Fused-ring drug conjugate, preparation method therefor, and use thereof

Abstract

A fused-ring drug conjugate, a preparation method therefor, and use thereof. The conjugate has a structure represented by the formula AB-[M-L-E-D]x. The conjugate has a relatively good drug-to-antibody conjugation ratio and has excellent targeted killing effects on solid tumors such as gastric cancer, breast cancer, lung cancer, and urothelial carcinoma.

Description

Cyclocyclic drug conjugates, their preparation methods and uses

[0001] This application is based on CN application number 202410580820.6, filed on May 10, 2024, and claims priority to the aforementioned application, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of targeted therapy, specifically to a cyclic drug conjugate, its preparation method, and its uses. Background Technology

[0003] Antibody-drug conjugates (ADCs) for cancer treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a cytotoxic agent that kills tumor cells), and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells. The ADC then enters the cancer cells via endocytosis. The bioactive molecule is then released within the cancer cells, killing them by inhibiting microtubules or damaging their DNA, thereby minimizing damage to normal tissue cells.

[0004] ErbB family receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. This family includes four members: epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4). Clinically, the anti-ErbB2 antibody trastuzumab (trade name Herceptin) is commonly used to treat breast cancer with high ErbB2 expression, but the clinical response rate is low. To improve treatment efficacy, in recent years, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansine (e.g., DM1), aurestatins (e.g., MMAE), or DNA topoisomerase I inhibitors (e.g., Dxd) have been used in clinical treatment (Trastuzumab emtansine, Disitamab vedotin, Trastuzumab deruxtecan).

[0005] With the widespread clinical application of the aforementioned ADC drugs in treating Her2-expressing tumors, safety issues and drug resistance problems, including neurotoxicity, hematologic toxicity, hepatotoxicity, and interstitial pneumonia, have gradually emerged (Pharmacology & Therapeutics 2019, 200, 110-125; Breast Cancer Research and Treatment 2020, 183, 23-39; JAMA Oncol. 2021, 7, 1873-1881; Drug Deliv. 2022, 29, 1335-1344; Cancers 2023, 15, 1130; Cancers 2023, 15, 1278).

[0006] Specifically, both Disitamab vedotin and Trastuzumab deruxtecan use maleimide linkers (MC) as the linker portion. Literature reports that under physiological conditions, MC linkers are prone to reverse Michael reaction and thiol exchange, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). Regarding the hydrophilicity and hydrophobicity of linkers, Disitamab vedotin and Trastuzumab deruxtecan used valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively, which are relatively hydrophobic. Literature reports that the hydrophilicity of linkers significantly affects the hydrophilicity of ADCs, thereby affecting the aggregation, pharmacokinetics and toxicity of ADCs (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3.). In terms of the bioactive molecules that kill cancer cells, Trastuzumab emtansine uses the microtubule inhibitor DM1 as a cytotoxin, which results in a weak bystander effect when combined with a non-cleavable linker; Disitamab vedotin uses the aurorastin toxin MMAE as a bioactive molecule, which is prone to problems such as neurotoxicity accumulation after continuous and repeated use; and both of the above ADC drugs are non-site-specific random conjugations with a drug loading ratio (DAR) of about 4, resulting in poor homogeneity.

[0007] This invention aims to improve upon the aforementioned problems in existing pharmaceutical technologies, specifically by providing a class of anti-Her2 antibody-drug conjugates for the treatment of Her2-expressing tumors. The antibody-drug conjugates exhibit good stability, homogeneity, hydrophilicity, efficacy, and safety. Summary of the Invention

[0008] This application relates to a compound having the structure shown in the general formula DEL-M'. The compound can be used to prepare antibody-drug conjugates, and the conjugates exhibit excellent targeted killing effects against tumors.

[0009] compound

[0010] In one aspect, this application provides a compound or a pharmaceutically acceptable salt thereof having the structure shown in formula DEL-M', wherein:

[0011] M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site;

[0012] L is the structural segment connecting connectors M and E;

[0013] E is a structural segment connecting L and D;

[0014] D is a cytotoxic drug fragment.

[0015] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1- 6-alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be substituted by one or more suitable substituents.

[0016] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1- 6-alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments.

[0017] In some implementations, Lg is selected from halogenated, substituted, or unsubstituted C. 1-6 Alkyl sulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH2).

[0018] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methanesulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH) or amino (-NH2).

[0019] In some implementations, Lg is selected from C 1-6 Alkyl sulfonyl or halophenoxy.

[0020] In some implementations, Lg is selected from methanesulfonyl or pentafluorophenoxy.

[0021] In some implementations, Lg is a methanesulfonyl group.

[0022] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0023] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0024] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0025] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0026] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0027] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0028] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0029] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0030] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic groups, -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Gl). u, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly- Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG,SEQ ID NO:41), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:42), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:43), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:44), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:45)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocyclic, -CH2NH-SO3H, -CH2N(C 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6 Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1- 6-alkylene-N + (C 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2- 6-alkylene-N + (C 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylenes -CO2H, -CH2N +(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, or polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or Nota (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein r is selected from an integer from 1 to 20 (e.g., an integer from 1 to 15, such as 1-12, 3-12, 1-10, 1-8, 3-8, ... Integers from 1 to 6, 1 to 4, 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); s is an integer from 1 to 20 (e.g., integers from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, integers from 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0031] In some implementations, "-NOTA" refers to

[0032] In some implementations, "-DOTA" refers to

[0033] In some implementations, "-DOTAGA" refers to

[0034] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6Alkylene, carbonyl, 9-12 member nitrogen-containing heterocyclic groups, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gl y-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys- Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Where s is selected from integers from 1 to 20;

[0035] In some implementations, the substructure of L is selected from the following fragments:

[0036] In some embodiments, L is selected from a divalent or unsubstituted structural fragment composed of one or more of the following groups: s is an integer selected from 1 to 20.

[0037] In some embodiments, L is selected from a divalent or unsubstituted structural fragment composed of one or more of the following groups: s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0038] In some embodiments, L is selected from a divalent or unsubstituted structural fragment composed of one or more of the following groups: s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0039] In some implementation schemes, L is selected from One of them and The structure consists of one or both of the following: a divalent substituted or unsubstituted structural fragment, where s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, or 8 to 10. For example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0040] In some implementation schemes, L is selected from One of them and The structure consists of one or both of the following: a divalent substituted or unsubstituted structural fragment, where s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, or 8 to 10. For example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0041] In some implementation schemes, L is selected from A divalent substituted or unsubstituted structural segment composed of one or both of them.

[0042] In some implementation schemes, L is selected from A divalent substituted or unsubstituted structural segment composed of one of the components.

[0043] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2-, or a structural fragment selected from the following substituted or unsubstituted segments:

[0044] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0045] In some implementations, E is substituted or unsubstituted -NH-CH2- or

[0046] In some implementations, E is a single bond or

[0047] In some implementations, E is a single bond, or a substituted or unsubstituted -NH-CH2-.

[0048] In some implementation schemes, Selected from the following substituted or unsubstituted structures: s is an integer selected from 1 to 20.

[0049] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0050] n is an integer selected from 1 to 20.

[0051] In some implementation schemes, Selected from the following substituted or unsubstituted structural fragments: n is an integer selected from 1 to 20.

[0052] In some implementation schemes, Selected from the following substituted or unsubstituted structural fragments:

[0053] n is an integer selected from 1 to 20, and preferably s is an integer selected from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0054] In some implementation schemes, Selected from the following substituted or unsubstituted structural fragments:

[0055] n is an integer selected from 1 to 20, and preferably s is an integer selected from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0056] In some embodiments, the cytotoxic agent is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors. In some embodiments, the anti-microtubule agent is an oliguriatin class, a maytansine class, or an eribulin class. In some embodiments, the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) class, trabectedin, or rubotecan. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin. In some embodiments, the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs.

[0057] In some embodiments, the cytotoxic drug is selected from PNU-159682 analogs.

[0058] The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), and secondary amino (-NR). a H), tertiary amino group (-NR) b R c ), where R a R b R c In this context, it refers only to non-hydrogen substituents on N, or mercapto groups (-SH), which can react with suitable functional groups in the rest of the conjugate to achieve linkage.

[0059] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary or tertiary amino group, or -SH group. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amino group on the cytotoxic drug.

[0060] In some embodiments, the cytotoxic drug has the structure shown in formula (I):

[0061] in, Indicates a single bond or no chemical bond;

[0062] R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C). 1-6 alkyl), -N(C) 1- 6-alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 aryl and 5-10 heteroaryl groups; wherein the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0063] R5 is -(C=O)-D2-D3-X6, where D2 either does not exist or is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1-6 Alkyl, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl group)2, -CN substituents; D3 is C 1-6 Alkylene, C 2-6 imide or C 2-6 Alkyne group; the alkylene group, alkenylene group, and alkyne group may optionally be selected from one or more groups selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; X6 is selected from hydrogen, halogen, -OH, C 1-6 Alkoxy, -SH, -SC 1- 6-alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C)1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups;

[0064] Y1 and Y2 are -C(R8)2-, where each R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C 1-6 alkyl), -N(C) 1- 6-alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl groups and 5-10 heteroaryl groups, or two R8 groups attached to the same carbon atom, together form =O;

[0065] X1, X2, and X5 are each independently selected from substituted or unsubstituted C. 1-6 Alkylene, -O-, -NR Y -or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl;

[0066] when When representing a single bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkylene, -O-, -NR Y -or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl;

[0067] when When there is no chemical bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkyl, -OR Y -N(R) Y )2 or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl;

[0068] X3 is selected from CR Z Or N, where R Z Selected from hydrogen or optionally by one or more elements selected from halogens, -OH, -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl.

[0069] In some implementation schemes, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C). 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl and C 1-6 Alkoxy group; the alkyl group or alkoxy group may optionally be selected from one or more groups selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0070] In some implementation schemes, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C. 1-6 Alkyl group; the alkoxy group is optionally surrounded by one or more elements selected from -CN, halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0071] In some implementation schemes, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, C, ... 1-6 Alkyl and C 1-6 Alkyl group.

[0072] In some implementation schemes, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C. 1-6 Alkyl group.

[0073] In some implementations, R2, R3, and R4 are all -OH.

[0074] In some implementation schemes, R1 and R7 are each independently selected from C. 1-6Alkyl groups, such as methoxy, ethoxy, n-propoxy, or isopropoxy. In some embodiments, both R1 and R7 are methoxy groups.

[0075] In some implementation schemes, R6 is selected from C. 1-6 Alkyl groups, such as methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R6 is methyl.

[0076] In some embodiments, R1 and R7 are both methoxy groups; R2, R3 and R4 are all -OH groups; and R6 is a methyl group.

[0077] In some implementations, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1- 6-alkyl; D3 is C 1-6 Alkylene; the alkylene group may optionally be composed of one or more elements selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1- 6-alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The substituents are aryl and 5-10 heteroaryl groups; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups;

[0078] In some implementations, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1- 6-alkyl; D3 is C 1-6 Alkylene; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2.

[0079] In some implementations, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen, methyl and ethyl; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2.

[0080] In some embodiments, R5 is -(C=O)-D2-D3-X6, wherein D2 is selected from -O-, -S-, -NH- and -N(CH3)-; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; and X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2.

[0081] In some implementations, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1- 3 alkyl groups; D3 is C 1-6 Alkylene; X6 is selected from -OH, -SH, -NH2.

[0082] In some implementation schemes, R5 is selected from:

[0083] In some implementations, Y1 and Y2 are -C(R8)2-, where R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy groups, or two R8 atoms bonded to the same carbon atom, together form =O;

[0084] Preferably, Y1 and Y2 are -(C=O)-.

[0085] In some implementations, X1, X2, and X5 are each independently selected from substituted or unsubstituted C. 1-6 Alkylene, -O- or -NR Y ;where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6-alkyl)2,-CN substituents of C 1-6 alkyl;

[0086] Preferably, X1, X2, X4 and X5 are all -O-.

[0087] In some implementation schemes, when When representing a single bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkylene, -O-; preferably, X4 is selected from -O-;

[0088] In some implementation schemes, when When there is no chemical bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkyl, -OR Y ;where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 Alkyl group; preferably, X4 is selected from -OH.

[0089] In some implementation schemes, X3 is selected from CR. Z Or N, where R Z C selected from hydrogen or optionally substituted with one or more substituents selected from halogens, -OH, -NH2, -CN. 1-6 alkyl;

[0090] In some implementations, X3 is N.

[0091] In some embodiments, the cytotoxic drug has the structure shown in formula (III)-1 or (III)-2:

[0092] Among them, X1~X5, R1~R7, Y1, and Y2 are as described above.

[0093] In some embodiments, the cytotoxic drug has a structure as shown in formula (IV)-1 or (IV)-2:

[0094] R5 is as described above.

[0095] All technical features disclosed in this specification, except for mutually exclusive technical features, can be combined in any way.

[0096] This invention covers compounds obtained by arbitrarily combining various embodiments.

[0097] In some embodiments, the cytotoxic agent is selected from the following compounds or compounds labeled with their isotopes:

[0098] In some embodiments, the cytotoxic agent is selected from the following compounds or compounds labeled with their isotopes:

[0099] In some embodiments, the cytotoxic drug fragment has the structure shown in formula (I'):

[0100] Wherein: X1~X5, Y1, Y2, D2, D3, R1~R4, R6, R7 are as described in any of the above; X6' is selected from -O-, -S-, -NH-, or -N(C 1-6 alkyl)-.

[0101] In some embodiments, the cytotoxic drug fragment has the following structure:

[0102] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, a compound in its free form as a "drug-linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking it to an antibody or its antigen-binding fragment) can be obtained, and then covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugates described in this application. Accordingly, in the free form of the "drug-linker," M' is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or an addition reaction.

[0103] In some embodiments, the free form of the "drug-linker" is selected from K-19 to K-37 below:

[0104] In some embodiments, the compound described above or a pharmaceutically acceptable salt thereof may optionally be replaced by one or more suitable substituents.

[0105] Coupled

[0106] A second aspect of the invention provides a coupling as shown in formula (II), wherein: Ab-[MLED]x Formula (II)

[0107] M, L, E, and D are as described in any of the preceding text; Ab is the target portion; x is 1 to 10.

[0108] In some preferred embodiments, the target of the targeted portion is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2. BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL- 6. ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN , ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR 1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2. .

