Topoisomerase inhibitor and application of conjugate thereof

Abstract

The invention relates to application of a topoisomerase inhibitor and a conjugate thereof. Specifically, the inventor provides a class of camptothecin derivatives, linker conjugates and antibody-drug conjugates with novel structures, and the camptothecin derivatives, linker conjugates and antibody-drug conjugates have excellent anti-tumor activity and good lipophilicity. In-vitro and in-vivo experiments show that the drug conjugate containing the camptothecin derivative disclosed by the invention has an improved anti-tumor curative effect and high safety, and has an application prospect in the field of cancer treatment.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511561535.0 (22) Application Date 2025.10.29 (71) Applicant Keyi (Zhejiang) Pharmaceutical Technology Co., Ltd. Address 313399 Room 302, Building 1, Zhejiang North Life and Health Micro-industry Park, Yuedu Middle Road, Dipu Street, Anji County, Huzhou City, Zhejiang Province (Self-declaration) (72) Inventors Cai Xianghai, Wu Guoxiang, Ma Rui (74) Patent Agency Shanghai Yiping Intellectual Property Agency Co., Ltd. 31266 Patent Attorney Xu Xun, Cui Jiajia (51) Int.Cl. C07D 491 / 22 (2006.01) A61K 31 / 4745 (2006.01) C07K 7 / 02 (2006.01) A61K 47 / 65 (2017.01) A61K 47 / 68 (2017.01) A61P 35 / 00 (2006.01) (54) Invention Title: Application of a Topoisomerase Inhibitor and its Conjugates (57) Abstract: This invention relates to the application of a topoisomerase inhibitor and its conjugates. Specifically, the inventors provide a class of novel camptothecin derivatives, linker conjugates, and antibody-drug conjugates with excellent antitumor activity and good lipophilicity. In vitro and in vivo experiments show that drug conjugates containing the camptothecin derivatives of this invention have enhanced antitumor efficacy and high safety, and have application prospects in the field of cancer treatment. Claims (8 pages), Description (30 pages), Sequence List (electronic publication), Drawings (8 pages), CN 121449623 A, 2026.02.03, CN 1 21 44 96 23 A 1. A compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the structure of the compound is as shown in formula (I) or formula (II): wherein Y is selected from the group consisting of: 2. The compound of claim 1, characterized in that the compound is selected from the group consisting of: Table A Claims (1 / 8 pages) 2 CN 121449623 A Claims (2 / 8 pages) 3 CN 121449623 A 3. A linker conjugate, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the general formula of the linker conjugate is as shown in formula (III) or formula (IV): Claims (3 / 8 pages) 4 CN 121449623 A wherein Y is selected from the group consisting of: L1 is a polypeptide residue selected from the following group: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-; L2 is a linker unit for linking with the antibody, and is 4. The linker conjugate according to claim 3, characterized in that the linker conjugate is a compound selected from Table B: Table B Claims 4 / 8 pages 5 CN 121449623 A Claims 5 / 8 pages 6 CN 121449623 A 5. An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, characterized in that the general formula of the antibody-drug conjugate is shown in formula (V) or formula (VI): Wherein, Y Selected from the following groups: L1 is a polypeptide residue selected from the following groups: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-; L2 is a linker unit for linking with the antibody, where Ab is the antibody or its antigen-binding fragment; n is an integer or decimal from 2 to 8. Claims 6 / 8, Page 7, CN 121449623 A 6. The antibody-drug conjugate (ADC) of claim 5, characterized in that the antibody-drug conjugate is a compound selected from Table C: Table C Claims 7 / 8, Page 8, CN 121449623 A 7. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises (a) the antibody-drug conjugate of claim 5 or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient. 8. Use of the antibody-drug conjugate of claim 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 7, in the preparation of a medicament for treating tumors or cancer. 9. The use of claim 8, characterized in that the tumor or cancer is selected from the group consisting of: colorectal cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, etc.Cellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, gastric cancer, glioma, mesothelioma. 10. A method for preparing the antibody-drug conjugate as described in claim 5, characterized in that it includes the steps of: (1) reacting an antibody with a reducing agent in a buffer solution to obtain a reduced antibody; (2) crosslinking (coupling) the reduced antibody obtained in step (1) with a compound of formula (III) or (IV) in a mixture of buffer solution and organic solvent to obtain the antibody-drug conjugate. Claims 8 / 8 pages 9 CN 121449623 A Application of a topoisomerase inhibitor and its conjugate in the field of technical field

[0001] This invention relates to the field of medicinal chemistry, specifically to the application of a topoisomerase inhibitor and its conjugate. Background Art

[0002] The basic components of an antibody-drug conjugate (ADC) include an antibody or antibody-like ligand, a linker, and a cytotoxic small molecule. Antibody-drug conjugates (ADCs) utilize the specific recognition of antigens by antibodies to transport small molecule drugs into or near target cells and effectively release toxic molecules to achieve therapeutic purposes. ADCs are characterized by suitable molecular weight, high stability, strong targeting, and low toxicity, making them promising anti-tumor drugs.

[0003] The cytotoxic small molecule in antibody-drug conjugates can be a camptothecin derivative, which exerts its anti-tumor effect by inhibiting topoisomerase I. Recently, Immunomedics developed a novel ADC drug, IMMU-132 (Sacituzumab Govitecan), which uses the camptothecin derivative SN-38 (the active metabolite of irinotecan) as a toxin molecule. It has shown good anti-tumor effects. IMMU-132 uses a pH-sensitive linker, which can release toxins near tumor cells to kill them. However, its stability is poor, and the released SN-38 is prone to phenolic glycosylation, thus producing unnecessary toxicity. Daiichi Sankyo and AstraZeneca jointly developed another camptothecin derivative, Dxd (an amide derivative formed from eczema and glycolic acid), as an ADC drug with a toxin molecule, DS-8201a (Enhertu). It also exhibited good antitumor effects. DS-8201a uses a tetrapeptide linker, -Gly-Gly-Phe-Gly-, which has good stability, but still suffers from poor viability, low safety, and tumor resistance.

[0004] Therefore, there is an urgent need in the art to develop camptothecin derivatives with superior antitumor activity to improve their safety and efficacy in ADC drug applications, thereby obtaining antitumor drugs with excellent efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide camptothecin derivatives, linker conjugates, and antibody-drug conjugates and their applications in antitumor therapy.

[0006] In a first aspect of the invention, a compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided, the structure of which is shown in formula (I) or formula (II):

[0007] Specification 1 / 30 page 10 CN 121449623 A

[0008] wherein, Y is selected from the group consisting of:

[0009] In another preferred embodiment, the structure of the compound is shown in formula (I).

[0010] In another preferred embodiment, Y is selected from the group consisting of:

[0011] In another preferred embodiment, the structure of the compound is as shown in formula (I), and Y is selected from the group consisting of:

[0012] In another preferred embodiment, the compound is selected from the group consisting of:

[0013] Table A

[0014] Specification 2 / 30 pages 11 CN 121449623 A

[0015] Specification 3 / 30 pages 12 CN 121449623 A

[0016]

[0017] In a second aspect of the invention, a linker conjugate, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided, the general formula of which is shown in formula (III) or formula (IV): Specification 4 / 30 pages 13 CN 121449623 A

[0018]

[0019] wherein Y is selected from the group consisting of:

[0020] L1 is a polypeptide residue selected from the group consisting of: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-;

[0021] L2 is a linker unit for linking with the antibody, and is

[0022] In another preferred embodiment, L1 is -Gly-Gly-Phe-Gly-.

[0023] In another preferred embodiment, the structure of the linker conjugate is as shown in formula (III).

[0024] In another preferred embodiment, the Y is selected from the group consisting of:

[0025] In another preferred embodiment, the structure of the linker conjugate is as shown in formula (III), and the Y is selected from the group consisting of: Specification 5 / 30 page 14 CN 121449623 A

[0026] In another preferred embodiment, the linker conjugate is a compound selected from Table B:

[0027] Table B

[0028] Specification 6 / 30 page 15 CN 121449623 A

[0029]

[0030] In a third aspect of the invention, an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof is provided, the general formula of said antibody-drug conjugate being shown in formula (V) or formula (VI): Specification 7 / 30 pages 16 CN 121449623 A

[0031]

[0032] wherein, Y is selected from the group consisting of:

[0033] L1 is a polypeptide residue selected from the group consisting of: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, - Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-;

[0034] L2 is a linker unit for connecting with the antibody, and

[0035] Ab is the antibody or its antigen-binding fragment;

[0036] n is an integer or decimal from 2 to 8.

[0037] In another preferred embodiment, L1 is -Gly-Gly-Phe-Gly-.

[0038] In another preferred embodiment, n is 2 to 5, preferably 3 to 4.

