An antibody-drug conjugate for treating tumors

Abstract

This invention relates to an antibody-drug conjugate for treating tumors, belonging to the field of biomedical technology. The invention provides an antibody-drug conjugate for treating tumors, with the general structural formula Ab-[L1-L2-L3-L4-D]. p In this formulation, Ab is a monoclonal antibody targeting tumor cell surface antigens, D is a bioactive molecule with inhibitory effects on tumor cells, p is the drug loading, and L1-L2-L3-L4 are linkers connecting the monoclonal antibody and the bioactive molecule. This antibody-drug conjugate exhibits good drug-like properties. Furthermore, this antibody-drug conjugate demonstrates significant in vitro cell-killing activity against NCI-H82 cells, SHP-77 cells, and DMS-53 cells. Moreover, it significantly reduces tumor volume in NCI-H82 and SHP-77 tumor-bearing mice, showing great promise for tumor treatment.

Description

Technical Field

[0001] This invention relates to an antibody-drug conjugate for the treatment of tumors, belonging to the field of biomedical technology. Background Technology

[0002] Antibody-drug conjugates (ADCs) are targeted anticancer drugs. Their core structure includes monoclonal antibodies that target tumor cell surface antigens, linkers, and cytotoxic compounds—bioactive molecules that inhibit tumor cells. The monoclonal antibody is conjugated to the bioactive molecule that inhibits tumor cells through a cleavable or non-cleavable linker, so as to directly deliver the bioactive molecule that inhibits tumor cells to the 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] Studies have shown that linkers play a crucial role in antibody-drug conjugates (ADCs), and their properties affect the efficacy and pharmacokinetic characteristics of ADCs. Stable linkers can maintain drug concentrations in the bloodstream and prevent the release of cytotoxic drugs before reaching the target site, thereby minimizing off-target effects and improving the safety of ADCs. Developing more linkers that can effectively enhance the efficacy and pharmacokinetic characteristics of ADCs is essential for their development and optimization. Summary of the Invention

[0004] To address the aforementioned problems, this 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 that targets tumor cell surface antigens;

[0006] D represents a bioactive molecule that inhibits tumor cells;

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

[0008] L1-L2-L3-L4 are linkers that connect monoclonal antibodies and bioactive molecules;

[0009] L1 is selected from the following structure:

[0010]

[0011] In L1, The numbers represent connection sites. Connection sites marked with number 1 are connected to Ab via S atoms, and connection sites marked with number 2 are connected to L2.

[0012] L2 is

[0013] In L2, The numbers 3 and 4 represent connection sites. Connection sites marked with digit 3 are connected to L1, and connection sites marked with digit 4 are connected to L3.

[0014] Z1 is selected from hydrogen atom, 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, aryl or heteroaryl; or, R1 and R2 together with the attached nitrogen atom form a 4- to 12-membered heterocyclic group; R3 is selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclic;

[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, hydroxy, or hydroxyalkyl groups; the atoms in the heterocyclic groups are each independently selected from C, N, O, or S atoms; and the heterocyclic group is substituted by R4; the value of 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, hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[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, hydroxy, or hydroxyalkyl groups; m is an integer from 1 to 24.

[0020] R5 is selected from hydrogen atoms, alkyl groups, cycloalkyl groups, or heterocyclic groups;

[0021] In R5, the alkyl, cycloalkyl, and heterocyclic groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxy, 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 takes the value of an integer from 1 to 10; j takes the value of an integer from 1 to 20; ** is the 4th bit;

[0025] L3 is selected from amino acid residues or short peptides composed of 2 to 10 amino acid residues;

[0026] In L3, the amino acid residues are either natural amino acid residues or non-natural amino acid residues.

[0027] L4 is selected from Or chemical bonds;

[0028] In L4, The numbers 5 and 6 represent connection sites. The connection site marked with the number 5 is connected to L3, and the connection site marked with the 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 monomethylauribulin E (MMAE), monomethylauribulin F (MMAF), eryribulin, 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, L1 is In L1, The numbers represent connection sites. Connection sites marked with number 1 are connected to Ab via S atoms, while connection sites marked with number 2 are connected to L2.

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

[0034] In L2, The numbers 3 and 4 represent connection sites. Connection sites marked with digit 3 are connected to L1, and connection sites marked with digit 4 are connected to L3.

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

[0036] In Z2, n takes the value of 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, i takes the value of an integer from 1 to 10; j takes the value of an integer from 1 to 20; ** is the 4th bit.

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

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

[0040] In one embodiment of the invention, each of the amino acid residues is independently substituted by a substituent selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, or cycloalkyl.

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

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

[0043]

[0044]

[0045]

[0046]

[0047]

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

[0049] In one embodiment of the present invention, the tumors expressing anti-δ-like ligand 3 include 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, 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 CDR1, CDR2 and CDR3 as shown in SEQ ID NO.1, and the heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO.4, CDR2 and CDR3 as shown in SEQ ID NO.5.

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

[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) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a) while retaining the biological function derived from the sequence.

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

[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) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (c) while retaining the biological function of the sequence from which it originated.

[0057] In one embodiment of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is 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; and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is 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.

[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) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (e) while retaining the biological function of the sequence from which it originated.

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

[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) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined by (g) while retaining the biological function of the sequence from which it originated.

[0064] In one embodiment of the present invention, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO. 24, SEQ ID NO. 27 or SEQ ID NO. 30; and the amino acid sequence of the heavy chain of the monoclonal antibody is shown in 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 linked by disulfide bonds.

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

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

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

[0069] This 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 In this formulation, Ab is a monoclonal antibody targeting tumor cell surface antigens, D is a bioactive molecule with inhibitory effects on tumor cells, p is the drug loading, and L1-L2-L3-L4 are linkers connecting the monoclonal antibody and the bioactive molecule. Experiments have demonstrated that the antibody-drug conjugate exhibits good drug-like properties. Furthermore, experiments have shown that the antibody-drug conjugate has significant in vitro cell-killing effects on NCI-H82 cells, SHP-77 cells, and DMS-53 cells. Moreover, the antibody-drug conjugate can significantly reduce the volume of tumors in NCI-H82 and SHP-77 tumor-bearing mice. Therefore, the antibody-drug conjugate shows great promise for application in tumor treatment. Attached Figure Description

[0070] Figure 1 Synthetic route of compound LP-1.

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

[0072] Figure 3 Synthetic route of compound LP-2.

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

[0074] Figure 5 Synthetic route of compound LP-3.

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

[0076] Figure 7 Synthetic route of compound LP-4.

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

[0078] Figure 9 Synthetic route of compound LP-5.

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

[0080] Figure 11 Synthetic route of compound LP-6.

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

[0082] Figure 13 Synthetic route of compound LP-7.

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

[0084] Figure 15 Synthetic route of compound LP-8.

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

[0086] Figure 17 Synthetic route of compound LP-9.

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

[0088] Figure 19 Synthetic route of compound LP-10.

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

[0090] Figure 21 Synthetic route of compound LP-11.

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

[0092] Figure 23 Synthetic route of compound LP-12.

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

[0094] Figure 25 Synthetic route of compound LP-13.

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

[0096] Figure 27 Synthetic route of compound LP-14.

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

[0098] Figure 29 Synthetic route of compound LP-15.

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

[0100] Figure 31 Synthetic route of compound LP-16.

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

[0102] Figure 33 Synthetic route of compound LP-17.

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

[0104] Figure 35 Synthetic route of compound LP-18.

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

[0106] Figure 37 Synthetic route of compound LP-19.

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

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

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

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

[0111] Figure 42 Detection of ADC antitumor activity in SHP-77 xenograft model.

[0112] Figure 43 Changes in body weight in SHP-77 xenograft mouse model.

[0113] Figure 44 Detection of ADC antitumor activity in NCI-H82 xenograft model.

[0114] Figure 45 Changes in body weight in NCI-H82 xenograft mouse model. Detailed Implementation

[0115] To facilitate understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly stated elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0116] Antibody: refers to immunoglobulin molecules and the immunologically active portion of immunoglobulin molecules, that is, molecules containing antigen-binding sites that specifically bind to immune antigens. Therefore, the term antibody not only covers the complete antibody molecule, but also fragments of the antibody and variants (including derivatives) of the antibody and antibody fragments.

[0117] Antibody heavy chain: contains the heavy chain variable region (abbreviated as "VH" in this article) and the heavy chain constant region containing three domains CH1, CH2, and CH3.

[0118] Antibody light chain: contains a light chain variable region (abbreviated as "VL" in this article) and a light chain constant region containing a structural domain CL.

[0119] FR (Frame Region): The term frame region refers to the four conserved polypeptide fragments 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 region within the light chain variable domain and heavy chain variable domain that primarily facilitates antigen binding. The light chain variable domain and heavy chain variable domain each contain three CDR regions: LCDR1, LCDR2, and LCDR3; and HCDR1, HCDR2, and HCDR3. The antibodies of this 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 this invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, gA1, and IgA2), or subclass of immunoglobulin.

[0121] Constant regions: The constant regions of an antibody are located near the carbon ends of the H chain and L chain. The constant regions of the antibody heavy chain are divided into CH1, CH2, and CH3, while the constant region of the light chain is CL. The CH3 region is involved in cell membrane surface receptor binding, and CH2 is involved in the complement activation pathway and is a complement binding site.

[0122] Antibody Acquisition: The antibodies of the present invention can be prepared using techniques well known in the field, such as hybridoma methods, recombinant DNA technology, phage display technology, synthesis technology or combinations thereof, or other techniques known in the field.

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

[0124] Antibody humanization: The mouse anti-human DLL3 monoclonal antibody in this invention is a monoclonal antibody with a human FR region formed by humanization engineering.

[0125] Antibody-drug conjugates (ADCs) are conjugates obtained by linking an antibody (or its antigen-binding fragment) directly or through a linker to a drug.

[0126] Linkers are chemical structural fragments or bonds that connect to an antibody at one end and a drug at the other. They can also connect to other linkers before being attached to an antibody or drug. Linker attachment to antibodies can be accomplished in various ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, release of cysteine ​​residues through reduced interchain disulfide bonds, modification of reactive cysteine ​​residues at specific sites, and tags containing acyl donor glutamine, or modification of peptides in the presence of transglutaminase and amines to induce reactive endogenous glutamine.

