Method for preparing antibody drug conjugates

Abstract

The present disclosure relates to processes for the preparation of compounds of structural formula (I) wherein PG is as defined herein for the preparation of antibody drug conjugates. The present disclosure also relates to processes for preparing related payload compounds, related linker-payload compounds, and compounds useful as synthetic intermediates for preparing compounds of formula (I). # imgabs0 #

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 430,898, filed on December 07, 2022, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] Provided herein are methods for preparing linker-payload moieties for use in the preparation of antibody-drug conjugates for the treatment of cancer. Also provided are methods for preparing related payload moieties, and compounds useful as synthetic intermediates for preparing the linker-payload moieties of the present disclosure. Background Art

[0004] Given the aging population and modern lifestyles, the global cancer burden has been continuously increasing and is expected to continue to increase in the coming decades. The American Cancer Society estimates that there will be 1.9 million new cancer diagnoses in 2022 and 609,360 cancer deaths in the United States alone. Nearly 200 existing types of cancer share the fundamental characteristics of uncontrolled growth and spread, due to the gradual acquisition by cancer cells of the ability to proliferate indefinitely and escape regulatory mechanisms.

[0005] Antibody-drug conjugates or ADCs are a class of highly potent biopharmaceutical drugs composed of an antibody linked to a bioactive drug or cytotoxic compound via a chemical linker. These targeted agents combine the unique and highly sensitive targeting ability of antibodies to distinguish between healthy and cancerous tissues with the cell-killing ability of cytotoxic drugs.

[0006] Because these agents are capable of directly delivering highly cytotoxic payloads to tumor cells, they can be used to achieve high lethality to targeted cancer cells while minimizing damage to healthy cells.

[0007] Camptothecin (CPT) is a topoisomerase inhibitor that was discovered in 1966 during a systematic screening of natural products for anticancer drugs. It was isolated from the bark and branches of Camptotheca acuminata. CPT showed anticancer activity in preliminary clinical trials, particularly against breast, ovarian, colon, lung, and gastric cancers. However, camptothecin has low solubility and there have been reports of adverse reactions during treatment. Therefore, synthetic and medicinal chemists have developed many synthetic methods for camptothecin, as well as various derivatives, to increase the benefits of this chemical substance and have achieved promising results. Currently, four CPT analogs have been approved for cancer chemotherapy: topotecan, irinotecan, belotecan, and trastuzumab deruxtecan, the latter being an ADC that uses the camptothecin derivative deruxtecan as a payload. Currently, many other camptothecin-based ADCs are being evaluated in human clinical trials, and several academic and industrial research groups are conducting further research on such molecules, including evaluating the clinical trials of ADCs incorporating the linker-payload moiety ("linker-payload A") described below:

[0008]

[0009] in which a sulfone-substituted pyrimidine group is used as a conjugation handle and is disclosed in International Publication No. WO 2020 / 0347075.

[0010] The success of targeting Trop-2 by antibody-drug conjugates (ADCs) in metastatic breast cancer (MBC) and urothelial cancer, as well as ongoing trials in NSCLC, has established Trop-2 targeting as an effective and productive strategy, and several Trop-2-targeted therapies have recently been developed for clinical use, such as anti-Trop-2 antibodies and Trop-2-targeted ADCs. Subsequently, multiple early clinical trials have shown that Trop-2-based ADCs have favorable safety profiles and clinical benefits in multiple tumor types. This includes clinical benefits and tolerability in tumor types with limited treatment options, such as triple-negative breast cancer, platinum-resistant urothelial cancer, and small cell lung cancer. An example of an ADC currently in the early clinical trial stage is immunoconjugate A, which incorporates linker-payload A:

[0011]

[0012] where Ab is an anti-Trop-2 antibody (sacituzumab), and n is an integer or decimal from 1 to 10, and it is described in U.S. Patent Publication No. 20200347075.

[0013] However, there is still a need for cost-effective synthetic routes to prepare such ADCs. This disclosure addresses this need. SUMMARY OF THE INVENTION

[0014] This disclosure relates to methods for preparing linker-payload moieties of formula (I) and methods for preparing ADCs incorporating these linker-payload moieties. The ADCs can be used for treating cancer. In one aspect, this disclosure provides a method for preparing a compound of structural formula (I) (or referred to herein as "Method A"):

[0015]

[0016] wherein PG is a primary amine protecting group,

[0017] The method comprises the steps of:

[0018] (A) In organic solvent A, contacting a compound of structural formula (i):

[0019]

[0020] with a dechlorinating agent at a certain temperature for a period of time sufficient to form a compound of structural formula (ii):

[0021]

[0022] wherein organic solvent A is selected from dichloromethane, toluene, THF, acetonitrile, and mixtures thereof; and

[0023] (B) In organic solvent B, in the presence of a base, contacting the product of step A with a compound of structural formula (iii):

[0024]

[0025] and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (iv):

[0026]

[0027] wherein organic solvent B is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and

[0028] (C) In organic solvent C, in the presence of a copper salt and in the presence of a source of azide anions, the product of step B is contacted with a compound of structural formula (v):

[0029]

[0030] at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein organic solvent C is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, dichloromethane, water, and mixtures thereof.

[0031] In another aspect, the present disclosure provides novel synthetic intermediates that can be used in the methods of the present disclosure.

[0032] Other embodiments, aspects, and features of the present disclosure are further described in or will be apparent from the following specification, examples, and appended claims. Detailed Description

[0033] The present disclosure relates to methods for preparing compounds of structural formula (I), and methods for using compounds of structural formula (I) as linkers to prepare ADCs. These ADCs can be used for the treatment of cancer.

[0034] Definitions and Abbreviations

[0035] For compounds used as reactants or reagents in the methods of the present disclosure (e.g., compounds (I), (II), (III), (i), (ii), (iii), etc.), a "stable" compound is a compound whose structure and properties remain or can remain substantially unchanged for a period of time sufficient to allow its use in the methods of the present disclosure to achieve the preparation of compounds of formula (I), (II), (III), etc. For compounds of formula (I), (II), (III) or immunoconjugate A, a "stable" compound is a compound that can be prepared and then isolated according to the methods of the present disclosure, and whose structure and properties remain or can remain substantially unchanged for a time sufficient for the compound to be used for its intended purpose; e.g., for therapeutic administration to a subject suffering from cancer, or in the case of a compound of formula (I), for linking a payload to an antibody as part of an antibody-drug conjugate.

[0036] As used herein, the term "salt" refers to acid salts formed with inorganic and / or organic acids, and base salts formed with inorganic and / or organic bases. Additionally, when the described compound contains both a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, carboxylic acid), zwitterions ("inner salts") can be formed, which are encompassed by the term "salt" as used herein. In one embodiment, the salt is a pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salt. In another embodiment, the salt is not a pharmaceutically acceptable salt. Salts of the compounds, starting materials, or synthetic intermediates of the present disclosure can be formed, for example, by reacting the compound, starting material, or synthetic intermediate with a quantity of acid or base (such as an equivalent amount) in a medium such as a medium for salt precipitation or an aqueous medium, and then freeze-drying.

[0037] Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates), and the like. Additionally, for example, P. Stahl et al., Camille G. (ed.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2nd ed., (2011) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Wermuth et al., The Practice of Medicinal Chemistry, 4th ed., (2015), Academic Press, New York; and The Orange Book (Food & Drug Administration, Washington, D.C., on its website) discuss the acids that are generally considered suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds. These disclosures are incorporated herein by reference.

