Exatecan derivatives and antibody-drug conjugates thereof

Abstract

Disclosed herein, in part, are novel exatecan derivatives having novel chemical linkers containing a cathepsin B-cleavable moiety conjugated to a targeting antibody. In preferred embodiments, the antibody is an anti-TROP2 antibody, an anti-EGFR antibody, or an anti-HER2 antibody.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 422,517, filed November 4, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) offer a mechanism for selectively delivering small molecule therapeutic payloads to antigen-positive cancer cells, thereby attenuating the systemic toxicity of cytotoxic drugs to antigen-negative normal cells. The three components of an ADC—the antibody, the cytotoxic payload, and the linker connecting them—are crucial for designing effective therapeutics. Despite vigorous development, challenges remain, such as the toxicity of antibodies binding to their targets in normal tissues and the dispersion of the cytotoxic payload in normal tissues due to the instability of the ADC linker. Therefore, many ADCs have not been successful in clinical trials due to insufficient safety and / or efficacy at tolerated doses.

[0003] Topoisomerase I plays a critical role in DNA replication in both normal and pathological conditions (e.g., cancer). Because inhibition of topoisomerase I leads to cell death, compounds that bind to and inhibit topoisomerase I may be useful as therapeutic agents.

[0004] Camptothecin is a natural product that has cytotoxic activity in various cell lines. Its active lactone ring binds to topoisomerase I, inhibiting DNA replication and resulting in cell apoptosis. However, limitations in its pharmaceutical development include its poor solubility in water and the equilibrium between the active lactone form and the inactive open-ring form.

[0005] Exatecan is a water-soluble camptothecin derivative. As a chemotherapy agent, exatecan mesylate failed to gain drug approval after several clinical trials due to ineffectiveness or high toxicity at the tested doses. Attempts to make exatecan clinically useful have been made by converting it into a prodrug form, in which exatecan is covalently attached to a carboxymethyldextran polyalcohol polymer via a peptidyl spacer (a substrate for intracellular cathepsin proteases). However, this prodrug was unsuccessful in clinical trials.

[0006] Therefore, there is a need for compounds that are amenable to clinical development and success in the treatment of human tumors. Furthermore, preferential delivery of topoisomerase I inhibitors to affected tissues via antibody-drug conjugates may lead to improved safety and efficacy, thereby providing treatment options to more patients and cancer types. Summary of the Invention

[0007] The present disclosure relates to compounds useful for treating cancer. The present disclosure relates, in part, to linker-payload constructs useful for attaching payloads to antibodies and exatecan-based drug conjugates. For example, provided herein are compounds that represent therapeutic payloads, linker-payload constructs, or drug conjugates.

[0008] For example, the present disclosure provides linker-payload constructs and drug conjugates, each comprising a therapeutic payload of the present disclosure. Further provided herein are uses of the compounds of the present disclosure as medicaments, processes for their preparation, and pharmaceutical compositions containing them as active ingredients, alone or in combination with other agents, as well as their use as medicaments for the treatment of cancer and / or in the manufacture of medicaments for the treatment of cancer.

[0009] For example, disclosed herein are compounds of formula IA or formula IB: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Lig is a targeting moiety, L is a linker moiety, R is -R 1 , -C(O)-R 1 -, -C(O)-OR 1 -, -C(O)-NH-R 1 -, -C(O)-C 0-3 Alkyl-C(O)-NH-R 1 -, -C(O)-(5-6 membered heteroaryl)-R 1 -, -CH2-(5-6 membered heteroaryl)-R 1 -, -C(O)-(4- to 6-membered heterocyclyl)-, -C(O)-(4- to 6-membered heterocyclyl)-C(O)-R 1 -, -C(O)-(4- to 6-membered heterocyclyl)-NH-R 1 -, -C(O)-C 3-4 Cycloalkyl-R 1 -, -C(S)-C 3-4 Cycloalkyl-R 1 - and -C(O)-O-phenyl-R 1 wherein any of the above heteroaryl, heterocyclyl, alkyl, and cycloalkyl are optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R 1 is selected from the group consisting of CH2-CH2O-, -CH2-O-, -NH-, -CH2ONH-, and -CH(CH3)-NH-; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0010] Also disclosed herein is a compound of formula II: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C 1-3 is alkyl, R is selected from the group consisting of —C(O)—C3 alkyl, —C(O)—C3 cycloalkyl, —C(O)—C4 cycloalkyl, —C1 alkyl, —C(O)-(pyrrolidinyl), and —C(O)-CH2ONH-C(O)-(pyrrolidinyl); where: -C(O)-C3 alkyl, -C(O)-C3 cycloalkyl, -C(O)-C4 cycloalkyl, or -C1 alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO, and -COOH; -C(O)-(pyrrolidinyl) and -C(O)-CHONH-C(O)-(pyrrolidinyl) may be optionally substituted on any available pyrrolidinyl with one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R 1 are joined together to form oxo, -C(O)-(pyrrolidinyl) may be optionally substituted on an available pyrrolidinyl nitrogen atom with -CHO, -C(O)CH3, or -C(O)CH2NH2.

[0011] Disclosed herein is a method for treating cancer, comprising administering to a patient in need thereof an effective amount of a compound of the present disclosure. For example, provided herein is a method for treating cancer to a patient in need thereof, comprising administering to the patient an effective amount of a therapeutic payload of the present disclosure, a linker-payload construct of the present disclosure, or a drug conjugate of the present disclosure.

[0012] Also described herein is a pharmaceutical composition, comprising at least one compound of the present disclosure and a pharmaceutically acceptable carrier.For example, provided herein is a pharmaceutically acceptable composition, comprising a compound of the present disclosure, for example, a therapeutic payload of the present disclosure, a linker-payload construct of the present disclosure, or a drug conjugate of the present disclosure, and a pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0013] [Figure 1] Figures A to C show the tumor-inhibitory effects of antibody-drug conjugates 3031, 3036, and 3038 on xenografts derived from the lung cancer cell line NCI-H292 in athymic nude mice. The compounds exemplified here show higher tumor inhibition than the control.

[0014] [Figure 2] Figures A and B show the tumor-inhibitory effects of antibody-drug conjugates 3058A and 3053B on xenografts derived from the human laryngeal carcinoma cell line FaDu in athymic nude mice. The compounds exemplified here exhibit greater tumor inhibition than the control.

[0015] [Figure 3] Figures A to D show the tumor-inhibitory effects of antibody-drug conjugates 3058A, 3058B, 3102, and 3053B on xenografts derived from the human lung cancer cell line H1975 in athymic nude mice. The compounds exemplified here exhibit greater tumor inhibition than the control.

[0016] [Figure 4A] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3053B on xenografts derived from the human breast cancer cell line MDA-MB-468 in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control. [Figure 4B]Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3058A on xenografts derived from the human breast cancer cell line MDA-MB-468 in NOD-SCID mice. The compounds exemplified herein exhibit greater tumor suppression than the control. [Figure 4C] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3102 on xenografts derived from the human breast cancer cell line MDA-MB-468 in NOD-SCID mice. The compounds exemplified herein exhibit greater tumor suppression than the control.

[0017] [Figure 5A] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3058A on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control. [Figure 5B] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3102 on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control. [Figure 5C] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3059B on xenografts derived from the human laryngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control. [Figure 5D] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3108 on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control. [Figure 5E] 1 shows the tumor suppression effect of antibody-drug conjugate 3110 on xenografts derived from the human laryngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified herein exhibit greater tumor suppression than the control. [Figure 5F] Figure 1 shows the tumor suppression effect of antibody-drug conjugate 3053B on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control.

[0018] [Figure 6] Figures A to D show the tumor-suppressing effects of antibody-drug conjugates 3058A, 3053B, 3102, and 3059B on xenografts co-inoculated with human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc in NOD-SCID mice. The compounds exemplified here exhibit greater tumor suppression than the control.

[0019] [Figure 7] Figures A-D show the tumor suppressive effects of antibody-drug conjugates 3058A, 3053B, 3102, and 3059B on co-inoculated xenografts of human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc in NOD-SCID mice. All four figures show luciferase signal expressed as total flux (photons per second) as a function of time after the start of antibody-drug conjugate administration compared to the vehicle control group. The compounds exemplified here demonstrate greater tumor suppression than the control.

[0020] [Figure 8] Figures A to D show the tumor-inhibitory effects of antibody-drug conjugates 3111, 3112, 3110, and 3109 (10 mg / kg, single dose) on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor inhibition than the control.

[0021] [Figure 9] Figures A to D show the tumor-inhibitory effects of antibody-drug conjugates 3111, 3112, 3110, and 3109 (3 mg / kg, single dose) on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor inhibition than the control.

[0022] [Figure 10]Figures A to D show the tumor-inhibitory effects of antibody-drug conjugates 3111, 3112, 3110, and 3109 (1 mg / kg, 3 doses) on xenografts derived from the human pharyngeal carcinoma cell line FaDu in NOD-SCID mice. The compounds exemplified here exhibit greater tumor inhibition than the control.

[0023] [Figure 11] Figures A to D show the tumor-inhibitory effects of antibody-drug conjugates 3111, 3112, 3110, and 3109 (3 mg / kg, single dose) on xenografts derived from the human breast cancer cell line MDA-MB-468 in NOD-SCID mice. The compounds exemplified here exhibit greater tumor inhibition than the control. DETAILED DESCRIPTION OF THE INVENTION

[0024] The features and other details of the present disclosure will now be described more specifically. Before further describing the present disclosure, certain terms employed in the specification, examples, and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0025] definition As used herein, the words "a" and "an" are meant to include one or more, unless otherwise specified. For example, the term "an agent" includes both a single agent and a combination of two or more agents.

[0026] As used herein, the term "alkenyl" refers to an unsaturated, straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. Exemplary alkenyl groups include straight-chain or branched groups of 2 to 6 or 3 to 4 carbon atoms (referred to herein as C , respectively). 2-6 Alkenyl, and C 3-4Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, and the like.

[0027] As used herein, the term "alkoxy" refers to a straight or branched chain alkyl group attached to oxygen (alkyl-O-). Exemplary alkoxy groups include alkoxy groups having 1 to 6 or 2 to 6 carbon atoms (respectively, C 1-6 Alkoxy, and C 2-6 Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, and the like.

[0028] As used herein, the term "alkoxyalkyl" refers to a straight or branched chain alkyl group attached to oxygen attached to a second straight or branched chain alkyl group (alkyl-O-alkyl-). Exemplary alkoxyalkyl groups include alkoxyalkyl groups in which each alkyl group independently contains 1 to 6 carbon atoms (referred to herein as C 1-6 Alkoxy-C 1-6 Exemplary alkoxyalkyl groups include, but are not limited to, methoxymethyl, 2-methoxyethyl, 1-methoxyethyl, 2-methoxypropyl, ethoxymethyl, 2-isopropoxyethyl, and the like.

[0029] As used herein, the term "alkoxycarbonyl" refers to a straight or branched alkyl group attached to an oxygen atom attached to a carbonyl group (alkyl-OC(O)-). Exemplary alkoxycarbonyl groups include alkoxycarbonyl groups of 1 to 6 carbon atoms (referred to herein as C 1-6 Exemplary alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and the like.

[0030] As used herein, the term "alkenyloxy" refers to a straight or branched chain alkenyl group attached to oxygen (alkenyl-O-). Exemplary alkenyloxy groups include groups having alkenyl groups of 3 to 6 carbon atoms (referred to herein as C 3-6 Exemplary "alkenyloxy" groups include, but are not limited to, allyloxy, butenyloxy, and the like.

[0031] The term "alkynyloxy" as used herein refers to a straight-chain or branched alkynyl group attached to oxygen (alkynyl-O). Exemplary alkynyloxy groups include, but are not limited to, groups having an alkynyl group of 3 to 6 carbon atoms (referred to herein as C3-6 alkynyloxy). Exemplary alkynyloxy groups include, but are not limited to, propynyloxy, butynyloxy, and the like.

[0032] As used herein, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon. Exemplary alkyl groups include straight or branched chain hydrocarbons of 1 to 6, 1 to 4, or 1 to 3 carbon atoms (respectively, C ). 1-6 Alkyl, C 1-4 Alkyl, and C 1-3 Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, and the like.

[0033] As used herein, the term "alkylcarbonyl" refers to a straight or branched alkyl group attached to a carbonyl group (alkyl-C(O)-). Exemplary alkylcarbonyl groups include alkylcarbonyl groups of 1 to 6 atoms (defined herein as C 1-6 Exemplary alkylcarbonyl groups include, but are not limited to, acetyl, propanoyl, isopropanoyl, butanoyl, and the like.

[0034] "Alkylene" means a linear or branched saturated aliphatic divalent radical having the specified number of carbon atoms. "Cycloalkylene" refers to a divalent radical of a carbocyclic saturated hydrocarbon group having the specified number of carbon atoms.

[0035] The term "alkynyl," as used herein, refers to an unsaturated, straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. Exemplary alkynyl groups include straight-chain or branched groups of 2 to 6 or 3 to 6 carbon atoms (referred to herein as C , respectively). 2-6 Alkynyl, and C 3-6 Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, and the like.

[0036] As used herein, the term "carbonyl" refers to the radical --C(O)--.

[0037] As used herein, the term "cyano" refers to the radical --CN.

[0038] As used herein, the term "cycloalkoxy" refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-). Exemplary cycloalkoxy groups include cycloalkoxy groups of 3 to 6 carbon atoms (defined herein as C 3-6Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclohexyloxy, and the like.

[0039] The term "cycloalkyl" or "carbocyclic group" as used herein refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3 to 6, or 4 to 6 carbons, and is used herein as C 3-6 Cycloalkyl or C 4-6 Exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl, or cyclopropyl.

[0040] The term "halo" or "halogen" as used herein refers to F, Cl, Br, or I.

[0041] As used herein, the term "heteroaryl" or "heteroaromatic group" refers to a monocyclic aromatic 5- or 6-membered ring system containing one or more heteroatoms, such as nitrogen, oxygen, and sulfur, e.g., 1 to 3 heteroatoms. Where possible, the heteroaryl ring may be linked to adjacent radicals through a carbon or nitrogen. Examples of heteroaryl rings include, but are not limited to, furan, thiophene, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine, or pyrimidine.

[0042] The terms "heterocyclyl" or "heterocyclic group" are art-recognized and refer to, for example, saturated or partially unsaturated 4- to 10-membered monocyclic or bicyclic ring structures, or 4- to 9- or 4- to 6-membered saturated ring structures, including, for example, bridged, fused, or spirocyclic rings, which ring structures contain one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, the heterocyclyl ring may be linked to adjacent radicals through a carbon or nitrogen atom. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran, or dihydrofuran.

[0043] The term "heterocyclyloxy" as used herein refers to a heterocyclyl group attached to oxygen (heterocyclyl-O-).

[0044] The term "heteroaryloxy," as used herein, refers to a heteroaryl group attached to oxygen (heteroaryl-O-).

[0045] As used herein, the terms "hydroxy" and "hydroxyl" refer to the radical --OH.

[0046] The term "oxo" as used herein refers to the radical =O.

[0047] "Pharmaceutically or pharmacologically acceptable" includes molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans, as appropriate. For human administration, preparations must meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0048] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide complementary, additional, or enhanced therapeutic functions.

[0049] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one compound disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0050] The terms "individual," "patient," or "subject" are used interchangeably and include any mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, and most preferably a human. The compounds of the present disclosure can be administered to mammals such as humans, but can also be administered to other mammals, such as animals requiring veterinary treatment, for example, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). "Modulation" includes antagonism (e.g., inhibition), agonism, partial antagonism, and / or partial agonism.

[0051] "Treating" includes any effect, eg, alleviation, reduction, modulation, or elimination, that results in an improvement of a condition, disease, disorder, or the like.

[0052] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a subject compound that elicits the biological or medical response in a tissue, system, or animal (e.g., a mammal or human) desired by a researcher, veterinarian, physician, or other clinician. The compounds of the present disclosure are administered in a therapeutically effective amount to treat a disease. Alternatively, a therapeutically effective amount of a compound is the amount necessary to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in weight loss.

[0053] As used herein, the term "pharmaceutically acceptable salt(s)" refers to salts of acidic or basic groups that may be present in compounds used in the compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, hydrogensulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds contained in the present compositions that are acidic in nature can form base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds contained in the present compositions that contain a basic or acidic moiety can also form pharmaceutically acceptable salts with various amino acids. Compounds of the present disclosure may contain both acidic and basic groups, for example, one amino group and one carboxylic acid group. In such cases, the compounds can exist as acid addition salts, zwitterions, or base salts.

[0054] As understood by one of ordinary skill in the art, "H" is the symbol for hydrogen, "N" is the symbol for nitrogen, "S" is the symbol for sulfur, and "O" is the symbol for oxygen. "Me" is an abbreviation for methyl. It is understood that this disclosure is to be interpreted in accordance with the laws and principles of chemical bonding.

[0055] The compounds of the present disclosure may contain one or more chiral centers and therefore may exist as stereoisomers. As used herein, "stereoisomers" consist of all enantiomers or diastereomers. These compounds may be designated with the symbols "(+)", "(-)", "R", or "S", depending on the configuration of substituents around the stereogenic carbon atom, although those of skill in the art will recognize that the structure may imply chiral centers. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers are designated with "(±)" in the nomenclature, although those of skill in the art will recognize that the structure may imply chiral centers.

[0056] The compounds of the present disclosure may contain one or more double bonds and, therefore, may exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. [ka] The symbol denotes a bond that may be a single bond, double bond, or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration, and the terms "Z" and "E" are used in accordance with IUPAC nomenclature. Unless otherwise specified, structures depicting double bonds encompass both the "E" and "Z" isomers. Substituents around a carbon-carbon double bond may be designated as "cis" or "trans," with "cis" referring to substituents on the same side of the double bond and "trans" referring to substituents on opposite sides of the double bond.

[0057] The compounds of the present disclosure may contain carbocyclic or heterocyclic rings and therefore may exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring is designated as the "Z" or "E" configuration, where the terms "Z" and "E" are used according to IUPAC nomenclature. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both the "Z" and "E" isomers. Substituents around a carbocyclic or heterocyclic ring may also be designated as "cis" or "trans," with the term "cis" referring to substituents on the same side of the plane of the ring and the term "trans" referring to substituents on opposite sides of the plane of the ring. Mixtures of compounds in which substituents are arranged on both the same and opposite sides of the plane of the ring are designated as "cis / trans."

[0058] Individual enantiomers and diastereomers of the compounds of the present disclosure can be prepared synthetically from commercially available starting materials containing asymmetric or stereoisomeric centers, or by preparing a racemic mixture followed by resolution methods well known to those skilled in the art. Examples of these resolution methods include (1) coupling the mixture of enantiomers to a chiral auxiliary, separating the resulting diastereomeric mixture by recrystallization or chromatography, and liberating the optically pure product from the auxiliary; (2) forming a salt with an optically active resolving agent; (3) directly separating the mixture of optical enantiomers on a chiral liquid chromatography column; or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well-known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective synthesis, i.e., chemical or enzymatic reactions that result in the formation of unequal stereoisomeric mixtures from a single reactant upon the formation of new stereocenters or the transformation of existing stereocenters, is well known in the art. Stereoselective synthesis encompasses both enantioselective and diastereoselective transformations and may involve the use of chiral auxiliaries. See, e.g., Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009.

[0059] The compounds disclosed herein can exist in solvated and unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the present disclosure is intended to encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form.

[0060] The present disclosure also encompasses isotopically labeled compounds of the present disclosure, which are identical to those listed herein, except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, 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 For example, compounds of the present disclosure may have one or more H atoms replaced with deuterium.

[0061] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C) are useful in compound and / or substrate tissue distribution assays. Tritium isotopes (i.e., 3 H) and carbon-14 isotopes (i.e., 14 C) is particularly preferred due to its ease of preparation and detectability. Furthermore, substitution with heavier isotopes, such as deuterium (i.e., 2H), may be preferred in some circumstances because it may offer certain therapeutic advantages (e.g., increased in vivo half-life or reduced required dosage) resulting from higher metabolic stability. Isotopically labeled compounds of the present disclosure can generally be prepared following procedures similar to those disclosed in the Examples herein, by using an isotopically labeled reagent instead of a non-isotopically labeled reagent.

[0062] The term "prodrug" refers to a compound that is converted in vivo to yield a compound of the present disclosure or a pharmaceutically acceptable salt, hydrate, or solvate of the compound. The conversion can occur at various locations (such as in the intestinal lumen or during passage through the intestine, blood, or liver) and by various mechanisms (such as esterases, amidases, phosphatases, oxidative and / or reductive metabolism). Prodrugs are well known in the art (see, e.g., Rautio, Kumpulainen, et al., Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the present disclosure or a pharmaceutically acceptable salt, hydrate, or solvate of the compound contains a carboxylic acid functional group, a prodrug can be prepared by replacing the hydrogen atom of the acidic group with a carboxylic acid functional group such as (C 1-8 ) alkyl, (C 2-12 ) alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkylcarbonyloxy)-ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolactone-4-yl, di-N,N-(C 1-2 ) Alkylamino(C 2-3 ) alkyl (β-dimethylaminoethyl, etc.), carbamoyl-(C 1-2 ) alkyl, N,N-di(C 1-2 ) alkylcarbamoyl-(C 1-2 ) alkyl and piperidino-, pyrrolidino- or morpholino (C 2-3 ) alkyl, etc.

[0063] Similarly, if a compound of the present disclosure contains an alcohol functional group, the prodrug may be a compound that converts a hydrogen atom of the alcohol group to a hydroxyl group such as (C1-6 ) alkylcarbonyloxymethyl, 1-((C 1-6 ) alkylcarbonyloxy) ethyl, 1-methyl-1-((C 1-6 ) alkylcarbonyloxy) ethyl (C 1-6 )alkoxycarbonyloxymethyl, N-(C 1-6 ) alkoxycarbonylaminomethyl, succinoyl, (C 1-6 ) alkylcarbonyl, α-amino (C 1-4 ) alkylcarbonyl, arylalkylcarbonyl and α-aminoalkylcarbonyl, or α-aminoalkylcarbonyl-α-aminoalkylcarbonyl, where each α-aminoalkylcarbonyl group is a group selected from the group consisting of natural L-amino acids, P(O)(OH), -P(O)(O(C 1-6 ) alkyl) 2 or glycosyl (a radical obtained from removal of the hydroxyl group of the hemiacetal form of a carbohydrate).

[0064] When an amine function is incorporated into a compound of the present disclosure, a prodrug can be formed, for example, by the formation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, an imine, or an enamine. In addition, secondary amines can be metabolically cleaved to generate biologically active primary amines, and tertiary amines can be metabolically cleaved to generate biologically active primary or secondary amines. See, e.g., Simplicio, et al., Molecules 2008, 13, 519 and references therein.

[0065] Procedures for making the compounds described herein are provided in the following working examples and may be supplemented or substituted by procedures known to those skilled in the art. The starting materials used in the working examples can be purchased or prepared by methods described in the chemical literature or by modifications thereof, using methods known to those skilled in the art. The order in which steps are performed may vary depending on the groups introduced and the reagents used, but will be apparent to those skilled in the art. The compounds of the present disclosure, or any of the intermediates described herein, can be further derivatized using one or more standard synthetic methods known to those skilled in the art.

[0066] Salts of the compounds disclosed herein can be prepared by reacting the compounds disclosed herein with an appropriate acid or base in a suitable solvent or mixture of solvents (such as an ether, e.g., diethyl ether, an alcohol, e.g., ethanol, or an aqueous solvent) using conventional procedures. Salts of the compounds disclosed herein can be exchanged for other salts by treatment using conventional ion exchange chromatography procedures.

[0067] compound Disclosed herein are compounds, for example, of formula IA or formula IB: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Lig is a targeting moiety, L is a linker moiety, R is -R 1 , -C(O)-R 1 -, -C(O)-OR 1 -, -C(O)-NH-R 1 -, -C(O)-C 0-3 Alkyl-C(O)-NH-R 1 -, -C(O)-(5-6 membered heteroaryl)-R 1 -, -CH2-(5-6 membered heteroaryl)-R 1-, -C(O)-(4- to 6-membered heterocyclyl)-, -C(O)-(4- to 6-membered heterocyclyl)-C(O)-R 1 -, -C(O)-(4- to 6-membered heterocyclyl)-NH-R 1 -, -C(O)-C 3-4 Cycloalkyl-R 1 -, -C(S)-C 3-4 Cycloalkyl-R 1 - and -C(O)-O-phenyl-R 1 wherein any of the above heteroaryl, heterocyclyl, alkyl, and cycloalkyl are optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R 1 is selected from the group consisting of CH2-CH2O-, -CH2-O-, -NH-, -CH2ONH-, and -CH(CH3)-NH-; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0068] In some embodiments, the drug conjugate of formula I of the present disclosure has formula IA: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein s is 1, 2, 3, 4, 5, 6, 7, or 8.

[0069] In some embodiments, Lig is a monoclonal antibody. For example, in some embodiments, Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, an anti-HER2 antibody, an anti-B7-H3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, and an anti-CD70 antibody. In other embodiments, Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody. In certain other embodiments, Lig is an anti-TROP2 antibody. In certain other embodiments, Lig is an anti-EGRF antibody. In still other embodiments, Lig is an anti-HER2 antibody. For example, in certain embodiments, Lig may be selected from the antibodies disclosed in Table 4. In further embodiments, s is 1 or 8. For example, in some embodiments, s is 1.

[0070] In some embodiments, L is, for example, [ka] is selected from the group consisting of where * represents the point of attachment to R.

[0071] In other embodiments, R is selected from the group consisting of -CHCHO-, -C(O)-CHONH-, -C(O)-O-CHCHO-, -C(O)-NH-CHCHO-, -C(O)-Calkyl-C(O)-NH-CHCHO-, -C(O)-Calkyl-C(O)-NH-CHCHO-, and -C(O)-CH(CH)-NH-.

[0072] In still other embodiments, R is selected from the group consisting of -C(O)-triazolyl-CH2CH2O-, -CH2-triazolyl-CH2CH2O-, and -C(O)-furanyl-CH2O-.

[0073] In further embodiments, R is selected from the group consisting of —C(O)—Ccycloalkyl-CHCHO—, —C(S)—Ccycloalkyl-CHCHO—, —C(O)—Ccycloalkyl-NH—, and —C(O)—O-phenyl-NH—.

[0074] In certain embodiments, R is selected from the group consisting of —C(O)-pyrrolidinyl- and —C(O)-pyrrolidinyl-C(O)—CH(CH)—NH—, wherein the pyrrolidinyl is optionally substituted with one or two fluoro atoms.

[0075] For example, in some embodiments, R is [ka] is selected from the group consisting of where ** represents the point of attachment to L.

[0076] For example, in some embodiments, the drug conjugates of the present disclosure are [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.

[0077] In certain embodiments, Lig is an anti-TROP2 antibody. In certain other embodiments, Lig is an anti-EGRF antibody, such as panitumumab, nimotuzumab, matuzumab, or cetuximab. In still other embodiments, Lig is an anti-HER2 antibody. In further embodiments, s is 1 or 8. For example, in some embodiments, s is 1.

[0078] For example, contemplated drug conjugates of the present disclosure are provided in Tables 1-3. [Table 1] [Table 2]

[0079] Table 3. EGFR antibody-drug conjugates Anti-EGFR IgG1 1 was used in compounds 3049 to 3107, anti-EGFR IgG1 2 was used in compound 3108, anti-EGFR IgG1 3 was used in compounds 3109 to 3110, anti-EGFR IgG1 4 was used in compounds 3111 and 3112, and anti-EGFR IgG1 5 was used in compound 3113. [Table 3-1] [Table 3-2]

[0080] Contemplated targets and corresponding exemplary antibodies of the present disclosure are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0081] Also disclosed herein is a compound of formula II: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C 1-3 is alkyl, R is selected from the group consisting of —C(O)—C3 alkyl, —C(O)O—C3 alkyl, —C(O)—C3 cycloalkyl, —C(O)—C4 cycloalkyl, —C1 alkyl, —C(O)-(pyrrolidinyl), and —C(O)-CH2ONH-C(O)-(pyrrolidinyl); where: -C(O)-C3 alkyl, -C(O)-C3 cycloalkyl, -C(O)O-C3 alkyl, -C(O)-C4 cycloalkyl or -C1 alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO, and -COOH; -C(O)-(pyrrolidinyl) and -C(O)-CHONH-C(O)-(pyrrolidinyl) may be optionally substituted on any available pyrrolidinyl with one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R 1 are joined together to form oxo, -C(O)-(pyrrolidinyl) may be optionally substituted on an available pyrrolidinyl nitrogen atom with -CHO, -C(O)CH3, or -C(O)CH2NH2.

[0082] In some embodiments, the therapeutic payload of formula II of the present disclosure has formula IIA: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0083] In some embodiments, Y is hydrogen. In other embodiments, Y is -C 1-3 For example, in certain embodiments, Y is -CH. In some embodiments, R is, for example, [ka] is selected from the group consisting of:

[0084] In some embodiments, the therapeutic payload of the present disclosure can be selected from, for example, any one of the compounds disclosed in Table 5, or a pharmaceutically acceptable salt or stereoisomer thereof. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0085] For example, in certain embodiments, the therapeutic payloads disclosed herein are [ka] [ka] or a pharmaceutically acceptable salt thereof. Further disclosed herein are compounds of formula IIIA or IIIB: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L is [ka] is selected from the group consisting of where * represents the point of attachment to R; R is [ka] is selected from the group consisting of where ** represents the point of attachment to L; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0086] In some embodiments, the linker payload construct of formula III of the present disclosure has formula IIIC: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein s is 1, 2, 3, 4, 5, 6, 7, or 8.

[0087] In some embodiments, R is, for example, [ka] is selected from the group consisting of where ** represents the point of attachment to L.

[0088] In certain embodiments, s is 1 or 8. In other embodiments, s is 1.

[0089] In some embodiments, a linker-payload construct of the present disclosure can be selected from, for example, any one of the compounds disclosed in Table 6, or a pharmaceutically acceptable salt or stereoisomer thereof. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0090] For example, in some embodiments, the linker-payload constructs contemplated herein comprise: [ka] [ka] [ka] [ka] [ka] may be selected from the group consisting of:

[0091] method Disclosed herein are, for example, methods of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a therapeutic payload disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0092] Also disclosed herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a linker-payload construct disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0093] Further disclosed herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a drug conjugate comprising any of the payloads disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0094] Also disclosed herein is a method of delivering a therapeutically effective amount of a therapeutic payload moiety to a patient in need thereof, comprising administering to the patient any one of the drug conjugates disclosed herein.

[0095] In certain embodiments, the patient is a human.

[0096] In certain embodiments, administering a compound of the present disclosure may include subcutaneous administration. In certain embodiments, administering a compound of the present disclosure may include intravenous administration. In certain embodiments, administering a compound of the present disclosure may include oral administration.

[0097] The methods of treatment provided may include administering a compound of the present disclosure once, twice, or three times daily; about every other day (e.g., every two days); twice a week (e.g., every third, fourth, fifth, sixth days, or, for example, with about 2 to about 3 days between doses); once a week; three times a week; every other week; twice a month; monthly; every other month; or even less frequently.

[0098] In particular, in certain embodiments, the present disclosure provides a method of treating one or more of the above medical indications, comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein.

[0099] In certain embodiments, the compound utilized by one or more of the methods disclosed herein is one of the general formula compounds, limited general formula compounds, or specific compounds described herein.

[0100] The compounds of the present disclosure can be administered to patients (animals and humans) in need of such treatment in dosages that provide optimal pharmaceutical efficacy. It will be understood that the dosage required for use in any particular application will vary from patient to patient, depending not only on the specific compound or composition selected, but also on the route of administration, the nature of the condition being treated, the patient's age and condition, any concomitant medications or special diets the patient is following, and other factors recognized by those skilled in the art, and that the appropriate dosage will ultimately be determined by the judgment of the attending physician. To treat the clinical conditions and diseases described herein, the compounds of the present disclosure can be administered orally, subcutaneously, topically, parenterally, by inhalation spray, or rectally in unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. Parenteral administration can include subcutaneous injection, intravenous or intramuscular injection, or infusion techniques.

[0101] Treatment can be continued for a long or short period as needed.Suitable treatment period can be, for example, at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 6 months, at least about 1 year, or indefinitely.The treatment period can be ended when the desired result is achieved.

[0102] Pharmaceutical compositions and kits Another aspect of the present disclosure provides pharmaceutical compositions comprising the compounds disclosed herein formulated with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising the compounds disclosed herein formulated with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), vaginal, or aerosol administration, although in any given case, the most suitable administration form will depend on the extent and severity of the condition being treated and the nature of the particular compound used. For example, the compositions of the present disclosure can be formulated as a unit dose and / or can be formulated for oral or subcutaneous administration.

[0103] For example, disclosed herein is a pharmaceutical composition comprising a therapeutic payload disclosed herein and a pharmaceutically acceptable excipient. Also disclosed herein is a pharmaceutical composition comprising a linker-payload construct disclosed herein and a pharmaceutically acceptable excipient. Further disclosed herein is a pharmaceutical composition comprising a drug conjugate disclosed herein and a pharmaceutically acceptable excipient.