[0109] In some preferred embodiments, Ab is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.

[0110] In some preferred embodiments, Ab is an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody, or multispecific antibody.

[0111] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof. In some embodiments, Ab is an antibody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0112] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0113] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0114] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0115] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0116] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0117] or,

[0118] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0119] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0120] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0121] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0122] or,

[0123] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0124] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0125] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0126] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0127] or,

[0128] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0129] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0130] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0131] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0132] In some embodiments, the antibody or its antigen-binding fragment comprises

[0133] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0134] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0135] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0136] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0137] or,

[0138] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0139] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0140] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0141] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0142] or,

[0143] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0144] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0145] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0146] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0147] or,

[0148] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0149] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0150] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0151] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0152] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0153] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0154] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0155] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0156] or,

[0157] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0158] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0159] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0160] or,

[0161] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0162] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0163] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0164] or,

[0165] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0166] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

[0167] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0168] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0169] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0170] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0171] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0172] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0173] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0174] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0175] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0176] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0177] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0178] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0179] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0180] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0181] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0182] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0183] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0184] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0185] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0186] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0187] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0188] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0189] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0190] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0191] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0192] In some embodiments, the N-terminal glutamine of the VH of the sequence shown in SEQ ID NO:1 or 3 or a variant thereof, or the N-terminal glutamine of the heavy chain of the sequence shown in SEQ ID NO:37 or 39 or a variant thereof, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0193] In some embodiments, the heavy chain constant region (CH) of the heavy chain as shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine.

[0194] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0195] In some implementations, M is linked to a thiol group (-SH) or an amino group (-NH2) on Ab.

[0196] In some implementations, M is linked to a thiol group (-SH) on Ab.

[0197] In some implementations, when Ab-[MLED] is present x In the couplings with the structure shown, MLED is formed from the compound shown in DEL-M', preferably by removing Lg from DEL-M', wherein the compound shown in DEL-M' is as defined above, Ab is as defined above, and x is 1-10.

[0198] In some implementations, when Ab-[MLED] is present x In the couplings with the structures shown, MLED is formed from compounds shown in K-25 to K-32, preferably by removing the fluorophenoxy group from the compounds, Ab is as defined above, x is 1-10, preferably x is 2.

[0199] In some implementations, when Ab-[MLED] is present x In the couplings with the structures shown, MLED is formed from compounds shown in K-19 to K-24 and K-33 to K-37, preferably by removing -SO2Me from the compounds, Ab is as defined above, x is 1-10, preferably x is 4.

[0200] In some embodiments, the conjugate is selected from ADC K-19 to ADC K-37 as shown below:

[0201] In each antibody-drug conjugate, HA represents the antibody or its antigen-binding fragment;

[0202] in, Or it may indicate the specific way in which the thiol group in the antibody or its antigen-binding fragment is linked to the M fragment;

[0203] This indicates the specific way in which the amino group in the antibody or its antigen-binding fragment is linked to the M fragment.

[0204] In some implementations, HA in each antibody-drug conjugate represents an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.

[0205] In some implementations, the antibody or its antigen-binding fragment in each antibody-drug conjugate is as defined above.

[0206] In some implementations, the HA includes:

[0207] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0208] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0209] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0210] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0211] or,

[0212] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0213] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0214] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0215] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0216] or,

[0217] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0218] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0219] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0220] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0221] or,

[0222] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0223] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0224] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0225] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0226] In some implementations, the HA includes

[0227] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0228] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0229] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0230] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0231] or,

[0232] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0233] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0234] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0235] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0236] or,

[0237] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0238] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0239] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0240] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0241] or,

[0242] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0243] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0244] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0245] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0246] In some implementations, the HA includes:

[0247] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0248] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0249] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0250] or,

[0251] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0252] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0253] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0254] or,

[0255] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0256] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0257] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0258] or,

[0259] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0260] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

[0261] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0262] In some implementations, the HA includes:

[0263] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0264] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0265] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0266] In some implementations, the HA includes:

[0267] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0268] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0269] In some implementations, the HA further includes:

[0270] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0271] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0272] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0273] In some embodiments, the HA comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0274] In some embodiments, the HA comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions).

[0275] In some embodiments, the HA includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0276] In some implementations, the HA includes:

[0277] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0278] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0279] In some implementations, the HA includes:

[0280] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0281] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0282] In some implementations, HA is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT / mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT / mAb-DB ID) of 80 in the IMGT database.

[0283] In some implementations, the HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.

[0284] In some implementations, the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:

[0285] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0286] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0287] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0288] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0289] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0290] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0291] In some embodiments, the N-terminal glutamine of the VH of the sequence shown in SEQ ID NO:1 or 3 or a variant thereof, or the N-terminal glutamine of the heavy chain of the sequence shown in SEQ ID NO:37 or 39 or a variant thereof, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0292] In some embodiments, the heavy chain constant region (CH) of the heavy chain as shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine.

[0293] In some implementations, x in the coupling shown as Ab-[MLED]x is 1-10, for example 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, or 4-10.

[0294] In some implementations, x in the coupling shown as Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0295] In some implementations, x in the coupling shown as Ab-[MLED]x is 1, 2, 3, or 4.

[0296] In some implementations, x in the coupling shown as Ab-[MLED]x is 3, 4, or 5.

[0297] In some implementations, x in the coupling shown as Ab-[MLED]x is 2 or 4.

[0298] In some embodiments, the conjugate described in this invention is an antibody-drug conjugate (ADC).

[0299] In some embodiments, the coupling compound of the present invention may optionally be replaced by one or more suitable substituents.

[0300] intermediate

[0301] In another aspect of this application, the following intermediate compounds are provided:

[0302] Wherein, each of PG1 is independently an H or amino protecting group, such as an alkoxycarbonyl amino protecting group, for example benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); preferably methoxycarbonyl (Fmoc) or allyloxycarbonyl (Alloc); each of PG2 is independently an H alkyl amino protecting group, such as triphenylmethyl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn); preferably triphenylmethyl (Trt); Lg is as defined in any of the preceding items.

[0303] In another aspect, this application provides the use of the intermediate compound as described above, or its salt, stereoisomer, tautomer, or isotopically labeled compound, in the preparation of the compound of the present invention or its pharmaceutically acceptable salt.

[0304] Composition

[0305] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0306] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0307] In some embodiments, the DAR of the ADC compositions described herein is about 1 to 8, for example about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 4.0 to 4.5, about about 4.5 to 5.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0.

[0308] In some embodiments, the DAR of the ADC composition described herein is about 1 to 5, such as about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 4.0 to 4.5, or about about 4.5 to 5.0.

[0309] In some embodiments, the DAR of the ADC composition described herein is about 1 to 3, such as about 1.0 to 1.5, about 1.0 to 2.0, about 1.0 to 2.5, about 1.0 to 3.0, about 1.5 to 2.0, about 1.5 to 2.5, about 1.5 to 3.0, about 2.0 to 2.5, about 2.0 to 3.0, or about 2.5 to 3.0.

[0310] In some embodiments, the DAR of the ADC compositions described herein is about 1.0 to 6.0, for example about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, about 2.0 to about 5.0, for example 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1 .25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1. 46, approximately 1.47, approximately 1.48, approximately 1.49, approximately 1.5, approximately 1.51, approximately 1.52, approximately 1.53, approximately 1.54, approximately 1.55, approximately 1.56, approximately 1.57, approximately 1.58, approximately 1.59, approximately 1.6, approximately 1.61, approximately 1.62, approximately 1.63, approximately 1.64, approximately 1.65, approximately 1.66, approximately 1.6 7, approximately 1.68, approximately 1.69, approximately 1.7, approximately 1.71, approximately 1.72, approximately 1.73, approximately 1.74, approximately 1.75, approximately 1.76, approximately 1.77, approximately 1.78, approximately 1.79, approximately 1.8, approximately 1.81, approximately 1.82, approximately 1.83, approximately 1.84, approximately 1.85, approximately 1.86, approximately 1.87, approximately 1.88 Approximately 1.89, approximately 1.9, approximately 1.91, approximately 1.92, approximately 1.93, approximately 1.94, approximately 1.95, approximately 1.96, approximately 1.97, approximately 1.98, approximately 1.99, approximately 2.0, approximately 2.01, approximately 2.02, approximately 2.03, approximately 2.04, approximately 2.05, approximately 2.06, approximately 2.07, approximately 2.08, approximately 2.09. Approximately 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2. .31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, approximately 2.53, approximately 2.54, approximately 2.55, approximately 2.56, approximately 2.57, approximately 2.58, approximately 2.59, approximately 2.6, approximately 2.61, approximately 2.62, approximately 2.63, approximately 2.64, approximately 2.65, approximately 2.66, approximately 2.67, approximately 2.68, approximately 2.69, approximately 2.7, approximately 2.71, approximately 2.72, approximately 2. 73, approximately 2.74, approximately 2.75, approximately 2.76, approximately 2.77, approximately 2.78, approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.9 4. Approximately 2.95, 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.1, 3.11, 3.12, 3.13, 3.14, 3.1 5. Approximately 3.16, 3.17, 3.18, 3.19, 3.2, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.3, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36 Approximately 3.37, 3.38, 3.39, 3.4, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.5, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57 Approximately 3.58, 3.59, 3.6, 3.61, 3.62, 3.63, 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.7, 3.71, 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78 Approximately 3.79, approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89, approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3.98, approximately 3.99, approximately 4.0, approximately 4.01, approximately 4.02, approximately 4.03, approximately 4.04, approximately 4.05, approximately 4.06, approximately 4.07, approximately 4.08, approximately 4.09, approximately 4.1, approximately 4.11, approximately 4.12, approximately 4.13, approximately 4.14, approximately 4.15, approximately 4.16, approximately 4.17, approximately 4.18, approximately 4.19, approximately 4.2, approximately 4.21, approximately 4.22, approximately 4.23, approximately 4.24, approximately 4.25, approximately 4.26, approximately 4.27, approximately 4.28, approximately 4.29, approximately 4.3, approximately 4.31, approximately 4.32, approximately 4.33, approximately 4.34, approximately 4.35, approximately 4.36, approximately 4.37, approximately 4.38, approximately 4.39, approximately 4.4, approximately 4.41, approximately 4.42, approximately 4.43, approximately 4.44, approximately 4.45, approximately 4.46, approximately 4.47, approximately 4.48, approximately 4.49, approximately 4.5, approximately 4.51, approximately 4.52, approximately 4.53, approximately 4.54, approximately 4.55, approximately 4.56, approximately 4.57, approximately 4.58, approximately 4.59, approximately 4.6, approximately 4.61, approximately 4.62, approximately 4.63, approximately 4.64, approximately 4.65, approximately 4.66, approximately 4.67, approximately 4.68, approximately 4.69, approximately 4.7, approximately 4.71, approximately 4.72, approximately 4.73, approximately 4.74, approximately 4.75, approximately 4.76, approximately 4.77, approximately 4.78, approximately 4.79, approximately 4.8, approximately 4.81, approximately 4.82, approximately 4.83, approximately 4.84, approximately 4.85, approximately 4.86, approximately 4.87, approximately 4.88, approximately 4.89, approximately 4.9, approximately 4.91, approximately 4.92, approximately 4.93, approximately 4.94, approximately 4.95, approximately 4.96, approximately 4.97, approximately 4.98, approximately 4.99, approximately 5.0.

[0311] Pharmaceutical Composition

[0312] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates, any of the compounds, or any of the drug-linkers described above, and one or more pharmaceutical excipients.

[0313] The antibody-drug conjugates, compounds, or drug-linkers described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, or intratumoral injection. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0314] application

[0315] The antibody-drug conjugates, drug-linkers, compositions, or drug compositions thereof described herein can be used to prevent or treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0316] Therefore, this application provides the use of any of the antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of medicaments for the prevention or treatment of Her2-expressing cancers.

[0317] Additionally, this application provides the use of any of the antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the thereof described above in a medicament for the prevention or treatment of Her2-expressing cancer.

[0318] Additionally, this application provides a method for preventing or treating Her2-expressing cancers, comprising administering an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the conjugates to a subject in need of such treatment.

[0319] In some embodiments, the antibody-drug conjugate, drug-linker, combination thereof, or pharmaceutical composition is sufficient (e.g., in a subject):

[0320] (1) Inhibits the proliferation of cells (such as tumor cells);

[0321] (2) Inhibits tumor growth;

[0322] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0323] (4) Inhibit HER2-mediated signal transduction;

[0324] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0325] (6) Any combination of (1)-(5) above.

[0326] In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

[0327] All technical features disclosed in this specification, such as the definitions of various functional groups, except for mutually exclusive technical features, can be combined in any way to obtain different general formula ranges or specific solutions. These ranges and solutions are all within the scope of this invention.

[0328] definition

[0329] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0330] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The amino acid assignment in each region or domain can follow various numbering systems known in the art. The term "antibody" also includes embodiments where the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or is a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into pyroglutamate. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise N-terminal glutamine or glutamic acid, or N-terminal amino acid cyclized into pyroglutamate.

[0331] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0332] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0333] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0334] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0335] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0336] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0337] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0338] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0339] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0340] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0341] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0342] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0343] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0344] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0345] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0346] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0347] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0348] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0349] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0350] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0351] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0352] The term "linker" refers to a structural segment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, the formula Ab-[MLED] x The -MLE- structure fragment in the text.

[0353] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described in this invention before they are linked to an antibody or its antigen-binding fragment. For example, "drug-linker" refers to M'-LED, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. The "drug-linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.

[0354] The "drug-linker" also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the "drug-linker" compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0355] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0356] As used in this article, This indicates the location where a structural segment connects to other parts of the molecule.

[0357] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkyl", C 1-10 Alkyl", C 1- 6-alkyl", C 1-4 Alkyl", C 1-3Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0358] The term "isotopically labeled compound" means that the compound is structurally identical to the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0359] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by one or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0360] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic group in this invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, for example, 4-6 membered nitrogen-containing heterocyclic groups and 4-6 membered oxygen-containing heterocyclic groups. Common heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0361] The term "oxygen-containing heterocycle" refers to a heterocycle as described above, in which one or more (e.g., 1, 2, or 3) ring atoms are oxygen atoms, such as 5-6 membered oxygen-containing heterocycles. Specific examples include, but are not limited to, ethylene oxide rings, tetrahydrofuran rings, furan rings, tetrahydropyran rings, and pyran rings. The term "nitrogen-containing heterocycle" as used in this invention refers to a heterocycle as described above, in which one or more (e.g., 1, 2, or 3) ring atoms are nitrogen atoms.

