Antibody drug conjugate for treating tumors

Abstract

The invention relates to an antibody drug conjugate for treating tumors, and belongs to the technical field of biological medicines. The invention provides an antibody drug conjugate for treating tumors. The structural general formula of the antibody drug conjugate is Ab-[L1-L2-L3-L4-D] p, wherein Ab is a monoclonal antibody targeting a tumor cell surface antigen, D is a bioactive molecule having an inhibition effect on tumor cells, p is drug loading capacity, and L1-L2-L3-L4 are linkers connecting the monoclonal antibody and the bioactive molecule. The antibody drug conjugate has good druggability. Meanwhile, the antibody drug conjugate has an obvious in-vitro cell killing effect on NCI-H82 cells, SHP-77 cells and DMS-53 cells, and the antibody drug conjugate can obviously reduce the volume of tumors in NCI-H82 tumor-bearing mice and SHP-77 tumor-bearing mice and has a great application prospect in tumor treatment.

Description

Technical Field

[0001] The present invention relates to an antibody-drug conjugate for treating tumors, and belongs to the technical field of biomedicine. Background Art

[0002] Antibody-Drug Conjugate (ADC) is a targeted anti-cancer drug whose core structure includes monoclonal antibodies that target tumor cell surface antigens, linkers (also called linkers), and cytotoxic compounds and other biologically active molecules that have inhibitory effects on tumor cells. Among them, the monoclonal antibody is coupled to the biologically active molecule that has inhibitory effects on tumor cells through a cleavable or non-cleavable linker to directly deliver the biologically active molecule that has inhibitory effects on tumor cells to tumor cells and achieve precise killing of tumor cells. This precise killing of tumor cells can significantly reduce damage to normal cells, thereby improving the efficacy of tumor treatment and reducing side effects.

[0003] Research has shown that linkers play a key role in antibody-drug conjugates (ADCs), with their properties influencing their efficacy and pharmacokinetic properties. Stable linkers can maintain the drug concentration of ADCs in the bloodstream and prevent the release of cytotoxic drugs before reaching their target, thereby minimizing off-target effects and improving the safety of ADCs. Providing more linkers that can effectively enhance the efficacy and pharmacokinetic properties of ADCs will be crucial for the development and optimization of ADCs. Summary of the Invention

[0004] To solve the above problems, the present invention provides an antibody-drug conjugate for treating tumors, wherein the general structural formula of the antibody-drug conjugate is Ab-[L1-L2-L3-L4-D] p ;

[0005] Among them, Ab is a monoclonal antibody targeting tumor cell surface antigens;

[0006] D is a bioactive molecule that has an inhibitory effect on tumor cells;

[0007] p is the drug loading capacity, and the value of p is an integer or decimal from 1 to 16;

[0008] L1-L2-L3-L4 is a linker connecting the monoclonal antibody and the biologically active molecule;

[0009] L1 is selected from the following structures:

[0010]

[0011] In L1, Represents the connection site, the connection site marked by number 1 is connected to Ab through the S atom, and the connection site marked by number 2 is connected to L2;

[0012] L2 is

[0013] In L2, Represents the connection site, the connection site marked by number 3 is connected to L1, and the connection site marked by number 4 is connected to L3;

[0014] Z1 is selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, cyano, amino, nitro or hydroxyl;

[0015] Z2 is selected from -NR1R2 or -OR3;

[0016] In Z2, R1 and R2 are each independently selected from -(CH2CH2O) m -CH3, hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl; or, R1 and R2 together with the nitrogen atom to which they are connected form a 4-12 membered heterocyclic group; R3 is selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclic group;

[0017] In R1, R2 and R3, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; the atoms in the heterocyclic group are each independently selected from C atoms, N atoms, O atoms or S atoms; and the heterocyclic group is substituted by R4; m is an integer from 1 to 24;

[0018] R4 is selected from -(CH2CH2O) m -CH3, -C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, -C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0019] In R4, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; m is an integer from 1 to 24;

[0020] R5 is selected from a hydrogen atom, an alkyl group, a cycloalkyl group or a heterocyclic group;

[0021] In R5, the alkyl, cycloalkyl and heterocyclic groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl;

[0022] Z3 is selected from -(CH2) i -C(=O)-**、-(CH2) i (OCH2CH2) j -C(=O)-**,

[0023] -(CH2) i -C(=O)-NH-(CH2CH2O) j -CH2CH2-C(=O)-**、-NH-(CH2) i -C(=O)-**、-NH-(CH2) i (OCH2CH2) j -C(=O)-**or chemical bond;

[0024] In Z3, i is an integer from 1 to 10; j is an integer from 1 to 20; ** is the 4th position;

[0025] L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues;

[0026] In L3, the amino acid residue is a natural amino acid residue or a non-natural amino acid residue;

[0027] L4 is selected from or chemical bonds;

[0028] In L4, Represents the connection site. The connection site marked with number 5 is connected to L3, and the connection site marked with number 6 is connected to D.

[0029] In one embodiment of the present invention, D is a cytotoxic compound, immunomodulator, enzyme or kinase inhibitor that has an inhibitory effect on tumor cells.

[0030] In one embodiment of the present invention, D is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Eribulin, exatecan, maytansine or SN-38.

[0031] In one embodiment of the present invention, the value of p is an integer or decimal of 1 to 3, 3 to 5, 5 to 7 or 7 to 9.

[0032] In one embodiment of the present invention, the L1 is In L1, Represents the connection site. The connection site marked with number 1 is connected to Ab through the S atom, and the connection site marked with number 2 is connected to L2.

[0033] In one embodiment of the present invention, L2 is

[0034] In L2, Represents the connection site. The connection site marked with number 3 is connected to L1, and the connection site marked with number 4 is connected to L3.

[0035] In one embodiment of the present invention, Z2 is selected from

[0036] In Z2, the value of n is an integer from 1 to 13.

[0037] In one embodiment of the present invention, Z3 is selected from -(CH2) i -C(=O)-** or -(CH2) i (OCH2CH2) j -C(=O)-**; in Z3, the value of i is an integer from 1 to 10; the value of j is an integer from 1 to 20; ** is the 4th position.

[0038] In one embodiment of the present invention, L3 is an amino acid residue, and L3 is selected from a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine ​​residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue or a valine residue;

[0039] Alternatively, the L3 is a short peptide consisting of 2 to 10 amino acid residues, and the amino acid residues in the L3 are each independently selected from a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine ​​residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue or a valine residue.

[0040] In one embodiment of the present invention, the amino acid residues are each independently substituted with a substituent selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy or cycloalkyl.

[0041] In one embodiment of the present invention, the L3 is selected from a tetrapeptide residue comprising glycine-glycine-phenylalanine-glycine, or a dipeptide residue comprising valine-citrulline or valine-alanine.

[0042] In one embodiment of the present invention, the L1-L2-L3-L4-D is selected from

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] In one embodiment of the present invention, the tumor is an anti-delta-like ligand 3 (DLL3) expressing tumor.

[0049] In one embodiment of the present invention, the tumor expressing anti-delta-like ligand 3 includes lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), squamous cell carcinoma (e.g., esophageal squamous cell carcinoma, cervical cancer), brain cancer (e.g., glioma, neuroblastoma), thyroid cancer (e.g., medullary thyroid carcinoma), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), hepatobiliary cancer (e.g., liver cancer, bile duct cancer, gallbladder cancer), pancreatic cancer, gastrointestinal cancer (e.g., gastric cancer, colorectal cancer), kidney cancer, ovarian cancer, endometrial cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer and / or melanoma.

[0050] In one embodiment of the present invention, Ab in the antibody-drug conjugate is a monoclonal antibody against delta-like ligand 3; the light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3, and the heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0051] In one embodiment of the present invention, the light chain variable region of the monoclonal antibody comprises:

[0052] (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.20 or SEQ ID NO.32;

[0053] (b) a derivative polypeptide derived from (a) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (a) and which retains the biological function derived from the sequence;

[0054] The heavy chain variable region of the monoclonal antibody comprises:

[0055] (c) a polypeptide with an amino acid sequence as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.31;

[0056] (d) A derivative polypeptide derived from (c) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (c) and retains the biological function of the sequence derived therefrom.

[0057] In one embodiment of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.20 or SEQ ID NO.32; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.31.

[0058] In one embodiment of the present invention, the light chain of the monoclonal antibody comprises:

[0059] (e) a polypeptide with an amino acid sequence as shown in SEQ ID NO. 24, SEQ ID NO. 27 or SEQ ID NO. 30;

[0060] (f) a derivative polypeptide derived from (a) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (e) and which retains the biological function of the sequence from which it is derived;

[0061] The heavy chain of the monoclonal antibody comprises:

[0062] (g) a polypeptide with an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28 or SEQ ID NO. 29;

[0063] (h) A derivative polypeptide derived from (c) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (g) and retains the biological function of the derived sequence.

[0064] In one embodiment of the present invention, the amino acid sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO.24, SEQ ID NO.27 or SEQ ID NO.30; the amino acid sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.28 or SEQ ID NO.29.

[0065] In one embodiment of the present invention, the light chain and heavy chain of the monoclonal antibody are connected by a disulfide bond.

[0066] In one embodiment of the present invention, the number of substituted, deleted or added amino acids in the derivative polypeptide does not exceed five.

[0067] In one embodiment of the present invention, the number of substituted, deleted or added amino acids in the derivative polypeptide does not exceed three.

[0068] The technical solution of the present invention has the following advantages:

[0069] The present invention provides an antibody-drug conjugate for treating tumors, wherein the general structural formula of the antibody-drug conjugate is Ab-[L1-L2-L3-L4-D] p Wherein, Ab is a monoclonal antibody targeting a tumor cell surface antigen, D is a bioactive molecule with an inhibitory effect on tumor cells, p is the drug loading, and L1-L2-L3-L4 is a linker connecting the monoclonal antibody and the bioactive molecule. Experiments have demonstrated that the antibody-drug conjugate has good drugability. Furthermore, experiments have shown that the antibody-drug conjugate has a significant in vitro cytotoxic effect on NCI-H82 cells, SHP-77 cells, and DMS-53 cells. Furthermore, the antibody-drug conjugate can significantly reduce tumor volume in NCI-H82 and SHP-77 tumor-bearing mice. Therefore, the antibody-drug conjugate has great application prospects in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 : The synthetic route of compound LP-1.

[0071] Figure 2 : Mass spectrometry results of compound LP-1.

[0072] Figure 3 : The synthetic route of compound LP-2.

[0073] Figure 4 : Mass spectrometry results of compound LP-2.

[0074] Figure 5 : The synthetic route of compound LP-3.

[0075] Figure 6 : Mass spectrometry results of compound LP-3.

[0076] Figure 7 : The synthetic route of compound LP-4.

[0077] Figure 8 : Mass spectrometry results of compound LP-4.

[0078] Figure 9 : The synthetic route of compound LP-5.

[0079] Figure 10 : Mass spectrometry results of compound LP-5.

[0080] Figure 11 : The synthetic route of compound LP-6.

[0081] Figure 12 : Mass spectrometry results of compound LP-6.

[0082] Figure 13 : The synthetic route of compound LP-7.

[0083] Figure 14 : Mass spectrometry results of compound LP-7.

[0084] Figure 15 : The synthetic route of compound LP-8.

[0085] Figure 16 : Mass spectrometry results of compound LP-8.

[0086] Figure 17 : The synthetic route of compound LP-9.

[0087] Figure 18 : Mass spectrometry results of compound LP-9.

[0088] Figure 19 : The synthetic route of compound LP-10.

[0089] Figure 20 : Mass spectrometry results of compound LP-10.

[0090] Figure 21 : The synthetic route of compound LP-11.

[0091] Figure 22 : Mass spectrometry results of compound LP-11.

[0092] Figure 23 : The synthetic route of compound LP-12.

[0093] Figure 24 : Mass spectrometry results of compound LP-12.

[0094] Figure 25 : The synthetic route of compound LP-13.

[0095] Figure 26 : Mass spectrometry results of compound LP-13.

[0096] Figure 27 : The synthetic route of compound LP-14.

[0097] Figure 28 : Mass spectrometry results of compound LP-14.

[0098] Figure 29 : The synthetic route of compound LP-15.

[0099] Figure 30 : Mass spectrometry results of compound LP-15.

[0100] Figure 31 : The synthetic route of compound LP-16.

[0101] Figure 32 : Mass spectrometry results of compound LP-16.

[0102] Figure 33 : The synthetic route of compound LP-17.

[0103] Figure 34 : Mass spectrometry results of compound LP-17.

[0104] Figure 35 : The synthetic route of compound LP-18.

[0105] Figure 36 : Mass spectrometry results of compound LP-18.

[0106] Figure 37 : The synthetic route of compound LP-19.

[0107] Figure 38 : Mass spectrometry results of compound LP-19.

[0108] Figure 39 : The endocytosis results of different antibodies in NCI-H82 cells.

[0109] Figure 40 : The endocytosis results of different antibodies in SHP-77 cells.

[0110] Figure 41 : Specificity detection results of JFab13b antibody.

[0111] Figure 42 :The anti-tumor activity of ADC was detected in SHP-77 transplanted tumor model.

[0112] Figure 43 : Body weight changes in SHP-77 transplanted tumor model mice.

[0113] Figure 44 :The anti-tumor activity of ADC was detected in the NCI-H82 xenograft tumor model.

[0114] Figure 45 : Body weight changes in mice with NCI-H82 transplanted tumor model. DETAILED DESCRIPTION

[0115] In order to make the present invention easier to understand, certain technical terms are specifically defined below. Unless otherwise explicitly stated elsewhere in this document, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0116] Antibody: refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only complete antibody molecules but also fragments of such antibodies and variants (including derivatives) of such antibodies and antibody fragments.

[0117] Antibody heavy chain: comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains: CH1, CH2, and CH3.

[0118] Antibody light chain: comprises a light chain variable region (abbreviated herein as "VL") and a light chain constant region containing one domain, CL.

[0119] FR (Framework Region): The term framework region refers to four conserved polypeptide segments within the light chain variable domain and the heavy chain variable domain.

