NOVEL GLUCOCORTICOID RECEPTOR AGONISTS AND

IMMUNOCONJUGATES THEREOF

Priority Claims and Related Patent Applications

[0001] This application claims the benefit of priority to PCT/CN2022/118982, filed September 15, 2022, the entire content of which is incorporated herein by reference.

Technical Field of the Invention

[0002] The invention generally relates to novel compounds and therapeutic uses thereof. More particularly, the invention provides novel glucocorticoid receptor agonists and immunoconjugates thereof, as well as pharmaceutical compositions and methods of preparation and use for treating various diseases and disorders (e.g., inflammatory disorders).

Background of the Invention

[0003] Glucocorticoids are a class of corticosteroids that bind to the glucocorticoid receptor. Glucocorticoids are part of the feedback mechanism in the immune system and affect cells by binding to the glucocorticoid receptor. The activated glucocorticoid receptor-glucocorticoid complex up-regulates the expression of anti-inflammatory proteins in the nucleus and represses the expression of proinflammatory proteins in the cytosol. Cortisol (or hydrocortisone) is an important human glucocorticoid. It regulates or supports a variety of important cardiovascular, metabolic, immunologic, and homeostatic functions. Glucocorticoids are used in medicine to treat diseases caused by an overactive immune system, such as allergies, asthma, autoimmune diseases, and sepsis. (Barnes, 2006 Eur. J. Pharmacol. 533, 2-14).

[0004] Significant adverse side effects, however, have limited the therapeutic potential of glucocorticoids. Side effects include suppression of hypothalamic- pituitary -adrenal axis, bone demineralization and osteoporosis, disruption of carbohydrate metabolism, ocular side effects such as glaucoma, cataracts, and growth retardation in children. (Rhen, et al. 2005 New Eng. J. Med. 353 (16): 1711-23.) [0005] Synthetic glucocorticoid receptor agonists, e.g., dexamethasone, prednisolone and budesonide, are small molecules used in the treatment of inflammatory disorders. Their utility in the treatment of chronic disease is limited due to severe side effects.

[0006] Despite significant progress in clinical development of ADCs in recent years, design and development of glucocorticoid receptor agonist-based ADCs involve many challenges including lack of stability, high aggregation propensity and limited bioavailability as well as limited numbers of potent glucocorticoid receptor agonists that suitable for development.

[0007] Novel glucocorticoid receptor agonists that are potent but with reduced side effects and suitable for development and immunoconjugates are highly desired.

Summary of the Invention

[0008] The invention provides novel glucocorticoid receptor agonists that possess high anti- inflammatory activity and favorable stability and other characteristics making them suitable for use alone or in immunoconjugates. The glucocorticoid receptor agonists disclosed herein are characterized by the placement of an anilino functionality at position(s) 17/16 for conjugation with a linker. This design is synergistically combined with further variations at position-20 for fine-tuning of the payload to suit different ADC constructs and applications The high potency, high stability, low immunogenicity, as well as satisfactory solubility render these compounds ideally suited as glucocorticoid receptor agonists and for development of immunoconjugates as novel therapeutics for inflammatory disorders.

[0009] In one aspect, the invention generally relates to a compound having the structural formula (I): or a pharmaceutically acceptable form thereof, wherein

R ¹ is H or halogen;

R ² is H or halogen;

R ³ is C _1-4 alkyl and R ⁴ is OC(O)-R ⁷ , wherein R ⁷ is a substituted or unsubstituted aniline, or

R ³ and R ⁴ , together with the carbon atoms they are bound to, form a 5-membered dioxolane substituted with W-R ⁶ , wherein W is a single bond or -phenyl-Q-, Q is selected from CH ₂ , O, S, S(O), S(O) ₂ , NH and NCH ₃ , and R ⁶ is a substituted or unsubstituted aniline; and

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered, unsubstituted or substituted heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl.

[0010] In certain embodiments of (I), a compound of the invention has the structural formula (II):

[0011] In certain embodiments, a compound of the invention has the structural formula (II ^a ): wherein each of R ^7a , R ^7b and R ^7c is selected from H and NR ^7x R ^7y , provided that only one of R ^7a , R ^7b and R ^7c is NR ^7x R ^7y and each of the others is H; each of R ^7x and R ^7y is independently selected from R, R ^7r and L ⁷ -R ^7z , provided that when one of R ^7x and R ^7y is L ⁷ -R ^7z or R ^7r , the other is R;

L ⁷ is a linker;

R ^7r is (C=O)-O-(CH ₂ ) _i -R ^7v or (C=O)-(CH ₂ ) _j -R ^7v ;

R ^7v is R, OR, NHR, NR ₂ , an aryl group or an amino acid; i is 0, 1, 2, 3, 4, 5 or 6; j is 0, 1, 2, 3, 4, 5 or 6;

R ^7z comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[0012] In certain embodiments of (I), R ³ and R ⁴ , together with the carbon atoms they are bound to, form a 5-membered dioxolane substituted with W-R ⁶ , having the structural formula:

[0013] In certain embodiments, W is a single bond and a compound of the invention has the structural formula (III): (III)

[0014] In certain embodiments, a compound of the invention has the structural formula (III ^a ): wherein each of R ^6a , R ^6b and R ^6c is selected from H and NR ^6x R ^6y , provided that only one of R ^6a , R ^6b and R ^6c is NR ^6x R ^6y and each of the others is H; each of R ^6x and R ^6y is independently selected from R, R ^6r and L ⁶ -R ^6z , provided that when one of R ^6x and R ^6y is L ⁶ -R ^6z or R ^6r , the other is R;

L ⁶ is a linker;

R ^6r is (C-O)-O-(CH ₂ ) _p -R ^6v or (C=O)-(CH ₂ ) _q -R ^6v ;

R ^6v is R, OR, NHR, NR?, an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6;

R ^6z comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[0015] In certain embodiments, W is -phenyl -Q-.

[0016] In certain embodiments, Q is CH ₂ , a compound of the invention has the structural formula (III ^b ):

wherein each of R ^6a' , R ^6b' and R ^6c'' is selected from H and NR ^6x' R ^{6y ’} , provided that only one of R ^6a' , R ^6b' and R ^6c'' is NR ^6x' R ^{6y ’} and each of the others is H; each of R ^6x' and R ^{6y ’} is independently selected from R, R ^{6r '} and L ^6' -R ^6z'' , provided that when one of R ^6x' and R ^{6y ’} is L ^6' -R ^6z'' or R ^{6r '} , the other is R’;

L ⁶ is a linker; R ^{6r '} is (C=O)-O-(CH ₂ ) _p -R ^6v'' or (C=O)-(CH ₂ ) _q -R ^6v'' ; R ^6v'' is R, OR, NHR, NR ₂ , an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6; R ^6z'' comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[0017] In yet another aspect, the invention generally relates to an immunoconjugate having the structural formula (IV): or a pharmaceutically acceptable form thereof, wherein Ab represents an antigen binding moiety;

R ¹ is H or halogen;

R ² is H or halogen;

R ³ is C _1-4 alkyl;

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl;

R ^7x is H or C _1-6 alkyl;

L ^Ab is a linker; and n is an integer in the range of 1 to about 20.

[0018] In yet another aspect, the invention generally relates to an immunoconjugate having the structural formula (V) or (VI): or or a pharmaceutically acceptable form thereof, wherein Ab represents an antigen binding moiety;

Q is selected from CH ₂ , O, S, S(O), S(O) ₂ , NH and NCH ₃ ;

R ¹ is H or halogen;

R ² is H or halogen;

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl;

R ^6x is H or C _1-6 alkyl;

L ^Ab is a linker; and n is an integer in the range of 1 to about 20.

[0019] In yet another aspect, the invention generally relates to a composition comprising a compound disclosed herein, such as according to any one of formulae (I)-(III ^b ) and in Table 1, or a pharmaceutically acceptable form thereof, and optionally a pharmaceutically acceptable excipient, carrier or diluent.

[0020] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an immunoconjugate disclosed herein, such as according to any one of formulae (IV)-(V), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

[0021 ] In yet another aspect, the invention generally relates to a method for treating or reducing a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate disclosed herein.

[0022] In certain embodiments, the disease or condition is an inflammatory disorder.

[0023] In yet another aspect, the invention generally relates to use of an immunoconjugate disclosed herein for the manufacture of a medicament.

[0024] In certain embodiments, an immunoconjugate disclosed herein is used for treating a disease or condition, wherein the disease or condition is an inflammatory disorder.

[0025] In yet another aspect, the invention generally relates to use of an immunoconjugate disclosed herein for use in treating inflammatory disorders.

[0026] In yet another aspect, the invention generally relates to a combination comprising a therapeutically effective amount of a compound or an immunoconjugate disclosed herein, and one or more therapeutically active co-agent(s) and/or adjuvant(s).

Detailed Description of the Invention

[0027] The invention is based in part on the discovery of novel glucocorticoid receptor agonists that possess favorable potency, stability and other profiles as payloads for immunoconjugates.

[0028] Key structural improvements to existing glucocorticoid receptor agonists include an anilino functional group at position(s) 17/16 for linkage to an antigen binding moiety. Further fine-tuning of the payload molecule can be achieved through modifications at position 20 to suit a wide range of ADC constructs and applications. The highly potent and stabile glucocorticoid receptor agonists also enjoy satisfactory solubility and low immunogenicity making them suitable for development as immunoconjugates and novel therapeutics for inflammatory disorders.

Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 2006.

[0030] The following terms, unless indicated otherwise according to the context wherein the terms are found, are intended to have the following meanings.

[0031] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 16 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0032] As used herein, “at least” a specific value is understood to be that value and all values greater than that value.

[0033] As used herein, “more than one” is understood as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, etc., or any value therebetween.

[0034] In this specification and the appended claims, the singular forms "a, " "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[0035] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0036] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0037] Any compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

[0038] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0039] The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of", when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of" refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of", when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0040] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, atropisomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. In certain embodiments, each asymmetric atom has at least 50 % enantiomeric excess, at least 60 % enantiomeric excess, at least 70 % enantiomeric excess, at least 80 % enantiomeric excess, at least 90 % enantiomeric excess, at least 95 % enantiomeric excess, or at least 99 % enantiomeric excess of either the R- or S-configuration. For optically active compounds, it is often preferred to use one enantiomer to the substantial exclusion of the other enantiomer. [0041] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures. [0042] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic methods well known in the art, and subsequent recovery of the pure enantiomers.

[0043] A mixture of isomers can be separated on the basis of the physicochemical differences of the constituents, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and/or fractional crystallization. [0044] Definitions of specific functional groups and chemical terms are described in more detail below. When a range of values is listed, it is intended to encompass each value and sub- range within the range. For example, “C _1-6 alkyl” is intended to encompass, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C _1-6 , C _1-5 , C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-5 , C _2-4 , C _2-3 , C _3-6 , C _3-5 , C _3-4 , C _4-6, C _4-5 , and C _5-6 alkyl. [0045] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -C(=O)-O- is equivalent to -O- C(=O)-.

[0046] Structures of compounds of the invention are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds that are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions (e.g., aqueous, neutral, and several known physiological conditions).

[0047] As used herein, the term “alkyl” refers to a straight, branched or cyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., C _1-10 alkyl). Whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range; e.g., "1 to 10 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated. In some embodiments, “alkyl” can be a C _1-6 alkyl group. In some embodiments, alkyl groups have 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, -methyl, - ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3- methylbutyl, 2-methylpentyl, 3 -methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4- methylhexyl, 5-methylhexyl, 2,3 -dimethylbutyl, and the like. The alkyl is attached to the parent molecule by a single bond. Unless stated otherwise in the specification, an alkyl group is optionally substituted by one or more of substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R ^a ) ₃ , -OR ^a , -SR ^a , -OC(O)-R ^a , -N(R ^a ) ₂ , - C(O)R _a , -C(O)OR ^a , -OC(O)N(R ^a ) ₂ , -C(O)N(R ^a ) ₂ , -N(R ^a )C(O)OR ^a , -N(R ^a )C(O)R ^a , - N(R ^a )C(O)N(R ^a ) ₂ , -N(R ^a )C(NR ^a )N(R ^a ) ₂ , -N(R ^a )S(O) _t N(R ^a ) ₂ (where t is 1 or 2), -P(=O)(R ^a )(R ^a ), or -O-P(=O)(OR ^a ) ₂ where each R ^a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. In a non-limiting embodiment, a substituted alkyl can be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3 -fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3- hydroxypropyl, benzyl, and phenethyl.

[0048] As used herein, the term “alkoxy” refers to the group -O-alkyl, including from 1 to 10 carbon atoms (C _1-10 ) of a straight, branched, saturated cyclic configuration and combinations thereof, attached to the parent molecular structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy, cyclohexyloxy and the like. "Lower alkoxy" refers to alkoxy groups containing one to six carbons. In some embodiments, C _1-3 alkoxy is an alkoxy group that encompasses both straight and branched chain alkyls of from 1 to 3 carbon atoms. Unless stated otherwise in the specification, an alkoxy group can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R ^a ) ₃ , -OR ^a , - SR ^a , -OC(O)-R ^a , -N(R ^a ) ₂ , -C(O)R _a , -C(O)OR ^a , -OC(O)N(R ^a ) ₂ , -C(O)N(R ^a ) ₂ , -N(R ^a )C(O)OR ^a , - N(R ^a )C(O)R ^a , -N(R ^a )C(O)N(R ^a ) ₂ , -N(R ^a )C(NR ^a )N(R ^a ) ₂ , -N(R ^a )S(O) _t N(R ^a ) ₂ (where t is 1 or 2), - P(=O)(R ^a )(R ^a ), or -O-P(=O)(OR ^a ) ₂ where each R ^a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein.

[0049] As used herein, the terms “aromatic” or “aryl” refer to a radical with 6 to 14 ring atoms (e.g., C _6-14 aromatic or C _6-14 aryl) that has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). In some embodiments, the aryl is a C _6-10 aryl group. For example, bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. In other embodiments, bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in"-yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene" to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Whenever it appears herein, a numerical range such as "6 to 14 aryl" refers to each integer in the given range; e.g., "6 to 14 ring atoms" means that the aryl group can consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicycles, tricycles, tetracycles, and the like. In a multi-ring group, only one ring is required to be aromatic, so groups such as indanyl are encompassed by the aryl definition. Non- limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl, indanyl, and the like. Unless stated otherwise in the specification, an aryl moiety can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R ^a ) ₃ , -OR ^a , - SR ^a , -OC(O)-R ^a , -N(R ^a ) ₂ , -C(O)R _a , -C(O)OR ^a , -OC(O)N(R ^a ) ₂ , -C(O)N(R ^a ) ₂ , -N(R ^a )C(O)OR ^a , - N(R ^a )C(O)R ^a , -N(R ^a )C(O)N(R ^a ) ₂ , -N(R ^a )C(NR ^a )N(R ^a ) ₂ , -N(R ^a )S(O) _t N(R ^a ) ₂ (where t is 1 or 2), - P(=O)(R ^a )(R ^a ), or -O-P(=O)(OR ^a ) ₂ where each R ^a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. [0050] As used herein, the terms “cycloalkyl” and “carbocyclyl” each refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and can be saturated or partially unsaturated. Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted cycloalkyl groups. Partially unsaturated cycloalkyl groups can be termed "cycloalkenyl" if the carbocycle contains at least one double bond, or "cycloalkynyl" if the carbocycle contains at least one triple bond. Cycloalkyl groups include groups having from 3 to 13 ring atoms (i.e., C _3-13 cycloalkyl). Whenever it appears herein, a numerical range such as "3 to 10" refers to each integer in the given range; e.g., "3 to 13 carbon atoms" means that the cycloalkyl group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, etc., up to and including 13 carbon atoms. The term "cycloalkyl" also includes bridged and spiro-fused cyclic structures containing no heteroatoms. The term also includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicycles, tricycles, tetracycles, and the like. In some embodiments, “cycloalkyl” can be a C _3-8 cycloalkyl radical. In some embodiments, “cycloalkyl” can be a C _3-5 cycloalkyl radical. Illustrative examples of cycloalkyl groups include, but are not limited to the following moieties: C _3-6 carbocyclyl groups include, without limitation, cyclopropyl (C ₃ ), cyclobutyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ), cyclohexenyl (C ₆ ), cyclohexadienyl (C ₆ ) and the like. Examples of C _3-7 carbocyclyl groups include norbomyl (C ₇ ). Examples of C _3-8 carbocyclyl groups include the aforementioned C _3-7 carbocyclyl groups as well as cycloheptyl (C ₇ ), cycloheptadienyl (C ₇ ), cycloheptatrienyl (C ₇ ), cyclooctyl (C ₈ ), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the like. Examples of C _3-13 carbocyclyl groups include the aforementioned C _3-8 carbocyclyl groups as well as octahydro-lH indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and the like. Unless stated otherwise in the specification, a cycloalkyl group can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R ^a ) ₃ , -OR ^a , - SR ^a , -OC(O)-R ^a , -N(R ^a ) ₂ , -C(O)R _a , -C(O)OR ^a , -OC(O)N(R ^a ) ₂ , -C(O)N(R ^a ) ₂ , -N(R ^a )C(O)OR ^a , - N(R ^a )C(O)R ^a , -N(R ^a )C(O)N(R ^a ) ₂ , -N(R ^a )C(NR ^a )N(R ^a ) ₂ , -N(R ^a )S(O) _t N(R ^a ) ₂ (where t is 1 or 2), - P(=O)(R ^a )(R ^a ), or -O-P(=O)(OR ^a ) ₂ where each R ^a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. The terms “cycloalkenyl" and "cycloalkynyl" mirror the above description of "cycloalkyl" wherein the prefix "alk" is replaced with "alken" or "alkyn" respectively, and the parent "alkenyl" or "alkynyl" terms are as described herein. For example, a cycloalkenyl group can have 3 to 13 ring atoms, such as 5 to 8 ring atoms. In some embodiments, a cycloalkynyl group can have 5 to 13 ring atoms.

