Drug conjugates and methods for making and using same

Abstract

The present invention provides novel linkers, linker conjugates, and drug conjugates thereof, as well as methods of making and using same for the treatment of various diseases and conditions.

Description

[Technical Field]

[0001] Priority claims and related patent applications This application claims the benefit of priority from PCT / CN2023 / 073383, filed January 20, 2023, the entire contents of which are incorporated herein by reference.

[0002] Technical field of the invention The present invention relates generally to novel compounds, their methods of manufacture and therapeutic applications. More particularly, the present invention provides novel linkers, linker conjugates, and drug conjugates thereof, as well as methods of manufacture and use thereof for the treatment of various diseases or conditions. [Background technology]

[0003] Background of the Invention Drug conjugation (e.g., antibody-drug conjugates (ADCs)) can provide an effective approach for delivering drugs to target sites in tissues or organs. Gemtuzumab ozogamicin (Mylotarg), the first ADC approved by the FDA in 2000, TM To date, 12 ADCs have been approved by the FDA, including the 12-ADC (see, e.g., Drago et al. 2021 Nature Reviews 18, 327-344; Mckertish et al. 2021 Biomedicines 9, 872; Khongorzui et al. 2020 Molecular Cancer Res. 18:3-19; Bross et al. 2001 Clin. Cancer Res. 7, 1490-1496; Hamann et al. 2002 Bioconjug. Chem. 13, 47-58; Lamb, 2017 Drugs 77, 1603-1610). Lessons learned from the development of these ADCs highlight the importance of optimizing drug-protein conjugation strategies. Cysteine ​​modification has gained popularity due to its high nucleophilicity, selectivity for electrophiles, and low natural abundance of sulfhydryl group-bearing amino acid residues in naturally occurring proteins.

[0004] Maleimide-based linkers are currently being used in several ADCs under investigation. This is due in part to the high reactivity and selectivity of maleimides toward cysteine ​​residues. One conventional method used in ADC design involves the use of self-hydrolyzing maleimides for cysteine ​​modification, as maleimides react rapidly and selectively with thiols. While maleimide conjugation has resulted in stable drug-protein conjugates, self-hydrolyzing maleimides produce acid species, which can have unexpected and detrimental effects on the properties of the resulting ADC. The maleimide moiety is directly related to the instability of maleimide-based ADCs in plasma. Furthermore, ADCs have been shown to exhibit a decreased drug-antibody ratio (DAR) in the presence of glutathione (see, e.g., WIPO2013 / 173337; Alley et al. 2008 Bioconjugate Chem. 19, 759). Summary of the Invention [Problem to be solved by the invention]

[0005] There remains a need for drug conjugates and conjugation methods that feature improved efficiency, stability, and drug loading. [Means for solving the problem]

[0006] Summary of the Invention Provided herein are drug conjugates comprising a targeting moiety, a linker, and a drug moiety, methods for making same, and methods for using same to treat and / or prevent conditions.

[0007] In particular, the present invention provides a linker strategy that significantly expands the available cysteine-targeting electrophilic species, which have been shown to exhibit high selectivity for thiols (e.g., cysteine) over other reactive functional groups, such as amines. The linker compounds and methods disclosed herein enable drug-linker conjugates with efficient conjugation at low pH (e.g., a pH of about 6.0), no retro-Michael unconjugation, and overall good stability at room temperature.

[0008] In some embodiments, the present invention provides a drug conjugate generally comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (I): [ka] [During the ceremony: Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 It is aryl. or a pharmaceutically acceptable salt thereof.

[0009] In other aspects, the invention generally relates to drug conjugates comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (II): [ka] [During the ceremony: Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; and Each Y is independently a peptide or antigen-binding moiety that contains a cysteine ​​moiety. or a pharmaceutically acceptable salt thereof.

[0010] In yet another aspect, the invention generally relates to compositions comprising the drug conjugates disclosed herein.

[0011] In yet another aspect, the invention generally relates to pharmaceutical compositions comprising a drug conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0012] In yet another aspect, the invention generally relates to unit dosage forms comprising the pharmaceutical compositions of the invention.

[0013] In yet another aspect, the present invention generally relates to compounds useful in forming linker moiety-drug moiety conjugates, targeting moiety-linker moiety conjugates, or targeting moiety-linker moiety-drug moiety conjugates, comprising a compound having structural formula (III): [ka] [During the ceremony, Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C2-6 Alkyne, C 6-10 aryl, and W; Each R 5 and R Y are independently H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 and W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; and R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 It is aryl. The present invention relates to a compound having a structure comprising:

[0014] In yet another aspect, the present invention generally relates to compositions comprising the compounds disclosed herein that are useful for forming linker moiety-drug moiety conjugates, targeting moiety-linker moiety conjugates, or targeting moiety-linker moiety-drug moiety conjugates.

[0015] In yet another aspect, the invention generally relates to a method of making a linker-drug conjugate, comprising: (a)Structural formula: [ka] preparing a compound having (b) reacting the compound with a precursor of the spacer, a precursor of the peptide, and / or a precursor of the self-immolative moiety to form a compound having the structural formula: [ka] [During the ceremony, Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; L Ais a spacer moiety or is absent; L B is a peptide moiety or is absent; L C is a self-sacrificing part or is absent; and D' is a drug moiety D or is absent. and forming a conjugate having

[0016] In yet another aspect, the present invention generally relates to a method of treating and / or preventing a condition in a subject in need thereof, comprising administering to said subject a drug conjugate of the present invention.

[0017] In yet another aspect, the invention generally relates to the use of a drug conjugate disclosed herein for the manufacture of a medicament.

[0018] In yet another aspect, the present invention relates generally to the use of the drug conjugates disclosed herein for the treatment of one or more autoimmune disorders, or infectious diseases. [Brief explanation of the drawings]

[0019] [Figure 1] Some exemplary RP-HPLC data is shown.

[0020] [Figure 2] Some exemplary RP-HPLC and HIC data are shown.

[0021] [Figure 3] Some exemplary LCMS data is shown. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention As shown herein, novel linkers, coupling methods and conjugates, and drug conjugates have been developed that have unexpected advantages over the prior art.

[0023] More notably, the linkers, linker conjugates, and conjugation strategies disclosed herein significantly expand the available cysteine-targeting electrophilic species, demonstrating high selectivity for thiols (e.g., cysteine) over other reactive functional groups, such as amines. The present invention offers the ability to efficiently perform conjugation at low pH (e.g., a pH of about 6.0) without retro-Michael unconjugation, thus leading to high stability. Furthermore, the drug-linker conjugates of the present invention have good overall stability at room temperature.

[0024] The present invention further defines drug conjugates, methods for their preparation, kits containing these drug conjugates and components thereof, and methods of using the drug conjugates and kits in the treatment of one or more diseases or conditions.

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. General principles of organic chemistry and specific functional moieties and reactivities are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 2006.

[0026] The following terms are intended to have the following meanings, unless otherwise indicated according to the context in which the term appears.

[0027] Ranges provided herein should be understood as abbreviations of all values ​​within that range. For example, a range of 1 to 16 should be understood to include any number, combination of numbers, or subrange of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0028] As used herein, "at least" a particular value should be understood to include that value and all values ​​greater than that value.

[0029] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0030] Unless otherwise specified or clear from context, the term "or" as used herein should be understood to be inclusive.

[0031] Any composition or method disclosed herein may be combined with one or more of the other compositions and methods provided herein.

[0032] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as a single group or combination of groups in that list. The recitation of an embodiment of a variable or aspect herein includes that embodiment as a single embodiment or in combination with any other embodiment or portion thereof.

[0033] The term "comprising," when used in defining compositions and methods, is intended to mean that the compositions and methods include the elements recited, but do not exclude other elements. The term "consisting essentially of," when used in defining compositions and methods, means that the compositions and methods include the elements recited, but exclude other elements of any essential significance to the compositions and methods. For example, "consisting essentially of" includes the administration of specified pharmacologically active agents, but excludes unspecified pharmacologically active agents. The term "consisting essentially of" does not exclude pharmacologically non-active or inactive agents, such as pharmaceutically acceptable additives, carriers, or diluents. The term "consisting of," when used in defining compositions and methods, means the exclusion of trace elements and substantial method steps of other ingredients. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0034] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, atropisomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as being within its scope. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are intended to be encompassed by the present invention. In certain embodiments, each asymmetric atom is in the R- or S-configuration in at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess. For optically active compounds, it is often preferable to use one enantiomer to the substantial exclusion of the other enantiomer.

[0035] Isomeric mixtures of any of a variety of isomer ratios can be utilized according to the present invention. For example, when combining only two isomers, mixtures containing isomer ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are contemplated by the present invention. Those skilled in the art will recognize that similar ratios are contemplated for more complex isomer mixtures.

[0036] For example, if a specific enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivatization with a chiral auxiliary, which separates the resulting diastereomeric mixture and cleaves the auxiliary group to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group such as amino, or an acidic functional group such as carboxyl, diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers so formed by fractional crystallization or chromatographic methods well known in the art, followed by recovery of the pure enantiomers.

[0037] Isomeric mixtures can be separated on the basis of the physical chemical differences of the constituents into pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0038] Definitions of specific functional groups and chemical terms are described in more detail below. When a range of values ​​is described, it is intended that each value and subrange within that range be encompassed. For example, "C 1-6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 It is intended to include alkyl.

[0039] When a substituent is specified by a conventional chemical formula written from left to right, it equally encompasses the chemically identical substituent resulting from writing the structure from right to left, e.g., -C(=O)-O- is equivalent to -OC(=O)-.

[0040] The structures of the compounds of the present invention are limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group can be substituted with one or more substituents, such substitutions are selected to result in compounds that conform to the principles of chemical bonding and that are not known to those skilled in the art as potentially being inherently unstable and / or unstable under environmental conditions (e.g., aqueous, neutral, and several known physiological conditions).

[0041] The use of numerical values ​​in the various quantitative values ​​specified herein, unless otherwise expressly stated, is described as approximations, such that the minimum and maximum values ​​within the stated range are preceded by the word "about." Although not always expressly stated, all numerical designations should be understood to be preceded by the word "about." Such range formats should be understood as being used for convenience and brevity and should be understood flexibly to include the numerical values ​​expressly specified as range limits, but also to include individual numerical values ​​or subranges within such ranges as if each numerical value and subrange were expressly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include not only the expressly recited limits of about 1 and about 200, but also individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc. Although not always expressly stated, it should also be understood that the reagents described herein are merely examples, and equivalents thereof are known in the art.

[0042] Unless otherwise specified or clear from the context, the term "about," as used herein, when referring to a measurable value such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0043] The terms "treatment" and "treating" as used herein with respect to a condition refer to partially or completely alleviating the condition; slowing the progression or progression of the condition; eliminating, alleviating, or slowing the progression of one or more symptoms associated with the condition; or increasing progression-free or overall survival of the condition.

[0044] Treatment may be directed at one or more of the effects or symptoms of a disease and / or the underlying pathology. Treatment may be any alleviation, including, but not limited to, the complete disappearance of the disease or symptoms of the disease. Thus, treating or treatment refers to any indicator of successful treatment or amelioration of an injury, disease, pathology, or condition, including any objective or subjective parameter, such as relief; remission; reduction of symptoms or making the injury, pathology, or condition more tolerable for the patient; slowing the rate of degeneration or decline; reducing the debilitating end point of degeneration; or improving the patient's physical or mental well-being. Treatment or amelioration of symptoms may be based on objective or subjective parameters, such as the results of a physical examination, neuropsychiatric examination, and / or psychological evaluation. Compared to comparable untreated controls, the degree of such reduction or improvement may be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique.

[0045] The treatment method includes administering a therapeutically effective amount of a compound described herein to a subject. The administration step can be a single administration or can include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the compound, the activity of the composition used for treatment, or a combination thereof. It is also recognized that the effective dosage of the agent used for treatment can increase or decrease over the course of a particular treatment regimen. The dosage can be modified and will be revealed by the results of standard diagnostic assays known in the art. In some cases, chronic administration may be necessary. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient.

[0046] The terms "prevention" and "preventing," as used herein with respect to a condition, refer to preventing the onset of a condition or reducing the likelihood of the onset or recurrence of a condition, including in subjects who may be predisposed to the condition but have not yet been diagnosed as having the condition.

[0047] As used herein, the terms "disease," "condition," or "disorder" are used interchangeably herein and refer to a pathological condition, e.g., one that can be identified by symptoms or other identifying factors as a deviation from a healthy or normal state. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic disorders.

[0048] The term "cancer" can refer to any accelerated cell proliferation, including solid tumors, ascites tumors, hematologic or lymphatic or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignancies, angiogenesis associated with malignancies, or other forms of cancer. Thus, the terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer.

[0049] The term "autoimmune disorder" can refer to a set of persistent, organ-specific or systemic clinical symptoms and signs associated with altered immune homeostasis manifested by qualitative and / or quantitative deficiencies in the expressed autoimmune repertoire.

[0050] The term "infectious disease" can refer to any disease caused by an infectious organism, such as a virus, bacterium, parasite, and / or fungus.

[0051] As used herein, the term "in need of" a treatment refers to a subject who would benefit biologically, medically, or in quality of life from such treatment.

[0052] The term "alkyl" describes an aliphatic hydrocarbon, including straight-chain and branched-chain groups.

[0053] The term "heteroalkyl" describes aliphatic hydrocarbons including straight- and branched-chain groups substituted with one or more atoms such as nitrogen, oxygen, and sulfur.

[0054] As used herein, the term "amino acid" refers to a molecule of the general formula NH2-CHR-COOH, where "R" is one of several different side chains or a residue in a peptide bearing the parent amino acid. Amino acids include naturally occurring amino acids where "R" is a substituent found in naturally occurring amino acids. "R" can also be a substituent not found in naturally occurring amino acids. The term "amino acid residue" refers to the portion of an amino acid that remains after loss of a water molecule when attached to another amino acid. The term "modified amino acid" refers to an amino acid bearing an "R" substituent that does not correspond to one of the 20 genetically encoded amino acids.

[0055] As used herein, the term "antibody" refers to an immunoglobulin molecule or immunologically active portion thereof that binds to a specific antigen, e.g., a cancer cell antigen, a viral antigen, or a microbial antigen. In embodiments where the targeting moiety is an antibody and the antibody is a full-length immunoglobulin molecule, the antibody has two heavy chains and two light chains, and each heavy and light chain contains three complementarity-determining regions (CDRs). In embodiments where the targeting moiety is an antibody and the antibody is an immunologically active portion of an immunoglobulin molecule, the antibody can be, for example, a Fab, Fab', Fv, F(ab')2, disulfide-linked Fv, scFv, single-domain antibody (dAb), bispecific antibody, triabody, tetrabody, or linear antibody. The antibody used as the targeting moiety can be, for example, a natural antibody, synthetic antibody, monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, multispecific antibody, bispecific antibody, dual-specific antibody, anti-idiotypic antibody, or fragment thereof that retains the ability to bind to a specific antigen.

[0056] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or using other methods commonly used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Salts include sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, p-toluenesulfonate, undecanoate, valerate, etc. In certain embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0057] Salts can be prepared in situ during the isolation and purification of the disclosed compounds, or separately, such as by reacting the free base or free acid of the parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed, where appropriate, with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. Organic bases which can form salts include, for example, primary, secondary, and tertiary amines, including naturally occurring substituted amines, substituted amines, cyclic amines, basic ion exchange resins, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, and others. In certain embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts. obtain.

[0058] As used herein, the term "pharmaceutically acceptable" excipient, carrier, or diluent refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, that is involved in the transport or transfer of a subject drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; additives such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0059] As used herein, the terms "protein" and "polypeptide" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within this definition. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, polypeptide can refer to proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate or accidental. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0060] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, a human, a non-human primate, a rodent, etc., that is the recipient of a particular treatment. Subjects to which administration is intended include humans (e.g., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or other non-human animals, e.g., commercially relevant mammals such as non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); cows, pigs, horses, sheep, goats, cats, and / or dogs), rodents (e.g., rats and / or mice). In some embodiments, non-human animals include, but are not limited to, mammals. Non-human animals can be male or female at any stage of development. Non-human animals can be transgenic animals. Typically, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0061] The ranges described herein are intended as continuous ranges, including every value between the minimum and maximum values ​​recited, and any ranges created by such values. Also disclosed herein are any and all ratios (and any such ratio ranges) that may be created by dividing a disclosed numerical value by any other disclosed numerical value. Accordingly, one of ordinary skill in the art will recognize that many such ratios, ranges, and ratio ranges may be derived uniquely from the numerical values ​​set forth herein, and in all cases, such ratios, ranges, and ratio ranges represent various embodiments of the present invention.

[0062] Targeting Moiety-Linker-Drug Conjugates In some embodiments, provided herein are drug conjugates comprising a linker, a drug moiety, and a targeting moiety. Also provided herein are components of these drug conjugates, including, for example, linkers, linker-drug moiety conjugates, and linker-targeting moiety conjugates.

[0063] In some embodiments, the present invention generally relates to a drug conjugate comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (I): [ka] [During the ceremony: Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 It is aryl. or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments, R 6 is unsubstituted or substituted C 1-6 It is alkyl.

[0065] In certain embodiments, the drug conjugate of (I) is: [ka] having a structural formula selected from:

[0066] In certain embodiments, the drug conjugate of (I) is: [ka] having a structural formula selected from:

[0067] (I)~(I 10 In certain embodiments of the drug conjugate of formula (I), R 2 , R 3 and R 4one of which is selected from the group consisting of NO2, CN, CF3 and F.

[0068] In some embodiments, R is H. In some embodiments, R is unsubstituted or substituted C 1-6 It is alkyl.

[0069] In some embodiments, R 2 , R 3 and R 4 One of them is NO2.

[0070] In some embodiments, R 2 , R 3 and R 4 One of them is CN.

[0071] In some embodiments, R 2 or R 4 One of them is CF3.

[0072] In some embodiments, R 4 is C(O)OR and R 2 is not CF3.

[0073] In some embodiments, R 2 is not CF3.

[0074] In some embodiments, R 2 or R 4 is F.

[0075] In certain embodiments of (I), the drug conjugate is: [ka] having a structural formula selected from:

[0076] In certain embodiments of (I), the drug conjugate is: [ka] having a structural formula selected from:

[0077] In some embodiments of (I), the linker moiety is (I 1 ) or (I 7 ) structural formula.