[0120] CDR (Complementarity Determining Region): The term "complementarity determining region" or "CDR" refers to the regions within the light chain variable domain and the heavy chain variable domain that primarily contribute to antigen binding. The light chain variable domain and the heavy chain variable domain each contain three CDR regions: LCDR1, LCDR2, LCDR3; and HCDR1, HCDR2, HCDR3. The antibodies of the present invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single-chain antibodies, scFV fragments, and Fab fragments. The antibody molecules of the present invention can be of any immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, gA1, and IgA2), or subclass.

[0121] Constant region: The constant region of an antibody is located near the carbon-terminus of the H chain and the L chain. The heavy chain constant region is divided into CH1, CH2, and CH3, while the light chain constant region is CL. The CH3 region is involved in binding to cell membrane receptors, while CH2 is involved in the complement activation pathway and serves as the complement binding site.

[0122] Antibody acquisition: The antibodies of the present invention can be prepared using techniques well known in the art, such as the hybridoma method, recombinant DNA technology, phage display technology, synthetic technology, or a combination of these technologies, or other techniques known in the art.

[0123] Antibody discovery: The monoclonal antibody of the present invention obtained by hybridoma method is preferably a mouse anti-human DLL3 monoclonal antibody.

[0124] Antibody humanization: The mouse anti-human DLL3 monoclonal antibody of the present invention is a monoclonal antibody with human FR regions formed by humanization engineering.

[0125] Antibody drug conjugate (ADC): A conjugate obtained by connecting an antibody (or its antigen-binding fragment) to a drug directly or through a linker.

[0126] Linker: A chemical structure or bond that is attached to an antibody at one end and to a drug at the other. Other linkers can also be attached to the antibody or drug. Linker attachment to antibodies can be accomplished in a variety of ways, such as via surface lysines, reductive coupling to oxidized carbohydrates, cysteine ​​residues released by reduction of interchain disulfide bonds, reactive cysteine ​​residues engineered at specific sites, and tags containing the acyl donor glutamine or endogenous glutamine made reactive by engineering the polypeptide in the presence of transglutaminase and an amine.

[0127] Drug loading: Also known as the drug-to-antibody ratio (DAR), this refers to the average number of drugs conjugated per antibody in an ADC. This can range, for example, from about 1 to about 10 drugs conjugated per antibody, and in certain embodiments, from about 1 to about 8 drugs conjugated per antibody. Drug loading can be expressed as p, which can be a decimal or integer. Drug loading can be measured using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, HIC, and RP-HPLC.

[0128] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0129] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0130] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (HNMR) and / or mass spectrometry (MS). The instrument used for nuclear magnetic resonance (HNMR) was a Bruker 400 MHz nuclear magnetic resonance, the measurement solvent was deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (d6-DMSO), and the internal standard was tetramethylsilane (TMS). All chemical shift values ​​(δ) are given in parts per million (ppm). The instrument used for mass spectrometry (MS) was a Thermo Fisher Vanquish & TSQ Quantis TM The instrument used for preparative liquid chromatography was a Gilson GX-281, and the preparative chromatography column was a Welch Xtimate C18 30×150 mm, 5 μm. The reaction was monitored by thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). The developing systems used included, but were not limited to, dichloromethane and methanol systems and petroleum ether and ethyl acetate systems. The volume ratio of the solvents was adjusted according to the polarity of the compound, including but not limited to the addition of triethylamine or acetic acid.

[0131] Example 1-1: A linker-payload and its preparation

[0132] This embodiment provides a linker-cytotoxin LP-1, wherein the linker-cytotoxin LP-1 has the following structure:

[0133]

[0134] The preparation method of the linker-cytotoxin LP-1 comprises the following steps:

[0135] Step 1: Dissolve 2-bromo-5-nitrophenol (0.94 g, 1.0 eq, CAS No. 52427-05-1) in N,N-dimethylformamide (40 mL) to obtain a solution. 1-Boc-4-methanesulfonyloxypiperidine (1.80 g, 1.5 eq, CAS No. 141699-59-4) and potassium carbonate (1.20 g, 2.0 eq) were added to the solution in sequence, stirred to mix thoroughly, and then stirred at 90°C for 3 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (40 mL × 1) and then concentrated under reduced pressure to obtain compound 1-1. Compound 1-1 was used directly in the synthesis of compound 1-2 without purification. Compound 1-1 is a yellow oil (1.70 g, yield 98%). The mass spectrum data of compound 1-1 is: ESI-MS (m / z): 401.08 [M+H] + .

[0136] Step 2: Compound 1-1 (1.70 g, 1.00 eq) was dissolved in N,N-dimethylformamide (10 mL) to obtain a solution; benzyl acrylate (1.37 g, 2.0 eq, CAS No.: 2495-35-4), palladium acetate (25 mg, 0.025 eq), triphenylphosphine (0.11 g, 0.1 eq) and sodium bicarbonate (0.54 g, 1.5 eq) were added to the solution in sequence, and the mixture was stirred and mixed, and then stirred at 100 ° C for 4 hours under a nitrogen atmosphere to obtain a reaction mixture. After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (20 mL × 4). The organic phase was collected and washed with water (40 mL × 2), then with saturated brine (40 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 1-2. Compound 1-2 was a yellow oil (1.86 g, yield 89%). The mass spectrometric data of compound 1-2 were: ESI-MS (m / z): 483.23 [M+H] + .

[0137] Step 3: Compound 1-2 (1.71 g, 1.00 eq) was dissolved in ethyl acetate (15 mL) to obtain a solution; after cooling the solution to 0°C, a solution of hydrogen chloride in ethyl acetate (4 mol / L, 13.5 mL, 15 eq) was added to the solution, and then stirred at 0°C for 24 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first concentrated under reduced pressure, then adjusted to pH 9 with saturated sodium carbonate aqueous solution, and then extracted with ethyl acetate (20 ml × 4), and the organic phase was taken; the organic phase was first washed with water (40 ml), then with saturated brine (40 ml), and then concentrated under reduced pressure to obtain compound 1-3. Compound 1-3 was not purified and was directly used in the synthesis of compound 1-4. Compound 1-3 is a yellow oil (1.35 g, yield 99%). The mass spectrometric data of compound 1-3 are: ESI-MS (m / z): 383.11 [M+H] + .

[0138] Step 4: Compound 1-3 (1.30 g, 1.00 eq) was dissolved in N, N-dimethylformamide (10 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (1.00 g, 1.05 eq, CAS No.: 81836-43-3) and N, N-diisopropylethylamine (1.32 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.55 g, 1.2 eq, CA S No.: 148893-10-1), and then stirred at 25°C for 16 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with saturated aqueous sodium carbonate (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 1-4. Compound 1-4 was obtained as a yellow oil 1-4 (1.73 g, yield 79%). The mass spectrometric data of compound 1-4 were: ESI-MS (m / z): 645.25 [M+H] + .

[0139] Step 5: Compound 1-4 (1.73 g, 1.00 eq) was dissolved in a mixture of methanol / dichloromethane / tetrahydrofuran (50 mL / 40 mL / 30 mL) to obtain a solution; 5% palladium carbon (0.30 g) was added to the solution, and the mixture was reacted at 25° C. under a hydrogen atmosphere for 24 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with methanol (5 mL) and the filtrate was obtained; the filtrate was first concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 1-5. Compound 1-5 was a yellow oil 1-5 (0.92 g, yield 65%). The mass spectrum data of compound 1-5 is: ESI-MS (m / z): 527.25 [M+H] + .

[0140] Step 6: Compound 1-5 (0.92 g, 1.0 eq) was dissolved in dichloromethane (20 mL) to obtain a solution. Triethylamine (0.35 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.32 g, 1.2 eq, CAS No. 55750-48-6) were added to the solution, and the mixture was heated to 45°C and reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 1-6. Compound 1-6 was obtained as a yellow oil (0.79 g, 74% yield). The mass spectrum data of compound 1-6 is: ESI-MS (m / z): 607.26 [M+H] + .

[0141] Step 7: Compound 1-6 (30 mg, 1.00 eq) was dissolved in N,N-dimethylformamide (1.5 mL) to obtain a solution; Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS No.: 1599440-12-6) (41 mg, 1.00 eq) and N,N-diisopropylethylamine (32 mg, 5.00 eq) were added to the solution, and then the temperature was first cooled to 0°C, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (37.6 mg, 2.00 eq) was added, and then the mixture was reacted at 25°C for 2 hours to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-1. Compound LP-1 is a yellow powdery solid (22 mg, yield 31%). The synthetic route of compound LP-1 is shown in Figure 1 The mass spectrometry results of compound LP-1 are shown in Figure 2 The mass spectrum data of compound LP-1 are: LCMS (ESI) [M+H] + :1429.55[M+H] + .

[0142] Example 1-2: A linker-payload and its preparation

[0143] This embodiment provides a linker-cytotoxin LP-2, wherein the linker-cytotoxin LP-2 has the following structure:

[0144]

[0145] The preparation method of the linker-cytotoxin LP-2 comprises the following steps:

[0146] Step 1: Dissolve compound 4-bromo-2-fluorobenzaldehyde (2.00 g, 1.00 eq) in N,N-dimethylformamide (20 mL) to obtain a solution; add N-Boc-piperazine (1.6 g, 1.2 eq) and potassium carbonate (1.2 g, 1.2 eq) to the solution in sequence, stir to mix, and then stir and react at 100°C for 24 hours to obtain a reaction solution; after thin layer chromatography shows that the reaction is complete, the reaction solution is first cooled to room temperature (25°C), then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase is taken; the organic phase is first washed with saturated brine (40 mL×1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate is increased from petroleum ether:ethyl acetate=95:5 to petroleum ether:ethyl acetate=0:100) to obtain compound 2-1. Compound 2-1 is a yellow oil (2.14 g, yield 78%). The mass spectrum data of compound 2-1 is: ESI-MS (m / z): 369.02 [M+H] + .

[0147] Step 2: Compound 2-1 (2.14 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.42 g, 1.50 eq, CAS No. 15097-38-8) was added to the solution, stirred evenly, and reacted at 20°C for 18 hours to obtain a reaction solution. After thin-layer chromatography indicated the reaction was complete, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 2-2. Compound 2-2 was obtained as a yellow oil (2.78 g, yield 96%). The mass spectrometric data of compound 2-2 were: ESI-MS (m / z): 501.12 [M+H] + .

[0148] Step 3: Compound 2-2 (2.78 g, 1.00 eq) was dissolved in toluene (30 mL) to obtain a solution; benzyl carbamate (0.94 g, 1.20 eq), palladium acetate (23 mg, 0.025 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.12 g, 0.05 eq, CAS No.: 564483-18-7) and potassium carbonate (0.85 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and heated at 1 The reaction was continued at 00°C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (30 mL × 1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 2-3. Compound 2-3 was obtained as a yellow oil (3.1 g, yield 96%). The mass spectrometric data of compound 2-3 were: ESI-MS (m / z): 572.23 [M+H] + .

[0149] Step 4: Compound 2-3 (3.1 g, 1.00 eq) was dissolved in ethyl acetate (10 mL) to obtain a solution. After cooling the solution to 0°C, a solution of hydrogen chloride in ethyl acetate (4 mol / L, 20 mL, 15 eq) was added to the solution, stirred evenly, and stirred at 0°C for 24 hours to obtain a reaction solution. After TLC indicated the reaction was complete, the reaction solution was filtered, and the filter cake was dissolved in 30 mL of a mixture of dichloromethane / methanol (dichloromethane:methanol = 10:1, v / v). The organic phase was collected and washed with saturated aqueous sodium carbonate (10 mL x 2) and then evaporated to dryness under reduced pressure to obtain compound 2-4. Compound 2-4 was used directly in the synthesis of compound 2-5 without purification. Compound 2-4 was obtained as a yellow oil (1.6 g, yield 62%). The mass spectrometric data of compound 2-4 were: ESI-MS (m / z): 472.25 [M+H] + .

[0150] Step 5: Compound 2-4 (0.8 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (0.52 g, 1.1 eq) and N, N-diisopropylethylamine (0.66 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C., and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.77 g, 1.2 eq) was added, and then The reaction was stirred at 25°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated the reaction was complete, the reaction solution was diluted with saturated aqueous sodium carbonate (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 2-5. Compound 2-5 was obtained as a yellow oil (0.95 g, yield 76%). The mass spectrometric data of compound 2-5 were: ESI-MS (m / z): 734.33 [M+H] + .

[0151] Step 6: Compound 2-5 (0.95 g, 1.00 eq) was dissolved in a mixture of dichloromethane and methanol (2 mL / 10 mL) to obtain a solution. 5% palladium on carbon (0.09 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 2-6. Compound 2-6 was obtained as a yellow oil (0.43 g, yield 65%). The mass spectrometric data of compound 2-6 were: ESI-MS (m / z): 512.21 [M+H] + .

[0152] Step 7: Compound 2-6 (0.15 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.06 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.06 g, 1.2 eq) were added to the solution, and the mixture was heated to 45°C and reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 2-7. Compound 2-7 was obtained as a yellow oil (0.12 g, yield 64%). The mass spectrometric data of compound 2-7 were: ESI-MS (m / z): 592.20 [M+H] + .

[0153] Step 8: Compound 2-7 (40 mg, 1.20 eq) was dissolved in N,N-dimethylformamide (0.5 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (50 mg, 1.00 eq) and N,N-diisopropylethylamine (22 mg, 3.00 eq) were added to the solution, and the mixture was cooled to 0°C. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (33 mg, 1.50 eq) was then added. The mixture was reacted at 25°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-2. Compound LP-2 was obtained as a yellow powdery solid (20 mg, 20% yield). The synthetic route of compound LP-2 is shown in Figure 3 The mass spectrometry results of compound LP-2 are shown in Figure 4 The mass spectrum data of compound LP-2 are: LCMS (ESI) [M+H] + :1414.40[M+H] + .

[0154] Example 1-3: A linker-payload and its preparation

[0155] This embodiment provides a linker-cytotoxin LP-3, wherein the linker-cytotoxin LP-3 has the following structure:

[0156]

[0157] The preparation method of the linker-cytotoxin LP-3 comprises the following steps:

[0158] Step 1: Dissolve the compound 4-bromo-2-fluorobenzaldehyde (1.5 g, 1.00 eq) in N,N-dimethylformamide (15 mL) to obtain a solution; add 4-(N-Boc-amino)piperidine (1.28 g, 1.2 eq, CAS No.: 73874-95-0) and potassium carbonate (0.88 g, 1.2 eq) to the solution in sequence, stir to mix, and then stir at 100 ° C for 24 hours to obtain a reaction solution; thin layer chromatography After the reaction was complete as shown by chromatography, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-1. Compound 3-1 was a yellow oil (2 g, yield 70%). The mass spectrometric data of compound 3-1 were: ESI-MS (m / z): 383.03 [M+H] + .

