz, CDCI ₃ ) δ 169.0, 133.2, 130.5, 130.1, 130.0, 128.6, 25.7; IR (KBr) 3035, 2927, 2855, 1799, 1499, 1447 LRMS (EI) m/z 323.0 (M ⁺ , 1), 209.0 (M ⁺ -C ₄ H ₄ N0 ₃ , 100); HRMS (EI) calcd for Ci ₄ H ₉ 0 ₂ (M ⁺ -C ₄ H ₄ N0 ₃ ) m/z 209.0597, found 209.0597.

Preparation of compound 66: [0287] To a solution of compound 65 (493 mg, 2 mmol) in DMF ( 10 mL) was added

H-Lys(Boc)-OH (646 mg, 2 mmol) and Et ₃ N (0.56 mL, 4 mmol). The reaction was heated at 60 °C for 1.5 day. DMF was removed and the mixture was redissolved in CH ₂ Q ₂ ( 10 mL). The mixture was washed by 1 M HC1 (2 ^χ 10 mL) and sat. NaHC0 ₃ (1 x 10 mL). The organic layer was collected and was filtered via a short-pad silica gel. The filtrate was concentrated to give product as yellow solid (612 mg, 67%).

[0288] Analytical TLC (silica gel 60), 20% MeOH in CHC1 _3, R/= 0.58; H NMR

(400 MHz, CDCI ₃ ) δ 7.95-7.88 (m, 3H), 7.67 (d, / = 7.4 Hz, 2H), 7.62 (d, / = 7.2 Hz, 2H), 7.54 (t, / = 7.2 Hz, 1H), 7.41 (t, / = 7.4 Hz, 2H), 5.02 (br s, 1H, NH), 4.53 (br s, 1H, NH), 2.97-2.87 (br m, 2H), 1.86-1.68 (br m, 2H), 1.33-1.18 (m, 13H); ¹³ C NMR (100 MHz, CDCI ₃ ) δ 195.8, 177.5, 167.2, 156.3, 139.9, 137.0, 136.9, 132.8, 130.0, 129.8, 128.4, 127.5, 79.1, 54.5, 40.0, 33.7, 31.1, 23.0. IR (KBr) 3035, 2927, 2855, 1799, 1499, 1447 cm ; LRMS (EI) m/z 323.0 (M ⁺ , 1), 209.0 (M ⁺ -C ₄ H ₄ N0 ₃ , 100); HRMS (EI) calcd for Ci ₄ H ₉ 0 ₂ (M ⁺ - C ₄ H ₄ NO ₃ ) m/z 209.0597, found 209.0597.

Preparation of compound 67:

[0289] To a solution of compound 66 (20 mg, 0.0445 mmol) in CH ₂ C1 ₂ (2 mL) EDC

(11 mg, 0.0579 mmol), HOBt (7.8 mg, 0.0579 mmol) and Et ₃ N (8 μί, 0.0579 mmol) were added. The resulting mixture was stirred for 1 h. Compound 59 (25 mg, 0.0668 mmol) was predissolved in CH ₂ CI ₂ (2 mL) and was added to the reaction mixture dropwisely. The reaction was stirred at room temperature for 1 day. When the reaction completed, the mixture was diluted with CH ₂ C1 ₂ (2 mL), washed with 1 M HC1 (2 ^χ 2 mL), 1 M K ₂ C0 ₃ (3 ^χ 2 mL) and brine ( 1 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by flash chromatography (10% EtOH in CH ₂ C1 ₂ ) to give purified product as yellow solid (24 mg, 62%).

[0290] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 _2, R/= 0.35; ¾ NMR

(300 MHz, CD ₃ OD) δ 8.02 (d, / = 8.3 Hz, 2H), 7.85 (d, / = 8.3 Hz, 2H), 7.79 (d, / = 7.2 Hz, 2H), 7.67 (t, / = 7.4 Hz, 1H), 7.55 (t, / = 7.4 Hz, 2H), 4.56 (dd, / = 8.5, 5.6 Hz, 1H), 4.48 (dd, / = 7.5, 4.8 Hz, 1H), 4.28 (dd, / = 7.6, 4.6 Hz, 1H), 3.61-3.57 (m, 6H), 3.53 (t, / = 5.4 Hz, 2H), 3.42 (t, / = 5.4 Hz, 2H), 3.36-3.35 (m, 2H), 3.21-3.15 (m, 1H), 3.06 (t, / = 5.5 Hz, 2H), 2.90 (dd, / = 12.7, 4.9 Hz, 1H), 2.68 (d, / = 12.7 Hz, 1H), 2.20 (t, / = 7.4 Hz, 2H), 1.93-1.78 (m, 2H), 1.75-1.47 (m, 8H), 1.45-1.41 (m, 11H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 197.7, 176.2, 176.1, 174.5, 169.2, 166.1, 141.5, 138.8, 138.4, 134.2, 131.1, 130.9, 129.7, 128.8, 79.8, 71.3, 70.6, 70.5, 63.4, 61.6, 57.0, 55.6, 41.0, 40.4, 40.3, 36.8, 32.8, 30.7, 30.6, 30.4, 29.8, 29.5, 28.8, 26.8, 24.4; IR (CH ₂ C1 ₂ ) 3035, 2927, 2855, 1799, 1499, 1447 cm ^"1 ; LRMS (ESI) m/z 811.2 (M ⁺ +H), 833.2 (M ⁺ +Na); HRMS (ESI) calcd for C ₄ iH ₅₉ N ₆ 0 ₉ S (M ⁺ +H) m/z

811.4064, found 811.4064.

Preparation of compound 68:

[0291] To a solution of compound 67 (24 mg) in CH2CI2 (2 mL) TFA (0.5 mL) was added dropwisely. After 1 h, the mixture was concentrated and TFA was pumped off at high vacuum. The product was obtained as yellow solid (20 mg, 100%).

[0292] Analytical TLC (silica gel 60), 20% MeOH in CH ₂ C1 _2, R/= 0.58; ¾ NMR

(400 MHz, CD ₃ OD) δ 8.02 (d, / = 8.3 Hz, 2H), 7.85 (d, / = 8.3 Hz, 2H), 7.79 (d, / = 7.2 Hz, 2H), 7.67 (t, / = 7.4 Hz, 1H), 7.55 (t, / = 7.4 Hz, 2H), 4.62-4.59 (m, 1H), 4.50-4.47 (m, 1H), 4.31-4.27 (m, 1H), 3.61-3.57 (m, 6H), 3.52 (t, / = 5.4 Hz, 2H), 3.44-3.42 (m, 2H), 3.35 (t, / = 2.1 Hz, 2H), 3.20-3.16 (m, 1H), 3.06 (t, / = 5.5 Hz, 2H), 2.97-2.89 (m, 3H), 2.68 (d, / = 12.7 Hz, 1H), 2.20 (dt, / = 7.3, 3.5 Hz, 2H), 1.96-1.85 (m, 2H), 1.72-1.40 (m, 10H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 197.6, 176.1, 174.2, 169.3, 166.1, 141.6, 138.6, 138.3, 134.2, 131.0, 130.9, 129.7, 128.7, 71.3 (2C), 70.5 (2C), 63.4, 61.6, 57.0, 55.3, 41.0, 40.5, 40.4, 40.3, 36.7, 32.5, 29.7, 29.5, 28.1, 26.9, 24.1; LRMS (ESI) m/z 711.2 (M ⁺ +H), 733.0 (M ⁺ +Na); HRMS (ESI) calcd for C ₃ 6H ₅₁ N ₆ 0 ₇ S (M ₊ +H) m/z 711.3540, found 711.3544.

Preparation of T129:

[0293] To a solution of compound 68 (20 mg, 0.0273 mmol) and compound 12 (13 mg, 0.0249) in CH ₂ C1 ₂ (4 mL) and MeOH (1 mL) was added Et ₃ N (4 μί, 0.0274 mmol) and DMAP (1 small crystal). The reaction was stirred for overnight. When TLC indicated reaction was complete, the reaction was transferred to a separatory funnel. The organic layer was washed with IN HC1 (1 x 2 mL), 1M K ₂ C0 ₃ (8 x 2 mL) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude mixture was purified by column chromatography using 10% MeOH in CH ₂ C1 ₂ to give purified product as yellow solid (16 mg, 60%).

[0294] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, %= 0.14; ¾ NMR

(500 MHz, CDC1 ₃ ) δ 8.83 (br s, 1H), 8.19 (br s, 1H), 8.04-7.99 (m, 2H), 7.83-7.77 (m, 4H), 7.63-7.59 (m, 1H), 7.52-7.48 (m, 2H), 6.78 (br s, 1H), 6.47 (br s, 1H), 5.81 (br s, 1H), 5.12 (br s, 1H), 4.75-4.46 (m, 3H), 4.33-4.28 (m, 1H), 4.10-4.01 (m, 3H), 3.92-3.89 (m, 1H), 3.71-3.32 (m, 14H), 3.28-3.25 (m, 1H), 3.18-3.06 (m, 3H), 2.92-2.81 (m, 2H), 2.72 (d, / = 13.0 Hz, 1H), 2.35-2.27 (m, 1H), 2.24-1,82 (m, 8H), 1.70-1.25 (m, 14H), 1.04 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 197.6, 196.1, 173.4, 173.2, 173.1, 172.6, 167.4, 166.9, 164.0, 159.5, 156.8, 140.3, 137.1, 136.6, 132.9, 130.1, 130.0, 129.9, 128.5, 127.6, 127.5, 125.7, 70.3, 70.1, 70.0, 69.9, 69.7, 69.6, 69.5, 69.4, 65.4, 63.8, 63.3, 62.0, 61.1, 60.9, 60.3, 60.1, 56.3, 55.8, 40.6, 40.5, 40.2, 39.5, 39.4, 39.3, 39.1, 35.4, 30.9, 30.5, 28.0, 25.6, 25.2, 24.3, 23.2, 17.1, 13.9; LRMS (ESI) m/z 1133.2 (M ⁺ +Na).

[0295] Scheme 16. Synthetic route to T132.

H,N NHBoc

[0296] Scheme 16 Reagents and conditions: (a) Boc ₂ 0, Et ₃ N, CHC1 ₃ , 0 °C, 16%;

(b) Biotin-NHS, Et ₃ N, CH ₂ C1 ₂ , 80%; (c)(i) cone. HC1, CH ₂ C1 ₂ , 0 °C (ii) 1M K ₂ C0 ₃ , EtOH extract, crude; (d)(i) EDC, HOBt, Et ₃ N, CH ₂ C1 ₂ ; (ii) 72, CH ₂ C1 ₂ , 51%; (e) TFA, CH ₂ C1 ₂ , crude; (f) Compound 12, Et ₃ N, DMAP, MeOH/CH ₂ Cl ₂ , 73%.

Preparation of compound 69:

[0297] To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (2.2 g, 10 mmol) in

CHC1 ₃ (100 mL) at 0 °C was added Boc ₂ 0 (2.18 g, 10 mmol) and Et ₃ N (1.4 mL, 10 mmol). The reaction was stirred at 0 °C for 40 min. The reaction was concentrated to give crude product. The crude product was purified by column chromatography using 2-6% EtOH in CH ₂ C1 ₂ to give purified product as slight yellow oil (516 mg, 16%). Note: the mono- protected amine must be freshly prepared. It undergoes gradual disproportionation upon storage. [0298] Analytical data matched that described in /. Am. Chem. Soc. 2003, 125, 2416-

2425.

Preparation of compound 70:

[0299] To a solution of compound 69 (86 mg, 0.268 mmol) in CH ₂ CI ₂ (1 mL) was added biotm-NHS (82 mg, 0.242 mmol) and Et ₃ N (38 0.268 mmol). The reaction was stirred at rt for 1 h. The mixture was diluted with CH ₂ CI ₂ (10 mL), washed with IN HC1 (2 ^χ 4 mL), 1M K ₂ CO3 (2 x 4 mL) and brine (1 x 4 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. Compound 70 (106 mg, 80%) was obtained as yellow solid.

[0300] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , R/ = 0.20; ¾ NMR

(400 MHz, CDCI ₃ ) δ 6.80 (br s, 1H, NH), 6.69 (s, 1H), 6.03 (s, 1H), 5.13 (br s, 1H, NH), 4.50 (dd, / = 7.4, 5.1 Hz, 1H), 4.30 (dd, / = 7.4, 5.1 Hz, 1H), 3.65-3.63 (m, 4H), 3.61-3.57 (m, 4H), 3.56-3.52 (m, 4H), 3.32 (q, / = 6.1 Hz, 2H), 3.22-3.12 (m, 3H), 2.89 (dd, / = 12.8, 4.9 Hz, 1H), 2.74 (d, / = 12.7 Hz, 1H), 2.19 (t, / = 6.4 Hz, 2H), 1.81-1.61 (m, 8H), 1.48-1.39 (m, 11H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 173.2, 164.1, 156.0, 78.8, 70.4 (2C), 70.1, 70.0, 69.7, 69.4, 61.7, 60.1, 55.7, 40.4, 38.4, 37.5, 36.0, 29.6, 28.9, 28.4, 28.2, 28.0, 25.7; LRMS (EI) m/z 546.4 (M ⁺ , 1), 472.3 (70); HRMS (EI) calcd for C ₂₅ H ₄₆ O ₇ N ₄ S (M ⁺ ) m/z 546.3082, found 546.3084.

Preparation of compound 71:

[0301] To a solution of compound 70 (81 mg, 0.148 mmol) in CH ₂ CI ₂ (3 mL) was added cone. HC1 (0.5 mL) at 0 °C. The reaction was stirred at 0 °C for 30 min. The mixture was basicified with 1M K ₂ CO ₃ solution and was concentrated. The solid residue was extracted with EtOH and was filtered. The filtrate was obtained and was concentrated to give crude compound 71 as a colorless oil.

[0302] Analytical TLC (silica gel 60), 20% MeOH in CH ₂ C1 ₂ , R/ = 0.10; H NMR

(400 MHz, CD ₃ OD) δ 4.49 (dd, / = 7.7, 4.6 Hz, 1H), 4.30 (dd, / = 7.7, 4.6 Hz, 1H), 3.65- 3.62 (m, 4H), 3.60-3.57 (m, 4H), 3.56-3.51 (m, 4H), 3.26 (t, / = 6.8 Hz, 2H), 3.24-3.18 (m, 1H), 2.93 (dd, / = 12.7, 5.0 Hz, 1H), 2.74-2.69 (m, 3H), 2.20 (t, / = 7.4 Hz, 2H), 1.78-1.61 (m, 3H), 1.46-1.42 (m, 2H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 175.9, 166.0, 71.5, 71.4, 71.2, 71.1, 70.4, 69.9, 63.4, 61.6, 57.0, 41.1, 40.1, 37.7, 36.8, 30.4, 29.8, 29.5, 26.9; LRMS (EI) m/z 446.3 (M , 1), 399.3 (16); HRMS (EI) calcd for C ₂ 0H38O5N ₄ S (M ⁺ ) m/z 446.2557, found 446.2547.

