WO2025167742A1 - Antibody-drug conjugate, method for preparing same, and use thereof - Google Patents

Abstract

Provided are an antibody-drug conjugate, a method for preparing same, and use thereof. The antibody-drug conjugate has a structure represented by formula Ab-[M-L-E-D] _x, a superior drug-to-antibody ratio, and an excellent targeted killing effect on various cancers or tumors.

Description

Antibody drug conjugates and preparation methods and uses thereof

This application is based on the application with CN application number 202410173874.0 and application date February 6, 2024, the application with PCT application number PCT/CN2024/092425 and application date May 10, 2024, the application with CN application number 202411554776.8 and application date November 1, 2024, and the application with CN application number 202411858078.7 and application date December 17, 2024, and claims its priority. The disclosed contents of the aforementioned applications are hereby introduced into this application as a whole.

Technical Field

The present application relates to the field of targeted therapy, and specifically to an antibody-drug conjugate and its preparation method and use.

Background Art

Antibody-drug conjugates (ADCs) for tumor therapy typically consist of a monoclonal antibody, a bioactive molecule (primarily a tumor-killing cytotoxin), 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, where it is internalized. The bioactive molecule is then released inside the cancer cells and kills them by inhibiting their microtubules or damaging their DNA, minimizing damage to normal tissue cells.

The ErbB family of receptor tyrosine kinases is an important mediator 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). In clinical practice, 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. In order to improve the therapeutic effect, in recent years, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansines (such as DM1) and auristatins (such as MMAE) or DNA topoisomerase I inhibitors (such as Dxd) (Trastuzumab emtansine, Disitamab vedotin, Trastuzumab deruxtecan) have been used for clinical treatment.

With the widespread use of the above-mentioned ADC drugs in the clinical treatment of Her2-expressing tumors, safety issues or drug resistance issues including neurotoxicity, hematotoxicity, 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.).

Specifically, the linker portion of Disitamab vedotin and Trastuzumab deruxtecan both use a maleimide linker (MC). Literature reports that the MC linker is prone to reverse Michael reaction and thiol exchange under physiological conditions, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). In terms of the hydrophilicity of the linker, Disitamab vedotin and Trastuzumab deruxtecan use the more hydrophobic valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively. Literature reports that the hydrophilicity of the linker significantly affects the hydrophilicity of the ADC, thereby affecting the aggregation, pharmacokinetic properties and toxicity of the ADC (Chemical Linkers in Antibody–Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3.). As for the biologically active molecules that kill cancer cells, Trastuzumab emtansine uses the microtubule inhibitor DM1 as a cytotoxin, which results in a weak bystander effect when paired with a non-cleavable linker. Disitamab vedotin uses the auristatin-like toxin MMAE as a biological effector molecule, which is prone to problems such as neurotoxic accumulation after continuous repeated use. In addition, both of the above ADC drugs are non-site randomly coupled, with a drug loading ratio (DAR) of approximately 4, and poor uniformity.

Summary of the Invention

The present invention aims to improve the above-mentioned problems in existing pharmaceutical technologies, specifically to provide a class of anti-Her2 antibody-drug conjugates that can be used to treat Her2-expressing tumors. The antibody-drug conjugates have good stability, homogeneity, hydrophilicity, efficacy, and safety.

The present application relates to an antibody-drug conjugate and exemplarily discloses an antibody-drug conjugate having a structure represented by the general formula Ab-[MLED] _x , with trastuzumab as the targeting moiety. The results show that the conjugate has a relatively good drug-antibody coupling ratio (e.g., 6.5 to 8.5), and the conjugate has excellent binding activity and proliferation inhibition effects on Her2-positive cells, and has a good targeted killing effect on Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, the present application provides an antibody-drug conjugate for treating Her2-expressing cancers, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating Her2-expressing cancers.

Antibody Drug Conjugates

In one aspect, the present application provides an antibody drug conjugate having a structure shown in the formula Ab-[MLED] _x , wherein:

Ab is an antibody or its antigen-binding fragment:

M is a linker connected to the antibody or its antigen-binding fragment;

L is the structure connecting the joints M and E;

E is the structure connecting L and D;

D is the cytotoxic drug moiety;

x is 1 to 10.

In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof through the "-M-L-E-" structure shown in this application.

In some embodiments, Ab-[MLED] _x is further HA-(MLED)x or HA-(ZMLED)x, wherein HA or HA-(Z-) represents an antibody or antigen-binding fragment thereof as described above, and -(Z-) represents the linking portion of the amino acid residues in the antibody or antigen-binding fragment thereof to M.

In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

In some embodiments, Z is S, NH, or O.

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, M is selected from the following substituted or unsubstituted structures:

In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following: C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclyl (e.g., substituted with one or more R'), -N(R')-, -NH(R'), -N(R') ₂ , carbonyl, -O-, natural or unnatural amino acids and their analogs (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(COCH ₂ CH ₂ (OCH ₂ CH ₂ )rOCH ₃ )), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Gly-Lys, 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-A la-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), Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:46), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:47)),
wherein R′ is composed of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) groups, including but not limited to hydrogen, C _1-6 alkyl, C _1-6 alkylene, amine, hydroxyl, carboxyl, acyl, —O—, —C _1-6 alkyleneCO ₂ H, —C _1-6 alkyleneSO ₃ H, —SO ₃ H, —PO ₃ H ₂ , —C _1-6 alkylene-NHC _1-6 alkyl, —C _1-6 alkylene-N(C _1-6 alkyl) ₂ , —CH ₂ N(C _1-6 alkyl)-C(═O)C _1-6 alkylene-heterocycle, —C _1-6 alkylene-heterocycle, —NHC _1-6 alkylene-SO ₃ H, -CH ₂ NH-SO ₃ H, -CH ₂ N(C _1-6 alkyl)-SO ₃ H, -CH ₂ NHC _1-6 alkylene-SO ₃ H, -CH ₂ N( _{C 1-6 alkyl} )C _1-6 alkylene-SO ₃ H, -CH ₂ N(C _1-6 alkylene-SO ₃ H) ₂ , -CH ₂ N ⁺ (C _1-6 alkylene-SO ₃ H) ₃ , -CH ₂ N ⁺ (C _1-6 alkyl) ₂ -C _1-6 alkylene-SO ₃ H, -CH ₂ N(C _1-6 alkyl)-C(═O)C _1-6 alkylene-N ⁺ (C _1-6 alkylene-SO ₃ H) ₃ , -CH ₂ NH-C(═O)C _1-6 alkylene-N ⁺ (C _1-6 alkylene-SO ₃ H) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)C _1-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ NH-C(＝O)C _1-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₂ -CH ₂ CO ₂ H, -CH 2 N(C 1-6 alkyl)-C _1-6 alkylene-CO ₂ H, -CH ₂ N + (C _1-6 alkyl) 2 -C 1-6 alkylene-CO 2 H, glucosyl, galactosyl, glucuronic acid group, galacturonic acid group, _{-CH 2} N(C 1-6 alkyl)-C(＝O) _{OC 2-6} alkylene-N ⁺ (C _1-6 alkyl) ₃ , -C _1-6 alkyl)-C(═O)-(CH ₂ CH ₂ O) _r -C _1-6 alkyl, -CH ₂ N(C _1-6 alkyl)-C(═O)-(OCH ₂ CH ₂ ) _r -OC _1-6 alkyl, -(CH ₂ N(Me)-C(═O)) _r -C _1-6 alkyl, a polyethylene glycol fragment containing 1-10 ethoxy (EO) units (i.e., -(CH ₂ CH ₂ O) _r -C _1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C _{-C 1-6} alkylene-N(C _1-6 alkyl)-DOTA, -C _1-6 alkylene-N(C _1-6 alkyl)-DOTAGA, -C _1-6 alkylene-N(C _1-6 alkyl)-NOTA or -C _1-6 alkylene-N(C _1-6 alkyl)-EDTA, wherein r is selected from an integer of 1-20, for example, an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from an integer of 1-20, for example, an integer of 1-15, 1-12, 3-12, 5-10, 8-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, "-EDTA" refers to

In some embodiments, "-NOTA" refers to

In some embodiments, "-DOTA" refers to

In some embodiments, "-DOTAGA" refers to

In some embodiments, Lys(R') refers to the structure

In some embodiments, Glu(R') refers to the structure

In some embodiments, s is represented by n.

In some embodiments, the structure of L contains the following fragment:

In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following: 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(R'), wherein R′ is a glucosyl group, a galactosyl group, a glucuronic acid group, a galacturonic acid group, a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), a DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl group), a NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid residue), an EDTA (ethylenediaminetetraacetic acid residue), a -C _1-6 alkylene-N(C _1-6 alkyl)-DOTA, a -C _1-6 alkylene-N(C _1-6 alkyl)-DOTAGA, a -C _1-6 alkylene-N(C _1-6 alkyl)-NOTA or a -C _1-6 alkylene-N(C _1-6 alkyl)-EDTA, wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following: 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,

In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:



s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is composed of one or more substituted or unsubstituted structures selected from Group I linked to one or more substituted or unsubstituted structures selected from Group II:

Group I:

Group II:


s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, Group 1 consists of composition.

In some embodiments, Group II consists of
s is selected from an integer of 1-20, for example, an integer of 1-15, 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following Group I and one or more substituted or unsubstituted structural fragments selected from the following Group II:

Group I:

Group II:

wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is composed of one structural fragment selected from the following group I linked to one, two or three structural fragments selected from the following group II:

Group I:

Group II:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably s is selected from 5, 8, 10.

In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following Group I and one or more substituted or unsubstituted structural fragments selected from the following Group II:

Group I:

Group II:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In some embodiments, L is composed of a structural fragment selected from the following group I linked to one or two structural fragments selected from the following group II:

Group I:

Group II:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, L is selected from the following substituted or unsubstituted structures:

(For example ), (For example ), (For example ), (For example ), s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, L is selected from the following substituted or unsubstituted structures:

(For example ), (For example ), (For example ), (For example s is an integer selected from 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH ₂ -, or a substituted or unsubstituted structure selected from the following:

In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH ₂ -,

In some embodiments, E is a single bond, substituted or unsubstituted -NH- _CH2- or

In some embodiments, E is a single bond, -NH- _CH2- or

In some embodiments, E is a single bond,

In some embodiments, E is substituted or unsubstituted -NH- _CH2- or

In some embodiments, Selected from the following substituted or unsubstituted structures:



n is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, Selected from the following substituted or unsubstituted structures:

(For example ), (For example ), (For example ), (For example ), (For example ),

s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, Selected from the following substituted or unsubstituted structures:

(For example ), (For example ), (For example ), (For example ), (For example );

s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, an RNA polymerase inhibitor, and a gene transcription inhibitor. In some embodiments, the microtubule inhibitor is an auristatin compound, a maytansine compound, a hemicycline compound, or an eribulin compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, an anthracycline compound, trabectedin, or rubectedin, and derivatives or analogs thereof. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belotecan, rubitecan, diflomotecan, lurtotecan, karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogs, duocarmycin, 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.

In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor (e.g., Camptothecin, Hydroxycamptothecin, 9-aminocamptothecin, SN-38, Irinotecan, Isotecan, Topotecan, Belotecan, Rubitecan, Diflomotecan, Lurtotecan, Karenitecin, Gimatecan, Namitecan, Simmitecan, Chimmitecan, Silatecan, or Elomotecan).

The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH ₂ ), secondary amine (-NR ₁ H), tertiary amine (-NR ₂ R ₃ ), wherein R ₁ , R ₂ , and R ₃ herein merely represent non-hydrogen substituents on N, or sulfhydryl (-SH), which can react with appropriate functional groups in the rest of the conjugate to achieve linkage.

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

In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, stereoisomers or isotope-labeled compounds thereof:

In some embodiments, the cytotoxic drug is selected from the following compounds of Formula III or pharmaceutically acceptable salts, stereoisomers or isotope-labeled compounds thereof:

wherein R ₅ and R ₆ are each independently selected from H, OH, -NH ₂ , -NH(C _1-6 alkyl), C _1-6 alkyl and halogen; the C _1-6 alkyl is optionally further substituted by one or more substituents selected from halogen, hydroxyl, C _1-6 haloalkyl, and C _3-6 cycloalkyl; or, R ₅ and R ₆ together with adjacent carbon atoms (ring-forming carbon atoms connecting R ₅ and R ₆ , respectively) form a five-membered oxygen-containing heterocyclic ring.

R ₇ is selected from H, -OH, -NH ₂ , -NH(C _1-6 alkyl), and -NH-CO-(C _1-6 alkylene)-OH; the C _1-6 alkyl and C _1-6 alkylene groups are optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, and C _3-6 cycloalkyl;

q is 1, 2, or 3, and in some embodiments is 1 or 2.

In some embodiments, R ₅ and R ₆ are each independently selected from H, OH, -NH ₂ , C _1-4 alkyl and halogen; the C _1-4 alkyl is optionally further substituted with one or more substituents selected from halogen, hydroxyl, C _1-4 haloalkyl, and C _3-6 cycloalkyl. In some embodiments, R ₅ and R ₆ together with adjacent carbon atoms (ring-forming carbon atoms connecting R ₅ and R ₆ , respectively) form a five-membered oxygen-containing heterocyclic ring.

In some embodiments, R ₇ is selected from -H, -NH ₂ , -NH(C _1-4 alkyl), and -NH-CO-(C _1-4 alkylene)-OH; the C _1-4 alkyl and C _1-4 alkylene are optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-4 haloalkyl, and C _3-6 cycloalkyl.

In some embodiments, R ₅ and R ₆ are each independently selected from H, OH, -NH ₂ , C _1-4 alkyl, and halogen.

In some embodiments, R ₅ is selected from H, OH, and methyl.

In some embodiments, R ₆ is selected from H and halogen.

In some embodiments, R ₇ is selected from -H, -NH ₂ , -NH(C _1-4 alkyl), and -NH-CO-(C _1-4 alkylene)-OH.

In some embodiments, R ₇ is H and -NH ₂ .

In some embodiments, the cytotoxic drug is selected from Compound 1-1, 1-2, or a pharmaceutically acceptable salt, stereoisomer, or isotope-labeled compound thereof.

In some embodiments, the cytotoxic drug is selected from Compound 2-2 or a pharmaceutically acceptable salt, stereoisomer or isotope-labeled compound thereof.

In some embodiments, the structure of D is shown in Formula III-A, Formula III-B or Formula III-C below:

wherein R _5A is selected from -O- or -N-; R _7A is selected from a chemical bond, -O-, -NH-, -N(C _1-6 alkyl)-, and -NH-CO-(C _1-6 alkylene)-O-; the C _1-6 alkyl group is optionally further substituted with one or more substituents selected from halogen, hydroxyl, C _1-6 haloalkyl, and C _3-6 cycloalkyl;

R ₅ , R ₆ , R ₇ and q are as described above.

In some embodiments, R _7A is selected from a chemical bond, -O-, -NH-, -N(C _1-4 alkyl)-, and -NH-CO-(C _1-4 alkylene)-O-; the C _1-4 alkyl is optionally further substituted with one or more substituents selected from halogen, hydroxyl, C _1-4 haloalkyl, and C _3-6 cycloalkyl. In some embodiments, R _7A is selected from -NH-.