[0039] In another preferred embodiment, the structure of the antibody-drug conjugate is shown in formula (V).

[0040] In another preferred embodiment, Y is selected from the group consisting of: Specification 8 / 30 page 17 CN 121449623 A

[0041] In another preferred embodiment, the structure of the antibody-drug conjugate is shown in formula (V), and Y is selected from the group consisting of:

[0042] In another preferred embodiment, the antibody is a double-chain antibody, a single-chain antibody, or a single-domain antibody / nanobody.

[0043] In another preferred embodiment, the antibody includes a bivalent antibody or a multivalent antibody.

[0044] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, or a chimeric antibody.

[0045] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0046] In another preferred embodiment, the antibody targets targets selected from the group consisting of: EGFR, HER2, HER3, BCMA, B7-H3, TF, CD70, CD73, AXL, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H6, GPC3, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, Trop2, TIGIT, LAG-3, TLR7, or combinations thereof, but is not limited thereto.

[0047] In another preferred embodiment, the sequence of the antibody is shown in SEQ ID NO:1.

[0048] In another preferred embodiment, the antigen-binding fragment comprises: (i) a Fab fragment; (ii) an F(ab')2 fragment; (iii) an Fd fragment; (iv) an Fv fragment; (v) a single-chain Fv (scFv) molecule; and (vi) a dAb fragment.

[0049] In another preferred embodiment, the antibody-drug conjugate is a compound selected from Table C:

[0050] Table C

[0051] Specification 9 / 30 pages 18 CN 121449623 A

[0052] Specification 10 / 30 pages 19 CN 121449623 A

[0053]

[0054] In a fourth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising (a) an antibody-drug conjugate as described in the third aspect of the invention or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

[0055] In a fifth aspect of the invention, the use of an antibody-drug conjugate as described in the third aspect of the invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the fourth aspect of the invention, in the preparation of a medicament for treating tumors or cancer is provided.

[0056] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: colorectal cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, gastric cancer, glioma, mesothelioma, but not limited thereto.

[0057] In a sixth aspect of the invention, a method for preparing an antibody-drug conjugate is provided, comprising the steps of:

[0058] (1) reacting an antibody with a reducing agent in a buffer solution to obtain a reduced antibody;

[0059] (2) crosslinking (conjugating) a compound of formula (III) or (IV) with the reduced antibody obtained in step (1) in a mixture of buffer solution and organic solvent to obtain the antibody-drug conjugate.

[0060] In another preferred embodiment, the present invention also provides a method for preparing an antibody-drug conjugate, specifically comprising the following steps:

[0061] (1) providing a reaction system, wherein a drug small molecule and a polypeptide residue are linked at a specific site, and a specific linker unit is attached to the N-terminus of the polypeptide residue, thereby obtaining a linker conjugate;

[0062] (2) providing another reaction system, wherein the reaction system comprises an antibody and a linker conjugate, wherein the linker conjugate comprises a drug small molecule, a polypeptide residue and a linker unit; Specification 11 / 30 pages 20 CN 121449623 A

[0063] (3) in the reaction system, the antibody and the linker conjugate are coupled to obtain the antibody-drug conjugate as described above.The antibody-drug conjugate as described in the third aspect of the invention.

[0064] In a seventh aspect of the invention, a method for treating a tumor or cancer is provided, comprising the steps of administering to a subject in need an antibody-drug conjugate as described in the third aspect of the invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the fourth aspect of the invention.

[0065] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: colorectal cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, gastric cancer, glioma, mesothelioma, but not limited thereto.

[0066] It should be understood that within the scope of the invention, the above-described technical features of the invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0067] Figure 1 shows the chromatogram of L-MY71 (DAD18, Sig = 214 nm).

[0068] Figure 2 shows the chromatogram of L-MY72 (DAD18, Sig = 214 nm).

[0069] Figure 3 shows the chromatogram of L-MY73 (DAD18, Sig = 214 nm).

[0070] Figure 4 shows the chromatogram of L-MY74 (DAD18, Sig = 214 nm).

[0071] Figure 5 shows the chromatogram of L-MY78 (DAD18, Sig = 214 nm).

[0072] Figure 6 shows the toxicity evaluation of the antibody-drug conjugate against colorectal cancer cells. ADC-1, ADC-2, ADC-3, and ADC-5 effectively killed HT29 (Fig. 6-A), HCT116 (Fig. 6-B), RKO (Fig. 6-C), SW480 (Fig. 6-D), LS1034 (Fig. 6-E), and COLO320 (Fig. 6-F) colorectal cancer cells.

[0073] Fig. 7 shows the cytotoxicity evaluation of antibody-drug conjugates against colorectal cancer cells. ADC-5 effectively killed ASPC1 (Fig. 7-A), BxPC3 (Fig. 7-B), PANC1 (Fig. 7-C), and SW1990 (Fig. 7-D) pancreatic cancer cells.

[0074] Fig. 8 shows the cytotoxicity evaluation of antibody-drug conjugate ADC-5 compared to ADC-7. ADC-5 was more effective than ADC-7 in killing ASPC-1 (Fig. 8-A), BxPC3 (Fig. 8-B), HCT-116 (Fig. 8-C), and NCI-H1975 (Fig. 8-D).

[0075] Figure 9 shows tumor growth in the HT29 xenograft mouse model (Figure 9-A) and mouse weight (Figure 9-B).

[0076] Figure 10 shows tumor growth in the RKO xenograft mouse model (Figure 10-A) and mouse weight (Figure 10-B).

[0077] Figure 11 shows tumor growth in a PANC-1 xenograft mouse model (Figure 11-A) and mouse body weight (Figure 11-B). Detailed Embodiments

[0078] Through extensive and in-depth research, the inventors have developed a class of camptothecin derivatives with novel structures, exhibiting superior antitumor activity, good lipophilicity, and membrane permeability compared to compounds with similar structures. Based on this, novel camptothecin derivative-linker conjugates and antibody-drug conjugates containing novel camptothecin derivatives have been further developed. In vitro and in vivo experiments have verified that antibody-drug conjugates containing novel camptothecin derivatives have enhanced antitumor efficacy and high safety. Based on this, the present invention has been completed.

[0079] Definitions

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Specification 12 / 30 pages 21 CN 121449623 A

[0081] As used herein, the terms “containing” or “comprising” can be open, semi-closed, and closed. In other words, the terms also include “consisting substantially of” or “consisting of”.

[0082] Hereinafter, “optionally” means that the events or conditions described below may, but are not necessarily, occur, and the description includes both the occurrence of the events or conditions and the absence of the events or conditions.

[0083] Unless otherwise stated, the structural formulas described in this invention are intended to include all isomers (e.g., enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R, S configurations containing an asymmetric center, (Z), (E) isomers of double bonds, etc. Therefore, individual stereochemical isomers of the compounds of this invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are within the scope of this invention.

[0084] As used herein, the term "tautomer" means that structural isomers with different energies can cross a low energy barrier and thus interconvert. Valence tautomers include interconversion through some bonding electron recombination.

[0085] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining specific embodiments with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art, with preferred embodiments including but not limited to the embodiments of this application.

[0086] Compounds

[0087] As used herein, the terms "small molecule drug," "small drug molecule," and "camptothecin derivative" are used interchangeably and all refer to compounds having the structure shown in formula (I) or formula (II).

[0088] In a preferred embodiment, the small molecule drug has the structure of formula (I), and Y is selected from the group consisting of:Preferably, Y is selected from the group below: More preferably, Y is

[0089] In a preferred embodiment, the small molecule drug has the structure of formula (II), and Y is

[0090] In a preferred embodiment, the small molecule drug is selected from compounds MY78, MY71, MY72, MY73, MY74, MY75, MY76, MY77, and MY79 in Table A.

[0091] The small molecule drug of the present invention can be used as the drug portion in an antibody-drug conjugate to exert antitumor effects as a topoisomerase I inhibitor.

[0092] In the present invention, the reference compound for the small molecule drug is Dxd, whose structural formula is shown below: Specification 13 / 30 pages 22 CN 121449623 A

[0093]

[0094] Linker conjugate

[0095] The small molecule drug provided by the present invention can be linked with a linker to form a conjugate.

[0096] In the present invention, the terms "linker" and "connector" are used interchangeably. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, tripeptides, or tetrapeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0097] Before being linked to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and the linking is achieved through the active reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide linked to the antibody via a thiosuccinimide.

[0098] In a preferred embodiment, the linker unit coupled to the antibody in the linker of the present invention has the following structure:

[0099] In a preferred embodiment, the peptide linker is selected from the group consisting of: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-; preferably -Gly-Gly-Phe-Gly-.

