TW202317088A - Preparation method of eribulin derivatives - Google Patents

Abstract

The present invention relates to a preparation method of eribulin derivatives. Specifically, the present invention relates to the preparation method of compound represented by formula (I). The preparation method has high yield, mild reaction conditions and suitable for industrial production.

Description

Preparation method of eribulin derivative

The disclosure belongs to the field of medicine, and relates to a preparation method of eribulin derivatives.

This application claims priority to Chinese patent application 202111196863.7 with a filing date of October 14, 2021. This application cites the full text of the above-mentioned Chinese patent application.

Microtubules are powerful, filamentous cytoskeletal proteins associated with a variety of cellular functions including intracellular migration and transport, cell signaling, and maintenance of cell shape. Microtubules also play a key role in mitosis, the division of cells by forming the mitotic spindle required for the division of chromosomes into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization kinetics through the reversible, noncovalent addition of α and β tubulin dimers to the ends of microtubules. This kinetic behavior and the resulting control of microtubule length are integral to the proper function of the mitotic spindle. Even small changes in microtubule dynamics implicate axial checkpoints, inhibit cell cycle progression during mitosis, and subsequently cause cell death (Mukhtar et al. (2014) Mol. Cancer Ther . 13: 275-84). Because of their rapid cell division, cancer cells are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Therefore, tubulin inhibitors and other microtubule-targeting agents hold promise as a class of drugs for the treatment of cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9: 790-803).

Halichondrin B (Halichondrin B) is a polyether macrolide compound containing only C, H, and O atoms isolated from the sponge Halichondria okadai by Japanese scientists Hirata and Uemura in 1986. tumor activity. The molecular structure of halichondrin B is very complex, including 32 chiral centers, with more than 4 billion isomers, and the synthesis is very difficult. Eribulin is a derivative of halichondrin B and is a tubulin inhibitor. WO9965894 discloses the structure and synthesis method of Eribulin for the first time. On November 15, 2010, the FDA approved Eribulin mesylate (Halaven) injection for the treatment of patients with metastatic breast cancer who have received at least two chemotherapy regimens.

PCT/CN2021/073314 relates to a new eribulin derivative and its drug conjugate. The compound and its drug conjugate have good antitumor activity. The structure of the compound is as follows:

The purpose of the present disclosure is to provide a new preparation method of eribulin derivatives.

其中，R ₁選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、環烷基（如C _3-8環烷基，包括但不限於環丙基、環戊基或環己基）、芳基和雜芳基，所述的烷基、環烷基、芳基和雜芳基各自獨立地任選被選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、烷氧基（如C _1-6烷氧基，包括但不限於甲氧基、乙氧基、丙氧基、異丙氧基）、鹵素（如氟、氯、溴）、氘、氨基、氰基、硝基、羥基、羥烷基、環烷基、雜環烷基、芳基和雜芳基中的一個或多個取代基所取代，優選C _1-6烷基或C _3-8環烷基，更優選甲基。 The present disclosure also provides a method for preparing the compound shown in formula (I), including the step of preparing the compound shown in formula (I-1) from the compound shown in formula (I-2), and the compound shown in formula (I-1) The step of compound deprotection group preparation compound shown in formula (I),

Among them, R ₁ is selected from alkyl (such as C _1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C _3-8 cycloalkyl, including but not limited to cycloalkyl propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C _1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C _1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl The substituent is substituted, preferably C _1-6 alkyl or C _3-8 cycloalkyl, more preferably methyl.

In certain embodiments, R _is selected from methyl, and the methylating agent used in the step is selected from methyl iodide, dimethyl sulfate or trimethyloxonium tetrafluoroborate, preferably trimethyloxonium Tetrafluoroborate.

In some embodiments, the molar ratio of the compound represented by the formula (I-2) to the methylating agent is 1:0.1~1:10, preferably 1:1~1:5, more preferably 1: 1~1:3.

In some embodiments, the reaction temperature is -50~-50°C, preferably -20~-5°C.

The removal of the amino-protecting group can be carried out by methods commonly used in the art. In some embodiments, the reagent for removing the amino protecting group is selected from ammonia water, methylamino alcohol solution, ethylenediamine and hydrazine, preferably methylamine ethanol solution.

In some embodiments, the method further includes the step of preparing the compound represented by formula (I-2) from the compound represented by formula (I-3),

Solvents used in the reactions described in the present disclosure may be conventional solvents such as dimethylformamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide, tetrahydrofuran, ethyl acetate, dioxane, toluene , dimethylsulfoxide, diethyl ether, isopropyl ether, methyl tertiary butyl ether, methylene chloride, chloroform, acetone, acetonitrile, methanol, ethanol, isopropanol, one or more of water, preferably tetrahydrofuran, ethyl acetate , dioxane, toluene, dimethyl oxide, diethyl ether, isopropyl ether, dichloromethane, chloroform, acetone, acetonitrile, methanol, ethanol, isopropanol in one or more.

