TW202434300A - Drug conjugates and methods of preparing and using the same - Google Patents

Abstract

The invention provides novel linkers, linker conjugates, and drug conjugates thereof, as well as methods of preparation and use thereof for treating various diseases and conditions.

Description

Drug conjugates and methods of preparation and use thereof

Related applications

This application claims priority to PCT/CN2023/073383 filed on January 20, 2023, the entire contents of which are incorporated herein by reference. Field of Invention

The present invention generally relates to novel compounds, methods of preparing the same, and therapeutic uses thereof. More particularly, the present invention provides novel linkers, linker conjugates, and drug conjugates thereof, as well as methods of preparing the same and uses thereof for treating various diseases or conditions.

Invention Background

Drug conjugates, such as antibody-drug conjugates (ADCs), can provide an effective method for delivering drugs to targeted sites in tissues or organs. To date, the FDA has approved twelve ADCs, including the first ADC approved by the FDA in 2000: gemtuzumab ozogamicin (Mylotarg™). (See, for example, 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.) The experience gained from the development of these ADCs highlights the importance of optimizing the method of linking drugs to proteins. Cysteine modification is becoming increasingly popular due to its high nucleophilicity, selectivity for electrophilic reagents, and the low abundance of sulfhydryl groups in naturally occurring proteins.

Cis-butenediamide-based linkers have been used in a variety of ADCs currently under investigation. This is due in part to the high reactivity and selectivity of cis-butenediamides for cysteine residues. One known approach used in ADC design involves the use of self-hydrolyzed cis-butenediamides for cysteine modification, as cis-butenediamides react rapidly and selectively with thiols. While cis-butenediamide conjugation has resulted in stable drug-protein conjugates, self-hydrolyzed cis-butenediamides generate acid species. Acid species can have unpredictable and adverse effects on the properties of the resulting ADC. The cis-butylenediimide moiety is directly related to the instability of cis-butylenediimide-based ADCs in plasma. In addition, it has been shown that the drug-antibody ratio (DAR) of ADCs is reduced in the presence of glutathione. (See, e.g., WIPO 2013/173337; Alley et al., 2008 Bioconjugate Chem. 19, 759.)

There remains a need for drug conjugates and conjugation methods that can provide improved efficacy, stability, and drug loading.

Summary of the invention

Provided herein are drug conjugates comprising a targeting moiety, a linker, and a drug moiety, methods of preparing the same, and methods of using the same to treat and/or prevent conditions.

In particular, the present invention provides a significantly expanded available cysteine-targeted electrophilic linker strategy with high selectivity for thiols (e.g., cysteine) over other reactive functional groups (e.g., amines). The linker compounds and methods disclosed herein can be efficiently conjugated at low pH (e.g., pH about 6.0) without retro-Michael deconjugation, and the drug-linker conjugates have overall good stability at room temperature.

In one aspect, 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 the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (I): (I) wherein: each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkyene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; ^R1 is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl group; R is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl group; or a pharmaceutically acceptable salt thereof.

In another aspect, 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 the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (II): (II) wherein: each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; ^R1 is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; R is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; and each Y is independently a peptide or an antigen-binding portion comprising a cysteine portion, or a pharmaceutically acceptable salt thereof.

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

In another aspect, the present invention generally relates to a pharmaceutical composition comprising a drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

In another aspect, the invention generally relates to a unit dosage form comprising the pharmaceutical composition of the invention.

In yet another aspect, the invention generally relates to a compound suitable for forming a linker moiety-drug moiety conjugate, a targeting moiety-linker moiety conjugate, or a targeting moiety-linker moiety-drug moiety conjugate, the compound having a structure comprising structural formula (III): (III) Each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; Each of R ⁵ and ^RY is independently selected from the group consisting of H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ , and W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; R ¹ is H, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl group; and R is H, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl group.

In yet another aspect, the invention generally relates to a composition comprising a compound disclosed herein suitable for forming a linker moiety-drug moiety conjugate, a targeting moiety-linker moiety conjugate, or a targeting moiety-linker moiety-drug moiety conjugate.

In another aspect, the present invention generally relates to a method for preparing a linker-drug conjugate, comprising: (a) providing a compound having the following structural formula: (III ¹ ) (b) reacting the compound with a promotor of a spacer, a promotor of a peptide and/or a promotor of a self-degradable moiety to form a complex having the following structural formula: , (III ² ) , (III ³ ) (III ⁴ ) (III ⁵ ) or (III ⁶ ) (III ⁷ ) wherein each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _{2-6 alkene, C 2-6 alkynyl} , C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; R ¹ is H, an unsubstituted or substituted C _{1-6 alkyl, C 2-6 alkene, C 2-6 alkynyl, or C 6-10 aryl; R is H, an unsubstituted or substituted C 1-6 alkyl, C 2-6 alkene , C 2-6 alkynyl} , or C _6-10 aryl; ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; ^LC is a self-degrading moiety or is absent; and D' is a drug moiety D or is absent.

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

In another aspect, the present invention generally relates to the use of the drug conjugates disclosed herein for the manufacture of a medicament.

In yet another aspect, the invention generally relates to the use of the drug conjugates disclosed herein for treating one or more of cancer, autoimmune disorders, or infectious diseases.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As described herein, novel linkers, linking methods and conjugates, and drug conjugates have been developed that have unexpected advantages over the prior art.

Specifically, the linkers, linker conjugates, and conjugation strategies disclosed herein significantly expand the available cysteine-targeted electrophilic chemistries with high selectivity for thiols (e.g., cysteine) over other reactive functional groups (e.g., amines). The present invention provides the ability to efficiently conjugate at low pH (e.g., pH of about 6.0) without reverse Michael deconjugation, thereby resulting in high stability. In addition, the drug-linker conjugates of the present invention exhibit overall good stability at room temperature.

The present disclosure further provides drug conjugates, methods of preparing the same, kits comprising the drug conjugates and components thereof, and methods of using the drug conjugates and kits in treating one or more diseases or conditions.

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

The following terms are intended to have the following meanings unless the context in which the terms appear indicates otherwise.

The ranges provided herein should be understood as shorthand for all values within the range. For example, a range of 1 to 16 should be understood to include any number, combination of numbers, or sub-range selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

As used herein, "at least" a particular value should be understood to mean that value and all values greater than that value.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless expressly stated or obvious from the context, as used herein, the term "or" should be construed as inclusive.

Any composition or method disclosed herein may be combined with one or more of any other compositions and methods provided herein.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

When used to define compositions and methods, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" shall mean that the compositions and methods include the recited elements and exclude other elements that are of any significance to the compositions and methods. For example, "consisting essentially of" refers to the administration of the pharmacologically active agents that are explicitly recited and excludes pharmacologically active agents that are not explicitly recited. The term essentially of does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" shall mean excluding trace elements of other ingredients and essential method steps. Embodiments defined by each of these transition terms are within the scope of the present invention.

Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention encompasses all such compounds that fall within the scope of the present invention, including cis and trans isomers, configurational isomers, R- and S-mirror isomers, non-mirror isomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included in the present invention. In certain embodiments, each asymmetric atom has an R-configuration or S-configuration with at least a 50% mirror image excess, at least a 60% mirror image excess, at least a 70% mirror image excess, at least an 80% mirror image excess, at least a 90% mirror image excess, at least a 95% mirror image excess, or at least a 99% mirror image excess. For photoactive compounds, it is often preferred to use one mirror image to the substantial exclusion of another mirror image.

Isomeric mixtures containing any of a variety of isomer ratios may be used in accordance with the present invention. For example, when only two isomers are combined, the present invention encompasses mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios. One of ordinary skill in the art will readily appreciate that more complex isomer mixtures encompass similar ratios.

For example, if a specific mirror image isomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, wherein the resulting non-mirror image isomer mixture is separated and the auxiliary group is cleaved to provide the pure desired mirror image isomer. Alternatively, in the case where the molecule contains a basic function (such as an amine group) or an acidic function (such as a carboxyl group), a non-mirror image isomer salt is formed from an appropriate optically active acid or base, and the non-mirror image isomer thus formed is subsequently resolved by fractional crystallization or chromatographic methods well known in the art, and the pure mirror image isomer is then recovered.

Isomeric mixtures can be separated into the pure or substantially pure geometric or optical isomers, non-mirror isomers, racemates on the basis of the physicochemical differences of the constituents, for example by separation by chromatography and/or fractional crystallization.

The definitions of specific functional groups and chemical terms are described in more detail below. When a range of values is listed, it is intended that each value and subrange be included within that range. For example, "C _1-6 alkyl" is intended to include C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C _1-6 , C _1-5 , C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-5 , C _2-4 , C _{2-3 ,} C _3-6 , C 3-5, C _3-4 , C _4-6 , C _4-5 , and C _5-6 alkyl.

Where substituents are specified by their conventional chemical formula written from left to right, this also covers chemically identical substituents resulting from writing the structure from right to left, e.g. -C(=O)-O- is equivalent to -O-C(=O)-.

The structures of the compounds of the present invention are constrained by chemical bonding principles known to those skilled in the art. Therefore, where a group may be substituted with one or more of a variety of substituents, such substitutions are selected so as to comply with chemical bonding principles and to yield compounds that are not inherently unstable and/or that are generally known to those skilled in the art to be unstable under environmental conditions (e.g., aqueous, neutral, and certain known physiological conditions).

Although the word "about" is placed before the minimum and maximum values in the stated range, the use of numerical values in the multiple quantitative values specified in this application is stated as approximate values unless otherwise expressly indicated. It should be understood that the term "about" is placed before all numerical value designations, although it may not always be expressly stated. It should be understood that such range format is used for convenience and brevity, and should be flexibly interpreted to include not only the values expressly specified as the limits of the range, but also all individual values or sub-ranges encompassed within that range, as if each value and sub-range were expressly specified. For example, ratios ranging from about 1 to about 200 should be understood to include not only the explicitly recited limits of about 1 and about 200, but also include individual ratios such as about 2, about 3, and about 4, as well as sub-ranges such as about 10 to about 50, about 20 to about 100, and the like. It should also be understood that, although not always explicitly recited, the reactants described herein are merely exemplary and that equivalents of such reactants are known in the art.

Unless expressly stated or obvious from the context, the term "about" as used herein when referring to a measurable value such as an amount or concentration and the like is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount.

As used herein, the terms "treat," "treating," and "treatment" with respect to a condition refer to partial or complete relief of the condition; delaying the progression or development of the condition; eliminating, reducing, or delaying the development of one or more symptoms associated with the condition; or increasing progression-free or overall survival of the condition.

Treatment may be directed at one or more effects or symptoms of a disease and/or potential pathology. Treatment may be any reduction in the disease or symptoms of a disease and may be, but is not limited to, complete removal. Treating or treatment refers to any sign of success in treating or improving an injury, disease, lesion, or condition, including any objective or subjective parameter, such as a reduction, alleviation, reduction, or rendering the injury, lesion, or condition more tolerable to the patient; slowing the rate of deterioration or decline; rendering the endpoint of deterioration less debilitating; improving the patient's physical or mental well-being. Treatment or amelioration of symptoms may be based on objective or subjective parameters; for example, the results of a physical examination, a neuropsychiatric examination, and/or a psychiatric evaluation. Such a reduction or improvement may be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% compared to an equivalent untreated control as measured by any standard technique.

The method of treatment comprises administering to a subject a therapeutically effective amount of a compound described herein. The administration step may be a single administration or may include a series of administrations. The duration of the treatment period depends on a variety of 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 should also be understood that the effective dose of the agent used for treatment may be increased or decreased during a particular treatment regimen. The dosage may be varied and made apparent by standard diagnostic assays known in the art. In some cases, long-term administration may be required. For example, the composition is administered to a subject in an amount and for a duration sufficient to treat the patient.

As used herein with respect to a condition, the terms "prevent," "preventing," and "prevention" mean preventing the onset of the condition or reducing the likelihood of the condition occurring or recurring, including in an individual who may be susceptible to the condition but has not yet been diagnosed as having the condition.

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 distinguishing factors that are different from a healthy or normal state. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative diseases, inflammatory diseases, immune diseases, metabolic diseases, infectious diseases, and ischemic diseases.

The term "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, hematologic or lymphatic or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease following 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 the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, liver tumors, breast cancer, colon cancer, and head and neck cancer.

The term "autoimmune disorder" may refer to a persistent set of organ-specific or systemic clinical symptoms and signs associated with altered immune homeostasis manifested by qualitative and/or quantitative defects in the expressed autoimmune spectrum.

The term "infectious disease" may refer to any disease caused by infectious organisms such as viruses, bacteria, parasites and/or fungi.

As used herein, the term "in need of" treatment refers to an individual who would benefit biologically, medically, or in terms of quality of life from such treatment.

The term "alkyl" describes an aliphatic hydrocarbon including both straight and branched chain groups.

The term "heteroalkyl" describes an aliphatic hydrocarbon group including straight and branched chain groups substituted with one or more atoms such as nitrogen, oxygen and sulfur.

As used herein, the term "amino acid" refers to a molecule of the general formula _NH2 -CHR-COOH, where "R" is one of a variety of different side chains, or a residue within a peptide having a parent amino acid. Amino acids include naturally occurring amino acids, where "R" is a substituent found in naturally occurring amino acids. "R" may also be a substituent not found in naturally occurring amino acids. The term "amino acid residue" refers to the portion of an amino acid that remains after the loss of a water molecule when joined to another amino acid. The term "modified amino acid" refers to an amino acid having an "R" substituent that does not correspond to one of the twenty genetically encoded amino acids.

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

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with tissues of an individual without undue toxicity, irritation, allergic reaction, and the like, and commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amine groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate Salt, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, apple acid salt, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, brown salt The invention relates to a kind of organic acid derivatives of the present invention. The organic acid derivatives of the present invention include acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

Such 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 suitable bases include alkali metals, alkali earth metals, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali metal or alkali earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins and their analogs, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt can be selected from ammonium, potassium, sodium, calcium and magnesium salts.

As used herein, the term "pharmaceutically acceptable" excipient, carrier or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or delivering the pharmaceutical agent of the invention 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 materials 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; excipients 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 glycerol, 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; ethanol; phosphate buffered solutions; and other nontoxic 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 colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives and antioxidants may also be present in the composition.

As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Therefore, peptides, oligopeptides, dimers, polymers and the like are included in the definition. Full-length proteins and fragments thereof are all covered by the definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation and similar modifications. In addition, a polypeptide may refer to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (usually of a conservative nature), as long as the protein maintains the desired activity. Such modifications may be intentional or may be accidental. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, which is the recipient of a particular treatment. Contemplated subjects for administration include but are not limited to humans (e.g., males or females of any age group, such as 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, such as non-human mammals (e.g., primates (e.g., cynomolgus macaques, Ganges monkeys); commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and/or dogs), rodents (e.g., rats and/or mice), etc. In certain embodiments, the non-human animal is a mammal. Non-human animals may be male or female at any stage of development. Non-human animals may be genetically modified animals. The terms "subject" and "patient" are often used interchangeably herein with respect to human subjects.

The ranges recited herein are intended to be continuous ranges, including every value between the recited minimum and maximum values, and any ranges that can be formed from such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that can be formed by dividing a disclosed numerical value by any other disclosed numerical value. Thus, one skilled in the art will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the numerical values set forth herein, and in all instances such ratios, ranges, and ranges of ratios represent multiple embodiments of the disclosure. Targeting Moiety - Linker - Drug Conjugates

Provided herein in certain embodiments are drug conjugates comprising a linker, a drug moiety, and a targeting moiety. Also provided herein are components of such drug conjugates, including, for example, linkers, linker-drug moiety complexes, and linker-targeting moiety complexes.

In one aspect, 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 the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (I): (I) wherein: each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of H, NO ₂ , CN, CF ₃ , F, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; ^R1 is H, unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; R is H, unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; or a pharmaceutically acceptable salt thereof.

In certain embodiments, R ⁶ is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments, the drug conjugate of (I) has a structural formula selected from the following: , (I ¹ ) , (I ² ) , (I ³ ) , (I ⁴ ) and (I ⁵ ) (I ⁶ )

In certain embodiments, the drug conjugate of (I) has a structural formula selected from the following: , (I ⁷ ) , (I ⁸ ) or (I ⁹ ) (I ¹⁰ )

In certain embodiments of the drug conjugates of (I) to (I ¹⁰ ), one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F.