[0362] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy groups, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention are optionally substituted by one or more substituents described in this invention.

[0363] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0364] As used herein, the term "suitable substituent" refers to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents. "Suitable substituents" include oxo (=O), halogen, cyano, and NR. 8 R 9 Carboxyl, thiol, hydroxyl, ester group (e.g., -C) 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1- 6-alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 R 9 Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, halogens, hydroxyl groups, carboxyl groups, and ester groups (e.g., -C) 1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0365] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. In some embodiments, each substituent is independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkyl (alkylene) group, C1-C6 alkoxy group, C2-C6 (alkenyl) group, C2-C6 (alkynyl) group, C3-C8 (cycloalkyl) group, 3-8 membered (heterocyclic) group, C6-C 10 (A)aryl and 5-10 membered (A)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 8 R 9-O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkylene group, C1-C6 alkoxy group, C2-C6 (alkenylene) group, C2-C6 (alkynylene) group, C3-C8 (cycloalkylene) group, 3-10 membered (heterocyclic) group, C6-C 10 (sub-)aryl and 5-10 quinone (sub-)heteroaryl, etc., among which R 8 R 9 R' is as defined above. For example, the substituent can be a suitable substituent as described above.

[0366] If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted. Detailed Implementation

[0367] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0368] Information about the sequences involved in this invention is described in the table below:

[0369] The abbreviations used in this article have the following meanings:

[0370] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0371] Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0372] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0373] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0374] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0375] Intermediate Example 1: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrica-31-carboxylic acid (INT-1):

[0376] Step 1: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrica-31-carboxylic acid (INT-1-2)

[0377] Using standard solid-phase synthesis methods:

[0378] 1) Resin preparation: 2-CTC resin (3.00 mmol, 3.70 g, 0.81 mmol / g), N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (3.00 mol, 933 mg, 3.00 equivalents), and DIPEA (4.00 equivalents) were added to dichloromethane (10.0 mL) and reacted under a nitrogen atmosphere for 2 hours. Then, MeOH (1.0 mL) was added to the resin over a nitrogen bubbling atmosphere over 30 minutes, and the resin was obtained by filtration.

[0379] 2) Coupling: Under nitrogen bubbling, a DMF solution (10.0 mL) of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (5.60 g, 6.00 equivalent) and HATU (6.58 g, 5.70 equivalent) was added to the resin. After adding DIPEA (6.00 equivalent), the mixture was bubbled under nitrogen at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

[0380] 3) Deprotection: Add 20% piperidine DMF solution (30.0 mL) to the resin and bubble with nitrogen at 20°C for 30 minutes. Then wash the resin with DMF (30.0 mL) × 5.

[0381] 4) Repeat steps 2 and 3 using the amino acids in Table 1: numbers 2-10 in Table 1.

[0382] 5) Then wash the resin with DMF (30.0 mL x 5) and MeOH (30.0 mL x 5), and then dry it under vacuum.

[0383] Table 1:

[0384] Peptide cleavage and purification:

[0385] 1) Add the lysis solution (TFA / DCM, 1 / 100, v / v, 200.0 mL) to a flask containing the side-chain protected peptide at room temperature and stir twice for 3 minutes each time.

[0386] 2) After filtration, combine the filtrates and concentrate them.

[0387] 3) The crude product was prepared and purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (1117.1 mg, TFA salt).

[0388] Its structural characterization data are as follows:

[0389] ESI-MS (m / z): 10¹⁰.⁴ (M+H) + .

[0390] The purification method is as follows:

[0391] Step 2: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1)

[0392] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatricarne-31-carboxylic acid (665 mg, 615.04 μmol) was added to water (7.5 mL) and acetonitrile (15 mL), followed by the addition of sodium periodate (1.32 g, 6.15 mmol) and ruthenium trichloride hydrate (51.03 mg, 246.02 μmol), and the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by reverse-phase reaction (acetonitrile / water (0.05% formic acid) = 0-25%) and freeze-dried to give the title compound (515 mg, 449.69 μmol).

[0393] Its structural characterization data are as follows:

[0394] ESI-MS (m / z): 1074.4 (M+H) + .

[0395] Intermediate Example 2: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-acid (INT-2)

[0396] Step 1: Preparation of methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-2-2)

[0397] The raw materials methyl 3,5-dibromobenzoate (720 mg, 2.45 mmol), 2-methylthiopyrimidin-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and water (4 mL). The reaction system was stirred at 90 °C for 3 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was filtered through diatomaceous earth, and water and ethyl acetate were added to the filtrate for extraction and concentration to obtain the crude product. The crude product was purified by column chromatography (EA / PE = 0-25%) to obtain 710 mg of methyl 3,5-di(2-(methylthio)pyrimidin-5-yl)benzoate.

[0398] Its structural characterization data are as follows:

[0399] ESI-MS (m / z): 385.1 [M+H] + .

[0400] Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-2-3)

[0401] Methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and H₂O (2 mL). The reaction was stirred at 25 °C for 2 hours, and the reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1 N HCl, and a large amount of solid precipitated. The filter cake was collected by filtration and dried to obtain 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

[0402] Its structural characterization data are as follows:

[0403] ESI-MS (m / z): 371.1 [M+H] + .

[0404] Step 3: Preparation of tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-olate (INT-2-4)

[0405] 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), followed by HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL). The reaction mixture was stirred at 60 °C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-oic acid (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[0406] Step 4: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-acid (INT-2-5)

[0407] 3.60 g (4.24 mmol) of tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-oic acid was dissolved in dichloromethane (30 mL), and TFA (15.3 g, 134 mmol, 10 mL) was added. The reaction mixture was stirred at 25 °C for 6 hours. Water (60 mL) and ethyl acetate (40 mL x 3) were added to the reaction mixture for extraction. After the organic phases were combined, they were dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-acid (2.93 g, 3.63 mmol).

[0408] Its structural characterization data are as follows:

[0409] ESI-MS (m / z): 794.3 [M+H] + .

[0410] The purification method is as follows:

[0411] Column: Phenomenex luna C18 (250mm*70mm*10μm)

[0412] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0413] Step 5: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-acid (INT-2)

[0414] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), followed by sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, and the mixture was stirred at 25 °C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-acid (155 mg).

[0415] Its structural characterization data are as follows:

[0416] ESI-MS (m / z): 858.3 [M+H] + .

[0417] Intermediate Example 3: Preparation of 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-3)

[0418] Step 1: Preparation of 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (INT-3-2)

[0419] Chlorosulfonyl isocyanate (2.14 g, 15.13 mmol, FR) was added to dichloromethane (50 mL), and the mixture was cooled in an ice-water bath for 10 minutes. Then, tert-butyl 2-hydroxyacetate (2 g, 15.13 mmol, FR) was added to the reaction mixture, and the mixture was stirred in an ice-water bath for 2 hours. Piperidine-4-carboxylic acid allyl ester hydrochloride (3.74 g, 18.16 mmol) and triethylamine (4.59 g, 45.40 mmol) were added to the reaction mixture, the ice-water bath was removed, and the mixture was allowed to return to room temperature naturally and stirred for another 3 hours. The reaction was quenched with water (100 mL), and the mixture was extracted three times with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / PE = 0-100%), yielding the title compound (1.87 g, 4.19 mmol).

[0420] Its structural characterization data are as follows:

[0421] 1 H NMR (400MHz, DMSO) δ11.64(s,1H),6.06-5.79(m,1H),5.35-5.17(m,2H),4.61-4.48(m,4H),4.03(q,J=7.1Hz,1H),3.5 9(dt,J=12.4,3.3Hz,2H),3.04-2.88(m,2H),1.93(dd,J=13.5,3.2Hz,2H),1.59(dd,J=13.4,3.6Hz,2H),1.43(s,9H).

[0422] Step 2: Preparation of 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-3)

[0423] 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (1.75 g, 4.31 mmol, FR) was added to a mixed solvent of trifluoroacetic acid (5 mL) and dichloromethane (10 mL) and reacted at 25 °C for 2 hours. The reaction mixture was concentrated to dryness, diluted with ethyl acetate, and the pH was adjusted to approximately 8 with sodium bicarbonate aqueous solution. Impurities were removed by extraction, and the pH was adjusted to approximately 3 with 3N dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (1.4 g, 4.00 mmol).

[0424] Its structural characterization data are as follows:

[0425] ESI-MS (m / z): 351.1 [M+H] + .

[0426] Intermediate Example 4: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-4)

[0427] Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-4-2)

[0428] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solution was concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0429] Its structural characterization data are as follows:

[0430] MS m / z (ESI): 782.2 [M+H] +

[0431] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-4-3)

[0432] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0433] Its structural characterization data are as follows:

[0434] MS m / z (ESI): 566.1 [M+Na] +

[0435] Step 3: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-4)

[0436] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, and p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (134.50 mg, 0.19 mmol).

[0437] Its structural characterization data are as follows:

[0438] MS m / z (ESI): 731.2 [M+Na] +

[0439] Intermediate Example 5: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (INT-5)

[0440] Step 1: Preparation of 2,5-dioxopyrrolidine-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-5-2)

[0441] 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (0.6 g, 1.62 mmol) was dissolved in THF (15 mL), and N-hydroxysuccinimide (278.89 mg, 2.43 mmol) was added, followed by dicyclohexylcarbodiimide (0.4 g, 1.94 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product of the title compound (1.5 g, 3.42 mmol), which was used directly in the next reaction without purification.

[0442] Step 2: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (INT-5-3)

[0443] 2,5-Dioxopyrrolidone-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (331.5 mg, 0.99 mmol) was dissolved in DMF (4 mL), and 1-amino-3,6,9,12,15-pentaoctadecane-18-carboxylic acid (0.3 g, 0.67 mmol) and DIPEA (588.24 mg, 4.56 mol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solution was obtained by rapid column chromatography (C18, water / acetonitrile = 0.5) to give the title compound (315.50 mg, 0.44 mmol).

[0444] Its structural characterization data are as follows:

[0445] MS m / z (ESI): 662.2 [M+H] +

[0446] Step 3: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (INT-5)

[0447] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosano-20-oleic acid (315.50 mg, 0.44 mmol) was dissolved in acetonitrile (3 mL) and water (1.5 mL), and sodium periodate (470.50 mg, 2.20 mmol) and ruthenium trichloride monohydrate (9.11 mg, 0.04 mmol) were added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (115.50 mg, 0.16 mmol).

[0448] Its structural characterization data are as follows:

[0449] MS m / z (ESI): 726.1 [M+H] +

[0450] Intermediate Example 6: Preparation of (9H-fluorene-9-yl)methyl((S)-1-((S)-1-((S)-1-((2-mercapto-2-methylpropyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (INT-6)

[0451] Step 1: Preparation of 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (INT-6-2) (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1 g, 2.62 mmol) was dissolved in dichloromethane (10 mL), N-hydroxysuccinimide (300.95 mg, 2.62 mmol) was added, followed by EDCI (551.43 mg, 2.88 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was washed directly with 10 mL x 2 saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product of the title compound (1.29 g, 2.42 mmol), which was used directly in the next reaction without purification.

[0452] Its structural characterization data are as follows:

[0453] MS m / z (ESI): 497.3 [M+H2O+H] +

[0454] Step 2: Preparation of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (INT-6-3)

[0455] 2,5-Dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1.19 g, 2.23 mmol) and L-alanine (199.00 mg, 2.23 mmol) were dissolved in tetrahydrofuran (10 mL) and water (5 mL), and sodium bicarbonate (375.29 mg, 4.47 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 20 mL of water was added to the reaction solution, and the pH was adjusted to 4–5 with 2N HCl aqueous solution, resulting in the precipitation of a large amount of white flocculent precipitate. The precipitate was extracted with 20 mL x 4 of ethyl acetate, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.23 g, 2.03 mmol), which was used directly in the next reaction without purification.

[0456] Its structural characterization data are as follows:

[0457] MS m / z (ESI): 454.1 [M+H] +

[0458] Step 3: Preparation of 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (INT-6-4)

[0459] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (400 mg, 882.05 μmol) and N-hydroxysuccinimide (152.27 mg, 1.32 mmol) were dissolved in THF (4 mL) and DMF (2 mL), and then EDCI (253.64 mg, 1.32 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was diluted with 100 mL of ethyl acetate, and then washed directly with 20 mL x 2 of saturated brine. After drying with anhydrous sodium sulfate, the crude product of the title compound (400 mg, 726.53 μmol) was concentrated under reduced pressure and used directly in the next reaction without purification.

[0460] Its structural characterization data are as follows:

[0461] MS m / z (ESI): 568.3 [M+H2O+H] +

[0462] Step 4: Preparation of (9H-fluorene-9-yl)methyl((S)-1-((S)-1-((S)-1-((2-mercapto-2-methylpropyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (INT-6-5)

[0463] 2,5-Dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine (200 mg, 363.27 μmol) was dissolved in THF (3 mL) and water (1 mL), and 1-amino-2-methylpropane-2-thiol (38.22 mg, 363.27 μmol) and sodium bicarbonate (61.03 mg, 726.53 μmol) were added. The mixture was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (5 mL x 3). The solution was washed with 5 mL of organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10–100%, with methanol / dichloromethane finishing at 5–10%) and concentrated under reduced pressure to give the title compound (93 mg, 172.01 μmol).

[0464] Its structural characterization data are as follows:

[0465] MS m / z (ESI): 541.3 [M+H] +

[0466] Preparation Example 1: (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-3)

[0467] Step 1: Preparation of (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (6-3-2)

[0468] (8S,10S)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetraphenyl-5,12-dione (157 mg, 244.69 μmol) was added to methanol (3 mL) and water (2 mL), followed by sodium periodate (78.51 mg, 367.04 μmol). After the addition was complete, the mixture was stirred at room temperature in the dark. After the reaction was complete, the mixture was concentrated under reduced pressure to remove the solvent, and then lyophilized to remove the solvent, yielding the title compound (142 mg, 210.42 μmol), which was used directly in the next step without purification.