。 The present disclosure also provides a method for preparing a compound represented by formula (LI), including the method for preparing a compound represented by formula (I) described in the present disclosure, wherein L is a linker,

.

In certain embodiments, the linker is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in a cell, eg, a cancer cell. In some embodiments, when the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC, the Eribulin Derivative drug moiety is cleaved from the antibody portion, and the cleavage releases the unmodified form of the Eribulin Derivative . In some embodiments, the linker comprises a cleavable moiety positioned such that no portion of the linker or the antibody portion remains bound to the eribulin derivative after cleavage.

In certain embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody:drug ratios, increased targeted killing of cancer cells, reduced non- Off-target killing of cancer cells, and/or high drug load. In some embodiments, addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker. In some embodiments, the increased potency and/or cytotoxicity is in cancers expressing intermediate levels of an antigen targeted by the antibody portion of the ADC (eg, intermediate FRA expression). In some embodiments, the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is one that is cleavable by an enzyme. In some embodiments, the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving. In certain embodiments, an enzymatically cleavable linker (eg, a cathepsin-cleavable linker) exhibits one or more of the improved properties described above compared to an alternative cleavage mechanism.

In certain embodiments, the linker comprises an amino acid unit preferably comprising from 2 to 7 members selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, silk A peptide residue composed of amino acids of amine, glutamic acid, aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala -Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valamine acid-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

In certain embodiments, the linker is selected from:

其中，R ₁選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、環烷基（如C _3-8環烷基，包括但不限於環丙基、環戊基或環己基）、芳基和雜芳基，所述的烷基、環烷基、芳基和雜芳基各自獨立地任選被選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、烷氧基（如C _1-6烷氧基，包括但不限於甲氧基、乙氧基、丙氧基、異丙氧基）、鹵素（如氟、氯、溴）、氘、氨基、氰基、硝基、羥基、羥烷基、環烷基、雜環烷基、芳基和雜芳基中的一個或多個取代基所取代，優選C _1-6烷基或C _3-8環烷基，更優選甲基。 The present disclosure also provides a method for preparing an antibody-drug conjugate represented by formula (ADC-I), including the step of preparing the antibody-drug conjugate represented by formula (LI) from a compound represented by formula (LI), and the step of preparing the compound represented by the formula (LI) described in the present disclosure, Ab-(LD) _k (ADC-I) wherein, Ab is an antibody or an antigen-binding fragment thereof, and L is a link for covalently linking Ab to D child, said L is as defined in any one of claim items 5-7; and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or any value between any two values), -D is as follows:

Among them, R ₁ is selected from alkyl (such as C _1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C _3-8 cycloalkyl, including but not limited to cycloalkyl propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C _1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C _1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl The substituent is substituted, preferably C _1-6 alkyl or C _3-8 cycloalkyl, more preferably methyl.

In some embodiments, the antibody in the antibody-drug conjugate (ADC) of the present disclosure is selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In some embodiments, the antibody or antigen-binding fragment thereof of the antibody-drug conjugate (ADC-I) is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, Anti-HER3 (ErbB3) Antibody, Anti-HER4 (ErbB4) Antibody, Anti-CD20 Antibody, Anti-CD22 Antibody, Anti-CD30 Antibody, Anti-CD33 Antibody, Anti-CD44 Antibody, Anti-CD56 Antibody, Anti-CD70 Antibody, Anti-CD73 Antibody, Anti-CD105 Antibody Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin- C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-folate receptor alpha (FRA) antibody or antigen-binding fragment thereof.

In some embodiments, the antibody in the antibody-drug conjugate (ADC-I) is a known antibody, selected from but not limited to Trastuzumab (Trastuzumab), Pertuzumab (Pertuzumab) , Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Vitin-Pinertuzumab ( Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, farletuzumab, and Glematumamab or antigen-binding fragments thereof .

或

， 其中D為

，優選Ab為farletuzumab，優選k選自2.0~2.5或2.5~3.5。 In some embodiments, the antibody-drug conjugate is

or

, where D is

, preferably Ab is farletuzumab, preferably k is selected from 2.0~2.5 or 2.5~3.5.

The method disclosed in the prior art can be used to prepare the compound represented by the formula (L-I) with the compound represented by the formula (I) as a reactant, or to prepare the antibody represented by the formula (ADC-I) with the compound represented by the formula (L-I) as a reactant- Drug conjugates, such as methods disclosed in PCT/CN2021/073314, are incorporated herein in their entirety.