In certain embodiments, R is H. In certain embodiments, R is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments, one of R ² , R ³ and R ⁴ is NO ₂ .

In certain embodiments, one of R ² , R ³ and R ⁴ is CN.

In certain embodiments, one of R ² or R ⁴ is CF ₃ .

In certain embodiments, R ⁴ is C(O)OR and R ² is not CF ₃ .

In certain embodiments, R ² is not CF ₃ .

In certain embodiments, R ² or R ⁴ is F.

In certain embodiments of (I), the drug conjugate has a structural formula selected from the following: , , or .

In certain embodiments of (I), the drug conjugate has a structural formula selected from the following: , , or .

In certain embodiments of (I), the linker moiety has the structural formula of (I ¹ ) or (I ⁷ ).

In certain embodiments of (I), the linker moiety has the formula (I ² ) or (I ⁸ ).

In certain embodiments of (I), the linker moiety has the structural formula of (I ³ ) or (I ⁹ ).

In certain embodiments of (I), the linker moiety has the structural formula of (I ⁴ ) or (I ¹⁰ ).

In certain embodiments of (I), the linker moiety has the structural formula of (I ⁵ ) or (I ⁶ ).

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

In certain embodiments of (I), the drug conjugate has a structural formula selected from the following: , (I ¹¹ ) , (I ¹² ) , (I ¹³ ) , (I ¹⁴ ) and (I ¹⁵ ) (I ¹⁶ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; and ^LC is a self-cleaving moiety or is absent.

In certain embodiments, the drug conjugate further has a drug moiety D: , (I ¹⁷ ) , (I ¹⁸ ) , (I ¹⁹ ) , (I ²⁰ ) or (I ²¹ ) (I ²² ) where D is the drug part.

In certain embodiments of (I), the drug conjugate has a structural formula selected from the following: , (I ²³ ) , (I ²⁴ ) and (I ²⁵ ) (I ²⁶ )

In certain embodiments of (I), the drug conjugate has a structural formula selected from the following: (I ²⁷ ) (I ²⁸ ) and (I ²⁹ ) (I ³⁰ )

In certain embodiments of (I ¹¹ ) to (I ³⁰ ), the spacer moiety is selected from the group consisting of an alkyl group, a heteroalkyl group, a polyethylene glycol (PEG), and a peptide.

In certain embodiments of (I ¹¹ ) to (I ³⁰ ), the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5 or 6) amino acid units.

In some embodiments of (I ¹¹ ) to (I ³⁰ ), one or more of the amino acids are natural amino acids. In some embodiments, one or more of the amino acids are non-natural amino acids.

In certain embodiments of (I ¹¹ ) to (I ³⁰ ), the self-degradable moiety is selected from the group consisting of: , , and .

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

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

In certain embodiments, the ratio of the targeting moiety to the drug moiety of the drug conjugate is 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).

In another aspect, 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 the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (II): (II) wherein: each R ² , R ³ , R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl, or W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from: , , , , , , , , ; R ¹ is H, unsubstituted or substituted C _{1-6 alkyl, C 2-6 alkene, C 2-6 alkynyl or C 6-10 aryl; R is H, unsubstituted or substituted C 1-6 alkyl, C 2-6 alkene , C 2-6 alkynyl} or C _6-10 aryl; and each Y is independently a peptide or an antigen-binding portion comprising a cysteine portion, or a pharmaceutically acceptable salt thereof.

In certain embodiments, R ⁶ is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments of (II), the drug conjugate has a structural formula selected from the following: , (II ¹ ) , (II ² ) , (II ³ ) , (II ⁴ ) and (II ⁵ ) (II ⁶ )

In certain embodiments of (II), the drug conjugate has a structural formula selected from the following: (II ⁷ ) (II ⁸ ) or (II ⁹ ) (II ¹⁰ )

In certain embodiments of (II) to (II ¹⁰ ), one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F.

In certain embodiments of (II) to (II ¹⁰ ), R is H. In certain embodiments, R is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments, one of R ² , R ³ and R ⁴ is NO ₂ .

In certain embodiments, one of R ² , R ³ and R ⁴ is CN.

In certain embodiments, R ² or R ⁴ is CF ₃ .

In certain embodiments, R ⁴ is C(O)OR and R ² is not CF ₃ .

In certain embodiments, R ² is not CF ₃ .

In certain embodiments, R ² or R ⁴ is F.

In certain embodiments of (II), the drug conjugate has a structural formula selected from the following: , , or .

In certain embodiments of (II), the drug conjugate has a structural formula selected from the following: , , or .

In certain embodiments of the drug conjugate, the linker moiety further has a spacer, a peptide moiety, and/or a self-immolative moiety.

In certain embodiments of (II), the drug conjugate has a structural formula selected from the following: , (II ¹¹ ) , (II ¹² ) , (II ¹³ ) (II ¹⁴ ) or (II ¹⁵ ) (II ¹⁶ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; and ^LC is a self-cleaving moiety or is absent.

In certain embodiments, the drug conjugate further comprises a drug moiety having a structural formula selected from the group consisting of: (II ¹⁷ ) (II ¹⁸ ) (II ¹⁹ ) (II ²⁰ ) and (II ²¹ ) (II ²² ) where D is the drug part.

In certain embodiments, the drug conjugate has a structural formula selected from the following: (II ²³ ) (II ²⁴ ) and (II ²⁵ ) (II ²⁶ )

In certain embodiments, the drug conjugate further comprises a drug moiety having a structural formula selected from the group consisting of: (II ²⁷ ) (II ²⁸ ) and (II ²⁹ ) (II ³⁰ )

In certain embodiments of (II ¹¹ ) to (II ³⁰ ), the spacer is selected from the group consisting of alkyl, heteroalkyl, PEG and peptide.

In certain embodiments of (II ¹¹ ) to (II ³⁰ ), the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5 or 6) amino acid units.

In certain embodiments of (II ¹¹ ) to (II ³⁰ ), one or more of the amino acids are natural amino acids. In certain embodiments, one or more of the amino acids are non-natural amino acids.

In certain embodiments of (II ¹¹ ) to (II ³⁰ ), the self-degradable moiety is selected from the group consisting of: , , and .

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

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

In certain embodiments, the ratio of the targeting moiety to the drug moiety of the drug conjugate is 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).

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

In another aspect, the present invention generally relates to a pharmaceutical composition comprising a drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

The present invention therefore provides a pharmaceutical preparation comprising a therapeutically effective amount of a compound or immunoconjugate according to the present invention.

Examples of applicable excipients include, but are not limited to, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, starch, cellulose and resin. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated into a pharmaceutical form for solid administration (e.g., tablets, capsules, buccal tablets, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide liquid forms, etc.), liquid administration (e.g., solutions, suspensions, emulsions, elixirs, lotions, ointments, etc.) or semisolid administration (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, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal routes. Different administration forms of the active ingredient, formulations to be used and revisions of their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (AR Gennaro, ed.), 20th edition, Williams & Wilkins PA, USA ( 2000 ). Examples of pharmaceutically acceptable carriers are known in the prior art and include phosphate buffered saline solutions, water, emulsions (such as oil/water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by known procedures in the prior art. Preservatives, stabilizers, dyes and even flavorings, antioxidants and/or suspending agents may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and p-hydroxybenzoic acid esters may be added as preservatives.

In another aspect, the present invention generally relates to a unit dosage form comprising a pharmaceutical composition of the present invention. Linker, Linker - Drug Conjugate, Targeting Moiety - Linker Conjugate

In yet another aspect, the invention generally relates to a compound suitable for forming a linker moiety-drug moiety conjugate, a targeting moiety-linker moiety conjugate, or a targeting moiety-linker moiety-drug moiety conjugate, the compound having a structure comprising structural formula (III): (III) each R ² , R ³ , and R ⁴ are independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; each R ⁵ and ^RY are independently selected from the group consisting of: H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ , and W; R ⁶ is unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from: , , , , , , , , ; R ¹ is H, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl; and R is H, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl.

In certain embodiments, R ⁶ is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments of (III), R ^Y is SO ₂ R ⁶ and the compound has the following structure: (III ¹ )

In certain embodiments of (III) or (III ¹ ), one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F.

In certain embodiments of (III) or (III ¹ ), R is H. In certain embodiments of (III) or (III ¹ ), R is unsubstituted or substituted C _1-6 alkyl.

In certain embodiments of (III) or (III ¹ ), one of R ² , R ³ and R ⁴ is NO ₂ .

In certain embodiments of (III) or (III ¹ ), one of R ² , R ³ , R ⁴ and R ⁵ is CN.

In certain embodiments of (III) or (III ¹ ), R ² or R ⁴ is CF ₃ .

In certain embodiments of (III) or (III ¹ ), R ⁴ is C(O)OR and R ² is not CF ₃ .

In certain embodiments of (III) or (III ¹ ), R ² is not CF ₃ .

In certain embodiments of (III) or (III ¹ ), R ² or R ⁴ is F.

In certain embodiments of (III) or (III ¹ ), one of R ² , R ³ , R ⁴ and R ⁵ is C(O)NHR′.

In certain embodiments of (III), the drug conjugate has a structural formula selected from the following: , (III ² ) , (III ³ ) (III ⁴ ) (III ⁵ ) and (III ⁶ ) (III ⁷ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; ^LC is a self-immolative moiety or is absent; R ⁶ and R' ⁶ may be the same or different and are unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; and D' is a drug moiety D or is absent.

In certain embodiments of (III), the drug conjugate has a structural formula selected from the following: , (III ⁸ ) , (III ⁹ ) (III ¹⁰ ) (III ¹¹ ) and (III ¹² ) (III ¹³ )

In certain embodiments, each of R ⁶ and R ' ⁶ is methyl.

In certain embodiments of (III ² ) to (III ¹³ ), the spacer is selected from the group consisting of alkyl, heteroalkyl, PEG and peptide.

In certain embodiments of (III ² ) to (III ¹³ ), the peptide portion comprises 1 to 6 (eg, 1, 2, 3, 4, 5 or 6) amino acid units.

In certain embodiments of (III ² ) to (III ¹³ ), one or more of the amino acids are natural amino acids. In certain embodiments, one or more of the amino acids are non-natural amino acids.

In certain embodiments of (III ¹¹ ) to (III ³⁰ ), the self-degradable moiety is selected from the group consisting of: , , and .

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

In another aspect, the present invention generally relates to a composition comprising a compound disclosed herein that is suitable for forming a linker moiety-drug moiety conjugate, a targeting moiety-linker moiety conjugate, or a targeting moiety-linker moiety-drug moiety conjugate .

In another aspect, the present invention generally relates to a method for preparing a linker-drug conjugate, comprising: (a) providing a compound having the following structural formula: (III ¹ ) (b) reacting the compound with a spacer pre-driver, a peptide pre-driver and/or a self-degrading part pre-driver to form a complex having the following structural formula: , (III ² ) , (III ³ ) (III ⁴ ) (III ⁵ ) or (III ⁶ ) (III ⁷ ) wherein each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of H, NO ₂ , CN, CF ₃ , F, unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; R ¹ is H, unsubstituted or substituted C _{1-6 alkyl, C 2-6 alkene, C 2-6 alkynyl, or C 6-10 aryl; R is H, unsubstituted or substituted C 1-6 alkyl, C 2-6 alkene, C 2-6 alkynyl , or C 6-10} aryl; ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; ^LC is a self-degrading moiety or is absent; and D' is a drug moiety D or is absent.

In certain embodiments, the combining step (b) is performed at a pH in the range of 5.5 to 6.5 (e.g., about 6.0).

In certain embodiments, ^LA is a spacer moiety, ^LB is a peptide moiety, ^LC is a self-immolative moiety, and D' is a drug moiety.

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

In certain embodiments of the method, the spacer is selected from the group consisting of alkyl, heteroalkyl, PEG, and peptide.

In certain embodiments, the peptide portion has 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) amino acid units.

In certain embodiments, one or more of the amino acids are natural amino acids. In certain embodiments, one or more of the amino acids are non-natural amino acids.

In certain embodiments, the self-degradable portion is selected from the group consisting of: , , and .

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

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

In certain embodiments, the ratio of the targeting moiety to the drug moiety of the drug conjugate is 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).

In some embodiments, the targeting moiety is an antibody comprising a cysteine residue, and the methods comprise reducing the cysteine residue to form a sulfhydryl group, and reacting the sulfhydryl group with one or more linker moieties described herein.

In certain embodiments, the targeting moiety is an antibody fragment comprising a cysteine residue, and the methods comprise reducing the cysteine residue to form a sulfhydryl group, and reacting the sulfhydryl group with one or more linker moieties described herein.

In certain embodiments, the targeting moiety is a protein ligand comprising cysteine, and the methods comprise reducing the cysteine residue to form a sulfhydryl group, and reacting the sulfhydryl group with one or more linker moieties described herein.

In certain embodiments, the targeting moiety is a protein backbone comprising cysteine, and the methods comprise reducing the cysteine residue to form a sulfhydryl group, and reacting the sulfhydryl group with one or more linker moieties described herein.

In certain embodiments, the targeting moiety is a small molecule comprising cysteine, and the methods comprise reducing the cysteine residue to form a sulfhydryl group, and reacting the sulfhydryl group with one or more linker moieties described herein.

In certain embodiments, the conjugation of the linker moiety to the targeting moiety does not produce harmful byproducts. Non-limiting examples of harmful byproducts include acids, bases, or combinations thereof.

In certain embodiments, the drug conjugate has a high drug load. For example, in some embodiments, the molar ratio of the targeting moiety to the 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.

In certain embodiments, the drug conjugate is stable in vivo (e.g., does not undergo deconjugation). Uses of compounds and drug conjugates

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

In some embodiments, the condition is cancer, an autoimmune disorder, or an infectious disease.

In some 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 disclosure, wherein after administration to the subject, the drug moiety is released from the drug conjugate. In certain embodiments, the drug moiety is released from the drug conjugate by autolytic cleavage of an autolytic moiety.

In some embodiments, a method for treating and/or preventing a condition comprises administering to a subject one or more drug conjugates of the present disclosure, wherein upon administration to the subject, the drug moiety is released from the drug conjugate according to Exemplary Schemes 1 to 5 , each of which has a different self-degrading moiety.

In some embodiments, proteolytic cleavage of the drug conjugate is performed according to Scheme 1 : Process 1

In some embodiments, proteolytic cleavage of the drug conjugate is performed according to Scheme 2 : Process 2  

In some embodiments, proteolytic cleavage of the drug conjugate is performed according to Scheme 3 : Process 3  

In some embodiments, proteolytic cleavage of the drug conjugate is performed according to Scheme 4 : Process 4

In some embodiments, proteolytic cleavage of the drug conjugate is performed according to Scheme 5 : Process 5

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

In some embodiments, the condition to be treated and/or prevented is cancer. In some of these embodiments, the cancer is adrenal cancer, anal cancer, basal cell and squamous cell skin cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors (e.g., astrocytoma, multiforme neuroblastoma, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumors, eye cancer (ocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumor, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, Kaposi's sarcoma (Kaposi sarcoma), kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumor, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer cancer), nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, tumors of the central nervous system (CNS), oral 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 intestinal cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor In some embodiments, the cancer is a leukemia or lymphoma, such as lymphoblastic lymphoma or B-cell Non-Hodgkin's lymphoma.

In some of these embodiments, the cancer is a hematologic malignancy. In some embodiments, the hematologic malignancy is: chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma, B-cell lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia, B-cell prolymphocytic leukemia, blastoid plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma (Burkitt's lymphoma), diffuse large B-cell 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 syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or proleukemia. In other embodiments, the cancer is a human hematological malignancy, such as myeloma, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML, acute leukemia of unidentified lineage, myeloproliferative neoplasms, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndrome (MDS) , myeloproliferative/myelodysplastic syndrome, chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, myelodysplastic syndrome (MDS), refractory anemia with ring sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated deletion (5q), unclassifiable MDS, myeloproliferative/myelodysplastic syndrome syndrome, chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, myeloproliferative/myelodysplastic syndrome, unclassifiable, lymphocytic neoplasm, progenitor lymphocytic neoplasm, B-lymphoblastic leukemia, B-lymphoblastic lymphoma, T-lymphoblastic leukemia, T-lymphoblastic lymphoma, mature B-cell neoplasm, diffuse large B-cell lymphoma, primary central nervous system lymphoma, primary septal B-cell lymphoma, Burkitt lymphoma/leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, marginal zone lymphoma, post-transplant lymphoproliferative disorder, HIV-related lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of unknown significance (MGUS), depression-type multiple myeloma, or solitary plasmacytoma (isolated skeletal and extramedullary).