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

[0128] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0129] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0130] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (HNMR) and / or mass spectrometry (MS). HNMR was performed using a Bruker 400 MHz NMR spectrometer, with deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (d6-DMSO) as the solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values ​​(δ) are given in parts per million (ppm). Mass spectrometry (MS) was performed using a Thermo Fisher Vanquish & TSQ Quantis instrument. TM The preparative liquid chromatography (HPLC) was performed using a Gilson GX-281 instrument and a Welch Xtimate C18 30×150mm, 5μm column. The reaction was monitored using 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 solvent 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-cytotoxin and its preparation

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

[0133]

[0134] The method for preparing the adapter-cytotoxin LP-1 includes the following steps:

[0135] Step 1: 2-Bromo-5-nitrophenol (0.94 g, 1.0 eq, CAS No.: 52427-05-1) was dissolved 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 sequentially to the solution. The mixture was stirred until homogeneous, and then stirred at 90 °C for 3 hours to obtain a reaction solution. After thin-layer chromatography showed complete 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. The organic phase was washed with saturated brine (40 mL × 1) and concentrated under reduced pressure to obtain compound 1-1. Compound 1-1 was not purified and was directly used in the synthesis of compound 1-2. Compound 1-1 is a yellow oily substance (1.70 g, 98% yield). The mass spectrometry data for compound 1-1 are: ESI-MS (m / z): 401.08 [M+H] + .

[0136] Step 2: Dissolve compound 1-1 (1.70 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution; add 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) to the solution in sequence, stir to mix well, and then stir at 100 °C for 4 hours under a nitrogen atmosphere to obtain the reaction product. After the reaction was complete as shown by thin-layer chromatography, the reaction solution was cooled to room temperature (25℃), 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 to ethyl acetate was increased from 95:5 to 0:100) to obtain compounds 1-2. Compounds 1-2 were yellow oily substances (1.86 g, yield 89%). The mass spectrometry data of compounds 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, ethyl hydrochloride solution (4 mol / L, 13.5 mL, 15 eq) was added, and the mixture was stirred at 0°C for 24 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was concentrated under reduced pressure, then the pH was adjusted to 9 with saturated sodium carbonate aqueous solution, and then extracted with ethyl acetate (20 mL × 4). The organic phase was collected. The organic phase was 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 was a yellow oil (1.35 g, 99% yield). The mass spectrometry data of compound 1-3 were: ESI-MS (m / z): 383.11 [M+H] + .

[0138] Step 4: Dissolve compounds 1-3 (1.30 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution; add 4,7,10,13,16-pentaheptadecanoic acid (1.00 g, 1.05 eq, CAS No.: 81836-43-3) and N,N-diisopropylethylamine (1.32 g, 3.00 eq) to the solution, cool to 0 °C, and then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.55 g, 1.2 eq, CA... S No.: 148893-10-1), and then stirred at 25℃ for 16 hours to obtain the reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was first diluted with saturated sodium carbonate aqueous solution (30 mL), and then extracted with ethyl acetate (30 mL × 3), and the organic phase was collected; the organic phase was first washed with saturated brine (30 mL × 3), then 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 compounds 1-4. Compound 1-4 is a yellow oily substance 1-4 (1.73 g, yield 79%). The mass spectrometry data of compound 1-4 is: ESI-MS (m / z): 645.25 [M+H] + .

[0139] Step 5: Dissolve compounds 1-4 (1.73 g, 1.00 eq) in a mixture of methanol / dichloromethane / tetrahydrofuran (50 mL / 40 mL / 30 mL) to obtain a solution; add 5% palladium on carbon (0.30 g) to the solution and react at 25 °C for 24 hours under a hydrogen atmosphere to obtain a reaction solution; after thin-layer chromatography shows that the reaction is complete, filter the reaction solution and collect the filter cake; wash the filter cake with methanol (5 mL) and collect the filtrate; concentrate the filtrate under reduced pressure and then purify it 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 compounds 1-5. Compounds 1-5 are yellow oily substances 1-5 (0.92 g, yield 65%). The mass spectrometry data for compounds 1-5 are as follows: ESI-MS (m / z): 527.25 [M+H] + .

[0140] Step Six: Compounds 1-5 (0.92 g, 1.0 eq) were 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 showed that the reaction was complete, the reaction solution was cooled to room temperature (25 °C), and then the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 compounds 1-6. Compounds 1-6 were yellow oily substances (0.79 g, yield 74%). The mass spectrometry data for compounds 1-6 are as follows: ESI-MS (m / z): 607.26 [M+H] + .

[0141] Step 7: Dissolve compounds 1-6 (30 mg, 1.00 eq) in N,N-dimethylformamide (1.5 mL) to obtain a solution; add Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., CAS No.: 1599440-12-6) (41 mg, 1.00 eq) and N,N-diisopropylethylamine (32 mg, 5.00 eq) to the solution, cool to 0℃, then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (37.6 mg, 2.00 eq), and react at 25℃ for 2 hours to obtain a reaction solution; after the reaction is complete as monitored by HPLC, purify the reaction solution by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v), and then freeze-dry to obtain compound LP-1. Compound LP-1 is a yellow powder (22 mg, yield 31%). The synthetic route for compound LP-1 is shown below. Figure 1 The mass spectrometry results for compound LP-1 are shown in [link to mass spectrometry results]. Figure 2 The mass spectrometry data for compound LP-1 are as follows: LCMS (ESI) [M+H] + 1429.55 [M+H] + .

[0142] Examples 1-2: A linker-cytotoxin and its preparation

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

[0144]

[0145] The method for preparing the adapter-cytotoxin LP-2 includes 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 well, and then stir at 100 °C for 24 hours to obtain a reaction solution; after thin-layer chromatography shows that the reaction is complete, cool the reaction solution to room temperature (25 °C), then dilute with water (40 mL), and extract with ethyl acetate (30 mL × 3) to obtain the organic phase; wash the organic phase with saturated brine (40 mL × 1), then concentrate under reduced pressure, and finally purify by silica gel column chromatography (during purification, the volume ratio of petroleum ether to 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 was a yellow oil (2.14 g, yield 78%). The mass spectrometry data for compound 2-1 were: 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, and the mixture was stirred until homogeneous. Finally, the reaction was carried out 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 to 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 a yellow oil (2.78 g, yield 96%). The mass spectrometry data of compound 2-2 were: ESI-MS (m / z): 501.12 [M+H] + .

[0148] Step 3: Dissolve compound 2-2 (2.78 g, 1.00 eq) in toluene (30 mL) to obtain a solution; add benzyl carbamate (0.94 g, 1.20 eq), palladium acetate (23 mg, 0.025 eq), 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.12 g, 0.05 eq, CAS No.: 564483-18-7), and potassium carbonate (0.85 g, 1.2 eq) to the solution in sequence, then heat to 100 °C under a nitrogen atmosphere and 1 The reaction was continued at 00℃ for 4 hours to obtain the reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was cooled to room temperature (25℃), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected. 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 to 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 a yellow oil (3.1 g, yield 96%). The mass spectrometry 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. The solution was cooled to 0°C, and then ethyl hydrochloride solution (4 mol / L, 20 mL, 15 eq) was added. The mixture was stirred until homogeneous, and then stirred at 0°C for 24 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was dissolved in 30 mL of a dichloromethane / methanol mixture (dichloromethane:methanol = 10:1, v / v). The organic phase was collected. The organic phase was washed with saturated sodium carbonate aqueous solution (10 mL × 2) and then evaporated to dryness under reduced pressure to obtain compound 2-4. Compound 2-4 was not purified and was directly used in the synthesis of compound 2-5. Compound 2-4 was a yellow oil (1.6 g, yield 62%). The mass spectrometry data of compound 2-4 were: ESI-MS (m / z): 472.25 [M+H]. + .

[0150] Step 5: Dissolve compound 2-4 (0.8 g, 1.00 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add 4,7,10,13,16-pentaheptadecanoic acid (0.52 g, 1.1 eq) and N,N-diisopropylethylamine (0.66 g, 3.00 eq) to the solution, cool to 0°C, then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.77 g, 1.2 eq), then... The reaction mixture was 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 diluted with saturated sodium carbonate aqueous solution (30 mL), and then extracted with ethyl acetate (30 mL × 3). The organic phase was collected. 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 a yellow oil (0.95 g, yield 76%). The mass spectrometry data of compound 2-5 were: ESI-MS (m / z): 734.33 [M+H] + .

[0151] Step Six: Compound 2-5 (0.95 g, 1.00 eq) was dissolved in a mixture of dichloromethane / 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 for 12 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed 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 a yellow oil (0.43 g, yield 65%). The mass spectrometry 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 thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 a yellow oil (0.12 g, yield 64%). The mass spectrometry 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. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (33 mg, 1.50 eq) was added, and the mixture was reacted at 25°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-2. Compound LP-2 was a yellow powder (20 mg, yield 20%). The synthetic route for compound LP-2 is shown below. Figure 3 The mass spectrometry results for compound LP-2 are shown in [link to mass spectrometry results]. Figure 4 The mass spectrometry data for compound LP-2 are as follows: LCMS (ESI) [M+H] + :1414.40[M+H] + .

[0154] Examples 1-3: A linker-cytotoxin and its preparation

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

[0156]

[0157] The method for preparing the adapter-cytotoxin LP-3 includes the following steps:

[0158] Step 1: Dissolve 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) sequentially to the solution, stir to mix well, and then stir at 100 °C for 24 hours to obtain a reaction solution; thin-layer chromatography... After the chromatographic analysis showed that the reaction was complete, the reaction solution was cooled to room temperature (25℃), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected. 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 to 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 spectrometry 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, and the mixture was stirred at 20 °C for 18 hours to obtain a reaction solution. Thin-layer chromatography showed that the reaction was complete. The reaction solution was then purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from 95:5 to 0:100) to obtain compound 3-2. Compound 3-2 was a yellow oil (2.57 g, 95% yield). The mass spectrometry data of compound 3-2 were: ESI-MS (m / z): 515.18 [M+H] + .