其中，R ₁選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、環烷基（如C _3-8環烷基，包括但不限於環丙基、環戊基或環己基）、芳基和雜芳基，所述的烷基、環烷基、芳基和雜芳基各自獨立地任選被選自烷基（如C _1-6烷基，包括但不限於甲基、乙基、異丙基）、烷氧基（如C _1-6烷氧基，包括但不限於甲氧基、乙氧基、丙氧基、異丙氧基）、鹵素（如氟、氯、溴）、氘、氨基、氰基、硝基、羥基、羥烷基、環烷基、雜環烷基、芳基和雜芳基中的一個或多個取代基所取代，優選C _1-6烷基或C _3-8環烷基，更優選甲基。 The present disclosure also provides compounds represented by formula (I-1),

Among them, R ₁ is selected from alkyl (such as C _1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C _3-8 cycloalkyl, including but not limited to cycloalkyl propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C _1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C _1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl The substituent is substituted, preferably C _1-6 alkyl or C _3-8 cycloalkyl, more preferably methyl.

The preparation method of the new eribulin derivatives described in this disclosure adopts a new amino protecting group, which avoids the selectivity problem in the methylation process, greatly reduces the generation of dimethylated impurities, and alleviates the The post-reaction treatment is difficult, and the reaction yield is improved, which is beneficial to large-scale industrial production. The product obtained by the existing preparation process has many dimethylated impurities, which are difficult to remove and ultimately affect the quality of the final product.

The "alkyl" mentioned in the present disclosure is preferably C ₁ -C ₆ alkyl. The "alkenyl" mentioned in the present disclosure is preferably a C ₂ -C ₆ alkenyl. The "alkynyl" in the present disclosure is preferably a C ₂ -C ₆ alkynyl. The "alkylene" in the present disclosure is preferably a C ₁ -C ₆ alkylene. The "alkenylene group" mentioned in the present disclosure is preferably a C ₂ -C ₆ alkenylene group. The "alkynylene group" mentioned in the present disclosure is preferably a C ₂ -C ₆ alkynylene group. The "alkoxy" in the present disclosure is preferably C ₁ -C ₆ alkoxy. The "alkylsulfide group" described in the present disclosure is preferably a C ₁ -C ₆ alkylsulfide group. The "cycloalkyl" mentioned in the present disclosure is preferably 3 to 12 membered, more preferably 3 to 6 membered cycloalkyl. The "fused cycloalkyl group" mentioned in the present disclosure is preferably 6 to 14 membered, more preferably 7 to 10 membered fused cycloalkyl group. The "heterocyclic group" mentioned in the present disclosure is preferably a 3- to 12-membered heterocyclic group, more preferably a 3- to 6-membered heterocyclic group. The "fused heterocyclic group" described in the present disclosure is preferably 6 to 14 membered, more preferably 7 to 10 membered condensed heterocyclic group. The "aryl group" mentioned in the present disclosure is preferably 6 to 14 membered, more preferably 6 to 10 membered aryl group. The "heteroaryl" mentioned in the present disclosure is preferably 5 to 12 membered, more preferably 5 to 10 membered heteroaryl.

Unless stated to the contrary, the terms used in the specification and claims for invention have the following meanings.

The term "drug" refers to a cytotoxic drug or an immunomodulator. Cytotoxic drugs can have strong chemical molecules in tumor cells that disrupt their normal growth. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, resulting in serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (e.g. At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu radioisotopes), toxic drugs, chemotherapy drugs, antibiotics and nucleolysin. Immunomodulators are inhibitors of immune checkpoint molecules.

The term "linker", "linker unit", "linker unit", "linker" or "linker segment" refers to a chemical structural segment or bond that is connected to a ligand at one end and a drug at the other end, and may also be connected after other linkers. Then connect with the drug. A joint may comprise one or more joint components. Exemplary linker building blocks include 6-maleiminocaproyl (MC), maleiminopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine- Phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP ), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4- iodo-acetyl)aminobenzoate (SIAB). Linkers can include Stretcher units, Spacer units, Amino acid units and Stretcher units. It can be synthesized by methods known in the art, such as described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid labile (e.g. hydrazone), protease sensitive (e.g. peptidase sensitive) linkers, photolabile linkers, dimethyl linkers or disulfide containing linkers can be used (Chari et al., Cancer Research 52: 127-131 ( 1992); U.S. Patent No. 5,208,020).

The term "stretch unit" refers to a chemical moiety that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.

The term "spacer unit" is a bifunctional chemical structural fragment that can be used to couple amino acid units and cytotoxic drugs to form antibody-drug conjugates. This coupling method can selectively link cytotoxic drugs to amines on the amino acid unit.