[0159] Step 2: Compound 3-1 (2 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.5 g, 1.60 eq) was added to the solution, and the mixture was stirred at 20°C for 18 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-2. Compound 3-2 was obtained as a yellow oil (2.57 g, yield 95%). The mass spectrometric data of compound 3-2 were: ESI-MS (m / z): 515.18 [M+H] + .

[0160] Step 3: Compound 3-2 (2.5 g, 1.00 eq) was dissolved in toluene (30 mL) to obtain a solution; benzyl carbamate (0.88 g, 1.20 eq), palladium acetate (27 mg, 0.025 eq), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.11 g, 0.05 eq) and potassium carbonate (0.8 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and reacted at 100 ° C for 4 hours to obtain a reaction solution; After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25°C) and then concentrated under reduced pressure to obtain a residue. The residue was diluted with water (40 mL) and extracted with dichloromethane (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3) and then concentrated under reduced pressure. Finally, it was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-3. Compound 3-3 was obtained as a yellow oil (2.27 g, yield 80%). The mass spectrometric data of compound 3-3 were: ESI-MS (m / z): 586.31 [M+H] + .

[0161] Step 4: Compound 3-3 (2.2 g, 1.00 eq) was dissolved in ethyl acetate (8 mL) to obtain a solution; after adding hydrogen chloride ethyl acetate solution (4 mol / L, 14 mL, 15 eq) to the solution, the temperature was lowered to 0°C and the reaction was continued at 0°C for 24 hours to obtain a reaction solution; after the reaction was completed by thin layer chromatography, the reaction solution was concentrated under reduced pressure to obtain a residue; the reaction solution was first diluted with saturated sodium carbonate aqueous solution (30 mL), then extracted with ethyl acetate (30 mL×3), and the organic phase was obtained; the organic phase was first washed with saturated brine (30 mL×3), and then evaporated to dryness under reduced pressure to obtain compound 3-4. Compound 3-4 was not purified and was directly used in the synthesis of compound 3-5. Compound 3-4 was a yellow oil (1.72 g, yield 94%). The mass spectrometric data of compound 3-4 were: ESI-MS (m / z): 486.24 [M+H] + .

[0162] Step 5: Compound 3-4 (1.7 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (1.1 g, 1.1 eq) and N, N-diisopropylethylamine (1.35 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.6 g, 1.2 eq) was added. The mixture was stirred and reacted at 25°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 3-5. Compound 3-5 was obtained as a yellow oil (1.2 g, yield 46%). The mass spectrometric data of compound 3-5 were: ESI-MS (m / z): 748.24 [M+H] + .

[0163] Step 6: Dissolve compound 3-5 (1.2 g, 1.00 eq) in methanol (10 mL) to obtain a solution; add 5% palladium carbon (0.1 g) to the solution, and react at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution; thin layer chromatography shows that the reaction is complete, filter the reaction solution, and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 3-6. Compound 3-6 is not purified and is directly used in the synthesis of compound 3-7. Compound 3-6 is a yellow oil (0.80 g, yield 95%). The mass spectrometry data of compound 3-6 are: ESI-MS (m / z): 526.30 [M+H] + .

[0164] Step 7: Compound 3-6 (0.2 g, 1.00 eq) was dissolved in dichloromethane (10 mL) to obtain a solution. Triethylamine (0.077 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.071 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 3-7. Compound 3-7 was obtained as a yellow oil (0.16 g, yield 69%). The mass spectrometric data of compound 3-7 were: ESI-MS (m / z): 606.20 [M+H] + .

[0165] Step 8: Compound 3-7 (38 mg, 1.10 eq) was dissolved in N,N-dimethylformamide (1 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (50 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 3.00 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (33 mg, 1.10 eq), and 1-hydroxybenzotriazole (8.5 mg, 1.10 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After completion of the reaction, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-3 as a yellow powdery solid (48 mg, 59% yield). The synthetic route of compound LP-3 is shown in Figure 5 The mass spectrometry results of compound LP-3 are shown in Figure 6 The mass spectrum data of compound LP-2 are: LCMS (ESI) [M+H] + :1428.45[M+H] + .

[0166] Example 1-4: A linker-payload and its preparation

[0167] This embodiment provides a linker-cytotoxin LP-4, wherein the linker-cytotoxin LP-4 has the following structure:

[0168]

[0169] The preparation method of the linker-cytotoxin LP-4 comprises the following steps:

[0170] Step 1: Dissolve 4-bromo-2-fluorobenzaldehyde (1.5 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution. Add diethylamine (0.66 g, 1.2 eq) and potassium carbonate (0.9 g, 1.2 eq) to the solution in sequence, stir to mix, and then react at 100°C for 24 hours to obtain a reaction solution. After thin-layer chromatography (TLC) shows the reaction is complete, cool the reaction solution to room temperature (25°C), dilute with water (40 mL), and extract with ethyl acetate (30 mL × 3). The organic phase is collected and washed with saturated brine (30 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate is increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-1. Compound 4-1 is a yellow oil (1.5 g, yield 79%). The mass spectrum data of compound 4-1 is: ESI-MS (m / z): 256.01 [M+H] + . 1 HNMR(400MHz,Chloroform-d)δ10.25(s,1H),7.67(d,J=8.3Hz,1H),7.29(d,J=1.7 Hz,1H),7.22(dd,J=8.3,1.7Hz,1H),3.22(q,J=7.1Hz,4H),1.11(t,J=7.1Hz,6H).

[0171] Step 2: Compound 4-1 (1.46 g, 1.00 eq) was dissolved in dichloromethane (20 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.5 g, 1.50 eq) was added to the solution, stirred evenly, and reacted at 20°C for 18 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-2. Compound 4-2 was obtained as a yellow oil (2.12 g, yield 95%). The mass spectrometric data of compound 4-2 were: ESI-MS (m / z): 388.11 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.06(d,J=16.2Hz,1H),7.48–7.30(m,7H),7.23–7.02(m, 2H), 6.43 (d, J = 16.2Hz, 1H), 5.28 (s, 2H), 3.07 (q, J = 7.1Hz, 4H), 1.03 (t, J = 7.1Hz, 6H).

[0172] Step 3: Compound 4-2 (2.12 g, 1.00 eq) was dissolved in toluene (20 mL) to obtain a solution; benzyl carbamate (1 g, 1.20 eq), palladium acetate (31 mg, 0.025 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.13 g, 0.05 eq) and potassium carbonate (0.91 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and the reaction was continued at 100 ° C for 4 hours to obtain After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25°C), concentrated under reduced pressure, diluted with water (40 mL), and finally extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-3. Compound 4-3 is a yellow oil (2.03 g, yield 80%). The mass spectrometric data of compound 4-3 are: ESI-MS (m / z): 459.18 [M+H] + .

[0173] Step 4: Dissolve compound 4-3 (2.35 g, 1.00 eq) in methanol (20 mL) to obtain a solution; add 5% palladium carbon (0.2 g) to the solution, and react at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution; after thin layer chromatography shows that the reaction is complete, filter the reaction solution and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 4-4. Compound 4-4 is not purified and is directly used in the synthesis of compound 4-5. Compound 4-4 is a yellow oil (1.15 g, yield 95%). The mass spectrometry data of compound 4-4 are: ESI-MS (m / z): 237.11 [M+H] + .

[0174] Step 5: Compound 4-4 (0.1 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.084 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.078 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 4-5. Compound 4-5 was obtained as a yellow oil (0.09 g, yield 69%). The mass spectrum data of compound 4-5 is: ESI-MS (m / z): 317.09 [M+H] + .

[0175] Step 6: Compound 4-5 (56 mg, 2.60 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (60 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-4. Compound LP-4 was obtained as a yellow powdery solid (20 mg, 25% yield). The synthetic route of compound LP-4 is shown in Figure 7 The mass spectrometry results of compound LP-4 are shown in Figure 8 The mass spectrum data of compound LP-4 are: LCMS (ESI) [M+H] + :1139.46[M+H] + .

[0176] Example 1-5: A linker-payload and its preparation

[0177] This embodiment provides a linker-cytotoxin LP-5, wherein the linker-cytotoxin LP-5 has the following structure:

[0178]

[0179] The preparation method of the linker-cytotoxin LP-5 comprises the following steps:

[0180] Step 1: Compound 4-bromo-2-fluorobenzaldehyde (2 g, 1.00 eq) was dissolved in N,N-dimethylformamide (20 mL) to obtain a solution; N-methylpiperazine (1.18 g, 1.2 eq) and potassium carbonate (1.63 g, 1.2 eq) were added to the solution in sequence, stirred to mix, and then stirred at 100°C for 24 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25°C), then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase was taken; the organic phase was first washed with saturated brine (30 mL×1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-1. Compound 5-1 is a yellow oil (2.17 g, yield 78%). The mass spectrum data of compound 5-1 is: ESI-MS (m / z): 283.01 [M+H] + .

[0181] Step 2: Compound 5-1 (2.14 g, 1.00 eq) was dissolved in dichloromethane (25 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (4.7 g, 1.50 eq) was added to the solution and stirred evenly. Finally, the mixture was reacted at 20°C for 18 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-2. Compound 5-2 was obtained as a yellow oil (2.12 g, yield 67%). The mass spectrometric data of compound 5-2 were: ESI-MS (m / z): 415.11 [M+H] + .

[0182] Step 3: Compound 5-2 (2.1 g, 1.00 eq) was dissolved in toluene (20 mL) to obtain a solution; benzyl carbamate (0.78 g, 1.20 eq), palladium acetate (48 mg, 0.05 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.2 g, 0.1 eq) and potassium carbonate (0.72 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and the reaction was continued at 100 ° C for 4 hours. , obtaining a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25°C), then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL × 3). The organic phase was collected; the organic phase was first washed with saturated brine (30 mL × 1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-3. Compound 5-3 was a yellow oil (1.08 g, yield 44%). The mass spectrometric data of compound 5-3 were: ESI-MS (m / z): 486.25 [M+H] + .

[0183] Step 4: Compound 5-3 (1.08 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution; 5% palladium carbon (0.2 g) was added to the solution, and the mixture was reacted at 25° C. under a hydrogen atmosphere for 12 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with methanol (5 mL) and the filtrate was obtained; the filtrate was evaporated to dryness under reduced pressure to obtain compound 5-4. Compound 5-4 was not purified and was directly used in the synthesis of compound 5-5. Compound 5-4 was a yellow oil (0.59 g, yield 100%). The mass spectrometry data of compound 5-4 was: ESI-MS (m / z): 264.15 [M+H] + .

[0184] Step 5: Compound 5-4 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution; triethylamine (0.038 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.035 g, 1.2 eq) were added to the solution, stirred and mixed, and then reacted at 45 ° C for 4 hours to obtain a reaction solution; after the reaction was completed by thin layer chromatography, the reaction solution was cooled to room temperature (25 ° C), and then evaporated to dryness under reduced pressure to obtain compound 5-5. Compound 5-5 was not purified and was directly used in the synthesis of compound 5-6. Compound 5-6 is a yellow oil (0.06 g, yield 92%). The mass spectrometry data is: ESI-MS (m / z): 344.09 [M + H] + .

[0185] Step 6: Compound 5-5 (65 mg, 2.70 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (60 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-5. Compound LP-5 was obtained as a yellow powdery solid (17 mg, yield 21%). The synthetic route of compound LP-5 is shown in Figure 9 The mass spectrometry results of compound LP-5 are shown in Figure 10 The mass spectrum data of compound LP-5 are: LCMS (ESI) [M+H] + :1166.47[M+H] + .

[0186] Example 1-6: A linker-payload and its preparation

[0187] This embodiment provides a linker-cytotoxin LP-6, wherein the linker-cytotoxin LP-6 has the following structure:

[0188]

[0189] The preparation method of the linker-cytotoxin LP-6 comprises the following steps:

[0190] Step 1: Compound 2-4 (0.2 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; N-fluorenylmethyloxycarbonyl-glycyl-glycine (0.18 g, 1.2 eq) and N, N-diisopropylethylamine (0.14 g, 2.5 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.19 g, 1 .2eq), and then stirred at 25°C for 4 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30mL), then extracted with ethyl acetate (30mL×3), and the organic phase was taken; the organic phase was first washed and diluted with saturated sodium bicarbonate solution (30mL), then washed with saturated brine (30mL×3), and then evaporated to dryness under reduced pressure to obtain compound 6-1. Compound 6-1 was not purified and was directly used in the synthesis of compound 6-2. Compound 6-1 was a yellow oil (0.36g, 100%). The mass spectrum data of compound 6-1 was: ESI-MS (m / z): 808.26 [M+H] + .

[0191] Step 2: Compound 6-1 (0.36 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. The solution was cooled to 0°C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.068 g, 1.00 eq) was added to the solution. The mixture was then reacted at 0°C for 1.5 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 6-2. Compound 6-2 was obtained as a yellow oil (0.21 g, yield 80%). The mass spectrometric data of compound 6-2 were: ESI-MS (m / z): 586.21 [M+H] + .

[0192] Step 3: Compound 6-2 (0.21 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (0.12 g, 1.2 eq) and N, N-diisopropylethylamine (0.12 g, 2.50 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.16 g, 1.2 eq) was added, and then the mixture was stirred at 2 ° C for 2 hours. The reaction was stirred at 5°C for 2 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 5). The organic phase was washed with water (30 mL) and then with saturated brine (30 mL), then concentrated under reduced pressure and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 6-3. Compound 6-3 was obtained as a yellow oil (0.19 g, yield 62.5%). The mass spectrometric data of compound 6-3 were: ESI-MS (m / z): 848.37 [M+H] + .

[0193] Step 4: Compound 6-3 (0.19 g, 1.00 eq) was dissolved in methanol (5 mL) to obtain a solution. 5% palladium on carbon (0.02 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 6-4. Compound 6-4 was obtained as a yellow oil (0.06 g, yield 43%). The mass spectrometric data of compound 6-4 were: ESI-MS (m / z): 626.39 [M+H] + .

