PNU anthracycline substance-derived linker-load, pharmaceutical composition and application of PNU anthracycline substance-derived linker-load

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 434,520, filed December 22, 2022, and U.S. Provisional Application No. 63 / 496,986, filed April 19, 2023, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present disclosure provides linker-load compounds comprising a PNU anthracycline load and their use as components of antibody-drug conjugates. The present disclosure also provides antibody-drug conjugates comprising linker-load compounds, compositions thereof, and methods for their use in treating cancer. Background Art

[0004] Anthracyclines are a class of drugs used in cancer chemotherapy that are extracted from Streptomyces bacteria and are one of the most effective anticancer treatments ever developed, demonstrating efficacy against more types of cancer than any other type of chemotherapeutic agent. These compounds are used to treat many cancers, including leukemia, lymphoma, breast cancer, gastric cancer, uterine cancer, ovarian cancer, bladder cancer, and lung cancer. However, the effectiveness of anthracyclines is limited by their toxicity, and therefore, only a few anthracyclines or related DNA intercalators are available for clinical use.

[0005] Antibody-drug conjugates represent an innovative therapeutic application that combines the uniqueness, high specificity, properties, and antitumor activity of tumor-specific but poorly cytotoxic monoclonal antibodies (mAbs) with the potent cell-killing activity of highly cytotoxic small molecule drugs (e.g., anthracyclines) that are not suitable for systemic administration alone.

[0006] Antibody-drug conjugates are three-component systems consisting of a cytotoxic payload linked to an antibody via a biodegradable linker. The antibody first binds to a specific marker (antigen or receptor) on the cancer cell surface. The intact antibody-drug conjugate is then internalized within the cancer cell, where the linker degrades and releases the payload.

[0007] Advances in linker technology are needed to provide improved control over drug pharmacokinetics and to improve the delivery and release of cytotoxic payloads in cancer cells. The present disclosure addresses this need.

[0008] Summary of the Invention

[0009] In one aspect, provided are compounds of formula (I):

[0010]

[0011] and pharmaceutically acceptable salts thereof, wherein:

[0012] When R 2 When R is H or C1-C6 alkyl, 1 Selected from -XYZR 3 ;

[0013] R 2 is H or C1-C6 alkyl, or R 1 and R 2 are connected to form a 4- to 6-membered monocyclic heterocycloalkylene group or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene group, each of which is substituted on a ring carbon atom by -XYZR 3 replaced by;

[0014] R 3 Selected from:

[0015]

[0016] X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、-(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5-NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-(CH2) n -N(R 4 )- and -R 5 -O-CH2N(R 4 )-;

[0017] Y is a bond or -C(O)C(R 4 )(R 6 )NH-;

[0018] Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-;

[0019] R 4 H, C 1-6 Alkyl or

[0020]

[0021] R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bridged bicyclic cycloalkylene, -NH-NH-, and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group;

[0022] R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain;

[0023] R 9 -CH2NHC(O)-(CH2CH2O) q -CH3;

[0024] m is 0, 1, or 2;

[0025] n is independently an integer from 0 to 4 at each occurrence; and

[0026] q is selected from 4, 8, 10 and 12.

[0027] The compounds of formula (I) and their pharmaceutically acceptable salts are useful as components of antibody-drug conjugates for the treatment and prevention of cancer. Without being bound by any particular theory, it is believed that the cargo moiety of the compound of formula (I) acts as an inhibitor of topoisomerase.

[0028] Thus, provided herein are antibody-drug conjugates comprising a compound of formula (I) as a payload / linker moiety. Also provided are methods for treating or preventing cancer in a patient comprising administering to the patient an effective amount of at least one antibody-drug conjugate comprising a compound of formula (I).

[0029] More details are set forth in the detailed description attached below.

[0030] Although any methods and materials similar to those described herein can be used in the practice or testing of compounds of formula (I) or antibody-drug conjugates comprising compounds of formula (I), exemplary methods and materials are now described. Other embodiments, aspects, and features are further described in or will be apparent from the following description, examples, and appended claims. Detailed Description of the Invention

[0032] The present disclosure relates to a class of PNU anthracycline drug-derived linker-payload compounds ("linker-payload compounds of the present disclosure"), wherein the linker structure contains a maleimide group, or a sulfone-substituted pyridyl group attached to a peptide linker, with variations in the amino acid sequence and optional incorporation of a PEG unit, and terminates in a connection to a PNU anthracycline-derived payload. One embodiment of the present disclosure relates to linker-payload compounds. Another embodiment of the present disclosure relates to antibody-drug conjugates ("ADCs of the present disclosure") comprising linker-payload compounds of the present disclosure. Yet another embodiment relates to novel linker portions of linker-payload compounds.

[0033] The terms used herein have their conventional meanings and the meaning of these terms is independent at each occurrence. Nevertheless, and unless otherwise stated, the following definitions apply to the entire specification and claims. Chemical name, common name and chemical structure can be used interchangeably to describe identical structures. If both chemical structure and chemical name are used to refer to a chemical compound, and there is ambiguity between the structure and the name, it should be understood that the structure shall prevail. Unless otherwise stated, no matter whether the term is used alone or in combination with other terms, these definitions apply. Therefore, the definition of "alkyl" is applicable to the "alkyl" part of "alkyl" and "hydroxyalkyl", "haloalkyl", "-O-alkyl" etc.

[0034] Unless otherwise stated, the following terms as used herein and throughout the disclosure shall be understood to have the following meanings.

[0035] The terms used herein have their conventional meanings, and the meanings of these terms are independent at each occurrence. Nevertheless and unless otherwise indicated, the following definitions apply to the entire specification and claims. Chemical name, common name and chemical structure can be used interchangeably to describe the same structure. If both chemical structure and chemical name are used to refer to a chemical compound, and there is ambiguity between the structure and the name, it should be understood that the structure shall prevail. Unless otherwise indicated, these definitions apply regardless of whether the term is used alone or in combination with other terms. Therefore, the definition of "alkyl" applies to the "alkyl" part of "alkyl" as well as "hydroxyalkyl", "haloalkyl", "-O-alkyl" etc.

[0036] Unless otherwise stated, the following terms as used herein and throughout the disclosure shall be understood to have the following meanings.

[0037] The term "anticancer agent" refers to a chemical compound that can be used to treat cancer. The definition also includes antihormonal agents that act to regulate, reduce, block or inhibit the effects of hormones that promote cancer growth, which are usually in the form of systemic or holistic therapy. The anticancer agent can be a hormone.

[0038] The term "compound of the present disclosure" refers to a chemical compound disclosed herein. Included within the definition of "compound of the present disclosure" are linker-load compounds of Formulas (I) to (XXII), linker-load compounds of Examples 1-36, and linker-load compounds comprising: (i) a linker selected from linkers numbered L-1 to L-12, and (ii) a load of Formula (XVI), all of which are collectively referred to herein as "linker-load compounds of the present disclosure." Also included within the definition of "compound of the present disclosure" are antibody-drug conjugates disclosed herein, including but not limited to compounds of Formulas (XXIII) to (LVII) and the antibody-drug conjugates of Examples 37-72, which are collectively referred to herein as "ADCs of the present disclosure."

[0039] A "patient" is a human or non-human mammal. In one embodiment, the patient is a human.

[0040] As used herein, the term "effective amount" refers to an amount of a subject compound and / or additional therapeutic agent, or a combination thereof, that is effective to produce the desired therapeutic, ameliorative, inhibitory, or preventive effect when administered. In the combination therapies described herein, an effective amount can be for each individual agent or for the combination as a whole, wherein the amounts of all agents administered are effective together, but wherein the component agents of the combination may not be present individually in an effective amount.

[0041] As used herein, the term "preventing" with reference to a cell proliferative disorder means reducing the likelihood of a cell proliferative disorder.

[0042] As used herein, the terms "treating" or "treatment" (e.g., of a disease, disorder or condition or associated symptoms, which together or individually may be referred to as an "indication") include: inhibiting the disease, disorder or condition, i.e., halting or reducing the development of the disease or its biological process or progression or its clinical symptoms; or alleviating the disease, i.e., causing regression of the disease or its biological process or progression and / or its clinical symptoms. As used herein, "treating" also refers to controlling, ameliorating or reducing the risk of a subject developing a disease, disorder or condition involving a tumor. As used herein, the terms "preventing" or "preventing" or "preventing" a disease, disorder or condition include: hindering the development or progression of clinical symptoms of a disease, disorder or condition in a mammal that may be exposed to or susceptible to the disease, disorder or condition but has not yet experienced or displayed symptoms of the disease, disorder or condition, etc.

[0043] As used herein, the term "DAR" or "drug-to-antibody ratio" refers to the average number of linker / drug moieties attached to an antibody in a composition comprising a plurality of ADCs of the present disclosure. For a composition comprising an ADC of the present disclosure, the DAR of the composition is the average of the DARs of all individual antibody-drug conjugate molecules present in the composition, and this average is expressed as a decimal. Thus, in some embodiments of a composition comprising an ADC of the present disclosure, the DAR of the composition is a decimal of 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 ADC of the present disclosure, the DAR of the composition is a decimal of 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 ADC of the present disclosure, the DAR of the composition is a decimal of 1 to 3, 2 to 4, 3 to 5, 4 to 6, 5 to 7, and 6 to 8. In further embodiments, for compositions comprising an ADC of the present disclosure, the DAR of the composition is a decimal of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and 7 to 8. The term "composition" as used above is understood to encompass pharmaceutical compositions.

[0044] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced by a bond. Alkyl groups can be straight or branched and contain from about 1 to about 20 carbon atoms. In one embodiment, the alkyl group contains from about 1 to about 10 carbon atoms. In various embodiments, the alkyl group contains from 1 to 10 carbon atoms ("C1-C 10Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl, and neohexyl. An alkyl group can be unsubstituted or substituted with one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. In one embodiment, an alkyl group is straight-chain. In another embodiment, an alkyl group is branched. Unless otherwise specified, an alkyl group is unsubstituted.

[0045] As used herein, term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having one of its hydrogen atoms replaced by a key. The alkenyl group can be straight or branched and contains approximately 2 to approximately 15 carbon atoms. In one embodiment, the alkenyl group contains approximately 2 to approximately 10 carbon atoms. In another embodiment, the alkenyl group contains approximately 2 to approximately 6 carbon atoms. The limiting examples of the alkenyl group include vinyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl and decenyl. An alkenyl group may be unsubstituted or substituted with one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. The term "C2-C 10 "Alkenyl" refers to an alkenyl group having 2 to 10 carbon atoms. Unless otherwise specified, an alkenyl group is unsubstituted.

[0046] As used herein, the term "alkynyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and having one of its hydrogen atoms replaced by a bond. The alkynyl group can be straight or branched and contains approximately 2 to approximately 15 carbon atoms. In one embodiment, the alkynyl group contains approximately 2 to approximately 10 carbon atoms. In another embodiment, the alkynyl group contains approximately 2 to approximately 6 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl. Alkynyl groups may be unsubstituted or substituted with one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. The term "C2-C 10 "Alkynyl" refers to an alkynyl group having 2 to 10 carbon atoms. Unless otherwise specified, an alkynyl group is unsubstituted.

[0047] As used herein, the term "alkylene" refers to an alkyl group as defined above in which one of the hydrogen atoms of the alkyl group has been replaced by a bond. Non-limiting examples of alkylene groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH(CH3)-, and -CH2CH(CH3)CH2-. In one embodiment, the alkylene group has 1 to about 10 carbon atoms. In another embodiment, the alkylene group has 1 to about 6 carbon atoms. In another embodiment, the alkylene group is branched. In another embodiment, the alkylene group is straight-chain. In one embodiment, the alkylene group is -CH2-. The term "C1-C6 alkylene" refers to an alkylene group with 1 to 6 carbon atoms.

[0048] As used herein, the term "alkenylene" refers to an alkenyl group as defined above in which one of the hydrogen atoms of the alkenyl group has been replaced by a bond. Non-limiting examples of alkenylene groups include -CH=CH-, -CH=CHCH2-, -CH2CH2CH=CH- and -CH2(CH3)C=CH-. In one embodiment, the alkenylene group has 2 to about 6 carbon atoms. In one embodiment, the alkenylene group has 2 to about 10 carbon atoms. In another embodiment, the alkenylene group is branched. In another embodiment, the alkenylene group is straight chain. The term "C2-C6 alkenylene" refers to an alkenylene group with 2 to 6 carbon atoms.

[0049] As used herein, the term "alkynylene" refers to an alkynyl group as defined above in which one of the hydrogen atoms of the alkynyl group has been replaced by a bond. Non-limiting examples of alkynylene groups include -C≡C-, -C≡CCH2-, and -C≡CCH(CH3)2-. In one embodiment, the alkynylene group has 2 to about 6 carbon atoms. In one embodiment, the alkynylene group has 2 to about 10 carbon atoms. In another embodiment, the alkynylene group is branched. In another embodiment, the alkynylene group is straight-chain. The term "C2-C6 alkynylene" refers to an alkynylene group having 2 to 6 carbon atoms. The term "C2-C6 10 "Alkyne" refers to an alkynylene group having 2 to 10 carbon atoms.

[0050] As used herein, the term "aminoalkyl" refers to an alkyl group as defined above in which one of the hydrogen atoms of the alkyl group has been replaced by -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl). In one embodiment, the aminoalkyl group has 1 to 6 carbon atoms. Non-limiting examples of aminoalkyl groups include -CH2NH2, -CH2N(CH3)2, -CH2CH2NH2, and -CH2NH(CH). The term "C1-C6 aminoalkyl" refers to an aminoalkyl group having 1 to 6 carbon atoms.

[0051] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity, provided that the antibody fragment has the required number of attachment sites for the drug-linker. The natural form of an antibody is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (V L and V H ) together are primarily responsible for binding to the antigen. The light chain and heavy chain variable domains are composed of framework regions separated by three hypervariable regions (also referred to as "complementary determining regions" or "CDRs"). The constant region can be recognized by the immune system and interacts therewith (see, for example, Janeway et al., 2001, Immuno.Biology, 5th Ed., Garland Publishing, New York). Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Antibodies can be derived from any suitable species. In some aspects, antibodies are of human or mouse origin. Antibodies can be, for example, human antibodies, humanized antibodies or chimeric antibodies.

[0052] As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic ring system containing from about 6 to about 14 carbon atoms. In one embodiment, the aryl group contains from about 6 to about 10 carbon atoms ("C6-C 10 The aryl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined below. In one embodiment, the aryl group can be optionally fused to a cycloalkyl or cycloalkanoyl group. Non-limiting examples of aryl groups include phenyl and naphthyl. Examples of aryl groups fused to a cycloalkyl ring include:

[0053]

[0054] In one embodiment, the aryl group is phenyl. In another embodiment, the aryl group is naphthalene. Unless otherwise specified, the alkyl group is unsubstituted.

[0055] As used herein, the term "arylene" refers to an aryl group as defined above in which one of the hydrogen atoms of the aryl group has been replaced by a bond. Non-limiting examples of arylene groups include:

[0056]

[0057] In one embodiment, the alkylene group has 1 to about 10 carbon atoms. In another embodiment, the alkylene group has 1 to about 6 carbon atoms. In another embodiment, the alkylene group is branched. In another embodiment, the alkylene group is straight-chain. In one embodiment, the alkylene group is -CH2-. The term "C1-C6 alkylene" refers to an alkylene group having 1 to 6 carbon atoms.

[0058] As used herein, the term "composition" is intended to encompass a product comprising an ADC or a pharmaceutically acceptable salt thereof of the present disclosure, together with one or more additional specified ingredients in specified amounts, as well as any product resulting directly or indirectly from the combination of specified ingredients in specified amounts. This term, in relation to pharmaceutical compositions, is intended to encompass a product comprising an active ingredient, including an ADC or a pharmaceutically acceptable salt thereof of the present disclosure, optionally together with one or more additional active ingredients, and an inert ingredient constituting a carrier, as well as any product resulting directly or indirectly from the combination, complexation or aggregation of any two or more ingredients, or from the dissociation of one or more ingredients, or from other types of reactions or interactions of one or more ingredients. Thus, the pharmaceutical compositions of the present disclosure encompass any composition prepared by mixing an ADC or a pharmaceutically acceptable salt thereof of the present disclosure and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0059] As used herein, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic ring system containing from about 3 to about 11 ring carbon atoms. In one embodiment, the cycloalkyl contains from about 5 to about 11 ring carbon atoms. In another embodiment, the cycloalkyl is monocyclic and contains from about 3 to about 7 ring atoms. In another embodiment, the cycloalkyl is monocyclic and contains from about 5 to about 6 ring atoms. In another embodiment, the cycloalkyl is bicyclic and contains from about 4 to 10 ring atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Non-limiting examples of polycyclic cycloalkyls include 1-decalinyl, norbornyl, and adamantyl. The cycloalkyl group may optionally be substituted with one or more "ring system substituents" which may be the same or different and are defined below. Unless otherwise indicated, the cycloalkyl group is unsubstituted. In one embodiment, the cycloalkyl group is unsubstituted. The term "3- to 7-membered monocyclic monocyclic cycloalkyl" refers to a monocyclic cycloalkyl group having 3 to 7 ring carbon atoms. The term "5- to 11-membered bicyclic cycloalkyl" refers to a bicyclic cycloalkyl group having 5 to 11 ring carbon atoms.

[0060] Polycyclic cycloalkyl groups can have fused rings, rings connected in a spirocyclic manner, and bridged rings. In one embodiment, the cycloalkyl group can be a spirocyclic cycloalkyl group ("C5-C6-C7-C8-C9-C10-C12-C18-C19-C20-C21-C22-C23-C24-C26-C27-C28-C29-C30-C31-C32-C33-C34-C36-C37-C38-C39-C39-C31-C31-C32-C33-C36-C38 ... 11 Spirocyclic cycloalkyl"). Illustrative examples of such bicyclic cycloalkyl groups include:

[0061]

[0062] In another embodiment, the cycloalkyl group can be a fused bicyclic cycloalkyl group having 5 to 11 ring carbon atoms ("C5-C 11 "fused bicyclic cycloalkyl"). Illustrative examples of such fused bicyclic cycloalkyl groups include:

[0063]

[0064] In another embodiment, the cycloalkyl group can be a bridged bicyclic cycloalkyl group having 5 to 11 ring carbon atoms ("C5-C 11 Illustrative examples of such bridged bicyclic and tricyclic heterocycloalkyl groups include:

[0065]

[0066] The ring carbon atoms of the cycloalkyl group can be functionalized as a carbonyl group. Illustrative examples of such cycloalkyl groups (also referred to herein as "cycloalkanoyl" groups) include, but are not limited to, cyclobutanoyl:

[0067]

[0068] The term "cycloalkylene" as used herein refers to a cycloalkyl group as defined above in which one of the hydrogen atoms of the cycloalkyl group has been replaced by a bond. In one embodiment, the cycloalkylene group is monocyclic and contains from about 3 to about 7 ring carbon atoms ("C3-C7 monocyclic cycloalkylene"). In another embodiment, the cycloalkylene group is bicyclic and contains from about 5 to 10 ring atoms (C5-C7). 10 Non-limiting examples of monocyclic cycloalkylenes include the following:

[0069]

[0070] Cycloalkylene groups may optionally be substituted with one or more "ring system substituents" which may be the same or different and are as defined below. Unless otherwise indicated, cycloalkylene is unsubstituted. In one embodiment, cycloalkylene is unsubstituted.

[0071] Polycyclic cycloalkylene groups can have fused rings, rings connected in a spirocyclic manner, and bridged rings. In one embodiment, the cycloalkylene group can be a bridged bicyclic cycloalkylene group with 5 to 11 ring carbon atoms. Illustrative examples of such bridged bicyclic heterocycloalkyl groups include, but are not limited to:

[0072]

[0073] The ring carbon atoms of the cycloalkylene group may be functionalized as carbonyl groups. Illustrative examples of such cycloalkylene groups include, but are not limited to,

[0074]

[0075] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing about 4 to about 10 ring carbon atoms and at least one bridged double bond. In one embodiment, the cycloalkenyl contains about 4 to about 7 ring carbon atoms. In another embodiment, the cycloalkenyl contains 5 or 6 ring atoms. Non-limiting examples of monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cyclohepta-1,3-dienyl, etc. The cycloalkenyl group may optionally be substituted with one or more "ring system substituents" which may be the same or different and are defined below. The ring carbon atoms of the cycloalkylene group may be functionalized as a carbonyl group. In one embodiment, the cycloalkenyl group is cyclopentenyl. In another embodiment, the cycloalkenyl group is cyclohexenyl. The term "4 to 6 membered cycloalkenyl" refers to a cycloalkenyl group having 4 to 6 ring carbon atoms.

[0076] As used herein, the term "halo" refers to -F, -Cl, -Br, or -I.