A Ala 丙胺酸 Alanine

V Val 纈胺酸 Valine

L Leu 白胺酸 Leucine

I Ile 異白胺酸 Isoleucine

F Phe 苯丙胺酸 Phenylalanine

W Trp 色胺酸 Tryptophan

Y Tyr 酪胺酸 Tyrosine

D Asp 天冬胺酸 Aspartic acid

H His 組胺酸 Histidine

N Asn 天冬醯胺 Asparagine

E Glu 麩胺酸 Glutamic acid

K Lys 離胺酸 Lysine

Q Gln 麩醯胺酸 Glutamine

C Cit 瓜胺酸 Citrulline

The term "amino acid" refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure. The general formula is H ₂ NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the position of the amino group connected to the carbon atom in the carboxylic acid, it can be divided into α, β, γ, δ, ε...-amino acids. In the biological world, the amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the α-carbon atom, that is, α-amino acids, including glycine (Glycine), alanine (Alanine) ), Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid acid), histidine, asparagine, glutamic acid, lysine, glutamine, methionine, Arginine, Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline. As is well known to those of ordinary skill in the art to which the invention pertains, unnatural amino acids do not constitute native proteins and thus do not participate in the synthesis of the antibodies of the present disclosure. The three-letter and one-letter codes for amino acids used in this disclosure are as described in J.biol.chem , 243, p3558 (1968). Abbreviation abbreviation name structure G Gly Glycine

A Ala Alanine

V Val Valine

L Leu Leucine

I Ile Isoleucine

f Phe Phenylalanine Phenylalanine

W Trp Tryptophan

Y Tyr Tyrosine Tyrosine

D. Asp Aspartic acidAspartic acid

h His Histidine

N Asn Asparagine

E. Glu Glutamic acidGlutamic acid

K Lys Lysine

Q Gln Glutamine Glutamine

C Cit Citrulline Citrulline

(VI)； 縮寫 接頭組件包括但不限於： MC=6-馬來醯亞氨基己醯基，結構如下：

Val-Cit或「vc」=纈胺酸-瓜胺酸(蛋白酶可切割接頭中的例示二肽) 瓜胺酸=2-胺基-5-脲基戊酸 PAB=對胺基苄氧羰基(「自我犧牲」接頭組件的例示) Me-Val-Cit=N-甲基-纈胺酸-瓜胺酸(其中接頭肽鍵已經修飾以防止其受到組織蛋白酶B的切割) MC(PEG) ₆-OH=馬來醯亞胺基己醯基-聚乙二醇(可附著於抗體半胱胺酸) SPP=N-琥珀醯亞氨基4-(2-吡啶基硫代)戊酸酯 SPDP=N-琥珀醯亞氨基3-(2-吡啶基二硫代)丙酸酯 SMCC=琥珀醯亞氨基-4-(N-馬來醯亞氨基甲基)環己烷-1-羧酸酯 IT=亞氨基硫烷 PBS=磷酸緩衝鹽溶液。 In the present disclosure, the extension unit is PAB, which has a structure such as a 4-iminobenzylcarbamoyl segment, and its structure is shown in formula (VI), which is connected to D,

(VI); Abbreviated linker components include but are not limited to: MC=6-maleiminocaproyl, the structure is as follows:

Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease cleavable linker) Citrulline = 2-amino-5-ureidovalanoic acid PAB = p-aminobenzyloxycarbonyl ( An illustration of a "self-sacrificing" linker component) Me-Val-Cit=N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent its cleavage by cathepsin B) MC(PEG) ₆ - OH=maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine) SPP=N-succinimidyl 4-(2-pyridylthio)pentanoate SPDP=N -Succinimidyl 3-(2-pyridyldithio)propionate SMCC=Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate IT= Iminothiolane PBS = Phosphate Buffered Saline.

The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug through a stable linker unit. In this disclosure, "antibody drug conjugate" (antibody drug conjugate, ADC) refers to linking a monoclonal antibody or antibody fragment with a toxic drug with biological activity through a stable linker unit.

The term "drug loading" can be expressed as the ratio of the amount of drug to the amount of antibody, that is, the average amount of drug conjugated to each antibody in the ADC. The range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) linked to each antibody (Ab). In the embodiments of the present disclosure, the drug loading is expressed as k, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean value. The average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.

The molecular size variant determination method (CE-SDS) of the disclosed monoclonal antibody can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia" 0542), quantitative determination of the purity of recombinant monoclonal antibody products.

The loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including: (1) Control the molar ratio of the linking reagent and the monoclonal antibody, (2) Control reaction time and temperature, (3) Choose different reagents.

The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are µ chain, δ chain, γ chain, α chain, and ε chain. The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain. The antibody described in the present disclosure is preferably a specific antibody against a cell surface antigen on a target cell, and the sequence of about 110 amino acids near the N-terminus of the heavy chain and light chain of the antibody varies greatly, which is a variable region (Fv region); The rest of the amino acid sequence near the C-terminal is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.

