Oretastatin linker-load, pharmaceutical composition and use thereof

Abstract

The present disclosure relates to linker-loadings, and pharmaceutically acceptable salts, solvates or stereoisomers thereof, comprising the structure of Formula I: # imgabs0. The disclosure also relates to pharmaceutical compositions comprising these compounds and the use of these compounds, intermediates and compositions thereof in the prevention or treatment of cancer and / or tumors.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 432,470, filed Dec. 14, 2022, and U.S. Provisional Application No. 63 / 497,887, filed Apr. 24, 2023, the entire contents of which are incorporated herein by reference. Background of the Invention

[0003] Antibody-drug conjugates (ADCs) are a therapeutic modality consisting of monoclonal antibodies conjugated to cytotoxic payloads and have made significant progress in serving as delivery vehicles that recognize and bind protein antigens expressed in tumor tissue. Local delivery and release of the payload within or near malignant cells allows for targeted delivery of potent cytotoxic agents to diseased tissue while reducing damage to normal tissue. The linker component of an ADC is an important feature in the development of optimized therapeutic agents with high activity at well-tolerated doses (see WO2021055865, WO2018025168, and US20130309256).

[0004] There remains a need for cytotoxin linker-payloads for conjugation to antibodies or other targeting moieties to produce antibody-drug conjugates (ADCs) or other targeted ligand conjugates, particularly for oncology indications.

[0005] Summary of the Invention

[0006] The present disclosure provides linkers and linker-payloads for conjugation with an antibody or other targeting moiety to produce ligand-drug conjugates (e.g., ADCs) for oncology and other indications. In particular, novel linker-payload constructs are disclosed that contain a maleimide or pyridylsulfone linked to a cleavable dipeptide linker and a para-aminobenzyl carbamate (PABC) linked to a cytotoxic payload (drug) such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or other cytotoxic payloads disclosed herein. The utility of these linker-payloads is demonstrated by conjugation with a cysteine residue in a ligand (e.g., an antibody or an antigen-binding fragment of an antibody) to produce a ligand-drug conjugate (e.g., an ADC) that exhibits favorable physicochemical properties and high target-mediated potency. The linker-payloads are used to generate effective and novel ligand-drug conjugates that are active in a variety of cancer cell lines and demonstrate broad utility in conjugation with several antibodies or other targeting moieties while still retaining favorable properties and efficacy. Accordingly, one aspect of the present disclosure is a ligand-drug candidate having a linker dipeptide sequence that provides enhanced exposure of the released free cytotoxic compound to tumor tissue compared to normal tissue and thus selective entry into target cells, and reduced exposure of normal tissue to the cytotoxic compound compared to tumor tissue.

[0007] Exemplary linker-payload compounds include, but are not limited to, the structures shown throughout the present disclosure. Exemplary ligand-drug conjugates (e.g., ADCs) formed using the linker-payloads described herein are also described. Other embodiments, aspects, and features of the present disclosure are further described in the following specification, examples, and appended claims or will be apparent from the following specification, examples, and appended claims.

[0008] In each embodiment described herein, each variable is selected independently of one another, unless otherwise specified.

[0009] In one embodiment, the present disclosure provides a dipeptide linker-payload (drug), and pharmaceutically acceptable salts, solvates, and stereoisomers thereof, comprising a structure of Formula I:

[0010]

[0011] Wherein:

[0012] R 1 Is selected from:

[0013]

[0014] The wavy line indicates the site of covalent attachment;

[0015] R2 is a cytotoxic drug;

[0016] R 3 and R 4 each independently represents C 1-3 alkyl or a naturally occurring or non-natural amino acid side chain; and

[0017] n is an integer from 1 to 4.

[0018] One embodiment of Formula I is achieved when n is 1.

[0019] One embodiment of Formula I is achieved when n is 2.

[0020] One embodiment of Formula I is achieved when n is 3.

[0021] One embodiment of Formula I is achieved when n is 4.

[0022] One embodiment of Formula I is when R 1 is is achieved.

[0023] Another embodiment of Formula I is when R 1 is is achieved.

[0024] Another embodiment of Formula I is when R 1 is is achieved.

[0025] Another embodiment of Formula I is when R 2 is a cytotoxic drug or payload selected from: anthracyclines, auristatins, camptothecin, duocarmycins, etoposide, maytansinoids, pyrrolobenzodiazepine dimers, DNA minor groove binders, taxanes, vinca alkaloids, enediynes, anti-tubulin and vinca alkaloids. A sub-embodiment of this aspect of the present disclosure is when R 2Achieved when selected from: auristatin T, auristatin E, auristatin F phenylenediamine, AEB, AEVB, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE), lexitropsins, duocarmycin, paclitaxel and docetaxel, T67 (Tularik), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, cyano-morpholino-doxorubicin, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analogs, calicheamicin, maytansine, dolastatin 10 analogs, rhizoxin, palytoxin, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansine analogs, combretastatin, discodermoide and eleutrobin. The cytotoxic agent or cytostatic agent can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., (paclitaxel), (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansine analogs, combretastatin, discodermoide and eleutrobin.

[0026] Another sub-embodiment of this aspect of the present disclosure is achieved when R 2 is an auristatin drug. One aspect of this sub-embodiment is when R 2 is an auristatin drug selected from: AE, auristatin F phenylenediamine (AFP), AEB, AEVB, MMAF and MMAE. A further aspect of this embodiment is when R 2 is MMAE. A further aspect of this embodiment is when R 2 is MMAF.

[0027] Another embodiment of Formula I is achieved when R 2 is a pyrrolo[2,3-c]phenazine dimer. A sub-embodiment of this aspect is when R 2It is achieved when it is tesirine.

[0028] One embodiment of Formula I is when R 3 and R 4 are independently selected from C 1-3 alkyl. A sub - embodiment of this aspect is when R 3 and R 4 are independently selected from - CH3, - CH2CH3, -(CH2)2CH3. A sub - embodiment of this aspect of Formula I is when R 3 and R 4 are both CH3. A sub - embodiment of this aspect of Formula I is when R 3 and R 4 are both - CH2CH3. A sub - embodiment of this aspect of Formula I is when R 3 and R 4 are both -(CH2)2CH3. A sub - embodiment of this aspect of Formula I is when one of R 3 and R 4 is CH3 and the other is selected from - CH2CH3 and -(CH2)2CH3.

[0029] One embodiment of Formula I is when R 3 and R 4 are independently a naturally occurring or non - naturally occurring amino acid side chain. A sub - embodiment of this aspect of Formula I is when R 3 and R 4 are independently selected from arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamate, aspartate, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine in L or D - configuration or side chains. Another embodiment of Formula I is when R 3 and R 4 are independently selected from naturally occurring or non - naturally occurring amino acid side chains, and the amino acid side chains are selected from the side chains of alanine, valine, leucine, and methionine.

[0030] In another embodiment, the present disclosure provides compounds of Formula II and their pharmaceutically acceptable salts, solvates, and stereoisomers, which comprise the following structure:

[0031]

[0032] wherein R 1 and R 2 are as described herein. One embodiment of Formula II is when R 2It is achieved when it is an auristatin drug. One aspect of this sub - embodiment is when R 2 is an auristatin drug selected from the following: AE, auristatin F phenylenediamine (AFP), AEB, AEVB, MMAF, and MMAE. A further aspect of Formula II is when R 2 is MMAE. A further aspect of Formula II is when R 2 is MMAF.

[0033] One embodiment of Formula II is when R 1 is One embodiment of Formula II is when R 1 is One embodiment of Formula II is when R 1 is Another embodiment of Formula II is when R

[0034] is 1 and R is 2 is MMAE.

[0035] Another embodiment of Formula II is when R 1 is and R 2 is MMAE.

[0036] Another embodiment of Formula II is when R 1 is and R 2 is MMAE.

[0037] Another embodiment of Formula II is when R 2 is pyrrolo - benzodiazepine dimer. A sub - embodiment of this aspect is when R 2 is tesirine.

[0038] Another embodiment of Formula II is represented by Structural Formula II’:

[0039]

[0040] In another embodiment, the present disclosure provides compounds of Formula III and their pharmaceutically acceptable salts, solvates, and stereoisomers, which comprise the following structure:

[0041]

[0042] Wherein L’ is a ligand (which is an antibody or an antigen-binding fragment of an antibody), p represents the average number of drug linker moieties in the ligand-drug conjugate composition, and R 2 、R 3 and R 4 are as described herein and R 1 ’ is selected from:

[0043]

[0044] The single wavy line indicates the site covalently linked to -(CH2) n ;

[0045] The double wavy line indicates the site covalently linked to the sulfur of the cysteine residue of L’. It should be understood that when L’ is an antibody, the sulfur atom S bonded to L’ in Formula III represents the sulfur of the side chain of the cysteine side chain of the antibody. P is a positive real number, including all fractional and decimal values. In some embodiments, p is a positive rational number from 1 to 24, 1 to 12, 1 to 8, or is 4 or 8, including fractional and decimal values. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, or fractional and decimal values between 1 and 24. In some embodiments, p is 2, 4, 6, or 8, or p is from 1 to 2, including fractional and decimal values between 2 and 8.

[0046] Another embodiment of Formula III is represented by Structural Formulas III’ or III”:

[0047]

[0048] Wherein L’, R 1 ’ and R 2 are as described herein.

[0049] One embodiment of Formulas III, III', and III'' is achieved when L' comprises a full monoclonal antibody, polyclonal antibody, monospecific antibody, multispecific antibody (e.g., bispecific antibody), and antibody fragment that exhibits the desired biological activity. It is further contemplated that the antibody-drug conjugates of the present disclosure can be replaced with any substance that specifically binds, reactively associates, or complexes with a receptor, antigen, or other acceptor moiety associated with a given target cell population. For example, instead of containing an antibody, the conjugates of the present disclosure can contain a targeting molecule that binds, complexes, or reacts with a receptor, antigen, or other acceptor moiety (e.g., small molecular weight protein, polypeptide or peptide, lectin, glycoprotein, non-peptide, vitamin, nutrient-transporting molecule (e.g., transferrin)) of the cell population sought to be therapeutically or otherwise biologically modified and any other cell-binding molecule or substance.

[0050] Examples of ligands L' for the present disclosure are disclosed herein and are selected from trastuzumab or mutants thereof, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody against the vitronectin receptor (a v b3), alemtuzumab, anti-HLA-DR antibodies including humanized anti-HLA-DR antibodies for the treatment of non-Hodgkin lymphoma, 121I lym-1, anti-HLA-Dr10 antibodies including murine anti-DLA-Dr10 antibodies for the treatment of non-Hodgkin lymphoma, anti-CD33 antibodies, anti-CD22 antibodies including humanized anti-CD22 mAb for the treatment of Hodgkin disease or non-Hodgkin lymphoma, labetuzumab, bevacizumab, tiuxetan (ibritumomab tiuxetan), ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, sacituzumab, cetuximab (ERBITUX), and gemtuzumab.

[0051] One aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formulas I, II, II', III, III', III'' or a pharmaceutically acceptable salt or solvate thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0052] Another aspect of the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula I, II, II’, III, III’ or III” as described herein, or a tautomer, meso form, racemate, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0053] Another aspect of the present disclosure relates to a compound of formula I, II, II’, III, III’ or III” as described herein, or a tautomer, meso form, racemate, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament or pharmaceutical ingredient.

[0054] Another aspect of the present disclosure relates to a compound of formula I, II, II’, III, III’ or III” as described herein, or a tautomer, meso form, racemate, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for use in the preparation of a medicament for treating or preventing tumors.

[0055] In another embodiment, the compounds of the present disclosure include those identified as examples in the following table herein and their pharmaceutically acceptable salts. DETAILED DESCRIPTION OF THE INVENTION

[0057] For each of the following embodiments, any variable not explicitly defined in the embodiment is as defined in formula (I). In each embodiment described herein, unless otherwise specified, each variable is selected independently of one another.

[0058] The compounds of the present disclosure may contain one or more asymmetric centers and may thus exist as racemates and racemic mixtures, single enantiomers, mixtures of diastereoisomers and individual diastereoisomers. Depending on the nature of the various substituents on the molecule, additional asymmetric centers may exist. Each such asymmetric center will independently give rise to two optical isomers, and all possible optical isomers and diastereoisomers in mixtures and as pure or partially purified compounds are intended to be included within the scope of the present disclosure. Unless a specific stereochemistry is indicated, the present disclosure is meant to cover all such isomeric forms of these compounds.