[0077] The term "haloalkyl" as used herein refers to an alkyl group as defined above in which one or more hydrogen atoms of the alkyl group have been replaced by halogen. In one embodiment, the haloalkyl group has 1 to 10 carbon atoms. In another embodiment, the haloalkyl group has 1 to 6 carbon atoms. In another embodiment, the haloalkyl group is substituted by 1 to 6 F atoms. In one class of this embodiment, the haloalkyl group is substituted by 1 to 3 F atoms. Non-limiting examples of haloalkyl groups include -CH2CHF2, –CH2F, –CHF2, –CF3, –CH2Cl and –CCl3. The term "C1-C6 haloalkyl" refers to a haloalkyl group with 1 to 6 carbon atoms.

[0078] As used herein, term "haloalkylene" refers to a haloalkylene group as defined above in which one or more hydrogen atoms of the haloalkylene group have been replaced by halogen. In one embodiment, the haloalkylene group has 1 to 10 carbon atoms. In another embodiment, the haloalkylene group has 1 to 6 carbon atoms. In another embodiment, the haloalkylene group is replaced by 1 to 6 F atoms. In a class of this embodiment, the haloalkylene group is replaced by 1 to 3 F atoms. The limiting examples of haloalkylene groups include-CH2CHF2,-CH2F,-CHF2,-CF3,-CH2Cl and-CCl3. The term "C1-C6 haloalkylene" refers to a haloalkylene group with 1 to 6 carbon atoms.

[0079] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined above in which one or more hydrogen atoms of the alkyl group have been replaced by an -OH group. In one embodiment, the hydroxyalkyl group has 1 to 10 carbon atoms. In another embodiment, the hydroxyalkyl group has 1 to 6 carbon atoms. Non-limiting examples of hydroxyalkyl groups include -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, and -CH2CH(OH)CH3. The term "C1-C 10 The term "hydroxyalkyl" refers to a hydroxyalkyl group having 1 to 10 carbon atoms.

[0080] As used herein, term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system comprising approximately 5 to approximately 14 ring atoms, wherein 1 to 4 ring atoms are independently O, N, or S and the remaining ring atoms are carbon atoms. In one embodiment, the heteroaryl group has 5 to 10 ring atoms. In another embodiment, the heteroaryl group is monocyclic and has 5 or 6 ring atoms ("5 or 6 yuan monocyclic heteroaryl"). In another embodiment, the heteroaryl group is bicyclic and has 8 to 10 ring atoms ("8 to 10 yuan bicyclic heteroaryl"). In another embodiment, the heteroaryl group is bicyclic and has 9 or 10 ring atoms ("9 or 10 yuan bicyclic heteroaryl"). The heteroaryl group can optionally be replaced by one or more ring substituents that may be identical or different and as defined below. The heteroaryl group is connected via annular carbon atoms and any nitrogen atom of the heteroaryl group can optionally be oxidized to the corresponding N-oxide. The term "heteroaryl" also encompasses heteroaryl groups as defined above fused to a benzene ring. Non-limiting examples of heteroaryl include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothiophenyl, quinolinyl, imidazolyl, benzimidazolyl, thienopyridinyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridinyl, imidazopyridinyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, benzothiazolyl, etc., and all isomeric forms thereof. The term "heteroaryl" also refers to partially saturated heteroaryl moieties, such as tetrahydroisoquinolinyl, tetrahydroquinolinyl, etc. In one embodiment, the heteroaryl group is a 5-membered heteroaryl group. In another embodiment, the heteroaryl group is a 6-membered heteroaryl group, such as pyridinyl.

[0081] In one embodiment, the 8- to 10-membered bicyclic heteroaryl group includes a fused bicyclic heterocyclic group in which one of the two fused rings is a phenyl group or a monocyclic heteroaryl group, for example:

[0082]

[0083] "9- to 14-membered tricyclic heteroaryl" includes 8- to 10-membered bicyclic heteroaryl groups in which the third ring is fused to one ring of the 8- to 10-membered bicyclic heteroaryl group. Such a third ring can be a cycloalkyl, heterocycloalkyl, or heteroaryl ring. Examples of 9- to 14-membered tricyclic heteroaryl groups include:

[0084]

[0085]

[0086] As used herein, the term "heterocycloalkyl" refers to a non-aromatic saturated monocyclic or polycyclic ring system comprising 3 to about 14 ring atoms, wherein 1 to 4 ring atoms are independently O, S, N or Si, and the remaining ring atoms are carbon atoms. The heterocycloalkyl group can be connected via ring carbon, ring silicon atom or ring nitrogen atom. In one embodiment, the heterocycloalkyl group is monocyclic. In one embodiment, the heterocycloalkyl group is monocyclic and has about 3 to about 7 ring atoms ("3 to 7 yuan monocyclic heterocycloalkyl"). In another embodiment, the heterocycloalkyl group is monocyclic and has 5 or 6 ring atoms ("5 or 6 yuan monocyclic heterocycloalkyl"). In one embodiment, the heterocycloalkyl group is bicyclic. In another embodiment, the heterocycloalkyl group is bicyclic and has about 5 to about 11 ring atoms ("5 to 11 yuan bicyclic heterocycloalkyl"). In another embodiment, the heterocycloalkyl group is tricyclic and has about 10 to about 14 ring atoms ("10 to 14 yuan tricyclic heterocycloalkyl"). There are no adjacent oxygen and / or sulfur atoms in the ring system. Any -NH group in a heterocycloalkyl ring may be protected, for example, as an -N(BOC), -N(CBz), -N(Tos) group, and the like; such protected heterocycloalkyl groups are considered part of this disclosure. A heterocycloalkyl group may be optionally substituted with one or more "ring system substituents," which may be the same or different, and are as defined below. The nitrogen or sulfur atom of a heterocycloalkyl group may be optionally oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. Non-limiting examples of monocyclic heterocycloalkyl rings include oxetanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, δ-lactam, δ-lactone, silacyclopentane, silapyrrolidine, and the like, and all isomers thereof. Non-limiting illustrative examples of silyl-containing heterocycloalkyl groups include:

[0087]

[0088] The ring carbon atoms of the heterocycloalkyl group may be functionalized as a carbonyl group. Illustrative examples of such heterocycloalkyl groups include, but are not limited to:

[0089]

[0090] The ring sulfur atom of the heterocycloalkyl group can also be functionalized as a sulfonyl group. Examples of such heterocycloalkyl groups are:

[0091]

[0092] In one embodiment, the heterocycloalkyl group is a 5-membered monocyclic heterocycloalkyl.In another embodiment, the heterocycloalkyl group is a 6-membered monocyclic heterocycloalkyl.

[0093] Polycyclic heterocycloalkyl groups can have fused rings, rings connected in a spirocyclic manner, and bridged rings. In one embodiment, heterocycloalkyl groups can be bicyclic spirocyclic heteroaryl groups with 1 to 11 ring atoms. Illustrative examples of such bicyclic heterocycloalkyl groups include:

[0094]

[0095] In another embodiment, the heterocycloalkyl group can be a fused bicyclic heterocycloalkyl group having 5 to 11 ring atoms ("5- to 11-membered fused bicyclic heterocycloalkyl"). Illustrative examples of such fused bicyclic heterocycloalkyl groups include:

[0096]

[0097] In another embodiment, the heterocycloalkyl group can be a bridged bicyclic heterocycloalkyl group having 5 to 11 ring atoms ("5- to 11-membered bridged bicyclic heterocycloalkyl"). Illustrative examples of such bridged bicyclic heterocycloalkyl groups include:

[0098]

[0099] As used herein, term "heterocycloalkylidene" refers to a heterocycloalkylidene group as defined above in which one of the hydrogen atoms of the heterocycloalkylidene group has been replaced by a bond. The heterocycloalkylidene group can be connected via a ring carbon or a ring nitrogen atom. In one embodiment, the heterocycloalkylidene group has 4 to 6 ring atoms ("4 to 6 yuan heterocycloalkylidene"). In another embodiment, the heterocycloalkenyl group is monocyclic and has 5 or 6 ring atoms. In another embodiment, the heterocycloalkenyl group is bicyclic and has 5 to 10 ring atoms ("5 to 10 yuan bicyclic heterocycloalkylidene"). The heterocycloalkylidene group can be optionally substituted by one or more ring system substituents, wherein "ring system substituents" are as defined above. The nitrogen or sulfur atom of the heterocycloalkylidene group can be optionally oxidized to corresponding N-oxide, S-oxide or S, S-dioxide. The ring carbon atom of the heterocycloalkylidene group can be functionalized to a carbonyl group. Non-limiting examples of monocyclic heterocycloalkylidene groups include:

[0100]

[0101] Polycyclic heterocyclic alkylene groups can have fused rings, rings connected in spirocyclic mode and bridged rings.In one embodiment, heterocyclic alkylene groups can be bicyclic spirocyclic heterocyclic alkylene groups (" 5 to 11 membered bicyclic spirocyclic heterocyclic alkylene groups ") with 5 to 11 ring atoms.The illustrative examples of such bicyclic spirocyclic alkylene groups include:

[0102]

[0103] In another embodiment, the heterocycloalkylene group can be a fused bicyclic heterocycloalkylene having 5 to 11 ring atoms ("5- to 11-membered fused bicyclic heterocycloalkylene"). Illustrative examples of such fused bicyclic heterocycloalkylenes include:

[0104]

[0105] In another embodiment, the heterocycloalkylene group can be a bridged heterocycloalkylene group having 5 to 11 ring atoms ("5- to 11-membered bridged bicyclic heterocycloalkylene"). Illustrative examples of such bridged bicyclic heterocycloalkylene groups include:

[0106]

[0107] As used herein, the term "heterocycloalkenyl" refers to a heterocycloalkyl group as defined above wherein the heterocycloalkyl group contains 4 to 10 ring atoms and at least one bridged carbon-carbon or carbon-nitrogen double bond. The heterocycloalkenyl group can be connected via a ring carbon or ring nitrogen atom. In one embodiment, the heterocycloalkenyl group has 4 to 6 ring atoms. In another embodiment, the heterocycloalkenyl group is monocyclic and has 5 or 6 ring atoms. In another embodiment, the heterocycloalkenyl group is bicyclic. The heterocycloalkenyl group can be optionally substituted by one or more ring system substituents, wherein "ring system substituents" are as defined above. The nitrogen or sulfur atom of the heterocycloalkenyl group can be optionally oxidized to corresponding N-oxides, S-oxides or S,S-dioxides. The ring carbon atoms of the heterocycloalkenyl group can be functionalized to carbonyl groups. The limiting examples of heterocycloalkenyl groups include 1,2,3,4-tetrahydropyridinyl, 1,2-dihydropyridinyl, 1,4-dihydropyridinyl, 1,2,3,6-tetrahydropyridinyl, 1,4,5,6-tetrahydropyrimidinyl, 2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, dihydroimidazolyl, dihydrooxazolyl, dihydrooxadiazolyl, dihydrothiazolyl, 3,4-dihydro-2H-pyranyl, dihydrofuranyl, fluorine-substituted dihydrofuranyl, 7-oxabicyclo [2.2.1] heptenyl, dihydrothienyl, dihydrothiopyranyl (dihydrothiopyranyl) etc. In one embodiment, the heterocycloalkenyl group is a 5-membered heterocycloalkenyl. In another embodiment, the heterocycloalkenyl group is a 6-membered heterocycloalkenyl. The term "4- to 6-membered heterocycloalkenyl" refers to a heterocycloalkenyl group having 4 to 6 ring atoms.

[0108] The term "substituted" means that one or more hydrogen atoms on the designated atom are replaced with a radical selected from the specified group, provided that the normal valency of the designated atom in the existing circumstances is not exceeded and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. A "stable compound" or "stable structure" is intended to mean a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an efficacious therapeutic agent.

[0109] As used herein, the term "substantially purified form" refers to the physical state of a compound after isolating the compound from a synthetic process (e.g., from a reaction mixture), a natural source, or a combination thereof. The term "substantially purified form" also refers to the physical state of a compound after obtaining the compound from one or more purification methods described herein or known to those skilled in the art (e.g., chromatography, recrystallization, etc.), and its purity is sufficient to be characterized by standard analytical techniques described herein or known to those skilled in the art.

[0110] It should also be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and tables herein are assumed to have the sufficient number of hydrogen atoms to satisfy the valences.

[0111] When a functional group in a compound is referred to as "protected," this means that the group is in a modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be well known to those of ordinary skill in the art and by reference to standard textbooks such as Greene et al., Protective Groups in Organic Synthesis, Wiley-Interscience, New York, (1999).

[0112] Examples of "ring system substituents" include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -alkylene-aryl, -arylene-alkyl, -alkylene-heteroaryl, -alkenylene-heteroaryl, -alkynylene-heteroaryl, -OH, hydroxyalkyl, haloalkyl, -O-alkyl, -O-haloalkyl, -alkylene-O-alkyl, -O-aryl, -O-alkylene-aryl, acyl, -C(O)-aryl, halo, -NO2, -CN, -SF5, -C(O)OH, -C(O)O-alkyl, -C(O)O-aryl, -C(O)O-alkylene-aryl, -S(O)-alkyl, -S(O)2-alkyl, -S(O)-aryl, -S(O)2-aryl, -S(O)-heteroaryl, -S(O)z-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -S-alkylene-aryl, -S-alkyleneheteroaryl, -S(O)2-alkylene-aryl, -S(O)2-alkylene-heteroaryl, -Si(alkyl)2, -Si(aryl)2, Si(heteroaryl)2-Si(alkyl)(aryl), -Si(alkyl)(cycloalkyl), -Si(alkyl)(heteroaryl), cycloalkyl, heterocycloalkyl, -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(=N-CN)-NH2, -C(=NH)-NH2, -C(=NH)-NH(alkyl), -N(Y 1 )(Y 2 ), -alkylene-N(Y 1 )(Y 2 )、-C(O)N(Y 1 )(Y 2 ) and -S(O)2N(Y 1 )(Y 2 ), where Y 1 and Y 2"Ring system substituents" can also refer to a single moiety that simultaneously replaces two available hydrogens (one H on each carbon) on two adjacent carbon atoms in the ring system. Examples of such moieties are methylenedioxy, ethylenedioxy, -C(CH 3 ) 2 -, and the like, which form, for example, the following moieties:

[0113]

[0114] When any substituent or variable (such as R 5 When (, n, etc.) occurs more than one time in any constituent or in formula (I), its definition on each occurrence is independent of its definition at every other occurrence, unless otherwise stated.

[0115] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results from combination of the specified ingredients in the specified amounts.

[0116] It should be understood that one of ordinary skill in the art can incorporate one or more silicon (Si) atoms into the compounds of the present disclosure in place of one or more carbon atoms to provide chemically stable compounds that can be easily synthesized from readily available starting materials by techniques known in the art. The covalent radii of carbon and silicon are different, resulting in differences in bond distances and spatial arrangement when similar C and Si element bonds are compared. These differences result in subtle changes in the size and shape of silicon-containing compounds when compared to carbon. One of ordinary skill in the art will appreciate that size and shape differences can result in subtle or significant changes in efficacy, solubility, lack of off-target activity, packaging properties, etc. (Diass, JO et al. Organometallics (2006) 5: 1188-1198; Showell, GA et al. Bioorganic & Medicinal Chemistry Letters (2006) 16: 2555-2558).

[0117] It will be understood that the substituents and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized from readily available starting materials by techniques known in the art and those described below. If a substituent is itself substituted with more than one group, it will be understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure results. The phrase "optionally substituted with one or more substituents" should be understood to mean that the group in question is unsubstituted or can be substituted with one or more substituents.

[0118] Where optional substitution of a moiety is described (e.g., "optionally substituted"), the term indicates that, if present, one or more of the substituents recited for a given substrate may be present in the bonding position on that substrate 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 an optional substituent may replace the default substituent.

[0119] Pharmaceutically acceptable esters of the compounds of the present invention include the following groups: (1) carboxylic acid esters obtained by esterifying the hydroxy group of a hydroxy compound, wherein the non-carbonyl portion of the carboxylic acid portion of the ester group is selected from a linear or branched alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, tert-butyl, sec-butyl or n-butyl), an alkoxyalkyl group (e.g., methoxymethyl), an aralkyl group (e.g., benzyl), an aryloxyalkyl group (e.g., phenoxymethyl), an aryl group (e.g., optionally substituted with, for example, halogen, C 1-4 Alkyl, -O-(C 1-4 (e.g., alkyl) or amino-substituted phenyl); (2) sulfonates, such as alkyl- or aralkylsulfonyl (e.g., methylsulfonyl); (3) amino acid esters (e.g., L-valyl or L-isoleucyl); (4) phosphonates, and (5) mono-, di-, or triphosphates. Phosphates can be prepared by, for example, C 1-20 Alcohol or its reactive derivatives or through 2,3-di(C 6-24 ) acylglycerols are further esterified.

[0120] One or more compounds of the present disclosure may exist in unsolvated form as well as in solvated form with pharmaceutically acceptable solvents such as water, ethanol, etc., and the present disclosure is intended to include both solvated and unsolvated forms. "Solvate" refers to the physical association of a compound of the present disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses solution phase and separable solvates. Non-limiting examples of solvates include ethanolates, methanolates, etc. "Hydrate" is a solvate in which the solvent molecule is water.

[0121] One or more compounds of the present disclosure may also be optionally 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 from water. Similar preparations of solvates, hemisolvates, hydrates, etc. are described in EC van Tonder et al, AAPS Pharm Sci Techours., 5 (1), article 12 (2004); and AL Bingham et al, Chem. Commun., 603-604 (2001). A typical non-limiting method comprises dissolving the compound of the present invention in a desired amount of a desired solvent (e.g., an organic solvent or water or a mixture thereof) at a temperature above room temperature, and cooling the solution at a rate sufficient to form crystals, followed by isolating the crystals by standard methods. Analytical techniques, such as IR spectroscopy, show that the solvent (or water) is present in the crystals as a solvate (or hydrate).

[0122] The linker-load compound can form salts, which are also within the scope of the present disclosure. As used herein, the term "pharmaceutically acceptable salt" or "salt" refers to a derivative in which the parent compound is modified by preparing an acid or base salt thereof. Salts in solid form may exist in more than one crystal structure and may also be in the form of hydrates.

[0123] Exemplary acid addition salts include acetate, ammonium, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate (also known as mesylate), naphthenate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also known as tosylate), and the like. Additionally, acids generally recognized as suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds are, for example, those described in P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2 ndRevised Ed. (2011) Zurich: Wiley-VCH;S.Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1)1-19;P.Gould, International J.of Pharmaceutics (1986) 33 201-217;Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York;and The Orange Book (Food & Drug Administration, Washington, DC Con their website). These disclosures are incorporated herein by reference. In one embodiment, the acidic salt is an ammonium salt or a diammonium salt.

[0124] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as dicyclohexylamine, tert-butylamine, choline, and salts with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups can be quaternized using reagents such as lower alkyl halides (e.g., methyl, ethyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl and dibutyl sulfate), long chain halides (e.g., decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and the like.

[0125] All such acid and base salts are intended to be pharmaceutically acceptable salts within the scope of the disclosure and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure.

[0126] Compounds of the present disclosure may contain one or more asymmetric centers and therefore may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Depending on the nature of the various substituents on the molecule, additional asymmetric centers may exist. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomers intended for mixtures and as pure or partially purified compounds are included within the scope of the present disclosure. Unless a specific stereochemistry is indicated, the present disclosure is meant to encompass all such isomeric forms of these compounds.

[0127] The independent synthesis of these diastereomers or their chromatographic separation can be realized as known in the art by the appropriate modification of the methodology disclosed herein. In addition to other methods, their absolute stereochemistry can be determined by the x-ray crystallography of crystalline products or crystalline intermediates (if necessary, derivatized with a reagent containing an asymmetric center of known absolute configuration). Diastereomeric mixtures can be separated into their individual diastereomers based on their physicochemical differences by methods well known to those skilled in the art (such as by chromatography and / or fractional crystallization). Enantiomeric mixtures can be converted into diastereomeric mixtures by reacting with appropriate optically active compounds (such as, chiral auxiliary agents, such as chiral alcohols or Mosher acyl chlorides), separating diastereomers and converting individual diastereomers (such as, hydrolyzing) into corresponding pure enantiomers to separate enantiomers. Stereochemically pure compounds can also be prepared by using chiral starting materials or by adopting salt resolution technology. In addition, some joint-loaded compounds can be atropisomers (such as, substituted biaryls) and are considered to be a part of the present disclosure. Chiral chromatographic techniques can also be used to separate enantiomers directly.

[0128] Linker-loaded compounds may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. For example, all keto-enol and imine-enamine forms of the compounds are included within the present disclosure.