The term "murine antibody" in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "Vbase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, E.A. et al. Al, 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid decreased immunogenicity and decreased activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991, and the methods of Winter and colleagues (Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)).

The term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody are all human, and the immunogen is removed sex and side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed by "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments, comprising (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature , 341: 544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally A combination of two or more isolated CDRs connected by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to link them via a synthetic linker, making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule chain (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science , 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci , USA 85: 5879 -5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art to which the invention pertains, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (which cleaves the amino acid residue at position 224 of the H chain), wherein the H chain N-terminal About half of the sides and the entire L chain are held together by disulfide bonds.

F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

In addition, the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" means an antibody comprising a heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules. Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444- 6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng . 8: 725-731; Choi et al. (2001), Eur.J. Immuno, 1.31: 94-106; Hu et al. (1996 ), Cancer Res. 56: 3055-3061; described by Kipriyanov et al. (1999), J. Mol. Biol . 293: 41-56 and Roovers et al. (2001), Cancer Immunol .

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991), Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the variable domain of the light chain, and to those of the variable domain of the heavy chain. CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3). Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997), JMB , 273: 927-948) and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist , 7, 132- 136 (1999); Lefranc, MP et al., Dev. Comp. Immunol ., 27, 55-77 (2003)) et al. Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

The term "antibody framework" refers to the portion of a variable domain VL or VH that serves as a scaffold for the complementarity determining regions (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G. E. Morris, Ed. (1996)).

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, antibodies bind with an affinity (KD) of less than about 10 ^"7M , eg, about less than 10 ^"8M , 10 ^"9M or ^10" 10M or less.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. Nucleic acids are "operably linked" when they are placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction, thereby following The host genome replicates together (eg, non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and Chapter 15. Antigen-binding fragments can also be prepared by conventional methods. In the antibody or antigen-binding fragment described in the invention, one or more human FR regions are added to the non-human CDR region by genetic engineering methods. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001 ISBN Obtained on 012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; the Bacillaceae, such as Bacillus subtilis, Pneumococcus , Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.

Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production. The culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. The bound antibody was then eluted by the pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.

Amino acid sequence "identity" means the alignment of the amino acid sequences and the introduction of gaps where necessary to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity, in the first sequence with The percentage of amino acid residues that are identical to the amino acid residues in the second sequence. For purposes of determining percent amino acid sequence identity, alignment can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art to which this invention pertains can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term "peptide" refers to a compound fragment between an amino acid and a protein. It is composed of two or more amino acid molecules connected to each other through a peptide bond. It is a structural and functional fragment of a protein, such as hormones, enzymes, etc. Classes, etc. are essentially peptides.

The term "sugar" refers to a biomacromolecule composed of three elements, C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.

The term "fluorescent probe" refers to a characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescent properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can vary with the nature of the environment, such as polarity A class of fluorescent molecules that can be sensitively changed by changes in refractive index, viscosity, etc., which interact non-covalently with nucleic acid (DNA or RNA), protein or other macromolecular structures to change one or several fluorescent properties. It can be used to study the properties and behavior of macromolecular substances.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, second-butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-di Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4 -Ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3 -Ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, di Butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Base, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Amylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, ring Alkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 12 carbon atoms, more preferably contains 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle" refers to the ring system in a cycloalkyl group.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine Piperyl, piperyl, morpholinyl, thiomorpholinyl, homopiperyl, etc., preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.

和

； 芳基可以是取代的或非取代的，當被取代時，取代基優選為一個或多個以下基團，其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、鹵素、氫硫基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、羧基或羧酸酯基，優選苯基。 The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e. rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 10 membered, having a conjugated π-electron system, such as benzene base and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, where the ring bonded to the parent structure is the aryl ring. "Aromatic ring" refers to a ring system in an aryl group. Non-limiting examples of aryl groups include:

and

; aryl may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio , alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

和

。 The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 12 membered, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyridyl group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring. "Heteroaryl" refers to a ring system in a heteroaryl group. Non-limiting examples of heteroaryl groups include:

and

.

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio group, heterocycloalkylthio group, carboxyl group or carboxylate group.

"Carboxyl protecting group" is a suitable group known in the art for carboxyl protection, see the carboxyl protecting group in the literature ("Protective Groups in Organic Synthesis", 5 ^Th Ed. TW Greene & PGM Wuts), as For example, the carboxyl protecting group can be a substituted or unsubstituted C _1-10 straight chain or branched chain alkyl, a substituted or unsubstituted C _2-10 straight chain or branched alkenyl or alkynyl, a substituted Or unsubstituted C _3-8 cyclic alkyl, substituted or unsubstituted C _5-10 aryl or heteroaryl, or (C _1-8 alkyl or aryl) ₃ silane, etc.