Preparation of compound 72:

[0303] To a solution of compound 66 (42 mg, 0.0933 mmol) in CH ₂ CI ₂ (5 mL) was added EDC (21 mg, 0.112 mmol), HOBt (15 mg, 0.112 mmol) and Et ₃ N (16 μί, 0.112 mmol). The reaction was stirred for 1 h. Compound 71 (50 mg, 0.112 mmol) was dissolved in CH ₂ CI ₂ (4 mL) and was added to the reaction. The reaction was further stirred for an overnight. When TLC indicated the reaction was complete, the reaction was transferred to a separatory funnel. The organic layer was washed with IM HCl (2 x 2 mL), IM K ₂ CO ₃ (2 x 2 mL) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and

concentrated. The crude mixture was purified by flash chromatography using 10% EtOH in CH ₂ CI ₂ to give purified product (42 mg, 51%) as yellow solid.

[0304] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, R/ = 0.38; ¾ NMR

(400 MHz, CD ₃ OD) δ 8.01 (d, / = 8.3 Hz, 2H), 7.84 (d, / = 8.3 Hz, 2H), 7.79 (d, / = 7.2 Hz, 2H), 7.67 (t, / = 7.4 Hz, 1H), 7.55 (t, / = 7.4 Hz, 2H), 4.56 (dd, / = 8.5, 5.6 Hz, 1H), 4.48 (dd, / = 7.5, 4.8 Hz, 1H), 4.28 (dd, / = 7.6, 4.6 Hz, 1H), 3.62-3.61 (m, 4H), 3.58-3.56 (m, 4H), 3.55-3.49 (m, 4H), 3.34-3.31 (m, 2H), 3.25 (q, / = 6.3 Hz, 2H), 3.20-3.15 (m, 1H), 3.06 (q, / = 6.1 Hz, 2H), 2.90 (dd, / = 12.7, 4.9 Hz, 1H), 2.68 (d, / = 12.7 Hz, 1H), 2.18 (t, / = 7.4 Hz, 2H), 1.92-1.82 (m, 2H), 1.81-1.70 (m, 5H), 1.68-1.57 (m, 3H), 1.57-1.48 (m, 3H), 1.45- 1.40 (m, 12H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 197.6, 175.9, 174.3, 169.2, 166.0, 158.9, 141.5, 138.8, 138.4, 134.2, 131.0, 130.9, 129.7, 128.7, 79.8, 71.5, 71.5, 71.2, 70.0, 69.9, 63.4, 61.6, 57.0, 55.7, 41.0, 38.0, 37.8, 36.9, 32.8, 30.6, 30.4, 30.3, 29.8, 29.5, 28.8, 26.9, 24.4; LRMS (ESI) m/z 883.3 (M ⁺ +H); HRMS (ESI) calcd for C ₄ 5H ₆₇ N ₆ O ₁₀ S (M ⁺ +H) m/z 883.4639, found 883.4636.

Preparation of compound 73:

[0305] To a solution of compound 72 (40 mg, 0.0453 mmol) in CH ₂ CI ₂ (5 mL) was added trifluoroacetic acid (0.5 mL). The reaction was stirred for 30 min. When TLC indicated the reaction was complete, the reaction was concentrated. The product was put under high vacuum for 1.5 h. The crude product as yellow solid (40 mg) was directly used for next step. [0306] Analytical TLC (silica gel 60), 20% MeOH in CH ₂ C1 ₂ „ R/ = 0.43; ¾ NMR

(300 MHz, CD ₃ OD) δ 8.01 (d, / = 8.3 Hz, 2H), 7.84 (d, / = 8.3 Hz, 2H), 7.78 (d, / = 7.2 Hz, 2H), 7.66 (t, / = 7.4 Hz, 1H), 7.54 (t, / = 7.4 Hz, 2H), 4.56 (dd, / = 8.5, 5.6 Hz, 1H), 4.48 (dd, / = 7.5, 4.8 Hz, 1H), 4.28 (dd, / = 7.6, 4.6 Hz, 1H), 3.60-3.48 (m, 12H), 3.34-3.15 (m, 4H), 2.97-2.88 (m, 3H), 2.69 (d, / = 12.7 Hz, 1H), 2.18 (t, / = 7.4 Hz, 2H), 1.95-1.87 (m, 2H), 1.85-1.39 (m, 14H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 197.7, 176.0, 174.1, 169.2, 166.1, 141.6, 138.6, 138.3, 134.2, 131.0, 130.9, 129.7, 128.7, 71.5, 71.4, 71.2, 69.9 (2C), 63.4, 61.7,

57.0, 55.4, 41.0, 40.5, 38.0, 37.8, 36.8, 32.5, 30.7, 30.4, 30.3, 29.7, 29.5, 28.1, 26.9, 24.1; LRMS (FAB) m/z 783.4 (M ⁺ +H); HRMS (ESI) calcd for C ₄ oH ₅₉ N ₆ 0 ₈ S (M ⁺ +H) m/z 783.4115, found 783.4110.

Preparation of T132:

[0307] To a solution of compound 42 (50 mg, 0.0639 mmol) and compound 12 (36 mg, 0.0703 mmol) in CH ₂ C1 ₂ (8 mL) and CH ₃ OH (2 mL) was added Et ₃ N (30 μί, 0.0703 mmol) and DMAP (1 small crystal). The reaction was stirred for overnight. When TLC indicated the reaction was complete, the reaction mixture was washed with 1 N HCl (2 mL x 1), 1 M K ₂ CC>3 (2 mL x 8) and brine (2 mL x 1). The mixture was dried over Na ₂ SC>4, filtered and concentrated. The crude mixture was purified by column chromatography using 10% MeOH in CH ₂ C1 ₂ to give purified product (41 mg, 73%).

[0308] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, R/= 0.28; H NMR

(500 MHz, CDCI ₃ +CD ₃ OD) δ 8.00 (dd, / = 2.7, 8.4 Hz, 2H), 7.84 (dd, / = 2.0, 8.4 Hz, 2H), 7.81-7.78 (m, 2H), 7.63 (t, / = 7.4 Hz, 1H), 7.51 (t, / = 7.9 Hz, 2H), 4.77-4.70 (m, 2H), 4.60-4.54 (m, 1H), 4.49 (dd, / = 5.0, 7.4 Hz, 1H), 4.34-4.30 (m, 1H), 4.09 (dd, / = 2.7 Hz, 1H), 4.10-3.98 (m, 2H), 3.91 (dd, / = 2.9, 4.0 Hz, 1H), 3.65-3.52 (m, 12H), 3.44-3.39 (m, 2H), 3.34-3.27 (m, 4H), 3.17-3.12 (m, 5H), 2.93-2.86 (m, 1H), 2.84-2.73 (m, 1H), 2.37- 2.32 (m, 1H), 2.26-2.11 (m, 4H), 2.06-2.04 (m, 1H), 1.97 (td, / = 1.7, 14.0 Hz, 1H), 1.89- 1.48 (m, 17H), 1.47-1.37 (3H), 1.33 (t, / = 7.0 Hz, 5H), 1.04 (s, 3H); ¹³ C NMR (125 MHz, CDCI ₃ +CD ₃ OD) δ 197.6, 196.4, 173.7, 173.5, 172.6, 172.3, 167.1, 166.9, 159.9, 157.0, 140.2, 137.2, 137.02, 133.1, 130.2, 130.1, 130.0, 128.5, 127.5, 125.6, 70.5, 70.40, 70.4, 70.2,

70.1, 70.02, 70.0, 69.9, 69.6, 69.2, 69.1, 65.4, 64.0, 63.3, 61.1, 60.9, 60.1, 56.4, 55.7, 55.6, 53.5, 45.9, 40.5, 37.2, 35.7, 35.4, 30.5, 29.7, 29.0, 28.2, 28.1, 25.8, 25.5, 24.2, 23.2, 22.7, 17.0, 13.9, 8.5; LRMS (ESI) m/z 1205.3 (M +Na); HRMS (ESI) calcd for CeiHygNeOieS (M ⁺ +H) m/z 1183.5273, found 1183.5294.

T143.

NHBoc

[0310] Scheme 17 Reagents and conditions: (a) (i) NaN ₃ , S0 ₂ C1 ₂ , CH ₃ CN, 0 °C to rt (ii) HC1 in EtOH, 97%; (b) 75, K ₂ C0 ₃ , CuS0 ₄ 5HC1, CH ₃ OH, rt, 100%; (c) (COCl) ₂ , cat. DMF, CH ₂ C1 ₂ , rt, crude; (d) H-Lys(Boc)-OMe, pyridine, CH ₂ C1 ₂ , 92%; (e) LiOH, THF, H ₂ 0, rt, 85%; (f) (i) HOBt, EDC, Et N, CH ₂ C1 ₂ , rt (ii) 71, 45%; (g) cone. HC1, CH ₂ C1 ₂ , 0 °C, then 1M K ₂ C0 ₃ , 100%; (h) 74, Et ₃ N, CH ₂ C1 ₂ , rt, 41%; (i) 5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 96%. Compound T138 was prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above.

Preparation of compound 74:

[0311] To a solution of ProC3-TN (112 mg, 0.313 mmol) in CH ₂ C1 ₂ (20 mL) was added 4-nitrophenylchloroformate (126 mg, 0.625 mmol) and pyridine (50 μί, 0.625 mmol). The reaction was stirred at rt overnight. When TLC indicated completion of reaction, the mixture was diluted with CH ₂ C1 ₂ (20 mL). The organic layer was washed with IN HC1 (5 mL x 2) and brine (5 mL* 1). The layer was dried over Na ₂ SC>4, filtered and concentrated to give crude product. The crude product was further purified by column chromatography using 80% EtOAc in «-hexane to give purified compound 74 as a white solid (90 mg, 55%).

[0312] Analytical TLC (silica gel 60), EtOAc: ra-hexane=5:5, R/= 0.125; ¾ NMR

(300 MHz, CDC1 ₃ ) S 8.28 (d, / = 9.1 Hz, 2H), 7.39 (d, / = 9.1 Hz, 2H), 7.08 (d, / = 4.6 Hz, 1H), 4.70-4.43 (m, 2H), 4.28 (t, / = 6.4 Hz, 2H), 3.83 (d, / = 4.7 Hz, 1H), 3.62 (d, / = 5.7 Hz, 1H), 2.75 (d, / = 13.5 Hz, 1H), 2.42-2.40 (m, 1H), 2.39-2.25 (m, 2H), 2.22-2.10 (m, 2H), 2.08-1.84 (m, 3H), 1.68 (dd, / = 12.5, 4.5 Hz, 1H), 1.27 (dt, / = 12.1, 6.0 Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 190.8, 173.1, 159.5, 155.4, 152.3, 145.4, 142.9, 140.0, 125.7 (2C), 125.3, 121.7 (2C), 69.9, 68.4, 64.2, 62.3, 60.9, 51.4, 40.8, 35.0, 30.0, 26.1, 25.9, 23.4, 17.0, 13.7; LRMS (ESI) m/z 546.1 (M ⁺ +Na). HRMS (EI) calcd for C20H22O6 (M ⁺ -p- nitrophenyl) m/z 358.1411, found 358.1416.

Preparation of compound 75:

[0313] For procedure and characterization data, refer to Org. Lett. 2007, 9, 3797-

3800.

Preparation of compound 76:

[0314] For procedure and characterization data, refer to Org. Lett. 2007, 9, 3797-

3800.

Preparation of compound 77:

[0315] Compound 77 was synthesized in accordance with or by slight modification of the method set forth in Groehn, V.; Frohlich, L.; Schmidt, H. H. H. W.; Pfleiderer, W. Helv. Chim. Acta 2000, 83, 2738. To a solution compound 77 (208 mg, 1.28 mmol) in CH ₂ C1 ₂ (13 mL) oxalyl chloride (0.11 mL, 1.28 mmol) was added slowly, followed by a drop of DMF. The reaction was stirred until there was no gas evolved. The solvent was evaporated and the crude product was put under high vacuum for 2 h before next step.

Preparation of compound 78:

[0316] Pretreatment of amine: H-Lys(Boc)-OMe was obtained from its HC1 salt. 1 g of HC1 salt was dissolved in CH2CI2 (40 mL) and was washed with 1M K ₂ CO3 (3 x 4 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated.

[0317] The crude compound 77 obtained from previous step was dissolved in CH2CI2

(13 mL). Pyridine (0.12 mL, 1.54 mmol) and H-Lys(Boc)-OMe (400 mg, 1.54 mmol) were added. The mixture was stirred for 1.5 h. When TLC indicated completion of reaction, the reaction mixture was diluted with CH ₂ CI ₂ (10 mL) and was washed with IN HC1 (2 x 5 mL), 1M K ₂ CO ₃ (2 x 5 mL) and brine (1 x 5 mL). The organic layer was dried over Na ₂ SC>4 and concentrated to give crude product. The crude product was purified by flash chromatography (30-40% EtOAc in «-hexane) to give purified product as white solid (476 mg, 92%). [0318] Analytical TLC (silica gel 60), 80% EtOAc in ra-hexane, R/ = 0.42; ¾ NMR

(400 MHz, CDC1 ₃ ) δ 7.77 (d, / = 8.5 Hz, 2H), 7.07 (d, / = 7.3 Hz, 1H), 6.98-6.95 (m, 2H), 4.77 (t, / = 5.8 Hz, 1H), 4.69 (q, / = 6.7 Hz, 1H), 3.70 (s, 3H), 3.05-3.02 (m, 2H), 1.91-1.84 (m, 1H), 1.77-1.72 (m, 1H), 1.49-1.42 (m, 2H), 1.38-1.34 (m, 11H); ¹³ C NMR (100 MHz, CDCI ₃ ) δ 173.0, 166.2, 156.0, 143.3, 130.1, 128.9, 118.7, 78.9, 52.4, 52.3, 39.8, 31.7, 29.5, 28.2, 22.5; LRMS (EI) m/z 405.2 (M ⁺ ), 349.2, 332.2; HRMS (EI) calcd for (M ⁺ ) m/z 405.2007, found 405.2001.