[0059] The independent synthesis or chromatographic separation of these diastereoisomers can be achieved by appropriate modification of the methodologies disclosed herein as known in the art. Among other methods, their absolute stereochemistry can be determined by x-ray crystallography of the crystalline product or crystalline intermediate, if desired, by derivatizing the crystalline product or crystalline intermediate with a reagent containing an asymmetric center of known absolute configuration.

[0060] If desired, the racemic mixture of the compound can be separated to isolate the individual enantiomers. The separation can be carried out by methods well known in the art, such as coupling the racemic mixture of the compound with an enantiomerically pure compound to form a mixture of diastereomers, and then separating the individual diastereomers by standard methods such as fractional crystallization or chromatography. The coupling reaction is usually the formation of a salt using an enantiomerically pure acid or base. Then, the diastereomeric derivative can be converted to the pure enantiomer by cleaving the added chiral residue. The racemic mixture of the compound can also be directly separated by a chromatographic method using a chiral stationary phase, which is well known in the art.

[0061] Alternatively, any enantiomer of the compound can be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

[0062] In the compounds of formula I, II, II’, III, III’ or III”, the atoms can exhibit their natural isotopic abundances, or one or more atoms can be artificially enriched to have specific isotopes with the same atomic number but different atomic masses or mass numbers from those predominantly found in nature. The present disclosure can include all suitable isotopic variants of the compounds of formula I, II, II’, III, III’ or III”. For example, different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H). Protium is the major hydrogen isotope found in nature. Enrichment of deuterium can provide certain therapeutic advantages, such as increased in vivo half-life or reduced dose requirements, or can provide compounds that can be used as standards for characterizing biological samples. For the purposes of the present disclosure, when a compound is referred to as “undeuterated”, it means that it is not enriched with deuterium beyond the background state. Isotopically enriched compounds within formula I, II, II’, III, III’ or III” can be prepared by conventional techniques well known to those skilled in the art or by methods similar to those described in the protocols and examples herein using appropriately isotopically enriched reagents and / or intermediates without undue experimentation.

[0063] When the compounds of the present disclosure can form tautomers, all such tautomeric forms are also included within the scope of the present disclosure. For example, a compound containing a carbonyl -CH2C(O)- group (keto form) can undergo tautomerization to form a hydroxy -CH=C(OH)- group (enol form). Both the keto and enol forms are included within the scope of the present disclosure when present.

[0064] When any variable (e.g., R 5When any moiety (etc.) appears more than once in any component, its definition at each occurrence is independent of its other occurrences. In addition, combinations of substituents and variables are permitted only if such combinations result in a stable compound. Lines drawn from substituents to the ring system indicate that the depicted bond can be attached to any ring atom that can be substituted. If the ring system is bicyclic, it means that the bond is attached to any suitable atom on either ring of the bicyclic moiety.

[0065] It should be understood that one or more silicon (Si) atoms can be incorporated into the compounds of the present disclosure in place of one or more carbon atoms to provide compounds that are chemically stable and can be readily synthesized from readily available starting materials by techniques known in the art. The covalent radii of carbon and silicon are different, resulting in differences in bond distances and spatial arrangements when comparing similar C-element and Si-element bonds. These differences result in minor changes in the size and shape of silicon-containing compounds when compared to carbon. One of ordinary skill in the art will understand that size and shape differences can result in minor or significant changes in potency, solubility, lack of off-target activity, packaging properties, etc. (Diass, J.O. et al. Organometallics (2006) 5:1188-1198; Showell, G.A. et al. Bioorganic & Medicinal Chemistry Letters (2006) 16:2555-2558).

[0066] It should be understood that the substituents and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized from readily available starting materials by techniques known in the art and those methods described hereinafter. If a substituent itself is substituted by more than one group, it should be understood that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure is produced. The phrase “optionally substituted by one or more substituents” should be understood to mean that the moiety in question is unsubstituted or can be substituted by one or more substituents.

[0067] Absolute stereochemistry is illustrated by the use of dashed and solid wedge bonds, as shown in Illus-I and Illus-II. Thus, the methyl group in Illus-I emerges from the page of the paper, and the ethyl group in Illus-II descends into the page, where the cyclohexene ring residue lies in the plane of the paper. It is assumed that the hydrogen on the same carbon as the methyl group in Illus-I descends into the page, and the hydrogen on the same carbon as the ethyl group in Illus-II emerges from the page. The convention is the same, where both the dashed and solid rectangles are attached to the same carbon as in Illus-III, the methyl group emerges from the plane of the paper, and the ethyl group descends into the plane of the paper, and the cyclohexene ring is in the plane of the paper.

[0068]

[0069] As is conventional, unless otherwise stated in the accompanying text, ordinary "bar" bonds or "wavy" bonds indicate the presentation of all possible stereochemistry, including pure compounds, mixtures of isomers, and racemic mixtures.

[0070] As used herein, unless otherwise specified, the following terms have the following meanings:

[0071] The phrase "at least one", as used in reference to the number of components of a composition, such as "at least one pharmaceutical excipient", means that one member of the specified group is present in the composition, and more than one may additionally be present. The components of a composition are typically aliquots of separate pure materials added to the composition, where the purity level of the separate materials added to the composition is the purity level commonly accepted for that type of reagent.

[0072] Whether used in reference to substituents on a compound or components of a pharmaceutical composition, the phrase "one or more" means the same as "at least one".

[0073] "Effective amount" or "therapeutically effective amount" is intended to describe an amount of at least one compound of the present disclosure or a composition comprising at least one compound of the present disclosure that is effective to treat or inhibit a disease or condition described herein and thus produces a desired therapeutic, ameliorating, inhibitory, or prophylactic effect. For example, when treating a central nervous system disease or disorder with one or more compounds described herein, an "effective amount" (or "therapeutically effective amount") refers to, for example, an amount of at least one compound of Formula I, Formula II, or Formula III that provides a therapeutic response in a patient suffering from a central nervous system disease or disorder ("condition"), the therapeutic response including a response suitable for controlling, alleviating, ameliorating, or treating the condition or a long-term stable response that alleviates, ameliorates, reduces, or eradicates one or more symptoms attributable to the condition and / or the condition; for example, it can be determined by analyzing pharmacodynamic markers or clinically evaluating patients suffering from the condition.

[0074] "Patient" and "subject" refer to an animal, such as a mammal (e.g., a human), and preferably a human.

[0075] "Prodrug" refers to, for example, a compound that is rapidly converted in vivo to the parent compound by hydrolysis in the blood; for example, converting a prodrug of Formula I, Formula II, or Formula III to a compound of Formula I, Formula II, or Formula III or a salt thereof; a detailed discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference; the scope of the present disclosure includes prodrugs of the novel compounds of the present disclosure.

[0076] The term "substituted" means that one or more of the recited substituents can occupy one or more of the bonding positions normally occupied by "-H" on a substrate, provided that such substitution does not exceed the normal valence rules of the atoms in the bonding configuration present in the substrate and that the substitution ultimately provides a stable compound, that is, such substitution does not provide a compound having mutually reactive substituents positioned pairwise or adjacent to each other; and wherein the substitution provides a compound robust enough to withstand separation from the reaction mixture to useful purity.

[0077] In the case of optional substitution in the description section (e.g., "optionally substituted"), the term means that if substituents are present, one or more of the recited substituents for the specified substrate can be present at the bonding positions on the substrate that are normally occupied by the default substituents that normally occupy that position. For example, the default substituent on a carbon atom of an alkyl moiety is a hydrogen atom, and an optional substituent can replace the default substituent.

[0078] As used herein, unless otherwise specified, the following terms used in the description section, whether including the entire definition of the variable portion of the structural representation of the compounds of the present disclosure or the substituents of the variable portion of the structural representation of the groups attached to the compounds of the present disclosure, have the following meanings, and unless otherwise specified, when the term is used alone or as a component (e.g., part or substituent) of another term, the definition of each term (i.e., part or substituent) applies (e.g., the definition of aryl is the same for aryl and for the aryl portion of arylalkyl, alkylaryl, arylalkynyl moieties, etc.); parts are equivalently described herein by structure, printed representation, or chemical terms, with no intention of making any distinction in meaning, e.g., an "acyl" substituent can be described herein by the term "acyl", by the printed representation "R'-(C=O)-" or "R'-C(O)-", or by the structural representation: Equivalently described, and likewise, the use of any or all of these representations does not imply a distinction.

[0079] The term "alkyl" (including alkyl moieties of other parts, such as trifluoromethyl-alkyl- and alkoxy-) means a straight-chain or branched-chain aliphatic hydrocarbon moiety containing up to about 20 carbon atoms (e.g., the designation "C 1-20 alkyl" means an aliphatic hydrocarbon moiety of 1 to 20 carbon atoms). In some embodiments, alkyl preferably includes up to about 10 carbon atoms, unless the term is modified by an indication considering shorter chains, e.g., an alkyl moiety of 1 to 8 carbon atoms is named "C 1-8 alkyl" herein. When the term "alkyl" is represented by two hyphens (i.e., "-alkyl-"), it means that the alkyl moiety is bonded in such a way that substituents are attached on either side of the alkyl moiety, e.g., "-alkyl-OH" means that the hydroxy moiety is attached to the alkyl moiety of the substrate.

[0080] As used herein, when the term "alkyl" is modified by "substituted" or "optionally substituted", it means that one or more C-H bonds in the alkyl moiety group are replaced by substituents bonded to the alkyl substrate as called out in the definition of the part or optionally can be replaced.

[0081] The term "solvate" refers to a pharmaceutically acceptable solvate formed by a compound of the present disclosure and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.

[0082] The term "halogen" refers to fluorine, chlorine, bromine or iodine; unless otherwise specified when using the term, the preferred halogens are fluorine, chlorine and bromine, and the substituents as halogen atoms are represented as -F, -Cl, -Br or -I, and "halo-" represents a fluoro-, chloro-, bromo- or iodo-substituent bonded to the defined moiety. For example, "haloalkyl" represents an alkyl group as defined above, wherein one or more bonding positions on the alkyl moiety that are normally occupied by hydrogen atoms are replaced by a halo group, and perhaloalkyl (or "fully halogenated" alkyl) represents that all bonding sites that do not participate in bonding the alkyl substituent to the substrate are occupied by halogen. For example, when the alkyl group is selected as methyl, the term perfluoroalkyl represents -CF3.

[0083] The terms "hydroxy" and "hydroxyl" refer to the HO- group, and "hydroxyalkyl" represents a substituent of the formula: "HO-alkyl-", wherein the alkyl group is bonded to the substrate and may be substituted or unsubstituted as defined above; preferred hydroxyalkyl moieties include lower alkyl; non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

[0084] The bonding sequence is represented by a hyphen, where the moiety is represented in text. For example, -alkyl indicates a single bond between the substrate and the alkyl moiety, -alkyl-X indicates that the alkyl group bonds the "X" substituent to the substrate, and in a structural representation, the bonding sequence is indicated by a wavy line terminated by a bond. For example: It indicates that the methylphenyl moiety is bonded to the matrix through the carbon atom adjacent to the methyl substituent, and the bond terminated by a wavy line and drawn into the structure without any specific indication of the atom to which it is bonded indicates that this moiety can be bonded to the matrix via any atom in this moiety, and it can be used for bonding as described in the above examples.

[0085] A line as a bond - generally represents a mixture of possible isomers (e.g., containing (R)- and (S)-stereochemical configurations) or either of them.

[0086] As used herein, the term "DAR" or "drug-to-antibody ratio" refers to the average number of linker / drug moieties attached to the antibodies present in the composition. For a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is the average of the "p" of all individual antibody-drug conjugate molecules present in the composition, and this average is expressed as a decimal. Thus, in some embodiments of a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 0 to 24, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, and 0 to 1. In additional embodiments, for a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 1 to 4, 2 to 5, 3 to 6, 4 to 7, 5 to 8, and 6 to 8. In other embodiments, for a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 1 to 3, 2 to 4, 3 to 5, 4 to 6, 5 to 7, and 6 to 8. In further embodiments, for a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and 7 to 8. In a specific embodiment, the DAR of the composition is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. As used above, the term "composition" is to be understood to encompass pharmaceutical compositions. The average DAR can be determined by various conventional means, such as UV spectroscopy, mass spectrometry, ELISA assays, radiometric assays, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0087] In all cases, the compound name accompanies the drawn structure, and each possible stereochemical arrangement is expected to be described based on the synthetic operations used in its preparation for a given structural isomer. A list of discrete stereoisomers is used in combination or indicates that the compound presented (e.g., 'Example number') has been isolated as a single stereoisomer, and the identity of that stereoisomer corresponds to one of the possible configurations listed. A list of discrete stereoisomers is used in combination and indicates that the compound presented has been isolated as a racemic mixture or a mixture of diastereomers.