[0160] Step 3: Dissolve compound 3-2 (2.5 g, 1.00 eq) in toluene (30 mL) to obtain a solution; add benzyl carbamate (0.88 g, 1.20 eq), palladium acetate (27 mg, 0.025 eq), 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.11 g, 0.05 eq) and potassium carbonate (0.8 g, 1.2 eq) to the solution in sequence, heat to 100 °C under a nitrogen atmosphere and continue to react at 100 °C for 4 hours to obtain a reaction solution; Thin-layer chromatography showed that after the reaction was complete, the reaction solution was first cooled to room temperature (25℃), then concentrated under reduced pressure, and the residue was collected. The residue was first diluted with water (40 mL), then extracted with dichloromethane (30 mL × 3), and the organic phase was collected. The organic phase was first washed with saturated brine (30 mL × 3), then concentrated under reduced pressure, and finally 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 3-3. Compound 3-3 was a yellow oily substance (2.27 g, yield 80%). The mass spectrometry 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. Ethyl hydrochloride solution (4 mol / L, 14 mL, 15 eq) was added to the solution, and the mixture was cooled to 0 °C and reacted at 0 °C for 24 hours to obtain a reaction solution. After thin-layer chromatography showed the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was collected. The reaction solution was first diluted with saturated sodium carbonate aqueous solution (30 mL), and then extracted with ethyl acetate (30 mL × 3) to obtain the organic phase. 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 spectrometry data of compound 3-4 were: ESI-MS (m / z): 486.24 [M+H] + .

[0162] Step 5: Dissolve compound 3-4 (1.7 g, 1.00 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add 4,7,10,13,16-pentaheptadecanoic acid (1.1 g, 1.1 eq) and N,N-diisopropylethylamine (1.35 g, 3.00 eq) to the solution, cool to 0°C, and then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.6 g, 1.2 eq). The reaction mixture was 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 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), 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 a yellow oil (1.2 g, yield 46%). The mass spectrometry data of compound 3-5 were: ESI-MS (m / z): 748.24 [M+H] + .

[0163] Step Six: Compound 3-5 (1.2 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 for 12 hours under a hydrogen atmosphere to obtain a reaction solution. Thin-layer chromatography showed complete reaction. The reaction solution was filtered, and the filter cake was collected. The filter cake was washed with methanol (5 mL), and the filtrate was collected. The filtrate was evaporated to dryness under reduced pressure to obtain compound 3-6. Compound 3-6 was not purified and was directly used in the synthesis of compound 3-7. Compound 3-6 was a yellow oil (0.80 g, 95% yield). The mass spectrometry data of compound 3-6 were: 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 3-7. Compound 3-7 was a yellow oil (0.16 g, yield 69%). The mass spectrometry 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-oxytripyrrolidinephosphine hexafluorophosphate (33 mg, 1.10 eq), and 1-hydroxybenzotriazole (8.5 mg, 1.10 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-3. Compound LP-3 was a yellow powder (48 mg, yield 59%). The synthetic route for compound LP-3 is shown in [link to synthetic route]. Figure 5 The mass spectrometry results for compound LP-3 are shown in [link to mass spectrometry results]. Figure 6 The mass spectrometry data for compound LP-2 are as follows: LCMS (ESI) [M+H] + 1428.45 [M+H] + .

[0166] Examples 1-4: A linker-cytotoxin and its preparation

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

[0168]

[0169] The method for preparing the adapter-cytotoxin LP-4 includes the following steps:

[0170] Step 1: 4-Bromo-2-fluorobenzaldehyde (1.5 g, 1.00 eq) was dissolved in N,N-dimethylformamide (10 mL) to obtain a solution. Diethylamine (0.66 g, 1.2 eq) and potassium carbonate (0.9 g, 1.2 eq) were added sequentially to the solution. The mixture was stirred until homogeneous, and then stirred at 100 °C for 24 hours to obtain a reaction solution. After thin-layer chromatography showed complete 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. 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 to ethyl acetate was increased from 95:5 to 0:100) to obtain compound 4-1. Compound 4-1 was a yellow oil (1.5 g, yield 79%). The mass spectrometry data for compound 4-1 are as follows: 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, and the mixture was stirred until homogeneous. The reaction was then carried out at 20 °C for 18 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, 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 95:5 to 0:100) to obtain compound 4-2. Compound 4-2 was a yellow oil (2.12 g, 95% yield). The mass spectrometry 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-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.13 g, 0.05 eq), and potassium carbonate (0.91 g, 1.2 eq) were added sequentially to the solution. The mixture was then heated to 100 °C under a nitrogen atmosphere and reacted at 100 °C for 4 hours to obtain the desired product. The reaction solution was cooled to room temperature (25°C) after thin-layer chromatography showed complete reaction, then 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 to ethyl acetate was increased from 95:5 to 0:100) to obtain compound 4-3. Compound 4-3 was a yellow oil (2.03 g, yield 80%). The mass spectrometry data of compound 4-3 were: ESI-MS (m / z): 459.18 [M+H]. + .

[0173] Step 4: Compound 4-3 (2.35 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.2 g) was added to the solution, and the mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with methanol (5 mL), and the filtrate was collected. The filtrate was evaporated to dryness under reduced pressure to obtain compound 4-4. Compound 4-4 was not purified and was directly used in the synthesis of compound 4-5. Compound 4-4 was a yellow oil (1.15 g, 95% yield). The mass spectrometry data of compound 4-4 were: 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, and the mixture was stirred until homogeneous. The reaction was then carried out 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), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 a yellow oily substance 4-5 (0.09 g, yield 69%). The mass spectrometry data for compounds 4-5 are as follows: ESI-MS (m / z): 317.09 [M+H] + .

[0175] Step Six: 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-oxytripyrrolidinephosphine hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0 °C and reacted at 0 °C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-4. Compound LP-4 was a yellow powder (20 mg, yield 25%). The synthetic route for compound LP-4 is shown in [link to synthetic route]. Figure 7 The mass spectrometry results for compound LP-4 are shown in [link to mass spectrometry results]. Figure 8 The mass spectrometry data for compound LP-4 are as follows: LCMS (ESI) [M+H] + 1139.46 [M+H] + .

[0176] Examples 1-5: A linker-cytotoxin and its preparation

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

[0178]

[0179] The method for preparing the adapter-cytotoxin LP-5 includes the following steps:

[0180] Step 1: Dissolve compound 4-bromo-2-fluorobenzaldehyde (2g, 1.00eq) in N,N-dimethylformamide (20mL) to obtain a solution; add N-methylpiperazine (1.18g, 1.2eq) and potassium carbonate (1.63g, 1.2eq) to the solution in sequence, stir to mix well, and then stir at 100℃ for 24 hours to obtain a reaction solution; after the reaction is complete as shown by thin-layer chromatography, cool the reaction solution to room temperature (25℃), then dilute with water (40mL), and extract with ethyl acetate (30mL×3) to obtain the organic phase; wash the organic phase with saturated brine (30mL×1), then concentrate under reduced pressure, and finally purify by silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate is increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-1. Compound 5-1 was a yellow oil (2.17 g, 78% yield). The mass spectrometry data for compound 5-1 were: 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 and stirred until homogeneous. The mixture was then reacted at 20 °C for 18 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, 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 95:5 to 0:100) to obtain compound 5-2. Compound 5-2 was a yellow oil (2.12 g, yield 67%). The mass spectrometry data of compound 5-2 were: ESI-MS (m / z): 415.11 [M+H] + .

[0182] Step 3: Dissolve compound 5-2 (2.1 g, 1.00 eq) in toluene (20 mL) to obtain a solution; add benzyl carbamate (0.78 g, 1.20 eq), palladium acetate (48 mg, 0.05 eq), 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.2 g, 0.1 eq), and potassium carbonate (0.72 g, 1.2 eq) to the solution in sequence, then heat to 100 °C under a nitrogen atmosphere and continue the reaction at 100 °C for 4 hours. The reaction solution was obtained. After the reaction was completed, the reaction solution was cooled to room temperature (25℃), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was collected. 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 to 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 spectrometry 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 on carbon (0.2 g) was added to the solution, and the mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with methanol (5 mL), and the filtrate was collected. 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, 100% yield). The mass spectrometry data of compound 5-4 were: 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, and the mixture was stirred until homogeneous. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After the reaction was complete as shown 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 used directly for the synthesis of compound 5-6. Compound 5-6 was a yellow oil (0.06 g, 92% yield). The mass spectrometry data were: ESI-MS (m / z): 344.09 [M+H] + .

[0185] Step Six: 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-oxytripyrrolidinephosphine hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-5. Compound LP-5 was a yellow powder (17 mg, yield 21%). The synthetic route for compound LP-5 is shown in [link to synthetic route]. Figure 9 The mass spectrometry results for compound LP-5 are shown in [link to mass spectrometry results]. Figure 10 The mass spectrometry data for compound LP-5 are as follows: LCMS (ESI) [M+H] + 1166.47 [M+H] + .

[0186] Examples 1-6: A linker-cytotoxin and its preparation

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

[0188]

[0189] The method for preparing the adapter-cytotoxin LP-6 includes the following steps:

[0190] Step 1: Dissolve compound 2-4 (0.2 g, 1.00 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add N-fluorenemethoxycarbonyl-glycyl-glycine (0.18 g, 1.2 eq) and N,N-diisopropylethylamine (0.14 g, 2.5 eq) to the solution, cool to 0 °C, and then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.19 g, 1 mL). 0.2 eq), and then stirred at 25℃ for 4 hours to obtain the reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30 mL), and then extracted with ethyl acetate (30 mL × 3), and the organic phase was collected; the organic phase was first washed with saturated sodium bicarbonate solution (30 mL), then washed with saturated brine (30 mL × 3), and then evaporated to dryness under reduced pressure to obtain compound 6-1. Compound 6-1 was not purified and was directly used for the synthesis of compound 6-2. Compound 6-1 was a yellow oil (0.36 g, 100%). The mass spectrometry data of compound 6-1 were: 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. After cooling the solution to 0 °C, 1,8-diazabicyclo[5.4.0]undec-7-ene (0.068 g, 1.00 eq) was added to the solution, and the mixture was reacted at 0 °C for 1.5 hours to obtain a reaction solution. After the reaction was completed as shown 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 dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 6-2. Compound 6-2 was a yellow oily substance 6-2 (0.21 g, yield 80%). The mass spectrometry data of compound 6-2 were: ESI-MS (m / z): 586.21 [M+H] + .