In some embodiments, the cancer comprises a solid tumor. In 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).

The methods of the present disclosure may further include administering one or more drug conjugates provided herein to treat and/or prevent cancer in combination therapy. For example, in certain embodiments, the combination therapy comprises administering one or more drug conjugates (simultaneously or sequentially) with a chemotherapeutic agent. In other embodiments, the combination therapy comprises administering one or more drug conjugates in combination with a second therapy (e.g., chemotherapeutic agent, radiation therapy, surgery, antibodies, or any combination thereof). In some embodiments, administration of one or more drug conjugates in combination with radiation therapy, antibody agents, and/or chemotherapy agents results in an enhancement of the radiation therapy, antibody agents, and/or chemotherapy agents, such that, for example, a smaller dose of radiation therapy, antibody therapy, and/or chemotherapy can be effective for treatment and/or prevention.

In some embodiments, the condition to be treated and/or prevented is an autoimmune disorder. In some of these embodiments, the autoimmune disorder is one or more of the following: Th2 lymphocyte disorder, Th1 lymphocyte disorder, activated B lymphocyte disorder, active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reaction, allergic rhinitis, Alport's syndrome, systemic allergy, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behcet's Disease, Bird fancier's lung, bronchial asthma, Caplan's syndrome, syndrome), cardiomyopathy, chylous diarrhea, Chagas' disease, chronic glomerulonephritis, Cogan's syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus, Dressler syndrome, Eaton-Lambert syndrome, echovirus infection, encephalomyelitis, endocrine eye disease, Epstein-Barr virus infection, equine heaves, erythematosus, Evans syndrome, Felty's syndrome, myofibral pain, Fuchs' heterochromic iridocyclitis heterochromatic iridocyclitis), gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerular nephritis, Goodpasture's syndrome, graft-versus-host disease, Graves' disease, Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes (type 1), Lambert-Eaton syndrome, syndrome), laminitis, tinea planus, lupus hepatitis, lupus, lymphocytopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, presenile dementia, essential agammaglobulinemia, primary biliary cirrhosis, eczema, eczema 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 I diabetes mellitus, ulcerative colitis, uveitis, vitiligo, Waldenstrom's macroglobulinemia, and granulomatosis with polyangiitis.

The methods of the present disclosure may further include administering one or more drug conjugates provided herein to treat and/or prevent autoimmune disorders in combination therapy. For example, in certain embodiments, the combination therapy comprises administering one or more drug conjugates (simultaneously or sequentially) with a known therapeutic agent for treating and/or preventing an autoimmune disorder.

In some embodiments, the condition to be 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.

In some embodiments, the one or more bacterial diseases include diphtheria, pertussis, occult bacteremia, urinary tract infection, gastroenteritis, cellulitis, epididymitis, tracheitis, adenoids, retropharyngeal abscess, abscess, acne, pneumonia, endocarditis, septic arthritis, pneumococcal infection, peritonitis, bacteremia, meningitis, acute purulent meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, is), anthrax, nocardiosis, salmonella, typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis, tularemia, cholera, the Black Death, tetanus, necrotizing enteritis, actinomycosis, mixed anaerobic infections, syphilis, recurrent fever, leptospirosis, Lyme disease, rat-bite fever, tuberculosis, lymphadenitis, measles, chlamydia infection, chlamydial pneumonia, trachoma, and/or inclusion conjunctivitis.

In some embodiments, the one or more systemic fungal diseases are selected from histoplasmosis, coccidioidomycosis, blastomycosis, sporotrichosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycetoma, and/or chromomycosis.

In some embodiments, the one or more rickettsial diseases are selected from typhus, Rocky Mountain spotted fever, ehrlichiosis, eastern tick-borne Rickettsioses, Rickettsialpox, Q fever, bartonellosis.

In some embodiments, the one or more parasitic diseases are selected from malaria, babesiosis, African sleeping sickness, chagas' disease, leishmaniasis, dum-dum fever, toxoplasmosis, meningoencephalitis, keratitis, amoebiasis, giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis, microsporidiosis, ascariasis, whipworm infection, hookworm infection, threadworm infection, ocular larvae migrans, larva migrans), trichinosis, guinea worm disease, lymphatic filariasis, loiasis, river blindness, canine filarial infection, schistosomiasis, swimmer's itch, oriental lung fluke, oriental liver fluke, fascioliasis, fasciolopsiasis, opisthorchiasis, tapeworm infection, hydatid disease, alveolar hydatid disease.

In some embodiments, the one or more viral diseases are selected from measles, subacute sclerosing panencephalitis, cold, mumps, rubella, roseola, fifth disease, chickenpox, respiratory syncytial virus infection, laryngitis, bronchitis, infectious mononucleosis, poliomyelitis, herpetic pharyngitis, hand, foot and mouth disease, Bornholm disease, genital herpes, genital warts, aseptic meningitis, myocarditis, pericarditis, gastroenteritis, acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV), Reye's syndrome, Kawasaki syndrome, influenza virus, bronchitis, viral "walking" pneumonia, acute febrile respiratory disease, acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus 1 herpes simplex virus-1 (hsv-1), herpes simplex virus-2 (hsv-2), herpes zoster, giant cell inclusion disease, rabies, progressive multifocal leukoencephalopathy, kuru, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstraann-Straussler-Scheinker disease, tropical spastic paraplegia, western equine encephalitis, California encephalitis, St. Louis encephalitis, yellow fever, dengue fever, lymphocytic plexus meningitis, Lassa fever, hemorrhagic fever, Hantavirus pulmonary syndrome, Marburg virus infection infections), Ebola virus infections, and/or smallpox.

The methods of the present disclosure may further include administering one or more drug conjugates provided herein to treat and/or prevent infectious diseases in combination therapy. For example, in certain embodiments, the combination therapy comprises administering one or more drug conjugates (simultaneously or sequentially) with a known therapeutic agent for treating and/or preventing infectious diseases.

Non-limiting examples of compounds of the present invention include: and .

Non-limiting examples of the compounds of the present invention also include: and . Spacer

In certain embodiments, the spacer moiety comprises an alkyl chain. In certain embodiments, the spacer moiety has the formula: -(CH ₂ ) _n , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the spacer moiety comprises a heteroalkyl chain. In certain embodiments, the spacer moiety has the formula: -(CH ₂ CH ₂ O) _n , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the alkyl group is a lower alkyl group having 1 to 4 carbon atoms (eg, methyl, ethyl, propyl, and butyl).

In certain embodiments, the spacer portion comprises a peptide. In certain embodiments, the peptide comprises two or more amino acids, such as a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In some of these embodiments, the spacer portion 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.

In some embodiments, the spacer moiety comprises a combination of alkyl, _{heteroalkyl, PEG, or a peptide. For example, the spacer moiety comprises -(CH2)n and a peptide, the spacer moiety comprises -(CH2CH2O ) n and a} peptide, the spacer moiety comprises PEG and _{a peptide, the spacer moiety comprises -(CH2)n and PEG, or the spacer moiety comprises -(CH2CH2O ) n and PEG} .

In some embodiments, the polypeptide portion comprises 1 to 6 amino acids. For example, the polypeptide may include 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids.

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

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). If the bond to the polypeptide is cleaved (e.g., by proteolytic cleavage), the self-immolative spacer can spontaneously separate from the drug moiety.

In some embodiments, the self-degradable moiety is selected from: , , and .

In some embodiments, a linker as provided herein is modified when conjugated to a drug moiety and/or targeting moiety, e.g., in a linker-drug moiety complex, linker-targeting moiety complex, or drug conjugate provided herein. For example, where the linker comprises a hydroxyl group, the hydroxyl group can react with a functional group on the drug moiety or the targeting moiety during the conjugation reaction, thereby producing a conjugate in which the linker no longer comprises a hydroxyl group. Drug Moiety

The drug moiety in the drug conjugates and their components provided herein can produce any compound or molecule that has a therapeutic effect, including both small molecules and biological agents. For example, the drug moiety can be a chemical agent, such as an antibiotic, an anticancer agent, a polypeptide or a nucleic acid.

In some embodiments, the drug moiety is a chemotherapeutic agent, an immunomodulatory agent, a tubulin binding agent, a DNA alkylating agent, an HSP90 inhibitor, a DNA topoisomerase, an anti-epigenetic agent, an HDAC inhibitor, an anti-metabolite, a proteasome inhibitor, a peptide, a peptidomimetic, a siRNA, and/or an antisense DNA.

In certain embodiments, the drug is a chemotherapeutic drug. Non-limiting examples of chemotherapeutic drugs include alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-metabolites, hormone therapy, kinase inhibitors and/or antibiotics.

In some embodiments, the alkylating agent is selected from one or more of the following: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard, mustard); CC-1065 (e.g., synthetic analogs of adozelesin, carzelesin, and bizelesin); duocarmycin (e.g., synthetic analogs KW-2189 and CBI-TMI); benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD) or tomaymycin, indolinolobenzodiazepine, imidazobenzothiazepine, or oxazolobenzodiazepine), nitrosoureas (e.g., carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine), sulfonyl benzoate (e.g., sulfonyl benzoate ... acid alkyl esters (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).

In some embodiments, the plant alkaloid is selected from one or more of the following: vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbin), taxoids (e.g., paclitaxel and docetaxol), maytansines (e.g., DM1, DM2, DM3, DM4, maytansine and ansamitocin), cyclopeptides (e.g., cryptophycin), 1 and cretoxin 8), epothilone, eleutherobin, discodermolide, bryostatin, dolostatin, auristatin, tubulysin, cephalostatins, pancratistatin, sarcodictyin and/or spongistatin.

In some embodiments, the DNA topoisomerase inhibitor is selected from one or more of the following: epipodophyllin (e.g., 9-aminocamptophyllin, camptophylline, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptophyllin (RFS 2000)) and/or mitomycin (e.g., mitomycin C).

In some embodiments, the anti-metabolite is selected from one or more of the following: antifolates, such as DHFR inhibitors (e.g., methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (e.g., mycophenolic acid); acid, tiazofurin, ribavirin, EICAR; ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine); pyrimidine analogs, such as uracil analogs (e.g., ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed) (e.g., Tomudex); cytosine analogs (e.g., cytarabine, cytosine arabinoside, fludarabine); purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine); and/or folic acid supplements (e.g., folinic acid).

In some embodiments, the hormonal therapy is one or more of the following: receptor antagonists, such as antiestrogens (e.g., megestrol, raloxifene, tamoxifen), LHRH agonists (e.g., goserelin, leuprolide), antiandrogens (e.g., bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, nilutamide, testolactone, trilostane and other androgen inhibitors), retinoids/vitamin D (e.g., vitamin D3 analogs: CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); photodynamic therapy (e.g., verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A) and interleukins (e.g., interferon-α, interferon-γ, tumor necrosis factor (TNF), human protein containing TNF domain).

In some embodiments, the kinase inhibitor is one or more of the following: BIBW 2992 (e.g., anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, 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.

In some embodiments, the antibiotic is an enediyne antibiotic (e.g., calicheamicin, particularly calicheamicin gamma 1, delta 1, alpha 1, and beta 1), a dynemicin (e.g., dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, and neocarcinogen and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, n), azoserine, bleomycin, actinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, N-hydroxy-doxorubicin, cranberries, cyano-N-pyrrolidino-cranberries, 2-pyrrolidino-cranberries and deoxy-cranberries, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, Potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and/or zorubicin.

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, beclometasone dipropionate, dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, and tacrolimus.

In some embodiments, the anti-autoimmune disease drug is selected from one or more of the following: polyketide compounds (e.g., acetyl arginine, such as bullatacin and bullatacinone), gemcitabine, epoxomicin (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, stimuvax, alovoxin-7 (allovectin-7), xegeva, provenge, yervoy, prenylation inhibitors (e.g., lovastatin), dopamine-stimulating neurotoxins (e.g., 1-methyl-4-phenylpyridinium), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g., bleomycin A2, bleomycin B2, pelomycin), anthracycline mycins (e.g., daunorubicin, cranberry, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors), Ca ²⁺ ATPase inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (e.g., Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A), thapsigargin, celecoxib, glitazone, epigallocatechin gallate gallate, disulfiram, halosporin A, antiadrenal, carbamate, siRNA, antisense drugs and/or ribozymes.

In certain embodiments, the drug is an infectious disease drug. Non-limiting examples of infectious disease drugs include aminoglycosides, amphenicols, ansamycins, carbapenems, cephems, glycopeptides, glycines, beta-lactamase inhibitors, lincosamides, lipopeptides, macrolides, monobactamides, oxazolidinones, penicillins, polypeptides, quinolinones, streptogramins, sulfonamides, steroidal antibacterial agents, tetracyclines, and/or antibiotics.

In some embodiments, the aminoglycoside is one or more of the following: amikacin, astromicin, gentamicin (e.g., netilmicin, sisomicin, and isepamicin), hygromycin B, kanamycin (e.g., amikacin, arbekacin, kanamycin B, dibekacin, and tobramycin), neomycin (e.g., neomycin B), framycetin, paromomycin and ribostamycin), netilmycin, spectinomycin, streptomycin, topromycin and/or verdamicin.

In some embodiments, the amide is one or more of: azidamfenicol, chloramphenicol, florfenicol, and/or thiamphenicol.

In some embodiments, the ansamycin is one or more of geldanamycin and/or herbimycin.

In some embodiments, the carbapenem is one or more of: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, and/or panipenem.

In some embodiments, the cephem is one or more of the following: carbacephem (e.g., loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin, or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir , cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpiramide ceftriaxone, cefuroxime, cefuroxime, cefazolin, cefotaxime, cefotaxime, cefozonam, cephamycins (e.g., cefoxitin, cefotetan, and cefmetazole) and/or oxacephems (e.g., flomoxef and latamoxef).

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

In some embodiments, the glycinylcycline is tigecycline.

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

In some embodiments, the lincosamide is one or more of clindamycin and/or lincomycin.

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

In some embodiments, the macrolide is one or more of the following: azithromycin, cethromycin, clarithromycin, diritromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, quinoline), midecamycin, miocamycin, oleandomycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, and/or telithromycin.

In some embodiments, the monocyclic lactam is selected from aztreonam and/or tigemonam.

In some embodiments, the oxazolidinone is linezolid.

In some embodiments, the penicillin is one or more of the following: amoxicillin, ampicillin (e.g., pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin The invention can include, for example, carindacillin, cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (e.g., pivmecillinam), mezlocillin, meticillin, nafcillin, oxacillin, penamcillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, and/or ticarcillin.

In some embodiments, the polypeptide is one or more of bacitracin, colistin and/or polymyxin B.

In some embodiments, the quinolinone is selected from one or more of the following: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, valerio ... trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, and/or trovafloxacin.

In some embodiments, the streptomycin is a pristinamycin, such as quinupristin and/or dalfopristin.

In some embodiments, the sulfonamide is one or more of the following: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim and/or trimethoprimsulfamethoxazole (co-trimoxazole).

In some embodiments, the steroidal antibacterial agent is fusidic acid.

In some embodiments, the tetracycline is one or more of: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocydine, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, and/or a glycinylcycline (e.g., tylosin).

In some embodiments, the anti-infective drug is an antibiotic selected from one or more of the following: annonacin, arsphenamine, bactoprenol inhibitors (e.g., bacitracin), DADAL/AR inhibitors (e.g., cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, iazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e.g., fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (e.g., rifampin), tazobactam tinidazole, and/or uvaricin. Targeting moiety

To date, a variety of unique antigens have been identified and can potentially be used as targets in antibody-based therapies. When selecting an antigen, several factors are generally considered. First, the target antigen should be highly expressed in tumors and not expressed or expressed at a low level in healthy cells. An example is the HER2 receptor, which is expressed nearly 100 times higher in tumor cells than in healthy cells. Second, the target antigen should be presented on the surface of tumor cells to be available to circulating monoclonal antibodies. In addition, the target antigen should have internalization properties because it facilitates the transport of the ADC into the cells, thereby enhancing the efficacy of the cytotoxic agent. Although some studies have demonstrated that non-internalizing ADC products targeting components of the tumor microenvironment can effectively dissociate their drugs in the extracellular space and confer strong therapeutic activity in some cases, ADCs often induce a strong "bystander effect". (Strohl WR 2018 Protein & Cell . 9(1):86-120; Damelin et al., 2015 Pharma. Res. 32(11):3494-507; Diamantis et al., 2016 British J. Cancer 114(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.)