[0129] All stereoisomers (e.g., geometric isomers, optical isomers, etc.) of the compounds of the invention (including salts, solvates, hydrates, esters and prodrugs of the compounds and salts, solvates and esters of the prodrugs), such as those that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotamer forms, atropisomers and diastereomeric forms, are contemplated as being within the scope of the present disclosure. If the linker-payload compound incorporates a double bond or a fused ring, both the cis- and trans- forms, as well as mixtures, are included within the scope of the present disclosure.

[0130] In all cases, the compound name accompanies the drawn structure and is intended to describe every stereochemical arrangement possible for a given structural isomer based on the synthetic procedures used in its preparation. A listing of discrete stereoisomers is used in conjunction with or indicates that the compound presented (e.g., 'Example Number') was isolated as a single stereoisomer, and the identity of that stereoisomer corresponds to one of the possible configurations listed. A listing of discrete stereoisomers is used in conjunction with and indicates that the compound presented was isolated as a racemic mixture or a mixture of diastereomers.

[0131] Specific absolute configurations are indicated by use of bold wedges or dashed wedges.Unless specific absolute configurations are indicated, the disclosure is intended to encompass all such stereoisomeric forms of these compounds.

[0132] In this specification, when multiple oxygen and / or sulfur atoms are present in a ring system, there cannot be any adjacent oxygen and / or sulfur atoms in the ring system.

[0133] Individual stereoisomers of the disclosed compounds may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other or other selected stereoisomers. The chiral centers of the disclosed compounds may have an S or R configuration as defined by the IUPAC 1974 Recommendations. The use of the terms "salt," "solvate," "ester," "prodrug," and the like is intended to apply equally to salts, solvates, esters, and prodrugs of enantiomers, stereoisomers, rotational isomers, tautomers, positional isomers, racemates, or prodrugs of the disclosed compounds.

[0134] In the compounds of formula (I), the atoms may exhibit their natural isotopic abundance, or one or more atoms may be artificially enriched to have a particular isotope with the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure is meant to include all suitable isotopic variants of the compounds of formula I. For example, different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H). Protium is the main hydrogen isotope found in nature. Enrichment with deuterium can provide certain therapeutic advantages, such as increasing half-life in vivo or reducing dosage requirements, or can provide compounds that can be used as standards for characterizing biological samples. The isotopically enriched compounds of Formula I can be prepared by conventional techniques well known to those skilled in the art or by methods similar to those described in the schemes and examples herein using appropriate isotopically enriched reagents and / or intermediates without excessive experimentation. In one embodiment, one or more hydrogen atoms of the compound of Formula (I) are replaced by deuterium.

[0135] Polymorphic forms of linker-payload compounds, as well as polymorphic forms of salts, solvates, hydrates, esters, and prodrugs of linker-payload compounds, are intended to be included in the present disclosure.

[0136] For all embodiments described herein, any variable not explicitly defined in the embodiment is defined as in Formula (I). In each embodiment described herein, unless otherwise indicated, each variable is selected independently of the others.

[0137] The following abbreviations are used below and have the following meanings:

[0138]

[0139]

[0140]

[0141] Compounds of the present disclosure

[0142] New linker-loaded compounds (compounds of formula (I)) containing a PNU anthracycline payload are described. Compounds of formula (I) are useful as components of antibody-drug conjugates for treating and preventing cancer. Antibody-drug conjugates containing compounds of formula (I), compositions containing such antibody-drug conjugates, and uses of such antibody-drug conjugates for treating or preventing cancer are also described.

[0143] Linker-Load Compounds of the Disclosure

[0144] In one aspect, the present disclosure provides linker-load compounds of formula (I), and pharmaceutically acceptable salts thereof, comprising a PNU anthracycline load and a cleavable linker:

[0145]

[0146] where R 1 and R 2 As defined above.

[0147] In one embodiment, R 1 Selected from -XYZR 3 .

[0148] In one embodiment, R 2 For H.

[0149] In another embodiment, R 2 It is a C1-C6 alkyl group.

[0150] In a specific embodiment, R 2 For H.

[0151] In a specific embodiment, R 2 It is a methyl group.

[0152] In one embodiment, R 1 -XYZR 3 , and R 2 is H or C1-C6 alkyl.

[0153] In one embodiment, R 1 and R 2 is connected to form a 4- to 6-membered monocyclic heterocycloalkylene group, which is replaced by -XYZR on a ring carbon atom.3 replaced.

[0154] In another embodiment, R 1 and R 2 is connected to form a 5- to 11-membered bicyclic heterocycloalkylene group, which is terminated on a ring carbon atom by -XYZR 3 replaced.

[0155] In a specific embodiment, R 1 and R 2 Connection formation:

[0156]

[0157] In one embodiment, R 3 for:

[0158]

[0159] In another embodiment, R 3 for:

[0160]

[0161] In another embodiment, R 3 for:

[0162]

[0163] In yet another embodiment, R 3 for:

[0164]

[0165] In a specific embodiment, R 3 for:

[0166]

[0167] In another specific embodiment, R 3 for:

[0168]

[0169] In another specific embodiment, R 3 for:

[0170]

[0171] In another specific embodiment, R 3 for:

[0172]

[0173] In yet another specific embodiment, R 3 for:

[0174]

[0175] In yet another specific embodiment, R 3 for:

[0176]

[0177] In another specific embodiment, R 3 for:

[0178]

[0179] In another specific embodiment, R 3 for:

[0180]

[0181] In one embodiment, X is -(CH2) n -N(R 4 )-.

[0182] In another embodiment, X is -(CH2) n -NH-, and n is 1 or 2.

[0183] In another embodiment, X is -(CH2) n -N(CH3)-, and n is 1 or 2.

[0184] In one embodiment, X is -NH-.

[0185] In another embodiment, X is -R 5 -NH-.

[0186] In one embodiment, X is -(CH2) n -N(R 4 )-C(O)R 5 -.

[0187] In another embodiment, X is -(CH2) n -N(R 4 )-(C1-C6 alkylene)-NH(R 4 )-.

[0188] In another embodiment, X is -(CH2) n -N(R 4 )-C(O)O(CH2) n -R 5 -N(R 4)-.

[0189] In another embodiment, X is -(CH2)3-NH-(CH2)3-NH-.

[0190] In another embodiment, X is a 5- or 6-membered monocyclic heterocycloalkylene.

[0191] In a specific embodiment, X is:

[0192]

[0193] In another embodiment, X is -(CH2) n -R 5 -.

[0194] In a particular embodiment, X is -CH2-R 5 -.

[0195] In another specific embodiment, X is:

[0196]

[0197] In yet another embodiment, X is -(CH2) n -R 5 -NH(R 4 )-.

[0198] In another embodiment, X is a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene.

[0199] In a specific embodiment, X is:

[0200]

[0201] In yet another embodiment, X is -(CH2) n -R 5 -NHC(O)OR 5 NH(R 4 )-.

[0202] In a further embodiment, X is -(CH2) n -NHC(O)O-(CH2) n -R 5 -NH(R 4 ).

[0203] In another embodiment, X is -(CH2) n -R 5 -OC(O)-NH(R 4 )-(C1-C6 alkylene)-NH(R 4 )-.

[0204] In one embodiment, X is -R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-.

[0205] In a specific embodiment, X is:

[0206]

[0207] In another embodiment, X is -(CH2) n -N(R 4 )C(O)NH-N(R 4 )-.

[0208] In a particular embodiment, X is: -CH2CH2NHC(O)NHNH-.

[0209] In another embodiment, X is -R 5 -N(R 4 )C(O)CH2N(R 4 )-.

[0210] In a specific embodiment, X is:

[0211]

[0212] In yet another embodiment, X is -(CH2) n -N(R 4 )-(CH2) n -N(R 4 )-.

[0213] In a particular embodiment, X is: -CH2CH2NH-CH2CH2NH-.

[0214] In a further embodiment, -R 5 -O-CH2N(R 4 )-.

[0215] In a specific embodiment, X is:

[0216]

[0217] In one embodiment, Y is a bond.

[0218] In another embodiment, Y is -C(O)C(R 4 )(R 6 )NH-.

[0219] In another embodiment, Y is -C(O)C(R 4 )(R 6 )NH-, where R 6 is H or isopropyl, and R 4 is H or methyl.

[0220] In one embodiment, Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-, where R 7 and R 8 Each is independently selected from H, methyl, isopropyl and -CH2CH2CH2NHC(O)NH2.

[0221] In one embodiment, R 5 C6-C 10 Aryl.

[0222] In another embodiment, R 5 It is a C3-C7 monocyclic cycloalkylene group.

[0223] In another embodiment, R 5 C5-C 11 Bicyclic cycloalkylene.

[0224] In another embodiment, R 5 It is -NHC(O)NH-.

[0225] In another embodiment, R 5 It is -NH-NH-.

[0226] In a specific embodiment, R 5 Selected from -NH-NH-, -NHC(O)NH-,

[0227]

[0228] In one embodiment, X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-(CH2) n -R 5 -N(R 4 )-、-(CH2)n -R 5 -、-(CH2) n -R 5 -NHC(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-;

[0229] R 4 Each occurrence is independently H, methyl or Fmoc; and R 5 Each occurrence is independently selected from -NH-NH-, -NHC(O)NH,

[0230]

[0231] In one embodiment, the group -XYZR 3 Selected from:

[0232]

[0233]

[0234] In one embodiment, the compound of formula (I) is a compound of formula (I'):

[0235]

[0236] and pharmaceutically acceptable salts thereof, wherein:

[0237] When R 2 When R is H or C1-C6 alkyl, 1Selected from -XYZR 3 ;

[0238] R 2 is H or C1-C6 alkyl, or R 1 and R 2 are connected to form a 4- to 6-membered monocyclic heterocycloalkylene group or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene group, each of which is substituted on a ring carbon atom by -XYZR 3 replaced by;

[0239] R 3 Selected from:

[0240]

[0241] X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -NHC(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )- and -(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-;

[0242] Y is a bond or -C(O)C(R 4 )(R 6 )NH-;

[0243] Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-;

[0244] R 4 H, C 1-6 Alkyl, or

[0245]

[0246] R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bicyclic cycloalkylene and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group;

[0247] R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2 and naturally occurring amino acid side chains;

[0248] m is 0, 1, or 2; and

[0249] n is independently an integer from 0 to 4 at each occurrence.

[0250] In one embodiment, the compound of formula (I) is represented by formula (II):

[0251]

[0252] where R 2 , X, Y, Z and m are as described herein for the compound of formula (I).

[0253] In another embodiment, the compound of formula (I) is represented by formula (III):

[0254]

[0255] wherein X, Y, Z, and M are as described herein; and A is a 4- or 6-membered monocyclic heterocycloalkylene group.

[0256] In another embodiment, the compound of formula (I) is represented by formula (IV):

[0257]

[0258] where R 2 , X, Y, Z and m are as described herein.

[0259] In yet another embodiment, the compound of formula (I) is represented by formula (V):

[0260]

[0261] wherein X, Y, Z, and M are as described herein; and A is a 4- or 6-membered monocyclic heterocycloalkylene group.

[0262] In a further embodiment, the compound of formula (I) is represented by formula (VI):

[0263]

[0264] where R 2 , X, Y, Z and m are as described herein.

[0265] In another embodiment, the compound of formula (I) is represented by formula (VII):

[0266]

[0267] wherein X, Y, Z, and M are as described herein; and A is a 4- or 6-membered monocyclic heterocycloalkylene group.

[0268] In another embodiment, the compound of formula (I) is represented by formula (VIII):

[0269]

[0270] where R 2 、R 9 , X, Y, Z and m are as described herein.

[0271] In another embodiment, the compound of formula (I) is represented by formula (IX):

[0272]

[0273] where R 9 , X, Y, Z and M are as described herein; and A is a 4- or 6-membered monocyclic heterocycloalkylene group.

[0274] In another embodiment, it may be desirable to synthesize a linker that is then conjugated to a drug or targeting moiety to provide an antibody-drug conjugate. In such an embodiment, the linker compound may serve as an intermediate compound. Exemplary linkers of the present disclosure include, but are not limited to, the following linker fragments (L-1 to L-12), wherein the attachment point of the payload to the linker is :express:

[0275]

[0276]

[0277] Linker fragments L-1 to L-12 can be linked to the payload using the following reactants L-1' to L-12', respectively, where Q is -OH or -Cl:

[0278]

[0279]

[0280] In one aspect, the present invention provides linker-load compounds having structural formula (X)-(XXI), wherein D is an anticancer agent:

[0281]

[0282]

[0283]

[0284] In one embodiment, the present disclosure provides a linker-load compound comprising a linker portion selected from L-1, L-2, L-1, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11 and L-12, which is conjugated to an anticancer agent having a reactive -OH group.

[0285] In one embodiment, the present disclosure provides a linker-load compound comprising a linker moiety selected from L-1, L-2, L-1, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11 and L-12, which is conjugated to an anticancer agent that is a PNU anthracycline.

[0286] In a specific embodiment, the present disclosure provides a linker-load compound comprising: (i) a linker (L) selected from L-1, L-2, L-1, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11, and L-12; and (ii) a load, which is a PNU anthracycline compound of formula (XXII):

[0287]

[0288] or a pharmaceutically acceptable salt thereof, wherein:

[0289] R 1’ H or C 1-6 alkyl;

[0290] R 2’ is a linker selected from linkers L-1 to L-12;

[0291] Or, R 1 and R 2The 3 to 7 membered monocyclic heterocycloalkyl group, the 5 to 11 membered bicyclic heterocycloalkyl group, the 5 to 11 membered fused heterocycloalkyl group and the 5 to 11 membered spirocyclic heterocycloalkyl group are each optionally and independently replaced by one or more R A and wherein a linker selected from linkers L-1 to L-12 is attached to a ring atom of (i), (ii), (iii) or (iv);

[0292] R A Each occurrence is independently selected from C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2, C1-C6 aminoalkyl, -(C1-C6 alkylene)-NHC(O)-(C1-C6 aminoalkyl), -O-(C 1-6 alkyl), -NHC(O)CH2OH and -(C1-C6 alkylene)-(3 to 7 membered monocyclic heterocycloalkyl), wherein the 3 to 7 membered monocyclic heterocycloalkyl group may be optionally and independently replaced by one or more R B substituted with a group; and

[0293] R B Each occurrence is independently selected from C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2, C1-C6 aminoalkyl, -O-(C 1-6 alkyl), -NHC(O)-(C1-C6 aminoalkyl), -(C1-C6 alkylene)-NHC(O)-(C1-C6 aminoalkyl), C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2、-O-(C 1-6 alkyl) and -NHC(O)CH2OH.

[0294] The payload of formula (XVI) can be attached to linkers of formula L-1, L-2, L-1, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11, and L-12 using methods described herein or methods well known in synthetic organic chemistry.

[0295] In one embodiment, the linker loading compounds of the present disclosure are in substantially purified form.

[0296] Non-limiting examples of linker-payload compounds of Formula (I) include Examples 1-36, and pharmaceutically acceptable salts thereof.

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] ADC of the present disclosure

[0308] The linker-load compounds of the present disclosure are used to conjugate with antibodies or other targeting moieties to produce antibody-drug conjugates or other targeted ligand conjugates for oncology indications. Thus, in one aspect, the present disclosure provides antibody-drug conjugates ("ADCs of the present disclosure") comprising a linker-load of the present disclosure attached to a ligand (e.g., an antibody).

[0309] Thus, one embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXIII):

[0310]

[0311] where R 2 、R 9 , X, Y and Z are as described above for the compound of formula (I); p is an integer from 1 to 8; L is a ligand, such as an antibody, or other targeting moiety; and

[0312] R 3’ Selected from:

[0313]

[0314] Where ** indicates R 3’ Attachment point to L.

[0315] In one embodiment, L is an antibody, and R3’ Attached to the sulfur atom of a cysteine ​​residue of the antibody.

[0316] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXIV):

[0317]

[0318] wherein X, Y, and Z are as described herein; A is a 4- or 6-membered monocyclic heterocycloalkylene group; p is an integer from 1 to 8; L is a ligand, such as an antibody, or other targeting moiety; and

[0319] R 3’ Selected from:

[0320]

[0321] Where ** indicates R 3’ Attachment point to L.

[0322] In one embodiment, L is an antibody and R3' is attached to the sulfur atom of a cysteine ​​residue of the antibody.

[0323] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXV):

[0324]

[0325] where R 2 , X, Y, Z and m are as described herein; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0326] Yet another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXVI):

[0327]

[0328] wherein X, Y, Z, and m are as described herein; A is a 4- or 6-membered monocyclic heterocycloalkylene group; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0329] A further embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXVII):

[0330]

[0331] where R 2 , X, Y, Z and m are as described herein; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0332] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXVIII):

[0333]

[0334] wherein X, Y, Z, and m are as described herein; A is a 4- or 6-membered monocyclic heterocycloalkylene group; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0335] Yet another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXIX):

[0336]

[0337] where R 2 , X, Y, Z and m are as described herein; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0338] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXX):

[0339]

[0340] wherein X, Y, Z, and m are as described herein; A is a 4- or 6-membered monocyclic heterocycloalkylene group; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0341] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXXI):

[0342]

[0343] where R 2 、R 9 , X, Y, and Z are as described herein; p is an integer from 1 to 8; and L is a ligand, such as an antibody, or other targeting moiety.

[0344] Another embodiment of the present disclosure is represented by an antibody-drug conjugate having structural formula (XXXII):

[0345]

[0346] where R 9 , X, Y and Z are as described herein; A is a 4- or 6-membered monocyclic heterocycloalkylene group; p is an integer from 1 to 8; and L is an A ligand, such as an antibody, or other targeting moiety.

[0347] In one embodiment, for compounds of Formula (XXIII) to (XXXII), L is an antibody and the linker is attached to the sulfur atom of the cysteine ​​group of the antibody.

[0348] In one embodiment, the antibody-drug conjugates of the present disclosure include the following compounds having structural formulas (XXXIII) to (LVII):

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357] wherein L is a ligand, and p is an integer from 0 to 8.

[0358] In one embodiment, the antibody-drug conjugates of the present disclosure are in substantially purified form.

[0359] Non-limiting examples of the disclosed antibody-drug conjugates include Examples 37-72, and pharmaceutically acceptable salts thereof, wherein L is Sacituzumab (S365C), and p is an integer from 1 to 8:

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] Other embodiments include the following:

[0371] (a) A composition comprising a mixture of ADCs of the present disclosure, wherein the DAR of the composition is a decimal from 0 to 8.

[0372] (b) A pharmaceutical composition comprising an effective amount of the ADC of the present disclosure, and a pharmaceutically acceptable carrier.

[0373] (c) The pharmaceutical composition of (a), further comprising a second therapeutic agent selected from anticancer agents.

[0374] (d) The pharmaceutical composition of (b), wherein the anticancer agent is an anti-human PD-1 antibody (or an antigen-binding fragment thereof).

[0375] (e) A pharmaceutical combination comprising: (i) an ADC of the present disclosure, and (ii) a second therapeutic agent selected from anticancer agents, wherein the ADC of the present disclosure and the second therapeutic agent are each used in an amount such that the combination is effective to inhibit cancer cell replication or treat cancer and / or reduce the likelihood or severity of cancer symptoms.

[0376] (f) The combination of (e), wherein the second therapeutic agent is an anti-human PD-1 antibody (or an antigen-binding fragment thereof).

[0377] (g) A method of inhibiting cancer cell replication in a subject in need thereof, comprising administering to the subject an effective amount of an ADC of the present disclosure.

[0378] (h) A method of treating cancer and / or reducing the likelihood or severity of symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of an ADC of the present disclosure.

[0379] (i) The method of (h), wherein the ADC of the present disclosure is administered in combination with an effective amount of at least one second therapeutic agent selected from anticancer agents.

[0380] (j) The method of (i), wherein the second therapeutic agent is an anti-human PD-1 antibody (or an antigen-binding fragment thereof).

[0381] (k) A method of inhibiting the replication of cancer cells in a subject in need thereof, comprising administering to the subject the composition of (a); the pharmaceutical composition of (b), (c) or (d); or the combination of (e) or (f).

[0382] (l) A method of treating cancer and / or reducing the likelihood or severity of symptoms of cancer in a subject in need thereof, comprising administering to the subject the composition of (a); the pharmaceutical composition of (b), (c) or (d) or the combination of (e) or (f).

[0383] Also described herein are ADCs of the present disclosure for use (i) in, (ii) as a medicament for, or (iii) in the preparation of a medicament for: (a) a drug; (b) inhibiting cancer cell replication; or (c) treating cancer and / or reducing the likelihood of cancer or the severity of cancer symptoms. In these uses, the ADCs of the present disclosure may optionally be used in combination with one or more additional therapeutic agents selected from anticancer agents.