[0469] Its structural characterization data are as follows:

[0470] MSm / z(ESI): 628.2 [M+H] +

[0471] Step 2: Preparation of (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-((((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-3)

[0472] Add (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (50 mg, 79.67 μmol) to DMF (2 mL), followed by 4-dimethylaminopyridine (38.93 mg, 318.68 μmol), EDCI (30.55 mg), and 2-aminoethanol (14 mg, 229.20 μmol). After the addition is complete, the mixture is heated to 35 °C / 45 °C under nitrogen protection for reaction. After the reaction was completed, the compound was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (2.04 mg, 2.89 μmol).

[0473] Its structural characterization data are as follows:

[0474] MSm / z(ESI): 671.4 [M+H] +

[0475] The separation and purification methods are as follows:

[0476] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0477] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0478] Preparation Example 2: (2-hydroxyethyl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid ester (6-6)

[0479] Add (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (50 mg, 79.67 μmol), potassium carbonate (28.06 mg, 203.06 μmol), and sodium iodide (12.17 mg, 81.22 μmol) to DMF (2 mL). After the addition is complete, stir the mixture at room temperature for 20 min. Finally, add 2-bromoethanol (20.30 mg, 162.45 μmol) and heat to 65 °C to react. After the reaction was completed, the compound was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5.95 mg, 8.42 μmol).

[0480] Its structural characterization data are as follows:

[0481] MSm / z(ESI): 672.2 [M+H] +

[0482] The separation and purification methods are as follows:

[0483] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0484] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0485] Preparation Example 3: S-(1-amino-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (6-7)

[0486] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-mercapto-2-methylpropyl)carbamate (6-7-2)

[0487] 1-Amino-2-methylpropane-2-thiol hydrochloride (0.5 g, 3.53 mmol) was dissolved in THF (10 mL) and water (2 mL), followed by the addition of sodium bicarbonate (593.01 mg, 7.06 mmol), and then 9-fluorenylmethyl-N-succinimide carbonate (1.13 g, 3.53 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.12 g, 3.43 mmol), which was used directly in the next reaction without purification.

[0488] Its structural characterization data are as follows:

[0489] MS m / z (ESI): 350.1 [M+Na] +

[0490] Step 2: Preparation of S-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (6-7-3)

[0491] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (86) (mg, 137.03 μmol) and DIPEA (35.42 mg, 274.06 μmol) and (9H-fluorene-9-yl)methyl(2-mercapto-2-methylpropyl)carbamate (67.30 mg, 205.55 μmol) were dissolved in DMF (4 mL). PyBOP (85.57 mg, 164.44 μmol) was added under nitrogen protection, and the mixture was stirred at room temperature for 0.5 hours. Water (5.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (3 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and then concentrated again under reduced pressure to obtain the title compound (41 mg, 43.76 μmol).

[0492] Its structural characterization data are as follows:

[0493] MS m / z (ESI): 937.3 [M+H] +

[0494] Step 3: Preparation of S-(1-amino-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (6-7)

[0495] S-(1-(9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (30 mg, 32.02 μmol) was dissolved in DMF (1.5 mL), and piperidine (8.18 mg, 96.05 μmol) in DMF (1.5 mL) was added dropwise under nitrogen protection. After the addition was complete, the system turned purple-red and was stirred at room temperature for 40 minutes. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (4.12 mg, 5.48 μmol).

[0496] Its structural characterization data are as follows:

[0497] MS m / z (ESI): 715.3 [M+H] +

[0498] 1 H NMR (400MHz, DMSO) δ14.07(s,1H),13.25(s,1H),7.96-7.93(m,2H),7.69-7.66(m,1H),5.45(s,1H),5.25(t,J=4.4Hz,1H),5.02(s,1H),4.58(d,J =2.0Hz,1H),4.23(d,J=2.0Hz,1H),4.21-4.13(m,1H),4.00(s,3H),3.93 (dd,J=6.0,1.6Hz,1H),3.66(t,J=8.8Hz,1H),3.51(dd,J=7.6,3.6Hz,1H) ,3.39(dd,J=13.2,7.2Hz,2H),3.31(s,3H),3.19(dd,J=13.2,6.0Hz,1H),3.03(dd,J=45.2,18.4Hz,2H),2.73-2.58(m,2H),2.29(dd,J=14.8,5.6 Hz,1H),2.20(d,J=13.6Hz,1H),2.00(dd,J=14.4,6.8Hz,1H),1.69(s,2H ),1.45(s,1H),1.28(d,J=5.6Hz,4H),1.23(s,2H),1.21(d,J=6.4Hz,3H).

[0499] The separation and purification methods are as follows:

[0500] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0501] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0502] Preparation Example 4: Preparation of S-(1-hydroxy-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (6-8)

[0503] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (60 mg, 95.60 μmol), DIPEA (24.71 mg, 191.21 μmol), and 2-mercapto-2-methylprop-1-ol (30.46 mg, 286.81 μmol) were dissolved in DMF (3 mL), and PyBOP (59.70 mg, 114.72 μmol) was added. The mixture was stirred at room temperature for 0.5 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5.74 mg, 7.22 μmol).

[0504] Its structural characterization data are as follows:

[0505] MS m / z (ESI): 716.3 [M+H] +

[0506] 1H NMR (400MHz, CDCl3) δ13.88(s,1H),13.29(s,1H),8.03(d,J=7.2Hz,1H),7.78(t,J=8.0Hz,1H),7.39(d,J=8.4Hz,1H),5.50(s,1 H),5.29(s,1H),5.05(s,1H),4.73(s,1H),4.52(s,1H),4.10(d,J=12.8Hz,5H),3.95(s,2H),3.80(q,J=11.6Hz,2H),3.60(d,J= 11.2Hz,1H),3.46(d,J=4.8Hz,3H),3.44-3.36(m,1H),3.29(d,J=18.8Hz,1H),3.09(d,J=18.8Hz,1H),2.88(s,1H),2.77(s,1H) ,2.59(d,J=14.8Hz,1H),2.12(dd,J=14.8,3.6Hz,1H),2.02(s,1H),1.82-1.75(m,2H),1.48(d,J=3.6Hz,5H),1.45-1.37(m,4H).

[0507] The separation and purification methods are as follows:

[0508] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0509] Mobile phase A: Acetonitrile; Mobile phase B: Water

[0510] Preparation Example 5: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(2-hydroxyethyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-9)

[0511] Step 1: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (6-9-2)

[0512] (8S,10S)-6,8,11-trihydroxy-10-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetraphenyl-5,12-dione (100 mg, 155.37 μmol) was added to a mixed solvent of methanol (4 mL) and water (2 mL), followed by the addition of sodium periodate (60 mg). The reaction mixture was reacted at room temperature for 2 hours. After removing methanol by vacuum concentration, the mixture was freeze-dried to obtain the crude title compound (80 mg, 120.71 μmol), which was used directly in the next step without purification.

[0513] Its structural characterization data are as follows:

[0514] MS m / z(ESI): 630.2 [M+1] +

[0515] Step 2: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(2-hydroxyethyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-9)

[0516] (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholinyl)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (13 mg, 23.82 μmol) was dissolved in DMF (2 mL), followed by the addition of 2-aminoethanol (3 mg), DIPEA (15 mg), and HATU (15 mg), and the reaction was carried out at room temperature for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (7.25 mg, 10.67 μmol).

[0517] Its structural characterization data are as follows:

[0518] MS m / z (ESI): 673.2 [M+1] +

[0519] 1H NMR (400MHz, DMSO) δ14.08(s,1H),13.26(s,1H),7.94(dd,J=11.7,5.3Hz,3H),7.71-7.64(m,1H),5.38(s,1H),5.30(s,1H ),4.98(s,1H),4.74(t,J=5.4Hz,1H),4.38(s,1H),4.09(s,1H),4.04-3.98(m,3H),3.79-3.71(m,1H),3.58(s,1H),3.49- 3.39(m,3H),3.26(s,3H),3.24-3.17(m,2H),2.99(dd,J=40.3,18.5Hz,2H),2.63(d,J=10.6Hz,1H),2.44(s,2H),2.25(dd ,J=14.3,5.3Hz,2H),2.11(d,J=13.7Hz,1H),1.83(dd,J=12.6,9.2Hz,1H),1.57(d,J=10.0Hz,1H),1.11(d,J=6.5Hz,3H).

[0520] Its preparation method is as follows:

[0521] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0522] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0523] Preparation Example 6: Preparation of (2S,4S)-2,5,12-trihydroxy-N-(2-hydroxyethyl)-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-10)

[0524] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (30 mg, 47.80 μmol) was dissolved in DMF (1 mL), and 2-(methylamino)ethane-1-ol (10 mg, 133.14 μmol) was added. The system turned black. DIPEA (12.36 mg, 95.60 μmol) and PyBOP (29.85 mg, 57.36 μmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (7.17 mg, 9.90 μmol).

[0525] Its structural characterization data are as follows:

[0526] MS m / z (ESI): 685.2 [M+1] +

[0527] 1 H NMR (400MHz, DMSO) δ14.06(s,1H),13.31(s,1H),7.95-7.88(m,2H),7.68-7.62(m,1H),5.71-5.69(m,1H),5.26-5.23(m,1H), 5.04-4.99(m,1H),4.84-4.80(m,1H),4.62-4.50(m,2H),4.23(d,J=2.0Hz,1H),4.10-4.02(m,1H),3.99(s,3H),3.93(d,J=6. 0Hz,1H),3.82-3.72(m,1H),3.70-3.60(m,2H),3.50(dd,J=7.6,3.6Hz,1H),3.40-3.36(m,4H),3.31(d,J=4.4Hz,6H),2.88(d ,J=17.6Hz,1H),2.79(s,1H),2.72-2.61(m,1H),2.45-2.35(m,2H),2.26-2.16(m,1H),1.69-1.64(m,1H),1.25-1.20(m,2H).

[0528] Its preparation method is as follows:

[0529] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0530] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0531] Preparation Example 7: Preparation of (2S,4S)-N-(2-aminoethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-11)

[0532] Step 1: Preparation of (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)ethyl)(methyl)carbamate tert-butyl ester (6-11-2)

[0533] (2-Aminoethyl)(methyl)carbamate tert-butyl ester (0.5 g, 2.87 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (967.99 mg, 2.87 mmol) were dissolved in THF (5 mL), followed by water (5 mL) and then sodium bicarbonate (241.07 mg, 2.87 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was diluted with 20 mL of ethyl acetate and then washed directly with 10 mL x 2 of brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product of the title compound (1.2 g, 2.18 mmol), which was used directly in the next reaction without purification.

[0534] Its structural characterization data are as follows:

[0535] MS m / z(ESI): 297.1 [M+1-Boc] +

[0536] Step 2: Preparation of (9H-fluorene-9-yl)methyl (2-(methylamino)ethyl)carbamate hydrochloride (6-11-3)

[0537] A hydrogen chloride / dioxane solution (4M, 10 mL) was added directly to (1.2 g, 3.03 mmol) tert-butyl 2-methylcarbamate (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)ethyl)(methyl)carbamate, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. 20 mL of water was added to the residue, the pH was adjusted to 4–5 with 4N HCl, and the mixture was extracted with 10 mL x 2 of ethyl acetate. The organic phase was discarded, and the aqueous phases were combined and freeze-dried to give the title compound (670 mg, 1.96 mmol), which required no further purification.

[0538] Its structural characterization data are as follows:

[0539] MS m / z(ESI): 297.3 [M+1] +

[0540] Step 3: Preparation of (9H-fluorene-9-yl)methyl(2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carbamoyl)ethyl)carbamate (6-11-4)

[0541] (9H-fluorene-9-yl)methyl(2-(methylamino)ethyl)carbamate hydrochloride, (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (50 mg, 79.67 μmol) was dissolved in DMF (1 mL), and DIPEA (30.89 mg, 239.01 μmol) and PyBOP (49.75 mg, 95.60 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was diluted with 5 mL of water, extracted with 5 mL x 3 of ethyl acetate, and the combined organic phases were washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 0–100%) and concentrated again under reduced pressure to give the title compound (18 mg, 19.87 μmol).

[0542] Its structural characterization data are as follows:

[0543] MS m / z(ESI): 906.3 [M+1] +

[0544] Step 4: Preparation of (2S,4S)-N-(2-aminoethyl)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamide (6-11)

[0545] Dissolve (9H-fluorene-9-yl)methyl(2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-N-methyl-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxamido)ethyl)carbamate (18 mg, 19.87 μmol) in DMF (2 mL), add piperidine (16.92 mg, 198.69 μmol) dropwise, and stir at room temperature for 5 minutes. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (2.61 mg, 3.74 μmol).

[0546] Its structural characterization data are as follows:

[0547] MS m / z (ESI): 684.3 [M+1] +

[0548] 1 H NMR (400MHz, DMSO) δ7.95-7.85(m,2H),7.68-7.60(m,1H),5.40-5.35(m,1H),5.27-5.20(m,1H),5.05-4.95( m,1H),4.58(s,1H),4.22(s,1H),4.17-4.03(m,1H),4.01-3.95(s,3H),3.92(d,J=4.0Hz,2H),3.66(t,J=12. 0Hz,2H),3.30(s,6H),3.06-2.97(m,2H),2.97-2.8(m,2H),2.80-2.73(m,1H),2.73-2.55(m,5H),2.34-2.25 (m,3H),2.25-2.20(m,1H),2.20-2.11(m,1H),2.08-1.94(m,1H),1.72-1.61(m,2H),1.25-1.19(d,J=8.0,2H)

[0549] Its preparation method is as follows:

[0550] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0551] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0552] Preparation Example 8: Preparation of 1-hydroxypropyl-2-yl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylate (6-12)

[0553] (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (30.0 mg, 47.8 μmol) and 2-bromopropanol (99.7 mg, 717.03 μmol) were dissolved in DMF (2 mL), potassium carbonate (99.10 mg, 717.03 μmol) was added, and the mixture was heated to 50 °C and reacted for 6 hours. The reaction solution was poured into dichloromethane (15 ml) and water (10 ml), the organic phase was separated, washed with saturated sodium chloride aqueous solution (2 ml), concentrated to obtain crude product, purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (3.50 mg, 4.95 μmol).

