Novel glucocorticoid receptor agonists and their immunoconjugates

Abstract

The present invention provides novel glucocorticoid receptor agonists and immunoconjugates thereof, as well as pharmaceutical compositions and methods of making and using them to treat various diseases and disorders (eg, inflammatory disorders).

Description

[Technical Field]

[0001] Priority claims and related patent applications This application claims the benefit of priority from PCT / CN2022 / 118982, filed September 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical field of the invention The present invention relates generally to novel compounds and their therapeutic uses. More particularly, the present invention provides novel glucocorticoid receptor agonists and immunoconjugates thereof, as well as pharmaceutical compositions and methods of manufacture and use for treating various diseases and disorders, such as inflammatory disorders. [Background technology]

[0003] Background of the Invention Glucocorticoids are a type of corticosteroid that binds to the glucocorticoid receptor. Glucocorticoids are part of a feedback mechanism in the immune system, affecting cells by binding to the glucocorticoid receptor. The activated glucocorticoid receptor-glucocorticoid complex upregulates the expression of anti-inflammatory proteins in the nucleus and suppresses the expression of pro-inflammatory proteins in the cytosol. Cortisol (or hydrocortisone) is an important human glucocorticoid. It regulates or supports a variety of important cardiovascular, metabolic, immune, 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] However, significant adverse side effects limit the therapeutic potential of glucocorticoids, including suppression of the hypothalamic-pituitary-adrenal axis, bone demineralization and osteoporosis, disruption of carbohydrate metabolism, ocular side effects such as glaucoma and cataracts, and developmental delay in children. (Rhen, et al. 2005 New Eng. J. Med. 353 (16): 1711-23.)

[0005] Synthetic glucocorticoid receptor agonists, such as dexamethasone, prednisolone, and budesonide, are small molecules used to treat inflammatory disorders. Their usefulness in treating chronic diseases is limited by severe side effects.

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

[0007] There is a great need for novel glucocorticoid receptor agonists and immunoconjugates that are potent yet have few side effects and are amenable to development. Summary of the Invention

[0008] Summary of the Invention The present invention provides novel glucocorticoid receptor agonists with high anti-inflammatory activity, favorable stability, and other properties suitable for use alone or in immunoconjugates. The glucocorticoid receptor agonists disclosed herein are characterized by the placement of an anilino functionality at the 17 / 16 position for linker attachment. This design is synergistically combined with additional variations at the 20 position to fine-tune the payload to suit various ADC constructs and applications. The high activity, high stability, low immunogenicity, and sufficient solubility make these compounds ideally suited for the development of immunoconjugates as glucocorticoid receptor agonists and as novel therapeutic agents for inflammatory disorders.

[0009] In some embodiments, the present invention generally relates to compounds of structural formula (I): [ka] [During the ceremony, R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 alkyl, and R 4 is OC(O)-R 7 where R 7 is a substituted or unsubstituted aniline, or R 3 and R 4 together with the carbon atoms to which they are attached, 6 wherein W is a single bond or -phenyl-Q-, Q is selected from CH, O, S, S(O), S(O), NH, and NCH, and R 6 is a substituted or unsubstituted aniline; and R 5 is NR 5a R 5b or CH2R 5c and R 5a and R 5beach independently represents H and C 1-6 alkyl, or R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered unsubstituted or substituted heterocyclic ring; and R 5c is H, halogen or OC 1-3 It is alkyl. or a pharmaceutically acceptable form thereof.

[0010] In certain embodiments of (I), the compounds of the present invention have structural formula (II): [ka] It has.

[0011] In certain embodiments, the compounds of the present invention have the structural formula (II a ): [ka] [During the ceremony, R 7a , R 7b and R 7c Each of 7x R 7y selected from, with the proviso that R 7a , R 7b and R 7c Only one of the 7x R 7y and each of the others is H; R 7x and R 7y Each of the groups independently represents R, R 7r and L 7 -R 7z selected from, with the proviso that R 7x and R 7y One of them is L 7 -R 7z or R 7r When , the other is R; L 7 is a linker; R7r is (C=O)-O-(CH2) i -R 7v or (C=O)-(CH2) j -R 7v and; R 7v is R, OR, NHR, NR2, 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 C1-C6 alkyl. It has.

[0012] In certain embodiments of (I), R 3 and R 4 together with the carbon atoms to which they are attached, 6 forming a five-membered dioxolane substituted with the structural formula: [ka] It has.

[0013] In certain embodiments, W is a single bond and the compounds of the present invention have structural formula (III): [ka] It has.

[0014] In certain embodiments, the compounds of the present invention have the structural formula (III a ): [ka] [During the ceremony, R 6a , R 6b and R 6c Each of 6x R 6y selected from, with the proviso that R 6a , R 6b and R6c Only one of the 6x R 6y and each of the others is H; R 6x and R 6y Each of the groups independently represents R, R 6r and L 6 -R 6z selected from, with the proviso that R 6x and R 6y One of them is L 6 -R 6z or R 6r When , the other is R; L 6 is a linker; R 6r is (C=O)-O-(CH2) p -R 6v or (C=O)-(CH2) q -R 6v and; R 6v is R, OR, NHR, NR2, 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 C1-C6 alkyl. It has.

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

[0016] In certain embodiments, Q is CH2 and the compounds of the present invention have the structural formula (III) b ): [ka] [During the ceremony, R 6a’ , R 6b’ and R 6c’ Each of 6x’ R 6y’ selected from, with the proviso that R6a’ , R 6b’ and R 6c’ Only one of the 6x’ R 6y’ and each of the others is H; R 6x’ and R 6y’ Each of the groups independently represents R, R 6r’ and L 6’ -R 6z’ selected from, with the proviso that R 6x’ and R 6y’ One of them is L 6’ -R 6z’ or R 6r’ when the other is R'; L 6’ is a linker; R 6r’ is (C=O)-O-(CH2) p -R 6v’ or (C=O)-(CH2) q -R 6v’ and; R 6v’ is R, OR, NHR, NR2, 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 C1-C6 alkyl. It has.

[0017] In yet another aspect, the present invention generally relates to a compound of structural formula (IV): [ka] [During the ceremony, Ab stands for antigen-binding moiety; R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 is alkyl; R5 is NR 5a R 5b or CH2R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b form, together with the N atom to which they are attached, a 4- to 7-membered heterocyclic ring; and R 5c is H, halogen or OC 1-3 alkyl; R 7x is H or C 1-6 alkyl; L Ab is the linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

[0018] In yet another aspect, the present invention generally relates to compounds of structural formula (V) or (VI): [ka] or [ka] [During the ceremony, Ab represents antigen-binding moiety; Q is selected from CH2, O, S, S(O), S(O)2, NH and NCH3; R 1 is H or a halogen; R 2 is H or a halogen; R 5 is NR 5a R 5b or CH2R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R5a and R 5b form, together with the N atom to which they are attached, a 4- to 7-membered heterocyclic ring; and R 5c is H, halogen or OC 1-3 is alkyl; R 6x is H or C 1-6 is alkyl; L Ab is the linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

[0019] In yet another aspect, the present invention generally relates to a compound of formula (I)-(III) b ) and any of Table 1, or a pharmaceutically acceptable form thereof, and optionally a pharmaceutically acceptable excipient, carrier, or diluent.

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

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

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

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

[0024] In certain embodiments, the immunoconjugates disclosed herein are used to treat a disease or condition, wherein the disease or condition is an inflammatory disorder.

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

[0026] In yet another aspect, the invention generally relates to combinations comprising a therapeutically effective amount of a compound or immunoconjugate disclosed herein and one or more therapeutically active co-agents and / or adjuvants. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention The present invention is based in part on the discovery of novel glucocorticoid receptor agonists that have advantageous potency, stability and other profiles as payloads for immunoconjugates.

[0028] Key structural improvements to existing glucocorticoid receptor agonists include an anilino functional group at positions 17 / 16 for attachment to the antigen-binding site. Further fine-tuning of the payload molecule can be achieved by modification at position 20 to accommodate a wide range of ADC constructs and applications. Highly potent and stable glucocorticoid receptor agonists also combine sufficient solubility and low immunogenicity, making them suitable for development as novel therapeutics for immune complex and inflammatory disorders.

[0029] definition Unless otherwise defined, 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 and specific functional moieties and reactivities are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 2006.

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

[0031] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 16 is understood to include any number, combination of numbers, or subrange 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 particular value is understood to refer to that value and all values ​​greater than that value.

[0033] As used herein, "greater than 1" is understood to mean 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 terms "a," "the," and similar terms include plural referents unless the context clearly dictates otherwise.

[0035] Unless otherwise specified or clear from the context, the term "about" used herein is understood to mean within the normal tolerance in the art, for example, within 2 standard deviations of the mean value. "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 the context, all numerical values ​​provided herein can be modified by the term "about".

[0036] Unless otherwise stated or apparent from context, the term "or" as used herein is understood to be inclusive.

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

[0038] The reference to a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of groups set forth in the list. The reference to an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0039] When used to define compositions and methods, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" is intended to mean that the compositions and methods include the recited elements and exclude other elements of essential importance to the compositions and methods. For example, "consisting essentially of" refers to the administration of a specified pharmacologically active substance and excludes pharmacologically active substances not specified. The term "consisting essentially of" does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" is intended to mean excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transitional phrases are within the scope of the present 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, racemic mixtures thereof, and other mixtures thereof, as being within its scope. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be encompassed by the present invention. In certain embodiments, each asymmetric atom has either the R- or S-configuration in 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 optically active compounds, it is often preferable to use one enantiomer to the substantial exclusion of the other.

[0041] Isomeric mixtures containing any of a variety of isomeric ratios can be utilized in accordance with the present invention. For example, when only two isomers are combined, mixtures containing isomeric ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are contemplated by the present invention. Those skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomeric mixtures.

[0042] For example, if a specific enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, in which case the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group, such as an amino group, or an acidic functional group, such as a carboxyl group, diastereomeric salts can be formed with an appropriate optically active acid or base, and the diastereomers so formed can then be separated by fractional crystallization or chromatographic methods well known in the art, followed by recovery of the pure enantiomer.

[0043] Mixtures of isomers can be separated on the basis of the physical chemical differences of the components into 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 detailed below. When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1-6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, 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 is intended to be included.

[0045] Where substituents are defined by a conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that would occur if the structure were written from right to left, e.g., -C(=O)-O- is equivalent to -OC(=O)-.

[0046] The structure of the compound of the present invention is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted with one or more of many substituents, such substitutions are selected to comply with the principles of chemical bonding and to obtain a compound that is known to those skilled in the art to be not inherently unstable and / or likely to be unstable under ambient conditions (for example, aqueous, neutral, and some known physiological conditions).

[0047] As used herein, the term "alkyl" refers to a straight-chain, branched, or cyclic hydrocarbon radical (e.g., C 1-10 In this specification, whenever a numerical range such as "1 to 10" appears, it refers to each integer within the specified range; for example, "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 this definition also extends to the occurrence of the term "alkyl" without specifying a numerical range. In some embodiments, "alkyl" refers to C 1-6It can be an alkyl group. In some embodiments, the alkyl group has 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representative saturated linear 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 an 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(NRa )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 -OP(=O)(OR a )2(where each R a are independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein. In non-limiting embodiments, the substituted alkyl can be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.

[0048] The term "alkoxy" as used herein refers to an alkyl group containing 1 to 10 carbon atoms (C 1-10 "Alkoxy" refers to an -O-alkyl group that is attached to the parent molecular structure through an oxygen, in a straight-chain, branched, saturated cyclic form, and combinations thereof, including alkoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy, cyclohexyloxy, and the like. "Lower alkoxy" refers to an alkoxy group containing 1 to 6 carbons. In some embodiments, C 1-3Alkoxy is an alkoxy group, including both straight and branched chain alkyls of 1 to 3 carbon atoms. Unless stated otherwise in the specification, alkoxy groups 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 -OP(=O)(OR a )2(where each R aare independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0049] As used herein, the term "aromatic" or "aryl" refers to a cyclic group of 6 to 14 ring atoms (e.g., C 6-14 Aromatic or C 6-14 In some embodiments, aryl refers to a radical having C 6-10It is an aryl group. For example, a divalent radical formed from a substituted benzene derivative and having a free valence at a ring atom is named a substituted phenylene radical. In another embodiment, a divalent radical derived from a monovalent polycyclic hydrocarbon radical whose name ends in "-yl" by removing one hydrogen atom from the carbon atom having the free valence is named by adding "-idene" to the name of the corresponding monovalent radical. For example, a naphthyl group with two attachment points is called naphthylidene. In this specification, whenever a numerical range such as "6 to 14 aryl" appears, it refers to each integer within the specified range; for example, "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 sharing adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicyclic, tricyclic, tetracyclic, etc. In polycyclic groups, only one ring need be aromatic, thus groups such as indanyl are included in the definition of aryl. 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 is any of 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 -OP(=O)(OR a )2(where each R a are independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0050] As used herein, the terms "cycloalkyl" and "carbocyclyl" refer to monocyclic or polycyclic radicals, respectively, that contain only carbon and hydrogen and may be saturated or partially unsaturated. Unless otherwise stated in the specification, the terms are intended to include both substituted and unsubstituted cycloalkyl groups. A partially unsaturated cycloalkyl group may be referred to as a "cycloalkenyl" if the carbocyclic ring contains at least one double bond, or a "cycloalkynyl" if the carbocyclic ring contains at least one triple bond. A cycloalkyl group is a group having from 3 to 13 ring atoms (i.e., C 3-13In this specification, whenever a numerical range such as "3 to 10" appears, it refers to each integer within the specified range; for example, "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 ring structures that do not contain heteroatoms. The term also includes monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicyclic, tricyclic, tetracyclic, etc. In some embodiments, "cycloalkyl" refers to C 3-8 It can be a cycloalkyl radical. "Cycloalkyl" refers to a C 3-5 Specific examples of cycloalkyl groups include, but are not limited to, the following moieties: C 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. 3-7 Examples of carbocyclyl groups include norbornyl (C7). 3-8 Examples of the carbocyclyl group include those described above in C 3-7 Carbocyclyl groups include, but are not limited to, cycloheptyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the like. 3-13 Examples of carbocyclyl groups are those listed above. 3-8Unless stated otherwise in the specification, cycloalkyl groups 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 -OP(=O)(OR a )2(where each R aare independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein. The terms "cycloalkenyl" and "cycloalkynyl" reflect the description of "cycloalkyl" above, where the prefix "alk" is replaced with "alkene" or "alkyne," respectively, and the core "alkenyl" or "alkynyl" terms are as described herein. For example, cycloalkenyl groups can have 3 to 13 ring atoms, e.g., 5 to 8 ring atoms. In some embodiments, cycloalkynyl groups can have 5 to 13 ring atoms.