[0384] It should also be understood that the embodiments of the compositions and methods provided above in (a) through (k) are understood to encompass all embodiments of the compounds, including such embodiments resulting from combinations of the embodiments.

[0385] ligand

[0386] In one embodiment, the linker-load compounds of the present disclosure can be conjugated to a ligand, such as an antibody, to provide an antibody-drug conjugate (ADC of the present disclosure). In these antibody-drug conjugates, the ligand is connected to the linker via a bond formed between a moiety on the ligand and a maleimide group or a sulfone-substituted pyridyl group on the linker.

[0387] The ligand can be any moiety with a free sulfur atom, including but not limited to an antibody, protein, peptide, polypeptide, or an engineered antibody modified to provide a free cysteine. One aspect of this is achieved when the ligand is an antibody, preferably a complete antibody. The role of the ligand is to target and present the 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 (such as, but not limited to, transferrin), or any other cell-binding molecule or substance, including small molecules and peptides. The ligand can be, for example, a non-antibody protein targeting agent.

[0388] When the conjugate comprises a non-immunoreactive protein, polypeptide or peptide ligand instead of an antibody, useful non-immunoreactive protein, polypeptide or peptide ligands include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factor ("TGF") such as TGF-α and TGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, somatostatin, lectins, and apoproteins from low-density lipoprotein.

[0389] Particularly preferred part is antibody, including complete antibody. In fact, in any embodiment as herein described, part can be antibody. Useful polyclonal antibody is the heterogeneous group of antibody molecules derived from the serum of immune animals. Useful monoclonal antibody is the homogeneous group of antibodies for specific antigenic determinants (for example, cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids or their fragments etc.). Monoclonal antibodies (mAbs) for antigens of interest can be prepared by using any technology known in the art, and the technology provides the production of antibody molecules by the continuous cell line in culture. In one embodiment, the part is an antibody, and is connected with a joint via a cysteine ​​group.

[0390] In addition, recombinant antibodies (such as chimeric and humanized monoclonal antibodies) comprising human and non-human parts that can be prepared using standard recombinant DNA techniques are useful antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those with variable regions and human immunoglobulin constant regions derived from murine monoclonals. (See, for example, U.S. Patent No. 4,816,567; and 4,816,397, the entire contents of which are incorporated herein by reference). Humanized antibodies are antibody molecules from non-human species, which have one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, for example, U.S. Patent No. 5,585,089, the entire contents of which are incorporated herein by reference). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, such as using the method described in International Publication No. WO 87 / 02671 and European Patent Publication No. 0184187, the entire contents of which are incorporated herein by reference.

[0391] Fully human antibodies are particularly desirable and can be produced using transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes. Antibodies include analogs and derivatives, which are either modified, i.e., by covalent attachment of any type of molecule, as long as this covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those that have been further modified, such as by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cell antibodies or other proteins, etc. Any of the many chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, analogs or derivatives can contain one or more non-natural amino acids.

[0392] In a specific embodiment, known antibodies for the treatment of cancer can be used. Antibodies having immunospecificity to cancer cell antigens can be commercially available or produced by any method well known to those skilled in the art, such as recombinant expression technology. The nucleotide sequence encoding the antibody having immunospecificity to cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by conventional cloning and sequencing.

[0393] In another specific embodiment, antibodies for treating autoimmune diseases are used according to the compositions and methods of the present disclosure. Antibodies immunospecific for the antigens of cells responsible for producing autoimmune antibodies can be obtained from any organization (e.g., university scientists or companies) or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques.

[0394] In another embodiment, it may be desirable to conjugate a component of the linker to the ligand (e.g., antibody) prior to attaching the payload component of the antibody-drug conjugate. For example, in embodiments where a thiol-containing substituent (e.g., cysteine) is used to attach the payload component, it may be desirable to conjugate a component of the linker to the ligand (e.g., antibody) prior to attaching the payload component of the antibody-drug conjugate.

[0395] Uses of the compounds disclosed herein

[0396] As an intermediate in ADC synthesis

[0397] The joint-load compound of the present disclosure is used as a component of an antibody-drug conjugate. In one embodiment, the joint-load compound of the present disclosure can be conjugated to a part (i.e., an antibody or antibody fragment) to prepare an ADC of the present disclosure. The maleimide group or the sulfone-substituted pyridyl group of the joint-load compound of the present disclosure can serve as a conjugation handle, as well as the attachment point of the part to the joint-load compound of the present disclosure. In a specific embodiment, the antibody is attached to the joint-load compound of the present disclosure via the sulfur atom of the cysteine ​​residue on the antibody.

[0398] In a specific embodiment, one or more engineered cysteine ​​groups on the antibody are reduced to provide free thiol groups, which can then undergo a conjugation reaction with a maleimide group or a sulfone-substituted pyridyl group on a linker-carrier compound of the present disclosure, thereby attaching the antibody to the linker portion of the linker-carrier compound of the present disclosure and forming an ADC of the present disclosure.

[0399] For the treatment and prevention of cancer

[0400] As noted above, additional embodiments of the present disclosure are each directed to a method for treating a disease, disorder or condition, or one or more symptoms thereof (an "indication"), comprising administering to a subject in need of such treatment a therapeutically effective amount of an ADC of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof.

[0401] One such embodiment provides a method of treating or preventing a cancer selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma) in a subject in need thereof, the method comprising administering to the subject in need of such treatment a therapeutically effective amount of an ADC of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the compound, salt, or solvate thereof. In one such embodiment, the subject is a human.

[0402] Another aspect of the present disclosure relates to a method for treating and / or preventing tumors, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the compound according to the present disclosure.

[0403] Combination therapy

[0404] Combinations with additional therapeutic agents are also contemplated in the present methods. For example, the ADCs disclosed herein are used in combination with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists for the treatment of certain malignancies. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors γ and δ. PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice (Arch. Ophthamol. 2001; 119: 709-717). Examples of PPAR-γ agonists and PPAR-γ / α agonists include, but are not limited to, thiazolidinediones (e.g., DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropanoic acid (disclosed in USSN 09 / 782,856) and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in USSN 60 / 235,708 and 60 / 244,697), or a pharmaceutically acceptable salt thereof.

[0405] Another embodiment of the present disclosure is the use of the ADC of the present disclosure in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies for treating cancer, see Hall et al. (Am. J. Hum. Genet. 61: 785-789, 1997) and Kufe et al. (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressor gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see, e.g., U.S. Pat. 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).

[0406] ADC of the present disclosure can also be used in combination with an inhibitor 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 pharmaceutically acceptable salts thereof.

[0407] The ADCs of the present disclosure may also be administered together with immune-enhancing drugs (eg, levamisole, isoprinosine, and Zadaxin or pharmaceutically acceptable salts thereof).

[0408] The ADCs of the present disclosure can also be used in combination with P450 inhibitors for the treatment or prevention of cancer. P450 inhibitors include: xenobiotics, quinidine, tyramine, ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem Terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir or pharmaceutically acceptable salts thereof.

[0409] The ADCs of the present disclosure can also be used in combination with Pgp and / or BCRP inhibitors for the treatment or prevention of cancer. Pgp and / or BCRP inhibitors include: cyclosporin A, PSC833, GF120918, cremophor EL, fumitremorgin C, Ko132, Ko134, Iressa, imatinib mesylate, EKI-785, C11033, novobiocin, diethylstilbestrol, tamoxifen, reserpine, VX-710, tryprostatin A, and sirolimus. A), flavonoids, 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.

[0410] The ADCs of the present disclosure can also be used in combination with bisphosphonates for the treatment or prevention of cancer, including bone cancer, including but not limited to etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate, and tiludronate, including any and all pharmaceutically acceptable salts, derivatives, hydrates, and mixtures thereof.

[0411] The ADCs of the present disclosure can also be used in combination with aromatase inhibitors for the treatment or prevention of breast cancer. Examples of aromatase inhibitors include, but are not limited to, anastrozole, letrozole, and exemestane, or pharmaceutically acceptable salts thereof.

[0412] The ADCs of the present disclosure can also be used in combination with siRNA therapeutics to treat or prevent cancer.

[0413] 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、WO2004 / 039800、WO 2004 / 039370、WO 2005 / 030731、WO 2005 / 014553、USSN 10 / 957,251、WO2004 / 089911、WO 02 / 081435、WO 02 / 081433、WO 03 / 018543、WO 2004 / 031137、WO The compounds of WO 2004 / 031139, WO 2004 / 031138, WO 2004 / 101538, WO 2004 / 101539 and WO 02 / 47671 (including LY-450139), or pharmaceutically acceptable salts thereof.

[0414] In one embodiment, specific anticancer agents that can be used in the combination therapy of the present invention include, but are not limited to: pembrolizumab Abarelix (Plenaxis ); Aldesleukin Aldesleukin Alemtuzumab Alitretinoin Allopurinol Hexamethylmelamine Amifostine Anastrozole Arsenic trioxide Asparaginase Azacitidine Bevacizumab Bexarotene capsules Bexarotene gel Bleomycin Bortezomib Busulfan intravenous Busulfan oral Captestosterone Capecitabine Carboplatin Carmustine Carmustine Carmustine and Polifeprosan 20 implant (Gliadel ); celecoxib Cetuximab Chlorambucil Cisplatin Cladribine Clofarabine Cyclophosphamide Cytoxan ); Cytoxan ); cytarabine Cytarabine liposomes Dacarbazine Dactinomycin, actinomycin Darbepoetin alfa Liposomal daunorubicin Daunorubicin, daunorubicin Daunorubicin, daunorubicin Denileukin Youlezoxin Docetaxel Doxorubicin (Adriamycin ); doxorubicin Doxorubicin (Adriamycin PFS ); liposomal doxorubicin Drostanolone Propionate Masterone Elliott's B solution ); Epirubicin Epoetin alfa Erlotinib Estramustine Etoposide phosphate Etoposide, VP-16 Exemestane Filgrastim Floxuridine (intra-arterial) Fludarabine Fluorouracil, 5-FU Fulvestrant Gefitinib Gemcitabine Gemtuzumab ozogamicin Goserelin acetate (Zoladex ); Goserelin acetate Histrelin acetate ), hydroxyurea Ibritumomab Tiuxetan Idabit Ifosfamide Imatinib mesylate Interferon α2a (Roferon ); Interferon α-2b (Intron irinotecan Lenalidomide Letrozole folinic acid Leuprorelin acetate Levamisole Lomustine, CCNU Meclorethamine, nitrogen mustard Megestrol acetate Melphalan, L-PAM Mercaptopurine, 6-MP Mesna Mesnex ); methotrexate Methoxsalen Mitomycin C Mitotane Mitoxantrone Nandrolone phenylpropionate Nalarabine Novozyme Oprelukin Oxaliplatin Paclitaxel Paclitaxel Paclitaxel protein-bound particles Palivmin Pamidronate Adagen (Pegademase Bovine) ); Pegaspargase Pegfilgrastim Pemetrexed disodium Pentostatin Pipobrom Plicamycin, mithramycin Porfimer sodium Procarbazine Quinacrine Rasburicase Rituximab Ridaforolimus; Sargramostim Sargramostim Sorafenib Streptozotocin Sunitinib maleate talc Tamoxifen Temozolomide Teniposide, VM-26 Testolactone Thioguanine, 6-TG Thiotepa Topotecan Toremifene Tositumomab Tositumomab / I-131 Tositumomab Trastuzumab Retinoic acid, ATRA Uracil Mustard ); valrubicin Vinblastine vincristine Vinorelbine Olaparib Vorinostat and zoledronate or a pharmaceutically acceptable salt thereof.

[0415] Thus, the scope of the present disclosure encompasses the use of the ADCs of the present disclosure in combination with a second compound selected from the group consisting of estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic / cytostatic agents, antiproliferative 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 intrinsic multidrug resistance, antiemetics, agents for treating anemia, agents for treating neutropenia, immunopotentiating drugs, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any of the therapeutic agents listed above.

[0416] Another example of the present disclosure is a method of treating cancer comprising administering a therapeutically effective amount of an ADC of the present disclosure in combination with paclitaxel or trastuzumab.

[0417] The therapeutic combinations disclosed herein can be used in combination with one or more other active agents, including but not limited to other anticancer agents for preventing, treating, controlling, improving or reducing the risk of a specific disease or condition (e.g., cell proliferative disorders). In one embodiment, the ADC of the present disclosure is used in combination with one or more other anticancer agents to prevent, treat, control, improve or reduce the risk of a specific disease or condition for which the ADC of the present disclosure is useful. Such other active agents can be administered before, simultaneously with, or sequentially with the ADC of the present disclosure by their commonly used routes and amounts.

[0418] The present disclosure also includes pharmaceutical compositions for treating or preventing cancer comprising a therapeutically effective amount of an ADC of the present disclosure and a second compound selected from the group consisting of estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic / cytostatic agents, antiproliferative 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 cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any of the therapeutic agents listed above.

[0419] The present disclosure also 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.

[0420] In a specific embodiment, the PD-1 antagonist is an anti-PD-1 antibody or an antigen-binding fragment thereof. In an alternative embodiment, the PD-1 antagonist is an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody independently selected from pembrolizumab, nivolumab, cemiplimab, sintilimab, tislelizumab, atezolizumab (MPDL3280A), camrelizumab and toripalimab. In other embodiments, the PD-L1 antagonist is an anti-PD-L1 antibody independently selected from atezolizumab, durvalumab and avelumab.

[0421] In one embodiment, the PD-1 antagonist is pembrolizumab. In certain subembodiments, the method comprises administering 200 mg of pembrolizumab to the patient approximately every three weeks. In other subembodiments, the method comprises administering 400 mg of pembrolizumab to the patient approximately every six weeks.

[0422] In a further subembodiment, the method comprises administering 2 mg / kg of pembrolizumab to the patient approximately every three weeks.In a specific subembodiment, the patient is a pediatric patient.

[0423] In some embodiments, the PD-1 antagonist is nivolumab. In certain subembodiments, the method comprises administering 240 mg of nivolumab to the patient approximately every two weeks. In other subembodiments, the method comprises administering 480 mg of nivolumab to the patient approximately every four weeks.

[0424] In some embodiments, the PD-1 antagonist is cemipril imab. In a specific embodiment, the method comprises administering 350 mg of cemipril imab to the patient approximately every three weeks.

[0425] In some embodiments, the PD-1 antagonist is atezolizumab. In a specific subembodiment, the method comprises administering 1200 mg of atezolizumab to the patient approximately every three weeks.

[0426] In some embodiments, the PD-1 antagonist is durvalumab. In certain subembodiments, the method comprises administering 10 mg / kg of durvalumab to the patient approximately every two weeks.

[0427] In some embodiments, the PD-1 antagonist is avelumab. In a specific subembodiment, the method comprises administering 800 mg of avelumab to the patient approximately every two weeks.

[0428] When the ADC of the present invention is administered in combination with an anti-human PD-1 antibody (or an antigen-binding fragment thereof), the anti-human PD-1 antibody (or an antigen-binding fragment thereof) can be administered simultaneously with the ADC of the present invention, or before or after the ADC of the present invention. Any of the anti-human PD-1 antibody (or its antigen-binding fragment) and / or the ADC of the present invention or a pharmaceutically acceptable salt thereof can be administered separately by the same or different routes of administration, or administered together in the same pharmaceutical composition as the other agents. The weight ratio of the anti-human PD-1 antibody (or its antigen-binding fragment) to the ADC of the present invention can vary and will depend on the therapeutically effective dose of each agent. Typically, a therapeutically effective dose of each will be used. A combination comprising at least one anti-human PD-1 antibody (or its antigen-binding fragment), an ADC of the present invention, and optional other active agents will typically include a therapeutically effective dose of each active agent. In such a combination, the anti-human PD-1 antibody (or its antigen-binding fragment), the ADC of the present invention, and the other active agents can be administered separately or in combination. In addition, the administration of one element can be before, simultaneously with, or after the administration of the other agent.

[0429] In one embodiment, the present disclosure provides an anti-human PD-1 antibody (or antigen-binding fragment thereof) and / or a compound of Formula IV and at least one other active agent as a combined preparation for simultaneous, separate or sequential use in treating cancer.

[0430] The present disclosure also provides the use of the ADC of the present disclosure for treating cancer, wherein the patient has previously (e.g., within 24 hours) been treated with an anti-human PD-1 antibody (or an antigen-binding fragment thereof). The present disclosure also provides the use of an anti-human PD-1 antibody (or an antigen-binding fragment thereof) for treating a cell proliferative disorder, wherein the patient has previously (e.g., within 24 hours) been treated with an antibody-linker-load compound (antibody-drug conjugate) ADC of the present disclosure.

[0431] The present disclosure also relates to a method of treating cancer, comprising administering to a subject in need thereof a combination therapy comprising (a) an ADC 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.

[0432] In addition, the present disclosure relates to a method for treating cancer, comprising administering to a subject in need thereof a combination therapy comprising: (a) an ADC of the present disclosure, and (b) an anti-human PD-1 antibody (or an antigen-binding fragment thereof). In a specific embodiment, the cancer occurs as one or more solid tumors or lymphomas. In a further embodiment, the cancer is selected from the group consisting of advanced or metastatic solid tumors and lymphomas. In a further embodiment, the cancer is selected from the group consisting of malignant melanoma, head and neck squamous cell carcinoma, MSI-H cancer, MMR-deficient cancer, non-small cell lung cancer, urothelial carcinoma, gastric or gastroesophageal junction adenocarcinoma, breast adenocarcinoma, 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, extranodal NK / T-cell lymphoma of nasal type, enteropathy-associated T-cell lymphoma, gamma / delta hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, and Hodgkin lymphoma. In certain embodiments, the cell proliferative disorder is a cancer that has metastasized, such as liver metastases from colorectal cancer. In other embodiments, the cell proliferative disorder is a cancer classified as a stage III cancer or a stage IV cancer. In these embodiments, the cancer is not surgically resectable.

[0433] In embodiments of the methods disclosed herein, the anti-human PD-1 antibody (or antigen-binding fragment thereof) is administered by intravenous infusion or subcutaneous injection.

[0434] In one embodiment, the present disclosure provides a composition comprising the ADC of the present disclosure, a pharmaceutically acceptable carrier, and an anti-human PD-1 antibody (or an antigen-binding fragment thereof).

[0435] In another embodiment, the present disclosure provides a composition comprising a compound of Formula IV, a pharmaceutically acceptable carrier, and pembrolizumab.

[0436] In one embodiment, the present disclosure provides a composition comprising a compound of Formula IV, a pharmaceutically acceptable carrier, and two additional therapeutic agents, one of which is an anti-human PD-1 antibody (or antigen-binding fragment thereof), and the other of which is independently selected from anticancer agents.

[0437] The ADCs of the present disclosure can be used in combination with antiemetics to treat nausea or vomiting, including acute, delayed, late, and anticipated vomiting, which can be caused by the use of the ADCs of the present disclosure alone or in combination with radiation therapy. To prevent or treat vomiting, the ADCs of the present disclosure can be used in combination with other antiemetics, particularly neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatesetron, GABAB receptor agonists, such as baclofen, corticosteroids, such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten, or others (e.g., in U.S. Pat. Nos. 2,789,118, 2,990,401, 3,04 8,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326, and 3,749,712), antidopaminergic drugs, such as phenothiazines (e.g., prochlorperazine, fluphenazine, thioridazine, and mesoridazine), metoclopramide, aprepitant, fosaprepitant, or dronabinol. In another example, a combination therapy using an antiemetic selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist, and a corticosteroid is disclosed for treating or preventing vomiting that may result from administration of the compounds of the present disclosure.

[0438] The ADCs of the present disclosure can also be administered with an agent useful for treating anemia. Such anemia therapeutic agents are, for example, continuous erythropoiesis receptor activators (e.g., epoetin alfa).

[0439] The ADCs of the present disclosure can also be administered with agents for treating neutropenia. Such neutropenia therapeutic agents are, for example, hematopoietic growth factors that regulate the production and function of neutrophils, such as human granulocyte colony-stimulating factor (G-CSF). Examples of G-CSF include filgrastim.

[0440] When co-administered with other treatment modalities (including but not limited to radiotherapy, surgery and gene therapy), the ADCs of the present disclosure may be useful. Therefore, in one embodiment, unless otherwise indicated, the methods for treating cancer described herein may optionally include administering an effective amount of radiotherapy. For radiotherapy, γ-radiation is preferred.

[0441] The methods of treating cancer described herein can optionally comprise administering an effective amount of radiation (ie, the methods of treating cancer described herein optionally comprise administering radiation therapy).