A targeting moiety can be any moiety that selectively binds to a cell-surface marker found on a targeted cell type. In general, antibodies should preferably have target specificity and deliver cytotoxic drugs to tumor cells and have target binding affinity, that is, high binding affinity for tumor cell surface antigens. In addition, antibodies should preferably have good retention, low immunogenicity, low cross-reactivity, and appropriate bond binding properties. (Peters et al., 2015 Bioscience Reports 35(4); Hughes B 2010 Nature Reviews Drug Discovery 9(9):665-7.)

In certain embodiments, the targeting moiety is an antibody.

In certain embodiments, the targeting moiety is a monoclonal antibody.

In certain embodiments, the targeting moiety is a chimeric antibody.

In certain embodiments, the targeting moiety is a humanized antibody.

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

In certain embodiments, the targeting moiety is an antibody fragment.

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

In certain embodiments, the targeting moiety is a peptide.

In certain embodiments, the targeting moiety is a small molecule ligand.

In some aspects, 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, such as a tumor-associated antigen.

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

Therefore, targeting moieties suitable for use in the immunoconjugates of the present 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 prepared for the production of antibodies using methods and information known in the art.

In an effort to discover effective cellular targets for cancer diagnosis and therapy, researchers seek to identify transmembrane peptides or other tumor-associated or tumor-specific peptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cells. Tumor-associated peptides are expressed more abundantly on the surface of cancer cells than on the surface of non-cancerous cells, while tumor-specific peptides are specifically expressed on the surface of one or more specific types of cancer cells but not on non-cancerous cells. Identification of such cell surface antigen peptides enables antibody-based therapies to specifically target cancer cells for destruction. (See, e.g., Liu et al., 2017 Eur. J. Cancer Care (Engl). 2017 Sep; 26(5), doi: 10.1111/ecc.12446; WO 2016/192527 A1.)

The tumor-associated antigen may be a cluster of differentiation factor (e.g., CD protein). In some aspects of the present invention, the targeting moiety of the present invention specifically binds to one antigen. In some aspects of the present invention, the targeting moiety of the present invention specifically binds to two or more antigens described herein, for example, the targeting moiety of the present invention is a bispecific or multispecific antibody or an antigen-binding fragment thereof.

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-epithelial calcification 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, anti-CD11 antibodies, and anti-CD125 antibodies. c 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-λ light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-τ antigen antibody, anti-fibroblast antibody, anti-keratin antibody and anti-Tn-antigen antibody.

Antibodies and antibody fragments suitable for use in the immunoconjugates of the present invention include modified antibodies or engineered antibodies, such as antibodies modified to introduce cysteine residues or other reactive amino acids (including Pel, pyrrolyl lysine, peptide tags and unnatural amino acids) to replace at least one amino acid in the native sequence, thereby providing a reactive site on the antibody or antigen-binding fragment to be conjugated with a cytotoxic agent.

The position of the drug moiety can be designed, controlled, and known. For example, cysteine amino acids can be engineered at reactive sites in an antibody and do not form intra- or intermolecular disulfide bonds. (Junutula et al., 2008 Nature Biotech . 26(8):925-932; Dornan et al., 2009 Blood 114(13):2721-2729; U.S. Patent No. 7,521,541 B2; U.S. Patent No. 7,723,485 B2; WO 2009/052249 A2.) The engineered cysteine thiol can react with a linker reagent or drug-linker reagent of the present invention having a thiol-reactive electrophilic group (such as cis-butylenediamide or α-halide amide) to form an ADC with the cysteine engineered antibody and the drug portion.

In addition, antibodies or antibody fragments can be modified to incorporate Pel or pyrrole lysine or unnatural amino acids as sites to be conjugated to drugs. 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; WO 2015/095301 A2; WO 2013/184514 A2. )

Antibodies and antibody fragments can be readily produced by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. (See, e.g., Carvalho et al., 2016 "Production Processes for Monoclonal Antibodies", DOI: 10.5772/64263 (https://www.intechopen.com/chapters/51512); Monoclonal Antibody Production, Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council, NATIONAL ACADEMY PRESS Washington, DC 1999 ; Jakobo vits 1998 Adv. Drug Del. Rev . 31:33-42; Marks et al., 1991 J. Mol. Biol . 222:581; Cole et al., 1985 Monoclonal Antibodies And Cancer Therapy 77-96; Teng et al., 1983 Proc. Natl. Acad. Sci. USA 80:7308-7312; Kozbor et al., 1983 Immunology Today 4:72-79; Olsson et al., 1982 Meth. Enzymol . 92:3-16; U.S. Patent No. 6,657,103 B2.)

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

In certain embodiments, the targeting moiety is a polypeptide, such as a protein ligand, a protein backbone or an antibody. In certain embodiments, the targeting moiety is a monoclonal antibody.

In some embodiments, the targeting moiety comprises 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), Bexxar (tositumomab), IMGN242, IMGN388, IMGN901, ^131I -labetuzumab, IMMU-102 ( ⁹⁰ Y-epratuzumab), IMMU-107 ( ^90Y -clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063 (hul4.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2), ^188Re -PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad ( ^F16-131I ), ^L19-131I , L19-TNF, PSMA-ADC, DI-Leul6-IL2, SAR3419, SGN-35 and/or CMC544, or target binding portions thereof.

In some embodiments, the targeting moiety comprises Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine, Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, AGS-16M8F, BIIB-015, BT-062, and/or IMGN-388, or a target binding portion thereof.

In certain embodiments, the present invention provides a kit comprising one or more of the pharmaceutical conjugates provided herein or their components. In certain embodiments, the kit further comprises instructions for use.

In some embodiments, the kits provided herein are used to prepare drug conjugates as disclosed herein. For example, the kits may include one or more of a linker, a drug moiety, and a targeting moiety, and may further include instructions for using the provided components to produce drug conjugates.

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

Isotopically labeled compounds are also within the scope of the present disclosure. As used herein, "isotopically labeled compounds" refers to compounds disclosed herein, including pharmaceutical salts and prodrugs thereof, wherein one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, respectively.

By isotopically labeling the compounds disclosed in the present invention, the compounds can be used in drug and/or substrate tissue distribution analysis. Tritiated ( ^3H ) and carbon-14 ( ^14C ) labeled compounds are particularly preferred due to their ease of preparation and detectability. In addition, substitution with heavier isotopes such as deuterium ( ^2H ) can provide certain therapeutic advantages caused by greater metabolic stability, such as extended in vivo half-life or reduced dosage requirements, and therefore may be preferred in certain circumstances. The isotopically labeled compounds disclosed in the present invention (including pharmaceutical salts, esters and prodrugs thereof) can be prepared by any means known in the art.

In addition, substitution of the normally abundant hydrogen ( ¹ H) with heavier isotopes such as deuterium may provide certain therapeutic advantages, for example due to improved absorption, distribution, metabolism and/or excretion (ADME) properties, resulting in drugs with improved efficacy, safety and/or tolerability. Substitution of the normally abundant ¹² C with ¹³ C may also provide benefits. (See WO 2007/005643, WO 2007/005644, WO 2007/016361 and WO 2007/016431.)

Thus, the present invention encompasses isotopically derived compounds having one or more hydrogen atoms (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, etc.) replaced with a deuterium atom. In certain embodiments, one hydrogen atom in an isotopically derived compound of the present invention is replaced with a deuterium atom.

Stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S mirror image isomers) of the compounds disclosed in the present invention, as well as racemic, non-mirror image isomers and other mixtures of such isomers are within the scope of the present disclosure.

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

Also included herein are solvates and polymorphs of the compounds of the present invention. Solvates of the compounds of the present invention include, for example, hydrates.

As can be understood from the above disclosure, the present invention has a wide variety of applications. The present invention is further illustrated by the following examples, which are merely illustrative and are not intended to limit the definition and scope of the present invention in any way . 6-( Methylthio )-5- nitropyridinecarboxylic acid INT-1

To a solution of 6-chloro-5-nitropyridinecarboxylic acid (500 mg, 2.47 mmol, 1.0 eq.) in MeOH (20 mL) was added sodium methanethiolate (CH ₃ SNa, 1.9 mL of 20% aqueous solution, 5.43 mmol, 2.2 eq.). The reaction mixture was stirred at room temperature for 4 hours. The methanol was removed under reduced pressure. Water (10 mL) was added, and the pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The solid precipitate 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] ⁺ . ¹ H NMR (DMSO-d ₆ , 600 MHz): δ 8.67 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 2.59 (s, 3H). 6-( Methylsulfonyl )-5- nitropyridinecarboxylic acid 1

To a solution of INT-1 (100 mg, 0.467 mmol, 1.0 eq.) in N,N-dimethylacetate (DMAc, 10 mL) was added 3-chloroperoxybenzoic acid (m-CPBA, 242 mg, 1.4 mmol, 3 eq.), 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-d ₆ , 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). 5- Cyano -6-( methylthio ) nicotinic acid INT-2

To a solution of 6-chloro-5-cyanonicotinic acid (150 mg, 0.82 mmol, 1.0 eq.) in MeOH (3 mL) was added CH ₃ SNa (0.58 mL of 20% aqueous solution, 1.65 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The 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] ⁺ 5- Cyano -6-( methylsulfonyl ) nicotinic acid 2

To a solution of INT-2 (19 mg, 0.1 mmol, 1.0 eq.) in DMAc (1 mL) was added m-CPBA (52 mg, 0.3 mmol, 3.0 eq.) at room temperature, 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] ⁺ 2-( Methylthio )-5- nitroisosonicotinic acid INT-3

To a solution of 2-chloro-5-nitroisonicotinic acid (1000 mg, 4.94 mmol, 1.0 eq.) in MeOH (20 mL) was added CH ₃ SNa (5.2 mL of 20% aqueous solution, 14.81 mmol, 3.0 eq.). 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] ⁺ 2-( Methylsulfonyl )-5- nitroisosonicotinic acid 3

To a solution of INT-3 (500 mg, 2.33 mmol, 1.0 eq.) in DMAc (20 mL) was added m-CPBA (1420 mg, 7.0 mmol, 4 eq.), and the reaction mixture was stirred at room temperature 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] ^{+ 1} H NMR (DMSO-d ₆ , 600 MHz): δ 9.44 (s, 1H), 8.37 (s, 1H), 3.41 (s, 3H). 6- Chloro -3- fluoro -2-( methylthio ) isosonicotinic acid INT-4

To a solution of 2,6-dichloro-3-fluoroisonicotinoic acid (500 mg, 2.38 mmol, 1.0 eq.) in MeOH (20 mL) was added CH ₃ SNa (2.5 mL of 20% aqueous solution, 7.14 mmol, 2.0 eq.). 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. The 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] ⁺ 6- Chloro -3- fluoro -2-( methylsulfonyl ) isosonicotinic acid 4

To a solution of INT-4 (90 mg, 0.41 mmol, 1.0 eq.) in THF (2 mL) was added potassium persulfate (500 mg, 0.814 mmol, 2 eq.) in H ₂ O (2 mL) at room temperature, 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 light white solid. LCMS (ESI): m/z 254.05 [M + H] ^{+ 1} H NMR (DMSO-d ₆ , 600 MHz): δ 8.19 (s, 1H), 3.44 (s, 3H). 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 eq.) in MeOH (3 mL) was added CH3SNa (0.4 mL of 20% aqueous solution, 1.1 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The methanol was removed under reduced pressure and the solid precipitate 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] ⁺ 6-( Methylsulfonyl )-5-( trifluoromethyl ) nicotinic acid 5

To a solution of INT-5 (100 mg, 0.421 mmol, 1.0 eq.) in DMAc (2 mL) was added m-CPBA (256 mg, 1.265 mmol, 3 eq.) at room temperature, and the reaction mixture was stirred at this temperature for 16 hours. DMAc was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 5 (37 mg, 31%) as a light white solid. LCMS (ESI): m/z 270.02 [M + H] ^{+ 1} H NMR (DMSO-d ₆ , 600 MHz): δ 9.36 (s, 1H), 8.72 (s, 1H), 3.53 (s, 3H). 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 eq.) in methanol (4 mL) was added CH ₃ SNa (0.86 mL of 20% aqueous solution, 2.45 mmol, 3.0 eq.). The reaction mixture was stirred at room temperature for 4 hours. 1.5 mL of 1N NaOH was added, and the solution was stirred at room temperature for 1 hour. The pH of the reaction mixture was adjusted to 4~5 with 3N HCl. Methanol was removed under reduced pressure and the solid precipitate 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] ⁺ . 5- Cyano -2,6- bis ( methylsulfonyl ) nicotinic acid 6

To a solution of INT-6 (171 mg, 0.712 mmol, 1.0 eq.) in DMF (6 mL) was added m-CPBA (6.14 mg, 3.56 mmol, 5.0 eq.) at room temperature, and the reaction mixture was stirred at this 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, yield: 47.5%) as a white solid. LCMS (ESI): m/z 305.23 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 600 MHz): δ 9.11 (s, 1H, Ar-H), 3.55 (s, 3H, -CH ₃ ), 3.47 (s, 3H, -CH ₃ ). 5- Cyano -6-( methylthio ) picolinic acid INT-7

To a solution of 6-chloro-5-cyanopicolinic acid (500 mg, 2.74 mmol, 1.0 eq.) in CH ₃ OH (4 mL) was added CH ₃ SNa (1.9 mL of 20% aqueous solution, 5.48 mmol, 2.0 eq.). 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. The methanol was removed under reduced pressure and the solid precipitate was filtered, washed with water and dried to give INT-7 (393 mg, yield: 72.8%) as a white solid. LCMS (ESI): m/z 195.16 [M + H] ⁺ . 5- Cyano -6-( methylsulfonyl ) picolinic acid 7

To a solution of INT-7 (193 mg, 0.995 mmol, 1.0 eq.) in DMAc (5 mL) was added m-CPBA (515 mg, 2.984 mmol, 3.0 eq.) at room temperature, 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, yield: 59.2%) as a white solid. LCMS (ESI): m/z 227.14 [M + H] ⁺ . 5- Fluoro -6-( methylthio ) nicotinic acid INT-8

To a solution of 6-chloro-5-fluoronicotinic acid (500 mg, 2.85 mmol, 1.0 eq.) in CH ₃ OH (10 mL) was added CH ₃ SNa (2.0 mL of 20% aqueous solution, 5.7 mmol, 2.0 eq.). 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. The methanol was removed under reduced pressure and the solid precipitate was filtered, washed with water and dried to give INT-8 (401 mg, yield: 85%) as a white solid. LCMS (ESI): m/z 188.15 [M + H] ⁺ . 5- Fluoro -6-( methylsulfonyl ) nicotinic acid 8

To a solution of INT-8 (100 mg, 0.535 mmol) in THF (3 mL) was added potassium persulfate (657 mg, 1.0695 mmol) in H ₂ O (3 mL) at room temperature 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-d ₆ , 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) 2-( Methylthio )-3- nitroisosonicotinic acid INT-9

To a solution of 2-chloro-3-nitroisonicotinic acid (500 mg, 2.47 mmol) in MeOH (10 mL) was added CH ₃ SNa (2.5 mL 20% aqueous solution, 7.2 mmol, 3 eq) under N _2. The reaction solution was stirred at room temperature for 2 hours. The reaction was completed 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 obtain a solid precipitate, which was filtered to obtain INT-9 (401 mg) as a white solid. LCMS (ESI): m/z 216.07 [M + H] ⁺ . 2-( Methylsulfonyl )-3- nitroisosonicotinic 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 room temperature for 16 hours. 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-d ₆ , 600 MHz): δ 9.11 (s, 1H), 8.29 (s, 1H), 3.46 (s, 3H). 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 eq.) in CH ₃ OH (4 mL) was added CH ₃ SNa (1.7 mL of 20% aqueous solution, 4.76 mmol, 2.0 eq.). 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. The methanol was removed under reduced pressure and the solid precipitate 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] ⁺ . 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 potassium persulfate (553 mg, 0.9 mmol) in H ₂ O (3 mL) at room temperature 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] ⁺ . 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 eq.) in CH ₃ OH (20 mL) was added CH ₃ SNa (3.2 mL of 20% aqueous solution, 9.2 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 4 hours. LCMS showed that the reaction was complete. 5 mL of 1N NaOH was added to the reaction solution and stirred at room temperature for 1 hour. The pH of the reaction mixture was adjusted to 4~5 with 3N HCl. Methanol was removed under reduced pressure and the solid precipitate was filtered, washed with water and dried to give INT-11 as a white solid (200 mg crude product). LCMS (ESI): m/z 209.14 [M + H] ⁺ . 5- Cyano -2- methyl -6-( methylsulfonyl ) nicotinic acid 11