[0192] Step 3: Dissolve compound 6-2 (0.21 g, 1.00 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add 4,7,10,13,16-pentaheptadecanoic acid (0.12 g, 1.2 eq) and N,N-diisopropylethylamine (0.12 g, 2.50 eq) to the solution, cool to 0°C, then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.16 g, 1.2 eq), and then... The reaction was carried out at 5℃ with stirring for 2 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30 mL), then extracted with ethyl acetate (30 mL × 5), and the organic phase was collected. The organic phase was first washed with water (30 mL), then washed with saturated brine (30 mL), then 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 6-3. Compound 6-3 was a yellow oily substance (0.19 g, yield 62.5%). The mass spectrometry 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 for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed 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 a yellow oily substance (0.06 g, yield 43%). The mass spectrometry 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was then 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 a yellow oil (0.02 g, yield 30%). The mass spectrometry data of compound 6-5 were: ESI-MS (m / z): 706.29 [M+H]. + .

[0195] Step Six: 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-oxytripyrrolidinephosphine hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-6. Compound LP-6 was a yellow powder (55 mg, yield 39%). The synthetic route for compound LP-6 is shown below. Figure 11 The mass spectrometry results for compound LP-6 are shown in [link to mass spectrometry results]. Figure 12 The mass spectrometry data for compound LP-6 are as follows: LCMS (ESI) [M+H] + 1528.64 [M+H] + .

[0196] Examples 1-7: A linker-cytotoxin and its preparation

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

[0198]

[0199] The method for preparing the adapter-cytotoxin LP-7 includes the following steps:

[0200] Step 1: Dissolve compound 2-4 (1.3 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution; add 4,7,10,13,16,19,22,25,28-nonoxanonocanoic acid (1.39 g, 1.1 eq) and N,N-diisopropylethylamine (1.07 g, 3.00 eq) to the solution, cool to 0°C, and then add 2-(7-azabenzotriazole)-N N,N',N'-Tetramethylurea hexafluorophosphate (1.26 g, 1.2 eq) was 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 sodium bicarbonate solution (30 mL), then with saturated brine (30 mL), and then evaporated under reduced pressure to obtain compound 7-1. Compound 7-1 was not purified and was used directly in the synthesis of compound 7-2. Compound 7-1 was a yellow oil (2.51 g, 100% yield). The mass spectrometry data of compound 7-1 were: 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 for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed 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 a yellow oily substance (1.6 g, yield 83%). The mass spectrometry 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 7-3. Compound 7-3 was a yellow oil (0.35 g, yield 89%). The mass spectrometry 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-oxytripyrrolidinephosphine hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-7. Compound LP-7 was a yellow powder (70 mg, yield 48%). The synthetic route for compound LP-7 is shown in [link to synthetic route]. Figure 13 The mass spectrometry results for compound LP-7 are shown in [link to mass spectrometry results]. Figure 14 The mass spectrometry data for compound LP-7 are as follows: LCMS (ESI) [M+H] + 1590.70 [M+H] + .

[0204] Examples 1-8: A linker-cytotoxin and its preparation

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

[0206]

[0207] The method for preparing the adapter-cytotoxin LP-8 includes the following steps:

[0208] Step 1: Dissolve compound 3-4 (1.97 g, 1.00 eq) in N,N-dimethylformamide (25 mL) to obtain a solution; add 4,7,10,13,16,19,22,25,28-nonoxanonocanoic acid (2.04 g, 1.1 eq) and N,N-diisopropylethylamine (1.73 g, 3.00 eq) to the solution, cool to 0°C, and then add 2-(7-azabenzotriazole)-N N,N',N'-Tetramethylurea hexafluorophosphate (1.85 g, 1.2 eq) was 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 sodium bicarbonate solution (30 mL), then with saturated brine (30 mL), and then evaporated under reduced pressure to obtain compound 8-1. Compound 8-1 was a yellow oil (3 g, yield 80%). Compound 8-1 was not purified and was used directly in the synthesis of compound 8-2. The mass spectrometry data of compound 8-1 were: ESI-MS (m / z): 924.42 [M+H] + .

[0209] Step 2: Compound 8-1 (3g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.3g) was added to the solution, and the mixture was reacted at 25°C for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed 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 a yellow oily substance (1.9g, yield 83%). The mass spectrometry 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, and the mixture was stirred until homogeneous. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was then 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 8-3. Compound 8-3 was a yellow oil (0.24 g, yield 72%). The mass spectrometry 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-oxytripyrrolidinephosphine hexafluorophosphate (45 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-8. Compound LP-8 was a yellow powder (75 mg, yield 58%). The synthetic route for compound LP-8 is shown below. Figure 15 The mass spectrometry results for compound LP-8 are shown in [link to mass spectrometry results]. Figure 16 The mass spectrometry data for compound LP-8 are as follows: LCMS (ESI) [M+H] + 1604.72 [M+H] + .

[0212] Examples 1-9: A linker-cytotoxin and its preparation

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

[0214]

[0215] The method for preparing the adapter-cytotoxin LP-9 includes 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-bromine (0.75 g, 1.1 eq) and potassium carbonate (0.44 g, 1.5 eq) were added to the solution, and the mixture was stirred until homogeneous. The temperature was then raised to 70 °C and the reaction was continued at 70 °C for 8 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3 times) to obtain the organic phase. The organic phase was washed with saturated brine (30 mL) 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 9-1. Compound 9-1 was a yellow oil (1.1 g, yield 73%). The mass spectrometry data for compound 9-1 are as follows: 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 for 22 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed 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 a yellow oily substance (0.35 g, yield 46%). The mass spectrometry 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 9-3. Compound 9-3 was a yellow oil (0.23 g, yield 98%). The mass spectrometry 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-oxytripyrrolylphosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored to be complete by HPLC, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-9. Compound LP-9 was a yellow powder (36 mg, yield 32%). The synthetic route of compound LP-9 is shown below. Figure 17 The mass spectrometry results for compound LP-9 are shown in [link to mass spectrometry results]. Figure 18 The mass spectrometry data for compound LP-9 are as follows: LCMS (ESI) [M+H] + 1386.60 [M+H] + .

[0220] Examples 1-10: A linker-cytotoxin and its preparation

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

[0222]

[0223] The method for preparing the adapter-cytotoxin LP-10 includes 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-nonaoxanonicosanoic acid (1.49 g, 1.2 eq) and N,N-diisopropylethylamine (0.88 g, 2.5 eq) to the solution, cool to 0 °C, and then add 2-(7-azabenzotriazole). -N,N,N',N'-Tetramethylurea hexafluorophosphate (1.28 g, 1.2 eq) was 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 times). The organic phase was collected and washed with water (30 mL), then with saturated brine (30 mL), and then evaporated under reduced pressure to obtain compound 10-1. Compound 10-1 was not purified and was used directly in the synthesis of compound 10-2. Compound 10-1 was a yellow oil (2.7 g, yield 96%). The mass spectrometry 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 for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with a mixture of dichloromethane / 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 a yellow oily substance (1.66 g, yield 75%). The mass spectrometry 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, and the mixture was stirred until homogeneous. The reaction was then carried out 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), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 10-3. Compound 10-3 was a yellow oil (0.02 g, yield 36%). The mass spectrometry data for compound 10⁻³ are as follows: 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-oxytripyrrolidinephosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-10. Compound LP-10 was a yellow powder solid (84 mg, yield 62%). The synthetic route for compound LP-10 is shown below. Figure 19 The mass spectrometry results for compound LP-10 are shown in [link to data]. Figure 20 The mass spectrometry data for compound LP-10 are as follows: LCMS (ESI) [M+H] + 1705.74 [M+H] + .

[0228] Examples 1-11: A linker-cytotoxin and its preparation

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

[0230]

[0231] The method for preparing the adapter-cytotoxin LP-11 includes 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 Biomedical 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-oxytripyrrolidinephosphine 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℃ and reacted at 0℃ for 1 hour to obtain a reaction solution; after the reaction was monitored by HPLC to ensure complete reaction, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % indicates v / v) and then lyophilized to obtain compound LP-11. Compound LP-11 is a yellow powder (39 mg, 65% yield). The synthetic route for compound LP-11 is shown below. Figure 21 The mass spectrometry results for compound LP-11 are shown in [link to mass spectrometry results]. Figure 22 The mass spectrometry data for compound LP-11 are as follows: LCMS (ESI) [M+H] + 1504.69 [M+H] + .

[0233] Examples 1-12: A linker-cytotoxin and its preparation

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

[0235]

[0236] The method for preparing the adapter-cytotoxin LP-12 includes 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 Biomedical Technology Co., Ltd.) (30 mg, 1.00 eq), N,N-diisopropylethylamine (12.8 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (22.8 mg, 1.10 eq), and 1-hydroxybenzotriazole (2.4 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-12. Compound LP-12 was a yellow powder (48 mg, yield 79%). The synthetic route for compound LP-12 is shown below. Figure 23 The mass spectrometry results for compound LP-12 are shown in [link to data]. Figure 24 The mass spectrometry data for compound LP-12 are as follows: LCMS (ESI) [M+H] + 1518.55 [M+H] + .

[0238] Examples 1-13: A linker-cytotoxin and its preparation

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

[0240]

[0241] The method for preparing the adapter-cytotoxin LP-13 includes the following steps:

[0242] Step 1: Dissolve compound N-[(1,1-dimethylethoxy)carbonyl]glycylglycyl-L-phenylalanyl-glycine (purchased from MedChemExpress, CAS No.: 187794-49-6) (0.25 g, 1.20 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add eczemab mesylate (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., CAS: 169869-90-3) (0.25 g, 1.00 eq) and N,N-dimethylformamide sequentially to the solution. Isopropyl ethylamine (0.216 g, 3.50 eq) and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.30 g, 1.20 eq) were reacted at 0 °C for 1 hour after cooling to 0 °C to obtain a reaction solution. After the reaction was monitored to completion by HPLC, the reaction solution was evaporated to dryness under reduced pressure, then redissolved in dichloromethane to the original volume, 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 11-1. Compound 11-1 is a yellow solid (0.4 g, yield 98%). The mass spectrometry data of compound 11-1 are: 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. Ethyl hydrochloride solution (4 mol / L, 3 mL, 25.6 eq) was added to the solution, and the mixture was stirred thoroughly. The reaction was then carried out at room temperature (25 °C) for 14 hours to obtain a reaction solution. After the reaction was complete as monitored by LC-MS, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with ethyl acetate (5 mL), and the filtrate was collected. 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-oxytripyrrolidinephosphine hexafluorophosphate (42 mg, 1.20 eq), and 1-hydroxybenzotriazole (4.5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-13. Compound LP-13 was a white powder (27 mg, yield 28%). The synthetic route of compound LP-13 is shown below. Figure 25 The mass spectrometry results for compound LP-13 are shown in [link to mass spectrometry results]. Figure 26 The mass spectrometry data for compound LP-13 are as follows: LCMS (ESI) [M+H] + 1517.69 [M+H] + .