In some embodiments, D is selected from the following structures:

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

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

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

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

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 the sequence from which it is derived, 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 is derived; preferably, the substitutions are conservative substitutions;

or,

(2) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

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

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

wherein the variant described in any one 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 the sequence from which it is derived, 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 is derived; preferably, the substitutions are conservative substitutions;

or,

(3) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

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

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

wherein the variant described in any one of (3a) and (3b) 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 is derived, 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 is derived; preferably, the substitutions are conservative substitutions;

or,

(4) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

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

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

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 compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

(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,

(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;

or,

(2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

(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

(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;

or,

(3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

(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

(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;

or,

(4) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system:

(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,

(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;

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

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

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

wherein 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 compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

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

(b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

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

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

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

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, wherein the variant has 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; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, wherein the variant has 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 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

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.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain comprising a VH sequence represented by SEQ ID NO: 1 and a heavy chain constant region (CH) represented by SEQ ID NO: 35, and a light chain comprising a VL sequence represented by SEQ ID NO: 2 and a light chain constant region (CL) represented by SEQ ID NO: 36; or

(2) A heavy chain comprising the VH 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 sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

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

(2) A heavy chain comprising the sequence shown in SEQ ID NO: 39, and a light chain comprising the sequence shown in SEQ ID NO: 40.

In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or lack a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

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

In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment 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, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification/storage.

In certain embodiments, the N-terminal glutamine of the VH or its variant as shown in SEQ ID NO: 1 or 3 or the heavy chain or its variant as shown in SEQ ID NO: 37 or 39 undergoes cyclization to form pyroglutamate or pyroglutamate salt.

In certain embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or its variant or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or its variant lacks a C-terminal lysine.

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

In some embodiments, the Ab is selected from trastuzumab, pertuzumab, a trastuzumab mutant, a pertuzumab mutant, or a bi-epitopic antibody or antigen-binding fragment thereof constructed from trastuzumab and pertuzumab.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab or Pertuzumab, and the amino acid sequence of Trastuzumab has a query accession number in the IMGT database (IMGT/mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has a query accession number in the IMGT database (IMGT/mAb-DB ID): 80.

In some embodiments, the antibody-drug conjugate has a structure as shown in Formula I-A1 or Formula I-A2:

Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof as described in any of the above items, preferably trastuzumab, -(S-) represents the specific connection method between the thiol group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and M and L are as defined in any of the above items.

In some embodiments, L is selected from the following structures:

In some embodiments, L is selected from the following structures:

In some embodiments, the antibody drug conjugate has a structure as shown in the following formula I-A1-1:

Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof as described in any of the above items, preferably trastuzumab, -(S-) represents the specific connection method between the thiol group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and L is as defined in any of the above items.

In some embodiments, L is composed of a structural fragment selected from the following group I linked to one or two structural fragments selected from the following group II:

Group I:

Group II:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, L is selected from the following structures:

(For example ), (For example ), s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, the antibody drug conjugate has a structure as shown in Formula I-E1 or Formula I-E2 below:

Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof as described in any of the above items, preferably trastuzumab, -(S-) represents the specific connection method between the thiol group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and M and L are as defined in any of the above items.

In some embodiments, L is selected from the following structures:

In some embodiments, the antibody drug conjugate has a structure as shown in Formula I-E3 below:

Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof as described in any of the above items, preferably trastuzumab, -(S-) represents the specific connection method between the thiol group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and M and L are as defined in any of the above items.

In some embodiments, L is composed of a structure selected from Group III linked to one or two structures selected from Group IV:

Group III:

Group IV:

wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, Group IV consists of composition.

In some embodiments, L is selected from the following structures:

In some embodiments, the antibody drug conjugate is selected from ADC A-1 to ADC A-9 and ADC E-1 to ADC E-6: ADC A-1:

ADC A-2:

ADC A-3:

ADC A-4:

ADC A-5:

ADC A-6:

ADC A-7:

ADC A-8:

ADC A-9:

ADC E-1:

ADC E-2:

ADC E-3:

ADC E-4:

ADC E-5:

ADC E-6:

Wherein HA-(S-) refers to any of the above antibodies or antigen-binding fragments thereof;

It indicates the specific connection method between the thiol group in the antibody or antigen-binding fragment thereof and the linker.

In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 1-10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 6-8.

In some embodiments, x is an integer.

In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is about 8.

In some embodiments, the antibody drug conjugate is selected from ADC A-1a, ADC A-2a, ADC A-4a:

ADC A-1a:

ADC A-2a:

ADC A-4a:

Wherein HA-(S- refers to the antibody or antigen-binding fragment thereof as described in any of the preceding items, -(S- represents the specific connection method between the thiol group from the antibody or antigen-binding fragment thereof and the pyrimidine group in ADC A-1a, ADC A-2a, or ADC A-4a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

In some embodiments, the antibody drug conjugate is selected from ADC A-1a, ADC A-2a, and ADC A-4a, HA-(S- comprises a VH with a sequence as shown in SEQ ID NO: 1 and a VL with a sequence as shown in SEQ ID NO: 2, or a VH with a sequence as shown in SEQ ID NO: 3 and a VL with a sequence as shown in SEQ ID NO: 4, -(S- represents the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the pyrimidine group in ADC A-1a, ADC A-2a, and ADC A-4a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8. In some embodiments, -(S- represents the specific connection mode between the thiol group of the cysteine residue in the antibody or its antigen-binding fragment and the pyrimidine group in ADC A-1a, ADC A-2a, and ADC A-4a.

In some embodiments, the antibody drug conjugate is selected from ADC A-1a, ADC A-2a, and ADC A-4a, HA-(S- comprises: a heavy chain comprising the VH 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 shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36, or a heavy chain comprising the VH 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 shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36, -(S- represents the specific connection method between the thiol group in the antibody or antigen-binding fragment thereof and the pyrimidine group in ADC A-1a, ADC A-2a, and ADC A-4a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

In some embodiments, the antibody drug conjugate is selected from ADC A-1b, ADC A-2b, ADC A-4b:

ADC A-1b:

ADC A-2b:

ADC A-4b:

wherein Ab is the antibody or antigen-binding fragment thereof as described above, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

In some embodiments, the antibody drug conjugate is selected from ADC A-1b, ADC A-2b, ADC A-4b, Ab comprises VH with the sequence shown in SEQ ID NO: 1 and VL with the sequence shown in SEQ ID NO: 2, or VH with the sequence shown in SEQ ID NO: 3 and VL with the sequence shown in SEQ ID NO: 4, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

In some embodiments, the antibody drug conjugate is selected from ADC A-1b, ADC A-2b, and ADC A-4b, and Ab comprises: a heavy chain comprising the VH 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 shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36, or a heavy chain comprising the VH 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 shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

In some embodiments, Ab is linked to form a conjugate through one or more sulfhydryl groups of a cysteine residue, one or more amino groups of a lysine residue, one or more hydroxyl groups of a threonine residue, or one or more hydroxyl groups of a serine residue in the antibody.

In some embodiments, Ab is linked through one or more sulfhydryl groups of cysteine residues in the antibody to form a conjugate.

In some embodiments, Ab is linked through one or more amino groups of lysine residues in the antibody to form a conjugate.

In some embodiments, Ab is linked through one or more hydroxyl groups of a threonine residue in the antibody to form a conjugate.

In some embodiments, Ab is linked through one or more hydroxyl groups of serine residues in the antibody to form a conjugate.

In some embodiments, Ab is linked to form a conjugate through x sulfhydryl groups of cysteine residues, x amino groups of lysine residues, x hydroxyl groups of threonine residues, or x hydroxyl groups of serine residues in the antibody.

In some embodiments, Ab is linked through x sulfhydryl groups of cysteine residues in the antibody.

In some embodiments, Ab is linked through x amino groups of lysine residues in the antibody.

In some embodiments, Ab is linked through x hydroxyl groups of a threonine residue in the antibody.

In some embodiments, Ab is linked through x hydroxyl groups of a serine residue in the antibody.

In some embodiments, the antibody drug conjugates described herein are optionally substituted with one or more suitable substituents.

Drug-linker

Those skilled in the art will appreciate that the antibody-drug conjugates described herein can be prepared modularly. For example, a pre-conjugated "drug linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking to the antibody or antigen-binding fragment thereof) is first obtained, and then covalently linked to the antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. The pre-conjugated "drug linker" can be a free form of the "drug linker." Accordingly, M' in the pre-conjugated "drug linker" is linked to one or more sulfhydryl (-SH), amino (-NH ₂ ) or carboxyl (-COOH) groups on the antibody or antigen-binding fragment thereof through a substitution reaction (e.g., removal of structures such as -SO ₂ Me or -Br thereon) or an addition reaction.

In one aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, which has a structure shown in formula M'-L-E-D, wherein:

M' is -M-Lg, wherein Lg is a leaving group or an addition reaction group for a nucleophilic substitution reaction, and M is a structure that binds to the antibody or its antigen-binding fragment;

L is the structure connecting M and E;

E is the structure connecting L and D;

D is a cytotoxic drug moiety.

In some embodiments, M' is selected from:

In some embodiments, Lg is selected from halogen (e.g., F, Cl, Br, I), halogenated C _1-6 alkyl, maleimide, halogenated maleimide, C _1-6 alkylsulfonyl, halogenated C _1-6 alkylsulfonyl, halogenated sulfonyl, C _1-6 alkylsulfonate, halogenated C 1-6 alkylsulfonate, C _1-6 alkylsulfinate, _{C 1-6 alkylsulfoxide} , halogenated phenoxy, hydroxyl (—OH), thiol (—SH), amino (—NH ₂ ), nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structures, wherein the halogenated C _1-6 alkyl, C _1-6 alkylsulfonyl, halogenated C _1-6 alkylsulfonyl, halogenated sulfonyl, C _{1-6 alkylsulfonate, halogenated C 1-6 alkylsulfonate} , C _1-6 alkylsulfinate, C _{The 1-6} alkylsulfoxide, halophenoxy, alkenyl, alkynyl and alkynyl-containing structures are optionally substituted with one or more suitable substituents.

In some embodiments, Lg is selected from halogen (e.g., F, Cl, Br, I), halogenated C _1-6 alkyl, maleimido, halogenated maleimido, C _1-6 alkylsulfonyl, halogenated C _1-6 alkylsulfonyl, halogenated sulfonyl, C _1-6 alkylsulfonate, halogenated C _1-6 alkylsulfonate, C _{1-6 alkylsulfinate, C 1-6 alkylsulfoxyl} , halogenated phenoxy, hydroxyl (—OH), thiol (—SH), amino (—NH ₂ ), nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structures.

In some embodiments, Lg is selected from halogen, substituted or unsubstituted C _1-6 alkylsulfonyl (C _1-6 alkyl-SO ₂ —), halophenoxy, hydroxyl (—OH), thiol (—SH), or amino (—NH ₂ ).

In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (—OH), thiol (—SH), or amino (—NH ₂ ).

In some embodiments, Lg is selected from C _1-6 alkylsulfonyl and halophenoxy.

In some embodiments, Lg is selected from methylsulfonyl.

In some embodiments, M' is selected from (For example ).

In some embodiments, M' is selected from (For example ).

In some embodiments, M' is

In some embodiments, M' is

In some embodiments, M' is

In some embodiments, the free form of the "drug linker" is selected from A-1 to A-9 and E-1 to E-6 shown below: A-1:

A-2:

A-3:

A-4:

A-5:

A-6:

A-7:

A-8:

A-9:

E-1:

E-2:

E-3:

E-4:

E-5:

E-6:

In some embodiments, the drug linkers described herein are optionally substituted with one or more suitable substituents.

Connection unit

In some embodiments, the present invention provides a linker unit of the formula -M-L-E-, wherein M, L, and E are as described above. In some embodiments, the linker unit is used to connect a cytotoxic drug and an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate. In some embodiments, in the linker unit, E is a linker for connecting a cytotoxic drug, and M is a linker connected to an antibody or an antigen-binding fragment thereof. In some embodiments, in the linker unit, E is used to connect to D described in any one of the present application, and M is used to connect to an antibody or an antigen-binding fragment thereof described herein.

In some embodiments, the linking unit represented by -M-L-E- is selected from the following structures:

(For example ), (For example ), (For example ), (For example ), (For example ),

s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10,

Preferably, the left end is connected to the antibody or antigen-binding fragment thereof, and the right end is connected to the cytotoxic drug;

Preferably, the antibody or antigen-binding fragment thereof is as defined above, and the cytotoxic drug is as defined above.

In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit represented by the structure -M-L-E-.

In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit of the structure represented by -M-L-E- and Ab and/or D, wherein the linker unit is connected to Ab and/or D; wherein Ab or D is as defined in any of the above items.

In some embodiments, the present invention provides a linker unit represented by the formula M'-L-E-, wherein M', L and E are as described above. In some embodiments, the linker unit is used to connect a cytotoxic drug and an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate. In some embodiments, in the linker unit, E is a linking portion for connecting a cytotoxic drug, and M' is a structure capable of reacting and connecting with an antibody or an antigen-binding fragment thereof. In some embodiments, in the linker unit, E is used to connect to D described in any one of the present application, and M' is used to react and connect with the antibody or antigen-binding fragment thereof described herein.

In some embodiments, the linking unit represented by M'-L-E- is selected from the following structures:

(For example ), (For example ), (For example ), (For example ), (For example ),

wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10,

The right end is connected to a cytotoxic drug; preferably, the cytotoxic drug is as defined in any one of the above items.

In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit of the structure represented by M'-L-E-.

In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit of the structure represented by M'-L-E- and D, wherein the linker unit is connected to D; wherein D is as defined in any of the above items.

In some embodiments, the present invention provides a linker unit of the formula -L-E-D, wherein L, E, and D are as described above. In some embodiments, the linker unit is used to connect an antibody or antigen-binding fragment thereof via a linker to obtain an antibody drug conjugate.

In some embodiments, the present invention provides a linker unit of the formula -M-L-E-D, wherein M, L, E, and D are as described above. In some embodiments, the linker unit is used to link an antibody or antigen-binding fragment thereof to obtain an antibody drug conjugate.

In some embodiments, the linking unit represented by -L-E-D is selected from the following structures:

(For example ), (For example ), (For example ), (For example ), (For example ),

wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10.

In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof is provided, which comprises a linker unit represented by a structure of -L-E-D or -M-L-E-D.

definition

Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of technology or substitutions of equivalent technology that would be apparent to those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing 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 is composed 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 is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of 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 also be subdivided into regions of high variability, 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 from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or 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 to a pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein 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, or have the N-terminal amino acid cyclized to a pyroglutamate.

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. The variable region of the heavy chain and light chain each contains three CDRs, designated 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; C (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 CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

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

The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and/or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, 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 produced by recombinant DNA techniques 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 (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab'fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab') ₂ fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

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

The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, e.g., 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, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2 _- VL-linker-VH-COOH or NH2 _- VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 48) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ (SEQ ID NO: 49) 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 the present 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 be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement, for example, a scFv comprising NH ₂ -VH-VH-COOH, NH ₂ -VL-VL-COOH.

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

Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen.

Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

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

The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent 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 x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted 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 bonds or hydrogen bonds, etc.). 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative 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).

The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present 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.

The term "linker" refers to a structural fragment that connects a cytotoxic drug to an antibody or antigen-binding fragment, for example, the -MLE- structural fragment in the formula Ab-[MLED] _x .

The term "drug linker" refers to the structure of the cytotoxic drug and linker described herein prior to linkage to the antibody or antigen-binding fragment thereof. For example, a "drug linker" refers to M'-L-E-D, where M' represents the structure of M prior to covalent linkage to the antibody or antigen-binding fragment thereof. The "drug linker" is covalently linked to the antibody or antigen-binding fragment thereof to produce the antibody-drug conjugate described herein.