[0100] The structure of the linker conjugate of the present invention is shown in formula (III) or (IV). In a preferred embodiment, the linker conjugate is selected from compounds L-MY78, L-MY71, L-MY72, L-MY73, L-MY74, L-MY75, L-MY76, L-MY77, and L-MY79 in Table B.

[0101] In this invention, the reference compound for the linker conjugate is Deruxtecan (MC-GGFG-DXD), purchased from MedChemExpress (MCE) and EO (from structure 11 in Hengrui patent WO2020063673A1). Specification 14 / 30 pages 23 CN 121449623 A

[0102]

[0103] Antibody-Drug Conjugate (ADC)

[0104] This invention also provides antibody-drug conjugates (ADCs) based on small molecule drugs or linker conjugates of this invention.

[0105] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetragonal protein of approximately 150,000 Daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also have regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0106] This invention includes not only complete antibodies but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0107] In this invention, antibodies include mouse, chimeric, and humanized antibodies prepared using techniques well known to those skilled in the art.Or fully human antibodies. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained through standard DNA recombination techniques.

[0108] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0109] In a preferred embodiment, the antibody of this invention is an anti-EGFR nanobody. In a preferred embodiment, the anti-EGFR nanobody is named KY-301-58, and its amino acid sequence is shown in SEQ ID NO:1:

[0110] EVQVVESGGGLVQAGGSLRLSCAASGSTFSSYAMGWYRQAPGKQRESVAFISSGGSTNVAESVKGRFT ISRDNAKNTVYLQMNNLKAEDTAVYYCNLHPRVNWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0111] The structure of the ADC of the present invention is shown in formula (V) or (VI). In a preferred embodiment, the ADC is selected from compounds 1 to 9 in Table C.

[0112] In this disclosure, DAR (drug antibody ratio) refers to the number of drug molecules conjugated to an antibody, for example, n in formula (V) or (VI). The number of drug molecules contained in the antibody-drug conjugate described herein can be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules conjugated to each antibody. "n is any value between 2 and 8" means that n can be any integer selected between 2 and 8 (including the endpoints 2 and 8) or any decimal selected between 2 and 8. Meanwhile, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches may not be exactly the same. For example, they may fluctuate within a range not exceeding 0.5 on or off page 15 / 30 of the specification, CN 121449623 A.

[0113] DAR can be determined by conventional means, such as mass spectrometry, ELISA, HIC, and HPLC. It can also be determined by...Quantitative distribution of ADC in the n aspect. In some cases, the separation, purification and verification of homogeneous ADCs with a certain n value from ADCs with other drug loadings can be achieved by means such as HIC, reversed-phase HPLC or electrophoresis.

[0114] Pharmaceutical composition and administration method

[0115] Since the antibody-drug conjugates of the present invention have excellent inhibitory activity against tumor cell proliferation, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used for the prevention and / or treatment (stabilization, mitigation or cure) of diseases related to tumor cell proliferation.

[0116] The pharmaceutical compositions of the present invention contain the compounds of the present invention and pharmaceutically acceptable excipients or carriers within a safe and effective range. Wherein, "safe and effective range" means that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 1-200 mg of the compound of the present invention per dose. Preferably, "one dose" is a capsule or tablet.

[0117] A “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. “Compatibility” here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g.), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0118] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous).

[0119] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, etc.(e) sodium carbonate; (f) a slowing agent, such as paraffin; (g) an absorption accelerator, such as a quaternary ammonium compound; (h) a wetting agent, such as cetyl alcohol and glyceryl monostearate; (i) an adsorbent, such as kaolin; and (v) a lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0120] Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such a composition may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0121] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0122] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0123] In addition to the active compound, the suspension may contain a suspending agent, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methacrylate and agar, or mixtures thereof.

[0124] The composition for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous water, dispersion, suspension or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0125] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0126] When administered in combination, the pharmaceutical composition further includes one or more (two, three, four or more) other pharmaceutically acceptable therapeutic agents. One or more (two, three, four, or more) of the other pharmaceutically acceptable therapeutic agents may be used simultaneously, separately, or sequentially with the compounds of the present invention for the prevention and / or treatment of cytokine and / or interferon-mediated diseases.

[0127] When using the pharmaceutical composition, a safe and effective amount of the compounds of the present invention is applied to the mammals requiring treatment.(e.g., human), where the dosage at administration is the pharmaceutically considered effective dosage. For a person weighing 60kg, the daily dosage is usually 1-2000mg, preferably 1-500mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0128] The beneficial effects of the present invention include:

[0129] 1. The various novel camptothecin derivatives provided by the present invention have significantly improved lipophilicity and membrane permeability compared with the reference compound Dxd.

[0130] 2. The ADC containing novel camptothecin derivatives provided by the present invention shows enhanced killing activity against tumor cells in in vitro and in vivo experiments compared with the control ADC.

[0131] 3. The ADC containing novel camptothecin derivatives provided by the present invention shows excellent tumor-suppressing effects against various solid tumors, such as colorectal cancer and pancreatic cancer, and has application prospects.

[0132] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, experimental methods in the following examples are generally performed under the conditions described in the standard conditions section or as recommended by the manufacturer. Percentages and fractions are weight percentages and weight fractions unless otherwise stated. Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C–35°C).

[0133] Example 1: Synthesis of L-MY71

[0134] 2-{[5-benzyl-18-(2,5-dioxane-2,5-dihydro-1H-pyrrole-1-yl)-1,4,7,10,13-pentoxane-3,6,9,12-tetraazaoctadecane-1-yl]amino}-2-(4-chlorophenyl)-N-[(10R,23R)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxane-4,15-diaza-8-oxahexane[14.7.1 .020,24.02,14.04, 13.06,11]tetracosyl-1(24),2(14),6(11) [12,15,17,19-Heptaene-23-yl]acetamide Specification 17 / 30 Page 26 CN 121449623 A

[0135]

[0136] Step 1: Add NHS (59.8mg,0.51mmol) and EDCI (98.61mg,0.51mmol) to a DCM (8mL) solution containing 1 (150mg,0.51mmol), stir and react at 20°C for 1 hour, then slowly add a DMF (8mL) solution containing 2 (273.86mg,0.51mmol) and TEA (0.071mL,0.51mmol), stir and react at 25°C for 16 hours, TLC showsThe reaction was completed. Then, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 × 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was directly purified by silica gel column chromatography (DCM:EA = 1:3) to obtain a yellow solid of 3130 mg, with a yield of 80.99%.

[0137] LCMS (ESI): tR = 2.088, tR = 2.183, m / z = 703.3 [M+H]+.

[0138] Second step: The obtained 3 (240.0 mg, 0.34 mmol) was redissolved in DCM (4 mL), and TFA (4 mL) was added. The mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by Prep-HPLC (H2O (0.1% TFA)-ACN) to obtain a yellow solid of 4180.0 mg, with a yield of 80.44%.

[0139] LCMS(ESI): tR=2.008, tR=2.312, m / z=603.2[M+H]+.

[0140] Step 3: Add NHS (21.07mg, 0.18mmol) and EDCI (34.74mg, 0.18mmol) to a DCM (1mL) solution containing 4 (86.34mg, 0.141mmol), stir and react at 20°C for 1 hour, then slowly add a DMF (2mL) solution containing MC-GGFG (96.94mg, 0.18mmol), stir and react at 25°C for 4 hours, and TLC shows that the reaction is complete. Subsequently, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 × 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was purified directly by silica gel column chromatography (DCM:EA = 1:3) to give 78.37 mg of yellow solid L-MY71, with a yield of 39.19%.

[0141] LCMS (ESI): tR=2.982, tR=3.131, m / z=1114.5[M+H]+.

[0142] HPLC: tR=5.24, tR=5.48, Purity: 99%.

[0143] 1HNMR (400MHz, DMSO‑d6) δ8.92(dd,J=67.0,8.5Hz,1H) ,8.65(dd,J=7.9,3.1Hz, 1H) ,8.22(dt,J=30.4,5.8Hz,1H) ,8.13‑8.00(m,2H) ,7.96(dt,J=14.4,5.6Hz,1H) ,7.79 (dd,J=11.0,8.0Hz,1H) ,7.52‑7.42(m,1H) ,7.36‑7.23(m,3H) ,7.23‑7.14(m,5H) ,6.98(s, 2H) ,5.58‑5.31(m,4H),5.24(d,J=12.8Hz,1H) ,5.05(d,J=19.0Hz,1H) ,4.48(qd,J=8.7, 4.3Hz ,1H) ,4.17(d ,J=18.9Hz ,3H) ,3.82(dd ,J=5.7 ,2.9Hz ,2H) ,3.73(dd ,J=16.6 ,5.6Hz,1H) ,3.65(t,J=5.4Hz,2H) ,3.58(dt,J=16.7,5.4Hz,1H) ,3.35(td,J=7.1,3.1Hz, 2H) ,3.27-3.07(m,2H) ,2.98(dd,J=13.8,4.1Hz,1H) ,2.74(dd,J=13.8,9.8Hz,1H) ,2.44- 2.33(m,3H) ,2.32-2.19(m,1H) ,2.08(q,J=7.9Hz,3H) ,1.86(hept,J=7.3Hz,2H) ,1.46(h, J=7.0Hz,4H) ,1.18(dtd,J=14.4,10.5,9.2,6.3Hz,2H) ,0.88(q,J=7.6Hz,3H) .