To a solution of INT-11 (200 mg, 0.562 mmol, 1.0 eq.) in DMAc (5 mL) was added m-CPBA (330 mg, 1.912 mmol, 3.4 eq.) at room temperature, and the reaction mixture was stirred at this 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-d ₆ , 600 MHz): δ 8.85 (s, 1H), 3.49 (s, 3H), 2.86 (s, 3H). 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 eq.) in MeOH (650 mL) was added CH ₃ SNa (100.3 mL 20% aqueous solution, 0.286 mol, 2 eq.). The reaction mixture was stirred at room temperature for 18 hours. The methanol was removed under reduced pressure. H ₂ O (600 ml) was added, and the pH of the reaction mixture was adjusted to 4~5 with 3N HCl. The solid precipitate was filtered, washed with water and dried to give INT-12-1 (27.8 g, 87.7%) as a light yellow solid. LCMS (ESI): m/z 215.07 [M + H] ⁺ . ¹ H NMR (CD ₃ OD, 600 MHz): δ 9.30 (d, J = 2.4 Hz, 1H), 8.82 (d, J = 2.4 Hz, 1H), 2.58 (s, 3H) 2-( Methylthio )-5- nitronicotinyl 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, and then DMF (5 mL) was added dropwise. The reaction solution was stirred at room temperature for 4 hours, the solvent was removed on a rotary evaporator to give INT-12-2 , DCM (50 mL) was added to prepare solution A 2,2 -Dimethyl -5-(2-( methylthio )-5- nitronicotinyl )-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), 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 hours. The reaction solution was monitored by MS. Water (100 mL) was added, stirred, separated, the organic phase was collected and the solvent was removed on a rotary evaporator to give 20.1 g of INT-12-3 . 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, water (100 mL) and DCM (100 mL) were added, stirred, separated, the organic phase was collected, washed with aqueous sodium hydroxide solution (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] ⁺ . (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 eq.) and 2-(aminooxy)acetic acid HCl salt (77 mg, 0.353 mmol, 1.7 eq) in MeOH (5 mL) was added AcONa (58 mg, 0.707 mmol, 3.5 eq.). The resulting mixture was stirred at 50 °C for 3 h. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. Methanol was removed under reduced pressure and the solid precipitate 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] ⁺ . (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 eq.) in DCM (5 mL) was added m-CPBA (224 mg, 1.18 mmol, 2.5 eq.) at room temperature, and the reaction mixture was stirred at this temperature for 16 hours. DCM was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 12 (85 mg, 57.0%) as a light white solid. LCMS (ESI): m/z 318.26 [M + H] ⁺ . 6-( Methylthio )-5- nitronicotinic acid INT-13

To a solution of 6-chloro-5-nitronicotinic acid (2.865 g, 14.10 mmol, 1.0 eq.) in MeOH (20 mL) was added CH ₃ SNa (74 mL 20% aqueous solution, 21.20 mmol, 1.5 eq.). 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. The methanol was removed under reduced pressure and the solid precipitate 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] ⁺ . 6-( Methylsulfonyl )-5- nitronicotinic acid 13

To a solution of INT-13 (100 mg, 0.47 mmol, 1.0 eq) in DMAc (5 mL) was added m-CPBA (243 mg, 3.450 mmol, 3.0 eq) at room temperature for 16 hours. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the crude product 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 (CDCl ₃ , 600 MHz): δ 14.35 (s, 1H), 9.34 (d, 1H), 8.94 (d,1H), 3.51 (s, 3H). 2-( Methylsulfonyl )-5- nitronicotinic acid 14

To a solution of INT-14 (100 mg, 0.47 mmol, 1.0 eq) in DMAc (5 mL) was added m-CPBA (243 mg, 3.45 mmol, 3.0 eq) at room temperature for 16 h. The reaction was complete as detected by LC-MS. The solvent was removed under reduced pressure and the crude product 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] ⁺ . 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 eq.) in MeOH (3 mL) was added CH ₃ SNa (0.4 mL of 20% aqueous solution, 1.1 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with 3N HCl. The methanol was removed under reduced pressure and the solid precipitate 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] ⁺ . 2-( Methylsulfonyl )-5-( trifluoromethyl ) nicotinic acid 15

To a solution of INT-15 (100 mg, 0.421 mmol, 1.0 eq.) in DMAc (2 mL) was added m-CPBA (256 mg, 1.26 mmol, 3 eq.) at room temperature, 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 light white solid. LCMS (ESI): m/z 270.02 [M + H] ⁺ . 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 eq.) in DCM (5 mL) under _N2 was added prop-2-yn-1-amine (610 mg, 11.2 mmol, 1.2 eq), followed by oxalyl chloride (11.8 g, 93.0 mmol, 10.0 eq.) and diisopropylethylamine (DIEA, 3.6 g, 27.9 mmol, 3.0 eq). The reaction mixture was stirred at room temperature for 2 hours. 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] ⁺ . 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 eq) in THF/water (1:1, V/V) (100 mL) was added potassium hydrogen persulfate (30.4 g, 49.5 mmol, 6.0 eq.) at room temperature, 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-d ₆ , 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). 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 eq.) in DCM (6 mL) was added prop-2-yn-1-amine (61 mg, 1.12 mmol, 1.2 eq), EDCI (530 mg) and DIEA (240 mg, 1.86 mmol, 2.0 eq) under N2. The reaction mixture was stirred at room temperature for 2 hours. 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] ⁺ . 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 eq.) in THF:H ₂ O (6 mL) was added potassium persulfate (1.44 mg, 2.34 mmol, 3 eq.) at room temperature, and the reaction mixture was stirred at room temperature for 16 hours. 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] ⁺ . 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 eq.) in THF (15 mL) and H ₂ O (15 mL) was added potassium persulfate (4.35 g, 7.068 mmol, 5.0 eq.). The reaction mixture was stirred at room temperature for 18 h. The solid was filtered 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 by silica gel column to give 18 (0.15 g, 43.5%) as a light yellow solid. LCMS (ESI): m/z 245.17 [M + H] ⁺ . 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 eq.) in THF (40 mL) was added _CH3SNa (5.5 mL 20% aqueous solution, 15.6 mmol, 1.5 eq.) under _N2. The reaction mixture was stirred at room temperature for 2 hours. The reaction was complete as detected by LC-MS. 3N hydrochloric acid was added to the reaction solution and the pH was adjusted to 4-5. The solvent was removed under reduced pressure. Water was added to the residue and filtered to give the crude product, which was purified by RP preparative HPLC to give two isomers as yellow solids. The late eluting peak was named INT-19-h (625 mg) and the early eluting peak was named INT-19-q (683 mg). The combined yield was 61.7%. The structure was assigned provisionally. INT-19-h : LCMS (ESI): m/z 205.07 [M + H] ⁺ (5.85 min) INT-19-q : LCMS (ESI): m/z 205.07 [M + H] ⁺ (5.44 min) 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 eq.) in THF (12 mL) under _N2 was added NaH (87 mg, 2.2 mmol, 1.5 eq) followed by INT-19-h (300 mg, 1.47 mmol, 1.0 eq). 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] ⁺ 6-( But -3- yn -1- yloxy )-2-( methylsulfonyl )-3 - nitropyridine19

To a solution of INT-19-1-h (80 mg, 0.32 mmol, 1.0 eq.) in DMF (10 mL) was added m-CPBA (160 mg, 0.96 mmol, 3 eq.) at room temperature, and the reaction mixture was stirred at this temperature for 16 hours. 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] ⁺ . 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 eq.) in THF (5 mL) under _N2 was added NaH (31 mg, 0.78 mmol, 1.5 eq) followed by INT-19-q (107 mg, 0.52 mmol, 1.0 eq). 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] ⁺ . 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 eq.) in DMF (10 mL) was added m-CPBA (160 mg, 0.96 mmol, 3 eq.) at room temperature, and the reaction mixture was stirred at room temperature for 16 hours. 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] ⁺ . (2-(6-( Methylthio )-5 -nitronicotinoylamino ) ethyl ) carbamic acid tertiary butyl ester (INT-21-1)

To a solution of tributyl (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 hours. The reaction was completed by LC-MS monitoring and purified by preparative HPLC to give INT-21-1 (6.820 g, 45.9%) as a light white solid. LCMS [MH] ^- : 355.4 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 h. The reaction was complete by LC-MS and concentrated to give INT-21-2 (4.9 g, yield: 100%) as a colorless solid. LCMS (ESI): m/z 257.2 [M + H] ⁺ . 2-(2-((2-(6-( methylthio )-5- nitronicotinoylamino ) 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) under _N2 , 1,4 -dioxane-2,6- dione (1.086 g, 9.365 mmol) and DIEA (2.215 g, 17.168 mmol) were added and stirred at room temperature for 16 hours. The reaction was complete as determined by LC-MS. 200 mL of water was added to the solution and stirred until a precipitate formed, filtered, and dried to give INT-21-34 (1.5 g, 51.7%) as a light white solid. LCMS (ESI): m/z 373.3 [M + H] ⁺ .

¹ H NMR (DMSO- d ₆ , 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). 2-(2-((2-(6-( methylsulfonyl )-5- nitronicotinoylamino ) ethyl ) amino )-2 -oxoethoxy ) acetic acid (21)

To a solution of INT-21-3 (100.0 mg, 0.269 mmol, 1.0 eq.) in DCM/DMAc (2 mL/2 mL) was added m-CPBA (139.0 mg, 0.807 mmol, 3.0 eq.). 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- d ₆ , 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). 6-( Methylthio )-5- nitro -N-(2-(2-(2- oxo -2-( prop -2- yn -1 -ylamino ) ethoxy ) acetamide (2- ( 4-(2-nitrophenyl) 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). It was stirred at room temperature for 2 hours. 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. 2-(2-((2-(6-( methylsulfonyl )-5- nitronicotinoylamino ) ethyl ) amino )-2 -oxoethoxy ) acetate prop -2- yn- 1-yl ester (22)

To a solution of INT-22-1 (900 mg, 2.198 mmol) in DMAc (10 mL) was added m-CPBA (1.327 g, 7.694 mmol) and stirred at room temperature for 2 hours. 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- d ₆ ) 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) . 6-( Methylsulfinyl )-5- nitronicotinic acid 23

To a solution of INT-13 (150 mg, 0.7 mmol) in THF (3 mL) and H ₂ O (3 mL) was added potassium persulfate (646 mg, 1.05 mmol) and stirred at room temperature for 2 hours. The reaction was complete as determined 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] ⁺ . 2-( Methylsulfinyl )-5- nitronicotinic acid 24

To a solution of INT-14 (150 mg, 0.7 mmol) in THF (3 mL) and H ₂ O (3 mL) was added potassium persulfate (646 mg, 1.05 mmol) and stirred at room temperature for 2 hours. 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] ⁺ . 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 eq.) in THF (30 mL) was added CH3SNa (4.7 mL of 20% aqueous solution, 15.2 mmol, 1.2 eq.). The suspended reaction mixture was stirred at room temperature for 1 hour. THF was removed under reduced pressure, filtered, washed with H2O (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). 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 eq.) in DIEA (15 mL) were added ethynyltrimethylsilane (464 mg, 4.72 mmol, 1.5 eq.), (Ph3P)2PdCl2 (110 mg, 0.16 mmol, 0.05 eq.), CuI (30 mg, 0.16 mmol, 0.05 eq.). The suspended reaction mixture was stirred at room temperature for 24 hours. It 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). 5- Ethynyl -2-( methylthio )-3- nitropyridine (INT-25-3)

To a solution of INT-25-2 (47 mg, 0.177 mmol, 1.0 eq.) in MeOH (1 mL) was added KOH (20 mg, 0.353 mmol, 2.0 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 2 hours. 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). 5- Ethynyl -2-( methylsulfonyl )-3- nitropyridine (25)

To a solution of INT-25-3 (18 mg, 0.093 mmol, 1.0 eq.) in DMAc (2 mL) was added m-CPBA (48 mg, 0.278 mmol, 3.0 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 2 hours. 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; ^1H 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). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl - 3-oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5- methyl -1 -oxohept -4- yl )( methyl ) 4 - ((9S,12S ) -9 - isopropyl - 2,2,5 - trimethyl - 4,7,10 - trioxo - 12- ( 3 - ureidopropyl ) -3 - oxa - 5,8,11 - triazatridecylamido ) benzyl INT - 26

To a solution of 2-((tert-butyloxycarbonyl)(methyl)amino)acetic acid (35 mg, 0.187 mmol, 2.1 eq.) in DMAc (5 mL) was added PyBOP (97 mg, 0.187 mmol, 2.1 eq.) followed by DIEA (65 µL, 0.374 mmol, 4.2 eq.) and stirred at room temperature for 20 min. Val-Cit-PAB-MMAE (CAS Reg. No. 644981-35-1) (100 mg, 0.0890 mmol, 1.0 eq.) 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 light white solid. LCMS (ESI): m/z 1296.14 [M + H] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2- yl )( methyl ) carbamic acid 4-((S)-2-((S)-3 -methyl -2-(2-( methylamino ) acetamido ) butyramido )-5- ureidopentanamido ) benzyl ester 26

To a solution of INT-26 (50 mg, 0.062 mmol, 1.0 eq.) in DCM (1 mL) was added 4N HCl/dioxane (1 mL) and the mixture was stirred at room temperature for 1 hour. The solvent was removed to give 26 as a white solid (76 mg, 100%) as the HCl salt. LCMS (ESI): m/z 1195.94 [M + H] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 - oxobutan - 2 - yl ) amino )-3- methyl - 1- oxobutan-2-yl)(methyl)carbamic acid 4 - ( ( S)-2-((S)-2-(2- azidoacetamido )-3- methylbutanamido )-5- ureidopentanamido ) benzyl ester 27

To a solution of Val-Cit-PAB-MMAE (100.05 mg, 0.0891 mmol, 1.0 eq.) in DMAc (2 mL) was added 2-aziroacetic acid (13.33 µL, 0.178 mmol, 2.0 eq.) and DIEA (46.53 µL, 0.267 mmol, 3.0 eq.), and PyBOP (92.69 mg, 0.178 mmol, 2.0 eq.) was added. 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] ⁺ . ¹ H NMR (DMSO- d ₆ , 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), 7.79-7.7 2 (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), 3.0 8-2.99 (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). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamate 4-((S)-2-((S)-3- methyl -2-(6-( methylsulfonyl )-5- nitropyridinecarboxamido ) butylamido )-5- ureidopentanamido ) benzyl ester 31