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

The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

As used herein, the "*" marked in a compound structural formula indicates that the marked carbon atom is a chiral carbon atom, and the present invention includes a pair of enantiomers formed by this chiral carbon atom. If a compound contains two different chiral carbon atoms, the present invention includes the four optical isomers formed by these chiral carbon atoms.

As used herein, Indicates where a structure is connected to the rest of the molecule.

The term "alkyl" is defined as a straight or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, for example 1 to 6, carbon atoms. For example, as used herein, the terms "C _1-6 alkyl" and "C _1-4 alkyl" refer to linear or branched groups of 1 to 6 carbon atoms and 1 to 4 carbon atoms, respectively (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl), which are optionally substituted with 1 or more (such as 1, 2, or 3) suitable substituents.

The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above. For example, the term "C _1-6 hydroxyalkyl" herein refers to a hydroxyalkyl group having 1 to 6 carbon atoms. Common hydroxyalkyl groups include (but are not limited to) -CH ₂ OH, -CH ₂ CH ₂ OH, -CH ₂ CH(OH) ₂ , and -(CH ₂ ) ₃ OH.

The term "aminoalkyl" refers to an alkyl group substituted with one or more amino groups, where alkyl is as defined above. For example, the term " _{C1-6aminoalkyl} " herein refers to an aminoalkyl group having 1 to 6 carbon atoms. Common aminoalkyl groups include, but are not limited to, -CH2 _{- NH2} , _{-CH2CH2 - NH2} , _-CH2CH ( _NH2 ) ₂ , and -( _CH2 ) ₃ - _NH2 .

The term "alkenyl" refers to a straight or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, " _C2-6 alkenyl,"" _C2-4 alkenyl," and the like. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, and the like.

The term "alkynyl" refers to a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. Examples include, for example, " _C2-6 alkynyl" and " _C4-6 alkynyl." Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and 1,4-hexadiynyl.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocyclic alkyl and bicyclic alkyl (such as spirocyclic alkyl, cycloalkyl and bridged cycloalkyl). The term " _C3-6 cycloalkyl" refers to a cycloalkyl group having 3 to 6 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may be optionally substituted with 1 or more (such as 1, 2 or 3) suitable substituents, for example, a methyl-substituted cyclopropyl group.

The term "carbocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon group connected through a ring carbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (for example, a monocyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a cyclononane ring, or a bicyclic ring, including a spirocyclic, fused or bridged system (such as a bicyclo[1.1.1]pentane ring, a bicyclo[2.2.1]heptane ring, a bicyclo[3.2.1]octane ring or a bicyclo[5.2.0]nonane ring, a decalin ring, etc.), which may be optionally substituted with one or more (such as one, two or three) suitable substituents. The term "3-6 membered carbocycle" refers to a carbocycle containing 3, 4, 5 or 6 ring-forming carbon atoms.

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

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 a 5-6 membered oxygen-containing heterocycle, a 5-membered oxygen-containing heterocycle, and specific examples include but are not limited to an oxirane ring, a tetrahydrofuran ring, a furan ring, a tetrahydropyran ring, a pyran ring, a 1,3-dioxolane ring, and the like.

The "nitrogen-containing heterocycle" described in the present invention refers to a heterocycle as described above in which one or more (eg, 1, 2 or 3) ring atoms are nitrogen atoms.

The term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1, 2, or 3) the same or different halogen atoms, wherein alkyl is as defined above. For example, the term "C _1-6 haloalkyl" as used herein refers to a haloalkyl group having 1 to 6 carbon atoms. Common haloalkyl groups include (but are not limited to) -CH ₂ F, -CHF ₂ , -CF ₃ , -CH ₂ CF ₃ , -CF ₂ CF ₃ , -CH ₂ CH ₂ CF ₃ , -CH ₂ Cl, and the like. The haloalkyl groups herein are optionally substituted with one or more substituents described herein.

The term "alkoxy" refers to a group having the structure "alkyl-O-," where alkyl is as defined above. Examples include _C1-6 alkoxy, _C1-4 alkoxy, _C1-3 alkoxy, and _C1-2 alkoxy. Common alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, and hexyloxy. The alkoxy groups herein are optionally substituted with one or more substituents described herein.

The term "alkoxyalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups, where alkoxy and alkyl are as defined above. For example, the term "C _1-6 alkoxyalkyl" as used herein refers to an alkyl group having 1-6 carbon atoms substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups. Common alkoxyalkyl groups include, but are not limited to, CH ₃ O—CH ₂ —, C ₂ H ₅ —O—CH ₂ —, and C ₂ H ₅ —O—CH ₂ CH ₂ —.

The term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

The term "nitrogen oxide" refers to an oxide (eg, mono- or di-oxide) of at least one nitrogen atom in the structure of the present invention. Nitrogen mono-oxides may exist as a single positional isomer or a mixture of positional isomers.

If a substituent is described as being "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more substituents from the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and/or collectively, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more substituents from the list of substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

If a functional group or structure is described as "substituted or unsubstituted," the functional group or structure may be (1) unsubstituted or (2) substituted.

As used herein, the term "suitable substituents" refers to modifications that one skilled in the art can make to a compound according to the needs of the compound's substituents. “Suitable substituents” include oxo (=O), halogen, cyano, NR ⁸ R ⁹ , carboxyl, thiol, hydroxy, ester (e.g., —C _1-6 alkylene-C(=O)—OC _1-6 alkyl), C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl-OC _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, 3-8 membered heterocyclyl, 5-10 membered heteroaryl, C _6-10 aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C _1-6 haloalkoxy, R ⁸ and R ⁹ are each independently selected from H, C _1-6 alkyl, C _3-6 cycloalkyl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, C _6-10 aryl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g., -C _1-6 alkylene-C(=O)-OC _1-6 alkyl).

The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms in a designated compound or structure with a substituent, provided that the designated atom's normal valence is not exceeded in the current context and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. For example, 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, -NH ₂ , -NO ₂ , -C(O)-, -CN, ＝O, C _1-6 alkylene, C _1-6 haloalkylene, C _1-6 alkoxy, C _2-6 alkenylene, C 2-6 alkynylene, C _3-8 cycloalkylene, 3-8 membered heterocyclyl, C _6-10 aryl and 5-10 membered heteroaryl, etc., wherein R ₈ ^, R ⁹ and R' are as defined above. For example, the substituents can be suitable substituents as described above. If a substituent is described as "independently selected from" a group of functional groups, each substituent is selected independently of the other. Therefore, each substituent can be the same as or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In a compound with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce a racemic mixture, a single enantiomer, a diastereomeric mixture, and a separate diastereomer. Specific individual molecules can also exist as geometric isomers (cis/trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

In this article, solid lines can be used , solid wedge or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

It should also be understood that certain compounds of the present invention may be used therapeutically in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs that, upon administration to a patient in need thereof, are capable of directly or indirectly providing a compound of the present invention or a metabolite or residue thereof. Therefore, when reference is made herein to a "compound of the present invention," such various derivative forms of the compound are also intended to be encompassed.

Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts and include aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, and the like.

Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts and the like.

Suitable salts are reviewed in Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The term "ester" refers to esters derived from the compounds of the general formula herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in the form of free acid or alcohol). The compounds of the present invention themselves may also be esters.

The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, and the like of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds produced by contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

The present invention further includes within its scope prodrugs of the compounds of the invention. Typically such prodrugs will be functional group derivatives of the compounds that are readily converted in vivo to the desired therapeutically active compound. Thus, in these instances, the term "administering" as used in the methods of treatment of the present invention shall include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but which are converted in vivo to the aforementioned compounds after administration to the individual. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985.

The present invention further includes within its scope isotopically labeled compounds of the invention which are identical to the compounds of the invention except that one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature.

The present invention also encompasses compounds of the invention containing protecting groups. During any process for the preparation of the compounds of the invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the invention. This may be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. The protecting groups may be removed at an appropriate subsequent stage using methods known in the art.

As used herein, the term "DAR" or "drug-to-antibody ratio" refers to: (a) the number of linkers/drug moieties attached to an antibody in a single antibody-drug conjugate molecule, which is an integer from 0 to 10, such as an integer from 1 to 10; or (b) the average number of linkers/drug moieties attached to an antibody in a composition comprising more than one antibody-drug conjugate molecule, which is an integer or decimal from 0 to 10, such as an integer or decimal from 1 to 10. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

Whether explicitly stated or not, the numerical values in this application are modified by the term “about.” The term “about” means within the range of ±10%, preferably within the range of ±5%, and more preferably within the range of ±2% of the numerical value.

intermediates

In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

in

PG ₁ is each independently H or an amino protecting group, such as an alkoxycarbonyl amino protecting group, for example, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, for example, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group, for example, trityl (Trt), C _1-6 alkyl-substituted trityl, p-methoxytrityl (MMT), dimethoxytrityl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

PG ₂ is each independently H or a carboxyl protecting group, such as C _1-6 alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

PG ₃ is each independently H or a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, trityl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl;

s1 is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

Lg is as defined above.

In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

In another aspect, the present application provides use of the aforementioned intermediate compound or its salt, stereoisomer, tautomer or isotope-labeled compound in preparing the compound of the present invention or a pharmaceutically acceptable salt thereof.

Composition

In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug linker as described herein, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug linkers. Thus, the composition is characterized in that the "drug-antibody" ratio (DAR) is 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 reverse phase chromatography or HPLC-MS.

For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 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 1 to 10. or about 9 to 10.

In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, 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 8.5, about 7.0 to 7.5.

In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 8, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, 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, about 7.5 to 8.0.

In some embodiments, the DAR of the ADC compositions described herein is between about 6.0 and 9.0, preferably between about 6.0 and 8.0, for example, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.30, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.40, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.50, about 6.51, about 6.52 3, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6.73, about 6.74, about 6.75, about 6.76, about 6.77, about 6.78, about 6.79, about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.9, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07 , about 7.08, about 7.09, about 7.1, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7.16, about 7.17, about 7.18, about 7.19, about 7.2, about 7.21, about 7.22, about 7.23, about 7.24, about 7.25, about 7.26, about 7.27, about 7.28, about 7.29, about 7.3, about 7.31, about 7.32, about 7.33, about 7.34, about 7.35, about 7.36, about 7.37, about 7.38, about 7.39, about 7.4, about 7.41, about 7.42, about 7.43, about 7.44, about 7.45, about 7. 46, about 7.47, about 7.48, about 7.49, about 7.5, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about 7.6, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.7, about 7.71, about 7.72, about 7.73, about 7.74, about 7.75, about 7.76, about 7.77, about 7.78, about 7.79, about 7.8, about 7.81, about 7.82, about 7.83, about 7.84, about 7.85, about 7.86, about 7.87, about 7.88, about 7.89, about 7.9, about 7.91, about 7.92, about 7.93, about 7.94, about 7.95, about 7.96, about 7.97, about 7.98, about 7.99, about 8.0, about 8.01, about 8.02, about 8.03, about 8.04, about 8.05, about 8.06, about 8.07, about 8.08, about 8.09, about 8.1, about 8.11, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.2 3, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33, about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, about 8.42, about 8.43, about 8.44, about 8.45, about 8.46, about 8.47, about 8.48, about 8.49, about 8.5, about 8.51, about 8.52, about 8.53, about 8.54, about 8.55, about 8.56, about 8.57, about 8.58, about 8.59, about 8.6, about 8.61, about 8 .62, about 8.63, about 8.64, about 8.65, about 8.66, about 8.67, about 8.68, about 8.69, about 8.7, about 8.71, about 8.72, about 8.73, about 8.74, about 8.75, about 8.76, about 8.77, about 8.78, about 8.79, about 8.8, about 8.81, about 8.82, about 8.83, about 8.84, about 8.85, about 8.86, about 8.87, about 8.88, about 8.89, about 8.9, about 8.91, about 8.92, about 8.93, about 8.94, about 8.95, about 8.96, about 8.97, about 8.98, about 8.99, and about 9.0.

In certain embodiments, the composition is composed of an antibody drug conjugate as shown in Ab-[MLED] _x . In certain embodiments, the composition has a DAR of 1-8, such as 5-8, such as about 7.1, 7.6, 7.8, or 8.0. In certain embodiments, antibody drug conjugates in which x is 8 account for greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate or drug linker described in any of the foregoing descriptions, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, and one or more pharmaceutically acceptable carriers.

The term "pharmaceutical composition" refers to a composition that can be used as a medicament, which comprises an active pharmaceutical ingredient (API) (or therapeutic agent) and, optionally, one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an excipient that is administered with a therapeutic agent and that is suitable, within the scope of sound medical judgment, for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The pharmaceutical compositions can act systemically and/or locally, which can be achieved by suitable dosage forms, including but not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

The pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof.

The present invention also provides a method for preparing the above-mentioned pharmaceutical composition or its corresponding formulation, which comprises combining at least one antibody-drug conjugate or drug linker of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof with one or more pharmaceutically acceptable carriers.

Medicine box products

In another aspect, the present invention provides a kit comprising:

a) at least one antibody-drug conjugate or drug-linker according to the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled, metabolite or prodrug thereof, or a combination thereof as a first therapeutic agent;

b) optionally at least one additional therapeutic agent as a second therapeutic agent, or a composition comprising an additional therapeutic agent; and

c) optional packaging and/or instructions.

The above-mentioned kit product may contain 0.01 mg to 1000 mg of at least one antibody-drug conjugate or drug linker of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof.

The present invention also provides a method for preparing the above-mentioned drug kit, which comprises combining at least one antibody-drug conjugate or drug linker of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, or the above-mentioned composition with optionally at least one other therapeutic agent or a composition comprising the other therapeutic agent, packaging and/or instructions.

Medical uses

The antibody-drug conjugate or drug linker of the present invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites or prodrugs thereof can exhibit a strong effect in inhibiting abnormal cell proliferation.

Therefore, the present application provides the antibody-drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites and prodrugs, the above-mentioned compositions, pharmaceutical compositions or the above-mentioned drug kit products, which are used to treat diseases related to abnormal cell proliferation.

In addition, the present application also provides the use of the antibody-drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites and prodrugs, the above-mentioned compositions, pharmaceutical compositions or the above-mentioned drug kit products in the preparation of drugs for treating diseases related to abnormal cell proliferation.

In some embodiments, the disorder involving abnormal cell proliferation includes, but is not limited to, cancer or a tumor, such as an advanced solid tumor.

The present application also provides the antibody drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug or the composition of the present invention or pharmaceutical composition in the preparation of a preparation, the preparation is used to inhibit the proliferation of tumor cells. In certain embodiments, the preparation is used for in vivo or in vitro administration. For example, the preparation can be administered to a subject to inhibit the proliferation of tumor cells in the subject; or, the preparation can be administered to in vitro cells (e.g., cell lines or cells from a subject) to inhibit the proliferation of tumor cells in vitro.

The cancer or tumor described in the present invention includes (but is not limited to): brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma.

Treatment

In another aspect, the present invention provides a method for treating diseases involving abnormal cell proliferation, comprising the following steps: administering a therapeutically effective amount of the antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, or the above-mentioned composition or pharmaceutical composition to an individual in need thereof.

In some embodiments, the disease is as described in any of the preceding items.

The term "effective amount" refers to a dose that can induce a biological or medical response in cells, tissues, organs or organisms (eg, individuals) and is sufficient to achieve the desired preventive and/or therapeutic effect.