Preparation of compound 79:

[0319] Compound 79 was synthesized in accordance with or by slight modification of the method set forth in: Ozaki, S.; Watanabe, Y.; Hirata, M.; Awaya, A.; (Mitsui Toatsu Chemicals, Inc., Japan). Application: WO9104258 (1991), p 139. Compound 75 (470 mg, 1.16 mmol) in THF (6 mL) and H ₂ 0 (6 mL) LiOH (46 mg, 1.28 mmol) was added. The reaction was stirred at rt. When TLC indicated completion of reaction, the mixture was acidified with IN HC1, followed by addition of EtOAc (30 mL). The organic layer was obtained and was washed with brine (5 mL ^χ 1). The organic layer was dried over NaiSO/ _t , filtered and concentrated to give crude product. The crude product was further purified by flash chromatography (4-6% EtOH in CH ₂ CI ₂ ) to give purified product as yellow solid (385 mg, 85%).

[0320] Analytical TLC (silica gel 60), 80% EtOAc in ra-hexane, _, R/= 0.13; ¾ NMR

(400 MHz, CDCI ₃ ) δ 9.61 (br s, 1H), 7.77 (d, / = 7.8 Hz, 1H), 7.73 (br s, 1H), 7.52 (d, / = 7.8 Hz, 1H), 6.90 (d, / = 8.5 Hz, 2H), 6.18 (br s, 0.4H), 4.93 (br s, 0.6H), 4.75 (br s, 1H), 3.06 (br s, 2H), 1.93-1.90 (m, 1H), 1.88-1.78 (br m, 1H), 1.41-1.35 (m, 13H); ¹³ C NMR (100 MHz, CDCI ₃ ) δ 175.1, 166.7, 156.6, 143.4, 129.7, 129.0, 118.7, 79.5, 52.6, 39.9, 31.4, 29.4, 28.2, 22.5; LRMS (EI) m/z 391.2 (M ⁺ ), 335.1; HRMS (EI) calcd for C ₁ 8H ₂ 5O5N5 (M ⁺ ) m/z

391.1850, found 391.1851.

Preparation of compound 80:

[0321] To a compound 79 (44 mg, 0.112 mmol) in CH ₂ C1 ₂ (5 mL) was added EDC

(26 mg, 0.134 mmol), HOBt (18 mg, 0.134 mmol) and Et ₃ N (19 The reaction was stirred for 1 h. Biotin-NH ₂ was pre-dissolved in DMF (1 mL) and was added to the reaction. When TLC indicated completion of reaction, the mixture was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (3 mL ^χ 2), brine (3 mL ^χ 1) and was dried over Na ₂ SC>4. The product was filtered and concentrated to give crude product. The crude product was purified by flash chromatography (10% EtOH in CH ₂ CI ₂ ) to give purified product as yellow solid (41 mg, 45%).

[0322] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, = 0.38; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.91 (d, / = 8.6 Hz, 2H), 7.15 (d, / = 8.6 Hz, 2H), 4.50-4.47 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 3.62-3.60 (m, 4H), 3.59-3.56 (m, 4H), 3.50 (q, / = 5.7 Hz, 4H), 3.30-3.23 (m, 4H), 3.22-3.17 (m, 1H), 3.07-3.02 (m, 2H), 2.91 (dd, / = 12.7, 5.0 Hz, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.19 (t, / = 7.4 Hz, 2H), 1.94-1.72 (m, 7H), 1.70-1.49 (m, 6H), 1.46-1.42 (m, 3H), 1.41 (s, 9H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.0, 175.9, 174.4, 169.0, 166.0, 145.0, 131.7, 130.5, 120.0, 79.8, 71.5 (2C), 71.2, 70.0, 69.9, 63.3, 61.6, 57.0, 55.5, 41.0, 37.9, 37.8, 36.9 (2C), 32.8, 30.6, 30.4, 30.3, 29.8, 29.5, 28.8, 26.9, 24.4; LRMS (ESI) m/z 820.2 (M ⁺ +H), 842.2 (M ⁺ +Na); HRMS (ESI) calcd for C38H6 ₂ N9O9S (M ⁺ +H) m/z 820.4391, found 820.4390.

Preparation of compound 81:

[0323] To a solution of compound 80 (41 mg, 0.05 mmol) in CH ₂ CI ₂ (4 mL) was added cone. HC1 (0.5 mL) slowly at 0 °C. The reaction time was 40 min. The reaction was then basicified with 1M K ₂ CO ₃ solution. The content was concentrated. The dried residue was extracted with EtOH repeatedly. The EtOH extract was concentrated to give crude product as yellow solid (41 mg, 100%).

[0324] Analytical TLC (silica gel 60), 20% MeOH in CH ₂ C1 ₂ „ Rf = 0.10; ^: H

NMR (400 MHz, CD ₃ OD) δ 7.93 (d, / = 8.6 Hz, 2H), 7.15 (d, / = 8.6 Hz, 2H), 4.50-4.46 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 3.62-3.60 (m, 4H), 3.59-3.57 (m, 4H), 3.51 (q, / = 5.7 Hz, 4H), 3.30-3.23 (m, 4H), 3.25 (t, / = 6.8 Hz, 2H), 3.22-3.17 (m, 1H), 2.92 (dd, / = 12.7, 5.0 Hz, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.64 (br s, 1H), 2.19 (t, / = 7.4 Hz, 2H), 1.94-1.72 (m, 7H), 1.70-1.55 (m, 4H), 1.53-1.46 (m, 3H), 1.44-1.38 (m, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.0, 174.5, 169.1, 166.0, 145.1, 131.8, 130.5, 120.0, 71.5, 71.4, 71.2, 69.9, 69.8, 63.4, 61.6, 57.0, 55.7, 42.3, 41.0, 37.9, 37.8, 36.8, 33.4, 32.9, 30.4, 30.3, 29.8, 29.5, 26.9, 24.6; LRMS (ESI) m/z 720.2 (M ⁺ +H), 742.3 (M ⁺ +Na); HRMS (ESI) calcd for C ₃₃ H ₅ 4N ₉ 0 ₇ S (M ⁺ +H) m/z 720.3867, found 720.3860.

Preparation of T143: [0325] To a solution of compound 81 (41 mg, 0.057 mmol) in CH ₂ Q ₂ (1 mL) the triptonide diepoxide intermediate was added (29 mg, 0.057 mmol), followed by Et3N (8 μί, 0.057 mmol). The reaction was stirred at rt overnight. The reaction was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HQ (3 mL ^χ 1), 1M K ₂ CO ₃ solution (3 mL portions repeatedly until the aqueous layer was colorless) and brine (3 mL ^χ 1). The organic layer was dried over Na ₂ SC>4, filtered and concentrated to give crude product. The crude product was purified by column chromatography using 15-20% EtOH in CH ₂ CI ₂ to give purified product as a yellow solid (26 mg, 41%).

[0326] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, = 0.33; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.90 (dd, / = 8.6, 1.7 Hz, 2H), 7.15 (d, / = 8.6 Hz, 2H), 4.87 (s, 2H), 4.50-4.46 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 3.97-3.95 (m, 2H), 3.62-3.60 (m, 4H), 3.59-3.57 (m, 4H), 3.51 (q, / = 5.7 Hz, 4H), 3.31-3.30 (m, 3H), 3.25 (t, / = 6.8 Hz, 2H), 3.22-3.17 (m, 1H), 3.11 (t, / = 6.5 Hz, 2H), 2.92 (dd, / = 12.7, 5.0 Hz, 1H), 2.87-2.83 (m, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.31-2.27 (m, 4H), 2.19 (t, / = 7.4 Hz, 2H), 2.17-2.03 (m, 1H), 1.99 (t, / = 14.0 Hz, 1H), 1.94-1.67 (m, 8H), 1.65-1.49 (m, 7H), 1.46-1.41 (m, 4H), 1.08 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 193.5, 176.2, 176.1, 174.6, 169.3, 166.2, 163.7, 159.2, 145.2, 144.3, 142.5, 131.9, 130.7, 125.8, 120.2, 72.2, 71.7, 71.6, 71.4, 70.1, 70.0, 65.4,

65.1, 63.5, 61.8, 57.1, 55.7, 53.2, 41.7, 41.4, 41.2, 38.1, 38.0, 37.0, 36.2, 32.9, 31.2, 30.7, 30.6, 30.5, 29.9, 29.7, 28.7, 27.1, 27.0, 24.5, 24.2, 18.1, 14.2; LRMS (ESI) m/z 1126.3 (M ⁺ +Na); HRMS (ESI) calcd for C54H74N9O14S (M ⁺ +H) m/z 1104.5076, found 1104.5059.

[0327] Compound T138, analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, =

0.33; ¾ NMR (400 MHz, CD ₃ OD) δ 7.92 (dd, / = 8.6, 1.7 Hz, 2H), 7.16 (d, / = 8.6 Hz, 2H), 4.87 (s, 2H), 4.50 (dd, / = 7.6, 4.4 Hz, 1H), 4.24 (d, / = 2.7 Hz, 1H), 4.04 (d, / = 2.7 Hz, 1H), 4.02-3.95 (m, 2H), 3.62-3.60 (m, 5H), 3.59-3.57 (m, 5H), 3.51 (q, / = 5.7 Hz, 4H), 3.43 (t, / = 5.2 Hz, 1H), 3.31-3.30 (m, 3H), 3.25 (t, / = 6.8 Hz, 2H), 3.22-3.17 (m, 1H), 3.11 (t, / = 6.5 Hz, 2H), 2.92 (dd, / = 12.7, 5.0 Hz, 1H), 2.87-2.83 (m, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.34- 2.21 (m, 2H), 2.18 (t, / = 7.4 Hz, 2H), 2.17-2.03 (m, 1H), 2.00-1.81 (m, 4H), 1.81-1.72 (m, 6H), 1.69-1.61 (m, 6H), 1.46-1.41 (m, 5H), 1.02 (s, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 199.9, 176.0, 174.4, 169.1, 166.2, 163.3, 145.1, 131.8, 130.5, 125.6, 125.6, 120.0, 72.0, 71.5 (2C), 71.2, 70.0, 69.9, 66.7, 65.2, 64.4, 63.4, 62.7, 62.4, 61.6, 58.3, 57.7, 57.0, 55.6, 41.7,

41.2, 41.0, 38.0, 37.8, 36.8, 36.4, 32.8, 31.5, 30.5, 30.4, 30.3, 29.8, 29.5, 26.9, 26.6, 25.3, 24.3, 23.9, 17.9, 14.3; LRMS (ESI) m/z 1 142.3 (M +Na); HRMS (ESI) calcd for C5 ₄ H7 ₄ N9O ₁ 5S (M ⁺ +H) m/z 1 120.5025, found 1 120.5020.

[0328] Scheme 18. Synthetic route to T144 and T145.

[0329] Scheme 18 Reagents and conditions: (a) /?-nitrophenylchloroformate, pyridine, CH ₂ C1 ₂ , rt, 55%; (b) DCC, NHS, DMF, 50 °C, 56%; (c) 69, Et ₃ N, CH ₂ C1 ₂ /DMF, rt, 62%; (d) cone. HC1, CH ₂ C1 ₂ , 0 °C, then 1M K ₂ C0 ₃ , 81%; (e) 75, K ₂ C0 ₃ , CuS0 ₄ 5HC1, CH ₃ OH, rt, 100%; (f) (COCl) ₂ , cat. DMF, CH ₂ C1 ₂ , rt, crude; (g) H-Lys(Boc)-OMe, pyridine, CH ₂ C1 ₂ , rt, 46%; (h) LiOH, THF, H ₂ 0, rt, 100%; (i) (i) EDC, HOBt, Et ₃ N, CH ₂ C1 ₂ (ii) 84, rt, 61%; (j) cone. HC1, CH ₂ C1 ₂ , 0 °C, then 1M K ₂ C0 ₃ , 100%; (k) 79, Et ₃ N, CH ₂ C1 ₂ , rt, 70%; (1) 5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 78%. Compound T145 was prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above.

Preparation of compound 82:

[0330] To a solution of desthiobiotin (214 mg, 1 mmol) in DMF (4 mL) was added

DCC (227 mg, 1.1 mmol) and NHS (127 mg, 1.1 mmol). The reaction was heated to 50 °C overnight. The reaction was then cooled in an ice-bath. The solid was filtered and the filtrate was concentrated. The crude compound 82 (173 mg, 56%) was a white solid. [0331] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/ = 0.53; ¾ NMR

(400 MHz, CD ₃ OD) δ 3.83 (quintet, / = 5.1 Hz, 1H), 3.71 (q, / = 6.9 Hz, 1H), 2.84 (s, 4H), 2.65 (t, / = 7.2 Hz, 2H), 1.75 (quintet, / = 6.9 Hz, 2H), 1.54-1.47 (m, 5H), 1.34-1.31 (m, 1H), 1.11 (d, / = 6.4 Hz, 1H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 172.0, 170.3, 166.2, 57.3, 52.7, 31.5, 30.6, 29.7, 26.9, 26.5, 25.6, 15.6; LRMS (EI) m/z 311.2 (M ⁺ ), 197.1; HRMS (EI) calcd for C ₁₄ H ₂₂ O ₅ N ₃ (M ⁺ ) m/z 311.1476, found 311.1476.

Preparation of compound 83:

[0332] Compound 69 (214 mg, 0.667 mmol) was pre-dissolved in CH ₂ Q ₂ (4 mL) was added to a solution of compound 82 (173 mg, 0.556 mmol) in DMF (1 mL). Et ₃ N (93 μί, 0.667 mmol) was added. The reaction was stirred for 1.5 h. When TLC indicated completion of reaction, the reaction was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (2 5 mL), 1M K ₂ C0 ₃ (2 5 mL) and brine (1 x 5 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by flash chromatography using 10% EtOH in CH ₂ CI ₂ to give purified product as white solid (178 mg, 62%).

[0333] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/= 0.35; H NMR

(400 MHz, CD ₃ OD) δ 3.82 (quintet, / = 5.1 Hz, 1H), 3.69 (q, / = 6.9 Hz, 1H), 3.64-3.63 (m, 4H), 3.59-3.58 (m, 4H), 3.52 (q, / = 3.5 Hz, 4H), 3.26 (t, / = 6.6 Hz, 2H), 3.12 (t, / = 6.0 Hz, 2H), 2.19 (t, / = 7.3 Hz, 2H), 1.77-1.71 (m, 4H), 1.65-1.60 (m, 2H), 1.52-1.30 (m, 15H), 1.10 (d, / = 6.4 Hz, 1H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.1, 166.1, 159.0, 79.8, 71.5, 71.2, 69.9 (2C), 57.4, 52.7, 38.7, 37.8, 37.0, 30.9, 30.7, 30.4, 30.2, 28.8, 27.1, 26.8, 15.6; LRMS (ESI) m/z 517.3 (M ⁺ +H), 539.2 (M ⁺ +Na); HRMS (ESI) calcd for C ₂₅ H ₄₉ N ₄ O ₇ (M ⁺ +H) m/z 517.3601, found 517.3600.