[0078] In some embodiments of (I), the linker moiety is (I 2 ) or (I 8 ) structural formula.

[0079] In some embodiments of (I), the linker moiety is (I 3 ) or (I 9 ) structural formula.

[0080] In some embodiments of (I), the linker moiety is (I 4 ) or (I 10 ) structural formula.

[0081] In some embodiments of (I), the linker moiety is (I 5 ) or (I 6 ) structural formula.

[0082] In some embodiments of (I), the linker moiety further comprises a spacer, a peptide moiety, and / or a self-immolative moiety. In some embodiments, the spacer, peptide moiety, and / or self-immolative moiety is R 2 , R 3 , R 4 and R 5 It is conjugated to one of the following.

[0083] In certain embodiments of (I), the drug conjugate is: [ka] [During the ceremony, L A is a spacer moiety or is absent; L B is a peptide moiety or is absent; and L C is a self-sacrificing part or does not exist. having a structural formula selected from:

[0084] In certain embodiments, the drug conjugate further comprises a drug moiety D: [ka] wherein D is a drug moiety. It has.

[0085] In certain embodiments of (I), the drug conjugate is: [ka] having a structural formula selected from:

[0086] In certain embodiments of (I), the drug conjugate is: [ka] having a structural formula selected from:

[0087] (I 11 )~(I 30 In certain embodiments of ), the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide.

[0088] (I 11 )~(I 30 In some embodiments, the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5, or 6) amino acid units.

[0089] (I 11 )~(I 30 In certain embodiments of (a), one or more of the amino acids are naturally occurring amino acids. In certain embodiments, one or more of the amino acids are unnatural amino acids.

[0090] (I 11 )~(I 30 In some embodiments of), the self-immolative moiety is: [ka] is selected from the group consisting of:

[0091] In certain embodiments, the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

[0092] In certain embodiments, the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

[0093] In certain embodiments, the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:16 (e.g., about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15 or about 1:16).

[0094] In other aspects, the invention generally relates to drug conjugates comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (II): [ka] [During the ceremony: Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 aryl, or W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] Selected from; R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; and Each Y is independently a peptide or antigen-binding moiety that contains a cysteine ​​moiety. or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, R 6 is unsubstituted or substituted C 1-6 It is alkyl.

[0096] In certain embodiments of (II), the drug conjugate is: [ka] having a structural formula selected from:

[0097] In certain embodiments of (II), the drug conjugate is: [ka] having a structural formula selected from:

[0098] (II)~(II 10 In some embodiments of 2 , R 3 and R 4 one of which is selected from the group consisting of NO2, CN, CF3 and F.

[0099] (II)~(II 10 In some embodiments of the formula (I), R is H. In some embodiments, R is unsubstituted or substituted C 1-6 It is alkyl.

[0100] In some embodiments, R 2 , R 3 and R 4 One of them is NO2.

[0101] In some embodiments, R 2 , R 3 and R 4 One of them is CN.

[0102] In some embodiments, R 2 or R 4 is CF3.

[0103] In some embodiments, R 4 is C(O)OR and R 2 is not CF3.

[0104] In some embodiments, R 2 is not CF3.

[0105] In some embodiments, R 2 or R 4 is F.

[0106] In certain embodiments of (II), the drug conjugate is: [ka] having a structural formula selected from:

[0107] In certain embodiments of (II), the drug conjugate is: [ka] having a structural formula selected from:

[0108] In some embodiments of the drug conjugate, the linker moiety further comprises a spacer, a peptide moiety, and / or a self-immolative moiety.

[0109] In certain embodiments of (II), the drug conjugate is: [ka] [During the ceremony, L A is a spacer moiety or is absent; L B is a peptide moiety or is absent; and L C is a self-sacrificing part or does not exist. having a structural formula selected from:

[0110] In certain embodiments, the drug conjugate further comprises: [ka] wherein D is a drug moiety. The drug moiety has a structural formula selected from:

[0111] In certain embodiments, the drug conjugate comprises: [ka] having a structural formula selected from:

[0112] In certain embodiments, the drug conjugate further comprises [ka] The drug moiety has a structural formula selected from:

[0113] (II 11 )~(II 30 In some embodiments of ), the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, PEG, and peptide.

[0114] (II 11 )~(II 30 In some embodiments, the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5, or 6) amino acid units.

[0115] (II 11 )~(II 30 In certain embodiments of (a), one or more of the amino acids are naturally occurring amino acids. In certain embodiments, one or more of the amino acids are unnatural amino acids.

[0116] (II 11 )~(II 30 In some embodiments of), the self-immolative moiety is: [ka] is selected from the group consisting of:

[0117] In certain embodiments, the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

[0118] In certain embodiments, the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

[0119] In certain embodiments, the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:16 (e.g., about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15 or about 1:16).

[0120] In yet another aspect, the invention generally relates to compositions comprising the drug conjugates disclosed herein.

[0121] In yet another aspect, the invention generally relates to pharmaceutical compositions comprising a drug conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0122] The present invention therefore provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound or immunoconjugate of the invention.

[0123] Examples of additives that may be useful include, but are not limited to, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine ​​hydrochloride, starch, cellulose, and gums. In a preferred embodiment, the pharmaceutical compositions of the present invention are formulated into pharmaceutical forms for administration as solids (e.g., tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide a liquid form, etc.), liquids (e.g., solutions, suspensions, emulsions, elixirs, lotions, ointments, etc.), or semisolids (gels, ointments, creams, and the like). The pharmaceutical compositions of the present invention may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intraspinal, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. A review of various dosage forms of the active principle, the additives used, and their preparation methods can be found in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20 thThe pharmaceutical compositions may be found in the "Pharmaceuticals and Pharmaceuticals, 2000" edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable vehicles are known in the art and include phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, various types of moisturizing agents, sterile solutions, etc. Compositions containing such vehicles may be formulated by conventional procedures known in the art. Preservatives, stabilizers, dyes and flavors, antioxidants, and / or suspending agents may be provided in the pharmaceutical compositions. For example, sodium benzoate, ascorbic acid, and p-hydroxybenzoic acid esters may be added as preservatives.

[0124] In yet another aspect, the invention generally relates to unit dosage forms comprising the pharmaceutical compositions of the invention.

[0125] Linkers, linker-drug conjugates, targeting moiety-linker conjugates In yet another aspect, the present invention generally relates to compounds useful in forming linker moiety-drug moiety conjugates, targeting moiety-linker moiety conjugates, or targeting moiety-linker moiety-drug moiety conjugates, comprising a compound having structural formula (III): [ka] (III) Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; Each R 5 and R Y are independently H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 and W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] Selected from; R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; and R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 It is aryl. The present invention relates to a compound having a structure comprising:

[0126] In some embodiments, R 6 is unsubstituted or substituted C 1-6 It is alkyl.

[0127] In some embodiments of (III), R Y is SO2R 6 and the compound has the structural formula: [ka] It has.

[0128] (III) or (III 1 In some embodiments of 2 , R 3 and R 4 one of which is selected from the group consisting of NO2, CN, CF3 and F.

[0129] (III) or (III 1 In some embodiments of (III), R is H. 1 In some embodiments of the formula (I), R is unsubstituted or substituted C 1-6It is alkyl.

[0130] (III) or (III 1 In some embodiments of 2 , R 3 and R 4 One of them is NO2.

[0131] (III) or (III 1 In some embodiments of 2 , R 3 , R 4 and R 5 One of them is CN.

[0132] (III) or (III 1 In some embodiments of 2 or R 4 is CF3.

[0133] (III) or (III 1 In some embodiments of 4 is C(O)OR and R 2 is not CF3.

[0134] (III) or (III 1 In some embodiments of 2 is not CF3.

[0135] (III) or (III 1 In some embodiments of 2 or R 4 is F.

[0136] (III) or (III 1 In some embodiments of 2 , R 3 , R 4 and R 5 One of them is C(O)NHR'.

[0137] In some embodiments of (III), the drug conjugate is: [ka] [During the ceremony, L A is a spacer moiety or is absent; L B is a peptide moiety or is absent; L C is a self-sacrificing part or is absent; R 6 and R' 6 may be the same or different, and may be unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; and D' is a drug moiety D or is absent. having a structural formula selected from:

[0138] In some embodiments of (III), the drug conjugate is: [ka] The compound has a structure selected from:

[0139] In some embodiments, R 6 and R' 6 Each of is methyl.

[0140] (III 2 )~(III 13 In some embodiments of ), the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, PEG, and peptide.

[0141] (III 2 )~(III 13 In some embodiments, the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5, or 6) amino acid units.

[0142] (III 2 )~(III 13In certain embodiments of (a), one or more of the amino acids are naturally occurring amino acids. In certain embodiments, one or more of the amino acids are unnatural amino acids.

[0143] (III 11 )~(III 30 In some embodiments of), the self-immolative moiety is: [ka] is selected from the group consisting of:

[0144] In certain embodiments, the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

[0145] In yet another aspect, the present invention generally relates to compositions comprising the compounds disclosed herein that are useful for forming linker moiety-drug moiety conjugates, targeting moiety-linker moiety conjugates, or targeting moiety-linker moiety-drug moiety conjugates.

[0146] Manufacturing method In yet another aspect, the invention generally relates to a method of making a linker-drug conjugate, comprising: (a)Structural formula: [ka] preparing a compound having (b) reacting the compound with a precursor of the spacer, a precursor of the peptide, and / or a precursor of the self-immolative moiety to form a compound having the structural formula: [ka] [During the ceremony, Each R 2 , R 3 , R 4 are independently H, NO2, CN, CF3, F, unsubstituted or substituted C 1-6 Alkyl, C 2-6Alkien, C 2-6 Alkyne, C 6-10 aryl, and W; R 5 are H, Cl, and SOR 6 , SO2R 6 , OR 7 , NRR 7 Or W; R 6 is unsubstituted or substituted C 1-6 Alkyl or C 6-10 is aryl; R 7 is unsubstituted or substituted C 2-6 Alkyne or C 2-6 is an alkyne; W is: [ka] selected from the group consisting of: R 1 is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; R is H, unsubstituted or substituted C 1-6 Alkyl, C 2-6 Alkien, C 2-6 Alkyne or C 6-10 is aryl; L A is a spacer moiety or is absent; L B is a peptide moiety or is absent; L C is a self-sacrificing part or is absent; and D' is a drug moiety D or is absent. and forming a conjugate having

[0147] In certain embodiments, the conjugation step (b) is carried out at a pH in the range of 5.5 to 6.5 (eg, about 6.0).

[0148] In one embodiment, L A is the spacer moiety, and L B is the peptide moiety, and L C is the self-immolating moiety and D' is the drug moiety.

[0149] In certain embodiments, the method further comprises reacting the resulting drug conjugate with a targeting moiety to form a targeting moiety-linker-drug conjugate.

[0150] In some embodiments of the method, the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, PEG, and peptide.

[0151] In some embodiments, the peptide moiety has 1 to 6 (eg, 1, 2, 3, 4, 5, or 6) amino acid units.

[0152] In certain embodiments, one or more of the amino acids are naturally occurring amino acids. In certain embodiments, one or more of the amino acids are unnatural amino acids.

[0153] In some embodiments, the self-immolative moiety is: [ka] is selected from the group consisting of:

[0154] In certain embodiments, the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

[0155] In certain embodiments, the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

[0156] In certain embodiments, the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:16 (e.g., about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15 or about 1:16).

[0157] In certain embodiments, the targeting moiety is an antibody that includes a cysteine ​​residue, and the method comprises reducing the cysteine ​​residue to form a sulfhydryl group and reacting the sulfhydryl group with one or more linker moieties described herein.

[0158] In certain embodiments, the targeting moiety is an antibody fragment containing a cysteine ​​residue, and the method comprises reducing the cysteine ​​residue to form a sulfhydryl group and reacting the sulfhydryl group with one or more linker moieties described herein.

[0159] In certain embodiments, the targeting moiety is a protein ligand that includes a cysteine ​​residue, and the method comprises reducing the cysteine ​​residue to form a sulfhydryl group and reacting the sulfhydryl group with one or more linker moieties described herein.

[0160] In certain embodiments, the targeting moiety is a protein scaffold that includes a cysteine, and the method comprises reducing the cysteine ​​residue to form a sulfhydryl group and reacting the sulfhydryl group with one or more linker moieties described herein.

[0161] In certain embodiments, the targeting moiety is a cysteine, including a cysteine, and the method comprises reducing the cysteine ​​residue to form a sulfhydryl group and reacting the sulfhydryl group with one or more linker moieties described herein.

[0162] In certain embodiments, the conjugation of the linker moiety and the targeting moiety does not produce harmful by-products. Non-limiting examples of harmful by-products include acids, bases, or combinations thereof.

[0163] In some embodiments, the drug conjugate has high drug loading. For example, in some embodiments, the molar ratio of targeting moiety to drug moiety is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, or about 1:16.

[0164] In certain embodiments, the drug conjugate is stable in vivo (eg, is not subject to the process of unconjugation).

[0165] Uses of Compounds and Drug Conjugates In yet another aspect, the present invention generally relates to a method of treating and / or preventing a condition in a subject in need thereof, comprising administering to said subject a drug conjugate of the present invention.

[0166] In certain embodiments, the condition is cancer, an autoimmune disorder, or an infectious disease.

[0167] In certain embodiments, a method of treating and / or preventing a condition in a subject in need thereof comprises administering to the subject one or more drug conjugates of the invention, wherein upon administration to the subject, the drug moiety releases the drug conjugate. In certain embodiments, the drug moiety releases the drug conjugate by self-immolative cleavage of the self-immolative moiety.

[0168] In certain embodiments, a method of treating and / or preventing a condition comprises administering to a subject one or more drug conjugates of the invention, wherein upon administration to a subject, the drug moiety releases the drug conjugate according to exemplary Schemes 1-5, each of which involves a different self-immolative moiety.

[0169] In certain embodiments, proteolytic cleavage of the drug conjugate proceeds according to Scheme 1: [ka]

[0170] In certain embodiments, proteolytic cleavage of the drug conjugate proceeds according to Scheme 2: [ka]

[0171] In certain embodiments, proteolytic cleavage of the drug conjugate proceeds according to Scheme 3: [ka]

[0172] In certain embodiments, proteolytic cleavage of the drug conjugate proceeds according to Scheme 4: [ka]

[0173] In certain embodiments, proteolytic cleavage of the drug conjugate proceeds according to Scheme 5: [ka]

[0174] In yet another aspect, the present invention generally relates to a method of treating or ameliorating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein.

[0175] In some embodiments, the condition being treated and / or prevented is cancer. In some of these embodiments, the cancer is selected from the group consisting of adrenal gland cancer, anal region cancer, basal and squamous cell skin cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors (e.g., astrocytoma, glioblastoma multiforme, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer (ocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumors, gastrointestinal stromal tumors (gist), gestational trophoblastic disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, pulmonary carcinoid tumor, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer, nasal cancer, and thyroid cancer. Cancers of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, neoplasms of the central nervous system (CNS), oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor (NET), penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, squamous cell carcinoma, cancer of unknown primary site (CUP), environmentally induced cancer, combinations of cancers, and metastatic lesions of cancer. In some embodiments, the cancer is leukemia or lymphoma, for example, lymphoblastic lymphoma or B-cell non-Hodgkin's lymphoma.

[0176] In some of these embodiments, the cancer is a hematological malignancy. In certain embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), T-cell lymphoma, B-cell lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, and leukemia. The primary risk factors for leukemia are: lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.In other embodiments, the cancer is a human hematological malignancy, e.g., myeloid neoplasm, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplasia-associated changes, therapy-related AML, acute leukemia of ambiguous lineage, myeloproliferative neoplasm, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndrome (MDS), myeloproliferative / myelodysplastic syndrome, chronic myelofibrosis, myeloproliferative neoplasm ... Myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, myelodysplastic syndromes (MDS), refractory anemia with ringed sideroblasts, refractory cytopenia with polycythemia vera, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated del(5q), unclassifiable MDS, myeloproliferative / myelodysplastic syndrome, chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, juvenile myelomonocytic leukemia Hematologic malignancies, unclassifiable myeloproliferative / myelodysplastic syndrome, lymphoid neoplasms, precursor lymphoid neoplasms, B lymphoblastic leukemia, B lymphoblastic lymphoma, T lymphoblastic leukemia, T lymphoblastic lymphoma, mature B-cell neoplasms, generalized large B-cell lymphoma, primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, Burkitt lymphoma / leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell precursor lymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, marginal zone lymphoma, post-transplant lymphoproliferative disorder, HIV-associated lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma, or solitary plasmacytoma (isolated bone and extramedullary).

[0177] In some embodiments, the cancer comprises a solid tumor, hi some embodiments, the solid tumor is lung cancer, colorectal cancer, breast cancer, pancreatic cancer, gallbladder cancer, brain and spinal cord cancer, head and neck cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, renal cell carcinoma (RCC), bladder cancer, and hepatocellular carcinoma (HCC).

[0178] The methods of the present invention may further comprise administering one or more drug conjugates provided herein to treat and / or prevent cancer in a combination therapy. For example, in certain embodiments, the combination therapy comprises administering one or more drug conjugates (simultaneously or sequentially) and a chemotherapeutic agent. In further embodiments, the combination therapy comprises administering one or more drug conjugates and a secondary treatment such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or any combination thereof. In certain embodiments, the administration of one or more drug conjugates in combination with radiation therapy, an antibody agent, and / or a chemotherapeutic agent results in the enhancement of the radiation therapy, antibody agent, and / or chemotherapeutic agent, such that a low dose of radiation, antibody therapy, and / or chemotherapy can be effective for treatment and / or prevention.