[0194] Step 5: Compound 6-4 (0.06 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.019 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.018 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 6-5. Compound 6-5 was obtained as a yellow oil (0.02 g, 30% yield). The mass spectrometric data of compound 6-5 were: ESI-MS (m / z): 706.29 [M+H] + .

[0195] Step 6: Compound 6-5 (120 mg, 2.20 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (75 mg, 1.00 eq), N,N-diisopropylethylamine (27.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After completion of the reaction, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-6. Compound LP-6 was obtained as a yellow powdery solid (55 mg, 39% yield). The synthetic route of compound LP-6 is shown in Figure 11 The mass spectrometry results of compound LP-6 are shown in Figure 12 The mass spectrum data of compound LP-6 are: LCMS (ESI) [M+H] + :1528.64[M+H] + .

[0196] Example 1-7: A linker-payload and its preparation

[0197] This embodiment provides a linker-cytotoxin LP-7, wherein the linker-cytotoxin LP-7 has the following structure:

[0198]

[0199] The preparation method of the linker-cytotoxin LP-7 comprises the following steps:

[0200] Step 1: Compound 2-4 (1.3 g, 1.00 eq) was dissolved in N, N-dimethylformamide (10 mL) to obtain a solution; 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (1.39 g, 1.1 eq) and N, N-diisopropylethylamine (1.07 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N , N,N',N'-tetramethyluronium hexafluorophosphate (1.26g, 1.2eq), and then reacted at 25°C for 8 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30mL), then extracted with ethyl acetate (30mL×3), and the organic phase was taken; the organic phase was first washed with sodium bicarbonate solution (30mL), then washed with saturated brine (30mL), and then evaporated to dryness under reduced pressure to obtain compound 7-1. Compound 7-1 was not purified and was directly used in the synthesis of compound 7-2. Compound 7-1 is a yellow oil (2.51g, yield 100%). The mass spectrometry data of compound 7-1 are: ESI-MS (m / z): 910.51 [M+H] + .

[0201] Step 2: Compound 7-1 (2.51 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.25 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 7-2. Compound 7-2 was obtained as a yellow oil (1.6 g, yield 83%). The mass spectrometric data of compound 7-2 were: ESI-MS (m / z): 688.31 [M+H] + .

[0202] Step 3: Compound 7-2 (0.35 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.103 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.095 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After TLC indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 7-3. Compound 7-3 was obtained as a yellow oil (0.35 g, yield 89%). The mass spectrometric data of compound 7-3 were: ESI-MS (m / z): 768.31 [M+H] + .

[0203] Step 4: Compound 7-3 (130 mg, 2.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (75 mg, 1.00 eq), N,N-diisopropylethylamine (27.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-7. Compound LP-7 was obtained as a yellow powdery solid (70 mg, 48% yield). The synthetic route of compound LP-7 is shown in Figure 13 The mass spectrometry results of compound LP-7 are shown in Figure 14 The mass spectrum data of compound LP-7 are: LCMS (ESI) [M+H] + :1590.70[M+H] + .

[0204] Example 1-8: A linker-payload and its preparation

[0205] This embodiment provides a linker-cytotoxin LP-8, wherein the linker-cytotoxin LP-8 has the following structure:

[0206]

[0207] The preparation method of the linker-cytotoxin LP-8 comprises the following steps:

[0208] Step 1: Compound 3-4 (1.97 g, 1.00 eq) was dissolved in N, N-dimethylformamide (25 mL) to obtain a solution; 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (2.04 g, 1.1 eq) and N, N-diisopropylethylamine (1.73 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N , N,N',N'-tetramethyluronium hexafluorophosphate (1.85g, 1.2eq), and then reacted at 25°C for 8 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30mL), then extracted with ethyl acetate (30mL×3), and the organic phase was taken; the organic phase was first washed with saturated sodium bicarbonate solution (30mL), then washed with saturated brine (30mL), and then evaporated to dryness under reduced pressure to obtain compound 8-1. Compound 8-1 is a yellow oil (3g, yield 80%). Compound 8-1 was not purified and was directly used in the synthesis of compound 8-2. The mass spectrometry data of compound 8-1 are: ESI-MS (m / z): 924.42 [M+H] + .

[0209] Step 2: Compound 8-1 (3 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.3 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 8-2. Compound 8-2 was obtained as a yellow oil (1.9 g, yield 83%). The mass spectrometric data of compound 8-2 were: ESI-MS (m / z): 702.39 [M+H] + .

[0210] Step 3: Compound 8-2 (0.3 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.08 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.086 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After TLC indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 8-3. Compound 8-3 was obtained as a yellow oil (0.24 g, yield 72%). The mass spectrometric data of compound 8-3 were: ESI-MS (m / z): 782.46 [M+H] + .

[0211] Step 4: Compound 8-3 (185 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (70 mg, 1.00 eq), N,N-diisopropylethylamine (25.5 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (45 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-8. Compound LP-8 was obtained as a yellow powdery solid (75 mg, 58% yield). The synthetic route of compound LP-8 is shown in Figure 15 The mass spectrometry results of compound LP-8 are shown in Figure 16 The mass spectrum data of compound LP-8 are: LCMS (ESI) [M+H] + :1604.72[M+H] + .

[0212] Example 1-9: A linker-payload and its preparation

[0213] This embodiment provides a linker-cytotoxin LP-9, wherein the linker-cytotoxin LP-9 has the following structure:

[0214]

[0215] The preparation method of the linker-cytotoxin LP-9 comprises the following steps:

[0216] Step 1: Compound 2-4 (1 g, 1.00 eq) was dissolved in N,N-dimethylformamide (10 mL) to obtain a solution. Methyl-pentaethylene glycol monobromide (0.75 g, 1.1 eq) and potassium carbonate (0.44 g, 1.5 eq) were added to the solution, stirred uniformly, then heated to 70°C and reacted at 70°C for 8 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (30 mL) and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 9-1. Compound 9-1 was obtained as a yellow oil (1.1 g, yield 73%). The mass spectrum data of compound 9-1 is: ESI-MS (m / z): 706.29 [M+H] + .

[0217] Step 2: Compound 9-1 (1.1 g, 1.00 eq) was dissolved in methanol (10 mL) to obtain a solution. 5% palladium on carbon (0.1 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 22 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 9-2. Compound 9-2 was obtained as a yellow oil (0.35 g, yield 46%). The mass spectrometric data of compound 9-2 were: ESI-MS (m / z): 484.31 [M+H] + .

[0218] Step 3: Compound 9-2 (0.2 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.08 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.08 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After TLC indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 9-3. Compound 9-3 was obtained as a yellow oil (0.23 g, yield 98%). The mass spectrometric data of compound 9-3 were: ESI-MS (m / z): 564.23 [M+H] + .

[0219] Step 4: Compound 9-3 (100 mg, 2.40 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq) and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution, and the temperature was lowered to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-9. Compound LP-9 is a yellow powdery solid (36 mg, yield 32%). The synthetic route of compound LP-9 is shown in Figure 17 The mass spectrometry results of compound LP-9 are shown in Figure 18 The mass spectrum data of compound LP-9 are: LCMS (ESI) [M+H] + :1386.60[M+H] + .

[0220] Example 1-10: A linker-payload and its preparation

[0221] This embodiment provides a linker-cytotoxin LP-10, wherein the linker-cytotoxin LP-10 has the following structure:

[0222]

[0223] The preparation method of the linker-cytotoxin LP-10 comprises the following steps:

[0224] Step 1: Dissolve compound 6-2 (1.6 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution; add 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (1.49 g, 1.2 eq) and N,N-diisopropylethylamine (0.88 g, 2.5 eq) to the solution, first cool to 0 ° C, and then add 2-(7-azabenzotriazole) -N,N,N',N'-tetramethyluronium hexafluorophosphate (1.28 g, 1.2 eq) was added and reacted at 25°C for 2 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 5). The organic phase was collected and washed with water (30 mL) and then with saturated brine (30 mL). It was then evaporated to dryness under reduced pressure to obtain compound 10-1. Compound 10-1 was used directly in the synthesis of compound 10-2 without purification. Compound 10-1 was a yellow oil (2.7 g, yield 96%). The mass spectrometric data of compound 10-1 were: ESI-MS (m / z): 1024.46 [M+H] + .

[0225] Step 2: Compound 10-1 (2.8 g, 1.00 eq) was dissolved in methanol (15 mL) to obtain a solution. 5% palladium on carbon (0.3 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with a mixture of dichloromethane and methanol (5 mL / 5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 10-2. Compound 10-2 was obtained as a yellow oil (1.66 g, yield 75%). The mass spectrometric data of compound 10-2 were: ESI-MS (m / z): 802.36 [M+H] + .

[0226] Step 3: Compound 10-2 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.013 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.012 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 10-3. Compound 10-3 was obtained as a yellow oil (0.02 g, yield 36%). The mass spectrum data of compound 10-3 is: ESI-MS (m / z): 882.35 [M+H] + .

[0227] Step 4: Compound 10-3 (150 mg, 2.30 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-10. Compound LP-10 was obtained as a yellow powdery solid (84 mg, 62% yield). The synthetic route of compound LP-10 is shown in Figure 19 The mass spectrometry results of compound LP-10 are shown in Figure 20 The mass spectrum data of compound LP-10 are: LCMS (ESI) [M+H] + :1705.74[M+H] + .

[0228] Example 1-11: A linker-payload and its preparation

[0229] This embodiment provides a linker-cytotoxin LP-11, wherein the linker-cytotoxin LP-11 has the following structure:

[0230]

[0231] The preparation method of the linker-cytotoxin LP-11 comprises the following steps:

[0232] Step 1: Compound 7-3 (92 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS No.: CAS2845164-91-0) (30 mg, 1.00 eq), N,N-diisopropylethylamine (12.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (22.8 mg, 1.10 eq) and 1-hydroxybenzotriazole (2.4 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-11. Compound LP-11 is a yellow powdery solid (39 mg, yield 65%). The synthetic route of compound LP-11 is shown in Figure 21 The mass spectrometry results of compound LP-11 are shown in Figure 22 The mass spectrum data of compound LP-11 are: LCMS (ESI) [M+H] + :1504.69[M+H] + .

[0233] Example 1-12: A linker-payload and its preparation

[0234] This embodiment provides a linker-cytotoxin LP-12, wherein the linker-cytotoxin LP-12 has the following structure:

[0235]

[0236] The preparation method of the linker-cytotoxin LP-12 comprises the following steps:

[0237] Step 1: Compound 8-3 (94 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (30 mg, 1.00 eq), N,N-diisopropylethylamine (12.8 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (22.8 mg, 1.10 eq), and 1-hydroxybenzotriazole (2.4 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-12. Compound LP-12 was obtained as a yellow powdery solid (48 mg, 79% yield). The synthetic route of compound LP-12 is shown in Figure 23 The mass spectrometry results of compound LP-12 are shown in Figure 24 The mass spectrum data of compound LP-12 are: LCMS (ESI) [M+H] + :1518.55[M+H] + .

[0238] Example 1-13: A linker-payload and its preparation

[0239] This embodiment provides a linker-cytotoxin LP-13, wherein the linker-cytotoxin LP-13 has the following structure:

[0240]

[0241] The preparation method of the linker-cytotoxin LP-13 comprises the following steps:

[0242] Step 1: The compound N-[(1,1-dimethylethoxy)carbonyl]glycylglycyl-L-phenylalanyl-glycine (purchased from MedChemExpress, CAS No.: 187794-49-6) (0.25 g, 1.20 eq,) was dissolved in N,N-dimethylformamide (5 mL) to obtain a solution; to the solution were added isotecan mesylate (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS: 169869-90-3) (0.25 g, 1.00 eq), N,N-dimethylformamide, and the like. Isopropylethylamine (0.216 g, 3.50 eq) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.30 g, 1.20 eq) were added, cooled to 0°C, and the reaction was continued at 0°C for 1 hour to obtain a reaction solution. After HPLC monitoring of the reaction completion, the reaction solution was evaporated to dryness under reduced pressure, then redissolved in dichloromethane to the original volume, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 11-1. Compound 11-1 was obtained as a yellow solid (0.4 g, 98% yield). The mass spectrometric data of compound 11-1 were: ESI-MS (m / z): 854.32 [M+H] + .

[0243] Step 2: Compound 11-1 (0.4 g, 1.00 eq) was dissolved in dioxane (2 mL) to obtain a solution; after adding hydrogen chloride ethyl acetate solution (4 mol / L, 3 mL, 25.6 eq) to the solution, the mixture was stirred evenly and then reacted at room temperature (25°C) for 14 hours to obtain a reaction solution; after LC-MS monitoring showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with ethyl acetate (5 mL) and the filtrate was obtained; the filtrate was dried to obtain compound 11-2. Compound 11-2 was a yellow solid (0.34 g, yield 92%). The mass spectrometry data of compound 11-2 were: ESI-MS (m / z): 754.30 [M+H] + .

[0244] Step 3: Compound 8-3 (156 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; compound 11-2 (50 mg, 1.00 eq), N,N-diisopropylethylamine (30 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.20 eq) and 1-hydroxybenzotriazole (4.5 mg, 0.50 eq) were added to the solution, and the temperature was lowered to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-13. Compound LP-13 is a white powdery solid (27 mg, yield 28%). The synthetic route of compound LP-13 is shown in Figure 25 The mass spectrometry results of compound LP-13 are shown in Figure 26 The mass spectrum data of compound LP-13 are: LCMS (ESI) [M+H] + :1517.69[M+H] + .

[0245] Example 1-14: A linker-payload and its preparation

[0246] This embodiment provides a linker-cytotoxin LP-14, wherein the linker-cytotoxin LP-14 has the following structure:

[0247]

[0248] The preparation method of the linker-cytotoxin LP-14 comprises the following steps:

[0249] Step 1: Dissolve compound 10-3 (100 mg, 2.50 eq) in N,N-dimethylformamide (1 mL) to obtain a solution; add compound 11-2 (36 mg, 1.00 eq), N,N-diisopropylethylamine (18 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (26 mg, 1.10 eq) and 1-hydroxybenzotriazole (3 mg, 0.50 eq) to the solution, cool to 0°C and continue the reaction at 0°C for 1 hour to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution is first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-14. Compound LP-14 is a white powdery solid (18 mg, yield 24%). The synthetic route of compound LP-14 is shown in Figure 27 The mass spectrometry results of compound LP-14 are shown in Figure 28The mass spectrum data of compound LP-14 are: LCMS (ESI) [M / 2+H] + :809.36[M / 2+H] + .