Preparation of compound 84:

[0334] To a solution of compound 82 (178 mg) in CH ₂ CI ₂ (5 mL) cone. HC1 (0.5 mL) was added at 0 °C. The reaction was stirred at 0 °C for 10 min. The mixture was then basicified with 1M K ₂ CO ₃ solution and was concentrated. The solid residue was extracted repeatedly with EtOH and the filtrate was concentrated to give compound 84 as a white solid (178 mg, 100%).

[0335] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/= 0.15; H NMR

(400 MHz, CD ₃ OD) δ 3.82 (quintet, / = 5.1 Hz, 1H), 3.69 (q, / = 6.9 Hz, 1H), 3.63-3.62 (m, 4H), 3.60-3.57 (m, 4H), 3.56-3.52 (m, 4H), 3.25 (t, / = 6.8 Hz, 2H), 3.13 (t, / = 6.7 Hz, 2H), 2.73 (t, / = 6.8 Hz, 2H), 2.19 (t, / = 7.4 Hz, 2H), 1.79-1.70 (m, 4H), 1.63 (quintet, / = 7.1 Hz, 2H), 1.50-1.30 (m, 6H), 1.10 (d, / = 6.4 Hz, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.1, 166.1, 71.5, 71.4, 71.2, 71.1, 70.4, 69.9, 57.4, 52.7, 40.1, 37.0, 33.4, 31.7, 30.7, 30.5, 30.2, 27.1, 26.8, 15.7; LRMS (ESI) m/z 417.1 (M ⁺ +H); HRMS (ESI) calcd for C ₂₀ H ₄₁ N ₄ O ₅ (M ⁺ +H) m/z 417.3077, found 417.3070.

Preparation of compound 85:

[0336] For procedure and characterization data, refer to Org. Lett. 2007, 9, 3797-

3800.

Preparation of compound 86:

[0337] Compound 86 was synthesized in accordance with or by slight modification of the method set forth in Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F. Bioorg. Chem. 1986, 14, 134. To a solution of m-azidobenzoic acid (446 mg, 2.74 mmol) in CH ₂ CI ₂ (30 mL) was added catalytic amount of DMF (Tl 13 drop). Oxalyl chloride (0.23 mL, 2.74 mmol) was slowly added. The reaction was stirred until no further gas bubbles given out. The reaction was concentrated and the product was subjected to high vacuum for 3 h. The crude product was directly used for next step without further purification.

Preparation of compound 87:

[0338] To a solution of compound 86 (in-situ generation, 2.74 mmol) in CH ₂ CI ₂ (30 mL) was added H-Lys(Boc)-OMe (856 mg, 3.29 mmol) and pyridine (0.27 mL, 3.29 mmol). When TLC indicated completion of reaction, the reaction was transferred to a separatory funnel. The organic layer was washed with IN HC1 (10 mL ^χ 2), 1M K2CO3 solution (10 mL x 2) and brine (10 mL ^χ 1). The organic layer was dried over Na ₂ SC>4, filtered via short-pad silica gel and be concentrated. The crude product was further purified by column

chromatography using 1-2% EtOH in CH ₂ CI ₂ to give purified product as white solid (509 mg, 46%).

[0339] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, R/= 0.70; ¾ NMR

(400 MHz, CDCI ₃ ) δ 7.51 (d, / = 7.7 Hz, 1H), 7.46 (s, 1H), 7.36 (t, / = 7.7 Hz, 1H), 7.10 (d, / = 7.8 Hz, 1H), 7.01 (d, / = 6.6 Hz, 1H), 4.73 (q, / = 6.6 Hz, 1H), 4.67 (br s, 1H), 3.74 (s, 3H), 3.07-3.02 (m, 2H), 1.95-1.88 (m, 1H), 1.82-1.75 (m, 1H), 1.51-1.43 (m, 2H), 1.40-1.36 (m, 11H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 172.9, 166.2, 156.1, 140.7, 135.6, 129.8, 123.2, 122.0, 118.1, 79.0, 52.5, 52.4, 39.9, 31.8, 29.6, 28.3, 22.5; LRMS (EI) m/z 405.2 (M ⁺ ), 305.2;

HRMS (EI) calcd for C ₁ 9H ₂ 7O5N5 (M ⁺ ) m/z 405.2007, found 405.2007.

Preparation of compound 88:

[0340] To a solution of compound 87 (509 mg, 1.26 mmol) in THF (10 mL) and H ₂ 0

(10 mL) Li OH (49 mg, 1.38 mmol) was added. The reaction was stirred at rt. When TLC indicated completion of reaction, the mixture was acidified with IN HC1, followed by addition of EtOAc (50 mL). The organic layer was obtained and was washed with brine (7 mL χ 1). The organic layer was dried over Na ₂ SO/ _t , filtered and concentrated to give crude product. The crude compound 88 was a yellow soid , and was pure enough for next step (494 mg, 100%).

[0341] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/ = 0.10; i NMR

(400 MHz, CDCI ₃ ) δ 10.90 (br s, 1H), 7.66 (d, / = 7.1 Hz, 1H), 7.49-7.32 (m, 2H), 7.24 (t, / = 7.7 Hz, 1H), 6.99 (dd, / = 7.8, 1.8 Hz, 1H), 4.97-4.76 (m, 2H), 3.05-2.99 (m, 2H), 1.98- 1.75 (m, 2H), 1.52-1.33 (m, 13H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 175.2, 166.6, 156.5, 140.3, 135.0, 129.6, 123.3, 121.9, 118.0, 79.4, 52.5, 39.8, 31.3, 29.3, 28.2, 20.8; LRMS (EI) m/z 391.2 (M ⁺ ), 347.2 (M ⁺ -C0 ₂ ); HRMS (EI) calcd for ^Η ₂₅ 0 ₃ Ν ₅ (M ⁺ -C0 ₂ ) m/z 347.1952, found 347.1944.

Preparation of compound 89:

[0342] Compound 88 (71 mg, 0.182 mmol) was dissolved in CH ₂ C1 ₂ ( 1 mL) . EDC

(42 mg, 0.219 mmol), HOBt (30 mg, 0.219 mmol) and Et ₃ N (31 μ_ϋ, 0.219 mmol) were added. The reaction was stirred for 40 min. Compound 82 (76 mg, 0.182 mmol) was pre-dissolved in CH ₂ C1 ₂ (3 mL) and DMF (1 mL) and was added to the reaction. The reaction was stirred at rt for 3 h. When TLC indicated completion of reaction, the reaction mixture was diluted with CH ₂ C1 ₂ (10 mL) and the organic layer was washed with IN HC1 (5 mL χ 2), H ₂ 0 (5 mL χ 2) and brine (5 mL χ 1). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by flash chromatography using 8-15% EtOH in CH ₂ C1 ₂ to give purified product as a white solid (88 mg, 61%).

[0343] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, R/= 0.38; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.67 (d, / = 7.8 Hz, 1H), 7.59 (t, / = 1.8 Hz, 1H), 7.49 (t, / = 7.9 Hz, 1H), 7.24 (ddd, / = 8.0, 2.3, 0.8 Hz, 1H), 4.50-4.46 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 3.63-3.60 (m, 4H), 3.59-3.56 (m, 4H), 3.50 (q, / = 5.7 Hz, 4H), 3.30-3.23 (m, 4H), 3.20 (quintet, / = 4.4 Hz, 1H), 3.04 (t, / = 6.6 Hz, 2H), 2.91 (dd, / = 12.7, 5.0 Hz, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.19 (t, / = 7.4 Hz, 2H), 1.93-1.72 (m, 7H), 1.70-1.49 (m, 6H), 1.46-1.42 (m, 3H), 1.40 (s, 9H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 176.0, 175.9, 174.4, 169.0, 166.0, 142.0, 137.1, 131.1, 125.1, 123.2, 119.2, 79.8, 71.5, 71.4, 71.2, 70.0, 69.9, 69.8, 63.4, 61.6, 57.0, 55.6, 41.0, 38.1, 37.9, 37.8, 36.9, 32.8, 30.6, 30.4, 30.3, 29.8, 29.5, 28.8, 26.9, 24.4; LRMS (ESI) m/z 820.2 (M ⁺ +H), 842.2 (M ⁺ +Na); HRMS (ESI) calcd for (M ⁺ +H) m/z 790.4827, found 790.4828.

Preparation of compound 90:

[0344] To a solution of compound 89 (88 mg) in CH ₂ C1 ₂ (4 mL) cone. HC1 (0.5 inL) was added at 0 °C. The reaction was stirred at 0 °C for 10 min. The mixture was then basicified with 1M K ₂ CO3 solution and was concentrated. The solid residue was extracted repeatedly with EtOH and the filtrate was concentrated to give product as a white solid (73 mg, 100%).

[0345] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/= 0.01; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.67 (d, / = 7.8 Hz, 1H), 7.59 (s, 1H), 7.49 (t, / = 7.9 Hz, 1H), 7.24 (d, / = 8.0 Hz, 1H), 4.50-4.46 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 3.69-3.67 (m, 1H), 3.63- 3.60 (m, 5H), 3.59-3.56 (m, 4H), 3.54-3.50 (m, 4H), 3.25 (t, / = 6.7 Hz, 2H), 3.20 (quintet, / = 4.4 Hz, 1H), 2.92 (dd, / = 12.7, 5.0 Hz, 1H), 2.72-2.66 (m, 3H), 2.19 (t, / = 7.4 Hz, 2H), 1.93-1.72 (m, 7H), 1.70-1.49 (m, 6H), 1.46-1.42 (m, 3H); ¹³ C NMR (100 MHz, D ₂ 0) δ 177.8, 175.3, 170.9, 166.6, 142.0, 131.8, 125.1, 124.1, 119.2, 73.0, 71.0, 70.8, 69.9, 69.7, 63.5, 61.6, 56.8, 56.3, 42.4, 41.3, 41.1, 37.8, 37.0, 32.2, 30.7, 29.7, 29.4, 29.1, 26.6, 24.0, 18.2; LRMS (ESI) m/z 720.2 (M ⁺ +H), 742.3 (M ⁺ +Na); HRMS (ESI) calcd for C ₃ 3H ₅₆ N ₉ 0 ₇ (M ⁺ +H) m/z 690.4303, found 690.4300.

Preparation of T144:

[0346] To a solution of compound 90 (37 mg, 0.0535 mmol) in CH ₂ C1 ₂ (2 mL) and

DMF (0.3 mL) was added compound 74 (28 mg, 0.0535 mmol), followed by Et ₃ N (7.5 μί, 0.0535 mmol). The reaction was stirred at rt for 3.5 h. When TLC indicated completion of reaction, the reaction mixture was diluted with CH ₂ C1 ₂ (20 mL) and the organic layer was washed with IN HC1 (5 mL ^χ 1), 1M K ₂ CC>3 (5 mL portion until the aqueous layer is colourless) and brine (5 mL ^χ 1). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by flash chromatography using 10-15% EtOH in CH ₂ CI ₂ to give purified product as a white solid (40 mg, 70%).

[0347] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, = 0.30; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.67 (d, / = 7.8 Hz, 1H), 7.58 (s, 1H), 7.49 (t, / = 7.9 Hz, 1H), 7.24 (dd, / = 8.0, 2.2 Hz, 1H), 7.15 (d, / = 8.6 Hz, 2H), 4.87 (s, 2H), 4.50-4.46 (m, 2H), 4.29 (dd, / = 6.8, 4.4 Hz, 1H), 4.20-3.95 (m, 3H), 3.62-3.60 (m, 4H), 3.59-3.57 (m, 5H), 3.51 (q, / = 5.7 Hz, 4H), 3.31-3.30 (m, 2H), 3.25-3.23 (m, 2H), 3.22-3.17 (m, 1H), 3.13-3.09 (m, 2H), 2.92 (dd, / = 12.7, 5.0 Hz, 1H), 2.87-2.83 (m, 1H), 2.70 (d, / = 12.7 Hz, 1H), 2.33-2.25 (m, 4H), 2.19 (t, / = 7.4 Hz, 2H), 1.99 (t, / = 14.0 Hz, 1H), 1.94-1.67 (m, 10H), 1.65-1.52 (m, 7H), 1.46-1.41 (m, 4H), 1.08 (s, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 193.3, 176.0 (2C), 174.4, 169.3, 166.2, 163.6, 159.2, 144.1, 142.3, 142.0, 137.1, 131.2, 125.7, 125.1, 123.2, 119.3, 72.0, 71.5 (2C), 71.2, 70.0, 69.9, 65.4, 64.9, 63.4, 61.6, 61.1, 57.0, 55.7, 53.0, 41.6, 41.3, 41.1, 38.0, 37.8, 36.9, 36.1, 32.7, 31.1, 30.6, 30.4, 30.3, 29.8, 29.5, 28.7, 27.0, 26.9, 24.3, 24.1, 17.9, 14.1; LRMS (ESI) m/z 1126.3 (M ⁺ +Na); HRMS (ESI) calcd for

C ₅₄ H ₇₆ N ₉ Oi ₄ (M ⁺ +H) m/z 1074.5512, found 1074.5510.

[0348] Compound T145, Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ Rf =

0.20; ¾ NMR (400 MHz, CDC1 ₃ ) δ 7.63 (t, / = 6.9 Hz, 1H), 7.60-7.59 (m, 1H), 7.57 (s, 1H), 7.40 (t, / = 7.9 Hz, 1H), 7.13 (ddd, / = 7.8, 2.1, 0.7 Hz, 1H), 4.70 (AB system, 2H), 4.60- 4.54 (m, 2H), 4.08 (d, / = 2.5 Hz, 1H), 4.07-3.95 (m, 2H), 3.89 (d, / = 2.5 Hz, 1H), 3.83 (quintet, / = 8.7 Hz, 1H), 3.71-3.50 (m, 14H), 3.43 (d, / = 5.3 Hz, 1H), 3.40-3.25 (m, 4H), 3.18-3.10 (m, 2H), 2.83-2.80 (m, 1H), 2.40-2.31 (m, 1H), 2.24-2.10 (m, 5H), 2.02-1.94 (m, 2H), 1.80-1.76 (m, 5H), 1.72-1.28 (m, 17H), 1.09 (dd, / = 15.3, 6.4 Hz, 3H), 1.06 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 200.1, 176.2, 176.1, 174.5, 169.2, 166.2, 163.4, 159.1, 142.2, 137.2, 131.3 125.8, 125.2, 123.4, 119.4, 72.1, 71.6 (2C), 71.3, 70.1 (2C), 70.0 (2C), 66.8, 65.3, 64.5, 62.8, 62.5, 57.8, 57.5, 55.9, 52.8, 41.7, 41.4, 38.1, 37.9, 37.2, 36.5, 32.8, 31.7, 30.8, 30.7, 30.6 (2C), 30.4, 30.3, 27.3, 27.0, 26.7, 25.5, 24.4, 24.0, 18.1, 15.8, 14.4; LRMS (ESI) m/z 1112.4 (M+Na); HRMS (ESI) calcd for C ₅ 4H ₇₆ N ₉ 0 ₁₅ (M ⁺ +H) m/z 1090.5041, found 1090.5040.