[0179] In some embodiments, the condition to be treated and / or prevented is an autoimmune disorder. In some of these embodiments, the autoimmune disorder is selected from the group consisting of Th2 lymphocyte disorder, Th1 lymphocyte disorder, activated B lymphocyte disorder, active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reaction, allergic rhinitis, Alport syndrome, anaphylaxis, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behcet's disease, bird fancier's lung, bronchial asthma, Kaplan's syndrome, cardiomyopathy, celiac disease, Chagas' disease, chronic glomerulonephritis, Cogan's syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, and cryoglobulinemia. Gushing syndrome, dermatomyositis, discoid lupus erythematosus, Dressler syndrome, Eaton-Lambert syndrome, echovirus infection, encephalomyelitis, endocrine ophthalmopathy, Epstein-Barr virus infection, equine chronic pulmonary emphysema, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs heterochromatic iridocyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (type 1), Lambert-Eaton syndrome, laminitis, lichen planus, lupoid hepatitis , lupus, lymphopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, presenile dementia, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, recurrent miscarriage, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, Samter's syndrome, schistosomiasis, Schmidt's syndrome, scleroderma, Shulman's syndrome, Sjögren's syndrome, stiff-person syndrome, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type 1 diabetes mellitus, ulcerative colitis, uveitis, leukoplakia, Waldenström's macroglobulinemia, and granulomatosis with polyangiitis.

[0180] The methods of the present invention can further include administering one or more drug conjugates provided herein to treat and / or prevent an autoimmune disorder in a combination therapy. For example, in certain embodiments, the combination therapy includes administering one or more drug conjugates (simultaneously or sequentially) and a known therapeutic agent for treating and / or preventing an autoimmune disorder.

[0181] In some embodiments, the condition being treated and / or prevented is an infectious disease. In some of these embodiments, the infectious disease is a bacterial disease, a systemic fungal disease, a rickettsial disease, a parasitic disease, and / or a viral disease.

[0182] In certain embodiments, the one or more bacterial diseases are diphtheria, whooping cough, occult bacteremia, urinary tract infection, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid hyperplasia, retropharyngeal abscess, impetigo, ecthyma, pneumonia, endocarditis, septic arthritis, pneumococcal disease, peritonitis, bacteremia, and meningitis. Includes acute suppurative meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella, typhoid, dysentery, conjunctivitis, sinusitis, brucellosis, tularemia, cholera, bubonic plague, tetanus, necrotizing enterocolitis, actinomycosis, mixed anaerobic infections, syphilis, relapsing fever, leptospirosis, Lyme disease, rat-bite fever, tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial pneumonia, trachoma, and / or inclusion conjunctivitis.

[0183] In certain embodiments, the one or more systemic fungal diseases are selected from histoplasmosis, coccidioidomycosis, blastomycosis, sporotrichosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycetoma, and / or dematiaceous fungal infection.

[0184] In certain embodiments, the one or more rickettsial diseases are selected from typhus, Rocky Mountain spotted fever, ehrlichiosis, eastern tick-borne rickettsiosis, rickettsial pox, Q fever, and bartonellosis.

[0185] In certain embodiments, the one or more parasitic diseases are selected from malaria, babesiosis, African sleeping sickness, Chagas disease, leishmaniasis, Dum Dum fever, toxoplasmosis, meningoencephalitis, keratitis, amebiasis, giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis, microsporidiosis, ascariasis, whipworm infection, hookworm infection, nematode infection, ocular larva migrans, trichinosis, dracunculiasis, lymphatic filariasis, loiasis, river blindness, heartworm infection, schistosomiasis, cercarial dermatitis, paragonimiasis, Clonorchiasis orientalis, fascioliasis, fascioliasis, opisthorchiasis, tapeworm infection, hydatid disease, and alveolar hydatid disease.

[0186] In some embodiments, the one or more viral diseases are measles, subacute sclerosing panencephalitis, common cold, mumps, rubella, roseola, erythema infectiosum, chickenpox, respiratory syncytial virus infection, croup, bronchiolitis, infectious mononucleosis, polio, herpangina, hand, foot and mouth disease, Bornholm disease, genital herpes, genital warts, aseptic meningitis, myocarditis, and pericarditis. The disease is selected from gastroenteritis, acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus (HIV), Reye's syndrome, Kawasaki syndrome, influenza, bronchitis, viral "walking" pneumonia, acute febrile respiratory illness, acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), herpes zoster, cytomegalic inclusion body disease, rabies, progressive multifocal leukoencephalopathy, kuru, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, tropical spastic paraparesis, western equine encephalitis, California encephalitis, St. Louis encephalitis, yellow fever, dengue, lymphocytic choriomeningitis, Lassa fever, hemorrhagic fever, hantavirus pulmonary syndrome, Marburg virus infection, Ebola virus infection, and / or smallpox.

[0187] The methods of the present invention can further include administering one or more drug conjugates provided herein to treat and / or prevent an infectious disease in a combination therapy. For example, in certain embodiments, the combination therapy includes administering one or more drug conjugates (simultaneously or sequentially) and a known therapeutic agent for treating and / or preventing an infectious disease.

[0188] Non-limiting examples of compounds of the present invention are: [ka] [ka] [ka] Includes.

[0189] Non-limiting examples of compounds of the present invention are also: [ka] [ka] [ka] Includes.

[0190] Spacer In some embodiments, the spacer moiety comprises an alkyl chain. In some embodiments, the spacer moiety has the formula: -(CH) n (wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the spacer moiety comprises a heteroalkyl chain. In some embodiments, the spacer moiety has the formula: -(CHCH0) n (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0191] In certain embodiments, the alkyl is a lower alkyl having 1 to 4 carbon atoms (eg, methyl, ethyl, propyl, and butyl).

[0192] In some embodiments, the spacer moiety comprises a peptide. In some embodiments, the peptide comprises two or more amino acids, e.g., a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some of these embodiments, the spacer moiety comprises Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, or a combination thereof.

[0193] In some embodiments, the spacer moiety comprises a combination of alkyl, heteroalkyl, PEG, or peptide. For example, the spacer moiety is -(CH) n and peptide, the spacer portion is -(CH2CH2O) n and peptide, the spacer moiety comprises PEG and peptide, the spacer moiety comprises -(CH2) n and PEG, or the spacer moiety is -(CH2CH2O) n and PEG.

[0194] In some embodiments, the polypeptide portion comprises 1 to 6 amino acids. For example, the polypeptide can comprise 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids.

[0195] The polypeptide moiety may comprise one or more natural amino acids and / or one or more unnatural amino acids. In some embodiments, the natural amino acids are one or more of the 20 common amino acids selected from one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. As used herein, the term "unnatural amino acid" refers to any amino acid, modified amino acid, and / or an amino acid analog that is not one of the 20 common naturally occurring amino acids. Non-limiting examples of unnatural amino acids include N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0196] Self-sacrifice part A "self-immolative moiety" refers to a chemical moiety that can covalently link two chemical moieties, e.g., a polypeptide moiety and a drug moiety. The self-immolative spacer can spontaneously detach from the drug moiety once the bond to the polypeptide is cleaved, e.g., by proteolytic cleavage.

[0197] In some embodiments, the self-immolative moiety is: [ka] is selected from.

[0198] In some embodiments, the linkers provided herein are modified when conjugated to a drug moiety and / or targeting moiety, e.g., in a linker-drug moiety conjugate, linker-targeting moiety conjugate, or drug conjugate provided herein. For example, if the linker contains a hydroxyl group, the hydroxyl group can react with a functional group on the drug moiety or targeting moiety during the conjugation reaction to produce a conjugate in which the linker no longer contains a hydroxyl group.

[0199] Drug portion The drug moiety in the drug conjugates and components thereof provided herein can be any compound or molecule that produces a therapeutic effect, including both small molecules and biologics. By way of example, the drug moiety can be a chemical agent, such as an antibiotic, an anticancer agent, a polypeptide, or a nucleic acid.

[0200] In certain embodiments, the drug moiety is a chemotherapeutic agent, an immunomodulator, a tubulin binding agent, a DNA alkylating agent, an HSP90 inhibitor, a DNA topoisomerase, an anti-epigenetic agent, an HDAC inhibitor, an antimetabolite, a proteasome inhibitor, a peptide, a peptidomimetic, an siRNA, and / or an antisense DNA.

[0201] In some embodiments, the drug is a chemotherapy drug. Non-limiting examples of chemotherapy drugs include alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antimetabolites, hormone therapy, kinase inhibitors, and / or antibiotics.

[0202] In some embodiments, the alkylating agent is chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, nobembine, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065 (e.g., adozelesin, carzelesin, and bizelesin synthetic analogs); duocarmycins (e.g., synthetic analogs, KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., pyrrolobenzodiazepines, dimers of benzodiazepines (PBDs) or tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidino-benzodiazepines), nitrosoureas (e.g., carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine), alkylsulfonates (e.g., busulfan, treosulfan, improsulfan, and piposulfan); triazenes (e.g., dacarbazine), platinum-containing compounds (e.g., carboplatin, cisplatin, oxaliplatin), and / or aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa).

[0203] In some embodiments, the plant alkaloid is chosen from one or more of vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbine), taxoids (e.g., paclitaxel and docetaxol), maytansinoids (e.g., DM1, DM2, DM3, DM4, maytansine, and ansamitocins), cryptophycins (e.g., cryptophycin 1 and cryptophycin 8), epothilones, eleutherobin, discodermolides, bryostatin, dolostatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictiin, and / or spongistatin.

[0204] In certain embodiments, the DNA topoisomerase inhibitor is chosen from one or more of epipodophyllins (e.g., 9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)) and / or mitomycins (e.g., mitomycin C).

[0205] In some embodiments, the antimetabolite is an antifolate, such as a DHFR inhibitor (e.g., methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or other folic acid analogs); an IMP dehydrogenase inhibitor (e.g., mycophenolic acid, tiazofurin, ribavirin, EICAR); a ribonucleotide reductase inhibitor (e.g., hydroxyurea, deferoxamine), a pyrimidine analog, such as a uracil analog: (e.g., ancitabine, azacitidine, and / or one or more of 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, raltitrexed (e.g., Tomudex), cytosine analogs (e.g., cytarabine, cytosine arabinoside, fludarabine), purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine), and / or folate replenishers (e.g., folinic acid).

[0206] In some embodiments, the hormone therapy is one of a receptor antagonist, such as an antiestrogen (e.g., megestrol, raloxifene, tamoxifen), an LHRH agonist (e.g., goserelin, leuprolide), an antiandrogen (e.g., bicalutamide, flutamide, calsterone, dromostanolone propionate, epitiostanol, mepitiostane, nilutamide, testolactone, trilostane, and other androgen inhibitors), a retinoid / deltoid (e.g., vitamin D3 analogs: CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); photodynamic therapy (e.g., verteporfin, phthalocyanines, photosensitizer Pc4, demethoxy-hypocrelin A), and a cytokine (e.g., interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), a human protein containing a TNF domain).

[0207] In some embodiments, the kinase inhibitor is BIBW 2992 (e.g., anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. One of vandetanib, E7080 (e.g., anti-VEGFR2), mubritinib, ponatinib (e.g., AP24534), bafetinib (e.g., INNO-406), bosutinib (e.g., SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, and / or ispinesib.

[0208] In certain embodiments, the antibiotic is an enediyne antibiotic (e.g., a calicheamicin, particularly calicheamicin gamma 1, delta 1, alpha 1, and beta 1), a dynemycin (e.g., dynemycin A and deoxydynemycin; esperamicin, kedarcidin, C-1027, maduropeptin, and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin , daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and / or zorubicin.

[0209] In some embodiments, the drug is an anti-autoimmune disease drug.Non-limiting examples of anti-autoimmune disease drugs include cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolic acid, prednisone, sirolimus, and tacrolimus.

[0210] In some embodiments, the anti-autoimmune disease drug is a polyketide (e.g., an acetogenin such as bullatacin and bullatacinone), gemcitabine, epoxomicin (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, stimuvax, allovectin-7, xegeva, provenzyl, yervoy, an isoprenylation inhibitor (e.g., lovastatin), ), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors), Ca 2+ The therapeutic agent is selected from one or more of an ATPase inhibitor (e.g., thapsigargin), a histone deacetylase inhibitor (e.g., vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD0103), belinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A), thapsigargin, celecoxib, a glitazone, epigallocatechin gallate, disulfiram, salinosporamide A, an antiadrenal agent, a urethane, an siRNA, an antisense drug, and / or a nuclease.

[0211] In some embodiments, the drug is an infectious disease drug. Non-limiting examples of infectious disease drugs include aminoglycosides, azidamfenicol, ansamycins, carbapenems, cephems, glycopeptides, glycylcyclines, β-lactamase inhibitors, lincosamides, lipopeptides, macrolides, monobactams, oxazolidinones, penicillins, polypeptides, quinolones, streptogramins, sulfonamides, steroid antibacterials, tetracyclines, and / or antibiotics.

[0212] In certain embodiments, the aminoglycoside is one or more of amikacin, astromycin, gentamicin (e.g., netilmicin, sisomicin, and isepamicin), hygromycin B, kanamycin (e.g., amikacin, arbekacin, bekanamycin, dibekacin, and tobramycin), neomycin (e.g., framycetin, paromomycin, and ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, and / or verdamycin.

[0213] In certain embodiments, the azidamfenicol is one of azidamfenicol, chloramphenicol, florfenicol, and / or thiamphenicol.

[0214] In certain embodiments, the ansamycin is one of geldanamycin and / or herbimycin.

[0215] In certain embodiments, the carbapenem is one or more of biapenem, doripenem, ertapenem, imipenem / cilastatin, meropenem, and / or panipenem.

[0216] In some embodiments, the cephem is a carbacephem (e.g., loracarbef), cephacetrile, cefaclor, cephradine, cefadroxil, cephalonium, cephaloridine, cephalothin or cephalocin, cephalexin, cephaloglycin, cefamandole, cephapirin, cefatrizine, cefazaflour, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamime cephalexin, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycins (e.g., cefoxitin, cefotetan, and cefmetazole), and / or oxacephems (e.g., flomoxef and latamoxef).

[0217] In some embodiments, the glycopeptide is one or more of bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), and ramoplanin.

[0218] In some embodiments, the glycylcycline is tigecycline.

[0219] In certain embodiments, the β-lactamase inhibitor is one of a penam (eg, sulbactam and tazobactam) and / or a clavam (eg, clavulanic acid).

[0220] In certain embodiments, the lincosamide is one or more of clindamycin and / or lincomycin.

[0221] In certain embodiments, the lipopeptide is one or more of daptomycin, A54145, and / or calcium-dependent antibiotic (CDA).

[0222] In certain embodiments, the macrolide is one or more of azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxthromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, and / or telithromycin.

[0223] In certain embodiments, the monobactam is selected from aztreonam and / or tigemonam.

[0224] In some embodiments, the oxazolidinone is linezolid.

[0225] In some embodiments, the penicillin is one or more of amoxicillin, ampicillin (e.g., pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (e.g., carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxillin, mecillinam (e.g., pivmecillinam), mezlocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, phenaticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, and / or ticarcillin.

[0226] In certain embodiments, the polypeptide is one or more of bacitracin, colistin, and / or polymyxin B.

[0227] In certain embodiments, the quinolone is chosen from one or more of alatrofloxacin, farofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, myoxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, and / or trovafloxacin.

[0228] In certain embodiments, the streptogramin is a pristinamycin, such as quinupristin and / or dalfopristin.

[0229] In certain embodiments, the sulfonamide is one or more of mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, and / or trimethoprim-sulfamethoxazole (co-trimoxazole).

[0230] In one embodiment, the steroid antibacterial is fusidic acid.

[0231] In certain embodiments, the tetracycline is one of doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocidine, methacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracidine, and / or a glycylcycline (e.g., tigecycline).

[0232] In some embodiments, the anti-infectious disease drug is an antibiotic selected from one or more of anonacin, arsphenamine, bactoprenol inhibitors (e.g., bacitracin), DADAL / AR inhibitors (e.g., cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e.g., fosmycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (e.g., rifampin), tazobactam tinidazole, and / or uvaricin.

[0233] Targeting section To date, numerous unique antigens have been identified and may have potential for use as targets in antibody-based therapies. Several factors are generally considered when selecting an antigen. First, the target antigen must be highly expressed in tumors and absent or low expression in healthy cells. An example is the HER2 receptor, which is approximately 100-fold more highly expressed in tumor cells than in healthy cells. Second, the target antigen must be presented on the tumor cell surface so that it is accessible to circulating monoclonal antibodies. Furthermore, the target antigen must possess internalization properties, as this facilitates the transport of ADCs into cells, which in turn enhances the efficacy of cytotoxic agents. Nevertheless, several studies have shown that non-internalizing ADC products targeting tumor microenvironment components efficiently shed drugs into the extracellular space, in some cases mediating potent therapeutic activity, and ADCs often induce potent "bystander effects" (Strohl WR 2018 Protein & Cell. 9(1):86-120; Damelin et al. 2015 Pharma. Res. 32(11):3494-507; Diamantis et al. 2016 British J. Cancer114(4):362-7; Tipton et al. 2015 Blood 125(12):1901-9; Donaghy et al. 2016 mAbs. 8(4):659-71; Casi et al. 2015 Molecular Pharmaceutics 12(6):1880-4).

[0234] The targeting moiety can be any moiety that selectively binds to the cell surface marker found on the target cell type. Generally, the antibody preferably has target specificity, delivers cytotoxic drugs to tumor cells, and has target binding affinity, i.e., high binding affinity to tumor cell surface antigens. Furthermore, the antibody preferably has good retention, low immunogenicity, low cross-reactivity, and suitable binding properties (Peters et al. 2015 Bioscience Reports 35(4); Hughes B 2010 Nature Reviews Drug Discovery 9(9):665-7).

[0235] In some embodiments, the targeting moiety is an antibody.

[0236] In some embodiments, the targeting moiety is a monoclonal antibody.

[0237] In some embodiments, the targeting moiety is a chimeric antibody.

[0238] In some embodiments, the targeting moiety is a humanized antibody.

[0239] In certain embodiments, the targeting moiety is a bispecific antibody.

[0240] In some embodiments, the targeting moiety is an antibody fragment.

[0241] In certain embodiments, the targeting moiety is a Fab fragment.

[0242] In some embodiments, the targeting moiety is a peptide.

[0243] In some embodiments, the targeting moiety is a small molecule ligand.

[0244] In some embodiments, the Ab is an antibody or antibody fragment (e.g., an antigen-binding fragment of an antibody) that specifically binds to an antigen that is primarily or preferentially found on the surface of cancer cells, e.g., a tumor-associated antigen.