[0144] The chromatogram of compound L-MY71 is shown in Figure 1. Instruction manual 18 / 30 pages 27 CN 121449623 A

[0145] Example 2: Synthesis of L-MY72

[0146] 5-{[5-benzyl-18-(2,5-dioxane-2,5-dihydro-1H-pyrrole-1-yl)-1,4,7,10,13-pentaoxane-3,6,9,12-tetraazaoctadecane-1-yl]amino}-N-[(10R,23R)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxane-4,15-diaza-8-oxahexane[14.7.1 .020,24.02,14.04,13.06,11]tetracosyl-1(24),2(14),6(11) ,12,15,17,19-Heptaeno-23-yl]-2-methoxybenzamide

[0147]

[0148] Step 1: Add NHS (62.61 mg, 0.53 mmol) and EDCI (103.24 mg, 0.53 mmol) to a DCM (8 mL) solution containing 1 (150 mg, 0.53 mmol), stir and react at 20 °C for 1 hour, then slowly add a DMF (8 mL) solution containing 2 (240.04 mg, 0.44 mmol) and TEA (0.148 mL, 0.533 mmol), stir and react at 25 °C for 16 hours.After 2 hours, TLC showed that the reaction was complete. Then, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 × 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was directly purified by silica gel column chromatography (DCM:EA = 1:3) to obtain 3130 mg of yellow solid, with a yield of 80.99%.

[0149] LCMS (ESI): tR = 0.913, m / z = 685.4 [M+H]+.

[0150] Second step: The obtained 3 (100.0 mg, 0.146 mmol) was redissolved in DCM (2 mL), and TFA (10.85 μ L) was added. The reaction was stirred at 0 °C for 2 hours. The reaction solution was directly purified by Prep-HPLC (H2O (0.1% TFA)-ACN) to obtain 480.0 mg of yellow solid, with a yield of 93.7%.

[0151] LCMS(ESI): tR=2.058, m / z=585.2[M+H]+.

[0152] Step 3: Add NHS (21.07mg, 0.18mmol) and EDCI (34.74mg, 0.18mmol) to a DCM (2mL) solution containing 4 (70mg, 0.12mmol), stir and react at 20°C for 1 hour, then slowly add a DMF (2mL) solution containing MC-GGFG (63.4mg, 0.12mmol), stir and react at 25°C for 4 hours, and TLC shows that the reaction is complete. Subsequently, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 x 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was purified directly by silica gel column chromatography (DCM:EA = 1:3) to obtain 30 mg of yellow solid L-MY72, with a yield of 22.86%.

[0153] LCMS (ESI): tR=2.982, tR=2.901, m / z=1096.5[M+H]+.

[0154] HPLC: tR=5.21, Purity: 99%.

[0155] 1H NMR (400MHz, DMSO-d6) δ9.81 (s, 1H), 8 .80(d,J=8.5Hz,1H),8.38(t,J=5.8Hz,1H),8.17(d,J=7.9Hz,1H),8.04(dt,J=11.5,5.6Hz,2H),7.94(d,J=2.7Hz,1H), 7.84‑7 .65(m ,2H) ,7 .31 (s, 1H), 7.30–7.13 (m, 5H), 7.09 (d, J = 9.0 Hz, 1H), 6.98 (s, 2H), Specification page 19 / 30, 28 CN 121449623 A6.51(s,1H) ,5.75(d ,J=6.5Hz,1H) ,5.52‑5.08(m,5H) ,4.49(d ,J=4.6Hz,1H) ,3.85(dd ,J=13.3,5.7Hz,2H) ,3.78(s ,3H) ,3.74(d ,J=5.7Hz ,1H) ,3.64(dd ,J=16.5,8.9Hz,4H) , 3.24‑3.15(m,2H) ,3.06(s,1H) ,2.83(s,1H) ,2.40(s,3H) ,2.35‑2.22(m,2H) ,2.09(t,J=7.4Hz,2H) ,1.93-1.76(m,2H) ,1.45(q,J=7.4Hz,4H) ,1.17(t,J=7.7Hz,2H) ,0.86(t,J=7.3Hz,3H) .

[0156] The chromatogram of compound L-MY72 is shown in Figure 2.

[0157] Example 3: Synthesis of L-MY73

[0158] 2-{[5-benzyl-18-(2,5-dioxane-2,5-dihydro-1H-pyrrole-1-yl)-1,4,7,10,13-pentoxane-3,6,9,12-tetraazaoctadecane-1-yl]amino}-N-[(10R,23R)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxane-4,15-diaza-8-oxahexane[14.7.1 .020,24.02,14.04,13.06,11]tetracosyl-1(24),2(14),6(11) ,12,15,17,19-Heptaene-23-yl]-4-phenylbutyramide

[0159]

[0160] Step 1: Add NHS (62.61 mg, 0.53 mmol) and EDCI (103.24 mg, 0.53 mmol) to a DCM (8 mL) solution containing 1 (148 mg, 0.53 mmol), stir and react at 20 °C for 1 hour, then slowly add a DMF (8 mL) solution containing 2 (240.04 mg, 0.44 mmol) and TEA (0.148 mL, 0.533 mmol), stir and react at 25 °C for 16 hours, and TLC shows that the reaction is complete. Then, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 × 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was purified directly by silica gel column chromatography (DCM:EA = 1:3) to give 3130 mg of a yellow solid, with a yield of 80.99%.

[0161] LCMS (ESI): tR = 0.913, m / z = 685.4 [M+H]+.

[0162] Step 2: The obtained 3 (101.7 mg, 0.146 mmol) was redissolved in DCM (2 mL), and TFA (10.85 μ L) was added. The mixture was stirred at 0 °C for 2 hours. The reaction solution was directly purified by Prep-HPLC (H2O (0.1% TFA)-ACN) to obtain 480.0 mg of yellow solid, with a yield of 93.7%.

[0163] LCMS(ESI): tR=2.058, m / z=585.2[M+H]+.

[0164] Step 3: Add NHS (21.07mg, 0.18mmol) and EDCI (34.74mg, 0.18mmol) to a DCM (2mL) solution containing 4 (71.6mg, 0.12mmol), stir and react at 20°C for 1 hour, then slowly add a DMF (2mL) solution containing MC-GGFG (63.4mg, 0.12mmol), stir and react at 25°C for 4 hours, and TLC shows that the reaction is complete. Subsequently, H2O (20 mL) and DCM (30 mL) were added for extraction. The organic phase was washed twice with H2O (2 x 20 mL), dried over anhydrous sodium sulfate, and the resulting solution was purified directly by silica gel column chromatography (DCM:EA = 1:3) to obtain 30 mg of yellow solid L-MY73, with a yield of 21.36%. Instruction manual, pages 20 / 30, CN 121449623 A

[0165] LCMS (ESI): tR = 1.364, m / z = 1109.3 [M+H]+.

[0166] HPLC: tR = 2.018, Purity: 99%.

[0167] 1H NMR (400MHz, dmso) δ 8.54 (d, J = 8.3Hz, 1H), 8.27 (t, J = 5.2Hz, 1H), 8.17–8.05 (m, 3H), 8.01 (t, J = 5.3Hz, 1H), 7.73 (d, J = 10.9Hz, 1H), 7.27–7.09 (m, 11H), 6.98 (s, 2H), 6.51 (s, 1H) ,5.61-5.49(m,1H) ,5.40(s,2H) ,5.19(dd,J=40.7,19.0Hz,2H) ,4.35(ddd,J=30.2,12.6,8.9Hz,2H) ,3.79-3.63(m,5H) ,3.56(dd,J=16.6,5.2Hz,1H) ,3.36(s,2H) ,3.15 (t,J=10.5Hz,2H) ,2.92-2.81(m,1H) ,2.63(ddd ,J=23.6,16.3,9.7Hz,2H) ,2.37(s,3H) , 2.26-1.77(m,9H),1.44(dt,J=14.1,7.0Hz,4H) ,1.17(dd ,J=14.7,7.8Hz,2H) ,0.85(t,J =7.2Hz,3H) .

[0168] The chromatogram of compound L-MY73 is shown in Figure 3.