[0051] As used herein, the terms “heterocycle”, “heterocyclic” or “heterocyclo” refer to fully saturated or partially unsaturated cyclic groups, for example, 3 to 7 membered monocyclic, 7 to 12 membered bicyclic, or 10 to 15 membered spirocyclic or tricyclic ring systems, which have at least one heteroatom (selected from the group consisting of N, O, and S) in at least one ring, wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quatemized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system. A heterocyclic group is optionally substituted. Examples of heterocyclic groups include, but not limited to, epoxy, azeiidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl, imidazolidinyl, imidazopyridinyl, thiazolidinyl, dithianyl, trithianyl, dioxolanyl, oxazolidinyl, oxazoli di nonyl, decahydroquinolinyl. piperidonyl, 4-piperidinonyL quinuclidinyl, thiomorpholinyl, thiomorpholinyl 1,1 dioxide, morpholinyl, azepanyl, oxazepanyl, azabicyclohexanyls, azabicycloheptanyl, azabicyclooctanyls, azabicyclononanyls (e.g., octahydroindolizinyl), azaspiroheptanyls, dihydro-1 H,3H,5H- oxazolo[3,4-c]oxazolyl, tetrahydro- 1'H,3'H- spiro[cyclopropane-l,2'-pyrrolizine], hexahydro- IH-pyrrolizinyl, hexahydro- lH-pyrrolo[2,l- c][l,4]oxazinyl, octahydroindolizinyl, oxaazaspirononanyls, oxaazaspirooctanyls, diazaspirononanyls, oxaazabiocycloheptanyls, hexahydropyrrolizinyl 4(lH)-oxide, and tetrahydro- 2H-thiopyranyl 1 -oxide and tetrahydro-2H- thiopyranyl 1,1 -di oxi de.

[0052] As used herein, the term “heterocycloalkyl” refers to a cycloalkyl radical, which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., O, N, S, P or combinations thereof. Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted heterocycloalkyl groups. Illustrative examples of heterocycloalkyl include 2-hydroxy-aziridin-l-yl, 3-oxo-l-oxacyclobutan-2-yl, 2,2-dimethyl- tetrahydrofuran-3-yl, 3-carboxy-morpholin-4-yl, l-cyclopropyl-4-methyl-piperazin-2-yl. 2- pyrrolinyl, 3-pyrrolinyl, dihydro-2H-pyranyl, 1,2,3,4-tetrahydropyridine, 3,4-dihydro-2H- [l,4]oxazine, etc.

[0053] As used herein, the term “halogen” refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). As used herein, the term “halide” or “halo”, means fluoro, chloro, bromo or iodo. The terms "haloalkyl," "haloalkenyl," "haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl, alkynyl and alkoxy groups are as defined herein and can be optionally further substituted as defined herein.

[0054] As used herein, the term “heteroatom” refers to oxygen (O), nitrogen (N), sulfur (S), and phosphorus (P).

[0055] As used herein, the term “heteroalkyl” refers to an alkyl radical, which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. A numerical range can be given, e.g., C _1-4 heteroalkyl, which refers to the chain length in total, which in this example is 4 atoms long. For example, a -CH ₂ OCH ₂ CH ₃ radical is referred to as a "C4" heteroalkyl, which includes the heteroatom center in the atom chain length description. Connection to the parent molecular structure can be through either a heteroatom or a carbon in the heteroalkyl chain. For example, an N-containing heteroalkyl moiety refers to a group in which at least one of the skeletal atoms is a nitrogen atom. One or more heteroatom(s) in the heteroalkyl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quatemized. For example, heteroalkyl also includes skeletal chains substituted with one or more nitrogen oxide (-O-) substituents. Exemplary heteroalkyl groups include, without limitation, ethers such as methoxyethanyl (-CH ₂ CH ₂ OCH ₃ ), ethoxymethanyl (-CH ₂ OCH ₂ CH ₃ ), (methoxymethoxy )ethanyl (-CH ₂ CH ₂ OCH ₂ OCH ₃ ), (methoxymethoxy) methanyl (-CH ₂ OCH ₂ OCH ₃ ) and (methoxyethoxy )methanyl (-CH ₂ OCH ₂ CH ₂ OCH ₃ ) and the like; amines such as (- CH ₂ CH ₂ NHCH ₃ , -CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ NHCH ₂ CH ₃ , -CH ₂ N(CH ₂ CH ₃ )(CH ₃ )) and the like. [0056] As used herein, the term “heteroaryl” or, alternatively, "heteroaromatic" refers to a refers to a radical of a 5-18 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic and the like) aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-6 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous and sulfur ("5-18 membered heteroaryl"). Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. Whenever it appears herein, a numerical range such as "5 to 18" refers to each integer in the given range; e.g., "5 to 18 ring atoms" means that the heteroaryl group can consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. In some instances, a heteroaryl can have 5 to 14 ring atoms. In some embodiments, the heteroaryl has, for example, bivalent radicals derived from univalent heteroaryl radicals whose names end in "-yl" by removal of one hydrogen atom from the atom with the free valence are named by adding "-ene" to the name of the corresponding univalent radical, e.g., a pyridyl group with two points of attachment is a pyridylene.

[0057] For example, an N-containing “heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. One or more heteroatom(s) in the heteroaryl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quatemized. Heteroaryl also includes ring systems substituted with one or more nitrogen oxide (-O-) substituents, such as pyridinyl N-oxides. The heteroaryl is attached to the parent molecular structure through any atom of the ring(s).

[0058] “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment to the parent molecular structure is either on the aryl or on the heteroaryl ring, or wherein the heteroaryl ring, as defined above, is fused with one or more cycloalkyl or heterocyclyl groups wherein the point of attachment to the parent molecular structure is on the heteroaryl ring. For polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl and the like), the point of attachment to the parent molecular structure can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, phosphorous, and sulfur. [0059] Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3 -benzodi oxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4] oxazinyl, 1,4- benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzopyranonyl, benzofurazanyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[ l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7- dihydro-5H-cyclopenta[4,5]thieno [2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6- dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H benzo[6,7]cyclohepta[ l,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, fu ranonyl, furo [3,2 -c]pyridinyl,

5.6.7.8.9.10-hexahydrocycloocta[d] pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,

5.6.7.8.9.10- hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano- 5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyri dinonyl, oxadiazolyl, 2- oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1 -phenyl - IH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4- d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5, 6,7,8- tetrahydrobenzo [4,5 ] thieno [2,3 -d]pyrimdinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno [2,3-d]pyrimidinyl, 5,6,7, 8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno [2,3-c]pridinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise in the specification, a heteroaryl moiety can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, -Si(R ^a ) ₃ , -OR ^a , - SR ^a , -OC(O)-R ^a , -N(R ^a ) ₂ , -C(O)R _a , -C(O)OR ^a , -OC(O)N(R ^a ) ₂ , -C(O)N(R ^a ) ₂ , -N(R ^a )C(O)OR ^a , - N(R ^a )C(O)R ^a , -N(R ^a )C(O)N(R ^a ) ₂ , -N(R ^a )C(NR ^a )N(R ^a ) ₂ , -N(R ^a )S(O) _t N(R ^a ) ₂ (where t is 1 or 2), - P(=O)(R ^a )(R ^a ), or -O-P(=O)(OR ^a ) ₂ where each R ^a is independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. [0060] As used herein, the terms “administer” and “administering” refer to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, inhalation, intraocular, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Suitable routes of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound.

[0061] Administration may be by any suitable route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0062] As used herein, the term “co-administer” refers to the presence of two pharmacological agents in a subject’s body (e.g., in the blood) at the same time. The two pharmacological agents can be administered concurrently or sequentially.

[0063] As used herein, the term "affinity" refers to the strength of interaction between an antigen binding moiety (e.g., antibody) and antigen at single antigenic sites.

[0064] As used herein, the term “agonist” refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor.

[0065] As used herein, the term “antagonist” refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of an agonist or inverse agonist on the receptor. However, an antagonist has no effect on constitutive receptor activity. [0066] As used herein, the term "amino acid" refers to a molecule of the general formula NH ₂ - CHR-COOH, wherein "R" is one of a number of different side chains, or a residue within a peptide bearing the parent amino acid. Amino acids include naturally occurring amino acids with "R" being a substituent found in naturally occurring amino acids. "R" can also be a substituent that is not found in naturally occurring amino acids. The term “amino acid residue" refers to the portion of the amino acid which remains after losing a water molecule when it is joined to another amino acid. The term "modified amino acid” refers to an amino acid bearing an "R“ substituent that does not correspond to one of the twenty genetically coded amino acids.

[0067] As used herein, the term “antigen” as used herein is meant any substance that causes the immune system to produce antibodies or specific cell-mediated immune responses against it. A disease associated antigen is any substance that is associated with any disease that causes the immune system to produce antibodies or a specific-cell mediated response against it. An antigen is capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. An antigen can have one or more epitopes (B- and/or T-cell epitopes). An antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.

[0068] As used herein, the term “antigen binding moiety” refers to a moiety capable of binding specifically to an antigen, and includes but is not limited to antibodies and antibody fragments, peptides and small molecule ligands.

[0069] As used herein, the term “antibody” refers to molecules that are capable of binding an epitope or antigenic determinant. The term is meant to include whole antibodies and antigen- binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fabs, Fvs, single-chain antibodies and single or multiple immunoglobulin variable chain or CDR domain designs as well as bispecific and multispecific antibodies. Antibodies can be from any animal origin. Preferably, the antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animals. Antibodies may recognize polypeptide or polynucleotide antigens. The term includes active fragments, including for example, an antigen binding fragment of an immunoglobulin, a variable and/or constant region of a heavy chain, a variable and/or constant region of a light chain, a complementarity determining region (cdr), and a framework region. The terms include polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F(ab) ₂ and F(ab) fragments; Fv molecules (for example, noncovalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments obtained from such molecules, wherein such fragments retain specific binding.

[0070] As used herein, the term “antigen binding fragment” refers to one or more portions of an antibody that retain the ability to specifically interact with, e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution, an epitope of an antigen.

[0071] Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the V _L , V _H , C _L and C _H I domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V _H and C _H I domains; a Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody; a dAb fragment (Ward et al. 1989 Nature 341:544- 546,), which consists of a V _H domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.

[0072] Additionally, the two domains of the Fv fragment, V _L and V _H can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain in which the V _L and V _H regions pair to form monovalent molecules, (known as single chain Fv ("scFv"); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al. 1988 Proc. Natl. Acad. Set. 85:5879-5883.) Such single chain antibodies are also intended to be encompassed within the term “antigen binding fragment.” These antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0073] Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. (See, e.g., Hollinger and Hudson, 2005 Nature Biotechnology 23:1 126-1136.) Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3). (See, e.g., U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies.) Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (V _H - C _H I-V _H -C _H I) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. (Zapata et al., 1995 Protein Eng. 8:1057-1062; U.S. Pat. No. 5,641 ,870.)

[0074] As used herein, the term “bispecific antibody” or “bispecific” refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes. The epitopes can be from the same antigen or from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. Alternatively, bispecific antibodies can be prepared using chemical linkage. Bispecific antibodies include bispecific antibody fragments. (See, e.g., Milstein et al. 1983 Nature 305:537- 39; Brennan et al. 1985 Science 229:81; Hollinger et al. 1994 Proc. Natl. Acad. Sci. U.S.A.

90:6444-48; Gruber et al. 1994 J. Immunol. 152:5368-74.)

[0075] As used herein, the term “chimeric antibody” or “chimeric” refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity

[0076] As used herein, the term “human antibody” refers to antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al. 2000 J. Mol. Biol. 296:57-86). Human antibodies may include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a substitution to promote stability or manufacturing.

[0077] As used herein, the term “humanized antibody” refers to antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. (See e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et al. 1989 Proc. Natl Acad. Sci. USA 86:10029-10033; ANTIBODY ENGINEERING: A PRACTICAL APPROACH, Oxford University Press 1996.)

[0078] As used herein, the term “monoclonal antibody”, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. “Monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by various methods known in the art, including the hybridoma method first described by Kohler et al. 1975 Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). “Monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. 1991 Nature 352: 624-628 and Marks et al. 1991 J. Mol. Biol. 222: 581-597, for example. These monoclonal antibodies will usually bind with at least a Kd of about 1 μM, more usually at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM.

[0079] As used herein, the term “biologically active” entity, or an entity having “biological activity,” is one having structural, regulatory, or biochemical functions of a naturally occurring molecule or any function related to or associated with a metabolic or physiological process. A biologically active polypeptide or fragment thereof includes one that can participate in a biological process or reaction and/or can produce a desired effect. The biological activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates biological activity when it participates in a molecular interaction with another molecule, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, or when it has diagnostic and/or prognostic value in determining the presence of a molecule. A biologically active protein or polypeptide can be naturally-occurring or it can be synthesized from known components, e.g., by recombinant or chemical synthesis and can include heterologous components.

[0080] As used herein, the terms "inflammatory disease" and "inflammatory disorder" refers to, without limitation, a disease resulting from the biological response of vascular tissues to harmful stimuli, including but not limited to such stimuli as pathogens, damaged cells, irritants, antigens and, in the case of autoimmune disease, substances and tissues normally present in the body. For example, an inflammatory disease or disorder may be an autoimmune, microbial, metabolic, neoplastic, and posttraumatic disease or disorder. Non-limiting examples of inflammatory disease or disorder include rheumatoid arthritis (RA), enthesitis-related arthritis (ERA), ankylosing spondylitis, psoriatic arthritis, atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplant rejection, and vasculitis.

[0081 ] As used herein, the term “cleavable” linker refers to a linker or linker component that connects two moieties by covalent connections, but breaks down to sever the covalent connection between the moieties under physiologically relevant conditions. Typically, a cleavable linker is severed in vivo more rapidly in an intracellular environment than when outside a cell, causing release of a payload to preferentially occur inside the targeted cell. Cleavage may be enzymatic or non-enzymatic. A payload is typically released from an antibody without degrading the antibody. Cleavage may leave some portion of a linker or linker component attached to the payload, or it may release the payload without any residual part or component of the linker (i.e., traceless release).

[0082] As used herein, the term “non-cleavable” linker refers to a linker or linker component that is not especially susceptible to breaking down under physiological conditions, i.e., it is at least as stable as the antibody or antigen binding fragment portion of the immunoconjugate. Such linkers are sometimes referred to as “stable,” meaning they are sufficiently resistant to degradation to keep the payload connected to the antigen binding moiety until the antigen binding moiety is itself at least partially degraded. In such a case, the degradation of Ab precedes cleavage of the linker in vivo. Degradation of the antibody portion of an immunoconjugate having a stable or non-cleavable linker may leave some or all of the linker, and one or more amino acid groups from an antibody, attached to the payload or drug moiety that is delivered in vivo.

[0083] As used herein, the term “cell” refers to any prokaryotic, eukaryotic, primary cell or immortalized cell line, any group of such cells as in, a tissue or an organ. Preferably the cells are of mammalian (e.g., human) origin and can be infected by one or more pathogens.

[0084] The term “payload” is used herein and refer to a compound or substance that inhibits or prevents or stops the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

[0085] As used herein, the terms “disease”, “condition” or “disorder” are used interchangeably herein and refer to a pathological condition, for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state. The term “disease” includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0086] As used here, the term “homology” or “homologous” refers to a sequence similarity between two polypeptides or between two polynucleotides. Similarity can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. If a given position of two polypeptide sequences is not identical, the similarity or conservativeness of that position can be determined by assessing the similarity of the amino acid of the position. A degree of similarity between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment of two sequences to determine their percent sequence similarity can be done using software programs known in the art, such as, for example, those described in Ausubel et al. 1999 Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD. The term “homologs” of to a given amino acid sequence or a nucleic acid sequence is intended to indicate that the corresponding sequences of the “homologs” having substantial identity or homology to the given amino acid sequence or nucleic acid sequence.

[0087] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0088] An example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al. 1977 Ntic. Acids Res. 25:3389-3402 and Altschul etal. 1990 J. Mol. Biol. 215:403-410, respectively. BLAST software is publicly available through the National Center for Biotechnology Information on the worldwide web at ncbi.nlm.nih.gov/. Both default parameters and other non- default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0089] As used herein, the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and oftentimes over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the full-length of an amino acid or nucleic acid sequences. [0090] The compound of the invention can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).

[0091 ] The compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, gels, for example, water or water/propylene glycol solutions.

[0092] The compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920;

5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, 1995 J. Biomater Set. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations (see, e.g., Gao 1995 Pharm. Res. 12:857-863); or, as microspheres for oral administration (see, e.g., Eyles 1997 J. Pharm. Pharmacol. 49:669-674).

[0093] As used herein, the term “in need of" a treatment refers to a subject that would benefit biologically, medically or in quality of life from such a treatment.

[0094] As used herein, the term "specifically binds" or "selectively binds," when used in the context of describing the interaction between an antigen (e.g., a protein or a glycan) and an antibody, antibody fragment, or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, e.g., in a biological sample, e.g., a blood, serum, plasma or tissue sample. Thus, under certain designated immunoassay conditions, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two (2) times the background and do not substantially bind in a significant amount to other antigens present in the sample. In embodiments, under designated immunoassay conditions, the antibody or binding agents with a particular binding specificity bind to a particular antigen at least ten (10) times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. As desired or appropriate, this selection may be achieved by subtracting out antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some embodiments, antibodies or antibody fragments are selected that cross-react with certain desired molecules.

[0095] A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein. (See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.) Typically, a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background.

[0096] As used herein, the term “therapeutically effective amount” refers to the dose of a therapeutic agent or agents sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be readily determined by a skilled physician, e.g., by first administering a low dose of the pharmacological agent(s) and then incrementally increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0097] The terms “immunoconjugate” and “antibody-drug-conjugate” are used interchangeably herein and refer to a compound with a linkage of an antigen binding moiety (e.g., an antibody or an antigen binding fragment thereof, a peptide or a small molecule ligand) with a cytotoxic agent or payload. The linkage can be covalent bonds or non-covalent interactions and can include chelation. Thus, the terms “immunoconjugate” and “antibody-drug- conjugate” include pepti de-drug-conjugates and small molecule-drug-conjugates.” Various linkers and linking strategies are known in the art and can be employed in order to form an immunoconjugate.