[0245] In some embodiments, an Ab is an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to a cell surface receptor protein or other cell surface molecule, a cell survival regulator, a cell growth regulator, a molecule associated with, or known or suspected to play a functional role in, tissue progression or differentiation, a lymphokine, a cytokine, a molecule involved in cell cycle control, a molecule involved in vasculogenesis, or a molecule associated with, or known or suspected to play a functional role in, angiogenesis.

[0246] Thus, targeting moieties useful in the immunoconjugates of the invention include, but are not limited to, antibodies against cell surface receptors and tumor-associated or tumor-specific antigens, which are well known in the art and can be produced for use in antibody production using methods and information known in the art.

[0247] In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated or tumor-specific polypeptides that are differentially expressed on the surface of one or more specific types of cancer cells compared to one or more normal, non-cancerous cells. Tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells, while tumor-specific polypeptides are differentially expressed on the surface of one or more specific types of cancer cells but not on non-cancerous cells. The identification of such cell surface antigen polypeptides has provided the ability to specifically target cancer cells for destruction via antibody-based therapies (see, e.g., Liu et al. 2017 Eur. J. Cancer Care (Engl). 2017 Sep; 26(5), doi: 10.1111 / ecc.12446; WO2016 / 192527A1).

[0248] The tumor-associated antigen is a cluster of differentiation antigen (e.g., CD protein). In some embodiments of the invention, a targeting moiety of the invention specifically binds to one antigen. In some embodiments of the invention, a targeting moiety of the invention specifically binds to two or more antigens described herein, e.g., a targeting moiety of the invention is a bispecific or multispecific antibody or antigen-binding fragment thereof.

[0249] Non-limiting examples of antibodies or antigen-binding fragments include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cahedrin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD1 antibodies, and anti-CD1 1c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody.

[0250] Antibodies and antibody fragments useful in the immunoconjugates of the invention include modified or engineered antibodies, e.g., antibodies modified to introduce a cysteine ​​residue or other reactive amino acid, including Pel, pyrrolysine, peptide tags, and unnatural amino acids, in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or antigen-binding fragment for conjugation with a cytotoxic agent.

[0251] The location of the drug moiety can be designed, controlled, and known. For example, a cysteine ​​amino acid can be engineered into a reactive site of an antibody and does not form intrachain or intermolecular disulfide bonds (Junutula, et al. 2008 Nature Biotech. 26(8):925-932; Dornan et al. 2009 Blood 114(13):2721-2729; U.S. Patent No. 7,521,541 B2; U.S. Patent No. 7,723,485 B2; WO2009 / 052249 A2). The engineered cysteine ​​thiol can react with a linker reagent or drug-linker reagent of the invention bearing a thiol-reactive, electrophilic group, such as a maleimide or alpha-haloamide, to form an ADC with a cysteine-engineered antibody and a drug moiety.

[0252] Additionally, the antibody or antibody fragment can be modified to incorporate Pel or pyrrolysine or an unnatural amino acid for conjugation with a drug. Peptide tags for enzymatic conjugation methods can be introduced into antibodies (Junutula et al. 2008 Nat. Biotechnol. 26:925-932; Ou et al. 2011 PNAS 108 (26), 10437-10442; Axup et al. 2012 Proc. Natl. Acad. Sci. USA, 109, 16101-16106; Liu et al. 2010 Annu. Rev. Biochem. 79, 413-444; Kim et al. 2013 Curr. Opin. Chem. Biol. 17, 412-419; Strop et al. 2013 Chem. Biol. 20(2):161-7; Rabuka 2010 Curr. Opin. Chem. Biol. 14(6):790-6; Rabuka et al. 2012 Nat. Protoc. 7(6): 1052-67; WO2015 / 095301A2; WO2013 / 184514A2).

[0253] Antibodies and antibody fragments can be readily produced by any method known in the art, including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, from hybridomas or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc. (see, e.g., arvalho et al. 2016 "Production Processes for Monoclonal Antibodies", DOI: 10.5772 / 64263 (https: / / www.intechopen.com / chapters / 51512); Monoclonal Antibody Production, Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council, NATIONAL ACADEMY PRESS Washington, DC 1999; Jakobovits 1998 Adv. Drug Del. Rev. 31:33-42; Marks et al. 1991 J. Mol. Biol. 222:581; Cole et al. 1985 Monoclonal Antibodies And Cancer Therapy 77-96; Teng et al. 1983 Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983 Immunology Today 4:72-79; Olsson et al. 1982 Meth. Enzymol. 92:3-16; see U.S. Patent 6,657,103 B2).

[0254] The targeting moiety of the drug conjugates and components thereof provided herein can be any compound or molecule capable of specifically binding to a target. For example, the targeting moiety can be a small molecule, peptide, polypeptide, or nucleic acid, such as an aptamer.

[0255] In certain embodiments, the targeting moiety is a polypeptide, such as a protein ligand, a protein scaffold, or an antibody. In certain embodiments, the targeting moiety is a monoclonal antibody.

[0256] In some embodiments, the targeting moiety is selected from the group consisting of HuM195-Ac-225, HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS 1409, Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307, CR011-vcMMAE, trastuzumab-DM1 (R3502), Bexar (tositumomab), IMGN242, IMGN388, IMGN901, 131 I-labetuzumab, IMMU-102( 90 Y-epratuzumab), IMMU-107( 90 Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063 (hul4.18-IL2), tucotuzumab celmoleukin (EMD 273066; huKS-IL2), 188 Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16- 131 I), L19- 131 I, L19-TNF, PSMA-ADC, DI-Leul6-IL2, SAR3419, SGN-35, and / or CMC544, or target-binding portions thereof.

[0257] In certain embodiments, the targeting moiety comprises brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, lorvotuzumab mertansine, glenbatumumab vedotin, SAR3419, moxetumomab pasudotox, AGS-16M8F, BIIB-015, BT-062, and / or IMGN-388, or a target-binding portion thereof.

[0258] kit In some embodiments, provided herein are kits comprising a drug conjugate provided herein or one or more of its components. In some embodiments, the kit further comprises instructions for use.

[0259] In some embodiments, the kits provided herein are for use in producing the drug conjugates disclosed herein. For example, the kits can include one or more of a linker, a drug moiety, and a targeting moiety, and can further include instructions for using the provided components to produce the drug conjugates.

[0260] In some embodiments, the kits provided herein are for use in the treatment methods disclosed herein. For example, the kits may include a drug conjugate or all of the components of a drug conjugate, and may further include instructions for preparing and / or administering the drug conjugate.

[0261] Isotopically labeled compounds are also within the scope of the present invention. As used herein, "isotopically labeled compounds" refers to presently disclosed compounds (including pharmaceutical salts and prodrugs thereof, each as described herein) in which one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into presently disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, respectively: 2 H, 3 H, 13 C. 14 C. 15 N, 18 O.17 O. 31 P, 32 P, 35 S, 18 F, and 36 Contains Cl.

[0262] Isotopic labeling of the presently disclosed compounds may make the compounds useful in drug and / or substrate tissue distribution assays. 3 H) and carbon-14 ( 14 C) labeled compounds are particularly preferred due to their ease of preparation and detectability. 2 Substitution with heavy isotopes, such as H, can offer certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements, due to greater metabolic stability, and therefore may be preferable in certain circumstances. Isotopically labeled presently disclosed compounds, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art.

[0263] Furthermore, the normally abundant hydrogen ( 1 Substitution of hydrogen (H) with heavier isotopes, such as deuterium, can offer certain therapeutic advantages due, for example, to improved absorption, distribution, metabolism, and / or excretion (ADME) properties, resulting in drugs with improved efficacy, safety, and / or tolerability. 12 C 13 Substitution at C may also be beneficial (see WO2007 / 005643, WO2007 / 005644, WO2007 / 016361, and WO2007 / 016431).

[0264] Thus, isotopically derivatized compounds having one or more (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, etc.) hydrogen atoms replaced with deuterium atoms are contemplated herein. In certain embodiments, isotopically derivatized compounds of the invention have one hydrogen atom replaced with a deuterium atom.

[0265] Stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the presently disclosed compounds, as well as racemic, diastereomeric and other mixtures of such isomers, are within the scope of the invention.

[0266] After their preparation, the compounds of the invention are preferably isolated and purified to obtain compositions containing greater than 95% by weight ("substantially pure"), which are then used or formulated as described herein. In certain embodiments, the compounds of the invention are greater than 99% pure.

[0267] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the invention include, for example, hydrates.

[0268] As can be appreciated from the above, the present invention has a wide variety of applications. The present invention is further described by the following examples, which are for illustrative purposes only and are not intended in any way to define or limit the scope of the invention. [Example]

[0269] synthesis [ka] 6-(methylthio)-5-nitropicolinic acid INT-1 To a solution of 6-chloro-5-nitropicolinic acid (500 mg, 2.47 mmol, 1.0 equiv) in MeOH (20 mL) was added sodium methanethiolate (CHSNa, 1.9 mL of a 20% aqueous solution, 5.43 mmol, 2.2 equiv). The reaction mixture was stirred at room temperature for 4 h. Methanol was removed under reduced pressure. Water (10 mL) was added, and the pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. The precipitated solid was filtered, washed with water, and dried to give INT-1 (528 mg, 99.8%) as a yellow solid. LCMS (ESI): m / z 215.12 [M + H] + . 1H NMR (DMSO-d6, 600 MHz): δ 8.67 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 2.59 (s, 3H).

[0270] [ka] 6-(Methylsulfonyl)-5-nitropicolinic acid 1 To a solution of INT-1 (100 mg, 0.467 mmol, 1.0 equiv) in N,N-dimethylacetate (DMAc, 10 mL) was added 3-chloroperoxybenzoic acid (m-CPBA, 242 mg, 1.4 mmol, 3 equiv) and the reaction mixture was stirred at 50 °C for 18 h. DMAc was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 1 (53.25 mg, 46.33%) as a yellow solid. LCMS (ESI): m / z 247.08 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 14.27 (s, 1H), 8.75 (d, J = 8.3 Hz, 1H), 8.50 (d, J = 8.3 Hz, 1H), 3.50 (s, 3H).

[0271] [ka] 5-Cyano-6-(methylthio)nicotinic acid INT-2 To a solution of 6-chloro-5-cyanonicotinic acid (150 mg, 0.82 mmol, 1.0 equiv.) in MeOH (3 mL) was added CHCl (0.58 mL 20% aqueous solution, 1.65 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 2 h. The pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. Methanol was removed under reduced pressure. The precipitated solid was filtered, washed with water, and dried to give INT-2 (27 mg, 16.9%) as a white solid. LCMS (ESI): m / z 195.16 [M + H] +

[0272] [ka] 5-Cyano-6-(methylsulfonyl)nicotinic acid 2 To a solution of INT-2 (19 mg, 0.1 mmol, 1.0 equiv.) in DMAc (1 mL) was added m-CPBA (52 mg, 0.3 mmol, 3.0 equiv.) at RT, and the reaction mixture was stirred at that temperature for 2 days. DMAc was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 2 (13 mg, 59%) as a white solid. LCMS (ESI): m / z 227.14 [M + H] + [ka]

[0273] 2-(Methylthio)-5-nitroisonicotinic acid INT-3 To a solution of 2-chloro-5-nitroisonicotinic acid (1000 mg, 4.94 mmol, 1.0 equiv.) in MeOH (20 mL) was added CHCl (5.2 mL 20% aqueous solution, 14.81 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure. The precipitated solid was filtered, washed with water, and dried to give INT-3 (874 mg, 82.7%) as a yellow solid. LCMS (ESI): m / z 215.15 [M + H] +

[0274] [ka] 2-(Methylsulfonyl)-5-nitroisonicotinic acid 3 To a solution of INT-3 (500 mg, 2.33 mmol, 1.0 equiv) in DMAc (20 mL) was added m-CPBA (1420 mg, 7.0 mmol, 4 equiv), and the reaction mixture was stirred at RT for 16 h. DMAc was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 3 (367 mg, 63.9%) as a yellow solid. LCMS (ESI): m / z 247.03 [M + H] + 1H NMR (DMSO-d6, 600 MHz): δ 9.44 (s, 1H), 8.37 (s, 1H), 3.41 (s, 3H).

[0275] [ka] 6-chloro-3-fluoro-2-(methylthio)isonicotinic acid INT-4 To a solution of 2,6-dichloro-3-fluoroisonicotinic acid (500 mg, 2.38 mmol, 1.0 equiv.) in MeOH (20 mL) was added CHCl (2.5 mL 20% aqueous solution, 7.14 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 4 h. The pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. Methanol was removed under reduced pressure. The precipitated solid was filtered, washed with water, and dried to give INT-4 (422 mg, 80%) as a white solid. LCMS (ESI): m / z 222.35 [M + H] +

[0276] [ka] 6-Chloro-3-fluoro-2-(methylsulfonyl)isonicotinic acid 4 To a solution of INT-4 (90 mg, 0.41 mmol, 1.0 equiv) in THF (2 mL) was added a solution of oxone (500 mg, 0.814 mmol, 2 equiv) in HO (2 mL) at RT, and the reaction mixture was stirred at that temperature for 16 h. THF was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 4 (53.11 mg, 51.4%) as a pale white solid. LCMS (ESI): m / z 254.05 [M + H] + 1 H NMR (DMSO-d6, 600 MHz): δ8.19 (s, 1H), 3.44 (s, 3H).

[0277] [ka] 6-(Methylthio)-5-(trifluoromethyl)nicotinic acid INT-5 To a solution of 6-chloro-5-(trifluoromethyl)nicotinic acid (250 mg, 1.1 mmol, 1.0 equiv.) in MeOH (3 mL) was added CHCl (0.4 mL 20% aqueous solution, 1.1 mmol, 1.0 equiv.). The reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-5 (286 mg, 99%) as a white solid. LCMS (ESI): m / z 238.06 [M + H] +

[0278] [ka] 6-(Methylsulfonyl)-5-(trifluoromethyl)nicotinic acid 5 To a solution of INT-5 (100 mg, 0.421 mmol, 1.0 equiv) in DMAc (2 mL) was added m-CPBA (256 mg, 1.265 mmol, 3 equiv) at RT, and the reaction mixture was stirred at that temperature for 16 h. DMAc was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 5 (37 mg, 31%) as a pale white solid. LCMS (ESI): m / z 270.02 [M + H] + 1 H NMR (DMSO-d6, 600 MHz): δ 9.36 (s, 1H), 8.72 (s, 1H), 3.53 (s, 3H).

[0279] [ka] 5-Cyano-2,6-bis(methylthio)nicotinic acid INT-6 To a solution of ethyl 2,6-dichloro-5-cyanonicotinate (200 mg, 0.816 mmol, 1.0 equiv.) in methanol (4 mL) was added CHCl (0.86 mL 20% aqueous solution, 2.45 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 4 h. 1.5 mL 1N NaOH was added, and the solution was stirred at RT for 1 h. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-6 (171 mg, yield: 87.2%) as a white solid. LCMS (ESI): m / z 241.22 [M + H] + .

[0280] [ka] 5-Cyano-2,6-bis(methylsulfonyl)nicotinic acid 6 To a solution of INT-6 (171 mg, 0.712 mmol, 1.0 equiv.) in DMF (6 mL) was added m-CPBA (6.14 mg, 3.56 mmol, 5.0 equiv.) at RT, and the reaction mixture was stirred at that temperature for 24 h. DMF was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 6 (104 mg, 47.5% yield) as a white solid. LCMS (ESI): m / z 305.23 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 9.11 (s, 1H, Ar-H), 3.55 (s, 3H, -CH3), 3.47 (s, 3H, -CH3).

[0281] [ka] 5-Cyano-6-(methylthio)picolinic acid INT-7 To a solution of 6-chloro-5-cyanopicolinic acid (500 mg, 2.74 mmol, 1.0 equiv.) in CHOH (4 mL) was added CHCl (1.9 mL 20% aqueous solution, 5.48 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-7 (393 mg, 72.8% yield) as a white solid. LCMS (ESI): m / z 195.16 [M + H] + .

[0282] [ka] 5-Cyano-6-(methylsulfonyl)picolinic acid 7 To a solution of INT-7 (193 mg, 0.995 mmol, 1.0 equiv) in DMAc (5 mL) was added m-CPBA (515 mg, 2.984 mmol, 3.0 equiv) at RT, and the reaction mixture was stirred at that temperature for 2 days. DMAc was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 7 (132 mg, 59.2% yield) as a white solid. LCMS (ESI): m / z 227.14 [M + H] + .

[0283] [ka] 5-Fluoro-6-(methylthio)nicotinic acid INT-8 To a solution of 6-chloro-5-fluoronicotinic acid (500 mg, 2.85 mmol, 1.0 equiv.) in CHOH (10 mL) was added CHSNa (2.0 mL 20% aqueous solution, 5.7 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-8 (401 mg, 85% yield) as a white solid. LCMS (ESI): m / z 188.15 [M + H] + .

[0284] [ka] 5-Fluoro-6-(methylsulfonyl)nicotinic acid 8 To a solution of INT-8 (100 mg, 0.535 mmol) in THF (3 mL) was added a solution of oxone (657 mg, 1.0695 mmol) in HO (3 mL) at RT for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure. The crude product was purified by RP preparative HPLC to give 8 (79 mg, 67.4%) as a white solid. LCMS (ESI): m / z 220.14 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 14.23 (s, 1H), 9.00 (s, 1H), 8.45 (dd, J = 10.0, 1.3 Hz, 1H) 3.40 (d, J = 108.3 Hz, 3H)

[0285] [ka] 2-(Methylthio)-3-nitroisonicotinic acid INT-9 To a solution of 2-chloro-3-nitroisonicotinic acid (500 mg, 2.47 mmol) in MeOH (10 mL) was added CHCl (2.5 mL 20% aqueous solution, 7.2 mmol, 3 equiv.) under N. The reaction solution was stirred at room temperature for 2 hours. The reaction was complete as monitored by LC-MS. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The solvent was removed under reduced pressure. Water was added to the residue to precipitate a solid, which was filtered to give INT-9 (401 mg) as a white solid. LCMS (ESI): m / z 216.07 [M + H] + .

[0286] [ka] 2-(Methylsulfonyl)-3-nitroisonicotinic acid 9 To a solution of INT-9 (200 mg, 0.934 mmol) in DCM (10 mL) was added m-CPBA (322 mg, 1.868 mmol) at RT for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure. The crude product was purified by RP preparative HPLC to give 9 (117 mg, 50.9%) as a white solid. LCMS (ESI): m / z 247.13 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 9.11 (s, 1H), 8.29 (s,1H), 3.46 (s, 3H).