[0051] As used herein, the terms "heterocycle," "heterocyclic," or "heterocyclo" refer to a fully saturated or partially unsaturated cyclic group, e.g., a 3- to 7-membered monocyclic, a 7- to 12-membered bicyclic, or a 10- to 15-membered spirocyclic or tricyclic ring system, having at least one heteroatom (selected from the group consisting of N, O, and S) in at least one ring, where zero, one, two, or three atoms in each ring may be substituted with a substituent. Each ring of a heteroatom-containing heterocyclic group may have one, two, three, or four heteroatoms selected from nitrogen, oxygen, and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heterocyclic group may be bonded at any heteroatom or carbon atom in the ring or ring system. The heterocyclic group may be optionally substituted. Examples of heterocyclic groups include epoxy, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl, imidazolidinyl, imidazopyridinyl, thiazolidinyl, dithianyl, trithianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4-piperidinonyl, quinuclidinyl, thiomorpholinyl, thiomorpholinyl 1,1 dioxide, morpholinyl, azepanyl, oxazepanyl, azabicyclohexanyl, azabicycloheptanyl, azabicyclooctanyl, aza, Examples of cyclononanyl include, but are not limited to, bicyclononanyl (e.g., octahydroindolizinyl), azaspiroheptanyl, dihydro-1H,3H,5H-oxazolo[3,4-c]oxazolyl, tetrahydro-1′H,3′H-spiro[cyclopropane-1,2′-pyrrolidine], hexahydro-1H-pyrrolidinyl, hexahydro-1H-pyrrolo[2,1-c][1,4]oxazinyl, octahydroindolizinyl, oxaazaspirononanyl, oxaazaspirooctanyl, diazaspirononanyl, oxazabiocycloheptanyl, hexahydropyrrolidinyl 4(1H)-oxide, and tetrahydro-2H-thiopyranyl 1-oxide and tetrahydro-2H-thiopyranyl 1,1-dioxide.

[0052] The term "heterocycloalkyl," as used herein, refers to a cycloalkyl radical having one or more skeletal atoms selected from atoms other than carbon, such as O, N, S, P, or a combination thereof. Unless otherwise stated in the specification, the term is intended to include both substituted and unsubstituted heterocycloalkyl groups. Specific examples of heterocycloalkyl include 2-hydroxyaziridin-1-yl, 3-oxo-1-oxacyclobutan-2-yl, 2,2-dimethyl-tetrahydrofuran-3-yl, 3-carboxy-morpholin-4-yl, 1-cyclopropyl-4-methyl-piperazin-2-yl, 2-pyrrolinyl, 3-pyrrolinyl, dihydro-2H-pyranyl, 1,2,3,4-tetrahydropyridine, 3,4-dihydro-2H-[1,4]oxazine, and the like.

[0053] The term "halogen" as used herein 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 substituted with one or more halo groups or combinations thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy" respectively include haloalkyl and haloalkoxy groups in which the halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl, alkynyl, and alkoxy groups is 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 having one or more skeletal atoms selected from atoms other than carbon, such as oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), or combinations thereof. For example, C 1-4 Numerical ranges can be given to refer to the overall chain length of a heteroalkyl, such as a 4-atom length. For example, the -CH2OCH2CH3 radical is referred to as a "C4" heteroalkyl, including the heteroatom center in describing the atom chain length. Attachment 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 backbone atoms is a nitrogen atom. One or more heteroatoms in a heteroalkyl radical can be optionally oxidized. If one or more nitrogen atoms are present, they can also be optionally quaternized. For example, heteroalkyl also includes a backbone chain substituted with one or more nitrogen oxide (-O-) substituents. Exemplary heteroalkyl groups include, but are not limited to, ethers such as methoxyethanyl (-CH2CHOCH3), ethoxymethanyl (-CHOCH2CH3), (methoxymethoxy)ethanyl (-CH2CHOCH2OCH3), (methoxymethoxy)methanyl (-CH2OCH2OCH3), and (methoxyethoxy)methanyl (-CHOCH2CH2OCH3); amines such as CH2CH2NHCH3, -CH2CH2N(CH3)2, -CH2NHCH2CH3, -CH2N(CH2CH3)(CH3).

[0056] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a radical of a 5- to 18-membered monocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic, etc.) aromatic ring system (e.g., having 6, 10, or 14 π-electrons shared in a cyclic arrangement) having ring carbon atoms and 1 to 6 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5- to 18-membered heteroaryl"). Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. Whenever a numerical range such as "5 to 18" appears herein, it refers to each integer within the specified range; for example, "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, heteroaryl has a divalent radical, e.g., derived from a monovalent heteroaryl radical whose name ends in "-yl" by removing a hydrogen atom from the atom having the free valence, and named by adding "-ene" to the name of the corresponding monovalent radical (e.g., a pyridyl group with two points of attachment is 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 heteroatoms in a heteroaryl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quaternized. Heteroaryl also includes ring systems substituted with one or more nitrogen oxide (-O-) substituents, such as pyridinyl N-oxide. A heteroaryl is attached to the parent molecular structure through any atom of the ring.

[0058] "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, where the point of attachment to the parent molecular structure is on either the aryl or heteroaryl ring, or a heteroaryl ring, as defined above, is fused to one or more cycloalkyl or heterocyclyl groups, where the point of attachment to the parent molecular structure is on the heteroaryl ring. In the case of polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment to the parent molecular structure can be on either ring, i.e., on either the ring containing a heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl). In some embodiments, the heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5- to 10-membered heteroaryl"). In some embodiments, heteroaryl groups are 5-8 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl groups are 5-6 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, phosphorus, and sulfur ("5-6 membered heteroaryl"). In some embodiments, 5-6 membered heteroaryls have 1-3 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. In some embodiments, 5-6 membered heteroaryls have 1-2 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. In some embodiments, 5-6 membered heteroaryls have 1 ring heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur.

[0059] Examples of heteroaryl are azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzopyranonyl, and benzofurazanyl. nyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-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[1,2-c]pyridazinyl, dibenzo Zofuranil, dibenzothiophenyl, furanyl, furazanyl, furanonyl, 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-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-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]pyrimidinyl, 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 is any of 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(Ra )2, -N(R a )S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a )(R a ), or -OP(=O)(OR a )2(where each R a are independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, each of which moieties can be optionally substituted as defined herein.

[0060] As used herein, the terms "administer" and "administration" refer to oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intracranial administration, inhalation administration, intraocular administration, intranasal administration, subcutaneous administration, or implantation of a sustained-release device (e.g., a mini-osmotic pump) to a subject. The route of administration appropriate for a particular patient will depend on the nature and severity of the disease or condition being treated, or the nature of the therapy and the nature of the active compound being used.

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

[0062] As used herein, the term "co-administration" refers to the simultaneous presence of two agents in a subject's body (e.g., in the blood). The two agents can be administered concurrently or sequentially.

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

[0064] The term "agonist" as used herein refers to a compound that can combine with a receptor to produce a cellular response. An agonist can be a ligand that directly binds to the receptor. Alternatively, an agonist can bind indirectly to the receptor, for example, (a) by forming a complex with another molecule that directly binds to the receptor, or (b) by modifying 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 the agonist or inverse agonist on the receptor. However, an antagonist does not affect constitutive receptor activity.

[0066] As used herein, the term "amino acid" refers to a molecule of the general formula NH2-CHR-COOH, where "R" is one of many different side chains or a residue in a peptide having a parent amino acid. Amino acids include naturally occurring amino acids in which "R" is a substituent found in naturally occurring amino acids. "R" can also be a substituent not found in naturally occurring amino acids. The term "amino acid residue" refers to the portion of an amino acid that remains after the amino acid loses a water molecule when it combines with another amino acid. The term "modified amino acid" refers to an amino acid with an "R" substituent that does not correspond to one of the 20 genetically encoded amino acids.

[0067] The term "antigen" as used herein refers to any substance that causes the immune system to produce antibodies or a specific cell-mediated immune response against it. A disease-associated antigen refers to any substance associated with any disease that causes the immune system to produce antibodies or a specific cell-mediated immune response against it. An antigen can be recognized by the immune system and / or can induce a humoral immune response and / or a cellular immune response that activates B lymphocytes and / or T lymphocytes. An antigen can have one or more epitopes (B cell epitopes and / or T cell epitopes). An antigen preferably reacts with a corresponding antibody or TCR, typically highly selectively, and does not react with many other antibodies or TCRs that may be elicited by other antigens. An antigen as used herein may also be a mixture of several individual antigens.

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

[0069] As used herein, the term "antibody" refers to a molecule capable of binding an epitope or antigenic determinant. The term is intended to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, Fabs, Fvs, single-chain antibodies, single or multiple immunoglobulin variable chain or CDR domain designs, and bispecific and multispecific antibodies. Antibodies can be derived from any animal. Preferably, the antibodies are mammalian, e.g., human, mouse, rabbit, goat, guinea pig, camel, horse, etc., or other suitable animal. Antibodies can recognize polypeptide or polynucleotide antigens. The term includes, for example, antigen-binding fragments of immunoglobulins, active fragments including the heavy chain variable and / or constant regions, light chain variable and / or constant regions, complementarity-determining regions (CDRs), and framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as preparations comprising hybrid antibodies, modified antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (e.g., non-covalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragment derived from such molecules, which fragment retains 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 an epitope of an antigen, e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution.

[0071] Examples of binding fragments are single chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, V L , V H , C L and C H a monovalent fragment consisting of one domain; an F(ab)2 fragment, a bivalent fragment containing two Fab fragments disulfide-bonded at the hinge region; a V H and C H Fd fragment consisting of one domain; V of a single arm of an antibodyL and V H Fv fragment consisting of domains; V H These include, but are not limited to, dAb fragments consisting of domains (Ward et al. 1989 Nature 341:544-546); as well as isolated complementarity determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0072] Furthermore, the two domains V of the Fv fragment L and V H used recombinant methods to L and V H The domains can be joined by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule (known as a single-chain Fv ("scFv"); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al. 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0073] Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005 Nature Biotechnology 23:1 126-1136). Antigen-binding fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see, e.g., U.S. Patent No. 6,703,199, which describes monobodies ... a pair of tandem Fv segments (V H -C H 1-V H -C H1), 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. Patent No. 5,641,870).

[0074] As used herein, the term "bispecific antibody" or "bispecific" refers to an antibody, typically a monoclonal antibody, that has binding specificities for at least two different antigenic epitopes. The epitopes may be from the same antigen or from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using coexpression of two immunoglobulin heavy / light chain pairs. Alternatively, bispecific antibodies can be prepared using chemical conjugation. Bispecific antibodies also 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. USA 90:6444-48; Gruber et al. 1994 J. Immunol. 152:5368-74).

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

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

[0077] The term "humanized antibody" as used herein refers to an antibody comprising non-human (e.g., murine) and human antibody sequences. Such antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region (Fc) (see, e.g., Cabilly U.S. Patent 4,816,567; Queen et al. 1989 Proc. Nat'l 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 substantially homogeneous population of antibodies (i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations). Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number 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, monoclonal antibodies used in accordance with the present invention can be produced by a variety of methods known in the art, including the hybridoma method first described by Kohler et al. 1975 Nature 256:495, or can be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries, using, for example, the techniques described in Clackson et al. 1991 Nature 352:624-628 and Marks et al. 1991 J. Mol. Biol.222:581-597. These monoclonal antibodies generally bind with a Kd of at least about 1 μM, more usually at least about 300 nM, typically at least about 30 nM, and preferably at least about 10 nM.

[0079] As used herein, the terms "biologically active" or "biologically active" refer to those that possess the structural, regulatory, or biochemical functions of naturally occurring molecules, or any function associated with or associated with a metabolic or physiological process. Biologically active polypeptides or fragments thereof include those that can participate in a biological process or reaction and / or produce a desired effect. Biological activity can include improved desired activity or decreased undesirable activity. For example, an entity exhibits biological activity if it participates in a molecular interaction with another molecule, has therapeutic value in alleviating a disease state, has preventative value in inducing an immune response, or has diagnostic and / or prognostic value in determining the presence of a molecule. Biologically active proteins or polypeptides can be naturally occurring, synthesized from known components (e.g., by recombinant or chemical synthesis), or can include heterologous components.

[0080] As used herein, the terms "inflammatory disease" and "inflammatory disorder" refer to, but are not limited to, diseases caused by the biological response of vascular tissue to harmful stimuli, including but not limited to, pathogens, damaged cells, irritants, antigens, and, in the case of autoimmune diseases, substances and tissues normally present in the body. For example, inflammatory diseases or disorders can be autoimmune, microbial, metabolic, neoplastic, and post-traumatic diseases or disorders. Non-limiting examples of inflammatory diseases or disorders include rheumatoid arthritis (RA), enthesitis-related arthritis (ERA), ankylosing spondylitis, psoriatic arthritis, atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, graft rejection, and vasculitis.

[0081] The term "cleavable" linker, as used herein, refers to a linker or linker component that covalently links two moieties but decomposes under physiologically relevant conditions to sever the covalent bond between the moieties. Typically, a cleavable linker is cleaved more rapidly in an intracellular environment than in an extracellular environment in vivo, with payload release occurring preferentially within the target cell. Cleavage can be enzymatic or non-enzymatic. The payload is typically released from the antibody without degrading the antibody. Cleavage can leave a portion of the linker or linker component attached to the payload, or it can result in payload release without any remaining linker portion or component (i.e., traceless release).

[0082] The term "non-cleavable" linker, as used herein, refers to a linker or linker component that is not particularly susceptible to degradation under physiological conditions, i.e., a linker that 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 that they are sufficiently resistant to degradation to keep the payload bound to the antigen-binding portion until the antigen-binding portion itself is at least partially degraded. In such cases, in vivo degradation of the Ab precedes cleavage of the linker. 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 the antibody attached to the payload or drug moiety to be delivered in vivo.