To a solution of 1 (8.77 mg, 0.036 mmol, 2.0 eq.) in dichloromethane (DCM, 1 mL) was added N,N-diisopropylcarbodiimide (DIC, 4.49 mg, 0.036 mmol, 2.0 eq.) followed by Val _- Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) under N2. The reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 31 (14.49 mg, 60.22%) as a light white solid. LCMS (ESI): m/z 1351.99 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 600 MHz): δ 10.08 (s, 1H), 8.76 (d, 1H), 8.66 (d, 1H), 8.48 (s, 1H), 8.46 (d, 1H), 8.27 (s, 1H), 8.03 (s, 1H), 7.87 (d, 1H), 7.60 (d, 1H), 7.56 (s, 2H), 7.32 (s, 1H), 7.31-7.22 (m, 4H), 7.16 (d, 1H), 5.97 (s, 1H), 5.00 (d, 4H), 4.72 (s, 1H), 4.49-4.38 (m, 4H), 4.26 (d, 1H), 4.22 (s, 1H), 3.98 (s, 4H), 3.60 (s, 3H), 3.48-3.42 (m, 2H), 3.30 (d, 1H), 3.22 (d, 3H), 3.18 (s, 2H), 3.16 (s, 1H), 3.10 (s, 2H), 2.98 (s, 2H), 2.95 (s, 1H), 2.87 (s, 2H), 2.83 (s, 1H), 2.63-2.60 (m, 1H), 2.41 (s, 1H), 2.38 (s, 1H), 2.36-2.33 (m, 1H), 2.29- 2.21 (m, 2H), 2.18 (dd, 1H), 2.14-2.07 (m, 2H), 1.95 (d, 2H), 1.70 (s, 5H), 1.65-1.40 (m, 6H), 1.37 (s, 2H), 1.03 (d, 2H), 0.99 (d, 2H), 0.96 (d, 1H), 0.93 (d, 2H), 0.87 (d, 3H), 0.83 (d, 2H), 0.80 (d, 3H), 0.78-0.71 (m, 6H). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamic acid 4-((S)-2-((S)-2-(5- cyano -6-( methylsulfonyl ) nicotinamido )-3 -methylbutylamido )-5- ureidopentanamido ) benzyl ester 32

To a solution of 2 (7 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) under _N2 was added Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) followed by DIC (5 mg, 0.036 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 2 h. It was then concentrated and the crude product was purified by RP preparative HPLC to give 32 (13 mg, 55.6%) as a light white solid. LCMS (ESI): m/z 1331.99 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 600 MHz): δ 9.76 (s, 1H), 9.29 (s, 1H), 8.93 (d, 1H), 8.36 (m, 3H), 8.28 (m, 1H) 7.59 (s, 2H), 7.42 - 7.22 (m, 7H), 6.0 0 (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). ((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- oxopropyl 4 - ( ( S ) -2 - ( ( S ) -3 - methyl - 2- ( 2- ( methylsulfonyl ) -5 - nitroisosonicotinamido ) butyrylamino ) -5 - ureidopentanamido ) benzyl ester 33 ​ ​ ​

To a solution of 3 (9 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) under _N2 was added Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) followed by DIC (5 mg, 0.036 mmol, 2.0 eq.). 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 light white solid. LCMS (ESI): m/z 1352.27 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 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). ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -2- ( 6 - chloro - 3 - fluoro - 2- ( methylsulfonyl ) isonicotinamido ) -3 - methylbutyramido ) -5 - ureidopentanamido ) benzyl ester 34

To a solution of 4 (9 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) was added Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) followed by DIC (5 mg, 0.036 mmol, 2.0 eq.) under N2. 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 light white solid. LCMS (ESI): m/z 1360.59 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 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). ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -3 - methyl - 2- ( 6- ( methylsulfonyl ) -5- ( trifluoromethyl ) nicotinamido ) butyrylamido ) -5 - ureidopentanamido ) benzyl ester 35 ​ ​

To a solution of 5 (9 mg, 0.032 mmol, 2.0 eq.) in DMAc (1 mL) under _N2 was added Val-Cit-PAB-MMAE (20 mg, 0.016 mmol, 1.0 eq.), followed by (benzotriazol-1-yloxy)-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyBOP, 13 mg, 0.024 mmol, 2.0 eq.) and 2,6-dimethylpyridine (3.5 mg, 0.032 mmol). 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 light white solid. LCMS (ESI): m/z 1375.93 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 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), 5.4 5 (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). ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -2- ( 5 - cyano - 2,6 - bis ( methylsulfonyl ) nicotinamido ) -3 - methylbutyramido ) -5 - ureidopentanamido ) benzyl ester 36 ​ ​

To a solution of 6 (16 mg, 0.010 mmol, 1.0 eq.) in DCM (3.0 mL) was added DIC (8.6 µL, 0.054 mmol, 2 eq.), Val-Cit-PAB-MMAE (30 mg, 0.027 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. DCM was removed under reduced pressure, and the crude product was purified by RP preparative HPLC to give 36 (13.0 mg, yield: 47.5%) as a white solid. LCMS (ESI): m/z 1410.70 [M + H] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamic acid 4-((S)-2-((S)-2-(5- cyano- 6-( methylsulfonyl ) pyridinylcarboxamido )-3 -methylbutylamido )-5- ureidopentanamido ) benzyl ester 37

To a solution of 7 (20 mg, 0.090 mmol, 2.0 eq.) in DCM (2 mL) was added DIC (11 µL, 0.090 mmol, 2 eq.), Val-Cit-PAB-MMAE (50 mg, 0.045 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. DCM was removed under reduced pressure, and the residue was purified by RP preparative HPLC to give 37 (33 mg, yield: 55.1%) as a white solid. LCMS (ESI): m/z 1332.11 [M + H] ⁺ . ¹ H NMR (MeOH-d ₄ , 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, 1 H), 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). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamic acid 4-((S)-2-((S)-2-(5- fluoro -6-( methylsulfonyl ) nicotinamido )-3 -methylbutylamido )-5- ureidopentanamido ) benzyl ester 38

To a solution of 8 (7.8 mg, 0.036 mmol, 2.0 eq.) in DCM (2 mL) was added DIC (5.3 µL, 0.036 mmol, 2.0 eq.), Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. DCM was removed under reduced pressure, and the residue was purified by RP-HPLC to give 38 (13 mg, yield: 54.6%) as a white solid. LCMS (ESI): m/z 1325.20 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 600 MHz): δ 10.04 (d, J = 10.1 Hz, 1H), 8.96 (s, 1H), 8.85 (d, J = 8.3 Hz, 1H), 8.48 (d, J = 10.5 Hz, 1H), 8.36 (d, J = 7.2 Hz, 1H), 8.30 (t, J = 16.0 Hz, 1H), 8.05 (d, J = 6.3 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.58 (s, 2H), 7.30 (tt, J = 19.6, 10.0 Hz, 6H), 7.20 - 7.14 (m, 1H), 5.99 (s, 1H), 5.40 (s, 2H), 5.13 - 4.95 (m, 2H), 4.54 - 4.38 (m, 4H), 4.31 - 3.93 (m, 5H), 3.78 (d, J = 7.2 Hz , 1H), 3.32 (d, J = 10.7 Hz, 1H), 3.22 (dd, J = 39.3, 16.1 Hz, 8H), 3.12 (s, 2H), 3.07 - 2.93 (m, 4H), 2.91 - 2.81 (m, 3H), 2.41 (d, J = 15.3 Hz, 1H), 2.27 (dd, J = 15.5, 10.9 Hz, 1H), 2.16 - 1.93 (m, 4H), 1.86 - 1.28 (m, 10H), 1.08 - 0.71 (m, 33H). ((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- oxopropyl 4 - ( ( S ) -2 - ( ( S ) -3 - methyl - 2- ( 2- ( methylsulfonyl ) -3 - nitroisosonicotinamido ) butyrylamino ) -5 - ureidopentanamido ) benzyl ester ​ ​ ​ ​

To a solution of 9 (8.8 mg, 0.036 mmol, 2.0 eq.) in DCM (2 mL) was added DIC (5.3 µL, 0.036 mmol, 2.0 eq.), Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. The solvent was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 39 (13.3 mg, yield: 54.1%) as a yellow solid. LCMS (ESI): m/z 1352.600 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 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, 4 H), 1.66 - 1.22 (m, 9H), 1.07 - 0.73 (m, 33H). ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -2- ( 2 - chloro - 5 - fluoro - 6- ( methylsulfonyl ) nicotinamido ) -3 - methylbutyramido ) -5 - ureidopentanamido ) benzyl 40 ​

To a solution of 10 (9.1 mg, 0.036 mmol, 2.0 eq.) in DCM (2 mL) was added DIC (5.3 µL, 0.036 mmol, 2.0 eq.), Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. The solvent was removed under reduced pressure and the residue was purified by RP preparative HPLC to give 40 (14 mg, yield: 57.6%) as a white solid. LCMS (ESI): m/z 1359.21 [M + H] ⁺ . ¹ H NMR (DMSO-d ₆ , 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), 8.0 5 (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). ((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 - ( ( S ) -2 - ( ( S ) -2- ( 5 - cyano - 2 - methyl - 6- ( methylsulfonyl ) nicotinamido ) -3 - methylbutyramido ) -5 - ureidopentanamido ) benzyl ester ​

To a solution of 11 (12.8 mg, 0.053 mmol, 2.0 eq.) in DCM (2 mL) was added DIC (8.3 µL, 0.053 mmol, 2 eq.), Val-Cit-PAB-MMAE (30 mg, 0.027 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 1 hour. The solvent was removed under reduced pressure and the residue was purified by RP-HPLC to give 41 (19 mg, yield: 52.3%) as a white solid. LCMS (ESI): m/z 1347.33 [M + H] ⁺ . ¹ H NMR (CDCl ₃ , 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, 4 H), 3.85 - 3.77 (m, 1H), 3.55 - 1.92 (m, 33H), 1.63 - 0.50 (m, 33H). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3- oxopropyl ) pyrrolidin- 1- yl )-3- methoxy -5- methyl -1 -oxohept -4- yl )( methyl ) amino )-3 - methyl- 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 ester

To a solution of 12 (7.5 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 eq.) under N2. After stirring at room temperature for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature 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] ⁺ . ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -3 - methyl - 2- ( 6- ( methylsulfonyl ) -5- ( trifluoromethyl ) nicotinamido ) butyrylamido ) -5 - ureidopentanamido ) benzyl ester ​ ​ ​

To a solution of 13 (8.9 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 eq.) under N2. After stirring at room temperature for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamate 4-((S)-2-((S)-3 -methyl -2-(2-( methylsulfonyl )-5- nitronicotinamido ) butylamido )-5- ureidopentanamido ) benzyl ester 44

To a solution of 14 (8.9 mg, 0.036 mmol, 2.0 eq.) in DCM (1 mL) was added DIC (5 mg, 0.036 mmol, 2.0 eq.) under N2. After stirring at room temperature for 30 min, Val-Cit-PAB-MMAE (20 mg, 0.018 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature 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] ⁺ . ((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 -oxoheptyl 4 - ( ( S ) -2 - ( ( S ) -3 - methyl - 2- ( 2- ( methylsulfonyl ) -5- ( trifluoromethyl ) nicotinamido ) butyrylamido ) -5 - ureidopentanamido ) benzyl ester ​ ​ ​

To a solution of 15 (9 mg, 0.0324 mmol, 2.0 eq.) in DMAc (1 mL) was added Val-Cit-PAB-MMAE (20 mg, 0.016 mmol, 1.0 eq.) followed by (PyBOP, 13 mg, 0.024 mmol, 2.0 eq.) and 2,6-dimethylpyridine (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 45 (12.2 mg, 55.7%) as a light white solid. LCMS (ESI): m/z 1375.35 [M + H] ⁺ . ((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 -oxoheptyl 4 - ( ( 2S ) -2 - ( ( 2S ) -3 - methyl - 2- ( 6- ( methylsulfinyl ) -5 - nitronicotinamido ) butyrylamino ) -5 - ureidopentanamido ) benzyl ester ​ ​ ​

To a solution of 21 (22 mg, 0.09 mmol, 1.1 eq.) and Val-Cit-PAB-MMAE (100 mg, 0.089 mmol, 1.0 eq.) in DMF (2.2 mL) were added HATU (50 mg, 0.133 mmol, 1.5 eq.) and DIEA (49 µL, 0.267 mmol, 3.0 eq.), and the reaction mixture was stirred at room temperature 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5 - methyl -1- oxohept -4 -yl )( methyl ) amino ) -3 - methyl -1 -oxobutyl - 2 - yl ) amino )-3- methyl - 1-oxobutyl-2 -yl )( methyl ) carbamic acid 4-((S)-2-((S)-3- methyl -2-(2-( methylthio ) -5- nitronicotinamido)butylamido ) -5- ureidopentanamido ) benzyl ester INT-47

To a solution of INT-12-1 (9.5 mg, 0.045 mmol, 1.0 eq.) in DMF (5 mL) were added HATU (25 mg, 0.0133 mmol, 1.5 eq.), DIEA (25 µL, 0.0267 mmol, 3.0 eq.) and Val-Cit-PAB-MMAE (50 mg, 0.045 mmol, 1.0 eq.), and the reaction mixture was stirred at room temperature for 4 hours. 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] ⁺ . ((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 -oxoheptyl 4 - ( ( 2S ) -2 - ( ( 2S ) -3 - methyl - 2- ( 2- ( methylsulfinyl ) -5 - nitronicotinamido ) butyrylamino ) -5 - ureidopentanamido ) benzyl ester ​ ​ ​

To a solution of INT-47 (10.0 mg, 0.0076 mmol) in THF (0.2 mL) and H ₂ O (0.2 mL) was added potassium persulfate (7.0 mg, 0.0114 mmol) at room temperature for 10 hours. 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1 -yl )-3- methoxy -5- methyl -1- oxohept -4- yl ) 4 - ((S)-2-((S)-3- methyl - 2- ( 2-(N- methyl -6-( methylsulfonyl )-5- nitronicotinoylamino )acetamido ) butyramido ) -5 - ureidopentanamido ) benzyl ( ( methyl ) amino )-3 - methyl - 1-oxobutyl - 2 - yl ) ( methyl ) carbamate

To a solution of 13 (30 mg, 0.122 mmol, 2.1 eq.) in DMAc (10 mL) was added PyBOP (63.56 mg, 0.122 mmol, 2.1 eq.) followed by DIEA (52.68 µL, 0.302 mmol, 5.2 eq.) and stirred at room temperature for 20 min. 26 (74 mg, 0.06 mmol, 1 eq.) 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 light yellow solid. LCMS (ESI): m/z: 1422.62 [M + H] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5- methyl -1- oxohept -4- yl )( methyl ) amino )-3 4 - ((S)-2-((S)-3- methyl - 2- ( 2- ( 4 - ( (6-( methylsulfonyl ) -5 - nitronicotinoylamino ) methyl ) -1H-1,2,3 - triazol - 1 - yl ) acetamido ) butyramido ) -5 - ureidopentanamido ) benzyl ester

Compound 27 (36.2 mg, 0.03 mmol, 1.0 eq.) and compound 16 (8.5 mg, 0.03 mmol, 1.0 eq.) were mixed with 6 mL of 1:1 DMSO/ _H2O solution. _CuSO4.5H2O (3 mg, 0.012 mmol, 0.4 eq.) and TBTA (6.3 mg, 0.012 mmol, 0.4 eq.) were added, followed by sodium ascorbate (4.7 mg, 0.0238 mmol, 0.8 eq.) and stirred at room temperature for 1 hour. 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5- methyl -1- oxohept -4- yl )( methyl ) amino )-3 4 - ((S)-2-((S)-3 - methyl - 2- ( 2- ( 4 - ( (2-( methylsulfonyl ) -5 - nitronicotinoylamino ) methyl ) -1H-1,2,3- triazol - 1- yl ) acetamido ) butyramido ) -5 - ureidopentanamido ) benzyl ester 50

Compound 27 (36.2 mg, 0.03 mmol, 1.0 eq.) and compound 17 (8.5 mg, 0.03 mmol, 1.0 eq.) were mixed with 6 mL of 1:1 DMSO/ _H2O solution. _CuSO4.5H2O (3 mg, 0.012 mmol, 0.4 eq.) and TBTA (6.3 mg, 0.012 mmol, 0.4 eq.) were added, followed by sodium ascorbate (4.7 mg, 0.0238 mmol, 0.8 eq.) and stirred at room temperature for 1 hour. 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3- oxopropyl ) pyrrolidin- 1- yl )-3- methoxy -5- methyl -1 -oxohept -4- yl )( methyl ) amino )-3 - methyl- 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 ) butyramido ) -5 - ureidopentanamido ) benzyl 51

Compound 27 (16.7 mg, 0.0138 mmol, 1.0 eq.) and compound 19 (3.8 mg, 0.0138 mmol, 1.0 eq.) were mixed with 3 mL of 1:1 DMSO/ _H2O solution. _CuSO4.5H2O (1.4 mg, 0.0055 mmol, 0.4 eq.) and TBTA (3 mg, 0.0055 mmol, 0.4 eq.) were added, followed by sodium ascorbate (2.2 mg, 0.0110 mmol, 0.8 eq.) and stirred at room temperature for 1 hour. 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3- oxopropyl ) pyrrolidin- 1- yl )-3- methoxy -5- methyl -1 -oxohept -4- yl )( methyl ) amino )-3 - methyl- 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 ) butyramido ) -5 - ureidopentanamido ) benzyl ester 52

Compound 27 (16.7 mg, 0.0138 mmol, 1.0 eq.) and compound 20 (3.8 mg, 0.0138 mmol, 1.0 eq.) were mixed with 3 mL of 1:1 DMSO/ _H2O solution. _CuSO4.5H2O (1.4 mg, 0.0055 mmol, 0.4 eq.) and TBTA (3 mg, 0.0055 mmol, 0.4 eq.) were added, followed by sodium ascorbate (2.2 mg, 0.0110 mmol, 0.8 eq.) and stirred at room temperature for 1 hour. 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] ⁺ . ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1- hydroxy - 1- phenylpropan- 2- yl ) amino )-1- methoxy -2- methyl -3 -oxopropyl ) pyrrolidin -1- yl )-3- methoxy -5- methyl -1 -oxohept -4- yl )( methyl ) amino )-3- methyl -1 4 - ((12S,15S)-12- isopropyl - 1- ( 6-( methylsulfonyl ) -5 - nitropyridin- 3 - yl ) -1,6,10,13 - tetraoxy -15- ( 3- ureidopropyl ) -8 - oxa- 2,5,11,14 - tetraazahexadecylamido ) benzyl ester ( 53 )

To a solution of 21 (20.0 mg, 0.049 mmol, 1.0 eq.) in DCM/DMAc (2 mL/2 mL) was added Val-Cit-PAB-MMAE (67.0 mg, 0.059 mmol, 1.2 eq.) followed by DIC (12.0 mg, 0.098 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 1 hour. 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] ⁺ .