The dosage regimen can be adjusted to provide the optimal desired response. For example, the drug may be administered in a single dose, divided doses may be administered over time, or the dosage may be proportionally reduced or increased based on the actual situation. It will be understood that for any particular individual, the specific dosage regimen should be adjusted according to the needs and the professional judgment of the person administering the composition or supervising the administration of the composition.

The dosage of the antibody drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug will depend on the individual situation, the severity of the disease or condition, the rate of administration, the disposal of the compound and the judgment of the prescribing physician. Generally speaking, the effective amount is about 0.001-10000 mg/kg subject body weight/day. In appropriate cases, the effective amount is about 0.01-1000 mg/kg subject body weight/day. About 0.01-1000 mg/kg subject body weight, typically about 0.1-500 mg/kg subject body weight, can be administered every day, every two days or every three days. An exemplary dosage regimen is once or more per day, or once or more per week, or once or more per month. During multiple administration, the intervals between single doses can generally be daily, weekly, monthly or annual. Alternatively, it can be administered in the form of a sustained-release formulation, in which case a lower frequency of administration is required. The dosage and frequency of administration may vary depending on the half-life of the drug in the subject, and may also vary depending on whether the application is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively infrequent intervals for a long period of time; in therapeutic applications, it is sometimes necessary to administer a relatively high dose at shorter intervals until the progression of the disease is slowed or stopped, preferably until the individual shows partial or complete improvement in the symptoms of the disease, after which prophylactic application may be employed.

The term "treat" refers to the alleviation or elimination of a targeted disease or condition. If a subject receives a therapeutic amount of an antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled, metabolite, and prodrug thereof, or a composition or pharmaceutical composition of the present invention, and at least one indicator or symptom of the subject exhibits observable and/or detectable relief and/or improvement, the subject has been successfully "treated." It is understood that treatment encompasses not only complete treatment but also includes achieving some biologically or medically relevant results while not achieving complete treatment.

The terms "administrate/administrating/administration" (or "dosage") refer to the process of applying a pharmaceutically active ingredient (such as an antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, and prodrug thereof) or a pharmaceutical composition comprising the pharmaceutically active ingredient (such as a pharmaceutical composition of the present invention) to an individual or a cell, tissue, organ, biological fluid, or other part thereof, so as to bring the pharmaceutically active ingredient or pharmaceutical composition into contact with the individual or a cell, tissue, organ, biological fluid, or other part thereof. Common routes of administration include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, and the like.

The term "in need of" refers to a physician's or other health care provider's judgment that an individual needs or will benefit from a preventive and/or therapeutic procedure, which judgment is based on various factors within the physician's or other health care provider's area of expertise.

The term "individual" (or subject) refers to a human or non-human animal. The individual of the present invention includes individuals (patients) suffering from a disease and/or condition and normal individuals. The non-human animals of the present invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Advantageous Effects of the Invention

The antibody-drug conjugates and/or drug linkers of the present invention have good anti-tumor activity, high safety, and good pharmacokinetic properties (e.g., suitable half-life and duration of action), and can be used to treat abnormal cell proliferation disorders, including but not limited to advanced solid tumors.

DETAILED DESCRIPTION

The present application is further described below by describing specific embodiments, but this is not intended to limit the present application. Those skilled in the art can make various modifications or improvements based on the teachings of the present application without departing from the basic idea and scope of the present application.

The information of the sequences involved in the present invention is described in the following table:

The abbreviations used in this invention have the following meanings:

The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance ( ¹ H NMR) or mass spectrometry (MS).

Nuclear magnetic resonance ( ¹ H NMR) measurements were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer; hexadeuterated dimethyl sulfoxide (DMSO-d ₆ ); and tetramethylsilane (TMS) as the internal standard.

The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

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

The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Intermediate Preparation Example 1: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (INT-1)

Step 1: Preparation of 1-amino-3,6,9,12,15-pentahydrooctadecane-18-oic acid (INT-1-2)

Dissolve 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatrioxane-23-oic acid (INT-1-1) (0.50 g, 1.22 mmol) in dichloromethane (3 mL). Add trifluoroacetic acid (3 mL) and stir at room temperature for 3 hours. After completion of the reaction, concentrate the reaction mixture under reduced pressure to obtain the title compound as a crude trifluoroacetate salt (650 mg, 1.21 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):310.2[M+H] ⁺

Step 2: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (INT-1)

Dissolve 1-amino-3,6,9,12,15-pentaoxoctadecane-18-oic acid trifluoroacetate (650 mg, 1.21 mmol) in DMF (5.0 mL), add DIPEA (781.61 mg, 6.05 mmol), and then add 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (CAS: 2813270-39-0, 441.91 mg, 1.21 mmol) portionwise. Stir at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly purified by reverse-phase column chromatography (acetonitrile/0.05% aqueous ammonium bicarbonate solution = 0-60%) and freeze-dried to obtain the title compound (646 mg, 1.15 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):560.3[M+H] ⁺

Intermediate Preparation Example 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

Step 1: Preparation of L-alanyl-L-alanine (INT-2-1)

Dissolve (tert-Butoxycarbonyl)-L-alanyl-L-alanine (A-40-1) (100 mg, 384.19 μmol) in dichloromethane (2 mL) and add trifluoroacetic acid (0.5 mL). The reaction is allowed to react at 20°C for 1 hour. After completion of the reaction, the reaction mixture is concentrated under reduced pressure to remove the solvent, yielding the light yellow title compound (160 mg, 370.93 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):161.1[M+H] ⁺

Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

Dissolve L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (150.58 mg, 412.14 μmol) in N,N-dimethylacetamide (2 mL). Add N,N-diisopropylethylamine (266.33 mg, 2.06 mmol). React at 20°C for 1 hour. The reaction mixture is purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (125 mg, 304.55 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):428.1[M+H ₂ O] ⁺

Intermediate Preparation Example 3: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (INT-3)

Dissolve glycylglycine (INT-3-1) (1 g, 7.57 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.77 g, 7.57 mmol) in dimethyl sulfoxide (10 mL). Stir the reaction at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (2.52 g, 6.59 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):383.2[M+H] ⁺

Intermediate Preparation Example 4: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

Step 1: Preparation of glycylglycyl-L-phenylalanine (INT-4-2)

Dissolve ((Benzyloxy)carbonyl)glycylglycylglycyla-L-phenylalanine (INT-4-1) (200 mg, 483.77 μmol) in methanol (8 mL). After nitrogen purge, add palladium on carbon (20 mg). Replace the atmosphere with hydrogen three times, and stir the reaction at room temperature for 3 hours. After completion of the reaction, remove the palladium on carbon by filtration. The filtrate is then concentrated to remove the solvent to yield the title compound (135 mg, 483.37 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):280.2[M+H] ⁺

Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

Glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (170.06 mg, 465.46 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (120.32 mg, 930.93 μmol) was added. The reaction was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (60 mg, 0.11 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):530.3[M+H] ⁺

Intermediate Preparation Example 5: Preparation of Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-5-2)

Add 6-nitroisobenzofuran-1(3H)-one (INT-5-1) (50.0 g, 279 mmol) to a 2M solution of methylamine in tetrahydrofuran (500 mL). Heat to 75°C and stir for 5 hours. Concentrate the reaction mixture to obtain the crude title compound (58.0 g), which is used in the next step without purification.

Its structural characterization data are as follows:

ESI-MS (m/z): 211.0 [M+H] ⁺ .

Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-5-3)

Dissolve 2-hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) in tetrahydrofuran (500 mL). Cool the reaction mixture to 0°C, then add a 10M borane-dimethyl sulfide solution (89.3 mL) dropwise to the mixture. After the addition is complete, heat the mixture to 70°C and stir for 5 hours. Cool the mixture again to 0°C, then add a 2M hydrogen chloride-methanol solution (100 mL) dropwise to the reaction mixture. Heat the mixture to 65°C and stir for 12 hours. Filter the reaction mixture, and concentrate the filtrate to obtain the crude title compound (44.3 g), which is used in the next step without purification.

Its structural characterization data are as follows:

ESI-MS (m/z): 197.0 [M+H] ⁺ .

Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (INT-5-4)

Dissolve (2-((Methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) in dichloromethane (800 mL), cool to 0°C, add imidazole (55.9 g, 822 mmol) and tert-butyldiphenylsilyl chloride (84.0 g, 308 mmol, 78.8 mL), return to 25°C, and continue stirring for 1 hour. Quench the reaction by adding water (200 mL) and extract with dichloromethane three times (300 mL x 3). The combined organic phases are washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain the crude product. Purify on a silica gel column (dichloromethane/methanol = 20/1 to 5/1) and concentrate again to obtain the title compound (59.7 g, 137 mmol).

Its structural characterization data are as follows:

ESI-MS (m/z): 435.1 [M+H] ⁺ .

Step 4: Preparation of allyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-5-5)

Dissolve 1-(2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (54.9 g, 126 mmol) in dichloromethane (550 mL), cool to 0°C, add DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL), return to 25°C, and continue stirring for 1 hour. Quench the reaction system with water (300 mL) and extract with dichloromethane three times (200 mL x 3). The combined organic phases are washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain the crude product. Purify on a silica gel column (petroleum ether/ethyl acetate = 20/1 to 3/1) and concentrate again to obtain the title compound (65.5 g, 126 mmol).

Its structural characterization data are as follows:

ESI-MS (m/z): 519.1 [M+H] ⁺ .

Step 5: Preparation of allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

Allyl (2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the temperature was raised to 80°C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted with dichloromethane three times (100 mL x 3). The organic phases were combined and washed with saturated aqueous sodium chloride solution (300 mL). After drying over anhydrous sodium sulfate, the mixture was filtered and concentrated to give the crude title compound (49.3 g).

Its structural characterization data are as follows:

ESI-MS (m/z): 511.7 [M+H] ⁺ .

Intermediate Preparation Example 6: Preparation of Allyl (5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-1)

Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in a mixed solvent of 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 mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0-90%) and concentrated again under reduced pressure to give the title compound (194.2 mg, 0.25 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):782.2[M+H] ⁺

Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamic acid allyl ester (INT-6-2)

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

Its structural characterization data are as follows:

MS m/z(ESI):566.1[M+Na] ⁺

Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-6-3)

Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), and DIPEA (81.3 mg, 0.63 mmol) and p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (134.50 mg, 0.19 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):731.2[M+Na] ⁺

Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-6-4)

(S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzene Depyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (Ixitecan, 165.50 mg, 0.38 mmol) was dissolved in DMF (5 mL). 1-Hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added and stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 0.7) and freeze-dried to obtain the title compound (114.70 mg, 0.12 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):1005.2[M+H] ⁺

Step 5: Preparation of allyl (5-((S)-2-aminopropionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-5)

Allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) was dissolved in DMF (3 mL), diethylamine (87.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, most of the solvent was removed from the reaction solution and then purified by flash column chromatography (C18, water/acetonitrile = 0.7), and freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):783.1[M+H] ⁺

Step 6: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-6-6)

(5-((S)-2-aminopropionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl ) allyl carbamate (1.0 g, 1.28 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (476.90 mg, 1.41 mmol) were dissolved in DMF (5 mL). HATU (631.06 mg, 1.66 mmol) and DIPEA (495.29 mg, 3.83 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was purified by flash preparative and freeze-dried to obtain the title compound (950 mg, 860.37 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1104.5[M+H] ⁺

The preparation method is as follows:

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

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

Step 7: Preparation of allyl (5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline) To the mixture of allyl (1,4-dimethoxy-1,4-dioxo-2-oxo ...

Its structural characterization data are as follows:

MS m/z(ESI):882.5[M+H] ⁺

Intermediate Preparation Example 7: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazadotriacontane-31-ynoic acid (INT-7)

Step 1: Preparation of allyl 3-((tert-butoxycarbonyl)amino)propionate (INT-7-2)

Dissolve 3-(tert-Butyloxycarbonyl)amino)propionic acid (INT-7-1) (2.3 g, 12.16 mmol) and 3-bromopropene (1.62 g, 13.37 mmol) in DMF (10 mL). Add potassium carbonate (5.04 g, 36.47 mmol) and stir at room temperature for 1 hour. Filter the reaction mixture, and concentrate the filtrate to obtain the crude title compound. Purify the mixture on a silica gel column (petroleum ether/ethyl acetate = 20/1 to 5/1) and reconcentrate to obtain the title compound (2.2 g, 9.6 mmol).

Its structural characterization data are as follows:

ESI-MS (m/z): 252.1 [M+Na] ⁺ .

Step 2: Preparation of allyl 3-aminopropionate (INT-7-3)

Dissolve allyl 3-((tert-butoxycarbonyl)amino)propionate (2.2 g, 9.6 mmol) in dichloromethane (10 mL). Add trifluoroacetic acid (5 mL) and stir at room temperature for 2 hours. The reaction mixture is concentrated to obtain the crude title compound (2.3 g), which is used in the next step without purification.

Its structural characterization data are as follows:

ESI-MS (m/z): 130.1 [M+H] ⁺ .

Step 3: Preparation of allyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide)propionate (INT-7-4)

Allyl 3-aminopropionate (2.0 g, 8.22 mmol) and N ² -(((9H-fluoren-9-yl)methoxy)carbonyl)-N ⁶ -(diphenyl(p-tolyl)methyl)-L-lysine (5.14 g, 8.22 mmol) were dissolved in DMF (20 mL). DIPEA (5.31 g, 41.12 mmol, 7.38 mL) and HATU (6.25 g, 16.45 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (4.5 g, 6.11 mmol).

Its structural characterization data are as follows:

ESI-MS (m/z): 436.4 [M+H] ⁺ .

Step 4: Preparation of (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamide)propionic acid allyl ester (INT-7-5)

Dissolve allyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide)propionate (3.0 g, 4.08 mmol) in DMF (10 mL). Add diethylamine (596.30 mg, 8.15 mmol, 828.20 μL) and stir at room temperature for 1 hour. After removing most of the solvent, the reaction mixture was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (1.8 g, 3.5 mmol).

Its structural characterization data are as follows:

ESI-MS (m/z): 514.3 [M+H] ⁺ .

Step 5: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazadotriacontane-31-ynoic acid allyl ester (INT-7-6)

Allyl (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexanamide)propionate (1.8 g, 3.5 mmol) and 2,5-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (1.69 g, 3.01 mmol) were dissolved in DMF (5 mL). DIPEA (1.85 g, 14.34 mmol, 2.57 mL) and HATU (2.18 g, 5.74 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction mixture was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (960 mg, 909.71 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1055.5[M+H] ⁺

Step 6: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazadotriacontane-31-ynoic acid (INT-7)

(S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazadotriacontane-31-ynoic acid allyl ester (960 mg, 909.71 μmol) was dissolved in DMF (5 mL). After nitrogen substitution three times, tetrakistriphenylphosphine palladium (525.61 mg, 454.85 μmol) and 1,3-dimethylbarbituric acid (710.21 mg, 4.55 mmol) were added and stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC and freeze-dried to obtain the title compound (390.0 mg, 384.15 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):1015.4[M+H] ⁺

The preparation method is as follows:

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

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

Intermediate Preparation Example 8: Preparation of Allyl (5-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8)

Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-1)

Allyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.0 g, 2.05 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-citrulline (1.22 g, 3.07 mmol) were dissolved in a mixed solvent of DCM (28 mL) and MeOH (7 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.52 g, 6.15 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0-90%) and concentrated again under reduced pressure to give the title compound (1.07 g, 1.23 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):868.2[M+H] ⁺

Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(hydroxymethyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-2)

(S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.07 g, 1.23 mmol, 90% purity) was dissolved in DMF (8 mL), and pyridine hydrofluoride (585.2 mg, 5.9 mmol) was added. The mixture was stirred at room temperature for 15 hours. After completion of the reaction, 50 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3), washed with 30 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound. The crude product was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (418.5 mg, 0.67 mmol, 92% purity).