"Amino-protecting group" is a suitable group known in the art for amino protection, see the amino-protecting group in the literature ("Protective Groups in Organic Synthesis", 5 ^Th . Ed. TW Greene & PGM Wuts), Preferably, the amino protecting group can be (C _1-10 alkyl or aryl) acyl group, for example: formyl, acetyl, benzoyl, etc.; can be (C _1-6 alkyl Or C _6-10 aryl) sulfonyl; it can also be (C _1-6 alkoxy or C _6-10 aryloxy) carbonyl, for example: Boc or Cbz; it can also be substituted or unsubstituted alkyl groups such as trityl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

The term "leaving group" refers to an atom or functional group that breaks away from a larger molecule in a chemical reaction. Representative leaving groups include halogen, substituted sulfonyloxy, phosphonyloxy, amino, (R _j ) ₃ N-, cyano, R _j S-, etc., wherein R _j is independently selected from From a hydrogen atom or a C ₁ ~C ₆ alkyl group. Substituted sulfonyloxy can be C ₁ ~C ₆ alkylsulfonyloxy, perfluoro C ₁ ~C ₆ alkylsulfonyloxy, arylsulfonyloxy, aralkylsulfonyloxy base oxygen etc. C ₁ ~C Specific examples of alkylsulfonyloxy groups include C ₁ ~C ₆ linear or branched alkylsulfonyloxy _groups , such as methylsulfonyloxy, ethylsulfonyloxy , n-propylsulfonyloxy, isopropylsulfonyloxy, n-butylsulfonyloxy, tertiary butylsulfonyloxy, n-pentylsulfonyloxy and n-hexyl Sulfonyloxy. Specific examples of perfluoro C ₁ ~C ₆ alkylsulfonyloxy groups include C ₁ ~C ₆ linear or branched perfluoroalkylsulfonyloxy groups, such as trifluoromethylsulfonyloxy groups, 1, 1, 2, 2, 2-pentafluoro-1-ethylsulfonyloxy, 1, 1, 2, 2, 3, 3, 3-heptafluoro-1-propylsulfonyloxy and 1, 1, 2, 2, 3, 3, 4, 4, 4-nonafluoro-1-butylsulfonyloxy. Examples of arylsulfonyloxy groups include: optionally having 1 to 3 alkyl groups selected from C ₁ to C ₆ straight or branched chains, C ₁ to C ₆ straight or branched chains on the benzene ring phenylsulfonyloxy and naphthylsulfonyloxy groups of substituents consisting of alkane, nitro and halogen atoms. Specific examples of phenylsulfonyloxy optionally having a substituent include phenylsulfonyloxy, 4-methylphenylsulfonyloxy, 2-methylphenylsulfonyloxy, 4-nitrophenylsulfonyloxy, 4-tolylsulfonyloxy, 2-nitrophenylsulfonyloxy, 3-chlorophenylsulfonyloxy and the like. Specific examples of naphthylsulfonyloxy include α-naphthylsulfonyloxy, β-naphthylsulfonyloxy and the like. Examples of aralkylsulfonyloxy groups include: phenyl (which optionally has 1 to 3 alkyl groups selected from C ₁ to C ₆ straight or branched chains on the benzene ring, C ₁ to C ₆ Straight chain or branched chain alkane, nitro and halogen atom substituents) substituted C ₁ ~C ₆ straight chain or branched alkylsulfonyloxy; and C ₁ ~C ₆ straight chain substituted by naphthyl Chain or branched alkylsulfonyloxy groups. Specific examples of alkylsulfonyloxy substituted by phenyl include benzylsulfonyloxy, 2-phenylethylsulfonyloxy, 4-phenylbutylsulfonyloxy, 4- Methylbenzylsulfonyloxy, 2-methylbenzylsulfonyloxy, 4-nitrobenzylsulfonyloxy, 4-methylbenzylsulfonyloxy, 3-chlorobenzyl Sulfonyloxy and the like. Specific examples of the alkylsulfonyloxy group substituted with naphthyl include α-naphthylmethylsulfonyloxy, β-naphthylmethylsulfonyloxy and the like.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .

」並未指定構型，即如果化學結構中存在構型異構，鍵「

」可以為「

」或「

」，或者同時包含「

」和「

」兩種構型。本公開所述化合物的化學結構中，鍵「

」並未指定構型，即可以為Z構型或E構型，或者同時包含兩種構型。 In the chemical structures of the compounds described in the present disclosure, the bond "

” does not specify configuration, i.e. if there is configurational isomerism in the chemical structure, the bond“

" can be "

"or"

, or both

"and"

"Two configurations. In the chemical structures of the compounds described in the present disclosure, the bond "

” does not specify the configuration, it can be the Z configuration or the E configuration, or contain both configurations at the same time.