[0083] As used herein, the term "cell" refers to any group of such cells in prokaryotes, eukaryotes, primary or immortalized cell lines, tissues or organs, etc. Preferably, the cells are of mammalian (e.g., human) origin and can be infected by one or more pathogens.

[0084] The term "payload" as used herein refers to a compound or substance that inhibits, blocks, or stops the expression activity, function, and / or causes destruction of a cell. The term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule 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 state, e.g., a state that can be identified by symptoms or other distinguishing factors as deviating from a healthy or normal state. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative diseases, inflammatory diseases, immune diseases, metabolic diseases, infectious diseases, and ischemic diseases.

[0086] As used herein, the term "homology" or "homology" refers to sequence similarity between two polypeptides or two polynucleotides. Similarity can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a given position in two polypeptide sequences is not identical, the similarity or conservation of that position can be determined by assessing the similarity of the amino acids at that position. The degree of similarity between sequences is a function of the number of matching or homologous positions shared by the sequences. Alignment of two sequences to determine percent sequence similarity can be performed using software programs known in the art, for example, as described in Ausubel et al. 1999 Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD. The term "homolog" with respect to a given amino acid or nucleic acid sequence is intended to indicate that the corresponding sequence of the "homolog" has substantial identity or homology with the given amino acid or nucleic acid sequence.

[0087] In sequence comparison, typically, one sequence serves as reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, partial sequence coordinate is designated, and sequence algorithm program parameter is designated.Preferably, default program parameter can be used, or alternative parameter can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameter.

[0088] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al. 1977 Nuc. Acids Res. 25:3389-3402 and Altschul et al. 1990 J. Mol. Biol. 215:403-410, respectively. BLAST software is publicly available on the World Wide Web through the National Center for Biotechnology Information at ncbi.nlm.nih.gov / . Both default and 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 default a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) uses as defaults an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0089] The term "identical" or percent "identity," as used herein in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides identical (i.e., about 70% identity over a specified region, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity, when compared and aligned for maximum correspondence over a comparison window or designated region) as determined by the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below, or by manual alignment and visual inspection. Such sequences are said to be "substantially identical." This definition also refers to or can apply to the complement of a test sequence. This definition includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the entire length of the amino acid or nucleic acid sequence.

[0090] The compounds of the present invention can be administered to a patient either alone or in combination. Co-administration is intended to include simultaneous or sequential administration (of more than one compound or agent), either alone or in combination. Thus, if desired (e.g., to reduce metabolic degradation), the formulation can also be combined with other active substances.

[0091] The compositions of the present invention can be delivered transdermally by topical routes by being formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral dosage forms include tablets, pills, powders, dragees, capsules, liquids, troches, cachets, gels, syrups, slurries, suspensions, and the like suitable for ingestion by patients. Solid dosage forms include powders, tablets, pills, capsules, cachets, suppositories, and granules. Liquid dosage forms include solutions, suspensions, and emulsions, gels, such as water or water / propylene glycol solutions.

[0092] The compositions of the present invention can further comprise components for providing sustained release and / or comfort.Such components include high molecular weight anionic mucus-mimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates.These components are further discussed in U.S. Patent 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 sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres for slow subcutaneous release (see Rao, 1995 J. Biomater Sci. 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 who would benefit biologically, medically, or in quality of life from such treatment.

[0094] As used herein, the terms "specifically bind" or "selectively bind," when used in the context of describing the interaction between an antigen (e.g., a protein or glycan) and an antibody, antibody fragment, or antibody-derived binder, refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, such as a biological sample (e.g., blood, serum, plasma, or tissue sample). Thus, under specified immunoassay conditions, an antibody or binder with a particular binding specificity binds to a specific antigen at least twice as much as background and does not bind significantly to other antigens present in the sample. In certain embodiments, under specified immunoassay conditions, an antibody or binder with a particular binding specificity binds to a specific antigen at least 10 times as much as background and does not bind significantly to other antigens present in the sample. To specifically bind to an antibody or binder under such conditions, the antibody or binder may need to be selected for its specificity for a particular protein. If desired or appropriate, this selection can be achieved by excluding antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some embodiments, an antibody or antibody fragment is selected that cross-reacts with a desired molecule.

[0095] A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact 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 measure specific immunoreactivity.) Typically, a specific or selective binding reaction produces a signal that is at least two times background, and more typically, at least 10 to 100 times background.

[0096] As used herein, the term "therapeutically effective amount" refers to a dose of a therapeutic agent or drug 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, for example, by administering a low dose of the drug initially and then gradually 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 having an antigen-binding moiety (e.g., an antibody or antigen-binding fragment thereof, a peptide, or a small molecule ligand) linked to a cytotoxic agent or payload. The linkage may be covalent or non-covalent, and may include chelation. Thus, the terms "immunoconjugate" and "antibody-drug conjugate" include peptide-drug conjugates and small molecule-drug conjugates. A variety of linkers and linking strategies are known in the art and can be used to form immunoconjugates.

[0098] As used herein, the terms "inhibition," "inhibit," and "inhibitory," etc., with respect to biological target-inhibitor interactions, refer to negatively affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In certain embodiments, inhibition refers to negatively affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In certain embodiments, suppression refers to the alleviation of a disease or disease symptoms. In certain embodiments, inhibition refers to a decrease in the activity of a specific protein target. Inhibition includes at least partially, partially, or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction or enzymatic activity or the amount of a protein. In certain embodiments, inhibition refers to a decrease in the activity of a target protein due to a direct interaction (e.g., an inhibitor binding to a target protein). In certain embodiments, inhibition refers to a decrease in the activity of a target protein resulting from an indirect interaction (e.g., an inhibitor binding to a protein that activates the target protein, thereby preventing activation of the target protein).

[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 natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis and high-performance liquid chromatography. The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0100] As used herein, the term "modulate" refers to directly or indirectly increasing or decreasing, stimulating, inhibiting, interfering, or blocking measured activity when compared to an appropriate control. A "modulator" of a polypeptide or polynucleotide refers to a substance that affects, for example, increases, decreases, stimulates, inhibits, interferes, or blocks, the measured activity of a polypeptide or polynucleotide when compared to an appropriate control. For example, a "modulator" can bind to a target with measurable affinity and / or activate or inhibit a target, or directly or indirectly affect the normal regulation of receptor activity.

[0101] As used herein, "pharmaceutically acceptable forms" of the disclosed compounds include, but are not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, isomers, prodrugs, and isotopically labeled derivatives thereof. In certain embodiments, "pharmaceutically acceptable forms" include, but are not limited to, pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives thereof. In some embodiments, "pharmaceutically acceptable forms" include, but are not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives thereof.

[0102] In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt.

[0103] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or the like, and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe 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 non-toxic acid addition salts include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, and malonic acid, or 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, hemisulfonate, heptanoate, hexanoate, hydroiodide, 2-hydroxybenzoate ... -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, 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.

[0104] Such salts can be prepared in situ during the isolation and purification of the disclosed compounds, or can be prepared separately, such as by reacting the free base or free acid of the parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-alkane salts. + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts, and amine cation salts, formed, where appropriate, with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. 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 selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0105] In certain embodiments, the pharmaceutically acceptable form is a "solvate" (e.g., a hydrate). As used herein, the term "solvate" refers to a compound that further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. The solvate may be of the disclosed compound or a pharmaceutically acceptable salt thereof. When the solvent is water, the solvate is a "hydrate". Pharmaceutically acceptable solvates and hydrates are complexes that may contain, for example, 1 to about 100, or 1 to about 10, or 1 to about 2, about 3, or about 4 solvent or water molecules. As used herein, the term "compound" is understood to include compounds and solvates of compounds, as well as mixtures thereof.

[0106] In certain embodiments, the pharmaceutically acceptable form is a prodrug. As used herein, the term "prodrug" refers to a compound that is converted in vivo to produce the disclosed compound or a pharmaceutically acceptable form of the compound. A prodrug may be inactive when administered to a subject, but is converted to an active compound in vivo, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical and / or delivery properties compared to the parent compound. A prodrug can improve the bioavailability of a compound when administered to a subject (e.g., by facilitating absorption into the blood after oral administration) or enhance delivery to the desired biological compartment (e.g., the brain or lymphatic system) compared to the parent compound. Exemplary prodrugs include derivatives of the disclosed compounds that have enhanced water solubility or active transport across the intestinal membrane relative to the parent compound.

[0107] Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms. (See, e.g., Bundgard, H. 1985 Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam; Higuchi et al. 1987 "Prodrugs as Novel Delivery Systems" ACS Symposium Series, Vol. 14, Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.)

[0108] Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms. (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 generally known in the art include well-known acid derivatives, such as esters prepared by reacting a parent acid with an appropriate alcohol, amides prepared by reacting a parent acid compound with an amine, and basic groups reacted to form acylated base derivatives. Other prodrug derivatives can be combined with other features disclosed herein to enhance bioavailability. Thus, those skilled in the art will understand that certain of the compounds disclosed herein having free amino, amide, hydroxy, or carboxyl 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.

[0109] Exemplary advantages of a prodrug include, but are not limited to, physical properties such as improved water solubility for parenteral administration at physiological pH, improved absorption from the gastrointestinal tract, or improved drug stability for long-term storage compared to the parent compound.

[0110] 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, that is involved in carrying or transporting a drug of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, 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, corn 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate, magnesium stearate, polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0111] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to polymers 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 this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, polypeptide may refer to proteins containing modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains the desired activity. These modifications can be intentional or 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.

[0112] As used herein, the term "receptor" refers to a protein, including a glycoprotein or fragment thereof, that can interact with another molecule called a ligand. A ligand is typically an extracellular molecule that, upon binding to a receptor, typically initiates a cellular response, such as the initiation of a signal transduction pathway. A receptor is not necessarily a membrane-bound protein. A ligand can belong to any class of biochemical or chemical compound.

[0113] The term "sample" as used herein refers to a sample of human or animal origin or a research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. A "sample" may be tested in vivo, e.g., without removal from a human or animal, or in vitro. A sample may also be tested after processing, e.g., by histological methods. A "sample" may also refer, for example, to cells comprising a fluid or tissue sample, or cells separated from a fluid or tissue sample. A "sample" may also refer to fresh cells, tissues, organs, or fluids freshly removed from a human or animal, or to cells, tissues, organs, or fluids that have been processed or preserved.

[0114] As used herein, the term "stimulate" or "stimulatory" refers to increasing, amplifying, enhancing, or enhancing a physiological activity, such as an immune response. Stimulation can be a positive change. For example, the increase can be a 5%, 10%, 25%, 50%, 75%, or 90-100% increase. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or 100-fold.

[0115] The term "subject" as used herein refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Subjects contemplated for administration include, but are not limited to, humans (e.g., males or females of any age, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)), and / or other non-human animals, e.g., non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cows, 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 male or female, regardless of the stage of development. The non-human animal may be a transgenic animal. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

[0116] As used herein, the term "inhibit" or "inhibitory" refers to decreasing, attenuating, reducing, halting, or stabilizing a physiological activity, such as an immune response. Inhibition can be a negative change. For example, the decrease can be a 5%, 10%, 25%, 50%, 75%, or 90-100% decrease. Exemplary decreases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or 100-fold decrease.

[0117] As used herein, the term "treatment" or "treating" of a disease or disorder refers to a method for alleviating, delaying, or ameliorating such a condition before or after it occurs. Treatment can be directed at one or more effects or symptoms of the disease and / or underlying pathology. Treatment can be any alleviation, including, but not limited to, complete elimination of the disease or symptoms of the disease. Thus, "treating" or "treatment" refers to any measure of success in treating or ameliorating an injury, disease, pathology, or condition, including any objective or subjective parameter, such as attenuation, remission, alleviation of symptoms, or making the injury, pathology, or condition more tolerable to the patient, slowing the rate of degeneration or decline, reducing the degree of debilitating end-point of degeneration, or improving the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on objective or subjective parameters, such as the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The degree of such reduction or improvement, as compared to an equivalent untreated control, can be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique.

[0118] The treatment method includes administering a therapeutically effective amount of a compound described herein to a subject. The administration step may be a single administration or may include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the compound, the activity of the composition used in the treatment, or a combination thereof. It is also understood that the effective amount of the agent used in the treatment may increase or decrease over the course of a particular treatment regimen. Changes in dosage can be guided and identified by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient.

[0119] Glucocorticoid receptor agonists A variety of novel glucocorticoid receptor agonists are disclosed herein.

[0120] In some embodiments, the present invention generally relates to compounds of structural formula (I): [ka] [During the ceremony, R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 alkyl, and R 4 is OC(O)-R 7 where R 7 is a substituted or unsubstituted aniline, or R 3 and R 4 together with the carbon atoms to which they are attached, 6 wherein W is a single bond or -phenyl-Q-, Q is selected from CH, O, S, S(O), S(O), NH, and NCH, and R 6 is a substituted or unsubstituted aniline; and R 5 is NR 5a R 5b or CH2R 5c and R 5aおよび R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b together with the N atom to which they are attached 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 OC 1-3 It is alkyl. or a pharmaceutically acceptable form thereof.

[0121] In certain embodiments, R 4 is OC(O)-R 7and the compound has structural formula (II): [ka] It has.

[0122] In certain embodiments, the compound has the structural formula (II a ):

[0123] [ka] [During the ceremony, R 7a , R 7b and R 7c Each of 7x R 7y selected from, with the proviso that R 7a , R 7b and R 7c Only one of the 7x R 7y and each of the others is H; R 7x and R 7y Each of the groups independently represents R, R 7r and L 7 -R 7z selected from, with the proviso that R 7x and R 7y One of them is L 7 -R 7z or R 7r When , the other is R; L 7 is a linker; R 7r is (C=O)-O-(CH2) i -R 7v or (C=O)-(CH2) j -R 7v and; R 7v is R, OR, NHR, NR2, 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 7zcomprises a functional or reactive group; and R is H or C1-C6 alkyl. It has.

[0124] In certain embodiments, R 7a is H and R 7b is NR 7x R 7y and R 7c is H.

[0125] In certain embodiments, R 7a is H and R 7b is H, and R 7c is NR 7x R 7y is.

[0126] In certain embodiments, R 7a is NR 7x R 7y and R 7b is H, and R 7c is H.