[0169] Example 4: Synthesis of L-MY74

[0170] 3-{[7-benzyl-20-(2,5-dioxonyl-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12,15-pentaoxonyl-2,5,8,11,14-pentazaeicosano-1-yl]oxy}-N-[(10R)-10-ethyl-10-hydroxy-5,9-dioxonyl-4,15-diaza-8-oxahexane[14.7.1 .020,24.02,14.04,13.06,11]tetracosyl-1(24),2(14),6(11),12,15,17,19-heptaen-19-yl]cyclobutaneformamide

[0171]

[0172] Step 1: Cs₂CO₃ (1285 mg, 3.95 mmol) was slowly added to a 12 mL solution of DMF containing 1 (764.65 mg, 6.585 mmol), and the mixture was stirred at 0 °C for 0.5 h. Then, BnBr (1237 mg, 7.23 mmol) was slowly added dropwise, and the mixture was stirred at 25 °C for 12 h. TLC (PE / EA = 3 / 1) showed the reaction was complete. The reaction mixture was then extracted with EtOAc (3 x 50 mL), and the organic phase was washed three times with saturated NH₄Cl (3 x 50 mL) and NaHCO₃ (3 x 50 mL) solutions. The solution was purified directly by silica gel column chromatography (EtOAc:PE = 1:4) to give 2888.05 mg of a white solid, with a yield of 65.39%.

[0173] Step 2: Dissolve the obtained 2 (1165.26 mg, 5.65 mmol) and 3 (1300 mg, 3.53 mmol) in THF (20 mL), and add Sc(OTf)3 (2171 mg, 4.41 mmol) and 4AMS (3714 mg). Stir the reaction overnight at room temperature. After the reaction is complete as detected by LCMS, the reaction solution is directly purified by silica gel column chromatography (EtOAc:PE = 1:1) to obtain a white solid 4 629.45 mg, with a yield of 21.65%.

[0174] Step 3: Dissolve the obtained 4 (370 mg, 0.67 mmol) in EtOH (10 mL) and EtOAc (5 mL), add Pd / C (36 mg, 10%), and purge the air with H2 three times. The reaction was then carried out at room temperature for 2 hours, and LCMS analysis confirmed the completion of the reaction. The reaction solution was used directly in the next step, yielding 5270 mg, with a yield of 87.38%.

[0175] Step 4: Dissolve the obtained 5 (233.45 mg, 0.55 mmol) and 6 (201 mg, 0.498 mmol) in DMF (5 mL) and bring to 0°C. Add DIPEA (193 mg, 1.49 mmol) and stir at 0°C for 5 minutes. Then add HATU (284 mg, 0.75 mmol) and stir at 0°C for 1 hour. After the reaction is complete as determined by LCMS, the reaction solution is directly purified by silica gel column chromatography (MeOH:DCM = 1:10) to obtain a white solid 7 335.68 mg, with a yield of 75.36%.

[0176] Step 5: Dissolve the obtained 7 (283 mg, 0.35 mmol) in DMF (5 mL) and bring to 0°C. Add piperidine (502 mg) and stir at 20°C for 30 minutes. After the reaction is complete as determined by LCMS, wash twice with METB (2 x 20 mL). The reaction solution is directly used for the next step of the reaction. 8 188 mg was obtained, with a yield of 91.26%.

[0177] Step 6: The obtained 8 (205 mg, 0.35 mmol) and MC-GGF (201 mg, 0.43 mmol) were dissolved in DMF (5 mL) and cooled to 0°C. DIPEA (137 mg, 1.06 mmol) was added and stirred at 0°C for 5 minutes. Then HATU (202 mg, 0.53 mmol) was added and stirred at 0°C for 1 hour. After the reaction was confirmed to be complete by LCMS, the reaction solution was directly purified by prep-HPLC to obtain a white solid L-MY74 53.5 mg, with a yield of 14.67%.

[0178] LCMS (ESI): tR=5.986, m / z=1042.3[M+H]+.

[0179] HPLC: tR=5.99, Purity: 93%.

[0180] 1H NMR (400MHz, DMSO-d6) δ9.68 (s, 1H) ,8.48(t,J=6.7Hz,1H) ,8.31(t,J=5.9Hz, 1H) ,8.15(d,J=8.0Hz,1H) ,8.07(dt,J=23.2,5.7Hz,2H) ,7.96‑7.82(m,2H) ,7.31(s,1H) , 7.25(d ,J=6.2Hz,4H) ,7.19(ddd ,J=8.6,5.7,2.4Hz,1H) ,6.98(s,1H) ,6.53(s,1H) ,5.43 (s,2H) ,5.26(s,2H) ,4.50(td ,J=8.5,3.7Hz,3H) ,4.00(p,J=7.8Hz,1H) ,3.79‑3.57(m, 7H) ,3.18‑3.03(m,4H) ,2.99‑2.75(m,5H),2.12(q,J=8.4,7.5Hz,4H) ,2.02(q,J=6.0Hz, 2H) ,1.87(hept,J=7.1Hz,2H) ,1.53-1.36(m,5H) ,1.25-1.14(m,3H) ,0.88(t,J=7.3Hz, 3H) .

[0181] The chromatogram of compound L-MY74 is shown in Figure 4.

[0182] Example 5: Synthesis of L-MY78

[0183] 1-{[7-benzyl-20-(2,5-dioxane-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12,15-pentoxane-2,5,8,11,14-pentazaeicosano-1-yl]oxy}-N-[(10R,23R)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxane-4,15-diaza-8-oxahexane[14.7.1.020,24.02,14.04,13.06,11]tetracosyl-1(24),2(14),6(11) ,12,15,17,19-Heptaene-23-yl]-3,3-Difluorocyclobutanecarboxamide

[0184]

[0185] Step 1: Slowly add Cs2CO3 (1285mg, 3.95mmol) to a DMF (12mL) solution containing 1 (1000mg, 6.585mmol), stir the reaction at 0℃ for 0.5 hours, then slowly add BnBr (1237mg, 7.23mmol), stir the reaction at 25℃ for 12 hours, and TLC (PE / EA=3 / 1) shows that the reaction is complete. The reaction solution was then extracted with EtOAc (3 x 50 mL), and the organic phase was washed with saturated NH4Cl (3 x 50 mL) and NaHCO3 (3 x 50 mL). The solution was then purified directly by silica gel column chromatography (EtOAc:PE = 1:4) to give 21340 mg of white solid, with a yield of 84.12%.

[0186] ¹H NMR (400MHz, CDCl₃) δ 7.50–7.34 (m, 5H), 5.33 (s, 2H), 3.52 (s, 1H), 3.29–3.20 (m, 2H), 2.94–2.81 (m, 2H).

[0187] Step 2: Dissolve the obtained 2 (1368 mg, 5.65 mmol) and 3 (1300 mg, 3.53 mmol) in THF (20 mL), and add Sc(OTf)₃ (2171 mg, 4.41 mg) and 4AMS (3714 mg). Stir the reaction overnight at room temperature. After the reaction is complete as detected by LCMS, the reaction solution is directly purified by silica gel column chromatography (EtOAc:PE = 1:1) to obtain white solid 4.210 mg, yield 10.81%.

[0188] LCMS (ESI): tR=3.228, m / z=573.95[M+Na]+.

[0189] Step 3: Dissolve the obtained 4 (370 mg, 0.67 mmol) in EtOH (10 mL) and EtOAc (5 mL), add Pd / C (36 mg, 10%), and purge the air with H2 three times. Then, react at room temperature for 2 hours, and the reaction is completed by LCMS. The reaction solution is directly used for the next step of the reaction to obtain 5270 mg, yield 87.38%.

[0190] LCMS (ESI): tR = 1.863, m / z = 483.81 [M+Na]+.

[0191] Step 4: Dissolve the obtained 5 (252 mg, 0.55 mmol) and 6 (217 mg, 0.498 mmol) in DMF (5 mL) at 0 °C, add DIPEA (193 mg, 1.49 mmol), stir at 0 °C for 5 minutes, then add HATU (284 mg, 0.75 mmol), stir at 0 °C for 1 hour, and after the reaction is complete as detected by LCMS, the reaction solution is directly purified by silica gel column chromatography (MeOH:DCM = 1:10) to obtain 7311 mg of white solid, yield 71.17%.

[0192] LCMS (ESI): tR = 3.044, m / z = 879.33 [M+H]+.

[0193] Step 5: Dissolve the obtained 7 (311 mg, 0.35 mmol) in DMF (5 mL) at 0 °C, add piperidine (502 mg), and stir at 20 °C for 30 minutes. The reaction was confirmed to be complete by LCMS. Wash twice with METB (2 x 20 mL), and the reaction solution was used directly for the next step. 8 220 mg was obtained, yield 94.83%.