[0349] Scheme 19. Synthetic route to T150.

[0350] Scheme 19 Reagents and conditions: (a) DMAP, CH ₂ C1 ₂ , rt, 33%.

Preparation of T150:

[0351] To a solution of compound 64 (11 mg, 0.01997 mmol) in CH ₂ Q ₂ (1 mL) were added compound 82 (6 mg, 0.01815 mmol) and one small crystal of DMAP. The reaction mixture was stirred at room temperature overnight, followed by concentration and purification by flash column chromatography to afford T150 as a colorless solid (5 mg, 33%).

[0352] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, Rf = 0.13; 1H NMR

(400 MHz, CDCI ₃ ) δ 6.30 (br s, 1H), 5.38 (br s, 1H), 5.09 (br s, 1H), 4.80-4.60 (m, 2H), 4.52 (br s, 1H), 4.20-3.95 (m, 2H), 3.90 (s, 1H), 3.89-3.75 (m, 1H), 3.70-3.50 (m, 9H), 3.48-3.43 (m, 3H), 3.42-3.30 (m, 3H), 2.80-2.60 (m, 1H), 2.40-2.28 (m, 2H), 2.26-2.05 (m, 5H), 2.04 (s, 1H), 1.96 (t, / = 14.1 Hz, 1H), 1.90-1.60 (m, 6H), 1.59-1.50 (m, 4H), 1.49-1.20 (m, 2H), 1.19-1.10 (m, 6H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 173.2, 173.1, 163.5, 159.9, 156.7, 125.6, 73.7, 70.2, 70.1, 70.0, 65.9, 64.3, 62.38, 60.6, 60.3, 56.5, 56.0, 55.5, 51.4, 40.9, 40.8, 40.4, 39.2, 36.2, 35.9, 35.1, 30.0, 29.7, 29.5, 28.8, 28.1, 25.3, 23.6, 23.3, 17.1, 15.8, 13.7; LRMS (ESI) m/z 769.0 (M ⁺ +Na); HRMS (ESI) calcd. for (M ⁺ +H) m/z 747.3811, found 747.3813.

[0353] Scheme 20. Synthetic route to T151. 110

T151

[0354] Scheme 20 Reagents and conditions: (a) EDC, HOBt, Et ₃ N, DMF, 78%; (b) piperidine, CH ₂ C1 ₂ , 72%; (c) Et ₃ N, CH ₂ C1 ₂ /DMF, 29%; (d) 5% H ₂ 0 ₂ , NaOH, MeOH, 0 °C, 92%; (e) K-selectride, THF/MeOH, -78 to -50 °C, 70%; (f) (i) cone. HC1, CH ₂ C1 ₂ , 0 °C; (ii) EtOH extract, crude; (g) 85, EDC, HOBt, DMF. Compound 95 was prepared under reaction conditions similar to compound 17 of Example 1, Scheme 1 and compound 94 was prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above. as described above.

Preparation of compound 91: [0355] Fmoc-Lys(Boc)-OH (62 mg, 0.132 mmol) was dissolved in DMF (1 mL).

EDC (30 mg, 0.158 mmol), HOBt (21 mg, 0.158 mmol) and Et ₃ N (22 μί, 0.158 mmol) were added. The reaction was stirred for 30 min. Compound 84 (55 mg, 0.132 mmol) was pre- dissolved in DMF ( 1 mL) and was added to the reaction. The reaction was stirred at rt for 4 h. When TLC indicated completion of reaction, the reaction mixture was diluted with CH ₂ CI ₂ (20 mL) and the organic layer was washed with IN HC1 (2 5 mL), 1M K ₂ C0 ₃ (2 5 mL), H ₂ 0 (2 x 5 mL) and brine ( 1 x 5 mL). The organic layer was dried over NaiSO/t, filtered and concentrated. The crude product was purified by flash chromatography using 10% EtOH in CH ₂ CI ₂ to give compound 91 as a white solid (89 mg, 78%).

[0356] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, = 0.48; ¾ NMR

(300 MHz, CDCI ₃ ) δ 7.82 (d, / = 7.6 Hz, 2H), 7.59 (d, / = 6.9 Hz, 2H), 7.37 (t, / = 7.3 Hz, 2H), 7.28 (t, / = 7.2 Hz, 2H), 7.20 (br s, 1H, NH), 6.61 (br s, 1H, NH), 6.22 (br s, 1H, NH), 5.74 (br s, 1H, NH), 4.99 (br s, 1H, NH), 4.87 (br s, 1H, NH), 4.36 (quintet, / = 6.8 Hz, 2H), 4.21-4.13 (m, 2H), 3.80 (quintet, / = 6.7 Hz, 1H), 3.69-3.62 (m, 1H), 3.56-3.40 (m, 12H), 3.42-3.29 (m, 4H), 3.13-3.00 (br m, 2H), 2.10 (t, / = 7.1 Hz, 2H), 1.88-1.68 (m, 5H), 1.68- 1.52 (m, 3H), 1.52-1.20 (m, 19H), 1.06 (d, / = 6.2 Hz, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 174.5, 173.4, 165.2, 157.8, 145.2, 142.6, 129.1, 128.5, 127.3, 126.5, 125.8, 121.3, 120.1, 80.4, 71.7, 71.6, 71.4, 71.3, 71.2, 71.0, 68.2, 57.4, 56.3, 52.8, 48.5, 44.8, 41.5, 39.0, 37.5, 33.7, 30.9 (2C), 30.3, 30.2, 29.8, 27.2, 26.7, 24.0, 17.1 ; LRMS (ESI) m/z 645.3 (M ⁺ +H), 667.2 (M ⁺ +Na); HRMS (ESI) calcd for C ₄ 6H ₇₁ N ₆ Oi ₀ (M ⁺ +H) m/z 867.5232, found 867.5230.

Preparation of compound 92:

[0357] To a solution of compound 90 (84 mg) in CH ₂ CI ₂ (4 mL) piperidine (0.3 mL) was added. The reaction was stirred at at for 1 h. When TLC indicated completion of reaction, the reaction was concentrated and was subjected under high vacuum for at least 30 min. The residue was re-dissolved in IN HC1 (2 mL) and was washed with CH ₂ CI ₂ (3 x 4 mL). The aqueous layer was basicified by the addition of solid K ₂ CO3 and was concentrated. The solid residue was extracted repeatedly with EtOH and was filtered. The filtrate was concentrated to give compound 92 as a white solid (45 mg, 72%).

[0358] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ Rf = 0.13; ¾ NMR

(400 MHz, CD ₃ OD) δ 3.81 (quintet, / = 6.7 Hz, 1H), 3.69 (q, / = 7.1 Hz, 1H), 3.66-3.62 (m, 4H), 3.62-3.57 (m, 4H), 3.52 (q, / = 6.0 Hz, 4H), 3.32-3.24 (m, 6H), 3.03 (t, / = 6.9 Hz, 1H), 2.65 (br s, 1H), 2.19 (t, / = 7.4 Hz, 2H), 1.76 (sextet, / = 6.8 Hz, 5H), 1.71-1.59 (m, 4H), 1.52-1.28 (m, 17H), 1.05 (d, / = 6.2 Hz, 3H); ¹³ C NMR (100 MHz, CD ₃ OD) δ 177.6, 176.1, 166.1, 157.8, 79.5, 71.5, 71.2, 69.9 (2C), 57.4, 56.1, 52.3, 42.2, 41.1, 37.7, 37.0, 36.3, 30.7, 30.4 (2C), 30.2, 28.8, 27.1, 26.8, 24.1, 24.0, 15.6; LRMS (ESI) m/z 645.3 (M ⁺ +H), 667.2 (M ⁺ +Na); HRMS (ESI) calcd for C ₃ iH ₆₁ N ₆ 0 ₈ (M ⁺ +H) m/z 645.4551, found 645.4550.

Preparation of compound 93:

[0359] To a solution of compound 92 (45 mg, 0.0698 mmol) in CH ₂ CI ₂ (1 mL) and

DMF (1 mL) was added the compound 74 (37 mg, 0.0698 mmol), followed by Et ₃ N (10 0.0698 mmol). The reaction was stirred at rt for 6 h. When TLC indicated completion of reaction, the reaction mixture was diluted with CH ₂ CI ₂ (10 mL) and the organic layer was washed with IN HC1 (1 x 5 mL), 1M K ₂ CO3 (5 mL portion until the aqueous layer is colourless) and brine (1 x 5 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by flash chromatography using 20% EtOH in CH ₂ CI ₂ to give compound 93 as a white solid (21 mg, 29%).

[0360] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ „ R/= 0.30; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.18 (d, / = 5.3 Hz, 1H), 4.05-4.00 (m, 2H), 3.97 (d, / = 6.3 Hz, 2H), 3.80 (quintet, / = 6.7 Hz, 1H), 3.69 (q, / = 7.1 Hz, 1H), 3.65-3.63 (m, 4H), 3.60-3.56 (m, 4H), 3.51 (t, / = 8.2 Hz, 4H), 3.30-3.23 (m, 4H), 3.10 (t, / = 8.4 Hz, 1H), 3.03 (t, / = 8.8 Hz, 1H), 2.92-2.87 (br m, 1H), 2.37-2.22 (m, 6H), 2.21-2.13 (m, 3H), 1.99 (t, / = 17.7 Hz, 1H), 1.80-1.70 (m, 8H), 1.67-1.55 (m, 5H), 1.53-1.32 (m, 16H), 1.11-1.08 (m, 6H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 193.5, 176.2, 176.1, 174.9, 166.3, 163.7, 158.6, 144.3, 142.5, 125.8,

80.0, 72.1, 71.7, 71.6, 71.4, 70.1, 65.4 (2C), 65.1, 64.4, 61.3, 57.5, 56.8, 53.2, 52.8, 41.7, 41.4, 41.2, 38.1, 37.9, 37.2, 36.2, 33.2, 31.2, 30.9, 30.7, 30.6, 30.5, 30.4 (2C), 29.0, 28.8, 27.3,

27.1, 27.0, 24.4, 24.2, 18.1, 15.8, 14.2; LRMS (ESI) m/z 1030.3 (M ⁺ +H), 1052.3 (M ⁺ +Na); HRMS (ESI) calcd for C5 ₂ H ₈ iN ₆ 0i5 (M ⁺ +H) m/z 1029.5760, found 1029.5755.

[0361] Compound 94, analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , _, R/ =

0.30; ¾ NMR (400 MHz, CD ₃ OD) δ 4.28-4.26 (m, 1H), 4.09-4.07 (m, 2H), 4.04-3.95 (m, 3H), 3.82 (quintet, / = 7.7 Hz, 1H), 3.80 (quintet, / = 6.5 Hz, 1H), 3.64-3.61 (m, 4H), 3.60- 3.58 (m, 4H), 3.53-3.50 (m, 4H), 3.46 (t, / = 5.2 Hz, 1H), 3.28-3.23 (m, 4H), 3.09 (t, / = 6.7 Hz, 1H), 3.02 (t, / = 6.7 Hz, 1H), 2.95-2.89 (br m, 1H), 2.37-2.22 (m, 3H), 2.19 (t, / = 7.4 Hz, 2H), 2.14-2.01 (m, 2H), 1.97 (t, / = 13.6 Hz, 2H), 1.78-1.70 (m, 8H), 1.66-1.60 (m, 3H), 1.53-1.32 (m, 18H), 1.10 (d, / = 6.4 Hz, 3H), 1.03 (s, 3H); ^lj C NMR (125 MHz, CD ₃ OD) δ 200.2, 176.2, 176.1, 175.0, 166.2, 163.4, 159.1, 158.5, 125.8, 80.0, 72.1 (2C), 71.6 (2C), 71.4, 70.1, 66.8, 65.8, 65.3, 64.6, 62.8, 62.6, 58.0, 57.5, 56.8, 54.9, 52.8, 41.7, 41.4, 41.2, 38.0, 37.9, 37.2, 36.5, 33.2, 31.7, 30.9, 30.7, 30.6, 30.4 (2C), 29.0, 27.3, 27.0, 26.8, 25.5, 24.4, 23.8,

18.1, 15.8, 14.4; LRMS (ESI) m/z 1045.3 (M ⁺ +H), 1067.3 (M ⁺ +Na); HRMS (ESI) calcd for C ₅₂ H ₈ iN ₆ 0i ₆ (M ⁺ +H) m/z 1045.5709, found 1045.5702.

Preparation of compound 95:

[0362] Compound 95, analytical TLC (silica gel 60), 10% Me OH in CH ₂ C1 ₂ , _, R/ =

0.30; ¹ H NMR (400 MHz, CD ₃ OD) δ 4.92-4.77 (m, 3H), 4.15-4.00 (m, 3H), 4.04-3.85 (m, 1H), 3.82 (quintet, / = 7.7 Hz, 1H), 3.73 (quintet, / = 6.5 Hz, 1H), 3.64-3.61 (m, 4H), 3.60- 3.58 (m, 4H), 3.53-3.50 (m, 4H), 3.39 (s, 1H), 3.30-3.25 (m, 4H), 3.09 (t, / = 6.7 Hz, 1H), 3.02 (t, / = 6.7 Hz, 1H), 2.79-2.76 (br m, 1H), 2.37-2.22 (m, 3H), 2.19 (t, / = 7.4 Hz, 2H), 2.14-2.01 (m, 2H), 1.97 (t, / = 13.6 Hz, 2H), 1.78-1.70 (m, 8H), 1.66-1.60 (m, 3H), 1.53- 1.32 (m, 18H), 1.10 (d, / = 6.4 Hz, 3H), 1.03 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 176.2, 176.1, 175.0, 164.2, 164.1, 158.7, 125.6, 125.5, 80.0, 74.3, 72.1, 71.7, 71.6, 71.4, 70.1, 66.2,

66.0, 65.6, 62.9, 62.2, 61.9, 57.7, 57.5, 57.2, 56.7, 54.9, 52.8, 41.7, 41.4, 41.2, 38.1, 38.0,

37.2, 37.1, 36.5, 33.2, 30.8, 30.7, 30.6, 30.4 (2C), 29.0, 27.3, 27.0, 26.8, 25.5, 24.4, 24.3, 24.1,

18.1, 15.8, 14.2; LRMS (ESI) m/z 1047.3 (M ⁺ +H), 1069.3 (M ⁺ +Na); HRMS (ESI) calcd for C ₅₂ H ₈ 3N ₆ Oi ₆ (M ⁺ +H) m/z 1047.5866, found 1047.5860.