[0554] Its structural characterization data are as follows:

[0555] MS m / z (ESI): 686.2 [M+H] +

[0556] 1H NMR (400MHz, DMSO-d6): δ14.06(s,1H),13.28(s,1H),7.93(m,2H),7.70-7.64(m,1H),5.24 (m,1H),4.96(m,1H),4.82-4.76(m,1H),4.59(d,J=2.0Hz,1H),4.23(d,J=1.6Hz,1H),4.19 -4.15(m,1H),4.12(dd,J=10.4,5.2Hz,1H),3.99(s,2H),3.95-3.85(m,2H),3.7 9-3.72(m,1H),3.69-3.63(m,1H),3.51-3.48(m,1H),3.33(s,3H),3.30(s,2H), 3.17(d,J=5.2Hz,3H),3.11-3.05(m,1H),3.00-3.95(m,1H),2.65-2.61(m,1H), 2.27-2.24(m,1H),1.67-1.62(m,2H),1.22(d,J=6.4Hz,3H),1.11-1.02(m,3H).

[0557] Its preparation method is as follows:

[0558] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0559] Mobile phase A: acetonitrile; Mobile phase B: 0.05% formic acid aqueous solution

[0560] Example 5: S-((S)-37-benzyl-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-44-methyl-1,29,32,35,38,41-hexaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39,42-hexaazatrapentadecane-44-yl)(2S,4S)-2,5 Preparation of 1,2-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-19)

[0561] Step 1: Preparation of 2-methyl-2-(triphenylthio)prop-1-amine (K-19-2)

[0562] 1-Amino-2-methylpropane-2-thiol (5.36 g, 50.98 mmol) was added to TFA (60 mL), and triphenylmethanol (14.6 g, 56.08 mmol) was added at room temperature. The mixture was stirred at room temperature for 7 days. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove trifluoroacetic acid. The residue was extracted with water, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a beige viscous substance. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 3:1) and then concentrated and dried again under reduced pressure to obtain a white solid (10.2 g, 29.35 mmol).

[0563] Its structural characterization data are as follows:

[0564] 1 H NMR (400MHz, DMSO) δ7.59-7.53(m,6H),7.36-7.29(dd,J=84,7.2Hz,6H),7.26-7.20(m,3H),5.37-5.02(s,2H),1.83-1.76(s,2H),1.03-0.91(s,6H).

[0565] Step 2: Preparation of (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (K-19-4)

[0566] Glycylglycyl-L-phenylalanine (2.91 g, 10.41 mmol) was dissolved in DMF (100 mL), and DIPEA (2.02 g, 15.61 mmol, 2.72 mL) and 9-fluorenylmethyl-N-succinimide carbonate (5.27 g, 15.61 mmol) were added. The mixture was stirred at room temperature for 24 hours. The reaction solution was added dropwise to 300 mL of water with stirring, resulting in the precipitation of a large amount of white solid. The solid was filtered, and the filtrate was simply extracted with MTBE, after which the organic phase was discarded. The aqueous phase was adjusted to pH 3-4 with 6N hydrochloric acid, resulting in the precipitation of a large amount of white solid. Filtration was ineffective, so the mixture was extracted three times with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Then, ethyl acetate was added, and the solid was dispersed by sonication. The mixture was filtered, and the filter cake was dried under reduced pressure to give the title compound (3.6 g, 7.18 mmol).

[0567] Its structural characterization data are as follows:

[0568] MS m / z (ESI): 502.3 [M+H] +

[0569] Step 3: Preparation of (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine tert-butyl ester (K-19-5)

[0570] (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (2.5 g, 4.98 mmol, FR) and glycine tert-butyl ester (686.56 mg, 5.23 mmol) were dissolved in DMF (25 mL), and DIPEA (1.29 g, 9.97 mmol) and HATU (1.99 g, 5.23 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with brine, and the organic phase was concentrated under reduced pressure to give the crude title compound (3.06 g, 4.98 mmol), which was used directly in the next reaction without purification.

[0571] Its structural characterization data are as follows:

[0572] MS m / z (ESI): 615.4 [M+H] +

[0573] Step 4: Preparation of (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine (K-19-6)

[0574] (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine tert-butyl ester (3.7 g, 6.02 mmol) was suspended in dichloromethane (20 mL), and trifluoroacetic acid (10 mL) was added dropwise. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to obtain the crude title compound (3.36 g, 6.02 mmol), which was used directly in the next reaction without purification.

[0575] Its structural characterization data are as follows:

[0576] MS m / z (ESI): 559.3 [M+H] +

[0577] Step 5: Preparation of (9H-fluorene-9-yl)methyl(S)-(10-benzyl-3,3-dimethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamate (K-19-7)

[0578] DMF (30 mL) was added to (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine) (3.3 g, 5.91 mmol), followed by DIPEA (1.53 g, 11.82 mmol) to make the reaction weakly basic. Then, 2-methyl-2-(triphenylthio)propyl-1-amine (2.05 g, 5.91 mmol) and HATU (2.24 g, 5.91 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was poured into 200 mL of water with stirring, resulting in the precipitation of a large amount of white solid. The solid was filtered and dried to obtain the title compound (5.8 g, 5.88 mmol).

[0579] Its structural characterization data are as follows:

[0580] MS m / z (ESI): 645.5 [M-243+H] +

[0581] Step Six: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((2-methyl-2-(triphenylthio)propyl)amino)-2-oxoethyl)-3-phenylpropionamide (K-19-8)

[0582] (9H-fluorene-9-yl)methyl(S)-(10-benzyl-3,3-dimethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamate (870 mg, 979.64 μmol) was dissolved in DMF (6 mL), and then diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to obtain the formate salt of the title compound (280 mg, 393.33 μmol).

[0583] Its structural characterization data are as follows:

[0584] MS m / z (ESI): 666.5 [M+H] + ; 1332.8 [2M+H] +

[0585] Step 7: Preparation of (S)-N-(10-benzyl-3,3-dimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-21,24,27,30,33,36,39,42-octaoxa-2-thia-5,8,11,14,17-pentazatetratetradecane-44-yl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (K-19-9)

[0586] 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-penta-2-azaeicosano-20-carboxylic acid (35 mg, 40.80 μmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((2-methyl-2-(triphenylthio)propyl)amino)-2-oxoethyl)-3-phenylpropionamide (27.16 mg, 40.80 μmol) were dissolved in DMF (1 mL), and DIPEA (10.54 mg, 81.59 μmol) and HATU (15.50 mg, 40.80 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, water was added to the reaction solution, and a white solid precipitated out. The solid was extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain the crude product of the title compound (60 mg, 39.85 μmol), which was used directly in the next reaction without purification.

[0587] Its structural characterization data are as follows:

[0588] MS m / z (ESI): 1505.3 [M+H] + ; 1263.5 [M-243+H] + ;

[0589] Step 8: Preparation of (S)-N-(35-benzyl-42-mercapto-42-methyl-27,30,33,36,39-pentoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34,37,40-pentazatetratetradecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (K-19-10)

[0590] (S)-N-(10-benzyl-3,3-dimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-21,24,27,30,33,36,39,42-octaoxa-2-thia-5,8,11,14,17-pentazatetratetradecane-44-yl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (60 mg, 39.85 μmol) was suspended in dichloromethane (2 mL), and TFA (0.5 mL) was added, resulting in a yellow color. Then, triisopropylsilane (18.93 mg, 119.54 μmol, 24.49 μL) was added dropwise, and the mixture was stirred at room temperature for 0.5 hours, during which the yellow color gradually faded. After the reaction was complete, the reaction solution was concentrated under reduced pressure at room temperature. The crude acetonitrile was dissolved and purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), followed by freeze drying to obtain the title compound (30 mg, 23.74 μmol).

[0591] Its structural characterization data are as follows:

[0592] MS m / z(ESI): 1264.0 [M+H] + 632.6 [M / 2+H] +

[0593] Step Nine: S-((S)-37-benzyl-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-44-methyl-1,29,32,35,38,41-hexaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39,42-hexaazatrapentadecane-44-yl)(2S,4S)-2,5, Preparation of 12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-19)

[0594] Directly to (S)-N-(35-benzyl-42-mercapto-42-methyl-27,30,33,36,39-pentoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34,37,40-pentazatetratetradecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (30 mg, 23.74 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10 A mixture of DMF (1 mL) and DIPEA (16 mg, 123.80 μmol) was added to (14.90 mg, 23.74 μmol) and HATU (18.05 mg, 47.49 μmol), and the reaction mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (8.08 mg, 4.23 μmol).

[0595] Its structural characterization data are as follows:

[0596] MS m / z (ESI): 1874.6 [M+H] +

[0597] Its preparation method is as follows:

[0598] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0599] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0600] Example 6: S-((S)-39-benzyl-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29,46-undecylmethyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40,43-pentadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,4 Preparation of 4-pentaazahexaoctadecan-46-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-20)

[0601] Step 1: Preparation of (S)-N-(10-benzyl-3,3,20,23,26,29,32,35,38,41,44-undecyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45-tetradecoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17,20,23,26,29,32,35,38,41,44-tetradecazahexadecane-46-yl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (K-20-1)

[0602] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (30 mg, 26.20 μmol) and (S)-2-(2 -(2-aminoacetamido)acetamido)-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (52.33 mg, 78.59 μmol) was dissolved in DMF (1 mL), and DIPEA (10.16 mg, 78.59 μmol) and HATU (9.95 mg, 26.20 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (20 mg, 11.15 μmol).

[0603] Its structural characterization data are as follows:

[0604] MS m / z (ESI): 1794.9 [M+H] + 1810.6 [M+H2O] +

[0605] Its preparation method is as follows:

[0606] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0607] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0608] Step 2: Preparation of (S)-N-(37-benzyl-44-mercapto-3,6,9,12,15,18,21,24,27,44-decamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecano-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecanoazatetraloctyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (K-20-2)

[0609] (S)-N-(10-benzyl-3,3,20,23,26,29,32,35,38,41,44-undecyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45-tetradecoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17,20,23,26,29,32,35,38,41,44) -Tetradecazatetrahexadecane-46-yl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (20 mg, 11.15 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.2 mL) was added dropwise. The system instantly turned yellow. Triisopropylsilane (5.30 mg, 33.46 μmol, 6.86 μL) was added, and the reaction was stirred at room temperature for 0.5 hours. The system gradually decolorized. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was dissolved in acetonitrile and purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), followed by freeze-drying to obtain the title compound (17 mg, 10.96 μmol).

[0610] Its structural characterization data are as follows:

[0611] MS m / z (ESI): 1551.8 [M+H] + 776.0 [M / 2+H] +

[0612] Step 3: S-((S)-39-benzyl-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29,46-undecylmethyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40,43-pentadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44 Preparation of -pentadecazahexadeca-46-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-20)

[0613] (S)-N-(10-benzyl-3,3,20,23,26,29,32,35,38,41,44-undecyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45-tetradecoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17,20,23,26,29,32,35,38,41,44-tetradecazahexadecane-46-yl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (17 mg, 10.96 μmol) and (2S,4S)-2,5 1,2-Trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (13.76 mg, 21.93 μmol) was dissolved in DMF (3 mL), PyBOP (17.11 mg, 32.89 μmol) was added, and finally DIPEA (16 mg, 123.80 μmol) was added dropwise. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was partially desolventized by lyophilization, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (8.57 mg, 3.91 μmol).

[0614] Its structural characterization data are as follows:

[0615] MS m / z (ESI): 1081.4 [M / 2+H] +

[0616] Its preparation method is as follows:

[0617] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0618] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0619] Example 7: S-((37S,40S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-37-isopropyl-40,44-dimethyl-1,29,32,35,38,41-hexaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39,42-hexaazapentadecane-44-yl)(2S,4 Preparation of 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K21)

[0620] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (K-21-1)

[0621] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (566.94 mg, 1.38 mmol) was dissolved in DMF (10 mL), and HATU (524.87 mg, 1.38 mmol) and DIPEA (225 mg, 1.74 mmol) were added. After stirring at room temperature for 0.5 hours, 2-methyl-2-(triphenylthio)propyl-1-amine (480 mg, 1.38 mmol, FR) was added, and the mixture was stirred at room temperature for another 0.5 hours. The reaction mixture was poured into water with stirring, and a large amount of white solid precipitated out. The solid was filtered and dried under reduced pressure to give the title compound (1.02 g, 1.38 mmol).

[0622] Its structural characterization data are as follows:

[0623] MS m / z (ESI): 762.4 [M+Na] +

[0624] Step 2: Preparation of (S)-2-amino-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (K-21-2)

[0625] (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (1.02 g, 1.38 mmol) was dissolved in DMF (5 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (713 mg, 1.38 mmol), which was used directly in the next reaction without purification.

[0626] Its structural characterization data are as follows:

[0627] MS m / z (ESI): 1037.7 [2M+H] +

[0628] Step 3: Preparation of (9H-fluorene-9-yl)methyl((7S,10S)-10-isopropyl-3,3,7-trimethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamate (K-21-3)

[0629] (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycine (485.96 mg, 1.37 mmol) was dissolved in DMF (8 mL), and HATU (521.13 mg, 1.37 mmol) and DIPEA (354.48 mg, 2.74 mmol) were added. After stirring at room temperature for half an hour, (S)-2-amino-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (710 mg, 1.37 mmol) was added, and the mixture was stirred at room temperature for another 0.5 hours. After the reaction was complete, the reaction solution was poured into water with stirring, and a large amount of white solid precipitated out. The solid was filtered and dried under reduced pressure to give the crude product of the title compound (1.17 g, 1.37 mmol), which was used directly in the next reaction without purification.

[0630] Its structural characterization data are as follows:

[0631] MS m / z (ESI): 877.5 [M+Na] + 612.4[M-243+H] +

[0632] Step 4: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (K-21-4)

[0633] (9H-fluorene-9-yl)methyl((7S,10S)-10-isopropyl-3,3,7-trimethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamate (1.17 g, 1.37 mmol) was dissolved in DMF (8 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with ammonium chloride and brine, dried, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane-(dichloromethane:methanol = 3:1) = 0-50%) and concentrated again under reduced pressure to give the title compound (430 mg, 680.57 μmol).

[0634] Its structural characterization data are as follows:

[0635] MS m / z(ESI): 390.3 [M-243+H] + ; 1264.7 [2M+H] +

[0636] Step 5: Preparation of N-((7S,10S)-10-isopropyl-3,3,7-trimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-21,24,27,30,33,36,39,42-octaoxa-2-thia-5,8,11,14,17-pentazatetratetradecane-44-yl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (K-21-5)

[0637] 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-acid (54.32 mg, 63.31 μmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (40 mg, 63.31 μmol) were dissolved in DMF (2 mL), and DIPEA (32.73 mg, 253.23 μmol) and PyBOP (32.95 mg, 63.31 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (93 mg, 63.19 μmol), which was used directly in the next reaction without purification.