[0194] LCMS (ESI): tR = 1.753, m / z = 657.04 [M+H]+.

[0195] Step 6: Dissolve the obtained 8 (232 mg, 0.35 mmol) and MC-GGF (201 mg, 0.43 mmol) in DMF (5 mL) and bring to 0°C. Add DIPEA (137 mg, 1.06 mmol) and stir at 0°C for 5 minutes. Then add HATU (202 mg, 0.53 mmol) and stir at 0°C for 1 hour. After the reaction is complete as detected by LCMS, the reaction solution is directly purified by prep-HPLC to obtain a white solid L-MY78 85 mg, with a yield of 21.64%.

[0196] LCMS (ESI): tR=2.423, m / z=1111.43[M+H]+.

[0197] HPLC: tR=6.625, Purity: 99%.

[0198] 1HNMR (400MHz, DMSO‑d6) δ9.01(d,J=8.8Hz,1H) ,8.80(t,J=6.8Hz,1H) ,8.23(t,J=6.0Hz,1H) ,8.02(d ,J=7.6Hz,2H) ,7.95(t,J=5.6Hz,1H) ,7 .75(d ,J=10.8Hz ,1H) , 7.30(s,1H) ,7.26‑7.07(m,5H) ,6.98(s,2H) ,6.50(s,1H) ,5.65‑5.59(m,1H) ,5.46‑5.37(m, 2H) ,5.17-5.04(m,2H) ,4.64‑4.60(m,1H) ,4.52‑4.50(m,1H) ,4.44‑4.39(m,1H) ,3.73‑3.63 (m,5H) ,3.57‑3.52(m,1H) ,3.39‑3.35(m,3H) ,3.26‑3.22(m,2H) ,3.17‑3.07(m,1H) ,3.02‑ 2.88(m ,3H) ,2.69‑2.63(m ,1H) ,2.35(s ,3H) ,2.30‑2.22(m ,2H) ,2.08(t,J=7 .6Hz,2H) , 1.90-1.79(m,2H) ,1.49-1.41(m,4H) ,1.24-1.12(m,2H) ,0.87(t,J=7.3Hz,3H) .

[0199] The chromatogram of compound L-MY78 is shown in Figure 5.

[0200] Example 6: Synthesis of ADC-1

[0201] At 37°C, EDTA aqueous solution (100mM,20μL) and TCEP aqueous solution (10mM,15.48μL) were added to the PBS buffer solution (16mg / mL,75μL) of anti-EGFR nanobody KY-301-58, and mixed well. PBS was added to 400μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0202] Compound L-MY71 was dissolved in 100 μL of DMSO, making it 9 times the equivalent of the antibody, and then added to the above solution. The solution was placed in a metal bath shaker and reacted at 25°C for 2 hours.

[0203] To the above solution, 7 times the equivalent of the antibody of L-cysteine ​​was added to terminate the coupling. Subsequently, ultrafiltration was performed using a 10 kDa ultrafiltration tube with a 10 mM histidine-hydrochloric acid buffer solution, repeated 25 times. The concentration was then detected, and the recovery rate was 66.19%.

[0204] The average value calculated by HPLC was n = 3.4.

[0205] Example 7: Synthesis of ADC-2

[0206] At 37°C, EDTA aqueous solution (100mM, 20μL) and TCEP aqueous solution (10mM, 15.48μL) were added to the PBS buffer solution (16mg / mL, 75μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, then allowed to return to room temperature.

[0207] Compound L-MY72 was dissolved in 100μL of DMSO to a concentration 9 times that of the antibody, and then added to the above solution. The mixture was placed in a metal bath shaker and shaken at 25°C for 2 hours.

[0208] In the above solution, L-cysteine, 7 times that of the antibody, was added to stop the coupling. Subsequently, ultrafiltration was performed using a 10kDa ultrafiltration tube with a 10mM histidine-hydrochloric acid buffer, repeated 25 times. The concentration was then detected, and the recovery rate was 60.97%.

[0209] The average value calculated by HPLC was n = 3.2.

[0210] Example 8: Synthesis of ADC-3

[0211] At 37°C, EDTA aqueous solution (100mM, 20μL) and TCEP aqueous solution (10mM, 15.48μL) were added to the PBS buffer solution (16mg / mL, 75μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0212] Compound L-MY73 was dissolved in 100μL of DMSO to make it 9 times the equivalent of the antibody, and then added to the above solution. The mixture was placed in a metal bath shaker and shaken at 25°C for 2 hours.

[0213] In the above solution, 7 times the equivalent of the antibody's L-cysteine ​​was added to stop the coupling. Subsequently, ultrafiltration was performed using a 10 kDa ultrafiltration tube with a 10 mM histidine-hydrochloric acid buffer solution, repeated 25 times. The concentration was then detected, and the recovery rate was 66.75%.

[0214] The average value calculated by HPLC was n = 3.7.

[0215] Example 9: Synthesis of ADC-4

[0216] At 37°C, EDTA aqueous solution (100 mM, 20 μL) and TCEP aqueous solution (10 mM, 15.48 μL) were added to the PBS buffer solution (16 mg / mL, 75 μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400 μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0217] Compound L-MY74 was dissolved in 100 μL of DMSO, making it 9 times the equivalent of the antibody, and then added to the above solution. The mixture was placed in a metal bath shaker and reacted with shaking at 25°C for 2 hours.

[0218] In the above solution, 7 times the equivalent of the antibody's L-cysteine ​​was added to stop the coupling. Then, ultrafiltration was performed using a 10 kDa ultrafiltration tube with 10 mM histidine-hydrochloric acid buffer, repeated 25 times. The concentration was then detected, and the recovery rate was 64.73%.

[0219] The average value calculated by HPLC was n = 3.0.

[0220] Example 10: Synthesis of ADC-5 24 / 30 pages 33 CN 121449623 A

[0221] At 37°C, EDTA aqueous solution (100 mM, 20 μL) and TCEP aqueous solution (10 mM, 15.48 μL) were added to the PBS buffer solution (16 mg / mL, 75 μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400 μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0222] Compound L-MY78 was dissolved in 100 μL of DMSO to a concentration 9 times that of the antibody. This solution was then added to the above solution, and the mixture was placed in a metal bath shaker and shaken at 25°C for 2 hours.

[0223] To the above solution, L-cysteine, at a concentration 7 times that of the antibody, was added to terminate the coupling. Subsequently, ultrafiltration was performed using a 10 kDa ultrafiltration tube with a 10 mM histidine-hydrochloric acid buffer solution, repeated 25 times. The concentration was then determined, and the recovery rate was 57.58%.

[0224] The average value calculated by HPLC was n = 3.6.

[0225] Example 11: Synthesis of ADC-6

[0226] At 37°C, EDTA aqueous solution (100mM, 20μL) and TCEP aqueous solution (10mM, 15.48μL) were added to the PBS buffer solution (16mg / mL, 75μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0227] The compound Deruxtecan (MC-GGFG-DXD, catalog number: HY-13631E, MedChemExpress) was dissolved in 100μL of DMSO to make it 9 times the equivalent of the antibody. This solution was then added to the above solution and placed in a metal bath shaker and shaken at 25°C for 2 hours.

[0228] In the above solution, 7 times the equivalent of the antibody's L-cysteine ​​was added to terminate the coupling. Subsequently, ultrafiltration was performed using a 10 kDa ultrafiltration tube with a 10 mM histidine-hydrochloric acid buffer solution, repeated 25 times. The concentration was then measured, and the recovery rate was 57.58%.

[0229] The average value calculated by HPLC was n = 3.6.

[0230] Example 12: Synthesis of ADC-7

[0231] At 37°C, EDTA aqueous solution (100mM, 20μL) and TCEP aqueous solution (10mM, 15.48μL) were added to the PBS buffer solution (16mg / mL, 75μL) of the anti-EGFR nanobody KY-301-58, mixed well, and PBS was added to 400μL. The mixture was placed in a metal bath shaker and shaken at 37°C for 4 hours, and then allowed to return to room temperature.

[0232] Compound EO (structure 11 in Hengrui patent WO2020063673A1) was dissolved in 100μL of DMSO to make it 9 times the equivalent of the antibody, and then added to the above solution. The mixture was placed in a metal bath shaker and shaken at 25°C for 2 hours.

[0233] In the above solution, L-cysteine ​​7 times the equivalent of the antibody was added to stop the coupling. Then, ultrafiltration was performed using a 10kDa ultrafiltration tube with 10mM histidine-hydrochloric acid buffer, and the reaction was repeated 25 times. The concentration was then detected, and the recovery rate was 53.62%.

[0234] The average value calculated by HPLC was n = 3.5.