[0127] In certain embodiments, R 7x is H or CH3, and R 7y is (C=O)-O-(CH2) i -R 7v where R 7v is R, OR, NHR, NR2, an aryl group, or an amino acid, and i is 0, 1, 2, or 3.

[0128] In certain embodiments, R 7x is H or CH3, and R 7y is (C=O)-(CH2) j -R 7v where R 7v is R, OR, NHR, NR2, an aryl group, or an amino acid, and j is 0, 1, 2, or 3.

[0129] In certain embodiments, R 3 is alkyl. In certain embodiments, R3 is methyl.

[0130] In certain embodiments of (I), R 3 and R 4 together with the carbon atoms to which they are attached, 6 forming a five-membered dioxolane substituted with the structural formula: [ka] It has.

[0131] In certain embodiments, W is a single bond and the compounds of the present invention have structural formula (III): [ka] It has.

[0132] In certain embodiments, the compound has the structural formula (III a ): [ka] [During the ceremony, R 6a , R 6b and R 6c Each of 6x R 6y selected from, with the proviso that R 6a , R 6b and R 6c Only one of the 6x R 6y and each of the others is H; R 6x and R 6y Each of the groups independently represents R, R 6r and L 6 -R 6z selected from, with the proviso that R 6x and R 6y One of them is L 6 -R 6z or R 6r When , the other is R; L 6 is a linker; R 6r is (C=O)-O-(CH2) p -R 6v or (C=O)-(CH2) q -R 6v and; R 6v is R, OR, NHR, NR2, 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 C1-C6 alkyl. It has.

[0133] In certain embodiments, R 6 has the S configuration.

[0134] In certain embodiments, R 6 has the R configuration.

[0135] In certain embodiments, R 6a is H and R 6b is NR 6x R 6y and R 6c is H.

[0136] In certain embodiments, R 6a is H and R 6b is H, and R 6c is NR 6x R 6y is.

[0137] In certain embodiments, R 6a is NR 6x R 6y and R 6b is H, and R 6c is H.

[0138] In certain embodiments, R 6x is H or CH3, and R6y is (C=O)-O-(CH2) p -R 6v where R 6v is R, OR, NHR, NR2, an aryl group or an amino acid, and p is 0, 1, 2 or 3.

[0139] In certain embodiments, R 7x is H or CH3, and R 7y is (C=O)-(CH2) q -R 7v where R 7v is R, OR, NHR, NR2, an aryl group or an amino acid, and q is 0, 1, 2 or 3.

[0140] In certain embodiments, W is -phenyl-Q-.

[0141] In certain embodiments, Q is CH2 and the compounds of the present invention have the structural formula (III) b ): [ka] [During the ceremony, R 6a’ , R 6b’ and R 6c’ Each of 6x’ R 6y’ selected from, with the proviso that R 6a’ , R 6b’ and R 6c’ Only one of the 6x’ R 6y’ and each of the others is H; R 6x’ and R 6y’ Each of the groups independently represents R, R 6r’ and L 6’ -R 6z’ selected from, with the proviso that R 6x’ and R 6y’ One of them is L 6’ -R 6z’ or R 6r’ when the other is R'; L 6’is a linker; R 6r’ is (C=O)-O-(CH2) p -R 6v’ or (C=O)-(CH2) q -R 6v’ and; R 6v’ is R, OR, NHR, NR2, 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 C1-C6 alkyl. It has.

[0142] In certain embodiments, WR 6 The carbon atom of the five-membered dioxolane bonded to has the S configuration.

[0143] In certain embodiments, WR 6 The carbon atom of the five-membered dioxolane bonded to has the R configuration.

[0144] In certain embodiments, R 6a’ is H and R 6b’ is NR 6x’ R 6y’ and R 6c’ is H.

[0145] In certain embodiments, R 6a’ is H and R 6b’ is H, and R 6c’ is NR 6x’ R 6y’ is.

[0146] In certain embodiments, R 6a’ is NR 6x’ R 6y’ and R 6b’ is H, and R 6c’ is H.

[0147] In certain embodiments, R 6x’ is H or CH3, and R 6y’ is (C=O)-O-(CH2) p -R 6v’ where R 6v’ is R, OR, NHR, NR2, an aryl group or an amino acid, and p is 0, 1, 2 or 3.

[0148] In certain embodiments, R 6x’ is H or CH3, and R 6y’ is (C=O)-(CH2) q -R 6v’ where R 6v’ is R, OR, NHR, NR2, an aryl group or an amino acid, and q is 0, 1, 2 or 3.

[0149] In certain embodiments, L 6 or L 7 , or L 6’ is a non-cleavable linker when present in a compound.

[0150] In certain embodiments, L 6 or L 7 , or L 6’ is a cleavable linker when present in the compound.

[0151] In certain embodiments, L 6 or L 7 , or L 6’ is an acid-labile or acid-sensitive linker when present in the compound.

[0152] In certain embodiments, L 6 or L 7 , or L 6’ is a protease-sensitive linker when present in a compound.

[0153] In certain embodiments, L 6 or L 7 , or L 6’is a lysosomal protease-sensitive linker when present in the compound.

[0154] In certain embodiments, L 6 or L 7 , or L 6’ is a β-glucuronide sensitive linker when present in the compound.

[0155] In certain embodiments, L 6 or L 7 , or L 6’ is a glutathione-sensitive disulfide linker when present in the compound.

[0156] In certain embodiments, R 6z or R 7z , or R 6z’ When present in a compound, -N3, -NR u C(=O)CH=CH2, -SH, -SSR t , -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NR u S(=O2)(CH=CH2), -NR u C(=O)CH2R w , -NR u C(=O)CH2Br, -NR u C(=O)CH2I, -NHC(=O)CH2Br, NHC(=O)CH2I, -ONH2, -C(=O)NHNH2, -CO2H, -NH2, -NCO, -NCS, [ka] [ka] wherein the functional or reactive group is selected from: R u is H or a C1-C6 alkyl group, R t is 2-pyridyl or 4-pyridyl, and R w teeth, [ka] is.

[0157] In certain embodiments, R 5 is NR 5a R 5b is.

[0158] In certain embodiments, R 5 is N(CH3)2.

[0159] In certain embodiments, R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered unsubstituted or substituted heterocyclic ring.

[0160] In certain embodiments, the heterocyclic ring is an unsubstituted 4-, 5-, or 6-membered heterocycle.

[0161] In certain embodiments, the heterocyclic ring is selected from the group consisting of OH and C 1-3 alkyl, wherein C 1-3 The alkyl may be optionally substituted with one or more OH groups.

[0162] In certain embodiments, R 5a and R 5b are independently H and C optionally substituted with one or more OH groups; 1-6 alkyl.

[0163] In certain embodiments, R 5a is H or methyl, and R 5b C is substituted with OH 1-6 It is alkyl.

[0164] In certain embodiments, R 5a is H or methyl, and R 5b C is substituted with two OH groups 2-6It is alkyl.

[0165] In certain embodiments, R 5 is CH2R 5c In certain embodiments, R 5c is F.

[0166] In certain embodiments, R 5c is Cl.

[0167] In certain embodiments, R 5c OC 1-3 It is alkyl.

[0168] In certain embodiments, R 1 and R 2 Each of is H.

[0169] In certain embodiments, R 1 and R 2 Each of is F.

[0170] In certain embodiments, R 1 and R 2 One of the is H and the other is F.

[0171] R 6z , R 7z or R 6z’ and / or L 6 , L 7 or L 6’ Further disclosure regarding linkers and reactive or functional groups that can be used in the components of is provided in the "Linkers and Linking Techniques" and "Linker-Antibody and Linker-Payload Conjugations" sections and the references cited therein, each of which is incorporated herein by reference.

[0172] The present invention also includes methods for synthesizing glucocorticoid receptor agonists, including intermediates or precursors thereof.

[0173] Non-limiting examples of glucocorticoid receptor agonists of the present invention include those listed in Table 1 or pharmaceutically acceptable forms thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [In the formula, R 1 and R 2 are each independently H or halogen (e.g., F).

[0174] In certain embodiments of the compounds of Table 1, R 1 and R 2 Each of is H.

[0175] In certain embodiments of the compounds of Table 1, R 1 and R 2 Each of is F.

[0176] In certain embodiments of the compounds of Table 1, R 1 and R 2 One of them is H and the other is F.

[0177] Methods for determining the 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).

[0178] immune complex A typical ADC is composed of an antigen-binding moiety (Ab), e.g., a monoclonal antibody, a linker (L), and a cytotoxic agent or payload (D), as depicted below: (D m -L) n -Ab wherein each of m and n is an integer. Payload D (e.g., a glucocorticoid receptor agonist disclosed herein) can be attached to a different portion of the Ab, typically via a cysteine ​​or lysine residue. Generally, more than one payload D molecule can be attached to each Ab. When a branched linker is used, more than one payload D moiety can be attached to each linker L. In some embodiments, n is in the range of 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 is in the range of 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.

[0179] The drug-to-antibody ratio (DAR) or drug loading can be characterized by conventional means such as UV, mass spectrometry, ELISA assay, HIC, HPLC, or electrophoresis. In exemplary embodiments, the DAR is in the range of 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.

[0180] The DAR of the immunoconjugate can be controlled by various methods, including limiting the molar excess of the payload-linker intermediate or linker reagent relative to the antigen-binding moiety, limiting the conjugation reaction time or temperature, varying the reductive conditions for cysteine ​​thiol modification, and altering the number and position of cysteine ​​residues and the position of the linker-payload bond (see, e.g., WO 2006 / 034488 A2).

[0181] In one aspect, the present invention generally relates to immunoconjugates comprising a compound disclosed herein covalently attached to an antigen-binding moiety.

[0182] In another aspect, the present invention generally relates to a compound of structural formula (IV): [ka] [During the ceremony, Ab stands for antigen-binding moiety; R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 is alkyl; R 5 is NR 5a R 5b or CH2R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b form, together with the N atom to which they are attached, a 4- to 7-membered (e.g., 4-, 5-, 6-, or 7-membered) heterocyclic ring; and R 5c is H, halogen or OC 1-3 is alkyl; R 7x is H or C 1-6 is alkyl; L Abis the linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

[0183] In certain embodiments, R 1 , R 2 , R 3 , R 5 and R 7x are the compounds of formulas (I), (II) and (II a ) can be selected as disclosed.

[0184] In certain embodiments, R 3 is alkyl. In certain embodiments, R 3 is methyl.

[0185] In certain embodiments, R 7x is H.

[0186] In certain embodiments, R 7x is C 1-6 It is alkyl. In yet another aspect, the present invention generally relates to compounds of structural formula (V) or (VI): [ka] or [ka] [During the ceremony, Ab represents antigen-binding moiety; Q is selected from CH2, O, S, S(O), S(O)2, NH and NCH3; R 1 is H or a halogen; R 2 is H or a halogen; R 5 is NR 5a R 5b or CH2R 5c and R 5a and R5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b form, together with the N atom to which they are attached, a 4- to 7-membered heterocyclic ring; and R 5c is H, halogen or OC 1-3 is alkyl; R 6x is H or C 1-6 is alkyl; L Ab is the linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

[0187] In certain embodiments, Q is CH2 and the immunoconjugate has the structural formula: [ka] It has.

[0188] In certain embodiments, Q is CH2 and the immunoconjugate has the structural formula: [ka] It has.

[0189] In certain embodiments, R 1 , R 2 , R 3 , R 5 and R 6x (I)~(III) b ) may be selected as disclosed herein.

[0190] In certain embodiments, R 6x is H.

[0191] In certain embodiments, R 6x is C 1-6 It is alkyl.

[0192] In certain embodiments, R 1 and R 2 Each of is H.

[0193] In certain embodiments, R 1 and R 2 Each of is F.

[0194] In certain embodiments, R 1 and R 2 One of the is H and the other is F.

[0195] In certain embodiments, R 5 is NR 5a R 5b is.

[0196] In certain embodiments, R 5 is N(CH3)2.

[0197] In certain embodiments, R 5 is CH2R 5c is.

[0198] In certain embodiments, R 5c is F.

[0199] In certain embodiments, R 5c is Cl.

[0200] In certain embodiments, R 5c is O.C. 1-3 It is alkyl.

[0201] In certain embodiments of Formulas (IV)-(VI), n is an integer ranging from 1 to 20. In certain embodiments, n is an integer ranging from 1 to 16. In certain embodiments, n is an integer ranging from 1 to 12. In certain embodiments, n is an integer ranging from 1 to 10. In certain embodiments, n is an integer ranging from 1 to 8. In certain embodiments, n is an integer ranging from 1 to 6. In certain embodiments, n is an integer ranging from 1 to 5. In certain embodiments, n is an integer ranging from 1 to about 4. In certain embodiments, n is an integer ranging from 1 to 3. In certain embodiments, n is 1 or 2. In certain embodiments, n is 1.

[0202] All substituents found in formulae (IV) to (VI), such as R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 is represented by the formulas (I) to (III b ) may be selected as described in the section entitled "Glucocorticoid Receptor Agonists" in connection with R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 Each and every combination of the Ab binding formulas (I) to (III) is incorporated herein in its entirety, including the resulting compounds. b ) containing immune complexes corresponding to the

[0203] In addition to immunoconjugates in which the antigen-binding moiety is an antibody or antibody fragment, the present invention also includes immunoconjugates in which the antigen-binding moiety is a peptide and immunoconjugates in which 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).

[0204] The present invention also includes methods for the synthesis of immunoconjugates, including intermediates or precursors thereof. The present invention further includes compositions comprising the immunoconjugates, intermediates or precursors thereof.

[0205] antigen binding part To date, numerous unique antigens have been identified and can be used as targets in antibody-based therapies. Several factors are generally considered when selecting an antigen. First, the target antigen should be highly expressed in the target tissue or cell and absent or low expression in healthy cells. Second, the target antigen should be expressed on the surface of the tissue or cell so that circulating monoclonal antibodies are available. Furthermore, the target antigen should possess internalization properties to facilitate intracellular transport of the ADC and enhance payload efficacy. However, several studies have shown that non-internalizing ADC formulations targeting components of the tumor microenvironment can efficiently release the drug in the extracellular space, potentially exerting potent therapeutic activity, and that ADCs often induce a strong "bystander effect." (Strohl WR 2018 Protein & Cell. 9(1):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).