[0088] Unless otherwise specified or implied by the context, as used herein, "natural amino acid" refers to a naturally occurring amino acid in the L or D-configuration, namely arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamate, aspartate, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine or their residues, unless otherwise specified or implied by the context.

[0089] Unless otherwise specified or implied by the context, as used herein, "unnatural amino acid" refers to an acid or its residue containing an α-amino group, which has the backbone structure of a natural amino acid but, for example, has a side chain group attached to the α-carbon that is not present in natural amino acids. Examples of unnatural amino acids are A', B', C', D', and E' containing amino acid side chains having the following structures:

[0090]

[0091] An example of an amino acid side chain is C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. Other examples of amino acid side chains are dimethyl, ethyl, propyl, butyl, -C≡C, and -CH2C≡C.

[0092] The ligand (L') can be any moiety having a free cysteine group, including but not limited to an antibody, protein, peptide, polypeptide, or an engineered antibody modified to provide a free cysteine. One aspect of this is achieved when the ligand is an antibody, preferably a whole antibody. The role of the ligand is to target and deliver the drug to a specific population of target cells with which the ligand interacts. Suitable ligands include, for example, antibodies such as full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, and colony-stimulating factors, vitamins, nutrient transport molecules (such as, but not limited to, transferrin), or any other cell-binding molecule or substance, including small molecules and peptides. The ligand can be, for example, a non-antibody protein targeting agent.

[0093] When the conjugate contains a non-immunoreactive protein, polypeptide, or peptide ligand rather than an antibody, useful non-immunoreactive proteins, polypeptides, or peptide ligands include but are not limited to transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factor ("TGF") such as TGF-α and TGF-β, vaccinia growth factor ("VGF"), insulin, and insulin-like growth factors I and II, somatostatin, lectin, and apolipoproteins from low-density lipoproteins.

[0094] Particularly preferred ligands (L’) are antibodies, including intact antibodies. In fact, in any of the embodiments described herein, the ligand can be an antibody. Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) directed against an antigen of interest can be prepared by using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0095] In addition, recombinant antibodies (e.g., chimeric and humanized monoclonal antibodies) containing human and non-human portions that can be prepared using standard recombinant DNA techniques are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from murine monoclonals and human immunoglobulin constant regions. (See, e.g., U.S. Patent Nos. 4,816,567; and 4,816,397, the entire contents of which are incorporated herein by reference). Humanized antibodies are antibody molecules from non-human species that have one or more complementarity-determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, e.g., U.S. Patent No. 5,585,089, the entire contents of which are incorporated herein by reference). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, such as using the methods described in International Publication No. WO 87 / 02671; European Patent Publication No. 0 184 187, the entire contents of each of which are incorporated herein by reference.

[0096] Fully human antibodies are particularly desirable and can be produced using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes.

[0097] Antibodies include modified analogs and derivatives, i.e., covalent attachments of any type of molecule, provided that such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell antibody or other protein, etc. Any of a number of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, an analog or derivative can contain one or more unnatural amino acids.

[0098] In one particular embodiment, known antibodies for treating cancer can be used. Antibodies that are immunospecific for cancer cell antigens can be obtained commercially or produced by any method known to those skilled in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies that are immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a database similar thereto, literature publications, or by conventional cloning and sequencing.

[0099] In another particular embodiment, antibodies for treating autoimmune diseases are used in the compositions and methods according to the present disclosure. Antibodies that are immunospecific for antigens of cells responsible for producing autoantibodies can be obtained from any source (e.g., university scientists or companies) or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques.

[0100] In another embodiment, it may be desirable to conjugate the components of the linker to a ligand (e.g., an antibody) prior to attaching the payload or drug component of the ADC. For example, in embodiments where a substituent containing a thiol (e.g., cysteine) is used to attach the drug component, it may be desirable to conjugate the components of the linker to the ligand L’ (e.g., an antibody) prior to attaching the drug component of the ADC.

[0101] In another embodiment, the compounds of the present disclosure include those identified herein as examples in the following table and their pharmaceutically acceptable salts.

[0102] Unless the context otherwise indicates or implies, as used herein, the term “antibody-drug conjugate” or ADC is a subset of the ligand-drug conjugates of Formula III, Formula III’, and Formula III”, and thus refers to a construct composed of an antibody ligand (L’) incorporating or corresponding to an antibody or an antigen-binding fragment thereof and a cytotoxic payload (commonly referred to as the free drug) incorporating or corresponding structurally to a bioactive compound, wherein L’ and R 2 are bonded to each other herein through a dipeptide linker unit, and wherein the antibody-drug conjugate is capable of selectively binding to the targeted antigen of the targeted cell through its targeting antibody ligand unit, which in some aspects is an antigen of abnormal cells (e.g., cancer cells).

[0103] In one aspect, the term antibody-drug conjugate (ADC) refers to a plurality of individual conjugate compounds (i.e., compositions), said individual conjugate compounds having a degree of sameness or difference in terms of the number of linker-payload moieties conjugated to each antibody ligand (L') and / or the position on the antibody ligand (L') to which the linker-payload moiety is conjugated. In some aspects, the term refers to a distribution or collection (i.e., population or plurality) of conjugate compounds having the same linker-payload moiety and antibody ligand (L'), allowing for mutant amino acid changes and variable glycosylation patterns as described herein to occur during the production of the antibody from cell culture, which in some aspects have a variable loading and / or distribution of linker-payload moieties linked to each antibody residue (e.g., when the number of linker-payload moieties in any two antibody-drug conjugate compounds among a plurality of such compounds is the same, but the position of the site linking the linker-payload moiety to the targeted antibody ligand (L') is different). In those cases, the antibody-drug conjugate is described by the average drug loading of the conjugate compounds.

[0104] In some aspects, the payload (drug) is a cytotoxic agent, typically a cytotoxic agent having a secondary aliphatic amine as a conjugation handle, and includes auristatin compounds as defined herein. Useful classes of cytotoxic agents include, for example, antimicrotubule agents, DNA minor groove binders, DNA replication inhibitors, chemosensitizers, and the like. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, enediynes, maytansinoids, etoposide, and vinca alkaloids. Exemplary cytotoxic agents include, for example, auristatin T, auristatin E, auristatin F phenylenediamine (AFP), monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE), DNA minor groove binders (e.g., enediynes and leishcidin), enediynes, maytansinoids, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), microtubulysin M, doxorubicin, morpholino-doxorubicin, and cyano-morpholino-doxorubicin.

[0105] The cytotoxic agent can be a chemotherapeutic agent such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analogue, calicheamicin, maytansine, a dolastatin 10 analogue, rhizoxin, or palytoxin.

[0106] Exemplary auristatins include, but are not limited to, auristatin E (AE), reacting auristatin E with p-acetylbenzoic acid or benzoylvaleric acid to produce benzoyl-auristatin E ester (AEB) and 5-benzoylvaleric acid-auristatin E ester (AEVB), auristatin F phenylenediamine (AFP), monomethyl auristatin F (MMAF) and monomethyl auristatin E (MMAE) and those further described in embodiments of the present disclosure. The synthesis and structure of auristatins are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, 2005-0009751, 2009-0111756 and 2011-0020343; International Patent Publication Nos. WO 04 / 010957, International Patent Publication No. WO 02 / 088172 and U.S. Patent Nos. 7,659,241 and 8,343,928. Their structures and their synthetic methods disclosed therein are hereby incorporated by reference in particular.

[0107] The cytotoxic agent can be a DNA minor groove binder (see, e.g., U.S. Patent No. 6,130,237). For example, the minor groove binder can be a CBI compound or an enediyne (e.g., calicheamicin).

[0108] The cytotoxic agent or cytostatic agent can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., (paclitaxel), (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine and vinorelbine). Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colchamine, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatin, discodermolide and halichondrin.

[0109] The cytotoxic agent can be a maytansinoid, another group of anti-tubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be maytansine or a maytansine-containing drug linker, such as DM-1 or DM-4 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.).

[0110] Unsaturated valences in the text, schemes, examples, structural formulas and any tables herein are assumed to have hydrogen atoms or a sufficient number of hydrogen atoms to saturate the valence.

[0111] One or more compounds of the present disclosure may also exist as solvates or optionally be converted to solvates. The preparation of solvates is generally known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93 (3) , 601 - 611 (2004) describes the preparation of the solvate of the antifungal fluconazole in ethyl acetate and from water. Similar preparations of solvates and hemisolvates, including hydrates (where the solvent is water or water-based), etc. are described in E.C. van Tonder et al., AAPS PharmSciTech., 5(1) , article 12 (2004); and A.L. Bingham et al., Chem. Commun., 603 - 604 (2001). A typical non-limiting method involves dissolving the compound of the invention in a desired amount of a desired solvent (e.g., an organic solvent, an aqueous solvent, water, or a mixture of two or more thereof) at a temperature above ambient temperature, and cooling the solution at a rate sufficient to form crystals in the presence or absence of an anti-solvent, and then separating the crystals by standard methods. Analytical techniques, such as I.R. spectroscopy, show the presence of the solvent (including water) in the crystal as a solvate (or a hydrate in the case where water is incorporated into the crystal form).

[0112] The present disclosure also includes the compounds of the present disclosure in isolated and purified forms obtained by conventional techniques. Polymorphic forms of the compounds of formula I, formula II, and formula II, as well as salts, solvates, and prodrugs of the compounds of formula I, formula II, and formula II are intended to be included in the present disclosure. Certain compounds of the present disclosure may exist in different isomeric forms (e.g., enantiomers, diastereomers, atropisomers). The compounds of the invention include all of their isomeric forms, including pure forms and mixtures of two or more, including racemic mixtures.

[0113] In the same way, unless otherwise indicated, any structural representation showing a tautomeric form of a compound exhibiting tautomerism is meant to include all such tautomeric forms of the compound. Thus, when the compounds of the present disclosure, their salts, and their solvates and prodrugs may exist in different tautomeric forms or in equilibrium between these forms, all of these forms of the compound are covered by the present disclosure and included within the scope of the present disclosure. Examples of such tautomers include, but are not limited to, keto / enol tautomeric forms, imine / enamine tautomeric forms, and, for example, heteroaromatic forms, such as the following moieties:

[0114]

[0115] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0116] As used herein, "pharmaceutically acceptable salts" refer to derivatives in which the parent compound is modified by formation of its acid or base salts. Salts in solid form may exist in more than one crystalline structure and may also be in the form of hydrates. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (e.g., amines); alkali metal salts or organic salts of acidic residues (e.g., carboxylic acids); and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as formic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like.

[0117] When the compounds of the present disclosure are basic, salts can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic and organic acids). Such acids include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. In one aspect of the present disclosure, the salts are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, fumaric acid, and tartaric acid. Similarly, salts of acidic compounds are formed by reaction with a suitable inorganic or organic base.

[0118] The term "cytotoxic agent" refers to a substance that inhibits or stops the function of cells and / or causes cell death or destruction. Cytotoxic agents include toxins and other compounds useful in the treatment of tumors.

[0119] The term "toxin" refers to any substance that can have a harmful effect on the growth or proliferation of cells. Toxins can be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as irinotecan, maytansinoids and their derivatives (CN101573384), such as DM1, DM3, DM4, auristatin F (AF) and its derivatives such as MMAF, MMAE, 3024 (WO 2016 / 127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, a-sarcin, Aleutites fordii toxin, dianthin, Phytolaca americana toxin, Momordica charantia inhibitor, curcin, crotin, Sapaonaria ojficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecene.