[0098] As used herein, the terms “inhibition,” “inhibit” and “inhibiting” and the like in reference to a biological target inhibitor interaction refers to negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

[0099] As used herein, the terms “isolated” or “purified” refer to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[00100] As used herein, the term “modulate” refers to the production, either directly or indirectly, of an increase or a decrease, a stimulation, inhibition, interference, or blockage in a measured activity when compared to a suitable control. A “modulator” of a polypeptide or polynucleotide refers to a substance that affects, for example, increases, decreases, stimulates, inhibits, interferes with, or blocks a measured activity of the polypeptide or polynucleotide, when compared to a suitable control. For example, a “modulator” may bind to and /or activate or inhibit the target with measurable affinity, or directly or indirectly affect the normal regulation of a receptor activity. [00101] As used herein, a “pharmaceutically acceptable form” of a disclosed compound includes, but is not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, isomers, prodrugs, and isotopically labeled derivatives thereof. In one embodiment, a "pharmaceutically acceptable form" includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs and isotopically labeled derivatives thereof. In some embodiments, a "pharmaceutically acceptable form" includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs and isotopically labeled derivatives thereof.

[00102] In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt.

[00103] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchlorate acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like.

[00104] The salts can be prepared in situ during the isolation and purification of the disclosed compounds, or separately, such as by reacting the free base or free acid of a parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt can be chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[00105] In certain embodiments, the pharmaceutically acceptable form is a “solvate” (e.g., a hydrate). As used herein, the term “solvate” refers to compounds that further include a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. The solvate can be of a disclosed compound or a pharmaceutically acceptable salt thereof. Where the solvent is water, the solvate is a “hydrate.” Pharmaceutically acceptable solvates and hydrates are complexes that, for example, can include 1 to about 100, or 1 to about 10, or 1 to about 2, about 3 or about 4, solvent or water molecules. It will be understood that the term “compound” as used herein encompasses the compound and solvates of the compound, as well as mixtures thereof.

[00106] In certain embodiments, the pharmaceutically acceptable form is a prodrug. As used herein, the term “prodrug” (or “pro-drug”) refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable form of the compound. A prodrug can be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical and/or delivery properties over the parent compound. Prodrugs can increase the bioavailability of the compound when administered to a subject (e.g., by permitting enhanced absorption into the blood following oral administration) or which enhance delivery to a biological compartment of interest (e.g., the brain or lymphatic system) relative to the parent compound. Exemplary prodrugs include derivatives of a disclosed compound with enhanced aqueous solubility or active transport through the gut membrane, relative to the parent compound.

[00107] The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism. (See, e.g., Bundgard, H. 1985 Design of Prodrugs, pp. 7- 9, 21-24, Elsevier, Amsterdam; Higuchi et al. 1987 "Pro-drugs as Novel Delivery Systems" ACS. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[00108] Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism. (See, e.g., Bundgard, Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992.) Prodrugs commonly known in the art include well-known acid derivatives, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative, etc. Other prodrug derivatives may be combined with other features disclosed herein to enhance bioavailability. As such, those of skill in the art will appreciate that certain of the presently disclosed compounds having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to any of the above substituents disclosed herein.

[00109] Exemplary advantages of a prodrug can include, but are not limited to, its physical properties, such as enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it can enhance absorption from the digestive tract, or it can enhance drug stability for long-term storage.

[00110] As used herein, the term “pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[00111] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. Furthermore, a polypeptide may refer to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[00112] As used herein, the term “receptor” refers to proteins, including glycoproteins or fragments thereof, capable of interacting with another molecule, called the ligand. The ligand is usually an extracellular molecule which, upon binding to the receptor, usually initiates a cellular response, such as initiation of a signal transduction pathway. The receptor need not necessarily be a membrane-bound protein. The ligand may belong to any class of biochemical or chemical compounds.

[00113] As used herein, the term “sample” refers to a sample from a human, animal, or to a research sample, e.g., a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. The “sample” may be tested in vivo, e.g., without removal from the human or animal, or it may be tested in vitro. The sample may be tested after processing, e.g., by histological methods. “Sample” also refers, e.g., to a cell comprising a fluid or tissue sample or a cell separated from a fluid or tissue sample. “Sample” may also refer to a cell, tissue, organ, or fluid that is freshly taken from a human or animal, or to a cell, tissue, organ, or fluid that is processed or stored.

[00114] As used herein, the terms “stimulate” or “stimulating” refer to increase, to amplify, to augment, to boost a physiological activity, e.g., an immune response. Stimulation can be a positive alteration. For example, an increase can be by 5%, 10%, 25%, 50%, 75%, or even 90- 100%. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100- fold.

[00115] As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. A subject to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example, non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), rodents (e.g., rats and/or mice), etc. In certain embodiments, the non- human animal is a mammal. The non-human animal may be a male or female at any stage of development. A non-human animal may be a transgenic animal. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[00116] As used herein, the terms “suppress” or “suppressing” refer to decrease, to attenuate, to diminish, to arrest, or to stabilize a physiological activity, e.g., an immune response.

Suppression can be a negative alteration. For example, a decrease can be by 5%, 10%, 25%, 50%, 75%, or even 90-100%. Exemplary decreases include 2-fold, 5-fold, 10-fold, 20-fold, 40- fold, or even 100-fold.

[00117] As used herein, the terms “treatment” or “treating” a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. Treating or treatment thus refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters, for example, the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. As compared with an equivalent untreated control, such reduction or degree of amelioration may be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

[00118] Treatment methods include administering to a subject a therapeutically effective amount of a compound described herein. The administering step may be a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the patient’s age, the concentration of the compound, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.

Glucocorticoid. Receptor Agonists

[00119] Various novel glucocorticoid receptor agonists are disclosed herein.

[00120] In one aspect, the invention generally relates to a compound having the structural formula (I): or a pharmaceutically acceptable form thereof, wherein

R ¹ is H or halogen;

R ² is H or halogen;

R ³ is C _1-4 alkyl and R ⁴ is OC(O)-R ⁷ , wherein R ⁷ is a substituted or unsubstituted aniline, or

R ³ and R ⁴ , together with the carbon atoms they are bound to, form a 5-membered dioxolane substituted with W-R ⁶ , wherein W is a single bond or -phenyl-Q-, Q is selected from CH ₂ , O, S, S(O), S(O) ₂ , NH and NCH ₃ , and R ⁶ is a substituted or unsubstituted aniline; and

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered (e.g., 4-, 5-, 6- or 7- membered), unsubstituted or substituted heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl.

[00121] In certain embodiments, R ⁴ is OC(O)-R ⁷ , and the compound has the structural formula (II): [00122] In certain embodiments, the compound has the structural formula (II ^a ): wherein each of R ^7a , R ^7b and R ^7c is selected from H and NR ^7x R ^7y , provided that only one of R ^7a , R ^7b and R ^7c is NR ^7x R ^7y and each of the others is H; each of R ^7x and R ^7y is independently selected from R, R ^7r and L ⁷ -R ^7z , provided that when one of R ^7x and R ^7y is L ⁷ -R ^7z or R ^7r , the other is R;

L ⁷ is a linker;

R ^7r is (C=O)-O-(CH ₂ ) _i -R ^7v or (C=O)-(CH ₂ ) _j -R ^7v ;

R ^7v is R, OR, NHR, NR ₂ , an aryl group or an amino acid; i is 0, 1, 2, 3, 4, 5 or 6; j is 0, 1, 2, 3, 4, 5 or 6;

R ^7z comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[00123] In certain embodiments, R ^7a is H, R ^7b is NR ^7x R ^7y and R ^7c is H.

[00124] In certain embodiments, R ^7a is H, R ^7b is H and R ^7c is NR ^7x R ^7y .

[00125] In certain embodiments, R ^7a is NR ^7x R ^7y , R ^7b is H and R ^7c is H.

[00126] In certain embodiments, R ^7x is H or CH ₃ and R ^7y is (C=O)-O-(CH ₂ ) _i -R ^7v , wherein R ^7v is R, OR, NHR, NR ₂ , an aryl group or an amino acid, and i is 0, 1, 2 or 3.

[00127] In certain embodiments, R ^7x is H or CH ₃ and R ^7y is (C=O)-(CH ₂ ) _j - R ^7v , wherein R ^7v is R, OR, NHR, NR ₂ , an aryl group or an amino acid and j is 0, 1, 2 or 3.

[00128] In certain embodiments, R ³ is an alkyl. In certain embodiments, R ³ is methyl.

[00129] In certain embodiments of (I), R ³ and R ⁴ , together with the carbon atoms they are bound to, form a 5-membered dioxolane substituted with W-R ⁶ , having the structural formula:

[00130] In certain embodiments, W is a single bond and a compound of the invention has the structural formula (III):

[00131] In certain embodiments, the compound has the structural formula (IIP): wherein each of R ^6a , R ^6b and R ^6c is selected from H and NR ^6x R ^6y , provided that only one of R ^6a , R ^6b and R ^6c is NR ^6x R ^6y and each of the others is H; each of R ^6x and R ^6y is independently selected from R, R ^6r and L ⁶ -R ^6z , provided that when one of R ^6x and R ^6y is L ⁶ -R ^6z or R ^6r , the other is R; L ⁶ is a linker;

R ^6r is (C=O)-O-(CH ₂ ) _p -R ^6v or (C=O)-(CH ₂ ) _q -R ^6v ;

R ^6v is R, OR, NHR, NR ₂ , an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6;

R ^6z comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[00132] In certain embodiments, R ⁶ has the S- configuration.

[00133] In certain embodiments, R ⁶ has the R- configuration.

[00134] In certain embodiments, R ^6a is H, R ^6b is NR ^6x R ^6y and R ^6c is H.

[00135] In certain embodiments, R ^6a is H, R ^6b is H and R ^6c is NR ^6x R ^6y .

[00136] In certain embodiments, R ^6a is NR ^6x R ^6y , R ^6b is H and R ^6c is H.

[00137] In certain embodiments, R ^6x is H or CH ₃ and R ^6y is (C=O)-O-(CH ₂ ) _p -R ^6v , wherein R ^6v is R, OR, NHR, NR ₂ , an aryl group or an amino acid, and p is 0, 1, 2 or 3.

[00138] In certain embodiments, R ^7x is H or CH ₃ and R ^7y is (C=O)-(CH ₂ ) _q -R ^7v , wherein R ^7v is R, OR, NHR, NR ₂ , an aryl group or an amino acid and q is 0, 1, 2 or 3.

[00139] In certain embodiments, W is -phenyl-Q-.

[00140] In certain embodiments, Q is CH ₂ , a compound of the invention has the structural formula (III ^b ): wherein each of R ^6a' , R ^6b' and R ^6c' is selected from H and NR ^6x' R ^{6y ’} , provided that only one of R ^6a' , R ^6b' and R ^6c' is NR ^6x' R ^{6y ’} and each of the others is H; each of R ^6x' and R ^{6y ’} is independently selected from R, R ^6r and L ⁶ -R ^6z' , provided that when one of R ^6x' and R ^{6y ’} is L ^6' -R ^6z' or R ^{6r '} , the other is R’; L ^6' is a linker; R ^{6r '} is (C=O)-O-(CH ₂ ) _p -R ^6v' or (C=O)-(CH ₂ ) _q -R ^6v' ; R ^6v' is R, OR, NHR, NR ₂ , an aryl group or an amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q is 0, 1, 2, 3, 4, 5 or 6; R ^6z' comprises a functional or reactive group; and

R is H or a C ₁ -C ₆ alkyl.

[00141] In certain embodiments, the carbon atom in 5-membered dioxolane linked to W-R ⁶ has the S- configuration.

[00142] In certain embodiments, the carbon atom in 5-membered dioxolane linked to W-R ⁶ has the R- configuration.

[00143] In certain embodiments, R ^6a' is H, R ^6b is NR ^6x' R ^{6y ’} and R ^6c' is H.

[00144] In certain embodiments, R ^6a is H, R ^6b' is H and R ^6c' is NR ^6x' R ^{6y ’} .

[00145] In certain embodiments, R ^6a is NR ^6x' R ^{6y ’} , R ^6b' is H and R ^6c' is H.

[00146] In certain embodiments, R ^6x' is H or CH ₃ and R ^{6y ’} is (C=O)-O-(CH ₂ ) _p -R ^6v' , wherein R ^6v' is R, OR, NHR, NR ₂ , an aryl group or an amino acid, and p is 0, 1, 2 or 3.

[00147] In certain embodiments, R ^6x' is H or CH ₃ and R ^{6y ’} is (C=O)-(CH ₂ ) _q -R ^6v' , wherein R ^6v' is R, OR, NHR, NR ₂ , an aryl group or an amino acid and q is 0, 1, 2 or 3.

[00148] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is a noncleavable linker.

[00149] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is a cleavable linker.

[00150] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is an acid- labile or acid-sensitive linker.

[00151] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is protease- sensitive linker.

[00152] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is lysosomal protease-sensitive linker.

[00153] In certain embodiments, L ⁶ or L ⁷ , or L ⁶ , when present in the compound, is β- glucuronide-sensitive linker.

[00154] In certain embodiments, L ⁶ or L ⁷ , or L ^6' , when present in the compound, is glutathione-sensitive disulfide linker.

[00155] In certain embodiments, R ^6z or R ^7z , or R ^6z' , when present in the compound, comprises a functional or reactive group selected from:

-N ₃ , -NR ^u C(=O)CH=CH ₂ , -SH, -SSR ^t , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), - NR ^u S(=O ₂ )(CH=CH ₂ ), -NR ^u C(=O)CH ₂ R ^w , -NR ^u C(=O)CH ₂ Br, -NR ^u C(=O)CH ₂ I, - NHC(=O)CH ₂ Br, NHC(=O)CH ₂ I, -0NH ₂ , -C(=O)NHNH ₂ , -CO ₂ H, -NH ₂ , -NCO, -NCS, wherein

R ^u is H or a C ₁ -C ₆ alkyl group, R ^t is 2-pyridyl or 4-pyridyl, and R ^w is

[00156] In certain embodiments, R ⁵ is NR ^5a R ^5b .

[00157] In certain embodiments, R ⁵ is N(CH ₃ ) ₂ .

[00158] In certain embodiments, R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered, unsubstituted or substituted heterocyclic ring.

[00159] In certain embodiments, the heterocyclic ring is an unsubstituted 4-, 5- or 6- membered heterocycle.

[00160] In certain embodiments, the heterocyclic ring is a 4-, 5- or 6-membered heterocycle substituted with a group selected from OH and C _1-3 alkyl, wherein the C _1-3 alkyl is optionally substituted with one or more OH groups.

[00161] In certain embodiments, R ^5a and R ^5b are independently selected from H and C _1-6 alkyl, optionally substituted with one or more OH groups.

[00162] In certain embodiments, R ^5a is H or methyl and R ^5b is C _1-6 alkyl substituted with an OH.

[00163] In certain embodiments, R ^5a is H or methyl and R ^5b is C _2-6 alkyl substituted with two OH groups.

[00164] In certain embodiments, R ⁵ is CH ₂ R ^5c . In certain embodiments, R ^5c is F.

[00165] In certain embodiments, R ^5c is Cl. [00166] In certain embodiments, R ^5c is O-C _1-3 alkyl.

[00167] In certain embodiments, each of R ¹ and R ² is H.

[00168] In certain embodiments, each of R ¹ and R ² is F.

[00169] In certain embodiments, one of R ¹ and R ² is H and the other is F.

[00170] Additional disclosures on linkers and reactive or functional groups that may be employed in R ^6z , R ^{7z 6z} and/or components of L ⁶ , L ⁷ or L ^6' , are provided in the sections

Linker and Linking Technologies” and “Linker-antibody and Linker-payload Attachments" and references cited therein, each of which is incorporated herein by reference.

[00171] The invention al so includes methods for synthesizing glucocorticoid receptor agonists, including intermediates or precursors thereof.

[00172] Non-limiting examples of glucocorticoid receptor agonists of the invention included those listed in Table 1.

Table 1. Example Compounds

wherein each of R ¹ and R ² is independently H or halogen (e.g., F), or a pharmaceutically acceptable form thereof.

[00173] In certain embodiments of the compounds in Table 1, each of R ¹ and R ² is H.

[00174] In certain embodiments of the compounds in Table 1, each of R ¹ and R ² is F.

[00175] In certain embodiments of the compounds in Table 1, one of R ¹ and R ² is H and the other is F.

[00176] Methods for determining binding affinity of a compound to tubulin are known in the art. (See, e.g., Muller et al. 2006 Anal. Chem. 78, 4390-4397; Hamel et al. 1995 Molecular Pharmacology 47 : 965-976; Hamel et al. 1990 J. Biological Chemistry 265:28, 17141-17149.)

Immunoconjugates

[00177] A typical ADC is comprised of an antigen binding moiety (Ab), e.g., a monoclonal antibody), a linker (L) and cytotoxic agent or payload (D), as represented below:

(D _m -L) _n -Ab wherein each m and n is an integer. The payload D (e.g., a glucocorticoid receptor agonist disclosed herein) can be conjugated to different parts of the Ab and is commonly attached via cysteine or lysine residues. Generally, more than one payload D molecules can be attached to each Ab. When a branched linker is employed, more than one payload D moieties can be attached to each linker L. In some embodiments, n ranges from 1 to 16, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, n ranges from 2 to 10, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, n is 1, 2, 3, 4, 5 or 6. In some embodiments, n is 2, 3 or 4. In some embodiments, L is an unbranched linker and m is 1. In some embodiments, L is a branched linker and m can range from 2 to 10, 2 to 8, 2 to 6, or 2 to 4. In some embodiments, m is 2, 3 or 4.