[0038] Exemplary base salts include ammonium salts, alkali metal salts such as sodium salts, lithium salts, and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with bases (e.g., organic amines) such as dicyclohexylamine, tert-butylamine, choline, and salts with amino acids such as arginine, lysine, etc. The basic nitrogen-containing group can be quaternized with reagents such as lower alkyl halides (e.g., chlorides, bromides, and iodides of methyl, ethyl, and butyl), dialkyl sulfates (e.g., sulfates of dimethyl, diethyl, and dibutyl), long-chain halides (e.g., chlorides, bromides, and iodides of decyl, lauryl, and stearyl), aromatic alkyl halides (e.g., bromides of benzyl and phenethyl), etc.

[0039] A mixture of diastereomers can be separated into its respective diastereomers according to their physicochemical differences by methods well known to those skilled in the art, e.g., by chromatography and / or fractional crystallization. A mixture of enantiomers can be separated by reacting with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride) to convert the mixture of enantiomers into a mixture of diastereomers, separating the diastereomers, and converting the individual diastereomers into the corresponding pure enantiomers (e.g., by hydrolysis). Stereochemically pure compounds can also be prepared by using chiral starting materials or by using salt resolution techniques.

[0040] The compounds, starting materials, and synthetic intermediates of the present disclosure can also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. For example, all keto-enol and imine-enamine forms of the compounds, starting materials, and synthetic intermediates of the present disclosure are included.

[0041] All stereoisomers (e.g., geometric isomers, optical isomers, etc.) of the compounds, starting materials, and synthetic intermediates of the present disclosure (including those of their salts, solvates, hydrates, and esters), such as those that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are within the scope of the present disclosure. If the compounds, starting materials, or synthetic intermediates of the present disclosure contain double bonds or fused rings, cis and trans forms as well as mixtures are included within the scope of the present disclosure.

[0042] The abbreviations used hereinafter have the following meanings: Ac is acetyl; anisole is methoxybenzene; Boc is tert-butoxycarbonyl; BOP is (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate); t-Bu is tert-butyl; Cbz is benzyloxycarbonyl; 545 is diatomaceous earth, flux-calcined; COMU is (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate; CuBr-SMe2 is copper(I) bromide dimethyl sulfide complex; CUNO-5 is a carbon resin; DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC is N,N'-dicyclohexylcarbodiimide; DCM is dichloromethane; DIEA or DIPEA is N,N-diisopropylethylamine; DMAc is dimethylacetamide; DMAP is dimethylaminopyridine; DMBCl is 2,2-dimethylbut-3-ynoyl chloride; DME is 1,2-dimethoxyethane; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; DPPA is diphenylphosphoryl azide; dppf is 1,1′-bis(diphenylphosphino)ferrocene; EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; EtOAc is ethyl acetate; Fmoc is fluorenylmethoxycarbonyl; HATU is (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HBTU is (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HPLC is high performance liquid chromatography; IPAc is isopropyl acetate; 2,6-lutidine is 2,6-dimethylpyridine; LC / MS is liquid chromatography / mass spectrometry; Me is methyl; MeCN is acetonitrile; Me-THF (and methyl THF) is 2-methyltetrahydrofuran; mesyl is mesyl; MMT is monomethoxytrityl; MTBE is tert-butyl methyl ether; NaMSA is sodium mesylate; NMP is N-methyl-2-pyrrolidone; [Pd2(dba)3] is tris(dibenzylideneacetone)dipalladium(0); 2-picoline is 2-methylpyridine; TBAF is tetrabutylammonium fluoride; TEA is triethylamine; THF is tetrahydrofuran; tosyl is tosyl; and TSTU is O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

[0043] The methods of the present disclosure

[0044] The present disclosure relates to methods for preparing compounds of structural formula (I), which compounds can be used as linkers in ADC molecules. One aspect of the present disclosure is the following method for preparing compounds of structural formula (I) (“Method A”):

[0045]

[0046] wherein PG is a primary amine protecting group, and the method comprises the steps of:

[0047] (A) In an organic solvent A, a compound of structural formula (i):

[0048]

[0049] is contacted with a dechlorinating agent at a certain temperature for a period of time sufficient to form a compound of structural formula (ii):

[0050]

[0051] wherein the organic solvent A is selected from dichloromethane, toluene, THF, acetonitrile, and mixtures thereof; and

[0052] (B) In an organic solvent B, in the presence of a base, the product of step A is contacted with a compound of structural formula (iii):

[0053]

[0054] and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (iv):

[0055]

[0056] and wherein the organic solvent B is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and

[0057] (C) In an organic solvent C, in the presence of a copper salt and a source of azide anions, the product of step B is contacted with a compound of structural formula (v):

[0058]

[0059] at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent C is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, dichloromethane, water, and mixtures thereof.

[0060] In one embodiment, for process A, the dechlorinating agent used in step (A) is selected from SOCl2, PCl3, PCl5, POCl3, oxalyl chloride, tosyl chloride, mesyl chloride, triphenylphosphine / CCl4, and triphenylphosphine / hexa chloroethane.

[0061] In another embodiment, for Method A, the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

[0062] In another embodiment, for Method A, the deoxychlorinating agent used in step (A) is SOCl2 or oxalyl chloride.

[0063] In one embodiment, for Method A, organic solvent A is dichloromethane or THF.

[0064] In one embodiment, for Method A, the product of step (A) is separated before performing step (B).

[0065] In another embodiment, for Method A, the product of step (A) is separated, purified, and dried before performing step (B).

[0066] In one embodiment, for Method A, the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU.

[0067] In another embodiment, for Method A, the amide coupling agent used in step (B) is selected from HATU, EDC, and DCC.

[0068] In one embodiment, for Method A, the base used in step (B) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine.

[0069] In another embodiment, for Method A, the base used in step (B) is K2CO3 or Na2CO3.

[0070] In one embodiment, for Method A, the primary amine protecting group in step (B) is selected from Boc, MMT, Cbz, or Fmoc.

[0071] In one embodiment, for Method A, organic solvent B is selected from methyl ethyl ketone, acetone, diethyl ether, THF, DCM, and NMP.

[0072] In one embodiment, for Method A, the product of step (B) is separated before performing step (C).

[0073] In another embodiment, for Method A, the product of step (B) is separated and purified before performing step (C).

[0074] In one embodiment, for Method A, the copper salt used in step (C) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2.

[0075] In one embodiment, for Method A, the azide anion source in step (C) is NaN3 or KN3.

[0076] In one embodiment, for Method A, the organic solvent C is dichloromethane, water, THF, or a mixture thereof.

[0077] In one embodiment, for Method A, the product of step (C) is separated.

[0078] In another embodiment, for Method A, the product of step (C) is separated and purified.