¹ H NMR (DMSO- d ₆ , 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). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R) -1- hydroxy - 1- phenylpropan -2- yl ) amino )-1- methoxy -2- methyl -3- oxopropyl ) pyrrolidin -1- yl )-3- methoxy ( 5 - methyl -1- oxohept -4- yl )( methyl ) amino )-3- methyl - 1-oxobutyl -2- yl ) amino )-3 -methyl - 1 -oxobutyl -2- yl )( methyl ) carbamate 4-((S)-2-((S)-3- methyl -2-(2-(4-( 1-(6-( Methylsulfonyl )-5- nitropyridin -3- yl )-1,6,10- trioxo -8- oxa -2,5,11- triazadecanoic acid ( 1,2 - dioxane -1H-1,2,3 -triazol -1- yl ) acetamido ) butyramido )-5- ureidopentanamido ) benzyl ester (54)

To a solution of 27 (26.45 mg, 0.0219 mmol, 1.0 eq.) in H ₂ O/DMSO (0.35 mL/1.00 mL) was added 22 (11.61 mg, 0.0263 mmol, 1.2 eq.) followed by CuSO ₄ .5H ₂ O (2.19 mg, 0.0088 mmol, 0.4 eq.), TBTA (4.65 mg 0.0088 mmol, 0.4 eq.) and sodium ascorbate (3.47 mg, 0.0175 mmol, 0.8 eq.) at room temperature, and the resulting mixture was stirred at room temperature for 1.0 h. The reaction mixture was then purified by preparative HPLC to give 54 (19.03 mg, 52.7%) as a white powder. LCMS (ESI): m/z: 1648.3 [M + H] ⁺ .

¹ H NMR (DMSO- d ₆ , 600 MHz): δ 10.05 (s, 1H), 9.28 (d, J = 1.5 Hz, 1H), 9.20 (t, J = 5.5 Hz, 1H), 8.90 (d, J = 1.5 Hz, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.43 (d, J = 8.5 Hz, 1H), 8.34 (d, J = 7.2 Hz, 1H), 8.26 (t, J = 5.9 Hz, 1H), 8.10 (s, 1H), 7.94-7.62 (m, 2H), 7.57 (s, 2H), 7.32-7.25 (m, 6H), 7.22-7.13 (m, 1H), 5.99 (s, 1H), 5.43 (s, 2H), 5.21-5.13 (m, 2H), 5.11-4.95 (m, 2H), 4.74 (s, 1H), 4.63 ( s, 1H), 4.48 (d, J = 6.1 Hz, 1H), 4.44-4.39 (m, 2H), 4.36 (d, J = 5.9 Hz, 2H), 4.32-4.23 (m, 2H), 3.98 (s , 3H), 3.95 (s, 3H), 3.50 (s, 4H), 3.43-3.38 (m, 3H), 3.37-3.33 (m, 3H), 3.23 (d, J = 9.6 Hz, 4H), 3.20 (s, 2H), 3.17 (s, 1H), 3.12 (s, 2H), 3.03-2.98 (m, 4H), 2.91-2.83 (m, 3H) , 2.41 (d, J = 19.1 Hz, 1H), 2.30-2.22 (m, 1H), 2.16-2.07 (m, 2H), 2.03-1.95 (m, 2H), 1.76-1.73 (m, 4H), 1.61 -1.40 (m, 4H), 1.35 (s, 2H), 1.06-0.96 (m, 6H), 0.91-0.72 (m, 24H). ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R) -1- hydroxy - 1- phenylpropan -2- yl ) amino )-1- methoxy -2- methyl -3- oxopropyl ) pyrrolidin -1- yl )-3- methoxy 5 - methyl -1- oxohept -4- yl )( methyl ) amino )-3- methyl - 1-oxobutyl -2- yl ) amino )-3- methyl -1- ( 2- (1 -hydroxybutyl ) -1 -yl ) ( methyl ) carbamic acid 4 -((S)-2-((S)-3 -methyl -2-(2-(4-(6-( methylsulfonyl )-5- nitropyridin -3- yl )-1H- 1,2,3- Triazol -1- yl ) acetamido ) butyramido )-5- ureidopentanamido ) benzyl ester (55)

To a solution of 25 (1.9 mg, 0.008 mmol, 1.0 eq.) in DMSO/H2O (2 mL, v/v=1:1) were added 27 (10 mg, 0.008 mmol, 1.0 eq.), TBTA (1.7 mg, 0.003 mmol, 0.4 eq.), CuSO4•5H2O (0.8 mg, 0.003 mmol, 0.4 eq.), sodium ascorbate (1.6 mg, 0.008 mmol, 1 eq.) at room temperature, and the reaction mixture was stirred at that temperature for 2 hours. The residue was then purified by preparative HPLC to give compound 53 (6.60 mg, yield: 57.4%, purity: 95.8%) as a white solid. LCMS[M+H]+:1433.1; ¹ H NMR (600 MHz, DMSO-d6) δ 10.02 (s, 1H), 9.45 (d, J = 1.8 Hz, 1H), 9.02 (s, 1H), 8.96 (s, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8 .35 (d, J = 7.5 Hz, 1H), 8.05 (d, J = 5.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 9.1 Hz, 1H), 7.57 (t, J = 7.0 Hz, 2H), 7.27 (m, 7H), 7. 18 (s, 2H), 7.09 (s, 1H), 7.01 (s, 1H), 6.01-5.95 (m, 1H), 5.40 (d, J = 16.6 Hz, 5H), 5.12-4.90 (m, 2H), 4.49 (d, J = 6.1 Hz, 1H), 4.38 (m, 3H), 4. 28 (d, J = 10.4 Hz, 1H), 3.78 (dd, J = 9.8, 2.3 Hz, 1H), 3.61-3.53 (m, 2H), 3.49 (s, 4H), 3.24 (d, J = 9.2 Hz, 6H), 3.20 (s, 3H), 3.17 (s, 2H ), 3.11 (s, 2H), 2.97 (s, 2H), 2.86 (dd, J = 22.0, 8.0 Hz, 4H), 2.44-2.37 (m, 3H), 2.31-2.22 (m, 1H), 2.15-1.99 (m, 4H), 1.75 (s, 1H), 1.35 (s, 1 H), 1.24 (s, 4H), 0.92-0.74 (m, 28H). (R)-2-((2- acetamido -2- carboxyethyl ) thio )-5- nitronicotinic acid 28 and / or 2-(((2S,4S)-1-( tributyloxy )-4- methyl -1 -oxohexyl -2- yl ) amino )-5- nitronicotinic acid 29

To a solution of N-acetyl-L-cysteine (21 mg, 0.13 mmol, 1.0 eq) and (2S,4S)-2-amino-4-methylhexanoic acid tributyl ester (29 mg, 0.13 mmol, 1.0 eq) in PBS buffer (6 mL, pH = 8.12) was added 24 (30 mg, 0.13 mmol, 1.0 eq) in DMSO (0.5 mL) dropwise at room temperature and stirred for one hour at room temperature. 24 was consumed and 28 was the major product. 29 was not detected. (R)-2-((2- acetamido -2- carboxyethyl ) thio )-5- nitronicotinic acid 28 and / or 2-(((2S,4S)-1-( tributyloxy )-4- methyl -1 -oxohexyl -2- yl ) amino )-5- nitronicotinic acid 29

To a solution of N-acetyl-L-cysteine (21 mg, 0.13 mmol, 1.0 eq) and (2S,4S)-2-amino-4-methylhexanoic acid tributyl ester (29 mg, 0.13 mmol, 1.0 eq) in PBS buffer (6 mL, pH = 8.12) was added 14 (32 mg, 0.13 mmol, 1.0 eq) in DMSO (0.5 mL) dropwise at room temperature and stirred for one hour at room temperature. 14 was consumed and 28 was the major product. 29 was not detected. Both reactions demonstrate the specificity of this chemotype for reacting with thiol nucleophiles. Exemplary compounds Compound Structure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four 25 26 27 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Bonding method

An exemplary conjugation method is exemplified below using Compound 39 and trastuzumab (DAR8): Reaction process

General conjugation protocol: ○ Prepare antibody at a concentration of approximately 15 mg/mL in 20 mM histidine (pH 6.0) ○ Adjust pH to 7.2 and adjust antibody concentration to approximately 12 mg/mL with 0.5 M sodium phosphate buffer + 50 mM EDTA, resulting in a final phosphate buffer concentration of 100 mM sodium phosphate + 10 mM EDTA ○ Reduce antibody by adding 8 to 10 molar equivalents of TCEP (stock solution: 10 mM in water) at 37°C for 2 hours ○ Monitor by RP-HPLC ○ Allow to stand at room temperature (20 to 25°C) for 10 minutes ○ Reconstitute by Zeba Desalt using a Zeba Spin column and change the buffer to 20 mM histidine at pH 6.5 to 8.7 (pH can be anywhere between 6.5 and 8.7, optimized for each drug linker) ○ Prepare a 10 mM stock solution of drug-linker in DMSO ○ Add 11 to 14 molar equivalents of drug-linker ○ Conjugate at room temperature and monitor by RP-HPLC ○ After completion (within 1 to 16 hours), quench by adding 20 equivalents of N-acetylcysteine and hold for 1 hour ○ Desalt using a Zeba Spin column and change the buffer to 20 mM histidine (pH 6.5 to 8.7) using a 30 kD amicon filter 6.0) to remove excess DL

Exemplary RP- HPLC results are shown in Figures 1-2 . DAR Stability

An exemplary DAR stability study of trastuzumab-32 and trastuzumab-38 in human plasma was performed. The trastuzumab conjugates showed minimal loss of DAR. Table X. DAR Stability (Trastuzumab- 32 and Trastuzumab- 38 ) Time (h) DAR ( % relative to T0 ) Trastuzumab -32 Trastuzumab -38 0 100 100 twenty four 101 103 72 101 99 168 98 93

Protocol for DAR stability analysis: ○ Plasma IgG depletion : Endogenous IgG was removed by filtration through recombinant protein A-Agarose gel. 10 mL of Agarose-A was used per 10 mL of plasma. Agarose-A was washed 3 times with PBS and the wash buffer was separated from the Agarose by centrifugation. The plasma was mixed with Agarose-A for 2 hours at 4°C and then the plasma was removed from the Agarose by centrifugation. ○ Incubation : ADC was added to depleted plasma from each test species and PBS to a concentration of ADC of 1 mg/mL. Samples were incubated at 37°C. Aliquots containing 20 μg of ADC were removed at designated time points. Freeze aliquots at -80°C until analysis. ○ Capture and Elution : Wash Protein-A beads twice with PBS and resuspend at the original volume. Add 15 μL of beads per well in a 96-well ultra-low attachment plate and incubate samples with 35 μL of PBS and 10 μL of ADC. Mix the plate for 1 hour at room temperature. Separate beads and collect supernatant and freeze at -80°C. Wash beads 3 times with 250 μL of PBS-T using PBS-T. Elute ADC from beads with 50 μL of 100 mM acetic acid, neutralize with 10 μL of 1.5 M Tris-HCl pH 8.5. Reduce ADC by adding 2 μL of 100 mM DTT, incubate at 37°C for 30 minutes. The beads were captured supernatant diluted 1:3 with acetonitrile and centrifuged at 17G for 10 minutes. The supernatant was removed from the pelleted protein and injected on MS. ○ Data analysis : DAR was determined by RP-MS and free payload was determined by MRM. Cytotoxicity data of representative conjugates

Test compounds included MC-VC-PABC-MMAE Seagen drug-linker, exemplary compounds and MMAE. Table Y. In vitro efficacy of representative conjugates of the present disclosure ADC DAR IC50 (nM) HCC1954 SK-BR-3 Trastuzumab-MC-VC-PABC-MMAE 4 0.004 0.002 Trastuzumab- 31 4 0.004 0.002 Trastuzumab- 33 4 0.004 0.002 Trastuzumab- 42 4 0.005 0.003 MMAE - 0.003 0.02 Analysis plan:

HCC1954 ductal breast carcinoma or SK-BR-3 cells (ATCC, Manassas, VA, USA) were seeded into 384-well white-walled culture plates and allowed to adhere for 2 to 4 hours. Cells were then treated with test articles in at least duplicate by adding 5-fold serial dilutions of the test articles prepared at 2× final concentration and incubated at 37°C for 120 hours. Cell viability after treatment was determined by 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 IC50 values were derived from nonlinear regression analysis using a 4-parameter logic equation.

The disclosure of the applicant is described herein with reference to the drawings in preferred embodiments, wherein like numbers represent the same or similar elements. Throughout this specification, the reference to "some embodiments" or "an embodiment" and similar language means that the specific characteristics, structures or features described in conjunction with the embodiment are included in at least one embodiment of the present invention. Therefore, throughout this specification, the phrases "in one embodiment", "in an embodiment" and similar language may (but not necessarily) all refer to the same embodiment.

In one or more embodiments, the described properties, structures or features of the applicant's disclosure may be combined in any suitable manner. In the description herein, many specific details are described to provide a thorough understanding of the embodiments of the present invention. However, those skilled in the art will recognize that the applicant's compositions and/or methods may be implemented without providing one or more specific details or by other methods, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid confusing the aspects of the present disclosure.

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. Although any methods and materials similar or equivalent to those described herein can also be used to implement or test the present disclosure, the preferred methods and materials are now described. The methods described herein can be performed in any order that is logically possible, except for the specific order disclosed. Incorporated by Reference

Other documents, such as patents, patent applications, patent publications, journals, books, papers, manuscripts, and web content, have been mentioned and cited in this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes. Any material or portion thereof that is allegedly incorporated by reference into this document but conflicts with existing definitions, language, or other disclosure material expressly set forth herein is incorporated only to the extent that there is no conflict between the incorporated material and the material in this disclosure. In the event of a conflict, the conflict shall be resolved in favor of this disclosure as the preferred disclosure. Equivalents

The representative examples are intended to help illustrate the invention and are not intended and should not be interpreted as limiting the scope of the invention. In fact, various modifications to the invention and many other embodiments thereof will be apparent to those skilled in the art based on the entire contents of this document (including the examples and references to scientific and patent literature included herein), in addition to the modifications and embodiments shown and described herein. The examples contain important additional information, examples, and guidance that can be applicable to the implementation of various embodiments of the invention and their equivalents.

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Figure 1 shows some exemplary RP-HPLC data.

Figure 2 shows some exemplary RP-HPLC and HIC data.

Figure 3 shows some exemplary LCMS data.