Its structural characterization data are as follows:

MS m/z(ESI):652.1[M+Na] ⁺

Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-3)

Dissolve allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (418.5 mg, 0.67 mmol, 92% purity) in DMF (5 mL), add DIPEA (172.3 mg, 1.34 mmol) and p-nitrophenyl chloroformate (161.8 mg, 0.81 mmol), and stir at room temperature for 2 hours. After completion of the reaction, purify by flash column chromatography (C18, water/acetonitrile = 3/2) and freeze-dry to obtain the title compound (416.10 mg, 0.52 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):817.2[M+Na] ⁺

Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-4)

(S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (416.10 mg, 0.52 mmol, 90% purity), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 H,13H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (331.50 mg, 0.78 mmol) was dissolved in DMF (8 mL). 1-Hydroxybenzotriazole (70.20 mg, 0.52 mmol), pyridine (61.5 mg, 0.78 mmol), and DIPEA (201.5 mg, 1.57 mmol) were added and stirred at room temperature for 2 hours. The reaction mixture was mostly solvent-free and purified by flash column chromatography (C18, water/acetonitrile = 0.7). The mixture was then freeze-dried to give the title compound (357.10 mg, 0.33 mmol, 89% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1091.2[M+H] ⁺

Step 5: Preparation of (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-5)

Allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (357.10 mg, 0.33 mmol, 89% purity) was dissolved in DMF (3 mL), diethylamine (73.8 mg, 1.0 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was freed of most of the solvent and purified by flash column chromatography (C18, water/acetonitrile = 0.6). The residue was freeze-dried to obtain the title compound (242.5 mg, 0.28 mmol, 95% purity).

Its structural characterization data are as follows:

MS m/z(ESI):869.1[M+H] ⁺

Step 6: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8-2)

Allyl (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (242.5 mg, 0.28 mmol, 95% purity), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (113. 6 mg, 0.34 mmol) was dissolved in DMF (3 mL), and HATU (160.3 mg, 0.42 mmol) and DIPEA (108.4 mg, 0.84 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, 80 mL of water was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate (30 mL x 3), washed with 50 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound. After purification by flash column chromatography (silica gel column, dichloromethane/methanol = 10/1), the title compound (266.6 mg, 0.23 mmol, 89% purity) was obtained after concentration.

Its structural characterization data are as follows:

MS m/z(ESI):1190.9[M+H] ⁺

Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-8)

Allyl (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (266.6 mg, 0.23 mmol, 89% purity) was dissolved in DMF (3 mL), diethylamine (50.4 mg, 0.69 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Most of the solvent was removed from the reaction solution, and the mixture was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (211.5 mg, 0.22 mmol, 92% purity).

Its structural characterization data are as follows:

MS m/z(ESI):968.3[M+H] ⁺

Intermediate Preparation Example 9: Preparation of N ⁶ -(diphenyl(p-tolyl)methyl)-N ² -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

Step 1: Preparation of N ⁶ -(diphenyl(p-tolyl)methyl)-L-lysine (INT-9-2)

Dissolve N ² -(((9H-fluoren-9-yl)methoxy)carbonyl)-N ⁶ -(diphenyl(p-tolyl)methyl)-L-lysine (INT-9-1) (150.0 mg, 0.24 mmol) in DMF (2 mL), add diethylamine (182.3 mg, 2.5 mmol), and stir at room temperature for 1 hour. After completion of the reaction, add 10 mL of a mixed solvent (EA/PE = 1/3) for recrystallization. Filter to obtain the title compound (90.1 mg, 0.22 mmol).

Step 2: Preparation of N ⁶ -(diphenyl(p-tolyl)methyl)-N ² -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

N ⁶ -(diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), and DIPEA (86.3 mg, 0.66 mmol) and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (160.6 mg, 0.44 mmol) were added. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 0.5) and freeze-dried to obtain the title compound (75.2 mg, 0.11 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):653.2[M+H] ⁺

Intermediate Preparation Example 10: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexatriacontane-35-ynoic acid (INT-10)

Step 1: Preparation of 5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-oic acid (INT-10-2)

Dissolve 1-(9H-fluoren-9-yl)-4,7,10,13,16,19,22,25,28,31-decamethyl-3,6,9,12,15,18,21,24,27,30-decaoxo-2-oxa-4,7,10,13,16,19,22,25,28,31-decaazatricarboxane-33-oic acid (INT-10-1) (1.0 g, 1.05 mmol) in DMF (5.0 mL). Add diethylamine (0.5 mL) and stir at room temperature for 0.5 hour. After completion of the reaction, the solvent was dried on a lyophilizer. Add ethyl acetate/petroleum ether (1/3, 50 mL) and stir for 1 hour. Remove the solvent to obtain the title compound (688.9 mg, 0.95 mmol, 85% purity).

Its structural characterization data are as follows:

MS m/z(ESI):729.2[M+H] ⁺

Step 2: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexatriacontane-35-ynoic acid (INT-10)

5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decaazanonatriacontane-31-oic acid (688.9 mg, 0.95 mmol, 85% purity) was dissolved in DMF (8 mL), and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (520.2 mg, 1.43 mmol) and DIPEA (367.7 mg, 2.9 mmol) were added. The mixture was stirred at room temperature under nitrogen for 2 hours. After completion of the reaction, the mixture was purified by flash preparative and freeze-dried to give the title compound (465.1 mg, 0.48 mmol, 98.0% purity).

Its structural characterization data are as follows:

MS m/z(ESI):979.3[M+H] ⁺

The preparation method is as follows:

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

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

Intermediate Preparation Example 11: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynyl)-L-valine (INT-11)

Step 1: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoyl)-L-valine tert-butyl ester (INT-11-1)

Dissolve 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (0.23 g, 0.41 mmol) and tert-butyl L-valine ester (0.95 g, 0.45 mmol) in DMF (3 mL). Add HATU (305.3 mg, 1.23 mmol) and DIPEA (106.2 mg, 0.82 mmol). Stir at room temperature for 3 hours. The reaction mixture is directly purified by preparative HPLC and then freeze-dried to obtain the title compound (142.0 mg, 198.6 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):715.8[M+H] ⁺

The preparation method is as follows:

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

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

Step 2: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynyl)-L-valine (INT-11)

Dissolve tert-butyl (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynyl)-L-valine (142.0 mg, 198.6 μmol) in DCM (5.0 mL). Add TFA (5.0 mL) and stir at room temperature for 2 hours. The reaction mixture was concentrated to obtain the crude product, which was purified by reverse-phase column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (60 mg, 91.08 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):659.7[M+H] ⁺

Example 1: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S, Preparation of triacetic acid (A-1)

Step 1: Preparation of (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indol[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazadotriacontane-30-ynamide)benzyl)(methyl)carbamic acid allyl ester (A-1-1)

Allyl (5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (194.9 mg, 0.22 mmol, 92% purity), 25-(2-(methylsulfonyl)- To the reaction mixture of 5-[(1-(2-pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (124.7 mg, 0.23 mmol, 90% purity) was dissolved in DMF (3 mL), and HATU (100.7 mg, 0.26 mmol) and DIPEA (85.2 mg, 0.66 mmol) were added, followed by stirring at room temperature for 1 hour. After most of the solvent was removed from the reaction solution, it was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and lyophilized to obtain the title compound (187.7 mg, 0.13 mmol, 87% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1423.9[M+H] ⁺

Step 2: Preparation of 4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatricarbon-30-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-1-2)

(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triaza To allyl (3,4-dimethyl-3-dotriacontane-3,0-ynamide)benzyl)(methyl)carbamate (187.7 mg, 0.13 mmol, 87% purity) was dissolved in DMF (3 mL), and 1,3-dimethylbarbituric acid (30.5 mg, 0.19 mmol) and tetrakistriphenylphosphine palladium (30.1 mg, 0.026 mmol) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at room temperature for 1 hour. After most of the solvent was removed from the reaction solution, it was purified by flash column chromatography (C18, water/acetonitrile = 0.5) and lyophilized to obtain the title compound (52.2 mg, 0.039 mmol, 80% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1339.3[M+H] ⁺

Step 3: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S, Preparation of triacetic acid (A-1)

4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatricarbon-30-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate ( The product (30.0 mg, 0.02 mmol, 87% purity) was dissolved in DMF (2 mL), and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (30.5 mg, 0.06 mmol) and DIPEA (12.9 mg, 0.1 mmol) were added. The mixture was stirred at room temperature under nitrogen for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (4.88 mg, 0.002 mmol, 93.22% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1726.9[M+H] ⁺

The preparation method is as follows:

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

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

Example 2: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-3- Preparation of 1-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

Step 1: Preparation of allyl (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indol[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrionedecane-30-ynamide)benzyl)(methyl)carbamate (A-2-1)

(5-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester ( To the mixture of 2,5-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (135.1 mg, 0.24 mmol, 90% purity) and HATU (125.7 mg, 0.33 mmol) and DIPEA (85.2 mg, 0.66 mmol) was added in DMF (3 mL), and the mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction mixture was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (199.2 mg, 0.13 mmol, 88% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1509.9[M+H] ⁺

Step 2: Preparation of 4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrionecontan-30-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-2-2)

(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6, Allyl (2,5-triazatrionecontan-30-ynamide)benzyl)(methyl)carbamate (199.2 mg, 0.13 mmol, 88% purity) was dissolved in DMF (3 mL), and 1,3-dimethylbarbituric acid (30.5 mg, 0.19 mmol) and tetrakistriphenylphosphine palladium (30.1 mg, 0.026 mmol) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 0.5) and freeze-dried to obtain the title compound (92.6 mg, 0.065 mmol, 86% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1425.9[M+H] ⁺

Step 3: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S Preparation of 1-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureidopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino A mixture of 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (47.5 mg, 0.09 mmol) and DIPEA (12.9 mg, 0.1 mmol) was added to the mixture. The mixture was stirred at room temperature for 2 hours under nitrogen protection. After completion of the reaction, the mixture was purified by rapid preparative purification and freeze-dried to give the title compound (28.08 mg, 0.015 mmol, 95.5% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1797.9[M+H] ⁺

The preparation method is as follows:

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

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

Example 3: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)- Preparation of 5-isopropyl-2-methylhydroxy-37-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxa-16,19,22,25,28-pentaoxa-3,6,9,12,31-pentaazaheptatriacontane-36-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-3)

Step 1: Preparation of (5-((11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-amide)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (A-3-1)

Allyl (5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.17 mmol, 90% purity), (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl Glycine (66.3 mg, 0.19 mmol) was dissolved in DMF (3 mL), and HATU (97.1 mg, 0.25 mmol) and DIPEA (65.8 mg, 0.51 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, 80 mL of water was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate (30 mL x 3), washed with 50 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound. After purification by flash column chromatography (silica gel column, dichloromethane/methanol = 10/1), the title compound (130.5 mg, 0.11 mmol, 87% purity) was obtained.

Its structural characterization data are as follows:

MS m/z(ESI):1218.9[M+H] ⁺

Step 2: Preparation of (5-((S)-2-((S)-2-(2-(2-aminoacetamide)acetamide)-3-methylbutanamide)propionamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (A-3-2)

(5-((11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-carboxamide)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexadecane) Allyl (1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (130.5 mg, 0.11 mmol, 87% purity) was dissolved in DMF (2 mL), and diethylamine (24.1 mg, 0.33 mmol) was added. The mixture was stirred at room temperature for 0.5 hour. The reaction mixture was directly purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (90.6 mg, 0.09 mmol, 91% purity).

Its structural characterization data are as follows:

MS m/z(ESI):996.8[M+H] ⁺

Step 3: Preparation of (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-37-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxo-16,19,22,25,28-pentaoxa-3,6,9,12,31-pentaazaheptatriacontane-36-ynamide)benzyl)(methyl)carbamic acid allyl ester (A-3-3)

Allyl (5-((S)-2-((S)-2-(2-(2-aminoacetamide)acetamide)-3-methylbutanamide)propionamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (90.6 mg, 0.09 mmol, 91% purity) was added. , 25-(2-(Methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (55.3 mg, 0.10 mmol, 90% purity) was dissolved in DMF (2 mL), HATU (51.3 mg, 0.14 mmol) and DIPEA (34.8 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (88.1 mg, 0.06 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1537.9[M+H] ⁺

Step 4: Preparation of 4-((2S,5S)-5-isopropyl-2-methyl-37-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxo-16,19,22,25,28-pentaoxa-3,6,9,12,31-pentaazatriacontane-36-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-3-4)

(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-37-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxo-16,19,22,25,28-pentaoxa-3,6,9,12, Allyl (31-pentaazaheptatriacontane-36-ynamide)benzyl)(methyl)carbamate (88.1 mg, 0.06 mmol, 90% purity) was dissolved in DMF (3 mL), and 1,3-dimethylbarbituric acid (14.1 mg, 0.09 mmol) and tetrakistriphenylphosphine palladium (13.9 mg, 0.012 mmol) were added. The atmosphere was purged with nitrogen three times and stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 0.55) and freeze-dried to obtain the title compound (35.3 mg, 0.024 mmol, 85% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1453.8[M+H] ⁺

Step 5: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5 Preparation of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxo-16,19,22,25,28-pentaoxa-3,6,9,12,31-pentaazaheptatriacontane-36-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-3)

4-((2S,5S)-5-isopropyl-2-methyl-37-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,32-pentaoxo-16,19,22,25,28-pentaoxa-3,6,9,12,31-pentaazaheptatriacontane-36-ynamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) Carbamate (35.3 mg, 0.024 mmol, 85% purity) was dissolved in DMF (2 mL), and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (36.1 mg, 0.07 mmol) and DIPEA (13.8 mg, 0.12 mmol) were added. The mixture was stirred at room temperature under nitrogen for 2 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to give the title compound (17.6 mg, 0.01 mmol, 99.5% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1841.1[M+H] ⁺

The preparation method is as follows:

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

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

Example 4: 2,2',2"-(10-((2S,5S,14S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)amino Preparation of triacetic acid (A-4)

Step 1: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (A-4-2)

(4-Aminophenyl)methanol (A-4-1) (300.0 mg, 2.44 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valyl-L-alanine (1.2 g, 2.93 mmol) were dissolved in DCM (28 mL) and MeOH (7 mL), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.81 g, 7.32 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to give a crude product. The product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0-90%) and concentrated again under reduced pressure to give the title compound (881.3 mg, 1.7 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):516.2[M+H] ⁺

Step 2: Preparation of (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (A-4-3)

(9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (200.0 mg, 0.39 mmol, 90% purity) was dissolved in DMF (3 mL). DIPEA (100.2 mg, 0.78 mmol) and p-nitrophenyl chloroformate (94.1 mg, 0.47 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 3/2) and freeze-dried to obtain the title compound (185.6 mg, 0.27 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):703.2[M+Na] ⁺

Step 3: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-4-4)

(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (185.6 mg, 0.27 mmol, 90% purity), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,1 5-Hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (172.13 mg, 0.41 mmol) was dissolved in DMF (5 mL). 1-Hydroxybenzotriazole (36.50 mg, 0.27 mmol), pyridine (21.5 mg, 0.27 mmol), and DIPEA (104.5 mg, 0.81 mmol) were added and stirred at room temperature for 2 hours. The reaction mixture was then stripped of most of the solvent and purified by flash column chromatography (C18, water/acetonitrile = 0.7). The title compound (158.30 mg, 0.16 mmol, 88% purity) was obtained by freeze-drying.