[0245] Examples 1-14: A linker-cytotoxin and its preparation

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

[0247]

[0248] The method for preparing the adapter-cytotoxin LP-14 includes the following steps:

[0249] Step 1: Compound 10-3 (100 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (1 mL) to obtain a solution. Compound 11-2 (36 mg, 1.00 eq), N,N-diisopropylethylamine (18 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (26 mg, 1.10 eq), and 1-hydroxybenzotriazole (3 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored by HPLC until complete, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-14. Compound LP-14 was a white powder (18 mg, yield 24%). The synthetic route of compound LP-14 is shown below. Figure 27 The mass spectrometry results for compound LP-14 are shown in [link to mass spectrometry results]. Figure 28The mass spectrometry data for compound LP-14 are as follows: LCMS (ESI) [M / 2+H] + 809.36 [M / 2+H] + .

[0250] Examples 1-15: A linker-cytotoxin and its preparation

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

[0252]

[0253] The method for preparing the adapter-cytotoxin LP-15 includes 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-dodecanoic 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, and then cool the solution. The mixture was heated to 0°C, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.62 g, 1.2 eq) was added. The reaction was then carried out at 25°C for 8 hours to obtain the 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 12-1. Compound 12-1 was not purified and was used directly in the synthesis of compound 12-2. Compound 12-1 was a yellow oil (1.4 g, 100% yield, theoretical value). The mass spectrometry 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 for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with a mixture of dichloromethane / methanol (dichloromethane:methanol = 1:1, v / v, 5 mL), and the filtrate was collected. The filtrate was evaporated to dryness under reduced pressure to obtain compound 12-2. Compound 12-2 was a yellow oily substance (1.1 g, 100% yield, theoretical value). The mass spectrometry 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was 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 12-3. Compound 12-3 was a yellow oil (0.6 g, 90% yield). The mass spectrometry 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-oxytripyrrolidinephosphine hexafluorophosphate (27 mg, 1.10 eq), and 1-hydroxybenzotriazole (3 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored to be complete by HPLC, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-15. Compound LP-15 was a yellow powder (45 mg, yield 58%). The synthetic route of compound LP-15 is shown below. Figure 29 The mass spectrometry results for compound LP-15 are shown in [link to data]. Figure 30 The mass spectrometry data for compound LP-15 are as follows: LCMS (ESI) [M / 2+H] +825.89 [M / 2+H] + .

[0258] Examples 1-16: A linker-cytotoxin and its preparation

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

[0260]

[0261] The method for preparing the adapter-cytotoxin LP-16 includes the following steps:

[0262] Step 1: Compound 12-3 (160 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (31 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (73 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored to be complete by HPLC, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-16. Compound LP-16 was a white powder (29 mg, yield 25%). The synthetic route of compound LP-16 is shown below. Figure 31 The mass spectrometry results for compound LP-16 are shown in [link to mass spectrometry results]. Figure 32 The mass spectrometry data for compound LP-16 are as follows: LCMS (ESI) [M+H] + 1649.47 [M+H] + .

[0263] Examples 1-17: A linker-cytotoxin and its preparation

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

[0265]

[0266] The method for preparing the adapter-cytotoxin LP-17 includes 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 Biomedical Technology Co., Ltd., CAS: 251459-32-2) (25 mg, 1.00 eq), N,N-diisopropylethylamine (9 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinephosphine 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℃ and reacted at 0℃ for 1 hour to obtain a reaction solution; after the reaction was monitored by HPLC to ensure complete reaction, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % indicates 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 for compound LP-17 is shown below. Figure 33 The mass spectrometry results for compound LP-17 are shown in [link to mass spectrometry results]. Figure 34 The mass spectrometry data for compound LP-17 are as follows: LCMS (ESI) [M+H] + 1798.81 [M+H] + .

[0268] Examples 1-18: A linker-cytotoxin and its preparation

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

[0270]

[0271] The method for preparing the adapter-cytotoxin LP-18 includes the following steps:

[0272] Step 1: Dissolve compound 2-4 (0.86 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-dodecanoic acid (1.0 g, 1.00 eq) and N,N-diisopropylethylamine (0.54 g, 2.50 eq) to the solution, cool to 0 °C, and then add 2 -(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.76 g, 1.2 eq) was 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), then evaporated under reduced pressure to obtain compound 13-1. Compound 13-1 was not purified and was directly used in the synthesis of compound 13-2. Compound 13-1 was a yellow oil (1.77 g, 100% yield, theoretical value). The mass spectrometry 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 for 16 hours under a hydrogen atmosphere to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was filtered, and the filter cake was collected. The filter cake was washed with methanol (5 mL), and the filtrate was collected. The filtrate was evaporated to dryness under reduced pressure to obtain compound 13-2. Compound 13-2 was a yellow oily substance (1.39 g, 100% yield, 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, and the mixture was stirred thoroughly. The reaction was then carried out at 45 °C for 4 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was cooled to room temperature (25 °C), and the pH was adjusted to 7 with trifluoroacetic acid. The solution was then 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 13-3. Compound 13-3 was a yellow oil (0.6 g, 90% yield). The mass spectrometry 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. To the solution, 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (32 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (48 mg, 1.30 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored to be complete by HPLC, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-18. Compound LP-18 was a yellow powder (14 mg, yield 12%). The synthetic route of compound LP-18 is shown below. Figure 35 The mass spectrometry results for compound LP-18 are shown in [link to mass spectrometry results]. Figure 36 The mass spectrometry data for compound LP-18 are as follows: LCMS (ESI) [M+H] + 1635.49 [M+H] + .

[0276] Examples 1-19: A linker-cytotoxin and its preparation

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

[0278]

[0279] The method for preparing the adapter-cytotoxin LP-19 includes 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 THF / H₂O mixture (80 mL / 80 mL) to obtain a solution. Slowly add the solution dropwise to a THF (80 mL) solution containing p-toluenesulfonyl chloride (40.00 g, 1.00 eq) cooled to 0 °C, and react at 0 °C for 3 hours to obtain a reaction solution. After the reaction is complete as shown by thin-layer chromatography, separate the reaction solution. Extract the aqueous layer with ethyl acetate (50 mL × 3), and collect the organic phase. Wash the organic phase with saturated brine (40 mL) and then evaporate to dryness under reduced pressure to obtain compound 14-1. Compound 14-1 is a colorless oil (58.20 g, yield 95%). The mass spectrometry data of compound 14-1 is: ESI-MS (m / z): 319.12 [M+H] + .

[0281] Step 2: Diethanolamine (13.32 g, 2.00 eq) was dissolved in DMF (54 mL) to obtain solution A; triphenylchloromethane (16.04 g, 1.00 eq) was dissolved in DCM (34 mL) to obtain solution B; after cooling solutions A and B to 0 °C, solution B was added dropwise to solution A, and the mixture was reacted at 0 °C for 24 hours to obtain the reaction solution; after the reaction was completed as shown by thin-layer chromatography, ether / water (30 mL / 40 mL) was added to the reaction solution, the mixture was stirred for 2 hours, and then filtered to obtain the filter cake; chloroform / petroleum ether (96 mL / 132 mL) was added to the filter cake, the mixture was stirred for 2 hours, and then concentrated to obtain compound 14-2. Compound 14-2 was a yellow oil (14.50 g, yield 72%). The mass spectrometry data for compound 14-2 are as follows: 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 sequentially to the solution, and the mixture was heated to 65 °C and reacted at 65 °C for 16 hours to obtain a reaction solution. After the reaction was complete as shown 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 to ethyl acetate was increased from 95:5 to 0:100) to obtain compound 14-3. Compound 14-3 was a yellow oil (1.63 g, yield 42%). The mass spectrometry 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: Dissolve compound 14-3 (1.73 g, 1.00 eq) in methanol (8 mL) to obtain a solution; add 5 mL of 5% (w / v, g / mL) hydrochloric acid aqueous solution dropwise to the solution at room temperature, and continue the reaction at room temperature for 30 minutes to obtain a reaction solution; after thin-layer chromatography shows that the reaction is complete, filter the reaction solution and collect the filter cake; wash the filter cake with methanol (5 mL) and collect the filtrate; concentrate the filtrate under reduced pressure, then dilute it with diethyl ether (20 mL), adjust the pH to 8 with saturated sodium carbonate solution, and finally evaporate the aqueous layer to dryness, collecting the residue; add dichloromethane (40 mL) to the residue, stir for 2 hours, then filter, and evaporate the dichloromethane to dryness to obtain compound 14-4. Compound 14-4 is a yellow oily substance (0.83 g, yield 74%). The mass spectrometry data of compound 14-4 is: ESI-MS (m / z): 412.31 [M+H] + .