[00178] The drug to antibody ratio (DAR) or drug loading may be characterized by conventional means such as UV, mass spectroscopy, ELISA assay, HIC, HPLC or electrophoresis. In exemplary embodiments, DAR ranges from 1 to 16, 2 to 8, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or about 1.

[00179] The DAR of an immunoconjugate may be controlled by various methods, including limiting the molar excess of payload-linker intermediate or linker reagent relative to antigen binding moieties; limiting the conjugation reaction time or temperature; varying reductive conditions for cysteine thiol modification; and modifying the number and positions of cysteine residues and positions of linker-payload attachments. (See, e.g., WO 2006/034488 A2.) [00180] In one aspect, the invention generally relates to an immunoconjugate comprising a compound disclosed herein covalently conjugated to an antigen binding moiety.

[00181] In another aspect, the invention generally relates to an immunoconjugate having the structural formula (IV): or a pharmaceutically acceptable form thereof, wherein

Ab represents an antigen binding moiety;

R ¹ is H or halogen;

R ² is H or halogen;

R ³ is C _1-4 alkyl;

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered (e.g., 4-, 5-, 6- or 7- membered) heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl;

R ^7x is H or C _1-6 alkyl;

L ^Ab is a linker; and n is an integer in the range of 1 to about 20.

[00182] In certain embodiments, R ¹ , R ² , R ³ , R ⁵ and R ^7x may be selected as disclosed herein, formulae (I), (II) and (II ^a ).

[00183] In certain embodiments, R ³ is alkyl. In certain embodiments, R ³ is methyl.

[00184] In certain embodiments, R ^7x is H.

[00185] In certain embodiments, R ^7x is C _1-6 alkyl.

[00186] In yet another aspect, the invention generally relates to an immunoconjugate having the structural formula (V) or (VI): or or a pharmaceutically acceptable form thereof, wherein

Ab represents an antigen binding moiety;

Q is selected from CH ₂ , O, S, S(0), S(O) ₂ , NH and NCH ₃ ;

R ¹ is H or halogen;

R ² is H or halogen;

R ⁵ is NR ^5a R ^5b or CH ₂ R ^5c , each of R ^5a and R ^5b is independently selected from H and C _1-6 alkyl, or R ^5a and R ^5b , together with the N atom they are bound to, form a 4- to 7-membered heterocyclic ring; and

R ^5c is H, halogen or O-C _1-3 alkyl;

R ^6x is H or C _1-6 alkyl;

L ^Ab is a linker; and n is an integer in the range of 1 to about 20.

[00187] In certain embodiments, Q is CH ₂ and the immunoconjugate has the structural formula of:

[00188] In certain embodiments, Q is CH ₂ and the immunoconjugate has the structural formula of:

[00189] In certain embodiments, R ¹ , R ² , R ³ , R ⁵ and R ^6x may be selected as disclosed herein in connection with (I)-(III ^b ).

[00190] In certain embodiments, R ^6x is H.

[00191] In certain embodiments, R ^6x is C _1-6 alkyl.

[00192] In certain embodiments, each of R ¹ and R ² is H.

[00193] In certain embodiments, each of R ¹ and R ² is F.

[00194] In certain embodiments, one of R ¹ and R ² is H and the other is F.

[00195] In certain embodiments, R ⁵ is NR ^5a R ^5b .

[00196] In certain embodiments, R ⁵ is N(CH ₃ ) ₂ .

[00197] In certain embodiments, R ⁵ is CH ₂ R ^5c .

[00198] In certain embodiments, R ^5c is F.

[00199] In certain embodiments, R ^5c is Cl.

[00200] In certain embodiments, R ^5c is O-C _1-3 alkyl.

[00201] In certain embodiments of formulae (IV)-(VI), n is an integer in the range of 1 to 20.

In certain embodiments, n is an integer in the range of 1 to 16. In certain embodiments, n is an integer in the range of 1 to 12. In certain embodiments, n is an integer in the range of 1 to 10. In certain embodiments, n is an integer in the range of 1 to 8. In certain embodiments, n is an integer in the range of 1 to 6. In certain embodiments, n is an integer in the range of 1 to 5. In certain embodiments, n is an integer in the range of 1 to about 4. In certain embodiments, n is an integer in the range of 1 to 3. In certain embodiments, n is 1 or 2. In certain embodiments, n is 1. [00202] All substitution groups, e.g., R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , found in formulae (IV)-(VI) can be selected as discussed in the section titled “Glucocorticoid Receptor Agonists” in connection with formulae (I)-(III ^b ) and is herein incorporated in its entirety, including each and all combinations of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ and the resulting compounds. The invention thus includes immunoconjugates corresponding to Ab-linked formulae (I)-(III ^b ). [00203] In addition to immunoconjugates wherein the antigen-binding moiety is an antibody or an antibody fragment, the invention additionally includes immunoconjugates wherein the antigen-binding moiety is a peptide and wherein the antigen-binding moiety is a small molecule ligand. (See, e.g., Zhuang et al. 2019 Eur. J. Med. Chem. 163, 883-895; Patel et al. 2021 New J. Chem. 45, 5291-5321.)

[00204] The invention also includes methods for synthesizing immunoconjugates, including intermediates or precursors thereof. The invention additionally includes a composition comprising an immunoconjugate, an intermediate or a precursor thereof.

Antigen Binding Moieties

[00205] To date, numerous unique antigens have been identified and may be potentially used in antibody-based therapy as a target. Several factors are generally considered when selecting an antigen. First, the target antigen should have high expression in the target tissue or cells and no or low expression in the healthy cell. Second, the target antigen should be displayed on the surface of the tissue or cells to be available to the circulated monoclonal antibody. In addition, the target antigen should possess internalization properties as it will facilitate the ADC to transport into the cell, which will in turn enhance the efficacy of the payload. Though some studies have demonstrated that non-intemalized ADC product directed against components of the tumor microenvironment can efficiently detach their drug in the extracellular space and arbitrate a potent therapeutic activity in some cases and that ADCs often induce a strong “bystander effect.” (Strohl WR 2018 Protein & Cell. 9(l):86-120; Damelin et al. 2015 Pharma. Res.

32(11):3494-507; Diamantis et al. 2016 British J. Cancer114(4):362-7; Tipton et al. 2015 Blood 125(12): 1901-9; Donaghy et al. 2016 mAbs. 8(4):659-71; Casi et al. 2015 Molecular Pharmaceutics 12(6): 1880-4.)

[00206] An antigen-binding moiety can be any moiety that selectively binds to a cell-surface marker found on a targeted cell type. In general, the antibody should preferably possess target specificity and deliver the payload to target tissue or cells and possess target binding affinity, i.e., a high binding affinity to the target tissue or cells. Additionally, the antibody should preferably possess good retention, low immunogenicity, low cross-reactivity, and appropriate linkage binding properties. (Peters et al. 2015 Bioscience Reports 35(4); Hughes B 2010 Nature Reviews Drug Discovery 9(9):665-7.)

[00207] In certain embodiments, Ab is an antibody. [00208] In certain embodiments, Ab is a monoclonal antibody.

[00209] In certain embodiments, Ab is a chimeric antibody.

[00210] In certain embodiments, Ab is a humanized antibody.

[00211] In certain embodiments, Ab is a bispecific antibody.

[00212] In certain embodiments, Ab is an antibody fragment.

[00213] In certain embodiments, Ab is a Fab fragment.

[00214] In certain embodiments, Ab is a peptide.

[00215] In certain embodiments, Ab is a small molecule ligand.

[00216] In some aspects, Ab is an antibody or antibody fragment (e.g. antigen binding fragment of an antibody).

[00217] In some aspects, Ab is an antibody or antibody fragment (e.g., antigen binding fragment) that specifically binds to a cell surface receptor protein or other cell surface molecules, a cell survival regulatory factor, a cell proliferation regulatory factor, a molecules associated with, known or suspected to contribute functionally to, tissue development or differentiation, a lymphokine, a cytokine, a molecule involved in cell cycle regulation, a molecule involved in vasculogenesis or a molecule associated with, known or suspected to contribute functionally to, angiogenesis.

[00218] Thus, antigen-binding moi eties useful in immunoconjugates of the invention include, but not limited to, antibodies against cell surface receptors and inflammation-associated antigens, which are well known in the art and can be prepared for use in generating antibodies using methods and information known in the art.

[00219] Non-limiting examples of antibodies or antigen binding fragments associated with inflammatory diseases and disorders include adalimumab, infliximab, certolizumab, afelimomab, nerelimomab, ozoralizumab, golimumab, and placulumab.

[00220] Antibodies and antibody fragments useful for the immunoconjugates of the invention include modified or engineered antibodies, such as an antibody modified to introduce a cysteine residue, or other reactive amino acid, including Pel, pyrrolysine, peptide tags, and non-natural amino acids, in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or antigen binding fragment for conjugation to a payload agent.

[00221] The location of the drug moiety may be designed, controlled and known. For example, cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or interm olecul ar disulfide linkages. (Junutula, et al. 2008 Nature Biotech. 26(8):925-932; Doman et al. 2009 Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541 B2; U.S. Pat. No. 7,723,485 B2; WO 2009/052249 A2.) The engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the drug moieties.

[00222] Additionally, the antibodies or antibody fragments can be modified to incorporate Pel or pyrrolysine or unnatural amino acids as sites for conjugation to a drug. Peptide tags for enzymatic conjugation methods can be introduced into an antibody. (Junutula et al. 2008 Nat. Biotechnol. 26:925-932; Ou et al. 2011 PNAS 108 (26), 10437-10442; Axup et al. 2012 Proc. Natl. Acad. Sci. USA, 109, 16101-16106; Liu et al. 2010 Annu. Rev. Biochem. 79, 413-444; Kim et al. 2013 Curr. Opin. Chem. Biol. 17, 412-419; Strop et al. 2013 Chem. Biol. 20(2): 161-7;

Rabuka 2010 Curr. Opin. Chem. Biol. 14(6):790-6; Rabuka et al. 2012 Nat. Protoc. 7(6): 1052- 67; WO 2015/095301 A2; WO 2013/184514 A2.)

[00223] Antibodies and antibody fragments can be readily produced by any methods known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. (See, e.g., Carvalho et al. 2016 “Production Processes for Monoclonal Antibodies”, DOI: 10.5772/64263 (https://www.intechopen.com/chapters/51512);

Monoclonal Antibody Production, Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council, NATIONAL ACADEMY PRESS Washington, DC 1999; Jakobovits 1998 Adv. Drug Del. Rev. 31:33-42; Marks et al. 1991 J. Mol. Biol. 222:581; Cole et al. 1985 Monoclonal Antibodies And Cancer Therapy 77-96; Teng et al. 1983 Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983 Immunology Today 4:72-79; Olsson et al. 1982 Meth. Enzymol. 92:3-16; U.S. Pat. No. 6,657,103 B2.)

Linkers and Linking Technologies

[00224] The payload agents disclosed herein are suitable for use as payloads in immunoconjugates. Glucocorticoid receptor agonists of the invention can be attached to a linker or directly to an antigen binding moiety. Linkers in ADCs are typically designed to achieve high stability in the circulation and, in the case of cleavable linkers, specific release of payload in the target tissue.

[00225] Suitable linkers and linking techniques for use in building an immunoconjugate are well known in the art and can be used in making the immunoconjugate conjugates of the invention. In general, a linker may be attached to the antigen binding moiety at any suitable available position on the antigen binding moiety, for examples, attached to an available amino nitrogen atom (e.g., a primary or secondary amine) or a hydroxylic oxygen atom, or to an available sulfhydryl, such as on a cysteine. The attachment of a linker to the glucocorticoid receptor agonists disclosed herein can be at the N-terminus or at the C-terminus of the payload agent.

[00226] Various linkers and linking strategies are known and can be employed in making immunoconjugates of the invention. (See, e.g., Kang et al. 2021 “Recent developments in chemical conjugation strategies targeting native amino acids in proteins and their applications in antibody-drug conjugates” Chemical Science Royal Soc. of Chem., DOI: 10.1039/dIsc02973h; Su et al. 2021 “Antibody-drug conjugates: Recent advances in linker chemistry” Acta Pharmaceutica Sinica B, https://doi.Org/10.1016/j.apsb.2021.03.042; Drago et al. 2021 Nature Reviews 18, 327-344; Mckertish et al. 2021 Biomedicines 9, 872; Bargh et al. 2019 “Cleavable linkers in antibody-drug conjugates” Chem. Soc. Rev. 48, 4361, DOI: 10.1039/c8cs00676h; Lash 2011 “Antibody -Drug Conjugates: the Next Generation of Moving Parts” Start-Up, Dec. 2011, 1-6; WO 2021/055865 Al; WO 2016/192527 Al ; WO 2015/095301 A2; WO 2011/097627 Al, WO 2004/010957 Al, U.S. Pub. No. 20060074008 A2, U.S. Pub. No. 20050238649 A2, and U.S. Pub. No. 20060024317 A2.)

[00227] A linker may be classified as either cleavable or non-cleavable. In the case of ADCs with noncleavable linkers, the release is typically via internalization of the ADC followed by degradation of the antibody in the lysosome, resulting in the release of the payload still attached via the linker to an antibody amino acid residue. Examples of noncleavable linker include maleimidoca-proyl (MC) and 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (MCC) linkers. Examples of cleavable linkers include Val-Cit, N-Succinimidyl-4-(2-pyridyldithio) butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP) and hydrazide.

[00228] For the immunoconjugates of comprising a cleavable linker, the linker is substantially stable in vivo until the immunoconjugate binds to or enters a cell, at which point either intracellular enzymes or intracellular chemical conditions (pH, reduction capacity) cleave the linker to free the payload.

[00229] Cleavable linkers may further be classified based on the cleavage mechanism into chemically cleavable linkers (such as acid-cleavable linkers, reducible disulfide linkers and exogeneous stimuli triggered linkers) and enzyme cleavable linkers (such as dipeptide Val-Cit - containing linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers). Acid cleavable linkers (a.k.a. pH-sensitive linkers) are designed to exploit the acidity of the endosomes (pH 5.5- 6.2) and lysosomes (pH 4.5-5.0), while maintaining stability in circulation at pH 7.4. An example of an acid-cleavable linkers is an acid-sensitive N-acyl hydrazine linkage that, upon acid catalysis, hydrolyses to a ketone and a hydrazide-payload. Acid cleavable linkers containing other functional groups have also been reported, such as a carbonate linker. Glycosidase- cleavable linkers include β-Glucuronidase-cleavable linkers, β-Galactosidase-cleavable linkers, phosphatase-cleavable linkers. (See, e.g., Bargh et al. 2019 “Cleavable linkers in antibody-drug conjugates” Chem. Soc. Rev. 48, 4361, DOI: 10.1039/c8cs00676h; Ducry, et al. 2010 Bioconiuqate Chem., vol. 21 , 5-13; Jeffrey et al. 2006 Bioconjugate Chem. 17, 831-840; Burke et al. 2009 Bioconjugate Chem. 20, 1242-1250; Kolodych et al. 2017 J. Med. Chem. 142, 376- 382; Kern et al. 2016 Bioconjugate Chem. 27, 2081-2088; Stenton et al. 2018 Chem. Sci. 9, 4185—4189; Pillow et al. 2017 Mol. Cancer Ther. 16, 871-878; Dubowchik et al. 1998 Bioorg. Med. Chem. Lett. 8, 3341-3346; Dubowchik et al. 1998 Bioorg. Med. Chem. Lett. 8, 3347-3352; WO 2021/055865 Al; WO 2016/192527 Al; WO 2015/095301 A2; US 2021/0138077 Al; WO 2013/173393 Al; WO 2011/097627 Al.)

Linker-antibody and Linker-payload Attachments

[00230] Various attachment strategies have been developed over the years including site- specific conjugation technologies, antibody engineering and chemical modifications.

[00231] Major attachment approaches include mal eimide attachment (e.g., N-alkyl mal eimide,

N-phenyl maleimide), bis(vinylsulfonyl)piperazine attachment, N-methyl-N- phenylvinylsulfonamide attachment, and Pt(II)-based attachment. (See, e.g., Su et al. 2021 “Antibody-drug conjugates: Recent advances in linker chemistry” Acta Pharmaceutica Sinica B, https://doi.Org/10.1016/i.apsb.2021.03.042; Mckertish et al. 2021 Biomedicines 9, 872; Patterson et al. 2015 Bioconjug. Chem. 26:2243e8; Lyu et al. 2018 ACS Chem. Biol. 13:958e64; Zhou 2017 Biomedicines 5:64; Christie et al. 2017 Antibodies (Basel) 6:20; Sun et al. 2019 Org. Biomol. Chem. 17: 2005el2; Huang et al. 2018 Org. Lett. 20: 6526e9; Sijbrandi et al. 2017 Cancer Res. 77: 257e67; Merkul et al. 2020 Angew Chem. hit. Ed. Engl. 60:3008el5; Merkul et al. 2019 Expert Opin. DrugDeliv. 16:783e93; WO 2015/095301 A2; US 2021/0138077 Al; WO 2013/173393 Al; WO 2016/192527 Al; WO 2021/055865 Al.)

[00232] Various linker-payload attachment strategies have been reported, such as carbamate attachment and carbonate attachment. (See, e.g., Wahby et al. 2020 Clin. Cancer Res. Available from: https://doi.l0.1158/1078-0432.CCR-20-3119; Perini et al. 2013 Biol. Ther. 3:15e23; Burke et al. 2016 Mol. Cancer Ther. 15:938e45; WO 2015/095301 A2; US 2021/0138077 Al; WO 2013/173393 Al; WO 2016/192527 Al; WO 2021/055865 Al.)

[00233] Non-limiting examples of attachment strategies and reactive groups are provided in Table 3. (See, e.g., WO 2015/095301 A2; US Pat. No. 9,988,420 B2.)