[0235] Example 13: DAR value detection

[0236] 20 μg of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, and ADC-7 were injected into an HPLC HIC column and eluted with mobile phases of different gradients. The chromatograms were recorded, and the DAR value was calculated according to DAR = Σ (relative peak area × number of loaded drugs) / 100.

[0237] The results are shown in Table 1 below.

[0238] Table 1 DAR Values ​​and Product Content

[0239] No. DAR1 DAR2 DAR3 DAR4 Average DAR ADC-1 0% 0% 57.5% 42.5% 3.4 ADC-2 0% 22.8% 20.4% 53.5% 3.2 Specification 25 / 30 pages 34 CN 121449623 A ADC-3 0% 0% 31.9% 68.1% 3.7 ADC-4 0% 31.2% 30% 35.9% 3.0 ADC-5 0% 0% 41% 59% 3.6 ADC-6 0% 0% 40% 60% 3.6 ADC-7 0% 0% 43% 57% 3.5

[0240] Example 14: Evaluation of the toxicity of antibody-drug conjugates to colorectal cancer cells

[0241] To detect the inhibitory activity of antibody-drug conjugates on the in vitro proliferation of HT29, HCT116, RKO, SW480, LS1034, and COLO320 colorectal cancer cells, cells were treated with different concentrations of antibody-drug conjugates and cultured for six days. The cells were then subjected to CTG (Cell Titer-Luminescent Cell Propagation Technology) assay.Cell Viability Assay (Promega) reagent was used to detect cell proliferation, and the in vitro activity of the drug was evaluated based on cell viability (%).

[0242] Experimental methods:

[0243] 1. HT29, HCT116, RKO, SW480, LS1034, and COLO320 colorectal cancer cells were cultured in 10% FBS RPMI-1640 / DMEM medium.

[0244] 2. HT29, HCT116, RKO, SW480, LS1034, and COLO320 colorectal cancer cells in logarithmic growth phase were washed once with PBS, and 2-3 mL of trypsin was added for digestion for 2-3 min. After digestion with cells, 15 mL of complete culture medium was added, the cells were pipetted, centrifuged, the supernatant was discarded, and 10 mL of complete culture medium was added to prepare a single-cell suspension.

[0245] 3. HT29, HCT116, RKO, SW480, LS1034, and COLO320 colorectal cancer cells were evenly seeded into 96-well plates with black walls and clear bottoms, 90 μL per well, with a cell count of 1000. The culture plates were incubated in an incubator for 18 hours.

[0246] 4. The antibody-drug conjugate was diluted to six concentration gradients: 50, 10, 2, 0.4, 0.08, and 0.016 μg / mL.

[0247] 5. 10 μL of the prepared antibody-drug conjugate at different concentrations was added to the culture plate, with two replicates for each concentration. The culture plates were incubated in an incubator for 6 days.

[0248] 6. 100 μL of CTG reagent was added to the culture plate, and the plates were incubated at room temperature for 10 minutes. Chemiluminescence was measured using a microplate reader. The data were processed and analyzed using Microsoft Excel and Graphpad Prism 7.

[0249] Experimental Results:

[0250] According to the results in Figure 6, the antibody-drug conjugates of the present invention have significant inhibitory activity against colorectal cancer tumor cells. Specifically:

[0251] HT29 (Figure 6-A) shows that ADC-1, ADC-2, ADC-3, and ADC-5 all have significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0252] HCT116 (Figure 6-B) shows that ADC-1, ADC-2, ADC-3, and ADC-5 all have significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0253] RKO (Figure 6-C) shows that ADC-1, ADC-2, ADC-3, and ADC-5 all have significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0254] As shown in SW480 (Figure 6-D), ADC-1, ADC-2, ADC-3, and ADC-5 all exhibit significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0255] As shown in LS1034 (Figure 6-E), ADC-1, ADC-2, ADC-3, and ADC-5 all exhibit significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0256] As shown in COLO320 (Figure 6-F), ADC-1, ADC-2, ADC-3, and ADC-5 all exhibit significant inhibitory activity at high, medium, and low concentrations, and are superior to ADC-6 (MC-GGFG-Dxd).

[0257] Example 15: Evaluation of the toxicity of antibody-drug conjugates to pancreatic cancer cells, 26 / 30 pages, 35 CN 121449623 A

[0258] To detect the inhibitory activity of antibody-drug conjugates on the in vitro proliferation of ASPC1, BxPC3, PANC1, and SW1990 pancreatic cancer cells, cells were treated with different concentrations of antibody-drug conjugates. After six days of culture, cell proliferation was detected using CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent. The in vitro activity of the drug was evaluated based on the cell viability (%).

[0259] Experimental methods:

[0260] 1. ASPC1, BxPC3, PANC1, and SW1990 pancreatic cancer cells were cultured in 10% FBS RPMI-1640 / DMEM medium.

[0261] 2. ASPC1, BxPC3, PANC1, and SW1990 pancreatic cancer cells in logarithmic growth phase were washed once with PBS, and 2-3 mL of trypsin was added for digestion for 2-3 min. After digestion with cells, 15 mL of complete culture medium was added, the cells were pipetted, centrifuged, and the supernatant was discarded. Then, 10 mL of complete culture medium was added to prepare a single-cell suspension.

[0262] 3. ASPC1, BxPC3, PANC1, and SW1990 pancreatic cancer cells were evenly spread into 96-well plates with black walls and transparent bottoms, 90 μL per well, with 1000 cells per well. The culture plates were cultured in an incubator for 18 hours.

[0263] 4. Dilute the antibody-drug conjugate to six concentration gradients: 50, 10, 2, 0.4, 0.08, and 0.016 μg / mL.

[0264] 5. Add 10 μL of the prepared antibody-drug conjugate at different concentrations to the culture plate, with two replicates for each concentration. Incubate the culture plate in an incubator for 6 days.

[0265] 6. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and measure the chemiluminescence using a microplate reader. (Use Microsoft...)Excel and Graphpad Prism 7 were used to process and analyze the data.

[0266] Experimental results:

[0267] According to the results in Figure 7, the antibody-drug conjugate of the present invention has significant inhibitory activity against pancreatic cancer tumor cells. Specifically:

[0268] ASPC1 (Figure 7-A) shows that ADC-5 has significant inhibitory activity at both high and medium concentrations.

[0269] BxPC3 (Figure 7-B) shows that ADC-5 has significant inhibitory activity at high, medium and low concentrations, and is superior to ADC-6 (MC-GGFG-Dxd).

[0270] PANC1 (Figure 7-C) shows that ADC-5 has significant inhibitory activity at both high and medium concentrations, and is superior to ADC-6 (MC-GGFG-Dxd).

[0271] SW1990 (Figure 7-D) shows that ADC-5 has significant inhibitory activity at high, medium and low concentrations.

[0272] Example 16: Cytotoxicity evaluation of antibody-drug conjugate ADC-5 compared with ADC-7

[0273] 1. ASPC1, BxPC3, HCT-116, and NCI-H1975 cells were cultured in 10% FBS RPMI-1640 / DMEM medium

[0274] 2. ASPC1, BxPC3, HCT-116, and NCI-H1975 cells in logarithmic growth phase were washed once with PBS, and 2-3 mL of trypsin was added for digestion for 2-3 min. After digestion with cells, 15 mL of complete culture medium was added, the cells were pipetted, centrifuged, and the supernatant was discarded. Then, 10 mL of complete culture medium was added to prepare a single-cell suspension.

[0275] 3. ASPC1, BxPC3, HCT-116, and NCI-H1975 cells were evenly seeded into 96-well plates with black walls and transparent bottoms, 90 μL per well, with 1000 cells per well. Incubate the culture plate in an incubator for 18 hours.

[0276] 4. Dilute the antibody-drug conjugate to 6 concentration gradients: 50, 10, 2, 0.4, 0.08, 0.016 μg / mL.

[0277] 5. Add 10 μL of the prepared antibody-drug conjugate at different concentrations to the culture plate, with two replicates for each concentration. Incubate the culture plate in an incubator for 6 days.

[0278] 6. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and measure the chemiluminescence using an ELISA reader. Process and analyze the data using Microsoft Excel and Graphpad Prism 7.

[0279] Experimental results: Instruction manual 27 / 30 pages 36 CN 121449623 A

[0280] According to the results in Figure 8, the antibody-drug conjugate ADC-5 of the present invention has a significantly greater inhibitory activity on cell proliferation than ADC-7. Specifically as follows:

[0281] ASPC1 (Figure 8-A) shows that ADC-5 has significantly more inhibitory activity than ADC-7 at both high and medium concentrations, and is superior to ADC-7.