[0250] Example 1-15: A linker-payload and its preparation

[0251] This embodiment provides a linker-cytotoxin LP-15, wherein the linker-cytotoxin LP-15 has the following structure:

[0252]

[0253] The preparation method of the linker-cytotoxin LP-15 comprises the following steps:

[0254] Step 1: Dissolve compound 3-4 (0.71 g, 1.00 eq) in N, N-dimethylformamide (5 mL) to obtain a solution; add 2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioctadecane-38-acid (0.8 g, 1.00 eq, CAS No.: 2135793-73-4) and N, N-diisopropylethylamine (0.79 g, 4.50 eq) to the solution, then cool down. The mixture was cooled to 0°C, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.62 g, 1.2 eq) was added. The mixture was then reacted at 25°C for 8 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3) and then evaporated to dryness under reduced pressure to obtain compound 12-1. Compound 12-1 was used directly in the synthesis of compound 12-2 without purification. Compound 12-1 was a yellow oil (1.4 g, 100% yield, theoretical value). The mass spectrometric data of compound 12-1 were: ESI-MS (m / z): 1056.58 [M+H] + .

[0255] Step 2: Compound 12-1 (1.4 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution; 5% palladium on carbon (0.3 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with a mixture of dichloromethane / methanol (dichloromethane:methanol = 1:1, v / v, 5 mL) and the filtrate was obtained; the filtrate was evaporated to dryness under reduced pressure to obtain compound 12-2. Compound 12-2 was a yellow oil (1.1 g, yield 100%, theoretical value). The mass spectrometric data of compound 12-2 were: ESI-MS (m / z): 834.50 [M+H] + .

[0256] Step 3: Compound 12-2 (0.6 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.15 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.13 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After TLC indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 12-3. Compound 12-3 was obtained as a yellow oil (0.6 g, yield 90%). The mass spectrometric data of compound 12-3 were: ESI-MS (m / z): 914.44 [M+H] + .

[0257] Step 4: Compound 12-3 (105 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; VA-PAB-Exatecan (35 mg, 1.00 eq), N,N-diisopropylethylamine (15 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (27 mg, 1.10 eq) and 1-hydroxybenzotriazole (3 mg, 0.50 eq) were added to the solution, and the temperature was lowered to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-15. Compound LP-15 is a yellow powdery solid (45 mg, yield 58%). The synthetic route of compound LP-15 is shown in Figure 29 The mass spectrometry results of compound LP-15 are shown in Figure 30 The mass spectrum data of compound LP-15 are: LCMS (ESI) [M / 2+H] +:825.89[M / 2+H] + .

[0258] Example 1-16: A linker-payload and its preparation

[0259] This embodiment provides a linker-cytotoxin LP-16, wherein the linker-cytotoxin LP-16 has the following structure:

[0260]

[0261] The preparation method of the linker-cytotoxin LP-16 comprises the following steps:

[0262] Step 1: Dissolve compound 12-3 (160 mg, 2.50 eq) in N,N-dimethylformamide (3 mL) to obtain a solution; add 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (31 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (73 mg, 1.10 eq) and 1-hydroxybenzotriazole (5 mg, 0.50 eq) to the solution, cool to 0°C and continue the reaction at 0°C for 1 hour to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution is first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-16. Compound LP-16 is a white powdery solid (29 mg, yield 25%). The synthetic route of compound LP-16 is shown in Figure 31 The mass spectrometry results of compound LP-16 are shown in Figure 32 The mass spectrum data of compound LP-16 are: LCMS (ESI) [M+H] + :1649.47[M+H] + .

[0263] Example 1-17: A linker-payload and its preparation

[0264] This embodiment provides a linker-cytotoxin LP-17, wherein the linker-cytotoxin LP-17 has the following structure:

[0265]

[0266] The preparation method of the linker-cytotoxin LP-17 comprises the following steps:

[0267] Step 1: Compound 12-3 (63 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; GGFG-PAB-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS: 251459-32-2) (25 mg, 1.00 eq), N,N-diisopropylethylamine (9 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (18 mg, 1.25 eq) and 1-hydroxybenzotriazole (2 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-17. Compound LP-17 is a white powdery solid (24 mg, yield 48%). The synthetic route of compound LP-17 is shown in Figure 33 The mass spectrometry results of compound LP-17 are shown in Figure 34 The mass spectrum data of compound LP-17 are: LCMS (ESI) [M+H] + :1798.81[M+H] + .

[0268] Example 1-18: A linker-payload and its preparation

[0269] This embodiment provides a linker-cytotoxin LP-18, wherein the linker-cytotoxin LP-18 has the following structure:

[0270]

[0271] The preparation method of the linker-cytotoxin LP-18 comprises the following steps:

[0272] Step 1: Compound 2-4 (0.86 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioctadecane-38-oic acid (1.0 g, 1.00 eq) and N, N-diisopropylethylamine (0.54 g, 2.50 eq) were added to the solution, and the mixture was cooled to 0 ° C, and then 2 -(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.76 g, 1.2 eq) was added and reacted at 25°C for 8 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (30 mL × 3) and then evaporated to dryness under reduced pressure to obtain compound 13-1. Compound 13-1 was used directly in the synthesis of compound 13-2 without purification. Compound 13-1 was a yellow oil (1.77 g, yield 100%, theoretical value). The mass spectrometric data of compound 13-1 were: ESI-MS (m / z): 1042.52 [M+H] + .

[0273] Step 2: Compound 13-1 (1.77 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution; 5% palladium on carbon (0.25 g) was added to the solution, and the mixture was reacted at 25° C. under a hydrogen atmosphere for 16 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with methanol (5 mL) and the filtrate was obtained; the filtrate was evaporated to dryness under reduced pressure to obtain compound 13-2. Compound 13-2 was a yellow oil (1.39 g, yield 100%, theoretical value). The mass spectrometry data of compound 13-2 were: ESI-MS (m / z): 820.51 [M+H] + .

[0274] Step 3: Compound 13-2 (0.6 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.15 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.14 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 13-3. Compound 13-3 was obtained as a yellow oil (0.6 g, yield 90%). The mass spectrometric data of compound 13-3 were: ESI-MS (m / z): 900.42 [M+H]+ .

[0275] Step 4: Compound 13-3 (170 mg, 2.70 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (32 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (48 mg, 1.30 eq) and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution, and the temperature was lowered to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-18. Compound LP-18 is a yellow powdery solid (14 mg, yield 12%). The synthetic route of compound LP-18 is shown in Figure 35 The mass spectrometry results of compound LP-18 are shown in Figure 36 The mass spectrum data of compound LP-18 are: LCMS (ESI) [M+H] + :1635.49[M+H] + .

[0276] Example 1-19: A linker-payload and its preparation

[0277] This embodiment provides a linker-cytotoxin LP-19, wherein the linker-cytotoxin LP-19 has the following structure:

[0278]

[0279] The preparation method of the linker-cytotoxin LP-19 comprises the following steps:

[0280] Step 1: Dissolve triethylene glycol monomethyl ether (34.68 g, 1.10 eq) and sodium hydroxide (11.52 g, 1.50 eq) in a mixture of THF / H2O (80 mL / 80 mL) to obtain a solution; slowly add the solution dropwise to a solution of p-toluenesulfonyl chloride (40.00 g, 1.00 eq) in THF (80 mL) cooled to 0°C, and react at 0°C for 3 hours to obtain a reaction solution; after thin-layer chromatography shows the reaction is complete, the reaction solution is separated, the aqueous layer is extracted with ethyl acetate (50 mL x 3), and the organic phase is collected; the organic phase is washed with saturated brine (40 mL) and then evaporated to dryness under reduced pressure to obtain compound 14-1. Compound 14-1 is a colorless oil (58.20 g, yield 95%). The mass spectrometric data of compound 14-1 are: ESI-MS (m / z): 319.12 [M+H] + .

[0281] Step 2: Dissolve diethanolamine (13.32 g, 2.00 eq) in DMF (54 mL) to obtain solution A; dissolve triphenylmethane (16.04 g, 1.00 eq) in DCM (34 mL) to obtain solution B; cool solution A and solution B to 0°C, then dropwise add solution B to solution A, and react at 0°C for 24 hours to obtain a reaction solution; after thin-layer chromatography shows the reaction is complete, add ether / water (30 mL / 40 mL) to the reaction solution, beat for 2 hours, and filter to obtain a filter cake; add chloroform / petroleum ether (96 mL / 132 mL) to the filter cake, beat for 2 hours, and then concentrate to obtain compound 14-2. Compound 14-2 is a yellow oil (14.50 g, yield 72%). The mass spectrum data of compound 14-2 is: ESI-MS (m / z): 348.22 [M+H] + .

[0282] Step 3: Compound 14-2 (2.10 g, 1.00 eq) was dissolved in THF (20 mL) to obtain a solution. 14-1 (7.70 g, 4.00 eq) and sodium hydride (2.70 g, 12.00 eq) were added to the solution in sequence. The temperature was raised to 65°C and the reaction was continued at 65°C for 16 hours to obtain a reaction solution. After the reaction was completed by thin-layer chromatography, the reaction solution was cooled to room temperature and then purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 14-3. Compound 14-3 was obtained as a yellow oil (1.63 g, yield 42%). The mass spectrometric data of compound 14-3 were: ESI-MS (m / z): 640.25 [M+H] + . 1 HNMR(400MHz,Chloroform-d)δ7.52(m,6H),7.26(m,6H),7.16(m,3H),3.72-3.48(m,28H),3.39(s,6H),2.57(t,J=7.0Hz,4H).

[0283] Step 4: Compound 14-3 (1.73 g, 1.00 eq) was dissolved in methanol (8 mL) to obtain a solution. 5 mL of 5% (w / v, g / mL) aqueous hydrochloric acid was added dropwise to the solution at room temperature, and the reaction was continued at room temperature for 30 minutes to obtain a reaction solution. After the reaction was complete by thin layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was obtained. The filtrate was first concentrated under reduced pressure, then diluted with ether (20 mL), and the pH was adjusted to 8 with saturated sodium carbonate solution. The aqueous layer was finally evaporated to dryness to obtain a residue. Dichloromethane (40 mL) was first added to the residue, and the mixture was stirred for 2 hours, then filtered, and the dichloromethane was evaporated to dryness to obtain compound 14-4. Compound 14-4 was a yellow oil (0.83 g, yield 74%). The mass spectrometric data of compound 14-4 were: ESI-MS (m / z): 412.31 [M+H] + .

[0284] Step 5: Compound 4-bromo-2-fluorobenzaldehyde (2.10 g, 1.00 eq) was dissolved in DMF (20 mL) to obtain a dissolving solution; compound 14-4 (5.60 g, 1.20 eq) and potassium carbonate (1.70 g, 1.20 eq) were added to the dissolving solution, stirred evenly, and then reacted at 105°C for 16 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature, then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase was taken; the organic phase was first washed with saturated brine (20 mL×3), and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane: methanol = 100:0 to dichloromethane: methanol = 90:10) to obtain compound 14-5. Compound 14-5 is a yellow oil (2.50 g, yield 38%). The mass spectrum data of compound 14-5 is: ESI-MS (m / z): 580.17 [M+H] + . 1 H NMR (400MHz, Chloroform-d) δ10.29(s,1H),7.65(m,1H),7.44(m,1H),7.25(m,1H),3.70-3.48(m,28H),3.45(t,J=5.6Hz,4H),3.39(s,6H).

[0285] Step 6: Compound 14-5 (2.50 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution; after cooling the solution to 0°C, benzyl (triphenylphosphine) acetate (2.67 g, 1.50 eq) was added, then stirred evenly, and reacted at 20°C for 18 hours to obtain a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was first concentrated under reduced pressure, then redissolved with methanol to the original volume, and then purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and finally lyophilized to obtain compound 14-6. Compound 14-6 was a yellow powdery solid (2.67 g, yield 87%). The mass spectrometric data of compound 14-6 were: ESI-MS (m / z): 712.18 [M+H] + .

[0286] Step 7: Compound 14-6 (2.50 g, 1.00 eq) was dissolved in toluene (25 mL) to obtain a solution; benzyl carbamate (0.64 g, 1.20 eq), palladium acetate (39 mg, 0.05 eq), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (0.17 g, 0.10 eq) and potassium carbonate (0.58 g, 1.20 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C. under a nitrogen atmosphere and continued to react at 100 ° C. for 4 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25 ° C.), then evaporated to dryness under reduced pressure, and then purified by silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane: methanol = 100: 0 to dichloromethane: methanol = 90: 10) to obtain compound 14-7. Compound 14-7 is a yellow oil (2.30 g, yield 44%). The mass spectrum data of compound 14-7 is: ESI-MS (m / z): 783.32 [M+H] + .

[0287] Step 8: Compound 14-7 (2.30 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.20 g) was added to the solution and the mixture was reacted at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 14-8. Compound 14-8 was obtained as a yellow oil (0.39 g, yield 23%). The mass spectrometric data of compound 14-8 were: ESI-MS (m / z): 561.27 [M+H] + .

[0288] Step 9: Compound 14-8 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution; triethylamine (18 mg, 2.00 eq) and N-methoxycarbonylmaleimide (17 mg, 1.20 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution was cooled to room temperature and then evaporated to dryness under reduced pressure to obtain compound 14-9. Compound 14-9 was not purified and was directly used in the synthesis of compound LP-19. Compound 14-9 was a yellow oil (0.04 g, yield 70%). The mass spectrometry data of compound 14-9 was: ESI-MS (m / z): 641.28 [M+H] + .

[0289] Step 10: Compound 14-9 (150 mg, 3.16 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-19. Compound LP-19 was obtained as a yellow powdery solid (36 mg, 33% yield). The synthetic route of compound LP-19 is shown in Figure 37 The mass spectrometry results of compound LP-19 are shown in Figure 38 The mass spectrum data of compound LP-19 are: LCMS (ESI) [M / 2+H] + :732.36[M / 2+H] + .