[0442] The methods of treating cancer described herein include methods of treating cancer comprising administering a therapeutically effective amount of an ADC of the present disclosure in combination with radiation therapy and / or in combination with a second compound selected from the group consisting of estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic / cytostatic agents, antiproliferative 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 intrinsic multidrug resistance, antiemetics, agents for treating anemia, agents for treating neutropenia, immune enhancing drugs, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase and / or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), agents that interfere with cell cycle checkpoints, and any additional therapeutic agents listed herein.

[0443] Other embodiments of the present disclosure include pharmaceutical compositions, combinations, uses and methods described above, wherein it should be understood that each embodiment can be combined with one or more other embodiments, as long as such combination is consistent with the description of the embodiment. It should also be understood that the embodiments provided above are understood to include all embodiments, including such embodiments resulting from combinations of the embodiments.

[0444] Reagent test kit

[0445] In one aspect, a kit is provided, comprising a therapeutically effective amount of an ADC of the present disclosure or a pharmaceutically acceptable salt, solvate or ester of the compound and a pharmaceutically acceptable carrier, vehicle or diluent.

[0446] On the other hand, a kit is provided, which includes an amount of an ADC of the present disclosure and an amount of at least one additional therapeutic agent listed above, wherein the amount of two or more active ingredients produces the 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.

[0447] Composition and administration

[0448] One aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of an ADC of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0449] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an ADC of the present disclosure or a pharmaceutically acceptable salt thereof, having a DAR that is a decimal from 0 to 8. In one aspect, the composition is a pharmaceutical composition and comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0450] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of an ADC of the present disclosure as 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.

[0451] Another aspect of the present disclosure relates to an ADC of the present disclosure as described herein, or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament or medicament component.

[0452] Another aspect of the present disclosure relates to an ADC as described herein, or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition for use in the preparation of a medicament for treating or preventing tumors.

[0453] In another embodiment, compounds of the present disclosure include those identified herein as examples in the table below, and pharmaceutically acceptable salts thereof.

[0454] In another embodiment, the present disclosure relates to a method of preparing a medicament for a subject, comprising combining an ADC of the present disclosure, or a pharmaceutically acceptable salt thereof, with a pharmaceutical carrier or diluent.

[0455] The ADC of the present disclosure can be administered orally, parenterally (e.g., intramuscularly, intraperitoneally, intravenously, ICV, intracisternal injection or infusion, subcutaneous injection or implantation), by inhalation spray, nasal, vaginal, rectal, sublingual, buccal or topical administration, and can be formulated, alone or together, into suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles suitable for each administration route. In addition to treating warm-blooded animals, the compounds of the present disclosure are effectively used in humans.

[0456] The pharmaceutical compositions for administering the disclosed compounds can be conveniently presented in dosage unit form and can be prepared by any method well known in the pharmaceutical field. All methods include the step of associating the active ingredient with a carrier constituting one or more auxiliary ingredients. Typically, the pharmaceutical composition is prepared by uniformly and tightly associating the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, the active compound is included in an amount sufficient to produce the desired effect on the course of the disease or condition.

[0457] Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, such as tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavorings, colorants, and preservatives to provide pharmaceutically aesthetically pleasing and palatable preparations. Tablets contain the active ingredient mixed with nontoxic, pharmaceutically acceptable excipients suitable for tablet preparation. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a longer-lasting effect. For example, a time-delaying material such as glyceryl monostearate or glyceryl distearate may be used. They may also be coated by the techniques described in U.S. Patents 4,256,108; 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlled release. Oral tablets may also be formulated for immediate release, such as fast melt tablets or wafers, rapid dissolve tablets or rapid dissolve films.

[0458] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0459] Aqueous suspensions contain the active material mixed with excipients suitable for preparing aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersants or wetting agents can be naturally occurring phospholipids such as lecithin, or condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives (e.g., ethyl parahydroxybenzoate or n-propyl parahydroxybenzoate), one or more colorants, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0460] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or in a mineral oil (e.g., liquid paraffin). Oily suspensions can contain thickeners such as beeswax, hard paraffin, or acetyl alcohol. Sweeteners (e.g., those mentioned above) and flavorings can be added to provide a palatable oral preparation. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

[0461] Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide a mixture of the active ingredient with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavorings, and coloring agents, may also be present.

[0462] Pharmaceutical compositions of the present disclosure can also be in the form of oil-in-water emulsions. The oil phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture thereof. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or gum tragacanth, naturally occurring phospholipids, such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of the partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweeteners and flavorings.

[0463] Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0464] The pharmaceutical composition can be in the form of a sterile injectable aqueous or oily suspension. The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents already mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenteral acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections.

[0465] The ADCs of the present disclosure can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0466] For topical use, creams, ointments, jellies, solutions or suspensions, etc. containing the ADCs of the present disclosure are used. Similarly, transdermal patches can also be used for topical administration.

[0467] The pharmaceutical compositions and methods of the present disclosure may also comprise other therapeutically active compounds as described herein, which are typically used to treat the aforementioned pathological conditions.

[0468] In the treatment, prevention, control, improvement or reduction of the risk of the conditions disclosed herein, the appropriate dosage level of the disclosed compounds is generally about 0.01 to 500 mg / kg patient body weight / day, which can be administered in single or multiple doses. Suitable dosage levels can be about 0.01 to 250 mg / kg / day, about 0.05 to 100 mg / kg / day, or about 0.1 to 50 mg / kg / day. Within this range, the dosage can be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg / kg / day. For oral administration, the composition can be provided in the form of tablets containing 1.0 to 1000 mg of 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 mg of active ingredient for symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times a day, or can be administered once or twice a day.

[0469] However, it should be understood that the specific dosage level and frequency of administration for any particular patient may vary and will depend on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, severity of the particular condition, and the host being treated.

[0470] Methods for preparing compounds of the present disclosure are illustrated in the following Schemes and Examples.Starting materials are prepared according to procedures known in the art or as illustrated herein.

[0471] Preparation Example

[0472] 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. Variants that are known per se to those of ordinary skill in the art but not mentioned in detail can also be utilized. By reviewing the following schemes and descriptions, those skilled in the art can readily understand the general procedures for preparing the compounds claimed in the present disclosure.

[0473] General experimental information:

[0474] Unless otherwise noted, all reactions were stirred magnetically. Unless otherwise noted, all reagents and solvents were purchased from commercial sources and used as is. When applicable, reaction progress and synthetic intermediate analysis were assessed by LCMS (UV detection, APCI or other mass detection using ESI) using a MeCN / water gradient with TFA, formic acid or NH4HCO3 modifier. Silica gel and reversed phase flash column chromatography were performed using commercially available pre-packed columns. Reverse phase preparative HPLC purification was performed using a MeCN / water gradient with TFA, formic acid or NH4OH modifier on a preparative HPLC instrument with UV and MS detection. The reaction mixture was collected at room temperature. 1 H NMR spectra, and chemical shifts are reported in ppm relative to the residual protein-solvent signal, and multiplicities, coupling constants (if applicable), and signal integrations are listed in parentheses. Unless otherwise stated, all EC 50 Data refer to the cytotoxicity assay described in the Bioassays section.

[0475] Synthetic Schemes, Intermediates and Examples

[0476] The compounds of the present disclosure can be prepared by methods known in the art of organic synthesis, which are illustrated in part by the following general synthetic schemes and specific preparative examples. Starting materials are commercially available or can be prepared by known methods.

[0477] Preparation of intermediate compounds

[0478] Preparation of intermediate compound i

[0479]

[0480] Step A - Synthesis of Compound ii

[0481] To a stirred mixture of (9H-fluoren-9-yl)methylhydrazinecarboxylate (i, 3.00 g, 11.8 mmol) in DMF (50.0 mL) was added bis(2,5-dioxopyrrolidin-1-yl)carbonate (3.02 g, 11.8 mmol) and the resulting reaction was stirred at 25° C. for 1 hour. The reaction was monitored using TLC (DCM:CH 3 OD = 10:1). The reaction mixture containing 1-((9H-fluoren-9-yl)methyl)2-(2,5-dioxopyrrolidin-1-yl)hydrazine-1,2-dicarboxylate (ii, 4.66 g, 11.8 mmol) in DMF was used without further purification.

[0482] Step B - Synthesis of Compound iii

[0483] To a stirred mixture of ii (4.66 g, 11.8 mmol) in DMF (50.0 mL) was added tert-butyl (2-aminoethyl) carbamate (1.89 g, 11.8 mmol), and the resulting reaction was stirred at 25 ° C for 16 hours. Water (50.0 mL) was added, and the mixture was extracted with ethyl acetate (2×40.0 mL). The combined organic extracts were washed with brine (50.0 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified using flash silica gel chromatography (5% MeOH / DCM) to give (9H-fluoren-9-yl)methyl 11,11-dimethyl-4,9-dioxo-10-oxa-2,3,5,8-tetraazadodecanoate (iii, 3.7 g, 8.40 mmol) as a solid. LCMS: MS (ESI) m / z: 441.3 [M+H] + .

[0484] Step C - Synthesis of Compound iv

[0485] To the stirred mixture of iii (3.0g, 6.8mmol) in DMF (10mL) is added piperidine (1.0mL, 10mmol), and the resulting reaction is stirred at 25 DEG C for 30 minutes. Use TLC (DCM:CH OD=10: 1) to monitor the reaction mixture. The reaction mixture is concentrated in a vacuum, and the residue obtained is purified using silica gel column flash chromatography (eluted with 0-10% MeOH / DCM) to obtain tert-butyl (2- (hydrazine formamido) ethyl) carbamate iv (1.10g, 5.04mmol) as an oil. 1 H NMR (500 MHz, CD3OD) δ = 3.28-3.19 (m, 2H), 3.18-3.11 (m, 2H), 1.50-1.36 (m, 9H) No exchangeable protons were reported.

[0486] Preparation of intermediate compound vi

[0487]

[0488] Step A - Synthesis of Compound VI

[0489] At 0 ℃, to the solution of tert-butyl (4-hydroxybenzyl) carbamate (v, 300mg, 1.344mmol) in DCM (10mL), add DIEA (0.235mL, 1.34mmol) and trichloromethyl chloroformate (0.380g, 1.92mmol).The reaction obtained was stirred 30 minutes at 0 ℃ and stirred 2 hours at 20 ℃.Reactant mixture is concentrated in a vacuum, obtain 4-(((tert-butoxycarbonyl) amino) methyl) phenyl chloroformate vi (300mg, 1.050mmol), use it without the need for further purification.

[0490] Preparation of intermediate compound x

[0491]

[0492] Step A - Synthesis of Compound viii

[0493] To a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-proline (vii, 3.4 g, 10 mmol) and HATU (3.83 g, 10.1 mmol) in DMF (30 mL) at 0° C. was added a solution of tert-butyl(2-aminoethyl)carbamate (1.61 g, 10.1 mmol) and DIEA (5.28 mL, 30.2 mmol). The mixture was stirred at 0° C. for 4 hours and then at 25° C. for an additional 17 hours. The crude 9H-fluoren-9-yl)methyl (S)-2-((2-((tert-butoxycarbonyl)amino)ethyl)carbamoyl)pyrrolidine-1-carboxylate (viii, 5 g, 5.73 mmol) was used directly without further purification.

[0494] Step B - Synthesis of Compound ix

[0495] To a solution of viii (4.0 g, 8.3 mmol) in DMF (10 mL) was added piperidine (1.0 mL, 8.3 mmol), and the resulting mixture was stirred for 1 hour at 20° C. The mixture was concentrated in vacuo and diluted with DCM (10 mL), filtered and lyophilized to give crude tert-butyl (S)-(2-(pyrrolidine-2-carboxamido)ethyl)carbamate (ix, 2.4 g, 6.5 mmol) as an oil which was used without further purification.

[0496] Step C - Synthesis of Compound x

[0497] To a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (2.70 g, 7.62 mmol) and HATU (4.35 g, 11.4 mmol) in DMF (10 mL) was added ix (1.96 g, 7.62 mmol) followed by DIEA (3.99 mL, 22.9 mmol). The resulting reaction was stirred at 0 ° C for 4 hours and then at room temperature for 17 hours, then concentrated in vacuo to give compound x, which was used without further purification. LCMS (ESI) m / z: 616.3 [M + Na + ].

[0498] Preparation of intermediate compound xiv

[0499]

[0500] Step A - Synthesis of Compound xiii

[0501] At room temperature, under an argon atmosphere, triethylamine (736 mg, 7.27 mmol), sodium triacetoxyborohydride (771 mg, 3.64 mmol) and tert-butyl 3-formyl azetidine -1- carboxylate (xii, 539 mg, 2.91 mmol) were added to a stirred mixture of azetidine -3- base carbamic acid benzyl ester (xi, 500 mg, 2.42 mmol) in DCM (20 mL) under an argon atmosphere. The resulting mixture was stirred for 16 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using flash silica gel column chromatography (0-100% EtOAc / petroleum ether) to give tert-butyl 3- ((3- (((benzyloxy) carbonyl) amino) azetidine -1- base) methyl) azetidine -1- carboxylate xiii as an oil. MS: m / z=376.25 [M+H] + .

[0502] Step B - Synthesis of Compound xiv

[0503] At room temperature, under a hydrogen atmosphere, 10% Pd / C (139 mg, 0.131 mmol) was added to a stirred mixture of xiii (490 mg, 1.31 mmol) in MeOH (10 mL). The resulting mixture was stirred for 1 hour, and the reaction mixture was then filtered and washed with MeOH (20 mL). The filtrate was concentrated in vacuo to give tert-butyl 3-((3-aminoazetidin-1-yl)methyl)azetidine-1-carboxylate xiv as a solid. MS: m / z=242.25[M+H] + . 1H NMR (400MHz, DMSO-d6) δ3.83 (s, 2H) 3.46-3.42 (m, 4H), 2.59 -2.56 (m, 2H), 1.84 (s, 4H), 1.36 (s, 9H).

[0504] Preparation of intermediate compound xviii

[0505]

[0506] Step A - Synthesis of Compound xvi

[0507] To a solution of (S)-2-amino-2,3-dimethylbutanoic acid (xv, 400 mg, 3.05 mmol) in THF (10 mL) was added a solution of potassium carbonate (843 mg, 6.10 mmol) in water (5 mL) followed by the addition of (9H-fluoren-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (1.54 g, 4.57 mmol). The resulting reaction was stirred at 20 ° C. for 17 hours, then the reaction mixture was concentrated in vacuo and acidified to pH = 2 to form a suspension, which was then filtered to give compound (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,3-dimethylbutanoic acid xvi as a solid, which was used without further purification. LCMS (ESI) m / z: 354.3 [M + H] + .

[0508] Step B - Synthesis of Compound xvii

[0509] To a solution of xvi (800 mg, 1.36 mmol) in DMF (5 mL) was added HATU (775 mg, 2.04 mmol) followed by tert-butyl (2- (methylamino) ethyl) carbamate (237 mg, 1.36 mmol) and DIEA (0.712 mL, 4.07 mmol). The reaction mixture was stirred at 20 ° C for 1 hour and then concentrated in vacuo. The resulting residue was purified using silica gel column chromatography (eluted with 0 to 50% EtOAc / petroleum ether) to give (9H-fluorenes-9-yl)methyl (S)-(1-((2-((tert-butoxycarbonyl)amino)ethyl)(methyl)amino)-2,3-dimethyl-1-oxobutane-2-yl)carbamate xvii as a solid. LCMS (ESI) m / z: 532.3 [M + Na] + .

[0510] Step C - Synthesis of Compound xviii

[0511] To a solution of xvii (400 mg, 0.785 mmol) in DMF (5 mL) was added piperidine (0.50 mL, 0.79 mmol), and the reaction mixture was stirred at 20 ° C. for 1 hour. The crude mixture was concentrated in vacuo and purified using HPLC (Boston Green ODS 150 × 30 mm × 5 μm, eluted with 15% to 35% MeCN / water (with 0.5% TFA as a modifier)) to give compound tert-butyl (S)-(2-(2-amino-N,2,3-trimethylbutyrylamino)ethyl)carbamate xviii as a solid. LCMS (ESI) m / z: 288.2 [M + H] + .

[0512] Preparation of intermediate compound xxiv

[0513]

[0514] Step A - Synthesis of Compound xx

[0515] To a stirred mixture of 3-aminopyrrolidin-2-one (xix, 50.0 mg, 0.499 mmol) and Boc2O (0.174 mL, 0.749 mmol) in MeOH (2 mL) was added TEA (0.209 mL, 1.50 mmol) and the resulting reaction was stirred at 70 ° C for 16 hours. The reaction was monitored using TLC (DCM: CH3OD = 10: 1). The solvent was removed in vacuo and the resulting residue was purified using flash silica gel chromatography (0-100% EtOAc / petroleum ether) to give tert-butyl (2-oxopyrrolidin-3-yl) carbamate xx. MS (ESI) m / z: 201.3 [M + H] + .

[0516] Step B - Synthesis of Compound xxi

[0517] At 0 ° C, to a stirred suspension of NaH (24 mg, 0.60 mmol) in THF (1 mL) was added xx (100 mg, 0.499 mmol) and the resulting reaction was stirred at 25 ° C for 30 minutes under N2 atmosphere. A solution of 2-bromoacetonitrile (71.9 mg, 0.599 mmol) and TBAI (36.9 mg, 0.100 mmol) in THF (1 mL) was added and the resulting reaction was stirred at 25 ° C for 3 hours. TLC (EtOAc) was used to monitor the reaction. Water (30 mL) was added and the resulting solution was extracted with ethyl acetate (100 mL×2). The combined organic extracts were washed with brine (100 mL×2), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified using flash silica gel chromatography (0-100% EtOAc / petroleum) to obtain tert-butyl (1-(cyanomethyl)-2-oxopyrrolidin-3-yl)carbamate xxi.

[0518] Step C - Synthesis of Compound xxii

[0519] To a stirred mixture of xxi (1.3 g, 5.4 mmol) in EtOH (15 mL) was added platinum (IV) oxide (0.247 g, 1.09 mmol) and the resulting reaction was stirred at 25 ° C. under an atmosphere of H2 for 16 hours. The reaction was monitored using TLC (EtOAc). The mixture was filtered and the filter cake was washed with ethanol (10 mL). The filtrate was concentrated in vacuo and the resulting residue was purified using preparative HPLC (Welch Xtimate C18 150×25 mm×5 um, 8 to 100% ACN:water (with TFA as a modifier)) and the solution was lyophilized to give tert-butyl (1-(2-aminoethyl)-2-oxopyrrolidin-3-yl)carbamate xxii. MS (ESI) m / z: 244.2 [M+H] + .

[0520] Step D - Synthesis of Compound xxiii

[0521] To a solution of xxii (400 mg, 1.64 mmol) in DCM (5 mL) was added TFA (1.0 mL, 13 mmol), and the resulting reaction was stirred at 0°C for 1 hour. The reaction mixture was filtered, concentrated in vacuo, and the resulting residue was lyophilized to afford 3-amino-1-(2-aminoethyl)pyrrolidin-2-one xxiii. LCMS (ESI) m / z: 144.1 [M+H] + .

[0522] Step E - Synthesis of Compound xxiv

[0523] To a solution of xxiii (200 mg, 1.40 mmol) in DCM (10 mL) was added TEA (0.389 mL, 2.79 mmol) at 0°C, followed by a solution of Boc2O (0.259 mL, 1.12 mmol) in DCM (10 mL), and the mixture was stirred at 0°C for 4 hours. The crude mixture was concentrated in vacuo to give the crude compound tert-butyl (2-(3-amino-2-oxopyrrolidin-1-yl)ethyl)carbamate xxiv, which was used in the next step without further purification. LCMS (ESI) m / z: 244.4 [M+H] + .

[0524] Preparation of intermediate compound xxvii

[0525]

[0526] Step A - Synthesis of Compound xxvi

[0527] To a solution of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanic acid (xxv, 500 mg, 1.01 mmol) in DMF (5 mL) was added tert-butyl(4-aminobenzyl)carbamate (224 mg, 1.01 mmol), TCFH (339 mg, 1.21 mmol) and 1-methylimidazole (248 mg, 3.02 mmol). The resulting reaction was stirred at 20° C. for 18 hours, and then the reaction mixture was concentrated in vacuo. The resulting residue was purified using preparative HPLC (Boston Green ODS 150×30 mm×5 μm, eluted with 48% to 78% acetonitrile / water (with 0.1% TFA as a modifier)) to give (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (xxvi). LCMS (ESI) m / z: 701.4 [M+H] + .

[0528] Step B - Synthesis of Compound xxvii

[0529] To a solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (xxvi, 260 mg, 0.371 mmol) in DMF (4 mL) was added piperidine (0.4 mL, 0.4 mmol). The resulting reaction was stirred at 20 °C for 1 hour, then the reaction mixture was concentrated in vacuo to give compound xxvii, which was used without purification. LCMS (ESI) m / z: 479.3 [M+H + ].