Its structural characterization data are as follows:

MS m/z(ESI):977.2[M+H] ⁺

Step 4: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-4-5)

4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (158.30 mg, 0.16 mmol, 88% purity) was dissolved in DMF (2 mL), diethylamine (23.8 mg, 0.32 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After most of the solvent was removed from the reaction solution, it was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (114.7 mg, 0.15 mmol, 95% purity).

Its structural characterization data are as follows:

MS m/z(ESI):755.3[M+H] ⁺

Step 5: Preparation of 4-((11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-amide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-4-6)

4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (114.7 mg, 0.15 mmol, 95% purity), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (62.3 mg, 0. 18mmol) was dissolved in DMF (3mL), and HATU (97.1mg, 0.25mmol) and DIPEA (65.8mg, 0.51mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, 80mL of water was added to the reaction solution, and the mixture was extracted 3 times with ethyl acetate (30mL x 3), washed with 50mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude title compound. After purification by flash column chromatography (silica gel column, dichloromethane/methanol = 10/1), the title compound (114.5mg, 0.1mmol, 89% purity) was obtained.

Its structural characterization data are as follows:

MS m/z(ESI):1091.3[M+H] ⁺

Step 6: Preparation of 4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-4-7)

4-((11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-carboxamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (114.5 mg, 0.1 mmol, 89% purity) was dissolved in DMF (2 mL), diethylamine (22.1 mg, 0.3 mmol) was added, and the mixture was stirred at room temperature for 0.5 hour. The reaction solution was freed from most of the solvent and purified by flash column chromatography (C18, water/acetonitrile = 0.6). The residue was freeze-dried to obtain the title compound (79.9 mg, 0.092 mmol, 91% purity).

Its structural characterization data are as follows:

MS m/z(ESI):869.2[M+H] ⁺

Step 7: Preparation of 4-((2S,5S,14S)-14-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaheneicosane-20-ynamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamate (A-4-8)

4-((S)-2-((S)-2-(2-(2-aminoacetamido)acetamido)-3-methylbutanamide)propionamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (79.9 mg, 0.092 mmol, 91% purity), N ⁶ -(diphenyl(p-tolyl)methyl)-N ² 5-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (72.1 mg, 0.11 mmol, 92% purity) was dissolved in DMF (3 mL), and HATU (51.3 mg, 0.14 mmol) and DIPEA (34.8 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After most of the solvent was removed from the reaction solution, it was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (96.8 mg, 0.06 mmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1503.9[M+H] ⁺

Step 8: Preparation of 4-((2S,5S,14S)-14-(4-aminobutyl)-5-isopropyl-2-methyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaheneicosane-20-ynamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-4-9)

4-((2S,5S,14S)-14-(4-((diphenyl(m-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaheneicosane-20-ynamide)benzyl((1S,9S)-9-ethyl-5-fluoro- 9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamate (96.8 mg, 0.06 mmol, 90% purity) was dissolved in DCM (5 mL), and formic acid (1 mL) was added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to give the title compound (37.4 mg, 0.03 mmol, 91% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1247.9[M+H] ⁺

Step 9: 2,2',2"-(10-((2S,5S,14S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)amino Preparation of triacetic acid (A-4)

4-((2S,5S,14S)-14-(4-aminobutyl)-5-isopropyl-2-methyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaheneicosane-20-ynamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (37 .4 mg, 0.03 mmol, 91% purity) was dissolved in DMF (2 mL), and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (47.5 mg, 0.09 mmol) and DIPEA (12.9 mg, 0.1 mmol) were added. The mixture was stirred at room temperature for 2 hours under nitrogen protection. The reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (22.34 mg, 0.013 mmol, 98.6% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1633.9[M+H] ⁺

The preparation method is as follows:

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

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

Example 5: 2,2',2"-(10-(2-((2-((((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5- Preparation of ((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (E-1)

Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (E-1-1)

2-(Methylsulfonyl)ethan-1-amine (204.97 g, 1.28 mmol) and allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (700 mg, 0.99 mmol) were added to a dichloromethane solution (50 mL). Triethylamine (199.89 mg, 1.98 mmol) and DMAP (96.53 mg, 0.79 mmol) were then added, and the mixture was stirred at 25°C for 18 hours. The product was purified by preparative HPLC and freeze-dried to give the title compound (620.0 mg, 894.9 μmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):693.7[M+H] ⁺

The preparation method is as follows:

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

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

Step 2: Preparation of allyl (S)-(5-(2-aminopropionamido)-2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (E-1-2)

Dissolve allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (180.0 mg, 0.26 mmol) in DMF (5 mL) and add diethylamine (57.01 mg, 0.78 mmol). Stir at room temperature for 3 hours. The reaction mixture was concentrated to dryness to obtain the crude title compound (115.0 mg, crude product), which was used in the next step without purification.

Its structural characterization data are as follows:

ESI-MS (m/z): 471.5 [M+H] ⁺ .

Step 3: Preparation of allyl (S)-methyl (2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5-(2-azidopropionamido)benzyl)carbamate (E-1-3)

Crude (S)-(5-(2-aminopropionamido)-2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (115.0 mg) was dissolved in methanol (3 mL) and purified water (1.8 mL). 1H-imidazole-1-sulfonyl azide hydrochloride (61.6 mg, 0.29 mmol), copper sulfate pentahydrate (6.10 mg, 0.02 mmol), and potassium carbonate (67.56 mg, 0.49 mmol) were added, and the mixture was stirred at 25°C for 16 hours. The crude product was concentrated, purified by preparative HPLC, and then freeze-dried to obtain the title compound (90.0 mg, 181.26 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):497.5[M+H] ⁺

The preparation method is as follows:

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

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

Step 4: Preparation of (S)-(2-((((chloromethyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5-(2-azidopropionamido)benzyl)(methyl)carbamic acid allyl ester (E-1-4)

Under nitrogen, allyl (S)-methyl(2-((((2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5-(2-azidopropionamido)benzyl)carbamate (90.0 mg, 181.26 μmol) was dissolved in dichloromethane (10 mL). The temperature was cooled to 0°C, and TMSCl (59.1 mg, 0.54 mmol) and paraformaldehyde (18.0 mg, 0.60 mmol) were added. The mixture was stirred at 40°C for 18 hours. The mixture was concentrated to obtain the crude title compound (70.0 mg, crude product).

Its structural characterization data are as follows:

ESI-MS(m/z):531.6[M+OH-Cl] ⁺ .

Step 5: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-azidopropionamido)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (E-1-5)

Crude (S)-allyl(2-((((chloromethyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5-(2-azidopropionamido)benzyl)(methyl)carbamate (70.0 mg, crude) was dissolved in DCM (5 mL), and (S)-9-ethyl-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (CAS No. 121062-62-2, 51.94 mg, 0.13 mmol) and 1,2,2,6,6-pentamethylpiperidine (45.8 mg, 0.30 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The crude product was concentrated, purified by preparative HPLC and freeze-dried again to give the title compound (35.0 mg, 38.34 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):913.9[M+H] ⁺

The preparation method is as follows:

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

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

Step 6: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropionamido)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (E-1-6)

2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-azidopropionamido)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (35.0 mg, 38.34 μmol) was dissolved in THF (5 mL). A solution of trimethylphosphine in tetrahydrofuran (14.99 mg, 0.20 mmol) was added, and the mixture was stirred at room temperature for 4 hours. The crude product was concentrated, purified by preparative HPLC, and then freeze-dried to give the title compound (18.0 mg, 20.29 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):887.9[M+H] ⁺

The preparation method is as follows:

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

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

Step 7: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (E-1-7)

2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropionamido)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)amino A 2-hydroxybenzoate (18.0 mg, 20.29 μmol) was dissolved in DMF (3 mL). (2,5-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynyl)-L-valine (13.37 mg, 20.29 μmol) and HATU (15.4 mg, 0.40 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The crude product was concentrated, purified by preparative HPLC, and then freeze-dried to obtain the title compound (12.0 mg, 7.85 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1528.7[M+H] ⁺

The preparation method is as follows:

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

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

Step 8: Preparation of 4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrionecontan-30-ynamide)-2-((methylamino)methyl)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (E-1-8)

2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexa 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamate (12.0 mg, 7.85 μmol) was dissolved in DMF (3 mL). Tetrakistriphenylphosphine palladium (13.37 mg, 20.29 μmol) and 1,3-dimethylbarbituric acid (3.68 mg, 23.56 μmol) were added and stirred at room temperature for 4 hours. The crude product was concentrated, purified by preparative HPLC, and then freeze-dried to give the crude title compound (11.34 mg, 7.85 μmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1444.6[M+H] ⁺

The preparation method is as follows:

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

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

Step 9: 2,2',2"-(10-(2-((2-((((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-5- Preparation of ((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (E-1)

4-((2S,5S)-5-Isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontane-30-ynamide)-2-((methylamino)methyl)benzyl((((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)methyl)(2-(methylsulfonyl)ethyl)amino The crude formate (11.34 mg, 7.85 μmol, 90% purity) was dissolved in DMF (3 mL). 2,2',2"-(10-(2-((2,5-dioxocyclopentyl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (13.31 mg, 26.60 μmol) and DIPEA (21.49 mg, 166.25 μmol) were added, and the mixture was stirred at room temperature for 4 hours. The crude product was concentrated, purified by preparative HPLC, and then freeze-dried to give the title compound (4.12 mg, 1.98 μmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1830.6[M+H] ⁺

The preparation method is as follows:

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

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

Example 6: 2,2',2"-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline Preparation of triacetic acid (A-5)

Step 1: Preparation of 4-((S)-2-((S)-2-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)hexanamido)-3-methylbutanamido)propanamido)benzyl((1S,9S)-9-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-5-1)

Benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (24 mg, 31.78 μmol) and INT-9 (22.80 mg, 34.96 μmol) were dissolved in D 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13.28 mg, 34.96 μmol) and DIPEA (12.30 mg, 95.34 μmol) were added to MF (1 mL) and stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 1/4) and freeze-dried to obtain the title compound (23 mg, 16.56 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1389.6[M+H] ⁺

Step 2: Preparation of ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamido)hexanamido)-3-methylbutanamido)propionamido)benzyl((1S,9S)-9-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-5-2)

4-((S)-2-((S)-2-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)hexanamido)-3-methylbutanamido)propionamido)benzyl((1S,9S)-9-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (23 mg, 16.56 μmol) was dissolved in DMF (1 mL), and formic acid (1 mL) was added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 1/2) and freeze-dried to give the title compound (15 mg, 13.24 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1133.3[M+H]+

Step 3: 2,2',2"-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline Preparation of triacetic acid (A-5)

((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamido)hexanamido)-3-methylbutanamido)propanamido)benzyl((1S,9S)-9-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (15m g, 13.24 μmol) was dissolved in DMF (1 mL), and DIPEA (5.12 mg, 39.72 μmol) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (19.90 mg, 39.72 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (4.29 mg, 2.79 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1519.7[M+H] ⁺

The preparation method is as follows:

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

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

Example 7: Preparation of 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-6)

Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (A-6-2)

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

Its structural characterization data are as follows:

MS m/z(ESI):782.2[M+H] ⁺

Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (A-6-3)

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

Its structural characterization data are as follows:

MS m/z(ESI):566.1[M+Na] ⁺

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 (A-6-4)

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

Its structural characterization data are as follows:

MS m/z(ESI):731.2[M+Na] ⁺

Step 4: Preparation of allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-6-5)

Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13 H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (165.50 mg, 0.38 mmol) was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water/acetonitrile = 0.7) and freeze-dried to obtain the title compound (114.70 mg, 0.12 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):1005.2[M+H] ⁺

Step 5: Preparation of allyl (5-((S)-2-aminopropionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-6-6)

Allyl(5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) was dissolved in DMF (3 mL), diethylamine (87.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the residue was purified by flash column chromatography (C18, water/acetonitrile = 0.7) and freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):783.1[M+H] ⁺

Step 6: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (A-6-7)

L-alanine (73.15 mg, 821.92 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (100.0 mg, 273.97 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (106.03 mg, 821.92 μmol) was added. The reaction was stirred at room temperature for 1 hour. After completion, the reaction was purified by flash column chromatography (C18, water/acetonitrile = 2/1) and freeze-dried to obtain the title compound (60 mg, 163.49 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):367.2[M+H]+

Step 7: Preparation of (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propionamido)benzyl)(methyl)carbamate (A-6-8)

(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (30 mg, 81.74 μmol), allyl(5-((S)-2-aminopropionamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl )benzyl)(methyl)carbamate (63.95 mg, 81.74 μmol) was dissolved in DMF (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34.17 mg, 89.91 μmol) and DIPEA (31.63 mg, 245.22 μmol) were added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 1/2) and freeze-dried to obtain the title compound (45 mg, 39.75 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1132.2[M+H] ⁺

Step 8: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butyramido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-6-9)

(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl) To the product was dissolved 45 mg of (amino)butyramide)propionamide)benzyl)methyl)carbamate (45 mg, 39.75 μmol) in DMF (1 mL), and tetrakistriphenylphosphine palladium (4.6 mg, 3.98 μmol) was added, along with 15 μL of formic acid and 30 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 4/1) and freeze-dried to obtain the title compound (27 mg, 25.76 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1048.4[M+H] ⁺

Step 9: Preparation of 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-6)

4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (27 mg, 25. The product was dissolved in DMF (1 mL) and DIPEA (9.97 mg, 77.28 μmol) was added. DIPEA and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (38.76 mg, 77.28 μmol) were added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (3.68 mg, 2.56 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1434.6[M+H] ⁺

The preparation method is as follows:

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

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

Example 8: Preparation of 2,2',2"-(10-(2-((2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-7)

Step 1: Preparation of allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-7-1)

Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (200.0 mg, 0.28 mmol), (1S,9S)-1-amino-9-ethyl-5-chloro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13 H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (253.0 mg, 0.56 mmol) was dissolved in DMF (6 mL). 1-Hydroxybenzotriazole (45.5 mg, 0.34 mmol), pyridine (24.5 mg, 0.31 mmol), and DIPEA (108.5 mg, 0.57 mmol) were added and stirred at room temperature for 2 hours. After completion of the reaction, the product was purified by flash column chromatography (C18, water/acetonitrile = 0.7) and freeze-dried to obtain the title compound (183.70 mg, 0.18 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):1022.2[M+H] ⁺

Step 2: Preparation of allyl (5-((S)-2-aminopropionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-7-2)

Allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (183.70 mg, 0.18 mmol) was dissolved in DMF (3 mL), diethylamine (131.8 mg, 1.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the residue was purified by flash column chromatography (C18, water/acetonitrile = 0.6) and freeze-dried to obtain the title compound (136.6 mg, 0.17 mmol).