[0079] In one embodiment, for Method A, the product of step C can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high surface area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0080] In another aspect, the present disclosure provides an alternative method (“Method B”) for preparing a compound of formula (I):

[0081]

[0082] wherein PG is a primary amine protecting group,

[0083] The method comprises the steps of:

[0084] (A) In an organic solvent D, contacting a compound of structural formula (vi):

[0085]

[0086] with a deoxychlorinating agent at a certain temperature for a period of time sufficient to form a compound of structural formula (vii):

[0087]

[0088] wherein the organic solvent D is selected from dichloromethane, toluene, THF, acetonitrile, and mixtures thereof;

[0089] (B) In an organic solvent E, contacting the product of step A with diglycolic anhydride (viii):

[0090]

[0091] at a certain temperature for a period of time sufficient to form a compound of structural formula (ix):

[0092]

[0093] wherein the organic solvent E is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, and mixtures thereof;

[0094] (C) In an organic solvent F, in the presence of a base, the product of step B is contacted with a compound of structural formula (iii):

[0095]

[0096] and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (iv):

[0097]

[0098] wherein the organic solvent F is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and

[0099] (D) In an organic solvent G, the product of step C is contacted with azide anions at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (x):

[0100]

[0101] Then, in the presence of a copper salt, the intermediate compound (x) is contacted with a compound of structural formula (v):

[0102]

[0103] at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent G is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water, and mixtures thereof.

[0104] In one embodiment, for process B, the dechlorinating agent used in step (A) is selected from SOCl2, PCl3, PCl5, POCl3, oxalyl chloride, tosyl chloride, mesyl chloride, triphenylphosphine / CCl4, and triphenylphosphine / hexa chloroethane.

[0105] In another embodiment, for process B, the dechlorinating agent used in step (A) is SOCl2 or oxalyl chloride.

[0106] In another embodiment, for Method B, the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

[0107] In one embodiment, for Method B, the organic solvent D is dichloromethane or THF.

[0108] In one embodiment, for Method B, before performing step (B), the product of step (A) is separated.

[0109] In another embodiment, for Method B, before performing step (B), the product of step (A) is separated and purified.

[0110] In one embodiment, for Method B, the organic solvent E is selected from methyl ethyl ketone, acetone, diethyl ether, THF, DCM, and NMP.

[0111] In one embodiment, for Method B, before performing step (C), the product of step (B) is separated.

[0112] In another embodiment, for Method B, before performing step (C), the product of step (B) is separated and purified.

[0113] In one embodiment, for Method B, the amide coupling agent used in step (C) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU.

[0114] In another embodiment, for Method B, the amide coupling agent used in step (C) is selected from HATU, EDC, and DCC.

[0115] In one embodiment, for Method B, the base used in step (C) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine.

[0116] In another embodiment, for Method B, the base used in step (C) is K2CO3 or Na2CO3.

[0117] In one embodiment, for Method B, the organic solvent F is selected from methyl ethyl ketone, acetone, diethyl ether, THF, DCM, and NMP.

[0118] In one embodiment, for Method B, before performing step (D), the product of step (C) is separated.

[0119] In another embodiment, for Method B, before performing step (D), the product of step (C) is separated and purified.

[0120] In one embodiment, for Method B, the source of the azide anion in step (D) is NaN3 or KN3.

[0121] In another embodiment, for Method B, the copper salt used in step (D) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2.

[0122] In one embodiment, for Method B, the organic solvent G is dichloromethane, water, THF, or a mixture thereof.

[0123] In one embodiment, for Method B, the intermediate compound (x) is not isolated during step (D).

[0124] In one embodiment, for Method B, the product of step D is isolated.

[0125] In another embodiment, for Method B, the product of step D is isolated and purified.

[0126] In one embodiment, for Method B, the product of step D can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high surface area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0127] In another aspect, the present disclosure provides another method (“Method C”) for preparing a compound of formula (I):

[0128]

[0129] wherein PG is a primary amine protecting group,

[0130] The method comprises the steps of:

[0131] (A) In an organic solvent H, contacting a compound of structural formula (vii):

[0132]

[0133] with an azide anion at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (xii):

[0134]

[0135] Then, in the presence of a copper salt, reacting the intermediate compound (xii) with a compound of structural formula (v):

[0136]

[0137] Contact at a certain temperature and for a period of time sufficient to form the compound of structural formula (xiii):

[0138]

[0139] wherein the organic solvent H is selected from THF, acetonitrile, DMF, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water and mixtures thereof, and

[0140] (B) In an organic solvent I, in the presence of an amide coupling agent and a base, reacting the product of step A with a compound of structural formula (xiv):

[0141]

[0142]

[0143] Contact at a certain temperature and for a period of time sufficient to form the compound of structural formula (I), wherein the organic solvent I is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane and mixtures thereof.

[0144] In one embodiment, for process C, the primary amine protecting group is selected from Boc, MMT, Cbz and Fmoc.

[0145] In one embodiment, for process C, the source of azide anion in step (A) is NaN3 or KN3.

[0146] In one embodiment, for process C, the copper salt used in step (A) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2.

[0147] In one embodiment, for process C, the organic solvent H is selected from dichloromethane, DMF, THF or mixtures thereof.

[0148] In one embodiment, for process C, the intermediate (xii) is not isolated during step A.

[0149] In one embodiment, for process C, the organic solvent H is selected from dichloromethane, DMF, THF and mixtures thereof.

[0150] In one embodiment, for process C, the product of step (A) is separated before carrying out step (B).

[0151] In another embodiment, for Method C, before performing step (B), the product of step (A) is separated and purified.

[0152] In one embodiment, for Method C, the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU.

[0153] In another embodiment, for Method C, the amide coupling agent used in step (B) is selected from HATU, EDC, and DCC.

[0154] In one embodiment, for Method C, the base used in step (B) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine.

[0155] In another embodiment, for Method C, the base used in step (B) is DBU, K2CO3, or Na2CO3.

[0156] In one embodiment, for Method C, organic solvent I is selected from THF, acetonitrile, and dichloromethane.

[0157] In one embodiment, for Method C, the product of step (B) is separated.

[0158] In another embodiment, for Method C, the product of step (B) is separated and purified.

[0159] In one embodiment, for Method C, the product of step B can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high-surface-area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0160] In another aspect, the present disclosure provides another alternative method (“Method D”) for preparing a compound of formula (I):

[0161]

[0162] wherein PG is a primary amine protecting group,

[0163] The method comprises the steps of:

[0164] (A) In organic solvent J, reacting a compound of structural formula (ii):

[0165]

[0166] Contact with azide anion at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (xv):

[0167]

[0168]

[0169] Then, in the presence of a copper salt, react the intermediate compound (xv) with a compound of structural formula (v):

[0170]

[0171] Contact at a certain temperature for a period of time sufficient to form a compound of structural formula (xvi):

[0172]

[0173] wherein the organic solvent J is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water and mixtures thereof, and

[0174] (B) In an organic solvent K, in the presence of an amide coupling agent and a base, react the product of step A with a compound of structural formula (iii):

[0175]

[0176] Contact at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent K is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane and mixtures thereof.

[0177] In one embodiment, for method D, the primary amine protecting group is selected from Boc, MMT, Cbz and Fmoc.

[0178] In one embodiment, for method D, the source of azide anion in step (A) is NaN3 or KN3.

[0179] In one embodiment, for method D, the copper salt used in step (A) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2.

[0180] In one embodiment, for method D, the organic solvent J is selected from dichloromethane, THF and acetonitrile.

[0181] In one embodiment, for method D, the intermediate (xv) is not isolated in step (A).

[0182] In one embodiment, for Method D, prior to performing step (B), the product of step (A) is separated.

[0183] In another embodiment, for Method D, prior to performing step (B), the product of step (A) is separated and purified.

[0184] In one embodiment, for Method D, the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU.

[0185] In another embodiment, for Method D, the amide coupling agent used in step (B) is selected from HATU, EDC, and DCC.

[0186] In one embodiment, for Method D, the base used in step (B) is selected from alkali carbonates, TEA, DIPEA, DBU, pyridine, and 2 - picoline.