[0530] Preparation of intermediate compound xxxi

[0531]

[0532] Step A - Synthesis of Compound xxix

[0533] To a solution of ((benzyloxy)carbonyl)-L-valyl-L-alanine (xxviii, 1.0 g, 3.1 mmol) in DMF (5 mL) was added HATU (1.30 g, 3.41 mmol). The mixture was stirred at room temperature for 5 minutes, and tert-butyl (4-aminobenzyl)carbamate (0.690 g, 3.10 mmol) was added. After stirring at room temperature for an additional 5 minutes, DIEA (1.63 mL, 9.31 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified using preparative HPLC (Boston UNI C18 150×40 mm×5 μm, eluting with 42% to 72% acetonitrile / water (with 0.1% TFA as a modifier)) to give benzyl ((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (xxix). LCMS (ESI) m / z: 527.3 [M+H] + .

[0534] Step B - Synthesis of Compound xxx

[0535] To a stirred mixture of xxix (550 mg, 1.044 mmol) in trifluoroethanol (15 mL) was added Pd-C (300 mg, 10% Pd), and the resulting reaction was stirred at 25 ° C. under a hydrogen atmosphere for 18 hours. The mixture was concentrated in vacuo to give tert-butyl (4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl)carbamate (xxx). LCMS (ESI) m / z: 393.4 [M+H] + .

[0536] Step C - Synthesis of Compound xxxi

[0537] To a solution of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (164 mg, 0.968 mmol) in DMF (4 mL) was added HATU (405 mg, 1.07 mmol). After stirring for 5 minutes, xxx (380 mg, 0.968 mmol) and DIEA (0.507 mL, 2.90 mmol) were added, and the resulting reaction was stirred at 20° C. for 2 hours. The resulting residue was purified using preparative HPLC (Boston Green ODS 150×30 mm×5 μm, eluted with 30% to 60% acetonitrile / water (with 0.1% TFA as a modifier)) to give tert-butyl (4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-3-methylbutyrylamino)propionamido)benzyl)carbamate (xxxi). LCMS (ESI) m / z: 544.3 [M+H] + .

[0538] Using the above methods and substituting appropriate reactants and / or reagents, the following intermediate compounds of the present disclosure were prepared:

[0539]

[0540] Preparation of intermediate compound xli

[0541]

[0542] Step A - Synthesis of Compound xxxiv

[0543] To a solution of 2-chloro-6-methylnicotinonitrile (xxxiii, 10 g, 66 mmol) in sulfuric acid (70.0 ml, 1310 mmol) was added chromic anhydride (19.7 g, 197 mmol) portionwise over 2 hours while maintaining the reaction mixture at 0° C. After stirring at room temperature for 18 hours, the mixture was poured into ice water and the resulting solid was collected by filtration to give crude 6-chloro-5-(aminocarbonyl)-2-pyridinecarboxylic acid xxxiv.

[0544] Step B - Synthesis of Compound xxxv

[0545] The mixture of xxxiv was dissolved in POCl3 (50 mL, 536 mmol), and the reaction mixture was heated to 115 ° C and stirred at this temperature for 30 minutes. The mixture was then concentrated in vacuo, and the resulting residue was diluted with ice water. The resulting solution was filtered, and the collected solid was dried to give 6-chloro-5-cyanopicolinic acid xxxv as a solid. LC / MS: MS (ESI) m / z: 183.2 [M+H] + .

[0546] Step C - Synthesis of Compound xxxvi

[0547] To a stirred mixture of xxxv (5.60 g, 30.7 mmol) in DMF (50 mL) was added sodium thiomethoxide (2.15 g, 30.7 mmol). The mixture was stirred at 20 ° C for 15 hours. The mixture was concentrated in vacuo and purified using flash silica gel column chromatography (0-50% MeOH / DCM) to give 5-cyano-6-(methylthio)picolinic acid xxxvi as a solid. LCMS (ESI) m / z: 195.2 [M+H] + .

[0548] Step D - Synthesis of Compound xxxvii

[0549] To a stirred mixture of xxxvi (200 mg, 1.03 mmol) in THF (5 mL) was added HATU (470 mg, 1.24 mmol), DIPEA (0.540 mL, 3.09 mmol) and tert-butyl 3-aminopropanoate (150 mmol, 1.03 mmol). The mixture was stirred at 20 ° C for 15 hours. The mixture was concentrated in vacuo and purified using flash silica gel column chromatography (0-100% petroleum ether / EtOAc) to give tert-butyl 3-(5-cyano-6-(methylthio)pyridine amido)propanoate xxxvii as an oil. LCMS (ESI): 266.2 [M-56+H] + .

[0550] Step E - Synthesis of Compound xxxviii

[0551] To a stirred mixture of xxxvii (200 mg, 0.678 mmol) in DCM (5 mL) was added m-CPBA (468 mg, 2.71 mmol). The mixture was stirred at 20 ° C for 18 hours. The mixture was concentrated in vacuo and purified using flash silica gel column chromatography (0-100% petroleum ether / EtOAc) to give tert-butyl 3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propanoate xxxviii as a solid. LCMS (ESI): 298.2 [M-56+H] + .

[0552] Step F - Synthesis of Compound xxxix

[0553] A solution of xxxviii (1.2 g, 3.4 mmol) in DCM (18 mL) and TFA (6 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo to give 3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propanoic acid xxxix as a solid, which was used without further purification. LCMS (ESI) m / z: 298.2 [M+H] + .

[0554] Step G - Synthesis of Compound XL

[0555] To a stirred mixture of xxxix (300 mg, 1 mmol) in DCM (6 mL) was added EDC (232 mg, 1.21 mmol) and N-hydroxysuccinamide (151 mg, 1.31 mmol). The mixture was stirred at 20 ° C for 5 hours. The solution was concentrated to give 2,5-dioxopyrrolidin-1-yl 3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propanoate xl as a solid, which was used without further purification. LCMS (ESI) m / z: 395.0 [M + H] + .

[0556] Step H - Synthesis of Compound xli

[0557] To a solution of xl (400 mg, 1 mmol) in DMF (10 mL) was added L-alanyl-L-alanine (179 mg, 1.12 mmol) and TEA (0.566 mL, 4.06 mmol). The resulting reaction was stirred at 25 ° C for 2 hours, and the reaction mixture was then concentrated in vacuo to give (methyl (3- (5-cyano -6- (methylsulfonyl) pyridine amido) propionyl) -L-alanyl-L-alaninate xli as a solid, which was used without purification. LCMS (ESI) m / z: 440.0 [M + H]+ .

[0558] Preparation of intermediate compound xlviii

[0559]

[0560] Step A - Synthesis of Compound xliii

[0561] Under N2 atmosphere, a solution of 5-bromo-6-hydroxypyridine-3-carboxylic acid (xlii, 600g, 2.59mol) and cuprous cyanide (231g, 2.59mol) in DMF (3.6L) was stirred at 125 ° C for 12 hours. The reaction mixture was cooled to room temperature and quenched with water (5.0L). The resulting mixture was filtered and the filter cake was dried under reduced pressure to give 5-cyano-6-hydroxypyridine-3-carboxylic acid (xliii). LCMS: (ESI, m / z): [MH] - =163.

[0562] Step B - Synthesis of Compound xliv

[0563] A solution of xliii (600 g, 3.37 mol) and phosphorus oxychloride (1.8 L) was stirred at 110 ° C for 1 hour. The reaction was monitored using LCMS and then concentrated in vacuo. The crude product was quenched with water, pre-cooled to 10 ° C, and the mixture was extracted with EtOAc (3 × 500 mL). The organic layer was washed with brine (500 mL) and dried over sodium sulfate. The mixture was filtered and concentrated in vacuo, and the residue obtained was purified using reverse phase flash chromatography (silica gel, 25% to 100% petroleum ether / EtOAc) to give 6-chloro-5-cyanopyridine-3-carboxylic acid (xliv). LCMS: (ESI, m / z): [MH] - =181.

[0564] Step C - Synthesis of Compound xlv

[0565] At 25 ° C, dimethylformamide (115.5 mL) and xliv (7.7 g, 42 mmol) were added to a 500 mL three-necked flask. The reaction was cooled to 10 ° C and (methylsulfanyl) sodium (7.39 g, 105 mmol) was added in batches. The resulting reaction was stirred at 25 ° C for 8 hours, and then the reaction mixture 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, and the resulting solution was filtered to obtain 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (xlv) as a solid. LCMS: (ESI, m / z): [MH] - =193.

[0566] Step D - Synthesis of Compound xlvi

[0567] To a solution of xlv (7.4 g, 38 mmol) in DCM (185 mL) was added m-CPBA (26.3 g, 152 mmol), and under a nitrogen atmosphere, the resulting reaction was stirred at 45 ° C for 24 hours. The reaction was quenched with saturated sodium bisulfite at 0 ° C and concentrated in vacuo. 2-methyltetrahydrofuran was added to the resulting residue, and the mixture was filtered and concentrated in vacuo. The resulting residue was purified using silica gel column chromatography (eluted with DCM / MeOH) to obtain compound xlvi. LCMS: (ESI, m / z): [M + H] + =227.05.

[0568] Step E - Synthesis of Compound xlvii

[0569] Intermediate xlvi (0.57 g, 2.5 mmol) and HATU (1.0 g, 2.7 mmol) were dissolved in 10 mL of DMF, and the resulting solution was stirred at 25°C for 30 minutes. Tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoate (0.65 g, 1.8 mmol) was added, and the resulting reaction was cooled to 10°C. DIPEA (0.873 mL, 5.00 mmol) was added dropwise to the reaction mixture at 10°C, and the reaction mixture was stirred at 25°C for 2 hours, then diluted with water and extracted with EtOAc (3 x 10 mL). The combined organic extracts were concentrated in vacuo, and the resulting residue was purified using silica gel column chromatography (eluting with 2:1 to 1:1 hexane:EtOAc) to provide compound xlvii. 1 H NMR (500MHz, 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).

[0570] Step F - Synthesis of Compound xlviii

[0571] Compound xlvii (80 g, 0.226 mmol) was dissolved in 1,4-dioxane (160 mL), and to the resulting mixture was added a 4M HCl solution in 1,4-dioxane (480 mL). The resulting reaction was stirred at 25 °C for 12 hours, then the reaction mixture was filtered and concentrated in vacuo to give compound xlviii, which was used without further purification. 1H NMR (500MHz, CD3OD) δ9.22 (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).

[0572] Preparation of intermediate compound 1

[0573]

[0574] Step A - Synthesis of Compound xlix

[0575] To a stirred mixture of 3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propanoic acid (xxxix, 1 g, 3 mmol) in THF (5 mL) was added HATU (1.54 g, 4.04 mmol), DIPEA (1.76 mL, 10.1 mmol) and tert-butyl glycylglycine ester (0.633 g, 3.36 mmol). The reaction was stirred at 20 ° C for 15 hours, then the reaction mixture was concentrated in vacuo and the resulting residue was purified using flash chromatography (0-100% petroleum ether / EtOAc) to give tert-butyl (3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propanoyl)glycylglycine ester xlix as a solid. LCMS (ESI): 468.2 [M+H] +

[0576] Step B - Synthesis of Compound 1

[0577] A solution of xlix (1.2 g, 2.6 mmol) in DCM (18 mL) and TFA (6 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo to give (3-(5-cyano-6-(methylsulfonyl)pyridinylamido)propionyl)glycylglycine 1 as a solid, which was used without further purification. LCMS (ESI) m / z: 412.1 [M+H] + .

[0578] Preparation of intermediate compound liv

[0579]

[0580] Step A - Synthesis of Compound lii

[0581] To a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (11, 1.5 g, 3.9 mmol) in DMF (30 mL) was added HATU (1.49 g, 3.92 mmol), and the resulting solution was stirred at room temperature for 10 minutes. tert-Butyl 3-(aminomethyl)azetidine-1-carboxylate (0.80 g, 4.3 mmol) was added, and the reaction was stirred for an additional 10 minutes. DIPEA (2.06 mL, 11.8 mmol) was added, and the reaction was stirred at 20° C. for an additional 3 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using preparative HPLC (Boston UNI C18 40×150×5 μm, eluting with acetonitrile / water (with 0.1% TFA as a modifier) ​​40% to 70% acetonitrile) to give tert-butyl 3-((5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundec-11-yl)azetidine-1-carboxylate (lii). LCMS (ESI) m / z: 551.2 [M+H] + .

[0582] Step B - Synthesis of Compound liii

[0583] To a solution of lii (2.37 g, 4.30 mmol) in DMF (15 mL) was added piperidine (1.5 mL, 15.15 mmol) and the resulting reaction was stirred at 25 ° C for 3 hours. The reaction mixture was dried using lyophilization to give tert-butyl 3-(((S)-2-((S)-2-aminopropionamido)propionamido)methyl)azetidine-1-carboxylate (liii), which was used without further purification. LCMS (ESI): 329.2 [M+H] + .

[0584] Step C - Synthesis of compound liv

[0585] To a solution of 3-(5-cyano-6-(methylsulfonyl)pyridinylamino)propanoic acid (xxxix, 400 mg, 1.35 mmol) in DMF (1.5 mL) was added HATU (767 mg, 2.02 mmol) and DIPEA (0.470 mL, 2.69 mmol), and the reaction was stirred at room temperature for 10 minutes. Compound liii (803 mg, 1.35 mmol) (55%) was added, and the reaction was stirred at 20° C. for another 30 minutes. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using preparative HPLC (Boston UNI C18 40×150×5 μm, eluting with acetonitrile / water (with 0.1% TFA as a modifier) ​​15% to 45% acetonitrile) to give tert-butyl 3-((4S,7S)-13-(5-cyano-6-(methylsulfonyl)pyridin-2-yl)-4,7-dimethyl-3,6,9,13-tetraoxo-2,5,8,12-tetraazatridecyl)azetidine-1-carboxylate (liv). LCMS (ESI): 608.2 [M+H] + .

[0586] Using the above methods and substituting appropriate reactants and / or reagents, the following intermediate compounds of the present disclosure were prepared:

[0587]

[0588] Preparation of intermediate compound lxv

[0589] Step A - Synthesis of Compound 1xiii

[0590] To a solution of (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine (1xii, 700 mg, 2.25 mmol) in DCM (10 mL) and MeOH (10 mL) was added (4-aminophenyl)methanol (332 mg, 2.70 mmol) followed by EEDQ (834 mg, 3.37 mmol). The resulting reaction was cooled to 20° C. and stirred at this temperature for 18 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using silica gel chromatography (0 to 10% MeOH in DCM) to give 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (1xiii). LCMS (ESI) m / z: 439.3 [M+Na]+.

[0591] Step B - Synthesis of Compound lxiv

[0592] To a solution of lxiii (200 mg, 0.48 mmol) in DMF (5 mL) was added bis (4-nitrophenyl) carbonate (161 mg, 0.528 mmol) and DIEA (0.109 mL, 0.624 mmol), and the resulting reaction was cooled to 20 ° C. and stirred at this temperature for 18 hours. The reaction mixture was diluted with water (30 mL), and the resulting solution was concentrated using freeze drying to give 4- ((S) -2- ((S) -2- (3- (2,5- dioxo -2,5- dihydro -1H- pyrrol-1-yl) propionamido) propionamido) benzyl (4- nitrophenyl) carbonate (lxiv). LCMS (ESI) m / z: 582.3 [M + H] + .

[0593] Step C - Synthesis of Compound 1xv

[0594] To a solution of lxiv (200 mg, 0.344 mmol) in DMF (5 mL) was added tert-butyl 3-(aminomethyl)azetidine-1-carboxylate (64.1 mg, 0.344 mmol), and the resulting reaction was stirred at 20° C. for 1 hour. The reaction mixture was directly purified using preparative-HPLC (Boston Green ODS 150×30 mm×5 μm, eluting with 40% to 60% MeCN / water with 0.01% TFA as a modifier) ​​to give tert-butyl 3-(((((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)propionamido)benzyl)oxy)carbonyl)amino)methyl)azetidine-1-carboxylate (lxv). LCMS (ESI) m / z: 529.3 [M+H + -Boc].

[0595] Preparation of intermediate compound lxxi

[0596]

[0597] Step A - Synthesis of Compound lxvii

[0598] At room temperature, to a solution of (4-aminophenyl)methanol (1xvi, 6.44 g, 52.3 mmol) in DCM / MeOH (2: 1) (60 mL) was added a stirred mixture of EEDQ (4.85 g, 19.6 mmol) and (((9H-fluorenes-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (5.0 g, 13 mmol). The resulting reaction was stirred at room temperature for 18 hours, and the reaction mixture was then concentrated in a vacuum. The resulting residue was suspended in TBME (80 mL) and stirred for 30 minutes, then the mixture was filtered, and the filter cake was washed with TBME (20 mL). The combined filtrate and washings were concentrated in vacuo to afford (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (lxvii).

[0599] Step B - Synthesis of Compound 1xviii

[0600] In room temperature, DIPEA (0.143mL, 0.820mmol) is joined in the stirred mixture of bis(4-nitrophenyl) carbonate (0.150g, 0.492mmol) and lxvii (0.2g, 0.410mmol) in DMF (2mL), and the reaction obtained is stirred at room temperature for 2 hours.Reaction mixture is poured into H in O (40mL), and the mixture obtained is filtered.Filter cake is washed with water (10mL), then dried under vacuum with toluene, obtain (9H-fluorenes-9-yl) methyl ((S) -1- (((S) -1- ((4- ((((4- nitrophenoxy) carbonyl) oxygen base) methyl) phenyl) amino) -1- oxo propyl-2-yl) amino) -1- oxo propyl-2-yl) carbamate (lxviii).LCMS(ESI)m / z:675.3[M+H] + .

[0601] Step C - Synthesis of Compound 1xix

[0602] By lxviii (2g, 3.06mmol), tert-butyl (2- (hydrazine formamido) ethyl) carbamate (0.803g, 3.68mmol) and HOBt (0.141g, 0.919mmol) solution in DMF (20mL) was stirred at room temperature 5 minutes.Pyridine (0.496mL, 6.13mmol) was added, the reaction obtained was stirred at room temperature 18 hours, and then the reaction mixture was concentrated in a vacuum.The residue obtained is purified using silica gel column flash chromatography (DCM / MeOH 0 to 30%), obtain 4- ((S) -2- ((((9H- fluorenes -9- yl) methoxy) carbonyl) amino) propionamido) propionamido) benzyl 11,11- dimethyl -4,9- dioxo -10- oxa- -2,3,5,8- tetraaza dodecanoate (lxix). LCMS (ESI) m / z: 732.4 [M+H] + .

[0603] Step D - Synthesis of Compound 1xx

[0604] Diethylamine (0.50 mL, 4.9 mmol) was added to a stirred mixture of lxix (1.1 g, 1.5 mmol) in DMF (10 mL) at room temperature, and the resulting reaction was stirred for 2 hours. The reaction mixture was then concentrated in vacuo to afford 4-((S)-2-((S)-2-aminopropionamido)propionamido)benzyl 11,11-dimethyl-4,9-dioxo-10-oxa-2,3,5,8-tetraazadodecanoate (lxx), which was used without further purification.

[0605] Step E - Synthesis of Compound 1xxi

[0606] DIPEA (0.679 mL, 3.89 mmol) was added to a stirred mixture of 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (414 mg, 1.55 mmol) and lxx (660 mg, 1.30 mmol) in DMF (8 mL) at room temperature, and the resulting reaction was stirred for 2 h. The reaction mixture was directly purified using preparative HPLC (Boston UNI C18 50×40 mm×5 μm, eluting with 19% to 49% acetonitrile / water (with 0.1% TFA as a modifier)) to give 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)propionamido)benzyl 11,11-dimethyl-4,9-dioxo-10-oxa-2,3,5,8-tetraazadodecanoate (lxxi). LCMS (ESI) m / z: 661.3 [M+H]+ .

[0607] Preparation of intermediate compound lxxv

[0608]

[0609] Step A - Synthesis of Compound 1xxiii

[0610] To a solution of (3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propionyl)glycylglycylglycine (1xxii, 0.50 g, 1.5 mmol) in DMF (5 mL) was added HATU (0.670 g, 1.76 mmol). The resulting solution was stirred at room temperature for 10 minutes, followed by the addition of a solution of tert-butylmethyl(2-(methylamino)ethyl)carbamate (0.304 g, 1.616 mmol) in DMF (0.1 mL), and the resulting reaction was stirred at room temperature for 10 minutes. DIEA (0.770 mL, 4.41 mmol) was added, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was directly purified using reverse phase HPLC (Boston Green ODS 150×30 mm×5 um, 23% to 53% acetonitrile / water (with 0.1% TFA as a modifier)) and concentrated in vacuo to give tert-butyl (15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecanyl) (methyl) carbamate (lxxiii, 0.5 g, 0.979 mmol).