[0290] Experimental Example 1: Preparation, extraction and identification of monoclonal antibodies against delta-like ligand 3 (DLL3)

[0291] 1. Mouse immunization

[0292] Five BALB / c mice and five C57bl / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) were immunized with human DLL3 target protein (purchased from Biopsy). The specific immunization procedure was as follows:

[0293] The mice were given the first immunization, the first booster immunization, the second booster immunization, and the third booster immunization on day 0, day 14, day 28, and day 56, respectively. During the immunization period, blood was collected from the mice 4 days before the first immunization, on day 21, and on day 35, and the serum was separated. The antibody titer in the mouse serum was detected by ELISA. Two mice with high antibody titers in the serum were selected for booster immunization to obtain δ-like ligand 3-immunized mice.

[0294] During the first immunization, the human DLL3 target protein was first diluted with physiological saline to obtain a dilution (dilution ratio of 1 mg: 1 mL); the dilution was then mixed with Freund's complete adjuvant (purchased from Shanghai Yisheng) at a volume ratio of 1:1 to obtain an immune preparation; the immune preparation was then subcutaneously injected into the mice at a dose of 50 μg protein per mouse to complete the first immunization; after the first immunization, Freund's incomplete adjuvant (purchased from Shanghai Yisheng) was used for subsequent booster immunizations, among which the first and third booster immunizations were intraperitoneal injections, and the second booster immunization was subcutaneous injection. The injection dose of the three booster immunizations was 25 μg protein per mouse.

[0295] 2. Spleen cell fusion

[0296] Two mice with high neutralizing antibody titers were selected based on serum titers. The mouse spleens were dissected and ground into single splenocytes. The single splenocytes were washed once using serum-free DMEM medium (purchased from Gibco). After washing, the cells were centrifuged at 300 g for 8 minutes, the supernatant was discarded, and the splenocyte pellet was collected. The splenocyte pellet was resuspended in serum-free DMEM medium, and the splenocytes were counted to obtain a splenocyte suspension. Mouse plasma cells were fused with sp20 cells (purchased from ATCC) to obtain fused cells (using the electrofusion protocol of Edward A. Greenfield, with a fusion efficiency of approximately one hybridoma per 200 B cells; for details, see the reference "Schmitt, JJ et al. "Efficient generation of stable antibody-forming hybridoma cells by electrofusion." Hybridoma vol. 8, 1 (1989): 107-15."). The fused cells were seeded into 384-well plates and cultured and screened using HAT complete medium (purchased from Gibco).

[0297] 3. Monoclonal Antibody Screening

[0298] The cell culture supernatant obtained by culture and screening was collected, and monoclonal antibody screening was performed by ELISA and flow cytometry to obtain the high-affinity antibody mJFab8.

[0299] The ELISA detection process is as follows: antigen A (human DLL3 protein, purchased from Biopsies, the complete amino acid sequence of human DLL3 protein is shown in SEQ ID NO. 16), antigen B (cynomolgus macaque DLL3 protein, purchased from Biopsies, the complete amino acid sequence of cynomolgus macaque DLL3 is shown in SEQ ID NO. 17) or antigen C (mouse DLL3 protein, purchased from Biopsies, the complete amino acid sequence of mouse DLL3 is shown in SEQ ID NO. 18) is coated with a coating buffer (Nanjing GenScript Co., Ltd.). NO.43) were diluted to a concentration of 0.5 μg / mL to obtain antigen dilution solutions; the antigen dilution solutions were added to the ELISA plate at an amount of 100 μL / well, incubated at 4°C overnight (16 h), and then the supernatant in the wells was discarded to obtain antigen-coated ELISA plates; blocking buffer (Nanjing GenScript Company) was added to the ELISA plate coated with the primary antibody at an amount of 100 μL / well, incubated at 37°C for 1 h, and then the supernatant in the wells was discarded to obtain blocked ELISA plates; human lovatuzumab ADC (purchased from Nanjing Pengbo Biological) and mouse lovatuzumab ADC (purchased from Nanjing Pengbo Biological) were used as positive controls, and human IgG (purchased from Nanjing Pengbo Biological) was used as a negative control. The antibody to be tested (i.e., the cell culture supernatant obtained by culture screening) was quantified and then gradiently diluted to obtain primary antibody dilution solutions of different concentrations; Different concentrations of primary antibody dilutions were added to the blocked ELISA plate at a volume of 100 μL / well, incubated at 37°C for 1 hour, and then the supernatant in the wells was discarded to obtain the ELISA plate incubated with the primary antibody; HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-human IgG (purchased from Jackson) was diluted to the recommended working concentration using the blocking buffer according to the instructions of the secondary antibody to obtain the secondary antibody dilution; the secondary antibody dilution was added to the antibody-incubated ELISA plate at a volume of 100 μL / well, incubated at 37°C for 330 minutes, and then the supernatant in the wells was discarded to obtain the ELISA plate incubated with the secondary antibody; the absorbance of each well in the ELISA plate incubated with the secondary antibody at 450 nm was detected using a multifunctional microplate reader (purchased from Beijing Kaiao Technology Co., Ltd.); according to the measured absorbance, GraphPad Prism was used to fit the EC50 values ​​of the test antibodies, which were used to measure their affinity for delta-like ligand 3. For ELISA assays, all test antibodies were initially diluted to a 10 mM concentration, followed by a three-fold serial dilution. The ELISA results are shown in Tables 1 and 2.

[0300] The flow cytometry detection process is as follows: CHO-K1 (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) is used as a negative cell line, and CHO-K1 / DLL3 overexpression cell line (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) is used as a positive cell line to detect each monoclonal antibody. During the detection, human lovatuzumab ADC and mouse lovatuzumab ADC are used as positive controls, human IgG is used as an isotype control, and PBS buffer is used as a negative control. The detection process is as follows: the monoclonal antibody sample is incubated with the above two cell lines at 4°C, washed, incubated with a secondary antibody, and then washed and detected by the machine (for the specific flow cytometry detection method, please refer to the literature "Tabatabaei, Mahdis Sadat, and Marya Ahmed. "Enzyme-Linked Immunosorbent Assay (ELISA)." Methods inmolecular biology (Clifton, NJ) vol. 2508 (2022): 115-134."). Among them, the secondary antibody of the monoclonal antibody to be tested and the human lovatuzumab ADC is detected using Alexa Fluor 500 antibody. 647AffiniPure goat anti-human IgG, Fcγ fragment specific antibody (Jackson, 109-605-098, 144613); the secondary antibody for mouse lovatuzumab ADC was Alexa 647AffiniPure goat anti-mouse IgG, Fcγ fragment-specific antibody (Nanjing Pengbo Biotechnology Co., Ltd.). Flow cytometry results are shown in Table 3.

[0301] As shown in Tables 1 and 2, a total of 10 hybridoma cell lines producing mouse-derived monoclonal antibodies against delta-like ligand 3 were screened by ELISA. The 10 hybridoma cell lines and the mouse-derived monoclonal antibodies against delta-like ligand 3 secreted by them were named mJFab1, mJFab2, mJFab3, mJFab4, mJFab5, mJFab6, mJFab7, mJFab8, mJFab9 and mJFab10, respectively. Among these monoclonal antibodies, except for mJFab5 which has a poor affinity, the other antibody samples have a high affinity for delta-like ligand 3.

[0302] As shown in Table 3 , among the monoclonal antibodies mJFab1, mJFab2, mJFab3, mJFab4, mJFab5, mJFab6, mJFab7, mJFab8, mJFab9, and mJFab10, mJFab5 bound weakly to CHO-K1 cells with high expression of δ-like ligands, while the remaining antibody samples could bind to CHO-K1 / human DLL3.

[0303] Table 1 ELISA test results

[0304]

[0305] Table 2 ELISA test results

[0306]

[0307] Table 3 Flow cytometry results

[0308]

[0309] 4. Antibody sequence determination

[0310] The hybridoma cell line mJFab8 obtained by screening was expanded and cultured, and total RNA was extracted using Trizol (purchased from ThermoFisher). Amplification was performed using mouse-Ig degenerate primers (purchased from Nanjing GenScript), and finally, sequencing was performed to obtain the antibody variable region CDR sequences of the monoclonal antibody mJFab8, as shown in Table 4. The amino acid sequence of the light chain variable region of the monoclonal antibody mJFab8 is shown in SEQ ID NO. 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 31.

[0311] Table 4 Antibody variable region CDR sequences

[0312] Functional domain Serial number Amino acid sequence LC CDR1 SEQ ID NO.1 RSSQSIVHSDGNTYLE LC CDR2 SEQ ID NO.2 KVSNRFS LC CDR3 SEQ ID NO.3 FQGSHLPFT HC CDR1 SEQ ID NO.4 TYGMS HC CDR2 SEQ ID NO.5 WINTYSGVPIYADDFKG HC CDR3 SEQ ID NO.6 WTVLTNWHFDV

[0313] 5. Antibody humanization

[0314] The monoclonal antibody mJFab8 was humanized using the bi0MOE plug-in. The CDR transplantation parameter was selected, that is, the CDR residues were fixed unchanged. The annotation method was selected as kabbat. Five humanized light chains and seven humanized heavy chains were generated. The variable region sequences of the humanized light chains and humanized heavy chains are shown in Table 5.

[0315] The five humanized light chains and seven humanized heavy chains were spliced ​​with the human IgG1 heavy chain constant region (amino acid sequence shown in SEQ ID NO. 21) and the human IgG kappa chain constant region (amino acid sequence shown in SEQ ID NO. 22), respectively, to form complete humanized antibody light and heavy chain sequences, thereby obtaining five humanized antibodies, namely, humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b. The light chain amino acid sequence of humanized antibody JFab4 is shown in SEQ ID NO. 24 (containing the VL-hum5 fragment), and the heavy chain amino acid sequence is shown in SEQ ID NO. 23 (containing the VH-hum6 fragment); the light chain amino acid sequence of humanized antibody JFab12 is shown in SEQ ID NO. 27 (containing the VL-hum2 fragment), and the heavy chain amino acid sequence is shown in SEQ ID NO. 25 (containing the VH-hum13 fragment); the light chain amino acid sequence of humanized antibody JFab13 is shown in SEQ ID NO. The amino acid sequence of the light chain of the humanized antibody JFab12b is shown in SEQ ID NO. 30 (containing the JFab12b / VL fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 28 (containing the VH-hum16 fragment); the amino acid sequence of the light chain of the humanized antibody JFab13b is shown in SEQ ID NO. 30 (containing the JFab13b / VL fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 29 (containing the JFab13b / VH fragment).

[0316] Table 5 Humanized sequences

[0317] Humanized sequence Serial number Sequence naming Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.11 VH-hum16 Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.12 VH-hum13 Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.13 VH-hum9 Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.14 VH-hum6 Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.15 VH-hum1 Humanized FR of the DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.18 VH-hum16 Humanized FR DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.19 JFab13b / VH Humanized FR DLL3 monoclonal antibody light chain variable region SEQ ID NO.7 VL-hum5 Humanized FR DLL3 monoclonal antibody light chain variable region SEQ ID NO.8 VL-hum4 Humanized FR DLL3 monoclonal antibody light chain variable region SEQ ID NO.9 VL-hum2 Humanized FR DLL3 monoclonal antibody light chain variable region SEQ ID NO.10 VL-hum1 Humanized FR DLL3 monoclonal antibody light chain variable region SEQ ID NO.20 JFab12b / VL,JFab13b / VL

[0318] 6. Humanized antibody screening

[0319] The humanized antibody coding sequence was derived, synthesized, and cloned into the PTT5 antibody expression vector (completed by Anhui General Biotechnology Co., Ltd.). The antibody light chain and heavy chain combination was expressed and purified (for details on the expression and purification methods, see the document "Kunert, Renate, and David Reinhart. "Advances in recombinant antibody manufacturing." Applied microbiology and biotechnology vol. 100, 8 (2016): 3451-61."), resulting in humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b. Affinity measurements were performed on the humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b using the monoclonal antibody mJFab8 as a control using Biacore T200 (Cytiva). The affinity determination results are shown in Tables 6 to 7 (in addition to JFab4, JFab12, JFab13, JFab12b and JFab13b, Tables 6 to 7 also show other humanized antibodies obtained from the same batch as JFab4, JFab12, JFab13, JFab12b and JFab13b as controls).

[0320] As shown in Tables 6 and 7, the equilibrium dissociation constant between the humanized antibody JFab13b and the DLL3 antigen is the lowest. This indicates that JFab13b has the best affinity for the DLL3 target among all the antibodies obtained through humanization.

[0321] Table 6 Affinity determination results

[0322]

[0323] Table 7 Affinity determination results

[0324]

[0325] 7. Expression and purification of humanized antibodies

[0326] The humanized antibody coding sequence was derived for synthesis and cloned into the PTT5 antibody expression vector (completed by Anhui General Biotechnology Co., Ltd.), and a recombinant vector expressing the antibody heavy chain and a recombinant vector expressing the antibody light chain were obtained. EXPI293F cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were subcultured using serum-free cell culture medium (purchased from Aupromycin) and the cell density was adjusted to 3×10 6After 100 cells / mL, the recombinant vector expressing the antibody heavy chain and the recombinant vector expressing the antibody light chain were transiently transfected into EXPI293F cells using a transfection reagent (purchased from Optum). After transfection, the cell density and viability were determined by trypan blue cell staining and counting. Six days after transfection, the cell culture supernatant was collected and affinity purified. The purified protein sample was analyzed by SEC HPLC. The SEC HPLC test results are shown in Table 8. Among them, the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab4 is shown in SEQ ID NO.34, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab4 is shown in SEQ ID NO.33; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab12 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab12 is shown in SEQ ID NO.35; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab13 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13 is shown in SEQ ID NO.37; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab12b is shown in SEQ ID NO.40, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab12b is shown in SEQ ID NO.38; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO. The nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO.40, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO.39.