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Claims (106)

A drug conjugate comprises a targeting moiety, a linker moiety and a drug moiety, wherein the drug moiety is conjugated to the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (I): (I) wherein: each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkyene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; ^R1 is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl group; R is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl group; or a pharmaceutically acceptable salt thereof. The drug conjugate of claim 1 comprises the following structural formula: , (I ¹ ) , (I ² ) , (I ³ ) , (I ⁴ ) or (I ⁵ ) (I ⁶ ) The drug conjugate of claim 1 or 2 comprises the following structural formula: , (I ⁷ ) , (I ⁸ ) or (I ⁹ ) (I ¹⁰ ) The drug conjugate of any one of claims 1 to 3, wherein one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F. The drug conjugate of any one of claims 1 to 4, wherein R is H. The drug conjugate of any one of claims 1 to 4, wherein R is an unsubstituted or substituted C _1-6 alkyl group. The drug conjugate of claim 4 or 5, wherein one of R ² , R ³ and R ⁴ is NO ₂ . The drug conjugate of claim 4 or 5, wherein one of R ² , R ³ and R ⁴ is CN. The drug conjugate of claim 4 or 5, wherein R ² or R ⁴ is CF ₃ . The drug conjugate of claim 4 or 5, wherein R ² is not CF ₃ . The drug conjugate of claim 4 or 5, wherein R ⁴ is C(O)OR and R ² is not CF ₃ . The drug conjugate of claim 4 or 5, wherein R ² or R ⁴ is F. The drug conjugate of any one of claims 1 to 4, comprising the following structural formula: , , or . The drug conjugate of any one of claims 1 to 4, comprising the following structural formula: , , or . The drug conjugate of any one of claims 1 to 12, wherein the linker moiety comprises structural formula (I ¹ ) or (I ⁷ ). The drug conjugate of any one of claims 1 to 12, wherein the linker moiety comprises structural formula (I ² ) or (I ⁸ ). The drug conjugate of any one of claims 1 to 12, wherein the linker moiety comprises structural formula (I ³ ) or (I ⁹ ). The drug conjugate of any one of claims 1 to 12, wherein the linker moiety comprises structural formula (I ⁴ ) or (I ¹⁰ ). The drug conjugate of any one of claims 1 to 12, wherein the linker moiety comprises structural formula (I ⁵ ) or (I ⁶ ). The drug conjugate of any one of claims 1 to 19, wherein the linker moiety further comprises a spacer, a peptide moiety and/or a self-immolative moiety. The drug conjugate of claim 20, wherein the spacer, the peptide portion and/or the self-immolative portion are conjugated to one of R ² , R ³ , R ⁴ and R ⁵ . The drug conjugate of claim 1 or 2 comprises the following structural formula: , (I ¹¹ ) , (I ¹² ) , (I ¹³ ) , (I ¹⁴ ) or (I ¹⁵ ) (I ¹⁶ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; and ^LC is a self-immolative moiety or is absent. The drug conjugate of claim 22 further comprises a drug moiety D, wherein the drug moiety D comprises the following structural formula: , (I ¹⁷ ) , (I ¹⁸ ) , (I ¹⁹ ) , (I ²⁰ ) or (I ²¹ ) (I ²² ) wherein D is a drug moiety. The drug conjugate of claim 3 comprises the following structural formula: , (I ²³ ) , (I ²⁴ ) or (I ²⁵ ) (I ²⁶ ) The drug conjugate of claim 24 further comprises a drug portion, wherein the drug portion comprises the following structural formula: (I ²⁷ ) (I ²⁸ ) or (I ²⁹ ) (I ³⁰ ) The drug conjugate of any one of claims 22 to 25, wherein the spacer moiety is selected from the group consisting of an alkyl group, a heteroalkyl group, polyethylene glycol (PEG) and a peptide. The drug conjugate of any one of claims 22 to 25, wherein the peptide portion comprises 1 to 6 amino acids. The drug conjugate of claim 27, wherein the amino acids are natural and/or unnatural amino acids. The drug conjugate of any one of claims 22 to 29, wherein the self-degradable portion is selected from the group consisting of: , , and . The drug conjugate of any one of claims 1 to 29, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anticancer agent, a steroid, a TLR7/TLR9 antagonist, a peptide, a protein and a nucleic acid. The drug conjugate of any one 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. The drug conjugate of claim 31, wherein the ratio of a targeting moiety to the drug moiety of the drug conjugate is about 1:1 to about 1:16. A drug conjugate comprises a targeting moiety, a linker moiety and a drug moiety, wherein the drug moiety is conjugated to the linker, the linker is conjugated to the targeting moiety, and wherein the linker moiety comprises structural formula (II): (II) wherein: each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from the group consisting of: , , , , , , , and ; ^R1 is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; R is H, an unsubstituted or substituted _C1-6 alkyl, _C2-6 alkene, _C2-6 alkynyl or _C6-10 aryl; and each Y is independently a peptide or an antigen-binding portion comprising a cysteine portion, or a pharmaceutically acceptable salt thereof. The drug conjugate of claim 33 comprises the following structural formula: , (II ¹ ) , (II ² ) , (II ³ ) (II ⁴ ) or (II ⁵ ) (II ⁶ ) The drug conjugate of claim 34 comprises the following structural formula: (II ⁷ ) (II ⁸ ) or (II ⁹ ) (II ¹⁰ ) The drug conjugate of any one of claims 33 to 35, wherein one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F. The drug conjugate of any one of claims 33 to 36, wherein R is H. The drug conjugate of any one of claims 33 to 36, wherein R is an unsubstituted or substituted C _1-6 alkyl group. The drug conjugate of claim 37 or 38, wherein one of R ² , R ³ and R ⁴ is NO ₂ . The drug conjugate of claim 37 or 38, wherein one of R ² , R ³ and R ⁴ is CN. The drug conjugate of claim 37 or 38, wherein R ² or R ⁴ is CF ₃ . The drug conjugate of claim 37 or 38, wherein R ² is not CF ₃ . The drug conjugate of claim 37 or 38, wherein R ⁴ is C(O)OR and R ² is not CF ₃ . The drug conjugate of claim 37 or 38, wherein ^R2 or ^R4 is F. The drug conjugate of any one of claims 33 to 36, comprising the following structural formula: , , or . The drug conjugate of any one of claims 33 to 36, comprising the following structural formula: , , or . The drug conjugate of any one of claims 33 to 46, wherein the linker moiety further comprises a spacer, a peptide moiety and/or a self-immolative moiety. The drug conjugate of claim 33 or 34, comprising the following structural formula: , (II ¹¹ ) , (II ¹² ) , (II ¹³ ) (II ¹⁴ ) or (II ¹⁵ ) (II ¹⁶ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; and ^LC is a self-immolative moiety or is absent. The drug conjugate of claim 48 further comprises a drug moiety, wherein the drug moiety comprises the following structural formula: (II ¹⁷ ) (II ¹⁸ ) (II ¹⁹ ) (II ²⁰ ) or (II ²¹ ) (II ²² ) wherein D is a drug moiety. The drug conjugate of claim 48 comprises the following structural formula: (II ²³ ) (II ²⁴ ) or (II ²⁵ ) (II ²⁶ ) The drug conjugate of claim 50 further comprises a drug moiety, wherein the drug moiety comprises the following structural formula: (II ²⁷ ) (II ²⁸ ) or (II ²⁹ ) (II ³⁰ ) The drug conjugate of any one of claims 48 to 51, wherein the spacer moiety is selected from the group consisting of an alkyl group, a heteroalkyl group, polyethylene glycol (PEG) and a peptide. The drug conjugate of any one of claims 48 to 52, wherein the peptide portion comprises 1 to 6 amino acids. The drug conjugate of claim 53, wherein the amino acids are natural and/or unnatural amino acids. The drug conjugate of any one of claims 48 to 54, wherein the self-degradable portion is selected from the group consisting of: , , and . The drug conjugate of any one of claims 48 to 55, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anticancer agent, a steroid, a TLR7/TLR9 antagonist, a peptide, a protein and a nucleic acid. The drug conjugate of any one of claims 48 to 56, wherein the targeting moiety is selected from the group consisting of an antibody, a small molecule, a peptide and a nucleic acid. The drug conjugate of claim 57, wherein the ratio of a targeting moiety to the drug moiety of the drug conjugate is about 1:1 to about 1:16. A composition comprising a drug conjugate as claimed in any one of claims 1 to 58. A pharmaceutical composition comprising a drug conjugate according to any one of claims 1 to 58 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent. A unit dosage form comprising a pharmaceutical composition as claimed in claim 60. A compound suitable for forming a linker moiety-drug moiety conjugate, a targeting moiety-linker moiety conjugate or a targeting moiety-linker moiety-drug moiety conjugate, the compound having a structure comprising a structural formula (III): (III) each R ² , R ³ , and R ⁴ are independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; each R ⁵ and ^RY are independently H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from: , , , , , , , , ; R ¹ is H, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl group; and R is H, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl or C _6-10 aryl group. The compound of claim 62, wherein R ^Y is SO ₂ R ⁶ , and the compound has the following structural formula: (III ¹ ) The compound of claim 62 or 63, wherein one of R ² , R ³ and R ⁴ is selected from the group consisting of NO ₂ , CN, CF ₃ and F. A compound as in any one of claims 62 to 64, wherein R is H. The compound of any one of claims 62 to 64, wherein R is an unsubstituted or substituted C _1-6 alkyl group. The compound of claim 65 or 66, wherein one of R ² , R ³ and R ⁴ is NO ₂ . The compound of claim 65 or 66, wherein one of R ² , R ³ and R ⁴ is CN. The compound of claim 65 or 66, wherein R ² or R ⁴ is CF ₃ . The compound of claim 65 or 66, wherein R ² is not CF ₃ . The compound of claim 65 or 66, wherein R ⁴ is C(O)OR and R ² is not CF ₃ . The compound of claim 65 or 66, wherein R ² or R ⁴ is F. The compound of claim 66, wherein one of R ² , R ³ , R ⁴ and R ⁵ is C(O)NHR'. The compound of claim 64 or 65 has the following structural formula: , (III ² ) , (III ³ ) (III ⁴ ) (III ⁵ ) or (III ⁶ ) (III ⁷ ) wherein ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; ^LC is a self-immolative moiety or is absent; ^R6 and ^R'6 may be the same or different and are an unsubstituted or substituted _C1-6 alkyl or _C6-10 aryl; and D' is a drug moiety D or is absent. The compound of claim 74 has the following structural formula: , (III ⁸ ) , (III ⁹ ) , (III ¹⁰ ) (III ¹¹ ) or (III ¹² ) (III ¹³ ) The compound of claim 74 or 75, wherein each of R ⁶ and R' ⁶ is methyl. The compound of any one of claims 74 to 76, wherein the spacer moiety is selected from the group consisting of an alkyl group, a heteroalkyl group, polyethylene glycol (PEG) and a peptide. The compound of any one of claims 74 to 77, wherein the peptide portion comprises 1 to 6 amino acids. The compound of claim 78, wherein the amino acids are natural and/or non-natural amino acids. The compound of any one of claims 74 to 79, wherein the self-immolative moiety is selected from the group consisting of: , , and . The compound of any one of claims 74 to 80, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anticancer agent, a steroid, a TLR7/TLR9 antagonist, a peptide, a protein and a nucleic acid. A composition comprising a compound as claimed in any one of claims 62 to 81. A method for preparing a linker-drug conjugate comprises: (a) providing a compound having the following structural formula: (III ¹ ) (b) reacting the compound with a promotor of a spacer, a promotor of a peptide and/or a promotor of a self-degradable moiety to form a complex having the following structural formula: , (III ² ) , (III ³ ) (III ⁴ ) (III ⁵ ) or (III ⁶ ) (III ⁷ ) wherein each of R ² , R ³ , and R ⁴ is independently selected from the group consisting of: H, NO ₂ , CN, CF ₃ , F, an unsubstituted or substituted C _1-6 alkyl, C _2-6 alkene, C _2-6 alkynyl, C _6-10 aryl, and W; R ⁵ is H, Cl, SOR ⁶ , SO ₂ R ⁶ , OR ⁷ , NRR ⁷ or W; R ⁶ is an unsubstituted or substituted C _1-6 alkyl or C _6-10 aryl; R ⁷ is an unsubstituted or substituted C _2-6 alkene or C _2-6 alkynyl; W is selected from: , , , , , , , , ; R ¹ is H, an unsubstituted or substituted C _{1-6 alkyl, C 2-6 alkene, C 2-6 alkynyl, or C 6-10 aryl; R is H, an unsubstituted or substituted C 1-6 alkyl, C 2-6 alkene , C 2-6 alkynyl} , or C _6-10 aryl; ^LA is a spacer moiety or is absent; ^LB is a peptide moiety or is absent; ^LC is a self-degrading moiety or is absent; and D' is a drug moiety D or is absent. The method of claim 83, wherein ^LA is a spacer moiety, ^LB is a peptide moiety, ^LC is a self-immolative moiety, and D' is a drug moiety. 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. The method of any one of claims 83 to 85, wherein the spacer moiety is selected from the group consisting of an alkyl group, a heteroalkyl group, polyethylene glycol (PEG), and a peptide. The method of any one of claims 83 to 86, wherein the peptide portion comprises about 1 to about 6 amino acids. The method of claim 87, wherein the amino acids are natural and/or unnatural amino acids. The method of any one of claims 83 to 88, wherein the self-decomposing portion is selected from the group consisting of: , , and . The method of any one of claims 83 to 89, wherein step (b) is performed at a pH in the range of 5.5 to 6.5. The method of any one of claims 83 to 89, wherein step (b) is performed at a pH of about 6.0. The method of any one of claims 83 to 91, wherein the drug moiety is a chemical agent selected from the group consisting of an antibiotic, an anticancer agent, a steroid, a TLR7/TLR9 antagonist, a peptide, a protein, and a nucleic acid. The method of any one of claims 83 to 92, wherein the targeting moiety is selected from the group consisting of: an antibody, a small molecule, a peptide and a nucleic acid. The method of claim 93, wherein the ratio of a targeting moiety to the drug moiety of the drug conjugate is about 1:1 to about 1:16. A method for treating and/or preventing a condition in a subject in need thereof, the method comprising administering to the subject a drug conjugate as claimed in any one of claims 1 to 58. The method of claim 95, wherein the condition is one or more of cancer, an autoimmune disorder, or an infectious disease. The method of claim 96, wherein the cancer is one or more of the following: adrenal cancer, anal cancer, basal cell and squamous cell skin cancer, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors (astrocytoma, multiforme neuroblastoma, meningioma), breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumors, eye cancer (ocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumor, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, Kaposi's sarcoma (Kaposi sarcoma), kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumor, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer cancer), nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, tumors of the central nervous system (CNS), oral cancer 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 intestinal cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor tumor), squamous cell carcinoma, cancer of unknown primary site (CUP), environmentally induced cancer, combinations of these cancers and/or metastatic lesions of these cancers. The method of claim 96, wherein the autoimmune disorder is one or more of the following: Th2 lymphocyte disorder, Th1 lymphocyte disorder, activated B lymphocyte disorder, active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reaction, allergic rhinitis, Alport's syndrome, systemic allergy, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behcet's Disease, Bird fancier's lung, bronchial asthma, Caplan's syndrome, syndrome), cardiomyopathy, chylous diarrhea, Chagas' disease, chronic glomerulonephritis, Cogan's syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus, Dressler syndrome, Eaton-Lambert syndrome, echovirus infection, encephalomyelitis, endocrine eye disease, Epstein-Barr virus infection, equine heaves, erythematosus, Evans syndrome, Felty's syndrome syndrome), myofibril pain, Fuchs heterochromatic iridocyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerular nephritis, Goodpasture's syndrome, graft-versus-host disease, Graves' disease, Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, 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, tinea planus, lupus hepatitis, lupus, lymphocytopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, presenile dementia, essential agammaglobulinemia, primary biliary cirrhosis, chloasma, chloasma 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, vitiligo, Waldenstrom's macroglobulinemia, and/or granulomatosis with polyangiitis. 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. A compound selected from the group consisting of: and . A compound selected from the group consisting of: and . A composition comprising a compound as claimed in claim 100 or 101. A use of a drug conjugate according to any one of claims 1 to 58 for the manufacture of a medicament. A use of a drug conjugate according to any one of claims 1 to 58 for treating cancer. A use of a drug conjugate according to any one of claims 1 to 58 for treating an autoimmune disease. A use of a drug conjugate according to any one of claims 1 to 58 for treating an infectious disease.