[0611] Step B - Synthesis of Compound 1xxiv

[0612] Compound lxxiii (200 mg, 0.392 mmol) was dissolved in a 3:1 mixture of DCM:TFA (4 mL), and the resulting solution was stirred for 1 hour at 20° C. The reaction mixture was concentrated in vacuo to give 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(5-methyl-6,9,12-trioxo-2,5,8,11-tetraazatridec-13-yl)propanamide (lxxiv), which was used without further purification.

[0613] Step C - Synthesis of Compound 1xxv

[0614] To a solution of lxxiv (100 mg, 0.244 mmol) in DCM (10 mL) was added DIEA (0.032 mL, 0.184 mmol) and 4-(((tert-butoxycarbonyl)amino)methyl)phenyl chloroformate (35 mg, 0.12 mmol). The resulting reaction was cooled to 0°C and stirred at this temperature for 30 minutes. The reaction mixture was warmed to room temperature and then stirred for an additional hour. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using reverse phase HPLC (Boston Green ODS 150×30 mm×5 um, 23% to 53% acetonitrile / water (with 0.1% TFA as a modifier)) and concentrated to give 4-(((tert-butoxycarbonyl)amino)methyl)phenyl(15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-methyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazapentadecanyl)(methyl)carbamate (lxxv). LCMS (ESI) m / z: 660.3 [M+H] + .

[0615] Preparation of intermediate compound lxxxiv

[0616]

[0617] Step A - Synthesis of Compound lxxvii

[0618] To a solution of lxxvi (10 g, 28 mmol) in THF (100 mL) and toluene (40 mL) was added lead tetraacetate (17.5 g, 39.5 mmol), and the resulting reaction was heated to 85 ° C. and allowed to stir at this temperature for 18 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using silica gel flash chromatography (EtOAc / petroleum ether 30% to 50%) to give lxxvii (11.2 g, 21.3 mmol) as a solid. LCMS (ESI) m / z: 391.2 [M + Na] + .

[0619] Step B - Synthesis of Compounds lxxviii

[0620] To a solution of lxxvii (2.5 g, 6.8 mmol) in DCM (30 mL) was added TFA (1.05 mL, 13.6 mmol) and tert-butyl 2-hydroxyacetate (4.48 g, 33.9 mmol). The resulting reaction was stirred at 25 ° C for 18 hours, then the reaction mixture was diluted with a saturated aqueous solution of NaHCO 3 (10 mL) and extracted with DCM (3×30 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated in vacuo, and the resulting residue was purified using silica gel column chromatography (EtOAc / petroleum ether 5% to 9%) to give lxxviii (1.02 g, 1.852 mmol) as a solid. LCMS (ESI) m / z: 463.1 [M+Na] + .

[0621] Step C - Synthesis of Compound 1xxix

[0622] To a solution of lxxviii (1.5 g, 3.4 mmol) in DCM (9 mL) was added TFA (3.0 mL, 39 mmol), and the resulting reaction was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo to give lxxix (1.2 g, 2.2 mmol) as an oil, which was used without further purification. LCMS (ESI) m / z: 407.1 [M+H + ].

[0623] Step D - Synthesis of Compounds 1xxx

[0624] To a solution of lxxix (1.0 g, 2.6 mmol) in DMF (0 mL) was added HATU (1.19 g, 3.12 mmol), and the resulting solution was stirred at room temperature for 5 minutes. Tert-butyl (3-aminobicyclo [1.1.1] pentane -1- yl) carbamate (0.671 g, 3.38 mmol) and DIEA (1.36 mL, 7.80 mmol) were added, and the reaction was stirred at 20 ° C for 2 hours. The reaction mixture was concentrated in vacuo, and the resulting residue was purified using preparative HPLC (Boston UNI C18 150 mm × 40 mm × 5 μm, eluted with acetonitrile / water (0.1% TFA), eluted from 37% to 67% at a flow rate of 60 mL / min to obtain lxxx (680 mg, 1.148 mmol) as a solid. LCMS (ESI) m / z: 565.4 [M + H] + .

[0625] Step E - Synthesis of Compounds lxxxi

[0626] To a solution of lxxx (370 mg, 0.655 mmol) in DMF (4 mL) was added piperidine (0.40 mL, 0.66 mmol). The resulting reaction was stirred at 20 ° C for 1 hour, and then the reaction mixture was concentrated in vacuo to give lxxxi (360 mg, 0.526 mmol) as a solid, which was used without further purification. LCMS (ESI) m / z: 343.2 [M + H] + .

[0627] Step F - Compound lxxxii Synthesis

[0628] To a solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (407 mg, 1.05 mmol) in DMF (5 mL) was added HATU (300 mg, 0.789 mmol), and the resulting solution was stirred at room temperature for 5 minutes. Compound lxxvi (360 mg, 0.526 mmol) and DIEA (0.275 mL, 1.58 mmol) were added, and the resulting reaction was stirred at 20 ° C for 1 hour. The reaction mixture was directly purified using preparative HPLC (Boston UNI C18 150 mm×40 mm×5 um, 45% to 75% MeCN / water (0.1% TFA) at a flow rate of 60 mL / min) to give lxxxii (180 mg, 0.253 mmol) as a solid. LCMS (ESI) m / z: 712.3 [M+H] + .

[0629] Step F - Synthesis of Compounds lxxxiii

[0630] To a solution of lxxxii (180 mg, 0.253 mmol) in DMF (2 mL) was added piperidine (0.20 mL, 0.25 mmol). The resulting reaction was stirred at 20 ° C for 30 minutes, and then the reaction mixture was concentrated in vacuo to give lxxxiii (150 mg, 0.184 mmol) as a solid, which was used without further purification. LCMS (ESI) m / z: 490.2 [M + H] + .

[0631] Step G - Synthesis of Compound lxxxiv

[0632] To a solution of (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)glycylglycine (226 mg, 0.797 mmol) in DMF (2 mL) was added HATU (202 mg, 0.531 mmol), and the resulting reaction was stirred at room temperature for 5 minutes. Compound lxxxiii (130 mg, 0.266 mmol) and DIEA (0.139 mL, 0.797 mmol) were added, and the resulting reaction was stirred at 20 ° C for 2 hours. The reaction mixture was then concentrated in vacuo, and the resulting residue was purified using preparative HPLC (Boston Prime C18 150 mm × 40 mm × 5 μm, eluted with MeCN / water (0.1% TFA) 20% to 40% at a flow rate of 25 mL / min) to give lxxxiv (80 mg, 0.079 mmol) as a solid. LCMS (ESI) m / z: 755.3 [M+H] + .

[0633] Preparation of intermediate compound lxxxvii

[0634]

[0635] Step A - Synthesis of Compounds lxxxvi

[0636] To a solution of lxxxv (420 mg, 1.02 mmol) in DMF (7 mL) was added HATU (466 mg, 1.23 mmol), and the resulting solution was stirred at room temperature for 5 minutes. Tert-butyl (3-aminobicyclo [1.1.1] pentane -1- yl) carbamate (202 mg, 1.02 mmol) and DIEA (0.535 mL, 3.06 mmol) were added, and the resulting reaction was stirred at 20 ° C for 2 hours. The reaction mixture was concentrated in vacuo to give lxxxvi (600 mg, 0.608 mmol) as a solid, which was used without further purification. LCMS (ESI) m / z: 592.3 [M + H] + .

[0637] Step B - Synthesis of Compounds lxxxvii

[0638] To a solution of lxxxvi (600 mg, 1.01 mmol) in DMF (6 mL) was added piperidine (0.60 mL, 1.0 mmol) and the resulting reaction was stirred at 20 ° C for 1 hour. The reaction mixture was concentrated in vacuo and the resulting residue was purified using preparative HPLC (Boston Prime C18 150 mm × 40 mm × 5 μm, eluted with MeCN / water (0.1% TFA) 5% to 35% at a flow rate of 25 mL / min) to afford lxxxvii (400 mg, 0.974 mmol) as a solid. LCMS (ESI) m / z: 370.3 [M + H] + .

[0639] Preparation of intermediate compound xciii

[0640]

[0641] Step A - Synthesis of Compound lxxxix

[0642] To a solution of lxxxviii (3.5 g, 17 mmol) in AcOH (30 mL) was added furan-2,5-dione (1.68 g, 17.1 mmol). The resulting reaction was stirred at 20 ° C for 18 hours, and then the reaction mixture was concentrated in vacuo to give lxxxix (5 g, 16.50 mmol) as a solid, which was used without further purification. LCMS (ESI) m / z: 203.2 [M + H-Boc] + .

[0643] Step B - Synthesis of Compound xc

[0644] To a solution of lxxxix (5.0 g, 16 mmol) in toluene (40 mL) was added dimethylacetamide (2 mL) and triethylamine (6.92 mL, 49.6 mmol). The resulting reaction was heated to 110 ° C and stirred at this temperature for 18 hours, then the reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was purified using preparative HPLC (Boston Green ODS 150 mm × 30 mm × 5 um, eluted at a flow rate of 25 mL / min with MeCN / water (0.1% TFA) 20% to 40% to obtain xc (3.08 g, 7.32 mmol) as a solid. LCMS (ESI) m / z: 229.1 [M + H-56] + .

[0645] Step C - Synthesis of Compound xci

[0646] To a solution of xc (3.08 g, 10.8 mmol) in DCM (15 mL) was added TFA (5.0 mL, 65 mmol), and the resulting reaction was stirred at 25 ° C for 3 hours. The reaction mixture was concentrated in vacuo to give xci (2.4 g) as an oil, which was used without further purification. LCMS (ESI) m / z: 185.2 [M+H] + .

[0647] Step D - Synthesis of Compound xcii

[0648] To a solution of xci (0.542 g, 2.94 mmol) in DMF (10 mL) was added 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxacohexacosa-26-oate (1.5 g, 2.9 mmol), followed by NMM (0.596 g, 5.89 mmol), and the resulting reaction was stirred at 20 ° C for 18 hours. The reaction mixture was directly purified using preparative HPLC (Boston UNI C18 150 mm × 40 mm × 5 um, eluted with MeCN / water (0.1% TFA) 5% to 35% at a flow rate of 60 mL / min) to give xcii (730 mg, 1.26 mmol) as an oil. LCMS (ESI) m / z: 579.3 [M + H] + .

[0649] Step E - Synthesis of Compound xciii

[0650] At 0 ° C, to a solution of xcii (0.20 g, 0.35 mmol) and lxxxvii (0.153 g, 0.415 mmol) in DMF (2 mL) was added NMM (0.105 g, 1.04 mmol) and HATU (0.131 g, 0.346 mmol) was slowly added. The resulting reaction was stirred at 0 ° C for 3 hours, and the reaction mixture was then concentrated in vacuo. The resulting residue was purified using preparative HPLC (Boston Prime C18 150 mm × 40 mm × 5 μm, eluted with MeCN / water (0.1% TFA) 15% to 45% at a flow rate of 25 mL / min) to give xciii (130 mg, 0.119 mmol) as a powder. LCMS (ESI) m / z: 930.7 [M + H] + .

[0651] Intermediate compound xcvii i Preparation

[0652]

[0653] Step A - Synthesis of Compound xciv

[0654] To a solution of ((benzyloxy)carbonyl)glycylglycine (1.0 g, 3.8 mmol) and N-hydroxysuccinimide (0.52 g, 4.5 mmol) in dioxane (7 mL) was slowly added a solution of DCC (0.930 g, 4.51 mmol) in anhydrous dioxane (7 mL). The reaction mixture was stirred at 20 ° C for 12 hours, and then the reaction mixture was concentrated in vacuo to give xciv (1.3 g, 2.147 mmol) as a solid, which was used without further purification. LCMS (ESI) m / z: 364.2 [M + H] + .

[0655] Step B - Synthesis of Compound xcv

[0656] The stirred mixture of xciv (1.3 g, 3.6 mmol) and sodium bicarbonate (0.361 g, 4.29 mmol) in water (10 mL) was added to a solution of L-phenylalanylglycine (0.954 g, 4.29 mmol) in dioxane (20 mL). The resulting reaction was stirred at 20 ° C for 18 hours, and the reaction mixture was then concentrated in vacuo. The resulting residue was purified using preparative HPLC (Boston UNI C18 150 mm × 40 mm × 5 μm, eluted with acetonitrile / water (0.1% TFA) from 15% to 45% at a flow rate of 60 mL / min) to obtain xcv (1.3 g, 2.76 mmol) as a solid. LCMS (ESI) m / z: 471.3 [M+H] + .

[0657] Step C - Synthesis of Compound xcvi

[0658] To a solution of xcv (500mg, 1.06mmol) in DMF (7mL) was added HATU (445mg, 1.17mmol), and the resulting solution was stirred at room temperature for 5 minutes. Tert-butyl (3-aminobicyclo [1.1.1] pentane -1- base) carbamate (211mg, 1.06mmol) and Hunig's base (Hunig's base) (0.557mL, 3.19mmol) were added, and the resulting reaction was stirred at 20 ° C for 2 hours. The reaction mixture was concentrated in a vacuum, and the resulting residue was purified using preparative HPLC (Boston Green ODS 150mm × 30mm × 5μm, eluted with acetonitrile / water (0.1% TFA) from 30% to 60% at a flow rate of 25mL / min) to obtain xcvi (645mg, 0.991mmol) as a solid. LCMS (ESI) m / z: 651.3 [M + H] + .

[0659] Step D - Synthesis of Compound xcvii

[0660] To a mixture of xcvi (645 mg, 0.991 mmol) in trifluoroethanol (15 mL) was added 10% Pd / C (300 mg), and the mixture was stirred at 25 ° C. under 1 atmosphere of hydrogen for 18 hours. The reaction mixture was filtered, and the filtrate was concentrated in vacuo to give xcvii (544 mg, 0.769 mmol) as an oil. LCMS (ESI) m / z: 517.3 [M+H] + .

[0661] Step E - Synthesis of Compound xcviii

[0662] To a solution of 3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propionic acid (49.1 mg, 0.290 mmol) in DMF (2 mL) was added HATU (121 mg, 0.319 mmol), and the resulting solution was stirred at room temperature for 5 minutes. Compound xcvii (150 mg, 0.290 mmol) and Hunig's base (0.152 mL, 0.871 mmol) were added, and the resulting reaction was stirred at 20 ° C for 2 hours, and the reaction mixture was then concentrated in vacuo. The resulting residue was purified using preparative HPLC (Boston Green ODS 150 mm × 30 mm × 5 μm, eluted with 23% to 53% acetonitrile / water (0.1% TFA) at a flow rate of 25 mL / min) to obtain xcviii (70 mg, 0.101 mmol) as a solid. LCMS (ESI) m / z: 668.3 [M + H] + .

[0663] Preparation of intermediate compound xcx

[0664]

[0665] To a solution of (8S,10S)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydronaphthacene-5,12-dione (xcix, 200 mg, 0.312 mmol) in methanol (5 mL) and water (5 mL) was added a solution of sodium periodate (80 mg, 0.37 mmol) in water (1 mL), and the resulting reaction was stirred at room temperature for 30 minutes. The solvent was gradually removed from the reaction mixture in vacuo over 18 hours at 25°C, and the resulting residue xcx was used without further purification. LCMS (ESI) m / z: 628.6 [M+H] + .

[0666] Example 1

[0667] Preparation of connector-load 1

[0668]

[0669] Step A - Synthesis of Compound I-1b

[0670] To a stirred solution of L-alanyl-L-alanine (I-1a, 1.00 g, 6.24 mmol) and TEA (1.74 mL, 12.5 mmol) in DCM (2 mL) was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (2.09 g, 7.87 mmol) at 25° C. The mixture was stirred at 25° C. for 18 hours. The mixture was concentrated in vacuo and purified using preparative HPLC (YMC-Triart Prep C18 150×40 mm×7 μm, eluted with 7% to 37% acetonitrile / water (with 0.1% TFA as a modifier)) to give (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine I-1b as a solid. LCMS (ESI) m / z: 312.1 [M+H] + .

[0671] Step B - Synthesis of Compound I-1c

[0672] To a stirred mixture of I-1b (300 mg, 0.964 mmol) in DMF (4 mL) was added HATU (733 mg, 1.93 mmol), and the resulting reaction was stirred for 10 minutes at 25° C. Then, tert-butyl (2-aminoethyl) carbamate (154 mg, 0.964 mmol) and DIPEA (0.505 mL, 2.89 mmol) in DMF (1 mL) were added to the above mixture, and the mixture was stirred at 25° C. for 25 minutes. The solution was purified using preparative HPLC (Boston UNI C18 40×150×5 μm, 8% to 100% acetonitrile / water with 0.1% TFA as a modifier) ​​and lyophilized to afford tert-butyl (2-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)propionamido)ethyl)(methyl)carbamate I-1c as an oil. LCMS (ESI) m / z: 490.0 [M+Na] + .

[0673] Step C - Synthesis of Compound I-1d

[0674] A solution of I-1c (300 mg, 0.642 mmol) and TFA (2 mL, 0.642 mmol) in DCM (6 mL) was stirred at 0 ° C for 1 hour. The reaction was monitored using LCMS, and the resulting reaction was concentrated in vacuo at 0 ° C to give compound 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-(methylamino)ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide I-1d as an oil, which was used in the next step without further purification. LCMS (ESI) m / z: 368.0 [M+H] + .

[0675] Step D - Synthesis of Compound 1

[0676] To a solution of xcx (150 mg, 0.239 mmol) in DMF (2 mL) was added HATU (182 mg, 0.478 mmol). After the reaction mixture was stirred for 10 minutes, I-1d (88 mg, 0.24 mmol) in DMF (0.5 mL) was added and the resulting reaction was stirred for 10 minutes. DIPEA (0.125 mL, 0.717 mmol) was added and the reaction mixture was stirred at 20°C for another hour. The product was purified by preparative HPLC (YMC-Triart Prep C18 The crude mixture was purified by eluting with 25% to 55% acetonitrile / water (using 7 mM ammonium formate as a modifier) ​​to afford (2S,4S)-N-(2-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)propionamido)ethyl)-2,5 ,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamide 1. 1 H NMR(400MHz,CDCl3)δ13.94(br s,1H),13.37(br s,1H),8.03(d,J=7.58Hz,1H),7.78(t,J=8.07Hz,1H),7.39(d,J=8.31Hz,1H),7.00-7.12(m,1H),6.77(br d,J=7.34Hz,1H),6.69(s,2H),6.17-6.33(m,1H),5.53(br s,1H),5.27-5.40(m,1H),4.70(s,1H),4.47(s,1H),4.21-4.44(m,2H),3.99-4.13(m,5H),3.78-3.96(m,3H),3.54- 3.72(m,2H),3.36-3.50(m,8H),2.93-3.11(m,2H),2.67-2.85(m,2H),2.30-2.65(m,4H),1.66-1.84(m,7H),1.38(br d,J=6.36Hz,6H),LCMS(ESI)m / z:977.3[M+H] + .

[0677] The following exemplary linker-load compounds of the present disclosure were prepared using the method described in Example 1 and substituting the appropriate intermediates and other reactants and / or reagents:

[0678]

[0679]

[0680]

[0681]

[0682] Using the procedures described in Example 1 (Steps B, C, and D) and substituting the appropriate intermediates and other reactants and / or reagents, the following exemplary embodiments of the present disclosure were prepared:

[0683]

[0684]

[0685] Using the procedures described in Example 1 (Steps C and D) and substituting the appropriate intermediates and other reactants and / or reagents, the following illustrative examples of the present disclosure were prepared:

[0686]

[0687]

[0688]

[0689]

[0690] Example 2

[0691] Preparation of linker-payload compound 34

[0692] To a solution of compound 19 (80 mg, 0.058 mmol) in DMF (0.3 mL) was added piperidine (0.010 mL, 0.058 mmol), and the resulting reaction was stirred at 0° C. for 10 minutes. The reaction mixture was purified using preparative HPLC (C18-1 150×30 mm×5 μm, eluting with 25% to 56% acetonitrile / water with 8 mM ammonium formate as a modifier) ​​to afford 5-cyano-N-((12S,15S)-12,15-dimethyl-1,11,14,17-tetraoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS ,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacene-2-yl)-2,6,10,13,16-pentaazanonadecan-19-yl)-6-(methylsulfonyl)picolinamide 34. 1 H NMR (400MHz, CD3OD) δ = 8.50 (dd, J = 8.1, 10.3Hz, 1H), 8.39 (s, 2H), 8.29 (dd, J = 3. 8,8.1Hz,1H),7.88(dd,J=2.1,7.4Hz,1H),7.81-7.73(m,1H),7.50(d,J=8.6Hz,1 H),5.31(q,J=5.0Hz,1H),5.14-5.06(m,1H),4.59(s,1H),4.31(s,1H),4.16-4. 03(m,3H),3.96(s,4H),3.77(td,J=6.1,11.9Hz,1H),3.63-3.55(m,2H),3.47(br d,J=11.7Hz,1H),3.42-3.36(m,5H),3.33(s,3H),3.31-3.25(m,2H),3.10-2.98(m,3H),2.95(br d,J=6.9Hz,4H),2.65(br s,2H),2.57-2.46(m,2H),2.28(br s,2H),1.93-1.77(m,5H),1.77-1.58(m,2H),1.31-1.20(m,9H).LCMS(ESI)m / z:1162.3[M+H] + .