[0104] Exemplary pharmaceutical compositions of the present disclosure can be used in the form of pharmaceutical preparations, for example, in solid, semi-solid, or liquid form, and contain one or more compounds of the present disclosure as an active ingredient in a mixture with organic or inorganic carriers or excipients suitable for topical, enteral, or parenteral use. The active ingredient can be formulated with conventional non-toxic pharmaceutically acceptable carriers for, for example, tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active subject compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect on the process or condition of a disease.

[0105] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present disclosure or a pharmaceutically acceptable non-toxic salt thereof. When these preformulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, thereby allowing the composition to be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.

[0106] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the subject compositions may be formulated with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any one of the following: (1) fillers or extenders, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, e.g., glycerol; (4) disintegrants. (5) solution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers for soft and hard gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0107] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersants. Molded tablets can be produced by molding a mixture of the subject compound moistened with an inert liquid diluent in a suitable machine. Tablets, as well as other solid dosage forms such as sugar-coated tablets, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical arts.

[0108] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the subject compositions, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, cyclodextrins, and mixtures thereof.

[0109] Suspensions may contain, in addition to the subject composition, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0110] Formulations for rectal or vaginal administration can be presented as suppositories, which can be prepared by mixing with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates, which are solid at room temperature but liquid at body temperature and will melt in the body cavity to release the active agent.

[0111] Dosage forms for transdermal administration of the subject compositions include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants which may be required.

[0112] Ointments, pastes, creams and gels may contain, in addition to the subject composition, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0113] Powders and sprays can contain, in addition to the subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0114] Alternatively, the compositions and compounds of the present disclosure can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the compound. Non-aqueous (e.g., fluorocarbon propellant) suspensions can also be used. Ultrasonic nebulizers may be used, as they minimize exposure of the agent to shear, which can lead to degradation of the compounds contained in the subject compositions. Typically, aqueous aerosols are made by formulating an aqueous solution or suspension of the subject compositions with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, amino acids such as oleic acid, lecithin, and glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0115] Pharmaceutical compositions of the present disclosure suitable for parenteral administration include the subject compositions in combination with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile solutions or dispersions for injection immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0116] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, and cyclodextrins. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0117] In another aspect, the present disclosure provides an enteral pharmaceutical formulation comprising a compound of the present disclosure, an enteric material, and a pharmaceutically acceptable carrier or excipient thereof. The enteric material refers to a polymer that is substantially insoluble in the acidic environment of the stomach and primarily dissolves in intestinal fluids at a specific pH. The small intestine is the portion of the gastrointestinal tract (intestine) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5, and the pH of the terminal ileum is about 7.5. Thus, the enteric material does not dissolve until a pH of, for example, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, or about 10.0. Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, cellulose acetate propionate, copolymers of methyl methacrylate and methyl methacrylate, copolymers of methyl acrylate, methyl methacrylate and methacrylic acid, copolymers of methyl vinyl ether and maleic anhydride (Gantrez Examples include natural resins such as ethyl methacrylate-methyl methacrylate-chlorotrimethylammonium ethyl acrylate copolymer, zein, shellac, and copal colophorium, as well as several commercially available enteric dispersions (e.g., Eudragit L30D55, Eudragit FS30D, Eudragit L100, Eudragit S100, Kollicoat EMM30D, Estacryl 30D, Coateric, and Aquateric). The solubility of each of the above materials is known or can be readily determined in vitro.While the above is a list of possible materials, one of ordinary skill in the art having the benefit of this disclosure will recognize that it is not exhaustive and that other enteric materials exist that would also meet the objectives of the present invention.

[0118] Advantageously, the present disclosure also provides kits for use by consumers in need of, for example, cancer treatment. Such kits include a suitable dosage form, such as those described herein, and instructions describing how to use such dosage form to relieve, alleviate, or prevent inflammation. The instructions would instruct the consumer or medical professional to administer the dosage form according to an administration mode known to those skilled in the art. Such kits can be advantageously packaged and sold in single or multiple kit units. An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used to package pharmaceutical unit dosage forms (tablets, capsules, etc.). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of preferably transparent plastic material. Recesses are formed in the plastic foil during the packaging process. The recesses have the size and shape of the tablets or capsules to be packed. The tablets or capsules are then placed in the recesses, and the foil is sealed with a sheet of relatively stiff material on the surface of the plastic foil opposite the recesses. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably, the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by applying manual pressure to the recesses, which form openings in the sheet at the locations of the recesses, through which the tablets or capsules can then be removed.

[0119] It may be desirable to provide a memory aid in the kit, for example, in the form of numbering next to the tablets or capsules, the numbers corresponding to the days in the regimen on which the tablets or capsules should be taken as specified. Another example of such a memory aid is a calendar printed on a card, for example, "Week 1: Monday, Tuesday, etc. ... Week 2: Monday, Tuesday, etc." Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or multiple tablets or capsules to be taken on a given day. Also, a daily dose of a first compound may consist of one tablet or capsule, while a daily dose of a second compound may consist of multiple tablets or capsules, or vice versa. The memory aid should reflect this. [Example]

[0120] The compounds described herein can be prepared in many ways based on the teachings contained herein and synthetic procedures known in the art. In the description of synthetic methods described below, it should be understood that all proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and workup procedures, can be selected to be standard conditions for the reaction unless otherwise specified. Those skilled in the art of organic synthesis understand that the functionality present on various portions of the molecule must be compatible with the proposed reagents and reactions. Substituents that are incompatible with the reaction conditions will be apparent to those skilled in the art, and alternative methods are therefore provided. The starting materials of the examples are commercially available or easily prepared by standard methods from known materials. At least some of the compounds identified herein as "intermediates" are contemplated as compounds of the present disclosure.

[0121] Unless otherwise noted, all reactions were carried out under argon in heat-gun dried glassware using standard septa techniques. All commercially available starting building blocks were purchased from commercial suppliers.

[0122] Example 1: General Methods Reactions were monitored by HPLC-MS analysis using a Shimadzu UFLC-MS-2020 system equipped with ESI and / or thin-layer chromatography (TLC) using silica gel 60 F254 plates (Merck) and visualized with UV at 254 nm. Purification was performed using an automated flash chromatography system (ECOM) with prepacked columns containing C18 or modified C18 silica gel (Interchim, PT-15C18AQ, 15 μm Puriflash 200, 5 g, 12 g, or 25 g). Semi-preparative HPLC was performed on the ECOM HPLC system using a modified C18 semi-preparative column (YMC-Actus, Triart Prep C18, 250 × 20 mm, S-10 μm, 12 nm). HPLC-MS analysis was performed on a Shimadzu UFLC-MS-2020 system equipped with ESI. Column: Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm. Solvent A: HO 0.1% HCOOH; Solvent B: MeCN + 0.1% HCOOH. Total flow rate: 0.6 ml / min. Total method time: 10 min. Mass spectra were recorded in both positive and negative modes over the m / z range of 100–3000 with an event time of 0.2 s. UV-Vis spectra were recorded over the 200–800 nm range using a Shimadzu SPD-M2OA Prominence diode array detector. NMR spectra were recorded on a 400 MHz Bruker AVANCE III spectrometer (1H at 400 MHz) and / or a Bruker AVANCE 500 (1H at 500.0 MHz) using >99% deuterated solvents. Chemical shifts (in ppm, δ scale) were taken from the solvent signal in the 1H spectrum. Intermediates and final products were lyophilized from water or mixtures of dioxane or acetonitrile and water using a Gregory Instruments Lyophilizer (Model L4-110). [Table 7]

[0123] Monoclonal antibody gene synthesis, antibody expression and purification The anti-HER2 antibody amino acid sequence was based on the heavy and light chain variable domains of the trastuzumab amino acid sequence in KEGG database entry D03257, and the anti-EGFR antibody was based on the heavy and light chain variable domains of the panitumumab amino acid sequence in KEGG database entry D05350, the cetuximab amino acid sequence in KEGG database entry D03255, the nimotuzumab amino acid sequence in DrugBank accession number DB06192, or the matuzumab amino acid sequence in DrugBank accession number DB06192.

[0124] Anti-HER2 light chain, SEQ ID NO: 1 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0125] Anti-HER2 IgG1 heavy chain, SEQ ID NO: 2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0126] Anti-EGFR IgG1 1 light chain, SEQ ID NO: 3 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0127] Anti-EGFR IgG1 1 heavy chain, SEQ ID NO: 4 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0128] Anti-EGFR IgG1 heavy chain, SEQ ID NO: 5 QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0129] Anti-EGFR IgG1 2 light chain, SEQ ID NO: 6 DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKL QITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0130] Anti-EGFR IgG1 triple heavy chain, SEQ ID NO: 7 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYAGRYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0131] Anti-EGFR IgG1 3 light chain, SEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0132] Anti-EGFR IgG1 4 heavy chain, SEQ ID NO: 9 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0133] Anti-EGFR IgG1 4 light chain, SEQ ID NO: 10 DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0134] Anti-EGFR IgG1 5 heavy chain, SEQ ID NO: 11 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0135] Anti-EGFR IgG1 5 light chain, SEQ ID NO: 12 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0136] Anti-EGFR IgG1 6 heavy chain (variant of anti-EGFR IgG1 5 heavy chain), SEQ ID NO: 13 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSEDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-EGFR IgG1 6 light chain, variant, SEQ ID NO: 14 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0137] Isotype antibody light chain, SEQ ID NO: 15 DIVLTQSPATLSVTPGNSVSLSCRASQSIGNDLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0138] Isotype antibody human IgG1 heavy chain, SEQ ID NO: 16 DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0139] Anti-TROP2 light chain, SEQ ID NO: 17 DIQMTQSPSSLSASVGDRVTITCQASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYITPLTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0140] Anti-TROP2 IgG1 heavy chain, SEQ ID NO: 18 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYGSSYWYFDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0141] The light and heavy chain protein sequences of anti-HER2, anti-TROP2, anti-EGFR, and IgG1 isotype control were generated by gene synthesis and codon-optimized for expression in mammalian cells (GeneUniversal). The heavy and light chain genes were cloned into separate mammalian expression vectors and then transiently co-transfected into EXPI293F cells (ThermoFisher). Antibody expression was performed in EXPI293F expression medium (ThermoFisher), and supernatants were harvested 5 days posttransfection. Each antibody was purified using MABSELECT Sure Resin (Cytiva) and buffer-exchanged into 1X phosphate-buffered saline (pH 7.4). Analytical size-exclusion chromatography (Cytiva, Superdex 200 10 / 300) data indicated that each antibody was approximately 99% monomeric.

[0142] Preparation of antibody-drug conjugates To prepare antibody-drug conjugates in which the linker-drug is conjugated to the cysteines between the heavy and heavy chains and between the heavy and light chains of an antibody, the linker-drug compound described herein was reacted with an anti-HER2, anti-TROP2, or anti-EGFR antibody to form a thioether bond to the cysteine ​​residues that form disulfide bond sites between the light and heavy chain antibody subunits and between the heavy and heavy chain antibody subunits, respectively. Deruxtecan (CAS number: 1599440-13-7) was obtained from ChemScene, Inc.

[0143] Step A: Anti-HER2 IgG1 monoclonal antibody (3.3 mg / ml, extinction coefficient at 280 nm: 215380 M-1 cm-1) was partially reduced by adding 40 molar equivalents of TCEP solution (BondBreaker, ThermoFisher) to 1x PBS (pH 7.4), 10% sucrose, and 5 mM EDTA for 60 min at 20 °C. Lyophilized powders of each linker-drug were dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture as 5-8% v / v DMSO solutions at 2-37 molar equivalents relative to the monoclonal antibody. The reaction solution was stirred at 20 °C for 2 h. Then, 20 mM N-acetylcysteine ​​solution (Sigma-Aldrich) was added at 9 molar equivalents relative to the antibody, and the reaction mixture was incubated at 20 °C for 20 min.

[0144] Step B: Anti-TROP2 IgG1 monoclonal antibody (3.8 mg / ml, extinction coefficient at 280 nm: 203460 M-1 cm-1) was partially reduced by adding 40 molar equivalents of TCEP (ThermoFisher) to 1x PBS (pH 7.4), 10% sucrose, and 5 mM EDTA for 60 min at 20 °C. Lyophilized powders of each linker-drug were dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture as 5-8% v / v DMSO solutions at 6-37 molar equivalents relative to the monoclonal antibody. The reaction solution was stirred for 2 h at 20 °C. Then, 20 mM N-acetylcysteine ​​(Sigma-Aldrich) was added at 9 molar equivalents relative to the antibody, and the reaction mixture was incubated at 20 °C for 20 min.

[0145] Step C: Anti-EGFR IgG1 monoclonal antibodies 1, 2, 3, 4, or 5 were partially reduced at 2.4–10 mg / ml in 1x PBS (pH 7.4), 10% sucrose, and 5 mM EDTA by adding 40 molar equivalents of TCEP (ThermoFisher) to the monoclonal antibody for 60 min at 20 °C. Lyophilized powders of each linker-drug were dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture as 2–37 molar equivalents of a 5–8% v / v DMSO solution. The reaction solution was stirred at 20 °C for 2 h. N-acetylcysteine ​​(Sigma-Aldrich) was then added at 1 molar equivalent relative to the linker-payload, and the reaction mixture was incubated at 20 °C for 20 min. The extinction coefficient at 280 nm of anti-EGFR IgG1 monoclonal antibody 1 is 232340 M-1 cm-1. The extinction coefficient at 280 nm of anti-EGFR IgG1 monoclonal antibody 2 is 217,440 M-1cm-1. The extinction coefficient at 280 nm of anti-EGFR IgG1 monoclonal antibody 3 is 227,360 M-1cm-1. The extinction coefficient at 280 nm of anti-EGFR IgG1 monoclonal antibody 4 is 227,360 M-1cm-1. The extinction coefficient at 280 nm of anti-EGFR IgG1 monoclonal antibody 5 is 217,440 M-1cm-1.

[0146] Removal of excess linker-drug from antibody drug conjugates The quenched excess linker-drug was separated from each antibody-drug conjugate by cation exchange chromatography (Cytiva, HiTrap SP HP resin). Buffer A consisted of 10% sucrose, 12.5 mM sodium acetate (pH 5.0), and 20 mM NaCl, while buffer B consisted of 10% sucrose, 12.5 mM sodium acetate (pH 5.0), and 1 M NaCl. The antibody-drug conjugate and linker-drug mixtures without conjugate in 1x PBS (pH 7.4) were diluted into 10% sucrose, 12.5 mM Na acetate (pH 5.0), and 0 mM NaCl buffer and loaded onto a HiTrap SP HP resin column (Cytiva). While the unconjugated linker-drug did not bind to the column, each antibody-drug conjugate bound to the column and was eluted with 10% sucrose, 100–150 mM NaCl, 12.5 mM Na-acetate (pH 5.0) buffer. Analytical size-exclusion chromatography (Cytiva Superdex 200 10 / 300 in 1x PBS (pH 7.4) buffer or Agilent AdvanceBio SEC 300 Å, 4.6 x 150 mm, 2.7 micron particle size column in 1x PBS (pH 7.4), 10% isopropanol buffer) showed that the antibody-drug conjugates were >90% monomeric.

[0147] Determination of drug linker / antibody ratio The drug linker to antibody ratio in the antibody-drug conjugate was determined using reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) of the antibody heavy and light chain subunit species, using approaches known in the art (Zhu X. et al. 2020. J Pharm Anal. 10(3):209-220; Firth D. et al. 2015 Analytical Biochemistry, 485, 34-42). Briefly, the antibody-drug conjugate was deglycosylated using PNGase F treatment (New Engl and Biolabs Rapid PNGAse), cysteine ​​disulfide bridges were reduced to separate the heavy and light chain subunits, and the samples were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS). Due to the reduction of cysteine ​​disulfide bridges and removal of N-linked glycans, the heavy and light chains eluted in separate peaks. Baseline-subtracted electrospray ionization spectra and deconvoluted mass spectra of the RPLC elution peaks were used to determine the mass and abundance (mass peak intensity) of the heavy and light chains of each antibody heavy and light chain subunit species. Heavy chain subunits were identified as having 0, 1, 2, or 3 linker-drug conjugates (referred to herein as H0, H1, H2, and H3), while light chain subunits were identified as having 0 or 1 linker-drug conjugate (referred to herein as L0 and L1). The data were used to determine the overall average linker-drug antibody conjugate ratio (DAR) for each antibody-drug conjugate. Reversed-phase liquid chromatography conditions were: mobile phase A: 0.05% TFA in water; mobile phase B: 0.05% trifluoroacetic acid in acetonitrile. The gradient program was 10–20% mobile phase B for 1 min, followed by 20–50% mobile phase B for 9 min, with a flow rate of 0.5 mL / min, post-column split, 2.1 × 50 mm Halo Diphenyl 2.7 μm column, and 80 °C. The mass spectrometer was a Waters Xevo G2-XS QTof equipped with an Acquity i-Class UPLC. Mass spectral data processing was performed using ProMass HR software for MassLyxn (Waters Limited).

[0148] For each antibody drug conjugate, the average number of drug molecules conjugated to the antibody was determined using the following formula: average linker payload conjugated to light chain = (mass peak intensity of L0 × 0 / (1 × mass peak intensity of L1 + 0 × mass peak intensity of L0)) + (mass peak intensity of L1 × 1 / (1 × mass peak intensity of L1 + 0 × mass peak intensity of L0)). Average of linker payloads conjugated to heavy chains = (H0 mass peak intensity × 0 / (0 × H0 mass peak intensity + 1 × H1 mass peak intensity + 2 × H2 mass peak intensity + 3 × H3 mass peak intensity)) + (H1 mass peak intensity × 1 / (0 × H0 mass peak intensity + 1 × H1 mass peak intensity + 2 × H2 mass peak intensity + 3 × H3 mass peak intensity)) + (H2 mass peak intensity × 2 / (0 × H0 mass peak intensity + 1 × H1 mass peak intensity + 2 × H2 mass peak intensity + 3 × H3 mass peak intensity)) + (H3 mass peak intensity × 3 / (0 × H0 mass peak intensity + 1 × H1 mass peak intensity + 2 × H2 mass peak intensity + 3 × H3 mass peak intensity)). The average number of drug molecules conjugated to an antibody composed of two light chain subunits and two heavy chain subunits = (2 x average linker payload conjugated to light chains + 2 x average linker payload conjugated to heavy chains).

[0149] The observed mass of the linker-drug-free anti-TROP2 antibody heavy chain (H0) was 49,208.8 Da (Daltons), and the observed mass of the linker-drug-free light chain (L0) was 23,343.8 Da. The observed mass of the linker-drug-free anti-HER2 antibody heavy chain (H0) was 49,123.9 Da, and the observed mass of the linker-drug-free light chain (L0) was 23,456.2 Da. The observed mass of the linker-drug-free anti-EGFR antibody 1 heavy chain (H0) was 48,897.5 Da, and the observed mass of the linker-drug-free light chain (L0) was 23,357.3 Da. The observed mass of the linker-drug-free anti-EGFR antibody triple heavy chain (H0) was 49,508 (Da), and the observed mass of the linker-drug-free light chain (L0) was 23,317 (Da). The observed mass of the linker-drug-free anti-EGFR antibody quadruple heavy chain (H0) was 49,552 (Da), and the observed mass of the linker-drug-free light chain (L2) was 23,317 (Da). The observed mass of the heavy chain with one linker-drug (H1) was (H0 + 1 × molecular weight of linker-drug), the observed mass of the heavy chain with two linker-drugs (H2) was (H0 + 2 × molecular weight of linker-drug), and the observed mass of the heavy chain with three linker-drugs (H3) was (H0 + 3 × molecular weight of linker-drug). The observed mass of a light chain (L1) having one linker-drug is (L0 + 1 × molecular weight of the linker-drug), where the molecular weight of each of the linker-drugs is provided in the synthesis procedure.

[0150] Additionally, DAR was determined by comparing the results with ADCs conjugated to the same linker-drug using reduced reverse-phase high-performance liquid chromatography (RP-HPLC). Parent (unconjugated) antibody samples were prepared in PBS (pH 7.4) at a sample concentration of 1 mg / mL. ADC samples (in the original sample buffer of 10 mM sodium acetate (pH 5.0), 130 mM NaCl, and 10% (wt. / vol.) sucrose) were prepared at a concentration of 1 mg / mL (protein ratio). To help raise the pH, a volume of 0.1 M Tris (pH 8.5) was added to a final concentration of 20 mM Tris. Dithiothreitol (DTT, No-Weigh™ Format ThermoScientific) was added to the antibody and ADC samples to a concentration of 50 mM, and the samples were incubated at 40°C for 5 minutes. After reduction with DTT, an equal volume of 0.1% (vol. / vol.) aqueous trifluoroacetic acid (TFA, Fisher Chemical) was immediately added to each antibody and ADC sample. Denaturing reverse-phase chromatography was performed on an Agilent 1260 HPLC system equipped with a thermostatted (10 °C) autosampler. Samples (5 mg total protein loaded) were analyzed on an Agilent AdvanceBio RP-mAb Diphenyl 2.1 x 50 mm column (3.5 mm particles with 450 Å surface pores) using a flow rate of 0.5 mL / min. Gradient elution was performed at 80 °C over 10 min using mobile phase "A" consisting of 30–70% mobile phase "B" (0.1% (vol. / vol.) TFA in acetonitrile) and 0.1% (vol. / vol.) TFA in water. UV detection was monitored at 220 nm, 280 nm, and 370 nm, and the signal at 405 nm (for 220 nm and 280 nm) or 600 nm (for 370 nm detection signal) was subtracted from the baseline. As is known in the art, retention time depends on the hydrophobicity of the compound. Compared with the heavy chain without linker-drug (H0), the heavy chains conjugated with one linker-drug (H1), two linker-drugs (H2), and three linker-drugs (H3) became progressively more hydrophobic.Compared with the light chain without a linker-drug (L0), the light chain with one linker-drug conjugate (L1) was more hydrophobic. For each ADC, the peaks detected in the RP-HPLC chromatogram were assigned to L0, L1, H0, H1, H2, and H3 by comparison with the retention times of L0 and H0 of the parent monoclonal antibody, comparison of the detection signals at 280 nm and 370 nm (the linker payload described here absorbs at 370 nm), and comparison with the known LC / MS DAR values ​​of the heavy and light chains. The heavy and light chains eluted in the following order: L0, L1, H0, H1, H2, and H3. For ADCs whose DARs were determined based on the RP-HPLC procedure, the peak areas of L0, L1, H0, H1, H2, and H3 detected at 280 nm were normalized by the extinction coefficients of each DAR species (L0, L1, H0, H1, H2, and H3) at 280 nm. As known in the art, the molar extinction coefficients of L0 and H0 were estimated based on the amino acid sequences of the monoclonal antibodies (M). -1 cm -1 The molar extinction coefficient of the linker-payload at 280 nm is 5000-6100 M according to Beer's law. -1 cm -1The molar extinction coefficient of L1 was estimated using the formula: molar extinction coefficient of L0 = (molar extinction coefficient of L0 + 1 × molar extinction coefficient of linker payload), molar extinction coefficient of H1 = (molar extinction coefficient of H0 + 1 × molar extinction coefficient of linker payload), molar extinction coefficient of H2 = (molar extinction coefficient of H0 + 2 × molar extinction coefficient of linker payload), molar extinction coefficient of H3 = (molar extinction coefficient of H0 + 3 × molar extinction coefficient of linker payload). For each antibody-drug conjugate, the average number of drug molecules conjugated to the antibody was determined using the following formula and the peak area normalized by each extinction coefficient: average linker payload conjugated to light chain = (peak area of ​​L0 × 0 / (1 × peak area of ​​L1 + 0 × peak area of ​​L0)) + (peak area of ​​L1 × 1 / (1 × peak area of ​​L1 + 0 × peak area of ​​L0)). Average linker payload conjugated to heavy chains = H0 peak area x 0 / (0 x H0 peak area + 1 x H1 peak area + 2 x H2 peak area + 3 x H3 peak area)) + (H1 peak area x 1 / (0 x H0 peak area + 1 x H1 peak area + 2 x H2 peak area + 3 x H3 area intensity)) + (H2 peak area x 2 / (0 x H0 peak area + 1 x H1 peak area + 2 x H2 peak area + 3 x H3 peak area)) + (H3 peak area x 3 / (0 x H0 peak area + 1 x H1 peak area + 2 x H2 peak area + 3 x H3 peak area)). Average number of drug molecules conjugated to an antibody composed of two light chain subunits and two heavy chain subunits = (2 x average linker drug conjugated to light chains + 2 x average linker drug conjugated to heavy chains).

[0151] Example 2: Synthesis of Compound 2 [ka] Intermediate 1. A mixture of ethanolamine (23 mg, 0.4142 mmol) and dimethoxysquarate (3 equiv., 177 mg, 1.242 mmol) was suspended in 10 mL of 1 M borate buffer (pH = 9), and the mixture was stirred at 55 °C for 16 h. 2 mL of DMF was added, and the solvent was evaporated under reduced pressure to a final volume of approximately 3 mL. The crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 → 50% ACN in HO). After lyophilization from water, the desired product was recovered as a white powder (46 mg, 65%). C7H in MS was 0.01%. 10 NO4 calculated value: 172.06, Found value: 172.25, [M+H] + .

[0152] Compound 2. Exatecan mesylate (20 mg, 0.0377 mmol) and the previously synthesized intermediate 1 (1.5 equivalents, 9.7 mg, 0.0564 mmol) were suspended in 5 mL of 1 M borate buffer (pH = 9), and the mixture was stirred at 55 °C for 16 h. 2 mL of DMF was added, and the solvent was evaporated under reduced pressure to a final volume of approximately 3 mL. The crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 → 50% ACN in HO). After lyophilization from water, the desired product was recovered as a white powder (12 mg, 57%). C in MS was 0.01%. 30 H 28 Calculated for FN4O7: 575.19, Found: 575.45, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 7.83 (d, J = 10.9 Hz, 1H), 7.78 (s, 1H), 7.32 (s, 1H), 5.79 (s, 1H), 5.42 (s, 2H), 5.29 (d, J = 7.5 Hz, 2H), 3.68 - 3.43 (m, 4H), 3.23 (d, J = 7.7 Hz, 2H), 2.42 (d, J = 1.9 Hz, 3H), 2.33 (td, J = 5.7, 4.8, 2.9 Hz, 1H), 1.96 - 1.78 (m, 2H), 1.76 (s, 1H), 1.26 - 1.15 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0153] Example 3: Synthesis of Compound 1001 [ka] Intermediate 1. Ethanolamine (100 mg, 1.637 mmol) and dimethoxysquarate (1.2 equiv., 1.964 mmol, 279 mg) were dissolved in 10 mL of 1 M borate buffer (pH 9). The reaction mixture was stirred at room temperature for 16 h. The solvent was evaporated under reduced pressure, and the resulting solid was redissolved in DMF and loaded directly onto the column. The product was purified by reverse-phase flash chromatography using a column containing 40 g of C18 and a gradient of ACN in water (0 to 20% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (205 mg, 73%). C7H in MS was 0.01%. 10 NO4 calculated value: 172.06, Found value: 172.17, [M + H] + .

[0154] Intermediate 2. Intermediate 1 (10 mg, 0.058 mmol) and the starting peptide FmocGGFG-OAc (1 eq., 0.058 mmol, 37 mg) were dissolved in 2 mL of anhydrous DMF under an argon atmosphere, and 100 μL of HCl (2 M in EtO) was added. The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto the column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 80% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (25 mg, 58%). C in MS was 0.058 mmol. 38 H 40 N6NaO 10 Calculated value: 763.27, Found value: 763.80, [M + Na] + .

[0155] Intermediate 3. Intermediate 2 (25 mg, 0.0338 mmol) and exatecan mesylate (1.5 equiv., 0.508 mmol, 27 mg) were suspended in 4 mL of 1 M borate buffer (pH 9), and the reaction mixture was stirred at 55° C. for 4 h. DMF (2 mL) was added, and the solvent was evaporated under reduced pressure to a final volume of approximately 2 mL. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water-dioxane, the desired product was recovered as a white solid (11 mg, 28%). C in MS was 0.01%. 61 H 59 FN9O 13 Calculated value: 1144.42, Measured value: 1144.01, [M + H] + .

[0156] Intermediate 4. Intermediate 3 (11 mg, 0.0096 mmol) was dissolved in 1 mL of DMF and morpholine (20 μL) was added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water-dioxane, the desired product was recovered as a yellowish solid (7.5 mg, 88%). C in MS was 0.01%. 46 H 50 FN9O 11 Calculated value: 923.36, Found value: 923.75, [M + H] + .

[0157] Compound 1001. Intermediate 4 (7.5 mg, 0.0081 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (2 equivalents, 0.0163 mmol, 5 mg) and DIPEA (20 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 80% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (7 mg, 77%). C in MS was 0.0163 mmol. 55 H 58 FN 10 O 15 Calculated value: 1117.41, Found value: 1117.44, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.62 (d, J = 10.9 Hz, 1H), 8.52 (s, 1H), 8.27 (s, 1H), 8.15 (d, J = 33.5 Hz, 1H), 8.02 (d, J = 16.0 Hz, 1H), 7.82 (d, J = 11.5 Hz, 1H), 7.67 (s, 1H), 7.36 - 7.20 (m, 4H), 7.19 - 7.12 (m, 2H), 6.68 (s, 1H), 6.05 (t, J = 20.7 Hz, 1H), 5.86 (s, 1H), 5.75 (s, 1H), 5.30 (d, J = 23.9 Hz, 1H), 5.18 (m, 2H), 4.85 (d, J = 11.1 Hz, 2H), 4.66 - 4.54 (m, 1H), 4.54 (s, 2H), 4.44 (m, 1H), 3.85 - 3.79 (m, 1H), 3.70 - 3.65 (m, 3H), 3.56 - 3.47 (m, 2H), 3.47 - 3.42 (m, 1H), 3.41 - 3.36 (m, 1H), 3.17 (s, 1H), 3.07 - 2.97 (m, 2H), 2.85 - 2.69 (m, 4H), 2.67 - 2.52 (m, 2H), 2.44 - 2.36 (m, 3H), 2.33 - 2.23 (m, 1H), 1.97 (m, 2H), 1.82 (d, J = 7.9 Hz, 2H), 1.23 (s, 2H), 0.87 - 0.81 (m, 3H).

[0158] Example 4: Synthesis of Compound 3002 Compound 3002 was prepared according to Procedure A in the General Methods section of Example 1, using 13 molar equivalents of linker-payload compound 1001 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 3.7, based on a linker-drug molecular weight of 1117 Da.

[0159] Example 5: Synthesis of compounds 3028A and 3028B Compound 3028A was prepared according to Procedure B in the General Methods section of Example 1, using 21 molar equivalents of linker-payload compound 1001 relative to an anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 4.21, based on a linker-drug molecular weight of 1117 Da. When prepared according to Procedure B, using 39 molar equivalents of linker-payload compound 1001 relative to an anti-TROP2 IgG1 monoclonal antibody, a DAR of 7.5 was achieved, yielding compound 3028B.

[0160] Example 6: Synthesis of Compound 3050 Compound 3050 was prepared according to Procedure C in the General Methods section of Example 1 using 13 molar equivalents of linker-payload compound 1001 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 4.5 based on a linker-drug molecular weight of 1117 Da.

[0161] Example 7: Synthesis of Compound 12 [ka] Intermediate 1. Exatecan mesylate (39 mg, 0.0737 mmol), malonic acid (5 equiv., 0.3687 mmol, 38 mg), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.3687 mmol, 102 mg) were dissolved in a 5:1 mixture of DMF and water (6 mL). Triethylamine (50 equiv., 3.6873 mmol, 514 μL) was added, and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA). After lyophilization from water, the desired product was recovered as a white powder (34 mg, 88%). C in MS was 0.01%. 27 H 23Calculated value of FN3O7: 520.15, Measured value: 520.49 [MH] - .

[0162] Intermediate 2. The previously synthesized Intermediate 1 (24 mg, 0.0461 mmol), 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (5 equiv., 0.2303 mmol, 48 μL), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.2303 mmol, 64 mg) were dissolved in a 5:1 mixture of DMF and water (6 mL). Triethylamine (50 equiv., 2.303 mmol, 321 μL) was added, and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 70% ACN in HO). After lyophilization from water-DMF, the desired product was recovered as a yellowish foam (7 mg, 22%). 35 H 44 Calculated value for FN4O7Si: 679.30, Found: 679.00, [M+H] + .

[0163] Compound 12. The previously synthesized intermediate 2 (7 mg, 0.0103 mmol) was suspended in 1% TFA (2 mL), and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was loaded directly onto a column and purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA). After lyophilization from water, the desired product was recovered as a yellowish powder (3 mg, 53%). C in MS was 0.0103 mmol. 29 H 30 Calculated for FN4O7: 565.21, Found: 565.70, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.6 Hz, 1H), 8.05 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 5.58 - 5.54 (m, 1H), 5.43 (s, 2H), 5.26 (d, J = 5.2 Hz, 2H), 3.38 (t, J = 6.3 Hz, 2H), 3.18 (s, 2H), 3.15 - 3.04 (m, 2H), 2.58 - 2.52 (m, 2H), 2.46 (m, 3H), 2.25 - 2.16 (m, 1H), 2.11 (s, 1H), 1.87 (m, 2H), 1.76 (s, 2H), 0.88 (t, J = 7.4 Hz, 3H).

[0164] Example 8: Synthesis of Compound 1005 [ka] Intermediate 1. FmocGGFG-N3 (23 mg, 0.0350 mmol) was dissolved in 2 mL of dioxane. Pd / C (10 w / w%, 5 mg) was suspended in the mixture and stirred at room temperature for 2 h while H2 was bubbled through the suspension using a balloon. The suspension was syringed, and the filtrate was passed through a 0.2 μm syringe filter directly into a flask containing the previously prepared solution of malonic acid (5 equiv., 0.1750 mmol, 18 mg), DMTMM (5 equiv., 0.1750 mmol, 48 mg), and DIPEA (100 μL) in ACN (2 mL) and water (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the resulting solid was redissolved in DMF and loaded directly onto the column. The product was purified by reversed-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 40% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (15 mg, 60%). 36 H 41 NO 10Calculated value: 715.27, Measured value: 715.55 [M - H] - .

[0165] Intermediate 2. Intermediate 1 (14 mg, 0.0195 mmol) was dissolved in a mixture of DMF (2 mL) and water (0.5 mL). Exatecan mesylate (1.5 eq., 0.0293 mmol, 16 mg), DMTMM (3 eq., 0.0587 mmol, 16 mg), and DIPEA (20 μL) were added to the solution, and the reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure, and the resulting solid was redissolved in DMF and loaded directly onto the column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water, the desired product was recovered as a yellow solid (15 mg, 66%). C in MS was 0.01%. 60 H 61 FN9O 13 Calculated value: 1134.44, Found value: 1134.40 [M + H] + .

[0166] Intermediate 3. Intermediate 2 (15 mg, 0.0132 mmol) was dissolved in 1 mL of DMF and morpholine (20 μL) was added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water-dioxane, the desired product was recovered as a yellow solid (10 mg, 83%). C in MS was 0.0132 mmol. 45 H 51 FN9O 11 Calculated value: 912.37, Found value: 912.91 [M + H] + .

[0167] Compound 1005. Intermediate 3 (10 mg, 0.0110 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (2 equivalents, 0.0219 mmol, 7 mg) and DIPEA (20 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 80% ACN in water). After lyophilization from water-dioxane, the desired product was recovered as a yellow solid (11 mg, 90%). C in MS was 0.01%. 54 H 60 FN 10 O 15 Calculated value: 1107.42, Found value: 1107.50 [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.67 (m, 1H), 8.61 (m,1H), 8.52 (m, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 8.12 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.67 (s, 1H), 7.28 (m, 4H), 7.25 - 7.24 (m, 2H), 7.212 (s, 1H), 7.15 (m, 1H), 6.68 (s, 1H), 6.09 - 6.00 (m, 1H), 5.58 - 5.47 (m, 1H), 5.19 - 5.08 (m, 2H), 4.88 (d, J = 11.8 Hz, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.50 (dd, J = 11.7, 6.6 Hz, 1H), 4.47 (m, 2H), 3.78 - 3.63 (m, 3H), 3.63 - 3.52 (m, 4H), 3.19 (m, 3H), 3.11 - 3.01 (m, 4H), 2.63 (p, J = 1.9 Hz, 5H), 2.43 - 2.36 (m, 6H), 2.24 - 2.14 (m, 1H), 1.75 (m, 2H), 1.23 (s, 2H), 0.85 (dd, J = 7.9, 6.3 Hz, 3H).

[0168] Example 9: Synthesis of Compound 3003 Compound 3003 was prepared according to Procedure A in the General Methods section of Example 1, using 2 molar equivalents of linker-payload compound 1005 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 2.1, based on a linker-drug molecular weight of 1107 Da.

[0169] Example 10: Synthesis of compounds 3029A and 3029B Compound 3029A was prepared according to Procedure B in the General Methods section of Example 1, using 2 molar equivalents of linker-payload compound 1005 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 1.2, based on a linker-drug molecular weight of 1107 Da. When 27 molar equivalents of linker-payload compound 1005 relative to the anti-TROP2 IgG1 monoclonal antibody were used, a DAR of 4.4 was achieved, yielding compound 3029B.

[0170] Example 11: Synthesis of Compounds 3051A and 3051B Compound 3051A was prepared according to Procedure C in the General Methods section of Example 1, using 2 molar equivalents of linker-payload compound 1005 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 1.9, based on a linker-drug molecular weight of 1107 Da. When 27 molar equivalents of linker-payload compound 1005 relative to anti-EGFR IgG1 monoclonal antibody 1 were used, a DAR of 3.14 was achieved, yielding compound 3051B.

[0171] Example 12: Synthesis of Compound 16 [ka] Intermediate 1. Exatecan mesylate (20 mg, 0.0376 mmol), succinic acid (5 equiv., 0.1881 mmol, 22 mg), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.1881 mmol, 53 mg) were dissolved in a 5:1 mixture of DMF and water (4 mL). Triethylamine (200 μL) was added, and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA). After lyophilization from water-dioxane, the desired product was obtained as a brown powder (18 mg, 89%). C in MS was 0.01%.28 H 25 Calculated value of FN3O7: 534.17, Measured value: 534.80 [MH] - .

[0172] Intermediate 2. The previously synthesized Intermediate 1 (15 mg, 0.0424 mmol), 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (3 equiv., 0.1272 mmol, 22 mg), and hexafluorophosphate azabenzotriazole tetramethyluronium (HATU, 3 equiv., 0.1272 mmol, 48 mg) were dissolved in DMF (1 mL). Diisopropylethylamine (50 μL) was added, and the reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 80% ACN in HO). After lyophilization from water-dioxane, the desired product was recovered as a yellow foam (22 mg, 74%). C in MS was 0.01%. 36 H 46 Calculated value for FN4O7Si: 693.31, Found: 693.55, [M+H] + .

[0173] Compound 16. The previously synthesized intermediate 2 (22 mg, 0.0317 mmol) was suspended in 1% TFA (2 mL), and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was loaded directly onto a column and purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA). After lyophilization from water, the desired product was recovered as a white powder (11 mg, 60%). C in MS was 0.01%. 30 H 32 Calculated for FN4O7: 579.23, Found: 579.25, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.7 Hz, 1H), 7.82 (m, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 5.56 (t, J = 4.8 Hz, 1H), 5.42 (s, 2H), 5.30 - 5.10 (m, 2H), 3.47 (m, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.18 (m, 2H), 3.07 (td, J = 6.1, 4.4 Hz, 2H), 2.56 - 2.52 (m, 2H), 2.42 - 2.32 (m, 5H), 2.12 (q, J = 5.3 Hz, 2H), 1.95 - 1.79 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0174] Example 13: Synthesis of Compound 1023 [ka] Intermediate 1. To a solution of FmocGGFG-N3 (1.0 equiv., 26 mg, 0.040 mmol) in dioxane (1.5 ml) was added Pd / C (10% w / w, 5 mg), and the resulting suspension was hydrogenated. The reaction mixture was hydrogenated (balloon) at room temperature for 1.5 hours. LC-MS analysis confirmed complete consumption of the starting material. The suspension was filtered to give Intermediate 1 as a solution in dioxane, which was used directly in the next step. C in MS 33 H 39 Calculated for N6O7: 631.29, Found: 631.30, [M+H] + .

[0175] Intermediate 2. To a mixture of exatecan mesylate (1.0 equiv., 20 mg, 0.037 mmol) and succinic anhydride (1.1 equiv., 4.1 mg, 0.041 mmol), DMF (0.5 mL) and diisopropylethylamine (2.2 equiv., 14 μL, 0.082 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Then, a solution of Intermediate 1 in dioxane (obtained in the previous step) was added to the reaction mixture, followed by DMTMM (1.0 equiv., 11 mg, 0.037 mmol), diisopropylethylamine (1.0 equiv., 7 μL, 0.037 mmol), and water (0.25 mL), and the resulting solution was stirred at room temperature for 1 hour. The mixture was concentrated on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / 0.1% TFA in water) to give the product intermediate 2 (15 mg, 35%) as a pale yellow solid after lyophilization. 61 H 63 FN9O 13 Calculated value: 1148.45, Found value: 1148.45, [M+H] + .

[0176] Compound 1023. To a solution of intermediate 2 (1.0 equiv., 15 mg, 0.013 mmol) in DMF (1 mL) was added morpholine (35 μL), and the reaction mixture was stirred at room temperature for 1.5 hours. DMF and excess morpholine were then evaporated using a rotary evaporator. The residue was redissolved in DMF (0.5 mL), followed by the addition of Mal-PEG-NHS ester (1.1 equiv., 4.4 mg, 0.014 mmol) and diisopropylethylamine (1.1 equiv., 2.5 μL, 0.014 mmol), and the resulting solution was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / 0.1% TFA in water) afforded the desired product, Compound 1023 (7 mg, 48%), as a pale yellow solid after lyophilization. C in MS indicated a C 52 H 62 FN 10 O 15 Calculated value: 1121.44, Found value: 1121.45, [M+H] +. 1 H NMR (500 MHz, DMSO-d6) δ: 8.53 - 8.42 (m, 2H), 8.29 (t, J = 5.9 Hz, 1H), 8.14 - 8.07 (m, 2H), 7.99 (t, J = 5.8 Hz, 1H), 7.89 (t, J = 5.2 Hz, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 7.27 - 7.20 (m, 4H), 7.20 - 7.12 (m, 1H), 6.99 (s, 2H), 5.55 (dt, J = 9.2, 4.8 Hz, 1H), 5.47 - 5.37 (m, 2H), 5.28 - 5.08 (m, 2H), 4.57 - 4.44 (m, 4H), 3.80 - 3.62 (m, 6H), 3.53 - 3.40 (m, 4H), 3.35 (t, J = 5.9 Hz, 2H), 3.16 (dq, J = 17.4, 5.8 Hz, 4H), 3.04 (dd, J = 13.9, 4.7 Hz, 1H), 2.87 - 2.73 (m, 2H), 2.43 - 2.35 (m, 7H), 2.32 (t, J = 6.5 Hz, 2H), 2.21 - 2.02 (m, 2H), 1.93 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0177] Example 14: Synthesis of Compound 3004 Compound 3004 was prepared according to Procedure A in the General Methods section of Example 1, using 17 molar equivalents of linker-payload compound 1023 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 4.4, based on a linker-drug molecular weight of 1121 Da.

[0178] Example 15: Synthesis of Compound 3030 Compound 3030 was prepared according to Procedure B in the General Methods section of Example 1 using 17 molar equivalents of linker-payload compound 1023 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.6 based on a linker-drug molecular weight of 1121 Da.

[0179] Example 16: Synthesis of Compound 3052 Compound 3052 was prepared according to Procedure C in the General Methods section of Example 1 using 17 molar equivalents of linker-payload compound 1023 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 6.6 based on a linker-drug molecular weight of 1121 Da.

[0180] Example 17: Synthesis of Compound 22 [ka] Intermediate 1. 2-[[tert-Butyl(dimethyl)silyl]oxy]ethanol (1.14 mmol, 200 mg) was dissolved in 4 mL of anhydrous dichloromethane under an argon atmosphere. The reaction mixture was cooled to 0 °C, and diisopropylethylamine (1.1 equiv., 1.25 mmol, 218 μL) was added, followed by triphosgene (1.2 equiv., 0.45 mmol, 135 mg). The reaction mixture was stirred at 0 °C for 2 h. Additional diisopropylethylamine (1.2 equiv., 1.25 mmol, 218 μL) was added, followed by 2-mercaptopyridine (1.1 equiv., 1.25 mmol, 155 mg). After an additional 2 h at 0 °C, the reaction mixture was diluted with 20 mL of dichloromethane, and saturated NH4Cl (10 mL) was added. The organic phase was washed with saturated NH4Cl (2 × 100 mL) and brine (100 mL). Silica gel flash chromatography using a gradient of EtOAc in cyclohexane (0 to 50% EtOAc in cyclohexane) gave the desired product (100 mg, 28%). 14 H 24 Calculated value of NO3SSi: 314.12, Measured value: 314.10, [M+H] +.

[0181] Compound 22. Exatecan mesylate (0.0376 mmol, 20 mg) and triethylamine (2 equivalents, 0.0752 mmol, 11 μL) were dissolved in 2 mL of anhydrous DMF under an argon atmosphere. Intermediate 1 (1.5 equivalents, 0.0752 mmol, 18 mg) was added, and the reaction mixture was stirred at room temperature for 48 hours. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 40% ACN in HO). The fractions containing the desired product (Intermediate 2) were evaporated, and the solid was resuspended in 1% TFA and stirred at room temperature for 1 hour. The desired product Sc-122 was repurified by reversed-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA) and recovered as a white powder after lyophilization from water (16 mg, 81% over two steps, calculated with exatecan). 27 H 27 Calculated for FN3O7: 524.18, Found: 524.20, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.43 (s, 2H), 5.32 - 5.16 (m, 2H), 4.11 (m, 2H), 3.33 (s, 1H), 3.25 (d, J = 6.0 Hz, 1H), 3.11 (d, J = 18.7 Hz, 1H), 2.68 (m, 1H), 2.55 (m, 2H), 2.39 - 2.29 (m, 4H), 2.24 - 2.12 (m, 2H), 1.87 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0182] Example 18: Synthesis of Compound 1007 [ka] Intermediate 1. Compound 22 (28 mg, 0.0535 mmol) and FmocGGFG-OAc (2 eq., 0.107 mmol, 67 mg) were dissolved in 1 mL of anhydrous DMF. HCl (100 μL, 2 M in EtO) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was directly loaded onto a column. The product was purified by reverse-phase flash chromatography using a column containing 25 g of C18 and a gradient of ACN in water (0 to 70% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (23 mg, 39%). C in MS was 0.01%. 58 H 58 FN8O 13 Calculated value: 1093.41, Found value: 1093.63, [M + H] + .

[0183] Intermediate 2. Intermediate 1 (23 mg, 0.0211 mmol) was dissolved in 1 mL of anhydrous DMF and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto the column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (16 mg, 85%). C in MS was 0.01%. 43 H 48 FN8O 11 Calculated value: 871.34, Found value: 871.44, [M + H] + .

[0184] Compound 1007. Intermediate 2 (16 mg, 0.0179 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (2 equivalents, 0.0358 mmol, 11 mg) and DIPEA (20 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (7 mg, 37%). C in MS was 0.01%. 52 H 57 FN9O 15 Calculated value: 1066.40, Found value: 1065.98, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.65 (d, J = 6.9 Hz, 1H), 8.58 - 8.49 (m, 1H), 8.45 (m, 1H), 8.37 (s, 1H), 8.31 - 8.22 (m, 1H), 8.15 - 8.08 (m, 2H), 8.07 - 7.96 (m, 1H), 7.91 (s, 1H), 7.79 (t, J = 11.5 Hz, 2H), 7.34 - 7.28 (m, 1H), 7.25 - 7.17 (m, 3H), 7.01 (d, J = 10.9 Hz, 2H), 6.67 (s, 1H), 6.61 - 6.46 (m, 1H), 5.42 (s, 1H), 5.39 - 5.28 (m, 1H), 5.26 - 5.23 (m, 2H), 5.15 (d, J = 7.3 Hz, 1H), 4.57 (m, 2H), 4.49 (d, J = 11.1 Hz, 2H), 4.18 (d, J = 4.8 Hz, 2H), 3.77 - 3.63 (m, 3H), 3.61 (m, 2H), 3.58 - 3.42 (m, 3H), 3.25 - 3.17 (m, 1H), 3.04 (s, 2H), 2.77 (dd, J = 8.5, 5.5 Hz, 1H), 2.47 - 2.29 (m, 4H), 2.20 - 2.11 (m, 1H), 2.00 (m, 1H), 1.86 (m, 2H), 1.23 (s, 2H), 0.97 - 0.82 (m, 3H).

[0185] Example 19: Synthesis of Compound 3005 Compound 3005 was prepared according to Procedure A in the General Methods section of Example 1, using 12 molar equivalents of linker-payload compound 1007 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 4.7, based on a linker-drug molecular weight of 1066 Da.

[0186] Example 20: Synthesis of Compound 3031 Compound 3031 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1007 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.2 based on a linker-drug molecular weight of 1066 Da.

[0187] Example 21: Synthesis of compounds 3053A and 3053B Compound 3053A was prepared according to Procedure C in the General Methods section of Example 1, using 12 molar equivalents of linker-payload compound 1007 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 3.5, based on a linker-drug molecular weight of 1066 Da. The molar ratio of mAb to linker-payload was adjusted to achieve a DAR of 7.7 (compound 3053B).

[0188] Example 22: Synthesis of Compound 42 [ka] Intermediate 1. 2-((tert-Butyldimethylsilyl)oxy)ethan-1-amine (21 mg, 0.121 mmol), triphosgene (0.95 equiv., 0.0383 mmol, 11 mg), and diisopropylethylamine (5 equiv., 0.605 mmol, 105 μL) were dissolved in dichloromethane (2 mL) under an argon atmosphere. The reaction mixture was stirred at room temperature for 1 h. Complete conversion to intermediate 1 was confirmed by LCMS analysis. The reaction product was used in the next step without further purification.

[0189] Compound 42. Exatecan mesylate (0.0602 mmol, 32 mg) and diisopropylethylamine (2 equivalents, 0.120 mmol, 21 μL) were dissolved in 1 mL of anhydrous DMF, and the solution was cooled to 0 °C. The previously prepared solution of isocyanate intermediate 1 (2 mL, 0.1150 mmol) in dichloromethane was added at 0 °C, and the reaction mixture was allowed to warm to room temperature and stirred for 1 h. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 → 60% ACN in HO). The fractions containing the desired reaction product (intermediate 2) were evaporated, and the solid was resuspended in 1% TFA and stirred at room temperature for 1 h. The desired product was repurified by reversed-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in 1% TFA (0 to 40% ACN in 1% TFA) and recovered as a white powder after lyophilization from water (15 mg, 48% over three steps, calculated from exatecan). 27 H 28 Calculated value for FN4O6: 523.20, Found: 523.25, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.76 (dd, J = 11.0, 2.0 Hz, 1H), 7.31 (s, 1H), 6.82 (d, J = 8.9 Hz, 1H), 6.61 (m, 1H), 5.43 (d, J = 2.6 Hz, 2H), 5.40 - 5.30 (m, 2H), 5.22 (s, 1H), 5.17 (s, 1H), 3.48 - 3.40 (m, 2H), 3.21 - 3.01 (m, 3H), 2.38 (d, J = 1.9 Hz, 3H), 2.23 - 2.06 (m, 2H), 1.96 - 1.80 (m, 2H), 1.76 (s, 2H), 0.90 (t, J = 7.3 Hz, 3H).

[0190] Example 23: Synthesis of Compound 1008 [ka] Intermediate 1. Compound 48 (17 mg, 0.0325 mmol) and FmocGGFG-OAc (3 eq, 0.0976 mmol, 61 mg) were dissolved in 1 mL of anhydrous DMF. HCl (100 μL, 2 M in EtO) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was directly loaded onto a column. The product was purified by reverse-phase flash chromatography using a column containing 25 g of C18 and a gradient of ACN in water (0 to 80% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (15 mg, 44%). C in MS was 0.05%. 58 H 59 FN9O 12 Calculated value: 1092.43, Measured value: 1093.03, [M + H] + .

[0191] Intermediate 2. Intermediate 1 (15 mg, 0.0135 mmol) was dissolved in 1 mL of anhydrous DMF and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto the column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (10 mg, 87%). C in MS was 0.0135 mmol. 43 H 49 FN9O 10 Calculated value: 870.36, Found value: 870.88, [M + H] - .

[0192] Compound 1008. Intermediate 2 (10 mg, 0.0118 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (2 equivalents, 0.0237 mmol, 7 mg) and DIPEA (20 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 μm syringe filter and loaded directly onto a column. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 and a gradient of ACN in water (0 to 100% ACN in water). After lyophilization from water, the desired product was recovered as a white solid (4 mg, 32%). C in MS was 0.01%. 52 H 58 FN 10 O 14 Calculated value: 1065.41, Found value: 1065.79, [M + H] + .

[0193] Example 24: Synthesis of Compound 3007 Compound 3007 was prepared according to Procedure A in the General Methods section of Example 1, using 9 molar equivalents of linker-payload compound 1008 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 4.9, based on a linker-drug molecular weight of 1065 Da.

[0194] Example 25: Synthesis of Compound 3032 Compound 3032 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1008 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.6 based on a linker-drug molecular weight of 1065 Da.

[0195] Example 26: Synthesis of Compound 3054 Compound 3054 was prepared according to Procedure C in the General Methods section of Example 1 using 9 molar equivalents of linker-payload compound 1008 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 4.4 based on a linker-drug molecular weight of 1065 Da.

[0196] Example 27: Synthesis of Compound 52 [ka] Intermediate 1. To a solution of methyl propiolate (1.0 equiv., 55 mg, 0.65 mmol), (2-azidoethoxy)(tert-butyl)dimethylsilane (1.15 equiv., 150 mg, 0.74 mmol), and tris[(1-benzyltriazol-4-yl)methyl]amine (0.15 equiv., 50 mg, 0.094 mmol) in DMF (3 mL) was added 1 M aqueous CuSO4.5HO (0.1 equiv., 0.06 mmol, 60 μL) and 2 M aqueous sodium ascorbate (0.2 equiv., 0.12 mmol, 60 μL), and the resulting mixture was stirred at room temperature for 2 h. DMF was evaporated, the residue was taken up in EtOAc, and the organic phase was washed with water, 0.2 M aqueous HCl, saturated aqueous NH4Cl, and brine, and dried over Na2SO4. Purification by flash chromatography (silica, 0% to 30% EtOAc / cyclohexane) afforded triazole intermediate 1 (158 mg, 85%) as a white solid.

[0197] Intermediate 2. To a solution of triazole intermediate 1 (1.0 equiv., 158 mg, 0.55 mmol) in MeOH (2 mL) was added 2 M aqueous NaOH (1.0 equiv., 0.55 mL), and the resulting mixture was stirred at room temperature overnight. The solvent was then evaporated, and the residue was re-evaporated twice with toluene, suspended in EtOAc, and filtered. The solid material was washed with EtO and dried under vacuum to give triazole intermediate 2 (135 mg, 84%) as a white solid.

[0198] Intermediate 3. To a suspension of exatecan mesylate (15 mg, 0.028 mmol), triazole intermediate 2 (2.1 equiv., 0.06 mmol, 17 mg), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.0 equiv., 0.056 mmol, 11 mg), and 1-hydroxybenzotriazole (2.0 equiv., 0.056 mmol, 8 mg) in DMF (1 mL), diisopropylethylamine (5.0 equiv., 0.14 mmol, 18 mg, 25 μL) was added under an argon atmosphere, and the mixture was stirred at room temperature for 5 h. LC-MS indicated complete consumption of the starting material. Purification of the mixture by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 75% ACN / HO) afforded triazole intermediate 3 (14 mg, 73%) as a white powder after lyophilization.

[0199] Compound 52. Triazole intermediate 3 (14 mg, 0.02 mmol) was dissolved in an ACN / 0.1% TFA aqueous mixture (1:1, 2 ml), followed by the addition of two drops of TFA, and the resulting mixture was stirred at room temperature for 2 hours. LC-MS showed that intermediate 1 was completely consumed. The solvent was evaporated under reduced pressure. The residue was purified by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→50% ACN / HO), and after lyophilization, compound 52 (10 mg, 85%) was obtained as a white powder. C in MS 29 H 28 Calculated for FN6O6: 575.20, Found: 575.25, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 8.63 (s, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.29 (s, 1H), 5.73 (dd, J = 7.7, 5.3 Hz, 1H), 5.36 (s, 2H), 5.14 (d, J = 3.7 Hz, 2H), 4.48 (t, J = 5.3 Hz, 2H), 3.81 (t, J = 5.4 Hz, 2H), 3.33 (s, 2H), 3.32 - 3.21 (m, 1H), 3.19 - 3.07 (m, 1H), 2.38 (d, J = 1.9 Hz, 3H), 2.34 - 2.20 (m, 2H), 1.93 - 1.76 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H).

[0200] Example 28: Synthesis of Compound 1010 [ka] Intermediate 1. A mixture of compound 52 (1.0 equiv., 30 mg, 0.052 mmol) and FmocGGFG-OAc (2.0 equiv., 0.104 mmol, 66 mg) was dissolved in anhydrous DMF (1.5 mL), followed by the addition of 2M HCl / EtO (150 μL). The reaction mixture was stirred at room temperature for 4 hours, during which time FmocGGFG-OAc was added in several portions (approximately 0.5 equiv. each) to the reaction mixture. The reaction mixture was then purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0%-75% ACN / H2O). The product-containing fractions were lyophilized, and the residue (product + coeluting impurities) was used directly in the next step. C in MS was 0.01%. 60 H 59 FN 11 O 12 Calculated value: 1144.43, Found value: 1144.40, [M+H] + .

[0201] Intermediate 2. To a solution of Intermediate 1 (obtained in the previous step) in anhydrous DMF (2 ml), morpholine (140 μL) was added, and the reaction mixture was stirred at room temperature for 1 h. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 60% ACN / HO), and after lyophilization, the product was obtained as a white solid (20 mg, 42%, 2 steps). MS showed C 45 H 49 FN 11 O 10 Calculated value: 922.36, Found value: 922.35, [M+H] + .

[0202] Compound 1010. Mal-PEG-NHS ester (1.0 equiv., 0.022 mmol, 6.7 mg) and DIPEA (2.4 equiv., 0.053 mmol, 7 mg, 9 μL) were added to a solution of intermediate 2 (1.0 equiv., 20 mg, 0.022 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0%-60% ACN / HO) afforded the desired product as a white solid (7.5 mg, 31%) after lyophilization. C in MS was 0.053 mmol. 54 H 58 FN 12 O 14 Calculated value: 1117.42, Found value: 1117.35, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ: 9.28 (t, J = 9.8 Hz, 1H), 8.65 (d, J = 2.1 Hz, 1H), 8.56 (t, J = 6.7 Hz, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.15 - 8.08 (m, 2H), 8.00 (t, J = 5.8 Hz, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.31 (d, J = 3.4 Hz, 1H), 7.28 - 7.21 (m, 5H), 7.20 - 7.15 (m, 1H), 7.00 (s, 1H), 6.51 (s, 1H), 5.78 - 5.71 (m, 1H), 5.41 - 5.33 (m, 2H), 5.25 - 5.11 (m, 2H), 4.63 - 4.60 (m, 2H), 4.58 (d, J = 6.7 Hz, 2H), 4.53 - 4.47 (m, 1H), 3.86 - 3.83 (m, 2H), 3.74 (td, J = 17.3, 5.8 Hz, 2H), 3.67 (d, J = 5.7 Hz, 2H), 3.64 - 3.50 (m, 5H), 3.46 (t, J = 5.7 Hz, 2H), 3.18 - 3.11 (m, 1H), 3.05 (dd, J = 14.0, 4.6 Hz, 1H), 2.84 - 2.75 (m, 1H), 2.40 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.30 - 2.23 (m, 3H), 1.93 - 1.78 (m, 2H), 0.90 - 0.83 (m, 3H).

[0203] Example 29: Synthesis of Compound 3006 Compound 3006 was prepared according to Procedure A in the General Methods section of Example 1, using 40 molar equivalents of linker-payload compound 1010 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.9, based on a linker-drug molecular weight of 1117 Da.

[0204] Example 30: Synthesis of Compound 103 [ka] Compound 103. To a suspension of exatecan mesylate (10 mg, 0.019 mmol), 5-(hydroxymethyl)furan-2-carboxylic acid (3 equiv., 8 mg, 0.057 mmol), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.5 equiv., 10 mg, 0.048 mmol), and 1-hydroxybenzotriazole (2.5 equiv., 7 mg, 0.048 mmol) in DMF (1 mL), diisopropylethylamine (5 equiv., 17 μL, 0.095 mmol) was added under an argon atmosphere, and the mixture was stirred at room temperature for 40 min. LC-MS showed that the starting material was completely consumed. Purification by reversed-phase flash chromatography (25 g, diol-modified C18, 0→70% ACN / HO) followed by another purification on a semi-preparative column (diol-modified C18, 0→70% ACN / HO) afforded the product (7 mg, 66%) as a yellowish powder after lyophilization. 30 H 27 Calculated value for FN3O7: 560.18, Found value: 560.15, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ: 8.90 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 7.17 (d, J = 3.4 Hz, 1H), 6.50 (s, 1H), 6.45 (d, J = 3.4 Hz, 1H), 5.77-5.67 (m, 1H), 5.40 - 5.33 (m, 3H), 5.18 (d, J = 18.9 Hz, 1H), 5.10 (d, J = 19.0 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 3.29 - 3.09 (m, 1H), 2.40 (d, J = 1.9 Hz, 3H), 2.24 (q, J = 6.2 Hz, 2H), 0.94-1.75 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H).

[0205] Example 31: Synthesis of Compound 1012 [ka] Intermediate 1. To a solution of FmocGGFGG-OAc (1.0 equiv., 50 mg, 0.079 mmol) and 5-(hydroxymethyl)furan-2-carboxylic acid (1.2 equiv., 14 mg, 0.095 mmol) in anhydrous DMF (0.5 mL), 2 M HCl / EtO (70 μL) was added, and the resulting mixture was stirred at room temperature for 2 h. The volatiles were removed, and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O) to give Intermediate 1 as a white solid (23 mg, 41%) after lyophilization. MS showed C 37 H 36 FN5O 10 Calculated value: 710.25, Measured value: 710.25, [MH] - .

[0206] Intermediate 2. To a mixture of Intermediate 1 (1.05 equiv., 23 mg, 0.032 mmol), exatecan mesylate (1.0 equiv., 16.3 mg, 0.031 mmol), and DMTMM (1.05 equiv., 8.9 mg, 0.032 mmol) was added DMF / water (5:1, 1.2 mL) and diisopropylethylamine (2.1 equiv., 12 μL, 0.065 mmol). The resulting mixture was stirred at room temperature for 40 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 2 as a white solid (27 mg, 77%) after lyophilization. MS analysis revealed a C 61 H 58 FN8O 13 Calculated value: 1129.41, Found value: 1129.45, [M+H] + .

[0207] Intermediate 3. To a solution of Intermediate 2 (1.0 equiv., 27 mg, 0.024 mmol) in anhydrous DMF (1 ml), morpholine (50 μL) was added, and the reaction mixture was stirred at room temperature for 1.5 h. LC-MS showed complete consumption of the starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O), and after lyophilization, the product was obtained as a white solid (15 mg, 69%). C in MS was 46 H 48 FN8O 11 Calculated value: 907.34, Found value: 907.35, [M+H] + .

[0208] Compound 1012. Mal-PEG-NHS ester (1.0 equiv., 0.017 mmol, 5.2 mg) and DIPEA (1.05 equiv., 0.0173 mmol, 3.05 μL) were added to a solution of intermediate 3 (1.0 equiv., 15 mg, 0.017 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1.5 hours, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→60% ACN / HO) afforded the desired product as a white solid (11 mg, 60%) after lyophilization. C in MS was 0.05 equiv., 0.0173 mmol, 3.05 μL. 55 H 57 FN9O 15 Calculated value: 1102.40, Found value: 1102.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 9.06 - 8.93 (m, 1H), 8.59 (t, J = 6.4 Hz, 1H), 8.31 (t, J = 5.9 Hz, 1H), 8.16 - 8.05 (m, 2H), 8.04 - 7.94 (m, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 7.27 - 7.11 (m, 7H), 6.99 (s, 1H), 6.59 (d, J = 3.4 Hz, 1H), 6.51 (s, 1H), 5.78 - 5.63 (m, 1H), 5.38 (s, 2H), 5.28 - 4.97 (m, 2H), 4.59 (d, J = 7.0 Hz, 2H), 4.53 - 4.37 (m, 3H), 3.84 - 3.63 (m, 5H), 3.63 - 3.48 (m, 5H), 3.45 (t, J = 5.8 Hz, 2H), 3.29 - 3.22 (m, 1H), 3.20 - 3.08 (m, 1H), 3.04 (dd, J = 13.9, 4.6 Hz, 1H), 2.79 (dd, J = 13.9, 9.6 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.6 Hz, 2H), 2.27 - 2.19 (m, 2H), 1.96 - 1.77 (m, 2H), 0.86 (t, J = 7.5 Hz, 3H).

[0209] Example 32: Synthesis of Compound 3008 Compound 3008 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1012 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.7, based on a linker-drug molecular weight of 1102 Da.

[0210] Example 33: Synthesis of Compound 3033 Compound 3033 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1012 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 4.5 based on a linker-drug molecular weight of 1102 Da.

[0211] Example 34: Synthesis of Compound 3055 Compound 3055 was prepared according to Procedure C in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1012 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 7.3 based on a linker-drug molecular weight of 1102 Da.

[0212] Example 35: Synthesis of Compound 105 [ka] Compound 105. To a suspension of exatecan mesylate (30 mg, 0.056 mmol) in DMF (1 mL), diisopropylethylamine (3.5 equiv., 0.196 mmol, 34 μL) and 2-bromoethanol (2 equiv., 0.112 mmol, 14 mg, 8 μL) were added under an argon atmosphere, and the mixture was heated to 80° C. for 2 days. LC-MS showed complete consumption of the starting material. Purification by reversed-phase flash chromatography on a semi-preparative column (diol-modified C18, 1% TFA in 0→50% ACN / HO) afforded the product (16 mg, 48%) as a white powder after lyophilization. C in MS indicated a 50% C+ / C ... 26 H 27 Calculated value for FN3O5: 480.19, Found value: 480.25, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 9.03 (s br, 1H), 8.80 (s br, 1H), 7.88 (d, J = 10.8 Hz, 1H), 7.34 (s, 1H), 6.57 (s, 1H), 5.57 - 5.36 (m, 4H), 5.30 (s, 1H), 5.16 - 5.02 (m, 1H), 3.69 (t, J = 5.5 Hz, 2H), 3.31 - 3.06 (m, 3H), 2.84 - 2.71 (m, 1H), 2.41 (s, 3H), 2.26 - 2.10 (m, 1H), 1.99 - 1.76 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). [ka]

[0213] Example 36: Synthesis of Compound 1013 [ka] Intermediate 1. A mixture of compound 105 (1.0 equiv., 20 mg, 0.034 mmol) and FmocGGFG-OAc (2.0 equiv., 0.068 mmol, 43 mg) was dissolved in anhydrous DMF (1 mL), followed by the addition of 2M HCl / EtO (100 μL). The reaction mixture was stirred at room temperature for 4 hours, during which time FmocGGFG-OAc was added in portions (approximately 0.5 equiv. each) to the reaction mixture. The reaction mixture was then purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / H2O). The product-containing fractions were lyophilized, and the residue (product + coeluting impurities) was used directly in the next step. C in MS was 0.01%. 57 H 58 FN8O 11 Calculated value: 1049.42, Found value: 1049.40, [M+H] + .

[0214] Intermediate 2. To a solution of Intermediate 1 (obtained in the previous step) in anhydrous DMF (2 ml), morpholine (80 μL) was added, and the reaction mixture was stirred at room temperature for 1.5 hours. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O), and after lyophilization, the product was obtained as a white solid (12 mg, 43%). C in MS 42 H 48 Calculated for FN8O9: 827.35, Found: 827.30, [M+H] + .

[0215] Compound 1013. Mal-PEG-NHS ester (1.0 equiv., 0.015 mmol, 4.3 mg) and DIPEA (2.2 equiv., 0.032 mmol, 4.2 mg, 5.7 μL) were added to a solution of intermediate 2 (12 mg, 0.015 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→60% ACN / HO) afforded the desired product as a white solid after lyophilization (6.3 mg, 41%). C in MS 51 H 57 FN9O 13 Calculated value: 1022.41, Measured value: 1022.40, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.65 (t, J = 6.7 Hz, 1H), 8.33 - 8.26 (m, 1H), 8.15 - 8.07 (m, 2H), 8.03 - 7.96 (m, 1H), 7.76 - 7.70 (m, 1H), 7.60 (s, 1H), 7.26 - 7.18 (m, 5H), 7.18 - 7.12 (m, 1H), 6.99 (s, 2H), 5.49 - 5.24 (m, 3H), 4.81 (d, J = 11.8 Hz, 1H), 4.71 - 4.55 (m, 3H), 4.48 (ddd, J = 9.6, 8.1, 4.5 Hz, 1H), 4.28 (t, J = 4.2 Hz, 1H), 3.81 - 3.67 (m, 3H), 3.65 (d, J = 5.7 Hz, 2H), 3.62 - 3.47 (m, 6H), 3.47 - 3.41 (m, 2H), 3.22 - 3.12 (m, 1H), 3.06 - 2.90 (m, 2H), 2.87 - 2.71 (m, 2H), 2.38 - 2.29 (m, 5H), 2.26 - 1.98 (m, 4H), 1.26 - 1.16 (m, 1H), 0.90 - 0.82 (m, 3H).

[0216] Example 37: Synthesis of Compound 3009 Compound 3009 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1013 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.5, based on a linker-drug molecular weight of 1022 Da.

[0217] Example 38: Synthesis of Compound 3034 Compound 3034 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1013 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.2 based on a linker-drug molecular weight of 1022 Da.

[0218] Example 39: Synthesis of Compound 3056 Compound 3056 was prepared according to Procedure C in the General Methods section of Example 1 using 4 molar equivalents of linker-payload compound 1013 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 3.9 based on a linker-drug molecular weight of 1022 Da.

[0219] Example 40: Synthesis of Compound 108 [ka] Diisopropylethylamine (2.5 equiv., 0.07 mmol, 9 mg, 13 μL) and propargyl bromide (2.5 equiv., 0.07 mmol, 8.5 mg, 9 μL 80% toluene solution) were added to a suspension of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol) in DMF (0.2 mL), and the resulting solution was stirred for 48 h. Then, 2-azidoethanol (5.0 equiv., 0.14 mmol, 12 mg, 11 μL), tris(benzyltriazolylmethyl)amine (1.5 equiv., 0.042 mmol, 22 mg), CuSO4.5HO (1.0 equiv., 0.028 mmol, 140 μL of a 2 M aqueous solution), and sodium ascorbate (2.0 equiv., 0.056 mmol, 56 μL of a 1 M aqueous solution) were added sequentially to the reaction mixture, and the solution was stirred overnight. The crude reaction mixture was loaded directly onto a column and purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0%-50% ACN / HO). After lyophilization, compound 108 (12 mg, 77%) was obtained as a white powder. C in MS was 0.056 mmol. 29 H 30 Calculated value for FN6O5: 561.23, Found value: 561.30, [M+H] + .1 H NMR (400 MHz, DMSO-d6) δ: 7.97 (s, 1H), 7.72 (d, J = 11.0 Hz, 1H), 7.29 (s, 1H), 5.43 (s, 2H), 5.28 (d, J = 19.0 Hz, 1H), 5.19 (d, J = 19.0 Hz, 1H), 4.39 (t, J = 5.5 Hz, 2H), 4.25 (t, J = 4.1 Hz, 1H), 3.97 (q, J = 13.9 Hz, 2H), 3.80 - 3.75 (m, 2H), 3.33 (s, 2H), 3.24 (ddd, J = 15.8, 10.4, 4.3 Hz, 1H), 3.01 (dt, J = 16.8, 4.8 Hz, 1H), 2.39 - 2.27 (m, 6H), 2.09 - 1.98 (m, 1H), 1.95 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0220] Example 41: Synthesis of Compound 1015 [ka] Intermediate 1. Compound 108 (1.0 equiv., 30 mg, 0.0536 mmol) and FmocGGFG-OAc (2.0 equiv., 0.107 mmol, 67 mg) were dissolved in anhydrous DMF (1.5 mL), followed by the addition of 2 M HCl / EtO (150 μL). The reaction mixture was stirred at room temperature for 4 hours, during which time FmocGGFG-OAc was added in portions (approximately 0.5 equiv. each) to the reaction mixture. The reaction mixture was then purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / H2O). The product-containing fractions were lyophilized, and the residue (product + coeluting impurities) was used directly in the next step. C in MS was 0.01%. 60 H 61 FN 11 O 11 Calculated value: 1130.45, Found value: 1130.40, [M+H] + .

[0221] Intermediate 2. To a solution of Sc-260-F1 Intermediate 1 (obtained in the previous step) in anhydrous DMF (2 ml), morpholine (100 μL) was added, and the reaction mixture was stirred at room temperature for 1 h. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 → 50% ACN / H2O), and after lyophilization, the product was obtained as a white solid (14 mg, 29% over two steps). MS showed that C 45 H 51 FN 11 Calculated for O9: 908.39, Found: 908.55, [M+H] + .

[0222] Compound 1015. Mal-PEG-NHS ester (1 equiv., 0.0154 mmol, 5 mg) and DIPEA (2.5 equiv., 0.039 mmol, 5 mg, 6.6 μL) were added to a solution of Sc-260-F1 intermediate 2 (1 equiv., 14 mg, 0.0154 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 30 minutes, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→100% CAN in HO) afforded the desired product as a white solid (7 mg, 41%) after lyophilization from water-acetonitrile. C in MS was 0.0154 mmol. 54 H 60 FN 12 O 13 Calculated value: 1103.44, Found value: 1103.61, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.54 (m, 1H), 8.30 (m, 1H), 8.12 (m, 2H), 8.01 (m, 1H), 7.97 (s, 1H), 7.73 (m, 1H), 7.65 (s, 1H), 7.29 (s, 1H), 7.25 - 7.17 (m, 4H), 7.17 - 7.12 (m, 1H), 7.01 (d, J = 5.9 Hz, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.43 (s, 1H), 5.34 - 5.06 (m, 2H), 4.55 (m, 2H), 4.51 (m, 1H), 4.49 - 4.43 (m, 1H), 4.25 (d, J = 17.1 Hz, 1H), 4.10 - 3.88 (m, 2H), 3.80 (q, J = 4.9 Hz, 2H), 3.76 - 3.63 (m, 5H), 3.45 (t, J = 5.9 Hz, 2H), 3.22 (d, J = 10.9 Hz, 1H), 3.04 - 2.98 (m, 2H), 2.87 (t, J = 6.0 Hz, 1H), 2.82 - 2.73 (m, 2H), 2.59 (s, 1H), 2.36 (m, 4H), 2.32 (m, 2H), 2.06 - 1.97 (m, 2H), 1.86 (m 1H), 1.29 - 1.21 (m, 2H), 0.86 (m, 3H).

[0223] Example 42: Synthesis of Compound 3010 Compound 3010 was prepared according to Procedure A in the General Methods section of Example 1, using 15 molar equivalents of linker-payload compound 1015 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.6, based on a linker-drug molecular weight of 1103 Da.

[0224] Example 43: Synthesis of Compound 3035 Compound 3035 was prepared according to Procedure B in the General Methods section of Example 1 using 15 molar equivalents of linker-payload compound 1015 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.3 based on a linker-drug molecular weight of 1103 Da.

[0225] Example 44: Synthesis of Compound 3057 Compound 3057 was prepared according to Procedure C in the General Methods section of Example 1 using 15 molar equivalents of linker-payload compound 1015 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 6.0 based on a linker-drug molecular weight of 1103 Da.

[0226] Example 45: Synthesis of Compound 111 [ka] Compound 111. Methyl 2-(hydroxymethyl)cyclopropane-1-carboxylate (25 mg, 0.175 mmol) was dissolved in 1 mL of methanol, and 870 μL of 1 M NaOH (1 equivalent) was added. The mixture was stirred at room temperature for 5 hours, then the solvent was evaporated, and the crude product was lyophilized from water. To the resulting solid, exatecan mesylate (46 mg, 0.5 equivalents, 0.874 mmol), DMTMM (48 mg, 1 equivalent, 0.175 mmol), and 10 mL of a 4:1 DMF / water mixture were added. The mixture was stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure to a final volume of approximately 2 mL. The crude reaction mixture was purified by reverse-phase flash chromatography using a column containing 25 g of diol-modified C18 and a gradient of ACN in water (0 to 60% ACN in HO). A second purification was performed using a semi-preparative column loaded with diol-modified C18 using a gradient of ACN in water (0 to 80% ACN in HO). Two isomers were separated during the semi-preparative purification. After lyophilization from water / dioxane, the products were recovered separately as white powders (42 mg total, 91%, calculated from exatecan). C in MS 29 H29 Calculated value for FN3O6: 534.20, Found value: 534.10, [M+H] + .

[0227] For Isomer A: 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.56 (q, J = 6.6 Hz, 1H), 5.42 (s, 2H), 5.16 (d, J = 2.9 Hz, 2H), 4.63 (t, J = 5.5 Hz, 1H), 3.48 - 3.38 (m, 1H), 3.31 - 3.25 (m, 2H), 3.21 - 3.08 (m, 1H), 2,30 (m, 2H), 2.24 - 2.07 (m, 2H), 1.96 - 1.77 (m, 2H), 1.58 (d, J = 4.4 Hz, 1H), 1.49 (m, 1H), 0.99 (dt, J = 8.4, 4.3 Hz, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.71 (m, 1H).

[0228] For Isomer B (containing 7% Isomer A based on NMR integral): 1H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.56 (m, 2H), 5.44 (s, 2H), 5.31 - 5.05 (m, 2H), 4.52 (dd, J = 6.1, 5.0 Hz, 1H), 3.45 (m 1H), 3.26 (m, 1H), 3.20 - 3.08 (m, 1H), 2.38 (m, 2H), 2.24 - 2.00 (m, 2H), 1.87 (m, 2H), 1.76 (s, 1H), 1.60 - 1.48 (m, 1H), 1.01 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.75 (m, 1H).

[0229] Example 46: Synthesis of Compound 1016 [ka] Intermediate 1. 2-((benzyloxy)methyl)cyclopropane-1-carboxylic acid (27.6 mg, 0.1338 mmol) was dissolved in 3 mL of anhydrous dioxane. Pd / C (10%) was added, and hydrogen was bubbled through the solution for 5 h while stirring at room temperature. The solution was filtered through a 0.2 μm syringe filter, and the flask was washed with acetonitrile. The filtrate was evaporated, redissolved in dioxane, and lyophilized overnight. The crude filtrate was redissolved in 2 mL of anhydrous DMF, and FmocGGFG-OAc (1 equiv., 0.1338 mmol, 90 mg) was added, followed by 200 μL of 2 M HCl solution in ethyl ether. The reaction mixture was stirred at room temperature for 1 h and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0 to 75% ACN / HO). The product-containing fractions were lyophilized from water (35 mg, 51%). C in MS 36 H 40 Calculated for N5O9: 686.28, Found: 686.66, [M+H] + .

[0230] Intermediate 2. To a solution of the previously prepared intermediate 1 (35 mg, 0.0505 mmol) in DMF (2 mL), exatecan mesylate (1 equiv., 0.0505 mmol, 27 mg), DMTMM (1.2 equiv., 0.0607 mmol, 17 mg), DIPEA (10 μL), and water (200 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0 to 60% ACN / H2O). The product-containing fractions were lyophilized from water (28 mg, 50%). C in MS was 0.01%. 60 H 60 FN8O 12 Calculated value: 1103.43, Found value: 1103.88, [M+H] + .

[0231] Intermediate 3. The previously prepared intermediate 2 (28 mg, 0.0254 mmol) was dissolved in DMF (2 mL) and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0 to 100% ACN / H2O). The product-containing fractions were lyophilized from water (11 mg, 51%). C in MS was obtained. 45 H 50 FN8O 10 Calculated value: 881.36, Found value: 881.12, [M+H] + .

[0232] Compound 1016. The previously prepared intermediate 3 (11 mg, 0.0130 mmol) was dissolved in 2 mL of anhydrous DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 0.0143 mmol, 4 mg) and DIPEA (1.1 equiv., 0.0143 mmol, 2.5 μL) were added, and the reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0 to 100% ACN / H2O). The product-containing fractions were lyophilized from water (7 mg, 50%). C in MS was 0.0143 mmol.54 H 59 FN9O 14 Calculated value: 1076.42, Found value: 1076.56, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.80 - 8.70 (m, 2H), 8.53 (m, 1H), 8.45 (m, 1H), 8.27 (m, 1H), 8.10 (m, 2H), 7.99 (m, 2H), 7.93 (d, J = 6.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.80 (m, 2H), 7.34 - 7.20 (m, 2H), 7.18 - 7.08 (m, 2H), 6.67 (s, 2H), 6.51 (s, 1H), 5.62 (d, J = 10.0 Hz, 1H), 5.55 (s, 2H), 5.43 (t, J = 5.7 Hz, 2H), 5.19 - 5.07 (m, 3H), 5.06 - 5.02 (m, 1H), 4.74 (d, J = 6.0 Hz, 1H), 4.61 - 4.44 (m, 3H), 3.72 (m, 2H), 3.57 (m, 2H), 3.16 (d, J = 8.7 Hz, 1H), 3.08 - 3.00 (m, 1H), 2.79 (m, 2H), 2.39 (m, 3H), 2.34 (d, J = 1.8 Hz, 3H), 2.24 - 2.07 (m, 2H), 1.96 - 1.77 (m, 2H), 1.55 (m, 1H), 1.51 - 1.44 (m, 1H), 0.99 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.83 - 0.71 (m, 1H).

[0233] Example 47: Synthesis of Compound 3012 Compound 3012 was prepared according to Procedure A in the General Methods section of Example 1, using 6 molar equivalents of linker-payload compound 1016 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-HER2 antibody-drug conjugate was 2, based on a linker-drug molecular weight of 1076 Da.

[0234] Example 48: Synthesis of Compound 3037 Compound 3037 was prepared according to Procedure B in the General Methods section of Example 1 using 6 molar equivalents of linker-payload compound 1016 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC was 1.45 based on a linker-drug molecular weight of 1076 Da.

[0235] Example 49: Synthesis of Compound 1017 [ka] Intermediate 1. To a solution of (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylic acid (1.0 equiv., 5.7 mg, 0.049 mmol), Fmoc-GE(OBn)VCit-NH-CH2-OAc (1.5 equiv., 62 mg, 0.074 mmol) in anhydrous DMF (0.7 ml), 2 M HCl / Et2O (100 μl) was added, and the resulting mixture was stirred at room temperature for 1 hour. LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→75% ACN / 0.1% HCl). The product-containing fractions (coeluting with an impurity) were lyophilized to give 31 mg of impure Intermediate 1, which was used directly in the next step. C in MS 46 H 56 FN7O 12 Calculated value: 898.40, Measured value: 898.40, [MH] - .

[0236] Intermediate 2. To a mixture of Intermediate 1 (1.0 equiv., 31 mg, 0.034 mmol), exatecan mesylate (0.9 equiv., 17 mg, 0.031 mmol), and DMTMM (1.0 equiv., 10 mg, 0.034 mmol), DMF / water (5:1, 1.2 mL) and diisopropylethylamine (2.0 equiv., 12 μL, 0.068 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 2 as a white solid (25 mg, 39% (2 steps)) after lyophilization. C in MS was 0.05%. 70 H 78 FN 10 O 15 Calculated value: 1317.56, Found value: 1317.55, [M+H] + .

[0237] Intermediate 3. To a solution of Intermediate 2 (1.0 equiv., 25 mg, 0.019 mmol) in a mixture of dioxane (1.0 ml) and DMF (0.5 ml), 10% Pd / C (5 mg) was added, and the reaction mixture was hydrogenated (balloon) at room temperature for 2 hours. LC-MS showed complete consumption of the starting material. The mixture was filtered through a pad of Celite, and the filtrate was concentrated on a rotary evaporator to remove dioxane. Morpholine (40 μl) was then added to the resulting solution, and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O) afforded the product Intermediate 3 as a white solid (8 mg, 42%) after lyophilization. C in MS was 0.05%. 48 H 62 FN 10 O 13 Calculated value: 1005.43, Found value: 1005.40, [M+H] + .

[0238] Compound 1017.Mal-PEG-NHS ester (1.0 equiv., 0.008 mmol, 2.5 mg) and DIPEA (1.05 equiv., 0.008 mmol, 1.5 μL) were added to a solution of intermediate 3 (1.0 equiv., 8 mg, 0.008 mmol) in anhydrous DMF (0.5 ml). The reaction mixture was stirred at room temperature for 1.5 hours, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / 0.1% TFA) afforded the desired product as a pale yellow solid (5 mg, 52%) after lyophilization. C in MS was 0.05 equiv., 0.008 mmol, 1.5 μL. 57 H 71 FN 11 O 17 Calculated value: 1200.50, Found value: 1200.50, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 12.09 (s br, 1H), 8.71 (d, J = 8.7 Hz, 1H), 8.58 - 8.49 (m, 1H), 8.05 (t, J = 5.2 Hz, 1H), 8.01 - 7.92 (m, 2H), 7.83 - 7.70 (m, 2H), 7.31 (s, 1H), 7.00 (s, 2H), 6.58 (s br, 1H), 5.92 (s, 1H), 5.62 - 5.49 (m, 1H), 5.50 - 5.33 (m, 2H), 5.28 - 5.12 (m, 2H), 4.60 - 4.40 (m, 2H), 4.39 - 4.25 (m, 1H), 4.21 - 4.02 (m, 2H), 3.77 - 3.62 (m, 3H), 3.31 - 3.19 (m, 2H), 3.20 - 3.09 (m, 1H), 3.04 - 2.82 (m, 2H), 2.40 (s, 3H), 2.35 - 2.26 (m, 2H), 2.27 - 2.10 (m, 4H), 1.99 - 1.79 (m, 4H), 1.76 - 1.64 (m, 1H), 1.63 - 1.41 (m, 4H), 1.40 - 1.23 (m, 3H), 1.05 - 0.93 (m, 1H), 0.92 - 0.84 (m, 3H), 0.84 - 0.75 (m, 6H), 0.75 - 0.69 (m, 2H).

[0239] Example 50: Synthesis of Compound 3013 Compound 3013 was prepared according to Procedure A in the General Methods section of Example 1, using 37 molar equivalents of linker-payload compound 1017 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.6, based on a linker-drug molecular weight of 1200 Da.

[0240] Example 51: Synthesis of Compound 3038 Compound 3038 was prepared according to Procedure B in the General Methods section of Example 1 using 37 molar equivalents of linker-payload compound 1017 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.6 based on a linker-drug molecular weight of 1200 Da.

[0241] Example 52: Synthesis of Compound 3059 Compound 3059A was prepared according to Procedure C in the General Methods section of Example 1, using 37 molar equivalents of linker-payload compound 1017 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 6.4, based on a linker-drug molecular weight of 1200 Da. The molar ratio of mAb to linker-payload was adjusted to achieve a DAR of 7.7 (compound 3059B).

[0242] Example 53: Synthesis of Compound 1025 [ka] Intermediate 1. FmocGGFG-OAc (1 equiv., 0.0892 mmol, 60 mg) was dissolved in 2 mL of DMF and (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylic acid (2 equiv., 0.1784 mmol, 21 mg) was added, followed by 10 μL of 4 M HCl in dioxane. The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / HO). The product-containing fractions were lyophilized from water (43 mg, 70%). C in MS was 0.01%. 36 H 40 Calculated for N5O9: 686.28, Found: 686.34, [M+H] + .

[0243] Intermediate 2. To a solution of the previously prepared intermediate 1 (43 mg, 0.0628 mmol) in DMF (2 mL), exatecan mesylate (1.1 equiv., 0.0691 mmol, 37 mg), DMTMM (1.2 equiv., 0.0754 mmol, 21 mg), DIPEA (20 μL), and water (400 μL) were added. The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / 0 / 1% TFA). The product-containing fractions were lyophilized from water (41 mg, 59%). C in MS was 0.01%. 60 H 60 FN8O 12 Calculated value: 1103.43, Found value: 1103.40, [M+H] + .

[0244] Intermediate 3. The previously prepared intermediate 2 (41 mg, 0.0372 mmol) was dissolved in DMF (2 mL) and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→100% ACN / 0.1% TFA). The product-containing fractions were lyophilized from water (14 mg, 44%). C in MS was 45 H 50 FN8O 10 Calculated value: 881.36, Found value: 881.65, [M+H] + .

[0245] Compound 1025. The previously prepared intermediate 3 (14 mg, 0.0160 mmol) was dissolved in 2 mL of anhydrous DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 0.0175 mmol, 5.4 mg) and DIPEA (2.5 μL) were added, and the reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→100% ACN / 0.1% TFA). The product-containing fractions were lyophilized from water (7 mg, 41%). C in MS was 0.0175 mmol. 54 H59 FN9O 14 Calculated value: 1076.42, Measured value: 1076.42, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.73 (t, J = 9.3 Hz, 1H), 8.42 (t, J = 6.6 Hz, 1H), 8.25 (t, J = 5.8 Hz, 1H), 8.10 (q, J = 7.5, 6.4 Hz, 2H), 7.99 (q, J = 6.7, 5.8 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.29 - 7.19 (m, 4H), 7.19 - 7.12 (m, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.56 (m, 1H), 5.49 - 5.38 (m, 2H), 5.19 (s, 2H), 5.16 (d, J = 4.7 Hz, 1H), 4.56 (d, J = 6.7 Hz, 1H), 4.52 (d, J = 6.7 Hz, 1H), 4.48 (m, 1H), 3.78 - 3.69 (m, 2H), 3.67 (d, J = 5.7 Hz, 2H), 3.57 (d, J = 5.8 Hz, 3H), 3.53 (t, J = 6.6 Hz, 2H), 3.46 (t, J = 5.8 Hz, 3H), 3.26 (m, 1H), 3.14 (m, 1H), 3.02 (m, 1H), 2.80 - 2.75 (m, 1H), 2.39 (d, J = 3.9 Hz, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.14 (m, 2H), 1.86 (m, 2H), 1.61 - 1.49 (m, 2H), 1.03 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.77 (p, J = 4.1 Hz, 1H).

[0246] Example 54: Synthesis of Compound 3011 Compound 3011 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1025 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 5.4, based on a linker-drug molecular weight of 1076 Da.

[0247] Example 55: Synthesis of Compound 3036 Compound 3036 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1025 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 6.4, based on a linker-drug molecular weight of 1076 Da.

[0248] Example 56: Synthesis of compounds 3058A and 3058B Compound 3058A was prepared according to Procedure C in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1025 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 7.1, based on a linker-drug molecular weight of 1076 Da. The molar ratio of mAb to linker-payload was adjusted to achieve a DAR of 3.1 (compound 3058B).

[0249] Example 57: Synthesis of Compound 130 [ka] Intermediate 1. To a mixture of exatecan mesylate (1 equiv., 40 mg, 0.075 mmol), 2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (1.2 equiv., 18 mg, 0.090 mmol), and DMTMM (1.2 equiv., 25 mg, 0.090 mmol), DMF / water (5:1, 2 mL) and diisopropylethylamine (2.2 equiv., 0.165 mmol, 29 μL) were added, and the resulting mixture was stirred at room temperature for 0.5 h. LC-MS indicated complete consumption of the starting material. Purification of the mixture by reverse-phase flash chromatography (diol-modified C18, 0→100% ACN / HO) afforded Intermediate 1 (43 mg, 94%) as a white powder after lyophilization.

[0250] Compound 130. Intermediate 1 (43 mg, 0.070 mmol) was dissolved in 4 M HCl / dioxane (2 ml), and the mixture was stirred at room temperature for 1.5 hours. The resulting suspension was filtered, and the solid was washed with dioxane and Et2O to give the hydrochloride salt of compound 130 (35 mg, 90%) as a yellow powder. 26 H 26 Calculated for FN4O6: 509.18, Found: 509.20, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ: 10.98 (s br, 3H), 8.89 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.62 (dt, J = 8.5, 4.2 Hz, 1H), 5.43 (s, 2H), 5.37 - 5.24 (m, 2H), 4.63 - 4.52 (m, 2H), 3.20 (dd, J = 7.9, 4.7 Hz, 2H), 2.41 (d, J = 1.9 Hz, 3H), 2.32 - 2.21 (m, 1H), 2.21 - 2.09 (m, 1H), 1.96 - 1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0251] Example 58: Synthesis of Compound 1018 [ka] Intermediate 1. To a mixture of compound 130 (1.0 equiv., 30 mg, 0.055 mmol), FmocGGFGGG-OH (1.25 equiv., 47 mg, 0.069 mmol), and DMTMM (1.25 equiv., 19 mg, 0.069 mmol), DMF / water (5:1, 2.4 mL) and diisopropylethylamine (2.25 equiv., 22 μL, 0.124 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 1 as a white solid (48 mg, 75%) after lyophilization. MS analysis revealed a C 60 H 60 FN 10 O 14 Calculated value: 1163.43, Found value: 1163.40, [M+H] + .

[0252] Intermediate 2. To a solution of Intermediate 1 (1.0 equiv., 48 mg, 0.041 mmol) in anhydrous DMF (1.5 ml), morpholine (100 μL) was added, and the reaction mixture was stirred at room temperature for 1 h. LC-MS showed complete consumption of the starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O), and after lyophilization, the product was obtained as a white solid (26 mg, 67%). C in MS was 45 H 50 FN 10 O 12 Calculated value: 941.36, Found value: 941.40, [M+H] + .

[0253] Compound 1018. Mal-PEG-NHS ester (1.0 equiv., 0.027 mmol, 8.4 mg) and DIPEA (1.1 equiv., 0.030 mmol, 5.2 μL) were added to a solution of intermediate 2 (1.0 equiv., 26 mg, 0.027 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, and LC-MS showed complete consumption of the starting material. DMF was removed, and the residue was concentrated from a mixture of 0.1% aqueous TFA and ACN. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→60% ACN / HO) afforded the desired product as a white solid (17 mg, 55%) after lyophilization. C in MS was 0.027 mmol. 54 H 59 FN 11 O 16 Calculated value: 1136.41, Found value: 1136.45, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ: 11.45 (s, 1H), 8.96 - 8.68 (m, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.22 - 8.04 (m, 3H), 8.04 - 7.93 (m, 2H), 7.87 - 7.75 (m, 1H), 7.31 (s, 1H), 7.23 (d, J = 6.9 Hz, 4H), 7.19 - 7.12 (m, 1H), 6.99 (s, 1H), 6.59 - 6.44 (m, 1H), 5.64 - 5.54 (m, 1H), 5.42 (s, 2H), 5.35 - 5.10 (m, 2H), 4.50 (td, J = 9.8, 9.1, 4.6 Hz, 1H), 4.45 - 4.28 (m, 2H), 3.79 - 3.39 (m, 17H), 3.25 - 3.10 (m, 2H), 3.09 - 2.99 (m, 1H), 2.88 - 2.72 (m, 1H), 2.39 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.27 - 2.02 (m, 2H), 1.96 - 1.78 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H).

[0254] Example 59: Synthesis of Compound 3015 Compound 3015 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1018 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 5, based on a linker-drug molecular weight of 1136 Da.

[0255] Example 60: Synthesis of Compound 3040 Compound 3040 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1018 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 4.8 based on a linker-drug molecular weight of 1136 Da.

[0256] Example 61: Synthesis of Compound 3061 Compound 3061 was prepared according to Procedure C in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1018 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 6.3 based on a linker-drug molecular weight of 1136 Da.

[0257] Example 62: Synthesis of Compound 1019 [ka] Intermediate 1. To a mixture of compound 130 (1.0 equiv., 35 mg, 0.064 mmol), FmocGGFGGP-OH (1.2 equiv., 55 mg, 0.077 mmol), and DMTMM (1.2 equiv., 22 mg, 0.077 mmol) was added DMF / water (5:1, 2.4 mL) and diisopropylethylamine (2.2 equiv., 25 μL, 0.141 mmol). The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 1 as a white solid (65 mg, 84%) after lyophilization. MS analysis revealed a C 63 H 64 FN 10 O 14 Calculated value: 1203.46, Found value: 1203.50, [M+H] + .

[0258] Intermediate 2. To a solution of Intermediate 1 (1.0 equiv., 65 mg, 0.054 mmol) in anhydrous DMF (1.7 ml), morpholine (130 μL) was added, and the reaction mixture was stirred at room temperature for 1 hour. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O), and after lyophilization, the product was obtained as a white solid (34 mg, 64%). C in MS 48 H 54 FN 10 O 12 Calculated value: 981.39, Found value: 981.40, [M+H] + .

[0259] Compound 1019. Mal-PEG-NHS ester (1.0 equiv., 0.035 mmol, 10.8 mg) and DIPEA (1.05 equiv., 0.037 mmol, 6.4 μL) were added to a solution of intermediate 2 (1.0 equiv., 34 mg, 0.035 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 hour, and LC-MS showed complete consumption of the starting material. DMF was removed, and the residue was concentrated from a mixture of 0.1% aqueous TFA and ACN. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→60% ACN / HO) afforded the desired product as a white solid (18 mg, 44%) after lyophilization. C in MS was 0.05 equiv. 57 H 63 FN 11 O 16 Calculated value: 1176.44, Found value: 1176.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 11.50 (s, 1H), 8.85 (d, J = 8.7 Hz, 1H), 8.31 - 8.25 (m, 1H), 8.16 - 8.02 (m, 3H), 7.96 (t, J = 5.7 Hz, 1H), 7.88 - 7.77 (m, 2H), 7.74 (t, J = 5.2 Hz, 1H), 7.35 - 7.28 (m, 2H), 7.28 - 7.18 (m, 5H), 7.18 - 7.11 (m, 1H), 6.99 (s, 1H), 6.57 - 6.46 (m, 1H), 5.70 - 5.58 (m, 1H), 5.43 (s, 2H), 5.35 - 5.11 (m, 3H), 4.54 - 4.46 (m, 1H), 4.32 (s, 2H), 4.08 - 4.03 (m, 1H), 3.90 - 3.39 (m, 14H), 3.24 - 3.10 (m, 2H), 3.03 (dd, J = 13.9, 4.4 Hz, 1H), 2.77 (dd, J = 13.9, 9.8 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.26 - 2.11 (m, 2H), 1.97 - 1.77 (m, 3H), 1.69 - 1.51 (m, 1H), 0.86 (t, J = 7.4 Hz, 3H).

[0260] Example 63: Synthesis of Compound 3014 Compound 3014 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1019 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 5.2, based on a linker-drug molecular weight of 1176 Da.

[0261] Example 64: Synthesis of Compound 3039 Compound 3039 was prepared according to Procedure B in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1019 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 7.5 based on a linker-drug molecular weight of 1176 Da.

[0262] Example 65: Synthesis of Compound 3060 Compound 3060 was prepared according to Procedure C in the General Methods section of Example 1 using 20 molar equivalents of linker-payload compound 1019 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC-MS was 7.8 based on a linker-drug molecular weight of 1176 Da.

[0263] Example 66: Synthesis of Compound 177 [ka] Intermediate 1. To a solution of compound 111-B (1.0 equiv., 20 mg, 0.036 mmol) and imidazole (2.5 equiv., 0.094 mmol, 6.4 mg) in DMF (2 mL), tert-butyl(chloro)diphenylsilane (2 equiv., 0.075 mmol, 19.5 μL) was added under argon atmosphere, and the mixture was stirred at room temperature overnight. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0→80% ACN / HO) afforded the product Intermediate 1 (22 mg, 79%) as an off-white powder after lyophilization.

[0264] Intermediate 2. To a solution of Intermediate 1 (22 mg, 0.029 mmol) in anhydrous pyridine (2 mL) was added tert-butyldimethylsilyl trifluoromethanesulfonate (0.436 mmol, 100 μL) under an argon atmosphere, and the mixture was heated to 80 °C for 4 h. It was then concentrated on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 → 100% ACN / HO) to give the product Intermediate 2 (23 mg, 89%) as a white powder after lyophilization.

[0265] Compound 177. To a mixture of intermediate 2 (23 mg, 0.026 mmol) and Lawesson's reagent (1.0 equiv., 0.026 mmol, 10.5 mg), anhydrous toluene (8 ml) was added, and the resulting mixture was heated to 100° C. for 1 hour. The reaction progress was monitored by LC-MS. The volatiles were then removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / H2O). The fractions containing intermediate 3 were combined and concentrated on a rotary evaporator. The residue was dissolved in DCM (2 ml) and trifluoroacetic acid (1 ml). The resulting mixture was stirred at room temperature for 48 hours, and the reaction progress was monitored by LC-MS. The solvent was then removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / HO). After lyophilization, the product, compound 177 (3.3 mg, 23%), was obtained as a yellow powder. 29 H 29 Calculated value for FN3O5S: 550.17, Found value: 550.15, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 10.63 (dd, J = 30.4, 8.6 Hz, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.32 (d, J = 9.4 Hz, 1H), 6.66 (s, 1H), 6.40 (dq, J = 14.0, 7.1 Hz, 1H), 5.42 (d, J = 5.7 Hz, 2H), 5.20 - 4.98 (m, 2H), 3.45 (dd, J = 11.5, 5.8 Hz, 1H), 3.35 (dd, J = 11.5, 6.2 Hz, 1H), 3.28 - 3.09 (m, 2H), 2.40 (s, 3H), 2.33 - 2.18 (m, 2H), 2.16 - 2.03 (m, 1H), 1.96 - 1.78 (m, 3H), 1.45 - 1.32 (m, 1H), 1.04 - 0.91 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H).

[0266] Example 67: Synthesis of Compound 1034 [ka] Intermediate 1. To a solution of compound 177 (1.0 equiv., 12 mg, 0.021 mmol) and FmocGGFG-NH-CH2-OAc (1.5 equiv., 21 mg, 0.032 mmol) in anhydrous DMF (1.0 ml), 2 M HCl / Et2O (30 μl) was added, and the resulting mixture was stirred at room temperature for 5 hours, during which time FmocGGFG-NH-CH2-OAc (0.5 equiv., 6 mg, 0.011 mmol) was added three additional times. The reaction mixture was then purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→80% ACN / 0.1% TFA in water), affording Intermediate 1 as an off-white solid (11 mg, 47%) after lyophilization. C in MS 60 H 60 FN8O 11 S calculated: 1119.41, Found: 1119.40, [M+H] + .

[0267] Intermediate 2. To a solution of Intermediate 1 (1.0 equiv., 11 mg, 0.010 mmol) in DMF (0.75 ml), morpholine (40 μl) was added and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 0.1% TFA in water) to give Intermediate 2 (4.5 mg, 45%) as a pale yellow solid. C in MS 45 H 50 Calculated value for FN8O9S: 897.34, Found: 897.35, [M+H] + .

[0268] Compound 1034. Mal-PEG-NHS ester (1.05 equiv., 1.45 mg, 0.0047 mmol) and DIPEA (2.1 equiv., 2.0 μl, 0.0095 mmol) were added to a solution of intermediate 2 (1.0 equiv., 4.5 mg, 0.0045 mmol) in anhydrous DMF (0.5 ml), and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / HO) afforded the product compound 1034 (4.2 mg, 85%) as a white solid after lyophilization. C in MS 54 H 59 FN9O 13 S calculated: 1092.39, Found: 1092.35, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 10.66 (d, J = 8.7 Hz, 1H), 8.48 (t, J = 6.7 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.14 - 8.06 (m, 2H), 8.00 (t, J = 5.9 Hz, 1H), 7.83 (d, J = 10.9 Hz, 1H), 7.35 - 7.30 (m, 1H), 7.28 - 7.12 (m, 5H), 7.00 (s, 2H), 6.52 (s, 1H), 6.45 - 6.34 (m, 1H), 5.42 (s, 1H), 5.21 - 5.02 (m, 3H), 4.58 - 4.52 (m, 2H), 4.52 - 4.44 (m, 1H), 3.78 - 3.63 (m, 5H), 3.62 - 3.50 (m, 6H), 3.46 (t, J = 5.8 Hz, 2H), 3.22 - 3.17 (m, 2H), 3.09 - 2.99 (m, 1H), 2.84 - 2.74 (m, 1H), 2.41 (s, 3H), 2.36 - 2.30 (m, 2H), 2.30 - 2.22 (m, 2H), 2.13 - 2.06 (m, 1H), 1.95 - 1.79 (m, 3H), 1.47 - 1.37 (m, 1H), 1.05 - 0.94 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H).

[0269] Example 68: Synthesis of Compound 3100 Compound 3100 was prepared according to Procedure A in the General Methods section of Example 1, using 6 molar equivalents of linker-payload compound 1034 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-HER2 antibody-drug conjugate was 3.6, based on a linker-drug molecular weight of 1092 Da.

[0270] Example 69: Synthesis of Compound 3101 Compound 3101 was prepared according to Procedure B in the General Methods section of Example 1 using 6 molar equivalents of linker-payload compound 1034 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC-MS was 3.7 based on a linker-drug molecular weight of 1092 Da.

[0271] Example 70: Synthesis of Compound 180 [ka] Intermediate 1. Exatecan mesylate (90 mg, 0.17 mmol), N-1-Fmoc-(2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (90 mg, 0.26 mmol), and DIPEA (100 μL) were dissolved in DMF (3 mL) and water (1 mL). DMTMM (70 mg, 0.26 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→50% ACN / HO (TFA)). The product-containing fractions were lyophilized from water (107 mg, 82%). C in MS was 0.01%. 44 H 40 FN4O8 + Calculated value: 771.28, Found value: 771.35 [M+H] + .

[0272] Compound 180. To a solution of intermediate 1 (33 mg, 0.0428 mmol) in DMF (2 ml) was added morpholine (100 μl), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 0.1% TFA), and after lyophilization, compound 180 (15 mg, 57%) was obtained as a white powder. 29 H 30 Calculated value for FN4O6: 549.21, Found value: 549.48 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 9.26 (d, J = 8.6 Hz, 1H), 8.81 (s, 1H), 7.83 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.67 (s, 1H), 5.61 (m, 1H), 5.43 (s, 2H), 5.30 (d, J = 18.7 Hz, 1H), 5.08 (d, J = 18.7 Hz, 1H), 4.42 (d, J = 4.7 Hz, 1H), 4.05 (s, 1H), 3.45 (s, 2H), 3.22 (d, J = 7.6 Hz, 2H), 2.42 (d, J = 1.8 Hz, 3H), 2.29 - 2.15 (m, 1H), 2.10 - 1.97 (m, 1H), 1.87 (m, 3H), 0.89 (t, J = 7.3 Hz, 3H).

[0273] Example 71: Synthesis of Compound 193 [ka] Compound 193. Compound 180 (10 mg, 0.018 mmol) and DIPEA (5 μL) were dissolved in DCM (2 mL). AcO (2.6 μL, 0.027) was added and the reaction was stirred at room temperature overnight. The mixture was evaporated and the residue was purified by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→50% ACN / H2O). The product-containing fractions were lyophilized from water (4.9 mg, 46%). C in MS was 0.018 mg. 31 H 32 FN4O7 + Calculated value: 591.22, Found value: 591.25 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.5 Hz, 1H), 7.81 (t, J = 10.4 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 6.52 (d, J = 5.4 Hz, 1H), 5.51 (m, 2H), 5.44 (m, 3H), 5.30 (d, J = 18.7 Hz, 1H), 5.20 (s, 2H), 4.25 (s, 1H), 4.17 (s, 1H), 3.58 (m, 2H), 3.17 (d, J = 8.1 Hz, 3H), 2.41 (m, 3H), 2.17 (s, 1H), 2.09 (d, J = 12.9 Hz, 1H), 1.88 (m, 3H), 1.76 (s, 1H), 0.90 (q, J = 7.3, 6.7 Hz, 3H).

[0274] Example 72: Synthesis of Compound 194 [ka] Compound 194. Formic acetic anhydride (2.2 equiv., 0.048 mg, 4.2 μL) and diisopropylethylamine (2.5 equiv., 0.055 mmol, 10 μL) were added to a solution of compound 180 (1.0 equiv., 15 mg, 0.027 mmol) in DCM (2 mL), and the reaction was stirred overnight at room temperature. The mixture was then evaporated, and the residue was purified by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→50% ACN / H2O). The product-containing fractions were lyophilized from water (5.5 mg, 46%). C in MS was 0.055 mmol. 30 H 30 FN4O7 + Calculated value: 577.20, Found value: 577.25 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J = 8.6 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.20 - 8.12 (m, 1H), 7.80 (m, 1H), 7.32 (d, J = 0.9 Hz, 2H), 6.67 (s, 1H), 5.62 - 5.50 (m, 1H), 5.22 (d, J = 7.9 Hz, 2H), 4.39 - 4.26 (m, 2H), 3.66 - 3.61 (m, 1H), 3.17 (d, J = 5.7 Hz, 4H), 2.41 (d, J = 1.8 Hz, 3H), 2.21 - 2.12 (m, 1H), 2.06 - 1.96 (m, 1H), 1.93 - 1.84 (m, 2H), 1.76 (s, 2H), 0.93 - 0.85 (m, 3H).

[0275] Example 73: Synthesis of Compound 195 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.0 equiv., 40 mg, 0.075 mmol), (1S,2S)-2-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid (1.2 equiv., 18 mg, 0.090 mmol), and DMTMM (1.2 equiv., 25 mg, 0.090 mmol) was added DMF / water (5:1, 1.8 mL) and diisopropylethylamine (2.2 equiv., 29 μL, 0.165 mmol). The resulting mixture was stirred at room temperature for 40 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 1 as a white solid (38 mg, 82%) after lyophilization. C in MS was 0.05%. 33 H 36 Calculated for FN4O7: 619.26, Found: 619.25, [M+H] + .

[0276] Compound 195. The obtained intermediate 1 (38 mg, 0.061 mmol) was dissolved in 4 M HCl / dioxane (3 ml), and the mixture was stirred at room temperature for 30 minutes. The resulting suspension was filtered, and the solid material was washed with dioxane and Et2O and dried under vacuum to give the hydrochloride salt of compound 195 (25 mg, 74%) as a yellow solid. C in MS 28 H 28 Calculated value for FN4O5: 519.20, Found: 519.20, [M+H] + The product is unstable and undergoes decomposition during preparation and further analysis.

[0277] Example 74: Synthesis of Compounds 1035 and 1036 [ka] Intermediate 1. Compound 195. To a mixture of HCl (1.0 equiv., 24 mg, 0.039 mmol), Fmoc-GGGFGGP-OH (1.0 equiv., 31 mg, 0.039 mmol), and DMTMM (1.1 equiv., 14 mg, 0.043 mmol) was added DMF / water (5:1, 1.5 mL) and diisopropylethylamine (2.1 equiv., 16 μL, 0.082 mmol). The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→80% ACN / 0.1% TFA aqueous solution). After lyophilization, Intermediate 1 (two separated isomers A and B) was obtained as a white solid (Isomer A: 19 mg, Isomer B: 21 mg, Total yield: 87%). MS analysis revealed that C 65 H 66 FN 10 O 13 Calculated value: 1213.48, Found value: 1213.45, [M+H] + .

[0278] Compound 1035. To a solution of Intermediate 1 Isomer A (1.0 equiv., 19 mg, 0.016 mmol) in DMF (1.5 ml) was added morpholine (60 μl), and the mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated to dryness, and the residue was redissolved in dry DMF (1 ml). Mal-PEG-NHS ester (1.1 equiv., 5.4 mg, 0.017 mmol) and DIPEA (1.1 equiv., 3.0 μl, 0.017 mmol) were then added, and the reaction mixture was stirred at room temperature for 1 h. Purification by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→65% ACN / HO) afforded the product 1035 as a white solid (10 mg, 54%) after lyophilization. C in MS was 0.017 mmol. 59 H 65 FN 11 O 15 Calculated value: 1186.46, Found value: 1186.40, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.30 - 8.18 (m, 1H), 8.14 - 8.02 (m, 3H), 7.96 (t, J = 5.7 Hz, 1H), 7.91 - 7.78 (m, 1H), 7.74 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 7.27 - 7.18 (m, 5H), 7.18 - 7.12 (m, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.72 - 5.56 (m, 1H), 5.47 - 5.36 (m, 2H), 5.27 - 5.03 (m, 2H), 4.49 (tt, J = 9.2, 5.4 Hz, 1H), 4.06 - 3.92 (m, 1H), 3.83 - 3.68 (m, 4H), 3.66 (d, J = 5.7 Hz, 2H), 3.62 - 3.50 (m, 7H), 3.45 (t, J = 5.8 Hz, 2H), 3.12 - 2.94 (m, 1H), 2.82 - 2.71 (m, 1H), 2.65 - 2.52 (m, 2H), 2.44 - 2.10 (m, 11H), 1.98 - 1.77 (m, 5H), 1.72 - 1.57 (m, 1H), 1.56 - 1.37 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H).

[0279] Compound 1036. The product was synthesized from Intermediate 1 Isomer B (1.0 equiv, 21 mg, 0.017 mmol) following the same procedure as for Isomer A. After lyophilization, Compound 1036 was obtained as a white solid (11 mg, 55%). 59 H 65 FN 11 O 15 Calculated value: 1186.46, Found value: 1186.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.69 (d, J = 8.8 Hz, 1H), 8.26 (t, J = 6.0 Hz, 1H), 8.11 - 8.06 (m, 2H), 8.02 - 7.92 (m, 1H), 7.84 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 7.27 - 7.19 (m, 5H), 7.19 - 7.12 (m, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.47 - 5.38 (m, 2H), 5.37 - 5.31 (m, 1H), 5.29 - 5.22 (m, 1H), 5.10 (d, J = 18.6 Hz, 1H), 5.00 (d, J = 18.6 Hz, 1H), 4.54 - 4.43 (m, 1H), 4.22 - 4.15 (m, 1H), 4.04 - 3.93 (m, 1H), 3.92 - 3.80 (m, 1H), 3.79 - 3.69 (m, 4H), 3.68 - 3.63 (m, 2H), 3.62 - 3.50 (m, 7H), 3.50 - 3.42 (m, 3H), 3.19 - 2.97 (m, 2H), 2.82 - 2.70 (m, 1H), 2.43 - 2.22 (m, 8H), 2.21 - 1.97 (m, 2H), 1.95 - 1.72 (m, 4H), 1.72 - 1.59 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H).

[0280] Example 75: Synthesis of Compound 3016 Compound 3016 was prepared according to Procedure A in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1035 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.5, based on a linker-drug molecular weight of 1186 Da.

[0281] Example 76: Synthesis of Compound 3041 Compound 3041 was prepared according to Procedure B in the General Methods section of Example 1 using 6 molar equivalents of linker-payload compound 1035 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC was 7.5, based on a linker-drug molecular weight of 1186 Da.

[0282] Example 77: Synthesis of Compounds 3062A and 3062B Compound 3062A was prepared according to Procedure C in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1035 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC was 7.5, based on a linker-drug molecular weight of 1186 Da. When 25 molar equivalents of linker-payload compound 1035 relative to anti-EGFR IgG1 monoclonal antibody 1 were used, a DAR of 4.7 was achieved, yielding compound 3062B.

[0283] Example 78: Synthesis of Compound 3017 Compound 3017 was prepared according to Procedure A in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1036 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.5, based on a linker-drug molecular weight of 1186 Da.

[0284] Example 79: Synthesis of Compound 3042 Compound 3042 was prepared according to Procedure B in the General Methods section of Example 1 using 6 molar equivalents of linker-payload compound 1036 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR by RPLC was 7.5 based on a linker-drug molecular weight of 1186 Da.

[0285] Example 80: Synthesis of Compound 3063 Compound 3063 was prepared according to Procedure C in the General Methods section of Example 1 using 6 molar equivalents of linker-payload compound 1036 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR by RPLC was 7.5 based on a linker-drug molecular weight of 1186 Da.

[0286] Example 81: Synthesis of Compound 1037 [ka] To a mixture of intermediate 1.195.HCl (1.0 equiv., 15 mg, 0.024 mmol), Fmoc-GGFG-OH (1.05 equiv., 14 mg, 0.025 mmol), and DMTMM (1.05 equiv., 6.9 mg, 0.025 mmol), DMF / water (5:1, 1.5 mL) and diisopropylethylamine (2.05 equiv., 8.6 μL, 0.049 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→80% ACN / 0.1% TFA in water), affording intermediate 1 as a pale yellow solid (12 mg, 47%) after lyophilization. C in MS was 58 H 56 FN8O 11 Calculated value: 1059.40, Found value: 1059.35, [M+H] + .

[0287] Compound 1037. To a solution of intermediate 1 (1.0 equiv., 12 mg, 0.011 mmol) in DMF (1.25 ml), morpholine (45 μl) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated to dryness, and the residue was redissolved in dry DMF (0.75 ml). Mal-PEG-NHS ester (1.0 equiv., 3.4 mg, 0.011 mmol) and DIPEA (1.05 equiv., 2.0 μl, 0.012 mmol) were then added, and the reaction mixture was stirred at room temperature for 1 h. Purification by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→65% ACN / H2O) afforded the product 1037 (mixture of isomers) as a white solid (6.6 mg, 58%) after lyophilization. C in MS was 0.05%. 52 H 55 FN9O 13 Calculated value: 1032.39, Found value: 1032.35, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.58 (d, J = 9.1 Hz, 0.4H), 8.43 (t, J = 6.0 Hz, 0.4H), 8.32 - 8.13 (m, 2.2H), 8.13 - 8.03 (m, 2H), 8.03 - 7.92 (m, 1H), 7.79 - 7.66 (m, 1H), 7.34 (s, 0.4H), 7.31 (s, 0.6H), 7.27 - 7.17 (m, 3H), 7.17 - 7.09 (m, 2H), 6.99 (s, 2H), 6.53 (s, 0.4H), 6.50 (s, 0.6H), 5.85 - 5.75 (m, 0.6H), 5.64 - 5.52 (m, 0.6H), 5.49 - 5.35 (m, 2H), 5.39 - 4.93 (m, 2.8H), 4.52 - 4.42 (m, 0.4H), 4.32 - 4.20 (m, 0.6H), 3.83 - 3.59 (m, 4H), 3.59 - 3.47 (m, 5H), 3.44 (t, J = 5.8 Hz, 2H), 3.18 - 2.92 (m, 2H), 2.90 - 2.57 (m, 2H), 2.45 - 2.26 (m, 7H), 2.26 - 2.04 (m, 1H), 1.92 - 1.75 (m, 3H), 1.30 - 1.20 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0288] Example 82: Synthesis of Compound 3018 Compound 3018 was prepared according to Procedure A in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1037 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.5, based on a linker-drug molecular weight of 1032 Da.

[0289] Example 83: Synthesis of Compound 3043 Compound 3043 was prepared according to Procedure B in the General Methods section of Example 1 using 30 molar equivalents of linker-payload compound 1037 relative to the anti-TROP2 IgG1 monoclonal antibody. The DAR based on RPLC was 7.5, based on a linker-drug molecular weight of 1032 Da.

[0290] Example 84: Synthesis of Compound 3064 Compound 3064 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of linker-payload compound 1037 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RPLC was 7.5 based on a linker-drug molecular weight of 1032 Da.

[0291] Example 85: Synthesis of Compound 196 [ka] Intermediate 1. To a solution of exatecan mesylate (1.0 equiv., 30 mg, 0.0564 mmol) and diisopropylethylamine (30 μL) in DMF (2 mL) was added bromoacetic acid (1.2 equiv., 0.0677 mmol, 9.4 mg), and the reaction mixture was stirred at room temperature overnight. The residue was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 0.1% TFA), affording Intermediate 1 (23 mg, 83%) as a white powder after lyophilization. C in MS 26 H 23 Calculated value of FN3O6: 492.16, Measured value: 492.11 [MH] - .

[0292] Compound 196. To a solution of intermediate 1 (1.0 equiv., 23 mg, 0.0466 mmol) in formic acid (1 ml), 37% aqueous formaldehyde (0.25 ml) was added, and the resulting mixture was stirred at 50° C. for 6 hours. Water was then added, and the mixture was concentrated on a rotary evaporator. The residue was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 0.1% TFA), affording 196 (14 mg, 61%) as a white powder after lyophilization. C in MS 27 H 25 Calculated value of FN3O6: 506.17, Measured value: 506.08 [MH] - . 1 H NMR (401 MHz, DMSO-d6) δ 7.73 (dd, J = 11.0, 3.8 Hz, 1H), 7.35 (d, J = 4.1 Hz, 1H), 7.31 (s, 1H), 5.54 - 5.43 (m, 2H), 5.43 (d, J = 2.1 Hz, 2H), 4.53 - 4.43 (m, 1H), 3.68 (s, 4H), 3.58 (s, 1H), 3.37 - 3.29 (m, 1H), 2.98 - 2.88 (m, 2H), 2.42 - 2.31 (m, 4H), 1.87 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

[0293] Example 86: Synthesis of Compound 197 [ka] Intermediate 1. Exatecan mesylate (1.0 equiv, 40 mg, 0.0753 mmol), Fmoc- LTo a mixture of 1.1 equiv. α-proline (28 mg, 0.0818 mmol) and DMTMM (23 mg, 0.0818 mmol), DMF / water (4:1, 2 mL) and diisopropylethylamine (30 μL) were added. The resulting mixture was stirred at room temperature for 1 h, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→80% ACN / HO), affording Intermediate 1 as a white solid (45 mg, 79%) after lyophilization. The C in MS analysis indicated complete consumption of the starting material. 44 H 39 Calculated for FN4O7: 755.29, Found: 755.56, [M+H] + .

[0294] Compound 197. To a solution of intermediate 1 (45 mg, 0.0595 mmol) in DMF (2 ml), morpholine (100 μl) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was purified by reverse-phase flash chromatography (semi-preparative column, diol-modified C18, 0→60% ACN / 0.1% TFA) to give 197 (26 mg, 82%) as a white solid. 29 H 29 Calculated value for FN4O5: 533.22, Found value: 533.08, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ: 8.65 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.53 (m, 1H), 5.43 (s, 2H), 5.17 (s, 1H), 5.06 (d, J = 18.8 Hz, 1H), 3.73 (m, 1H), 3.73 (m, 2H), 2.93 (m, 2H), 2.39 (s, 3H), 2.18 (m, 3H), 1.88 (m, 3H), 1.72 (m, 1H), 0.89 (t, J = 7.35Hz, 3H).

[0295] Example 87: Synthesis of Compound 198 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.0 equiv., 45 mg, 0.084 mmol), Fmoc-d-proline (1.1 equiv., 31 mg, 0.092 mmol), and DMTMM (1.1 equiv., 26 mg, 0.092 mmol), DMF / water (5:1, 3 mL) and diisopropylethylamine (2.1 equiv., 30 μL, 0.176 mmol) were added. The resulting mixture was stirred at room temperature for 40 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→80% ACN / HO), affording Intermediate 1 as a white solid (59 mg, 93%) after lyophilization. MS analysis revealed a C 44 H 39 Calculated for FN4O7: 755.29, Found: 755.25, [M+H] + .

[0296] Compound 198. To a solution of Intermediate 1 (59 mg, 0.078 mmol) in DMF (3 ml) was added morpholine (270 μl), and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was concentrated on a rotary evaporator, followed by the addition of EtOAc. The precipitated product was isolated by filtration, washed with EtOAc, and dried under high vacuum to give 198 (31 mg, 75%) as a white solid. C in MS 29 H 29 Calculated value for FN4O5: 533.22, Found value: 533.20, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 8.56 (d, J = 9.2 Hz, 1H), 7.75 (d, J = 10.9 Hz, 1H), 7.29 (s, 1H), 6.50 (s, 1H), 5.58 - 5.46 (m, 1H), 5.42 (s, 2H), 5.20 (d, J = 19.0 Hz, 1H), 5.06 (d, J = 18.8 Hz, 1H), 3.67 (dd, J = 8.9, 6.0 Hz, 1H), 3.24 - 3.04 (m, 2H), 2.85 (td, J = 6.5, 2.6 Hz, 2H), 2.36 (s, 3H), 2.20 - 1.99 (m, 3H), 1.84 (m, 3H), 1.63 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H).

[0297] Example 88: Synthesis of Compound 199 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.1 equiv., 38 mg, 0.0711 mmol), Fmoc-3-fluoro-d-proline (1 equiv., 23 mg, 0.0647 mmol), and DMTMM (1.5 equiv., 27 mg, 0.0971 mmol), DMF / water (4:1, 2 mL) and diisopropylethylamine (30 μL) were added. The resulting mixture was stirred at room temperature for 30 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→80% ACN / 0.1% TFA), affording Intermediate 1 as a white solid (40 mg, 73%) after lyophilization. MS analysis revealed a C 44 H 39 Calculated for F2N4O7: 773.28, Found: 773.25, [M+H] + .

[0298] Compound 199. To a solution of intermediate 1 (40 mg, 0.0518 mmol) in DMF (2 ml) was added morpholine (100 μl), and the mixture was stirred at room temperature for 1 h. The residue was purified by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 0.1% TFA), affording 199 (20 mg, 69%) as a white powder after lyophilization. 29 H 29 Calculated value for F2N4O5: 551.21, Found value: 551.18 [M+H] + . 1 H NMR (401 MHz, DMSO-d6) δ 9.22 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H), 5.71 - 5.62 (m, 2H), 5.53 - 5.48 (m, 1H), 5.41 (s, 1H), 5.39 (d, J = 23.3 Hz, 2H), 5.24 (d, J = 19.0 Hz, 1H), 4.50 (d, J = 3.8 Hz, 1H), 4.43 (d, J = 3.8 Hz, 1H), 3.26 (m, 1H), 3.18 - 3.05 (m, 1H), 2.42 (d, J = 1.9 Hz, 3H), 2.37 - 2.28 (m, 1H), 2.24 (m, 2H), 2.19 - 2.14 (m, 1H), 1.97 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0299] Example 89: Synthesis of Compounds 200 and 201 [ka] Intermediate 1. To a mixture of exatecan mesylate (1 equiv., 40 mg, 0.0752 mmol), Fmoc-3,3-difluoro-L-proline (1.2 equiv., 18 mg, 0.0903 mmol), and DMTMM (1.2 equiv., 25 mg, 0.0903 mmol), DMF / water (4:1, 2 mL) and diisopropylethylamine (30 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN in 0.1% TFA), affording Intermediate 1 as a yellow solid (45 mg, 76%) after lyophilization. MS analysis of C 44 H 38 Calculated value for F3N4O7: 791.27, Found: 791.35, [M+H] + .

[0300] Compounds 200 and 201. To a solution of intermediate 1 (45 mg, 0.0570 mmol) in DMF (2 mL) was added morpholine (100 μL), and the mixture was stirred at room temperature for 1 h. The residue was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN in 0.1% TFA). The two isomers, 200 (16 mg) and 201 (15 mg), were completely separated and lyophilized (95% combined yield).

[0301] Compound 200. C in MS 29 H 28 Calculated value for F3N4O5: 569.20, Found value: 569.19 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.58 (dt, J = 8.9, 4.5 Hz, 1H), 5.43 (s, 2H), 5.29 (d, J = 19.1 Hz, 1H), 5.10 (d, J = 19.1 Hz, 1H), 3.81 (m, 1H), 3.58 (s, 1H), 3.24 - 3.09 (m, 4H), 2.94 (m, 1H), 2.41 (d, J = 1.9 Hz, 3H), 2.29 - 2.19 (m, 2H), 1.87 (hept, J = 7.1 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0302] Compound 201.MSにおけるC 29 H 28 Calculated value of F3N4O5: 569.20, measured value: 569.44 [M+H] + . 1 H NMR (401 MHz, DMSO-d6) δ 9.38 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 10.9 Hz, 1H), 7.34 (s, 1H), 6.57 (s, 1H), 5.68 (dt, J = 7.8, 3.5 Hz, 1H), 5.44 (s, 1H), 5.44 - 5.22 (m, 2H), 4.44 (m, 1H), 3.60 - 3.52 (m, 2H), 3.38 - 3.22 (m, 3H), 3.15 - 3.02 (m, 1H), 2.68 - 2.52 (m, 1H), 2.43 (d, J = 1.9 Hz, 3H), 2.25 (m, 1H), 2.20 - 2.06 (m, 1H), 1.95 - 1.83 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0303] Example 90: Synthesis of Compound 202

change

[0304] Example 91: Synthesis of Compound 203 [ka] Compound 203. To a mixture of exatecan mesylate (1 equiv., 19 mg, 0.0354 mmol), Boc-Glu(OtBu)-OH (1.5 equiv., 16 mg, 0.0531 mmol), and DMTMM (1.5 equiv., 15 mg, 0.0531 mmol), DMF / water (4:1, 2 mL) and diisopropylethylamine (10 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, at which point LC-MS analysis indicated complete consumption of the starting material. The solvent was removed under reduced pressure, and the solid was redissolved in 2 mL of dioxane. HCl (4 M in dioxane, 2 mL) was added, and the solution was stirred at room temperature for 2 hours. Upon completion of the reaction, as indicated by LC-MS analysis, the crude reaction mixture was redissolved in DMF and purified by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→50% ACN / 0.1% TFA) to give 203 (12 mg, 62%) as a yellowish powder after lyophilization. The final product consists of two isomers in equilibrium with each other. C in MS 29 H 30 Calculated value for FN4O7: 565.21, Found value: 565.88 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (m, 1H), 8.26 (m, 2H), 7.84 (m, 1H), 7.34 (d, J = 7.6 Hz, 1H), 5.62 (m, 1H), 5.44 (d, J = 7.3 Hz, 2H), 5.43 - 5.26 (m, 2H), 5.06 (d, J = 18.8 Hz, 1H), 3.82 - 3.73 (m, 1H), 3.51 - 3.38 (m, 2H), 3.19 (d, J = 11.6 Hz, 1H), 2.42 (d, J = 2.0 Hz, 3H), 2.35 (m, 1H), 2.31 (s, 1H), 2.21 - 2.08 (m, 1H), 1.91 (m, 4H), 0.89 (t, J = 7.3 Hz, 3H).

[0305] Example 92: Synthesis of Compound 204 [ka] Compound 204. To a mixture of exatecan mesylate (20 mg, 0.038 mmol), L-pyroglutamic acid (7.3 mg, 0.056 mmol), and DMTMM (21 mg, 0.076 mmol), DMF / water (4:1, 2.5 mL) and diisopropylethylamine (50 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN in 0.1% TFA), affording 204 as a yellow solid (18.5 mg, 89%) after lyophilization. MS analysis of C 29 H 28 Calculated for FN4O6: 547.19, Found: 547.10, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.6 Hz, 1H), 7.83 (s, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.57 (dt, J = 9.0, 4.8 Hz, 1H), 5.43 (d, J = 1.4 Hz, 2H), 5.20 (q, J = 18.8 Hz, 2H), 4.08 (dd, J = 8.4, 4.8 Hz, 1H), 3.58 (s, 3H), 3.19 (t, J = 6.3 Hz, 2H), 2.41 (d, J = 1.9 Hz, 3H), 2.29 - 2.22 (m, 1H), 2.21 - 2.12 (m, 2H), 2.00 (m, 1H), 1.86 (h, J = 7.0 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0306] Example 93: Synthesis of Compound 1038 [ka] Intermediate 1. To a mixture of exatecan mesylate (1 equiv., 40 mg, 0.0753 mmol), Boc-Glu(OBn)-OH (1.5 equiv., 38 mg, 0.113 mmol), and DMTMM (1.5 equiv., 31 mg, 0.113 mmol), DMF / water (3:1, 4 mL) and diisopropylethylamine (25 μL) were added. The resulting mixture was stirred at room temperature for 3 hours, at which point LC-MS analysis indicated complete consumption of the starting material. The solvent was removed under reduced pressure, and the solid was redissolved in 3 mL of dioxane. HCl (4 M in dioxane, 3 mL) was added, and the solution was stirred at room temperature for 2 hours. Upon completion of the reaction, as indicated by LC-MS analysis, the crude reaction mixture was redissolved in DMF and purified by reverse-phase flash chromatography (diol-modified C18, 0→100% ACN / 0.1% TFA) to give the desired intermediate 1 (43 mg, 87%) as a yellowish powder after lyophilization. 36 H 36 Calculated for FN4O7: 655.26, Found: 655.33 [M+H] + .

[0307] Intermediate 2. A solution of intermediate 1 (40 mg, 0.0611 mmol), Fmoc-GGGFGG-OH (1.1 equiv., 41 mg, 0.0673 mmol), DMTMM (1.1 equiv., 19 mg, 0.0673 mmol), and diisopropylethylamine (25 μL) in DMF / water (3:1, 4 mL) was stirred at room temperature for 2 hours. Upon completion of the reaction, as indicated by LC-MS analysis, the crude reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 0→100% ACN / 0.1% TFA) to afford, after lyophilization, the desired intermediate 2 (52 mg, 71%) as a yellow powder. C in MS 66 H 64 FN8O 13 Calculated value: 1195.46, Found value: 1195.77 [M+H] + .

[0308] Intermediate 3. The previously prepared intermediate 2 (52 mg, 0.0435 mmol) was dissolved in dioxane (10 mL), and Pd / C (10%, 10 mg) was suspended in the solution. Hydrogen was bubbled through the reaction mixture for 3 hours, after which LC-MS analysis indicated complete benzoyl deprotection. The mixture was filtered through a syringe filter (0.2 μm), and the dioxane was evaporated under reduced pressure. The crude reaction product was redissolved in DMF (2 mL), after which morpholine (100 μl) was added, and the mixture was stirred at room temperature for 1 hour. Upon completion of the reaction, as indicated by LC-MS analysis, the crude reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / 0.1% TFA), affording the desired intermediate 3 (17 mg, 44%) as a yellowish solid after lyophilization. C in MS 44 H 48 FN8O 11 Calculated value: 883.34, Found value: 883.01 [M+H] + .

[0309] Compound 1038. A solution of the previously prepared intermediate 2 (17 mg, 0.0193 mmol), Mal-PEGNHS ester (1.2 equivalents, 7 mg, 0.0217 mmol), and diisopropylethylamine (2 μL) in DMF (2 mL) was stirred at room temperature for 2 hours, after which LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / 0.1% TFA). After lyophilization, compound 1038 was recovered as a white powder (11 mg, 53%). C in MS was 0.0193 mmol. 53 H 57 FN9O 15 Calculated value: 1078.40, Found value: 1078.67 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.63 (d, J = 12.7 Hz, 1H), 8.47 (m, 1H), 8.10 (m, 3H), 8.03 (m, 2H), 7.90 (m, 1H), 7.82 - 7.72 (m, 1H), 7.31 (s, 1H), 7.14 - 7.06 (m, 4H), 7.01 (s, 2H), 6.50 (m, 2H), 5.75 (m, 2H), 5.55 (m, 1H), 5.47 - 5.30 (m, 3H), 5.11 - 4.93 (m, 2H), 4.52 - 4.22 (m, 1H), 4.20 (s, 1H), 3.83 - 3.59 (m, 4H), 3.56 (m, 3H), 3.23 (m, 2H), 3.15 (tt, J = 11.7, 4.3 Hz, 2H), 3.10 - 2.92 (m, 3H), 2.90 - 2.57 (m, 2H), 2.42 - 2.38 (m, 4H), 2.06 (m, 1H), 1.94 (m, 1H), 1.87 (m, 1H), 1.22 - 1.06 (m, 1H), 0.92 (m, 3H).

[0310] Example 94: Synthesis of Compound 205 [ka] N-Boc-GlyProOH. To a solution of N-Boc-Gly NHS ester (277 mg, 1.02 mmol) and L-Pro (117 mg, 1.02 mmol) in DMF (3 ml), DIPEA (300 μl) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN), and after lyophilization, N-Boc-GlyProOH was obtained as a white solid (270 mg, 97%). C in MS 12 H 21 Calculated value for N2O5: 273.14, Found value: 273.20, [M+H] + .

[0311] To a mixture of compound 205.130.HCl (15 mg, 0.028 mmol), N-Boc-GlyProOH (22 mg, 0.081 mmol), and DMTMM (25 mg, 0.089 mmol), DMF / water (4:1, 2.5 mL) and DIPEA (30 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, at which point LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / 0.1% TFA), and the product-containing fractions were evaporated. The residue was dissolved in dioxane (1 mL), and HCl in dioxane (4 M; 2 mL) was added. The resulting mixture was stirred at room temperature for 3 hours. The products were then separated by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN, 0.1% TFA) to give 205 as a yellow solid after lyophilization (7.0 mg, 38%). 33 H 36 Calculated for FN6O8: 663.25, Found: 663.25, [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.83 (d, J = 8.6 Hz, 1H), 8.04 (m, 2H), 7.81 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (m, 1H), 5.44 (m, 2H), 5.37 - 5.19 (m, 2H), 4.45 - 4.30 (m, 2H), 4.13 (dd, J = 8.7, 3.7 Hz, 1H), 3.36 (t, J = 6.8 Hz, 2H), 3.19 (d, J = 6.3 Hz, 3H), 2.53 - 2.52 (m, 2H), 2.41 (d, J = 1.8 Hz, 2H), 2.28 - 2.17 (m, 2H), 2.01 - 1.92 (m, 1H), 1.87 (m, 2H), 1.72 (dd, J = 10.8, 5.4 Hz, 2H), 1.58 (m, 1H), 0.88 (td, J = 7.4, 3.8 Hz, 3H).

[0312] Example 95: Synthesis of Compound 206 [ka] To a mixture of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol), (R)-3-hydroxybutanoic acid (1.1 equiv., 3.2 mg, 0.030 mmol), and DMTMM (1.1 equiv., 8.5 mg, 0.030 mmol), DMF / water (5:1, 1.2 mL) and diisopropylethylamine (2.1 equiv., 10 μL, 0.059 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / HO), affording 206 as a white solid (10 mg, 69%) after lyophilization. C in MS indicated complete consumption of the starting material. 28 H 29 Calculated for FN3O6: 522.20, Found: 522.20, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ: 8.41 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.54 (dt, J = 9.3, 5.0 Hz, 1H), 5.42 (s, 2H), 5.28 - 5.10 (m, 2H), 4.66 (d, J = 4.6 Hz, 1H), 4.03 (dddd, J = 14.8, 7.3, 5.5, 4.4 Hz, 1H), 3.23 - 3.09 (m, 2H), 2.39 (d, J = 1.9 Hz, 3H), 2.30 (dd, J = 14.0, 7.5 Hz, 1H), 2.21 (dd, J = 13.9, 5.7 Hz, 1H), 2.17 - 2.05 (m, 2H), 1.93 - 1.79 (m, 2H), 1.09 (d, J = 6.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0313] Example 96: Synthesis of Compound 207 [ka] To a 4:1 DMF / water mixture (4 mL) was added exatecan mesylate (20 mg, 0.038 mmol), cis-3-hydroxycyclobutane-1-carboxylic acid (1.25 equiv., 6 mg, 0.048 mmol), DMTMM (2.0 equiv., 21 mg, 0.076 mmol), and diisopropylethylamine (20 μL). The resulting solution was stirred at room temperature for 1 h, and LC-MS showed complete consumption of the starting material. The mixture was directly purified by reverse-phase HPLC chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / HO). After lyophilization, the desired product 207 was obtained as a white powder (16 mg, 79%). C in MS indicated a 0.05% C NMR (δ 1.0–1.5 δ, 1.0 δ, ... 29 H 29 Calculated for FN3O6: 534.20, Found: 534.45, [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.49 (s, 1H), 5.60 - 5.51 (m, 1H), 5.42 (s, 2H), 5.11 (m, 2H), ), 5.07 (d, J = 6.2 Hz, 1H), 4.37 (m, 1H), 3.63 - 3.55 (m, 2H), 3.21 - 3.10 (m, 2H), 2.38 (d, J = 1.8 Hz, 3H), 2.29 (t, J = 5.2 Hz, 1H), 2.19 - 2.01 (m, 4H), 1.88 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0314] Example 97: Synthesis of Compound 1021 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.0 equiv., 50 mg, 0.094 mmol), FmocGE(OBn)VCitAP-OH (1.25 equiv., 111 mg, 0.118 mmol), and DMTMM (1.25 equiv., 33 mg, 0.118 mmol), DMF / water (5:1, 3.0 mL) and diisopropylethylamine (2.25 equiv., 37 μL, 0.212 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 1 as a white solid (126 mg, 98%) after lyophilization. MS analysis revealed a C 72 H 81 FN 11 O 15 Calculated value: 1358.59, Found value: 1358.60, [M+H] + .

[0315] Intermediate 2. To a solution of Intermediate 1 (1.0 equiv., 126 mg, 0.092 mmol) in a mixture of DMF (1.0 ml) and dioxane (1.5 ml), 10% Pd / C (15 mg) was added, and the reaction mixture was hydrogenated (balloon) at room temperature for 3 hours. LC-MS showed complete consumption of the starting material. The mixture was filtered through a pad of Celite, and the filtrate was concentrated on a rotary evaporator. Morpholine (175 μl) was then added to the resulting solution, and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O) afforded the product Intermediate 2 as a white solid (58 mg, 60%) after lyophilization. C in MS was 0.05%. 50 H 65 FN 11 O 13 Calculated value: 1046.47, Found value: 1046.50, [M+H] + .

[0316] Compound 1021. Mal-PEG-NHS ester (1.0 equiv., 0.055 mmol, 17 mg) and DIPEA (1.05 equiv., 0.058 mmol, 10 μL) were added to a solution of intermediate 2 (1.0 equiv., 58 mg, 0.055 mmol) in anhydrous DMF (1.5 ml). The reaction mixture was stirred at room temperature for 1.5 hours, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / 0.1% TFA) afforded the desired product as a pale yellow solid (38 mg, 56%) after lyophilization. C in MS was 0.058 mmol. 59 H 74 FN 12 O 17 Calculated value: 1241.53, Found value: 1241.50, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ: 8.45 (d, J = 8.5 Hz, 1H), 8.12 - 7.87 (m, 5H), 7.86 - 7.70 (m, 2H), 7.30 (s, 1H), 7.00 (s, 2H), 5.94 (s, 1H), 5.55 - 5.46 (m, 1H), 5.46 - 5.35 (m, 2H), 5.33 - 5.07 (m, 2H), 4.55 - 4.08 (m, 6H), 3.77 - 3.59 (m, 3H), 3.59 - 3.51 (m, 6H), 3.47 (t, J = 4.9 Hz, 2H), 3.22 - 3.12 (m, 2H), 2.99 - 2.88 (m, 2H), 2.39 (s, 4H), 2.35 - 2.28 (m, 2H), 2.28 - 2.16 (m, 2H), 2.16 - 2.04 (m, 2H), 2.05 - 1.93 (m, 1H), 1.93 - 1.78 (m, 6H), 1.79 - 1.66 (m, 1H), 1.67 - 1.52 (m, 1H), 1.47 - 1.26 (m, 3H), 1.10 (d, J = 6.6 Hz, 3H), 0.94 - 0.74 (m, 9H).

[0317] Example 98: Synthesis of Compound 3024 Compound 3024 was prepared according to Procedure A in the General Methods section of Example 1, using 24 molar equivalents of linker-payload compound 1021 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.3, based on a linker-drug molecular weight of 1241 Da.

[0318] Example 99: Synthesis of Compound 3048 Compound 3048 was prepared according to Procedure B in the General Methods section of Example 1, using 24 molar equivalents of linker-payload compound 1021 relative to the anti-TROP2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-TROP2 antibody-drug conjugate based on RPLC-MS was 7.2, based on a linker-drug molecular weight of 1241 Da.

[0319] Example 100: Synthesis of Compound 1022 [ka] Intermediate 1. (S,S)-3-Fluoropyrrolidine-2-carboxylic acid (62.5 mg, 0.47 mmol) was dissolved in 1,4-dioxane (1 mL) and HO (3 mL) and cooled to 0 °C. KCO (162 mg, 1.18 mmol) was added, followed by Fmoc-Cl (115 mg, 0.45 mmol). The mixture was stirred at room temperature overnight, and HO (10 mL) was added. The mixture was acidified to pH 2-3 with aqueous HCl (1 M) and extracted with DCM (2 × 10 mL). The combined organic layers were dried over NaSO and concentrated to dryness to give the product as a white solid (122 mg, 76% yield). C in MS 20 H 19 Calculated value for FNO4: 356.12, Found value: 356.22, [M+H] + .

[0320] Intermediate 2. Exatecan mesylate (30 mg, 0.056 mmol), previously prepared Intermediate 1 (5 eq., 100 mg), and DIPEA (200 μL) were dissolved in a 5:1 mixture of DMF and water (3 mL). DMTMM (5 eq., 78 mg) was added, and the reaction mixture was stirred at room temperature for 30 minutes and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→70% ACN / HO). The product-containing fractions were lyophilized from water (35 mg, 81%). C in MS was 0.01%. 44 H 39 F2N4O8 + Calculated value: 789.27, Found value: 789.43, [M+H] - .

[0321] Intermediate 3. The previously prepared intermediate 2 (35 mg, 0.044 mmol) was dissolved in DMF (2 mL) and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (25 g, diol-modified C18, 0→50% ACN / H2O). The product-containing fractions were lyophilized from water (19 mg, 76%). C in MS was 29 H 29 F2N4O6 + Calculated value: 567.20, Found value: 567.57, [M+H] + .

[0322] Intermediate 4. The previously prepared intermediate 3 (19 mg, 0.035 mmol), Fmoc-GGGFGG-COOH (2 equiv., 47 mg), and DIPEA (150 μL) were dissolved in a 5:1 mixture of DMF and water (3 mL). DMTMM (2 equiv., 19.4 mg) was added, and the reaction mixture was stirred at room temperature for 30 minutes and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (semi-preparative, diol-modified C18, 0→50% ACN / HO). The product-containing fractions were lyophilized from water (32 mg, 80%). C in MS was 0.01%. 61 H 60 F2N9O 12 +Calculated value: 1149.43, Found value: 1149.25, [M+H] + .

[0323] Intermediate 5. The previously prepared intermediate 4 (70 mg, 0.061 mmol) was dissolved in DMF (2 mL) and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→50% ACN / H2O). The product-containing fractions were lyophilized from water (21 mg, 38%). C in MS was 46 H 50 F2N9O 10 + Calculated value: 926.36, Found value: 926.78, [M+H] + .

[0324] Compound 1022. The previously prepared intermediate 5 (21 mg, 0.023 mmol) was dissolved in 1.5 mL of anhydrous DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 9.6 mg) and DIPEA (10 μL) were added, and the reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→50% ACN / H2O). The product-containing fractions were lyophilized from water (10 mg, 39%). C in MS was 0.05%. 54 H 59 FN9O 14 Calculated value: 1122.42, Found value: 1122.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.50 (d, J = 8.5 Hz, 1H), 8.29 (t, J = 5.9 Hz, 1H), 8.13 - 8.07 (m, 2H), 7.97 (t, J = 5.7 Hz, 1H), 7.84 - 7.76 (m, 2H), 7.34 (s, 1H), 7.24 (m, 5H), 7.17 (m, 1H), 7.00 (s, 2H), 6.52 (d, J = 4.1 Hz, 1H), 5.55 (dt, J = 8.5, 4.2 Hz, 1H), 5.51 - 5.39 (m, 2H), 5.38 - 5.32 (m, 1H), 5.26 - 5.23 (m, 2H), 4.57 - 4.45 (m, 2H), 4.07 (m, 1H), 3.86 (m, 1H), 3.75 (m, 3H), 3.67 (d, J = 5.6 Hz, 2H), 3.61 (d, J = 5.7 Hz, 1H), 3.55 (m, 4H), 3.46 (t, J = 5.8 Hz, 2H), 3.21 - 3.14 (m, 1H), 3.04 (m, 2H), 2.79 (m, 1H), 2.45 - 2.40 (m, 4H), 2.33 (t, J = 6.6 Hz, 3H), 2.21 (m, 1H), 2.15 (d, J = 5.0 Hz, 1H), 2.11 - 2.04 (m, 1H), 1.87 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H).

[0325] Example 101: Synthesis of Compound 3021 Compound 3021 was prepared according to Procedure A in the General Methods section of Example 1, using 24 molar equivalents of linker-payload compound 1022 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.9, based on a linker-drug molecular weight of 1121 Da.

[0326] Example 102: Synthesis of Compound 3045 Compound 3045 was prepared according to Procedure B in the General Methods section of Example 1, using 24 molar equivalents of linker-payload compound 1022 relative to the anti-TROP2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-TROP2 antibody-drug conjugate based on RPLC-MS was 4.5, based on a linker-drug molecular weight of 1121 Da.

[0327] Example 103: Synthesis of Compound 3066 Compound 3066 was prepared according to Procedure C in the General Methods section of Example 1, using 24 molar equivalents of linker-payload compound 1022 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC-MS was 7.4, based on a linker-drug molecular weight of 1121 Da.

[0328] Example 104: Synthesis of Compound 1028 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.0 equiv., 30 mg, 0.056 mmol), FmocGGFGGP-OH (1.5 equiv., 60 mg, 0.084 mmol), and DMTMM (1.5 equiv., 24 mg, 0.084 mmol), DMF / water (5:1, 2.4 mL) and diisopropylethylamine (2.5 equiv., 25 μL, 0.141 mmol) were added. The resulting mixture was stirred at room temperature for 40 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO), affording Intermediate 1 as a white solid (68 mg, 98%) after lyophilization. MS analysis revealed a C 61 H 61 FN9O 12 Calculated value: 1130.44, Found value: 1130.40, [M+H] + .

[0329] Intermediate 2. To a solution of Intermediate 1 (1.0 equiv., 62 mg, 0.055 mmol) in anhydrous DMF (2.0 ml), morpholine (140 μL) was added, and the reaction mixture was stirred at room temperature for 1 hour. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0→60% ACN / H2O), and after lyophilization, the product was obtained as a white solid (45 mg, 90%). C in MS 46 H 51 FN9O 10 Calculated value: 908.37, Found value: 908.40, [M+H] + .

[0330] Compound 1028. Mal-PEG-NHS ester (1.0 equiv., 0.05 mmol, 15.5 mg) and DIPEA (1.5 equiv., 0.075 mmol, 13 μL) were added to a solution of intermediate 2 (1.0 equiv., 45 mg, 0.05 mmol) in anhydrous DMF (2 ml). The reaction mixture was stirred at room temperature for 1 hour, and LC-MS showed complete consumption of the starting material. DMF was removed, and the residue was concentrated from a mixture of 0.1% aqueous TFA and ACN. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→60% ACN / HO) afforded the desired product as a white solid (20 mg, 37%) after lyophilization. C in MS was 0.05 mmol. 55 H 60 FN 10 O 14 Calculated value: 1103.43, Found value: 1103.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.51 (d, J = 8.7 Hz, 1H), 8.29 (t, J = 5.9 Hz, 1H), 8.18 - 8.03 (m, 2H), 7.98 (t, J = 5.7 Hz, 1H), 7.91 - 7.67 (m, 3H), 7.33 - 7.28 (m, 1H), 7.24 (d, J = 4.3 Hz, 5H), 7.21 - 7.10 (m, 2H), 6.99 (s, 2H), 6.57 - 6.47 (m, 1H), 5.56 - 5.46 (m, 1H), 5.46 - 5.37 (m, 2H), 5.28 - 5.00 (m, 2H), 4.59 - 4.44 (m, 1H), 4.37 - 4.26 (m, 1H), 4.00 (dd, J = 17.1, 5.7 Hz, 1H), 3.84 (dd, J = 17.1, 4.9 Hz, 1H), 3.79 - 3.64 (m, 5H), 3.65 - 3.49 (m, 6H), 3.45 (t, J = 5.8 Hz, 2H), 3.26 - 2.99 (m, 2H), 2.86 - 2.74 (m, 1H), 2.38 (s, 3H), 2.32 (t, J = 6.6 Hz, 2H), 2.26 - 1.98 (m, 3H), 1.97 - 1.77 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0331] Example 105: Synthesis of Compound 3019 Compound 3019 was prepared according to Procedure A in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1028 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 6.9, based on a linker-drug molecular weight of 1103 Da.

[0332] Example 106: Synthesis of Compound 1029 [ka] Intermediate 1. Exatecan mesylate (50 mg, 0.0941 mmol), Fmoc-Val-Cit-PAB-PNP (50 mg, 0.0652 mmol, 0.7 equiv.), and diisopropylethylamine (50 L) were dissolved in 1.3 mL of anhydrous DMF. The reaction mixture was stirred at room temperature for 12 hours and then loaded directly onto the column. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0→75% ACN / HO) afforded the product as a white solid (47 mg, 69%) after lyophilization from water-acetonitrile. C in MS was 0.01%. 58 H 60 FN8O 11 Calculated value: 1063.44, Found value: 1063.40, [M+H] + .

[0333] Intermediate 2. To a solution of the previously prepared intermediate 1 (53 mg, 0.0443 mmol) in anhydrous DMF (1 ml), morpholine (100 μL) was added, and the reaction mixture was stirred at room temperature for 1 hour. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase HPLC (semi-preparative HPLC, diol-modified C18, 0→100% ACN / H2O), and after lyophilization, the product was obtained as a white solid (20.5 mg, 55%). C in MS 43 H 50 Calculated for FN8O9: 841.37, Found: 841.30, [M+H] + .

[0334] Compound 1029. Mal-PEG-NHS ester (1.0 equiv., 0.0244 mmol, 7.6 mg) and DIPEA (2 equiv., 0.0488 mmol, 6.4 mg, 8.7 μL) were added to a solution of the previously prepared intermediate 2 (20 mg, 0.0244 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0→100% ACN / HO) afforded the desired product as a white solid (8 mg, 32%) after lyophilization. C in MS was 0.0244 mmol. 52 H59 FN9O 13 Calculated value: 1036.42, Measured value: 1036.60, [M + H] + . 1 H NMR (500 MHz, DMSO-d6) δ 9.99 (d, J = 5.7 Hz, 2H), 8.10 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 7.29 - 7.23 (m, 2H), 7.02 (s, 2H), 6.67 (s, 1H), 6.52 (s, 1H), 5.98 (t, J = 6.0 Hz, 1H), 5.45 (s, 2H), 5.44 - 5.40 (m, 3H), 5.29 (d, J = 7.1 Hz, 2H), 5.08 (s, 2H), 4.38 (q, J = 7.4 Hz, 2H), 4.21 (t, J = 7.4 Hz, 2H), 3.57 (m, 3H), 3.48 (m, 2H), 3.02 (m, 1H), 2.95 (m, 1H), 2.81 (s, 2H), 2.60 (s, 1H), 2.43 (m, 1H), 2.39 - 2.32 (m, 3H), 2.19 (m, 2H), 1.97 (m, 1H), 1.87 (m, 1H), 1.76 (s, 1H), 1.70 (m, 1H), 1.60 (m, 1H), 1.49 - 1.35 (m, 1H), 1.17 - 1.06 (m, 1H), 0.91 - 0.79 (m, 3H).

[0335] Example 107: Synthesis of Compound 3025 Compound 3025 was prepared according to Procedure A in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1029 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-HER2 antibody-drug conjugate was 7, based on a linker-drug molecular weight of 1036 Da.

[0336] Example 108: Synthesis of Compound 3069 Compound 3069 was prepared according to Procedure C in the General Methods section of Example 1, using 20 molar equivalents of linker-payload compound 1029 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC-MS was 7, based on a linker-drug molecular weight of 1036 Da.

[0337] Example 109: Synthesis of Compounds 1030 and 1032 [ka] Compounds 1030 and 1032. Mal-PEG-GGFGG-OH (1.2 equivalents, 37 mg, 0.0633 mmol), compounds 200 and 201 ( L Isomers and D To a solution of the unresolved mixture of isomers (1.0 equiv., 30 mg, 0.0528 mmol) and DMTMM (1.2 equiv., 18 mg, 0.0633 mmol) was added DMF / water (4:1, 2.0 mL) and diisopropylethylamine (1. equiv., 9.3 μL, 0.0528 mmol). The resulting mixture was stirred at room temperature for 1 h, after which LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / 0.1% TFA). The two L Isomers and DIsomers 1030 and 1032 were separately separated and repurified by a second flash chromatography run using a semi-preparative column (diol-modified C18, 0→100% ACN / 0.1% TFA). After lyophilization, the two isomers were obtained as yellowish solids (16 mg and 15 mg, combined yield 52%).

[0338] Compound 1030.C in MS 55 H 58 F3N 10 O 14 Calculated value: 1139.41, Found value: 1139.30, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 9.02 (d, J = 8.2 Hz, 1H), 8.36 - 8.28 (m, 1H), 8.11 (m, 4H), 7.99 (t, J = 5.7 Hz, 2H), 7.92 (t, J = 5.5 Hz, 1H), 7.83 (m, 1H), 7.34 (d, J = 17.8 Hz, 1H), 7.27 - 7.18 (m, 2H), 7.00 (s, 2H), 5.64 - 5.48 (m, 2H), 5.43 (s, 2H), 5.39 - 5.30 (m, 2H), 4.99 (d, J = 19.1 Hz, 1H), 4.59 - 4.45 (m, 4H), 4.07 (m, 2H), 3.86 - 3.72 (m, 5H), 3.65 - 3.53 (m, 2H), 3.47 (d, J = 5.8 Hz, 2H), 3.24 - 3.01 (m, 2H), 2.80 (m, 2H), 2.42 (s, 3H), 2.34 (m, 3H), 2.20 (m, 1H), 2.08 (m, 1H), 1.86 (m, 3H), 1.24 (d, J = 5.4 Hz, 2H), 0.89 (q, J = 6.9 Hz, 3H).

[0339] Compound 1032. C in MS 55 H 58 F3N 10 O 14Calculated value: 1139.41, Found value: 1139.67, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 9.09 (d, J = 8.3 Hz, 1H), 8.27 (t, J = 5.8 Hz, 1H), 8.09 (t, J = 7.8 Hz, 3H), 7.96 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 10.9 Hz, 1H), 7.77 (t, J = 5.3 Hz, 1H), 7.36 (s, 1H), 7.24 (m, 5H), 7.18 (m, 1H), 7.00 (s, 2H), 6.54 (s, 2H), 5.56 (m, 1H), 5.49 - 5.39 (m, 2H), 5.26 (d, J = 8.4 Hz, 1H), 4.59 - 4.49 (m, 3H), 4.11 (m, 1H), 4.05 - 3.91 (m, 2H), 3.80 - 3.67 (m, 4H), 3.61 - 3.54 (m, 4H), 3.46 (m, 2H), 3.14 - 3.03 (m, 1H), 2.79 (m, 2H), 2.66 - 2.56 (m, 2H), 2.42 (s, 3H), 2.33 (m, 1H), 2.13 (m, 1H), 1.95 - 1.82 (m, 3H), 1.24 (d, J = 5.2 Hz, 2H), 0.89 (q, J = 7.4 Hz, 3H).

[0340] Example 110: Synthesis of Compound 3022 Compound 3022 was prepared according to Procedure A in the General Methods section of Example 1, using 23 molar equivalents of linker-payload compound 1030 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.4, based on a linker-drug molecular weight of 1139 Da.

[0341] Example 111: Synthesis of Compound 3046 Compound 3046 was prepared according to Procedure B in the General Methods section of Example 1, using 23 molar equivalents of linker-payload compound 1030 relative to the anti-TROP2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-TROP2 antibody-drug conjugate was 6.5, based on a linker-drug molecular weight of 1139 Da.

[0342] Example 112: Synthesis of Compound 3067 Compound 3067 was prepared according to Procedure C in the General Methods section of Example 1, using 31 molar equivalents of linker-payload compound 1030 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC was 7.4, based on a linker-drug molecular weight of 1139 Da.

[0343] Example 113: Synthesis of Compound 3023 Compound 3023 was prepared according to Procedure A in the General Methods section of Example 1, using 27 molar equivalents of linker-payload compound 1032 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.4, based on a linker-drug molecular weight of 1139 Da.

[0344] Example 114: Synthesis of Compound 3047 Compound 3047 was prepared according to Procedure B in the General Methods section of Example 1, using 27 molar equivalents of linker-payload compound 1032 relative to the anti-TROP2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-TROP2 antibody-drug conjugate was 7.4, based on a linker-drug molecular weight of 1139 Da.

[0345] Example 115: Synthesis of Compound 3068 Compound 3068 was prepared according to Procedure C in the General Methods section of Example 1, using 27 molar equivalents of linker-payload compound 1032 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC was 7.4, based on a linker-drug molecular weight of 1139 Da.

[0346] Example 116: Synthesis of Compound 1031 [ka] Compound 1031. Mal-PEG-GGFGG-OH (1.0 equiv., 22 mg, 0.037 mmol), Sc-598 (1.0 equiv., 20 mg, 0.037 mmol), and DMTMM (1.1 equiv., 12 mg, 0.041 mmol) were added to a solution of DMF / water (5:1, 2.0 mL) and diisopropylethylamine (1.1 equiv., 7.2 μL, 0.041 mmol). The resulting mixture was stirred at room temperature for 50 minutes, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / HO), affording 1031 as a white solid (29 mg, 71%) after lyophilization. C in MS indicated a 0.05% C NMR spectrum. 55 H 60 FN 10 O 14 Calculated value: 1103.43, Found value: 1103.30, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 8.53 (d, J = 8.6 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.15 - 8.02 (m, 2H), 7.96 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.67 (t, J = 5.1 Hz, 1H), 7.34 (s, 1H), 7.27 - 7.20 (m, 4H), 7.20 - 7.13 (m, 1H), 6.99 (s, 2H), 6.52 (s, 1H), 5.60 - 5.36 (m, 4H), 5.14 (d, J = 19.2 Hz, 1H), 4.58 - 4.46 (m, 1H), 4.26 (dd, J = 8.1, 4.2 Hz, 1H), 4.04 (dd, J = 17.0, 5.7 Hz, 1H), 3.87 (dd, J = 17.1, 4.4 Hz, 1H), 3.78 (t, J = 5.8 Hz, 1H), 3.73 (dd, J = 16.9, 5.8 Hz, 1H), 3.66 (d, J = 5.7 Hz, 2H), 3.60 (d, J = 5.1 Hz, 1H), 3.58 - 3.49 (m, 6H), 3.45 (t, J = 5.8 Hz, 2H), 3.22 - 3.06 (m, 2H), 3.03 (dd, J = 13.8, 4.5 Hz, 1H), 2.77 (dd, J = 13.8, 9.7 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.18 - 2.01 (m, 4H), 1.99 - 1.79 (m, 4H), 0.89 (t, J = 7.1 Hz, 3H).

[0347] Example 117: Synthesis of Compound 3020 Compound 3020 was prepared according to Procedure A in the General Methods section of Example 1, using 31 molar equivalents of linker-payload compound 1031 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.4, based on a linker-drug molecular weight of 1103 Da.

[0348] Example 118: Synthesis of Compound 3044 Compound 3044 was prepared according to Procedure B in the General Methods section of Example 1, using 31 molar equivalents of linker-payload compound 1031 relative to the anti-TROP2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the RPLC-based anti-TROP2 antibody-drug conjugate was 7.4, based on a linker-drug molecular weight of 1103 Da.

[0349] Example 119: Synthesis of Compound 3065 Compound 3065 was prepared according to Procedure C in the General Methods section of Example 1, using 31 molar equivalents of linker-payload compound 1031 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC was 7.4, based on a linker-drug molecular weight of 1103 Da.

[0350] Example 120: Synthesis of Compound 1033 [ka] Intermediate 1. To a mixture of exatecan mesylate (1.0 equiv., 25 mg, 0.0470 mmol), Fmoc-Val-Ala-OH (1.2 equiv., 23 mg, 0.0564 mmol), and DMTMM (1.2 equiv., 16 mg, 0.0564 mmol), DMF / water (4:1, 1.8 mL) and diisopropylethylamine (25 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g diol-modified C18, 0→100% ACN / 0.1% TFA in water), affording Intermediate 1 as a yellow solid (37 mg, 95%) after lyophilization. MS analysis revealed a C 47 H 47 Calculated for FN5O8: 828.34, Found: 828.45, [M+H] + .

[0351] Intermediate 2. The previously prepared intermediate 1 (37 mg, 0.0447 mmol) was dissolved in 2 mL of DMF and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour, and the crude reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / 0.1% TFA). After lyophilization, the product was obtained as a yellow solid (25 mg, 92%). C in MS 32 H 37 Calculated for FN5O6: 606.27, Found: 606.24, [M+H] + .

[0352] Compound 1033. The previously prepared intermediate 2 (1.05 equiv., 15 mg, 0.0248 mmol) was dissolved in 2 mL of DMF, MI-amide-PEG8-NHS ester (1 equiv., 0.0236 mmol, 16 mg) and diisopropylethylamine (1.1 equiv., 0.0260 mmol, 4.54 μL) were added, and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→70% ACN / HO). After lyophilization, product 1033 was obtained as a white solid (4.2 mg, 85%). C in MS was 0.05%. 58 H 79 FN7O 18 Calculated value: 1180.55, Found value: 1180.10, [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ: 8.40 (d, J = 8.6 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1H), 8.00 (t, J = 5.7 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.53 (dt, J = 9.2, 4.9 Hz, 1H), 5.42 (s, 2H), 5.23 (d, J = 18.7 Hz, 1H), 5.11 (d, J = 18.9 Hz, 1H), 4.27 (p, J = 7.0 Hz, 1H), 4.12 (dd, J = 8.6, 6.6 Hz, 1H), 3.58 (t, J = 7.3 Hz, 2H), 3.56 - 3.41 (m, 32H), 3.36 (t, J = 5.9 Hz, 2H), 3.23 - 3.10 (m, 4H), 2.47 - 2.38 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 2.19 - 2.05 (m, 2H), 1.96 - 1.79 (m, 2H), 1.27 (d, J = 7.0 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H), 0.79 (dd, J = 6.9, 3.6 Hz, 6H).

[0353] Example 121: Synthesis of Compound 3026 Compound 3026 was prepared according to Procedure A in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1033 relative to the anti-HER2 IgG1 monoclonal antibody. The drug-to-antibody ratio (DAR) of the anti-HER2 antibody-drug conjugate based on RPLC-MS was 7.5, based on a linker-drug molecular weight of 1180 Da.

[0354] Example 122: Synthesis of Compound 3070 Compound 3070 was prepared according to Procedure C in the General Methods section of Example 1, using 30 molar equivalents of linker-payload compound 1033 relative to anti-EGFR IgG1 monoclonal antibody 1. The drug-to-antibody ratio (DAR) of the anti-EGFR antibody-drug conjugate based on RPLC was 7.5, based on a linker-drug molecular weight of 1180 Da.

[0355] Example 123: Synthesis of Compound 71 [ka] Intermediate 1. Bromoacetic acid (0.715 g, 10 mmol) was dissolved in 5 mL of water, and then sodium azide (0.696 g, 5 mmol) was added, and the solution was stirred at room temperature overnight. The solution was acidified with HCl until pH = 1, and then the desired product was extracted with diethyl ether. The solvent was dried over Na2SO4, filtered, and evaporated to give the reaction product, which was used in the next step without further purification.

[0356] Intermediate 2. Exatecan mesylate (100 mg, 0.188 mmol), 2-azidoacetic acid (1.1 equiv., 0.207 mmol, 21 mg), DMTMM (1.3 equiv., 0.244 mmol, 68 mg), and DIPEA (50 μL) were dissolved in 5 mL of a 4:1 mixture of DMF and water. The reaction mixture was stirred at room temperature for 1 hour. The mixture was directly purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→100% ACN in water). After lyophilization, the desired product was obtained as a white powder (79 mg, 74%). C in MS was 0.01%. 26 H 24 Calculated value for FN6O5: 519.18, Found value: 519.41, [M+H] + .

[0357] Compound 71. The previously prepared intermediate 2 (10 mg, 0.0193 mmol), propargyl alcohol (1.2 equiv., 0.0232 mmol, 1.35 μL), and catalyst CpRu(COD)Cl (10%, 0.7 mg) were suspended in anhydrous DCM (2 mL) under an argon atmosphere. The mixture was stirred at 40 °C for 16 h. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semi-preparative diol-modified C18, 0 → 100% ACN in water). After lyophilization from water-ACN, the desired product was obtained as a yellowish powder (9 mg, 90%). MS showed C 29 H 28 Calculated for FN6O6: 575.21, Found: 575.14, [M+H] + .

[0358] Example 124: Synthesis of Compound 72 [ka] Compound 72. Intermediate 2 (10 mg, 0.0193 mmol) from Example 63, previously prepared, propargyl alcohol (1.2 equiv., 0.0232 mmol, 1.35 μL), sodium ascorbate (0.2 equiv., 0.00386 mmol, 2 M in water, 1.93 μL), copper sulfate pentahydrate (0.1 equiv., 0.00193 mmol, 1 M in water, 1.93 μL), and TBTA (0.15 equiv., 0.0029 mmol, 1.5 mg) were dissolved in 2 mL of a 4:1 mixture of DMF / water. The reaction mixture was stirred at room temperature for 2 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semi-preparative diol-modified C18, 0→100% ACN in water). After lyophilization from water-ACN, the desired product was obtained as a yellowish powder (10 mg, 95%). C in MS was 0.00386 mmol. 29 H 28 Calculated for FN6O6: 575.21, Found: 575.55, [M+H] + .

[0359] Example 125: Synthesis of Compound 73 [ka] Compound 73. Intermediate 2 (10 mg, 0.0193 mmol) from Example 63, previously prepared, 3-butyn-1-ol (1.2 equivalents, 0.0232 mmol, 1.50 μL), and catalyst CpRu(COD)Cl (10%, 0.7 mg) were suspended in anhydrous DCM (2 mL) under an argon atmosphere. The mixture was stirred at 40° C. for 16 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semi-preparative diol-modified C18, 0→100% ACN in water). After lyophilization from water-ACN, the desired product was obtained as a yellowish powder (6 mg, 54%). C in MS was 0.0193 mmol. 30 H 30 Calculated for FN6O6: 589.22, Found: 589.23, [M+H] + .

[0360] Example 126: Synthesis of Compound 74 [ka] Compound 74. Intermediate 2 (10 mg, 0.0193 mmol) from Example 63, previously prepared, 3-butyn-1-ol (1.2 equiv., 0.0232 mmol, 1.50 μL), sodium ascorbate (0.2 equiv., 0.00386 mmol, 2 M in water, 1.93 μL), copper sulfate pentahydrate (0.1 equiv., 0.00193 mmol, 1 M in water, 1.93 μL), and TBTA (0.15 equiv., 0.0029 mmol, 1.5 mg) were dissolved in 2 mL of a 4:1 mixture of DMF / water. The reaction mixture was stirred at room temperature for 2 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semi-preparative diol-modified C18, 0→100% ACN in water). After lyophilization from water-ACN, the desired product was obtained as a white powder (7 mg, 62%). C in MS was 0.00386 mmol. 30 H 30 Calculated value for FN6O6: 589.22, Found value: 589.05, [M+H] + .

[0361] Example 127: Synthesis of Compound 208 [ka] Intermediate 1. To a 4:1 DMF / water mixture (4 mL) was added exatecan mesylate (20 mg, 0.0376 mmol), 3-(1,3-dioxolan-2-yl)propanoic acid (2 equiv., 0.0753 mmol, 11 mg), DMTMM (1.5 equiv., 0.0564 mmol, 16 mg), and diisopropylethylamine (20 μL). The resulting solution was stirred at room temperature for 1 hour, and LC-MS showed complete consumption of the starting material. The mixture was directly purified by reverse-phase HPLC chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / HO). After lyophilization, the desired product was obtained as a white powder (19 mg, 89%). C in MS was 0.0564 mmol. 30 H 31 Calculated for FN3O7: 564.21, Found: 564.34, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.56 (dt, J = 9.3, 5.0 Hz, 1H), 5.43 (s, 2H), 5.31 - 5.11 (m, 2H), 4.83 (t, J = 4.5 Hz, 1H), 3.89 - 3.80 (m, 2H), 3.79 - 3.69 (m, 2H), 3.24 - 3.10 (m, 2H), 2.40 (d, J = 1.9 Hz, 3H), 2.26 (t, J = 7.6 Hz, 2H), 2.14 (q, J = 7.3, 6.6 Hz, 2H), 1.98 - 1.77 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0362] Compound 208. Intermediate 1 (20 mg) was dissolved in 1 mL of 0.1% aqueous TFA, and the resulting mixture was stirred at room temperature for 5 hours, after which LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase HPLC chromatography using a semi-preparative column (diol-modified C18, 0→100% ACN / 1% TFA). After lyophilization, the desired product was obtained as a white powder (17 mg, 94%). C in MS 28 H 27 Calculated value for FN3O6: 520.19, Found value: 520.31, [M+H] + Due to the instability of the material, NMR spectra could not be obtained.

[0363] Example 128: Synthesis of Compound 209 [ka] Intermediate 1. To a solution of (S)-1-(benzyloxy)propan-2-ol (1.0 equiv., 100 mg, 0.6 mmol) and bis(4-nitrophenyl)carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (4 mL) was added diisopropylethylamine (1.1 equiv., 115 μL, 0.66 mmol), and the resulting mixture was stirred at room temperature for 2 h. The DMF was removed by rotary evaporation, and the residue was purified by flash chromatography (SiO, 0–20% EtOAc / cyclohexane) to give the product Intermediate 1 (135 mg, 68%) as a clear, viscous oil.

[0364] Intermediate 2. To a mixture of Intermediate 1 (1.0 equiv., 40 mg, 0.12 mmol) and exatecan mesylate (1.0 equiv., 65 mg, 0.12 mmol), DMF (1.5 mL) and diisopropylethylamine (3.0 equiv., 0.36 mmol, 62 μL) were added, and the resulting solution was stirred at room temperature overnight. Purification by reverse-phase flash chromatography (diol-modified C18, 0→60% ACN / 10 mM NHOAc in water) afforded the product Intermediate 2 (55 mg, 73%) as a white solid after lyophilization.

[0365] Compound 209. To a solution of intermediate 2 (1.0 equiv., 20 mg, 0.032 mmol) in dioxane / water (2:1, 1 mL) was added 10% Pd / C, and the resulting mixture was hydrogenated (H in a balloon) for 2 h. The suspension was then filtered, and the filtrate was evaporated on a rotary evaporator. Purification of the residue by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 10 mM NHOAc in water) afforded compound 209 (9 mg, 53%) as a white solid after lyophilization. C in MS 28 H 29 Calculated value for FN3O7: 538.20, Found value: 538.10, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ: 7.90 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 10.9 Hz, 1H), 7.28 (s, 1H), 6.50 (s, 1H), 5.41 (s, 2H), 5.30 - 5.20 (m, 2H), 5.06 (d, J = 19.0 Hz, 1H), 4.86 - 4.76 (m, 2H), 3.53 - 3.40 (m, 2H), 3.30 - 3.19 (m, 1H), 3.17 - 3.00 (m, 1H), 2.34 (d, J = 1.8 Hz, 3H), 2.29 - 2.18 (m, 1H), 2.15 - 2.00 (m, 1H), 1.93 - 1.77 (m, 2H), 1.26 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0366] Example 129: Synthesis of Compound 1039 [ka] Intermediate 1. Compound 209 (80 mg, 0.15 mmol) and Fmoc-GGFG-OAc (70 mg, 0.11 mmol) were co-evaporated three times from anhydrous DMF and then redissolved in anhydrous DMF (4 mL). HCl (4 M in dioxane, 30 μL) was added, and the reaction mixture was stirred at 50° C. for 60 minutes. The DMF was then removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→50% ACN / 10 mM NHOAc aqueous solution) to give product Intermediate 1 (30 mg, 25%) as a white solid. C in MS 59 H 60 FN8O 13 Calculated value: 1107.42, Found value: 1107.45, [M + H] + .

[0367] To a solution of Intermediate 1 (30 mg, 0.027 mmol) in 2.2 mL of anhydrous DMF, morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then the DMF was removed on a rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→100% ACN / 0.1% TFA) to give the product Intermediate 2 (15 mg, 63%) as a white solid. C in MS: 44 H 49 FN8O 11 Calculated value: 885.35, Found value: 885.25, [M + H] + .

[0368] Compound 1039. Intermediate 2 (15 mg, 0.017 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equiv., 0.025 mmol, 7.9 mg) and DIPEA (5 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 (0→100% ACN / 10 mM NHOAc in water). After lyophilization from water, the desired product was recovered as a white solid (11 mg, 60%). C in MS was 0.05%. 53 H 59 FN9O 15 Calculated value: 1080.40, Measured value: 1080.35, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.54 (t, J = 6.7 Hz, 1H), 8.31 (t, J = 5.9 Hz, 1H), 8.14 - 8.08 (m, 2H), 8.00 (dd, J = 7.3, 4.1 Hz, 2H), 7.76 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.25 (t, J = 4.4 Hz, 4H), 7.19 (m, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.42 (d, J = 2.8 Hz, 2H), 5.30 - 5.24 (m, 2H), 5.13 (d, J = 19.0 Hz, 1H), 4.95 (m, 1H), 4.61 (m, 2H), 4.54 - 4.46 (m, 1H), 3.75 (m, 3H), 3.68 (s, 1H), 3.58 - 3.53 (m, 5H), 3.49 - 3.45 (m, 4H), 3.29 - 3.22 (m, 1H), 3.13 - 3.03 (m, 2H), 2.81 (dd, J = 13.9, 9.6 Hz, 1H), 2.39 - 2.36 (m, 3H), 2.33 (t, J = 6.6 Hz, 2H), 2.29 - 2.23 (m, 1H), 2.13 - 2.06 (m, 1H), 1.87 (m, 2H), 1.28 (d, J = 6.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0369] Example 130: Synthesis of Compound 210

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[0370] Intermediate 2. To a mixture of Intermediate 1 (1.0 equiv., 50 mg, 0.15 mmol) and exatecan mesylate (1.0 equiv., 80 mg, 0.15 mmol), DMF (3 mL) and diisopropylethylamine (200 μL) were added, and the resulting solution was stirred at room temperature overnight. Purification by reverse-phase flash chromatography (diol-modified C18, 0→100% ACN / 10 mM NHOAc in water) afforded the product Intermediate 2 (67 mg, 72%) as a white solid after lyophilization.

[0371] Compound 210. To a solution of intermediate 2 (67 mg, 0.11 mmol) in dioxane (5 mL) was added 10% Pd / C, and the resulting mixture was hydrogenated (H in a balloon) for 4 hours. The suspension was filtered, and the filtrate was evaporated on a rotary evaporator. Purification of the residue by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 10 mM NHOAc in water) afforded compound 210 (40 mg, 70%) as a white solid after lyophilization. C in MS 28 H 29 Calculated value for FN3O7: 538.20, Found: 538.25, [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.43 (s, 2H), 5.31 - 5.20 (m, 2H), 5.15 (d, J = 19.1 Hz, 1H), 4.87 - 4.74 (m, 2H), 3.54 - 3.45 (m, 1H), 3.32 - 3.20 (m, 1H), 3.15 - 3.03 (m, 1H), 2.36 (d, J = 1.8 Hz, 3H), 2.27 - 2.18 (m, 1H), 2.17 - 2.08 (m, 1H), 1.88 (m, 2H), 1.21 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H).

[0372] Example 131: Synthesis of Compound 1040 [ka] Intermediate 1. Compound 210 (80 mg, 0.15 mmol) and Fmoc-GGFG-OAc (70 mg, 0.11 mmol) were coevaporated three times from anhydrous DMF and then redissolved in anhydrous DMF (2 mL). HCl (4 M in dioxane, 80 μL) was added, and the reaction mixture was stirred at 50° C. for 60 minutes. The DMF was then removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→50% ACN / 10 mM NHOAc aqueous solution) to give product Intermediate 1 (64 mg, 52%) as a white solid. C in MS 59 H 60 FN8O 13 Calculated value: 1107.42, Found value: 1107.35, [M + H] + .

[0373] To a solution of Intermediate 1 (52 mg, 0.047 mmol) in 2.2 mL of anhydrous DMF, morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then the DMF was removed on a rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→100% ACN / 0.1% TFA) to give the product Intermediate 2 (31 mg, 76%) as a white solid. C in MS: 44 H 49 FN8O 11 Calculated value: 885.35, Found value: 885.24, [M + H] - .

[0374] Compound 1040. Intermediate 2 (31 mg, 0.017 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equivalents, 0.025 mmol, 7.9 mg) and DIPEA (5 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 (0→100% ACN / 10 mM aqueous NH4OAc). The product was purified by reverse-phase flash HPLC using 0→100% ACN / 0.1% TFA followed by 0→100% ACN / 10 mM aqueous NH4OAc. After lyophilization from water, the desired product 1040 was recovered as a white solid (12 mg, 31%). It contained 5% of compound 209. C in MS 53 H 59 FN9O 15 Calculated value: 1080.40, Measured value: 1080.66, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.50 (t, J = 6.2 Hz, 1H), 8.25 (m, 1H), 8.12 - 8.04 (m, 2H), 8.01 - 7.91 (m, 2H), 7.86 (s, 1H), 7.25 - 7.11 (m, 6H), 7.00 (s, 2H), 6.56 (s, 1H), 5.44 (m, 2H), 5.35 - 5.25 (m, 2H), 5.11 (m, 1H), 4.95 (m, 1H), 4.60 (m, 2H), 4.50 (m, 1H), 3.75 - 3.65 (m, 4H), 3.56 - 3.43 (m, 8H), 3.29 - 3.19 (m, 2H), 3.06 (m, 2H), 2.71 (m, 1H), 2.40 (m, 3H), 2.29 (m, 2H), 2.23 (m, 1H), 2.12 - 2.00 (m, 1H), 1.80 (m, 2H), 1.27 (d, J = 6.2 Hz, 3H), 0.89 (t, J = 7.7 Hz, 3H).

[0375] Example 132: Synthesis of Compound 211 [ka] Intermediate 1. To a solution of (S)-2-(benzyloxy)propan-1-ol (1.0 equiv., 100 mg, 0.6 mmol) and bis(4-nitrophenyl)carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (2 mL) was added diisopropylethylamine (1.5 equiv., 157 μL, 0.9 mmol), and the resulting mixture was stirred at room temperature for 2 h. The DMF was removed on a rotary evaporator, and the residue was purified by flash chromatography (SiO, 0–20% EtOAc / cyclohexane) to give the product Intermediate 1 (186 mg, 93%) as a clear, viscous oil.

[0376] Intermediate 2. To a mixture of Intermediate 1 (1.2 equiv., 60 mg, 0.18 mmol) and exatecan mesylate (1.0 equiv., 80 mg, 0.15 mmol), DMF (5 mL) and diisopropylethylamine (100 μL) were added, and the resulting solution was stirred at room temperature overnight. The DMF was removed by rotary evaporation, and purification by reverse-phase flash chromatography (diol-modified C18, 0 → 50% ACN / 10 mM NHOAc aqueous solution) afforded the product Intermediate 2 (90 mg, 96%) as a white solid after lyophilization. C in MS was 35 H 35 Calculated value for FN3O7: 628.24, Found value: 628.20 [M+H] + .

[0377] Compound 211. To a solution of intermediate 2 (1.0 equiv., 90 mg, 0.14 mmol) in dioxane (5 mL) was added 10% Pd / C, and the resulting mixture was hydrogenated (H in a balloon) for 5 h. The suspension was then filtered, and the filtrate was evaporated on a rotary evaporator. Purification of the residue by reversed-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→50% ACN / 10 mM NHOAc in water) afforded compound 211 (71 mg, 92%) as a white solid after lyophilization. C in MS 28 H 29 Calculated value for FN3O7: 538.19, Found value: 508.15, [M+H] + .

[0378] Example 133: Synthesis of Compound 1041 [ka] Intermediate 1. Compound 211 (70 mg, 0.13 mmol) and Fmoc-GGFG-OAc (65 mg, 0.10 mmol) were co-evaporated three times from anhydrous DMF and then redissolved in anhydrous DMF (4 mL). HCl (4 M in dioxane, 30 μL) was added, and the reaction mixture was stirred at 50° C. for 2 hours. The DMF was then removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→50% ACN / 10 mM NHOAc aqueous solution) to give product Intermediate 1 (32 mg, 29%) as a white solid. C in MS 59 H 60 FN8O 13 Calculated value: 1107.42, Found value: 1107.30, [M + H] + .

[0379] To a solution of Intermediate 1 (32 mg, 0.029 mmol) in 2.2 mL of anhydrous DMF, morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then the DMF was removed on a rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→100% ACN / 0.1% TFA) to give the product Intermediate 2 (15 mg, 59%) as a white solid. C in MS: 44 H 49 FN8O 11 Calculated value: 885.35, Found value: 885.20, [M + H] - .

[0380] Compound 1041. Intermediate 2 (15 mg, 0.017 mmol) was dissolved in 1 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equiv., 0.025 mmol, 7.9 mg) and DIPEA (5 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 (0→100% ACN / 10 mM NHOAc in water). After lyophilization from water, the desired product was recovered as a white solid (11 mg, 60%). C in MS was 0.01%. 53 H 59 FN9O 15 Calculated value: 1080.40, Measured value: 1080.35, [M + H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.49 (t, J = 6.6 Hz, 1H), 8.28 (q, J = 5.1 Hz, 1H), 8.10 (q, J = 4.7, 3.7 Hz, 2H), 7.99 (q, J = 7.0, 5.8 Hz, 2H), 7.78 (d, J = 10.8 Hz, 1H), 7.34 - 7.31 (m, 1H), 7.26 - 7.15 (m, 5H), 7.00 (s, 1H), 6.52 (s, 1H), 5.43 (d, J = 2.8 Hz, 2H), 5.25 (q, J = 6.9, 6.3 Hz, 3H), 4.66 - 4.61 (m, 2H), 4.48 (q, J = 8.8, 8.3 Hz, 1H), 4.12 - 4.00 (m, 1H), 3.98 (m, 1H), 3.82 (m, 1H), 3.77 - 3.63 (m, 5H), 3.63 - 3.48 (m, 5H), 3.46 (t, J = 5.8 Hz, 2H), 3.29 - 3.04 (m, 1H), 3.03 (m, 1H), 2.78 (dt, J = 13.8, 8.8 Hz, 1H), 2.38 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.23 - 2.12 (m, 2H), 1.95 - 1.81 (m, 2H), 1.13 (t, J = 6.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H).

[0381] Example 134: Synthesis of Compound 1042

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[0382] Intermediate 2. To a mixture of exatecan mesylate (30 mg, 0.056 mmol), Intermediate 1 (45 mg, 0.065 mmol), and DMTMM (31 mg, 0.113 mmol), DMF / water (5:1, 2.5 mL) and diisopropylethylamine (30 μL) were added. The resulting mixture was stirred at room temperature for 1 hour, and LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→100% ACN / 0.1% TFA), affording Intermediate 2 as a white solid (30 mg, 48%) after lyophilization. C in MS indicated a 0.01% C NMR spectrum. 58 H 56 F3N8O 12 Calculated value: 1113.39, Found value: 1113.40, [M+H] + .

[0383] To a solution of intermediate 2 (30 mg, 0.027 mmol) in 3.2 mL of anhydrous DMF, morpholine (150 μL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then the DMF was removed on a rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0→50% ACN / 0.1% TFA) to give product intermediate 3 (10 mg, 42%) as a white solid. C in MS:43 H 46 F3N8O 10 Calculated value: 891.32, Found value: 891.30, [M + H] + .

[0384] Compound 1042. Intermediate 3 (10 mg, 0.011 mmol) was dissolved in 1.5 mL of DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (0.011 mmol, 3.5 mg) and DIPEA (4 μL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC using a semi-preparative column containing a diol-modified C18 (0→100% ACN / 10 mM NHOAc in water). After lyophilization from water, the desired product was recovered as a white solid (6 mg, 50%). C in MS was 0.011 mmol. 52 H 55 F3N9O 14 Calculated value: 1086.37, Found value: 1086.40, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 9.50 (d, J = 8.4 Hz, 1H), 8.66 (t, J = 6.8 Hz, 1H), 8.32 (t, J = 5.8 Hz, 1H), 8.10 (dd, J = 6.9, 4.5 Hz, 2H), 7.98 (t, J = 5.8 Hz, 1H), 7.81 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 7.27 - 7.20 (m, 4H), 7.20 - 7.11 (m, 1H), 7.00 (s, 2H), 6.53 (s, 1H), 5.60 (dt, J = 8.5, 5.5 Hz, 1H), 5.49 - 5.38 (m, 2H), 5.29 - 5.05 (m, 2H), 4.72 (m, 2H), 4.51 (m, 1H), 4.04 - 3.91 (m, 2H), 3.76 - 3.72 (m, 2H), 3.66 (s, 1H), 3.61 - 3.57 (m, 3H), 3.52 (s, 1H), 3.46 (t, J = 5.5 Hz, 2H), 3.24 - 3.12 (m, 2H), 3.05 (m, 1H), 2.79 (m, 1H), 2.40 (d, J = 1.9 Hz, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.26 - 2.15 (m, J = 6.8 Hz, 2H), 1.87 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0385] Example 135: Synthesis of Compound 1044

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[0386] Intermediate 2. To a solution of Intermediate 1 (83 mg, 0.098 mmol) in anhydrous DMF (2 ml), morpholine (300 μL) was added, and the reaction mixture was stirred at room temperature for 1 h. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 50% ACN / H2O), and after lyophilization, the product was obtained as a white solid (34 mg, 56%). 31 H 32 Calculated for F2N5O7: 624.61, Found: 624.25, [M+H] + .

[0387] Intermediate 3. To a mixture of Intermediate 2 (34 mg, 0.055 mmol), FmocGGFGG-OH (37 mg, 0.06 mmol), and DMTMM (16.6 mg, 0.06 mmol), DMF / water (3:1, 2 mL) and diisopropylethylamine (60 μL) were added, and the resulting mixture was stirred at room temperature for 60 minutes. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0.50% ACN / HO). After lyophilization, Intermediate 3 was obtained as a white solid (43 mg, 64%). MS analysis revealed a C 63 H 62 F2N 10 O 14Calculated value: 1221.94, Found value: 1221.40, [M+H] + .

[0388] Intermediate 4. To a solution of intermediate 3 (25 mg, 0.020 mmol) in anhydrous DMF (1.5 ml), morpholine (40 μL) was added and the reaction mixture was stirred at room temperature for 1 h. LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 50% ACN / H2O), and after lyophilization, the product was obtained as a white solid (15 mg, 75%). 48 H 53 F2N 10 O 12 Calculated value: 999.00, Measured value: 999.35, [M+H] + .

[0389] Compound 1044. Mal-PEG-NHS ester (15 mg, 0.014 mmol) and DIPEA (20 μL) were added to a solution of intermediate 4 (4.2 mg, 0.014 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 hour, and LC-MS showed that the starting material was completely consumed. The mixture was directly purified by reverse-phase flash HPLC using a semi-preparative column (diol-modified C18, 0.50% ACN / H2O), and after lyophilization, compound 1044 was obtained as a white solid (2.5 mg, 15%). C in MS 57 H 62 F2N 11 O 16 Calculated: 1194.17, Found: 1194.45, 15-54 [M+H] + .

[0390] Example 136: Synthesis of Compound 1045 [ka] Intermediate 1. FmocGGFG-OAc (1 equiv., 0.0878 mmol, 54 mg) was dissolved in 2 mL of DMF and 5-(hydroxymethyl)-1H-pyrazole-3-carboxylic acid (1.1 equiv., 0.0966 mmol, 14 mg) was added, followed by 10 μL of 4 M HCl in dioxane. The reaction mixture was stirred at room temperature for 2 hours and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / HO). The product-containing fractions were lyophilized from water. The product contained some impurities due to peptide degradation (72 mg). C in MS was 0.01%. 36 H 38 Calculated for N7O9: 712.27, Found: 712.24, [M+H] + .

[0391] Intermediate 2. To a solution of the previously prepared intermediate 1 (72 mg, 0.101 mmol) in DMF (2 mL) was added exatecan mesylate (1 equiv., 0.101 mmol, 54 mg), DMTMM (1.3 equiv., 0.132 mmol, 36 mg), DIPEA (50 μL), and water (1 mL). The reaction mixture was stirred at room temperature for 1 hour and then loaded directly onto a purification column. Purification was carried out by reverse-phase flash chromatography (25 g, diol-modified C18, 0→100% ACN / 0 / 1% TFA). The product-containing fractions were lyophilized from water (52 mg, 46%). C in MS was 0.01%. 60 H 58 FN 10 O 12 Calculated value: 1129.42, Found value: 1129.51, [M+H] + .

[0392] Intermediate 3. The previously prepared intermediate 2 (52 mg, 0.0461 mmol) was dissolved in DMF (2 mL) and morpholine (100 μL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→100% ACN / 0.1% TFA). The product-containing fractions were lyophilized from water (20 mg, 48%). C in MS was 45 H48 FN 10 O 10 Calculated value: 907.35, Found value: 907.69, [M+H] + .

[0393] Compound 1045. The previously prepared intermediate 3 (20 mg, 0.0221 mmol) was dissolved in 2 mL of anhydrous DMF. 2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.2 equiv., 0.0265 mmol, 8.2 mg) and DIPEA (5 μL) were added, and the reaction mixture was stirred at room temperature for 1 h. Purification was carried out by reverse-phase HPLC chromatography (semi-preparative, diol-modified C18, 0→100% ACN / 0.1% TFA). The product-containing fractions were lyophilized from water (13 mg, 53%). C in MS was 0.01%. 54 H 57 FN 11 O 14 Calculated value: 1102.41, Found value: 1102.43, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 9.13 (d, J = 8.5 Hz, 1H), 8.90 - 8.81 (m, 2H), 8.61 (t, J = 6.3 Hz, 1H), 8.31 (m, 3H), 8.09 (m, 2H), 7.97 (m, 2H), 7.82 (q, J = 10.7, 9.8 Hz, 1H), 7.32 (s, 1H), 7.21 (m, 3H), 7.00 (s, 2H), 6.52 (s, 2H), 5.87 (dd, J = 13.3, 6.6 Hz, 1H), 5.78 - 5.68 (m, 2H), 5.48 (m, 2H), 5.39 (d, J = 4.0 Hz, 1H), 5.29 - 5.13 (m, 2H), 4.68 (s, 1H), 4.54 - 4.47 (m, 1H), 4.40 (s, 2H), 3.75 (m, 4H), 3.66 (d, J = 5.7 Hz, 3H), 3.62 - 3.48 (m, 2H), 3.16 - 2.97 (m, 2H), 2.78 (td, J = 13.9, 9.6 Hz, 1H), 2.40 (d, J = 6.4 Hz, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.28 - 2.24 (m, 1H), 1.85 (dq, J = 14.1, 7.1 Hz, 2H), 1.24 (s, 1H), 0.87 (t, J = 7.5 Hz, 3H).

[0394] Example 137: Synthesis of Compound 1046

change

[0395] Example 138: Synthesis of Compound 1047

change

[0396] Intermediate 3. To a solution of Intermediate 2 (1.0 equiv., 47 mg, 0.038 mmol) in DMF (1.75 ml), morpholine (125 μL) was added and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated on a rotary evaporator and purified by reverse-phase flash chromatography (diol-modified C18, 0→50% ACN / 0.1% TFA in water), affording Intermediate 3 as a pale yellow solid (27 mg, 64%) after lyophilization. MS analysis revealed a C 50 H 59 FN9O 12 Calculated value: 996.43, Found value: 996.30, [M+H] + .

[0397] Intermediate 4. A solution of Intermediate 3 (1.0 equiv., 27 mg, 0.024 mmol), Intermediate 5 (1.0 equiv., 7 mg, 0.024 mmol), and diisopropylethylamine (2.1 equiv., 9.5 μl, 0.050 mmol) in anhydrous DMF (0.75 ml) was stirred at room temperature for 40 minutes, after which LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash chromatography (diol-modified C18, 0→60% ACN / 10 mM aqueous NHOAc) afforded the product Intermediate 4 as a white solid (20 mg, 73%) after lyophilization. The C in MS was 0.05%. 58 H 69 FN9O 14 Calculated value: 1134.49, Found value: 1134.35, [M+H] + .

[0398] Compound 1047. To a solution of Dess-Martin periodinane (1.3 equiv., 9.7 mg, 0.023 mmol) in anhydrous acetonitrile (2.5 ml) was added a solution of intermediate 4 (1.0 equiv., 20 mg, 0.018 mmol) in anhydrous DMF (1 ml). The resulting mixture was stirred at room temperature for 3 hours, at which point LC-MS showed complete consumption of the starting material. The reaction mixture was concentrated on a rotary evaporator and purified by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→60% ACN / 10 mM NHOAc aqueous solution). After lyophilization, compound 1047 was obtained as a white solid (15 mg, 74%). C in MS was 0.001%. 58 H 67 FN9O 14 Calculated value: 1132.48, Found value: 1132.35, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 8.71 (d, J = 8.8 Hz, 1H), 8.42 (t, J = 6.8 Hz, 1H), 8.25 (t, J = 5.9 Hz, 1H), 8.14 (t, J = 5.8 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.95 (t, J = 5.7 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 7.25 - 7.17 (m, 4H), 7.15 (t, J = 6.9 Hz, 1H), 6.51 (s, 1H), 5.55 (q, J = 6.1 Hz, 1H), 5.49 - 5.37 (m, 2H), 5.18 (s, 2H), 4.59 - 4.41 (m, 3H), 3.77 - 3.65 (m, 5H), 3.64 - 3.53 (m, 4H), 3.46 (dd, J = 10.7, 5.9 Hz, 1H), 3.36 (t, J = 6.0 Hz, 2H), 3.29 - 3.08 (m, 4H), 3.02 (dd, J = 14.0, 4.5 Hz, 1H), 2.81 - 2.66 (m, 2H), 2.43 - 2.35 (m, 5H), 2.31 and 2.28 (s, 3H), 2.21 - 2.07 (m, 3H), 1.94 - 1.78 (m, 2H), 1.61 - 1.46 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H), 0.87 (m, 4H), 0.81 - 0.72 (m, 1H).

[0399] Example 139: Synthesis of compound 3113: Compound 3113 was prepared according to Procedure C in the General Methods section of Example 1 using 34 molar equivalents of linker payload compound 1025 relative to anti-EGFR IgG1 monoclonal antibody 5. The DAR based on RP-HPLC was 7.6.

[0400] Example 140: Synthesis of Compound 3053B Compound 3053B was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1007 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 7.6.

[0401] Example 141: Synthesis of Compound 3102 Compound 3102 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1046 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 7.7.

[0402] Example 142: Synthesis of Compound 3103 Compound 3103 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1047 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 3.6.

[0403] Example 143: Synthesis of Compound 3104 Compound 3104 was prepared according to Procedure C in the General Methods section of Example 1 using 25 molar equivalents of the linker payload of compound 1045 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 7.8.

[0404] Example 144: Synthesis of Compound 3105 Compound 3105 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1044 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC-MS was 7.6 based on the drug's molecular weight of 1194.17.

[0405] Example 145: Synthesis of Compound 3107 Compound 3107 was prepared according to Procedure C in the General Methods section of Example 1 using 25 molar equivalents of the linker payload of compound 1042 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 7.7.

[0406] Example 146: Synthesis of Compound 3059B Compound 3059B was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1017 relative to anti-EGFR IgG1 monoclonal antibody 1. The DAR based on RP-HPLC was 7.7.

[0407] Example 147: Synthesis of Compound 3108 Compound 3108 was prepared according to Procedure C in the General Methods section of Example 1 using 22 molar equivalents of the linker payload of compound 1025 relative to anti-EGFR IgG1 monoclonal antibody 2. The DAR based on RP-HPLC was 7.5.

[0408] Example 148: Synthesis of Compound 3110 Compound 3110 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1025 relative to anti-EGFR IgG1 monoclonal antibody 3. The DAR based on RP-HPLC was 7.8.

[0409] Example 149: Synthesis of Compound 3111 Compound 3111 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1025 relative to the anti-EGFR IgG1 monoclonal antibody 4. The DAR based on RP-HPLC was 7.6 and the DAR based on RPHPLC-MS was 7.8.

[0410] Example 150: Synthesis of Compound 3109 Compound 3109 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1007 relative to anti-EGFR IgG1 monoclonal antibody 3. The DAR based on RP-HPLC was 7.7.

[0411] Example 151: Synthesis of Compound 3112 Compound 3112 was prepared according to Procedure C in the General Methods section of Example 1 using 30 molar equivalents of the linker payload of compound 1007 relative to the anti-EGFR IgG1 monoclonal antibody 4. The DAR based on RP-HPLC was 7.6 and the DAR based on RPHPLC-MS was 7.8.

[0412] Example 152: Synthesis of Compound 1043: [ka] Intermediate 1. To a solution of Fmoc-GE(OBn)VCit-NH-CH2-OAc (1.0 equiv., 220 mg, 0.260 mmol) and ethylene glycol (3.0 equiv., 45 μL, 0.782 mmol) in anhydrous DMF (6 mL), 2M HCl / Et2O (450 μL) was added, and the resulting mixture was stirred at room temperature for 2 hours. LC-MS analysis indicated complete consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 0→60% ACN / 0.1% HCl), affording Intermediate 1 as a white solid (120 mg, 55%) after lyophilization. MS analysis revealed a C 43 H 56 N7O 11 Calculated value: 846.40, Found value: 846.35, [M+H] + .

[0413] Intermediate 2. A solution of 4-nitrophenyl chloroformate (3.0 equiv., 86 mg, 0.426 mmol) in anhydrous THF (1.5 ml) was added dropwise to an ice-cold solution of Intermediate 1 (1.0 equiv., 120 mg, 0.142 mmol) in anhydrous pyridine (4 ml). The resulting mixture was stirred at 0 °C for 1 h, at which point LC-MS analysis indicated complete consumption of the starting material. The solvent was removed on a rotary evaporator, and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 0 → 75% ACN / 10 mM aqueous NHOAc) to give Intermediate 2 as a pale yellow solid (50 mg, 35%) after lyophilization. C in MS was 0.01%. 50 H 59 N8O 15 Calculated value: 1011.41, Found value: 1011.35, [M+H] + .

[0414] Intermediate 3. Exatecan mesylate (1.0 equiv., 0.049 mmol, 26 mg) and DIPEA (4.0 equiv., 0.049 mmol, 34 μL) were added to a solution of Intermediate 2 (1.0 equiv., 50 mg, 0.049 mmol) in anhydrous DMF (3 ml). The reaction mixture was stirred at room temperature for 40 hours, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash chromatography (diol-modified C18, 0→75% ACN / 10 mM NHOAc aqueous solution) afforded Intermediate 3 as a white solid (48 mg, 75%) after lyophilization. The C in MS showed no significant difference. 68 H 76 FN 10 O 16 Calculated value: 1307.54, Found value: 1307.55, [M+H] + .

[0415] Intermediate 4. To a solution of intermediate 3 (1.0 equiv., 48 mg, 0.036 mmol) in a mixture of dioxane / water (2:1, 2.5 ml) was added 10% Pd / C (5 mg), and the reaction mixture was hydrogenated (balloon) at room temperature for 1 hour. The mixture was then filtered through a pad of Celite, the solid was washed with DMF, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was dissolved in DMF (1.5 ml), followed by the addition of morpholine (150 μl). The resulting solution was stirred at room temperature for 40 minutes. Purification by reverse-phase flash chromatography (diol-modified C18, 0→50% ACN / 10 mM NHOAc in water) afforded intermediate 4 as a white solid (15 mg, 42%) after lyophilization. C in MS was 0.05%. 46 H 60 FN 10 O 14 Calculated value: 995.43, Found value: 995.35, [M+H] + .

[0416] Compound 1043. Mal-PEG-NHS ester (1.05 equiv., 0.016 mmol, 4.9 mg) and DIPEA (2.1 equiv., 0.032 mmol, 5.6 μL) were added to a solution of intermediate 4 (1.0 equiv., 15 mg, 0.015 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 hour, and LC-MS showed complete consumption of the starting material. Purification by reverse-phase flash chromatography using a semi-preparative column (diol-modified C18, 0→50% ACN / 10 mM NHOAc) afforded 1043 as a white solid after lyophilization (12 mg, 67%). C in MS was 0.05 equiv. 55 H 69 FN 11 O 18 Calculated value: 1190.48, Found value: 1190.45, [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ: 12.05 (s, 1H), 8.63 (t, J = 6.7 Hz, 1H), 8.08 - 7.96 (m, 4H), 7.77 (t, J = 10.1 Hz, 2H), 7.31 (s, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.96 - 5.92 (m, 1H), 5.47 - 5.33 (m, 4H), 5.29 - 5.16 (m, 2H), 4.57 (ddd, J = 34.3, 10.5, 6.7 Hz, 2H), 4.33 (td, J = 8.1, 5.2 Hz, 1H), 4.24 - 4.07 (m, 4H), 3.78 - 3.63 (m, 2H), 3.62 - 3.51 (m, 6H), 3.46 (t, J = 5.8 Hz, 2H), 3.30 - 3.18 (m, 1H), 3.18 - 3.03 (m, 1H), 3.01 - 2.86 (m, 2H), 2.37 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.28 - 2.10 (m, 4H), 2.03 - 1.79 (m, 4H), 1.78 - 1.66 (m, 1H), 1.66 - 1.55 (m, 1H), 1.56 - 1.44 (m, 1H), 1.45 - 1.23 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H), 0.82 (dd, J = 11.6, 6.7 Hz, 6H).

[0417] Example 153: Antibody extracts, including HER2, TROP2 and EGFR, show killing effect on homogeneous cells. To determine the relative cell killing potency of the disclosed ADCs compared to a similar ADC (DXD), cell killing assays were performed in multiple cell lines expressing HER2, TROP2, and EGFR. NCI-N87 and SK-BR-3 cell lines were used to test the anti-HER-2 ADCs. MDA-MB-468 and FaDu cell lines were used to test the anti-TROP2 ADCs. MDA-MB-468, as well as HCC827, NCI-H292, FaDu, and OVCAR3 cell lines were used to test the anti-EGFR ADCs.

[0418] NCI-N87, MDA-MB-468, HCC827, NCI-H292, and OVCAR3 cells were cultured in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Corning). SK-BR-3 cells were maintained in McCoys 5A medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Corning) at 37°C in a humidified incubator containing 5% CO. FaDu cells were maintained in EMEM medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Corning) at 37°C in a humidified incubator containing 5% CO.

[0419] Cancer cell viability in the presence of ADCs was measured in a series of in vitro assays. HER2- and TROP2-expressing cells were plated in 384-well white flat-bottom plates (Corning) at 0.5 × 10 cells per well in 30 μL of medium. 3 For EGFR-expressing cells, cells were plated at 2 × 10 per well in 100 μL of medium in a 96-well white flat-bottom plate (Corning). 3Cells were plated at 100°C / 1000°C. ADCs were added in quadruplicate at a concentration range of eight serial dilutions. After an additional 6 days of incubation at 37°C and 5% CO2, cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using a SpectraMax iD3 plate reader (Molecular Devices). Luminescence values ​​were plotted against the logarithmic concentration of the test compound, and cell viability was calculated by dividing the luminescence values ​​at different antibody-drug conjugate concentrations by the luminescence value at zero antibody concentration. Dose-response IC50 values ​​of the ADCs were calculated as the best fit using a four-parameter dose-response curve fit in GraphPad Prism. R-squared values ​​ranged from 0.97 to 0.999. Each IC50 value from two to nine independent experiments was averaged, and the respective means and respective standard deviations between independent experiments were calculated. Each minimum cell viability from two to nine independent experiments was averaged, and the respective means and respective standard deviations between independent experiments were calculated. The calculated average IC50 values ​​and average cell killing rates are shown in Tables 7 to 9 together with their respective standard deviations.

[0420] Table 7A shows the percent cell viability of NCI-N87 cells after exposure to different anti-HER2 ADCs of the present disclosure. Table 7B shows the percent cell viability of SK-BR-3 cells after exposure to different anti-HER2 ADCs of the present disclosure. Table 7C shows the percent cell viability of NCI-N87 cells after exposure to various exatecan-releasing anti-HER2 ADCs described herein. Table 7D shows the percent cell viability of SK-BR-3 cells after exposure to various exatecan-releasing anti-HER2 ADCs described herein. IC50 ADC (nM) was calculated based on the molecular weight of the monoclonal antibody (mAb) and the linker payload conjugated to the mAb, where ADC molecular weight (g / mol) = mAb molecular weight (g / mol) + DAR × linker payload molecular weight (g / mol). IC50 linker payload (nM) was calculated from the concentration of linker payload delivered by the ADC (calculated as ADC (nM) multiplied by DAR). [Table 8] [Table 9] [Table 10] [Table 11]

[0421] Table 8A shows the percent cell viability of FaDu cells after exposure to different anti-TROP2 ADCs of the present disclosure. Table 8B shows the percent viability of MDA-MB-468 cells after exposure to different anti-TROP2 ADCs of the present disclosure. Table 8C shows the percent cell viability of FaDu cells after exposure to various exatecan-releasing anti-TROP2 ADCs described herein. Table 8D shows the percent cell viability of MDA-MB-468 cells after exposure to various exatecan-releasing anti-TROP2 ADCs described herein.

[0422] The IC50 ADC (nM) was calculated based on the molecular weight of the monoclonal antibody (mAb) and the linker payload conjugated to the mAb: ADC molecular weight (g / mol) = mAb molecular weight (g / mol) + DAR × linker payload molecular weight (g / mol). The IC50 linker payload (nM) was calculated from the concentration of linker payload delivered by the ADC (calculated as ADC (nM) multiplied by DAR). [Table 12] [Table 13] [Table 14] [Table 15]

[0423] Table 9A shows the percent cell viability of MDA-MB-468 cells after exposure to different anti-EGFR ADCs of the present disclosure. Table 9B shows the percent viability of HCC827 cells after exposure to different anti-EGFR ADCs of the present disclosure. Table 9C shows the percent cell viability of MDA-MB-468 cells after exposure to various exatecan-releasing anti-EGFR ADCs described herein. Table 9D shows the percent cell viability of HCC827 cells after exposure to various exatecan-releasing anti-EGFR ADCs described herein. Table 9E shows the percent cell viability of cell lines NCI-H292, OVCAR3, and FaDu cells after exposure to different anti-EGFR ADCs of the present disclosure. The IC50 (nM) of an ADC was calculated based on the molecular weight (MW) of the monoclonal antibody (mAb) and the linker payload, where ADC molecular weight (g / mol) = mAb molecular weight (g / mol) + DAR × linker payload molecular weight. [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0424] Example 154: Cytotoxicity of Therapeutic Payloads (nmol / L) cell line Human tumor cell lines, SK-BR-3, NCI-H292, HT-29, MCF-7, NCI-N87, and FaDu, were obtained from ATCC. NCI-H292, HT-29, MCF-7, NCI-N87, MDA-MB-468, and HCC827 cells were cultured in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Corning). FaDu cells were maintained in EMEM medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Corning) at 37°C in a humidified incubator containing 5% CO. SK-BR-3 cells were maintained in McCoys 5A medium (Gibco, Life Technologies) supplemented with 10% v / v heat-inactivated FBS (Corning) at 37°C in a humidified incubator containing 5% CO .

[0425] Preparation of compounds For compounds 71, 72, 73, 74, 180, 193, 194, 195, 208, and 130, the lyophilized compounds were dissolved in 100% dehydrated DMSO, and aliquots were frozen and stored at -80 °C. The concentrations of the DMSO stock solutions of the compounds were determined by RP-HPLC on an Agilent 1100 platform equipped with a 1200 DAD and a SofTA ELSD detector. A Shimadzu 3.0 mm x 30 mm XR ODS 2.2 μm column was run at 50 °C and 1.5 mL / min. Solvent A: 0.1% formic acid / water, Solvent B: 0.08% formic acid / methanol gradient: 5% to 100% B in 3.0 min, 100% solvent B in 0.3 min. The concentrations of the compounds in 100% DMSO determined by ELS ranged from 1 to 6 mM. For compounds 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, the lyophilized compounds were dissolved in 100% dehydrated DMSO, and aliquots were frozen and stored at −80° C. Concentrations were calculated as W / V.

[0426] Cytotoxicity assay Cells were plated in 96-well white flat-bottom plates (Corning) at 2.0 × 10 cells per well in 100 μL of medium. 3 Alternatively, cells were plated at 0.5 × 10 cells per well in 30 μL of medium in a 384-well white flat-bottom plate (Corning). 3 After 24 hours of incubation, test compounds were added in duplicate or triplicate at a concentration range of 10-point serial dilutions. After an additional 6 days of incubation at 37°C and 5% CO2, cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using a GloMax instrument (Promega). Luminescence values ​​were plotted against the logarithmic concentration of test compound, and IC50 values ​​were calculated using GraphPad Prism 9 as the best fit using a four-parameter dose-response curve fit. R-squared values ​​ranged from 0.97 to 0.999. For a subset of payloads, each treatment was independently replicated two to eight times, and IC50 values ​​were averaged.

[0427] Table 10 shows the IC50 cytotoxicity values ​​(nmol / L) of the therapeutic payloads described herein against multiple tumor cell lines. The mean and standard deviation (stdev) of IC50 values ​​were determined using replicate treatments from two to eight independent experiments. The standard deviation for a single treatment is indicated as (N / A). [Table 21-1] [Table 21-2]

[0428] Example 155: Tumor growth inhibitory effects of anti-TROP2 antibody-drug conjugates 3038, 3036, and 3031 on human lung cancer NCI-H292 The NCI-H292 human lung cancer cell line was purchased from ATCC. Six to eight-week-old female athymic nude mice (Jackson Labs NU / J #2019) were inoculated into the hind flank with 5 × 10 cells suspended 1:1 with Matrigel in serum-free medium. 6 NCI-H292 cells were subcutaneously inoculated. 3 Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 8). Each anti-TRO2 antibody-drug conjugate was administered intravenously to the tail of each mouse at a single dose of 3 mg / kg. (In the figure, the day of administration initiation is designated as day 0. Arrows indicate the timing and frequency of dose administration.) The control group received phosphate-buffered saline (PBS). Mean tumor volumes were plotted for each group until either the first death or the end of the study. Error bars represent the standard error of the mean. Figure 1A shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3031 administration compared to the vehicle control group. Figure 1B shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3036 administration. Figure 1C shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3038 administration.

[0429] Example 156: Tumor growth inhibitory effect of anti-EGFR antibody-drug conjugates 3058A and 3053B on the human pharyngeal carcinoma cell line FaDu The FaDu human laryngeal carcinoma cell line was purchased from ATCC. Six to eight-week-old female athymic nude mice (Jackson Labs NU / J #2019) were inoculated into the hind flank with 5 × 10 cells suspended 1:1 with Matrigel in serum-free medium. 6 NCI-H292 cells were subcutaneously inoculated. 3Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 10). Each anti-EGFR antibody-drug conjugate was administered intravenously to the tail of each mouse at a dose of 3 mg / kg, a total of three times at weekly intervals (the first day of administration is designated as day 0 in the figure; arrows indicate the timing and frequency of dose administration). The control group received phosphate-buffered saline (PBS). Mean tumor volumes were plotted for each group until either the first death or the end of the study. Error bars represent the standard error of the mean. Figure 2A shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3058A administration compared to the vehicle control group. In the antibody-drug conjugate 3058A group, tumors were not observed in 5 / 5 mice. Figure 2B shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3053B administration. In the antibody-drug conjugate 3053B group, tumors were not observed in 5 / 5 mice.

[0430] Example 157: Tumor growth inhibitory effects of anti-EGFR antibody-drug conjugates 3058A, 3058B, 3102, and 3053B on human lung cancer cell line NCI-H1975 The NCI-H1975 human lung cancer cell line was purchased from ATCC. Six to eight-week-old female athymic nude mice (Jackson Labs NU / J #2019) were inoculated into the hind flank with 5 × 10 cells suspended 1:1 with Matrigel in serum-free medium. 6 NCI-H1975 cells were subcutaneously inoculated. 3Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 15). Each anti-EGFR antibody-drug conjugate was administered intravenously to the tail of each mouse at a dose of 10 mg / kg, a total of three times at weekly intervals (the day of administration is designated as day 0 in the figure; arrows indicate the timing and frequency of dose administration). The control group received phosphate-buffered saline (PBS). Mean tumor volumes were plotted for each group until either the first death or the end of the study. Error bars represent the standard error of the mean. Figure 3A shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3058A administration compared to the vehicle control. In the antibody-drug conjugate 3058A group, tumors were absent in 3 / 5 mice. Figure 3B shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3058B administration compared to the vehicle control. In the antibody drug conjugate 3058B group, 1 / 5 mice were tumor-free. Figure 3C shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3102 administration compared to vehicle control. In the antibody drug conjugate 3102 group, 3 / 5 mice were tumor-free. Figure 3D shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3053B administration compared to vehicle control. In the antibody drug conjugate 3053B group, 2 / 5 mice were tumor-free.

[0431] Example 158: Tumor growth inhibitory effects of anti-EGFR antibody-drug conjugates 3053B, 3058A, and 3102 on the human breast cancer cell line MDA-MB-468 The MDA-MB-468 human breast cancer cell line was purchased from Accegen. Six to eight-week-old female NOD-SCID mice (Charles River Labs, strain code #394) were implanted in the hind flank with 5 × 10 cells suspended in 10% Matrigel in serum-free medium. 6 MDA-MB-468 cells were subcutaneously inoculated. 3Once the mice reached 12 days of age, they were randomly assigned to each group (5 mice per group, day 12). Each anti-EGFR antibody-drug conjugate was administered intravenously to the tail of mice once at a dose of 3 mg / kg. The day of administration is designated as day 0, as indicated by the arrow in the figure. The control group received phosphate-buffered saline (PBS). Mean tumor volume was plotted for each group until either the first death or the end of the study. Error bars represent the standard error of the mean. Figure 4A shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3053B administration compared to the vehicle control. In the antibody-drug conjugate 3053B group, tumors were absent in 4 / 5 mice. Figure 4B shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3058A administration compared to the vehicle control. In the antibody-drug conjugate 3058A group, tumors were absent in 5 / 5 mice. Figure 4C shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3102 administration compared to vehicle control. In the antibody drug conjugate 3102 group, 3 / 5 mice were tumor-free.

[0432] Example 159: Tumor growth inhibitory effects of anti-EGFR antibody-drug conjugates 3058A, 3102, 3059B, 3108, 3110, and 3053B on the human pharyngeal carcinoma cell line FaDu The FaDu human laryngeal carcinoma cell line was purchased from ATCC. 6- to 8-week-old female NOD-SCID mice (Charles River Labs, strain code #394) were inoculated into the hind flank with 5 × 10 cells suspended in 10% Matrigel in serum-free medium. 6 FaDu cells were subcutaneously inoculated. The tumor size was approximately 100-200 mm. 3Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 9). Each anti-EGFR antibody-drug conjugate was intravenously administered to each mouse in the tail at a dose of 1 mg / kg, a total of three times at weekly intervals (the day of administration is designated as day 0 in the figure; arrows indicate the timing and frequency of dose administration). The control group received phosphate-buffered saline (PBS). Mean tumor volumes were plotted for each group until either the first death or the end of the study. Error bars represent the standard error of the mean. Figure 5A shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3058A administration compared to the vehicle control. Figure 5B shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3102 administration compared to the vehicle control. Figure 5C shows the mean tumor volume as a function of time after initiation of antibody-drug conjugate 3059B administration compared to the vehicle control. Figure 5D shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3108 administration compared to vehicle control. Figure 5E shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3110 administration compared to vehicle control. Figure 5F shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3053B administration compared to vehicle control. The compounds exemplified herein show greater tumor inhibition than the controls.

[0433] Example 160: Tumor growth inhibitory effects of anti-EGFR antibody drug conjugates 3058A, 3053B, 3102, and 3059B on an in vivo bystander model using the human breast cancer cell line MDA-MB-468 co-inoculated with the human colon adenocarcinoma cell line SW620-luc MDA-MB-468 human breast cancer cell line was purchased from Accegen. SW620-luc (luciferase) cells were purchased from PhoenixBIO. Six to eight-week-old female NOD-SCID mice (Charles River Labs, strain code #394) were implanted in the right hind flank with 5 × 10 cells suspended in 10% Matrigel in serum-free medium. 6 5 x 10 MDA-MB-468 cells and 5 x 10 6SW620-luc cells were inoculated subcutaneously. 5 × 10 cells suspended in 10% Matrigel in serum-free medium were inoculated subcutaneously. 6 SW620-luc cells were subcutaneously inoculated into the left hind flank. The tumor size in the right hind flank was approximately 100-200 mm. 3 Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 9). Each anti-EGFR antibody-drug conjugate was administered intravenously to the tail of the mice at a dose of 3 mg / kg. The day of administration is designated as day 0, as indicated by the arrow in the figure. The control group received phosphate-buffered saline (PBS). Mean tumor volume was measured with a caliper and plotted until the first death or the end of the study in each group. Error bars represent the standard error of the mean. Bioluminescence imaging was performed weekly from the start of administration. Each mouse was intraperitoneally injected with 15 mg of VivoGlo luciferin (Promega) and imaged using an IVIS Lumina S5 System (Perkin Elmer). Bioluminescence signals were analyzed using Living Image Software (Perkin Elmer), and total flux was expressed as photons per second. Figure 6A shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3058A administration compared to vehicle control. In the antibody drug conjugate 3058A group, 5 / 5 mice were tumor-free. Figure 6B shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3053B administration compared to vehicle control. In the antibody drug conjugate 3053B group, 4 / 5 mice were tumor-free. Figure 6C shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3102 administration compared to vehicle control. In the antibody drug conjugate 3102 group, 5 / 5 mice were tumor-free. Figure 6D shows the mean tumor volume as a function of time after initiation of antibody drug conjugate 3059B administration compared to vehicle control. In the antibody drug conjugate 3059B group, 3 / 5 mice were tumor-free.

[0434] Figure 7A shows the change in luciferase activity as a function of time after initiation of administration of antibody drug conjugate 3058A compared to a vehicle control. Figure 7B shows the change in luciferase activity as a function of time after initiation of administration of antibody drug conjugate 3053B compared to a vehicle control. Figure 7C shows the change in luciferase activity as a function of time after initiation of administration of antibody drug conjugate 3102 compared to a vehicle control. Figure 7D shows the change in luciferase activity as a function of time after initiation of administration of antibody drug conjugate 3059B compared to a vehicle control.

[0435] Example 161: Tumor growth inhibitory effects of anti-EGFR antibody-drug conjugates 3111, 3112, 3110, and 3109 on the human pharyngeal carcinoma cell line FaDu The FaDu human laryngeal carcinoma cell line was purchased from ATCC. 6- to 8-week-old female NOD-SCID mice (Charles River Labs, strain code #394) were inoculated into the hind flank with 5 × 10 cells suspended in 10% Matrigel in serum-free medium. 6 FaDu cells were subcutaneously inoculated. The tumor size was approximately 100-200 mm. 3 Once the mice reached maturity, they were randomly assigned to each group (5 mice per group, day 9). Each anti-EGFR antibody-drug conjugate was administered intravenously to the tail of each mouse at a dose of 10, 3, or 1 mg / kg, once or three times at weekly intervals (in the figure, the first day of administration is designated as day 0; arrows indicate the timing and frequency of dose administration). The control group re...

Claims

1. Formula IA or Formula IB: 【Chemistry 1】 or a pharmaceutically acceptable salt or stereoisomer thereof. (In the formula, Lig is a targeting moiety; L is a linker moiety; R is -R １ , —C(O)—R １ -, -C(O)-OR １ -, -C(O)-NH-R １ -, -C(O)-C ０－３ Alkyl-C(O)-NH-R １ -, -C(O)-(5- to 6-membered heteroaryl)-R １ -, -CH ２ -(5- to 6-membered heteroaryl)-R １ -, -C(O)-(4- to 6-membered heterocyclyl)-, -C(O)-(4- to 6-membered heterocyclyl)-C(O)-R １ -, -C(O)-(4- to 6-membered heterocyclyl)-NH-R １ -, -C(O)-C ３－４ Cycloalkyl-R １ -, -C(S)-C ３－４ Cycloalkyl-R １ -, and -C(O)-O-phenyl-R １ -, wherein any of said heteroaryl, heterocyclyl, alkyl, and cycloalkyl may be optionally substituted with one, two, or three substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R １ is CH ２ -CH ２ O-, -CH ２ -O-, -NH-, -CH ２ ONH- and -CH(CH ３ )—NH—; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, 5, 6, 7, or 8).

2. The drug conjugate of claim 1 , wherein Lig is a monoclonal antibody.

3. The drug conjugate of claim 1 or 2, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, an anti-HER2 antibody, an anti-B7-H3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, and an anti-CD70 antibody.

4. The drug conjugate of any one of claims 1 to 3, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.

5. The drug conjugate of any one of claims 1 to 4, wherein s is 1 or 8.

6. L, 【Chemistry 2】 is selected from the group consisting of where * represents the point of attachment to R. The drug conjugate according to any one of claims 1 to 5.

7. R is -CH ２ CH ２ O-, -C(O)-CH ２ ONH-, -C(O)-O-CH ２ CH ２ O-, -C(O)-NH-CH ２ CH ２ O-, -C(O)-C １ Alkyl-C(O)-NH-CH ２ CH ２ O-, -C(O)-C ３ Alkyl-C(O)-NH-CH ２ CH ２ O-, and -C(O)-CH(CH ３ 7. The drug conjugate of claim 1, wherein the compound is selected from the group consisting of —NH—.

8. R is —C(O)-triazolyl-CH ２ CH ２ O-, -CH ２ -triazolyl-CH ２ CH ２ O—, and —C(O)-furanyl-CH ２ The drug conjugate of any one of claims 1 to 6, wherein the conjugate is selected from the group consisting of: O-

9. R is —C(O)—C ３ Cycloalkyl-CH ２ CH ２ O-, -C(S)-C ３ Cycloalkyl-CH ２ CH ２ O-, -C(O)-C ３ The drug conjugate of any one of claims 1 to 6, wherein the conjugate is selected from the group consisting of cycloalkyl-NH-, and -C(O)-O-phenyl-NH-.

10. R is —C(O)-pyrrolidinyl- and —C(O)-pyrrolidinyl-C(O)—CH(CH ３ 7. The drug conjugate of any one of claims 1 to 6, wherein the pyrrolidinyl is selected from the group consisting of -NH-, wherein the pyrrolidinyl is optionally substituted with one or two fluoro atoms.

11. R is, 【Transformation 3】 is selected from the group consisting of wherein ** represents the point of attachment to L. The drug conjugate according to any one of claims 1 to 10.

12. the drug conjugate is 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 [Chemistry 4-5] [Chemistry 4-6] or a pharmaceutically acceptable salt or stereoisomer thereof; wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody. The drug conjugate according to any one of claims 1 to 11.

13. The drug conjugate according to any one of claims 1 to 12, wherein Lig is an anti-TROP2 antibody.

14. The drug conjugate of any one of claims 1 to 12, wherein Lig is an anti-EGRF antibody.

15. The drug conjugate of any one of claims 1 to 12, wherein Lig is an anti-HER2 antibody.

16. Formula II: 【Transformation 5】 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or —C １－３ is alkyl, R is —C(O)—C ３ Alkyl, —C(O)O—C ３ Alkyl, —C(O)—C ３ Cycloalkyl, —C(O)—C ４ Cycloalkyl, —C １ Alkyl, —C(O)—(pyrrolidinyl), and —C(O)—CH ２ ONH—C(O)—(pyrrolidinyl), where: -C(O)-C ３ Alkyl, —C(O)O—C ３ Alkyl, —C(O)—C ３ Cycloalkyl, —C(O)—C ４ Cycloalkyl or -C １ Alkyl is hydroxyl, -NH ２ , —CHO, and —COOH; —C(O)—(pyrrolidinyl) and —C(O)—CH ２ ONH-C(O)-(pyrrolidinyl) may be optionally substituted on any available pyrrolidinyl carbon atom with one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R １ are joined together to form oxo, -C(O)-(pyrrolidinyl) is a group selected from the group consisting of -CHO, -C(O)CH on an available pyrrolidinyl nitrogen atom. ３ , or —C(O)CH ２ NH ２ and optionally substituted by

17. R is, 【Transformation 6】 selected from the group consisting of 17. The therapeutic payload of claim 16.

18. the therapeutic payload is 【Chemistry 7-1】 【Chemistry 7-2】 or a pharmaceutically acceptable salt thereof; 18. The therapeutic payload of claim 16 or 17.

19. Formula IIIA or Formula IIIB: 【Transformation 8】 or a pharmaceutically acceptable salt or stereoisomer thereof. (In the formula, L is, 【Chemistry 9】 is selected from the group consisting of where * represents the point of attachment to R; R is 【Chemistry 10】 is selected from the group consisting of where ** represents the point of attachment to L; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 0, 1, 2, 3, 4, 5, 6, 7, or 8).

20. R is, 【Chemistry 11】 is selected from the group consisting of wherein ** represents the point of attachment to L.

20. The linker-payload construct of claim 19.

21. 21. The linker payload construct of claim 19 or 20, wherein s is 1 or 8.

22. the linker-payload construct comprising: 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】 selected from the group consisting of A linker payload construct according to any one of claims 19 to 21.

23. A pharmaceutical composition comprising the drug conjugate of any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

24. A pharmaceutical composition comprising a therapeutic payload according to any one of claims 16 to 18 and a pharmaceutically acceptable excipient.

25. A pharmaceutical composition comprising the linker-payload construct of any one of claims 16 to 18 and a pharmaceutically acceptable excipient.

26. 16. A method for delivering a therapeutically effective amount of a therapeutic payload moiety to a patient in need thereof, the method comprising administering to the patient a drug conjugate according to any one of claims 1 to 15.

27. A method for treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a drug conjugate described in any one of claims 1 to 15.

28. 19. A method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a therapeutic payload according to any one of claims 16 to 18.

29. 29. The method of claim 27 or 28, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

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