[0120] The term "chemotherapeutic agent" refers to chemical compounds that can be used to treat tumors. This definition also includes antihormonal agents that act to regulate, reduce, block, or inhibit the action of hormones that promote cancer growth, which are usually in the form of systemic or whole-body therapy. It can be a hormone. Examples of chemotherapeutic agents include alkylating agents such as thiotepa; cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benaodopa, carboquone, meturedopa, and uredopa; aziridines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitrobin hydrochloride; melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, chromomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin; streptozocin, tuberculocidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiomethopterin, thioguanopterin; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone;Anti-adrenaline, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pintostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; dibromodulcitol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel ( Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. The definition also includes antihormonal agents that modulate or inhibit the effect of hormones on tumors, such as antiestrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LYll7018, onapristone and Fareston; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0121] As used herein, the term "treating" or "treatment" (e.g., of a disease, disorder or condition or related symptoms, which together or separately may be referred to as an "indication") includes: inhibiting a disease, disorder or condition, i.e., arresting or reducing the development of the disease or its biological processes or progression or the development of its clinical symptoms; or alleviating a disease, i.e., causing regression of the disease or its biological processes or progression and / or its clinical symptoms. As used herein, "treatment" also refers to controlling, ameliorating or reducing the risk of a subject suffering from a disease, disorder or condition involving a tumor. As used herein, the terms "preventing" or "prevention" or "prophylaxis" of a disease, disorder or condition includes: impeding the development or progression of the clinical symptoms of a disease, disorder or condition in a mammal that may be exposed to or is susceptible to the disease, disorder or condition but has not yet experienced or shown symptoms of the disease, etc.

[0122] As will be apparent to those skilled in the art, the subjects treated by the methods described herein are generally mammals, including humans and non-human animals (e.g., laboratory animals and companion animals).

[0123] As used herein, the term "composition" is intended to cover a product comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, together with a specified amount of one or more additional specified ingredients, as well as any product directly or indirectly resulting from the combination of the specified amounts of the specified ingredients. Such a term related to a pharmaceutical composition is intended to cover a product comprising an active ingredient, which includes a compound of the present disclosure or a pharmaceutically acceptable salt thereof, optionally together with one or more additional active ingredients, and an inert ingredient constituting a carrier, as well as any product directly or indirectly resulting from the combination, complexation or aggregation of any two or more ingredients, or from the dissociation of one or more ingredients, or from other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present disclosure cover any composition prepared by mixing a compound of the present disclosure or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not harmful to its recipient.

[0124] As described above, further embodiments of the present disclosure each relate to a method for treating a disease, disorder or condition or one or more symptoms thereof ("indications"), the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or its salt.

[0125] In another embodiment, the present disclosure relates to a method of preparing a medicament for a subject, which comprises combining a compound of the present disclosure, or a pharmaceutically acceptable salt thereof with a pharmaceutical carrier or diluent.

[0126] One such embodiment provides a method of treating or preventing cancer in a subject in need thereof, the cancer being selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or anaplastic large cell lymphoma), the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the compound, its salt or solvate. In one such embodiment, the subject is a human.

[0127] Another aspect of the present disclosure relates to a method for treating and / or preventing tumors, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound according to the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a compound according to the present disclosure.

[0128] Combinations with additional therapeutic agents are also contemplated in the method. For example, combinations of the compounds of the present disclosure with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful for treating certain malignancies. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors gamma and delta. PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; troglitazone and rosiglitazone maleate both inhibit the development of neovascularization in the mouse retina (Arch. Ophthamol. 2001; 119:709-717). Examples of PAR-γ agonists and PPAR-γ / α agonists include, but are not limited to, thiazolidinediones (e.g., DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropanoic acid (disclosed in USSN 09 / 782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in USSN 60 / 235,708 and 60 / 244,697) or a pharmaceutically acceptable salt thereof.

[0129] Another embodiment of the present disclosure is the use of the compounds of the present disclosure in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies for treating cancer, see Hall et al. (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al. (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressor gene. Examples of such genes include, but are not limited to, p53, which can be delivered by recombinant virus-mediated gene transfer (see, for example, U.S. Patent No. 6,069,134), uPA / uPAR antagonists ("Adenovirus-Mediated Delivery of a uPA / uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998; 5(8):1105-13), and interferon gamma (J. Immunol. 2000; 164:217-222).

[0130] The compounds of the present disclosure can also be administered in combination with inhibitors of intrinsic multi-drug resistance (MDR), particularly MDR associated with high levels of expression of transporters. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853, and PSC833 (valspodar), or pharmaceutically acceptable salts thereof.

[0131] The compounds of the present disclosure can also be administered together with immunostimulating drugs (e.g., levamisole, isoprinosine, and Zadaxin or pharmaceutically acceptable salts thereof).

[0132] The compounds of the present disclosure can also be used in combination with P450 inhibitors for the treatment or prevention of cancer, said P450 inhibitors including: xenobiotics, quinidine, tyramine, ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone, and nelfinavir or pharmaceutically acceptable salts thereof.

[0133] The compounds of the present disclosure can also be used in combination with Pgp and / or BCRP inhibitors to treat or prevent cancer, and the Pgp and / or BCRP inhibitors include: cyclosporin A, PSC833, GF120918, cremophor EL, fumitremorgin C, Ko132, Ko134, Iressa, imatinib mesylate, EKI-785, C11033, novobiocin, diethylstilbestrol, tamoxifen, resperpine, VX-710, tryprostatin A, flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, ketoconazole, nifidepine, FK506, amiodarone, XR9576, indinavir, amprenavir, cortisol, testosterone, LY335979, OC144-093, erythromycin, vincristine, digoxin, and talinolol, or a pharmaceutically acceptable salt thereof.

[0134] The compounds of the present disclosure can also be used in combination with bisphosphonates to treat or prevent cancer, including bone cancer, and the bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate, or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate, and tiludronate, including any and all pharmaceutically acceptable salts, derivatives, hydrates, and mixtures thereof.

[0135] The compounds of the present disclosure can also be used in combination with aromatase inhibitors to treat or prevent breast cancer. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole, and exemestane, or a pharmaceutically acceptable salt thereof.

[0136] The compounds of the present disclosure can also be used in combination with siRNA therapeutics to treat or prevent cancer.

[0137] The compounds of the present disclosure can also be administered in combination with a γ-secretase inhibitor and / or an inhibitor of NOTCH signaling. Such inhibitors include the compounds described in WO 01 / 90084, WO 02 / 30912, WO 01 / 70677, WO 03 / 013506, WO 02 / 36555, WO 03 / 093252, WO 03 / 093264, WO 03 / 093251, WO 03 / 093253, WO2004 / 039800, WO 2004 / 039370, WO 2005 / 030731, WO 2005 / 014553, USSN 10 / 957,251, WO2004 / 089911, WO 02 / 081435, WO 02 / 081433, WO 03 / 018543, WO 2004 / 031137, WO 2004 / 031139, WO 2004 / 031138, WO 2004 / 101538, WO 2004 / 101539, and WO 02 / 47671 (including LY-450139), or a pharmaceutically acceptable salt thereof.

[0138] In one embodiment, specific anti-cancer agents useful in the combination therapies of the present invention include, but are not limited to: pembrolizumab abarelix (Plenaxis ); aldesleukin aldesleukin alemtuzumab alitretinoin allopurinol altretamine amifostine anastrozole arsenic trioxide asparaginase azacitidine bevacizumab bexarotene capsules bexarotene gel bleomycin bortezomib busulfan intravenous busulfan oral calusterone capecitabine carboplatin carmustine carmustine carmustine with Polifeprosan20 implant (Gliadel ); celecoxib cetuximab Chlorambucil Cisplatin Cladribine Clofarabine Cyclophosphamide Cyclophosphamide (Cytoxan );Cyclophosphamide (Cytoxan );Cytarabine Liposomal Cytarabine Dacarbazine Dactinomycin, Actinomycin D Darbepoetin alfa Liposomal Daunorubicin Daunorubicin, Adriamycin Daunorubicin, Adriamycin Denileukin diftitox Dexrazoxane Docetaxel Doxorubicin (Adriamycin );Doxorubicin Doxorubicin (Adriamycin PFS );Liposomal Doxorubicin Drostanolone propionate Drostanolone propionate Elliott’s B solution (Elliott’s B );Epirubicin Epoetin alfa Erlotinib Estramustine Etoposide phosphate Etoposide, VP-16 Exemestane Filgrastim Floxuridine (intra-arterial) Fludarabine Fluorouracil, 5-FU Fulvestrant Gefitinib Gemcitabine Gemtuzumab ozogamicin Goserelin acetate (Zoladex );Goserelin acetate Histrelin acetate (Histrelin );Hydroxycarbamide Ibritumomab tiuxetan Idarubicin Ifosfamide Imatinib mesylate Interferon alfa-2a (Roferon );Interferon alfa-2b (Intron );Irinotecan Lenalidomide Letrozole Leucovorin Leuprorelin acetate Levamisole Lomustine, CCNU Mechlorethamine, nitrogen mustard (nitrogen mustard) Megestrol acetate Melphalan, L-PAM Mercaptopurine, 6-MP Mesna Mesna (Mesnex );Methotrexate Methoxsalen Mitomycin C Mitotane Mitoxantrone Nandrolone phenylpropionate Nelarabine Nofetumomab Oprelvekin Oxaliplatin Paclitaxel Paclitaxel Paclitaxel protein-bound particles Palifermin Pamidronate Pegaspargase (Adagen (Pegademase Bovine) );Pegaspargase Pegfilgrastim Pemetrexed disodium Pentostatin Pipobroman Plicamycin, mithramycin Porfimer sodium Procarbazine Quinacrine Rasburicase Rituximab Ridaforolimus; Sargramostim Sargramostim Sorafenib Streptozocin Sunitinib malate Talc Tamoxifen Temozolomide Teniposide, VM-26 Testolactone Tioguanine, 6-TG Thiotepa Topotecan Toremifene Tositumomab Tositumomab / I-131 Tositumomab Trastuzumab Tretinoin, ATRA Uracil Mustard Valrubicin Vinblastine Vincristine Vinorelbine Olaparib Vorinostat and Zoledronate or a pharmaceutically acceptable salt thereof.

[0139] Accordingly, the scope of the present disclosure encompasses the use of a compound of the present disclosure in combination with a second compound selected from: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents / cytostatic agents, anti-proliferative agents, isoprenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, PPAR-γ agonists, PPAR-δ agonists, inhibitors of intrinsic multi-drug resistance, anti-emetics, agents for treating anemia, agents for treating neutropenia, immunopotentiating drugs, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any of the therapeutic agents listed above.

[0140] Another example of the present disclosure is a method of treating cancer, which comprises administering a therapeutically effective amount of a compound of the present disclosure in combination with paclitaxel or trastuzumab.

[0141] The therapeutic combinations disclosed herein can be used in combination with one or more other active agents, including but not limited to other anti-cancer agents for preventing, treating, controlling, ameliorating or reducing the risk of a particular disease or condition (e.g., a cell proliferation disorder). In one embodiment, the compounds of the present disclosure are used in combination with one or more other anti-cancer agents for preventing, treating, controlling, ameliorating or reducing the risk of a particular disease or condition for which the compounds of the present disclosure are useful. Such other active agents can be administered by their usual routes and in their usual amounts, before, simultaneously with or sequentially to the compounds of the present disclosure.

[0142] The present disclosure also includes a pharmaceutical composition for treating or preventing cancer, which comprises a therapeutically effective amount of a compound of the present disclosure and a second compound selected from the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents / cytostatic agents, antiproliferative agents, isoprenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, PPAR-γ agonists, PPAR-δ agonists, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any of the therapeutic agents listed above.

[0143] The present disclosure also relates to a method of treating cancer in a human patient, which comprises administering a PD-1 antagonist to the patient. The compound of the present disclosure and the PD-1 antagonist can be administered simultaneously or sequentially.

[0144] In certain embodiments, the PD-1 antagonist is an anti-PD-1 antibody or an antigen-binding fragment thereof. In alternative embodiments, the PD-1 antagonist is an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody independently selected from pembrolizumab, nivolumab, cemiplimab, sintilimab, tislelizumab, atezolizumab (MPDL3280A), camrelizumab, and toripalimab. In other embodiments, the PD-L1 antagonist is an anti-PD-L1 antibody independently selected from atezolizumab, durvalumab, and avelumab.

[0145] In one embodiment, the PD-1 antagonist is pembrolizumab. In certain sub-embodiments, the method comprises administering 200 mg of pembrolizumab to the patient approximately every three weeks. In other sub-embodiments, the method comprises administering 400 mg of pembrolizumab to the patient approximately every six weeks.

[0146] In further sub-embodiments, the method comprises administering 2 mg / kg of pembrolizumab to the patient approximately every three weeks. In certain sub-embodiments, the patient is a pediatric patient.

[0147] In some embodiments, the PD-1 antagonist is nivolumab. In certain sub-embodiments, the method comprises administering 240 mg of nivolumab to a patient approximately every two weeks. In other sub-embodiments, the method comprises administering 480 mg of nivolumab to a patient approximately every four weeks.

[0148] In some embodiments, the PD-1 antagonist is cemiplimab. In certain embodiments, the method comprises administering 350 mg of cemiplimab to a patient approximately every three weeks.

[0149] In some embodiments, the PD-1 antagonist is atezolizumab. In certain sub-embodiments, the method comprises administering 1200 mg of atezolizumab to a patient approximately every three weeks.

[0150] In some embodiments, the PD-1 antagonist is durvalumab. In certain sub-embodiments, the method comprises administering 10 mg / kg of durvalumab to a patient approximately every two weeks.

[0151] In some embodiments, the PD-1 antagonist is avelumab. In certain sub-embodiments, the method comprises administering 800 mg of avelumab to a patient approximately every two weeks.

[0152] When the compounds of the present disclosure are administered in combination with an anti-human PD-1 antibody (or antigen-binding fragment thereof), the anti-human PD-1 antibody (or antigen-binding fragment thereof) can be administered simultaneously with the compounds of the present disclosure, or before or after the compounds of the present disclosure. Either the anti-human PD-1 antibody (or antigen-binding fragment thereof) and / or the compounds of the present disclosure or a pharmaceutically acceptable salt thereof can be administered separately by the same or different routes of administration, or together in the same pharmaceutical composition with other reagents. The weight ratio of the anti-human PD-1 antibody (or antigen-binding fragment thereof) to the compounds of the present disclosure can vary and will depend on the therapeutically effective dose of each reagent. Generally, the respective therapeutically effective doses will be used. Combinations comprising at least one anti-human PD-1 antibody (or antigen-binding fragment thereof), the compounds of the present disclosure, and optionally other active agents will generally comprise a therapeutically effective dose of each active agent. In such combinations, the anti-human PD-1 antibody (or antigen-binding fragment thereof), the compounds, and the other active agents can be administered separately or in combination. Additionally, the administration of one element can be before, simultaneous with, or after the administration of the other reagents.

[0153] In one embodiment, the present disclosure provides an anti-human PD-1 antibody (or antigen-binding fragment thereof) and / or the compounds of the present disclosure and at least one other active agent as a combination preparation for simultaneous, separate, or sequential use in the treatment of cancer.

[0154] The present disclosure also provides the use of the compounds of the present disclosure for the treatment of cancer, wherein the patient has been previously treated (e.g., within 24 hours) with an anti-human PD-1 antibody (or an antigen-binding fragment thereof). The present disclosure also provides the use of an anti-human PD-1 antibody (or an antigen-binding fragment thereof) for the treatment of a cell proliferative disorder, wherein the patient has been previously treated (e.g., within 24 hours) with an antibody-linker-payload compound (ADC) of the present disclosure.

[0155] The present disclosure also relates to a method of treating cancer, the method comprising administering to a subject in need thereof a combination therapy comprising (a) a compound of the present disclosure, and (b) an anti-human PD-1 antibody (or an antigen-binding fragment thereof); wherein the anti-human PD-1 antibody (or an antigen-binding fragment thereof) is administered once every 21 days.

[0156] Additionally, the present disclosure relates to a method of treating cancer, the method comprising administering to a subject in need thereof a combination therapy comprising: (a) a compound of the present disclosure, and (b) an anti-human PD-1 antibody (or an antigen-binding fragment thereof). In certain embodiments, the cancer occurs as one or more solid tumors or lymphomas. In further specific embodiments, the cancer is selected from the group consisting of advanced or metastatic solid tumors and lymphomas. In still further specific embodiments, the cancer is selected from the group consisting of: malignant melanoma, head and neck squamous cell carcinoma, MSI-H cancer, MMR-deficient cancer, non-small cell lung cancer, urothelial cancer, gastric or gastroesophageal junction adenocarcinoma, breast cancer, and lymphoma. In additional embodiments, the lymphoma is selected from the group consisting of: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mediastinal large B-cell lymphoma, splenic marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (malt), nodal marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, primary effusion lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma (primary cutaneous type), anaplastic large cell lymphoma (systemic type), peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, adult T-cell lymphoma / leukemia, nasal-type extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, γ / δ hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, and Hodgkin lymphoma. In certain embodiments, the cell proliferative disorder is a cancer that has metastasized, such as liver metastases from colorectal cancer. In additional embodiments, the cell proliferative disorder is a cancer classified as stage III cancer or stage IV cancer. In the case of these embodiments, the cancer is not surgically resectable.

[0157] In embodiments of the methods disclosed herein, the anti-human PD-1 antibody (or an antigen-binding fragment thereof) is administered by intravenous infusion or subcutaneous injection.

[0158] In one embodiment, the present disclosure provides a composition comprising a compound of the present disclosure, a pharmaceutically acceptable carrier, and an anti-human PD-1 antibody (or an antigen-binding fragment thereof).

[0159] In another embodiment, the present disclosure provides a composition comprising a compound of the present disclosure, a pharmaceutically acceptable carrier, and pembrolizumab.

[0160] In one embodiment, the present disclosure provides a composition comprising a compound of the present disclosure, a pharmaceutically acceptable carrier, and two additional therapeutic agents, one of which is an anti-human PD-1 antibody (or an antigen-binding fragment thereof), and the other of which is independently selected from the group consisting of anti-cancer agents.

[0161] The compounds of the present disclosure can be used in combination with antiemetics to treat nausea or vomiting, including acute, delayed, late, and anticipatory vomiting, which can be caused by the use of the compounds of the present disclosure alone or in combination with radiotherapy. To prevent or treat vomiting, the compounds of the present disclosure can be used in combination with other antiemetics, particularly neurokinin-1 receptor antagonists, 5HT3 receptor antagonists such as ondansetron, granisetron, tropisetron, and zatosetron, GABAB receptor agonists such as baclofen, corticosteroids such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten, or others (such as those disclosed in U.S. Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326, and 3,749,712), antidopaminergic drugs such as phenothiazines (such as prochlorperazine, fluphenazine, thioridazine, and mesoridazine), metoclopramide, aprepitant, fosaprepitant, or dronabinol. In another example, combination therapies using antiemetics selected from neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, and corticosteroids are disclosed for treating or preventing vomiting that may be caused by the administration of the compounds of the present disclosure.

[0162] The compounds of the present disclosure can also be administered together with a reagent useful for treating anemia. Such an anemia therapeutic agent is, for example, a continuous erythropoietin receptor activator (such as epoetin alfa).

[0163] The compounds of the present disclosure can also be administered together with a reagent for treating neutropenia. Such a neutropenia therapeutic agent is, for example, a hematopoietic growth factor that regulates the production and function of neutrophils, such as human granulocyte colony-stimulating factor, (G-CSF). Examples of G-CSF include filgrastim.

[0164] When co-administered with other treatment modalities (including but not limited to radiotherapy, surgery, and gene therapy), the compounds of the present disclosure can be useful. Thus, in one embodiment, unless otherwise indicated, the methods for treating cancer described herein can optionally include administering an effective amount of radiotherapy. For radiotherapy, γ-radiation is preferred.

[0165] The methods for treating cancer described herein can optionally include administering an effective amount of radiation (i.e., the methods for treating cancer described herein optionally include administering radiotherapy).

[0166] The methods for treating cancer described herein include methods for treating cancer that include administering a therapeutically effective amount of a compound of formula III or formula III' or formula III" in combination with radiotherapy and / or in combination with a second compound selected from: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents / cytostatic agents, anti-proliferative agents, isoprenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, PPAR-γ agonists, PPAR-δ agonists, inhibitors of intrinsic multi-drug resistance, anti-emetics, reagents for treating anemia, reagents for treating neutropenia, immunopotentiating drugs, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, reagents that interfere with receptor tyrosine kinases (RTKs), reagents that interfere with cell cycle checkpoints, and any additional therapeutic agents listed herein.

[0167] Additional embodiments of the present disclosure include the pharmaceutical compositions, combinations, uses, and methods described above, where it should be understood that each embodiment can be combined with one or more other embodiments, provided that such combination is consistent with the description of the embodiment. It should also be understood that the embodiments provided above are understood to include all embodiments, including such embodiments resulting from the combination of embodiments.

[0168] Kit

[0169] In one aspect, a kit is provided that includes a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or ester of the compound and a pharmaceutically acceptable carrier, vehicle, or diluent.

[0170] In another aspect, a kit is provided that includes an amount of a compound of the present disclosure and an amount of at least one additional therapeutic agent listed above, wherein the amounts of two or more active ingredients produce a desired therapeutic effect. In one embodiment, the compound of the present disclosure and one or more additional therapeutic agents are provided in the same container. In one embodiment, the compound of the present disclosure and one or more additional therapeutic agents are provided in separate containers.

[0171] The present disclosure includes within its scope prodrugs of the compounds of the present disclosure. Generally, such prodrugs will be functional derivatives of the compounds of the present disclosure that are readily convertible in vivo to the desired compound. Accordingly, in the methods of treatment of the present disclosure, the term "administering" or "effecting an administration" of a compound shall cover treatment of the various conditions with the specifically disclosed compound or with a compound that may not be specifically disclosed but that is convertible in vivo to the designated compound upon administration to a patient. Conventional methods for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Metabolites of these compounds include the active substances that are produced when the compounds of the present disclosure are introduced into a biological environment.

[0172] The compounds or pharmaceutically acceptable salts and / or solvates described herein can be administered alone, in combination with other compounds of the present disclosure, and / or in mixtures in combination with other therapeutic agents. The selection of therapeutic agents that can be co-administered with the compounds of the present disclosure will depend in part on the condition being treated.

[0173] The compounds of the present disclosure can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intracisternal injection or infusion, subcutaneous injection or implantation), by inhalation spray, nasal, vaginal, rectal, sublingual, buccal, or topical routes of administration, and can be formulated singly or together as suitable unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for each route of administration. In addition to treating warm-blooded animals, the compounds of the present disclosure are effective for use in humans.

[0174] The pharmaceutical compositions for administering the compounds of the present disclosure can conveniently exist in dosage unit forms and can be prepared by any methods well-known in the pharmaceutical field. All methods include the step of associating the active ingredient with a carrier which constitutes one or more accessory ingredients. Generally, the pharmaceutical compositions are prepared by uniformly and intimately associating the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical compositions, the active compound is included in an amount sufficient to produce a desired effect on the course or condition of the disease. As used herein, the term "composition" is intended to cover a product containing a specific amount of specific ingredients, as well as any product directly or indirectly resulting from the combination of specific amounts of the specific ingredients.

[0175] The pharmaceutical compositions containing the active ingredient can be in forms suitable for oral use, such as tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft gelatin capsules, or syrups or elixirs. The compositions intended for oral use can be prepared by any methods known in the art for preparing pharmaceutical compositions, and such compositions can contain one or more reagents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets contain the active ingredient admixed with non-toxic pharmaceutically acceptable excipients suitable for the preparation of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets can be uncoated, or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a longer-lasting effect. For example, delayed-release materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated by the techniques described in U.S. Patents 4,256,108; 4,166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release. Oral tablets can also be formulated for immediate release, such as fast-dissolving tablets or wafers, fast-dissolving tablets or fast-dissolving films.

[0176] The preparations for oral use can also exist as hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules, in which the active ingredient is mixed with a water or oil medium (such as peanut oil, liquid paraffin, or olive oil).

[0177] The aqueous suspension contains an active substance mixed with excipients suitable for preparing an aqueous suspension. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic; the dispersing agent or wetting agent can be a naturally occurring phospholipid, such as lecithin, or a condensation product of an alkylene oxide and a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide and a long-chain aliphatic alcohol, such as heptadecaethyleneoxycetanol, or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitan monooleate, or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride, such as polyvinyl sorbitan monooleate. The aqueous suspension may also contain one or more preservatives (such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate), one or more colorants, one or more flavoring agents and one or more sweetening agents (such as sucrose or saccharin).

[0178] The oily suspension can be formulated by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspension may contain a thickening agent, such as beeswax, hard paraffin or acetylated alcohols. Sweetening agents (such as those mentioned above) and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by adding an antioxidant such as ascorbic acid.

[0179] The dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide a mixture of the active ingredient with a dispersing agent or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing agents or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients may also be present, such as sweetening agents, flavoring agents and colorants.

[0180] The pharmaceutical compositions of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof. Suitable emulsifying agents can be naturally occurring gums, such as gum arabic or tragacanth gum, naturally occurring phospholipids, such as soy lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening agents and flavoring agents.

[0181] Syrups and elixirs can be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain demulcents, preservatives and flavoring agents and colorants.

[0182] The pharmaceutical composition can be in the form of a sterile injectable aqueous or oily suspension. This suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile fixed oil is commonly used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic mono- or di-glycerides of fatty acids. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

[0183] The compounds of the present disclosure can also be administered in the form of suppositories for rectal administration of drugs. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient, which is solid at ordinary temperature but liquid at rectal temperature and will thus melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0184] For topical use, creams, ointments, jellies, solutions, or suspensions containing the compounds of the present disclosure are used. Similarly, transdermal patches can also be used for topical administration.

[0185] The pharmaceutical compositions and methods of the present disclosure can also contain other therapeutically active compounds as described herein, which are commonly used for treating the above-mentioned pathological conditions.

[0186] In the treatment, prevention, control, improvement, or reduction of the risk of the conditions disclosed herein, a suitable dosage level of the compounds of the present disclosure is generally about 0.01 to 500 mg / kg patient body weight per day, which can be administered in a single dose or multiple doses. Suitable dosage levels can be about 0.01 to 250 mg / kg / day, about 0.05 to 100 mg / kg / day, or about 0.1 to 50 mg / kg / day. Within this range, the dosage can be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg / day. For oral administration, the composition can be provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for symptomatic adjustment of the dosage for the patient to be treated. The compound can be administered in a regimen of 1 to 4 times a day, or can be administered once or twice a day.

[0187] However, it should be understood that the specific dosage levels and administration frequencies for any particular patient may vary and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, age, body weight, general health status, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder and the host undergoing therapy.

[0188] The methods for preparing the compounds of the present disclosure are illustrated in the following schemes and examples. Starting materials are prepared according to procedures known in the art or as shown herein.

[0189] Preparation Examples

[0190] The compounds of the present disclosure can be prepared using readily available starting materials, reagents, and conventional synthetic procedures according to the following schemes and specific examples or modifications thereof. Variations that are known to those of ordinary skill in the art but not detailed herein can also be utilized. By viewing the following schemes and descriptions, those skilled in the art can readily understand the general procedures for preparing the compounds claimed in the present disclosure. Abbreviations used in the experiments can include, but are not limited to, the following:

[0191]

[0192]

[0193] Preparation of Intermediate Compounds

[0194] The following Preparation Examples 1-3 describe the synthesis of intermediate compounds for preparing exemplary compounds of the present disclosure.

[0195] Preparation Example 1

[0196] Preparation of Intermediate L-Alanyl-L-alanine tert-Butyl Ester (iii)

[0197]

[0198] Step A - Synthesis of Compound ii

[0199] At room temperature, PyBOP (281 g, 540 mmol, 1.2 eq) was added to ((9H-fluoren-9-yl)methoxy(carbonyl)-L-alanine (i) (140 g, 450 mmol) dissolved in DCM (4500 mL). The reaction mixture was stirred for 20 minutes. L-Alanine tert-butyl ester HCl (85.6 g, 471 mmol) was added, and the solution was cooled to 0 °C. Then, DIEA (175 g, 1.35 mol) was added dropwise to the reaction over a period of 1 hour, and then the mixture was stirred at 20 °C overnight. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate, and washed with aqueous sodium carbonate (1 M), potassium bisulfate (1 M), water, and brine. Then, the organic phase was concentrated in vacuo and evaporated to 25% of the original solvent volume. MTBE (1400 mL) was added dropwise, and the mixture was stirred for 5 hours. The suspension was filtered, and the solid was washed with MTBE to give ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine tert-butyl ester (ii). LCMS: (ESI, m / z): [M+H] + = 439.2.

[0200] Step B - Synthesis of compound iii

[0201] TEA (2000 mL) was added to ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine tert-butyl ester (ii) (172 g, 392 mmol) dissolved in DCM (2000 mL). The reaction mixture was warmed to 40 °C and stirred for 16 hours. Then, the reaction mixture was concentrated in vacuo and purified using silica gel chromatography (DCM: 4 M NH4OH in MeOH 5:1) to give L-alanyl-L-alanine tert-butyl ester (iii). LCMS: (ESI, m / z): [M+H] + = 217.1.

[0202] Preparation Example 2

[0203] Preparation of intermediate compound 3-(5-cyano-6-methylsulfonyl)nicotinamido)propanoic acid (x)

[0204] Step A - Synthesis of compound v

[0205] Under N2 atmosphere at 165 °C, a solution of 5-bromo-6-hydroxypyridine-3-carboxylic acid (iv, 42.8 g, 196 mmol) and cuprous cyanide (35.2 g, 393 mmol) in NMP (430 mL) was stirred for 2 hours. The mixture was concentrated in vacuo, and the crude product was purified by reverse-phase flash chromatography (AQ C18 silica gel, ACN 0% to 20% gradient in water (with 0.5% NH3 as modifier)) to obtain the crude product. The resulting mixture was filtered, and the filter cake was washed with H2O. The filtrate was concentrated in vacuo to obtain 5-cyano-6-hydroxypyridine-3-carboxylic acid (v). LCMS: (ESI, m / z): [M-H] - = 163.

[0206] Step B - Synthesis of Compound vi

[0207] At 110 °C, a solution of 5-cyano-6-hydroxypyridine-3-carboxylic acid (v) (25.7 g, 157 mmol) and phosphorus oxychloride (130 mL) was stirred for 2 hours. The reaction was monitored by LCMS and then concentrated in vacuo. Water and EtOAc cooled to 10 °C were added, and the solid was filtered out. The mixture was extracted with ethyl acetate, dried, and concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (column AQ silica gel, 0% to 15% acetonitrile / sodium bicarbonate (aq.)). The pH of the aqueous layer was adjusted to 2 - 3 with 1M HCl, and then the aqueous layer was extracted with ethyl acetate (3×), dried, and concentrated in vacuo to obtain 6-chloro-5-cyanopyridine-3-carboxylic acid (vi). LCMS: (ESI, m / z): [M-H] - = 181.

[0208] Step C - Synthesis of Compound vii

[0209] At 25 °C, under nitrogen atmosphere, dimethylformamide (115 mL) and 6-chloro-5-cyanopyridine-3-carboxylic acid (vi) (7.7 g, 42 mmol) were added to a 500 mL three-necked flask. At 0 °C, sodium (methylthio) (7.39 g, 105 mmol) was added to the above mixture in portions. The resulting mixture was stirred at 25 °C for 8 hours. The reaction was monitored by LCMS. The solution was slowly transferred to H2O (1200 mL), and then extracted with ethyl acetate (1×700 mL). The pH of the aqueous layer was adjusted to 2 - 3 with 1M HCl. The solid was collected by filtration to obtain 5-cyano-6-(methylthio)pyridine-3-carboxylic acid (vii). LCMS: (ESI, m / z): [M-H] - = 193.

[0210] Step D - Synthesis of Compound viii

[0211] Under a nitrogen atmosphere at 45 °C, a solution of 5-cyano-6-(methylthio)nicotinic acid (vii) (7.4 g, 38 mmol) in DCM (185 mL) was treated with m-CPBA (26.3 g, 152 mmol) for 24 h. The reaction was monitored by LCMS and then quenched with saturated sodium bisulfite at 0 °C and concentrated in vacuo. 2-Methyltetrahydrofuran was added to the residue and then the mixture was filtered and concentrated in vacuo to give the crude product. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH, to give 5-cyano-6-(methylsulfonyl)nicotinic acid (viii). LCMS: (ESI, m / z): [M+H] + = 227.05.

[0212] Step E - Synthesis of compound ix

[0213] 5-Cyano-6-(methylsulfonyl)nicotinic acid (viii) (0.57 g, 2.5 mmol) and HATU (1.0 g, 2.7 mmol) were dissolved in 10 mL of DMF and stirred at 25 °C for 30 min. Then, tert-butyl 3-aminopropionate (0.42 g, 2.8 mmol) was added and the mixture was cooled to 10 °C. At 10 °C, DIPEA (0.873 ml, 5.00 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred at 25 °C for 2 h and then diluted with water and extracted with EtOAc (3×). The organic phase was concentrated in vacuo and the residue was purified by silica gel column chromatography, eluting with hexane:EtOAc 2:1 to 1:1, to give tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propionate (ix). 1 1H NMR (500 MHz, CD3OD) δ 8.49 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 4.12 (s, 3H), 2.92 (t, J = 6.9 Hz, 2H), 1.88 (t, J = 6.9 Hz, 2H), 0.74 (s, 9H).

[0214] Step F - Synthesis of compound x

[0215] Dissolve tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propionate (ix) (0.10 g, 0.28 mmol) in 10 mL of 1,4-dioxane. Then, add 4M HCl in 1,4-dioxane (10 mL) to the mixture. Stir the resulting mixture at 25 °C for 16 h, and filter the reaction mixture. Wash the solid with n-heptane and dry under nitrogen for 5 h to obtain 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propionic acid (x). 1 H NMR (500 MHz, D3OD) δ 9.22 (d, J = 1.9 Hz, 1H), 8.80 (d, J = 1.9 Hz, 1H), 3.66 (t, J = 5.8 Hz, 2H), 3.44 (s, 3H), 2.67 (t, J = 6.8 Hz, 2H).

[0216] Preparation Example 3

[0217] Preparation of intermediate compound 4-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv)

[0218]

[0219] Step A - Synthesis of compound xii

[0220] A solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xi) (13 g, 27 mmol, 1 eq) in DMF (130 mL) was added to a 500 mL four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. Then, at 20 °C, 2 mol% of DIEA (0.534 mmol) and bis(4-nitrophenyl) carbonate (16.3 g, 53.6 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was warmed to 45 °C and stirred for 16 h. The reaction mixture was cooled to room temperature and purified using reverse-phase flash column chromatography (30% to 60%, MeCN / H2O, with 0.05% TFA as the modifier) to afford (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xii). LCMS: (ESI, m / z): [M+H] + = 653.2.

[0221] Step B - Synthesis of Compound xiii

[0222] A solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xii) (8.30 g, 12.7 mmol) in DMF (83 mL) was added to a 250 mL four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. Then, HOBt (340 mg, 2.5 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Then, MMAE (9 g, 12.7 mmol) was added to the reaction mixture and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by reverse-phase flash column chromatography (20% to 50% MeCN / water with 0.05% TFA as modifier) to afford 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiii). LCMS: (ESI, m / z): [M+H] + = 1231.7.

[0223] Step C - Synthesis of Compound xiv

[0224] 4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiii) (0.092 g, 0.075 mmol) was dissolved in DCM (0.5 mL), and TEA (0.5 mL) was added to the reaction mixture. The reaction mixture was stirred at 40 °C for 16 h. The reaction mixture containing 4-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv) was used directly without further purification or concentration. LC-MS: (ESI, m / z): [M+H] + = 1009.8.

[0225] Preparation of Exemplary Compounds

[0226] The following Examples 1-3 describe the synthesis of exemplary compounds of the present disclosure.

[0227] Example 1 - 4 - ((S)-2 - ((S)-2-(3-(2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)propanamido yl)propanamido)benzyl((S)-1 - (((S)-1 - (((3R,4S,5S)-1 - ((S)-2 - ((1R,2R)-3 - (((1S,2R)-1 - hydroxy - 1 - phenylpropan - 2 - yl)amino)-1 - methoxy - 2 - methyl - 3 - oxopropyl)pyrrolidin - 1 - yl)-3 - methoxy - 5 - methyl - 1 - oxohept - 4 - yl)(methyl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)(methyl)carbamate (1)

[0228]

[0229] Step A - Synthesis of Compound I1-b

[0230] At 0 °C, p-aminobenzyl alcohol (154 g, 125 mmol) was added to a stirred mixture of ethyl 2-ethoxy-2H-quinoline-1-carboxylate (103 g, 418 mmol) and (2S)-2-[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanamido]propanoic acid I1-a (100 g, 261 mmol) in DCM:MeOH (2:1) (3000 mL). The reaction mixture was stirred at room temperature for 18 h and monitored by LCMS. The solvent was evaporated in vacuo and the residue was diluted with TBME (2000 mL) and stirred for 30 min. The solid was collected by filtration, washed with TBME (1000 mL) and dried in vacuo to give 9H-fluoren-9-ylmethyl N-[(1S)-1-{[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamate (I1-b). LCMS: (ES, m / z): [M+H] + = 488.

[0231] Step B - Synthesis of compound I1-c

[0232] At room temperature, diethylamine (756 g, 10.3 mol) was added to a stirred mixture of 9H-fluoren-9-ylmethyl N-[(1S)-1-{[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamate (I1-b) (140 g, 287 mmol) in DMF (1.4 L). The mixture was stirred at room temperature for 18 h. The reaction was monitored by LCMS. The solvent was evaporated and the residue was diluted with EtOAc (500 mL) and stirred for 30 min. The solid was collected by filtration, washed with EtOAc (300 mL) and dried in vacuo to give (2S)-2-[(2S)-2-aminopropanamido]-N-[4-(hydroxymethyl)phenyl]propanamide (I1-c). LCMS: (ES, m / z): [M+H] + = 266.

[0233] Step C - Synthesis of compound I1-d

[0234] A mixture of 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxopyrrol-1-yl)propionate (55.2 g, 207 mmol), I1-c (55.0 g, 207 mmol), and DIPEA (40.2 g, 311 mmol) in DMF (550 mL) was stirred at room temperature for 16 h. The reaction mixture was added to H2O (600 mL) with stirring. The solid was collected by filtration, washed with water (500 mL), and dried in vacuo to give (2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d), which was used directly in the following step.

[0235] Step D - Synthesis of Compound I1-e

[0236] At room temperature, DIPEA (30.7 g, 237 mmol) was added to a stirred mixture of bis(4-nitrophenyl) carbonate (60.3 g, 198 mmol) and (2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d) (55 g, 32 mmol) in DMF (550 mL). The mixture was stirred for 16 h and monitored by LCMS. The reaction solution was added to H2O (400 mL) with stirring. The mixture was filtered, and the filter cake was washed with water (1×100 mL). The solid was collected by filtration and purified using reverse-phase chromatography (dynamic axial column C-18, eluting with 15% to 65% ACN / water (with 0.1% ammonium acetate (NH4OAc) as modifier)). The resulting mixture was concentrated in vacuo, and the solid was dried in vacuo at 45 °C to give {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]propanamido]propanamido]phenyl}methyl 4-nitrophenyl carbonate (I1-e). LC-MS: (ES, m / z): [M+H] + = 582.

[0237] Step E-1 Synthesis

[0238] Under N2, add {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]propanamido]propanamido]phenyl}methyl 4-nitrophenyl carbonate (I1-e) (3.00 g, 5.15 mmol) and DMF (45 mL) to a 60 mL round-bottom flask. Then, add HOBt (140 mg, 1.03 mmol). Stir the reaction mixture at 25 °C for 10 minutes. After this time, at 25 °C, add MMAE (4.00 g, 5.57 mmol). Stir the reaction mixture at 25 °C for 16 hours. Then, purify the reaction using reverse-phase column chromatography (AQ C18 30% to 60% MeCN / water, with 0.1% ammonium acetate as the modifier). Concentrate the MeCN in vacuo, extract the remaining aqueous mixture with ethyl acetate (300 mL × 3), and concentrate the combined organic matter in vacuo. Run four batches in parallel, combine them, and dissolve in 200 mL of MeCN. Add 400 mL of water, freeze the solution, and then lyophilize to obtain 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (1). LC-MS: (ES, m / z): [M+H] + = 1160. 1 H NMR (400 MHz, CD3OD) δ 7.53 (br d, J = 8.19 Hz, 2H), 7.03 - 7.32 (m, 7H), 6.67 (s, 2H), 5.21 - 5.29 (m, 2H), 4.90 - 5.14 (m, 2H), 4.28 - 4.66 (m, 4H), 4.04 - 4.22 (m, 4H), 3.52 - 3.81 (m, 4H), 3.24 - 3.46 (m, 6H), 3.17 (s, 2H), 2.97 - 3.09 (m, 2H), 2.73 - 2.90 (m, 4H), 2.28 - 2.47 (m, 4H), 1.43 - 2.20 (m, 8H), 1.16 - 1.38 (m, 10H), 1.00 - 1.12 (m, 6H), 0.61 - 0.94 (m, 18H). Not all exchangeable protons are reported.

[0239] Example 2: 4 - ((S)-2 - ((S)-2-(3-(5 - cyano - 6 - (methylsulfonyl)pyridinecarboxamido)propanamido yl)propanamido)propanamido)benzyl((S)-1 - (((S)-1 - (((3R,4S,5S)-1 - ((S)-2 - ((1R,2R)-3 - (((1S,2R)-1 - hydroxy - 1 - phenylpropan - 2 - yl)amino)-1 - methoxy - 2 - methyl - 3 - oxopropyl)pyrrolidin - 1 - yl)- 3 - methoxy - 5 - methyl - 1 - oxohept - 4 - yl)(methyl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)(methyl)carbamate (2).

[0240]

[0241] Step A - Synthesis of Compound I-2b

[0242] Dissolve 6-chloro-5-cyanopicolinic acid (I-2a) (80 g, 0.44 mol) in DMF (5000 mL), and add sodium methanethiolate (77 g, 1.1 mol) to the mixture in portions. Stir the reaction mixture at 25 °C for 16 h. Then, dilute the reaction mixture with ethyl acetate and add it to water. Extract the mixture with ethyl acetate and adjust the aqueous phase to pH 5 with 10% citric acid. Extract the mixture with ethyl acetate three times and concentrate the combined organic matters in vacuo to obtain 5-cyano-6-(methylthio)picolinic acid (I-2b). LC-MS: (ES, m / z): [M+H] + = 195.

[0243] Step B - Synthesis of Compound I-2c

[0244] Add HATU (155 g, 0.41 mol) to 5-cyano-6-(methylthio)picolinic acid (I-2b) (66 g, 0.34 mol) dissolved in THF (3000 mL). Stir the resulting mixture at 25 °C for 30 min. Add tert-butyl 3-aminopropionate HCl salt (67.7 g, 0.37 mol) to the mixture and then cool it to 10 °C. At 10 °C, add DIEA (175 g, 1.35 mol) dropwise over a period of 1 h. Stir the reaction mixture at 25 °C for 16 h, concentrate it in vacuo and dilute it with ethyl acetate. Wash the resulting solution three times with water and concentrate the combined organic matters in vacuo. Purify the residue by silica gel column chromatography (ethyl acetate:petroleum ether 1:2) to obtain tert-butyl 3-(5-cyano-6-(methylthio)pyridinamido)propionate (I-2c). LC-MS: (ES, m / z): [M+Na] + = 344.

[0245] Step C - Synthesis of Compound I-2d

[0246] Dissolve tert-butyl 3-(5-cyano-6-(methylsulfanyl)pyridinecarboxamido)propionate (I-2c) (96 g, 0.30 mol) in DCM (1500 mL), and dropwise add a solution of m-CPBA (206 g, 1.19 mol) in DCM (1500 mL) at 0 °C. Stir the reaction mixture at room temperature for 16 h. Dilute the resulting reaction mixture with DCM and pour it into ice water. Wash the organic phase 4 times with an aqueous solution of 10% sodium bicarbonate and dry and concentrate the combined organic matter in vacuo to obtain tert-butyl 3-(5-cyano-6-(methylsulfonyl)pyridinecarboxamido)propionate (I-2d). LC-MS: (ES, m / z): [M+Na] + = 376.

[0247] Step D - Synthesis of Compound I-2e

[0248] Add 4M HCl in 1,4-dioxane (2500 mL) to tert-butyl 3-(5-cyano-6-(methylsulfonyl)pyridinecarboxamido)propionate (I-2d) (90 g, 0.25 mol) dissolved in 1,4-dioxane (1000 mL). Stir the resulting mixture at 25 °C for 16 h and then filter. Wash the solid with n-heptane and dry under nitrogen to obtain 3-(5-cyano-6-(methylsulfonyl)pyridinecarboxamido)propionic acid (I-2e). LC-MS: (ES, m / z): [M+Na] + = 320.

[0249] Step E - Synthesis of Compound I-2f

[0250] Dissolve tert-butyl L-alanyl-L-alaninate (iii) (50 g, 0.23 mol) in THF (2300 mL). Add 3-(5-cyano-6-(methylsulfonyl)pyridinecarboxamido)propionic acid (I-2e) (69 g, 0.23 mol) and HATU (106 g, 0.28 mol) to the mixture. Stir the reaction mixture for 30 min and then cool to 10 °C. Then, dropwise add DIEA (90 g) over 30 min. Stir the reaction mixture at 20 °C overnight. Then, concentrate the reaction mixture in vacuo and dilute the resulting residue with ethyl acetate. Wash the combined organic matter 3 times with water, dry, and concentrate in vacuo. Purify the resulting residue by silica gel chromatography with DCM:MeOH (2:1) to obtain tert-butyl (3-(5-cyano-6-(methylsulfonyl)pyridinecarboxamido)propanoyl)-L-alanyl-L-alaninate (I-2f). LC-MS: (ES, m / z): [M+Na] + = 518.

[0251] Synthesis of Compound I-2g

[0252] 4M HCl in 1,4-dioxane (1800 mL) was added to (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alanine tert-butyl ester (I-2f) (90 g, 0.18 mol) dissolved in MeCN (2000 mL). The reaction mixture was stirred at 25 °C for 16 h and then concentrated in vacuo. The resulting solid was washed with MTBE and filtered. The solid was air-dried overnight to give (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alanine (I-2g). LC-MS: (ES, m / z): [M+Na] + = 462.

[0253] Synthesis of Compound I-2h

[0254] (3-(5-Cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alanine (I-2g) (30 g, 0.65 mol), DMF (300 mL) and HATU (31.2 g, 0.780 mol) were added to a 500 mL four-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. The reaction mixture was stirred at room temperature for 30 min. Then, (4-aminophenyl)methanol (9.0 g, 0.068 mmol) was added at 20 °C and the reaction mixture was cooled to 10 °C. At 10 °C, DIEA (90 g, 0.19 mmol) was added dropwise to the reaction mixture over 30 min and the resulting mixture was stirred at 25 °C for 5 h. Then, bis(4-nitrophenyl) carbonate (42 g, 0.13 mmol) was added to the reaction mixture at 25 °C and the mixture was stirred at 25 °C for 1 h. The reaction mixture was purified using C-18 flash column chromatography (30% to 60% ACN / water (with 0.05% TFA as modifier)) to give 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)propanamido)benzyl (4-nitrophenyl) carbonate (I-2h). LC-MS: (ES, m / z): [M+Na] + = 732.

[0255] Synthesis of Compound 2

[0256] To 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)propanamido)benzyl (4-nitrophenyl) carbonate (I-2h) (5.6 g, 7.9 mmol) and 1H-benzotriazol-1-ol (0.213 g, 1.58 mmol) was added DMF (56.0 mL). At 20 °C, MMAE (5.67 g, 7.89 mmol) was added to the reaction and the reaction mixture was stirred at 40 °C for 16 h. The resulting mixture was purified by preparative HPLC (10 - 95% MeCN / water, 0.05% TFA) to afford 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (2). 1 H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.14 (t, J = 6.0 Hz, 1H), 8.79 (d, J = 8.1 Hz, 1H), 8.38 (d, J = 8.1 Hz, 1H), 8.33–7.97 (m, 3H), 7.96–7.51 (m, 3H), 7.41–6.87 (m, 7H), 6.09 (s, 1H), 5.37 (dd, J = 26.2, 5.0 Hz, 1H), 5.04 (tt, J = 25.6, 12.6 Hz, 2H), 4.69 (d, J = 44.2 Hz, 1H), 4.59–4.16 (m, 5H), 4.15–3.90 (m, 2H), 3.80–3.43 (m, 8H), 3.36–2.95 (m, 9H), 2.94–2.58 (m, 2H), 2.50–2.20 (m, 3H), 2.19–1.79 (m, 3H), 1.65–1.37 (m, 3H), 1.32–1.18 (m, 9H), 1.06–0.96 (m, 7H), 0.89–0.59 (m, 20H).