[0327] Table 8 SEC HPLC test results

[0328] Serial number Antibody name Purity (HPLC) 1 JFab4 96.26％ 2 JFab12 96.99％ 3 JFab13 95.07％ 4 JFab12b 100％ 5 JFab13b 98.57％ 6 mJFab8 98.62％

[0329] 8. Humanized Antibody Internalization Detection

[0330] The endocytic activity of the antibodies was detected using human small cell lung cancer SHP-77 (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) and human small cell lung cancer NCI-H82 (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences). The endocytic activity assay method was as follows: the antibody to be tested was diluted to a concentration of 15 μg / mL in PBS buffer containing 2% (v / v) fetal bovine serum (FBS) to obtain the antibody dilution solution to be tested; for each antibody, 4 aliquots of 1.5×10 5After the cells were plated in a 96-well U-shaped plate, the supernatant was discarded by centrifugation; the antibody dilution to be tested was added to the 96-well U-shaped plate at a volume of 200 μL / well, incubated on ice for 45 minutes, and after the incubation, the supernatant was discarded by centrifugation at 4°C; PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to the 96-well U-shaped plate at a volume of 150 μL / well, the supernatant was discarded by centrifugation, and the operation was repeated twice; after the cells in the 96-well U-shaped plate were resuspended with 200 μL / well PBS buffer, 3 aliquots of cells were taken out and placed in a 37°C cell culture incubator for 4 hours, 1 hour, and 0 hours respectively, and the remaining aliquot of cells was placed on ice for 4 hours. After the incubation time was over, all sample cells were placed at 4°C and centrifuged and the supernatant was discarded; Alexa Fluor647 labeled goat anti-human IgG (Jackson, catalog number 109-605-098, 2% FBS-PBS 1:800 dilution) was added to a 96-well U-shaped plate at a volume of 100 μL / well and incubated on ice for 30 minutes. After the incubation, the cells were centrifuged at 4°C and the supernatant was discarded. PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to a 96-well U-shaped plate at a volume of 150 μL / well and the supernatant was discarded. The operation was repeated twice. The cells in the 96-well U-shaped plate were resuspended with 200 μL / well PBS buffer containing 2% (v / v) FBS (fetal bovine serum) and the cells were washed with water. The average fluorescence intensity in the wells was detected by NxT (purchased from Thermo), and the internalization rate of different test antibodies in SHP-77 cells or NCI-H82 cells was calculated according to the formula: internalization rate (%) = 100-(MFI of the sample incubated at 37°C at that time point / MFI of the control sample incubated at 4°C at that time point) × 100, thereby evaluating the internalization effect of the test antibodies in small cell lung cancer cells with high expression of delta-like ligand 3. The statistics of the fluorescence values ​​are shown in Tables 9 and 10, and the internalization rate (%) is shown in Tables 1 and 2. Figures 39-40 .

[0331] The cell surface antibody loading can be expressed as mean fluorescence intensity (MFI), and the fluorescence intensity decreases as the incubation time increases, indicating that the antibody is internalized by the cells, the cell surface antibody loading decreases, and the fluorescence intensity decreases. Figures 39-40 It can be seen that the humanized antibodies JFab12, JFab13, JFab12b and JFab13b can all be internalized by small cell lung cancer cells with high expression of delta-like ligand 3 (DLL3) and the internalization efficiency is high.

[0332] Table 9 Fluorescence results of antibody internalization detected by flow cytometry (NCI-H82)

[0333] Antibody 0 hours 1 hour 4 hours JFab12 1816 809 762 JFab12b 2278 831 662 JFab13 2030 933 820 JFab13b 2305 935 835 mJFab8 2888 1268 1181

[0334] Table 10 Fluorescence results of flow cytometry detection of antibody internalization (SHP-77)

[0335] Antibody 0 hours 1 hour 4 hours JFab12 1150 451 435 JFab12b 2566 800 755 JFab13 1950 739 753 JFab13b 2617 787 730 mJFab8 2835 1042 993

[0336] 9. Study on the specificity of humanized antibodies

[0337] Immunoblotting assays were performed using the JFab13b antibody with human DLL1 recombinant protein (purchased from Yisheng Biotechnology, with the complete amino acid sequence shown as SEQ ID NO.41), human DLL3 recombinant protein (purchased from Biopsies, with the complete amino acid sequence shown as SEQ ID NO.16), or human DLL4 recombinant protein (purchased from Huamei Biotechnology, with the complete amino acid sequence shown as SEQ ID NO.42) to determine the specificity of the antibody (for details of immunoblotting assays, see the reference "Hirano, Seishiro. "Western blot analysis." Methods in molecular biology (Clifton, NJ) vol. 926 (2012): 87-97."). In immunoblotting assays, all antigens contained a histidine His tag, and anti-His tag antibodies were also used for immunoblotting detection. JFab13b and anti-His tag antibody (purchased from Mitaka Bio) were used as primary antibodies and combined with rabbit anti-human-IgG-HRP (purchased from Solebol) and rabbit anti-mouse-IgG-HRP (purchased from Solebol) for color development. The color development results are shown in Figure 41 .

[0338] Depend on Figure 41 It can be seen that JFab13b only binds to human DLL3 recombinant protein, and does not bind to DLL1 recombinant protein and DLL4 recombinant protein, proving that JFab13b specifically binds to DLL3 recombinant protein.

[0339] Example 2-1: A monoclonal antibody against delta-like ligand 3

[0340] This example provides an anti-δ-like ligand 3 monoclonal antibody JFab4, the amino acid sequence of the light chain of the monoclonal antibody JFab4 is shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.23.

[0341] Example 2-2: A monoclonal antibody against delta-like ligand 3

[0342] This example provides an anti-δ-like ligand 3 monoclonal antibody JFab12, the amino acid sequence of the light chain of the monoclonal antibody JFab12 is shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.25.

[0343] Example 2-3: A monoclonal antibody against delta-like ligand 3

[0344] This example provides an anti-δ-like ligand 3 monoclonal antibody JFab13, the amino acid sequence of the light chain of the monoclonal antibody JFab13 is shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.26.

[0345] Example 2-4: A monoclonal antibody against delta-like ligand 3

[0346] This example provides an anti-δ-like ligand 3 monoclonal antibody JFab12b, the amino acid sequence of the light chain of the monoclonal antibody JFab12b is shown in SEQ ID NO.30, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.28.

[0347] Example 2-5: A monoclonal antibody against delta-like ligand 3

[0348] This example provides an anti-δ-like ligand 3 monoclonal antibody JFab13b, the amino acid sequence of the light chain of the monoclonal antibody JFab13b is shown in SEQ ID NO.30, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.29.

[0349] Example 3-1: An antibody-drug conjugate and its preparation

[0350] This embodiment provides an antibody-drug conjugate, and the preparation method of the antibody-drug conjugate is as follows:

[0351] Method 1: Different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were exchanged into PBS (0.012 M PBS buffer, pH 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain exchange solutions. An aqueous solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) (7.5 eq) was added to the exchange solutions, and the mixture was reacted in a 37°C water bath for 1.5 hours to completely open the disulfide bonds between the antibodies, yielding reaction solution A. Reaction solution A was cooled to 25°C in a water bath. Deruxtecan (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd., CAS No.: 1599440-13-7, the linker used is MC-GGFG peptide) and the compounds of Example 1-1 to Example 1-19 (LP-1 to LP-19) were dissolved in DMSO to prepare a 10mM solution, and then added to reaction solution A in an amount of 12.0eq, and DMSO was added to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain reaction solution B. Reaction solution B was purified using an AmiconUltra 30K ultrafiltration tube or ProteinA4FF agarose purification resin (purchased from Sangon) to remove uncoupled small molecules to obtain the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR value) was detected using the RP-HPLC / MS method. The purity was detected using the SEC-HPLC method.

[0352] Method 2: Different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were exchanged into PBS (0.012 M PBS buffer, pH 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain exchange solutions. An aqueous solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) (2.5 eq) was added to the exchange solutions, and the mixture was reacted in a 37°C water bath for 1.5 hours to completely open the disulfide bonds between the antibodies, yielding reaction solution A. Reaction solution A was cooled to 25°C in a water bath. MC-VC-PAB-MMAE (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., the linker used is MC-Val-Cit-PAB), MC-VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., the linker used is MC-Val-Ala-PAB) and the compounds of Examples 1-1 to 1-19 (LP-1 to LP-19) were dissolved in DMSO to prepare a 10mM solution, and then added to the reaction solution A in an amount of 6.0eq, and DMSO was added to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain reaction solution B. Reaction solution B was purified using an Amicon Ultra 30K ultrafiltration tube or ProteinA4FF agarose purification resin (purchased from Biopharmaceuticals) to remove uncoupled small molecules to obtain the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR value) was calculated using the RP-HPLC / MS method. The purity was detected using the SEC-HPLC method.

[0353] Method 3: The different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were replaced with PBS solution (0.012M PBS buffer at pH = 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain replacement solution. An aqueous solution (3.4 eq) containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the replacement solution, and the solution was placed in a 37°C water bath for 1.5 hours to completely open the disulfide bonds between the antibodies to obtain reaction solution A. The reaction solution A was cooled to 25°C in a water bath. The compounds of Examples 1-1 to 1-19 (LP-1 to LP-19) were dissolved in DMSO to prepare 10 mM solutions, and then 10.0 eq of each solution was added to reaction solution A. DMSO was added to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain reaction solution B. Reaction solution B was purified using Amicon Ultra 30K ultrafiltration tubes or ProteinA4FF agarose purification resin (purchased from Bio-Pharma) to remove unconjugated small molecules to obtain the corresponding ADC, which was stored at -80°C. The antibody-drug conjugate ratio (DAR) was calculated using RP-HPLC / MS. Purity was determined using SEC-HPLC.

[0354] Among them, the antibody-drug coupling ratio (DAR value) detection method is as follows:

[0355] The sample was diluted to a concentration of 1 mg / mL with 25 mM ammonium bicarbonate aqueous solution, and 1 μL of N-glycosidase F (PNGase F) was added. The reaction was allowed to react at room temperature for 2 hours. After the reaction was completed, dithiothreitol (DTT) was added to a final concentration of 20 mM. The reaction was allowed to react at room temperature for 1 hour before analysis was performed directly on the instrument. Instrument model: Thermo Fisher Vanquish & QE-HF-X. Liquid chromatography parameters are shown in Table 11. Mass spectrometry conditions were: ESI ion source, spray voltage 3.8 kV, sheath gas (N2) flow rate 40 arb, scan range (m / z) 400-6000; data acquisition was performed in positive ion mode using data-dependent ionization (DDA) mode.

[0356] Purity detection method is as follows:

[0357] SEC Purity Analysis - SEC-HPLC (Size Exclusion Chromatography). Instrument: Agilent 1260 Liquid Chromatograph. Sample Preparation: Dilute the sample to approximately 1 mg / mL with mobile phase, centrifuge at 12,000 rpm for 5 minutes, and sample the supernatant for analysis. Chromatographic conditions are shown in Table 12.

[0358] Table 11 Liquid phase parameters

[0359]

[0360] Table 12 Chromatographic conditions

[0361] Chromatographic columns TOSOH, TSKge1 G3000WXL, 5μm, 7.8mm×300mm Mobile phase 100 mM PPB + 200 mM arginine hydrochloride, 5% (v / v) isopropanol (pH 6.8) flow rate 0.6mL / min Column temperature 25℃ Injection volume 20 μL Elution time 25min Elution gradient Isocratic elution

[0362] Experimental Example 2: Evaluation of the drugability of antibody-drug conjugates

[0363] The drugability of the antibody-drug conjugate of Example 3-1 was evaluated (drugability evaluation was performed by measuring the DAR value by RP-HPLC / MS and the purity by SEC-HPLC, as described in the patent application publication number CN116271079A). The evaluation results are shown in Table 13 (in Table 13, ADC-1 to ADC-12, ADC-13 to ADC-18, ADC-21-8, ADC-22, ADC-23, ADC-24-8, ADC-25, ADC-26, ADC-27-8, and ADC-28 were prepared using method 1 of Example 3-1; ADC-MMAE, ADC-19, ADC-20, ADC-21-4, ADC-24-4, and ADC-27-4 were prepared using method 2 of Example 3-1; and ADC-27-6 was prepared using method 3 of Example 3-1).

[0364] As can be seen from Table 13, at an appropriate drug loading amount, the JFab12b antibody can be normally coupled to Deruxtecan, LP-1, LP-2, LP-3, LP-4, LP-5, LP-6, LP-7, LP-8, LP-9, LP-10, and MC-Vc-PAB-MMAE; the JFab13 antibody can be normally coupled to LP-3 and LP-7; the JFab13b antibody can be normally coupled to LP-7, LP-8, LP-11, LP-12, Deruxtecan, LP-13, LP-14, LP-15, LP-16, LP-17, LP-18, and LP-19; the drug-antibody coupling ratio and aggregation degree obtained by coupling meet the expected requirements; however, the aggregation degree after coupling of JFab12b and JFab13b antibodies with MC-VA-PAB-Exatecan exceeds the acceptable range even at the lowest drug loading amount. It can be seen that except for the MC-VA-PAB-Exatecan coupling, the druggability of other studied cases is feasible.