[0284] Step 5: Dissolve compound 4-bromo-2-fluorobenzaldehyde (2.10 g, 1.00 eq) in DMF (20 mL) to obtain a solution; add compound 14-4 (5.60 g, 1.20 eq) and potassium carbonate (1.70 g, 1.20 eq) to the solution, stir well, and then react at 105 °C for 16 hours to obtain a reaction solution; after the reaction is complete as shown by thin-layer chromatography, cool the reaction solution to room temperature, dilute with water (40 mL), and extract with ethyl acetate (30 mL × 3) to obtain the organic phase; wash the organic phase with saturated brine (20 mL × 3), and then purify it 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 14-5. Compound 14-5 is a yellow oil (2.50 g, yield 38%). The mass spectrometry data for compound 14-5 are: 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 Six: Compound 14-5 (2.50 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. The solution was cooled to 0 °C, and benzyl (triphenylphosphine) acetate (2.67 g, 1.50 eq) was added first, followed by stirring until homogeneous. The mixture was then reacted at 20 °C for 18 hours to obtain a reaction solution. After thin-layer chromatography showed complete reaction, the reaction solution was concentrated under reduced pressure, then redissolved in methanol to the original volume. Purification was then performed by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v), and finally lyophilized to obtain compound 14-6. Compound 14-6 was a yellow powder (2.67 g, yield 87%). The mass spectrometry 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-dicyclohexylphosphine-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 reacted at 100 °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), then evaporated to dryness under reduced pressure, and then purified by 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 14-7. Compound 14-7 was a yellow oil (2.30 g, yield 44%). The mass spectrometry data for compound 14-7 were: 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 for 12 hours under a hydrogen atmosphere to obtain a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was filtered, and the filter cake was collected; the filter cake was washed 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 a yellow oily substance (0.39 g, yield 23%). The mass spectrometry 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, and the mixture was stirred thoroughly. The mixture was then reacted at 45 °C for 4 hours to obtain a reaction solution. After the reaction was monitored by HPLC until complete, 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 used directly 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 were: 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-oxytripyrrolidinephosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was then cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was monitored to be complete by HPLC, the reaction solution was purified by reversed-phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % v / v) and then lyophilized to obtain compound LP-19. Compound LP-19 was a yellow powder (36 mg, yield 33%). The synthetic route for compound LP-19 is shown below. Figure 37 The mass spectrometry results for compound LP-19 are shown in [link to data]. Figure 38 The mass spectrometry data for compound LP-19 are as follows: 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 Bismuth Substances). The specific immunization schedule was as follows:

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

[0294] For the initial immunization, the human DLL3 target protein was first diluted with physiological saline to obtain a diluted solution (1 mg: 1 mL). Then, the diluted solution was mixed with Freund's complete adjuvant (purchased from Shanghai Yisheng) at a volume ratio of 1:1 to obtain an immunizing agent. The immunizing agent was then administered subcutaneously to mice at a dose of 50 μg protein per mouse to complete the initial immunization. After the initial immunization, subsequent booster immunizations were performed using Freund's incomplete adjuvant (purchased from Shanghai Yisheng). The first and third booster immunizations were administered via intraperitoneal injection, while the second booster immunization was administered via subcutaneous injection. The injection dose for all 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 spleens of these mice were dissected and ground to obtain individual spleen cells. The individual spleen cells were washed once with serum-free DMEM medium (purchased from Gibco). After washing, the cells were centrifuged at 300g for 8 minutes, the supernatant was discarded, and the spleen cell pellet was collected. The spleen cell pellet was resuspended in serum-free DMEM medium, and the spleen cells were counted to obtain a spleen cell suspension. Mouse plasma cells were fused with sp20 cells (purchased from ATCC) to obtain fused cells (the Edward A. Greenfield electroporation fusion protocol was used, with a fusion efficiency of approximately one hybridoma per 200 B cells; see the literature "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 from the culture 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 Peptasys, the complete amino acid sequence of human DLL3 protein is shown in SEQ ID NO. 16), Antigen B (cynomolgus monkey DLL3 protein, purchased from Peptasys, the complete amino acid sequence of cynomolgus monkey DLL3 is shown in SEQ ID NO. 17), or Antigen C (mouse DLL3 protein, purchased from Peptasys, the complete amino acid sequence of mouse DLL3 is shown in SEQ ID NO. 17) are coated with a coating buffer (Nanjing Genscript Biotech Co., Ltd.). As shown in NO.43, the antigen was diluted to a concentration of 0.5 μg / mL to obtain antigen dilution buffer. The antigen dilution buffer was added to each well at a rate of 100 μL / well, and the plates were incubated overnight (16 h) at 4°C. The supernatant was then discarded to obtain antigen-coated ELISA plates. Blocking buffer (Nanjing Genscript Biotech) was added to each well at a rate of 100 μL / well, and the plates were incubated at 37°C for 1 h. The supernatant was then discarded to obtain blocked ELISA plates. Human lovastatin ADC (purchased from Nanjing Pengbo Biotech) and mouse lovastatin ADC (purchased from Nanjing Pengbo Biotech) were used as positive controls, and human IgG (purchased from Nanjing Pengbo Biotech) was used as a negative control. The antibody to be tested (i.e., the cell culture supernatant obtained from culture screening) was quantified and then serially diluted to obtain primary antibody dilution buffers of different concentrations. Different concentrations of primary antibody diluent were added to the blocked ELISA plates at a rate of 100 μL / well. The plates were incubated at 37°C for 1 hour, and the supernatant was discarded to obtain the ELISA plates incubated with the primary antibody. HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-human IgG (purchased from Jackson) were diluted to the recommended working concentration using blocking buffer according to the secondary antibody instructions. The secondary antibody diluent was added to the blocked ELISA plates at a rate of 100 μL / well. The plates were incubated at 37°C for 330 minutes, and the supernatant was discarded to obtain the ELISA plates incubated with the secondary antibody. The absorbance at 450 nm in each well of the ELISA plates incubated with the secondary antibody was measured using a multi-mode microplate reader (purchased from Beijing Kai'ao Technology Co., Ltd.). Based on the measured absorbance, the ELISA plates were analyzed using GraphPad. Prism fitting was used to obtain the EC50 value of the test antibody, and the EC50 value was used to measure the affinity of the test antibody for δ-like ligand 3. For ELISA detection, all test antibodies were initially concentrated at 10 mM and serially diluted 3-fold. ELISA results are shown in Tables 1 and 2.

[0300] The flow cytometry assay was performed as follows: CHO-K1 (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) was used as the negative cell line, and the CHO-K1 / DLL3 overexpression cell line (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) was used as the positive cell line. Each monoclonal antibody was tested separately. Human lovatozumab ADC and mouse lovatozumab ADC were used as positive controls, human IgG as the isotype control, and PBS buffer as the negative control. The assay procedure was as follows: the monoclonal antibody samples were incubated with the two cell lines at 4°C, washed, incubated with secondary antibodies, washed again, and then analyzed by the flow cytometry instrument (for specific flow cytometry assay methods, please refer to the literature "Tabatabaei, Mahdis Sadat, and Marya Ahmed. 'Enzyme-Linked Immunosorbent Assay (ELISA).' Methods in molecular biology (Clifton, NJ) vol. 2508 (2022): 115-134."). The secondary antibodies for the tested monoclonal antibodies and human lovatozumab ADC were prepared using Alexa. 647AffiniPure goat anti-human IgG, Fcγ fragment specific antibody (Jackson, 109-605-098, 144613); secondary antibody for mouse lovatozumab ADC was developed using Alexa. 647AffiniPure goat anti-mouse IgG, Fcγ fragment specific antibody (Nanjing Pengbo Biotechnology). Flow cytometry results are shown in Table 3.

[0301] As shown in Tables 1 and 2, ELISA screening identified 10 hybridoma cell lines producing murine monoclonal antibodies against delta-like ligand 3. These 10 hybridoma cell lines and their secreted murine monoclonal antibodies against delta-like ligand 3 were named mJFab1, mJFab2, mJFab3, mJFab4, mJFab5, mJFab6, mJFab7, mJFab8, mJFab9, and mJFab10, respectively. Among these monoclonal antibodies, except for mJFab5 which had relatively poor affinity, the other antibody samples all showed 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 had a weaker binding to CHO-K1 cells with high expression of δ-like ligands, while the other antibody samples could all 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 sequencing

[0310] The selected hybridoma cell line mJFab8 was amplified and cultured. Total RNA was extracted using Trizol (Thermo Fisher Scientific), and amplified using mouse-Ig degenerate primers (Nanjing Genscript Biotech). Finally, sequencing was performed to obtain the CDR sequence of the antibody variable region of monoclonal antibody mJFab8, as shown in Table 4. The amino acid sequence of the light chain variable region of 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 Sequence

[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 plugin. The parameters were selected as CDR transplantation, i.e., the CDR residues were fixed and unchanged. The annotation method was selected as kabbat, which generated 5 humanized light chains and 7 humanized heavy chains. The variable region sequences of the humanized light chains and humanized heavy chains are shown in Table 5.

[0315] Five humanized light chains and seven humanized heavy chains were spliced ​​with the constant regions of the human IgG1 heavy chain (amino acid sequence as shown in SEQ ID NO. 21) and the human IgG Kappa chain (amino acid sequence as shown in SEQ ID NO. 22), respectively, to form complete humanized antibody light and heavy chain sequences, resulting in five humanized antibodies: JFab4, JFab12, JFab13, JFab12b, and JFab13b. Specifically, the amino acid sequence of the light chain of humanized antibody JFab4 is shown in SEQ ID NO. 24 (containing the VL-hum5 fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 23 (containing the VH-hum6 fragment); the amino acid sequence of the light chain of humanized antibody JFab12 is shown in SEQ ID NO. 27 (containing the VL-hum2 fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 25 (containing the VH-hum13 fragment); the amino acid sequence of the light chain of humanized antibody JFab13 is shown in SEQ ID NO. 24. As shown in SEQ ID NO. 27 (containing the VL-hum2 fragment), the amino acid sequence of the heavy chain is shown in SEQ ID NO. 26 (containing the VH-hum9 fragment); 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 DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.11 VH-hum16 Humanized FR DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.12 VH-hum13 Humanized FR DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.13 VH-hum9 Humanized FR DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.14 VH-hum6 Humanized FR DLL3 monoclonal antibody heavy chain variable region SEQ ID NO.15 VH-hum1 Humanized FR 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. Screening of humanized antibodies

[0319] The coding sequence of the humanized antibody was derived, synthesized, and cloned into the PTT5 antibody expression vector (completed by Anhui General Biotechnology Co., Ltd.). The antibody light and heavy chains were combined for expression and purification (for specific expression and purification methods, please refer to the literature "Kunert, Renate, and David Reinhart. 'Advances in recombinant antibody manufacturing.' Applied microbiology and biotechnology vol. 100, 8(2016): 3451-61."), yielding humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b. Using the monoclonal antibody mJFab8 as a control, the affinity of the humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b was determined using a Biacore T200 (Cytiva). The affinity assay results are shown in Tables 6 and 7 (in addition to JFab4, JFab12, JFab13, JFab12b and JFab13b, Tables 6 and 7 also show other humanized antibodies obtained in the same batch as JFab4, JFab12, JFab13, JFab12b and JFab13b as controls).