Table 3. Exemplary Reactive Groups and Moieties

Pharmaceutical Compo sitions and. Methods of Use

Pharmaceutical Compositions

[00234] In another aspect, the invention generally relates to a composition comprising a compound disclosed herein, such as according to any one of formulae (I)-(III ^b ) and in Table 1, or a pharmaceutically acceptable form thereof, and optionally a pharmaceutically acceptable excipient, carrier or diluent.

[00235] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an immunoconjugate disclosed herein, such as according to any one of formulae (IV)-(V), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

[00236] The invention thus provides a pharmaceutical preparation comprising a therapeutically effective amount of a compound or immunoconjugate according to the invention. [00237] Examples of excipients that may be useful include, but not limited to, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, starches, celluloses and gums. In a preferred embodiment, the pharmaceutical composition of the invention is formulated in a pharmaceutical form for administration as a solid (for example tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide liquid forms, etc.), liquid (for example solutions, suspensions, emulsions, elixirs, lotions, unguents, etc.) or semi-solid (gels, ointments, creams and similar). The pharmaceutical compositions of the invention can be administered by any route, including, without limitation, oral, intravenous, intramuscular, intraarterial, intramedullary, intratecal, intraventricular, transdermic, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal route. A revision of the different forms of administration of active principles, the excipients to be used and their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 20 ^th edition, Williams & Wilkins PA, USA (2000) Examples of pharmaceutically acceptable vehicles are known in the state of the technique and include saline solutions buffered with phosphate, water, emulsions, such as oil/water emulsions, different types of humidifying agents, sterile solutions, etc. The compositions comprising said vehicles can be formulated by conventional procedures known in the state of the technique. Preservatives, stabilizers, dyes and even flavoring agents, antioxidants and/or suspending agents can be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives.

[00238] The invention also contemplates a kit comprising at least an immunoconjugate disclosed herein and a syringe and/or vial or ampoule in which the immunoconjugate and/or pharmaceutical composition is disposed.

Methods of Use [00239] In yet another aspect, the invention generally relates to a method for treating or reducing a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate disclosed herein.

[00240] In certain embodiments, the disease or condition is an inflammatory disorder.

[00241] In certain embodiments, the method further comprises administering one or more of anti-inflammatory agents to the subject.

[00242] In yet another aspect, the invention generally relates to use of an immunoconjugate disclosed herein for the manufacture of a medicament.

[00243] In certain embodiments, an immunoconjugate disclosed herein is used for treating a disease or condition, wherein the disease or condition is an inflammatory disorder.

[00244] In yet another aspect, the invention generally relates to use of an immunoconjugate disclosed herein for use in treating inflammatory disorders.

[00245] Exemplary inflammatory disease or disorder include RA, ERA, ankylosing spondylitis, psoriatic arthritis, atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplant rejection, and vasculitis.

[00246] In certain embodiments, the inflammatory disorder is an autoimmune disease.

[00247] In certain embodiments, the inflammatory disorder is an inflammatory disorder of the joints.

[00248] In certain embodiments, the inflammatory disorder is an inflammatory disorder of the cardiovascular system.

[00249] In certain embodiments, the inflammatory disorder is an inflammatory lung and airway.

[00250] In certain embodiments, the inflammatory disorder is an inflammatory intestine.

[00251] In certain embodiments, the inflammatory disorder is dermatitis.

[00252] In certain embodiments, the inflammatory disorder is acne vulgaris.

[00253] In certain embodiments, the inflammatory disorder is psoriasis.

[00254] In certain embodiments, the inflammatory disorder is rheumatoid arthritis.

[00255] In certain embodiments, the inflammatory disorder is cardiovascular disease.

[00256] In certain embodiments, the inflammatory disorder is atherosclerosis.

[00257] In certain embodiments, the inflammatory disorder is Type I diabetes. [00258] In certain embodiments, the inflammatory disorder is Lupus.

[00259] In one embodiment the inflammatory disorder is psoriatic arthritis.

[00260] In one embodiment the inflammatory disorder is an inflammatory bowel disease.

[00261] In certain embodiments, the inflammatory disorder is asthma.

[00262] In certain embodiments, the inflammatory disorder is cystic fibrosis.

[00263] Immunoconjugates may generally be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subcutaneous route, or by the oral route. Immunoconjugates are typically administered intravenously into the blood stream of a subject in order to avoid gastric acids or proteolytic enzymes degradation of the antibody. In some embodiments, the composition comprising the immunoconjugates disclosed herein will be administered several times, in a sequential manner.

Combination Therapies

[00264] In yet another aspect, the invention generally relates to a combination comprising a therapeutically effective amount of a compound or an immunoconjugate disclosed herein, and one or more therapeutically active co-agent(s) and/or adjuvant(s).

[00265] Co-agents include, but are not limited to, anti-inflammatory agents, antimicrobial agents, anti-angiogenesis agents, immunosuppressants, antibodies, steroids, or combinations thereof. Anti-inflammatory agents include, but are not limited to, non-steroidal anti- inflammatory drugs (NSAIDs), non-specific and COX-2 specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptors antagonists, immunosuppressants and methotrexate.

[00266] Non-limiting examples of anti-inflammatory agents include methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluromethalone acetate, fluromethalone alcohol, lotoprendol etabonate, medrysone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone acetate, lodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flubiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocorticoids, diclofenac, and any combination thereof. In one embodiment, an active compound or its salt or composition as described herein is combined with one or more non- steroidal anti-inflammatory drugs (NSAIDs) selected from naproxen sodium (Anaprox), celecoxib (Celebrex), sulindac (Clinoril), oxaprozin (Daypro), salsalate (Disalcid), diflunisal (Dolobid), piroxicam (Feldene), indomethacin (Indocin), etodolac (Lodine), meloxicam (Mobic), naproxen (Naprosyn), nabumetone (Relafen), ketorolac tromethamine (Toradol), naproxen/esomeprazole (Vimovo), and diclofenac (Voltaren), and combinations thereof.

[00267] Non-limiting examples of other co-agents include amikacin, anecortane acetate, anthracenedione, anthracycline, an azole, amphotericin B, bevacizumab, camptothecin, cefuroxime, chloramphenicol, chlorhexidine, chlorhexidine digluconate, clortrimazole, a clotrimazole cephalosporin, corticosteroids, dexamethasone, desamethazone, econazole, eftazidime, epipodophyllotoxin, fluconazole, flucytosine, fluoropyrimidines, fluoroquinolines, gatifloxacin, glycopeptides, imidazoles, itraconazole, ivermectin, ketoconazole, levofloxacin, macrolides, miconazole, miconazole nitrate, moxifloxacin, natamycin, neomycin, nystatin, ofloxacin, polyhexamethylene biguanide, prednisolone, prednisolone acetate, pegaptanib, platinum analogues, polymicin B, propamidine isethionate, pyrimidine nucleoside, ranibizumab, squalamine lactate, sulfonamides, triamcinolone, triamcinolone acetonide, triazoles, vancomycin, anti-vascular endothelial growth factor (VEGF) agents, VEGF antibodies, VEGF antibody fragments, vinca alkaloid, timolol, betaxolol, travoprost, latanoprost, bimatoprost, brimonidine, dorzolamide, acetazolamide, pilocarpine, ciprofloxacin, azithromycin, gentamycin, tobramycin, cefazolin, voriconazole, gancyclovir, cidofovir, foscamet, diclofenac, nepafenac, ketorolac, ibuprofen, indomethacin, fluoromethalone, rimexolone, anecortave, cyclosporine, methotrexate, tacrolimus and combinations thereof.

[00268] Isotopically-labeled compounds are also within the scope of the present disclosure. As used herein, an "isotopically-labeled compound" refers to a presently disclosed compound including pharmaceutical salts and prodrugs thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ C1, respectively.

[00269] By isotopically-labeling the presently disclosed compounds, the compounds may be useful in drug and/or substrate tissue distribution assays. Tritiated ( ³ H) and carbon-14 ( ¹⁴ C) labeled compounds are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium ( ² H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds presently disclosed, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art.

[00270] Further, substitution of normally abundant hydrogen ( ¹ H) with heavier isotopes such as deuterium can afford certain therapeutic advantages, e.g., resulting from improved absorption, distribution, metabolism and/or excretion (ADME) properties, creating drugs with improved efficacy, safety, and/or tolerability. Benefits may also be obtained from replacement of normally abundant ¹² C with ¹³ C. (See, WO 2007/005643, WO 2007/005644, WO 2007/016361, and WO 2007/016431.)

[00271] Thus, isotope derivative compounds having one or more hydrogen atoms (e.g., 1 , 2, 4, 5, 6, 7, 8, 9, 10, etc.) replaced with deuterium atoms are contemplated in the presented invention. In certain embodiments, isotope derivative compounds of the invention have one hydrogen atom replaced with a deuterium atom.

[00272] Stereoisomers (e.g., cis and trans isomers) and all optical isomers of a presently disclosed compound (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers are within the scope of the present disclosure.

[00273] Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% (“substantially pure”), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 99% pure.

[00274] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the present invention include, for example, hydrates. [00275] The following examples are meant to be illustrative of the practice of the invention and not limiting in any way.

Examples

Synthesis

[00276] To a solution of (6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-( 2- hydroxy acetyl)-6a, 8 a, 10, 10-tetramethyl- 1 ,2, 6a, 6b, 7, 8, 8a, 8b, 11 a, 12, 12a, 12b-dodecahydro-4H- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one (300 mg, 0.69 mmol), methanesulfonic anhydride (360 mg, 2.07 mmol) in DCM stirred under nitrogen at 0°C was added triethylamine (276 mg, 2.76 mmol) dropwise. The reaction mixture was stirred at 25°C for 0.5h, then concentrated directly. The residue was purified by FCC (eluted with PE/EA=1 : 1) to give INT-1 (180 mg, 48.5%) as a white solid. LCMS (ESI): m/z 513.2 [M+H] ⁺ .

[00277] To a solution of 2-((6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-6b-fluoro-7-hydroxy-

6a, 8a, 10, 10-tetramethyl-4-oxo- 1 ,2, 4, 6a, 6b, 7, 8, 8a, 11 a, 12, 12a, 12b-dodecahydro-8bH- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-8b-yl)-2-oxoethyl methanesulfonate INT-1 (180 mg, 0.35 mmol), LiCl (110 mg, 2.60 mmol) in DMF (4 mL) was stirred under nitrogen at 60°C for 2 h. The reaction mixture was diluted with EtOAC (20 mL), washed with water (3 x 20 mL), dried MgSO4, filtered and concentrated in vacuo. The residue was purified by FCC (eluted with PE/EA=l/3) to give INT-2 (120 mg, 75.2%) as a white solid. LCMS (ESI): m/z 453.2 [M+H] ⁺ . INT-2

[00278] To a solution of ((6aS,6bR,7S,8aS,8bS,10R,l laR, 12aS, 12bS)-10-(3-aminophenyl)- 8b-(2-chloroacetyl)-6b-fluoro-7-hydroxy-6a,8a-dimethyl- 1 ,2, 6a, 6b, 7,8, 8 a, 8b, 11 a, 12, 12a, 12b- dodecahydro-4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4- one (100 mg, 0.22 mmol), tert- butyl N-(3-formylphenyl)carbamate (73 mg, 0.33 mmol) and l-Butyl-3-MethyliMidazoliuM Hexafluoro-Phosphate (627 mg, 2.21 mmol) in DCM (5 mL) stirred at 25°C was added a solution of HC1O4 (221 mg, 2.21 mmol) dropwise. The reaction mixture was stirred at 25°C for 10 min, It was purified via FCC first, then purified by prep-HPLC (MeCN/H ₂ O, 0.05% TFA) and give compound 1 (3.8 mg, 3.4%) and compound 2 (32.5 mg, 28.6%). LCMS (ESI): m/z 516.1 [M+H] ⁺ . 1: ¹ HNMR (400 MHz, DMSO) δ 7.25 (d, J= 10.1 Hz, 1H), 6.94 (t, J= 7.8 Hz, 1H), 6.52 - 6.33 (m, 2H), 6.20 (dd, J= 10.1, 1.7 Hz, 1H), 5.98 (d, J= 11.6 Hz, 2H), 5.22 (d, J= 6.8 Hz, 1H), 4.31 (s, 2H), 4.13 (d, J= 9.4 Hz, 1H), 2.29 (s, 2H), 2.05-2.95 (m, 2H), 1.85-1.78 (m, 2H), 1.68-1 ,62(m, 2H), 1.44 (s, 3H), 1.11 (t, J = 7.2 Hz, 3H), 0.83 (s, 3H). 2: ¹ H NMR (400 MHz, DMSO) δ 7.31 (d, J= 10.1 Hz, 1H), 7.20 (t, J= 7.7 Hz, 1H), 6.96 - 6.79 (m, 3H), 6.24 (dd, J= 10.1, 1.7 Hz, 1H), 6.04 (s, 1H), 5.49 (s, 1H), 5.00 - 4.89 (m, 2H), 4.55 (d, J= 17.3 Hz, 1H), 4.19 (d, J= 9.5 Hz, 1H), 2.69-2.63(m, 1H), 2.38 (s, 1H), 2.15-2.07(m, 2H), 1.91-1.82(m, 1H), 1.78-1.63 (m, 3H), 1.50 (s, 3H), 1.41 (d, J= 7.5 Hz, 1H), 1.28 - 1.12 (m, 1H), 0.87 (s, 3H).

[00279] To a solution of (6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a, 10, 1 O-tetramethyl-1 ,2, 6a, 6b, 7, 8, 8a, 8b, 11 a, 12, 12a, 12b-dodecahydro-4H- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one (200 mg, 0.46 mmol), Triethylamine trihydrofluoride (148 mg, 0.90 mmol) and PBSF (417 mg, 1.38mmol) in ACN (10 mL) was added triethylamine (278 mg, 1.05 mmol) at 25 °C. The resulting mixture was warmed up to 50 °C and stirred for 17 h. It was concentrated and the residue was purified by FCC (eluted with CH ₂ Cl ₂ /MeOH= 10:1) to give INT-3 (150 mg, 74.2%) as a white solid. LCMS (ESI): m/z 437.2 [M+H] ⁺ .

[00280] To a solution of (6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-6b-fluoro-8b-(2- fluoroacetyl)-7-hydroxy-6a,8a,10, 10-tetramethyl-l,2,6a,6b,7,8,8a,8b, 1 la,12,12a,12b- dodecahydro-4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4- one INT-3 (20 mg, 0.045 mmol) in dry DCM (10 ml) was added tert-butyl N-(3-formylphenyl)carbamate(15 mg, 0.07 mmol), followed by l-butyl-3-methylimidazolium hexaflurophosphate (130 mg, 0.45 mmol) and HCIO ₄ (46 mg, 0.45 mmol). The mixture was stirred at 25 °C for 2 h. The solvent was removed off under reduced pressure. The residue was purified by FCC (C-l 8, H ₂ O: ACN = 95:5 ~ 30:70, v/v) to afford the product (20 mg). It was separated by chiral HPLC (40% IP A (NH ₄ OH 0.2%)) to give compound 3 (11 mg, 48.9%) as a white solid. LCMS (ESI): m/z 500.1 [M+H] ⁺ . ¹ H NMR (400 MHz, CD3CN) δ 7.26 (d, J= 10.2 Hz, 1H), 7.11 (t, J= 7.7 Hz, 1H), 6.74-6.71 (m, 2H), 6.71 - 6.66 (m, 1H), 6.25 (dd, J= 10.1, 1.9 Hz, 1H), 6.06 (d, J= 1.6 Hz, 1H), 5.48 (d, J= 17.4 Hz, 0.5H), 5.46(s, 1H), 5.37 (d, J= 17.4 Hz, 0.5H), 5.22 (d, J= 17.4 Hz, 0.5H), 5.10 (d, J= VIA Hz, 0.5H), 5.01 (d, J= 5.0 Hz, 1H), 4.38-4.31 (m, 1H), 2.75 - 2.51 (m, 2H), 2.42-2.35 (m, 1H), 2.32 - 2.19 (m, 2H), 1.93- 1.88 (m, 1H), 1.84 - 1.67 (m, 3H), 1.58 - 1.51 (m, 4H), 0.98 (s, 3H).

[00281] To a solution of (2S,6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-2,6b-difluoro-7-hydroxy- 8b-(2-hydroxyacetyl) -6a,8a,10,10-tetramethyl-l,2,6a,6b,7,8,8a,8b,l la,12,12a,12b-dodecahydro- 4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one (1000 mg, 1.21 mmol) and K ₂ CO ₃ (611 mg, 4.42 mmol) in MeOH (40 mL) was added H ₂ O2 (752 mg, 22.1 mmol) . The mixture was stirred at 25 °C for 16 h. MeOH was removed off under reduced pressure, the mixture was acidified with 2 N HC1 to pH 1 and the resulting precipitate was collected by filtration, washed with water and dried to afford INT-4 as a white solid (900 mg, 92.1%). LCMS (ESI): m/z 439.1 (M+H) ⁺ .

[00282] A solution of (2S,6aS,6bR,7S,8aS,8bS,llaR,12aS,12bS)-2,6b-difluoro-7-hydro xy-

6a, 8a, 10, 10- tetramethyl -4-oxo- 1,2, 4, 6a, 6b, 7, 8, 8a, 1 la,12,12a,12b-dodecahydro-8bH- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxole-8b-carboxylic acid INT-4 (100 mg, 0.23 mmol), HOB _t (40 mg, 0.30 mmol), EDCI (57 mg, 0.30 mmol) and 2,6-lutidine (73 mg, 0.68 mmol) in dry DMF (3 mL) was stirred 10 min at 25 °C, then dimethylamine hydrochloride (37 mg, 0.46 mmol) was added. The reaction mixture was stirred at 40 °C for 24 h. The mixture was diluted with H ₂ O (100 mL), extracted with EtOAc (30 mL*3). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated to afford INT-5 as a white solid (90 mg, 83.2%). LCMS (ESI): m/z 466.3 (M+H) ⁺ .