[0287] [ka] 2-Chloro-5-fluoro-6-(methylthio)nicotinic acid INT-10 To a solution of 2,6-dichloro-5-fluoronicotinic acid (500 mg, 2.38 mmol, 1.0 equiv.) in CHOH (4 mL) was added CHSNa (1.7 mL 20% aqueous solution, 4.76 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-10 (392 mg, 74.3%) as a white solid. LCMS (ESI): m / z 222.15 [M + H] + .

[0288] [ka] 2-Chloro-5-fluoro-6-(methylsulfonyl)nicotinic acid 10 To a solution of INT-10 (100 mg, 0.45 mmol) in THF (3 mL) was added a solution of oxone (553 mg, 0.9 mmol) in HO (3 mL) at RT for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure. The crude product was purified by RP preparative HPLC to give 10 (74.2 mg, 65%) as a white solid. LCMS (ESI): m / z 254.15 [M + H] + .

[0289] [ka] 5-Cyano-2-methyl-6-(methylthio)nicotinic acid INT-11 To a solution of ethyl 6-chloro-5-cyano-2-methylnicotinate (1000 mg, 4.463 mmol, 1.0 equiv.) in CHOH (20 mL) was added CHSNa (3.2 mL 20% aqueous solution, 9.2 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 4 h. LCMS showed the reaction was complete. 5 mL 1N NaOH was added to the reaction solution and stirred for 1 h at RT. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-11 (200 mg crude) as a white solid. LCMS (ESI): m / z 209.14 [M + H] + .

[0290] [ka] 5-Cyano-2-methyl-6-(methylsulfonyl)nicotinic acid 11 To a solution of INT-11 (200 mg, 0.562 mmol, 1.0 equiv) in DMAc (5 mL) was added m-CPBA (330 mg, 1.912 mmol, 3.4 equiv) at RT, and the reaction mixture was stirred at that temperature for 2 days. The solvent was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 11 (70 mg, yield: 51.9%) as a white solid. LCMS (ESI): m / z 241.10 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 8.85 (s, 1H), 3.49 (s, 3H), 2.86 (s, 3H).

[0291] [ka] 2-(Methylthio)-5-nitronicotinic acid INT-12-1 To a solution of 2-chloro-5-nitronicotinic acid (30.0 g, 0.148 mol, 1.0 equiv.) in MeOH (650 mL) was added CHCl (100.3 mL of a 20% aqueous solution, 0.286 mol, 2 equiv.). The reaction mixture was stirred at room temperature for 18 hours. Methanol was removed under reduced pressure. HO (600 mL) was added, and the pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The precipitated solid was filtered, washed with water, and dried to give INT-12-1 (27.8 g, 87.7%) as a pale yellow solid. LCMS (ESI): m / z 215.07 [M + H] + . 1 H NMR (CD3OD, 600 MHz): δ 9.30 (d, J = 2.4 Hz, 1H), 8.82 (d, J = 2.4 Hz, 1H), 2.58 (s, 3H)

[0292] [ka] 2-(Methylthio)-5-nitronicotinoyl chloride INT-12-2 INT-12-1 (10.0 g, 0.0467 mol) was added to DCM (100 mL) and cooled to 0-5 °C in an ice bath. Oxalyl chloride (12 mL, 0.140 mol) was added dropwise, followed by DMF (5 mL). The reaction solution was stirred at room temperature for 4 h, and the solvent was removed on a rotary evaporator to give INT-12-2. DCM (50 mL) was added to give the solution A.

[0293] [ka] 2,2-Dimethyl-5-(2-(methylthio)-5-nitronicotinoyl)-1,3-dioxane-4,6-dione INT-12-3 2,2-Dimethyl-1,3-dioxane-4,6-dione (7.40 g, 0.0514 mol), DMAP (5.70 g, 0.047 mol), and TEA (14.17 g, 0.140 mol) were added to DCM (50 mL) and cooled to 0-5 °C in an ice bath. Solution A (INT-12-2 in DCM) was added dropwise, and the mixture was stirred at room temperature for 16 h. The reaction solution was monitored by MS. Water (100 mL) was added, stirred, and separated. The organic phase was collected and the solvent removed on a rotary evaporator to give 20.1 g of INT-12-3.

[0294] [ka] 1-(2-(methylthio)-5-nitropyridin-3-yl)ethan-1-one INT-12-4 Ethanol (150 mL) and 2N dilute hydrochloric acid (20 mL) were added to INT-12-3, and the solution was heated to 80° C. and stirred for 72 hours. The reaction solution was monitored by MS. The solvent was removed on a rotary evaporator, and water (100 mL) and DCM (100 mL) were added, stirred, separated, and the organic phase was collected. The organic phase was washed with aqueous sodium hydroxide (80 mL), then with water (80 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed on a rotary evaporator to give INT-12-4 (5.98 g, 60%) as a yellow solid. LCMS (ESI): m / z 213.08 [M + H] + .

[0295] [ka] (E)-2-(((1-(2-(methylthio)-5-nitropyridin-3-yl)ethylidene)amino)oxy)acetic acid INT-12-5 To a solution of INT-12-4 (50 mg, 0.2 mmol, 1.0 equiv.) and 2-(aminooxy)acetic acid HCl salt (77 mg, 0.353 mmol, 1.7 equiv.) in MeOH (5 mL) was added AcONa (58 mg, 0.707 mmol, 3.5 equiv.). The resulting mixture was stirred at 50 °C for 3 h. The pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-12-5 (35 mg, 52.2%) as an off-white solid. LCMS (ESI): m / z 286.25 [M + H] + .

[0296] [ka] (E)-2-(((1-(2-(methylsulfonyl)-5-nitropyridin-3-yl)ethylidene)amino)oxy)acetic acid 12 To a solution of INT-12-5 (135 mg, 0.47 mmol, 1.0 equiv) in DCM (5 mL) was added m-CPBA (224 mg, 1.18 mmol, 2.5 equiv) at RT, and the reaction mixture was stirred at that temperature for 16 h. DCM was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 12 (85 mg, 57.0%) as a pale white solid. LCMS (ESI): m / z 318.26 [M + H] + .

[0297] [ka] 6-(Methylthio)-5-nitronicotinic acid INT-13 To a solution of 6-chloro-5-nitronicotinic acid (2.865 g, 14.10 mmol, 1.0 equiv.) in MeOH (20 mL) was added CHCl (74 mL 20% aqueous solution, 21.20 mmol, 1.5 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-13 (2000 mg, 64.5%) as a yellow solid. LCMS (ESI): m / z 215.22 [M + H] + .

[0298] [ka] 6-(Methylsulfonyl)-5-nitronicotinic acid 13 To a solution of INT-13 (100 mg, 0.47 mmol, 1.0 equiv) in DMAc (5 mL) was added m-CPBA (243 mg, 3.450 mmol, 3.0 equiv) at RT for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the crude material was purified by RP preparative HPLC to give 13 (24.6 mg, 21.7%) as a yellow solid. LCMS (ESI): m / z 247.18 [M + H] + . 1 H NMR (CDCl3, 600 MHz): δ14.35 (s, 1H), 9.34 (d, 1H), 8.94 (d,1H), 3.51 (s, 3H).

[0299] [ka] 2-(Methylsulfonyl)-5-nitronicotinic acid 14 To a solution of INT-14 (100 mg, 0.47 mmol, 1.0 equiv) in DMAc (5 mL) was added m-CPBA (243 mg, 3.45 mmol, 3.0 equiv) at RT for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the crude material was purified by RP preparative HPLC to give 14 (35.5 mg, 31.3%) as a yellow solid. LCMS (ESI): m / z 247.18 [M + H]+ .

[0300] [ka] 2-(Methylthio)-5-(trifluoromethyl)nicotinic acid INT-15 To a solution of 2-chloro-5-(trifluoromethyl)nicotinic acid (250 mg, 1.1 mmol, 1.0 equiv.) in MeOH (3 mL) was added CHCl (0.4 mL 20% aqueous solution, 1.1 mmol, 1.0 equiv.). The reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with 3 N HCl. Methanol was removed under reduced pressure, and the precipitated solid was filtered, washed with water, and dried to give INT-15 (255 mg, 89%) as a white solid. LCMS (ESI): m / z 238.06 [M + H] + .

[0301] [ka] 2-(Methylsulfonyl)-5-(trifluoromethyl)nicotinic acid 15 To a solution of INT-15 (100 mg, 0.421 mmol, 1.0 equiv) in DMAc (2 mL) was added m-CPBA (256 mg, 1.26 mmol, 3 equiv) at RT, and the reaction mixture was stirred at that temperature for 16 h. DMAc was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 15 (35 mg, 29%) as a pale white solid. LCMS (ESI): m / z 270.02 [M + H] + .

[0302] [ka] 6-(methylthio)-5-nitro-N-(prop-2-yn-1-yl)nicotinamide INT-16 To a solution of 6-(methylthio)-5-nitronicotinic acid INT-13 (2.0 g, 9.3 mmol, 1.0 equiv.) in DCM (5 mL) was added prop-2-yn-1-amine (610 mg, 11.2 mmol, 1.2 equiv.) followed by oxalyl dichloride (11.8 g, 93.0 mmol, 10.0 equiv.) and diisopropylethylamine (DIEA, 3.6 g, 27.9 mmol, 3.0 equiv.) under N2. The reaction mixture was stirred at room temperature for 2 h. Volatiles were removed under reduced pressure and the crude product was purified by RP preparative HPLC to give INT-16 (2.07 g, 86.7%) as a yellow solid. LCMS (ESI): m / z 252.25 [M + H] + .

[0303] [ka] 6-(Methylsulfonyl)-5-nitro-N-(prop-2-yn-1-yl)nicotinamide 16 To a solution of INT-16 (2.07 g, 8.2 mmol, 1 equiv.) in THF / water (1:1, V / V) (100 mL) was added Oxone (30.4 g, 49.5 mmol, 6.0 equiv.) at RT, and the reaction mixture was stirred at that temperature for 16 h. THF was removed under reduced pressure, and the residue was purified by RP-HPLC to give 16 (1.18 g, 50.6%) as a yellow solid. LCMS (ESI): m / z 284.25 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ 9.13 (s, 1H), 8.56 (s, 1H), 8.06 (s, 1H), 4.27 (s, 2H), 3.37 (s, 3H), 2.37 (s, 1H).

[0304] [ka] 2-(methylthio)-5-nitro-N-(prop-2-yn-1-yl)nicotinamide INT-17 To a solution of INT-12-1 (200 mg, 0.93 mmol, 1.0 equiv) in DCM (6 mL), prop-2-yn-1-amine (61 mg, 1.12 mmol, 1.2 equiv), EDCI (530 mg), and DIEA (240 mg, 1.86 mmol, 2.0 equiv) were added under N. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give INT-17 (197 mg, 84.5%) as a yellow solid. LCMS (ESI): m / z 252.22 [M + H] + .

[0305] [ka] 2-(Methylsulfonyl)-5-nitro-N-(prop-2-yn-1-yl)nicotinamide 17 To a solution of INT-17 (197 mg, 0.78 mmol, 1.0 equiv) in THF:HO (6 mL) was added oxone (1.44 mg, 2.34 mmol, 3 equiv) at RT, and the reaction mixture was stirred at RT for 16 h. THF was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 17 (204 mg, 92.1%) as a yellow solid. LCMS (ESI): m / z 284.22 [M + H] + .

[0306] [ka] 1-(2-(methylsulfonyl)-5-nitropyridin-3-yl)ethan-1-one 18 To a solution of INT-12-4 (0.3 g, 1.41 mmol, 1.0 equiv.) in THF (15 mL) and HO (15 mL) was added oxone (4.35 g, 7.068 mmol, 5.0 equiv.). The reaction mixture was stirred at room temperature for 18 h. The solid was filtered off from the solution. THF was removed from the filtrate, and the aqueous solution was extracted with ethyl acetate (10 mL × 3). After concentration, the crude product was purified on a silica gel column to give 18 (0.15 g, 43.5%) as a pale yellow solid. LCMS (ESI): m / z 245.17 [M + H] + .

[0307] [ka] 2-Chloro-6-(methylthio)-3-nitropyridine INT-19-q and 6-chloro-2-(methylthio)-3-nitropyridine INT-19-h To a solution of 2,6-dichloro-3-nitropyridine (2.0 g, 10.4 mmol, 1.0 equiv.) in THF (40 mL) was added CHCl (5.5 mL 20% aqueous solution, 15.6 mmol, 1.5 equiv.) under N. The reaction mixture was stirred at room temperature for 2 h. The reaction was complete as determined by LC-MS. 3N hydrochloric acid was added to the reaction solution to adjust the pH to 4-5. The solvent was removed under reduced pressure. Water was added to the residue, and filtration gave the crude product, which was purified by RP preparative HPLC to give two isomers as a yellow solid. The later-eluting peak is designated INT-19-h (625 mg), and the earlier-eluting peak is designated INT-19-q (683 mg). The combined yield was 61.7%. The structures were tentatively assigned. INT-19-h:LCMS (ESI): m / z 205.07 [M + H] + (5.85 minutes) INT-19-q:LCMS (ESI): m / z 205.07 [M + H] + (5.44 minutes)

[0308] [ka] 6-(but-3-yn-1-yloxy)-2-(methylthio)-3-nitropyridine INT-19-1-h To a solution of but-3-yn-1-ol (125 mg, 1.77 mmol, 1.2 equiv) in THF (12 mL) was added NaH (87 mg, 2.2 mmol, 1.5 equiv), followed by INT-19-h (300 mg, 1.47 mmol, 1.0 equiv) under N. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the crude product was purified by RP preparative HPLC to give INT-19-1-h (134 mg, 38.4%) as a yellow solid. LCMS (ESI): m / z 239.20 [M + H] +

[0309] [ka] 6-(But-3-yn-1-yloxy)-2-(methylsulfonyl)-3-nitropyridine 19 To a solution of INT-19-1-h (80 mg, 0.32 mmol, 1.0 equiv.) in DMF (10 mL) was added m-CPBA (160 mg, 0.96 mmol, 3 equiv.) at RT, and the reaction mixture was stirred at that temperature for 16 h. DMF was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 19 (17.8 mg, 19.6%) as a white solid. LCMS (ESI): m / z 271.24 [M + H] + .

[0310] [ka] 2-(but-3-yn-1-yloxy)-6-(methylthio)-3-nitropyridine INT-20-q To a solution of but-3-yn-1-ol (44 mg, 0.63 mmol, 1.2 equiv) in THF (5 mL) was added NaH (31 mg, 0.78 mmol, 1.5 equiv), followed by INT-19-q (107 mg, 0.52 mmol, 1.0 equiv) under N. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give INT-20-q (80 mg, 65.0%) as a yellow solid. LCMS (ESI): m / z 239.19 [M + H] + .

[0311] [ka] 2-(But-3-yn-1-yloxy)-6-(methylsulfonyl)-3-nitropyridine 20 To a solution of INT-20-q (80 mg, 0.32 mmol, 1.0 equiv.) in DMF (10 mL) was added m-CPBA (160 mg, 0.96 mmol, 3 equiv.) at RT, and the reaction mixture was stirred at RT for 16 h. DMF was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 20 (38 mg, 41.3%) as a yellow solid. LCMS (ESI): m / z 271.19 [M + H] + .

[0312] [ka] tert-Butyl (2-(6-(methylthio)-5-nitronicotinamido)ethyl)carbamate (INT-21-1) To a solution of tert-butyl (2-aminoethyl)carbamate (6.670 g, 41.7 mmol) in DCM (100 mL) was added INT-13 (5.940 g, 28.0 mmol) and EDCI (8.050 g, 41.7 mmol) under N2. The reaction suspension was stirred at room temperature for 16 h. The reaction was complete as monitored by LC-MS and purified by preparative HPLC to give INT-21-1 (6.820 g, 45.9%) as a pale white solid. LCMS [MH] - : 355.4

[0313] [ka] N-(2-aminoethyl)-6-(methylthio)-5-nitronicotinamide (INT-21-2) To a solution of INT-12-1 (6.820 g, 19.2 mmol) in DCM (68 mL) was added TFA (13 mL), and the mixture was stirred at room temperature for 3 hours. The reaction was complete as detected by LC-MS, and concentrated to give INT-21-2 as a colorless solid (4.9 g, 100% yield). LCMS (ESI): m / z 257.2 [M + H] + .

[0314] [ka] 2-(2-((2-(6-(methylthio)-5-nitronicotinamido)ethyl)amino)-2-oxoethoxy)acetic acid (INT-21-3) To a solution of INT-12-2 (2.0 g, 7.804 mmol) in DMF (20 mL), 1,4-dioxane-2,6-dione (1.086 g, 9.365 mmol) and DIEA (2.215 g, 17.168 mmol) were added under N2 and stirred at room temperature for 16 hours. The reaction was complete as detected by LC-MS. 200 mL of water was added to the solution, stirred until a precipitate formed, filtered, and dried to give INT-21-34 (1.5 g, 51.7%) as a pale white solid. LCMS (ESI): m / z 373.3 [M + H] + .

[0315] 1 H NMR (DMSO-d6, 600 MHz): δ 12.77 (brs, 1H), 9.19 (ds, 1H), 8.99 (t, 1H), 8.96 (ds, 1H), 8.07 (t, 1H), 4.10 (s, 2H), 3.96 (s, 2H), 3.41-3.48 (m, 2H), 3.34-3.30 (m, 2H), 2.60 (s, 3H).

[0316] [ka] 2-(2-((2-(6-(methylsulfonyl)-5-nitronicotinamido)ethyl)amino)-2-oxoethoxy)acetic acid (21) To a solution of INT-21-3 (100.0 mg, 0.269 mmol, 1.0 equiv.) in DCM / DMAc (2 mL / 2 mL) was added m-CPBA (139.0 mg, 0.807 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was purified by preparative HPLC to give 21 (38.4 mg, yield: 35.36%, purity: 97.30%) as a white powder. LCMS [M+H] + : 405.1. 1 H NMR (DMSO-d6, 600 MHz): δ 12.83 (s, 1H), 9.28 (s, 1H), 9.15 (t, 1H), 8.90 (s, 1H), 8.08 (t, 1H), 4.11 (s, 2H), 3.97 (s, 2H), 3.51 (s, 3H), 3.42-3.88 (m, 2H), 3.35-3.31 (m, 2H).