[0187] In another embodiment, for Method D, the base used in step (B) is DBU, K2CO3, or Na2CO3.

[0188] In one embodiment, for Method D, the organic solvent K is selected from THF, DCM, and acetonitrile.

[0189] In one embodiment, for Method D, the product of step (B) is separated.

[0190] In another embodiment, for Method D, the product of step (B) is separated and purified.

[0191] In one embodiment, for Method D, the product of step B can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high - surface - area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0192] In another aspect, the present disclosure provides another alternative method (“Method E”) for preparing a compound of formula (I):

[0193]

[0194] wherein PG is a primary amine protecting group,

[0195] The method comprises the steps:

[0196] (A) In an organic solvent L, a compound of structural formula (xv):

[0197]

[0198] with a compound of structural formula (iii):

[0199]

[0200] wherein PG is a primary amine protecting group, and contacting with an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (x):

[0201]

[0202] wherein PG is a primary amine protecting group, and the organic solvent L is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and

[0203] (B) In an organic solvent M, in the presence of a copper salt, reacting the product of step A with a compound of structural formula (v):

[0204]

[0205] contacting at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent M is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof.

[0206] In one embodiment, for process E, the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

[0207] In one embodiment, for process E, the organic solvent L in step (A) is THF.

[0208] In one embodiment, for process E, the product of step (A) is not isolated before carrying out step (B).

[0209] In one embodiment, for process E, the product of step (A) is prepared in solution and used in solution in step (B).

[0210] In one embodiment, for method E, the amide coupling agent used in step (A) is selected from EEDQ, HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU.

[0211] In another embodiment, for method E, the amide coupling agent used in step (A) is EEDQ.

[0212] In one embodiment, for method E, the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

[0213] In another embodiment, for method E, the primary amine protecting group is Boc.

[0214] In one embodiment, for method E, in step (B), the organic solvent M is THF.

[0215] In one embodiment, for method E, the copper salt used in step (B) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2.

[0216] In another embodiment, for method E, the copper salt used in step (B) is CuBr-SMe2.

[0217] In one embodiment, for method E, the product of step (B) is isolated.

[0218] In one embodiment, for method E, the product of step (B) is isolated and purified.

[0219] In one embodiment, the product of step B can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high surface area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0220] In another aspect, the present disclosure provides a method (“method F”) for preparing the linker-payload moiety of formula (II):

[0221]

[0222] wherein G is a cytotoxic drug payload and PG is a primary amine protecting group

[0223] The method comprises the steps of:

[0224] (A) In an organic solvent N, in the presence of a base, a drug containing an -OH group represented by (xvii):

[0225] Drug - OH

[0226] (xvii)

[0227] is contacted with triphosgene at a certain temperature for a period of time sufficient to form a compound of structural formula (xviii):

[0228]

[0229] wherein the organic solvent N is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, propylene carbonate, DME, 1,3 - dimethyl - 3,4,5,6 - tetrahydro - 2(1H) - pyrimidinone, 2 - methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile and mixtures thereof; and

[0230] (B) In an organic solvent O, in the presence of a base, the product of step A is contacted with a compound of formula (I):

[0231]

[0232] at a certain temperature for a period of time sufficient to form a compound having formula (II), wherein the organic solvent O is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3 - dimethyl - 3,4,5,6 - tetrahydro - 2(1H) - pyrimidinone, 2 - methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile and mixtures thereof.

[0233] In one embodiment, for method F, the base used in step (A) is selected from DMAP, TEA, pyridine and 2,6 - dimethylpyridine.

[0234] In one embodiment, for method F, it is selected from dichloromethane, DMF, THF and mixtures thereof.

[0235] In one embodiment, for method F, before carrying out step (B), the product of step (A) is not separated.

[0236] In another embodiment, for method F, before carrying out step (B), the product of step (A) is separated.

[0237] In another embodiment, for method F, before carrying out step (B), the product of step (A) is separated and purified.

[0238] In another embodiment, for method F, the reaction mixture of step (A) is directly used in step (B).

[0239] In one embodiment, for method F, the base used in step (B) is selected from DMAP, TEA, pyridine, and 2,6-lutidine.

[0240] In one embodiment, for method F, the organic solvent O is the organic solvent M selected from dichloromethane, DMF, THF, and mixtures thereof.

[0241] In another embodiment, the organic solvent N is the same as the organic solvent O.

[0242] In one embodiment, for method F, the product of step (B) is separated.

[0243] In another embodiment, for method F, the product of step (B) is separated and purified.

[0244] In one embodiment, for method F, the product of step B can be adsorbed onto a solid support for use in subsequent chemical reactions. In another embodiment, the solid support is a high-surface-area microporous solid. In a specific embodiment, the solid support is diatomaceous earth or cellulose. In a preferred embodiment, the solid support is diatomaceous earth.

[0245] In one embodiment, for method F, in step A, compound xvii has structure (xix):

[0246]

[0247] In step B, compound xviii has structure (xx):

[0248]

[0249] And the product of step B is compound (xxi):

[0250]

[0251] In one embodiment, the compound of formula (xxi) is deprotected to provide linker-payload A:

[0252]

[0253] The compounds of formula (II) can be deprotected using well-known methods and then conjugated to an antibody to convert them into the corresponding antibody-drug conjugates by using well-known conjugation methods, including those disclosed in International Publication Nos. WO 2018 / 025168, WO 2019 / 11466, and WO 2020 / 0347075.

[0254] In one embodiment, the compound of formula (II) can be deprotected to provide a compound of formula (xxii):

[0255]

[0256] wherein G is a drug, and then conjugated to an antibody or antibody fragment to prepare an antibody-drug conjugate of formula (III):

[0257]

[0258] wherein n is a fraction from 0 to 8, Ab is an antibody or antibody fragment, G is a cytotoxic drug payload, and the sulfur atom in formula (III) is from a thiol group on the antibody.

[0259] In one embodiment, the compound of formula (xxi) can be deprotected and then conjugated to a monoclonal antibody to prepare an antibody-drug conjugate of formula (xxiii):

[0260]

[0261] wherein PG is a primary amine protecting group, n is an integer from 1 to 8, Ab is an antibody or antibody fragment, and the sulfur atom in formula (xxiii) is from a thiol group on the antibody.

[0262] The compound (xix) can be prepared using the method disclosed in International Publication No. WO 2020 / 0347075 or using the methods described herein.

[0263] In one embodiment, for the antibody-drug conjugate of formula (xxiii)

[0264] In another aspect, the present disclosure provides a method (“Method G”) for preparing a payload compound of formula (xix):

[0265]

[0266]

[0267] The method includes, in solvent P, in the presence of an acid, a metal salt, and an oxidizing agent, reacting a compound of structural formula (xxiv) (camptothecin):

[0268]

[0269] with the compound of structural formula (xxv):

[0270]

[0271] contacted at a certain temperature and for a period of time sufficient to form a payload compound of structural formula xix, wherein the solvent P is selected from water, MeCN, acetic acid, trifluoroethanol, NMP, propylene glycol, trifluoroacetic acid, and mixtures thereof.

[0272] In one embodiment of method G, the acid used is selected from H2SO4, TFA, phosphoric acid, sulfonic acid, and methanesulfonic acid.

[0273] In one embodiment of method G, the metal salt used in step (A) is selected from Fe2SO4, V(acac)3, V2O3, NaVO3, FeCl3, and FeO.

[0274] In one embodiment, for method G, the oxidizing agent used is selected from H2O2, persulfate, peracetic acid, cumene hydroperoxide, 2-butanone peroxide, potassium peroxymonosulfate (oxone), urea hydrogen peroxide, sodium perborate, di-tert-butyl peroxide, dibenzoyl peroxide, and sodium hypochlorite.

[0275] In one embodiment of method G, the solvent P is water.

[0276] In one embodiment of method G, the product is isolated.

[0277] In another embodiment of method G, the product is isolated and purified.

[0278] Synthetic intermediate

[0279] The present disclosure also provides compounds that can be used as synthetic intermediates in the claimed methods for preparing the compounds of the present disclosure.

[0280] Examples of such synthetic intermediates include, but are not limited to, the following compounds:

[0281]

[0282] Methods for implementing the methods of the present disclosure

[0283] The methods described herein, including starting materials and synthetic intermediates, can be carried out using starting materials that are known or readily prepared, according to methods well known to those skilled in the art of organic synthesis. Methods for implementing the methods of the present disclosure are set forth herein. Alternative synthetic routes and similar structures will be apparent to those skilled in the art of organic synthesis.

[0284] Those skilled in the art of organic synthesis will recognize that the methods of the present disclosure may require protection of certain functional groups (i.e., derivatization for chemical compatibility with specific reaction conditions). Suitable protecting groups for the various functional groups of the starting materials, intermediates, and products used in the methods of the present disclosure, as well as methods for installing and removing these protecting groups, are well known in the field of organic chemistry. A summary of many of these methods can be found in Wuts et al., Protective Groups in Organic Synthesis, Wiley-Interscience, New York, 5th edition, (2014).

[0285] Those skilled in the art of organic synthesis will also realize that, depending on the choice of the attached substituents, one of the methods disclosed herein may be more desirable.

[0286] In addition, those skilled in the art will realize that, in certain cases, the reaction sequence of the methods of the present disclosure may be different from the sequence described herein to avoid incompatibility of functional groups, and thus the synthetic route is adjusted accordingly.

[0287] If desired, conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography (i.e., HPLC), etc., can be used to separate and purify the starting materials and synthetic intermediates prepared using the methods described herein. These materials can be characterized using conventional means, including physical constants and spectral data (i.e., NMR, LCMS).

[0288] Embodiments

[0289] General Methods

[0290] Commercially available solvents, reagents, and intermediates were used as received. Non-commercially available reagents and intermediates were prepared by the methods described below. 1H NMR spectra were obtained on a Bruker Avance 500 (500 MHz), reported in ppm downfield from Me4Si, with the number of protons, multiplicity, and coupling constants in hertz in parentheses. In cases where LC / MS data was provided, analysis was performed using a Waters Acquity SQD LCMS instrument, which used an ACQUITY UPLC BEH C18 column, 1 1.7 μm, 1 mm X 50 mm, and mobile phase A (0.1% aqueous formic acid) and mobile phase B (0.1% formic acid in MeCN) with a gradient flow at 50 °C at 0.300 mL / min. Gradient: 90% A to 1% A from 0 - 1.6 minutes, held at 1% from 1.6 to 3.0 minutes. 1.7 μm, 1 mm X 50 mm, and mobile phase A (0.1% aqueous formic acid) and mobile phase B (0.1% formic acid in MeCN) with a gradient flow at 50 °C at 0.300 mL / min. Gradient: 90% A to 1% A from 0 - 1.6 minutes, held at 1% from 1.6 to 3.0 minutes.

[0291] Embodiment 1

[0292] Preparation of Intermediate Compound v

[0293]

[0294] For example, Intermediate Compound v can be prepared using the method described in U.S. Patent Publication No. 2020 / 0347075.

[0295] Embodiment 2

[0296] Alternative Preparation of Intermediate Compound v

[0297]

[0298] Step A - Synthesis of Compound 2b

[0299] At room temperature, Compound 2a (100.0 g), Compound 2d (60.0 g) and Cs2CO3 (794.0 g) were placed in DMAc (1000 mL) and placed under a nitrogen atmosphere. CuI (2.79 g), Pd2(dba)3 (0.45 g), dppf (0.54 g) and LiCl (20.67 g) were added to the resulting solution, and nitrogen gas was bubbled through the resulting reaction mixture (3x), then heated to 55 - 65 °C and stirred at this temperature for 20 hours. Water (1000 mL) was added to the reaction mixture, and then it was stirred until the reaction mixture reached a temperature of 15 - 25 °C. HCl (4M, 900 g) and water (2000 mL) were added to this solution, and the resulting solution was cooled to 10 - 20 °C and stirred at this temperature for 1 hour. The solution was filtered, and the filter cake was dried at 45 - 55 °C for 8 hours to afford Compound 2b as a solid, which was used directly without further purification.

[0300] Step B - Synthesis of Compound 2c

[0301] A solution of Compound 2b (121.2 g) was placed in DMAc (500 mL), and the resulting solution was stirred at room temperature for 30 minutes. CDI (116.67 g) was added, and the resulting reaction mixture was cooled to 0 - 10 °C and stirred at this temperature for 16 hours. Propargylamine (1e, 35.0 g) was added, and the resulting reaction mixture was stirred at 0 - 10 °C for 1 hour. Water (1000 mL) was added, and the resulting solution was stirred at 0 - 10 °C for 1 hour. More water (500 mL) was added, and the resulting solution was stirred at 0 - 10 °C for 1 hour. Then the solution was filtered, and the filter cake was dried at 45 - 55 °C for 40 hours to afford Compound 2c as a solid.

[0302] Purification of compound 2c

[0303] Compound 2c (100.10 g) was placed in DCM (1500 mL), and to the resulting solution was added C941. The resulting solution is cooled to 0-10 ° C, stirred at this temperature for 2 hours, then filtered. The wet cake is washed with DCM (200 mL x 3). At 25-35 ° C, the filtrate is concentrated to a volume of 300-400 mL in a vacuum, and n-heptane (1500 ml) is added. The resulting solution is cooled to 0-10 ° C, stirred at this temperature for 2 hours, then filtered. The wet cake is washed with n-heptane (200 mL x 2), and dried at 45-55 ° C for 32 hours to provide compound 2c as a solid.

[0304] Step C – Synthesis of Compound v

[0305] At 5-15 ℃, compound 2c (86.86g) and potassium peroxymonosulfonate (402.66g) were added to a solution of MeCN (800mL) and water (800mL). The resulting reactant was stirred at 5-15 ℃ for 16 hours, then ascorbic acid (1040g, 20% aqueous solution, precooled to 0-10 ℃) was added and the resulting reactant was stirred at 0-10 ℃ for 1 hour. The reaction mixture was filtered, and the wet cake was washed with DCM (500mL), the filtrate was combined and water (1600mL) and DCM (400mL) were added to the washings. The organic layer was diluted with DCM (400mL) and washed with water (2x 800mL), and then the aqueous layer was extracted with DCM (400mL). The organic layer and the organic extract were combined to provide compound Int-1 and the resulting solution was cooled to 20-30 ℃, and Ecosorb C941 (8g) was added. The obtained solution was stirred for 30 minutes, then extra Ecosorb941 (8g) was added, and the solution was stirred for 1 hour. The solution was filtered, and wet cake was washed with DCM (160mL x 2). It was 240mL to combine filtrate and washings and to be concentrated to volume under vacuum. The solution of normal heptane (400mL) and DCM (80mL) was added, and the obtained solution was stirred for 30 minutes. The obtained solution was filtered, and wet cake was washed with normal heptane (2x 160mL), then dried in a vacuum to provide compound v. 11H NMR (500 MHz, DMSO) δ 9.12 (s, 2H), 8.31 (t, J = 5.2 Hz, 1H), 3.86 (dd, J = 5.5, 2.5 Hz, 2H), 3.41 (s, 3H), 3.08 (t, J = 2.5 Hz, 1H), 2.56 (t, J = 7.1 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.82 (p, J = 7.2 Hz, 2H).

[0306] Embodiment 3

[0307] Preparation of Intermediate Compound xv

[0308]

[0309] Step A – Preparation of Compound 3b

[0310] Place compound 3a (100.0 g) in toluene (346 g), and add DBU (95.5 g) to the resulting solution. Stir the resulting mixture at room temperature for 1 hour. In a separate container, place DPPA (145.5 g) in toluene (260 g), and stir the resulting mixture at room temperature for 1 hour. Then add the DPPA solution to the solution of compound A and heat at 100 °C. Then, mix the reactant with a solution of NaCl (201 g) in water (1800 g), and stir the resulting mixture at room temperature for 1 hour. Extract the aqueous mixture with toluene (3 x 500 mL). Wash the combined organic extracts with 10% aqueous NaCl solution (2 x 500 mL), and then concentrate to about 600 mL in vacuo. Use the solution containing compound 3b directly in the next step.

[0311] Step B – Preparation of Compound 3c

[0312] To a solution of compound 3b (573 g, prepared in Step A) was added additional toluene (2.3 L), followed by a solution of sulfuric acid (46.4 g) in water (232 mL). Over a 2-hour period, triphenylphosphine (67.7 g) was added to the resulting solution while maintaining the reaction temperature below 30 °C. The reaction mixture was then vented to release gas and then stirred at room temperature for 16 hours. The reaction mixture was partitioned and the aqueous phase was washed with DCM (2 x 1.1 L). To the combined organic phase and organic extracts was added 35% aqueous K3PO4 (954 g) until the pH of the solution was 12 - 13. To this solution was added KH2PO4 (90 g) and THF (5.7 L), and the resulting solution was partitioned. The organic phase was collected and concentrated in vacuo to approximately 2 L, then THF (2.9 L) was added and the resulting solution was concentrated in vacuo to approximately 2 L. This addition and concentration step of THF was repeated two more times, and then additional THF (2.9 L) was added to the final solution to provide compound 3c in solution, which was used in the next step.

[0313] Step C - Preparation of Compound xv

[0314] At room temperature, diethylene glycol anhydride (15.88 g, exothermic) was slowly added to a solution of compound 4c (291.9 g, prepared in Step B), and the resulting reaction mixture was stirred at room temperature for 10 minutes. DCM (1.5 L) and water (1.5 L) were added to the reaction mixture. 10% aqueous K3PO4 was added to the resulting solution until the pH of the solution was 7 - 8. The resulting solution was partitioned, the organic phase was discarded, and the aqueous phase was diluted with DCM (1.5 L). 1N H2SO4 was added to the resulting solution to adjust the solution to pH 2 - 3. The resulting solution was partitioned, and the organic phase was concentrated in vacuo to approximately 600 mL to provide the intermediate compound xv as a concentrated oil, which was used in the next step. 1 H NMR (400 MHz, CD3CN) δ 11.1 (broad peak, 1H), 7.14 (s, 1H), 4.18 (s, 2H), 4.09 (s, 2H), 3.68–3.49 (m, 32H), 3.40 (p, J = 5.1 Hz, 4H).

[0315] Compound xv is also commercially available (CAS 846549-37-9).

[0316] Embodiment 4

[0317] Preparation of Intermediate Compound iii

[0318]

[0319] For example, the intermediate compound iii can be prepared using the method described in U.S. Patent Publication No. 2020 / 0347075.

[0320] Embodiment 5

[0321] Preparation of Payload Compound XIX

[0322]

[0323] For example, the payload compound XIX can be prepared using the method described in U.S. Patent Publication No. 2020 / 0347075.

[0324] Embodiment 6

[0325] Alternative Preparation of Payload Compound XIX

[0326]

[0327] A vial containing xxiv ((S)-camptothecin, 87 mg, 0.25 mmol) was charged with water (0.75 mL) and concentrated sulfuric acid (0.95 mL), and the resulting solution was cooled to 0 °C. To this was added 0.25 mL of an FeSO4·7H2O solution (prepared by placing 120 mg of FeSO4·7H2O in 1 mL of water), followed by an aqueous solution of xxv (0.75 mmol, 3 eq) in water (1 mL). Hydrogen peroxide (30 wt%, 100 μL, 4 eq) was added dropwise to the resulting mixture, and the resulting reaction mixture was stirred at 0 °C for 1 hour. The reaction mixture was then warmed to room temperature, and dichloromethane (25 volumes) was added. Water (12 volumes) was added dropwise to the resulting mixture, then the purified compound XIX was inoculated into the resulting solution, and the mixture was stirred at room temperature for 4 hours. Water (10 volumes) was added dropwise to the resulting solution, and the resulting mixture was stirred at room temperature for 8 hours. The resulting solution was filtered, and the collected solid was washed successively with water (2 x 2 volumes), DCM (2 x 2 volumes), and EtOH (2 x 2 volumes), and then dried at 50 °C to afford compound XIX. 1H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.0 Hz, 1H), 8.19 (dd, J = 8.5, 1.0 Hz, 1H), 7.94–7.84 (m, 1H), 7.78 (ddd, J = 8.2, 6.9, 1.3 Hz, 1H), 7.35 (s, 1H), 6.52 (s, 1H), 5.43 (m, 4H), 3.98 (m, 1H), 3.56–3.34 (m, 4H), 2.99 (s, 3H), 1.88 (m, 2H), 1.18–1.11 (m, 6H), 0.89 (t, J = 7.3 Hz, 3H).

[0328] Embodiment 7

[0329] Preparation of Linker Compound 1

[0330]

[0331] Step A - Synthesis of Compound x

[0332] Under a nitrogen atmosphere, iii (8.02 g, 23.0 mmol, 1.2 equiv.) was added to a solution of xv (10.6 g, 19.2 mmol, 1.00 equiv.) in THF (53 mL) at room temperature, and then EEDQ (5.22 g, 21.1 mmol, 1.10 equiv.) was added. The resulting reaction mixture was heated to 40 °C and stirred at this temperature for 16 h. Then, the reaction mixture was filtered, and the collected solid was washed with THF (17 mL). The combined filtrate and washings were diluted with additional THF (15 mL) to provide a solution containing x, which was used directly in the next step.

[0333] Step B - Synthesis of Linker Compound 1

[0334] At room temperature, v (6.45 g, 21.1 mmol, 1.10 equiv.) was added to the solution of x obtained in Step A, and the reaction vessel was purged with nitrogen for 5 min. CuBr•SMe2 (197 mg, 0.959 mmol, 0.05 equiv.) and sodium ascorbate (190 mg, 0.959 mmol, 0.05 equiv.) were added sequentially, and the resulting reaction mixture was stirred at room temperature for 16 h. Then, the reaction mixture was diluted with THF (16 mL) and MeOH (342 mL) to a 3:1 MeOH / THF ratio, and Cuno-5 (11.4 g, 50 wt% relative to Compound v) was added. The resulting reaction mixture was stirred at room temperature for 3 h, and then the reaction mixture was filtered through a CELITE pad and the filtrate was collected to provide a solution of Compound 1 in THF (<10 ppm Cu). Then, 180 mL of the filtrate (40% of the total material) was purified using silica gel column chromatography (0% to 15% MeOH / DCM) to provide Linker Compound 1 as an oil. 11H NMR (400 MHz, CD3CN) δ 8.90 (s, 2H), 8.80 (s, 1H), 7.72 (s, 1H), 7.52 (dd, J = 13.4, 8.3 Hz, 3H), 7.27 (d, J = 8.6 Hz, 2H), 7.17 (s, 1H), 7.00 (s, 1H), 5.39 (s, 1H), 4.51 (d, J = 3.2 Hz, 2H), 4.49 - 4.41 (m, 3H), 4.39 (d, J = 5.7 Hz, 2H), 4.07 - 3.99 (m, 4H), 3.86 - 3.76 (m, 2H), 3.58 - 3.47 (m, 30H), 3.37 (q, J = 5.6 Hz, 2H), 3.29 (s, 4H), 3.00 (q, J = 6.5 Hz, 2H), 2.54 (t, J = 7.1 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 1.90 (q, J = 7.2 Hz, 3H), 1.74 (dq, J = 9.4, 4.1 Hz, 1H), 1.38 (s, 14H). MS: Calcd: [M+H] + 1193.6, Found: 1193.7

[0335] Step C (Optional) – Adsorption of Linker Compound 1 onto the Solid Support

[0336] To a solution of Linker Compound 1 in MeCN (2.5 V relative to the solution of Compound 1) was added IPAc (3x volume of MeCN). The resulting suspension was stirred at room temperature and Celite-545 (300 wt% relative to Compound 1) was added, followed by the dropwise addition of additional IPAC (2.5 V relative to Compound 1) over 15 minutes. Stirring was stopped and the resulting slurry was aged at room temperature for 30 minutes and then filtered. The collected solid was washed with 2-MeTHF (3x 4V relative to Compound 1) and then dried in vacuo under a nitrogen atmosphere to afford Linker Compound 1 adsorbed on diatomaceous earth.

[0337] Embodiment 8

[0338] Preparation of Linker-Payload A

[0339]

[0340] Step A – Synthesis of Compound xxi

[0341] Payload xix (485 mg, 0.948 mmol) and 5 mL of MeCN were added to a 40 mL vial. The resulting solution was cooled to -20 °C, and triphosgene (93 mg, 0.313 mmol) was added, followed by the dropwise addition of pyridine (192 μl, 2.370 mmol). The mixture was stirred at -20 °C for 45 minutes, and then a solution of linker compound 1 (1301 mg, 1.090 mmol) in 5 mL of MeCN was added dropwise. The resulting reaction mixture was stirred at -20 °C for 30 minutes, the cold bath was removed, and the reaction mixture was warmed to room temperature and then stirred overnight. Then, the resulting reaction mixture was filtered, and the filtrate was concentrated in vacuo. The residue obtained was purified by column chromatography (DCM eluent containing 5% MeOH) to afford compound xxi as a solid. Calculated exact mass: 1729.73. Observed mass: 1730.73 (M+H).

[0342] Step B - Deprotection of xxi

[0343] A solution of compound xxi (at a concentration of 100 mg / mL) was prepared in MeCN solution. Methanesulfonic acid (100 mg, 67.3 μL) was added to 3 mL of this solution, and the resulting reaction mixture was aged at 25 °C for 2 hours. Then, 5-ethyl-2-methylpyridine (167 mg, 182 μL) was added, and the resulting mixture was cooled to room temperature. The reaction mixture was purified directly by HPLC (Kromasil 60-10 diol resin, eluted with a mixture of MeCN, tBuOH, and MeOH). The HPLC fraction containing 216 mg of compound A in the mixture of acetonitrile:tert-butanol:methanol was concentrated and the solvent was converted to 1,3-dioxolane to a concentration of 94 mg / mL. The resulting solution was added dropwise to tert-butyl acetate (21 mL) to precipitate the product, and the resulting slurry was stirred at room temperature for 3 hours. Then, the slurry was filtered, and the collected solid was dried under a stream of nitrogen to afford the linker-payload A as a solid. The product identity was confirmed by HPLC using a true product standard. Observed mass: 1631.1 (M+H).

[0344] Embodiment 9

[0345] Alternative preparation of linker-payload A on a diatomaceous earth support

[0346]

[0347] Step A - Synthesis of payload intermediate xx

[0348] The payload compound xix (21.4 g) was slurried in MeCN (298 mL), pyridine (7.6 mL) was added to the resulting slurry, and the resulting mixture was cooled to -10 °C. Triphosgene (4.1 g) was added and the resulting reaction mixture was aged at -10 °C for 4 hours. The resulting solution containing compound xx was used directly in the next step.

[0349] Step B - Synthesis of Payload / Linker Intermediate xxi

[0350] A solution of linker compound 1 (33.1 g) in MeCN (139 mg / mL, KF <200 ppm) was added to a solution of payload intermediate xx (prepared in Step A), the resulting reaction mixture was aged at -10 °C for about 18 hours, and the reaction was quenched with water (2.5 mL). The resulting mixture was warmed to 20 °C, filtered, and washed with MeCN (33 mL) and EtOAc (33 mL). The filtrate was diluted with EtOAc (264 mL) and washed successively with 5% aqueous NaMSA (2 x 265 mL) and water (132 mL). The EtOAc phase (containing linker / payload intermediate compound xxi) was collected and the solvent was converted to MeCN by distillation to a final volume of 297 mL.

[0351] Step C - Synthesis of Payload / Linker Compound xxi on Celite

[0352] A MeCN solution containing compound 8a (prepared in Step B) was diluted with MTBE (132 mL), and Celite (165 g) was added to the resulting solution. Additional MTBE (462 mL) was added slowly and the resulting slurry was filtered. The collected solid was washed with a mixture of MTBE:MeCN (248 mL MTBE, 99 mL MeCN) and dried in vacuo to afford the drug / linker compound xxi adsorbed on a Celite support, 203 g, 18.6 wt%.

[0353] While the foregoing specification teaches the principles of the present disclosure and provides examples for purposes of illustration, the practice of the methods of the present disclosure includes all common variations, adaptations, and / or modifications that fall within the scope of the appended claims. The entire contents of all publications, patents, and patent applications cited herein are hereby incorporated by reference into the present disclosure.

Claims

1. A method for preparing a compound of structural formula (I): wherein PG is a primary amine protecting group, the method comprising the steps of: (A) In an organic solvent A, a compound of structural formula (i): is contacted with a deoxychlorinating agent at a certain temperature for a period of time sufficient to form a compound of structural formula (ii): wherein the organic solvent A is selected from dichloromethane, toluene, THF, acetonitrile, and mixtures thereof; (B) In an organic solvent B, in the presence of a base, the product of step A is contacted with a compound of structural formula (iii): and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (iv): The organic solvent B is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and (C) In an organic solvent C, in the presence of a copper salt and a source of azide anions, the product of step B is contacted with a compound of structural formula (v): at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent C is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, dichloromethane, water, and mixtures thereof.

2. The method according to claim 1, wherein the deoxychlorinating agent used in step (A) is selected from SOCl2, PCl3, PCl5, POCl3, oxalyl chloride, tosyl chloride, mesyl chloride, triphenylphosphine / CCl4, and triphenylphosphine / hexa chloroethane; the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU; the base used in step (B) is selected from carbonate salts, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine; the copper salt used in step (C) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2; the source of azide anions in step (C) is NaN3 or KN3; and the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

3. A method for preparing a compound of structural formula (I): wherein PG is a primary amine protecting group, the method comprising the steps of: (A) In an organic solvent D, a compound of structural formula (vi): is contacted with a deoxychlorinating agent at a certain temperature for a period of time sufficient to form a compound of structural formula (vii): wherein the organic solvent D is selected from dichloromethane, toluene, THF, acetonitrile, and mixtures thereof; (B) In an organic solvent E, the product of step A is contacted with diglycolic anhydride (viii): at a certain temperature for a period of time sufficient to form a compound of structural formula (ix): wherein the organic solvent E is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, and mixtures thereof; (C) In an organic solvent F, in the presence of a base, the product of step B is contacted with a compound of structural formula (iii): and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula (iv): wherein the organic solvent F is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and (D) In an organic solvent G, the product of step C is contacted with azide anions at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (x): Then, in the presence of a copper salt and a source of azide anions, the intermediate compound (x) is contacted with a compound of structural formula (v): at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent G is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water, and mixtures thereof.

4. The method according to claim 3, wherein: the dechlorinating agent used in step (A) is selected from SOCl2, PCl3, PCl5, POCl3, oxalyl chloride, tosyl chloride, mesyl chloride, triphenylphosphine / CCl4, and triphenylphosphine / hexa chloroethane; the amide coupling agent used in step (C) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU; the base used in step (C) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine; the source of azide anions in step (D) is NaN3 or KN3; the copper salt used in step (D) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2; and the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

5. A method for preparing a compound of formula (I): wherein PG is a primary amine protecting group, the method comprising the steps of: (A) In an organic solvent J, a compound of structural formula (ii): is contacted with azide anions at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (xv): Then, in the presence of a copper salt, the intermediate compound (xv) is contacted with a compound of structural formula (v): at a certain temperature for a period of time sufficient to form a compound of structural formula (xvi): wherein the organic solvent J is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water, and mixtures thereof, and (B) In organic solvent K, in the presence of an amide coupling agent and a base, the product of step A is contacted with a compound of structural formula (iii): at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent K is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, and mixtures thereof.

6. The method according to claim 5, wherein: the source of the azide anion in step (A) is NaN3 or KN3; the copper salt used in step (A) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, and Cu3(PO4)2; the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU; the base used in step (B) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine; and the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

7. A method for preparing a compound of formula (I): wherein PG is a primary amine protecting group, the method comprising the steps of: (A) In organic solvent H, a compound of structural formula (xi): is contacted with an azide anion at a certain temperature for a period of time sufficient to form an intermediate compound of structural formula (xii): Then, in the presence of a copper salt, the intermediate compound (xii) is contacted with a compound of structural formula (v): at a certain temperature for a period of time sufficient to form a compound of structural formula (xiii): wherein the organic solvent H is selected from THF, acetonitrile, DMF, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, water, and mixtures thereof, and (B) In organic solvent I, in the presence of an amide coupling agent and a base, the product of step A is contacted with a compound of structural formula (xiv): at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent I is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, ethyl acetate, dichloromethane, and mixtures thereof.

8. The method according to claim 7, wherein: the source of the azide anion in step (A) is NaN3 or KN3; the copper salt used in step (A) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2; the amide coupling agent used in step (B) is selected from HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU; the base used in step (B) is selected from carbonate bases, TEA, DIPEA, DBU, pyridine, and 2-methylpyridine; and the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

9. A method for preparing a compound of structural formula (I): wherein PG is a primary amine protecting group, the method comprising the steps of: (A) In an organic solvent L, a compound of structural formula xv: is contacted with a compound of structural formula iii: wherein PG is a primary amine protecting group, and an amide coupling agent at a certain temperature for a period of time sufficient to form a compound of structural formula x: The organic solvent A is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, DMF, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and (B) In an organic solvent M, in the presence of a copper salt, the product of step A is contacted with a compound of structural formula v: at a certain temperature for a period of time sufficient to form a compound of structural formula (I), wherein the organic solvent M is selected from THF, acetonitrile, toluene, methyl THF, isopropyl acetate, dichloromethane, water, and mixtures thereof.

10. The method according to claim 9, wherein the amide coupling agent used in step (A) is selected from EEDQ, HATU, EDC, DCC, HBTU, BOP, COMU, and TSTU; the copper salt used in step (B) is selected from CuCl, CuBr, CuBr-SMe2, CuI, Cu(OAc), Cu(OAc)2, CuSO4, Cu3(PO4)2; and the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc.

11. A method for preparing a compound of structural formula xix: The method comprises, in a solvent P, in the presence of an acid, a metal salt, and an oxidizing agent, contacting a compound of structural formula (xx): with a compound of structural formula xxv: at a certain temperature for a period of time sufficient to form a compound of structural formula (xix), wherein the solvent P is selected from water, MeCN, acetic acid, trifluoroethanol, NMP, propylene glycol, trifluoroacetic acid, and mixtures thereof.

12. The method according to claim 11, wherein: the acid used is selected from H2SO4, TFA, phosphoric acid, sulfonic acid, and methanesulfonic acid; the metal salt used is selected from Fe2SO4, V(acac)3, V2O3, NaVO3, FeCl3, and FeO; and the oxidizing agent used is selected from H2O2, persulfate, peracetic acid, cumene hydroperoxide, 2-butanone peroxide, potassium peroxymonosulfate, urea hydrogen peroxide, sodium perborate, di-tert-butyl peroxide, dibenzoyl peroxide, and sodium hypochlorite.

13. A method for preparing linker-payload A: The method comprises the steps of: (A) In an organic solvent N, in the presence of a base, contacting a compound of structural formula xix: with phosgene or triphosgene at a certain temperature for a period of time sufficient to form a compound of structural formula xx: wherein the organic solvent N is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and (B) in an organic solvent O, reacting the product of step A with a compound of structural formula (I): contacting at a certain temperature for a period of time sufficient to form a compound of structural formula xxi: wherein the organic solvent N is selected from methyl ethyl ketone, acetone, dichloroethane, dimethyl ether, diethyl ether, methyl isobutyl ketone, toluene, THF, DCM, MTBE, propylene carbonate, DME, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 2-methyltetrahydrofuran, xylene, ethyl acetate, NMP, anisole, isopropyl acetate, acetonitrile, and mixtures thereof; and (C) removing the protecting group from the product of step B to provide linker-payload A:

14. The method according to claim 13, wherein: the base used in step A is selected from carbonate bases, TEA, DIPEA, pyridine, and 2-methylpyridine; the primary amine protecting group is selected from Boc, MMT, Cbz, and Fmoc; and TFA or methanesulfonic acid is used in step (C) to remove the primary amine protecting group.

15. The method according to any one of claims 1-10, wherein the compound of formula (I) is adsorbed onto a solid support.

16. The method according to claim 13 or claim 14, wherein the compound of formula xxi is adsorbed onto a solid support.

17. The method according to claim 13 or claim 14, wherein linker-payload A is adsorbed onto a solid support.

18. The method according to any one of claims 15-17, wherein the solid support is diatomaceous earth.

19. A compound selected from: or a salt thereof.

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