[0320] As shown in Tables 6 and 7, the humanized antibody JFab13b has the lowest equilibrium dissociation constant with the DLL3 antigen. Therefore, JFab13b is the antibody with the best affinity for the DLL3 target among all humanized antibodies.

[0321] Table 6. Results of Affinity Measurement

[0322]

[0323] Table 7. Affinity Measurement Results

[0324]

[0325] 7. Expression and purification of humanized antibodies

[0326] The humanized antibody coding sequence was derived, synthesized, and cloned into the PTT5 antibody expression vector (completed by Anhui General Biotechnology Co., Ltd.), resulting in recombinant vectors expressing the antibody heavy chain and light chain. EXPI293F cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were passaged in serum-free cell culture medium (purchased from OPMA) and the cell density was adjusted to 3 × 10⁻⁶ cells / year. 6After cell density was reduced to 800 mL, recombinant vectors expressing the antibody heavy chain and recombinant vectors expressing the antibody light chain were simultaneously and transiently transfected into EXPI293F cells using transfection reagent (purchased from OPMA). Cell density and viability were assessed by trypan blue staining and counting after transfection. Cell culture supernatant was collected 6 days after transfection and subjected to affinity purification. The purified protein samples were analyzed by SEC HPLC. The SEC HPLC results are shown in Table 8. The nucleotide sequences of the genes encoding the light chain variable region of the humanized antibody JFab4 are shown in SEQ ID NO. 34, and the nucleotide sequences of the genes encoding the heavy chain variable region of the humanized antibody JFab4 are shown in SEQ ID NO. 33; the nucleotide sequences of the genes encoding the light chain variable region of the humanized antibody JFab12 are shown in SEQ ID NO. 36, and the nucleotide sequences of the genes encoding the heavy chain variable region of the humanized antibody JFab12 are shown in SEQ ID NO. 35; the nucleotide sequences of the genes encoding the light chain variable region of the humanized antibody JFab13 are shown in SEQ ID NO. 36, and the nucleotide sequences of the genes encoding the heavy chain variable region of the humanized antibody JFab13 are shown in SEQ ID NO. 37; the nucleotide sequences of the genes encoding the light chain variable region of the humanized antibody JFab12b are shown in SEQ ID NO. 40, and the nucleotide sequences of the genes encoding the heavy chain variable region of the humanized antibody JFab12b are shown in SEQ ID NO. 38; the nucleotide sequences of the genes encoding the light chain variable region of the humanized antibody JFab13b are shown in SEQ ID NO. 38. As shown in NO.40, the nucleotide sequence of the gene encoding the variable region of the humanized antibody JFab13b heavy chain is shown in SEQ ID NO.39.

[0327] Table 8 SEC HPLC Detection 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. Detection of humanized antibody endocytosis

[0330] The endocytic activity of antibodies was determined using human small cell lung cancer SHP-77 (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) and human small cell lung cancer NCI-H82 (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences). The method for detecting endocytic activity was as follows: the antibody to be tested was diluted to a concentration of 15 μg / mL using PBS buffer containing 2% (v / v) fetal bovine serum (FBS) to obtain the antibody dilution; four aliquots of 1.5 × 10⁻⁶ were dispensed for each antibody. 5Cells were cultured in 96-well U-plates and centrifuged, then the supernatant was discarded. 200 μL of the antibody dilution solution was added to each well of the 96-well U-plate, and the plates were incubated on ice for 45 min. After incubation, the plates were centrifuged at 4°C, and the supernatant was discarded. 150 μL of PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to each well of the 96-well U-plate, and the plates were centrifuged, then the supernatant was discarded. This process was repeated twice. Cells were resuspended in 200 μL of PBS buffer in each well of the 96-well U-plate. Three aliquots of cells were incubated at 37°C for 4 h, 1 h, and 0 h, respectively. The remaining aliquot was incubated on ice for 4 h. After incubation, all cell samples were centrifuged at 4°C, 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 96-well U-plates at a volume of 100 μL / well and incubated on ice for 30 min. After incubation, the cells were centrifuged at 4°C and the supernatant was discarded. Then, PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to 96-well U-plates at a volume of 150 μL / well, centrifuged, and the supernatant was discarded. This process was repeated twice. The cells in the 96-well U-plates were resuspended in PBS buffer containing 2% (v / v) FBS (fetal bovine serum) at a volume of 200 μL / well. The average fluorescence intensity within the NxT (purchased from Thermo) detection wells was used to calculate the endocytosis rate of different test antibodies in SHP-77 cells or NCI-H82 cells using the formula: endocytosis rate (%) = 100 - (MFI of the sample incubated at 37℃ at this time point / MFI of the control sample incubated at 4℃ at this time point) × 100. This was used to evaluate the endocytosis effect of the test antibodies in small cell lung cancer cells with high expression of δ-like ligand 3. The fluorescence values ​​are statistically analyzed in Tables 9 and 10, and the endocytosis rate (%) is shown in Table 10. Figures 39-40 .

[0331] Cell surface antibody load can be represented by mean fluorescence intensity (MFI), and the fluorescence intensity decreases with prolonged incubation time, indicating that antibodies are endocytosed by cells, reducing the cell surface antibody load and thus leading to a decrease in fluorescence intensity. (See Tables 9-10 and...) Figures 39-40 It is known that humanized antibodies JFab12, JFab13, JFab12b and JFab13b can all be endocytosed by small cell lung cancer cells with high expression of delta-like ligand 3 (DLL3) and the endocytosis 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 antibody internalization detected by flow cytometry (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. Specificity study of humanized antibodies

[0337] The JFab13b antibody was used to perform immunoblotting experiments with recombinant human DLL1 protein (purchased from Yisheng Biotechnology, complete amino acid sequence shown in SEQ ID NO. 41), recombinant human DLL3 protein (purchased from Bipsy Biotech, complete amino acid sequence shown in SEQ ID NO. 16), or recombinant human DLL4 protein (purchased from Huamei Biotechnology, complete amino acid sequence shown in SEQ ID NO. 42) to obtain the antibody specificity (for details of the immunoblotting experiment, please refer to the literature "Hirano, Seishiro. "Westernblot analysis." Methods in molecular biology (Clifton, NJ) vol. 926 (2012): 87-97."). In the immunoblotting experiment, all antigens contain histidine His tags, and anti-His tag antibodies can also be used for immunoblotting detection. JFab13b and anti-His tag antibody (purchased from Sanying Biotechnology) were used as primary antibodies, respectively, and bound to rabbit anti-human-IgG-HRP (purchased from Solarebo) and rabbit anti-mouse-IgG-HRP (purchased from Solarebo), respectively, for colorimetric development. The colorimetric results are shown in the figure. 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 or 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 embodiment provides a monoclonal antibody JFab4 against delta-like ligand 3. 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 embodiment provides a monoclonal antibody JFab12 against delta-like ligand 3. 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] Examples 2-3: A monoclonal antibody against delta-like ligand 3

[0344] This embodiment provides a monoclonal antibody JFab13 against delta-like ligand 3. 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] Examples 2-4: A monoclonal antibody against delta-like ligand 3

[0346] This embodiment provides a monoclonal antibody JFab12b against delta-like ligand 3. 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] Examples 2-5: A monoclonal antibody against delta-like ligand 3

[0348] This embodiment provides a monoclonal antibody JFab13b against delta-like ligand 3. 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 Example 1 were replaced with PBS solution (0.012M PBS buffer, pH = 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using an Amicon Ultra 30K ultrafiltration tube to obtain replacement solutions. An aqueous solution (7.5 eq) containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the replacement solution, and the mixture was incubated 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 then cooled to 25°C using a water bath. Derextecan (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd., CAS No.: 1599440-13-7, using MC-GGFG peptide as the linker) and compounds (LP-1 to LP-19) from Examples 1-1 to 1-19 were dissolved in DMSO to prepare 10 mM solutions. These solutions were then added to reaction solution A at a rate of 12.0 eq, with DMSO added to bring the final concentration to 10% (v / v) to obtain the reaction system. The reaction system was reacted in a water bath at 25°C for 2 hours to obtain reaction solution B. Reaction solution B was purified using an Amicon Ultra 30K ultrafiltration tube or Protein A4FF agarose purification resin (purchased from Sangon Biotech) to remove uncoupled small molecules, yielding the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR value) was determined using RP-HPLC / MS. Purity was determined using SEC-HPLC.

[0352] Method 2: Different monoclonal antibodies (purity greater than 95%) obtained in Experiment 1 were replaced with PBS solution (0.012M PBS buffer, pH = 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using an Amicon Ultra 30K ultrafiltration tube to obtain replacement solutions. An aqueous solution (2.5 eq) containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the replacement solution, and the mixture was incubated 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 then cooled to 25°C using a water bath. MC-VC-PAB-MMAE (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., using MC-Val-Cit-PAB connector), MC-VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., using MC-Val-Ala-PAB connector), and compounds from Examples 1-1 to 1-19 (LP-1 to LP-19) were dissolved in DMSO to prepare 10 mM solutions. These solutions were then added to reaction solution A at a rate of 6.0 eq, with DMSO added to bring the final concentration to 10% (v / v) to obtain the reaction system. The reaction system was incubated in a water bath at 25°C for 2 hours to obtain reaction solution B. Reaction solution B was purified using an Amicon Ultra 30K ultrafiltration tube or Protein A4FF agarose purification resin (purchased from Sangon Biotech) to remove uncoupled small molecules, yielding the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR value) was determined using RP-HPLC / MS. Purity was detected using SEC-HPLC.

[0353] Method 3: Different monoclonal antibodies (purity greater than 95%) obtained in Experiment 1 were replaced with PBS solution (0.012M PBS buffer, pH = 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using an Amicon Ultra 30K ultrafiltration tube to obtain replacement solutions. An aqueous solution (3.4 eq) containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the replacement solution, and the mixture was incubated 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 then cooled to 25°C in a water bath. The compounds (LP-1 to LP-19) from Examples 1-1 to 1-19 were dissolved in DMSO to prepare 10 mM solutions, and then added to reaction solution A at a concentration of 10.0 eq. DMSO was then added to bring the final concentration to 10% (v / v), yielding the reaction system. The reaction system was incubated 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 Protein A4FF agarose purification resin (purchased from Sangon Biotech) to remove unconjugated small molecules, yielding the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR value) was determined using RP-HPLC / MS. Purity was determined using SEC-HPLC.