[0317] [ka] 6-(methylthio)-5-nitro-N-(2-(2-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)acetamido)ethyl)nicotinamide (INT-22-1) To a solution of INT-21-3 (1.5 g, 4.028 mmol) in DMF (15 mL) was added prop-2-yn-1-amine (332 mg, 6.043 mmol), followed by HATU (2.298 g, 6.043 mmol) and DIEA (1.562 g, 12.086 mmol). The mixture was stirred at rt for 2 h. The reaction mixture was purified by preparative HPLC to give INT-22-1 (1.0 g, 60.6%) as a white solid. LCMS [M+H] + : 410.2.

[0318] [ka] Prop-2-yn-1-yl 2-(2-((2-(6-(methylsulfonyl)-5-nitronicotinamido)ethyl)amino)-2-oxoethoxy)acetate (22) To a solution of INT-22-1 (900 mg, 2.198 mmol) in DMAc (10 mL), m-CPBA (1.327 g, 7.694 mmol) was added and stirred at rt for 2 h. The reaction mixture was purified by preparative HPLC to give 22 (841 mg, 86.7%) as a white solid. LCMS [M+H] + : 422.2. 1 H NMR (600 MHz, DMSO-d6) δ 9.27 (s, 1H), 9.18 (t, 1H), 8.89 (s, 1H), 8.43 (t, 1H), 8.23 ​​(t, 1H), 3.98 (s, 2H), 3.95 (s, 2H), 3.91 (dd, 2H), 3.51 (s, 3H), 3.45-3.38 (m, 2H), 3.37-3.33 (m, 2H), 3.10 (d, 1H).

[0319] [ka] 6-(Methylsulfinyl)-5-nitronicotinic acid 23 To a solution of INT-13 (150 mg, 0.7 mmol) in THF (3 mL) and HO (3 mL) was added oxone (646 mg, 1.05 mmol) and stirred at RT for 2 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 23 (101 mg, 61%) as a yellow solid. LCMS (ESI): m / z 231.19 [M + H] + .

[0320] [ka] 2-(Methylsulfinyl)-5-nitronicotinic acid 24 To a solution of INT-14 (150 mg, 0.7 mmol) in THF (3 mL) and HO (3 mL) was added oxone (646 mg, 1.05 mmol) and stirred at RT for 2 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 24 (95 mg, 59%) as a yellow solid. LCMS (ESI): m / z 231.19 [M + H] + .

[0321] [ka] 5-Bromo-2-(methylthio)-3-nitropyridine (INT-25-1) To a solution of 5-bromo-2-chloro-3-nitropyridine (3.00 g, 12.6 mmol, 1.0 equiv.) in THF (30 mL) was added CHClSNa (4.7 mL of a 20% aqueous solution, 15.2 mmol, 1.2 equiv.). The suspension reaction mixture was stirred at room temperature for 1 h. The THF was removed under reduced pressure, filtered, washed with HO (20 mL), and dried to give INT-25-1 (2.78 g, yield: 88.7%, purity: 100%) as a yellow solid. 1 H NMR (600 MHz, DMSO-d6) δ 9.01 (d, J = 2.1 Hz, 1H), 8.82 (d, J = 2.1 Hz, 1H), 2.54 (s, 3H).

[0322] [ka] 2-(Methylthio)-3-nitro-5-((trimethylsilyl)ethynyl)pyridine (INT-25-2) To a solution of INT-25-1 (780 mg, 3.15 mmol, 1.0 equiv.) in DIEA (15 mL), ethynyltrimethylsilane (464 mg, 4.72 mmol, 1.5 equiv.), (PhP)PdCl (110 mg, 0.16 mmol, 0.05 equiv.), and CuI (30 mg, 0.16 mmol, 0.05 equiv.) were added. The suspension reaction mixture was stirred at room temperature for 24 hours. The mixture was then evaporated, and the residue was purified by preparative HPLC to give INT-21-2 (47 mg, yield: 5.6%, purity: 100%) as a brown solid. LCMS [M+H]: 267.1; 1 H NMR (600 MHz, DMSO-d6) δ 8.89 (d, J = 1.9 Hz, 1H), 8.57 (d, J = 1.9 Hz, 1H), 2.56 (s, 3H), 0.27 (d, J = 3.5 Hz, 9H).

[0323] [ka] 5-ethynyl-2-(methylthio)-3-nitropyridine (INT-25-3) To a solution of INT-25-2 (47 mg, 0.177 mmol, 1.0 equiv.) in MeOH (1 mL), KOH (20 mg, 0.353 mmol, 2.0 equiv.) was added at rt, and the reaction mixture was stirred at that temperature for 2 h. The reaction mixture was then purified by preparative HPLC to give INT-21-3 (18 mg, yield: 52.3%, purity: 100%) as a yellow solid. LCMS [M+H]: 195.1; 1 H NMR (600 MHz, DMSO-d6) δ 8.94 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 4.64 (s, 1H), 2.56 (s, 3H).

[0324] [ka] 5-Ethynyl-2-(methylsulfonyl)-3-nitropyridine (25) To a solution of INT-25-3 (18 mg, 0.093 mmol, 1.0 equiv.) in DMAc (2 mL) was added m-CPBA (48 mg, 0.278 mmol, 3.0 equiv.) at rt, and the reaction mixture was stirred at that temperature for 2 h. The reaction mixture was then purified by preparative HPLC to give 25 (2.3 mg, yield: 11%, purity: 96%) as a white solid. LCMS [M+H]: 227.0; 1 H NMR (600 MHz, DMSO-d6) δ 9.08 (d, J = 1.7 Hz, 1H), 8.79 (d, J = 1.7 Hz, 1H), 4.98 (s, 1H), 3.46 (s, 3H).

[0325] [ka] 4-((9S,12S)-9-Isopropyl-2,2,5-trimethyl-4,7,10-trioxo-12-(3-ureidopropyl)-3-oxa-5,8,11-triazatridecamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate INT-26 To a solution of 2-((tert-butoxycarbonyl)(methyl)amino)acetic acid (35 mg, 0.187 mmol, 2.1 equiv.) in DMAc (5 mL), PyBOP (97 mg, 0.187 mmol, 2.1 equiv.) was added, followed by DIEA (65 μl, 0.374 mmol, 4.2 equiv.) and stirred at room temperature for 20 min. Val-Cit-PAB-MMAE (CAS No. 644981-35-1) (100 mg, 0.0890 mmol, 1.0 equiv.) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give INT-26 (93.3 mg, 81.3%) as a pale white solid. LCMS (ESI): m / z 1296.14 [M + H] + .

[0326] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(methylamino)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 26 To a solution of INT-26 (50 mg, 0.062 mmol, 1.0 equiv) in DCM (1 mL) was added 4N HCl / dioxane (1 mL) and the mixture was stirred at RT for 1 h. The solvent was removed to give a white solid 26 as the HCl salt (76 mg, 100%). LCMS (ESI): m / z 1195.94 [M + H] + .

[0327] [ka] 4-((S)-2-((S)-2-(2-azidoacetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 27 To a solution of Val-Cit-PAB-MMAE (100.05 mg, 0.0891 mmol, 1.0 equiv.) in DMAc (2 mL), 2-azidoacetic acid (13.33 μL, 0.178 mmol, 2.0 equiv.) and DIEA (46.53 μL, 0.267 mmol, 3.0 equiv.) were added, followed by PyBOP (92.69 mg, 0.178 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was then purified by preparative HPLC to give 27 (88 mg, 81.9%) as a white powder. LCMS (ESI): m / z 1207.1 [M + H] + . 11H NMR (DMSO-d6, 600 MHz): δ 10.01 (d, J = 12.9 Hz, 1H), 8.32 (d, J = 7.5 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.79 - 7.72 (m, 1H), 7.59 (s, 2H), 7.37 - 7.23 (m, 6H), 7.17 (dd, J = 15.0, 7.7 Hz, 1H), 6.02 (s, 1H), 5.08 - 5.01 (m, 3H), 4.74 (s, 1H), 4.63 (s, 1H), 4.48 (t, J = 7.8 Hz, 1H), 4.43 (d, J = 6.6 Hz, 1H), 4.42 - 4.35 (m, 1H), 4.29 - 4.22 (m, 2H), 4.04 - 3.91 (m, 2H), 3.90 (d, J = 1.1 Hz, 2H), 3.81 - 3.74 (m, 1H), 3.61 - 3.53 (m, 1H), 3.47 (s, 1H), 3.31 (d, J = 10.1 Hz, 1H), 3.24 (d, J = 9.6 Hz, 4H), 3.20 (s, 2H), 3.17 (s, 1H), 3.12 (s, 2H), 3.08 - 2.99 (m, 2H), 2.99 - 2.92 (m, 2H), 2.88 - 2.81 (m, 3H), 2.41 (d, J = 17.7 Hz, 1H), 2.29 - 2.21 (m, 1H), 2.16 - 2.08 (m, 2H), 2.01 - 1.93 (m, 2H), 1.85 - 1.66 (m, 4H), 1.59 - 1.46 (m, 4H), 1.38 - 1.31 (m 2H), 1.06 - 0.96 (m, 6H), 0.90 - 0.73 (m, 24H).

[0328]

Chem.

[0329] <~

Chem.

[0330] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(methylsulfonyl)-5-nitroisonicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 33 To a solution of 3 (9 mg, 0.036 mmol, 2.0 equiv) in DCM (1 mL) was added Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 equiv) followed by DIC (5 mg, 0.036 mmol, 2.0 equiv) under N. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the crude product was purified by RP preparative HPLC to give 33 (14 mg, 57.5%) as a pale white solid. LCMS (ESI): m / z 1352.27 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ10.10 (s, 1H), 9.41 (s, 1H), 9.17 (d, 1H), 8.38 (d, 1H), 8.07 (d, 2H), 7.87 (d, 2H), 7.68 - 7.03 (m, 7H), 6.00 (s, 1H), 5.45 (m, 3H), 5.03 (s, 1H), 4.64 - 4.08 (m, 8H), 4.03 -3.29 (m, 13H), 3.28 - 3.11 (m, 6H), 3.02 (s, 2H), 2.95 (m, 4H), 2.52 - 2.44 (m, 2H), 2.35 -1.70 (d, 7H), 1.65 (m, 4H), 1.55 (m, 2H), 1.38 (m, 3H), 1.33 (m, 3H), 1.33 - 0.79 (m, 24H).

[0331] [ka] 4-((S)-2-((S)-2-(6-chloro-3-fluoro-2-(methylsulfonyl)isonicotinamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 34 To a solution of 4 (9 mg, 0.036 mmol, 2.0 equiv) in DCM (1 mL) was added Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 equiv) followed by DIC (5 mg, 0.036 mmol, 2.0 equiv) under N. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the crude product was purified by RP preparative HPLC to give 34 (15 mg, 61.3%) as a pale white solid. LCMS (ESI): m / z 1360.59 [M + H] + . 1H NMR (DMSO-d6, 600 MHz): δ 10.08 (s, 1H), 8.96 (s, 1H), 8.41 (m, 2H), 8.06 (m, 1H), 7.69 - 7.51 (m, 2H), 7.43 - 7.10 (m, 7H), 6.00 (s, 1H), 5.45 (m, 3H), 5.03 (s, 1H), 4.64 - 4.08 (m, 8H), 4.03 -3.29 (m, 13H), 3.28 - 3.11 (m, 6H), 3.02 (s, 2H), 2.95 (m, 4H), 2.52 - 2.44 (m, 2H), 2.35 -1.70 (d, 7H), 1.65 (m, 4H), 1.55(m, 2H), 1.38 (m, 3H), 1.33 (m, 3H), 1.33 - 0.79 (m, 24H).

[0332] [ka] 4-((S)-2-((S)-3-methyl-2-(6-(methylsulfonyl)-5-(trifluoromethyl)nicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 35 To a solution of 5 (9 mg, 0.032 mmol, 2.0 equiv.) in DMAc (1 mL), Val-Cit-PAB-MMAE (20 mg, 0.016 mmol, 1.0 equiv.) was added, followed by (benzotriazol-1-yloxy)-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP, 13 mg, 0.024 mmol, 2.0 equiv.) and 2,6-lutidine (3.5 mg, 0.032 mmol) under N2. The reaction mixture was stirred at room temperature for 2 h. DMAc was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 35 (10.11 mg, 46.5%) as a pale white solid. LCMS (ESI): m / z 1375.93 [M + H] + . 1 H NMR (DMSO-d6, 600 MHz): δ9.41 (s, 1H), 9.17 (d, 1H), 8.38 (d, 2H), 8.07 (d, 2H), 7.87 (d, 2H), 7.68 - 7.03 (m, 7H), 6.00 (s, 1H), 5.45 (m, 3H), 5.03 (s, 1H), 4.64 - 4.08 (m, 8H), 4.03 -3.29 (m, 13H), 3.28 - 3.11 (m, 6H), 3.02 (s, 2H), 2.95 (m, 4H), 2.52 - 2.44 (m, 2H), 2.35 -1.70 (d, 7H), 1.65 (m, 4H), 1.55(m, 2H), 1.38 (m, 3H), 1.33 (m, 3H), 1.33 - 0.79 (m, 24H).

[0333] [ka] 4-((S)-2-((S)-2-(5-cyano-2,6-bis(methylsulfonyl)nicotinamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 36 To a solution of 6 (16 mg, 0.010 mmol, 1.0 equiv) in DCM (3.0 mL) was added DIC (8.6 μL, 0.054 mmol, 2 equiv) and Val-Cit-PAB-MMAE (30 mg, 0.027 mmol, 1 equiv) at RT, and the reaction mixture was stirred at that temperature for 1 h. DCM was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 36 (13.0 mg, 47.5% yield) as a white solid. LCMS (ESI): m / z 1410.70 [M + H] + .

[0334] [ka] 4-((S)-2-((S)-2-(5-cyano-6-(methylsulfonyl)picolinamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 37 7 (20 mg, 0.090 mmol, 2.0 equivalent) solution in DCM (2 mL), DIC (11 μL, 0.090 mmol, 2 equivalent), Va l-Cit-PAB-MMAE (50 mg, 0.045 mmol, 1 equivalent) was added at RT, reacted with the mixture, and stirred at temperature for 1 time. DCM was removed under reduced pressure, and the residue was separated by RP and purified by HPLC. 37 (33 mg, yield: 55.1%) was obtained as a white solid. LCMS (ESI): m / z 1332.11 [M + H] + . 1 H NMR (MeOH-d4, 600 MHz): δ 8.73 - 8.65 (m, 2H), 8.58 (dd, J = 7.3, 4.2 Hz, 1H), 8.46 (dd, J = 8.1, 2.1 Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.57 (d, J = 6.2 Hz, 2H), 7.40 - 7.26 (m, 5H), 7.19 (dd, J = 15.0, 7.7 Hz, 2H), 5.36 - 5.32 (m, 1H), 5.18 (dd, J = 21.7, 10.9 Hz, 3H), 5.10 - 5.04 (m, 1H), 4.57 - 4.48 (m, 3H), 4.26 - 4.14 (m, 3H), 4.09 - 4.03 (m, 1H), 3.86 (dd, J = 9.1, 2.1 Hz, 1H), 3.75 - 3.63 (m, 3H), 3.53 (d, J = 2.4 Hz, 3H), 3.45 - 3.38 (m, 2H), 3.22 - 3.06 (m, 6H), 2.93 (dd, J = 12.9, 6.3 Hz, 3H), 2.53 - 2.43 (m, 3H), 2.29 - 2.16 (m, 4H), 1.64 - 1.51 (m, 5H), 1.38 - 1.25 (m, 10H), 1.21 - 0.66 (m, 30H).

[0335]

change

[0336] [Chemical formula] 4-((S)-2-((S)-3-methyl-2-(2-(methylsulfonyl)-3-nitroisonicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 39 To a solution of 9 (8.8 mg, 0.036 mmol, 2.0 equiv.) in DCM (2 mL) was added DIC (5.3 μL, 0.036 mmol, 2.0 equiv.) and Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1 equiv.) at RT, and the reaction mixture was stirred at that temperature for 1 h. The solvent was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 39 (13.3 mg, 54.1% yield) as a yellow solid. LCMS (ESI): m / z 1352.600 [M + H] + . 11H NMR (DMSO-d6, 600 MHz): δ 10.08 (d, J = 16.3 Hz, 1H), 9.21 (d, J = 8.9 Hz, 1H), 9.08 - 9.04 (m, 1H), 8.38 (d, J = 7.3 Hz, 1H), 8.06 (t, J = 9.2 Hz, 1H), 8.02 (dd, J = 4.8, 1.7 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.59 (s, 2H), 7.37 - 7.24 (m, 6H), 7.17 (dt, J = 14.9, 7.1 Hz, 1H), 6.00 (s, 1H), 5.13 - 4.96 (m, 2H), 4.68 (d, J = 77.0 Hz, 1H), 4.54 - 4.38 (m, 4H), 4.27 (dd, J = 23.2, 10.8 Hz, 1H), 3.97 (ddd, J = 15.8, 14.8, 9.3 Hz, 3H), 3.32 (d, J = 10.8 Hz, 1H), 3.22 (dd, J = 39.6, 16.1 Hz, 8H), 3.12 (s, 2H), 3.05 (dt, J = 13.9, 7.0 Hz, 2H), 2.98 (s, 2H), 2.87 (dd, J = 29.5, 13.0 Hz, 3H), 2.44 - 2.39 (m, 1H), 2.28 (dt, J = 15.5, 8.2 Hz, 1H), 2.16 - 2.04 (m, 3H), 1.99 (ddt, J = 32.0, 20.7, 7.3 Hz, 1H), 1.86 - 1.68 (m, 4H), 1.66 - 1.22 (m, 9H), 1.07 - 0.73 (m, 33H).