[0693] Using the methods described in the above examples and substituting the appropriate intermediates and other reactants and / or reagents, the following exemplary linker-load compounds of the present disclosure were prepared:

[0694]

[0695]

[0696] Example 3

[0697] Preparation of linker-payload compound 36

[0698]

[0699] Step A - Synthesis of Compound I-36b

[0700] To a solution of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (669 mg, 1.82 mmol)) in DCM (10 mL) was added TFA (0.466 mL, 6.05 mmol) and 3-aminobicyclo[1.1.1]pentan-1-ol (I-36a, 120 mg, 1.21 mmol). The resulting reaction was stirred at 25 ° C. for 18 hours, then the reaction mixture was concentrated in vacuo and the resulting residue was purified using preparative HPLC (Boston Prime C18 150 mm × 40 mm × 5 μm, eluted with 15% to 45% acetonitrile / water (0.1% TFA) at a flow rate of 25 mL / min) to give I-36b (230 mg, 0.524 mmol) as a solid. LCMS (ESI) m / z: 408.3 [M + H] + .

[0701] Step B - Synthesis of Compound I-36c

[0702] To a solution of xcx (200 mg, 0.319 mmol) in DMF (4 mL) was added HATU (145 mg, 0.382 mmol), and the resulting solution was stirred at room temperature for 10 minutes. A solution of compound I-36b (130 mg, 0.319 mmol) in DMF (0.4 mL) was added, and the resulting solution was stirred at room temperature for 10 minutes. Hunig's base (124 mg, 0.956 mmol) was added, and the resulting reaction was stirred at room temperature for 20 minutes. The reaction mixture containing compound I-36c (300 mg, 0.147 mmol) in DMF was used without further purification.

[0703] Step C - Synthesis of Compound I-36d

[0704] To a solution of I-36c (300 mg, 0.147 mmol) in DMF (4.4 mL) was added piperidine (0.4 mL, 4.04 mmol), and the resulting reaction was stirred at 20 ° C for 10 minutes. The reaction mixture was directly purified using preparative HPLC (Phenomenex Gemini-NX 150 mm × 30 mm × 5 μm; 23% to 53% acetonitrile / water (7 mM HCOONH4) at a flow rate of 25 mL / min) to give I-36d (50 mg, 0.057 mmol) as a solid. LCMS (ESI) m / z: 795.5 [M + H] + .

[0705] Step D - Synthesis of Compound 36

[0706] To a solution of (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)glycylglycine (27 mg, 0.094 mmol) in DMF (0.2 mL) was added HATU (47.8 mg, 0.126 mmol), and the resulting solution was stirred at room temperature for 10 minutes. Compound I-36d (50 mg, 0.063 mmol) was added, and the mixture was stirred at room temperature for 10 minutes, followed by the addition of Hunig's base (0.033 mL, 0.189 mmol), and the resulting reaction was stirred at 25 ° C. for 30 minutes. The reaction mixture was directly purified using preparative HPLC (Phenomenex Gemini-NX 150 mm × 30 mm × 5 μm; 23% to 53% acetonitrile / water (7 mM HCOONH4) at a flow rate of 25 mL / min) to give 36 (14.7 mg, 0.014 mmol) as a solid. 1 H NMR (400MHz, DMSO-d6)δ=8.49-8.38(m,2H),8.20(br s,1H),8.06(br s,2H),7.86(br s,2H),7.60(br s,1H),6.93(s,2H),5.31(br s,1H),5.19(br s,1H),4.94(br s,1H),4.51(br s,3H),4.16(br s,1H),4.05(br s,1H),3.93(br s,4H),3.71-3.50(m,10H),3.44(m,2H),2.97-2.77(m,2H),2.61(br s,1H),2.35(br s,4H),2.28-2.18(m,3H),2.06(br s,6H),1.60(br s,2H),1.14(m,3H).LCMS(ESI)m / z:1060.6[M+H+ ].

[0707] Example 4

[0708] Preparation of Antibody-Drug Conjugates Antibody Conjugation Schemes for Examples 37-68

[0709] Exemplary linker-carrier complexes of the present disclosure were conjugated to anti-TROP2 antibodies using the following conjugation scheme:

[0710] The antibody (humanized × [TACSTD2_H] mAb (sacitozumab ((S375C)) IgG1 / κ) (20 mg) was used as is in Hist (10 mM pH 6.5) 9% sucrose. The antibody was diluted to ~7 mg / ml in 90% buffer / 10% DMSO. A 10 mM solution of the linker-load compound (3.1 eq.) in DMSO was added, and the resulting conjugation reaction was stirred at room temperature for 2 hours. The reaction mixture was then purified by exchange into 10 mM histidine pH 6.5 using a desalting column on a chromatography system, followed by the addition of 9% sucrose. Using this method, the following antibody-drug conjugates of the present disclosure were prepared:

[0711]

[0712]

[0713] Example 5

[0714] Preparation of Antibody-Drug Conjugates Antibody Conjugation Schemes for Examples 69-70

[0715] Exemplary linker-load compounds of the present disclosure were conjugated to anti-TROP2 antibodies using the following conjugation scheme to provide antibody-drug conjugate Examples 69-71, respectively:

[0716] The antibody (humanized × [TACSTD2_H] mAb (sacitozumab (S375C)) IgG1 / κ) (20 mg) was exchanged into 40 mM Tris-acetate, 1 mM EDTA, pH 8.3. The antibody was diluted to ~10 mg / ml in 90% buffer / 10% DMF. A 10 mM solution of the exemplary linker-load compound (5.5 Eq) in DMF was added, and the resulting conjugation reaction was stirred at room temperature overnight. The reaction mixture was then purified using a desalting column exchange into 10 mM histidine pH 6.5 on an AKTA chromatography system, followed by the addition of 9% sucrose.

[0717] Using this method, the following antibody-drug conjugates of the present disclosure were prepared:

[0718] Example mAbs Drugs / connectors 69 Sacilotuzumab (S375C) Example 32 70 Sacilotuzumab (S375C) Example 33

[0719] Example 6

[0720] TROP2 BxPC-3 Cytotoxicity Assay Protocol for Examples 37-41, 43-52, 57-58, and 60-70

[0721] The exemplary TROP2 antibody-drug conjugates of the present disclosure (Examples 37-41, 43-53, 57-58, 60-68) were subjected to a cell-based cytotoxicity assay (BxPC-3 cells, 2.0 Cytotoxicity Assay):

[0722] BxPC-3 cells were cultured in T75 flasks supplemented with 10% FBS (Gibco TM RPMI 1640 medium (Gibco 26140-079) TM The cells were cultured in PBS (without calcium or magnesium) and then 2 ml of 0.25% trypsin-EDTA (Thermo Fisher Scientific) was added. TM catalog#25200056) and incubate the flask at 37°C for ~3 minutes. Next, add 10 mL of cell culture medium and pipette up and down several times to dissociate the cells. Transfer the cell mixture to a 15-mL conical tube and centrifuge at 300g for 5 minutes. Resuspend the cell pellet in 2 mL of cell culture medium and count the cells using Vi-CELL. Seed the cells in 96-well plates (Corning Microplates) at 90 μl of cell culture medium per well (3,000 cells per well). TM catalog #3904). After 24 hours, serial dilutions of the small molecule and antibody-drug conjugates in cell culture medium were prepared and 10 μL of the dilution was added to each well (total volume of 100 μL / well). Only the middle 60 wells on the plate were used for drug treatment and untreated controls. The plates were incubated in a cell culture incubator for 96 hours and then equilibrated at room temperature for approximately 30 minutes. CellTiter-Glo TM Thaw the buffer and allow it to equilibrate to room temperature. Add an appropriate volume of CellTiter-Glo TM Transfer the buffer to a medium containing CellTiter-Glo TM Amber bottles of substrate were used to reconstitute the lyophilized enzyme / substrate mix (Promega TM catalog#G7573). Next, add 100 μL of CellTiter-Glo to each well.TM The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. TM Luminescence was recorded on the ELISA kit using GraphPad Prism 8 [four-parameter nonlinear regression, y = bottom + (top - bottom) / (1 + (IC 50 / x) HillSlope )]Analyze the data.

[0723] Exemplary embodiments of the present disclosure were tested, and the results are provided below:

[0724]

[0725]

[0726] Example 7

[0727] TROP2 BxPC-3 Cytotoxicity Assay Protocol for Examples 42, 54, and 59

[0728] Exemplary TROP2 antibody-drug conjugates of the present disclosure (Examples 31, 41, and 46) were subjected to a cell-based cytotoxicity assay (BxPC-3 cells, 2.0 Cytotoxicity Assay):

[0729] Step 1: Seed 384-well plates for assay on day 0 (45 μl per well)

[0730] BxPC-3 cells (in sample vials) are quickly thawed in frozen vials by incubating in 37°C water baths for <1 min until only a small amount of ice is left in the sample vials. The vials are taken out from the water bath and wiped with 70% ethanol. The cells are transferred from the vials to a sterile centrifuge tube containing 8mL of pre-warmed cell culture medium (RPMI-1640 (Cat#30-2001)+10% FBS+1% P / S). The vials are rinsed with another 1mL culture medium to ensure that the cells are fully transferred to the centrifuge tube. Then, the cells are centrifuged (150 × g) for 5 minutes. The supernatant is aspirated and the cell pellet is resuspended in a 10-20ml cell culture medium (RPMI-1640 (Cat#30-2001)+10% FBS+1% P / S). Vi-cell is used to count the cells and prepare 1500 cells / well (in 45 μL of culture medium). Then, 45 μl / well of cells were added to the The plate was centrifuged (150×g) for 30 seconds in a 384-well Low Flange White Flat Bottom Polystyrene TC-treated microplate (Corning, Cat#3570).

[0731] Step 2: Add Antibody-Drug Conjugate on Day 1

[0732] Take out the centrifuge tube containing exemplary antibody-drug conjugate and reference storage solution, and thaw it at room temperature.Then the tube is centrifuged (2000 × g) 30 seconds.Use Bravo liquid processor, use special buffer (10mM pH 6.5 histidine 9% sucrose buffer) to carry out serial dilution to prepare 10 × intermediate assay plate (Waters plate, Cat#186002632). Culture medium (cell-free) is used for Max_E.Then, use Bravo liquid processor (using extremely slow speed so that cell monolayer is not disturbed) 5 μ L of 10X storage solution is added to the assay plate from the intermediate plate.The plate is spun centrifuged (150 × g) 30 seconds.

[0733] Step 3: CellTiter-Glo 2.0 assay (Promega, Cat# G9242) on day 5 (TROP2)

[0734] CellTiter-Glo TM 2.0 reagent was thawed at 4°C overnight (during this time, the reagent was not exposed to temperatures above 25°C). The kit was equilibrated to room temperature for approximately 30 minutes, and then 20uL of CellTiter-Glo™ 2.0 reagent was added to 50uL of culture medium containing cells using a Standard Cassette Combi. The contents were mixed on an orbital shaker for 2-3 minutes to induce cell lysis, and the plate was spun (150×g) for 30 seconds. The plate was then incubated at room temperature for 5 minutes to stabilize the luminescent signal. Luminescence was recorded to calculate EC 50 values, using an integration time of 0.25–1 s per well as a guide.

[0735] Exemplary embodiments of the present disclosure were tested, and the results are provided below:

[0736] Example <![CDATA[BxPC3 EC 50 (nM)]]> 42 0.51 54 0.54 59 0.62

Claims

1. A compound having structural formula I: and pharmaceutically acceptable salts thereof, wherein: When R 2 When R is H or C1-C6 alkyl, 1 Selected from -XYZR 3 ; R 2 is H or C1-C6 alkyl, or R 1 and R 2 are connected to form a 4- to 6-membered monocyclic heterocycloalkylene group or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene group, each of which is substituted on a ring carbon atom by -XYZR 3 replaced by; R 3 Selected from: X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、-(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-(CH2) n -N(R 4 )- and -R 5 -O-CH2N(R 4 )-; Y is a bond or -C(O)C(R 4 )(R 6 )NH-; Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-; R 4 H, C 1-6 Alkyl or R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bridged bicyclic cycloalkylene, -NH-NH- and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group; R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain; R 9 -CH2NHC(O)-(CH2CH2O) q -CH3; m is 0, 1, or 2; n is independently an integer from 0 to 4 at each occurrence; and q is selected from 4, 8, 10 and 12.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 3 for:

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 3 for:

4. The compound according to claim 2 or 3, wherein m is 1.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R 1 -XYZR 3 , and R 2 is H or C1-C6 alkyl.

6. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are connected to form a 4- to 6-membered monocyclic heterocycloalkylene group or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene group, each of which is substituted on a ring carbon atom by -XYZR 3 replaced.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R 2 For H.

8. The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein X is a 5- or 6-membered monocyclic heterocycloalkylene group or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene group.

9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -、-(CH2) n -R 5 -NHC(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-;R 4 Each occurrence is independently H, methyl or Fmoc; and R 5 Each occurrence is independently selected from -NH-NH-, -NHC(O)NH, 10. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein Y is a bond.

11. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein Y is -C(O)C(R 4 )(R 6 )NH-, where R 6 is H, benzyl or isopropyl, and R 4 is H or methyl.

12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-, and R 7 and R 8 Each is independently selected from H, methyl, isopropyl and -CH2CH2CH2NHC(O)NH2.

13. The compound of any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein the group -XYZR 3 Selected from:

14. A compound, or a pharmaceutically acceptable salt thereof, selected from:

15. An antibody-drug conjugate having structural formula (XXIII): or a pharmaceutically acceptable salt thereof, wherein: L is antibody; R 2 is H or C1-C6 alkyl; R 3’ Selected from: Where ** indicates R 3’ Attachment point to L; X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、-(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-; Y is a bond or -C(O)C(R 4 )(R 6 )NH-; Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-; R 4 H, C 1-6 Alkyl or R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bridged bicyclic cycloalkylene, -NH-NH- and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group; R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain; R 9 -CH2NHC(O)-(CH2CH2O) q -CH3; m is 0, 1, or 2; n is independently an integer from 0 to 4 at each occurrence; and q is selected from 4, 8, 10 and 12.

16. An antibody-drug conjugate having structural formula (XXIV): or a pharmaceutically acceptable salt thereof, wherein: A is a 4- to 6-membered monocyclic heterocycloalkylene or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene; L is antibody; R 3’ Selected from: Where ** indicates R 3’ Attachment point to L; X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、-(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-; Y is a bond or -C(O)C(R 4 )(R 6 )NH-; Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-; R 4 H, C 1-6 Alkyl or R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bridged bicyclic cycloalkylene, -NH-NH- and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group; R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain; R 9 -CH2NHC(O)-(CH2CH2O) q -CH3; m is 0, 1, or 2; n is independently an integer from 0 to 4 at each occurrence; and q is selected from 4, 8, 10 and 12.

17. The antibody-drug conjugate of claim 15, which is selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein: X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、-(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-; Y is a bond or -C(O)C(R 4 )(R 6 )NH-; Z is -C(O)CH(R 7 )NHC(O)CH(R 8 )NH-; R 4 H, C 1-6 Alkyl or R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, 5- to 11-membered bicyclic spirocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 Bridged bicyclic cycloalkylene, -NH-NH- and -NHC(O)NH-, wherein the 4- to 6-membered monocyclic heterocycloalkylene may be optionally substituted on a ring carbon atom with an oxo group; R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain; R 9 -CH2NHC(O)-(CH2CH2O) q -CH3; m is 0, 1, or 2; n is independently an integer from 0 to 4 at each occurrence; and q is selected from 4, 8, 10 and 12.

18. The antibody-drug conjugate of claim 16, which is selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein: A is a 4- to 6-membered monocyclic heterocycloalkylene or a 5- to 11-membered bicyclic spirocyclic heterocycloalkylene; X is selected from -(CH2) n -N(R 4 )-、-R 5 -N(R 4 )-、-(CH2) n -N(R 4 )-C(O)R 5 -、-(CH2) n -N(R 4 )-(C1-C6 alkylene)-N(R 4 )-, 5- or 6-membered monocyclic heterocycloalkylene, -(CH2) n -R 5 -N(R 4 )-, 5- to 11-membered bicyclic spirocyclic heterocyclic alkylene, -(CH2) n -R 5 -、(CH2) n -R 5 -N(R 4 )C(O)O-(CH2) n R 5 -N(R 4 )-、-(CH2) n -NHC(O)O-(CH2) n -R 5 -N(R 4 )-、-(CH2) n -R 5 -OC(O)-N(R 4 )-(C1-C6 alkylene)-N(R 4 )-、-R 5 -NHC(O)CH2OCH2N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )C(O)NH-N(R 4 )-、-R 5 -N(R 4 )C(O)CH2N(R 4 )-、-(CH2) n -N(R 4 )-CH2-N(R 4 )- and -R 5 -O-CH2N(R 4 )-; Y is a bond or -C(O)CH(R 7 )NH-; Z is -C(O)CH(R 6 )NHC(O)CH(R 7 )NH-; R 2 is H or C1-C6 alkyl; R 4 H or C 1-6 alkyl; R 5 Selected from C6-C 10 Arylene, 4- to 6-membered monocyclic heterocycloalkylene, C3-C7 monocyclic cycloalkylene, C5-C 11 bridged bicyclic cycloalkylenes, -NH-NH-, and -NHC(O)NH-; R 6 、R 7 and R 8 Each is independently selected from H, -CH2CH2CH2NHC(O)NH2, benzyl, and a naturally occurring amino acid side chain; R 9 -CH2NHC(O)-(CH2CH2O) q -CH3; m is 0, 1, or 2; n is independently an integer from 0 to 4 at each occurrence; and q is selected from 4, 8, 10 and 12.

19. The antibody-drug conjugate of any one of claims 15 to 18, wherein L is an antibody.

20. A linker-load compound comprising: (i) a payload, which is a compound of formula (XXII): or a pharmaceutically acceptable salt thereof, wherein: R 1’ H or C 1-6 alkyl; R 2’ is a linker selected from linkers L-1 to L-12; Or, R 1 and R 2 The 3 to 7 membered monocyclic heterocycloalkyl group, the 5 to 11 membered bicyclic heterocycloalkyl group, the 5 to 11 membered fused heterocycloalkyl group and the 5 to 11 membered spirocyclic heterocycloalkyl group are each optionally and independently replaced by one or more R A and wherein a linker selected from linkers L-1 to L-12 is attached to a ring atom of (i), (ii), (iii) or (iv); R A Each occurrence is independently selected from C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2, C1-C6 aminoalkyl, -(C1-C6 alkylene)-NHC(O)-(C1-C6 aminoalkyl), -O-(C 1-6 alkyl), -NHC(O)CH2OH and -(C1-C6 alkylene)-(3 to 7 membered monocyclic heterocycloalkyl), wherein the 3 to 7 membered monocyclic heterocycloalkyl group may be optionally and independently replaced by one or more R B substituted with a group; and R B Each occurrence is independently selected from C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2, C1-C6 aminoalkyl, -O-(C 1-6 alkyl), -NHC(O)-(C1-C6 aminoalkyl), -(C1-C6 alkylene)-NHC(O)-(C1-C6 aminoalkyl), C 1-6 Alkyl, halo, -CN, -OR 3 、-N(R 3 )2、-O-(C 1-6 alkyl) and -NHC(O)CH2OH; and (ii) a linker selected from the group consisting of: wherein the connector is connected to the load R 2 group, and the point of attachment on the linker is determined by express.

21. A linker-load compound having a formula selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein D is a cargo selected from anticancer agents.

22. A pharmaceutical composition comprising: (a) the antibody-drug conjugate of any one of claims 15 to 19, or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier.

23. Use of: (a) the antibody-drug conjugate of any one of claims 15 to 19, or a pharmaceutically acceptable salt thereof, or (b) the pharmaceutical composition of claim 22 in the preparation of a medicament for treating or preventing cancer or tumors.

24. A method of treating or preventing a cancer selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma) in a subject in need thereof, the method comprising administering to the subject in need of such treatment a therapeutically effective amount of: (a) the antibody-drug conjugate of any one of claims 15-19, or a pharmaceutically acceptable salt thereof, or (b) the pharmaceutical composition of claim 22.

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