[00283] To a solution of (2S,6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-2,6b-difluoro-7-hydroxy-

N,N,6a,8a,10,10- hexamethyl-4-oxo-l,2,4,6a,6b,7,8,8a,l la,12,12a,12b-dodecahydro-8bH- naphtho[2', 1':4,5]indeno[l,2-d][l,3]dioxole-8b-carboxamide INT-5 (90 mg, 0.19 mmol) in dry DCM (15 ml) was added tert-butyl N-(3-formylphenyl)carbamate (128 mg, 0.58 mmol), followed by l-butyl-3-methylimidazolium hexaflurophosphate (605 mg, 1.94 mmol) and HCIO ₄ (194 mg, 1.94 mmol). The mixture was stirred at 25 °C for 2 h. Triethylamine was added to neutralize the acid and the solvent was removed off under reduced pressure. ACN was added until the mixture all dissolved, purified by prep-HPLC (ACN-H ₂ O (0.1%TFA), 30%-50%) to give compound 4 (33.5 mg, 21.5%) and 5 (32.5 mg, 20.5%) as white solid. 4: LCMS (ESI): m/z 529.2 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7 . 4 6 -.55 (m, 3H), 7.36 - 7.30 (m, 2H), 6.28 - 6.34 (m, 2H), 5.60-5.66 (m, 0.5H), 5.56 (s, 1H), 5.49-5.54 (m, 0.5H), 5.39(t, J= 4.0, 1H), 4.29 (d, J= 8.6 Hz, 1H), 3.29 (s, 3H), 3.02 (s, 3H), 2.68-2.75 (m, 1H), 2.25 - 2.41 (m, 3H), 1.81 (d, J = 10.6 Hz, 3H), 1.61-1.68 (m, 1H), 1.59 (s, 3H), 1.10 (s, 3H). 5: LCMS (ESI): m/z 529.3 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.50 - 7.55 (m, 1H), 7.45 (d, J= 7.8 Hz, 1H), 7.39 - 7.29 (m, 3H), 6.29 - 6.38 (m, 3H), 5.69 (d, J= 6.4 Hz, 1H), 5.61-5.65 (m, 0.5H), 5.49-5.53 (m,

O.5H), 4.32 (d, J= 9.8 Hz, 1H), 3.07 (s, 3H), 2.66 (s, 3H), 2.36 - 2.44 (m, 1H), 2.27-2.66 (m, 2H), 1.66 - 1.93 (m, 5H), 1.59 (s, 3H), 1.07 (s, 3H).

[00284] To a stirred solution (8S,9R,10S,l lS,13S,14S,16R,17R)-9-fluoro-l 1,17-dihydroxy- 17-(2-hydroxyacetyl)- 10, 13, 16-trimethyl-6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one (1000 mg, 2.55 mmol) and triethylamine(515 mg, 5.10mmol) in DCM (30 mL) was added methanesulfonic anhydride (489 mg, 2.80 mmol). The mixture was stirred at room temperature for 4h. It was concentrated under vacuum. The residue was purified by Flash Chromatography (eluted with CH ₂ Cl ₂ /MeOH=20/l) to give INT-6 (800 mg, 68.2%) as a white solid. LCMS (ESI): m/z 471.1 [M+H] ⁺ .

[00285] To a solution of2-((8S,9R,10S,l lS,13S,14S,16R,17R)-9-fluoro-l l,17-dihydroxy- 10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethyl methanesulfonate INT-6 (300 mg, 0.63 mmol) in DMF (5 mL) was added LiCl (81 mg, 1.90 mmol). The resulting mixture was stirred at 80 °C for 17 h. It was diluted with DCM (50 mL), washed with H ₂ O, brine, dried over Na ₂ SO ₄ filtrated and concentrated. This resulted in INT-7 290 mg as a white solid which was used in next step without further purification., LCMS (ESI): m/z 411.1 [M+H] ⁺ .

[00286] To a mixture of 4-(dimethylamino)pyridine (594 mg, 4.80 mmol) and

(8 S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)- 17-(2-chloroacetyl)-9-fluoro- 11,17-dihydroxy- 10,13,16- trimethyl-6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro-3H-cyclopenta[a]phenanthren-3 -one INT-7 (200 mg, 0.49 mmol) in DCM (20 mL) stirred at 0 °C was added 3 -nitrobenzoyl chloride (548 mg, 2.94 mmol) portion-wise. After the addition, the resulting mixture was stirred at room temperature for 17 h. The solvent was removed off under reduced pressure and the residue was purified by FCC (eluted with CH ₂ Cl ₂ /MeOH= 30/1) to give INT-8 (70 mg, 11.2%) as an off- white solid. LCMS (ESI): m/z 560.1 [M+H]+.

[00287] To a solution of ((8S,9R,10S,l lS,13S,14S,16R,17R)-17-(2-chloroacetyl)-9-fluoro- l l-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren- 17-yl 3 -nitrobenzoate INT-8 (70 mg, 0.13 mmol) in EtOH (1 mL) and AcOH (1 mL) was added Zn dust (48 mg, 0.75 mmol). The mixture was stirred for 4 h at 25 °C. The solid was filtered off and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (ACN-H2O (0.1% TFA), 50%-60%) to give compound 6 (2.7 mg, 4.2 %). LCMS (ESI): m/z 559.0 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.61 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 1.9 Hz, 1H), 7.42-7.34 (m, 2H), 7.25 (dd, J = 8.0, 1.4 Hz, 1H), 6.28 (dd, J = 10.1, 1.9 Hz, 1H), 6.08 (s, 1H), 4.33-4.26 (m, 3H), 2.72 (td, J = 14.0, 5.7 Hz, 1H), 2.61 - 2.27 (m, 5H), 1.96-1.88 (m, 2H), 1.76 (d, J = 14.0 Hz, 1H), 1.57 (s, 3H), 1.39 - 1.30 (m, 1H), 1.11 (d, J = 8.0 Hz, 3H), 0.87 (d, J = 7.1 Hz, 3H).

[00288] To a solution of(6S,8S,9R,10S,HS,13S,14S,16R,17R)-6,9-difluoro-l l,17- dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10 ,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-3-one (1000 mg, 2.436 mmol) and K ₂ CO ₃ (673 mg, 4.873 mmol) in MeOH (40 mL) was added H ₂ O ₂ (829 mg, 24.36 mmol) . The mixture was stirred at 25 °C for 36 h. MeOH was removed off under reduced pressure, the mixture was acidified with 2 N HCl to pH 1.0 and the resulting precipitate was filtered, washed with water, and dried in air to afford INT-9 as a white solid (900 mg, 92%). LCMS (ESI): m/z 397.2 (M+H) ⁺ .

[00289] To a suspension of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-ll,17- dihy droxy- 10,13,16-trimethyl-3 -oxo-6, 7, 8, 9, 10, 11 , 12, 13 , 14, 15, 16, 17 -dodecahy dro-3H- cyclopenta[a]phenanthrene-17-carboxylic acid INT-9 (600 mg, 1.5135 mmol) and furan-2- carbonyl chloride (593 mg, 4.5405 mmol) in acetone (10 mL) was added triethylamine (460 mg, 4.5405 mmol). The reaction was stirred 2 h at 25 °C, then diethylamine (664 mg, 9.081 mmol) was added to the solution. The reaction mixture was stirred at 25 °C for 4 h. The mixture was acidified with 2 N HCl to pH 2.0 and the resulting precipitate was filtered, washed with water, and dried in air to afford INT-10 as a white solid (600 mg, 80%). LCMS (ESI): m/z 491.1 (M+H) ⁺ .

[00290] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-17-((furan-2- carbonyl)oxy)- 11 -hydroxy- 10, 13 , 16-trimethyl-3 -oxo-6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-10 (130 mg, 0.265 mmol) in dry DCM (5 mL) was added SOCl ₂ (2 mL) under N ₂ . The reaction mixture was stirred at 50 °C for 2 h. Removed off DCM and SOCl ₂ under vacuum. l-(2-aminoethoxy)-4-nitrobenzene hydrochloride (125 mg, 0.572 mmol) and DIEA (111 mg, 0.858 mmol) in DCM (3 mL) was added to the mixture. The reaction mixture was stirred at 25 °C for 2 h. Then DCM was removed off under reduced pressure and the residue was purified by Flash Chromatography (DCM: EtOAc = 2:1, v/v) to afford INT-11 as a yellow solid (45 mg, 26%). LCMS (ESI): m/z 655.0 (M+H) ⁺ .

[00291] To a solution of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-l 1-hydroxy-

10, 13 , 16-trimethyl- 17-((2-(4-nitrophenoxy)ethyl)carbamoyl)-3 -oxo-

6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17 -dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl furan-2- carboxylate INT-11 (30 mg, 0.0229 mmol) in EtOH (5 ml) and AcOH (3 mL) was added Zn dust (15 mg, 0.229 mmol). The mixture was stirred for 4 h at 25 °C. The Zn dust was filtered, and the mixture solution was purified by prep-HPLC (ACN-H2O (0.1%TFA), 50%-60%) to give compound 7 (10 mg, 34%) as a white solid. LCMS (ESI): m/z 625.2 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.83 (d, J= 7.7 Hz, 1H), 7.73 (s, IH), 7.53 (t, J= 7.9 Hz, IH), 7.32-7.38 (m, 2H), 6.36 - 6.28 (m, 2H), 5.62-5.46 (m, IH), 4.46 (q, J= 16.5 Hz, 2H), 4.29 (d, J= 10.3 Hz, IH), 2.65 - 2.51 (m, IH), 2.43 - 2.14 (m, 6H), 1.91-2.04 (m, 2H), 1.61-1.74 (m, IH), 1.57 (s, 3H), 1.29-1.35 (m, IH), 1.15 (s, 3H), 1.09 (t, J= 7.6 Hz, 3H), 0.98 (d, J= 7.1 Hz, 3H).

[00292] A mixture of (6S,8S,9R, IOS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro-l 1, 17-dihydroxy- 17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one INT-9 (500 mg, 1.26 mmol), 3 -nitrobenzoyl chloride (702 mg, 3.78 mmol) and triethylamine (383 mg, 3.78 mmol) in acetone(5 mL) was stirred 2 h at 25 °C, then it was quenched with diethylamine (461 mg, 6.31 mmol). The reaction mixture was stirred at 25°C for Ih. The solvent was removed off under reduced pressure. The residue was purified by Flash Chromatography (ACN-H2O, 30%-40%) to afford INT-12 as a white solid (500 mg, 65.2%). LCMS (ESI): m/z 546.0 (M+H) ⁺ .

[00293] To solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy- 10, 13, 16-trimethyl-l 7-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9, 10, 11,12, 13,14, 15,16, 17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (200 mg, 0.37 mmol) in DCM (6 mL) was added SOCl ₂ (3 mL) dropwise. The resulting mixture was stirred at 40 °C for 2h under N ₂ . The volatile phase was removed off under reduced pressure. The residue was dissolved in THE (4 mL), Dimethylamine (2M in THF, 2 mL) was added dropwise to the mixture, which was stirred at 25°C for 16 h. It was concentrated under reduced pressure and the residue was purified by Prep-TLC (DCM: EtOAc = 2:1, v/v) to afford INT-13 as a white solid (30 mg, 13.1%). LCMS (ESI): m/z 572.8 (M+H) ⁺ .

[00294] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-17-(dimethylcarbamoyl)-6,9- difluoro-l l-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro- 3H-cyclopenta[a]phenanthren- 17-yl 3 -nitrobenzoate INT-13 (15 mg, 0.026 mmol) in EtOH (1 ml) and AcOH (1 mL) was added Zn dust (17 mg, 0.26 mmol). The mixture was stirred at 25 °C for 4h. The solid was filtrated off and the filtrate was concentrated. The residue was purified by prep-HPLC (ACN-H2O (0.1%TFA), 50%-60%) to give compound 8 (2.3 mg, 16.2%) as a white solid. LCMS (ESI): m/z 543.3 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.70 (d, J - 7.9 Hz, 1H), 7.64 (s, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.35 (dd, J = 10.1, 4.0 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 6.38 - 6.31 (m, 2H), 5.62-5.66 (m, 0.5H), 5.50-5.54 (m, 0.5H), 4.37 (d, J = 8.6 Hz, 1H), 2.94 (s, 6H), 2.73 - 2.33 (m, 5H), 1.81-1.97 (m, 2H), 1.65-1.72 (m, 1H), 1.60 (s, 3H), 1.32 - 1.26 (m, 1H), 1.19 (s, 3H), 0.91 (d, J = 7.2 Hz, 3H).

[00295] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy-

10,13,16-trimethyl-17-((4-nitrobenzoyl)oxy)-3-oxo-6,7,8,9 ,10,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-14 (100 mg, 0.18 mmol) in DCM (6 mL) and SOCl ₂ (3 mL) was stirred at 40 °C for 2 h. The volatile phase was removed off under reduced pressure. The residue was dissolved in THE (2 mL), then dimethylamine (2M in THF, 2 mL) was added dropwise to the mixture, which was stirred at 25 °C for 16 h. It was concentrated under reduced pressure and the residue was purified by Prep-TLC (DCM/EtOAc = 2/1) to afford INT-15 as a white solid (20 mg, 19.2%). LCMS (ESI): m/z 613.1 (M+ACN) ⁺ .

[00296] To a solution of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-17-(dimethylcarbamoyl)-6, 9- difluoro- 11 -hydroxy- 10, 13 , 16-trimethyl-3 -oxo-6,7,8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro- 3H-cyclopenta[a]phenanthren- 17-yl 4-nitrobenzoate INT-15 (20 mg, 0.035 mmol) in EtOH (1 mL) and AcOH (1 mL) was added Zn dust (23 mg, 0.35 mmol). The mixture was stirred for 4 h at 25 °C. The solid was filtered off and the filtrate was concentrated. The residue was purified by prep-HPLC (ACN-H2O (0.1%TFA), 50%-60%) to give compound 9 (2.3 mg, 16.2%) as a white solid. LCMS (ESI): m/z 543.3 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.72 (d, J= 8.8 Hz, 2H), 7.36 (dd, J= 10.0, 4.0 Hz, 1H), 6.69 (d, J= 8.7 Hz, 2H), 6.39 - 6.33 (m, 2H), 5.61-5.66 (m, 0.5H), 5.49-5.54 (m, 0.5H), 4.36 (d, J= 8.6 Hz, 1H), 2.93 (d, J= 3.0 Hz, 6H), 2.69 - 2.33 (m, 5H), 1.92 - 1.77 (m, 2H), 1.63-1.73 (m, 1H), 1.60 (s, 3H), 1.23-1.27 (m, 1H), 1.16 (s, 3H), 0.91 (d, J = 7.2 Hz, 3H).

[00297] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10,13,16-trimethyl- 17 -((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (200 mg, 0.3666 mmol) in DCM (6 mL) and SOCl ₂ (3 mL) was stirred under N ₂ at 40 °C for 2 h. Removed off DCM and SOCl ₂ under vacuum. THF (2 mL) and methylamine (2 M in THF, 2 mL) was added to the mixture. The reaction mixture was stirred at 25 °C for 16 h. Then THF was removed off under reduced pressure. DMF was added until the mixture all dissolved, purified by flash (ACN-H ₂ O, 60%-70%) to afford INT-16 as a white solid (75 mg, 36%). LCMS (ESI): m/z 559.1 (M+H) ⁺ .

[00298] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy-

10, 13 , 16-trimethyl- 17-(methylcarbamoyl)-3-oxo-6,7,8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro- 3H-cyclopenta[a]phenanthren- 17-yl 3 -nitrobenzoate INT-16 (55 mg, 0.099 mmol), tetrahydroxydiboron (26 mg, 0.296 mmol) and 4,4'-Bipyridine (0.15 mg, 0.001 mmol) in DMF (3 mL) was stirred at 25 °C for 30 min. The mixture was filtered and purified by prep-HPLC (ACN-H ₂ O (0.1% TFA), 50%-60%) to give compound 10 (35 mg, 67%) as a white solid. LCMS (ESI): m/z 529.2 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.72 (d, J= 7.5 Hz, 1H), 7.65 (s, 1H), 7.47 (t, J= 7.9 Hz, 1H), 7.34 (m, 2H), 6.39 - 6.31 (m, 2H), 5.64-5.50 (m, 1H), 4.34 (d, J= 8.3 Hz, 1H), 2.77 (s, 3H), 2.72 - 2.32 (m, 5H), 1.98 (q, J= 11.5 Hz, 1H), 1.81 (d, J= 13.7 Hz, 1H), 1.65-1.75 (m, 1H), 1.60 (s, 3H), 1.36 - 1.28 (m, 1H), 1.14 (s, 3H), 0.97 (d, J= 7.1 Hz, 3H).

[00299] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10, 13, 16-trimethyl- 17-((4-nitrobenzoyl)oxy)-3-oxo-6,7,8,9, 10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (200 mg, 0.37 mmol) in DCM (6 mL) and SOCl ₂ (3 mL) was stirred at 40 °C for 2 h under N ₂ atmosphere. The volatile phase was removed off under vacuum. The residue was dissolved in THF (2 mL), then dimethylamine (2 M in THF, 2 mL) was added dropwise to the mixture. The reaction mixture was stirred at 25 °C for 16 h. It was concentrated under reduced pressure. The residue was purified by FCC (ACN-H ₂ O, 60%-70%) to afford INT-17 as a white solid (90 mg, 43.3%). LCMS (ESI): m/z 559.2 (M+H) ⁺ .