[0337] ​​​​​​4-((S)-2-((S)-2-(2-chloro-5-fluoro-6-(methylsulfonyl)nicotinamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 40 To a solution of 10 (9.1 mg, 0.036 mmol, 2.0 equiv.) in DCM (2 mL) was added DIC (5.3 μL, 0.036 mmol, 2.0 equiv.) and Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1 equiv.) at RT, and the reaction mixture was stirred at that temperature for 1 h. The solvent was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 40 (14 mg, 57.6% yield) as a white solid. LCMS (ESI): m / z 1359.21 [M + H] + . 1H NMR (DMSO-d6, 600 MHz): δ 10.08 (d, J = 15.9 Hz, 1H), 8.83 (d, J = 10.0 Hz, 1H), 8.34 (d, J = 7.3 Hz, 1H), 8.30 (dd, J = 8.7, 1.5 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.59 (s, 2H), 7.36 - 7.25 (m, 6H), 7.20 - 7.14 (m, 1H), 6.00 (s, 1H), 5.43 (s, 2H), 5.13 - 4.96 (m, 2H), 4.52 - 4.42 (m, 4H), 4.27 (dd, J = 23.5, 10.7 Hz, 1H), 4.05 - 3.93 (m, 3H), 3.32 (d, J = 10.6 Hz, 1H), 3.27 - 2.81 (m, 19H), 2.27 (dd, J = 14.9, 9.8 Hz, 1H), 2.15 - 2.06 (m, 3H), 2.02 - 1.93 (m, 1H), 1.84 - 1.69 (m, 4H), 1.65 - 1.37 (m, 6H), 1.06 - 0.74 (m, 34H).

[0338] [ka] 4-((S)-2-((S)-2-(5-cyano-2-methyl-6-(methylsulfonyl)nicotinamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 41 To a solution of 11 (12.8 mg, 0.053 mmol, 2.0 equiv.) in DCM (2 mL) was added DIC (8.3 μL, 0.053 mmol, 2 equiv.) and Val-Cit-PAB-MMAE (30 mg, 0.027 mmol, 1 equiv.) at RT, and the reaction mixture was stirred at that temperature for 1 h. The solvent was removed under reduced pressure, and the residue was purified by RP-HPLC to give 41 (19 mg, 52.3% yield) as a white solid. LCMS (ESI): m / z 1347.33 [M + H] + . 1 H NMR (CDCl3, 600 MHz): δ 8.08 - 7.92 (m, 12H), 7.26 (d, J = 7.2 Hz, 2H), 4.62 (d, J = 4.1 Hz, 8H), 4.44 (d, J = 5.0 Hz, 1H), 4.37 - 4.24 (m, 4H), 3.85 - 3.77 (m, 1H), 3.55 - 1.92 (m, 33H), 1.63 - 0.50 (m, 33H).

[0339] [ka] 4-((8S,11S,E)-8-isopropyl-2-(2-(methylsulfonyl)-5-nitropyridin-3-yl)-6,9-dioxo-11-(3-ureidopropyl)-4-oxa-3,7,10-triazadodec-2-en-12-amido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(( (1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 42 To a solution of 12 (7.5 mg, 0.036 mmol, 2.0 equiv) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 equiv) under N2. After stirring at RT for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 equiv) was added. The reaction mixture was stirred at RT for 2 h. DCM was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 42 (16.5 mg, 64.4%) as an off-white solid. LCMS (ESI): m / z 1423.79 [M + H] + .

[0340] [ka] 4-((S)-2-((S)-3-methyl-2-(6-(methylsulfonyl)-5-(trifluoromethyl)nicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 43 To a solution of 13 (8.9 mg, 0.036 mmol, 2.0 equiv) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 equiv) under N2. After stirring at RT for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 equiv) was added. The reaction mixture was stirred at RT for 2 h. DCM was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 43 (14.7 mg, 60%) as a yellow solid. LCMS (ESI): m / z 1354.15 [M + H] + .

[0341] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(methylsulfonyl)-5-nitronicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 44 To a solution of 14 (8.9 mg, 0.036 mmol, 2.0 equiv) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 equiv) under N2. After stirring at RT for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 equiv) was added. The reaction mixture was stirred at RT for 2 h. DCM was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 44 (18.5 mg, 75.5%) as a yellow solid. LCMS (ESI): m / z 1354.15 [M + H] + .

[0342] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(methylsulfonyl)-5-(trifluoromethyl)nicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 45 To a solution of 15 (9 mg, 0.0324 mmol, 2.0 equiv) in DMAc (1 mL) was added Val-Cit-PAB-MMAE (20 mg, 0.016 mmol, 1.0 equiv), followed by (PyBOP, 13 mg, 0.024 mmol, 2.0 equiv) and 2,6-lutidine (3.5 mg, 0.032 mmol) under N. The reaction mixture was stirred at room temperature for 2 h. DMAc was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 45 (12.2 mg, 55.7%) as a pale white solid. LCMS (ESI): m / z 1375.35 [M + H] + .

[0343] [ka] 4-((2S)-2-((2S)-3-methyl-2-(6-(methylsulfinyl)-5-nitronicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 46 To a solution of 21 (22 mg, 0.09 mmol, 1.1 equiv) and Val-Cit-PAB-MMAE (100 mg, 0.089 mmol, 1.0 equiv) in DMF (2.2 mL) was added HATU (50 mg, 0.133 mmol, 1.5 equiv) and DIEA (49 μl, 0.267 mmol, 3.0 equiv), and the reaction mixture was stirred at RT for 3 h. The solution was purified by RP preparative HPLC to give 46 (35 mg, 30%) as a yellow solid. LCMS (ESI): m / z 1336.85 [M + H] + .

[0344] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(methylthio)-5-nitronicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate INT-47 To a solution of INT-12-1 (9.5 mg, 0.045 mmol, 1.0 equiv.) in DMF (5 mL) was added HATU (25 mg, 0.0133 mmol, 1.5 equiv.), DIEA (25 μl, 0.0267 mmol, 3.0 equiv.), and Val-Cit-PAB-MMAE (50 mg, 0.045 mmol, 1.0 equiv.), and the reaction mixture was stirred at RT for 4 h. The solution was purified by RP preparative HPLC to give INT-47 (41 mg, 69%) as a white solid. LCMS (ESI): m / z 1319.75 [M + H] + .

[0345] [ka] 4-((2S)-2-((2S)-3-methyl-2-(2-(methylsulfinyl)-5-nitronicotinamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 47 To a solution of INT-47 (10.0 mg, 0.0076 mmol) in THF (0.2 mL) and HO (0.2 mL) was added oxone (7.0 mg, 0.0114 mmol) at rt for 10 h. The solution was then purified by RP preparative HPLC to give 47 (2.8 mg, 27%) as a yellow solid. LCMS (ESI): m / z 1335.89 [M + H] + .

[0346] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(N-methyl-6-(methylsulfonyl)-5-nitronicotinamido)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 48 To a solution of 13 (30 mg, 0.122 mmol, 2.1 equiv) in DMAc (10 mL) was added PyBOP (63.56 mg, 0.122 mmol, 2.1 equiv), followed by DIEA (52.68 μl, 0.302 mmol, 5.2 equiv) and stirred at room temperature for 20 min. 26 (74 mg, 0.06 mmol, 1 equiv) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give 48 (47 mg, 55%) as a pale yellow solid. LCMS (ESI): m / z: 1422.62 [M + H] + .

[0347] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(4-((6-(methylsulfonyl)-5-nitronicotinamido)methyl)-1H-1,2,3-triazol-1-yl)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((( 1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 49 Compound 27 (36.2 mg, 0.03 mmol, 1.0 equiv.) and compound 16 (8.5 mg, 0.03 mmol, 1.0 equiv.) were mixed with 6 mL of a 1:1 DMSO / HO solution. CuSO4.5HO (3 mg, 0.012 mmol, 0.4 equiv.) and TBTA (6.3 mg, 0.012 mmol, 0.4 equiv.) were added, followed by sodium ascorbate (4.7 mg, 0.0238 mmol, 0.8 equiv.), and the mixture was stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give 49 (29.5 mg, 66%) as a yellow solid. LCMS (ESI): m / z: 1489.79 [M + H] + .

[0348] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(4-((2-(methylsulfonyl)-5-nitronicotinamido)methyl)-1H-1,2,3-triazol-1-yl)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((( 1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 50 Compound 27 (36.2 mg, 0.03 mmol, 1.0 equiv.) and compound 17 (8.5 mg, 0.03 mmol, 1.0 equiv.) were mixed with 6 mL of a 1:1 DMSO / HO solution. CuSO4.5HO (3 mg, 0.012 mmol, 0.4 equiv.) and TBTA (6.3 mg, 0.012 mmol, 0.4 equiv.) were added, followed by sodium ascorbate (4.7 mg, 0.0238 mmol, 0.8 equiv.) and stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give 50 (32.6 mg, 72%) as a yellow solid. LCMS (ESI): m / z: 1489.60 [M + H] + .

[0349] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(4-(2-((6-(methylsulfonyl)-5-nitropyridin-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 51 Compound 27 (16.7 mg, 0.0138 mmol, 1.0 equiv.) and compound 19 (3.8 mg, 0.0138 mmol, 1.0 equiv.) were mixed with 3 mL of a 1:1 DMSO / HO solution. CuSO4.5HO (1.4 mg, 0.0055 mmol, 0.4 equiv.) and TBTA (3 mg, 0.0055 mmol, 0.4 equiv.) were added, followed by sodium ascorbate (2.2 mg, 0.0110 mmol, 0.8 equiv.), and the mixture was stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give 51 (11.2 mg, 55%) as a yellow powder. LCMS (ESI): m / z: 1476.82 [M + H] + .

[0350] [ka] 4-((S)-2-((S)-3-methyl-2-(2-(4-(2-((6-(methylsulfonyl)-3-nitropyridin-2-yl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetamido)butanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate 52 Compound 27 (16.7 mg, 0.0138 mmol, 1.0 equiv.) and compound 20 (3.8 mg, 0.0138 mmol, 1.0 equiv.) were mixed with 3 mL of a 1:1 DMSO / HO solution. CuSO4.5HO (1.4 mg, 0.0055 mmol, 0.4 equiv.) and TBTA (3 mg, 0.0055 mmol, 0.4 equiv.) were added, followed by sodium ascorbate (2.2 mg, 0.0110 mmol, 0.8 equiv.), and the mixture was stirred at room temperature for 1 h. The reaction mixture was purified by RP preparative HPLC to give 52 (12.3 mg, 60%) as a yellow powder. LCMS (ESI): m / z: 1477.96 [M + H] + .

[0351] [ka] 4-((12S,15S)-12-Isopropyl-1-(6-(methylsulfonyl)-5-nitropyridin-3-yl)-1,6,10,13-tetraoxo-15-(3-ureidopropyl)-8-oxa-2,5,11,14-tetraazahexadecaamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-( ((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (53) To a solution of 21 (20.0 mg, 0.049 mmol, 1.0 equiv) in DCM / DMAc (2 mL / 2 mL) was added Val-Cit-PAB-MMAE (67.0 mg, 0.059 mmol, 1.2 equiv), followed by DIC (12.0 mg, 0.098 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by preparative HPLC to give 53 (14.5 mg, yield: 19.59%, purity: 97.30%) as a white powder. LCMS (ESI): m / z: 1510.1 [M + H] +. 11H NMR (DMSO-d6, 600 MHz): δ 10.04 (d, 1H), 9.27 (d, 1H), 9.16 (t, 1H), 8.89 (d, 1H), 8.36 - 8.23 (m, 2H), 8.14 - 8.01 (m, 1H), 7.91 - 7.82 (m, 2H), 7.57 (s, 2H), 7.36 - 7.23 (m, 6H), 7.19 - 7.15 (m, 1H), 6.00 (s, 1H), 5.13 - 4.94 (m, 3H), 4.74 (s, 1H), 4.63 (s, 1H), 4.52 - 4.45 (m, 2H), 4.43 - 4.36 (m, 3H), 4.30 - 4.22 (m, 3H), 4.05 - 3.93 (m, 7H), 3.78 (d, 1H), 3.58 - 3.54 (m, 1H), 3.49 - 3.47 (m, 3H), 3.42 - 3.37 (m, 2H), 3.34 - 3.30 (m, 2H), 3.24 - 3.16 (m, 5H), 3.12 (s, 1H), 3.07 - 2.91 (m, 4H), 2.89 - 2.81 (m, 2H), 2.42 - 2.38 (m, 1H), 2.30 - 2.22 (m, 1H), 2.17 - 1.89 (m, 4H), 1.85 - 1.65 (m, 4H), 1.62 - 1.41 (m, 4H), 1.39 - 1.18 (m, 4H), 1.07 - 0.72 (m, 30H).

[0352]

Chem.

[0353]

Chem.

[0354] [ka] (R)-2-((2-acetamido-2-carboxyethyl)thio)-5-nitronicotinic acid 28 and / or 2-(((2S,4S)-1-(tert-butoxy)-4-methyl-1-oxohexan-2-yl)amino)-5-nitronicotinic acid 29 To a solution of N-acetyl-L-cysteine ​​(21 mg, 0.13 mmol, 1.0 equiv.) and tert-butyl (2S,4S)-2-amino-4-methylhexanoate (29 mg, 0.13 mmol, 1.0 equiv.) in PBS buffer (6 mL, pH = 8.12) was added a solution of 24 (30 mg, 0.13 mmol, 1.0 equiv.) in DMSO (0.5 mL) dropwise at RT and stirred for 1 h at RT. 24 was consumed, and the major product was 28. 29 was not detected.

[0355] [ka] (R)-2-((2-acetamido-2-carboxyethyl)thio)-5-nitronicotinic acid 28 and / or 2-(((2S,4S)-1-(tert-butoxy)-4-methyl-1-oxohexan-2-yl)amino)-5-nitronicotinic acid 29 To a solution of N-acetyl-L-cysteine ​​(21 mg, 0.13 mmol, 1.0 equiv.) and tert-butyl (2S,4S)-2-amino-4-methylhexanoate (29 mg, 0.13 mmol, 1.0 equiv.) in PBS buffer (6 mL, pH = 8.12) was added a solution of 14 (32 mg, 0.13 mmol, 1.0 equiv.) in DMSO (0.5 mL) dropwise at RT and stirred for 1 h at RT. 14 was consumed, and the major product was 28. 29 was not detected. Both reactions demonstrate the specificity of this species for reaction with thiol nucleophiles.

[0356] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0357] Conjugation Methods An exemplary conjugation method is illustrated below with compound 39 and trastuzumab (DAR8): [ka] Reaction scheme

[0358] General conjugation protocol: Prepare the antibody at approximately 15 mg / mL in 20 mM histidine, pH 6.0. Adjust the pH to 7.2 with 0.5M sodium phosphate buffer + 50mM EDTA, adjust the antibody concentration to approximately 12mg / mL, and make the final phosphate buffer concentration 100mM sodium phosphate + 10mM EDTA. Reduce the antibody with 8-10 molar equivalents of TCEP (stock solution: 10 mM aqueous solution) at 37°C for 2 hours. Monitor by RP-HPLC Bring to room temperature (20-25°C) in 10 minutes o Buffer exchange to 20 mM histidine pH 6.5-8.7 (pH can be anywhere between 6.5 and 8.7 as optimized for each drug linker) by desalting on a Zeba Spin desalting column Prepare a 10 mM stock solution of drug-linker in DMSO Add 11-14 molar equivalents of drug-linker Conjugate at RT and monitor by RP-HPLC After completion (within 1-16 hours), quench by adding 20 equivalents of N-acetylcysteine ​​and let stand for 1 hour. ○ Excess DL is desalted through a Zeba Spin desalting column and buffer exchanged into 20 mM histidine pH 6.0 using a 30 kD Amicon filter.

[0359] Exemplary RP-HPLC results are shown in Figures 1-2.

[0360] DAR Stability An exemplary DAR stability study of trastuzumab-32 and trastuzumab-38 in human plasma was performed. DAR loss was minimal for the trastuzumab-conjugates. [Table 8]

[0361] DAR Stability Assay Protocol: Plasma IgG depletion: Endogenous IgG was removed by recombinant protein A-Sepharose gel filtration. 10 mL of Sepharose-A was used per 10 mL of plasma. The Sepharose-A was washed three times with PBS, and centrifugation was used to separate the wash buffer from the Sepharose. Plasma was mixed with Sepharose-A for 2 hours at 4°C, after which the plasma was removed from the Sepharose by centrifugation. Incubation: ADC was spiked with depleted plasma from each test species and PBS to obtain a 1 mg / mL ADC concentration. Samples were incubated at 37°C. Aliquots containing 20 μg ADC were taken at designated time points. Aliquots were frozen at -80°C until analysis. Capture and elution: Protein-A beads were washed twice with PBS and resuspended at the original volume. 15 μL of beads were added per well to a 96-well ultra-low binding plate along with 35 μL PBS and 10 μL of ADC-incubated sample. The plate was mixed for 1 hour at room temperature. The beads were separated, and the supernatant was collected and frozen at -80°C. The beads were washed three times with PBS-T and 250 μL PBS-T. The ADC was eluted from the beads with 50 μL of 100 mM acetic acid and neutralized with 10 μL of 1.5 M Tris-HCl pH 8.5. The ADC was reduced with 2 μL of 100 mM DTT and incubated for 30 minutes at 37°C. The bead-captured supernatant was diluted 1:3 with acetonitrile and centrifuged at 17 g for 10 minutes. The supernatant was removed from the pelleted protein and injected into the MS. Data analysis: DAR was determined by RP-MS and free payload by MRM.

[0362] Cytotoxicity data of representative conjugates Test compounds included the MC-VC-PABC-MMAE Seagen drug-linker, example compound, and MMAE. [Table 9]

[0363] Assay Protocol: HCC1954 breast ductal carcinoma or SK-BR-3 cells (ATCC, Manassas, VA, USA) were seeded into 384-well white-walled culture plates and allowed to attach for 2–4 hours. Cells were then treated with 5-fold serial dilutions of test articles prepared in at least duplicate at 2× final concentration and incubated at 37°C for 120 hours. Post-treatment cell viability was determined using the Cell Titer Glo 2.0 Assay (Promega, Madison, WI, USA) and normalized to untreated controls. Dose-response relationships were analyzed using GraphPad Prism (La Jolla, CA, USA) and IC values ​​were calculated. 50 Values ​​were derived by nonlinear regression analysis using a four-parameter logistic equation.

[0364] Applicant's disclosure is now described in preferred embodiments with reference to the figures, in which like numbers indicate identical or similar elements. Throughout this specification, the use of "one embodiment," "an embodiment," or similar phrases means that a particular feature, structure, or configuration described in connection with that embodiment is included in at least one embodiment of the invention. Thus, throughout this specification, the use of "one embodiment," "an embodiment," and similar phrases may, but do not necessarily, refer to the same embodiment.