The present disclosure will be explained in detail below in conjunction with specific examples, so that those with ordinary knowledge in the technical field of the present invention can more fully understand that the specific examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the present disclosure in any way.

The known starting materials of the present disclosure can be used or synthesized according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Darui Chemicals and other companies. Unless otherwise specified in the examples, the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere. The hydrogenation reaction is usually vacuumized and filled with hydrogen, and the operation is repeated 3 times. Unless otherwise specified in the examples, the solution refers to an aqueous solution. Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20°C to 30°C.

Example 1

Step 1: Synthesis of Compound 1-2 Weigh sodium bicarbonate (2.0g, 24 mmol) in the reaction flask, add 10 mL of purified water, dissolve compound 1-3 (4.95 g, 6.00 mmol) in 130 mL of purified water, add it to the reaction flask, weigh N-ethoxycarbonylphthalimide (5.3 g, 24 mmol) was added to the above reaction solution, after the addition was completed, the reaction was vacuumed under argon protection, and the reaction was stirred at room temperature until the end. DCM was added to the reaction liquid for extraction, the aqueous phase was extracted with DCM, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, filtered with suction, concentrated, and dried to obtain a crude product. Column chromatography obtained 4.89 g of compound 1-2, yield: 93%.

Step 2: Synthesis of compound 1-1 Weighing: trimethyloxonium tetrafluoroborate (13.4 g, 90.5 mmol) and 1,8-bisdimethylaminonaphthalene (19.4 g, 90.5 mmol) in a reaction flask, after addition, vacuum argon protection, Add 100 mL of anhydrous DCM, cool to -10 °C; dissolve 1-2 (4.86 g, 5.66 mmol) in 150 mL of DCM, add to the above reaction solution, and keep the reaction overnight. Control until the basic reaction of the raw materials is complete, and then stop the reaction. Suction filtration, concentrate the reaction solution to dryness, add 200 mL methyl tertiary butyl ether, filter, add 100 mL 1M potassium bisulfate aqueous solution to wash the mother liquor three times, and wash the aqueous phase twice with methyl tertiary butyl ether; phase, dried over anhydrous magnesium sulfate, concentrated, and dried to obtain a crude product. Column chromatography obtained 4.74 g of compound 1-1; yield: 96%, HPLC purity 98%.

Step 3: Synthesis of Compound 1 Weigh 6.0 g of compound 1-1 into a reaction flask, add 180 mL of methylamine in ethanol, under argon protection, stir at room temperature overnight, and then stop the reaction until the reaction of the raw materials is complete. Concentrate to dryness under reduced pressure, add 200 mL DCM and 100 mL purified water for extraction, wash the organic phase 3 times with 100 mL purified water, combine the aqueous phase, extract 2 times with DCM, combine the organic phase, dry over anhydrous magnesium sulfate, and concentrate to obtain the crude product . Column chromatography obtained 4.2 g of compound 1; total yield: 86.7%.

Example 2

Add compound 2 (17.6g, prepared according to PCT/CN2021/073314 method) and 126 ml DMF to the reaction flask, stir to dissolve, add sodium bicarbonate, stir at 20-25°C, dissolve compound 1 in 63 ml DMF, and use constant pressure Add the dropping funnel dropwise to the above reaction solution, after the dropwise addition, continue to react at 20-25°C for 2.5-3h, TLC detects that the reaction is complete, add 1134 ml of ethyl acetate/isobutanol (v:v=9:1), 1134 ml drinking water, 110ml saturated NaCl, extraction and separation, the remaining water phase was extracted twice with ethyl acetate/isobutanol (v:v=9:1), the combined organic phase was washed with saturated NaCl, dried over anhydrous sodium sulfate, Concentrate to dryness under reduced pressure to obtain the crude product. The column chromatography of the crude product gave 16.9 g of compound L-1, yield: 74%.