[00300] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l 1-hydroxy-

10, 13 , 16-trimethyl- 17-(methylcarbamoyl)-3-oxo-6,7,8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro- 3H-cyclopenta[a]phenanthren- 17-yl 4-nitrobenzoate (70 mg, 0.13 mmol), tetrahydroxydiboron (34 mg, 0.38 mmol) and 4,4'-bipyridine(l mg, 0.0063 mmol) in DMF (4 mL) was stirred at 25 °C for 30 min. It was purified by prep-HPLC (ACN-H ₂ O (0.1%TFA), 50%-60%) to give compound 11 (45 mg, 67%) as a white solid. LCMS (ESI): m/z 529.2 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) 5 7.72 (d, J= 8.7 Hz, 2H), 7.36 (dd, J= 10.0, 1.3 Hz, 1H), 6.71 (d, J= 20.0 Hz, 2H), 6.39 - 6.31 (m, 2H), 5.68 - 5.60 (m, 0.5H), 5.54 - 5.48 (m, 0.5H), 4.34 (d, J= 9.4 Hz, 1H), 2.75 (s, 3H), 2.66 - 2.29 (m, 5H), 1.95 (q, J= 10.9 Hz, 1H), 1.64-1.80 (m, 2H), 1.60 (s, 3H), 1.30 (m, 1H), 1.11 (s, 3H), 0.97 (d, J= 7.2 Hz, 3H).

[00301] To a solution of (8S,9R,10S,l lS,13S,14S,16R,17R)-9-fluoro-l l,17-dihydroxy-17-(2- hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one (2000 mg, 5.1 mmol) and K ₂ CO ₃ (1410 mg, 102 mmol) in MeOH (20 mL) stirred at 0 °C was added H ₂ O ₂ (50 mL). The reaction mixture was stirred at 25 °C for 3 h. MeOH was removed off under reduced pressure, the mixture was acidified with 2 N HC1 to 1.0 pH and the resulting precipitate was filtered, washed with water, and dried in air to afford INT-18 as a white solid (1200 mg, 56.9%). LCMS (ESI): m/z 379.2 (M+H) ⁺ .

[00302] A suspension of (8S,9R,10S,l lS,13S,14S,16R,17R)-9-fluoro-l l,17-dihydroxy-

10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthrene-17-carboxylic acid INT-18 (400 mg, 1.06 mmol), 3 -nitrobenzoyl chloride (588 mg, 3.17 mmol) and triethylamine (321 mg, 3.17 mmol) in acetone (10 mL) was stirred 2 h at 25 °C, then diethylamine (387 mg, 5.29 mmol) was added to the solution. The reaction mixture was stirred at 25 °C for 4 h and concentrated. DMF and water were added until the mixture all dissolved, purified by Flash Chromatography (ACN-H ₂ O, 65%) to afford INT-19 as a white solid (500 mg, 65%). LCMS (ESI): m/z 528.0 (M+H) ⁺ .

[00303] A solution of (8 S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-9-fluoro- 1 1 -hydroxy- 10, 13,16- trimethyl- 17-((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7, 8, 9, 10, 11 , 12, 13 , 14, 15 , 16, 17 -dodecahy dro-3H- cyclopenta[a]phenanthrene-17-carboxylic acid INT-19 (20 mg, 0.04 mmol) and BTFFH (14 mg, 0.05 mmol) in dry DCM (2 mL) under argon in dry microwave vial. DIEA (15 mg, 0. 11 mmol) was subsequently added to the reaction was stirred under argon for 30 min. Dimethylamine (2M, THF, ImL) was added to the mixture vial was sealed and heated in an oil bath at 40 °C for 16 h. Then the solution was removed under reduced pressure and the residue was purified by Prep- TLC (DCM: EtOAc = 2: 1, v/v) to afford INT-20 as a white solid (2 mg, 9.5%). LCMS (ESI): m/z 555.2 (M+H) ⁺ .

[00304] A solution of (8S,9R,10S,l lS,13S,14S,16R,17R)-17-(dimethylcarbamoyl)-9-fluoro- l l-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren- 17-yl 3 -nitrobenzoate (20 mg, 0.03 mmol), 4,4'-bipyridine (0.3 mg, 0.002 mmol) and tetrahydroxyboron (9 mg, 0.10 mmol) in dry DMF (2 mL) was stirred at 25 °C for 30 min. The mixture solution was purified by prep-HPLC (ACN-H ₂ O (0. 1% TFA), 55%) to give compound 12 (10 mg, 52.4%) as a white solid. LCMS (ESI): m/z 525.2 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.74-7.60 (m, 2H), 7.49-7.40 (m, 2H), 7.29 (d, J= 7.8 Hz, 1H), 6.32 (dd, J = 10.1, J= 1.8 Hz, 1H), 6.13 (s, 1H), 4.37 (d, J= 9.8 Hz, 1H), 2.94 (s, 6H), 2.80-2.72 (m, 1H), 2.65-2.30 (m, 5H), 2.02-1.94 (m, 1H), 1.91-1.78 (m, 2H), 1.62 (s, 3H), 1.60-1.53 (m, 1H), 1.29- 1.23 (m, 1H), 1.19 (s, 3H), 0.90 (d, J = 7.2 Hz, 3H).

[00305] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difhioro-l l-hydroxy-

10,13,16-trimethyl- 17 -((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (100 mg, 0.183 mmol) in dry DCM (5 mL) was added SOCl ₂ (1.5 mL), the reaction was stirred at 40 °C for 2 h, concentrated, then the residue was dissolved into DCM (3 mL), azetidine (21 mg, 0.367 mmol) was added. The reaction mixture was stirred under nitrogen at 40 °C for 2 h. The solvent was removed off under reduced pressure and the crude product was purified by FCC (Cl 8, ACN/H ₂ O 10%-50%) to give INT-21 (40 mg, 37.4%) as a white solid. LCMS (ESI): m/z 585.2 [M+H] ⁺ .

[00306] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-17-(azetidine-l-carbonyl)-

6,9-difluoro- 11 -hydroxy- 10, 13 , 16-trimethyl-3-oxo-6,7,8,9, 10, 11 , 12, 13 , 14, 15, 16, 17- dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl 3-nitrobenzoate INT-21 (40 mg, 0.068 mmol) and 4,4 -Bipyridine (1 mg, 0.007 mmol) in dry DMF (1.5 mL) was added tetrahydroxydiboron (18 mg, 0.200 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 0.5 h. The crude material was purified by Prep-HPLC (ACN-H ₂ O (0.1% TFA)) to give the product (8 mg, 21.1%) as a yellow solid. LCMS (ESI): m/z 555.3 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.64 - 7.45 (m, 2H), 7.40 - 7.08 (m, 3H), 6.31 - 6.15 (m, 2H), 5.59 - 5.50 (m, 0.5H), 5.42 (dd, J = 9.9, 6.9 Hz, 0.5H), 4.54 - 3.54 (m, 5H), 2.52 (ddd, J= 16.1, 12.6, 5.9 Hz, 1H), 2.34 - 2.11 (m, 5H), 2.01 - 1.73 (m, J= 17.9 Hz, 3H), 1.62 - 1.41 (m, 4H), 1.19 (s, 1H), 1.10 (s, 3H), 0.87 (d, J = 7.1 Hz, 3H).

[00307] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy- 10,13,16-trimethyl-17-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9,10 ,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (100 mg, 0.183 mmol) in dry DCM (5 mL) was added SOCl ₂ (1.5 mL), the reaction was stirred at 40 °C for 2 h, concentrated, then the residue was dissolved into DCM (3 mL), pyrrolidine (26 mg, 0.367 mmol) was added. The reaction mixture was stirred under nitrogen at 40 °C for 2 h. The solvent was removed off under reduced pressure and the crude product was purified by FCC (Cl 8, ACN/H ₂ O 10%-50%) to give INT-12 (40 mg, 36.4%) as a white solid. LCMS (ESI): m/z 599.2 [M+H] ⁺ .

[00308] To a solution of tert-butyl ((S)-l-(((S)-l-(((S)-4-hydroxy-4-methyl-l 1-morpholino- 3,14-dioxo-3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizi no[l,2-b]quinolin-9-yl)amino)-l- oxopropan-2-yl)amino)-l-oxopropan-2-yl)carbamate INT-22 (40 mg, 0.0668 mmol) and tetrahydroxyboron (18 mg, 0.2004 mmol) in dry DMF (1.5 mL) was added 4, 4' -bipyridine (1 mg, 0.0066 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 0.5 h. The crude material was purified by Prep-HPLC (ACN-H ₂ O (0.1% TEA)) to give compound 14 (5.8 mg, 14.97%) as a yellow solid. LCMS (ESI): m/z 569.1 [M+H] ⁺ .

[00309] ¹ H NMR (400 MHz, MeOD) δ 7.75 (d, J= 7.9 Hz, 1H), 7.68 (s, 1H), 7.49 (t, J= 7.9 Hz, 1H), 7.39 - 7.29 (m, 2H), 6.36 - 6.28 (m, 2H), 5.69 - 5.44 (m, 1H), 4.32 (t, J= 11.0 Hz, 1H), 3.84 - 3.68 (m, 2H), 3.54 - 3.45 (m, 2H), 2.66 - 2.32 (m, 5H), 1.97 - 1.90 (m, 1H), 1.78 - 1.66 (m, 6H), 1.58 (s, 3H), 1.27 (t, J = 9.6 Hz, 1H), 1.17 (s, 3H), 0.90 (d, J= 7.1 Hz, 3H).

[00310] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy-

10,13,16-trimethyl-17-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9 ,10,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (100 mg, 0.18 mmol) in DCM (3 mL) and SOCl ₂ (2 mL) was stirred 2 h under N ₂ at 40 °C. Removed off DCM and SOCl ₂ under vacuum. DCM (2 mL) and morpholine (32 mg, 0.3666 mmol) was added to the mixture. The reaction mixture was stirred at 25 °C for 3 h and concentrated. The residue was purified by flash (ACN-H ₂ O, 60%) to give INT-23 (20 mg, 16.0%) as a white solid. LCMS (ESI): m/z 615 [M+H] ⁺ .

[00311] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10, 13, 16-trimethyl- 17-(morpholine-4-carbonyl)-3-oxo-6,7,8,9, 10, 11,12, 13,14, 15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-nitrobenzoate INT-23 (20 mg, 0.03 mmol), 4,4'-bipyridine (0.3 mg, 0.002 mmol) and tetrahydroxyboron (9 mg, 0.10 mmol) in dry DMF (2 mL) was stirred for 30 min at 25 °C. The reaction mixture was purified by HPLC (ACN-H ₂ O (0.1% TFA), 50%) to give compound AD-407 (10 mg, 51.1%) as a white solid. LCMS (ESI): m/z 585.2 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.56-7.48 (m, 2H), 7.39-7.33 (m, 2H), 7.17 (d, J= 8.2 Hz, 1H), 6.38-6.32 (m, 2H), 5.55-5.50 (m, 1H), 4.37 (d, J= 9.3 Hz, 1H), 3.91-3.47 (m, 8H), 2.73-2.58 (m, 1H), 2.54-2.30 (m, 4H), 1.92 (q, J= 11.6 Hz, 1H), 1.81-1.64 (m, 2H), 1.61 (s, 3H), 1.27 (d, J= 12.1 Hz, 1H), 1.21 (s, 3H), 0.92 (d, J= 7.1 Hz, 3H).

[00312] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10,13,16-trimethyl- 17 -((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (200 mg, 0.3666 mmol) in dry DCM (5 mL) was added SOCl ₂ (3 mL), the mixture was stirred at rt for 2 h, concentrated, the residue was dissolved into DCM (5 mL), 3-azetidinol (27 mg, 0.3666 mmol) was added. The reaction mixture was stirred under nitrogen at 40 °C for 1 h. The solvent was removed off under reduced pressure and the crude material was purified by purified by FCC (Cl 8, ACN/H ₂ O 10%- 50%) to give INT-24 (120 mg, 49.05%) as a white solid. LCMS (ESI): m/z 601.3 [M+H] ⁺ .

[00313] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-17- (3-hydroxyazetidine-l-carbonyl)-10,13,16-trimethyl-3-oxo-6,7 ,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl 3-nitrobenzoate INT-24 (50 mg, 0.0832 mmol) and tetrahydroxyboron (23 mg, 0.2496 mmol) in dry DMF (2 mL) was added 4,4'- bipyridine (2 mg, 0.0083 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 0.5 h. The solvent was removed off under reduced pressure and the crude material was purified by Prep-HPLC (ACN-H ₂ O (0.1% TFA)) to give compound 16 (11.8 mg, 23.56%) as a white solid. LCMS (ESI): m/z 571.3 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO) δ 7.29 (d, J = 10.2 Hz, 1H), 7.26 - 7.21 (m, 2H), 7.19 - 7.11 (m, 1H), 6.98 - 6.87 (m, 1H), 6.32 (d, J = 10.1 Hz, 1H), 6.13 (s, 1H), 5.73 - 5.61 (m, 1H), 4.26 (s, 6H), 2.39 - 1.97 (m, 4H), 1.95 - 1.65 (m, 3H), 1.63 - 1.55 (m, 1H), 1.51 (s, 3H), 1.27 - 1.14 (m, 1H), 1.05 (s, 3H), 0.81 (s, 3H). OH

[00314] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10,13,16-trimethyl- 17 -((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (200 mg, 0.367 mmol) in dry DCM (10 mL) was added SOCl ₂ (3 mL), the reaction was stirred at 40 °C for 2 h, concentrated, then the residue was dissolved into DCM (5 mL), azeti din-3 -ylmethanol (32 mg, 0.367 mmol) was added. The reaction mixture was stirred under nitrogen at 40 °C for 2 h. The solvent was removed off under reduced pressure and the crude product was purified by FCC (Cl 8, ACN/H ₂ O 10%-50%) to give INT-25 (60 mg, 26.7%) as a white solid.

[00315] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-17-(azetidine-l-carbonyl)-

6,9-difluoro-l l-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl 3-nitrobenzoate INT-25 (50 mg, 0.081 mmol) and 4,4'-bipyridine (1 mg, 0.007 mmol) in dry DMF (2 mL) was added tetrahydroxydiboron (22 mg, 0.244 mol). The reaction mixture was stirred under nitrogen at 25 °C for 30 min, quenched by water (2 mL), purified by Prep-HPLC (ACN-HzO, 0.1%TFA) to give the product (13.7 mg, 28.8%) as a white solid. LCMS (ESI): m/z 585.3 [M+H] ⁺ . ^r H NMR (400 MHz, DMSO) δ 7.29 (d, J= 9.8 Hz, 1H), 7.22 (t, J= 7.7 Hz, 2H), 7.13 (s, 1H), 6.91 (s, 1H), 6.31 (dd, J= 10.2, 1.6 Hz, 1H), 6.13 (s, 1H), 5.71 (d, J= 6.2 Hz, 0.5H), 5.59 (d, J= 9.4 Hz, 0.5H), 4.31 - 4.11 (m, 5H), 3.57 - 3.45 (m, 2H), 2.31 - 2.07 (m, 4H), 1.98 - 1.68 (m, 3H), 1.62 - 1.44 (m, 4H), 1.29 - 1.10 (m, 2H), 1.04 (s, 3H), 0.81 (s, 3H).

[00316] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l 1-hydroxy-

10,13,16-trimethyl- 17 -((3 -nitrobenzoyl)oxy)-3 -oxo-6, 7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (300 mg, 0.55 mmol) in DCM (20 mL) and SOCl ₂ (4 mL) was stirred 1 h under N ₂ at 40 °C. Removed off DCM and SOCl ₂ under vacuum. DCM (10 mL) and {2-[(tert-butyldimethylsilyl)oxy]ethyl}(methyl)amine (312 mg, 1.65 mmol) was added to the mixture. The reaction mixture was stirred at 25 °C for 6 h and concentrated. The residue was purified by flash (ACN-H ₂ O, 70%) to give compound 3 (70 mg, 16.8%) as a white solid. LCMS (ESI): m/z 717.3 [M+H] ⁺ .

[00317] To a solution of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-17-((2-((tert- buty ldimethylsilyl)oxy)ethy l)(methyl)carbamoy l)-6,9-difluoro- 11 -hydroxy- 10, 13 , 16-trimethyl-3 - oxo-6, 7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17-dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl 3 - nitrobenzoate INT-26 (50 mg, 0.07 mmol) in diy DCM (5 mL) was added 3HF.Et ₃ N (1 mL). The reaction mixture was stirred at 25 °C for 1 h. The mixture was extracted with DCM (3* 20 mL) and the combined organic extracts were washed with water (50 mL), brine (50 mL), dried (Na ₂ SO ₄ ) and concentrated in vacuo to give INT-27 (40 mg, 93.3%) as a white solid. LCMS (ESI): m/z 603.2 [M+H] ⁺ .

[00318] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy- 17-((2- hydroxyethyl)carbamoyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10, l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-aminobenzoate INT-27 (40 mg, 0.07 mmol), 4,4'-bipyridine (0.5 mg, 0.003 mmol) and tetrahydroxydiboron (18 mg, 0.20 mmol) in dry DMF (2 mL) was stirred at 25 °C for 30 min. The reaction mixture was purified by HPLC (ACN-H ₂ O (0.1% TFA), 45%) to give compound 18 (35 mg, 82.8%) as a white solid. LCMS (ESI): m/z 573.1 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.76-7.64 (m, 2H), 7.49 (t, J= 7.9 Hz, 1H), 7.41- 7.29 (m, 2H), 6.41-6.30 (m, 2H), 5.55-5.49 (m, 1H), 4.37 (d, J= 10.0 Hz, 1H), 3.91-3.82 (m, 1H), 3.81-3.73 (m, 2H), 3.71-3.62 (m, 1H), 3.01 (s, 3H), 2.72-2.33 (m, 5H), 1.97-1.84 (m, 2H), 1.74-1.64 (m, 1H), 1.61 (s, 3H), 1.33-1.25 (m, 1H), 1.22 (s, 4H), 0.91 (d, J= 7.1 Hz, 4H).

[00319] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy-

10,13,16-trimethyl-17-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9 ,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (300 mg, 0.55 mmol) in DCM (30 mL) and SOCl ₂ (4 mL) was stirred 2 h under N ₂ at 40 °C. Removed off DCM and SOCl ₂ under vacuum. 3 -aminopropane- 1 ,2-diol (336 mg, 5.5 mmol) and DCM (5mL) was added to the mixture. The reaction mixture was stirred at 25 °C for 4 h and concentrated. The residue was purified by flash (ACN-H ₂ O, 45%) to give INT-28 (150 mg, 44.0%) as a white solid. LCMS (ESI): m/z 589.1 [M+H] ⁺ .