Its structural characterization data are as follows:

MS m/z(ESI):800.1[M+H] ⁺

Step 3: Preparation of (2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propionamido)benzyl)(methyl)carbamate (A-7-3)

(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (30 mg, 81.74 μmol), (5-((S)-2-aminopropionamido)-2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl Allyl)(methyl)carbamate (65.23 mg, 81.74 μmol) was dissolved in DMF (1 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34.17 mg, 89.91 μmol) and DIPEA (31.63 mg, 245.22 μmol) were added. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 1/2) and freeze-dried to obtain the title compound (35 mg, 30.50 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1147.4[M+H] ⁺

Step 4: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butyramido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-7-4)

(2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl) To the product was added 1,4-diamino-3,4-butyrylamino-3,4-propionylamino-3,4-benzylamino-3,4-di ...

Its structural characterization data are as follows:

MS m/z(ESI):1063.4[M+H] ⁺

Step 5: Preparation of 2,2',2"-(10-(2-((2-(((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-7))

4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propionamido)-2-((methylamino)methyl)benzyl((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (18 mg, 16. 93 μmol) was dissolved in DMF (1 mL), DIPEA (6.55 mg, 50.78 μmol) was added, and 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (25.47 mg, 50.78 μmol) was added. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (2.77 mg, 2.56 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1451.0[M+H] ⁺

The preparation method is as follows:

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

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

Example 9: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methane Preparation of triacetic acid (A-8)

Step 1: Preparation of allyl (2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-18-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadec-17-ynyl)benzyl)(methyl)carbamate (A-8-3)

Allyl (5-((S)-2-aminopropionamido)-2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (35.3 mg, 0.04 mmol) and (6-(2-(methylsulfonyl)pyrimidinyl- To the mixture of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19 mg, 0.05 mmol) and DIPEA (19.35 mg, 0.15 mol) was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (19 mg, 0.05 mmol) and DIPEA (19.35 mg, 0.15 mol). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 1/2) and freeze-dried to give the title compound (30 mg, 24.07 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1246.4[M+H]+

Step 2: Preparation of 4-((2S,5S)-5-isopropyl-2-methyl-18-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecane-17-ynamino)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (A-8-4)

(2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-18-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13-tetraoxo- Allyl 3,6,9,12-tetraazaoctadec-17-ynyl)benzyl)(methyl)carbamate (30 mg, 24.07 μmol) was dissolved in DMF (1 mL), and tetrakistriphenylphosphine palladium (2.1 mg, 2.41 μmol) was added, along with 15 μL of formic acid and 30 μL of N-methylmorpholine. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, the mixture was purified by flash column chromatography (C18, water/acetonitrile = 4/1) and freeze-dried to give the title compound (25 mg, 21.51 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1162.2[M+H] ⁺

Step 3: 2,2',2"-(10-(2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl Preparation of 5-((2S,5S)-5-isopropyl-2-methyl-18-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecane-17-ynamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-8)

4-((2S,5S)-5-isopropyl-2-methyl-18-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadec-17-ynylamino)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate The product (25 mg, 21.51 μmol) was dissolved in DMF (1 mL), and DIPEA (8.32 mg, 64.53 μmol) was added, followed by 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (32.36 mg, 64.53 μmol). After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (13.42 mg, 8.58 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1549.6[M+H] ⁺

The preparation method is as follows:

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

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

Example 10: Preparation of (S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butanamide)-5-ureidopentanamide)benzyl)ethane-1,2-diylbis(methylcarbamate) (E-3)

Step 1: Preparation of (S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl(4-nitrophenyl)carbonate (E-3-2)

(S)-9-Ethyl-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (100 mg, 247.27 μmol), bis(4-nitrophenyl) carbonate (165.49 mg, 544.00 μmol), and DIPEA (31.96 mg, 247.27 μmol) were added to DMF (5 mL) and stirred at 25°C for 3 hours. The reaction solution was used directly in the next step without purification.

Its structural characterization data are as follows:

MS m/z(ESI):570.2[M+H] ⁺

Step 2: Preparation of (S)-tert-butyl (9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo [de] pyrano [3', 4': 6,7] indolizino [1,2-b] quinolin-4-yl) ethane-1,2-diyl bis (methylcarbamate) (E-3-3)

(S)-9-Ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl(4-nitrophenyl)carbonate (140 mg, 245.82 μmol) was dissolved in DMF (5 mL). Tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (138.84 mg, 737.47 μmol) and DIPEA (95.31 mg, 737.47 μmol) were added to the mixture. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, the reaction mixture was purified by HPLC to obtain the title compound (106 mg, 171.33 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):619.3[M+H] ⁺

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

Step 3: Preparation of (S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-ylmethyl(2-(methylamino)ethyl)carbamate (E-3-4)

(S)-tert-Butyl(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (106 mg, 171.33 μmol) was added to DCM (4 mL) and trifluoroacetic acid (1 mL) and stirred at 25°C for 1 hour. The reaction was concentrated to give the crude product (108 mg, 170.73 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):519.2[M+H] ⁺

Step 4: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (E-3-5)

(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-ylmethyl(2-(methylamino)ethyl)carbamate (108 mg, 170.73 μmol), (9H-fluoren-9-yl)methyl((S)-3-methyl-1- (((S)-1-((4-((((4-Nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (261.83 mg, 341.46 μmol) and DIPEA (110.32 mg, 853.64 μmol) were added to DMF (4 mL) and stirred at 25°C for 2 hours. The reaction solution was purified by reverse phase purification (ACN/H2O = 0-65%, 0.05% formic acid) and lyophilized to obtain a light yellow solid (99 mg, 86.37 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1147.4[M+H] ⁺

Step 5: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentanamido)benzyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (E-3-6)

4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (30.00 mg, 26.17 μmol) and 1,8-diazobisspiro[5.4.0]undec-7-ene (11.95 mg, 78.52 μmol) were added to DMF (5 mL) and stirred at 25°C for 30 min. The product was used directly in the next step without purification.

Its structural characterization data are as follows:

MS m/z(ESI):924.4[M+H] ⁺

Step 6: Preparation of (S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butanamide)-5-ureidopentanamide)benzyl)ethane-1,2-diylbis(methylcarbamate) (E-3)

4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)ethane-1,2-diylbis(methylcarbamate) (24.00 mg, 25.97 μmol), 6-( 2-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoic acid (17.42 mg, 64.93 μmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (22.97 mg, 77.92 μmol) and DIPEA (16.78 mg, 129.87 μmol) were added to DMF (5 mL), and the mixture was stirred at 25 ° C for 2 hours. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (15 mg, 12.65 μmol).

Its structural characterization data are as follows:

MS m/z(ESI):1174.4[M+H] ⁺

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

Example 11: Preparation of 2,2'-((2-((2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butanamide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)azanediyl)diacetic acid (A-9)

Weigh 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)butanamide)propionamide)-2-((methylamino)methyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2 A mixture of 2-[(2,6-dioxomorpholin-4-yl)ethyl]morpholine-2,6-dione (14.67 mg, 57.24 μmol) and 4-[2-(2,6-dioxomorpholin-4-yl)ethyl]morpholine-2,6-dione (14.67 mg, 57.24 μmol) was dissolved in DMF (1 mL). Diisopropylethylamine (11.10 mg, 85.87 μmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (4.5 mg, 3.06 μmol, 90% purity).

Its structural characterization data are as follows:

MS m/z(ESI):1322.3[M+H]+

The preparation method is as follows:

Column: Waters SunFire Prep C18 OBD (5μm*45mm*450mm)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

Coupling Example

1. Preparation of Example Trastuzumab-A-1

To 0.41 mL of trastuzumab antibody (24.4 mg/mL) was added 21.0 μL _of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.6, 38.0 μL) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of the antibody-based solution of A-1 (147.0 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-A-1). The DAR value, determined by mass spectrometry, was 7.1.

2. Preparation of Example Trastuzumab-A-2

To 1.025 mL of trastuzumab antibody (24.4 mg/mL) was added 51.2 μL of 20 mM PB + 0.1 _M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.8 μL, pH 7.6) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-2 dissolved in dimethyl sulfoxide (216.0 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-A-2). The DAR value was determined by mass spectrometry to be 8.0.

3. Preparation of Example Trastuzumab-A-3

To 1.025 mL of trastuzumab antibody (24.4 mg/mL) was added 66.0 μL of 20 mM PB + 0.1 _M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.8 μL, pH 7.6) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-3 dissolved in dimethyl sulfoxide (208.0 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-A-3). The DAR value was determined by mass spectrometry to be 8.0.

4. Preparation of Example Trastuzumab-A-4

To 1.025 mL of trastuzumab antibody (24.4 mg/mL) was added 66.0 μL of 20 mM PB + 0.1 _M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.8 μL, pH 7.6) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-4 dissolved in dimethyl sulfoxide (210.0 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-A-4). The DAR value was determined by mass spectrometry to be 8.0.

5. Preparation of Example Trastuzumab-A-5

1.7 mL of trastuzumab antibody (14.7 mg/mL) was diluted with 85 μL _of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M _Na₂HPO₄ solution. 95 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A solution of A-5 (208.8 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) (12 times the amount of the antibody) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-5). The DAR value, determined by mass spectrometry, was 7.8.

6. Preparation of Example Trastuzumab-A-8

2.027 mL of trastuzumab antibody (14.8 mg/mL) was diluted with 101 μL of 20 mM PB + 0.1 _M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M _Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 113.6 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A solution of A-8 (250.5 μL, 10 mM) dissolved in dimethyl sulfoxide (DMSO) (12 times the amount of the antibody) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., trastuzumab-A-8). The DAR value, determined by mass spectrometry, was 8.0.

7. Preparation of Example Trastuzumab-A-9

To 0.957 mL of trastuzumab antibody (20.9 mg/mL) was added 48.0 μL _of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-9 dissolved in dimethyl sulfoxide (192.0 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-A-9). The DAR value, determined by mass spectrometry, was 7.6.

8. Preparation of Example Trastuzumab-E-1

To 0.833 mL of trastuzumab antibody (24 mg/mL) was added 52.0 μL _of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.6, solution, 75.8 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of E-1 dissolved in dimethyl sulfoxide (183.7 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-E-1). The DAR value, determined by mass spectrometry, was 8.0.

9. Preparation of Example Trastuzumab-E-3

To 0.667 mL of trastuzumab antibody (22.5 mg/mL) was added 33.3 μL _of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.6 with 1 M _Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.6, solution, 56.8 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. An 11-fold amount of E-3 dissolved in dimethyl sulfoxide (114.8 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (trastuzumab-E-3). The DAR value, determined by mass spectrometry, was 7.8.

Biological data

1. Evaluation of the Antibody Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

The preparations containing the ADC of the present invention were administered via tail vein injection to a CDX mouse model subcutaneously transplanted with human breast cancer cells JIMT-1. The tumor volume and animal body weight changes were measured once a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

Test drug

Take an appropriate amount of ADC and administer at 1.5 mg/kg and 3 mg/kg, as detailed below. Dilute the stock solution with 0.9% NaCl injection to prepare the dosing solution. Use 0.9% NaCl injection as the vehicle control (Vehicle).

Experimental animals and cell lines

NOD SCID mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., production license number: SCXK (Su) 2023-0009)

Human breast cancer cells JIMT-1 (Nanjing Kebai)

Experimental grouping and evaluation methods

Tumor-bearing mice with an average tumor volume of approximately 150 ^mm³ were randomly divided into groups (the number of groups was determined based on the sample size). Groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and ADC, with dosing frequency as described in the specific examples. The administration method was tail vein injection, with a volume of 10 ml/kg. Tumor diameters were measured weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × ^b² , where a and b represent the major and minor diameters of the tumor, respectively. Animal mortality was recorded daily.

The following formula was used to calculate the tumor growth inhibition rate (TGI) to evaluate the tumor inhibition efficacy of ADC:
_VTend > _VT0 , TGI (%) = [1-( _VTend - _VT0 )/( _VCend - _VC0 )]*100% or _VTend≤VT0 , _TGI (%) = [1-( _VTend - _VT0 )/
V _T0 ]*100%.

Wherein, V _{T end} : mean tumor volume at the end of the experiment in the treatment group;

V _T0 : mean tumor volume at the start of drug administration in the treatment group;

V _{C end} : mean tumor volume of negative control group at the end of the experiment;

V _C0 : mean tumor volume of the negative control group at the beginning of drug administration;

The following formula was used to calculate the relative tumor proliferation rate T/C (%), which was used to evaluate the tumor inhibition efficacy of ADC:
T/C (%) = ( _{VT end} / _VTO ) / ( _{VC end} / _VCO ) * 100%.

(1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the JIMT-1 model

JIMT-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% _CO₂ . JIMT-1 cells were harvested during the exponential growth phase, resuspended in PBS containing 50% Matrigel to an appropriate concentration, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume reached approximately 150 ^mm³ , the mice were randomly divided into the following groups based on tumor size: a vehicle control group (i.e., negative control, vehicle group), a 1.5 mg/kg group receiving trastuzumab-A-1 of the present invention, a 1.5 mg/kg group receiving trastuzumab-A-2, a 1.5 mg/kg group receiving trastuzumab-A-4, and a control group receiving DS8201 at 3 mg/kg (Note: DS8201 is a human HER2-targeting ADC developed by Daiichi Sankyo; the sample used in this study was manufactured by Kelun Biotech). The mice were administered once on Day 0.

The ADC of this invention demonstrated significant tumor growth inhibition in the JIMT-1 breast cancer xenograft model. On Day 20, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The mice tolerated the ADC of this invention well during treatment. Specific results are shown in Table 1.