[0206] The antigen-binding moiety can be any moiety that selectively binds to a cell surface marker found on the target cell type. Generally, the antibody should preferably have target specificity, deliver the payload to the target tissue or cell, and have target binding affinity, i.e., high binding affinity to the target tissue or cell. More preferably, the antibody should have good retention, low immunogenicity, low cross-reactivity, and appropriate tethering binding properties. (Peters et al. 2015 Bioscience Reports 35(4); Hughes B 2010 Nature Reviews Drug Discovery 9(9):665-7.)

[0207] In certain embodiments, the Ab is an antibody.

[0208] In certain embodiments, the Ab is a monoclonal antibody.

[0209] In certain embodiments, the Ab is a chimeric antibody.

[0210] In certain embodiments, the Ab is a humanized antibody.

[0211] In certain embodiments, the Ab is a bispecific antibody.

[0212] In certain embodiments, the Ab is an antibody fragment.

[0213] In certain embodiments, the Ab is a Fab fragment.

[0214] In certain embodiments, the Ab is a peptide.

[0215] In certain embodiments, the Ab is a small molecule ligand.

[0216] In some embodiments, the Ab is an antibody or antibody fragment (eg, an antigen-binding fragment of an antibody).

[0217] In some embodiments, the Ab is an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to a cell surface receptor protein or other cell surface molecule, a cell survival regulator, a cell proliferation regulator, a molecule associated with tissue development or differentiation, a molecule known or suspected to functionally contribute to tissue development or differentiation, a lymphokine, a cytokine, a molecule involved in cell cycle regulation, a molecule involved in vasculogenesis, a molecule associated with angiogenesis, or a molecule known or suspected to functionally contribute to angiogenesis.

[0218] Thus, antigen-binding moieties useful in the immunoconjugates of the invention include, but are 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.

[0219] Non-limiting examples of antibodies or antigen-binding fragments relevant to inflammatory diseases and disorders include adalimumab, infliximab, certolizumab, afelimomab, nerelimomab, ozoralizumab, golimumab, and placulumab.

[0220] Antibodies and antibody fragments useful in the immunoconjugates of the invention include modified or engineered antibodies, such as antibodies modified to introduce a cysteine ​​residue or other reactive amino acid, including Pel, pyrrolysine, peptide tags and unnatural 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 attachment to a payload agent.

[0221] The location of the drug moiety can be designed, controlled, and known. For example, cysteine ​​amino acids can be engineered into reactive sites of antibodies so that they do not form intrachain or intermolecular disulfide bonds. (Junutula, et al. 2008 Nature Biotech. 26(8):925-932; Dornan et al. 2009 Blood 114(13):2721-2729; U.S. Patent No. 7,521,541 B2; U.S. Patent No. 7,723,485 B2; WO 2009 / 052249 A2.) The engineered cysteine ​​thiols can be reacted with linker reagents or drug-linker reagents of the invention bearing thiol-reactive electrophilic groups, such as maleimides or alpha-haloamides, to form ADCs bearing cysteine-engineered antibodies and drug moieties.

[0222] Furthermore, antibodies or antibody fragments can be modified to incorporate Pel or pyrrolysine or unnatural amino acids as drug attachment sites. Peptide tags for enzymatic conjugation can be introduced into antibodies. (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. )

[0223] Antibodies and antibody fragments can be readily produced by any method known in the art, including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. (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. Patent No. 6,657,103 B2.)

[0224] Linkers and Linking Technologies The payload agents disclosed herein are suitable for use as payloads in immunoconjugates.The glucocorticoid receptor agonists of the present invention can be linked to a linker or directly linked to an antigen-binding moiety.The linker of ADCs is typically designed to achieve high stability in circulation and, in the case of cleavable linkers, to specifically release the payload in target tissues.

[0225] Linkers and linking techniques suitable for constructing immunoconjugates are well known in the art and can be used when producing the immunoconjugates of the present invention. Generally, the linker can be attached to the antigen-binding moiety at any suitable available position on the antigen-binding moiety, for example, to an available amino nitrogen atom (e.g., primary or secondary amine) or a hydroxyl oxygen atom or to an available sulfhydryl, such as on a cysteine. The linker can be attached to the N-terminus or C-terminus of the payload agent disclosed herein.

[0226] A variety of linkers and linking strategies are known and can be used in preparing the immunoconjugates of the invention. (For example, 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 / d1sc02973h; 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, pp. 1-6; WO 2021 / 055865 A1; WO 2016 / 192527 A1; WO 2015 / 095301 A2; WO 2011 / 097627 A1, WO 2004 / 010957 A1, U.S. Patent Publication No. 20060074008 A2, U.S. Patent Publication No. 20050238649 A2, and U.S. Patent Publication No. 20060024317 A2.

[0227] Linkers are classified as either cleavable or non-cleavable.For ADCs with non-cleavable linkers, release typically occurs through the internalization of ADCs and then antibody degradation in lysosomes, resulting in the release of the payload that remains linked to the amino acid residue of the antibody via the linker.Examples of non-cleavable linkers include maleimidocaproyl (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.

[0228] In the case of immunoconjugates containing 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, reducing potential) cleave the linker and liberate the payload.

[0229] Based on the cleavage mechanism, cleavable linkers can be further classified into chemically cleavable linkers (e.g., acid-cleavable linkers, reducible disulfide linkers, and exogenous stimulus-induced linkers) and enzymatically cleavable linkers (e.g., dipeptide Val-Cit-containing linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers). Acid-cleavable linkers (also known as pH-sensitive linkers) are designed to take advantage of the acidity of endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0) while maintaining stability in the blood circulation at pH 7.4. An example of an acid-cleavable linker is the acid-sensitive N-acylhydrazine bond, which hydrolyzes to ketone and hydrazide payloads upon acid catalysis. Acid-cleavable linkers containing other functional groups have also been reported, such as carbonate linkers. Glycosidase-cleavable linkers include β-glucuronidase-cleavable linkers, β-galactosidase-cleavable linkers, and phosphatase-cleavable linkers.(For example, 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. 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, WO 2021 / 055865 A1; WO 2016 / 192527 A1; WO 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; WO 2011 / 097627 A1. ).

[0230] Linker-antibody and linker-payload conjugation A variety of conjugation strategies have been developed over the years, including site-specific conjugation techniques, antibody engineering, and chemical modification.

[0231] The main linking techniques include maleimide linkages (e.g., N-alkylmaleimides, N-phenylmaleimides), bis(vinylsulfonyl)piperazine linkages, N-methyl-N-phenylvinylsulfonamide linkages, and Pt(II)-based linkages. (For example, 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; 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: 2005e12; Huang et al. 2018 Org. Lett. 20: 6526e9; Sijbrandi et al. 2017 Cancer Res. 77: 257e67; Merkul et al. 2020 Angew Chem. Int. Ed. Engl. 60:3008e15; Merkul et al. 2019 Expert Opin. Drug Deliv. 16:783e93; WO See 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; WO 2016 / 192527 A1; WO 2021 / 055865 A1. )

[0232] Various linker-payload conjugation strategies have been reported, including carbamate and carbonate conjugations (see, e.g., Wahby et al. 2020 Clin. Cancer Res. Available from: https: / / doi.10.1158 / 1078-0432.CCR-20-3119Perini et al. 2013 Biol. Ther. 3:15e23; Burke et al. 2016 Mol. Cancer Ther. 15:938e45; WO 2015 / 095301 A2; US 2021 / 0138077 A1; WO 2013 / 173393 A1; A1; see WO 2021 / 055865 A1. )

[0233] Non-limiting examples of conjugation strategies and reactive groups are provided in Table 3. (See, e.g., WO 2015 / 095301 A2; U.S. Pat. No. 9,988,420 B2.) [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0234] Pharmaceutical Compositions and Methods of Use Pharmaceutical Composition In another aspect, the present invention generally relates to a compound of formula (I)-(III) b ) and any one of Table 1, or a pharmaceutically acceptable form thereof, and optionally a pharmaceutically acceptable excipient, carrier, or diluent.

[0235] In yet another aspect, the present invention generally relates to a pharmaceutical composition comprising an immunoconjugate disclosed herein, such as according to any one of Formulas (IV)-(V), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0236] Accordingly, the present invention provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound or immunoconjugate according to the invention.

[0237] Examples of excipients that may be useful include, but are not limited to, water, saline, glucose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine ​​hydrochloride, starch, cellulose, and gum. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated into a pharmaceutical form for administration as a solid (e.g., tablet, capsule, lozenge, granule, suppository, crystalline or amorphous sterile solid that can be reconstituted to provide a liquid form, etc.), liquid (e.g., solution, suspension, emulsion, elixir, lotion, ointment, etc.), or semisolid (gel, ointment, cream, etc.). The pharmaceutical composition of the present invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Various dosage forms of active ingredients, excipients to be used, and their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable vehicles are known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of moisturizing agents, sterile solutions, etc. Compositions containing such vehicles can be prepared by conventional procedures known in the art. Preservatives, stabilizers, dyes, as well as 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.

[0238] The present invention also contemplates kits comprising at least the immunoconjugates disclosed herein and syringes and / or vials or ampoules into which the immunoconjugates and / or pharmaceutical compositions are disposed.

[0239] How to use In yet another aspect, the present disclosure generally relates to a method of treating or ameliorating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate disclosed herein.

[0240] In certain embodiments, the disease or condition is an inflammatory disorder.

[0241] In certain embodiments, the method further comprises administering to the subject one or more anti-inflammatory agents.

[0242] In yet another aspect, the invention generally relates to the use of an immunoconjugate disclosed herein for the manufacture of a medicament.

[0243] In certain embodiments, the immunoconjugates disclosed herein are used to treat a disease or condition, wherein the disease or condition is an inflammatory disorder.

[0244] In yet another aspect, the invention generally relates to the use of the immunoconjugates disclosed herein for use in the treatment of inflammatory disorders.

[0245] Exemplary inflammatory diseases or disorders include RA, ERA, ankylosing spondylitis, psoriatic arthritis, atherosclerosis, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity disorders, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, graft rejection, and vasculitis.

[0246] In certain embodiments, the inflammatory disorder is an autoimmune disease.

[0247] In certain embodiments, the inflammatory disorder is an inflammatory disorder of the joints.

[0248] In certain embodiments, the inflammatory disorder is an inflammatory disorder of the cardiovascular system.

[0249] In certain embodiments, the inflammatory disorder is inflamed lung and inflamed airways.

[0250] In certain embodiments, the inflammatory disorder is inflammatory bowel.

[0251] In certain embodiments, the inflammatory disorder is dermatitis.

[0252] In certain embodiments, the inflammatory disorder is acne vulgaris.

[0253] In certain embodiments, the inflammatory disorder is psoriasis.

[0254] In certain embodiments, the inflammatory disorder is rheumatoid arthritis.

[0255] In certain embodiments, the inflammatory disorder is a cardiovascular disease.

[0256] In certain embodiments, the inflammatory disorder is atherosclerosis.

[0257] In certain embodiments, the inflammatory disorder is type I diabetes.

[0258] In certain embodiments, the inflammatory disorder is lupus.

[0259] In certain embodiments, the inflammatory disorder is psoriatic arthritis.

[0260] In certain embodiments, the inflammatory disorder is inflammatory bowel disease.

[0261] In certain embodiments, the inflammatory disorder is asthma.

[0262] In certain embodiments, the inflammatory disorder is cystic fibrosis.

[0263] The immunoconjugate can generally be administered systemically, particularly intravenously, intramuscularly, intradermally, intraperitoneally, or subcutaneously, or orally. The immunoconjugate is typically administered intravenously into the subject's bloodstream to avoid degradation of the antibody by stomach acid or proteolytic enzymes. In some embodiments, the composition comprising the immunoconjugate disclosed herein is administered several times in a sequential manner.

[0264] Combination Therapy In yet another aspect, the invention generally relates to combinations comprising a therapeutically effective amount of a compound or immunoconjugate disclosed herein and one or more therapeutically active co-agents and / or adjuvants.

[0265] Co-agents include, but are not limited to, anti-inflammatory agents, antibacterial agents, anti-angiogenic agents, immunosuppressants, antibodies, steroids, or combinations thereof. Anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), nonspecific cyclooxygenase enzyme inhibitors and COX-2 specific cyclooxygenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptor antagonists, immunosuppressants, and methotrexate.

[0266] Non-limiting examples of anti-inflammatory agents include methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, flurometholone acetate, flurometholone alcohol, lotoprendol etabonate, medrysone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone acetate, lodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flurbiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocorticoids, diclofenac, and any combination thereof. In certain embodiments, an active compound or salt thereof or composition described herein is combined with one or more nonsteroidal 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.

[0267] Non-limiting examples of other co-agents include amikacin, anecortane acetate, anthracenedione, anthracyclines, azoles, amphotericin B, bevacizumab, camptothecin, cefuroxime, chloramphenicol, chlorhexidine, chlorhexidine digluconate, clotrimazole, clotrimazole cephalosporins, corticosteroids, dexamethasone, desamethasone, econazole, ceftazidime, 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 analogs, polymyxin B B), propamidine isethionate, pyrimidine nucleosides, ranibizumab, squalamine lactate, sulfonamides, triamcinolone, triamcinolone acetonide, triazoles, vancomycin, anti-vascular endothelial growth factor (VEGF) agents, VEGF antibodies, VEGF antibody fragments, vinca alkaloids, timolol, betaxolol, travoprost, latanoprost, bimatoprost, brimonidine, dorzolamide, acetazolamide, pilocarpine, ciprofloxacin, azithromycin, gentamicin, tobramycin, cefazolin, voriconazole, ganciclovir, cidofovir, foscarnet, diclofenac, nepafenac, ketorolac, ibuprofen, indomethacin, fluorometholone, rimexolone, anecortave, cyclosporine, methotrexate, tacrolimus, and combinations thereof.

[0268] Isotopically labeled compounds are also within the scope of the present disclosure.As used herein, " isotope-labeled compounds " refers to the compounds disclosed herein, including pharmaceutical salts and prodrugs thereof, in which one or more atoms are replaced with atoms that have different atomic mass or mass number from the atomic mass or mass number that is normally found in nature.The examples of isotopes that can be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, for example, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F and 36 Contains Cl.

[0269] By isotopically labeling the compounds disclosed herein, the compounds may be useful in drug and / or substrate tissue distribution assays. 3 H) and carbon-14 ( 14 C) labeled compounds are particularly preferred due to their ease of preparation and detection. 2 Substitution with heavier isotopes, such as H, can offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced required dosage, and therefore may be preferred in some circumstances. The isotopically-labeled compounds disclosed herein (including pharmaceutical salts, esters, and prodrugs thereof) can be prepared by any means known in the art.

[0270] Furthermore, the normally abundant hydrogen ( 1 Substitution of hydrogen (H) with heavier isotopes, such as deuterium, can confer certain therapeutic advantages, for example, improved absorption, distribution, metabolism, and / or excretion (ADME) properties, resulting in drugs with improved efficacy, safety, and / or tolerability. 12 C 13Advantages may also be obtained by replacing C. (See WO 2007 / 005643, WO 2007 / 005644, WO 2007 / 016361, and WO 2007 / 016431.)

[0271] Thus, isotopically derivatized compounds having one or more hydrogen atoms (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, etc.) replaced with a deuterium atom are contemplated herein. In certain embodiments, isotopically derivatized compounds of the invention have one hydrogen atom replaced with a deuterium atom.

[0272] Stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the compounds disclosed herein, as well as racemates, diastereomers and other mixtures of such isomers, are within the scope of this disclosure.

[0273] After preparation, the compounds of the present invention are preferably isolated and purified to obtain compositions containing an amount equal to or greater than 95% by weight ("substantially pure"), which are then used or formulated as described herein. In certain embodiments, the compounds of the present invention are greater than 99% pure.

[0274] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the invention include, for example, hydrates.

[0275] The following examples are intended to illustrate the practice of the present invention and are not intended to be limiting in any way. [Example]

[0276] [Table 3]

[0277] synthesis [ka] Triethylamine (276 mg, 2.76 mmol) was added dropwise to a stirred DCM solution of (6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (300 mg, 0.69 mmol) and methanesulfonic anhydride (360 mg, 2.07 mmol) under nitrogen at 0 °C. The reaction mixture was stirred at 25 °C for 0.5 h and then directly concentrated. 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] + .

[0278] [ka] A solution of 2-((6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl methanesulfonate INT-1 (180 mg, 0.35 mmol) and LiCl (110 mg, 2.60 mmol) in DMF (4 mL) was stirred at 60 °C under nitrogen for 2 h. The reaction mixture was diluted with EtOAc (20 mL), washed with water (3 x 20 mL), dried over MgSO, filtered, and concentrated in vacuo. The residue was purified by FCC (eluted with PE / EA = 1 / 3) to give INT-2 (120 mg, 75.2%) as a white solid. LCMS (ESI): m / z 453.2 [M+H] + .

[0279] [ka] A solution of (6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-aminophenyl)-8b-(2-chloroacetyl)-6b-fluoro-7-hydroxy-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (100 mg, 0.22 mmol), tert-butyl N-(3-formylphenyl)carbamate (73 mg, 0.33 mmol), and 1-butyl-3-methylimidazolium hexafluorophosphate (627 mg, 2.21 mmol) in DCM (5 mL) was stirred at 25 °C. To the resulting solution (2 mL) of HClO (221 mg, 2.21 mmol) was added dropwise. The reaction mixture was stirred at 25 °C for 10 min and purified first by FCC and then by prep-HPLC (MeCN / HO, 0.05% TFA) to 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 H NMR (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: 1H 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).

[0280] [ka] To a solution of (6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (200 mg, 0.46 mmol), triethylamine trihydrofluoride (148 mg, 0.90 mmol), and PBSF (417 mg, 1.38 mmol) in ACN (10 mL) was added triethylamine (278 mg, 1.05 mmol) at 25 °C. The resulting mixture was warmed to 50 °C and stirred for 17 h. It was concentrated, and the residue was purified by FCC (eluted with CH2Cl2 / MeOH = 10:1) to give INT-3 (150 mg, 74.2%) as a white solid. LCMS (ESI): m / z 437.2 [M+H] + .

[0281] [ka] To a solution of (6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-6b-fluoro-8b-(2-fluoroacetyl)-7-hydroxy-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]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 hexafluorophosphate (130 mg, 0.45 mmol) and HClO (46 mg, 0.45 mmol) was added. The mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure. The residue was purified by FCC (C-18, HO: ACN = 95:5 to 30:70, v / v) to give the product (20 mg). This was separated by chiral HPLC (40% IPA (NH4OH 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 = 17.4 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).

[0282] [ka] To a solution of (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (1000 mg, 1.21 mmol) and KCO (611 mg, 4.42 mmol) in MeOH (40 mL) was added HO (752 mg, 22.1 mmol). The mixture was stirred at 25 °C for 16 h. The MeOH was removed under reduced pressure, the mixture was acidified to pH 1 with 2N HCl, and the resulting precipitate was collected by filtration, washed with water, and dried to give INT-4 as a white solid (900 mg, 92.1%). LCMS (ESI): m / z 439.1 (M+H). + .

[0283] [ka] A solution of (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-8b-carboxylic acid INT-4 (100 mg, 0.23 mmol), HOBt (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 for 10 min at 25 °C, followed by dimethylamine hydrochloride (37 mg, 0.46 mmol) was added. The reaction mixture was stirred at 40° C. for 24 hours. The mixture was diluted with H2O (100 mL) and extracted with EtOAc (30 mL*3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give INT-5 as a white solid (90 mg, 83.2%). LCMS (ESI): m / z 466.3 (M+H) + .

[0284] [ka] (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-N,N,6a,8a,10,10-hexamethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-8b-carboxamide. To a solution of INT-5 (90 mg, 0.19 mmol) in dry DCM (15 ml), tert-butyl N-(3-formylphenyl)carbamate (128 mg, 0.58 mmol) was added, followed by l-butyl-3-methylimidazolium hexafluorophosphate (605 To the resulting mixture, HClO (194 mg, 1.94 mmol) was added. The mixture was stirred at 25°C for 2 hours. Triethylamine was added to neutralize the acid, and the solvent was removed under reduced pressure. ACN was added until the mixture was completely dissolved, and the mixture was purified by prep-HPLC (ACN-HO (0.1% TFA), 30%-50%) to give compound 4 (33.5 mg, 21.5%) and compound 5 (32.5 mg, 20.5%) as a white solid. 4: LCMS (ESI): m / z 529.2 [M+H] + . 1 H NMR (400 MHz, MeOD) δ 7.46-7.55(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] + . 1H NMR (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, 0.5H), 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).

[0285] [ka] To a stirred solution of (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-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, 5.10 mmol) in DCM (30 mL) was added methanesulfonic anhydride (489 mg, 2.80 mmol). The mixture was stirred at room temperature for 4 h. It was then concentrated in vacuo. The residue was purified by flash chromatography (eluted with CHCl / MeOH = 20 / 1) to give INT-6 (800 mg, 68.2%) as a white solid. LCMS(ESI): m / z 471.1 [M+H] + .

[0286] [ka] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-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)-2-oxoethyl methanesulfonate. To a solution of 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 HO, brine, dried over NaSO, filtered, and concentrated. This resulted in 290 mg of INT-7 as a white solid, which was used in the next step without further purification. LCMS (ESI): m / z 411.1 [M+H] + .

[0287] [ka] To a mixture of 4-(dimethylamino)pyridine (594 mg, 4.80 mmol) and (8S,9R,10S,11S,13S,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) was added 3-nitrobenzoyl chloride (548 mg, 2.94 mmol) in portions while stirring at 0° C. After the addition, the resulting mixture was stirred at room temperature for 17 hours. The solvent was removed under reduced pressure, and the residue was purified by FCC (eluted with CH2Cl2 / MeOH = 30 / 1) to give INT-8 (70 mg, 11.2%) as an off-white solid. LCMS (ESI): m / z 560.1 [M+H]+.

[0288] [ka] (8S,9R,10S,11S,13S,14S,16R,17R)-17-(2-chloroacetyl)-9-fluoro-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. To a solution of INT-8 (70 mg, 0.13 mmol) in EtOH (1 mL) and AcOH (1 mL) was added zinc dust (48 mg, 0.75 mmol). The mixture was stirred for 4 h at 25 °C. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (ACN-HO (0.1% TFA), 50%-60%) to give compound 6 (2.7 mg, 4.2 mmol). %). LCMS(ESI): m / z 559.0 [M+H] + . 1 H NMR (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).

[0289] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-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, 2.436 mmol) and KCO (673 mg, 4.873 mmol) in MeOH (40 mL) was added HO (829 mg, 24.36 mmol). The mixture was stirred at 25 °C for 36 h. The MeOH was removed under reduced pressure, the mixture was acidified to pH 1.0 with 2N HCl, and the resulting precipitate was filtered, washed with water, and air-dried to give INT-9 as a white solid (900 mg, 92%). LCMS (ESI): m / z 397.2 (M+H). + .

[0290] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-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. To a suspension of 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 at 25 °C for 2 h, after which 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 to pH 2.0 with 2N HCl and the resulting precipitate was filtered, washed with water, and air-dried to give INT-10 as a white solid (600 mg, 80%). LCMS (ESI): m / z 491.1 (M+H). + .

[0291] [ka] (6S,8S,9R,10S,11S,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. To a solution of INT-10 (130 mg, 0.265 mmol) in dry DCM (5 mL) was added SOCl (2 mL) under N. The reaction mixture was stirred at 50 °C for 2 h. DCM and SOCl were removed under vacuum. 1-(2-aminoethoxy)-4-nitrobenzene hydrochloride (125 mg, 0.572 mmol) and DIEA (111 mg, 0.858 mmol) in DCM (3 mL) were added to the mixture. The reaction mixture was stirred at 25° C. for 2 hours. Then, DCM was removed under reduced pressure, and the residue was purified by flash chromatography (DCM: EtOAc = 2:1, v / v) to give INT-11 as a yellow solid (45 mg, 26%). LCMS (ESI): m / z 655.0 (M+H) + .

[0292] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 zinc dust (15 mg, 0.229 mmol). The mixture was stirred at 25 °C for 4 h. The zinc dust was filtered off, and the mixture was purified by prep-HPLC (ACN-HO (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, 1H), 7.53(t, J = 7.9 Hz, 1H), 7.32-7.38(m, 2H), 6.36-6.28(m, 2H), 5.62-5.46(m, 1H), 4.46(q, J = 16.5 Hz, 2H), 4.29(d, J = 10.3 Hz, 1H), 2.65-2.51(m, 1H), 2.43-2.14(m, 6H), 1.91-2.04(m, 2H), 1.61-1.74(m, 1H), 1.57(s, 3H), 1.29-1.35(m, 1H), 1.15(s, 3H), 1.09(t, J = 7.6 Hz, 3H), 0.98(d, J = 7.1 Hz, 3H).

[0293] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-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. A mixture of 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 at 25 °C for 2 h, after which it was quenched with diethylamine (461 mg, 6.31 mmol). The reaction mixture was stirred at 25 °C for 1 h. The solvent was removed under reduced pressure. The residue was purified by flash chromatography (ACN-H2O, 30%-40%) to give INT-12 as a white solid (500 mg, 65.2%). LCMS (ESI): m / z 546.0 (M+H) + .

[0294] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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. To a solution of INT-12 (200 mg, 0.37 mmol) in DCM (6 mL) was added SOCl (3 mL). The resulting mixture was stirred at 40 °C under N for 2 h. The volatile phase was removed under reduced pressure. The residue was dissolved in THF (4 mL), and dimethylamine (2 M in THF, 2 mL) was added dropwise to the mixture, which was then 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 give INT-13 as a white solid (30 mg, 13.1%). LCMS (ESI): m / z 572.8 (M+H) + .

[0295] [ka] (6S,8S,9R,10S,11S,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 3-nitrobenzoate. To a solution of INT-13 (15 mg, 0.026 mmol) in EtOH (1 mL) and AcOH (1 mL) was added zinc dust (17 mg, 0.26 mmol). The mixture was stirred at 25 °C for 4 h. The solid was removed by filtration, and the filtrate was concentrated. The residue was purified by prep-HPLC (ACN-HO (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 H NMR (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).

[0296] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 INT-14 (100 mg, 0.18 mmol) in DCM (6 mL) and SOCl2 (3 mL) was stirred at 40 °C for 2 h. The volatile phase was removed under reduced pressure. The residue was dissolved in THF (2 mL), and then dimethylamine (2 M 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 give INT-15 as a white solid (20 mg, 19.2%). LCMS (ESI): m / z 613.1 (M+ACN). + .

[0297] [ka] (6S,8S,9R,10S,11S,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. To a solution of INT-15 (20 mg, 0.035 mmol) in EtOH (1 mL) and AcOH (1 mL) was added zinc dust (23 mg, 0.35 mmol). The mixture was stirred at 25 °C for 4 h. The solid was removed by filtration, and the filtrate was concentrated. The residue was purified by prep-HPLC (ACN-HO (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] + . 1H NMR (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).

[0298] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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. A solution of INT-12 (200 mg, 0.3666 mmol) in DCM (6 mL) and SOCl2 (3 mL) was stirred at 40 °C under N2 for 2 h. DCM and SOCl2 were removed under vacuum. THF (2 mL) and methylamine (2 M in THF, 2 mL) were added to the mixture. The reaction mixture was stirred at 25 °C for 16 h. Then, THF was removed under reduced pressure. DMF was added until the mixture was completely dissolved, and purified by flash (ACN-HO, 60%-70%) to give INT-16 as a white solid (75 mg, 36%). LCMS (ESI): m / z 559.1 (M+H). + .

[0299] [ka] A solution of (6S,8S,9R,10S,11S,13S,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-HO (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 H NMR (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).

[0300] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 SOCl2 (3 mL) was stirred at 40 °C under a N2 atmosphere for 2 h. The volatile phase was removed under vacuum. The residue was dissolved in THF (2 mL) and then added dropwise to a mixture of dimethylamine (2 M in THF, 2 mL). The reaction mixture was stirred at 25 °C for 16 h. It was then concentrated under reduced pressure. The residue was purified by FCC (ACN-HO, 60%-70%) to give INT-17 as a white solid (90 mg, 43.3%). LCMS (ESI): m / z 559.2 (M+H) + .

[0301] [ka] A solution of (6S,8S,9R,10S,11S,13S,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 4-nitrobenzoate (70 mg, 0.13 mmol), tetrahydroxydiboron (34 mg, 0.38 mmol), and 4,4'-bipyridine (1 mg, 0.0063 mmol) in DMF (4 mL) was stirred at 25 °C for 30 min. The resulting mixture was purified by prep-HPLC (ACN-HO (0.1% TFA), 50%-60%) to give compound 11 (45 mg, 67%) as a white solid. LCMS (ESI): m / z 529.2 [M+H] + . 1H NMR (400 MHz, MeOD) δ 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).

[0302] [ka] To a stirred solution of (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-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 (2000 mg, 5.1 mmol) and KCO (1410 mg, 102 mmol) in MeOH (20 mL) was added HO (50 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 3 h. The MeOH was removed under reduced pressure, then the mixture was acidified to 1.0 pH with 2N HCl, and the resulting precipitate was filtered, washed with water, and air-dried to give INT-18 as a white solid (1200 mg, 56.9%). LCMS (ESI): m / z 379.2 (M+H). + .

[0303] [ka] A suspension of (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-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-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 at 25 °C for 2 h, after which 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 was completely dissolved, and purified by flash chromatography (ACN-HO, 65%) to give INT-19 as a white solid (500 mg, 65%). LCMS (ESI): m / z 528.0 (M+H) + . [ka] (8S,9R,10S,11S,13S,14S,16R,17R)-9-Fluoro-11-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. A solution of INT-19 (20 mg, 0.04 mmol) and BTFFH (14 mg, 0.05 mmol) in dry DCM (2 mL) was placed in a dry microwave vial under argon. DIEA (15 mg, 0.11 mmol) was then added to the reaction mixture and stirred under argon for 30 min. Dimethylamine (2M, THF, 1 mL) was added to the mixture vial, which was then sealed and heated in an oil bath at 40° C. for 16 hours. The solvent was then removed under reduced pressure, and the residue was purified by prep-TLC (DCM: EtOAc = 2:1, v / v) to give INT-20 as a white solid (2 mg, 9.5%). LCMS (ESI): m / z 555.2 (M+H). + .

[0304] [ka] A solution of (8S,9R,10S,11S,13S,14S,16R,17R)-17-(dimethylcarbamoyl)-9-fluoro-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 (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 was purified by prep-HPLC (ACN-HO (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).

[0305] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (100 mg, 0.183 mmol) in dry DCM (5 mL) was added SOCl (1.5 mL), and the reaction was stirred at 40 °C for 2 h and concentrated. The residue was then dissolved in DCM (3 mL) and 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 under reduced pressure, and the crude product was purified by FCC (C18, ACN / HO 10%-50%) to give INT-21 (40 mg, 37.4%) as a white solid. LCMS (ESI): m / z 585.2 [M+H] + .

[0306] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-(azetidine-1-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. To a solution of 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-HO (0.1% TFA)) to give the product (8 mg, 21.1%) as a yellow solid. LCMS (ESI): m / z 555.3 [M+H] + . 1H 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).

[0307] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (100 mg, 0.183 mmol) in dry DCM (5 mL) was added SOCl (1.5 mL). The reaction mixture was stirred at 40 °C for 2 h and concentrated. The residue was then dissolved in DCM (3 mL) and 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 under reduced pressure, and the crude product was purified by FCC (C18, ACN / HO 10%-50%) to give INT-12 (40 mg, 36.4%) as a white solid. LCMS (ESI): m / z 599.2 [M+H] + .

[0308] [ka] tert-Butyl ((S)-1-(((S)-1-(((S)-4-hydroxy-4-methyl-11-morpholino-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate. To a solution of 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-HO (0.1% TFA)) to give compound 14 (5.8 mg, 14.97%) as a yellow solid. LCMS (ESI): m / z 569.1 [M+H] + . 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).

[0309] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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. A solution of INT-12 (100 mg, 0.18 mmol) in DCM (3 mL) and SOCl2 (2 mL) was stirred at 40 °C for 2 h under N2. DCM and SOCl2 were removed under vacuum. DCM (2 mL) and morpholine (32 mg, 0.3666 mmol) were added to the mixture. The reaction mixture was stirred at 25 °C for 3 h and concentrated. The residue was purified by flash (ACN-H2O, 60%) to give INT-23 (20 mg, 16.0%) as a white solid. LCMS (ESI): m / z 615 [M+H] + .

[0310] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 at 25 °C for 30 min. The reaction mixture was purified by HPLC (ACN-HO (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] + . 1H 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).

[0311] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (5 mL) in dry DCM (3 mL) was added SOCl (3 mL). The mixture was stirred at room temperature (rt) for 2 h, concentrated, and the residue was dissolved in DCM (5 mL) and 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 under reduced pressure and the crude material was purified by FCC (C18, ACN / HO 10% to 50%) to give INT-24 (120 mg, 49.05%) as a white solid. LCMS (ESI): m / z 601.3 [M+H] + .

[0312] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-(3-hydroxyazetidine-1-carbonyl)-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. To a solution of 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 under reduced pressure, and the crude material was purified by Prep-HPLC (ACN-HO (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).

[0313] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (3 mL). The reaction mixture was stirred at 40 °C for 2 h and concentrated. The residue was then dissolved in DCM (5 mL) and azetidin-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 under reduced pressure, and the crude product was purified by FCC (C18, ACN / H2O 10% to 50%) to give INT-25 (60 mg, 26.7%) as a white solid.

[0314] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-(azetidine-1-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. To a solution of 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 with water (2 mL), and purified by prep-HPLC (ACN-HO, 0.1% TFA) to give the product (13.7 mg, 28.8%) as a white solid. LCMS (ESI): m / z 585.3 [M+H] + . 1H 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).

[0315] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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. A solution of INT-12 (300 mg, 0.55 mmol) in DCM (20 mL) and SOCl2 (4 mL) was stirred at 40 °C under N2 for 1 h. DCM and SOCl2 were removed under vacuum. DCM (10 mL) and {2-[(tert-butyldimethylsilyl)oxy]ethyl}(methyl)amine (312 mg, 1.65 mmol) were added to the mixture. The reaction mixture was stirred at 25° C. for 6 hours and concentrated. The residue was purified by flash (ACN—H2O, 70%) to give compound 3 (70 mg, 16.8%) as a white solid. LCMS (ESI): m / z 717.3 [M+H] + .

[0316] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-((2-((tert-butyldimethylsilyl)oxy)ethyl)(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-cyclopenta[a]phenanthren-17-yl 3-nitrobenzoate INT-26 (50 mg, 0.07 mmol) in dry DCM (5 mL) was added 3HF.EtN (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 (Na2SO4) and concentrated in vacuo to give INT-27 (40 mg, 93.3%) as a white solid. LCMS (ESI): m / z 603.2 [M+H] + .

[0317] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-hydroxyethyl)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-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-HO (0.1% TFA), 45%) to give compound 18 (35 mg, 82.8%) as a white solid. LCMS (ESI): m / z 573.1 [M+H] + . 1H NMR (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).

[0318] [ka] (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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. A solution of INT-12 (300 mg, 0.55 mmol) in DCM (30 mL) and SOCl2 (4 mL) was stirred at 40 °C for 2 h under N2. DCM and SOCl2 were removed under vacuum. 3-Aminopropane-1,2-diol (336 mg, 5.5 mmol) and DCM (5 mL) were added to the mixture. The reaction mixture was stirred at 25 °C for 4 h and concentrated. The residue was purified by flash (ACN-H2O, 45%) to give INT-28 (150 mg, 44.0%) as a white solid. LCMS (ESI): m / z 589.1 [M+H] + .

[0319] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-hydroxyethyl)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-HO (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).

[0320] [ka] To a solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (3 mL). The mixture was stirred at room temperature for 2 h and concentrated. The residue was dissolved in DCM (5 mL) and 3-amino-1,2-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 under reduced pressure and the crude material was purified on a C18 column (ACN / HO 10% to 50%) to give INT-29 (100 mg, 20.11%) as a white solid. LCMS (ESI): m / z 633.3 [M+H] + .

[0321] [ka] Step 4: (6S,8S,9R,10S,11S,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-cyclopenta[a]phenanthren-17-yl 3-aminobenzoate (6S,8S,9R,10S,11S,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-cyclopenta[a]phenanthren-17-yl 3-nitrobenzoate. To a solution of 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-HO (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 H NMR (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).

[0322] [ka] A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-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 (300 mg, 0.55 mmol) in DCM (30 mL) and SOCl (4 mL) was stirred under N at 40 °C for 2 h. DCM and SOCl were removed under vacuum. EtN (556 mg, 5.50 mmol), 3-aminopropane-1,2-diol (150 mg, 1.65 mmol), and DCM (5 mL) were 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 O, 45%) to give compound 3 (80 mg, 22.4%) as a white solid. LCMS (ESI): m / z 619.2 [M+H] + .

[0323] [ka] A solution of (6S,8S,9R,10S,11S,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-HO (0.1% TFA), 45%) to give compound 21 (45 mg, 76.5%) as a white solid. LCMS (ESI): m / z 589.1 [M+H] + . 1H 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).

[0324] [ka] A solution of (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]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 to NEt3 (2.09 g, 0.0034 mol). g, 0.0207 mol) was added. The reaction mixture was stirred under nitrogen at 50° C. for 17 hours. The solvent was removed under reduced pressure, and the crude material was purified by Prep-HPLC (ACN-HO (0.1% TFA)) to give INT-31 (1.3 g, 78.26%) as a white solid. LCMS (ESI): m / z 437.2 [M+H] + .

[0325] [ka] To a solution of (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-8b-(2-fluoroacetyl)-7-hydroxy-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one INT-31 (500 mg, 1.15 mmol) and 1-butyl-3-methylimidazolium hexafluorophosphate (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 under reduced pressure, and the crude material was purified on a C18 column (ACN / HO 10% to 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).

[0326] [ka] A solution of (6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]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 at 25 °C for 10 min, after which 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 (Na2SO4) and concentrated in vacuo to give INT-33 as a white solid (80 mg, 89.9%). LCMS (ESI): m / z 430.0 (M+H) + .

[0327] [ka] (6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-N,N,6a,8a,10,10-hexamethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-8b-carboxamide. To a solution of 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 HClO (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-HO, 40%) to give compound 23 (32 mg, 22.6%) and compound 24 (10 mg, 7.1%). LCMS (ESI): m / z 582.9 (M+H). + .twenty three: 1 H NMR (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). twenty four: 11H 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).

[0328] [Chemical formula] A solution of (6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]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 to NEt3 (1.46 g, 0.0144 mol). mol) was added. The reaction mixture was stirred under nitrogen at 50°C for 17 h. The solvent was quenched with 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 (Na2SO4), and concentrated in vacuo. The crude material was purified on a C18 column (ACN / H2O 10% to 50%) to give INT-35 (0.9 g, 79.17%) as a white solid. LCMS (ESI): m / z 419.2 [M+H] + .

[0329] [ka] (6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-fluoroacetyl)-7-hydroxy-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one INT-35 (150 mg, 0.3584 mmol), 1-butyl-3-methylimidazolium hexafluorophosphate (204 mg, 0.7168 mmol), and tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (145 mg, 0.4659 mmol) in dry DCM (5%). To the (mL) solution 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 under reduced pressure, and the crude material was purified by prep-HPLC (ACN-HO (0.1% TFA)) to give 25 (18.0 mg, 8.81%) as a yellow solid. LCMS (ESI): m / z 572.2 [M+H] + . 1 H NMR (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). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0330] biological activity GR reporter assay : The assay is a pBind assay following the general principles outlined below. 4 HEK293 cells / 96-well plate are transiently transfected with pG5luc and GR LBD at a 1:1 ratio at 50 ng / well. The GR LBD (ligand-binding domain) is a luciferase fusion protein (fused to yeast GAL4) that, upon binding to a test substance, binds to a luciferase reporter and activates luciferase transcription. Upon addition of substrate, luciferase activity can be measured and corresponds to the activity of the test substance. The assay is performed in agonist mode. [Table 5]

[0331] HEK293 cell reporter assay of ADC activity : This assay is a pBind assay: typically, cells are transiently transfected with pG5luc and pBind-GR at a 1:1 DNA ratio. The GR LBD (ligand binding domain) is a fusion protein (fused to yeast GAL4) that, upon binding to a ligand, e.g., dexamethasone, or a test substance, binds to a luciferase reporter and activates luciferase transcription. Upon addition of substrate, luciferase activity can be measured and corresponds to the activity of the test substance. The assay is performed in agonist mode.

[0332] To enable TNFα-mediated uptake of adalimumab-ADC, human TNFα was transiently overexpressed in the HEK293 cells used in this pBind assay. A TACE-resistant hTNFα(77-88del) construct was used to enable optimal membrane-associated TNFα expression. [Table 6]

[0333] Applicant's disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent identical or similar elements. References throughout this specification to "one embodiment" or similar language mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, appearances of "one embodiment" and similar language throughout this specification do not necessarily all refer to the same embodiment.

[0334] The described features, structures, or characteristics of the applicant's disclosure can be combined in any suitable manner in one or more embodiments. Numerous specific details are described herein to provide a thorough understanding of the embodiments of the present invention. However, those skilled in the relevant art will recognize that the applicant's compositions and / or methods can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other embodiments, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present disclosure.

[0335] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Any method and material similar or equivalent to those described herein can also be used in the implementation or testing of this disclosure, but preferred method and material are described herein.The method described herein can be carried out in any logically possible order, in addition to the specific order disclosed.

[0336] Citation by reference In this disclosure, references and citations are made to other documents, such as patents, patent applications, patent publications, journals, books, papers, manuscripts, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is incorporated herein by reference that conflicts with existing definitions, descriptions, or other disclosure material expressly set forth herein is incorporated only to the extent that no conflict arises between the incorporated material and the present disclosure material. In the event of a conflict, the conflict will be resolved in favor of the present disclosure.

[0337] equivalent The representative examples are intended to be illustrative of the invention and are not intended to, and should not be construed as, limiting 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 entire contents of the specification, including the examples contained herein and references to the scientific and patent literature. 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.

Claims

1. Structural formula (I): 【Chemical 1】 [During the ceremony, R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 alkyl, and R 4 is OC(O)-R 7 where R 7 is a substituted or unsubstituted aniline, or R 3 and R 4 together with the carbon atoms to which they are attached, 6 where W is a single bond or -phenyl-Q- and Q is CH 2 , O, S, S(O), S(O) 2 , NH and NCH 3 is selected from, and R 6 is a substituted or unsubstituted aniline; and R 5 is NR 5a R 5b or CH 2 R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered unsubstituted or substituted heterocyclic ring; and R 5c is H, halogen or OC 1-3 It is alkyl. or a pharmaceutically acceptable form thereof.

2. R 4 OC(O)-R 7 and structural formula (II): 【Chemistry 2】 2. The compound of claim 1, having the formula:

3. Structural formula (II a ): 【Chemistry 3】 [During the ceremony, R 7a , R 7b and R 7c Each of 7x R 7y selected from, with the proviso that R 7a , R 7b and R 7c Only one of the 7x R 7y and each of the others is H; R 7x and R 7y Each of the groups independently represents R, R 7r and L 7 -R 7z selected from, with the proviso that R 7x and R 7y One of them is L 7 -R 7z or R 7r and 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 and R 7v are R, OR, NHR, and 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 C 1 -C 6 It is alkyl.

3. The compound of claim 2, having the formula:

4. R 7a is H and R 7b NR 7x R 7y and R 7c 4. The compound of claim 3, wherein is H.

5. R 7a is H and R 7b is H and R 7c NR 7x R 7y 4. The compound of claim 3, wherein:

6. R 7a NR 7x R 7y and R 7b is H and R 7c 4. The compound of claim 3, wherein is H.

7. R 7x is H or CH 3 and R 7y (C=O)-O-(CH 2 ) i -R 7v where R 7v R, OR, NHR, NR 2 , an aryl group or an amino acid, and i is 0, 1, 2 or 3.

8. R 7x is H or CH 3 and R 7y (C=O)-(CH 2 ) j -R 7v where R 7v R, OR, NHR, NR 2 7. The compound of claim 4, wherein j is 0, 1, 2, or 3.

9. R 3 The compound of any one of claims 1 to 8, wherein is alkyl.

10. R 3 10. The compound of claim 9, wherein is methyl.

11. R 3 and R 4 together with the carbon atoms to which they are attached, 6 forming a 5-membered dioxolane substituted with the structural formula: 【Chemistry 4】 2. The compound of claim 1, having the formula:

12. W is a single bond, and the compound of formula (III): 【Chemistry 5】 12. The compound of claim 11, having the formula:

13. Structural formula (III a ): 【Chemistry 6】 [During the ceremony, R 6a , R 6b and R 6c Each of 6x R 6y selected from, with the proviso that R 6a , R 6b and R 6c Only one of the 6x R 6y and each of the others is H; R 6x and R 6y Each of the groups independently represents R, R 6r and L 6 -R 6z selected from, with the proviso that R 6x and R 6y One of them is L 6 -R 6z or R 6r and 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 and R 6v are R, OR, NHR, and 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 C 1 -C 6 It is alkyl.

13. The compound of claim 12, having the formula:

14. R 6 14. The compound of claim 12 or 13, wherein has the S configuration.

15. R 6 14. The compound of claim 12 or 13, wherein has the R configuration.

16. R 6a is H and R 6b NR 6x R 6y and R 6c The compound of any one of claims 13 to 15, wherein is H.

17. R 6a is H and R 6b is H and R 6c NR 6x R 6y The compound according to any one of claims 13 to 15, wherein

18. R 6a NR 6x R 6y and R 6b is H and R 6c The compound of any one of claims 13 to 15, wherein is H.

19. R 6x is H or CH 3 and R 6y (C=O)-O-(CH 2 ) p -R 6v where R 6v are R, OR, NHR, and NR 2 , an aryl group, or an amino acid, and p is 0, 1, 2, or 3.

20. R 6x is H or CH 3 and R 6y (C=O)-(CH 2 ) q -R 6v where R 6v are R, OR, NHR, and NR 2 , an aryl group, or an amino acid, and q is 0, 1, 2, or 3.

21. 12. The compound of claim 11, wherein W is -phenyl-Q-.

22. Q is CH 2 and the structural formula (III b ): 【Chemistry 7】 [During the ceremony, R 6a’ , R 6b’ and R 6c’ Each of 6x’ R 6y’ selected from, with the proviso that R 6a’ , R 6b’ and R 6c’ Only one of the 6x’ R 6y’ and each of the others is H; R 6x’ and R 6y’ Each of the groups independently represents R, R 6r’ and L 6’ -R 6z’ selected from, with the proviso that R 6x’ and R 6y’ One of them is L 6’ -R 6z’ or R 6r’ and 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’ and R 6v’ are R, OR, NHR, and 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 C 1 -C 6 It is alkyl.

22. The compound of claim 21, having the formula:

23. WR 6 23. The compound of claim 21 or 22, wherein the carbon atom of the five-membered dioxolane bonded to has an S configuration.

24. WR 6 23. The compound of claim 21 or 22, wherein the carbon atom of the five-membered dioxolane bonded to has the R configuration.

25. R 6a’ is H and R 6b’ NR 6x’ R 6y’ and R 6c’ The compound of any one of claims 22 to 24, wherein is H.

26. R 6a’ is H and R 6b’ is H and R 6c’ NR 6x’ R 6y’ The compound according to any one of claims 22 to 24, wherein

27. R 6a’ NR 6x’ R 6y’ and R 6b’ is H and R 6c’ The compound of any one of claims 22 to 24, wherein is H.

28. R 6x’ is H or CH 3 and R 6y’ (C=O)-O-(CH 2 ) p -R 6v’ where R 6v’ are R, OR, NHR, and NR 2 , an aryl group or an amino acid, and p is 0, 1, 2 or 3.

29. R 6x’ is H or CH 3 and R 6y’ (C=O)-(CH 2 ) q -R 6v’ where R 6v’ are R, OR, NHR, and NR 2 , an aryl group, or an amino acid, and q is 0, 1, 2, or 3.

30. L 6 or L 7 , or L 6’ 30. The compound of any one of claims 3 to 29, wherein, when present, is a non-cleavable linker.

31. L 6 or L 7 , or L 6’ 30. The compound of any one of claims 3 to 29, wherein, when present, is a cleavable linker.

32. L 6 or L 7 , or L 6’ 32. The compound of claim 30 or 31, wherein, when present, is an acid-labile or acid-sensitive linker.

33. L 6 or L 7 , or L 6’ 32. The compound of claim 30 or 31, wherein, when present, is a protease-sensitive linker.

34. L 6 or L 7 , or L 6’ 32. The compound of claim 30 or 31, wherein, when present, is a lysosomal protease-sensitive linker.

35. L 6 or L 7 , or L 6’ 32. The compound of claim 30 or 31, wherein, when present, is a β-glucuronide-sensitive linker.

36. L 6 or L 7 , or L 6’ 32. The compound of claim 30 or 31, wherein, when present, is a glutathione-sensitive disulfide linker.

37. R 6z or R 7z , or R 6z’ But when there exists -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、-ONH 2 、-C(=O)NHNH 2 、-CO 2 H、-NH 2 、-NCO、-NCS、 【Chemistry 8】 【Chemistry 9】 wherein the functional or reactive group is selected from: R u is H or C 1 -C 6 is an alkyl group, R t is 2-pyridyl or 4-pyridyl, and R w teeth, 【Chemistry 10】 That is, 37. The compound of any one of claims 3 to 36.

38. R 5 NR 5a R 5b 38. The compound of any one of claims 1 to 37, wherein

39. R 5 is N(CH 3 ) 2 39. The compound of claim 38, wherein:

40. R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered unsubstituted or substituted heterocyclic ring.

41. 41. The compound of claim 40, wherein the heterocyclic ring is an unsubstituted 4-, 5-, or 6-membered heterocycle.

42. The heterocyclic ring is a ring containing OH and C 1-3 alkyl, wherein C 1-3 41. The compound of claim 40, wherein the alkyl is optionally substituted with one or more OH groups.

43. R 5a and R 5b C, which is optionally substituted with H and one or more OH groups, 1-6 39. The compound of claim 38, wherein the alkyl is selected from:

44. R 5a is H or methyl, and R 5b C is substituted with OH 1-6 33. The compound of claim 32, wherein the compound is alkyl.

45. R 5a is H or methyl, and R 5b C is substituted with two OH groups 2-6 33. The compound of claim 32, wherein the compound is alkyl.

46. R 5 But CH 2 R 5c 38. The compound of any one of claims 1 to 37, wherein

47. R 5c 47. The compound of claim 46, wherein is F.

48. R 5c 47. The compound of claim 46, wherein is Cl.

49. R 5c But, O.C. 1-3 47. The compound of claim 46, wherein the compound is alkyl.

50. R 1 and R 2 50. The compound of any one of claims 1 to 49, wherein each of is H.

51. R 1 and R 2 50. The compound of any one of claims 1 to 49, wherein each of is F.

52. R 1 and R 2 50. The compound of any one of claims 1 to 49, wherein one of is H and the other is F. 【Request 53】 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 [In the formula, R 1 and R 2 Each of is independently H or halogen. or a pharmaceutically acceptable form thereof.

54. 54. An immunoconjugate comprising a compound of any one of claims 1 to 53 covalently linked to an antigen-binding moiety.

55. Structural formula (IV): 【Chemistry 11】 [During the ceremony, Ab stands for antigen-binding moiety; R 1 is H or a halogen; R 2 is H or a halogen; R 3 is C 1-4 is alkyl; R 5 is NR 5a R 5b or CH 2 R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered heterocyclic ring; and R 5c is H, halogen or OC 1-3 is alkyl; R 7x is H or C 1-6 is alkyl; L Ab is a linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

56. R 3 The immunoconjugate of claim 55, wherein is alkyl.

57. R 3 The immunoconjugate of claim 56, wherein is methyl.

58. R 7x The immune complex of any one of claims 55 to 57, wherein is H.

59. R 7x But C 1-6 The immunoconjugate of any one of claims 55 to 57, which is alkyl.

60. Structural Formula (V) or (VI): 【Chemistry 12】 or 【Chemistry 13】 [During the ceremony, Ab stands for antigen-binding moiety; Q is CH 2 , O, S, S(O), S(O) 2 , NH and NCH 3 Selected from: R 1 is H or a halogen; R 2 is H or a halogen; R 5 is NR 5a R 5b or CH 2 R 5c and R 5a and R 5b each independently represents H and C 1-6 alkyl, or R 5a and R 5b together with the N atom to which they are attached form a 4- to 7-membered heterocyclic ring; and R 5c is H, halogen or OC 1-3 is alkyl; R 6x is H or C 1-6 is alkyl; L Ab is a linker; and and n is an integer ranging from 1 to about 20. or a pharmaceutically acceptable form thereof.

61. Q is CH 2 and has the structural formula: 【Chemistry 14】 61. The compound of claim 60, having the formula:

62. Q is CH 2 and has the structural formula: 【Chemistry 15】 61. The compound of claim 60, having the formula:

63. R 6x The immune complex of any one of claims 60 to 62, wherein is H.

64. R 6x But C 1-6 The immunoconjugate of any one of claims 60 to 62, which is alkyl.

65. R 1 and R 2 The immune complex of any one of claims 55 to 64, wherein each of is H.

66. R 1 and R 2 The immune complex of any one of claims 55 to 64, wherein each of is F.

67. R 1 and R 2 The immune complex of any one of claims 55 to 64, wherein one of is H and the other is F.

68. R 5 But NR 5a R 5b The immune complex according to any one of claims 55 to 67,

69. R 5 But N(CH 3 ) 2 69. The immune complex of claim 68, wherein

70. R 5 But CH 2 R 5c The immune complex according to any one of claims 55 to 67,

71. R 5c The immune complex of claim 70, wherein

72. R 5c The immune complex of claim 70, wherein is Cl.

73. R 5c But, O.C. 1-3 71. The immunoconjugate of claim 70, wherein the alkyl is

74. The immune complex of any one of claims 54 to 73, wherein Ab is an antibody.

75. 75. The immune complex of claim 74, wherein the antibody is a monoclonal antibody.

76. 75. The immune complex of claim 74, wherein the antibody is a chimeric antibody.

77. 75. The immunoconjugate of claim 74, wherein the antibody is a humanized antibody.

78. 75. The immune complex of claim 74, wherein the antibody is a bispecific antibody.

79. The immune complex of any one of claims 54 to 73, wherein the Ab is an antibody fragment.

80. 80. The immune complex of claim 79, wherein the Ab is a Fab fragment.

81. The immune complex of any one of claims 54 to 73, wherein the Ab is a peptide.

82. The immunoconjugate of any one of claims 54 to 73, wherein the Ab is a small molecule ligand.

83. 54. A pharmaceutical composition comprising a compound according to any one of claims 1 to 53 and a pharmaceutically acceptable excipient, carrier or diluent.

84. A pharmaceutical composition comprising the immunoconjugate of any one of claims 54 to 82 and a pharmaceutically acceptable excipient, carrier or diluent.

85. A combination comprising a therapeutically effective amount of a compound according to any one of claims 1 to 53 and one or more therapeutically active co-agents or adjuvants.

86. A combination comprising a therapeutically effective amount of the immunoconjugate of any one of claims 54 to 82 and one or more therapeutically active co-agents or adjuvants.

87. 54. A method of treating or ameliorating a disease or condition, said method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-53.

88. 83. A method of treating or ameliorating a disease or condition, said method comprising administering to a subject in need thereof a therapeutically effective amount of the immunoconjugate of any one of claims 54 to 82.

89. 89. The method of claim 87 or 88, wherein the disease or condition is an inflammatory disorder.

90. Use of a compound according to any one of claims 1 to 53 for the manufacture of a medicament.

91. Use of an immunoconjugate according to any one of claims 54 to 82 for the manufacture of a medicament.

92. 54. Use of a compound according to any one of claims 1 to 53 for treating an inflammatory disorder.

93. Use of an immunoconjugate according to any one of claims 54 to 82 for treating an inflammatory disorder.

94. 54. A compound according to any one of claims 1 to 53 for use in the treatment of an inflammatory disorder.

95. 83. The immunoconjugate of any one of claims 54 to 82 for use in the treatment of an inflammatory disorder.

96. A composition comprising a compound according to any one of claims 1 to 53.

97. A composition comprising an immunoconjugate according to any one of claims 54 to 83.

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