[0282] BxPC3 (Figure 8-B) shows that ADC-5 has significantly more inhibitory activity than ADC-7 at high, medium, and low concentrations, and is superior to ADC-7.

[0283] HCT-116 (Figure 8-C) shows that ADC-5 has significantly more inhibitory activity than ADC-7 at both high and medium concentrations, and is superior to ADC-7.

[0284] NCI-H1975 (Figure 8-D) shows that ADC-5 has significantly more inhibitory activity than ADC-7 at both high and medium concentrations, and is superior to ADC-7.

[0285] Example 17: Hydrophilicity and hydrophobicity detection of antibody-drug conjugates

[0286] To evaluate the hydrophilicity and hydrophobicity of antibody-drug conjugates, 20 μg of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, and ADC-7 were injected into an HPLC HIC column and eluted with mobile phases of different gradients. The retention times (tR) were recorded. At the same time, the LogP values ​​of L-MY71, L-MY72, L-MY73, L-MY74, L-MY78, and L-MY710 were calculated using compound LogP calculation software.

[0287] Table 2. HIC tR values ​​of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, and ADC-6

[0288] Number DAR1 DAR2 DAR3 DAR4 ADC-1 0 0 20.82 21.90 ADC-2 0 17.22 18.06 18.75 ADC-3 0 0 20.9 21.83 ADC-4 0 14.77 15.37 16.02 ADC-5 0 0 18.27 18.97 ADC-6 0 0 16.31 16.92 ADC-7 0 0 16.91 17.68

[0289] *Values ​​are in min

[0290] Table 3. LogP calculated values ​​of L-MY71, L-MY72, L-MY73, L-MY74, L-MY78, and L-MY710

[0291]

[0292]

[0293] According to the results in Tables 2 and 3, the compounds L-MY71, L-MY72, L-MY73, L-MY74, and L-MY78 of the present invention have good lipophilicity.

[0294] Example 18: Antitumor efficacy of antibody-drug conjugates against HT29 xenograft mouse model 28 / 30 pages 37 CN 121449623 A

[0295] To assess the in vivo antitumor activity of the antibody-drug conjugate, 5 x 10⁶ human colorectal cancer cells (HT29) were subcutaneously inoculated into the right rib area of ​​Balb / c nude mice. Five mice were divided into groups on day 0, with an average tumor volume of approximately 178.9 mm³. The drugs were administered via tail vein injection three times, on days 0, 14, and 27. Tumor volume and body weight were measured and recorded every two days.

[0296] The formula for calculating tumor volume (V) is: V = 1 / 2 * L length * L width 2

[0297] Relative volume: RTV = VT / V0

[0298] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0299] Table 4 % tumor inhibition rate of antibody-drug conjugate on HT29 xenograft mouse model

[0300]

[0301] According to the experimental results in Figure 9, the antibody-drug conjugate ADC-5 in the present invention has significantly better tumor inhibition activity than the control ADC-6 in the human colorectal cancer HT29 tumor-bearing mouse model (Figure 9-A), and the mouse weight does not decrease significantly (Figure 9-B).

[0302] Example 19: Antitumor efficacy of antibody-drug conjugate against RKO xenograft mouse model

[0303] To detect the in vivo tumor-suppressive activity of the antibody-drug conjugate, human colorectal cancer cells RKO cells (3x106) were subcutaneously inoculated in the right rib area of ​​Balb / c nude mice. Five mice were divided into groups on day 0, with an average tumor volume of approximately 132.96 mm3.

[0304] The drugs were administered via tail vein injection twice, on day 1 and day 7.

[0305] Tumor volume and body weight were measured every two days, and the data were recorded.

[0306] The formula for calculating tumor volume (V) is: V = 1 / 2 * L length * L width 2

[0307] Relative volume: RTV = VT / V0

[0308] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0309] Table 5 % tumor inhibition rate of antibody-drug conjugate on RKO xenograft mouse model

[0310]

[0311] According to the experimental results in Figure 10, the antibody-drug conjugate ADC-5 in the present invention showed significantly better tumor inhibition activity than the control ADC-6 in the human colorectal cancer RKO tumor-bearing mouse model (Figure 10-A); the weight of the mice decreased within a reasonable range after treatment (Figure 10-B).

[0312] Example 20: Antitumor efficacy of antibody-drug conjugate against a mouse model of PANC-1 xenograft tumor

[0313] To detect the in vivo tumor-suppressive activity of the antibody-drug conjugate, human colorectal cancer cell PANC-1 cells (5x106) were subcutaneously inoculated in the right rib area of ​​Balb / c nude mice. The mice were divided into groups of 6 on day 0, with an average tumor volume of approximately 137.76 mm3.

[0314] The drug was administered via tail vein injection twice, on day 1 and day 7 respectively.

[0315] Tumor volume and body weight were measured every two days and the data were recorded.

[0316] The formula for calculating tumor volume (V) is: V = 1 / 2 * L length * L width 2

[0317] Relative volume: RTV = VT / V0

[0318] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0319] Table 6. % tumor inhibition rate of antibody-drug conjugate on PANC-1 xenograft mouse model

[0320]

[0321] According to the experimental results in Figure 11, the antibody-drug conjugate ADC-5 in this invention showed significantly better tumor inhibition activity than the control ADC-6 in the human colorectal cancer

[0322] PANC-1 tumor-bearing mouse model (Figure 11-A); compared with the solvent group, the body weight of the mice in the administration group did not decrease significantly (Figure 11-B).

[0323] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Instruction manual 30 / 30 pages 39 CN 121449623 A Figure 1 Figure 2 Figure 3 Instruction manual drawing 1 / 8 pages 40 CN 121449623 A Figure 4 Figure 5 Instruction manual drawing 2 / 8 pages 41 CN 121449623 A Figure 6 Instruction manual drawing 3 / 8 pages 42 CN 121449623 A Figure 7 Instruction manual drawing 4 / 8 pages 43 CN 121449623 A Figure 8 Instruction manual drawing 5 / 8 pages 44 CN 121449623 A Figure 9 Instruction manual drawing 6 / 8 pages 45 CN 121449623 A Figure 10 Instruction manual drawing 7 / 8 pages 46 CN 121449623 A Figure 11 Instruction manual drawing 8 / 8 pages 47 CN 121449623 A Abstract The invention relates to the application of a topoisomerase inhibitor and a conjugate thereof. Specifically, the inventor provides a class of camptothecin derivatives, linker conjugates and antibody-drug conjugateswith novel structures, and the camptothecin derivatives, linker conjugates and antibody-drug conjugates have excellent anti-tumor activity and good lipophilicity. In-vitro and in-vivo experiments show that the drug conjugate containing the camptothecin derivative disclosed by the invention has an improved anti-tumor curative effect and high safety, and has an application prospect in the field of cancer treatment.

Claims

1. A compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the compound is shown in formula (I) or formula (II): Among them, Y is selected from the following group:

2. The compound according to claim 1, characterized in that, The compounds are selected from those in Table A: Table A 3. A linker conjugate, or its stereoisomer, or its pharmaceutically acceptable salt, characterized in that, The general formula of the connecting subconjugate is shown in formula (III) or formula (IV): Among them, Y is selected from the following group: L1 is a polypeptide residue selected from the following group: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-; L2 is a connector unit used for linking with antibodies.

4. The linker conjugate as described in claim 3, characterized in that, The linker conjugates are compounds selected from Table B: Table B 5. An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, characterized in that, The general formula of the antibody-drug conjugate is shown in formula (V) or formula (VI): Among them, Y is selected from the following group: L1 is a polypeptide residue selected from the following group: -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Thr-Asp-Gly-, -Glu-Thr-Gly-, -Glu-Ala-Gly-, -Glu-Val-Gly-, -Gly-Gly-Val-Ala-; L2 is a connector unit used for linking with antibodies. Ab represents an antibody or its antigen-binding fragment; n is an integer or decimal between 2 and 8.

6. The antibody-drug conjugate (ADC) as described in claim 5, characterized in that, The antibody-drug conjugate is a compound selected from Table C: Table C 7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises (a) the antibody-drug conjugate as claimed in claim 5 or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

8. Use of an antibody-drug conjugate as claimed in claim 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 7, in the preparation of a medicament for treating tumors or cancer.

9. The use as described in claim 8, characterized in that, The tumors or cancers mentioned are selected from the following group: colorectal cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, gastric cancer, glioma, and mesothelioma.

10. A method for preparing the antibody-drug conjugate as described in claim 5, characterized in that, Including the following steps: (1) The antibody was reacted with a reducing agent in a buffer solution to obtain the reduced antibody; (2) The antibody-drug conjugate is obtained by cross-linking (coupling) the reduced antibody obtained in step (1) with the compound shown in formula (III) or (IV) in a mixture of buffer and organic solvent.

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