室溫下，將1-2’ (190 mg, 0.179 mmol)，三甲基氧四氟硼酸鹽(212 mg, 1.433 mmol)，1,8-雙二甲氨基萘(306 mg, 1.43 mmol)加入到反應瓶中；加入4.5 mL的二氯甲烷，然後氮氣球保護在室溫下攪拌至反應結束，加20 mL的MTBE稀釋，過濾，用約20 mL 的MTBE洗濾餅；合併濾液用1 N鹽酸洗滌；有機相用無水硫酸鈉乾燥；過濾，濾液減壓蒸乾；經矽膠柱(PE:EtOAc= 1:1）純化後得到產物1-1’ (154 mg)，收率80%，HPLC純度92%。 Comparative example 1

At room temperature, 1-2' (190 mg, 0.179 mmol), trimethyloxytetrafluoroborate (212 mg, 1.433 mmol), 1,8-bisdimethylaminonaphthalene (306 mg, 1.43 mmol) were added into a reaction flask; add 4.5 mL of dichloromethane, then stir at room temperature under nitrogen balloon protection until the reaction is complete, add 20 mL of MTBE to dilute, filter, and wash the filter cake with about 20 mL of MTBE; combine the filtrates with 1 N Wash with hydrochloric acid; dry the organic phase with anhydrous sodium sulfate; filter, and evaporate the filtrate to dryness under reduced pressure; purify by silica gel column (PE:EtOAc= 1:1) to obtain product 1-1' (154 mg), yield 80%, HPLC 92% purity.

Since this disclosure has been described in terms of specific embodiments thereof, certain modifications and equivalents will be apparent to those skilled in the art to which this invention pertains and are intended to be encompassed within the scope of this disclosure.

none

none

Claims (13)

A method for preparing a compound shown in formula (I), comprising the steps of preparing a compound shown in formula (I-1) from a compound shown in formula (I-2), and preparing a compound shown in formula (I-1) by deprotecting The step of compound shown in formula (I),

Among them, R ₁ is selected from alkyl (such as C _1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C _3-8 cycloalkyl, including but not limited to cycloalkyl propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C _1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C _1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl The substituent is substituted, preferably C _1-6 alkyl or C _3-8 cycloalkyl, more preferably methyl. The preparation method as described in claim item 1, wherein _R is selected from methyl, and the methylating reagent used in the step is selected from methyl iodide, dimethyl sulfate or trimethyloxonium tetrafluoroborate, preferably three Methyloxonium tetrafluoroborate. The preparation method as described in claim item 2, wherein the molar ratio of the compound represented by the formula (I-2) to the methylating reagent is 1:0.1~1:10, preferably 1:1~1:5, More preferably 1:1~1:3. The preparation method according to any one of claims 1-3, wherein the preparation method further comprises the step of preparing the compound represented by formula (I-3) from the compound represented by formula (I-2),

. A preparation method of the compound shown in formula (LI), including the preparation method of the compound shown in formula (I) described in any one of claim items 1-4, wherein L is a linker,

. The preparation method as described in claim item 5, wherein the linker comprises an amino acid unit, and the amino acid unit preferably comprises 2 to 7 selected from the group consisting of phenylalanine, glycine, valine, and lysine. , citrulline, serine, glutamic acid, aspartic acid amino acid residues, more preferably valine-citrulline (Val-Cit), alanine-alanine-aspartate Amide (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val -Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly). The preparation method as claimed in item 5, wherein the linker is selected from:

. A method for preparing an antibody-drug conjugate shown in formula (ADC-I), comprising the step of preparing the antibody-drug conjugate shown in formula (ADC-I) from a compound shown in formula (LI), and claim item 5 -7 The step of preparing the compound represented by formula (LI), Ab-(LD) _k (ADC-I) wherein, Ab is an antibody or an antigen-binding fragment thereof, and L is covalently linking Ab to D The linker, said L is as defined in any one of claim items 5-7; and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 or any value between any two values), -D is as follows:

Among them, R ₁ is selected from alkyl (such as C _1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C _3-8 cycloalkyl, including but not limited to cycloalkyl propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C _1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C _1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl The substituent is substituted, preferably C _1-6 alkyl or C _3-8 cycloalkyl, more preferably methyl. The preparation method according to claim 8, wherein the antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies. The preparation method according to claim 8 or 9, wherein the antibody or antigen-binding fragment thereof in the antibody-drug conjugate (ADC-I) is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7 -H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 Antibody, Anti-CD73 Antibody, Anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody , anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-folate receptor alpha (FRA) antibody or an antigen-binding fragment thereof. The preparation method as described in claim item 8, wherein the antibody in the antibody-drug conjugate (ADC-I) is a known antibody, selected from but not limited to Trastuzumab (Trastuzumab), Pertuzumab Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Vitin-Pina Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, farletuzumab, and Glematumamab or an antigen-binding fragment thereof. The preparation method as described in claim item 8, wherein the antibody-drug conjugate is

or

, where D is

, preferably Ab is farletuzumab, preferably k is selected from 2.0~2.5 or 2.5~3.5. A compound shown in formula (I-1),

Among them, R1 is selected from alkyl (such as C1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C1-6 alkyl, including But not limited to methyl, ethyl, isopropyl), alkoxy (such as C1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen ( Such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, substituted by one or more substituents, It is preferably C1-6 alkyl or C3-8 cycloalkyl, more preferably methyl.