[0365] Table 13 Evaluation of drugability of antibody-drug conjugates (ADCs)

[0366] ADC Name Antibody name Linker-Cytotoxin No. Concentration mg / mL DAR Aggregation ADC-1 JFab12b Deruxtecan 3.37 7.36 5.2％ ADC-2 JFab12b LP-1 2.20 7.42 2.9％ ADC-3 JFab12b LP-2 2.20 7.61 1.9％ ADC-4 JFab12b LP-3 2.53 7.73 3.0％ ADC-5 JFab12b LP-4 1.40 7.53 7.2％ ADC-6 JFab12b LP-5 0.39 7.69 5.2％ ADC-7 JFab12b LP-6 1.74 7.58 2.3％ ADC-8 JFab12b LP-7 2.04 7.66 1.6％ ADC-9 JFab12b LP-8 2.21 7.54 1.2％ ADC-10 JFab12b LP-9 1.18 7.44 2.9％ ADC-11 JFab12b LP-10 2.24 7.70 2.1％ ADC-12 JFab12b LP-11 2.99 7.48 ＞10％ ADC-MMAE JFab12b MC-VC-PABC-MMAE 2.65 4.31 0.9％ ADC-13 JFab13 LP-3 4.15 7.67 1.6％ ADC-14 JFab13 LP-7 1.85 7.34 1.3％ ADC-15 JFab13b LP-7 5.99 7.73 1.8％ ADC-16 JFab13b LP-8 2.43 7.62 1.7％ ADC-17 JFab13b LP-11 3.63 7.53 ＞10％ ADC-18 JFab13b Deruxtecan 2.21 8.00 5.3％ ADC-19 JFab13b LP-11 1.84 4.25 8.8％ ADC-20 JFab13b MC-VA-PABC-Exatecan 2.65 3.64 ＞10％ ADC-21-4 JFab13b LP-12 4.15 4.34 4.0％ ADC-21-8 JFab13b LP-12 4.15 7.82 6.0％ ADC-22 JFab13b LP-13 3.69 8.00 0.1％ ADC-23 JFab13b LP-16 4.13 8.00 0.1％ ADC-24-4 JFab13b LP-17 1.37 4.52 1.1％ ADC-24-8 JFab13b LP-17 1.38 7.63 ＞10％ ADC-25 JFab13b LP-18 3.86 8.00 0.1％ ADC-26 JFab13b LP-14 4.2 7.85 0.1％ ADC-27-4 JFab13b LP-15 4.4 4.82 4.4％ ADC-27-6 JFab13b LP-15 1.86 6.27 6.2％ ADC-27-8 JFab13b LP-15 4.3 7.26 4.8％ ADC-28 JFab12b LP-19 1.63 7.24 0.2％

[0367] Experimental Example 3: Evaluation of in vitro cytotoxic activity of antibody-drug conjugates

[0368] The antibody drug conjugate of Example 3-1 was evaluated for in vitro cytotoxic activity using NCI-H82 cells, SHP-77 cells, and DMS-53 cells (all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences). The in vitro cytotoxic activity evaluation method is as follows: a cell suspension was prepared using a cell culture medium containing 10% (v / v) fetal bovine serum, and 135 μL of the cell suspension was added to a 96-well plate at each well. No cells were plated in the first and twelfth columns, and only 135 μL of culture medium was added. The cells were then incubated at 37°C in 5% (v / v) carbon dioxide for 16 hours. The ADC sample to be tested was prepared into a first-well working solution (10× concentration) using PBS buffer, and then graded diluted with PBS buffer according to the corresponding multiples. 15 μL of a 10× concentration ADC solution was added to each well, and the cells were incubated at 37°C in 5% carbon dioxide for 5 days. Add 15 μL of CCK-8 solution (APExBIO) to each well and incubate in the dark at 37°C in 5% CO2 for 1–4 hours. Read absorbance at 450 nm using a microplate reader and analyze data using GraphPad Prism 5. See Table 14 for different cell plating densities, initial working solution concentrations (10× concentration), and ADC sample dilution factors. See Table 15 for in vitro cytotoxic activity evaluation results.

[0369] As shown in Table 15, with the exception of ADC-20, which exhibited relatively weak in vitro cytotoxic activity, the remaining ADCs in Example 3-1 all exhibited significant in vitro cytotoxic activity against NCI-H82 cells. Furthermore, their cytotoxicity was not significantly different from that of ADC-1 and ADC-18, which were conjugated to deruxtecan. This indicates that the antibody-drug conjugates in Example 3-1 exhibited strong target cell cytotoxic activity, specifically against NCI-H82 cells expressing delta-like ligand 3 (DLL3).

[0370] As shown in Table 15, the antibody-drug conjugates of Example 3-1 all exhibited significant in vitro cytotoxicity against SHP-77 cells. Furthermore, when conjugated with JFab12b, ADC-8 exhibited a lower IC50 compared to ADC-1 conjugated with deruxtecan. When conjugated with JFab13b, ADC-15, ADC-27-6, and ADC-27-8 exhibited lower IC50s compared to ADC-18 conjugated with deruxtecan. ADC-16, ADC-25, and ADC-27-4 showed no significant differences. The remaining ADCs tested exhibited higher IC50s. Consequently, ADC-1, ADC-8, ADC-9, ADC-15, ADC-16, ADC-18, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited potent target cell cytotoxicity, capable of killing SHP-77 cells expressing delta-like ligand 3 (DLL3).

[0371] As shown in Table 15, the antibody-drug conjugates of Example 3-1 all exhibited significant in vitro cytotoxicity against DMS-53 cells. Furthermore, when conjugated with JFab13b, compared to ADC-18 conjugated with Deruxtecan, ADC-16, ADC-24-4, ADC-24-8, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited lower IC50 values, while ADC-25 showed no significant difference. The remaining ADCs tested exhibited higher IC50 values. Consequently, ADC-16, ADC-18, ADC-24-4, ADC-24-8, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited potent target cell cytotoxicity, capable of killing DMS-53 cells expressing delta-like ligand 3 (DLL3).

[0372] Table 14 Plating density of different cells, concentration of working solution in the first well (10× concentration) and dilution multiple of ADC samples

[0373]

[0374] Table 15 In vitro killing activity of antibody drug conjugates (ADC) (NCI-H82, SHP-77, DMS-53 cells)

[0375] ADC H82 IC50(nM) SHP77 IC50(nM) DMS53 IC50 (nM) ADC-1 0.89 613.27 -- ADC-2 3.26 -- -- ADC-3 0.9 -- -- ADC-4 0.98 553.15 -- ADC-5 1.12 -- -- ADC-7 0.96 -- -- ADC-8 0.4 -- -- ADC-9 0.58 804.08 -- ADC-10 1.72 -- -- ADC-11 1.43 ＞900 -- ADC-12 0.36 -- -- ADC-13 2.83 -- -- ADC-14 2.35 -- -- ADC-15 1 135.86 -- ADC-16 1.2 376.83 3.6 ADC-17 1.35 -- -- ADC-18 0.99 407.24 9.00 ADC-19 3.74 -- -- ADC-20 18.78 -- -- ADC-22 0.78 >1000nM 30.69 ADC-23 0.69 >1000nM 34.50 ADC-24-4 1.81 -- 3.54 ADC-24-8 0.62 -- 2.54 ADC-25 0.31 385.75 16.62 ADC-26 0.47 -- 21.61 ADC-27-4 6.60 331.50 12.15 ADC-27-6 2.46 281.68 4.97 ADC-27-8 1.95 210.45 4.15 ADC-28 2.56 -- --

[0376] In Table 15, “--” means not detected.

[0377] Experimental Example 4: In vivo efficacy evaluation of antibody-drug conjugates

[0378] Experiment 1: NCI-H82 tumor-bearing mice

[0379] 1. Test drugs and materials

[0380] Blank control group: PBS;

[0381] ADC-15 (treatment group): low dose 2 mg / kg; high dose 5 mg / kg;

[0382] ADC-25 (treatment group): dose 5 mg / kg;

[0383] ADC-27-8 (treatment group): dose 5 mg / kg;

[0384] JFab13b (naked antibody group): dose 5 mg / kg;

[0385] Female Balb / c nude mice aged 6 to 8 weeks (purchased from Beijing Weitonglihua Company) were injected subcutaneously on the right back with 100 μL of 5×10 50% (v / v) Matrigel.6 Human small cell lung cancer cells (NCI-H82) were grown to an average volume of 150 mm 3 Around 6:00 p.m., the mice were randomly divided into 5 groups according to tumor size and mouse body weight, with 5 animals in each group. The day of group administration was defined as day 0.

[0386] Administration was via the tail vein, once weekly for a total of two doses. The experiment was terminated after 21 days of dosing or when tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice weekly, and animal survival was observed. Data were recorded using Excel statistical software and plotted using GraphPad Prism software. Following the experiment, mice were euthanized, and the tumor inhibition rate (TGI) (%) was calculated. Tumor volume (V) was calculated as follows: V = 1 / 2 * L. 长 *L 短 2 The calculation formula of tumor inhibition rate (TGI%) is: TGI%=(1-(T i -T0) / (V i -V0))); where T i is the mean tumor volume of the treatment group on the i-th day of administration, T0 is the mean tumor volume of the treatment group on the 0th day of administration, V i is the mean tumor volume of the blank control group on the i-th day of administration, and V0 is the mean tumor volume of the blank control group on the 0th day of administration.

[0387] 2. Data Analysis

[0388] Data statistics were calculated using Excel 2021. The experimental results are shown in Table 16 and Figures 42 and 43 shown.

[0389] 3. Conclusion

[0390] The antibody-drug conjugate of Example 3-1 can significantly reduce tumor volume and has good in vivo anti-tumor activity. At the same time, there is no death in the test mice and no significant weight loss, indicating that the test drug has good safety.

[0391] Experiment 2: SHP-77 tumor-bearing mice

[0392] 1. Test drugs and materials

[0393] Blank control group: PBS;

[0394] ADC-15 (treatment group): low dose 3 mg / kg; medium dose 6 mg / kg; high dose 9 mg / kg;

[0395] ADC-27-8 (treatment group): 6 mg / kg;

[0396] Female Balb / c nude mice aged 6 to 8 weeks (purchased from Beijing Weitonglihua Company) were injected subcutaneously on the right back with 100 μL of 1×10 7 Human small cell lung cancer cells (SHP-77, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were grown to an average volume of 150 mm. 3 Around 6:00 p.m., the mice were randomly divided into 4 groups according to tumor size and mouse body weight, with 5 animals in each group. The day of group administration was defined as day 0.

[0397] Administration was via the tail vein, once weekly for a total of two doses. The experiment was terminated after 21 days of dosing or when tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice weekly, and animal survival was observed. Data were recorded using Excel statistical software and plotted using GraphPad Prism software. Following the experiment, mice were euthanized, and the tumor inhibition rate (TGI) (%) was calculated. Tumor volume (V) was calculated as follows: V = 1 / 2 * L. 长 *L 短 2 The calculation formula of tumor inhibition rate (TGI%) is: TGI%=(1-(T i -T0) / (V i -V0))); where Ti is the mean tumor volume of the treatment group on the i-th day of administration, T0 is the mean tumor volume of the treatment group on the 0th day of administration, V i is the mean tumor volume of the blank control group on the i-th day of administration, and V0 is the mean tumor volume of the blank control group on the 0th day of administration.

[0398] 2. Data Analysis

[0399] Data statistics were calculated using Excel 2021. The experimental results are shown in Table 17 and Figures 44 and 45 shown.

[0400] 3. Conclusion

[0401] The antibody-drug conjugate of Example 3-1 can significantly reduce tumor volume and has good in vivo anti-tumor activity. At the same time, there is no death in the test mice and no significant weight loss, indicating that the test drug has good safety.

[0402] Table 16 Efficacy of ADC on NCI-H82 xenograft tumors in nude mice

[0403] ADC dosage TGI on day 17 (%) ADC-15 2mg / kg 108 ADC-15 5mg / kg 110 ADC-25 5mg / kg 110 ADC-27-8 5mg / kg 110

[0404] Table 17 Efficacy of ADC on SHP-77 xenografts in nude mice

[0405] ADC dosage TGI on day 14 (%) ADC-15 3mg / kg 87 ADC-15 6mg / kg 104 ADC-15 9mg / kg 105 ADC-27-8 6mg / kg 108

[0406] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An antibody-drug conjugate for treating tumors, characterized in that: The general structural formula of the antibody drug conjugate is Ab-[L1-L2-L3-L4-D] p ; Among them, Ab is a monoclonal antibody targeting tumor cell surface antigens; D is a bioactive molecule that has an inhibitory effect on tumor cells; p is the drug loading capacity, and the value of p is an integer or decimal from 1 to 16; L1-L2-L3-L4 is a linker connecting the monoclonal antibody and the biologically active molecule; L1 is selected from the following structures: In L1, Represents the connection site, the connection site marked by number 1 is connected to Ab through the S atom, and the connection site marked by number 2 is connected to L2; L2 is In L2, Represents the connection site, the connection site marked by number 3 is connected to L1, and the connection site marked by number 4 is connected to L3; Z1 is selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, cyano, amino, nitro or hydroxyl; Z2 is selected from -NR1R2 or -OR3; In Z2, R1 and R2 are each independently selected from -(CH2CH2O) m -CH3, hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl; or, R1 and R2 together with the nitrogen atom to which they are connected form a 4-12 membered heterocyclic group; R3 is selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclic group; In R1, R2 and R3, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; the atoms in the heterocyclic group are each independently selected from C atoms, N atoms, O atoms or S atoms; and the heterocyclic group is substituted by R4; m is an integer from 1 to 24; R4Select-(CH2CH2O) m -CH3, -C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, -C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group; In R4, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; m is an integer from 1 to 24; R5 is selected from a hydrogen atom, an alkyl group, a cycloalkyl group or a heterocyclic group; In R5, the alkyl, cycloalkyl and heterocyclic groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; Z3 is selected from -(CH2) i -C(=O)-**、-(CH2) i (OCH2CH2) j -C(=O)-**, -(CH2) i -C(=O)-NH-(CH2CH2O) j -CH2CH2-C(=O)-**、-NH-(CH2) i -C(=O)-**、-NH-(CH2) i (OCH2CH2) j -C(=O)-**or chemical bond; In Z3, i is an integer from 1 to 10; j is an integer from 1 to 20; ** is the 4th position; L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues; In L3, the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; L4 is selected from or chemical bonds; In L4, Represents the connection site. The connection site marked with number 5 is connected to L3, and the connection site marked with number 6 is connected to D.

2. The antibody-drug conjugate according to claim 1, wherein The D is a cytotoxic compound, immunomodulator, enzyme or kinase inhibitor that has an inhibitory effect on tumor cells.

3. The antibody-drug conjugate according to claim 1, wherein The L1 is In L1, Represents the connection site. The connection site marked with number 1 is connected to Ab through the S atom, and the connection site marked with number 2 is connected to L2.

4. The antibody-drug conjugate according to claim 1, wherein The L2 is In L2, Represents the connection site. The connection site marked with number 3 is connected to L1, and the connection site marked with number 4 is connected to L3.

5. The antibody-drug conjugate according to claim 4, wherein The Z2 is selected from In Z2, the value of n is an integer from 1 to 13.

6. The antibody-drug conjugate according to claim 4, wherein Said Z3 is selected from -(CH2) i -C(=O)-** or -(CH2) i (OCH2CH2) j -C(=O)-**; in Z3, the value of i is an integer from 1 to 10; the value of j is an integer from 1 to 20; ** is the 4th position.

7. The antibody-drug conjugate according to claim 1, wherein L3 is an amino acid residue, and L3 is selected from a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine ​​residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue or a valine residue; Alternatively, the L3 is a short peptide consisting of 2 to 10 amino acid residues, and the amino acid residues in the L3 are each independently selected from a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine ​​residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue or a valine residue.

8. The antibody-drug conjugate according to claim 1, wherein The L1-L2-L3-L4-D is selected from 9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein: The tumor is a tumor expressing anti-delta-like ligand 3.

10. The antibody-drug conjugate according to claim 9, wherein The Ab in the antibody-drug conjugate is a monoclonal antibody against delta-like ligand 3; the light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3, and the heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

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