[0257] Example 3: 4 - ((S)-2 - ((S)-2-(3-(5 - cyano - 6 - (methylsulfonyl)nicotinamido)propanamido)pro panamido)propanamido)benzyl((S)-1 - (((S)-1 - (((3R,4S,5S)-1 - ((S)-2 - ((1R,2R)-3 - (((1S, 2R)-1 - hydroxy - 1 - phenylpropan - 2 - yl)amino)-1 - methoxy - 2 - methyl - 3 - oxopropyl)pyrrolidin - 1 - yl)-3 - methoxy yl - 5 - methyl - 1 - oxohept - 4 - yl)(methyl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)amino)-3 - methyl - 1 - oxo butan - 2 - yl)(methyl)carbamate (3)

[0258]

[0259] Dissolve 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoic acid (x) (0.027 g, 0.090 mmol) and HATU (0.037 g, 0.097 mmol) in 0.4 mL of DMF and stir for 10 minutes. Then, add xiv dropwise, followed by DIEA (0.034 mL, 0.19 mmol). Stir the reaction for 1.5 hours and then purify using reverse-phase chromatography (Waters CSH-C18 column, 19×250 mm×5 um, 35-70% acetonitrile in water (with 0.1% formic acid as the modifier)) to obtain 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (3). LC-MS: (ESI, m / z): [M+H] + = 1288.665. 11H NMR (600 MHz, CD3CN) δ 9.12 (s, 1H), 8.68 (s, 1H), 8.63 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.4 - 7.06 (m, 9H), 6.66 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 7.75 Hz, 1H), 5.18 (d, J = 12.18 Hz, 1H), 5.04 (d, J = 12.18 Hz, 1H), 4.71 (d, J = 4.16 Hz, 1H), 4.63 (m, 1H), 4.45 (q, J = 7.3 Hz, 1H), 4.32 - 4.00 (m, 4H), 3.9 - 3.73 (m, 3H), 3.67 (m, 2H), 3.58 - 3.38 (m, 3H), 3.36 (s, 3H), 3.35 (s, 3H), 3.28 (s, 3H), 3.18 (m, 1H), 3.01 (s, 3H), 2.86 (br, 3H), 2.63 (m, 1H), 2.52 (m, 1H), 2.46 (br, 2H), 2.17 (br, 2H), 1.99 (m, 1H), 1.9 - 1.6 (m, 4H), 1.59 (br, 1H), 1.48 (d, J = 7.38 Hz, 3H), 1.36 (m, 4H), 1.12 (d, J = 6.81 Hz, 3H), 1.02 (d, J = 6.95 Hz, 3H), 0.98 (m, 4H), 0.92 - 0.67 (m, 15H).

[0260] Example 4 - Conjugation Scheme

[0261] Scheme 1: Using the literature procedure (WO2017072662, incorporated herein by reference), decap the sacituzumab with engineered Cys residues (S375C - two heavy chains) and dilute it to 5 mg / mL with PBS. DMSO was added to the monoclonal antibody (mAb) solution to obtain a 90% buffer 10% DMSO (v / v) solution. A 10 mM solution of Example 1 (3.1 Eq) in DMSO was added and the solution was mixed at rt for 2 hr. The resulting ADC was purified via AKTA TM (desalting column, histidine pH 6.5 buffer, monitored at 280 nm) and characterized by LCMS (Agilent PLRP - S column, 5 μm, 15 - 90% MeCN / H2O containing 0.1% formic acid, 80 °C column temperature) and SEC (Acquity UPLC Protein BEH SEC, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA added to the mobile phase for hydrophobic ADC).

[0262] Protocol 2: Use the literature procedure (WO2017072662) to de-capitate sacituzumab with an engineered Cys residue (S375C) in the heavy chain and dilute it to 5 mg / mL with PBS. Exchange the antibody (20 mg) into 40 mM Tris-acetate, 1 mM EDTA, pH 8.3. Dilute the mAb to ~10 mg / ml in 90% buffer / 10% DMF. Add a 10 mM solution of Example 3 (5.5 Eq) in DMF and mix the solution overnight at rt. Purify the ADC via AKTA TM (desalting column, histidine pH 6.5 buffer, monitored at 280 nm) and characterize it by LCMS (Agilent PLRP-S column, 5 μm, 15 - 90% MeCN / H2O containing 0.1% formic acid, 80 °C column temperature) and SEC (Acquity UPLC Protein BEH SEC, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA added to the mobile phase for hydrophobic ADC).

[0263] ADC Data

[0264] Table 1 below confirms the average drug - antibody ratio (DAR) and percentage aggregation of the ADCs using the exemplary compounds of the present invention and the above conjugation protocols:

[0265] Table 1

[0266]

[0267] ADC N87 Cytotoxicity Assay Protocol

[0268] Perform a cell - based cytotoxicity assay (NCI - N87 cells) on the exemplary ADCs of the present disclosure using the following protocol:

[0269] Step 1: Seed a 384 - well plate for the assay on Day 0 (45 μl per well)

[0270] Thaw the cells (NCI-N87) quickly in cryovials as follows: Incubate it in a 37 °C water bath for <1 min until only a small amount of ice remains in the vial. Immediately remove the vial and wipe it with 70% ethanol. Transfer the cells from the vial to a sterile centrifuge tube containing 8 mL of pre-warmed cell culture medium (RPMI-1640 (Cat#30-2001) + 10% FBS + 1% P / S). Rinse the vial with an additional 1 mL of medium to ensure complete transfer of the cells to the centrifuge tube. Then, centrifuge the cells at 150 g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in 10 - 20 ml of cell culture medium (RPMI-1640 (Cat#30-2001) + 10% FBS + 1% P / S). Count the cells using Vi-cell and prepare 1,500 cells / 45 μl / well. Then, using a Standard Cassette Combi, add 45 μl / well of the cells to a 384-well LowFlange White Flat Bottom Polystyrene TC-treated microplate (Corning, Cat#3570) (if needed, use medium speed to dispense 1 false plate at 20 μl to help normalize the Combi). Spin the plate at 150 g for 30 seconds.

[0271] Step 2: Add the ADC on Day 1. Remove the ADC vial and reference stock solution and allow them to thaw at RT. Centrifuge the tube at 2000 g for 30 seconds. Prepare a 10× intermediate assay plate (Waters plate, Cat#186002632) using a Bravo liquid handler. Perform serial dilutions using a buffer of 10 mM pH 6.5 histidine 9% sucrose buffer. Medium (cell-free) is used for Max_E. Then, using a Bravo liquid handler, add 5 μL of the 10X stock solution from the intermediate plate to the assay plate at an extremely slow speed so that the cell monolayer is not disturbed. Spin the plate at 150 g for 30 seconds.

[0272] Step 3: On Day 7, the CellTiter-Glo 2.0 assay (Promega, Cat#G9242) (CellTiter-Glo kit stored at -70 °C) will thaw the 2.0 reagent overnight at 4 °C (the reagent is not exposed to temperatures higher than 25 °C). Equilibrate the kit to RT for approximately 30 minutes. Using a Standard Cassette Combi, 2.0 reagent (20 uL) was added to 50 uL of cell-containing medium. The contents were mixed on an orbital shaker for 2 - 3 minutes to induce cell lysis. The plate was spun at 150 g for 30 seconds. The plate was incubated at RT for 5 minutes to stabilize the luminescence signal. Luminescence was recorded to calculate the EC 50 value, using an integration time of 0.25 - 1 second per well as a guide. Exemplary ADCs of the present disclosure were tested in the NCI-N87 cytotoxicity assay described above, and the results are provided in Table 2 below:

[0273] Table 2

[0274] ADC Examples <![CDATA[EC 50 (nM)]]> 4 0.28 5 0.40 6 0.44

[0275] It should be understood that the various features, functions, or alternatives discussed above and others can be desirably combined into many other different systems or applications. Various presently unforeseen or unexpected substitutions, modifications, variations, or improvements that may be made by those skilled in the art are also intended to be encompassed by the appended claims.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: Wherein: R 1 Selected from: The wavy line indicates the site of covalent linkage; R 2 is a cytotoxic drug; R 3 and R 4 each independently represents C 1-3 alkyl or a naturally occurring or non-naturally occurring amino acid side chain; and n is an integer from 1 to 4.

2. The compound according to claim 1, wherein R 1 is 3. The compound according to claim 1, wherein R 1 is 4. The compound according to claim 1, wherein R 1 is 5. A compound according to any one of claims 1 to 4, wherein R 2 is selected from anthracyclines, auristatins, camptothecins, esperamicins, etoposides, maytansinoids, pyrrolobenzodiazepines dimers, DNA minor groove binders, taxanes, vinca alkaloids, enediynes, cytotoxic agents against tubulin and vinca alkaloids.

6. A compound according to any one of claims 1 to 5, wherein R 2 is selected from auristatin T, auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, monomethyl auristatin F, lipophilic monomethyl auristatin F, monomethyl auristatin E, leishptin, esperamicin, paclitaxel and docetaxel, T67 (Tularik), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), microtubulysin M, doxorubicin, morpholino-doxorubicin, cyano-morpholino-doxorubicin, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analogs, calicheamicin, maytansine, analogs of dolastatin 10, rhizoxin, palytoxin, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colchamine, estramustine, cryptophycin, simadotin, maytansine analogs, combretastatin, discodermolide, tesmilifene and sarcodictyin.

7. A compound according to any one of claims 1 to 6, wherein R 2 is an auristatin drug.

8. A compound according to any one of claims 1 to 7, wherein R 2 is an auristatin drug selected from auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-auristatin E ester, monomethyl auristatin F and monomethyl auristatin E.

9. A compound according to any one of claims 1 to 8, wherein R 2 is monomethyl auristatin E.

10. A compound according to any one of claims 1 - 5, wherein R 2 is a pyrrolobenzodiazepine dimer.

11. A compound according to any one of claims 1 to 10, wherein R 3 and R 4 are independently selected from C 1-3 alkyl.

12. A compound according to any one of claims 1 to 11, wherein R 3 and R 4 are both CH3.

13. A compound according to any one of claims 1 to 10, wherein R 3 and R 4 are independently a naturally occurring or non-naturally occurring amino acid residue.

14. The compound according to claim 1 and its pharmaceutically acceptable salt, solvate or stereoisomer, including the structure of formula II: wherein R 1 and R 2 are as described herein.

15. The compound according to claim 14, wherein R 2 is an auristatin drug selected from auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, monomethyl auristatin F, monomethyl auristatin E, or a pyrrolobenzodiazepine dimer.

16. A compound of formula III, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: Wherein R 1 Selected from: The single wavy line indicates the site covalently linked to -(CH2) n ; The double wavy line indicates the site covalently linked to the sulfur of the cysteine residue of L’; R 2 is a cytotoxic drug; R 3 and R 4 each independently represents C 1-3 alkyl or a naturally occurring or non-naturally occurring amino acid side chain; n is an integer from 1 to 4; L’ is a ligand which is an antibody or an antigen-binding fragment of an antibody; and p is a positive rational number from 1 to 24, including fractions and decimals.

17. The compound according to claim 16, wherein R 2 is an auristatin drug selected from auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, monomethyl auristatin F, monomethyl auristatin E or pyrrolobenzodiazepine dimer, and R 3 and R 4 are both CH3.

18. A compound according to any one of claims 16 and 17, wherein R 2 is monomethyl auristatin E.

19. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is 20. The compound according to claim 16, or a pharmaceutically acceptable salt or solvate thereof, selected from: Wherein L’ is an antibody and p is an integer from 1 to 8.

21. The compound according to claim 19, or a pharmaceutically acceptable salt or solvate thereof, wherein p is an integer from 1 to 2.

22. A pharmaceutical composition comprising the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

23. Use of the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 22 in the manufacture of a medicament for the treatment or prevention of cancer or tumor.

24. A method for treating or preventing cancer in a subject in need thereof, said cancer being selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or recurrent anaplastic large cell lymphoma), said method comprising administering to a subject in need of such treatment a therapeutically effective amount of the compound according to claims 1-21 or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising said compound, its salt or solvate.

25. A method for treating and / or preventing a tumor, comprising administering to a patient in need thereof an effective amount of the compound according to claims 1-21 or a pharmaceutical composition comprising said compound, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 22.

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