Table 1 Human breast cancer cell JIMT-1 CDX model

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

Claims (22)

An antibody-drug conjugate having a structure represented by the formula Ab-[MLED] _x , wherein: Ab is an antibody or its antigen-binding fragment: M is a linker connected to the antibody or its antigen-binding fragment; L is the structure connecting the joints M and E; E is the structure connecting L and D; D is the cytotoxic drug moiety; x is 1 to 10. The antibody drug conjugate of claim 1, wherein M is selected from the following substituted or unsubstituted structures:
Preferably, M is selected from the following substituted or unsubstituted structures:
The antibody drug conjugate of claim 1 or 2, wherein L is selected from a substituted or unsubstituted structure consisting of one or more of the following: C _1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclic group (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R') ₂ , carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as 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(COCH ₂ CH ₂ (OCH ₂ CH ₂ )rOCH ₃ )), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly(DGGFG), Glu-Gly-Gly-Phe-Gly(EGGFG), 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-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Va l-Lys-Gly, Gly-Gly-Phe-Gly(GGFG), Gly-Gly-Val-Ala(GGVA), Gly-Phe-Leu-Gly(GFLG), Glu-Ala-Ala-Ala(EAAA), Gly-Gly-Gly-Gly-Gly(GGGGG)), wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C _1-6 alkyl, C _1-6 alkylene, amine, hydroxyl, carboxyl, acyl, -O-, -C _1-6 alkyleneCO ₂ H, -C _1-6 alkyleneSO ₃ H, -SO ₃ H, -PO ₃ H ₂ , -C _1-6 alkylene-NHC _1-6 alkyl, -C _1-6 alkylene-N(C _1-6 alkyl) ₂ , -CH ₂ N(C _1-6 alkyl)-C(＝O)C _1-6 alkylene-heterocycle, -C _1-6 alkylene-heterocycle, -NHC _1-6 alkylene-SO ₃ H, _-CH2NH -SO3H, _-CH2N ( _C1-6alkyl )-SO3H, -CH2NHC1-6alkylene-SO3H, -CH2N _{( C1-6alkyl} ) _C1-6alkylene -SO3H, _-CH2N (C1-6alkylene-SO3H) ₂ , _{-CH2N +} ( _C1-6alkylene - _SO3H ) ₃ , _-CH2N ⁺ (C1-6alkyl) _{2- C1-6alkylene} -SO3H, _-CH2N ( _C1-6alkyl ) _{-C ( =} O) _C1-6alkylene -N ⁺ ( _C1-6alkylene - _SO3H ) ₃ , _-CH2NH - _C (=O) _C1-6alkylene - ^{N+ (} _{C1-6alkylene - SO3H ) 3 , -CH2N} ( _C1-6alkyl ) _- -C _1-6 alkyl)-C(＝O)C _1-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ NH-C(＝O)C _1-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₃ , -CH ₂ N(C _1-6 alkyl)-C(＝O)OC _2-6 alkylene-N ⁺ (C _1-6 alkyl) ₂ -CH ₂ CO ₂ H, -CH ₂ N(C _1-6 alkyl)-C _1-6 alkylene-CO ₂ H, -CH ₂ N ⁺ (C _1-6 alkyl) ₂ -C _1-6 alkylene-CO ₂ H, glucosyl, galactosyl, glucuronic acid group, galacturonic acid group, -CH ₂ N(C 1-6 alkyl) -C _1-6 alkyl)-C(═O)-(CH ₂ CH ₂ O) _r -C _1-6 alkyl, -CH ₂ N(C _1-6 alkyl)-C(═O)-(OCH ₂ CH ₂ ) _r -OC _1-6 alkyl, -(CH ₂ N(Me)-C(═O)) _r -C _1-6 alkyl, a polyethylene glycol fragment containing 1-10 ethoxy (EO) units (i.e., -(CH ₂ CH ₂ O) _r -C _1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), EDTA (ethylenediaminetetraacetic acid residue), -C -C _1-6 alkylene-N(C _1-6 alkyl)-DOTA, -C _1-6 alkylene-N(C _1-6 alkyl)-DOTAGA, -C _1-6 alkylene-N(C _1-6 alkyl)-NOTA or -C _1-6 alkylene-N(C _1-6 alkyl)-EDTA, wherein r is selected from an integer of 1-20, for example, an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from an integer of 1-20, for example, an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from the following substituted or unsubstituted structures:
(For example ), (For example ), (For example ), (For example ); wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10; Alternatively, L is selected from the following substituted or unsubstituted structures: The antibody drug conjugate according to any one of claims 1 to 3, wherein E is selected from a single bond, a substituted or unsubstituted -NH-CH ₂ -, or the following substituted or unsubstituted structures:
The antibody drug conjugate according to any one of claims 1 to 4, wherein the cytotoxic drug is selected from a topoisomerase inhibitor, a microtubule inhibitor, a DNA intercalator, an RNA polymerase inhibitor and a gene transcription inhibitor; preferably, the microtubule inhibitor is an auristatin compound, a maytansine compound, a hemicycline compound or an eribulin compound; the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, an anthracycline compound, trabectedin or rubectin and its derivatives or analogs; the topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, Belotecan, Rubitecan, Diflomotecan, Lurtotecan, Karenitecin, Gimatecan, Namitecan, Simmitecan, Chimmitecan, Silatecan or Elomotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogs, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin; the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters and analogs; Preferably, the cytotoxic drug is selected from the following compounds of Formula III or pharmaceutically acceptable salts, stereoisomers or isotope-labeled compounds thereof:
wherein R ₅ and R ₆ are each independently selected from H, OH, -NH ₂ , -NH(C _1-6 alkyl), C _1-6 alkyl and halogen; the C _1-6 alkyl is optionally further substituted by one or more substituents selected from halogen, hydroxyl, C _1-6 haloalkyl, and C _3-6 cycloalkyl; or, R ₅ and R ₆ together with adjacent carbon atoms (ring-forming carbon atoms connecting R ₅ and R ₆ , respectively) form a five-membered oxygen-containing heterocyclic ring. R ₇ is selected from H, -OH, -NH ₂ , -NH(C _1-6 alkyl), and -NH-CO-(C _1-6 alkylene)-OH; the C _1-6 alkyl and C _1-6 alkylene groups are optionally further substituted with one or more substituents selected from halogen, hydroxy, C _1-6 haloalkyl, and C _3-6 cycloalkyl; q is 1, 2 or 3, for example, 1 or 2; Preferably, R ₅ and R ₆ are each independently selected from H, OH, -NH ₂ , C _1-4 alkyl and halogen; Preferably, R ₅ is selected from H, OH and methyl; Preferably, R ₆ is selected from H and halogen; Preferably, R ₇ is selected from -H, -NH ₂ , -NH(C _1-4 alkyl), and -NH-CO-(C _1-4 alkylene)-OH; Preferably, the cytotoxic drug is selected from compound 1-1, 1-2 or a pharmaceutically acceptable salt, stereoisomer or isotope-labeled compound thereof,
or, The cytotoxic drug is selected from compound 2-2 or a pharmaceutically acceptable salt, stereoisomer or isotope-labeled compound thereof,
Preferably, D is a monovalent structure obtained by losing one H from -OH, _-NH2 or a secondary amine group on the cytotoxic drug; Preferably, the structure of D is shown in the following formula III-A or formula III-B:
wherein R _5A is selected from -O- or -N-; R _7A is selected from -NH-; R ₅ , R ₆ , R ₇ , and q are as described in the claims; More preferably, D is selected from the following structures:
The antibody-drug conjugate of claims 1-5, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase, and preferably comprises: (1) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; 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 the sequence from which it is derived, 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 is derived; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one 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 the sequence from which it is derived, 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 is derived; preferably, the substitutions are conservative substitutions; or, (3) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (3a) and (3b) 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 is derived, 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 is derived; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; 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 the sequence from which it is derived, 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 is derived; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) VH shown in SEQ ID NO: 1 or a variant thereof, and/or VL shown in SEQ ID NO: 2 or a variant thereof; or (b) VH shown in SEQ ID NO: 3 or a variant thereof, and/or VL shown in SEQ ID NO: 4 or a variant thereof; wherein 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 compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; More preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence represented by SEQ ID NO: 1 and a heavy chain constant region (CH) represented by SEQ ID NO: 35, and a light chain comprising a VL sequence represented by SEQ ID NO: 2 and a light chain constant region (CL) represented by SEQ ID NO: 36; or (2) a heavy chain comprising the VH sequence of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 36; Even more preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the sequence shown in SEQ ID NO: 37, and a light chain comprising the sequence shown in SEQ ID NO: 38; or (2) a heavy chain comprising the sequence shown in SEQ ID NO: 39, and a light chain comprising the sequence shown in SEQ ID NO: 40; Even more preferably, the antibody is a trastuzumab antibody, a pertuzumab antibody, or a bi-epitope antibody composed thereof. The antibody-drug conjugate of claim 1 has a structure as shown in Formula I-A1, Formula I-A2, Formula I-E1, or Formula I-E2:
Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof according to claim 6, preferably trastuzumab, -(S-) represents the specific connection mode between the sulfhydryl group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, 6-8, M is as defined in claim 2, and L is as defined in claim 3; Preferably, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following group I and one or more substituted or unsubstituted structural fragments selected from the following group II: Group I:
Group II:

wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from the following structures:
Alternatively, preferably, L is formed by connecting one or more substituted or unsubstituted structural fragments selected from the following group I and one or more substituted or unsubstituted structural fragments selected from the following group II: Group I:
Group II:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from the following structures:
Alternatively, the structure of the antibody-drug conjugate is shown in Formula I-E3 below:
Wherein, HA-(S-) is the antibody or antigen-binding fragment thereof according to claim 6, preferably trastuzumab, -(S-) represents the specific connection mode between the sulfhydryl group from the antibody or antigen-binding fragment thereof and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, 6-8, M is as defined in claim 2, and L is as defined in claim 3; Preferably, L is formed by linking a structure selected from Group III and one or two structures selected from Group IV: Group III:
Group IV:
wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10; Preferably, L is selected from the following structures:
The antibody drug conjugate according to any one of claims 1 to 7, wherein Selected from the following substituted or unsubstituted structures:
(For example ), (For example ), (For example ), (For example ), (For example ), s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10; or Selected from the following substituted or unsubstituted structures:
The antibody-drug conjugate of claim 1, which is selected from ADC A-1 to ADC A-9 and ADC E-1 to ADC E-6:




The HA-(S-) is the antibody or antigen-binding fragment thereof according to claim 6, preferably trastuzumab, pertuzumab, or a bi-epitope antibody composed thereof. The antibody-drug conjugate according to claim 1 has the following structure:

Wherein HA-(S-) is the antibody or antigen-binding fragment thereof according to claim 6, preferably trastuzumab, -(S- represents the specific connection mode between the thiol group from the antibody or antigen-binding fragment thereof and the pyrimidine group in ADC A-1a, ADC A-2a, or ADC A-4a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, having a structure represented by the formula M'-L-E-D, wherein: M' is -M-Lg, wherein Lg is a leaving group or an addition reaction group for a nucleophilic substitution reaction, and M is a structure that binds to the antibody or its antigen-binding fragment; L is the structure connecting M and E; E is the structure connecting L and D; D is the cytotoxic drug moiety; Preferably, M, L, E, and D are as defined in any one of claims 1-10. The compound of claim 11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled form, metabolite or prodrug thereof, wherein M' is selected from:
Wherein, Lg is selected from halogen (such as F, Cl, Br, I), halogenated C _1-6 alkyl, maleimide, halogenated maleimide, C _1-6 alkylsulfonyl, halogenated C _{1-6 alkylsulfonyl, halogenated sulfonyl, C 1-6 alkylsulfonate, halogenated C 1-6 alkylsulfonate ,} C _1-6 alkylsulfinate, C _1-6 alkylsulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH ₂ ), nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structures, and the halogenated C _1-6 alkyl, C _1-6 alkylsulfonyl, halogenated C _1-6 alkylsulfonyl, halogenated sulfonyl, C _{1-6 alkylsulfonate, halogenated C 1-6 alkylsulfonate} , C _1-6 alkylsulfinate, C _1-6 alkylsulfoxide, halophenoxy, alkenyl, alkynyl and alkynyl-containing structures are optionally substituted with one or more suitable substituents; preferably, Lg is selected from halogen, substituted or unsubstituted C _1-6 alkylsulfonyl (C _1-6 alkyl-SO ₂ -), halophenoxy, hydroxyl (-OH), thiol (-SH) or amino (-NH ₂ ), more preferably selected from C _1-6 alkylsulfonyl (e.g., methylsulfonyl); Preferably, M' is selected from (For example ). The compound of claim 11 or 12, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled form, metabolite, or prodrug thereof, having structures A-1 to A-9 and E-1 to E-6:



A composition comprising one or more antibody drug conjugates according to any one of claims 1 to 10, wherein the DAR value (drug-antibody conjugate ratio) of the composition is about 1-10, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 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, and for example, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6. 1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6.73, about 6.74, about 6.75, about 6.76, about 6. 77, about 6.78, about 6.79, about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.9, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99 , about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, about 7.1, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7.16, about 7.17, about 7.18, about 7.19, about 7.2, about 7.21, about 7 .22, about 7.23, about 7.24, about 7.25, about 7.26, about 7.27, about 7.28, about 7.29, about 7.3, about 7.31, about 7.32, about 7.33, about 7.34, about 7.35, about 7.36, about 7.37, about 7.38, about 7.39, about 7.4, about 7.41, about 7.42, about 7.43, about 7. 44, about 7.45, about 7.46, about 7.47, about 7.48, about 7.49, about 7.5, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about 7.6, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66 , about 7.67, about 7.68, about 7.69, about 7.7, about 7.71, about 7.72, about 7.73, about 7.74, about 7.75, about 7.76, about 7.77, about 7.78, about 7.79, about 7.8, about 7.81, about 7.82, about 7.83, about 7.84, about 7.85, about 7.86, about 7.87, about 7.88, about 7.89, about 7.9, about 7.91, about 7.92, about 7.93, about 7.94, about 7.95, about 7.96, about 7.97, about 7.98, about 7.99, about 8.0, about 8.01, about 8.02, about 8.03, about 8.04, about 8.05, about 8.06, about 8.07, about 8.08, about 8.09, about 8.1, about 8.1 1, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.23, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33 , about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, about 8.42, about 8.43, about 8.44, about 8.45, about 8.46, about 8.47, about 8.48, about 8.49, about 8.5, about 8.51, about 8.52, about 8.53, about 8.54, about 8.55, about 8.56, about 8.57, about 8.58, about 8.59, about 8.6, about 8.61, about 8.62, about 8.63, about 8.64, about 8.65, about 8.66, about 8.67, about 8.68, about 8.69, about 8.7, about 8.71, about 8.72, about 8.73, about 8.74, about 8.75, about 8.76, about 8.77, about 8. 78, about 8.79, about 8.8, about 8.81, about 8.82, about 8.83, about 8.84, about 8.85, about 8.86, about 8.87, about 8.88, about 8.89, about 8.9, about 8.91, about 8.92, about 8.93, about 8.94, about 8.95, about 8.96, about 8.97, about 8.98, about 8.99, about 9.0. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 10, the compound of any one of claims 11 to 13, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, or the composition of claim 14, and one or more pharmaceutically acceptable carriers. A medicine kit product, comprising: a) at least one antibody-drug conjugate according to any one of claims 1 to 10, a compound according to any one of claims 11 to 13, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled, metabolite, or prodrug thereof, or a composition according to claim 14 or 15 as a first therapeutic agent; b) optionally at least one additional therapeutic agent as a second therapeutic agent, or a composition comprising an additional therapeutic agent; and c) optional packaging and/or instructions. Use of the antibody-drug conjugate according to any one of claims 1 to 10, the compound according to any one of claims 11 to 13, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, and prodrugs thereof, the composition according to claim 14 or 15, or the kit according to claim 16 in the preparation of a medicament for treating diseases involving abnormal cell proliferation; Preferably, the disease is cancer or a tumor, such as an advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma. A method for treating a disease involving abnormal cell proliferation, comprising the steps of: administering a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 10, the compound of any one of claims 11 to 13, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, and prodrug thereof, or the composition of claim 14 or 15, or the kit of claim 16 to an individual in need thereof; Preferably, the disease is cancer or a tumor, such as an advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma. The antibody-drug conjugate of any one of claims 1 to 10, the compound of any one of claims 11 to 13, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, and prodrugs thereof, the composition of claim 14 or 15, or the kit of claim 16, for use in treating diseases involving abnormal cell proliferation; Preferably, the disease is cancer or a tumor, such as an advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma. A linking unit represented by the formula -MLE-, wherein M, L and E are as described in any one of claims 1 to 10, preferably selected from the following structures:
(For example ), (For example ), (For example ), (For example ), (For example ), s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 5, 8, 10. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, comprising the linker unit of claim 20 and at least one of Ab and/or D, wherein the linker unit is linked to Ab and/or D; wherein Ab and D are as defined in any one of claims 1 to 10. The compound shown below or its salt, stereoisomer, tautomer or isotope-labeled compound:




in PG ₁ is each independently H or an amino protecting group, such as an alkoxycarbonyl amino protecting group, for example, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, for example, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group, for example, trityl (Trt), C _1-6 alkyl-substituted trityl, p-methoxytrityl (MMT), dimethoxytrityl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn); PG ₂ is each independently H or a carboxyl protecting group, such as C _1-6 alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl; PG ₃ is each independently H or a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, trityl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl; s1 is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Lg is as defined in any one of claims 11 to 13; Preferably, the compound is selected from: