Auristatin linker-payload, pharmaceutical composition and its use

Abstract

The present disclosure relates to a linker-payload comprising the structure of formula I: (I) and pharmaceutically acceptable salts, solvates or stereoisomers thereof. The present disclosure also relates to pharmaceutical compositions comprising these compounds, and to the use of these compounds, their intermediates, and compositions in the prevention or treatment of cancer and / or tumors. 【Chemical 1】 TIFF2025522671000048.tif70150

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 432,470, filed on December 14, 2022, and U.S. Provisional Application No. 63 / 497,887, filed on April 24, 2023, the disclosures of which are hereby incorporated by reference in their entireties.

Background Art

[0002] Antibody - drug conjugates (ADCs) are therapies consisting of monoclonal antibodies conjugated to cytotoxic payloads and have made significant progress in serving as transport vehicles that recognize and bind to protein antigens expressed in tumor tissues. Local delivery and release of the payload into or near malignant cells enables targeted delivery of potent cytotoxic drugs to diseased tissues while reducing damage to normal tissues. The linker component of an ADC is one important feature in developing highly active optimized therapeutic agents at well - tolerated doses (see International Publication No. WO 2021 / 055865, International Publication No. WO 2018 / 025168, and U.S. Patent Application Publication No. US 2013 / 0309256).

[0003] There is still a need for conjugation to antibodies or other targeting moieties to generate antibody - drug conjugates (ADCs) or other targeted ligand conjugates, particularly cytotoxic linker - payloads useful for tumor indications.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure provides linkers and linker - payloads useful for conjugation to antibodies or other targeting moieties for the production of ligand - drug conjugates (e.g., ADCs) useful for tumor indications and other indications. Specifically, a novel linker - payload structure is disclosed that includes maleimide or pyridylsulfone conjugated to a cleavable dipeptide linker and a p - aminobenzyl carbamate (PABC) connection 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 to cysteine residues in a ligand (e.g., an antibody, or an antigen - binding fragment of an antibody) for obtaining a ligand - drug conjugate (e.g., an ADC), which exhibits favorable physicochemical properties and high target - mediated efficacy. The linker - payloads are used to obtain potent and novel ligand - drug conjugates active across multiple cancer cell lines and exhibit broad utility for conjugation to 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 enhances the exposure of the released free cytotoxic compound to tumor tissue compared to normal tissue, and thus results in selective penetration into target cells and reduces the exposure of normal tissue to the cytotoxic compound compared to tumor tissue.

[0006] Exemplary linker-payload compounds include, but are not limited to, the structures shown throughout this disclosure. Exemplary ligand drug conjugates (e.g., ADCs) formed using the linkers and payloads described herein are also described. Other embodiments, aspects, and features of the disclosure will be described in more detail in the following description, examples, and appended claims, or will become apparent therefrom.

[0007] In each embodiment described herein, unless otherwise specified, each variable is selected independently of the others.

[0008] In one embodiment, the disclosure provides a structure of Formula I:

Chemical formula

Chemical formula

Chemical formula

[0009] Embodiments of Formula I are realized when n is 1.

[0010] Embodiments of Formula I are realized when n is 2.

[0011] Embodiments of Formula I are realized when n is 3.

[0012] Embodiments of Formula I are realized when n is 4.

[0013] One embodiment of Formula I is where R 1 is

Chemical formula

[0014] Another embodiment of Formula I is where R 1 is

Chemical formula

[0015] Another embodiment of Formula I is where R 1 is

Chemical formula

[0016] Another embodiment of Formula I is where R 2 is a cytotoxic drug or payload selected from anthracyclines, auristatins, camptothecins, duocarmycins, etoposides, maytansinoids, pyrrolobenzodiazepine dimers, DNA minor groove binders, taxanes, vinca alkaloids, enediynes, anti-tubulins, and vinca alkaloids. Sub - embodiments of this aspect of the present disclosure are where R 2is achieved when selected from auristatin T, auristatin E, auristatin F phenylenediamine, AEB, AEVB, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE), lexitropsin, duocarmycin, paclitaxel and docetaxel, T67 (tularik), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analog, calicheamicin, maytansine, a drastatin 10 analog, lysocine, paritoxin, baccatin derivative, taxane analog (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, semadotin, maytansinoid, combretastatin, discodermoid and eleutherobin. The cytotoxic or cytostatic drug can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., are mentioned. Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, semadotin, maytansinoid, combretastatin, discodermoid and eleutherobin.

[0017] Another sub-embodiment of this aspect of the present disclosure is R 2 is achieved when the R is an auristatin drug. One aspect of this sub-embodiment is that 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 that R 2is realized when it is MMAE. A further aspect of this embodiment is R 2 is realized when it is MMAF.

[0018] Another embodiment of Formula I is where R 2 is realized when it is a pyrrolobenzodiazepine dimer. A sub - embodiment of this aspect is where R 2 is realized when it is teslin.

[0019] One embodiment of Formula I is where R 3 and R 4 are independently selected from C 1~3 alkyl. A sub - embodiment of this aspect of Formula I is where R 3 and R 4 are independently selected from -CH3, -CH2CH3, -(CH2)2CH3. A sub - embodiment of this aspect of Formula I is where R 3 and R 4 are both CH3. A sub - embodiment of this aspect of Formula I is where R 3 and R 4 are both -CH2CH3. A sub - embodiment of this aspect of Formula I is where R 3 and R 4 are both -(CH2)2CH3. A sub - embodiment of this aspect of Formula I is where one of R 3 and R 4 is CH3 and the other is selected from -CH2CH3 and -(CH2)2CH3.

[0020] An embodiment of Formula I is where R 3 and R 4 are independently a naturally occurring or non - natural amino acid side chain. A sub - embodiment of this aspect of Formula I is where R 3 and R 4is realized when selected independently from arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine or side chains in the L or D configuration. Another embodiment of formula I is where R 3 and R 4 are selected independently from naturally occurring or non-natural amino acid side chains selected from the side chains of alanine, valine, leucine, and methionine.

[0021] In another embodiment, the present disclosure

Chemical Formula

[0022] One embodiment of formula II is when R 1 is

Chemical Formula

Chemical Formula

Chem.

[0023] Another embodiment of Formula II is when R 1 is

Chem.

[0024] Another embodiment of Formula II is when R 1 is

Chem.

[0025] Another embodiment of Formula II is when R 1 is

Chem.

[0026] Another embodiment of Formula II is realized when R 2 is a pyrrolobenzodiazepine dimer. A sub - embodiment of this aspect is realized when R 2 is tesirine.

[0027] Another embodiment of Formula II is Structural Formula II’:

Chem.

[0028] In another embodiment, the present disclosure is

Chem.

Chemical formula

Chemical formula

Chemical formula

[0029] Another embodiment of Formula III is Structural Formula III’ or III”:

Chemical formula

[0030] Embodiments of Formulas III, III', and III'' are realized when L' comprises an intact monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), and an antibody fragment that exhibits the desired biological activity. It is further envisioned that the antibody-drug conjugates of the present disclosure can be replaced with any that specifically bind, reactively associate, or complex with a receptor, antigen, or other receptor 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 to or reacts with a complex of a receptor, antigen, or other receptor moiety (e.g., a low molecular weight protein, polypeptide or peptide, lectin, glycoprotein, non-peptide, vitamin, nutrient transport molecule (e.g., transferrin)) of a cell population that is required to be biologically modified therapeutically or otherwise, and any other cell-binding molecule or substance.

[0031] Examples of ligands L' useful in the present disclosure are disclosed herein.

[0032] Trastuzumab or variants thereof, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody against the vitronectin receptor (a v b3), alemtuzumab, anti-HLA-DR antibodies including a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin lymphoma, 121I Lym-1, anti-HLA-Dr10 antibodies including a murine anti-DLA-Dr10 antibody for the treatment of non-Hodgkin lymphoma, anti-CD33 antibodies, anti-CD22 antibodies including a humanized anti-CD22 mAb for the treatment of Hodgkin's disease or non-Hodgkin lymphoma, lebrikizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, sacituzumab, cetuximab (ERBITUX), and gemtuzumab.

[0033] One aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, II, II’, III, III’, III”, or a pharmaceutically acceptable salt or solvate thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0034] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, II, II’, III, III’, or III” described herein, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0035] Another aspect of the present disclosure relates to a compound of Formula I, II, II’, III, III’, or III” described herein, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, for use as a drug or drug ingredient.

[0036] Another aspect of the present disclosure relates to a compound of Formula I, II, II’, III, III’, or III” described herein, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, in the preparation of a medicament for treating or preventing a tumor.

[0037] In another embodiment, the compounds of the present disclosure include those specifically identified herein as examples in the following table, and pharmaceutically acceptable salts thereof.

Mode for Carrying Out the Invention

[0038] 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.

[0039] The compounds of the present disclosure may contain one or more chiral centers and, accordingly, may exist as racemates and racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers. Depending on the nature of the various substituents on the molecule, additional chiral centers may be present. Each such chiral center independently gives rise to two optical isomers, and all possible optical isomers and diastereomers in the mixture, as well 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 encompass all such isomers of these compounds.

[0040] The independent synthesis of these diastereomers or their chromatographic separation can be achieved as known in the art by appropriate modification of the methods disclosed herein. Their absolute stereochemistry can be determined, among other methods, by X-ray crystallography of crystalline products or crystalline intermediates derivatized as necessary using reagents containing chiral centers of known absolute configuration.

[0041] If desired, the racemic mixture of the compound may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, for example, by coupling the racemic compound mixture to enantiomerically pure compounds to form a diastereomer mixture, followed by separation of the individual diastereomers by standard methods such as fractional crystallization or chromatography. The coupling reaction is often the formation of a salt using an enantiomerically pure acid or base. The diastereomer derivative can then be converted to the pure enantiomer by cleavage of the added chiral residue. The racemic mixture of the compound can also be separated directly by chromatography methods utilizing chiral stationary phases well known in the art.

[0042] Alternatively, any enantiomer of the compound can be obtained by stereoselective synthesis by methods well known in the art using optically pure starting materials or reagents of known configuration.

[0043] In the compounds of formula I, II, II', III, III' or III", the atoms may exhibit their natural isotopic abundances, or one or more atoms may be artificially enriched with a specific isotope having the same atomic number but a different atomic mass or mass number than that found predominantly in nature. The present disclosure may include all suitable isotopic variants of the compounds of general 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. Enriching deuterium may provide certain therapeutic advantages, such as an extended in vivo half-life or a reduced required dose, or may result in a compound useful as a standard for the characterization of biological samples. In the present disclosure, when a compound is said to be "not deuterated", it means that deuterium is not enriched beyond the background level. Isotopically enriched compounds within general formula I, II, II', III, III' or III" can be prepared using conventional techniques well known to those skilled in the art without undue experimentation or by processes similar to those described in the schemes and examples herein using suitable isotopically enriched reagents and / or intermediates.

[0044] If 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 hydroxyl -CH=C(OH)- group (enol form). Both the keto and enol forms, if present, are included within the scope of the present disclosure.

[0045] For any variable in any component (e.g., R 5When such as) appears two or more times, the definition in each occurrence is independent of each other occurrence. Also, combinations of substituents and variables are only permitted if such combinations result in stable compounds. A line drawn from a substituent to a ring system represents that the indicated bond can be attached to any of the ring atoms that can be substituted. When the ring system is bicyclic, the bond is intended to be attached to any of the appropriate atoms on any of the rings of the bicyclic moiety.

[0046] It is understood that in order to provide a compound that is chemically stable and can be readily synthesized by techniques known in the art from readily available starting materials, one or more silicon (Si) atoms can be incorporated into the compounds of the present disclosure in place of one or more carbon atoms. Carbon and silicon have different covalent radii, and when comparing bonds of similar C and Si elements, the bond distances and steric arrangements are different. These differences result in subtle changes in the size and shape of silicon-containing compounds when compared to carbon. Those skilled in the art will understand that differences in size and shape can result in subtle or dramatic changes such as efficacy, 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).

[0047] It is understood that the substituents and substitution patterns on the compounds of the present disclosure can be selected by those skilled in the art so as to provide compounds that can be readily synthesized from readily available starting materials, are chemically stable, and by techniques known in the art and the methods shown below. When a substituent itself is substituted with two or more groups, it is understood that these multiple groups can be on the same carbon or on different carbons as long as a stable structure is obtained. The phrase "optionally substituted with one or more substituents" should be understood to mean that the group in question can be unsubstituted or optionally substituted with one or more substituents.

[0048] Absolute stereochemistry is illustrated by the use of hash wedges and solid wedges. As shown in Illus-I and Illus-II. Thus, the methyl group in Illus-I projects out of the plane of the paper, the ethyl group in Illus-II descends into the plane of the paper, and the cyclohexene ring is in the plane of the paper. And it is considered that the hydrogen on the same carbon as the methyl group in Illus-I descends into the plane of the paper, and the hydrogen on the same carbon as the ethyl group in Illus-II projects out of the plane of the paper. The convention is the same, with both a hash rectangle and a solid rectangle added to the same carbon in Illus-III, the methyl group projecting out of the plane of the paper, the ethyl group descending into the plane of the paper, and having a cyclohexene ring in the plane of the paper. [Chem.]

[0049] As is conventional, unless otherwise specified in the accompanying text, normal "bar" bonds or "wavy" bonds indicate that all possible stereochemistries are represented, including pure compounds, mixtures of isomers, and racemic mixtures.

[0050] As used herein, unless otherwise specified, the following terms have the following meanings.

[0051] The phrase "at least one", when used with respect to the number of components in a composition, for example, the phrase "at least one pharmaceutical excipient" means that, for example, one member of a particular group is present in the composition and two or more may further be present. The components of a composition are typically aliquots of isolated pure substances added to the composition, and the purity level of the isolated substances added to the composition is the purity level normally acceptable for that type of reagent.

[0052] The phrase "one or more" means the same as "at least one", whether used with respect to a compound or a substituent on a component of a pharmaceutical composition.

[0053] "Effective amount" or "therapeutically effective amount" means an amount of at least one compound of the present disclosure or an amount of 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 results in 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") means, for example, administering an amount of at least one compound of Formula I, Formula II, or Formula III that results in a therapeutic response in a patient suffering from a central nervous system disease or disorder ("condition"), and the therapeutic response can be determined, for example, by analysis of pharmacodynamic markers or clinical evaluation of the patient suffering from the condition, a response suitable for managing, alleviating, ameliorating, or treating the condition, or a response suitable for alleviating, ameliorating, reducing, or eradicating one or more symptoms resulting from the condition, and / or long-term stabilization of the condition.

[0054] "Patient" and "subject" mean an animal such as a mammal (e.g., a human), preferably a human.

[0055] "Prodrug" means a compound that is rapidly converted in vivo to the parent compound, for example, by hydrolysis in the blood, for example, the conversion of a prodrug of Formula I, Formula II, or Formula III to a compound of Formula I, Formula II, or Formula III or to a salt thereof, and 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, and the scope of the present disclosure includes prodrugs of the novel compounds of the present disclosure.

[0056] The term "substituted" means that one or more of the listed substituents occupy one or more of the bonding positions on the substrate that are typically occupied by "-H", provided that such substitution does not exceed the normal valence rules of the atoms of the bonding arrangement presented by the substrate and that the substitution ultimately results in a stable compound, i.e., such substitution does not result in a compound in which mutually reactive substituents are geminally or vicinally located, and that the substitution results in a compound that is sufficiently robust to withstand isolation from the reaction mixture to a useful degree of purity.

[0057] When any substitution of a moiety is described (e.g., "optionally substituted"), this term means that, when a substituent is present, one or more of the substituents listed for a particular substrate may be present on the substrate at the bonding positions normally occupied by the default substituent that normally occupies that position. For example, the default substituent on a carbon atom of an alkyl moiety is a hydrogen atom, and any substituent can replace the default substituent.

[0058] As used herein, unless otherwise specified, the following terms used to describe a moiety, whether or not they include the entire definition of a variable portion of the structural representation of a compound of the present disclosure or a substituent added to a variable portion of the structural representation of a group of compounds of the present disclosure, have the following meanings, and unless otherwise specified, the definition of each term (i.e., moiety or substituent) applies whether the term is used individually or as a component of another term (e.g., the definition of aryl is the same for aryl and for aryl moieties such as arylalkyl, alkylaryl, arylalkynyl moieties, etc.). A moiety is described equivalently herein, without intending to distinguish meaning, by structure, typographic notation, or chemical terms. For example, an "acyl" substituent is represented by the term "acyl", by the printed notation "R'-(C=O)-" or "R'-C(O)-", or by the structural notation:

Chemical formula

[0059] The term "alkyl" (including alkyl moieties of other moieties such as trifluoromethyl-alkyl- and alkoxy-) means a straight-chain or branched aliphatic hydrocarbon moiety containing up to about 20 carbon atoms (e.g., the name "C 1-20 -alkyl" indicates an aliphatic hydrocarbon moiety of 1 to 20 carbon atoms). In some embodiments, alkyl preferably contains up to about 10 carbon atoms, and unless the term is modified by a statement that shorter chains are intended, for example, an alkyl moiety of 1 to 8 carbon atoms is referred to herein as "C 1~8 -alkyl". When the term "alkyl" is shown using two hyphens (i.e., "-alkyl-" indicates that the alkyl moiety is bonded in a form connecting the substituents on both sides thereof, for example, "-alkyl-OH" indicates an alkyl moiety connecting a hydroxyl moiety to a substrate.

[0060] 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 are substituted or may be substituted by substituents bonded to the alkyl group called when defining the moiety.

[0061] 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.

[0062] The term "halogen" means fluorine, chlorine, bromine, or iodine, and the preferred halogens, unless otherwise specified, when this term is used, are fluorine, chlorine, and bromine. A substituent that is a halogen atom means -F, -Cl, -Br, or -I, and "halo" means a fluoro, chloro, bromo, or iodo substituent attached to the defined moiety. For example, "haloalkyl" means an alkyl as defined above, where one or more of the bonding positions normally occupied by hydrogen atoms on the alkyl moiety are instead occupied by halo groups. Perhaloalkyl (or "fully halogenated" alkyl) means that all bonding positions not involved in the attachment of the alkyl substituent to the substrate are occupied by halogen. For example, if methyl is selected as the alkyl, the term perfluoroalkyl means -CF3.

[0063] The terms "hydroxyl" and "hydroxy" mean an HO- group, and "hydroxyalkyl" means a substituent of the formula: "HO-alkyl-", where the alkyl group is attached 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.

[0064] Bond sequences are indicated by hyphens and moieties are represented by letters. For example, -alkyl indicates a single bond between the substrate and the alkyl moiety, -alkyl-X indicates that the alkyl group attaches an "X" substituent to the substrate, and in structural notations, it is indicated by a wavy line that terminates the bond notation. For example,

Chemical formula

[0065] The line that is a bond generally represents a mixture of possible isomers including, for example, (R)- and (S)-stereochemical configurations, or any of them.

[0066] 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 "p" for all individual antibody-drug conjugate molecules present in the composition, and this average is expressed as a decimal. Thus, in some embodiments for 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 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 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 certain embodiments, 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. The term "composition" as used above is understood to encompass pharmaceutical compositions. The average DAR can be determined by various conventional means such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric methods, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0067] In all cases, the (one or more) compound names are associated with the depicted structure and are intended to capture each of the possible stereochemical permutations for a given structural isomer based on the synthetic operations used in its preparation. A list of individual stereoisomers joined by "or" indicates that the presented compound (e.g., "Example number") is isolated as a single stereoisomer and that stereoisomer corresponds to one of the possible configurations in the list. A list of individual stereoisomers joined by "and" indicates that the presented compound is isolated as a racemic mixture or a mixture of diastereomers.

[0068] As used herein, "natural amino acid" refers to an amino acid that occurs naturally, namely arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, or residues thereof, in the L or D configuration, unless otherwise specified or implied by the context.

[0069] As used herein, "unnatural amino acid" refers to an alpha - amino acid - containing acid or a residue thereof that has the backbone structure of a natural amino acid but has, for example, a side - chain group not present in natural amino acids attached to the alpha - carbon. Examples of unnatural amino acids are A’, B’, C’, D’ and E’ having amino acid side chains with the structure:

Chemical formula

[0070] Examples of amino acid side chains are C 1~6 alkyl, C 2~6 alkenyl and C 2~6 alkynyl. Further examples of amino acid side chains are dimethyl, ethyl, propyl, butyl, - C≡C and - CH2C≡C.

[0071] The ligand (L’) can be any moiety having a free cysteine group, such as, but not limited to, an antibody, protein, peptide, polypeptide, or engineered antibody modified to yield a free cysteine. This aspect is realized when the ligand is an antibody, preferably an intact antibody. The ligand acts to target a drug to a specific target cell population with which the ligand interacts. Suitable ligands include, for example, antibodies such as full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules (e.g., but not limited to, transferrin), or any other cell-binding molecule or substance such as small molecules and peptides. The ligand can be, for example, a non-antibody protein targeting agent.

[0072] When the conjugate contains a non-immunoreactive protein, polypeptide or peptide ligand instead of 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”), e.g., TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, somatostatin, lectin and apolipoproteins derived from low density lipoprotein.

[0073] Particularly preferred ligands (L’) are antibodies such as 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 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) against the antigen of interest can be prepared by using any technique known in the art that enables the production of antibody molecules by continuous cell lines in culture.

[0074] Furthermore, recombinant antibodies such as chimeric and humanized monoclonal antibodies that contain both human and non-human portions and can be produced using standard recombinant DNA techniques are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, e.g., molecules having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions (see, e.g., U.S. Pat. Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules derived 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. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, e.g., using the methods described in International Publication No. 87 / 02671 and European Patent Application Publication No. 0184187, which are incorporated herein by reference in their entirety, respectively.

[0075] 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.

[0076] Antibodies include analogs and derivatives, all of which are modified, i.e., modified by such covalent bonds as long as the covalent bonds of any type of molecule enable the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those further modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cell antibodies or other proteins, etc. Any of many 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. Further, an analog or derivative can contain one or more unnatural amino acids.

[0077] In a specific embodiment, known antibodies for the treatment of cancer can be used. Antibodies immunospecific for cancer cell antigens can be obtained commercially or can be prepared by any method known to those skilled in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, a published literature, or by routine cloning and sequencing.

[0078] In another specific embodiment, antibodies for the treatment of autoimmune diseases are used according to the compositions and methods of the present disclosure. Antibodies immunospecific for antigens of cells responsible for the production of autoantibodies can be obtained from any tissue (e.g., university researchers or companies) or can be prepared by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques.

[0079] In another embodiment, it may be desirable to conjugate the linker component to a ligand (e.g., an antibody) before attaching the payload component or drug component of the ADC. For example, in embodiments where a thiol-containing substituent, such as cysteine, is used to attach the drug component, it may be desirable to conjugate the linker component to a ligand L’ (e.g., an antibody) before attaching the drug component of the ADC.

[0080] In another embodiment, the compounds of the present disclosure include those specifically identified herein as examples in the following table, and pharmaceutically acceptable salts thereof.

[0081] “Antibody-drug conjugate” or ADC, as the term is used herein, unless otherwise specified or implied by context, is a subset of the ligand-drug conjugates of Formulas III, III’ and III”, and thus comprises an antibody or antigen-binding fragment thereof incorporating or corresponding antibody ligand (L’), and often a biologically active compound, often referred to as a free drug, incorporating or structurally corresponding cytotoxic payload, where L’ and R herein 2 are linked to each other via a dipeptide linker unit, and the antibody-drug conjugate can selectively bind, via the targeted antibody ligand unit, to a targeted antigen of a targeted cell, which in some embodiments is an antigen of an abnormal cell such as a cancer cell.

[0082] The term "antibody-drug conjugate" (ADC), in one aspect, refers to a number of linker-payload moieties conjugated to each antibody ligand (L'), and / or a plurality of individual conjugate compounds (i.e., compositions) in which the position of the linker-payload moiety conjugated to the antibody ligand (L') is the same or somewhat different. 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'), which allows for the mutational amino acid mutations and various glycosylation patterns described herein that occur during the production of the antibody from cell culture, which in some aspects results in variously different loadings and / or distributions of the linker-payload moiety attached to each antibody residue (e.g., the number of linker-payload moieties of any two antibody-drug conjugate compounds in a plurality of such compounds is the same, but the positions of those binding sites of the linker-payload moiety relative to the targeting antibody ligand (L') are different). In those cases, the antibody-drug conjugate is represented by the average drug loading of the conjugate compounds.

[0083] In some embodiments, the payload (drug) is a cytotoxic drug, typically one having a secondary aliphatic amine as a conjugation handle, and includes an auristatin compound as defined herein. Useful classes of cytotoxic drugs include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, and the like. Other exemplary classes of cytotoxic drugs include anthracyclines, auristatins, camptothecins, duocarmycins, etoposides, maytansinoids, and vinca alkaloids. Exemplary cytotoxic drugs 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 lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0084] The cytotoxic drug can be, for example, a chemotherapeutic agent such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The drug can also be a CC-1065 analog, calicheamicin, maytansine, dolastatin 10, risoxin, or a paritoxin analog.

[0085] Exemplary auristatins include auristatin E (AE), auristatin F phenylenediamine (AFP), monomethyl auristatin F (MMAF), and monomethyl auristatin E (MMAE), which react with para-acetylbenzoic acid or valeryl benzoic acid to form benzoyl-auristatin E ester (AEB) and 5-valeryl benzoic acid-auristatin E ester (AEVB), respectively, and those further described in embodiments of the present disclosure, but are not limited thereto. 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 Publication Nos. 04 / 010957, 02 / 088172, and U.S. Patents Nos. 7,659,241 and 8,343,928. Their structures and their synthesis methods disclosed therein are specifically incorporated herein by reference.

[0086] The cytotoxic drug can be a DNA minor groove binder (see, for example, U.S. Patent No. 6,130,237). For example, the minor groove binder can be a CBI compound or an enediyne (e.g., calicheamicin).

[0087] The cytotoxic or cytostatic drug can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (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 colcemid, estramustine, cryptophycin, sermadotin, maytansinoids, combretastatin, discodermoid, and eleutherobin.

[0088] The cytotoxic drug can be a maytansinoid, another group of anti-tubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be a maytansine containing a drug linker such as maytansine or DM-1 or DM-4 (see also ImmunoGen, Inc., Chari et al., 1992, Cancer Res.).

[0089] In the text, schemes, examples, structural formulas, and all tables of this specification, unsatisfied valences are assumed to have a sufficient number of hydrogen atoms or multiple atoms to satisfy the valences.

[0090] One or more compounds of the present disclosure may also exist as solvates or may be converted into 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 solvates of the antifungal fluconazole in ethyl acetate and also from water. Similar preparations of solvates including hydrates (where the solvent is water or an aqueous system) and hemisolvates 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 includes dissolving the desired amount of the compound of the present invention in a desired solvent (e.g., an organic solvent, an aqueous solvent, water, or a mixture of two or more thereof) at a temperature higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals that are later isolated by standard methods, regardless of the presence or absence of an antisolvent. For example, analytical techniques such as IR spectroscopy indicate the presence of the solvent (including water) in the crystal as a solvate (or hydrate if water is incorporated into the crystal form).

[0091] 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, diastereoisomers, atropisomers). The compounds of the invention include all of their isomers, both in pure form and in mixtures of two or more such as racemic mixtures.

[0092] Similarly, unless otherwise indicated, showing the structural representation of any tautomeric form of a compound that exhibits tautomerism means including all such tautomeric forms of the compound. Thus, if the compounds of the present disclosure, their salts, and their solvates and prodrugs can exist in different tautomeric forms or in equilibrium between such forms, all such forms of the compounds are encompassed by the present disclosure and are within the scope of the present disclosure. Examples of such tautomers include keto / enol tautomeric forms, imine-enamine tautomeric forms, and, for example, the following moieties:

Chemical formula

[0093] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, without undue toxicity, irritation, allergic reaction, or other problems or complications, are commensurate with a reasonable benefit / risk ratio and are suitable for use in contact with human and animal tissues within the scope of sound medical judgment.

[0094] As used herein, "pharmaceutically acceptable salts" refer to derivatives in which the parent compound is modified by making its acid or base salts. Salts in solid form may exist in two or more crystal structures 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 such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids or quaternary ammonium salts. 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 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, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, etc. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron, iron, lithium, magnesium, manganese salts, manganese, potassium, sodium, zinc, etc.

[0095] When the compounds of the present disclosure are basic, the salts can be prepared from pharmaceutically acceptable non-toxic acids including inorganic acids 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, etc. 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 appropriate inorganic or organic bases.

[0096] The term "cytotoxic drug" refers to a substance that inhibits or halts the function of cells and / or causes cell death or destruction. Toxic drugs include toxins and other compounds that can be used in tumor treatment.

[0097] The term "toxin" refers to any substance that can have a harmful effect on the growth or proliferation of cells. Toxins can be camptothecin derivatives, such as exatecan, maytansinoids and their derivatives (CN101573384), such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (International Publication No. WO2016 / 127790, Compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, a-sarcin, Aleutites fordii toxic protein, dianthin toxic protein, Phytolaca americana toxic protein, Momordica charantia inhibitor, cucurbitacin, crotonin, Sapaonaria ojficinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecene, etc., which can be small molecule toxins and their derivatives derived from bacteria, fungi, plants or animals.

[0098] The term "chemotherapeutic agent" refers to a chemical compound that can be used to treat tumors. This definition includes antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and these are often in the form of systemic or total therapy. They can be hormones. Examples of chemotherapeutic agents include alkylating agents such as thiotepa; cyclic amides (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benayodopa, carbocone, meturedopa, and uredopa; methylmelamines such as aziridine, and altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, nitrobicin hydrochloride; melphalan, nobenbitin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, aclarubicin, aza-irin, bleomycin, cactinomycin C, calicheamicin, carabicin, chromomycin, cardinophilin, chromomycin, actinomycin D, daunorubicin, doxorubicin, 6-diazo-5-oxy-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rhodomycin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; pterin analogs such as fludarabine, 6-mercaptopterin, thiomethopterin, thioguanopterin;Pyrimidine analogues such as ansitavim, azacitidine, 6-azuridine, carmoful, cytarabine, dideoxyuridine, doxitluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamidic glycosides; aminolevulinic acid; amsacrine; bestrabseal; biastrench; edatraxate; defofamine; demecolcine; diazicon; erfomitin; elliptinium acetate; etoglucide; nitrate Gallium acetate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pintostatin;Phenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK®;Razoxane;Sizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2"-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Dibromodulcitol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Cyclophosphamide;Thiotepa;Paclitaxel (TAXOL®, Bristol-Myers Squibb Taxanes such as cyclosporine (Trifluoroethylene, Rhone-Pou-lenc 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; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylomitin (DMFO); esperamicin retinoic acid; capecitabine;And pharmaceutically acceptable salts, acids or derivatives of any of the above substances are included. This definition also includes antihormonal agents that can modulate or inhibit the effects of hormones on tumors, such as antiestrogens like tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LYll 7018, onapristone and fairston, and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.;

[0099] As used herein, the term "treating" or "treatment" (e.g., of a disease, disorder, or condition or related symptoms, which may sometimes be referred to together or individually as an "indication") means inhibiting a disease, disorder or condition, i.e., arresting or reducing the onset or progression of a disease or its biological process or clinical symptoms, or alleviating a disease, i.e., causing regression of a disease or its biological process or progression and / or its clinical symptoms. "Treatment" as used herein also refers to the control, amelioration, or reduction of risk in a subject afflicted with a disease, disorder or condition involving a tumor. As used herein, the terms "preventing" or "prevention" or "prophylaxis" of a disease, disorder or condition means preventing the occurrence or progression of clinical symptoms of a disease, disorder or condition in a mammal that is exposed to or predisposed to the disease, disorder or condition but does not yet have or exhibit symptoms of the disease.

[0100] As will be apparent to those skilled in the art, the subjects treated by the methods described herein are generally mammals (e.g., laboratory animals and companion animals).

[0101] As used herein, the term "composition" is intended to encompass a product containing a compound of the present disclosure or a pharmaceutically acceptable salt thereof with one or more additional specified ingredients in specified amounts, as well as any product directly or indirectly resulting from a combination of specified amounts of specified ingredients. Such terms related to pharmaceutical compositions are intended to encompass products containing a compound of the present disclosure or a pharmaceutically acceptable salt thereof, which may include (one or more) active ingredients together with (one or more) inactive ingredients constituting one or more additional active ingredients and carriers, as well as any product directly or indirectly obtained from a 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 encompass any composition prepared by mixing a compound of the present disclosure or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not harmful to the recipient.

[0102] As described above, additional embodiments of the present disclosure are each a method of treating a disease, disorder, or condition, or one or more symptoms thereof (a "medical indication"), 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 such compound or salt.

[0103] In another embodiment, the present disclosure relates to a method of manufacturing a medicament for use in a subject, the method comprising combining a compound of the present disclosure or a pharmaceutically acceptable salt thereof with a pharmaceutical carrier or diluent.

[0104] One such embodiment is a method of treating or preventing cancer selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach 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 lymphocytic 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, recurrent) in a subject in need thereof, 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 such compound, salt, or solvate. In one such embodiment, the subject is human.

[0105] Another aspect of the present disclosure relates to a method of treating and / or preventing a tumor, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a compound according to the present disclosure.

[0106] This method also contemplates combinations with additional therapeutic agents. 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 in the treatment of certain malignancies. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors gamma and delta, respectively. PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro, and both troglitazone and rosiglitazone maleate inhibit the development of retinal angiogenesis in mice (Arch. Ophthamol. 2001;119:709-717). Examples of PPAR-γ agonists and PPAR-γ / α agonists include 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-methylpropionic acid (disclosed in U.S. Patent Application No. 09 / 782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchroman-2-carboxylic acid (disclosed in U.S. Patent Application Nos. 60 / 235,708 and 60 / 244,697), or a pharmaceutically acceptable salt thereof, but are not limited thereto.

[0107] 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, pp876-889, BC Decker, Hamilton 2000). Any tumor suppressor gene can be delivered using gene therapy. Examples of such genes include, but are not limited to, p53, which can be delivered via 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).

[0108] The compounds of the present disclosure can also be administered in combination with inhibitors of intrinsic multidrug resistance (MDR), particularly MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853, and PSC833 (valspodar), or a pharmaceutically acceptable salt thereof.

[0109] The compounds of the present disclosure can also be administered with immunopotentiating agents such as levamisole, isoprinosine, and zidaxine, or a pharmaceutically acceptable salt thereof.

[0110] The compounds of the present disclosure may also be useful in treating or preventing cancer in combination with P450 inhibitors such as foreign substances, quinidine, tyramine, ketoconazole, testosterone, quinine, methylrapone, caffeine, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycin, cocaine, fraxillin, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir, or pharmaceutically acceptable salts thereof.

[0111] The compounds of the present disclosure may also be useful in treating or preventing cancer in combination with Pgp and / or BCRP inhibitors such as cyclosporin A, PSC833, GF120918, cremophor EL, fumitremorgin C, Ko132, Ko134, Iressa, imatinib mesylate, EKI-785, Cl1033, novobiocin, diethylstilbestrol, tamoxifen, reserpine, VX-710, triptolide A, flavonoid, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, ketoconazole, nifedipine, FK506, amiodarone, XR9576, indinavir, amprenavir, cortisol, testosterone, LY335979, OC144-093, erythromycin, vincristine, digoxin and talinolol, or pharmaceutically acceptable salts thereof.

[0112] The compounds of the present disclosure may also be useful in the treatment or prevention of cancer such as bone cancer, in combination with bisphosphonates such as, but not limited to, etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or simadronate, clodronate, EB-1053, minodronate, neridronate, pyridronate and tiludronate (including all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof).

[0113] The compounds of the present disclosure may also be useful in treating or preventing breast cancer in combination with an aromatase inhibitor. Examples of aromatase inhibitors include, but are not limited to, anastrozole, letrozole and exemestane, or pharmaceutically acceptable salts thereof.

[0114] The compounds of the present disclosure may also be useful in treating or preventing cancer in combination with an siRNA therapeutic agent.

[0115] The compounds of the present disclosure may also be administered in combination with a γ-secretase inhibitor and / or an inhibitor of NOTCH signaling. Such inhibitors include those 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, WO 2004 / 039800, WO 2004 / 039370, WO 2005 / 030731, WO 2005 / 014553, USSN 10 / 957,251, WO 2004 / 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 (such as LY-450139), or a pharmaceutically acceptable salt thereof.

[0116] In one embodiment, specific anti-cancer agents useful in the combination therapy include pembrolizumab (Keytruda (registered trademark)), abarelix (Plenaxis depot (registered trademark)), aldesleukin (Prokine (registered trademark)); aldesleukin (Proleukin (registered trademark)); alemtuzumab (Campath (registered trademark)); alitretinoin (Panretin (registered trademark)); allopurinol (Zyloprim (registered trademark)); altretamine (Hexalen (registered trademark)); amifostine (Ethyol (registered trademark)); anastrozole (Arimidex (registered trademark)); arsenic trioxide (Trisenox (registered trademark)); asparaginase (Elspar (registered trademark)); azacitidine (Vidaza (registered trademark)); bevacizumab (Avastin (registered trademark)); bexarotene capsule (Targretin (registered trademark)); bexarotene gel (Targretin (registered trademark)); bleomycin (Blenoxane (registered trademark)); bortezomib (Velcade (registered trademark)); busulfan intravenous (Busulfex (registered trademark)); busulfan oral (Myleran (registered trademark)); carmustine (BCNU (registered trademark), BiCNU (registered trademark)); carmustine (Gliadel (registered trademark)); carmustine (Gliadel Wafer (registered trademark)) containing polifeprosan 20 implant; celecoxib (Celebrex (registered trademark)); cetuximab (Erbitux (registered trademark)); chlorambucil (Leukeran (registered trademark)); cisplatin (Platinol (registered trademark)); cladribine (Leustatin (registered trademark), 2-CdA (registered trademark)); clofarabine (Clolar (registered trademark)); cyclophosphamide (Cytoxan (registered trademark), Neosar (registered trademark)); cyclophosphamide (Cytoxan Injection (registered trademark)); cyclophosphamide (Cytoxan Tablet (registered trademark)); cytarabine (Cytosar-U (registered trademark)); cytarabine liposome (DepoCyt (registered trademark));Dacarbazine (DTIC-Dome®); Dactinomycin, Actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®); Daunorubicin liposome (DanuoXome®); Daunorubicin, Daunomycin (Daunorubicin®); Daunorubicin, Daunomycin (Cerubidine®); Denileukin diftitox (Ontak®); Dexrazoxane (Zinecard®); Docetaxel (Taxotere®); Doxorubicin (Adriamycin PFS®); Doxorubicin (Adriamycin®, Rubex®); Doxorubicin (Adriamycin PFS Injection®); Doxorubicin liposome (Doxil®); Drostanolone propionate (Dromostanolone®); Drostanolone propionate (Masterone injection®); Elliott's B Solution (Elliott’s B Solution®); Epirubicin (Ellence®); Epoetin alfa (epogen®); Erlotinib (Tarceva®); Estramustine (Emcyt®); Etoposide phosphate (Etopophos®); Etoposide, VP-16 (Vepesid®); Exemestane (Aromasin®); Filgrastim (Neupogen®); Floxuridine (intra-arterial) (FUDR®); Fludarabine (Fludara®); Fluorouracil, 5-FU (Adrucil®); Fulvestrant (Faslodex®); Gefitinib (Iressa®); Gemcitabine (Gemzar®); Gemtuzumab ozogamicin (Mylotarg®); Goserelin acetate (Zoladex Implant®); Goserelin acetate (Zoladex®); Histrelin acetate (Histrelin implant®); Hydroxyurea (Hydrea®);Ibritumomab tiuxetan (Zevalin®); Idarubicin (Idamycin®); Ifosfamide (IFEX®); Imatinib mesylate (Gleevec®); Interferon alpha-2a (Roferon A®); Interferon alpha-2b (Intron A®); Irinotecan (Camptosar®); Lenalidomide (Revlimid®); Letrozole (Femara®); Leucovorin (Wellcovorin®, Leucovorin®); Leuprolide acetate (Eligard®); Levamisole (Ergamisol®); Lomustine, CCNU (CeeBU®); Mechlorethamine, Nitrogen mustard (Mustargen®); Megestrol acetate (Megace®); Melphalan, L-PAM (Alkeran®); Mercaptopurine, 6-MP (Purinol®); Mesna (Mesnex®); Mesna (Mesnex tabs®); Methotrexate (Methotrexate®); Methoxsalen (Uvadex®); Mitomycin C (Mutamycin®); Mitotane (Lysodren®); Mitoxantrone (Novantrone®); Nandrolone phenylpropionate (Durabolin-50®); Nelarabine (Arranon®); Nofetumomab (Verluma®); Oprelvekin (Neumega®); Oxaliplatin (Eloxatin®); Paclitaxel (Paxene®); Paclitaxel (Taxol®); Paclitaxel protein-bound particles (Abraxane®); Palifermin (Kepivance®); Pamidronate (Aredia®); Pegademase (Adagen (Pegademase Bovine)®); Pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®);Pemetrexed disodium (Alimta (registered trademark)); Pentostatin (Nipent (registered trademark)); Pipobroman (Vercyte (registered trademark)); Plicamycin, Mitracin (Mithracin (registered trademark)); Porfimer sodium (Photofrin (registered trademark)); Procarbazine (Matulane (registered trademark)); Quinacrine (Atabrine (registered trademark)); Rasburicase (Elitek (registered trademark)); Rituximab (Rituxan (registered trademark)); Ridafolimus; Sargramostim (Leukine (registered trademark)); Sargramostim (Prokine (registered trademark)); Sorafenib (Nexavar (registered trademark)); Streptozocin (Zanosar (registered trademark)); Sunitinib maleate (Sutent (registered trademark)); Talc (Sclerosol (registered trademark)); Tamoxifen (Nolvadex (registered trademark)); Temozolomide (Temodar (registered trademark)); Teniposide, VM-26 (Vumon (registered trademark)); Testolactone (Teslac (registered trademark)); Thioguanine, 6-TG (Thioguanine (registered trademark)); Thiotepa (Thioplex (registered trademark)); Topotecan (Hycamtin (registered trademark)); Toremifene (Fareston (registered trademark)); Tositumomab (Bexxar (registered trademark)); Tositumomab / I-131 tositumomab (Bexxar (registered trademark)); Trastuzumab (Herceptin (registered trademark)); Tretinoin, ATRA (Vesanoid (registered trademark)); Uracil Mustard Capsules (registered trademark); Valrubicin (Valstar (registered trademark)); Vinblastine (Velban (registered trademark)); Vincristine (Oncovin (registered trademark)); Vinorelbine (Navelbine (registered trademark)); Olaparib (Lynparza (registered trademark)), Vorinostat (Zolinza (registered trademark)) and Zoledronic acid (Zometa (registered trademark)), or a pharmaceutically acceptable salt thereof, including but not limited to these.;

[0117] Accordingly, the scope of the present disclosure includes the use of the present disclosure in combination with a second compound selected from estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic / cell growth inhibitors, anti-proliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, PPAR-γ agonists, PPAR-δ agonists, inhibitors of multidrug innate resistance, antiemetics, agents useful for the treatment of anemia, agents useful for the treatment of neutropenia, immunostimulants, inhibitors of cell growth 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.

[0118] Yet another example of the present disclosure is a method of treating cancer comprising administering a therapeutically effective amount of a compound of the present disclosure in combination with paclitaxel or trastuzumab.

[0119] The therapeutic combinations disclosed herein can be used in combination with one or more other active agents, such as, but not limited to, other anti-cancer agents used for the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition (e.g., a cell proliferative disorder). In one embodiment, the compounds of the present disclosure are combined with one or more other anti-cancer agents for use in the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition for which the compounds of the present disclosure are useful. Such other active agents can be administered before, simultaneously with, or sequentially to the compounds of the present disclosure by the generally used routes and amounts therefor.

[0120] The present disclosure also includes a pharmaceutical composition useful for treating or preventing cancer, comprising a therapeutically effective amount of a compound of the present disclosure and a second compound selected from an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic / cytostatic agent, an anti-proliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, a γ-secretase and / or NOTCH inhibitor, an agent that interferes with a receptor tyrosine kinase (RTK), an agent that interferes with a cell cycle checkpoint, and any of the aforementioned therapeutic agents.

[0121] The present disclosure further relates to a method of treating cancer in a human patient, comprising 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.

[0122] 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, semipilimab, 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.

[0123] In one embodiment, the PD-1 antagonist is pembrolizumab. In certain sub-embodiments, the method comprises administering 200 mg of pembrolizumab to the patient every about 3 weeks. In other sub-embodiments, the method comprises administering 400 mg of pembrolizumab to the patient every about 6 weeks.

[0124] In a further lower embodiment, the method includes administering pembrolizumab to the patient at 2 mg / kg every about three weeks. In certain lower embodiments, the patient is a pediatric patient.

[0125] In some embodiments, the PD-1 antagonist is nivolumab. In certain lower embodiments, the method includes administering 240 mg of nivolumab to the patient every about two weeks. In other lower embodiments, the method includes administering 480 mg of nivolumab to the patient every about four weeks.

[0126] In some embodiments, the PD-1 antagonist is semiprimab. In certain embodiments, the method includes administering 350 mg of semiprimab to the patient every about three weeks.

[0127] In some embodiments, the PD-1 antagonist is atezolizumab. In certain lower embodiments, the method includes administering 1200 mg of atezolizumab to the patient every about three weeks.

[0128] In some embodiments, the PD-1 antagonist is durvalumab. In certain lower embodiments, the method includes administering 10 mg / kg of durvalumab to the patient every about two weeks.

[0129] In some embodiments, the PD-1 antagonist is avelumab. In certain lower embodiments, the method includes administering 800 mg of avelumab to the patient every about two weeks.

[0130] 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 pharmaceutically acceptable salts thereof can be administered separately, by the same or different routes of administration, or incorporated into the same pharmaceutical composition with one or more other agents and administered together. 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 depends on the therapeutically effective dosage of each agent. Generally, the therapeutically effective dosage of each is 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 generally include the therapeutically effective dosage of each active agent. In such combinations, the anti-human PD-1 antibody (or antigen-binding fragment thereof), the compound, and the other active agents can be administered separately or in combination. Further, the administration of one element can be before, simultaneous with, or after the administration of one or more other agents.

[0131] In one embodiment, the present disclosure provides a combination preparation for the simultaneous, separate, or sequential use of 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 in the treatment of cancer.

[0132] The present disclosure also provides the use of the compounds of the present disclosure for the treatment of cancer in cases where the patient has been previously treated (e.g., within 24 hours) with an anti-human PD-1 antibody (or antigen-binding fragment thereof). The present disclosure also provides the use of an anti-human PD-1 antibody (or antigen-binding fragment thereof) for the treatment of a cell proliferative disorder in cases where the patient has been previously treated (e.g., within 24 hours) with an antibody-linker-payload compound (ADC) of the present disclosure.

[0133] The present disclosure further relates to a method for treating cancer, 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.

[0134] Furthermore, the present disclosure relates to a method for treating cancer, 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 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 further 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, gamma / delta hepatosplenic T-cell lymphoma, subcutaneous sebaceous gland-like T-cell lymphoma, mycosis fungoides, and Hodgkin lymphoma. In certain embodiments, the proliferative disorder is metastatic cancer, for example, liver metastasis from colorectal cancer. In further embodiments, the proliferative disorder is cancer classified as stage III cancer or stage IV cancer. In examples of these embodiments, the cancer is not surgically resectable.

[0135] In an embodiment of the method disclosed herein, the anti-human PD-1 antibody (or an antigen-binding fragment thereof) is administered by intravenous injection or subcutaneous injection.

[0136] 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).

[0137] In another embodiment, the present disclosure provides a composition comprising a compound of the present disclosure, a pharmaceutically acceptable carrier, and pembrolizumab.

[0138] 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, wherein one therapeutic agent is an anti-human PD-1 antibody (or an antigen-binding fragment thereof) and the other therapeutic agent is independently selected from the group consisting of anti-cancer agents.

[0139] The compounds of the present disclosure can be used in combination with an antiemetic for treating nausea or vomiting such as acute, delayed, late, and anticipatory vomiting that can result from the use of the compounds of the present disclosure alone or in combination with radiotherapy. For the prevention or treatment of vomiting, the compounds of the present disclosure can be combined with other antiemetics, particularly, neurokinin-1 receptor antagonists, 5HT3 receptor antagonists such as ondansetron, granisetron, tropisetron, and zacisetron, GABAB receptor agonists such as baclofen, corticosteroids such as decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten, or others as 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, phenothiazines (e.g., prochlorperazine, fluphenazine, thioridazine, and mesoridazine), metoclopramide, aprepitant, fosaprepitant, or antidopaminergic agents such as dronabinol. In another example, a combination therapy using an antiemetic selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist, and a corticosteroid for treating or preventing vomiting that may occur upon administration of the compounds of the present disclosure is disclosed.

[0140] The compounds of the present disclosure can also be administered together with an agent useful for the treatment of anemia. Such anemia-treating agents include, for example, continuous erythropoietin receptor activators (e.g., epoetin alpha).

[0141] The compounds of the present disclosure can also be administered together with an agent useful for the treatment of neutropenia. Such neutropenia-treating agents are hematopoietic growth factors that regulate the production and function of neutrophils, such as, for example, human granulocyte colony-stimulating factor (G-CSF). Examples of G-CSF include filgrastim.

[0142] The compounds of the present disclosure may be useful when co-administered with other therapies such as, but not limited to, radiation therapy, surgery, and gene therapy. Thus, in one embodiment, the method of treating cancer described herein may include administration of an effective amount of radiation therapy, unless otherwise specified. Gamma rays are preferred as the radiation therapy.

[0143] The method of treating cancer described herein may include administration of an effective amount of radiation (i.e., the method of treating cancer described herein may include radiation therapy).

[0144] The method of treating cancer described herein includes administering a therapeutically effective amount of a compound of Formula III or Formula III' or Formula III" in combination with radiation therapy and / or an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic / cytostatic agent, an anti-proliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonist, an inhibitor of multidrug innate resistance, an antiemetic, an agent useful for the treatment of anemia, an agent useful for the treatment of neutropenia, an immunopotentiator, an inhibitor of cell growth and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, a γ-secretase inhibitor and / or an agent that interferes with NOTCH, a receptor tyrosine kinase (RTK), an agent that interferes with a cell cycle checkpoint, and a cancer treatment method including administration in combination with a second compound selected from any of the additional therapeutic agents listed herein is included.

[0145] It should be understood that further embodiments of the present disclosure include the pharmaceutical compositions, combinations, uses, and methods described above, and that each embodiment can be combined with one or more other embodiments, provided that such combinations are consistent with the description of the embodiments. It should be further understood that the embodiments provided above include all embodiments, including embodiments that arise from combinations of the embodiments.

[0146] Kit 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.

[0147] 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 the 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.

[0148] The present disclosure includes prodrugs of the compounds of the present disclosure within its scope. Generally, such prodrugs are functional derivatives of the compounds of the present disclosure that are readily convertible in vivo to the required compound. Thus, in the therapeutic methods of the present disclosure, the term “administering” or “administration” of a compound includes the treatment of the various conditions described using a compound that is specifically disclosed or that may not be specifically disclosed but that converts in vivo to a particular compound after administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985. The metabolites of these compounds include the active species produced when the compounds of the present disclosure are introduced into a biological environment.

[0149] The compounds described herein, or pharmaceutically acceptable salts and / or solvates thereof, can be administered alone, in combination with other compounds of the present disclosure, and / or as a cocktail in combination with other therapeutic agents. The selection of therapeutic agents that can be co-administered with the compounds of the present disclosure depends, in part, on the condition being treated.

[0150] 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 implant), inhalation spray, nasal, vaginal, rectal, sublingual, buccal, or topical routes of administration, alone or together, and formulated as appropriate dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles suitable for each route of administration. In addition to the treatment of warm-blooded animals, the compounds of the present disclosure are effective for use in humans.

[0151] The pharmaceutical compositions for administering the compounds of the present disclosure can be provided in unit dosage form for convenience and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with a carrier that constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. The pharmaceutical compositions contain the active compound in an amount sufficient to produce the desired effect on the process or symptoms of the disease. As used herein, the term "composition" is intended to encompass the product containing the specified amount of the specified ingredients, as well as any product that results directly or indirectly from the combination of the specified amounts of the specified ingredients.

[0152] A pharmaceutical composition containing an active ingredient can be in a form suitable for oral use, such as, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more substances selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preservatives to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture 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 lubricating agents such as magnesium stearate, stearic acid or talc. Tablets can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, time delay materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated by the techniques described in U.S. Patent Nos. 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.

[0153] Formulations for oral use can also be provided 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.

[0154] The aqueous suspension contains an active material, mixed with an excipient suitable for the manufacture of an aqueous suspension. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic, and the dispersing agent or wetting agent is a natural phosphatide 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 sorbitol monooleate, or a condensation product of ethylene oxide and a partial ester derived from a fatty acid and a hexitol anhydride such as polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives such as ethyl 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.

[0155] 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 a mineral oil such as liquid paraffin. The oily suspension may contain a thickening agent such as beeswax, hard paraffin or acetyl alcohol. The sweetening agents and flavoring agents as described above can be added to provide an orally acceptable oral formulation. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

[0156] Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient, mixed 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 described above. Additional excipients such as sweetening agents, flavoring agents and colorants may also be present.

[0157] The pharmaceutical composition of the present disclosure may be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof. Suitable emulsifiers may be naturally occurring gums, such as gum arabic or tragacanth gum, naturally occurring phosphatides, such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening agents and flavoring agents.

[0158] Syrups and elixirs can be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain demulcents, preservatives as well as flavoring and coloring agents.

[0159] 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 the appropriate dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils have conventionally been used as solvents or suspending media. For this purpose, any sterile fixed oil, such as synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids, such as oleic acid, are used in the preparation of injectables.

[0160] 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 that is solid at normal temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0161] For local use, creams, ointments, jellies, solutions, or suspensions containing the compounds of the present disclosure are used. Similarly, transdermal patches can also be used for local administration.

[0162] The pharmaceutical compositions and methods of the present disclosure may further comprise other therapeutically active compounds described herein that are commonly applied for the treatment of the aforementioned pathological conditions.

[0163] In the treatment, prevention, control, amelioration, or risk reduction of the conditions disclosed herein, appropriate dosage levels of the compounds of the present disclosure are generally about 0.01 - 500 mg / kg patient body weight per day and can be administered as a single dose or multiple doses. Appropriate dosage levels can be about 0.01 - 250 mg / kg / day, about 0.05 - 100 mg / kg / day, or about 0.1 - 50 mg / kg / day. Within this range, the dosage can be 0.05 - 0.5, 0.5 - 5, or 5 - 50 mg / kg / day. For oral administration, the composition can be provided in the form of tablets containing 1.0 - 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 to the patient being treated. The compound can be administered in a regimen of 1 - 4 times per day or can be administered once or twice a day.

[0164] However, it will be understood that the specific dosage levels and frequency of administration for any particular patient may vary and depend on various factors such as the activity of the specific compound being used, the metabolic stability and duration of action of that compound, age, body weight, general health status, gender, diet, mode and time of administration, rate of excretion, drug combinations, the severity of the specific condition, and the host being treated.

[0165] Methods for preparing the compounds of the present disclosure are shown in the following schemes and examples. Starting materials are prepared according to procedures known in the art or as shown herein.

[0166] Preparation Example The compounds of the present disclosure can be prepared according to the following schemes and specific examples, or modifications thereof, using readily available starting materials, reagents, and conventional synthetic procedures. It is also possible to use modifications known per se to those skilled in the art but not mentioned in detail. The general procedures for preparing the compounds claimed in the present disclosure can be readily understood by those skilled in the art by considering the following schemes and descriptions. Abbreviations used in the experiments may include, but are not limited to, the following.

[0167] [Table 1]

[0168] Preparation of Intermediate Compounds The following Preparation Examples 1 to 3 describe the synthesis of intermediate compounds useful for preparing exemplary compounds of the present disclosure.

[0169] Preparation Example 1 Preparation of Intermediate tert-Butyl L-Alanyl-L-alaninate (iii) [Chemical formula]

[0170] Step A - Synthesis of Compound ii To (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (i) (140 g, 450 mmol) dissolved in DCM (4500 mL), PyBOP (281 g, 540 mmol, 1.2 eq) was added at room temperature. The reaction mixture was stirred for 20 minutes. HCl salt of tert-butyl L-alaninate (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 1 hour and then this was stirred at 20 °C overnight. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate and washed with aqueous sodium carbonate solution (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 tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (ii). LCMS: (ESI, m / z): [M+H] + = 439.2.

[0171] Step B - Synthesis of Compound iii To tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (ii) (172 g, 392 mmol) dissolved in DCM (2000 mL), TEA (2000 mL) was added. 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 (4 M NH3 in DCM:MeOH 5:1) to give tert-butyl L-alanyl-L-alaninate (iii). LCMS: (ESI, m / z): [M+H] + = 217.1.

[0172] Preparation Example 2 Preparation of Intermediate Compound 3-(5-cyano-6-methylsulfonyl)nicotinamide)propanoic acid (x) [Chemical Structure Diagram]

[0173] Synthesis of Engineering A - Compound v 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 at 165 °C for 2 hours under a N2 atmosphere. The mixture was concentrated in vacuo, and the crude product was purified using reverse - phase flash chromatography (AQ C18 silica gel, ACN 0% - 20% gradient in water (using 0.5% NH3·H2O 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.

[0174] Synthesis of Engineering B - Compound vi A solution of 5 - cyano - 6 - hydroxypyridine - 3 - carboxylic acid (v) (25.7 g, 157 mmol) and phosphorus oxychloride (130 mL) was stirred at 110 °C 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 off. The mixture was extracted with ethyl acetate, dried, and concentrated in vacuo. The residue was purified using reverse - phase flash chromatography (column AQ silica gel, 0% - 15% acetonitrile / sodium bicarbonate (aq.)). The pH value of the aqueous layer was adjusted to 2 - 3 with 1M HCl, then the aqueous layer was extracted with ethyl acetate (3 times), dried, and concentrated in vacuo to obtain 6 - chloro - 5 - cyanopyridine - 3 - carboxylic acid (vi). LCMS: (ESI, m / z): [M - H] - = 181.

[0175] Synthesis of Engineering C - Compound vii Into a 500 mL three-necked flask under a nitrogen atmosphere, dimethylformamide (115 mL) and 6-chloro-5-cyanopyridine-3-carboxylic acid (vi) (7.7 g, 42 mmol) were added at 25 °C. To the above mixture, (methylsulfanyl)sodium (7.39 g, 105 mmol) was added portionwise at 0 °C. The resulting mixture was stirred at 25 °C for 8 hours. The reaction was monitored by LCMS. This solution was slowly transferred to H2O (1200 mL), and then extracted with ethyl acetate (1 × 700 mL). The pH value of the aqueous layer was adjusted to 2 - 3 with 1M HCl. The solid was recovered by filtration to obtain 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (vii). LCMS: (ESI, m / z): [M-H] - =193.

[0176] Step D - Synthesis of Compound viii A solution of 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (vii) (7.4 g, 38 mmol) in DCM (185 mL) was treated with m-CPBA (26.3 g, 152 mmol) at 45 °C for 24 hours under a nitrogen atmosphere. The reaction was monitored by LCMS, 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 obtain the crude product. The residue was purified using silica gel column chromatography eluting with DCM / MeOH to obtain 5-cyano-6-(methylsulfonyl)nicotinic acid (viii). LCMS: (ESI, m / z): [M+H] + =227.05.

[0177] Step E - Synthesis of Compound ix 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 minutes. Then, tert-butyl 3-aminopropanoate (0.42 g, 2.8 mmol) was added and the mixture was cooled to 10 °C. DIPEA (0.873 mL, 5.00 mmol) was added dropwise to the reaction mixture at 10 °C. The reaction mixture was stirred at 25 °C for 2 hours, then diluted with water and extracted with EtOAc (3×). The organic phase was concentrated in vacuo and the residue was purified using silica gel column chromatography eluting with 2:1 to 1:1 hexane:EtOAc to give tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoate (ix). 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).

[0178] Step F - Synthesis of Compound x tert-Butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoate (ix) (0.10 g, 0.28 mmol) was dissolved in 10 mL of 1,4-dioxane. Then, 4M HCl in 1,4-dioxane (10 mL) was added to the mixture. The resulting mixture was stirred at 25 °C for 16 hours and the reaction mixture was filtered. The solid was washed with n-heptane and dried under nitrogen for 5 hours to give 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoic acid (x). 1H NMR (500 MHz, CD3OD) 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).

[0179] Preparation Example 3 Preparation of Intermediate Compound 4-((S)-2-((S)-2-aminopropanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv) [Chemical formula]

[0180] Step A - Synthesis of Compound xii 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 equiv) in DMF (130 mL) was added to a 500 mL four-necked round-bottom flask purged and maintained under an inert atmosphere of nitrogen. Then, 2 mol% DIEA (0.534 mmol) and bis(4-nitrophenyl) carbonate (16.3 g, 53.6 mmol, 2 equiv) were added to the reaction mixture at 20 °C. 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% - 60% MeCN / H2O containing 0.05% TFA as a modifier) to obtain (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.

[0181] Step B - Synthesis of Compound xiii 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 atmosphere of nitrogen. 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 using reverse-phase flash column chromatography (20% - 50% MeCN / H2O containing 0.05% TFA as modifier) to obtain 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)propanamide)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-oxoheptan-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.

[0182] Step C - Synthesis of Compound xiv 4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)propanamide)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-oxoheptan-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-aminopropanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv) was used as such without further purification or concentration. LC-MS: (ESI, m / z): [M+H] + = 1009.8.

[0183] Preparation of Exemplary Compounds Examples 1 to 3 below illustrate the synthesis of exemplary compounds of the present disclosure.

[0184] Example 1 - 4 - ((S)-2 - ((S)-2-(3-(2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)propanamide)propanamide)propanamide)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 - oxoheptan - 4 - yl)(methyl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)(methyl)carbamate (1)

Chemical formula

[0185] Step A - Synthesis of Compound I1 - b 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}propanamide]propanoic acid I1 - a (100 g, 261 mmol) in DCM:MeOH (2:1) (3000 mL) at 0 °C. 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.

[0186] Step B - Synthesis of Compound I1 - c 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) at room temperature. 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-aminopropanamide]-N-[4-(hydroxymethyl)phenyl]propanamide (I1-c). LCMS: (ES, m / z): [M+H] + =266.

[0187] Step C - Synthesis of Compound I1-d A mixture of 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxopyrrol-1-yl)propanoate (55.2 g, 207 mmol), I1-c (55.0 g, 207 mmol) and DIPEA (40.2 g, 311 mmol) in DMF (550 mL) at room temperature was stirred 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)propanamide]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d), which was used as such in the next step.

[0188] Step D - Synthesis of Compound I1-e 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)propanamide]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d) (55 g, 32 mmol) in DMF (550 mL) at room temperature. 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 chromatography column C-18 eluting with 15% - 65% ACN / water containing 0.1% ammonium acetate (NH4OAc) as modifier).

[0189] 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)propanamide]propanamide]propanamide]phenyl}methyl 4-nitrophenyl carbonate (I1-e). LC-MS: (ES, m / z): [M+H] + = 582.

[0190] Synthesis of Step E-1 To a 60 mL round-bottom flask under N2, {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamide]propanamide]propanamide]phenyl}methyl-4-nitrophenyl carbonate (I1-e) (3.00 g, 5.15 mmol) and DMF (45 mL) were added, followed by the addition of HOBt (140 mg, 1.03 mmol). The reaction mixture was stirred at 25 °C for 10 minutes, and then MMAE (4.00 g, 5.57 mmol) was added at 25 °C. The reaction mixture was stirred at 25 °C for 16 hours. Subsequently, the reaction product was purified using reverse-phase column chromatography (AQ C18 30% - 60% MeCN / water containing 0.1% ammonium acetate as a modifier). MeCN was concentrated in vacuo, and the remaining aqueous mixture was extracted with ethyl acetate (300 ml × 3), and the combined organics were concentrated in vacuo. Four batches were run in parallel, combined, and dissolved in 200 mL of MeCN. 400 mL of water was added, the solution was frozen, and then lyophilized to obtain 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)propanamide)propanamide)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-oxoheptan-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; 1H 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 recorded.

[0191] Example 2: 4-((S)-2-((S)-2-(3-(5-Cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (2)

Chemical Structure

[0192] Step A - Synthesis of Compound I-2b 6-Chloro-5-cyanopicolinic acid (I-2a) (80 g, 0.44 mol) was dissolved in DMF (5000 mL), and sodium methanethiolate (77 g, 1.1 mol) was added to the mixture in one batch. The reaction mixture was stirred at 25 °C for 16 hours. Then, the reaction mixture was diluted with ethyl acetate and added to water. The mixture was extracted with ethyl acetate, and the aqueous phase was adjusted to pH 5 with 10% citric acid. The mixture was extracted three times with ethyl acetate, and the combined organic matter was concentrated in vacuo to obtain 5-cyano-6-(methylthio)picolinic acid (I-2b). LC-MS: (ES, m / z): [M+H] + =195.

[0193] Step B - Synthesis of Compound I-2c To 5-cyano-6-(methylthio)picolinic acid (I-2b) (66 g, 0.34 mol) dissolved in THF (3000 mL), HATU (155 g, 0.41 mol) was added. The resulting mixture was stirred at 25 °C for 30 minutes. tert-Butyl 3-aminopropanoate HCl salt (67.7 g, 0.37 mol) was added to the mixture, and then it was cooled to 10 °C. DIEA (175 g, 1.35 mol) was added dropwise at 10 °C over 1 hour. The reaction mixture was stirred at 25 °C for 16 hours, concentrated in vacuo, and diluted with ethyl acetate. The resulting solution was washed three times with water, and the combined organic matter was concentrated in vacuo. The residue was purified using silica gel column chromatography (ethyl acetate: petroleum ether 1:2) to obtain tert-butyl 3-(5-cyano-6-(methylthio)picolinamido)propanoate (I-2c). LC-MS: (ES, m / z): [M+Na] + =344

[0194] Step C - Synthesis of Compound I-2d tert-Butyl 3-(5-cyano-6-(methylthio)picolylamide)propanoate (I-2c) (96 g, 0.30 mol) was dissolved in DCM (1500 mL), and a solution of m-CPBA (206 g, 1.19 mol) in DCM (1500 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The resulting reaction mixture was diluted with DCM and poured into ice water. The organic phase was washed 4 times with 10% aqueous sodium bicarbonate, the combined organic phases were dried, and concentrated in vacuo to afford tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolylamide)propanoate (I-2d). LC-MS: (ES, m / z): [M+Na] + =376

[0195] Step D - Synthesis of Compound I-2e To tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolylamide)propanoate (I-2d) (90 g, 0.25 mol) dissolved in 1,4-dioxane (1000 mL) was added 4 M HCl in 1,4-dioxane (2500 mL). The resulting mixture was stirred at 25 °C for 16 h, then filtered. The solid was washed with n-heptane and dried under nitrogen to afford 3-(5-cyano-6-(methylsulfonyl)picolylamide)propanoic acid (I-2e). LC-MS: (ES, m / z): [M+Na] + =320

[0196] Step E - Synthesis of Compound I-2f tert-Butyl L-alanyl-L-alaninate (iii) (50 g, 0.23 mol) was dissolved in THF (2300 mL). To this mixture, 3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoic acid (I-2e) (69 g, 0.23 mol) and HATU (106 g, 0.28 mol) were added. The reaction mixture was stirred for 30 minutes and then cooled to 10 °C. Then, DIEA (90 g) was added dropwise over 30 minutes. The reaction mixture was stirred at 20 °C overnight. Then, the reaction mixture was concentrated in vacuo, and the resulting residue was diluted with ethyl acetate. The combined organics were washed three times with water, dried, and concentrated in vacuo. The resulting residue was purified using silica gel chromatography with DCM:MeOH (2:1) to obtain tert-butyl (3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alaninate (I-2f). LC-MS: (ES, m / z): [M+Na] + =518

[0197] Step F - Synthesis of Compound I-2g 4M HCl in 1,4-dioxane (1800 mL) was added to tert-butyl (3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alaninate (I-2f) (90 g, 0.18 mol) dissolved in MeCN (2000 mL). The reaction mixture was stirred at 25 °C for 16 hours and then concentrated in vacuo. The resulting solid was washed with MTBE and filtered. The solid was air-dried overnight to obtain (3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alanine (I-2g). LC-MS: (ES, m / z): [M+Na] + =462

[0198] Step G - Synthesis of Compound I-2h Into a 500 mL four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, (3-(5-cyano-6-(methylsulfonyl)picolinamide)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. The reaction mixture was stirred at room temperature for 30 minutes. Then, (4-aminophenyl)methanol (9.0 g, 0.068 mmol) was added at 20 °C and the reaction mixture was cooled to 10 °C. DIEA (90 g, 0.19 mmol) was added dropwise to the reaction mixture at 10 °C over 30 minutes and the resulting mixture was stirred at 25 °C for 5 hours. Then, bis(4-nitrophenyl) carbonate (42 g, 0.13 mmol) was added to the reaction mixture at 25 °C and stirred at 25 °C for 1 hour. The reaction mixture was purified using C-18 flash column chromatography (30% - 60% ACN / water (containing 0.05% TFA as modifier)) to obtain 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)benzyl (4-nitrophenyl) carbonate (I-2h). LC-MS: (ES, m / z): [M+Na] + =732

[0199] Synthesis of Compound 2 in Step H 4-((S)-2-((S)-2-(3-(5-Cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)benzyl (4-nitrophenyl) carbonate (I-2h) (5.6 g, 7.9 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol (0.213 g, 1.58 mmol) were added to DMF (56.0 mL). MMAE (5.67 g, 7.89 mmol) was added to the reaction mixture at 20 °C, and the reaction mixture was stirred at 40 °C for 16 hours. The resulting mixture was purified using Prep-HPLC (10~95% MeCN / water, 0.05% TFA) to obtain 4-((S)-2-((S)-2-(3-(5-Cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (2). 1H 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).

[0200] Example 3: 4-((S)-2-((S)-2-(3-(5-Cyano-6-(methylsulfonyl)nicotinamide)propanamide)propanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3)

Chem.

[0201] 3-(5-Cyano-6-(methylsulfonyl)nicotinamide)propanoic acid (x) (0.027 g, 0.090 mmol) and HATU (0.037 g, 0.097 mmol) were dissolved in 0.4 mL of DMF and stirred for 10 minutes. Then, xiv was added dropwise, followed by the dropwise addition of DIEA (0.034 ml, 0.19 mmol). The reaction mixture was stirred for 1.5 hours and then purified by reverse-phase chromatography (Waters CSH-C18 column, 19×250 mm×5 um, using an aqueous solution of 35 - 70% acetonitrile containing 0.1% formic acid as a modifier) to obtain 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)nicotinamide)propanamide)propanamide)propanamide)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3).LC-MS: (ESI, m / z): [M+H]+ = 1288.665. 1H 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).

[0202] Example 4 - Conjugation Protocol Protocol 1: Trastuzumab with a heavy-chain engineered Cys residue (S375C - heavy chain) was de-capped using the literature procedure (WO 2017 / 072662, which is incorporated herein by reference) and diluted to 5 mg / mL in PBS. DMSO was added to the monoclonal antibody (mAb) solution to reach a 90% buffer 10% DMSO (v / v) solution. A 10 mM solution (3.1 equivalents) of Example 1 in DMSO was added and the solution was mixed at room temperature for 2 hours. The resulting ADC was purified by AKTA™ (desalting column, histidine pH 6.5 buffer, monitoring at 280 nm) and characterized by LCMS (Agilent PLRP-S column, 1000 Å, 5 μm, 15 - 90% MeCN / H2O containing 0.1% formic acid, column temperature 80 °C) and SEC (Acquity UPLC Protein BEH SEC, 200 Å, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA added to the mobile phase for hydrophobic ADCs).

[0203] Protocol 2: Trastuzumab with a heavy-chain engineered Cys residue (S375C) was de-capped using the literature procedure (WO 2017 / 072662) and diluted to 5 mg / mL in PBS. The antibody (20 mg) was exchanged into 40 mM Tris-acetate, 1 mM EDTA, pH 8.3. The mAb was placed in 90% buffer / 10% DMF and diluted to approximately 10 mg / ml. A 10 mM solution (5.5 equivalents) of Example 3 in DMF was added and the solution was mixed overnight at room temperature. The ADC was purified by AKTA™ (desalting column, histidine pH 6.5 buffer, monitoring at 280 nm) and characterized by LCMS (Agilent PLRP-S column, 1000 Å, 5 μm, 15 - 90% MeCN / H2O containing 0.1% formic acid, column temperature 80 °C) and SEC (Acquity UPLC Protein BEH SEC, 200 Å, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% IPA added to the mobile phase for hydrophobic ADCs).

[0204] ADC Data Table 1 below shows the average drug-to-antibody ratio (DAR) and percent aggregation for exemplary compounds of the invention and ADCs utilizing the conjugation protocol described above. [Table 2]

[0205] ADC N87 Cell Cytotoxicity Assay Protocol Exemplary ADCs of the present disclosure were subjected to a cell-based cytotoxicity assay (NCI-N87 cells) utilizing the following protocol.

[0206] Step 1: Seed 384-well assay plate on day 0 (45 uL / well) Cells (NCI-N87) were rapidly thawed in cryovials by incubating them in a 37 °C water bath for less than 1 minute until a small amount of ice remained in the vial. The vial was immediately removed and wiped with 70% ethanol. The cells were transferred from the vial to a sterile centrifuge tube containing 8 mL of pre-warmed cell culture medium (RPMI-1640 (catalog number 30-2001) + 10% FBS + 1% P / S). An additional 1 mL of medium was run through the vial to ensure complete transfer of the cells to the centrifuge tube. The cells were then centrifuged at 150 g for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 10 - 20 mL of cell culture medium (RPMI-1640 (catalog number 30-2001) + 10% FBS + 1% P / S). Cells were counted using Vi-cell and 1,500 cells / 45 uL per well were prepared. Then, 45 uL / well of cells were added to a Corning® 384-well low-flange white flat-bottom polystyrene TC-treated microplate (Corning, catalog number 3570) using a Standard Cassette Combi (dispense 20 uL into one dummy plate to help normalize the Combi at medium speed if necessary). The plate was spun down at 150 g for 30 seconds.

[0207] Procedure 2: Add ADC on the 1st day. Take out the ADC vial and the reference stock, and thaw them at RT. Centrifuge the tube at 2000 g for 30 seconds. Prepare a 10-fold intermediate assay plate (Waters plate, catalog number 186002632) using a Bravo liquid handler. Perform serial dilutions using a buffer of 10 mM pH 6.5 histidine 9% sucrose buffer. Use medium (without cells) for Max_E. Then, add 5 μL of the 10X stock from the intermediate plate to the assay plate using a Bravo liquid handler at a very slow speed so as not to disrupt the cell monolayer. Spin down the plate at 150 g for 30 seconds.

[0208] Procedure 3: CellTiter-Glo 2.0 assay on the 7th day (Promega, catalog number G9242) (CellTiter-Glo kit stored at -70 °C) Thaw the CellTiter-Glo® 2.0 reagent at 4 °C overnight (the reagent was not exposed to temperatures exceeding 25 °C). Equilibrate this kit at RT for about 30 minutes. Add 20 μL of the CellTiter-Glo® 2.0 reagent to 50 μL of cell-containing medium using a Standard Cassette Combi. Mix the contents on an orbital shaker for 2 - 3 minutes to induce cell lysis. Spin down the plate at 150 g for 30 seconds. Incubate the plate at RT for 5 minutes to stabilize the luminescence signal. Record the luminescence and calculate the EC 50 values using an integration time of 0.25 - 1 second per well as a guideline.

[0209] Test the exemplary ADCs of the present disclosure in the above NCI-N87 cytotoxicity assay, and the results are shown in Table 2 below.

Table 3

[0210] Of course, various ones of the features and functions described above and others, or alternatives thereto, can desirably be combined in many other different systems or applications. It is also intended that various presently unforeseeable or unexpected alternatives, modifications, variations or improvements that may be made later by those skilled in the art are also encompassed by the following claims.

Claims

1. A compound of formula I 【Chemical 1】 wherein R １ is [Chemical 2] selected from [Chemical Formula 3] the wavy line indicates the covalent bond site, R ２ is a cytotoxic drug, R ３ and R ４ each independently represents C １～３ alkyl or a naturally occurring or non-natural amino acid side chain, n is an integer from 1 to 4), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

2. R １ is 【Chemical Formula 4】 The compound according to claim 1, wherein

3. R １ is 【Chemical Formula 5】 The compound according to claim 1, wherein

4. R １ is [Chemical Formula 6] The compound according to claim 1, wherein

5. R ２ The compound according to any one of claims 1 to 4, wherein R is a cytotoxic drug selected from anthracycline, auristatin, camptothecin, duocarmycin, etoposide, maytansinoid, pyrrolobenzodiazepine dimer, DNA minor groove binder, taxane, vinca alkaloid, enediyne, anti-tubulin and vinca alkaloid.

6. R ２ is a compound according to any one of claims 1 to 5, 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, lexitropsin, duocarmycin, paclitaxel and docetaxel, T67 (turapliq), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analog, calicheamicin, maytansine, analog of dolastatin 10, lysocine, parythoxin, baccatin derivative, taxane analog (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, semadotin, maytansinoid, combretastatin, discodermoid, tesmilifene and eleutherobin.

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

8. R ２ The compound according to any one of claims 1 to 7, wherein R 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. R ２ The compound according to any one of claims 1 to 8, wherein R is monomethyl auristatin E.

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

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

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

13. R ３ and R ４ are each independently an amino acid residue that is either naturally occurring or non-naturally occurring, the compound according to any one of claims 1 to 10.

14. The structure of formula II: 【Chemical 7】 (wherein, R １ and R ２ are as described herein) The compound according to claim 1 and a pharmaceutically acceptable salt, solvate or stereoisomer thereof, comprising

15. R ２ The compound according to claim 14, wherein R 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: 【Chemical 8】 wherein R １ is 【Chemical Formula 9】 selected from 【Chemical Formula 10】 The single wavy line indicates the covalent bonding site to - (CH ２ ) ｎ and 【Chemical 11】 the double wavy line indicates the site bonded 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-natural amino acid side chain, n is an integer from 1 to 4, L' is a ligand that is an antibody or an antigen-binding fragment of an antibody, p is a positive rational number from 1 to 24 including fractions and decimals), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

17. 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, and R ３ and R ４ are both CH ３ The compound according to claim 16, wherein is

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

19. 【Fig. 12】 wherein the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

20. 【Figure 13】 wherein L' is an antibody and p is an integer from 1 to 8) selected from the compounds according to claim 16, or pharmaceutically acceptable salts or solvates.

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

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 for the manufacture of a medicament for treating or preventing cancer or a tumor, or use of the pharmaceutical composition according to claim 22.

24. A method for treating or preventing cancer 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) in a subject in need thereof, the method comprising administering to a subject in need of such treatment an effective amount of the compound according to claims 1 to 21, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising said compound, salt or solvate thereof.

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

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