[0365] The described properties, structures, or characteristics disclosed by applicants may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth to provide a thorough understanding of embodiments of the present invention. However, those skilled in the relevant art will recognize that applicants' compositions and / or methods may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the present disclosure.

[0366] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. The methods described herein can be performed in any order that is logically possible in addition to the specific order disclosed.

[0367] Incorporation by Reference Other documents, such as patents, patent applications, patent publications, journals, textbooks, articles, manuscripts, web content, etc., are mentioned and cited in this disclosure. All such documents are incorporated herein by reference in their entirety and for all purposes. Any matter, or portion thereof, that is indicated as being incorporated herein by reference but that contradicts existing definitions, descriptions, or other disclosures is incorporated by reference only to the extent that the incorporated matter does not contradict the matters of this disclosure. In the event of a conflict, the conflict will be resolved in favor of the present disclosure as the preferred disclosure.

[0368] equivalent The representative examples are intended to aid in the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will be apparent to those skilled in the art from the entire contents of this document, including the examples, and by reference to the scientific and patent literature contained therein. The examples contain important additional information, exemplification, and guidance that can be used to adapt the practice of this invention to various embodiments and equivalents thereof.

Claims

1. A drug conjugate comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (I): 【Chemistry 1】 [In the ceremony: Each R ２ , R ３ , R ４ are independently H and NO ２ , C.N., C.F. ３ , F, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne, C ６－１０ aryl, and W; R ５ is H, Cl, SOR ６ , S.O. ２ R ６ , OR ７ , N.R.R. ７ or W; R ６ is unsubstituted or substituted C １－６ Alkyl or C ６－１０ is aryl; R ７ is unsubstituted or substituted C ２－６ Alkyne or C ２－６ is an alkyne; W is: 【Chemistry 2】 selected from the group consisting of: R １ is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; R is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ It is aryl. or a pharmaceutically acceptable salt thereof.

2. Structural formula: 【Transformation 3】 The drug conjugate of claim 1, comprising:

3. Structural formula: 【Chemistry 4】 The drug conjugate of claim 1 or 2, comprising:

4. R ２ , R ３ and R ４ One of them is NO ２ , C.N., C.F. ３ The drug conjugate of any one of claims 1 to 3, wherein the compound is selected from the group consisting of:

5. 5. The drug conjugate of claim 1, wherein R is H.

6. R is unsubstituted or substituted C １－６ The drug conjugate of any one of claims 1 to 4, wherein the aryl group is alkyl.

7. R ２ , R ３ and R ４ One of them is NO ２ The drug conjugate of claim 4 or 5,

8. R ２ , R ３ and R ４ The drug conjugate of claim 4 or 5, wherein one of is CN.

9. R ２ or R ４ is CF ３ The drug conjugate of claim 4 or 5,

10. R ２ is CF ３ The drug conjugate of claim 4 or 5, which is not

11. R ４ is C(O)OR, and R ２ is CF ３ The drug conjugate of claim 4 or 5, which is not

12. R ２ or R ４ The drug conjugate of claim 4 or 5, wherein is F.

13. Structural formula: 【Transformation 5】 The drug conjugate of any one of claims 1 to 4, comprising:

14. Structural formula: 【Transformation 6】 The drug conjugate of any one of claims 1 to 4, comprising:

15. The linker moiety has the structural formula (I １ ) or (I ７ 13. The drug conjugate of any one of claims 1 to 12, comprising:

16. The linker moiety has the structural formula (I ２ ) or (I ８ 13. The drug conjugate of any one of claims 1 to 12, comprising:

17. The linker moiety has the structural formula (I ３ ) or (I ９ 13. The drug conjugate of any one of claims 1 to 12, comprising:

18. The linker moiety has the structural formula (I ４ ) or (I １０ 13. The drug conjugate of any one of claims 1 to 12, comprising:

19. The linker moiety has the structural formula (I ５ ) or (I ６ 13. The drug conjugate of any one of claims 1 to 12, comprising:

20. The drug conjugate of any of claims 1 to 19, wherein the linker moiety further comprises a spacer, a peptide moiety, and / or a self-immolative moiety.

21. The spacer, peptide moiety, and / or self-immolative moiety may be R ２ , R ３ , R ４ and R ５ 21. The drug conjugate of claim 20, wherein the drug conjugate is conjugated to one of:

22. Structural formula: 【Transformation 7】 [During the ceremony, L Ａ is a spacer moiety or is absent; L Ｂ is a peptide moiety or is absent; and L Ｃ is a self-sacrificing part or does not exist. The drug conjugate of claim 1 or 2, comprising:

23. Further structural formula: 【Transformation 8】 wherein D is a drug moiety.

23. The drug conjugate of claim 22, comprising a drug moiety D comprising:

24. Structural formula: 【Chemistry 9】 The drug conjugate of claim 3, comprising:

25. Further structural formula: 【Chemistry 10】 25. The drug conjugate of claim 24, comprising a drug moiety comprising:

26. The drug conjugate of any of claims 22 to 25, wherein the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide.

27. The drug conjugate of any of claims 22 to 25, wherein the peptide portion comprises 1 to 6 amino acids.

28. 28. The drug conjugate of claim 27, wherein the amino acid is a natural and / or unnatural amino acid.

29. The self-sacrifice part: 【Chemistry 11】 The drug conjugate of any of claims 22 to 29, selected from the group consisting of:

30. 30. The drug conjugate of any of claims 1-29, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

31. 31. The drug conjugate of any of claims 1 to 30, wherein the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

32. 32. The drug conjugate of claim 31, wherein the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:

16.

33. A drug conjugate comprising a targeting moiety, a linker moiety, and a drug moiety, wherein the drug moiety is conjugated to a linker that is conjugated to the targeting moiety, and wherein the linker moiety has structural formula (II): 【Chemistry 12】 [In the ceremony: Each R ２ , R ３ , R ４ are independently H, NO ２ , C.N., C.F. ３ , F, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne, C ６－１０ aryl, and W; R ５ H, Cl, SOR ６ , S.O. ２ R ６ , OR ７ , N.R.R. ７ or W; R ６ is unsubstituted or substituted C １－６ Alkyl or C ６－１０ is aryl; R ７ is unsubstituted or substituted C ２－６ Alkyne or C ２－６ is an alkyne; W is: 【Chemistry 13】 selected from the group consisting of: R １ is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; R is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; and Each Y is independently a peptide or antigen-binding moiety that contains a cysteine ​​moiety. or a pharmaceutically acceptable salt thereof.

34. Structural formula: 【Chemistry 14】 34. The drug conjugate of claim 33, comprising:

35. Structural formula: 【Chemistry 15】 35. The drug conjugate of claim 34, comprising:

36. R ２ , R ３ and R ４ One of them is NO ２ , C.N., C.F. ３ The drug conjugate of any of claims 33 to 35, wherein the compound is selected from the group consisting of:

37. 37. The drug conjugate of any of claims 33 to 36, wherein R is H.

38. R is unsubstituted or substituted C １－６ The drug conjugate of any of claims 33 to 36, wherein the group is alkyl.

39. R ２ , R ３ and R ４ One of them is NO ２ 39. The drug conjugate of claim 37 or 38, wherein:

40. R ２ , R ３ and R ４ 39. The drug conjugate of claim 37 or 38, wherein one of is CN.

41. R ２ or R ４ is CF ３ 39. The drug conjugate of claim 37 or 38, wherein:

42. R ２ is CF ３ 39. The drug conjugate of claim 37 or 38, which is not

43. R ４ is C(O)OR, and R ２ is CF ３ 39. The drug conjugate of claim 37 or 38, which is not

44. R ２ or R ４ The drug conjugate of claim 37 or 38, wherein is F.

45. Structural formula: 【Chemistry 16】 The drug conjugate of any of claims 33 to 36, comprising:

46. Structural formula: 【Chemistry 17】 The drug conjugate of any of claims 33 to 36, comprising:

47. The drug conjugate of any of claims 33 to 46, wherein the linker moiety further comprises a spacer, a peptide moiety, and / or a self-immolative moiety.

48. Structural formula: [Chemistry 18] [During the ceremony, L Ａ is a spacer moiety or is absent; L Ｂ is a peptide moiety or is absent; and L Ｃ is a self-sacrificing part or does not exist.

35. The drug conjugate of claim 33 or 34, comprising:

49. Further structural formula: 【Chemistry 19】 wherein D is a drug moiety.

49. The drug conjugate of claim 48, comprising a drug moiety comprising:

50. Structural formula: 【Chemistry 20】 49. The drug conjugate of claim 48, comprising:

51. Further structural formula: 【Chemistry 21】 51. The drug conjugate of claim 50, comprising a drug moiety comprising:

52. 52. The drug conjugate of any of claims 48 to 51, wherein the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide.

53. The drug conjugate of any of claims 48 to 52, wherein the peptide portion comprises 1 to 6 amino acids.

54. 54. The drug conjugate of claim 53, wherein the amino acid is a natural and / or unnatural amino acid.

55. The self-sacrifice part: 【Chemistry 22】 The drug conjugate of any of claims 48 to 54, selected from the group consisting of:

56. 56. The drug conjugate of any of claims 48-55, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

57. 57. The drug conjugate of any of claims 48-56, wherein the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

58. 58. The drug conjugate of claim 57, wherein the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:

16.

59. A composition comprising the drug conjugate of any of claims 1 to 58.

60. 59. A pharmaceutical composition comprising the drug conjugate of any one of claims 1 to 58, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

61. 61. A unit dosage form comprising the pharmaceutical composition of claim 60.

62. 1. Compounds useful for forming linker moiety-drug moiety conjugates, targeting moiety-linker moiety conjugates, or targeting moiety-linker moiety-drug moiety conjugates, comprising a compound having structural formula (III): 【Chemistry 23】 [In the formula, each R ２ , R ３ , R ４ are independently H, NO ２ , C.N., C.F. ３ , F, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne, C ６－１０ aryl, and W; Each R ５ and R Ｙ are independently H, Cl, SOR ６ , S.O. ２ R ６ , OR ７ , N.R.R. ７ or W; R ６ is unsubstituted or substituted C １－６ Alkyl or C ６－１０ is aryl; R ７ is unsubstituted or substituted C ２－６ Alkyne or C ２－６ is an alkyne; W is: 【Chemistry 24】 Selected from: R １ is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; and R is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ It is aryl. A compound having a structure comprising:

63. R Ｙ is SO ２ R ６ and has the structural formula: 【Chemistry 25】 63. The compound of claim 62 having the formula:

64. R ２ , R ３ and R ４ One of them is NO ２ , C.N., C.F. ３ 64. The compound of claim 62 or 63, wherein the compound is selected from the group consisting of:

65. 65. The compound of any of claims 62-64, wherein R is H.

66. R is unsubstituted or substituted C １－６ 65. The compound of any one of claims 62 to 64, which is alkyl.

67. R ２ , R ３ and R ４ One of them is NO ２ 67. The compound of claim 65 or 66, wherein:

68. R ２ , R ３ and R ４ 67. The compound of claim 65 or 66, wherein one of is CN.

69. R ２ or R ４ is CF ３ 67. The compound of claim 65 or 66, wherein:

70. R ２ is CF ３ 67. The compound of claim 65 or 66, which is not

71. R ４ is C(O)OR, and R ２ is CF ３ 67. The compound of claim 65 or 66, which is not

72. R ２ or R ４ 67. The compound of claim 65 or 66, wherein is F.

73. R ２ , R ３ , R ４ and R ５ 67. The compound of claim 66, wherein one of is C(O)NHR'.

74. Structural formula: 【Chemistry 26】 [During the ceremony, L Ａ is a spacer moiety or is absent; L Ｂ is a peptide moiety or is absent; L Ｃ is a self-sacrificing part or is absent; R ６ and R' ６ may be the same or different, and unsubstituted or substituted C １－６ Alkyl or C ６－１０ is aryl; and D' is a drug moiety D or is absent.

66. The compound of claim 64 or 65, having the formula:

75. Structural formula: 【Chemistry 27】 75. The compound of claim 74, having the formula:

76. R ６ and R' ６ 76. The compound of claim 74 or 75, wherein each of is methyl.

77. 77. The compound of any of claims 74-76, wherein the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide.

78. 78. The compound of any of claims 74-77, wherein the peptide portion comprises 1 to 6 amino acids.

79. 79. The compound of claim 78, wherein the amino acid is a natural and / or unnatural amino acid.

80. The self-sacrifice part: 【Chemistry 28】 80. The compound of any of claims 74 to 79, selected from the group consisting of:

81. 81. The compound of any of claims 74-80, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

82. A composition comprising a compound according to any one of claims 62 to 81.

83. 1. A method of making a linker-drug conjugate, comprising: (a) Structural formula: 【Chemistry 29】 providing a compound having (b) reacting the compound with a precursor of the spacer, a precursor of the peptide, and / or a precursor of the self-immolative moiety to form a compound of the structural formula: 【Transformation 30】 [During the ceremony, Each R ２ , R ３ , R ４ are independently H, NO ２ , C.N., C.F. ３ , F, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne, C ６－１０ aryl, and W; R ５ H, Cl, SOR ６ , S.O. ２ R ６ , OR ７ , N.R.R. ７ or W; R ６ is unsubstituted or substituted C １－６ Alkyl or C ６－１０ is aryl; R ７ is unsubstituted or substituted C ２－６ Alkyne or C ２－６ is an alkyne; W is: 【Chemistry 31】 Selected from: R １ is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; R is H, unsubstituted or substituted C １－６ Alkyl, C ２－６ Alkien, C ２－６ Alkyne or C ６－１０ is aryl; L Ａ is a spacer moiety or is absent; L Ｂ is a peptide moiety or is absent; L Ｃ is a self-sacrificing portion or is absent; and D' is a drug moiety D or is absent. forming a conjugate having

84. L Ａ is the spacer moiety, and L Ｂ is the peptide moiety, and L Ｃ 84. The method of claim 83, wherein D is a self-immolative moiety and D' is a drug moiety.

85. 85. The method of claim 83 or 84, further comprising reacting the resulting drug conjugate with a targeting moiety to form a targeting moiety-linker-drug conjugate.

86. 86. The method of any of claims 83-85, wherein the spacer moiety is selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide.

87. 87. The method of any of claims 83-86, wherein the peptide portion comprises from about 1 to about 6 amino acids.

88. 88. The method of claim 87, wherein the amino acids are natural and / or unnatural amino acids.

89. The self-sacrifice part: 【Chemistry 32】 89. The method of any of claims 83 to 88, wherein the method is selected from the group consisting of:

90. 90. The process of any of claims 83 to 89, wherein step (b) is carried out at a pH in the range of 5.5 to 6.

5.

91. 90. The method of any of claims 83-89, wherein step (b) is carried out at a pH of about 6.

0.

92. 92. The method of any of claims 83-91, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anti-cancer drug, a steroid, a TLR7 / TLR9 antagonist, a peptide, a protein, and a nucleic acid.

93. 93. The method of any of claims 83-92, wherein the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide, and a nucleic acid.

94. 94. The method of claim 93, wherein the drug conjugate has a targeting moiety to drug moiety ratio of about 1:1 to about 1:

16.

95. A method for treating and / or preventing a condition in a subject in need thereof, comprising administering to said subject a drug conjugate of any of claims 1-58.

96. 96. The method of claim 95, wherein the condition is one or more of cancer, an autoimmune disorder, or an infectious disease.

97. Cancers include adrenal gland cancer, anal region cancer, basal and squamous cell skin cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors (astrocytoma, glioblastoma multiforme, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's family tumors, eye cancer (ocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumors, gastrointestinal stromal tumors (gist), gestational trophoblastic disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, pulmonary carcinoid tumors, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, and neuroblastoma. , non-small cell lung cancer, neoplasms of the central nervous system (CNS), oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (NETs), penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, squamous cell carcinoma, cancer of unknown primary site (CUP), environmentally induced cancer, a combination of cancers, and / or a metastatic lesion of cancer.

98. Autoimmune disorders include Th2 lymphocyte disorders, Th1 lymphocyte disorders, activated B lymphocyte disorders, active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reactions, allergic rhinitis, Alport syndrome, anaphylaxis, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behcet's disease, bird fancier's lung, bronchial asthma, Kaplan's syndrome, cardiomyopathy, celiac disease, Chagas' disease, chronic glomerulonephritis, Cogan's syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, and cryoglobulinemia. Gushing syndrome, dermatomyositis, discoid lupus erythematosus, Dressler syndrome, Eaton-Lambert syndrome, echovirus infection, encephalomyelitis, endocrine ophthalmopathy, Epstein-Barr virus infection, equine chronic pulmonary emphysema, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs heterochromatic iridocyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (type 1), Lambert-Eaton syndrome, laminitis, lichen planus, lupoid hepatitis, lupus 97. The method of claim 96, wherein the condition is one of lymphopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, presenile dementia, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, recurrent miscarriage, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, Samter's syndrome, schistosomiasis, Schmidt's syndrome, scleroderma, Shulman's syndrome, Sjogren's syndrome, stiff-person syndrome, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type 1 diabetes mellitus, ulcerative colitis, uveitis, leukoplakia, Waldenstrom's macroglobulinemia, and / or granulomatosis with polyangiitis.

99. 97. The method of claim 96, wherein the infectious disease is one or more of a bacterial disease, a systemic fungal disease, a rickettsial disease, a parasitic disease, and / or a viral disease. 【Request Item 100】 【Chemistry 33】 【Transformation 34】 【Chemistry 35】 A compound selected from the group consisting of: 【Request Item 101】 【Chemistry 36】 【Chemistry 37】 【Transformation 38】 A compound selected from the group consisting of:

102. 102. A composition comprising a compound of claim 100 or 101.

103. 60. Use of a drug conjugate of any of claims 1 to 58 for the manufacture of a medicament.

104. 60. Use of the drug conjugate of any of claims 1 to 58 for the treatment of cancer.

105. 60. Use of the drug conjugate of any of claims 1 to 58 for the treatment of an autoimmune disorder.

106. 60. Use of the drug conjugate of any of claims 1 to 58 for the treatment of an infectious disease.

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