[0638] Its structural characterization data are as follows:

[0639] MS m / z (ESI): 1471.5 [M+H] + 1488.7 [M+H2O] + ;1231.5[M-243+H] +

[0640] Step Six: Preparation of N-((35S,38S)-35-isopropyl-42-mercapto-38,42-dimethyl-27,30,33,36,39-pentoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34,37,40-pentazatetrazotetrachetyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (K-21-6)

[0641] N-((7S,10S)-10-isopropyl-3,3,7-trimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-21,24,27,30,33,36,39,42-octaoxa-2-thia-5,8,11,14,17-pentazatetratetradecane-44-yl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (93 mg, 63.19 μmol) was dissolved in dichloromethane (2 mL), and TFA (1 mL) was added. The system turned yellow. Triisopropylsilane (100.06 mg, 631.90 μmol, 129.45 μL) was added at room temperature, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, then purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and freeze-dried to obtain the title compound (60 mg, 48.80 μmol).

[0642] Its structural characterization data are as follows:

[0643] MS m / z (ESI): 1231.5 [M+H] +

[0644] Step 7: S-((37S,40S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-37-isopropyl-40,44-dimethyl-1,29,32,35,38,41-hexaoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36,39,42-hexaazatrapentadecane-44-yl)(2S,4S Preparation of 2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-21)

[0645] N-((35S,38S)-35-isopropyl-42-mercapto-38,42-dimethyl-27,30,33,36,39-pentoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34,37,40-pentazatetrazotetrachetyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (68 mg, 55.31 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS, 9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (34.71 mg, 55.31 μmol) was dissolved in DMF (2 mL), PyBOP (43.17 mg, 82.96 μmol) was added, and then DIPEA (32.00 mg, 247.60 μmol) was added dropwise. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (43.10 mg, 22.55 μmol).

[0646] Its structural characterization data are as follows:

[0647] MS m / z (ESI): 1839.8 [M+H] + 920.1 [M / 2+H]+

[0648] Its preparation method is as follows:

[0649] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0650] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0651] Example 8: S-((39S,42S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-39-isopropyl-2,5,8,11,14,17,20,23,26,29,42,46-dodecylmethyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40,43-pentadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,3 Preparation of 8,41,44-pentadecazaheptadecane-46-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-22)

[0652] Step 1: Preparation of N-((7S,10S)-10-isopropyl-3,3,7,20,23,26,29,32,35,38,41,44-dodecylmethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45-tetradecoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17,20,23,26,29,32,35,38,41,44-tetradecazahexadecane-46-yl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (K-22-1)

[0653] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (65 mg, 56.76 μmol) and (S)-2-(2- (2-Aminoacetamido)acetamido)-3-methyl-N-((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)butyramide (35.86 mg, 56.76 μmol) was dissolved in DMF (2 mL), and PyBOP (32.49 mg, 62.43 μmol) and DIPEA (32 mg, 247.60 μmol) were added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to give the title compound (90 mg, 51.16 μmol).

[0654] Its structural characterization data are as follows:

[0655] MS m / z (ESI): 1759.5 [M+H] + ;1516.7[M-243+H] +

[0656] Step 2: Preparation of N-((37S,40S)-37-isopropyl-44-mercapto-3,6,9,12,15,18,21,24,27,40,44-undecyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecoxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecazatetraloctyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (K-22-2)

[0657] N-((7S,10S)-10-isopropyl-3,3,7,20,23,26,29,32,35,38,41,44-dodecylmethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45-tetradecoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17,20,23,26,29,32,35,3 8,41,44-Tetraazahexahexadecane-46-yl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (90 mg, 48.05 μmol) was dissolved in dichloromethane (2 mL), and TFA (1.0 mL) was added dropwise. Then, triisopropylsilane (76.09 mg, 480.50 μmol, 98.43 μL) was added, and the mixture was stirred for 1 hour. The yellow color of the system gradually faded. After the reaction was complete, the reaction solution was concentrated under reduced pressure, then purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-60%), and then freeze-dried to give the title compound (56 mg, 36.92 μmol).

[0658] Its structural characterization data are as follows:

[0659] MS m / z (ESI): 1517.7 [M+H] + 759.0 [M / 2+H] +

[0660] Step 3: S-((39S,42S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-39-isopropyl-2,5,8,11,14,17,20,23,26,29,42,46-dodecylmethyl-1,4,7,10,13,16,19,22,25,28,31,34,37,40,43-pentadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38 Preparation of 41,44-pentaazaheptadecane-46-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-22)

[0661] N-((37S,40S)-37-isopropyl-44-mercapto-3,6,9,12,15,18,21,24,27,40,44-undecyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecano-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecanoazatetraloctyl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (46 mg, 30.33 μmol) and (2S,4S)-2,5,12-trihydroxy-7 -Methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (19.03 mg, 30.33 μmol) was dissolved in DMF (1 mL), PyBOP (18.94 mg, 36.39 μmol) was added, followed by DIPEA (16.00 mg, 123.80 μmol). The system was deep red, and the reaction was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was lyophilized to remove part of the solvent, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (27.0 mg, 12.44 μmol).

[0662] Its structural characterization data are as follows:

[0663] MS m / z(ESI): 720.9 [M / 3+H] + 1064.2 [M / 2+H] +

[0664] Its preparation method is as follows:

[0665] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0666] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0667] Example 9: -S-(1-(((4-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methyl(propyl)amino)methyl)benzyl)oxy)carbonyl)amino)-2-methylpropyl- Preparation of 2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (K-24)

[0668] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (K-23-1)

[0669] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (50 mg, 70.55 μmol) and 2-methyl-2-(triphenylthio)prop-1-amine (28 mg, 80.57 μmol) were dissolved in DMF (1 mL). DIPEA (18.24 mg, 141.10 μmol) and HOBt (9.53 mg, 70.55 μmol) were added. The system turned yellow instantaneously, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, and concentrated under reduced pressure to obtain the crude product of the title compound (64 mg, 69.78 μmol), which was used directly in the next reaction without purification.

[0670] Its structural characterization data are as follows:

[0671] MS m / z (ESI): 934.6 [M+H2O] +

[0672] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (K-23-2)

[0673] Crude (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (100 mg, 109.04 μmol) was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was removed by lyophilization to obtain crude (75 mg, 107.93 μmol) of the title compound, which was used directly in the next step of the reaction without purification.

[0674] Step 3: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((((2-methyl-2-(triphenylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (K-23-3)

[0675] Dissolve (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (36.63 mg, 107.93 μmol) in DMF (2 mL), add HATU (41.01 mg, 107.93 μmol) and DIPEA (13.95 mg, 107.93 μmol), and stir at room temperature for 0.5 hours. Then add this reaction solution to (S)-(5-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (75 mg, 107.93 μmol), and stir for 1 hour. After the reaction is complete, the reaction solution can be used directly for the next reaction without further treatment.

[0676] Its structural characterization data are as follows:

[0677] MS m / z (ESI): 1016.5 [M+H] +

[0678] Step 4: Preparation of allyl (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl))(methyl)carbamate (K-23-4)

[0679] Diethylamine (0.5 mL) was added directly to the reaction solution from the previous step, which theoretically contained (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((((2-methyl-2-(triphenylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (109 mg, 107.26 μmol) and DMF (2 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was added to water, extracted with ethyl acetate, washed with brine, and the organic phase was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane-(dichloromethane:methanol = 3:1) = 0–50%) and then concentrated again under reduced pressure to obtain the title compound (52 mg, 65.49 μmol).

[0680] Its structural characterization data are as follows:

[0681] MS m / z (ESI): 794.5 [M+H] +

[0682] Step 5: Preparation of (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexaane-26-amido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (K-23-5)

[0683] 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-penta-2-azaeicosano-20-carboxylic acid (52.88 mg, 62.97 μmol) and allyl(5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl))(methyl)benzyl carbamate (50 mg, 62.97 μmol) were dissolved in DMF (2 mL), and DIPEA (32 mg, 247.60 μmol) and HATU (23.93 mg, 62.97 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was added to water, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain the crude product of the title compound (94 mg, 62.59 μmol), which was used directly in the next reaction without purification.

[0684] Its structural characterization data are as follows:

[0685] MS m / z (ESI): 1518.7 [M+H2O] + 1523.6 [M+Na] +

[0686] Step Six: Preparation of (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexaane-26-amido)-2-((((2-mercapto-2-methylpropyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (K-23-6)

[0687] (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-penta-2,21,24-triazahexa-26-amido)-2-((((2-methyl-2-(triphenylmethylthio)propyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (50 mg, 33.29 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (75.93 mg, 665.88 μmol) and triisopropylsilane (31.63 mg, 199.76 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure at room temperature, dissolved in acetonitrile, purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), and then freeze-dried to obtain the trifluoroacetate of the title compound (50 mg, 36.40 μmol).

[0688] Its structural characterization data are as follows:

[0689] MS m / z (ESI): 1277.5 [M+H2O] +

[0690] Step 7: S-(1-((((2-(((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3-(2-hydrosulfinylpyrimidin-5-yl)-5-(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-penta-2,21,24-triazahexacosane-26-acylamino)benzyl)oxy)carbonyl) Preparation of (amino)-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-23-7)

[0691] Allyl (50 mg, 39.70 μmol) of (5-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexadecane-26-amido)-2-((((2-mercapto-2-methylpropyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1 S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (24.91 mg, 39.70 μmol) was dissolved in DMF (2 mL), PyBOP (20.66 mg) was added, followed by pyridine (47.10 mg, 595.49 μmol). The mixture was stirred at room temperature for 1 hour, but the starting material did not react. PyBOP (20.66 mg) and DIPEA (32 mg, 247.60 μmol) were then added, and the color turned dark red. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (25 mg, 13.38 μmol).

[0692] Its structural characterization data are as follows:

[0693] MS m / z (ESI): 1871.7 [M+H] + 943.5 [M / 2+H] +

[0694] Its preparation method is as follows:

[0695] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0696] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0697] Step 8: S-(1-(((4-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl)oxy)carbonyl)amino)-2-methylpropyl-2-yl (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (K-23-8) and S-(1-(((4-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methyl(propyl)amino)methyl)benzyl)oxy)carbonyl)amino)-2-methylpropyl-2-yl Preparation of (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbonate (K-24)

[0698] S-(1-((((2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((22S,25S)-1-(3-(2-hydrosulfinylpyrimidin-5-yl)-5-(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-acylamino)benzyl)oxy)carbonyl)amino)-2-methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S, 9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarboxylate (25 mg, 13.38 μmol) was dissolved in DMF (1 mL), and tetraphenylphosphine palladium (15.46 mg, 13.38 μmol), formic acid (24.40 mg, 530.14 μmol), and N-methylmorpholine (27.60 mg, 272.87 μmol) were added. After nitrogen purging, the mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was lyophilized to remove part of the solvent and then purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compounds K-23-8 (7 mg, 3.92 μmol) and K-24 (2.11 mg, 1.13 μmol), of which K24 was prepared and purified twice.

[0699] The structural characterization data for K23-8 are as follows:

[0700] MS m / z (ESI): 1786.6 [M+H] + 893.5 [M / 2+H] +

[0701] The structural characterization data of K24 are as follows:

[0702] MS m / z(ESI): 1826.7 [M+H] + ; 913.0 [M / 2+H] +

[0703] The first preparation method is as follows:

[0704] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0705] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0706] The second preparation method is as follows:

[0707] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0708] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0709] Example 10: 2,2',2”-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-((methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((((2-methyl-2-(((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R, Preparation of 4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carbonyl)thio)propyl)carbamoyl)oxy)methyl)benzyl))methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (K-23)

[0710] S-(1-(((4-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl)oxy)carbonyl)amino)-2 -methylpropyl-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydro Tetraphenyl-2-thiocarbonate (7.00 mg, 3.92 μmol) and 2,2',2”-(10-(2-((2,5-dioxocyclopentyl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (5.90 mg, 11.76 μmol) were dissolved in DMF (2 mL), and DIPEA (16 mg, 12) was added. 3.80 μmol) was added, and the mixture was stirred at room temperature for 2 hours. Then, 2,2',2”-(10-(2-((2,5-dioxocyclopentyl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (5.90 mg, 11.76 μmol) and DIPEA (16 mg, 123.80 μmol) were added, and the mixture was stirred for another hour. After the reaction was complete, the reaction solution was partially lyophilized and purified by preparative high-performance liquid chromatography (HPLC), followed by freeze-drying to obtain the title compound (4.62 mg, 2.02 μmol).

[0711] Its structural characterization data are as follows:

[0712] MS m / z (ESI): 724.5 [M / 3+H] +

[0713] Its preparation method is as follows:

[0714] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0715] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0716] Example 11: 2,2',2”-(10-((7S,10S,13S,16S)-16-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3,3,7,10,13-pentamethyl-1,6,9,12,15,22-hexaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)) Preparation of 9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14,21-pentazatrisane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (K-33)

[0717] Step 1: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (K-33-1)

[0718] (9H-fluorene-9-yl)methyl ((7S,10S,13S)-3,3,7,10-tetramethyl-6,9,12-trioxo-1,1,1-triphenyl-2-thia-5,8,11-triazaester (tetradecanoate (13-yl)carbamate (3.8 g, 4.85 mmol, FR)) was dissolved in DMF (20 mL), and diethylamine (4 mL) was added. The mixture was stirred at room temperature for 1.0 h. After removing part of the solvent directly under reduced pressure, the mixture was purified by reversed-phase silica gel column chromatography (acetonitrile-0.05% ammonium bicarbonate aqueous solution = 0–65%) and then freeze-dried to give the title compound (2.2 g, 3.92 mmol).

[0719] Its structural characterization data are as follows:

[0720] MS m / z (ESI): 173.4 [M+H] +

[0721] Step 2: Preparation of (9H-fluorene-9-yl)methyl((7S,10S,13S,16S)-16-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3,3,7,10,13-pentamethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazaeicosano-20-yl)carbamate (K-33-2)

[0722] N6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2 -(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (60 mg, 76.45 μmol) and (S)-2-amino-N-((S)-1-(((S)-1-((2-methyl-2-(triphenylthio)propyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (51.44 mg, 91.74 μmol) were dissolved in DMF (3 mL), PyBOP (59.67 mg, 114.67 μmol) was added, followed by DIPEA (32 mg, 247.60 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was added dropwise to water, and a large amount of white solid precipitated out. The solid was filtered, washed with water, and dried to obtain the crude title compound (100 mg, 75.32 μmol), which could be used directly in the next reaction without further purification.

[0723] Its structural characterization data are as follows:

[0724] MS m / z (ESI): 1085.4 [M-243+H] + 1349.5 [M+Na] +

[0725] Step 3: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S,15S)-15-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoylamino)-2-mercapto-2,6,9,12-tetramethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazanonadecan-19-yl)carbamate (K-33-3)

[0726] Dichloromethane (2 mL) was added to (9H-fluorene-9-yl)methyl((7S,10S,13S,16S)-16-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-3,3,7,10,13-pentamethyl-6,9,12,15-tetraoxo-1,1,1-triphenyl-2-thia-5,8,11,14-tetraazaeicosano-20-yl)carbamate (100 mg, 75.32 μmol), followed by the addition of trifluoroacetic acid (0.1 mL). The system instantly turned yellow. Triisopropylsilane (119.28 mg, 753.25 μmol) was then added dropwise, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, and then purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and freeze-dried to obtain the title compound (50 mg, 46.07 μmol).

[0727] Its structural characterization data are as follows:

[0728] MS m / z (ESI): 1085.4 [M+H] +

[0729] Step 4: S-((9S,12S,15S,18S)-9-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluorene-9-yl)-12,15,18,22-tetramethyl-3,10,13,16,19-pentoxo-2-oxa-4,11,14,17,20-pentazatridecane-22-yl)(2S,4S)-2, Preparation of 5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-33-4)

[0730] The carbamate ((9H-fluorene-9-yl)methyl((6S,9S,12S,15S)-15-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoylamino)-2-mercapto-2,6,9,12-tetramethyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazanonadecan-19-yl)carbamate (50 mg, 46.07 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1 43.37 mg (69.11 μmol) of methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (43.37 mg, 69.11 μmol) was dissolved in DMF (3 mL), PyBOP (35.96 mg, 69.11 μmol) was added, and finally DIPEA (32 mg, 247.60 μmol) was added dropwise. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution did not require further treatment and was used directly for the next reaction.

[0731] Its structural characterization data are as follows:

[0732] MS m / z (ESI): 1695.7 [M+H] + 847.9 [M / 2+H] +

[0733] Step 5: S-((3S,6S,9S,12S)-3-(4-aminobutyl)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-6,9,12,16-tetramethyl-1,4,7,10,13-pentoxo-2,5,8,11,14-pentazaheptadecane-16-yl)(2S,4S)-2,5,12-trihydroxy-7 Preparation of 1-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-33-5)

[0734] Directly to the theoretically contained S-((9S,12S,15S,18S)-9-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluorene-9-yl)-12,15,18,22-tetramethyl-3,10,13,16,19-pentoxo-2-oxa-4,11,14,17,20-pentazatridecane-22-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4a Diethylamine (67.32 mg, 920.43 μmol) was added to the reaction solution of (S,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (78 mg, 46.02 μmol) and DMF (3 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was lyophilized to remove some solvent, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (40 mg, 27.16 μmol).

[0735] Its structural characterization data are as follows:

[0736] MS m / z (ESI): 1474.4 [M+H] + 736.9 [M / 2+H] +

[0737] Its preparation method is as follows:

[0738] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0739] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0740] Step Six: 2,2',2”-(10-((7S,10S,13S,16S)-16-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3,3,7,10,13-pentamethyl-1,6,9,12,15,22-hexaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)- Preparation of 9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14,21-pentazatoridecane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (K-33)

[0741] S-((3S,6S,9S,12S)-3-(4-aminobutyl)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-6,9,12,16-tetramethyl-1,4,7,10,13-pentoxo-2,5,8,11,14-pentazaheptadecane-16-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6, 11-Hexahydrotetraphenyl-2-thiocarboxylate (20 mg, 13.58 μmol) was dissolved in DMF (6 mL), and then 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (13.62 mg, 27.16 μmol) was added. Then, DIPEA (16 mg, 123.80 μmol) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was partially lyophilized and purified by preparative high-performance liquid chromatography (HPLC), followed by freeze-drying to obtain the title compound (11.43 mg, 5.53 μmol).

[0742] Its structural characterization data are as follows:

[0743] MS m / z (ESI): 1861.7 [M+H] + 930.1 [M / 2+H] + 620.5 [M / 3+H]+

[0744] Its preparation method is as follows:

[0745] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0746] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0747] Example 12: 2,2',2”-(10-((10S,19S)-10-benzyl-19-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3,3-dimethyl-1,6,9,12,15,18,25-heptaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methyl Preparation of oxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14,17,24-hexaazahexa-26-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (K-34)

[0748] Step 1: Preparation of (9H-fluorene-9-yl)methyl((10S,19S)-10-benzyl-19-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-3,3-dimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17-pentazatrisane-23-yl)carbamate (K-34-1)

[0749] N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (70 mg, 89.19 μmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((2-methyl-2-(triphenylthio)propyl)amino)-2-oxoethyl)-3-phenylpropionamide (59.39 mg, 89.19 μmol) were dissolved in DMF (3 mL), PyBOP (51.05 mg, 98.11 μmol) was added, followed by DIPEA (23.05 mg, 178.38 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was added to water, extracted with ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (127 mg, 88.64 μmol), which was used directly in the next reaction without purification.

[0750] MS m / z (ESI): 1190.4 [M-243+H] +

[0751] Step 2: Preparation of (9H-fluorene-9-yl)methyl ((9S,18S)-9-benzyl-18-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoamide)-2-mercapto-2-5,8,11,14,17-pentoxo-4,7,10,13,16-pentazadocosane-22-yl)carbamate (K-34-2)

[0752] Dichloromethane (2 mL) was added to (9H-fluorene-9-yl)methyl((10S,19S)-10-benzyl-19-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-3,3-dimethyl-6,9,12,15,18-pentoxo-1,1,1-triphenyl-2-thia-5,8,11,14,17-pentazatocetane-23-yl)carbamate (127 mg, 88.64 μmol), followed by the addition of trifluoroacetic acid (0.2 mL). The system turned yellow instantly. Triisopropylsilane (140.37 mg, 886.45 μmol) was then added, and the yellow color gradually disappeared. The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, and then purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%) and freeze-dried to obtain the title compound (72 mg, 60.49 μmol).

[0753] Its structural characterization data are as follows:

[0754] MS m / z (ESI): 1191.5 [M+H] +

[0755] Step 3: S-((9S,18S)-18-benzyl-9-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluorene-9-yl)-25-methyl-3,10,13,16,19,22-hexaoxo-2-oxa-4,11,14,17,20,23-hexaazahexacosane-25-yl)(2S,4S)-2,5,1 Preparation of 2-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-34-3)

[0756] Directly to (9H-fluorene-9-yl)methyl ((9S,18S)-9-benzyl-18-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoamide)-2-mercapto-2-5,8,11,14,17-pentoxo-4,7,10,13,16-pentazadocosane-22-yl)carbamate (73 mg, 61.33 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)) DMF (3 mL) was added to a mixture of 9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (46.18 mg, 73.59 μmol) and PyBOP (41.49 mg, 79.72 μmol), followed by the addition of DIPEA (32 mg, 247.60 μmol). The mixture was stirred at room temperature for 1 hour. (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-carboxylic acid (46.18 mg, 73.59 μmol), PyBOP (41.49 mg, 79.72 μmol), and DIPEA (32 mg, 247.60 μmol) were added, and the reaction was continued with stirring at room temperature for 2 hours. After the reaction was completed, the reaction solution did not require further treatment and was used directly for the next reaction.

[0757] Its structural characterization data are as follows:

[0758] MS m / z (ESI): 1801.2 [M+H] + 901.5 [M / 2+H] +

[0759] Step 4: S-((3S,12S)-3-(4-aminobutyl)-12-benzyl-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-19-methyl-1,4,7,10,13,16-hexaoxo-2,5,8,11,14,17-hexaazaeicosane-19-yl)(2S,4S)-2,5,12-trihydroxy-7 Preparation of 1-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (K-34-4)

[0760] Directly to the theoretically contained S-((9S,18S)-18-benzyl-9-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-1-(9H-fluorene-9-yl)-25-methyl-3,10,13,16,19,22-hexaoxo-2-oxa-4,11,14,17,20,23-hexaazahexacosane-25-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS, Diethylamine (111.74 mg, 1.53 mmol) was added dropwise to the reaction solution of 9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (110 mg, 61.11 μmol) and DMF (3 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was lyophilized to remove some solvent, purified by preparative high performance liquid chromatography, and then lyophilized to obtain the formate salt of the title compound (25 mg, 15.40 μmol).

[0761] Its structural characterization data are as follows:

[0762] MS m / z (ESI): 1577.7 [M+H] + 790.0 [M / 2+H] +

[0763] Its preparation method is as follows:

[0764] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0765] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0766] Step 5: 2,2',2”-(10-((10S,19S)-10-benzyl-19-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-3,3-dimethyl-1,6,9,12,15,18,25-heptaoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy Preparation of 1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14,17,24-hexaazahexa-26-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (K-34)

[0767] S-((3S,12S)-3-(4-aminobutyl)-12-benzyl-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-19-methyl-1,4,7,10,13,16-hexaoxo-2,5,8,11,14,17-hexaazaeicosano-19-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11 The formate of hexahydrotetraphenyl-2-thiocarbamate (25 mg, 15.40 μmol) was dissolved in DMF (2 mL), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (23.16 mg, 46.19 μmol) was added, followed by DIPEA (32 mg, 247.60 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was lyophilized to remove some of the solvent, purified by preparative high performance liquid chromatography, and then lyophilized to obtain the title compound (8.92 mg, 4.22 μmol).

[0768] Its structural characterization data are as follows:

[0769] MS m / z(ESI): 1964.8 [M+H] + 982.7 [M / 2+H] + 655.5 [M / 3+H] +

[0770] Its preparation method is as follows:

[0771] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0772] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0773] Example 13: 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H- Preparation of pyranyl[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenyl ester (K-32)

[0774] Step 1: Preparation of 1-(N-((2-(tert-butoxy)-2-oxoethyl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (K-32-1)

[0775] Reactor 1: Glycine tert-butyl ester (2.0 g, 15.25 mmol) was dissolved in dichloromethane (200 mL), cooled to -25 °C under nitrogen protection, and chlorosulfonic acid isocyanate (2.37 g, 16.77 mmol, 1.46 mL) was added dropwise under an ice bath. The mixture was kept at this temperature with stirring for 1 hour, then allowed to rise naturally to room temperature with stirring for another hour. Reactor 2: 4-Piperidinyl allyl ester hydrochloride (3.12 g, 15.25 mmol) was suspended in dichloromethane (200 mL), and triethylamine (4.63 g, 45.74 mmol) was added. The mixture was then cooled to -25 °C under nitrogen protection. The reaction solution from Reactor 1 was transferred to a dropping funnel and added dropwise to Reactor 2 at -25 °C. After the addition was complete, the mixture was kept at this temperature for 1 hour, then allowed to rise naturally overnight. After the reaction was complete, the reaction solution was extracted with water and dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-60%), followed by concentration under reduced pressure to give the title compound (2.37 g, 5.85 mmol).

[0776] Its structural characterization data are as follows:

[0777] MS m / z (ESI): 350.2 [M+H-56] +

[0778] Step 2: Preparation of (((4-((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)glycine (K-32-2)

[0779] 1-(N-((2-(tert-butoxy)-2-oxoethyl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (2.37 g, 5.85 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (10 mL) was added dropwise. The mixture was stirred at room temperature for 4.5 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure, and water and ethyl acetate were added. Then, saturated sodium bicarbonate was added to adjust the pH to 8-9, and the organic phase was separated. The aqueous phase was adjusted to pH 4-5 with 6N HCl, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title compound (800 mg, 2.29 mmol).

[0780] Its structural characterization data are as follows:

[0781] MS m / z (ESI): 350.2 [M+H] + 699.3 [2M+H] +

[0782] Step 3: 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran) Preparation of [4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (K-32-3)

[0783] S-(1-((S)-2-((S)-2-((S)-2-aminopropamido)propamido)propamido)-2-methylprop-2-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2 3,4,6,11-hexahydrotetraphenyl-2-thiocarbamate (100 mg, 107.76 μmol) and (((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)glycine (41.41 mg, 118.53 μmol)) were dissolved in DMF (3 mL), DIPEA (48.00 mg, 371.40 μmol) was added, followed by PyBOP (61.68 mg, 118.53 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (62 mg, 49.23 μmol).

[0784] Its structural characterization data are as follows:

[0785] MS m / z (ESI): 1259.7 [M+H] + 630.5 [M / 2+H] +

[0786] Its preparation method is as follows:

[0787] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0788] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0789] Step 4: Preparation of 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid (K-32-4)

[0790] 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyranyl[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo 60 mg (47.64 μmol) of allyl 4-(1,2,3,4,6,11-hexafluorotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidin-4-carboxylate (3 mL) was dissolved in DMF, followed by the addition of tetrahydropyrrole (6.78 mg, 95.29 μmol), and then Pd(PPh3)4 (5.51 mg, 4.76 μmol). After nitrogen purging, the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate. The organic phase was discarded, and the aqueous phase was partially lyophilized, purified by preparative high-performance liquid chromatography, and then freeze-dried to obtain the title compound (15 mg, 12.30 μmol).

[0791] Its structural characterization data are as follows:

[0792] MS m / z (ESI): 1220.5 [M+H] +

[0793] Its preparation method is as follows:

[0794] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0795] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0796] Step 5: 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyridine) Preparation of 2-(4',3':4,5)oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenyl ester (K-32)

[0797] 1-(N-(((7S,10S,13S)-3,3,7,10,13-pentamethyl-1,6,9,12,15-pentoxo-1-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyran[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo- 1,2,3,4,6,11-hexahydrotetraphenyl-2-yl)-2-thia-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)aminosulfonyl)piperidin-4-carboxylic acid (15 mg, 12.30 μmol) was dissolved in DMF (2 mL), followed by the addition of 2,3,4,5,6-pentafluorophenol (22.64 mg, 123.02 μmol), and then EDCI (11.79 mg, 61.51 μmol). The mixture was stirred at room temperature for 2 hours after the addition was complete. After the reaction was complete, the reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (1.69 mg, 1.12 μmol).

[0798] Its structural characterization data are as follows:

[0799] MS m / z (ESI): 1385.6 [M+H] +

[0800] Its preparation method is as follows:

[0801] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0802] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0803] Other compounds of the present invention can be prepared by referring to the synthesis method of the above embodiments.

[0804] II. Detection of the inhibitory effect of compounds on tumor cell proliferation

[0805] 1. Inhibitory effect of the compound on the proliferation of NCI-N87 cells

[0806] (1) Cell plating: First, NCI-N87 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 3.

[0807] Table 3. Tumor cell origin

[0808] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0809] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Tables 4-1, 4-2, 4-3, and 4-4.

[0810] (2) Data Results

[0811] Table 4-1. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0812] Table 4-2. Inhibitory activity of compounds against NCI-N87 cell proliferation

[0813] Table 4-3. Inhibitory activity of compounds against NCI-N87 cell proliferation

[0814] Table 4-4. Inhibitory activity of compounds on NCI-N87 cell proliferation

[0815] The test results show that the compounds of the present invention listed in Tables 4-1, 4-4, 4-3 and 4-4 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

[0816] 2. Inhibitory effect of the compound on the proliferation of HT29 cells

[0817] (1) Cell plating: First, HT29 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 5.

[0818] Table 5. Tumor cell origin

[0819] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0820] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Tables 6-1, 6-2, 6-3, and 6-4.

[0821] (2) Data Results

[0822] Table 6-1. Inhibitory activity of compounds on HT29 cell proliferation

[0823] Table 6-2. Inhibitory activity of compounds against HT29 cell proliferation

[0824] Table 6-3. Inhibitory activity of compounds against HT29 cell proliferation

[0825] Table 6-4. Inhibitory activity of compounds against HT29 cell proliferation

[0826] The test results show that the compounds of the present invention in Tables 6-1, 6-2, 6-3 and 6-4 have a strong inhibitory effect on the proliferation of HT29 colon cancer cells.

[0827] 3. Inhibitory effect of the compound on the proliferation of HCC1806 cells

[0828] (1) Cell plating: First, HCC1806 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 7.

[0829] Table 7. Tumor cell origin

[0830] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0831] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 8.

[0832] (2) Data Results

[0833] Table 8. Inhibitory activity of compounds on HCC1806 cell proliferation

[0834] The test results show that the compounds of the present invention listed in Table 8 have a significant inhibitory effect on the proliferation of HCC1806 human breast squamous cell carcinoma cells.

[0835] 4. Inhibitory effect of the compound on SKOV-3 cell proliferation

[0836] (1) Cell plating: First, SKOV-3 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 9.

[0837] Table 9. Tumor cell origin

[0838] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0839] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 10.

[0840] (2) Data Results

[0841] Table 10. Inhibitory activity of compounds against SKOV-3 cell proliferation

[0842] The test results show that the compounds of the present invention listed in Table 10 have a significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

[0843] 5. Inhibitory effect of the compound on the proliferation of NCI-H358 cells

[0844] (1) Cell plating: First, NCI-H358 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 11.

[0845] Table 11. Tumor cell origin

[0846] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0847] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 12.

[0848] (2) Data Results

[0849] Table 12. Inhibitory activity of compounds on NCI-H358 cell proliferation

[0850] The test results show that the compounds of the present invention listed in Table 12 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[0851] 6. Inhibitory effect of the compound on KPL-4 cell proliferation

[0852] (1) Cell plating: First, KPL-4 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 13.

[0853] Table 13. Tumor cell origin

[0854] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0855] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 14.

[0856] (2) Data Results

[0857] Table 14. Inhibitory activity of compounds against KPL-4 cell proliferation

[0858] The test results show that the compounds of the present invention listed in Table 14 have a significant inhibitory effect on the proliferation of KPL-4 human breast cancer cells.

[0859] 7. Inhibitory effect of the compound on the proliferation of HCC827 cells

[0860] (1) Cell plating: First, HCC827 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 15.

[0861] Table 15. Tumor cell origin

[0862] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0863] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 16.

[0864] (2) Data Results

[0865] Table 16. Inhibitory activity of compounds on the proliferation of HCC827 cells

[0866] The test results show that the compounds of the present invention listed in Table 16 have an inhibitory effect on the proliferation of HCC827 human non-small cell lung cancer cells.

[0867] 8. Inhibitory effect of the compound on the proliferation of HCC1954 cells

[0868] (1) Cell plating: First, HCC1954 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 17.

[0869] Table 17. Tumor cell origin

[0870] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0871] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 18.

[0872] (2) Data Results

[0873] Table 18. Inhibitory activity of compounds on the proliferation of HCC1954 cells

[0874] The test results show that the compounds of the present invention listed in Table 18 have an inhibitory effect on the proliferation of HCC1954 human non-small cell lung cancer cells.

[0875] 9. Inhibitory effect of the compound on the proliferation of NCI-H1975 cells

[0876] (1) Cell plating: First, NCI-H1975 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 19.

[0877] Table 19. Tumor cell origin

[0878] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[0879] In vitro cell viability assay: After incubation, add 50 μL of Cell Counting-Lite™ 2.0 reagent (Vazyme / Novozymes) to each well, mix thoroughly by shaking in the dark, and react for 10 min before detection. Read the values ​​using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Background RLU was obtained using Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained using Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. RLU (relative light unit): relative light unit. The detection results are shown in Table 20.

[0880] (2) Data Results

[0881] Table 20. Inhibitory activity of compounds on the proliferation of NCI-H1975 cells

[0882] The test results show that the compounds of the present invention listed in Table 20 have an inhibitory effect on the proliferation of NCI-H1975 human non-small cell lung cancer cells.

[0883] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, M'-LED formula (I) in: M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site; L is the structural segment connecting connectors M and E; E is a structural segment connecting L and D; D is a cytotoxic drug fragment.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, M is selected from the following substituted or unsubstituted structural segments:

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, M is selected from the following substituted or unsubstituted structural segments:

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein, L is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6 Alkyl groups, 6-10 aryl groups, 5-6 heteroaryl groups, 9-12 nitrogen-containing heterocyclic groups, -N(R')-, -NH(R'), -N(R')2, carbonyl groups, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac)). Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-V al-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1- 6-alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocyclic, -CH2NH-SO3H, -CH2N(C 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6 Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1- 6-alkylene-SO3H)3、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1- 6-alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylenes -CO2H, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, or polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or Nota (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein r is selected from integers from 1 to 20; s is selected from integers from 1 to 20; Preferably, L is selected from a divalent or unsubstituted structural fragment composed of one or more of the following groups: s is an integer selected from 1 to 20.

5. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, E is a single bond, substituted or unsubstituted -NH-CH2-, or selected from the following substituted or unsubstituted structural segments: Preferably, E is a single bond, substituted or unsubstituted -NH-CH2-, or 6. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein, Selected from the following substituted or unsubstituted structures: Selected from the following substituted or unsubstituted structures: s is an integer selected from 1 to 20; or Selected from the following substituted or unsubstituted structures:

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein, The cytotoxic drugs are selected from anti-microtubule agents, DNA intercalation agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors. Preferably, the anti-microtubule agent is an oligurianin, maytansine, or eribulin; the DNA intercalating agent is a pyrrolobenzodiazepine (PBD), trabectedin, or rubotedin; the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin; and the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogues. Preferably, the cytotoxic drug has the structure shown in formula (I): in, Indicates a single bond or no chemical bond; R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C). 1-6 alkyl), -N(C) 1- 6-alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 aryl and 5-10 heteroaryl groups; wherein the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more of -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; R5 is -(C=O)-D2-D3-X6, where D2 either does not exist or is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1-6 Alkyl, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl group)2, -CN substituents; D3 is C 1-6 Alkylene, C 2-6 imide or C 2-6 Alkyne group; the alkylene group, alkenylene group, and alkyne group may optionally be selected from one or more groups selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; X6 is selected from hydrogen, halogen, -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups; Y1 and Y2 are -C(R8)2-, where each R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl groups and 5-10 heteroaryl groups, or two R8 groups attached to the same carbon atom, together form =O; X1, X2, and X5 are each independently selected from substituted or unsubstituted C. 1-6 Alkylene, -O-, -NR Y -or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl; when When representing a single bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkylene, -O-, -NR Y -or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl; when When there is no chemical bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkyl, -OR Y -N(R) Y )2 or -S-; where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1- 6-alkyl)2,-CN substituents of C 1-6 alkyl; X3 is selected from CR Z Or N, where R Z Selected from hydrogen or optionally by one or more elements selected from halogens, -OH, -NH2, -NH(C 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl; More preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C) 1- 6-alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl and C 1-6 Alkoxy group; the alkyl group or alkoxy group may optionally be selected from one or more groups selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6- 10 Substituents of aryl and 5-10 heteroaryl groups; More preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C. 1-6 Alkyl group; the alkoxy group is optionally surrounded by one or more elements selected from -CN, halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; More preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, C, ... 1-6 Alkyl and C 1-6 Alkoxy More preferably, R1, R2, R3, R4, R6, and R7 are each independently selected from hydrogen, -OH, and C. 1-6 Alkoxy; More preferably, R2, R3, and R4 are all -OH; More preferably, R1 and R7 are each independently selected from C 1-6 Alkyl groups, such as methoxy, ethoxy, n-propoxy, or isopropoxy; More preferably, R1 and R7 are both methoxy groups; More preferably, R6 is selected from C 1-6 Alkyl groups, such as methyl, ethyl, n-propyl, or isopropyl; More preferably, R6 is a methyl group; More preferably, R1 and R7 are both methoxy groups; R2, R3 and R4 are all -OH groups; and R6 is a methyl group. More preferably, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1-6 Alkyl; D3 is C 1-6 Alkylene; the alkylene group may optionally be composed of one or more elements selected from -CN, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The substituents are aryl and 5-10 heteroaryl groups; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, the C 1-6 Alkyl groups may be optionally surrounded by one or more elements selected from halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, -CN, C 1-6 Substitution of alkoxy groups; More preferably, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1-6 Alkyl; D3 is C 1-6 Alkylene; X6 is selected from -OH, C 1-6 Alkoxy, -SH, -SC 1-6 Alkyl, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2; More preferably, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen, methyl, and ethyl; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)-, and -CH2-C(CH3)2-; X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3), and -N(CH3)2; More preferably, R5 is -(C=O)-D2-D3-X6, wherein D2 is selected from -O-, -S-, -NH- and -N(CH3)-; D3 is selected from methylene, ethylene, -(CH2)3, -CH2-CH(CH3)- and -CH2-C(CH3)2-; and X6 is selected from -OH, methoxy, ethoxy, -SH, -S-CH3, -NH2, -NH(CH3) and -N(CH3)2; More preferably, R5 is -(C=O)-D2-D3-X6, where D2 is -O-, -S-, or -NR. X -, where R X Selected from hydrogen or C 1-3 Alkyl; D3 is C 1-6 Alkylene; X6 is selected from -OH, -SH, -NH2; More preferably, R5 is selected from: More preferably, Y1 and Y2 are -C(R8)2-, wherein each of R8 is independently selected from hydrogen, -CN, halogen, -OH, -NH2, and -NH(C 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Alkoxy groups, or two R8 atoms bonded to the same carbon atom, together form =O; Preferably, Y1 and Y2 are -(C=O)-; More preferably, X1, X2, and X5 are each independently selected from substituted or unsubstituted C. 1-6 Alkylene, -O- or -NR Y ;where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 alkyl)2, -CN substituents of C 1-6 alkyl; More preferably, X1, X2, X4 and X5 are all -O-; More preferably, when When representing a single bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkylene, -O-; preferably, X4 is selected from -O-; More preferably, when When there is no chemical bond, X4 is selected from substituted or unsubstituted C. 1-6 Alkyl, -OR Y ;where R Y Each time it appears, it is independently selected from hydrogen or optionally selected from one or more halogens, -OH, -NH2, -NH(C) 1-6 alkyl), -N(C) 1- 6-alkyl)2,-CN substituents of C 1-6 Alkyl group; preferably, X4 is selected from -OH; More preferably, X3 is selected from CR Z Or N, where R Z C selected from hydrogen or optionally substituted with one or more substituents selected from halogens, -OH, -NH2, -CN. 1-6 alkyl; More preferably, X3 is N; More preferably, the cytotoxic drug has the structure shown in formula (III)-1 or (III)-2: More preferably, the cytotoxic drug has the structure shown in formula (IV)-1 or (IV)-2: Preferably, the cytotoxic drug is selected from the following compounds or compounds labeled with their isotopes: Preferably, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group thereon.

8. The compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from the following structures:

9. A coupling as shown in formula (II), wherein: Ab-[MLED]x Equation (II) Ab is the target portion; M, L, E, and D are as described in any one of claims 1-8; x is between 1 and 10.

10. The conjugate of claim 9, wherein the Ab is an antibody.

11. A coupling as shown in formula (II), wherein: Ab-[MLED]x Equation (II) M, L, E and D are as described in any one of claims 1-8; Ab is the targeted component, and its targets are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2;. x is between 1 and 10.

12. The coupling agent according to any one of claims 9 to 11, wherein the coupling agent is selected from: in, HA is an antibody or antigen-binding fragment; This indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the M fragment; This indicates the specific way in which the amino group in the antibody or its antigen-binding fragment is linked to the M fragment.

13. A composition comprising one or more conjugates according to any one of claims 9-12, wherein the composition has a DAR (drug-antibody conjugate ratio) of 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.

0.

14. A pharmaceutical composition comprising a compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, a conjugate of any one of claims 9-12, or a composition of claim 13, and one or more pharmaceutical excipients.

15. Use of the compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 9-12, the composition of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating cancer.

16. The use of claim 15, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

17. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 9-12, the composition of claim 13, or the pharmaceutical composition of claim 14, for the treatment of cancer.

18. The compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, the conjugate of any one of claims 9-12, the composition of claim 13, or the pharmaceutical composition of claim 14, for the treatment of solid tumors or hematologic malignancies, for example selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and urothelial carcinoma.

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