Preparation of compound 96:

[0363] To a solution of compound 95 (14 mg) in CH ₂ CI ₂ (2 mL) was added cone. HC1

(0.4 mL) slowly at 0 °C. The reaction time was 25 min. The reaction was then basicified with 1M K ₂ CO ₃ solution. The content was concentrated. The dried residue was extracted with EtOH repeatedly. The EtOH extract was concentrated to give crude compound 96 as slurry of solid (10 mg).

[0364] Characterization was not performed for this crude product.

Preparation of T151:

[0365] To a solution of compound 85 (2 mg, 0.0106 mmol) in DMF (1 mL) was added EDC (2.5 mg, 0.0127 mmol), HOBt (1.7 mg, 0.0127 mmol) and Et ₃ N (1.8 μί, 0.0127 mmol). The reaction was stirred for 1 h. Compound 96 (10 mg, 0.0106 mmol) was added to the reaction. When TLC indicated completion of reaction, the mixture was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (2 x 3 mL), brine (1 x 3 mL) and was dried over Na ₂ SC>4. The product was filtered and concentrated to give crude product. The crude product was purified by flash chromatography (10% EtOH in CH ₂ CI ₂ ) to give

T151 (2 mg).

[0366] Note: since only 2 mg of product was obtained, only LC-MS can be used to characterize the product. The amount is too little for NMR analysis.

[0368] Scheme 21 Reagents and conditions: (a) Et ₃ N, DMAP, CH ₂ C1 ₂ , DMF, 37%.

[0369] To a solution of compound 71 (24 mg, 0.058 mmol) in CH ₂ Q ₂ (1 mL) and

DMF (0.3 mL) was added compound 74 (23 mg, 0.058 mmol), Et ₃ N (7 uL, 0.0484 mmol) and DMAP (0.5 mg, 0.00484 mmol). The reaction was stirred overnight. The reaction was then diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (2 x 2 mL), 1M K ₂ CO ₃ solution (2 mL portion until aqueous was colorless) and brine (1 x 2 mL). The mixture was dried over Na ₂ SC>4, filtered and concentrated. The crude product was taken for column chromatography using 12% EtOH to give purified product as a white solid (18 mg, 37%).

[0370] Analytical TLC (silica gel 60), 60% acetone in CH ₂ C1 ₂ , R/= 0.39; ¾ NMR

(400 MHz, CD ₃ OD) δ 7.18 (d, / = 4.4 Hz, 1H), 4.49 (dd, / = 7.8, 5.0 Hz, 1H), 4.31 (dd, / = 7.8, 4.4 Hz, 1H), 4.00 (t, / = 6.3 Hz, 2H), 3.97 (d, / = 4.7 Hz, 1H), 3.64-3.63 (m, 4H), 3.60- 3.56 (m, 5H), 3.52 (t, / = 6.2 Hz, 4H), 3.26 (t, / = 6.8 Hz, 2H), 3.20-3.17 (m, 3H), 2.96-2.91 (m, 2H), 2.70 (d, / = 12.7 Hz, 1H), 2.35-2.28 (m, 4H), 2.20 (t, / = 7.4 Hz, 2H), 2.00 (t, / = 13.8 Hz, 1H), 1.79-1.54 (m, 12H), 1.47-1.40 (m, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 193.5, 176.1 (2C), 166.2, 163.7, 159.1, 72.2, 71.7, 71.4, 70.1, 70.0, 65.4, 65.1, 64.4, 63.5, 61.8, 61.3, 57.1, 53.2, 47.7, 41.8, 41.2, 39.3, 38.0, 37.0, 36.2, 31.2, 31.0, 30.6, 29.9, 28.7, 27.1, 27.0,

24.2, 18.1 ; LRMS (ESI) m/z 831.4 (M ⁺ +H); HRMS (ESI) calcd for C ₄₁ H58N ₄ O ₁₂ S (M ⁺ +H) m/z 831.3845, found 831.3834.

[0372] Scheme 22 Reagents and conditions: (a) p-nitrophenylchloroformate, pyridine, CH ₂ C1 ₂ , 45%; (b) 95, Et ₃ N, DMAP, CH ₂ C1 ₂ /DMF, 91%; (c) K-selectride, THF, -78 °C, 37%; (d) p-nitrophenylchloroformate, pyridine, CH ₂ C1 ₂ , 73%; (e) 96, Et ₃ N, DMAP, CH ₂ C1 ₂ /DMF, 72%. Compound 98 was prepared under reaction conditions similar to compound 17 of Example 1, Scheme 1 as described above.

Preparation of compound 97:

[0373] To a solution of T125 (20 mg, 0.0483 mmol) in CH ₂ C1 ₂ (1.5 inL) was added

4-nitrophenylchloroformate (19 mg, 0.0966 mmol) and pyridine (8 0.0966 mmol). The mixture was stirred overnight at rt. The mixture was diluted with CH ₂ C1 ₂ (7 mL). The organic layer was washed with IN HC1 (1 x 2 mL), sat. NaHC0 ₃ (2 2 mL) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by column chromatography using 50% EtOAc in «-hexane to give purified compound 97 as a slight yellow solid (12 mg, 45%).

[0374] Analytical TLC (silica gel 60), 60% EtOAc in ra-hexane, = 0.47; ¾ NMR

(400 MHz, CDCI ₃ ) δ 8.31-8.27 (m, 2H), 7.41-7.37 (m, 2H), 4.76-4.65 (m, 2H), 4.29 (t, / = 6.4 Hz, 2H), 4.06 (d, / = 2.8 Hz, 1H), 3.88 (d, / = 4.7 Hz, 1H), 3.44 (d, / = 5.4 Hz, 1H), 2.84-2.81 (m, 1H), 2.39-2.35 (m, 1H), 2.26-2.13 (m, 2H), 2.04-1.94 (m, 2H), 1.79-1.75 (m, 3H), 1.63-1.50 (m, 4H), 1.32 (dt, / = 12.1, 5.9 Hz, 1H), 1.08 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 197.2, 172.9, 159.3, 155.5, 152.5, 145.4, 125.8, 125.3, 121.8, 69.9, 68.9, 65.3, 63.3, 61.2, 60.6 (2C), 56.0, 40.6, 35.4, 30.6, 28.3, 28.2, 23.3, 20.7, 17.1, 13.8; LRMS (EI) m/z

553.2 (M ⁺ , 5); HRMS (EI) calcd for m/z 553.1579, found 553.1579.

Preparation of T249:

[0375] To a solution of compound 96 (11 mg, 0.0247 mmol) in DMF (0.3 mL) was added a solution of compound 97 (12 mg, 0.0217 mmol) in CH ₂ Q ₂ (1 mL), followed by Et ₃ N (3.6 0.026 mmol) and DMAP (0.3 mg, 0.00217 mmol). The reaction was stirred at rt for 3 h. The mixture was diluted with CH2CI2 (10 mL). The organic layer was washed with IN HC1 (1 x 2 mL), 1M K ₂ CO3 (2 mL portion until aqueous was colorless) and brine (2 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The purity of T249 (16 mg, 91%) was analyzed by LC-MS. The product was appeared as colorless solid.

[0376] Analytical TLC (silica gel 60), 15% MeOH in CH ₂ C1 ₂ , R/= 0.56; ¾ NMR

(400 MHz, CD ₃ OD) δ 4.92-4.82 (m, 2H), 4.50 (dd, / = 7.8, 4.9 Hz, 1H), 4.31 (dd, / = 7.8,

4.5 Hz, 1H), 4.23 (d, / = 2.6 Hz, 1H), 4.04-4.00 (m, 3H), 3.65-3.63 (m, 4H), 3.60-3.59 (m, 4H), 3.53 (t, / = 6.1 Hz, 4H), 3.42 (d, / = 5.4 Hz, 1H), 3.26 (t, / = 6.8 Hz, 2H), 3.23 (dd, / = 8.5, 4.1 Hz, 1H), 3.18 (t, / = 6.7 Hz, 2H), 2.95-2.91 (m, 2H), 2.70 (d, / = 12.7 Hz, 1H), 2.37- 2.26 (m, 2H), 2.20 (t, / = 7.4 Hz, 2H), 2.16-2.06 (m, 1H), 2.01-1.88 (m, 2H), 1.79-1.73 (m, 6H), 1.69-1.52 (m, 8H), 1.48-1.40 (m, 2H), 1.34-1.31 (m, 1H), 1.04 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 200.2, 176.1, 166.2, 163.5, 159.3, 125.8, 72.2, 71.7, 71.4 (2C), 70.1, 70.0, 66.7, 65.5, 64.7, 63.5, 62.8 (2C), 62.7, 62.4, 61.8, 57.7, 57.2, 41.7, 41.2, 39.3, 38.0, 37.0, 36.6, 31.7, 31.0, 30.6, 30.2, 30.0, 29.7, 29.6, 29.5, 27.0, 24.1, 22.2, 18.1, 14.4; LRMS (ESI) m/z 861.2 (M ⁺ +H), 883.3 (M ⁺ +Na); HRMS (ESI) m/z calcd for C42H60N4O13S (M ⁺ +H) 861.3950, found 861.3932.

[0377] Compound 98, analytical TLC (silica gel 60), 60% EtOAc in «-hexane, R/ =

0.24; ¾ NMR (400 MHz, CDC1 ₃ ) δ 4.69 (AB system, 2H), 3.90 (d, / = 3.1 Hz, 1H), 3.64 (t, / = 6.1 Hz, 2H), 3.44 (d, / = 2.6 Hz, 1H), 3.41-3.34 (m, 2H), 2.77 (d, / = 10.3 Hz, 1H), 2.73- 2.70 (m, 1H), 2.35-2.30 (m, 1H), 2.21-2.10 (m, 3H), 1.97 (t, / = 14.1 Hz, 1H), 1.71 (br s, 1H), 1.61-1.40 (m, 6H), 1.21 (dt, / = 12.3, 6.2 Hz, 1H), 1.11 (s, 3H); ¹³ C NMR (100 MHz, CDCI ₃ ) δ 173.2, 159.9, 125.5, 73.7, 69.9, 66.0, 62.7, 62.4, 60.5, 60.2, 56.6, 55.4, 40.4, 35.8, 32.4, 31.2,29.8, 23.6, 19.7, 17.0, 13.6; LRMS (EI) m/z 390.2 (M ⁺ ); HRMS (EI) calcd for C ₂₁ H ₂₆ O ₇ (M ⁺ ) m/z 390.1673, found 390.1673.

Preparation of compound 99:

[0378] To a solution of compound 98 (24 mg, 0.0615 mmol) in CH ₂ Q ₂ (1 mL) was added 4-nitrophenylchloroformate (28 mg, 0.139 mmol) and pyridine (10 0.123 mmol). The mixture was stirred overnight at rt. The mixture was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (2 x 2 mL), H ₂ 0 (1 x 2 mL) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SO/t, filtered and concentrated. The crude product was purified by column chromatography using 50-60% EtOAc in «-hexane to give compound 99 as a white solid (25 mg, 73%).

[0379] Analytical TLC (silica gel 60), 80% EtOAc in ra-hexane, = 0.44; ¾ NMR

(300 MHz, CDCI ₃ ) δ 8.28 (d, / = 9.0 Hz, 2H), 7.38 (d, / = 9.0 Hz, 2H), 4.69 (AB system, 2H), 4.29 (t, / = 6.3 Hz, 2H), 3.91 (d, / = 2.9 Hz, 1H), 3.45 (d, / = 2.7 Hz, 1H), 3.40-3.34 (m, 2H), 2.75 (d, / = 10.3 Hz, 1H), 2.74-2.69 (m, 1H), 2.36-2.30 (m, 1H), 2.23-2.14 (m, 3H), 1.98 (t, / = 14.0 Hz, 1H), 1.78 (quintet, / = 7.1 Hz, 2H), 1.64-1.47 (m, 4H), 1.22 (dt, / = 12.3, 6.2 Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 173.2, 159.8, 155.5, 152.5, 145.4, 125.6, 125.3, 121.8, 73.7, 69.9, 69.0, 66.0, 62.5, 60.5, 60.2, 56.5, 55.4, 40.4, 35.8, 31.1, 29.8, 28.8, 23.6, 19.8, 17.0, 13.6; LRMS (EI) m/z 555.2 (M ⁺ ); HRMS (EI) calcd for C ₂₈ H ₂₉ O ₁₁ N (M ⁺ ) m/z 555.1735, found 555.1730.

Preparation of T255:

[0380] To a solution of compound 99 (13.4 mg, 0.0241 mmol) and compound 96

(15.8 mg, 0.0354 mmol) in CH ₂ C1 ₂ ( 1 mL) and DMF (0.25 mL) was added Et ₃ N (5 μί, 0.0362 mmol) and DMAP (0.3 mg, 0.00241 mmol). The reaction was stirred at rt for 1.5 h. When TLC indicated the reaction was complete, the mixture was diluted with CH ₂ CI ₂ ( 10 mL). The organic layer was washed with IN HC1 (2 x 2 mL), 1M K ₂ CO3 (2 mL portion until aqueous was colorless) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SO/t, filtered and concentrated. The DMF residue was removed by azeotropic distillation with toluene, CHCI ₃ and MeOH. T255 ( 15 mg, 72%) was obtained as colorless solid.