[00320] A solution of (6S,8S,9R, 1 OS, 11 S, 13 S, 14S, 16R, 17R)-6,9-difluoro- 11 -hydroxy- 17-((2- hydroxy ethyl )carbamoyl)- 10, 13 , 16-trimethyl-3 -oxo-6,7,8,9, 10, 11,12, 13,14, 15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-nitrobenzoate INT-28 (200 mg, 0.34 mmol), 4,4'-bipyridine (0.5 mg, 0.003 mmol) and tetrahydroxydiboron (91 mg, 1.02 mmol)in dry DMF (3 mL) was stirred at 25 °C for 30 min. The reaction mixture was purified by HPLC (ACN-H ₂ O (0.1%TFA), 45%) to give compound 19 (150 mg, 75.1%) as a white solid. LCMS (ESI): m/z 559.2 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.77 (d, J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.68 (s, 0.5H), 7.50 (t, J= 7.9 Hz, 1H), 7.36 (d, J= 9.0 Hz, 2H), 6.40-6.30 (m, 2H), 5.67-5.49 (m, 1H), 4.35 (d, J= 8.2 Hz, 1H), 3.70-3.61 (m, 2H), 3.49-3.38 (m, 2H), 3.28 (s, 1H), 2.73-2.57 (m, 1H), 2.49-2.32 (m, 3H), 1.99 (q, J= 11.3 Hz, 1H), 1.85 (d, J= 14.1 Hz, 1H), 1.77-1.63 (m, 1H), 1.61 (s, 3H), 1.38-1.29 (m, 1H), 1.17 (s, 3H), 0.97 (d, J= 7.1 Hz, 3H).

[00321] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-6,9-difluoro-l l-hydroxy-

10,13,16-trimethyl-17-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9 ,10,l l,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (200 mg, 0.3666 mmol) in dry DCM (5 mL) was added SOCl ₂ (3 mL), the mixture was stirred at rt for 2h, concentrated, the residue was dissolved into DCM (5 mL), 3-amino-l,2-propanediol (34 mg, 0.4766 mmol) was added. The reaction mixture was stirred under nitrogen at 40 °C for 2 h. The solvent was removed off under reduced pressure and the crude material was purified by purified by Cl 8 column (ACN/H ₂ O 10%-50%) to give INT-29 (100 mg, 20.11%) as a white solid. LCMS (ESI): m/z 633.3 [M+H] ⁺ .

Step4: (6S,8S,9R,1OS,11S,13S,14S,16R,17R)-17-((2,3-dihydroxypropyl) (methyl)carbamoyl)-6, 9-difluoro-l l-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,l 5,16,17-dodecahydro- 3H-cyclopenta[a]phenanthren-l 7-yl 3-aminobenzoate

[00322] To a solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-17-((2,3- dihydroxypropyl)(methyl)carbamoyl)-6,9-difluoro- 11 -hydroxy- 10, 13 , 16-trimethyl-3 -oxo- 6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17 -dodecahydro-3H-cy clopenta[a]phenanthren- 17-yl 3- nitrobenzoate INT-29 (100 mg, 0.1581 mmol) and tetrahydroxydiboron (43 mg, 0.4743 mmol) in dry DMF (2 mL) was added 4,4'-bipyridine (8 mg, 0.4743 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 0.5 h. The crude material was purified by Prep-HPLC (ACN- H ₂ O (0.1% TFA)) to give compound 20 (44.0 mg, 41.56%) as a white solid. LCMS (ESI): m/z 603.4 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 7.36 - 7.29 (m, 2H), 7.18 - 7.09 (m, 2H), 7.02 (d, J= 8.1 Hz, 1H), 6.33 - 6.26 (m, 2H), 5.66 - 5.43 (m, 1H), 4.35 - 4.25 (m, 1H), 3.94 - 3.84 (m, 1H), 3.51 - 3.30 (m, 2H), 3.13 - 2.86 (m, 2H), 2.81 - 2.71 (m, 1H), 2.66 (s, 3H), 2.48 - 2.25 (m, 3H), 2.12 - 1.98 (m, 1H), 1.93 - 1.78 (m, 1H), 1.58 (s, 4H), 1.36 (s, 3H), 1.14 (d, J= 2.9 Hz, 1H), 0.95 (d, J= 7.0 Hz, 1H), 0.59 (s, 3H). [00323] A solution of (6S,8S,9R,10S,l l S,13S,14S,16R,17R)-6,9-difluoro-l 1-hydroxy- 10,13,16-trimethyl-17-((3-nitrobenzoyl)oxy)-3-oxo-6,7,8,9,10 ,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid INT-12 (300 mg, 0.55 mmol) in DCM (30 mL) and SOCl ₂ (4 mL) was stirred 2 h under N ₂ at 40 °C. Removed off DCM and SOCl ₂ under vacuum. Et ₃ N (556 mg, 5.50 mmol), 3 -aminopropane- 1,2-diol (150 mg, 1.65 mmol) and DCM (5 mL) was added to the mixture. The reaction mixture was stirred at 25 °C for 4 h and concentrated. The residue was purified by flash (ACN-H ₂ O, 45%) to give compound 3 (80 mg, 22.4%) as a white solid. LCMS (ESI): m/z 619.2 [M+H] ⁺ .

[00324] A solution of (6S,8S,9R,10S,l lS,13S,14S,16R,17R)-17-((2,3- dihydroxypropyl)carbamoyl)-6,9-difluoro- 11 -hydroxy- 10,13,16-trimethyl-3-oxo- 6,7, 8,9, 10, 11 , 12, 13 , 14, 15, 16, 17 -dodecahydro-3H-cyclopenta[a]phenanthren- 17-yl 3- nitrobenzoate INT-30 (60 mg, 0.10 mmol), 4,4'-bipyridine (0.2 mg, 0.0009 mmol) and tetrahydroxydiboron (26 mg, 0.29 mmol)in dry DMF (2 mL) was stirred at 25 °C for 30 min. The reaction mixture was purified by HPLC (ACN-H ₂ O (0.1% TFA), 45%) to give compound 21 (45 mg, 76.5%) as a white solid. LCMS (ESI): m/z 589.1 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.78 (d, J= 7.8 Hz, 1H), 7.71 (s, 1H), 7.65 (s, 1H), 7.50 (t, J= 7.9 Hz, 1H), 7.42-7.33 (m, 2H), 6.40-6.30 (m, 2H), 5.56-5.48 (m, 1H), 4.35 (d, J= 8.3 Hz, 1H), 3.83-3.74 (m, 1H), 3.62-3.50 (m, 2H), 3.47-3.34 (m, 2H), 3.30-3.18 (m, 1H), 2.76-2.55 (m, 1H), 2.50-2.32 (m, 3H), 1.99 (q, J = 11.2 Hz, 1H), 1.91-1.80 (m, 1H), 1.78-1.63 (m, 1H), 1.61 (s, 3H), 1.39-1.29 (m, 1H), 1.18 (s, 3H), 0.98 (d, J= 7.0 Hz, 3H).

[00325] To a solution of (2S,6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-2,6b-difluoro-7-hydroxy- 8b-(2-hydroxyacetyl)-6a, 8a, 10, 10-tetramethyl- 1 ,2, 6a, 6b, 7, 8,8a, 8b, 11 a, 12, 12a, 12b-dodecahydro- 4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one (1.5 g, 0.0034 mol), nonafluorobutanesulfonyl fluoride (2.08 g, 0.0069 mol) and triethylamine trihydrofluoride (1.11 g, 0.0069 mol) in dry ACN (60 mL) was added NEt ₃ (2.09 g, 0.0207 mol). The reaction mixture was stirred under nitrogen at 50 °C for 17 h. The solvent was removed off under reduced pressure and the crude material was purified by Prep-HPLC (ACN-H ₂ O (0.1%TFA)) to give INT-31 (1.3 g, 78.26%) as a white solid. LCMS (ESI): m/z 437.2 [M+H] ⁺ .

[00326] To a solution of (2S,6aS,6bR,7S,8aS,8bS,l laR,12aS,12bS)-2,6b-difluoro-8b-(2- fluoroacetyl)-7-hydroxy-6a,8a,10,10-tetramethyl-l,2,6a,6b,7, 8,8a,8b,l la,12,12a,12b- dodecahydro-4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4- one INT-31 (500 mg, 1.15 mmol) and l-butyl-3-methylimidazolium hexafluoro-phosphate (654 mg, 2.3 mmol) in dry DCM (10 mL) was added tert-butyl (3-formylphenyl)carbamate (331 mg, 1.495 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 0.2 h. The solvent was removed off under reduced pressure and the crude material was purified by purified by C18 column (ACN/H ₂ O 10%-50%) to give compound 22 (300 mg, 46.96 %) as a white solid. LCMS (ESI): m/z 518.3 [M+H] ⁺ . ¹ H NMR (400 MHz, MeOD) δ 7.30 (dd, J= 10.0, 1.4 Hz, 1H), 7.08 (t, J= 7.9 Hz, 1H), 6.82 - 6.68 (m, 3H), 6.36 - 6.24 (m, 2H), 5.65 - 5.46 (m, 1H), 5.42 (s, 1H), 5.41 - 5.04 (m, 2H), 4.99 (d, J= 4.8 Hz, 1H), 4.34 - 4.25 (m, 1H), 2.78 - 2.58 (m, 1H), 2.44 - 2.30 (m, 2H), 2.28 - 2.17 (m, 1H), 1.84 - 1.74 (m, 2H), 1 .72 - 1.61 (m, 2H), 1.56 (s, 3H), 0.98 (s, 3H).

[00327] A solution of (6aR,6bS,7S,8aS,8bS,l laR,12aS,12bS)-7-hydroxy-6a,8a,10,10- tetramethyl-4-oxo-l,2,4,6a,6b,7,8,8a,l la,12,12a,12b-dodecahydro-8bH- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxole-8b-carboxylic acid INT-32 (150 mg, 0.37 mmol), HOBt (65 mg, 0.48 mmol), EDCI (93 mg, 0.48 mmol) and DIEA (145 mg, 1.12 mmol) in dry ACN (2 mL) was stirred 10 min at 25 °C, then dimethylamine (50 mg, 0.11 mmol) was added to the solution. The reaction mixture was stirred at 45 °C for 16 h. The mixture was extracted with

EA (3* 20 mL) and the combined organic extracts were washed with water (50 mL), brine (50 mL), dried (Na ₂ SO ₄ ) and concentrated in vacuo to afford INT-33 as a white solid. (80 mg, 89.9%). LCMS (ESI): m/z 430.0 (M+H) ⁺ .

[00328] To a solution of (6aR,6bS,7S,8aS,8bS,l laR,12aS,12bS)-7-hydroxy-N,N,6a,8a,10,10- hexamethyl-4-oxo-l,2,4,6a,6b,7,8,8a, 1 la,12,12a,12b-dodecahydro-8bH- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxole-8b-carboxamide INT-33 (100 mg, 0.33 mmol) in dry DCM (5 mL) was added tert-butyl(3-formylphenyl)carbamate (662 mg, 2.33 mmol) and HCIO ₄ (234 mg, 2.33 mmol). The reaction mixture was stirred at 25 °C for 8 h. Triethylamine was added to neutralize the acid. The solvent was removed in vacuo and the crude product was purified by prep-HPLC (ACN-H ₂ O, 40%) to give compound 23 (32 mg, 22.6%) and 24 (10 mg, 7.1%). LCMS (ESI): m/z 582.9 (M+H) ⁺ . 23: ¹ HNMR (400 MHz, MeOD) δ 7.48-7.38 (m, 4H), 7.33 (d, J= 7.6 Hz, 1H), 7.25 (d, J= 8.0 Hz, 2H), 7.17 (d, J= 7.9 Hz, 1H), 7.14 (s, 1H), 6.25 (dd, J= 10.1, J= 1.8Hz, 1H), 6.02 (s, 1H), 5.46 (s, 1H), 5.31 (d, J= 4.6 Hz, 1H), 4.42 (d, J= 2.9 Hz, 1H), 4.05 (s, 2H), 3.27 (s, 3H), 3.00 (s, 3H), 2.74-2.63 (m, 1H), 2.39 (d, J= 10.4 Hz, 1H), 2.32-2.13 (m, 2H), 2.00 (dd, J= 13.3, J= 3.3 Hz, 1H), 1.93-1.73 (m, 4H), 1.51 (s, 3H), 1.17-1.07 (m, 4H), 1.02 (dd, J= 11.2, J= 3.5 Hz, 1H). 24: ¹ H NMR (400 MHz, MeOD) δ 7.46 (d, J= 10.1 Hz, 1H), 7.39 (t, J= 7.8 Hz, 1H), 7.27-7.17 (m,5H), 7.14-7.07 (m, 2H), 6.25 (dd, J= 10.1, J = 1.9 Hz, 1H), 6.15 (s, 1H), 6.03 (s, 1H), 5.62 (d, J= 4.3 Hz, 1H), 4.42 (d, J= 2.8 Hz, 1H), 4.01 (s, 2H), 3.02 (s, 3H), 2.73-2.66 (m, 1H), 2.64 (s,3H), 2.40 (d, J= 13.7 Hz, 1H), 2.26-2.13 (m, 2H), 2.03 (dd, J= 13.4 Hz, J= 3.4 Hz, 1H), 1.87-1.72 (m, 4H), 1.50 (s, 3H), 1.23-1.14 (m, 1H), 1.11 (dd, J= 11.3, J= 3.6 Hz, 1H), 1.07 (s, 3H).

[00329] To a solution of (6aR,6bS,7S,8aS,8bS,l laR,12aS,12bS)-7-hydroxy-8b-(2- hydroxy acetyl)-6a, 8 a, 10, 10-tetramethyl- 1 ,2, 6a, 6b, 7, 8, 8a, 8b, 11 a, 12, 12a, 12b-dodecahydro-4H- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one INT-34 (1 g, 0.0024 mol), Nonafluorobutanesulfonyl fluoride (1.45 g, 0.0048 mol) and triethylamine trihydrofluoride (0.77 g, 0.0048 mol) in dry ACN (15 mL) was added NEt ₃ (1.46 g, 0.0144 mol). The reaction mixture was stirred under nitrogen at 50 °C for 17 h. The solvent was quenched by ice water (30 mL), then the mixture was extracted with EA (3*500 mL) and the combined organic extracts were washed with water (50 mL), dried (Na ₂ SO ₄ ) and concentrated in vacuo. The crude material was purified by purified by C18 column (ACN/H2O 10%-50%) to give INT-35 (0.9 g, 79.17 %) as a white solid. LCMS (ESI): m/z 419.2 [M+H] ⁺ .

[00330] To a solution of (6aR,6bS,7S,8aS,8bS,l laR,12aS,12bS)-8b-(2-fluoroacetyl)-7- hydroxy-6a, 8a, 10, 10-tetramethyl - 1 ,2, 6a, 6b, 7, 8,8a, 8b, 11 a, 12, 12a, 12b-dodecahydro-4H- naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxol-4-one INT-35 (150 mg, 0.3584 mmol), l-butyl-3- methylimidazolium hexafluoro-phosphate (204 mg, 0.7168 mmol) and tert-butyl(3-(4- formylbenzyl)phenyl)carbamate (145 mg, 0.4659 mmol) in dry DCM (5 mL) was added perchloric acid (360 mg, 3.584 mmol). The reaction mixture was stirred under nitrogen at 25 °C for 2 h. The solvent was removed off under reduced pressure and the crude material was purified by prep-HPLC (ACN-H ₂ O (0.1%TFA)) to give 25 (18.0 mg, 8.81%) as a yellow solid. LCMS (ESI): m/z 572.2 [M+H] ⁺ . ¹ HNMR (400 MHz, MeOD) δ 8.81 (d, J= 2.3 Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 7.76 (dd, J= 9.1, 2.4 Hz, 1H), 7.64 (s, 1H), 5.59 (d, J= 16.2 Hz, 1H), 5.50 (s, 2H), 5.39 (d, J= 16.2 Hz, 1H), 4.62 (q, J= 7.2 Hz, 1H), 4.08 - 3.95 (m, 5H), 3.59 (d, J= 4.2 Hz, 4H), 1.97 (dt, J= 11.8, 7.0 Hz, 2H), 1.59 (d, J= 7.1 Hz, 3H), 1.54 (d, J= 7.2 Hz, 3H), 1.01 (t, J= 7.4 Hz, 3H).

Biological Activity GR Reporter Assay:

[00331] The assay is a pBind assay foliowing the general principle outlined below. 2xl0 ⁴ HEK293 cells/96-well are transiently transfected with pG5luc and GR LBD 1: 1 at 50ng/well. GR LBD (ligand binding domain) is a luciferase fusion protein (fused to yeast GAL4) and upon binding to the test article will bind the luciferase reporter and activate transcription of luciferase. Upon addition of a substrate, the luciferase activity can be measured and corresponds to the activity of the test article. The assay is performed in agonist mode.

HEK293 Cell Reporter Assay for ADC activity:

[00332] The assay is a pBind assay: In general, cells are transiently transfected with pG51uc and pBind-GR at a 1 : 1 DNA ratio. The GR LBD (ligand binding domain) is a fusion protein (fused to yeast GAL4) and upon binding to the ligand, e.g. dexamethasone or test article, will bind the luciferase reporter and activate transcription of luciferase. Upon addition of a substrate, the luciferase activity can be measured and corresponds to the activity of the test article. The assay is performed in agonist mode.

[00333] To enable TNFa-mediated uptake of the Adalimumab-ADCs, we had to transiently overexpress human TNFa in the HEK293 used for this pBind assay. We used the T ACE-resistant hTNFa(77-88del) construct, to allow for optimal membrane-associated TNFa expression.

[00334] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[00335] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description, herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that Applicant’s composition and/or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[00336] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference

[00337] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, manuscripts, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

Equivalents

[00338] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.