[0381] Analytical TLC (silica gel 60), 15% MeOH in CH ₂ C1 ₂ , R/ = 0.44; H NMR

(400 MHz, CD ₃ OD) δ 4.88-4.76 (m, 2H), 4.49 (dd, / = 7.8, 5.0 Hz, 1H), 4.31 (dd, / = 7.8, 4.5 Hz, 1H), 4.01 (t, / = 6.3 Hz, 2H), 3.94 (d, / = 3.0 Hz, 1H), 3.65-3.63 (m, 4H), 3.60-3.58 (m, 4H), 3.54-3.51 (m, 4H), 3.47 (d, / = 2.5 Hz, 1H), 3.38-3.35 (m, 2H), 3.26 (t, / = 6.8 Hz, 2H), 3.23-3.16 (m, 3H), 2.93 (dd, / = 12.8, 5.1 Hz, 1H), 2.79-2.76 (m, 1H), 2.70 (d, / = 12.8 Hz, 1H), 2.29-2.18 (m, 4H), 2.12-2.02 (m, 2H), 1.94 (t, / = 13.5 Hz, 1H), 1.79-1.71 (m, 5H), 1.69-1.56 (m, 5H), 1.52-1.42 (m, 6H), 1.33 (dt, / = 12.3, 6.2 Hz, 1H), 1.09 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 176.3, 176.1, 166.2, 164.2, 159.3, 125.6, 74.3, 72.1, 71.7, 71.4, 70.1, 70.0, 67.9, 66.3, 65.7, 63.5, 63.3, 62.2, 61.9, 61.8, 57.3, 57.2 (2C), 41.8, 41.2, 39.3, 38.0, 37.1, 37.0, 32.5, 31.4, 31.0, 30.9, 30.6, 30.4, 30.0, 29.7, 27.0, 24.6, 21.0, 18.1, 14.2; LRMS (ESI) m/z 863.2 (M ⁺ +H), 885.2 (M ⁺ +Na); HRMS (ESI) calcd for (M ⁺ ) m/z

863.4107, found 863.4098.

[0382] Scheme 23. Synthetic route to T285.

[0384] To a solution of compound 99 (6 mg, 0.0108 mmol) in CH ₂ Q ₂ (1 mL) was added propargylamine (0.9 mg, 0.0162 mmol). The reaction was stirred at rt overnight. The mixture was diluted with CH ₂ CI ₂ (5 mL). The organic layer was washed with IN HC1 (2 x 1 mL), 1M K ₂ CO3 solution (1 mL portion until aqueous is colorless) and brine (l x l mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was further purified by column chromatography using 30-40% acetone in «-hexane to give T285 (4 mg, 78%) as a white solid.

[0385] Analytical TLC (silica gel 60), 10% MeOH in CH ₂ C1 ₂ , R/ = 0.43; ¾ NMR

(400 MHz, CD ₃ OD) δ 4.89-4.81 (m, 2H), 4.04 (t, / = 6.2 Hz, 2H), 3.94 (d, / = 3.1 Hz, 1H), 3.85 (d, / = 2.3 Hz, 2H), 3.46 (d, / = 2.9 Hz, 1H), 2.28-2.26 (m, 2H), 2.79-2.76 (m, 1H), 2.56 (s, 1H), 2.29-2.22 (m, 2H), 2.18-2.10 (m, 2H), 1.94 (t, / = 14.1 Hz, 1H), 1.70-1.60 (m, 2H), 1.52-1.39 (m, 4H), 1.36-1.29 (m, 2H), 1.09 (s, 3H); ¹³ C NMR (125 MHz, CD ₃ OD) δ 176.2, 164.1, 137.7, 125.6, 79.6, 74.3, 72.1 (2C), 66.3, 66.0, 63.3, 62.2, 61.9, 57.3, 57.2, 54.9, 41.7, 37.1, 32.5, 31.1, 30.9, 30.2, 24.6, 21.0, 18.1, 14.2; LRMS (ESI) m/z 494.2 (M ⁺ +Na); HRMS (EI) calcd for CzsHzgNOgNa (M ⁺ ) m/z 494.1785, found 494.1779.

[0386] Scheme 24. Synthetic route to T296.

HO.

0 ,ΝΗΒοο ^► [ _| ,NHBoc

0,N

2-(2-aminoet oxy)et anol 100

[0387] Scheme 24 Reagents and conditions: (a) Boc ₂ 0, CH ₂ C1 ₂ , rt, 100%; (b) 4- nitrophenylchloroformate, pyridine, CH ₂ CI ₂ , rt, 73%; (c) N3CH ₂ CH ₂ CH ₂ NH ₂ , CH ₂ CI ₂ , rt, 100%; (d) cone. HCl, CH ₂ C1 ₂ , 0 °C, then 1M K ₂ C0 ₃ , EtOH extract, crude; (e) DCC, NHS, DMF, crude; (f) 103, Et ₃ N, CH ₂ C1 ₂ , 83%; (g) cone. HCl, CH ₂ C1 ₂ , 0 °C, then 1M K ₂ C0 ₃ , EtOH extract, crude; (h) 99, Et ₃ N, DMAP, DMF, 98%.

Preparation of compound 100:

[0388] To a solution of 2-(2-aminoethoxy)ethanol (2.10 g, 20 mmol) in CH ₂ CI ₂ (40 mL) was added di-teri-butyl dicarbonate (4.36 g, 20 mmol) at rt. The reaction was monitored by TLC. When the reaction completed, the content was concentrated and was purified by column chromatography (50-70% EtOAc in «-hexane) to yield compound 100 as a colorless oil (4.21 g, 100%). [0389] Analytical TLC (silica gel 60), 80% EtOAc in ra-hexane _, Rf = 0.30; ¾ NMR

(400 MHz, CDC1 ₃ ) δ 5.28 (br s, 1H), 3.74 (br t, / = 4.1 Hz, 2H), 3.59-3.54 (m, 4H), 3.32- 3.28 (m, 2H), 3.08 (br s, 1H), 1.44 (s, 9H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 156.1, 79.2, 72.2, 70.2, 61.5, 40.3, 28.3; IR (CH ₂ C1 ₂ ) 3228 (br), 3035, 2927, 2855, 1523 cm ^"1 ; LRMS (ESI) m/z 228.0 (M ⁺ +Na); HRMS (ESI) calcd for C ₉ H ₂₀ NO ₄ (M ⁺ +H) m/z 206.1392, found 206.1390. Preparation of compound 101:

[0390] To a solution of compound 100 (2.05 g, 10 mmol) in CH ₂ CI ₂ (50 mL) was added 4-nitrophenyl chloroformate (2.02 g, 10 mmol), DMAP (122 mg, 1 mmol) and pyridine (0.81 mL, 10 mmol). The reaction was stirred at rt for 4 h. When the reaction completed, the reaction mixture was washed with 1 M HCl (2 x 10 mL), sat. NaHCC>3 (1 x 10 mL) and the organic layer was finally dried over NaiSOzt. The organic layer was filtered and crude product was obtained. The crude product was purified by flash chromatography (30- 35% EtOAc in «-hexane) to yield compound 101 as a white solid (2.70 g, 73%).

[0391] Analytical TLC (silica gel 60), 80% EtOAc in ra-hexane _, = 0.65; ¾ NMR

(400 MHz, CDCI ₃ ) δ 8.26 (d, / = 8.6 Hz, 2H), 7.38 (d, / = 8.6 Hz, 2H), 4.91 (br s, 1H), 4.43- 4.19 (br m, 2H), 3.76-3.75 (br m, 2H), 3.57 (t, / = 4.9 Hz, 2H), 3.34-3.30 (br m, 2H), 1.43 (s, 9H); ¹³ C NMR ( 100 MHz, CDC1 ₃ ) δ 155.9, 155.4, 152.4, 145.4, 125.3, 121.7, 79.4, 70.4, 68.3, 68.1, 40.2, 28.3; IR (CH ₂ C1 ₂ ) 3035, 2927, 2855, 2230, 1523 cm ^"1 ; LRMS (ESI) m/z 393.0 (M ⁺ +Na); HRMS (ESI) calcd for C ₁₆ H ₂₂ N ₂ 0 ₈ (M ⁺ ) m/z 370.1376, found 370.1376.

Preparation of compound 102:

[0392] To a solution of compound 101 (370 mg, 1 mmol) in CH ₂ CI ₂ ( 1 mL) was added 3-azidopropylamine (200 mg, 2 mmol) and Et ₃ N (0.28 mL, 2 mmol). The reaction was stirred at rt for 1.5 h. The reaction was transferred to a separatory funnel and diluted with CH ₂ CI ₂ (20 mL). The organic layer was washed with IN HCl (2 x 5 mL), sat. NaHCC>3 (2 x 5 mL) and brine ( 1 x 5 mL). The organic layer was dried over Na ₂ SC>4, filtered and

concentrated. The crude product was purified by column chromatography using 45-60% EtOAc in «-hexane to give compound 102 (348 mg, 100%) as colorless oil.

[0393] Analytical TLC (silica gel 60), 5% MeOH in CH ₂ C1 ₂ , R/ = 0.44; ¾ NMR (500

MHz, CDCI ₃ ) δ 5.07 (br s, 1H, NH), 4.98 (br s, 1H, NH), 4.22 (apparent br s, 2H), 3.65 (apparent br s, 2H), 3.55 (t, / = 5.2 Hz, 2H), 3.32 (t, / = 5.1 Hz, 2H), 3.29-3.26 (m, 4H), 1.80 (quintet, / = 6.6 Hz, 2H), 1.45 (s, 9H); ^lj C NMR (125 MHz, CDC1 ₃ ) δ 156.4, 156.0, 79.3, 70.1, 69.4, 63.9, 49.0, 40.3, 38.4, 28.4; LRMS (ESI) m/z 332.2 (M ⁺ +H); HRMS (ESI) calcd for Ci ₃ H ₂₆ N ₅ 0 ₅ (M ⁺ +H) m/z 332.1934, found 332.1933.

Preparation of compound 103:

[0394] To a solution of compound 102 (56 mg, 0.169 mmol) in CH ₂ CI ₂ (1 mL) was added cone. HC1 (0.3 mL). The reaction was stirred at rt for 1 h. After completion of reaction, the mixture was basicified with 1M K ₂ CO3 solution and concentrated. The dried residue was extracted with EtOH repeatedly and filtered. The filtrate was concentrated to give crude product 103 as slight yellow oil (24 mg,61%), which was directly use to next step.

Analytical TLC (silica gel 60), 5% MeOH in CH ₂ C1 ₂ , R/= 0.05; ¾ NMR (500 MHz, CDC1 ₃ ) δ 4.18 (t, / = 4.4 Hz, 2H), 3.65 (t, / = 4.4 Hz, 2H), 3.51 (t, / = 5.1 Hz, 2H), 3.35 (t, / = 6.6 Hz, 2H), 3.18 (t, / = 6.7 Hz, 2H), 2.77 (apparent br s, 2H), 1.75 (quintet, / = 6.6 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 159.0, 73.5, 70.4, 65.0, 49.9, 42.1, 39.0, 30.1.

Mass analysis not perform for this crude product.

Preparation of compound 104:

[0395] To a solution of L-photoleucine (49 mg, 0.202 mmol) in DMF (1 mL) was added DCC (42 mg, 0.202 mmol) and NHS (23 mg, 0.202 mmol). The reaction was stirred at rt overnight. The reaction was then filtered and concentrated to give crude product, which was unstable on silica. The crude product was taken to next step.

Preparation of compound 105:

[0396] To a solution of compound 103 (62 mg, 0.25 mmol) in CH ₂ CI ₂ (0.5 mL) was added compound 104 (34 mg, 0.1 mmol) and Et ₃ N (35 μί, 0.25 mmol). The reaction was stirred overnight. After the completion of reaction, the mixture was diluted with CH ₂ CI ₂ (10 mL). The organic layer was washed with IN HC1 (2 x 2 mL), 1M K ₂ CO ₃ (2 x 2 mL) and brine (1 x 2 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated. The crude product was purified by column chromatography using 2-15% acetone in CH ₂ CI ₂ to give compound 105 (38 mg, 83%) as colorless oil.

[0397] Analytical TLC (silica gel 60), 10% acetone in CH ₂ C1 ₂ , R/= 0.42; ¾ NMR

(500 MHz, CDCI ₃ ) δ 6.78 (br s, 1H, NH), 5.49 (br s, 1H, NH), 5.14 (br s, 1H, NH), 4.24-4.18 (m, 2H), 4.08 (br s, 1H), 3.65 (br t, 2H), 3.57 (br t, 2H), 3.45-3.44 (br m, 2H), 3.39 (t, / = 6.5 Hz, 2H), 3.29 (q, / = 6.2 Hz, 2H), 2.07-2.04 (br m, 1H), 1.82 (quintet, / = 6.4 Hz, 2H), 1.54- 1.47 (m, 10 H), 1.07 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 170.9, 156.5, 155.4, 80.5, 69.4, 69.2, 63.7, 50.8, 49.0, 39.2, 38.4, 37.4, 29.1, 28.3, 23.8, 19.8; LRMS (ESI) m/z 457.3 (M ⁺ +H); HRMS (ESI) calcd for C ₁₈ H ₃₃ N ₈ 0 ₆ (M ⁺ +H) m/z 457.2523, found 457.2523.

Preparation of compound 106:

[0398] To a solution of compound 105 (9 mg, 0.0197 mmol) in CH ₂ Q ₂ (0.5 mL) was added cone. HC1 (0.2 mL). The reaction was stirred for 35 min. When TLC indicated completion of reaction, the mixture was basicified with 1M K ₂ CO ₃ and concentrated. The dried residue was extracted with EtOH and filtered. The filtrate was concentrated to give crude compound 106 as yellow oil (8 mg). The crude product was directly used for next step.

[0399] Analytical TLC (silica gel 60), 10% acetone in CH ₂ C1 ₂ , R/= 0.05; ¾ NMR

(500 MHz, CD ₃ OD) δ 4.17 (apparent t, 2H), 3.92 (t, / = 7.1 Hz, 1H), 3.66 (apparent t, 2H), 3.62 (apparent t, 2H), 3.50 (td, / = 9.4, 4.7 Hz, 1H), 3.40 (td, / = 8.3, 5.9 Hz, 1H), 3.36 (t, / = 6.5 Hz, 2H), 3.19 (t, / = 6.5 Hz, 2H), 1.95 (dd, / = 14.9, 6.7 Hz, 1H), 1.76-1.68 (m, 3H), 1.12 (s, 3H).

¹³ C and mass analysis not perform for this crude product.

Preparation of T296:

[0400] To a solution of compound 106 (8 mg, 0.0208 mmol) in DMF (0.3 mL) was added compound 99 (8.8 mg, 0.0162 mmol), followed by Et ₃ N (2.9 μί, 0.0208 mmol) and DMAP (0.25 mg, 0.00208 mmol). The reaction was stirred overnight. The mixture was then diluted with CH ₂ Q ₂ ( 10 mL). The organic layer was washed with IN HQ (1 x 2 mL), 1M K ₂ CO ₃ (2 x 2 mL) and brine (1 x 2 mL). The combined organic layer was extracted with CH ₂ Cl ₂ EtOH 9: 1 (2 x 2 mL). The combined organic layer was dried over Na ₂ SO/t, filtered and concentrated. The crude product was purified by column chromatography using 5% EtOH in CH ₂ CI ₂ to give purified T296 (12 mg, 98%) as white solid.

[0401] Analytical TLC (silica gel 60), 10% EtOH in CH ₂ C1 ₂ , = 0.50; ¾ NMR (500

MHz, CD ₃ OD) δ 4.82 (AB system, 2H), 4.18-4.15 (m, 2H), 4.12-4.02 (m, 2.5 H), 3.94 (d, / = 2.9 Hz, 1H), 3.64 (t, / = 4.6 Hz, 2H), 3.56-3.53 (m, 2.5 H), 3.47 (d, / = 2.8 Hz, 1H), 3.39- 3.33 (m, 6H), 3.19 (t, / = 6.8 Hz, 2H), 2.79-2.76 (m, 1H), 2.28-2.23 (m, 2H), 2.16-2.06 (m, 2H), 1.97-1.91 (m, 2H), 1.75 (quintet, / = 6.7 Hz, 2H), 1.68-1.60 (m, 2H), 1.54-1.42 (m, 5H), 1.33 (td, / = 12.0, 5.8 Hz, 1H), 1.09 (s, 3H), 1.05 (s, 3H); ¹³ C NMR ( 125 MHz, CD ₃ OD) δ 176.1, 173.8, 164.0, 158.4, 153.8, 125.5, 72.0, 70.4, 70.3, 66.1, 66.0, 65.1, 63.2, 62.7, 62.1, 61.7, 57.1, 57.0, 52.6, 50.0, 41.6, 40.5, 39.1, 38.1, 37.0, 33.6, 32.5, 33.3, 30.7, 30.2, 30.1, 24.9, 24.4, 20.9, 20.8, 19.9, 17.9, 14.1 ; LRMS (ESI) m/z 773.3 (M ⁺ +H); HRMS (ESI) calcd for C ₃₅ H ₄₉ N ₈ Oi ₂ (M ⁺ +H) m/z 773.3470, found 773.3470.

[0403] Reagent and condition: (a) N ₃ CH ₂ CH ₂ CH ₂ NH ₂ , Et ₃ N, DMAP, CH ₂ C1 ₂ , rt,

91%.

To a solution of compound 74 (70 mg, 0.134 mmol) in CH ₂ CI ₂ (2 mL) was added 3- azidopropylamine (27 mg, 0.267 mmol), Et ₃ N (37 μί, 0.267 mmol) and DMAP (1.6 mg, 0.0134 mmol). The reaction was stirred at rt for 1 h. When TLC indicated the reaction was complete, CH ₂ CI ₂ (20 mL) was added. The mixture was transferred to a separating funnel. The organic layer was washed with IN HC1 (2 x 4 mL), 1M K ₂ CO3 (4 mL x 8) and brine (1 x 4 mL). The organic layer was dried over Na ₂ SC>4, filtered and concentrated to give crude product. The crude product was purified by flash chromatography to give T225 (59 mg, 91%) as white solid.

[0404] Analytical TLC (silica gel 60), 5% MeOH in CH ₂ C1 ₂ , R/ = 0.53; ¾ NMR (300

MHz, CDC1 ₃ ) δ 7.02 (d, / = 4.6 Hz, 1H), 5.00 (apparent br t, 1H, NH), 4.71 (s, 2H), 4.04 (t, / = 5.8 Hz, 2H), 3.81 (d, / = 4.7 Hz, 1H), 3.61 (d, / = 5.8 Hz, 1H), 3.38 (t, / = 6.6 Hz, 2H), 3.26 (q, / = 6.4 Hz, 2H), 2.77-2.72 (m, 1H), 2.41-2.18 (m, 5H), 2.04 (dd, / = 15.6, 10.6, 1H), 1.84-1.65 (m, 6H), 1.31 (dt, / = 1 1.5, 6.0 Hz, 1H), 1.13 (s, 3H); ¹³ C NMR (100 MHz, CDC1 ₃ ) δ 190.7, 173.1, 159.5, 156.5, 143.6, 139.4, 125.8, 70.0, 64.2, 63.9, 62.3, 59.1, 51.6, 49.0, 40.8, 38.4, 35.0, 30.1, 29.1, 27.2, 26.2, 23.3, 17.1, 13.7; LRMS (EI) m/z 484.2 (M ⁺ ); HRMS (EI) calcd for C ₂₄ H ₂₈ N ₄ O ₇ (M ⁺ ) m/z 484.1958, found 484.1958.

[0405] Scheme 26. Synthetic route to T124:

[0406] Scheme 26 Reagents and conditions: (a) ethanolamine, Et ₃ N, DMAP,

CH ₂ C1 ₂ , 71%; (b) Dess-Martin periodinane, CH ₂ C1 ₂ , 98%; (c) OXONE ^® , NaHC0 ₃ ,

MeCN/H ₂ 0, crude; (d) 5% H ₂ 0 ₂ , 1M NaOH, MeOH, 50% over 2 steps. Compound T124 was prepared under reaction conditions similar to compound T68 of Example 1, Scheme 4 as described above.

Preparation of compound 107:

[0407] To a mixture of compound 74 (100 mg, 0.191 mmol) and ethanolamine (13 μί,

0.2103 mmol) in CH ₂ Q ₂ (10 mL) were added Et ₃ N (30 μί, 0.214 mmol) and one small crystal of DMAP. The reaction mixture was stirred at room temperature for 2 h under argon. When TLC analysis (CH ₂ Cl ₂ :CH ₃ OH=9: 1, v/v) indicated the reaction was complete, CH ₂ CI ₂ (50 mL) was added to the reaction mixture, and the organic layer was washed with saturated NaHC0 ₃ solution until the water layer was colorless. The organic layer was dried over anhydrous Na ₂ SC>4 and the solvent was then removed to give yellow oil, which was subjected to flash chromatography to give compound 107 as a white solid (60 mg, 71% yield).

[0408] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, R/= 0.25; ¾ NMR

(300 MHz, CDC1 ₃ ) δ 7.03 (d, / = 4.4 Hz, 1H), 5.30 (br s, 1H), 4.80-4.60 (m, 2H), 4.05 (s, 2H), 3.82 (d, / = 4.6 Hz, 1H), 3.72-3.68 (m, 2H), 3.61 (d, / = 5.6 Hz, 1H), 3.40-3.25 (m, 2H), 2.75 (d, / = 13.2 Hz, 1H), 2.66 (br s, OH), 2.45-2.10 (m, 5H), 2.03 (t, / = 13.8 Hz, 1H), 1.85-1.60 (m, 4H), 1.31 (td, / = 5.9, 12.0 Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 190.8, 173.1, 159.6, 156.4, 143.6, 139.5, 125.8, 70.0, 64.2, 64.0, 62.4, 62.2, 59.2, 51.6, 43.5, 40.8, 35.0, 30.1, 27.3, 26.2, 23.3, 17.1, 13.7; LRMS (ESI) m/z 468.0 (M ⁺ +Na).

Preparation of compound 108:

[0409] Compound 107 (60 mg, 0.135 mmol) was dissolved in CH ₂ Q ₂ (15 mL) and was treated with Dess-Martin periodinane (172 mg, 0.405 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirring was continued for 3 h. The reaction was quenched with a mixture (340 mg) in aqueous saturated NaHCC>3 (5 mL). The resulting mixture was stirred until the organic layer turned clear. The organic layer was separated and the aqueous layer was extracted with dichloromethane (50 mL ^χ 3). The combined organic layers were dried over anhydrous NaiSO/ _t , filtrated, and concentrated. The residue was purified by flash column chromatography to afford the desired compound 108 (59 mg, 98% yield).

[0410] Analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 9: 1, Rf = 0.53; *H NMR

(300 MHz, CDCI ₃ ) δ 9.65 (s, 1H), 7.03 (d, / = 4.5 Hz, 1H), 5.48 (br s, lH), 4.80-4.60 (m, 2H), 4.20-4.00 (m, 4H), 3.81 (d, / = 4.6 Hz, 1H), 3.61 (d, / = 5.6 Hz, 1H), 2.75 (d, / = 13.0 Hz, 1H), 2.40-2.10 (m, 5H), 2.03 (t, / = 14.1 Hz, 1H), 1.81-1.74 (m, 2H), 1.68 (dd, / = 5.1, 12.2 Hz, 1H), 1.31 (td, / = 6.1, 12. Hz, 1H), 1.12 (s, 3H); ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 196.8, 190.7, 173.1, 159.6, 156.4, 143.4, 139.6, 125.8, 70.0, 64.5, 64.2, 62.3, 61.5, 59.2, 51.6, 40.9, 35.0, 30.1, 27.1, 26.1, 23.3, 17.1, 13.7; LRMS (ESI) m/z 466.0 (M ⁺ +Na).

Preparation of compound 109:

[0411] To a compound 108 (60 mg, 0.135 mmol) and NaHC0 ₃ (68 mg, 0.8124 mmol) in acetonitrile (10 mL) and water (2 ml), was added a solution of OXONE ^® (168 mg, 0.271 mmol, dissolved in 0.5 mL water) in portions over 0.5 h at room temperature. After stirring for anotherl 2.5 h, the pH of the reaction mixture was adjusted to 2 by adding hydrochloric acid (3 M) and then extracted with CH ₂ CI ₂ (50 mL ^χ 3). The organic layers were washed with water and brine, dried over anhydrous NaiS04, filtered and concentrated under reduced pressure. The residue was used in the next step without further purification. [0412] Compound T124, analytical TLC (silica gel 60), CH ₂ C1 ₂ : CH ₃ OH= 5 : 1, Rf =

0.23; ¾ NMR (500 MHz, CDC1 ₃ ) ^ 5.53 (br s, 1H), 4.77-4.66 (m, 2H), 4.16-4.06 (m, 3H), 4.01 (dd, / = 5.9, 12.8 Hz, 2H), 3.90 (d, / = 2.5 Hz, 1H), 3.44 (d, / = 5.5 Hz, 1H), 2.82 (d, / = 13.3 Hz, 1H), 2.36 (d, / = 14.0 Hz, 1H), 2.26-2.04 (m, 3H), 1.98 (t, / = 13.8, 13.8 Hz, 1H), 1.82-1.65 (m, 3H), 1.60 (dd, / = 5.4, 12.3 Hz, 1H), 1.33 (td, / = 5.8, 12.3, 12.3 Hz, 1H), 1.06 (s, 3H); ¹³ C NMR (125 MHz, CDC1 ₃ ) S 197.2, 173.2, 173.2, 159.5, 156.6, 125.8, 70.0, 65.5, 64.9, 63.2, 61.2, 60.8, 56.3, 53.4, 42.5, 40.6, 35.4, 30.6, 26.2, 24.1, 23.2, 17.1, 13.8; LRMS (ESI) m/z 498.0 (M ⁺ +Na).

Example 2: Biological Activity

[0413] Cell Culture. HCT116, Hela, MDCK, Jurkat, and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's media (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. HCT116 cells were cultured in McCoy's 5A media (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in a humidified 37 °C incubator with 5% CO ₂ .

[0414] The Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. In the viability assay, HCT116, Hela, MDCK, Jurkat, or NIH 3T3 cells were seeded and allowed to attach for 12 hours and then treated with triptolide or its analogs at varying concentrations (from 5 nM to 10 uM ) in 0.1% DMSO or in 0.1% DMSO alone (control) for 48 hours. Thereafter, CellTiter-Glo® Reagent for Luminescent Cell Viability Assay was added, and then viable cells were characterized as luminescent signal detected by using a luminometer. The data was further analyzed using GraphPadPrism to determine the compound concentration at which 50% cells were viable (IC ₅₀ values). The results were summarized in Table 1.

In vitro cytotoxicity of triptolide and triptolide analogs against tumor cell

No. and/or structure IC50 (μΜ)

HCT116 HeLa MDCK Jurkat NIH3T3 T144

T145

T149

T150

T151

T225 ~7

T234

T249

T255

T285

T296

In Vitro Cytotoxicity of Triptolide and Selected Expoxide Compounds against ODCA Tumor Cell Line Panel (MTS assay after 72 hour drug exposure)

IC50 (μΜ)

Tumor cell line TL T52 T59 T62 T67 T68 T69 T70 T71

Human ovarian A2780/ 0.00879 0.56016 0.47699 0.28379 0.00680 0.08233 0.03932 0.08529 0.07321 cancer DDP-S

Human ovarian A2780/ 0.00654 0.83237 0.55138 1.11662 0.00704 0.06020 0.02942 0.06108 0.08152 cancer DDP-R

human breast SKBR3 0.07648 2.86212 7.68933 3.75791 0.13184 >2.7500 0.62542 1.85550 1.53331 cancer

Human prostate PC3 0.03991 3.20106 5.33225 2.66108 0.21879 >2.7500 0.60595 1.96390 0.58004 cancer

human colon HCT11 0.01162 0.50538 0.50073 0.53733 0.00839 0.09113 0.02614 0.07069 0.07474 cancer 6

Human hepatic LX-1 0.02326 0.62183 1.76735 0.47706 0.02914 0.30060 0.11399 0.20718 0.16851 stellate

human K562 0.03366 0.57555 1.08672 0.32877 0.04362 0.78822 0.12557 0.17005 0.29305 chronic

myelogenous

leukemia

adult bovine ABAE 0.01766 0.53618 0.60962 0.33373 0.01441 0.14999 0.07092 0.11991 0.12655 aortic

endothelial

human colon HT-29 0.01340 1.08292 0.48022 1.30934 0.00637 0.07729 0.2550 0.08628 0.11537 adenocarcinoma

human colon HCT- 0.03250 1.24092 2.41697 0.89047 0.05219 >2.7500 1.30073 2.19370 0.93462 carcinoma 15 human breast MDA- 0.03968 0.51326 1.34981 0.24657 0.02753 1.15615 0.17603 0.40152 0.27337 cancer MB- 231

human colon WiDr 0.03831 2.57952 0.88847 2.53376 0.01658 0.84811 0.12734 0.11269 0.28548 carcinoma

EQUIVALENTS

[0415] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms 'comprising,' 'including,' 'containing,' etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase 'consisting essentially of will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase 'consisting of excludes any element not specified.

[0416] The present disclosure is not to be limited in terms of the particular

embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent compositions, apparatuses, and methods within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims.

The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or

biological systems, which can, of course, vary. It is also to be understood that the

terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0417] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0418] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as 'up to,' 'at least,' 'greater than,' 'less than,' and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0419] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.