[0354] The antibody-drug conjugate ratio (DAR value) detection method is as follows:

[0355] The sample was diluted with 25 mM ammonium bicarbonate aqueous solution to a concentration of 1 mg / mL, and then 1 μL of N-glycoamide enzyme F (PNGase F) was added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, dithiothreitol (DTT) was added to a final concentration of 20 mM, and the reaction was carried out at room temperature for 1 hour before direct analysis. Instrument model: Thermo Fisher Vanquish & QE-HF-X. Liquid chromatography parameters are shown in Table 11. Mass spectrometry conditions: 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 (DDA) mode.

[0356] The purity testing method is as follows:

[0357] SEC purity analysis - SEC-HPLC (size exclusion chromatography). Instrument model: Agilent 1260 liquid chromatograph. Sample preparation: The sample was diluted with the mobile phase to approximately 1 mg / mL, centrifuged at 12000 rpm for 5 minutes, and the supernatant was injected for analysis. Chromatographic conditions are shown in Table 12.

[0358] Table 11 Liquid Phase Parameters

[0359]

[0360] Table 12 Chromatographic conditions

[0361] chromatographic column TOSOH, TSKge1 G3000WXL, 5μm, 7.8mm×300mm mobile phase 100 mM MPB + 200 mM arginine hydrochloride, 5% (v / v) isopropanol (pH 6.8) Flow rate 0.6 mL / min Column temperature 25℃ Injection volume 20μL Washout time 25min elution gradient Isocratic elution

[0362] Experimental Example 2: Drugability Evaluation of Antibody-Drug Conjugates

[0363] The drug-drug conjugates of Example 3-1 were evaluated for drug-likeness (the evaluation consisted of DAR value determination by RP-HPLC / MS and purity determination by SEC-HPLC, referring to the patent application text with 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 shown in Table 13, under appropriate drug loading, JFab12b antibody can be normally conjugated with 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. JFab13 antibody can be normally conjugated with LP-3 and LP-7. JFab13b antibody can be normally conjugated with 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 conjugated drug-antibody conjugation ratio and aggregation degree meet the expected requirements. However, even at the lowest drug loading, the aggregation degree of JFab12b and JFab13b antibody with MC-VA-PAB-Exatecan exceeds the acceptable range. It is evident that, except for the infeasibility of MC-VA-PAB-Exatecan conjugation, the drug-making feasibility of the other studied cases is feasible.

[0365] Table 13 Drugability Evaluation of Antibody-Drug Conjugates (ADCs)

[0366] ADC Name Antibody name Connector-Cytoxin Number Concentration mg / mL DAR Clustering 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 the in vitro cytotoxic activity of antibody-drug conjugates

[0368] The in vitro cytotoxic activity of the antibody-drug conjugates of Example 3-1 was evaluated using NCI-H82, SHP-77, and DMS-53 cells (all purchased from the Shanghai Cell Bank, Chinese Academy of Sciences). The in vitro cytotoxic activity evaluation method was as follows: Cell suspensions were prepared using cell culture medium containing 10% (v / v) fetal bovine serum. 135 μL of the cell suspension was added to each well of a 96-well plate. The first and twelfth columns were left unplatened with only 135 μL of culture medium. The plates were incubated at 37°C for 16 hours with 5% (v / v) carbon dioxide. The ADC samples to be tested were prepared as working solutions (10× concentration) using PBS buffer, and then serially diluted with PBS buffer at the corresponding folds. 15 μL of 10× concentration ADC solution was added to each well, and the plates were incubated at 37°C for 5 days with 5% carbon dioxide. Add 15 μL of LCCK-8 solution (APExBIO) to each well, incubate at 37°C in the dark with 5% CO2 for 1–4 hours, and read the absorbance at 450 nm using a microplate reader. Process the data using GraphPad Prism5. Table 14 shows the cell plating density, initial working solution concentration (10× concentration), and ADC sample dilution factor. Table 15 shows the results of in vitro cytotoxicity evaluation.

[0369] As shown in Table 15, except for ADC-20, which exhibits relatively weak in vitro cytotoxic activity, the other ADCs in Example 3-1 all demonstrate significant in vitro cytotoxic activity against NCI-H82 cells. Furthermore, their cytotoxic effects are not significantly different from those of ADC-1 and ADC-18 conjugated with Deruxtecan. Therefore, the antibody-drug conjugates in Example 3-1 possess strong target cell killing activity and can kill NCI-H82 cells expressing δ-like ligand 3 (DLL3).

[0370] As shown in Table 15, all antibody-drug conjugates in Example 3-1 exhibited significant in vitro cytotoxic activity against SHP-77 cells. Furthermore, when conjugated with JFab12b, ADC-8 showed a lower IC50 compared to ADC-1 conjugated with Deruxtecan; when conjugated with JFab13b, ADC-15, ADC-27-6, and ADC-27-8 showed lower IC50 compared to ADC-18 conjugated with Deruxtecan, while ADC-16, ADC-25, and ADC-27-4 showed no significant difference, and the remaining ADCs showed higher IC50. Therefore, ADC-1, ADC-8, ADC-9, ADC-15, ADC-16, ADC-18, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 possess strong target cell killing activity and can kill SHP-77 cells expressing δ-like ligand 3 (DLL3).

[0371] As shown in Table 15, all antibody-drug conjugates in Example 3-1 exhibited significant in vitro cytotoxic activity against DMS-53 cells. Furthermore, when conjugated with JFab13b, ADC-16, ADC-24-4, ADC-24-8, ADC-27-4, ADC-27-6, and ADC-27-8 showed lower IC50 values ​​compared to ADC-18 conjugated with Deruxtecan, while ADC-25 showed no significant difference. The remaining ADCs exhibited higher IC50 values. Therefore, ADC-16, ADC-18, ADC-24-4, ADC-24-8, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 possess strong target cell killing activity and can kill 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 factor of ADC samples.

[0373]

[0374] Table 15. In vitro cytotoxic activity of antibody-drug conjugates (ADCs) (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, "--" indicates that no detection was performed.

[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 6–8 weeks (purchased from Beijing Vital River Pharmaceutical Co., Ltd.) were subcutaneously injected with 100 μL of 50% (v / v) Matrigel at 5 × 10⁻⁶ doses in the right back.6 Personal small cell lung cancer cells (NCI-H82) were used until the tumor grew to an average volume of 150 mm. 3 Around 10:00 AM, mice were randomly divided into 5 groups based on tumor size and body weight, with 5 animals in each group. The day of administration for grouping was defined as day 0.

[0386] The drug was administered via tail vein, once a week for a total of two weeks. The experiment ended after 21 days of administration or when the tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice a week, and animal survival status was observed throughout the experiment. Data were recorded using Excel and plotted using GraphPad Prism software. After the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated. The tumor volume (V) was calculated using the formula: V = 1 / 2 * L 长 *L 短 2 The formula for calculating tumor inhibition rate (TGI%) is: TGI% = (1 - (T...) i -T0) / (V i -V0)));where, T i V represents the mean tumor volume in the treatment group on day i after drug administration, T0 represents the mean tumor volume in the treatment group on day 0 after drug administration, and V i Vi represents the mean tumor volume of the blank control group on day i after drug administration, and V0 represents the mean tumor volume of the blank control group on day 0 after drug administration.

[0387] 2. Data Analysis

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

[0389] 3. Conclusion

[0390] The antibody-drug conjugate of Example 3-1 can significantly reduce tumor volume and has good in vivo antitumor activity. At the same time, no mice died and no weight decreased significantly, 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 6–8 weeks (purchased from Beijing Vital River Pharmaceutical Co., Ltd.) were subcutaneously injected with 100 μL of 1×10⁻⁶ dextrose solution containing 50% (v / v) Matrigel into the right back. 7 Personally derived small cell lung cancer cells (SHP-77, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were used until the tumors grew to an average volume of 150 mm. 3 Around 10:00 AM, mice were randomly divided into 4 groups of 5 animals each, based on tumor size and body weight. The day of administration was defined as day 0.

[0397] The drug was administered via tail vein, once a week for a total of two weeks. The experiment ended after 21 days of administration or when the tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice a week, and animal survival status was observed throughout the experiment. Data were recorded using Excel and plotted using GraphPad Prism software. After the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated. The tumor volume (V) was calculated using the formula: V = 1 / 2 * L 长 *L 短 2 The formula for calculating 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 day i after drug administration, T0 is the mean tumor volume of the treatment group on day 0 after drug administration, and V i Vi represents the mean tumor volume of the blank control group on day i after drug administration, and V0 represents the mean tumor volume of the blank control group on day 0 after drug administration.

[0398] 2. Data Analysis

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

[0400] 3. Conclusion

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

[0402] Table 16. Efficacy of ADCs on NCI-H82 xenografts in tumor-bearing nude mice.

[0403] ADC dose Day 17 TGI (%) 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 tumor-bearing nude mice

[0405] ADC dose Day 14 TGI (%) 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 illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this 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 ; Wherein, Ab is a monoclonal antibody against delta-like ligand 3; the light chain variable region of the monoclonal antibody includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO.1, and the heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO.4, CDR2 and CDR3 as shown in SEQ ID NO.5, and the heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO.6; D represents a bioactive molecule that inhibits 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 are linkers that connect monoclonal antibodies and bioactive molecules; L1 is selected from the following structure: , , , or ; In L1, The numbers represent connection sites. Connection sites marked with number 1 are connected to Ab via S atoms, and connection sites marked with number 2 are connected to L2. L2 is ; In L2, The numbers 3 and 4 represent connection sites. Connection sites marked with digit 3 are connected to L1, and connection sites marked with digit 4 are connected to L3. Z1 is selected from hydrogen atom, 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, aryl or heteroaryl; or, R1 and R2 together with the attached nitrogen atom form a 4- to 12-membered heterocyclic group; R3 is selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclic; 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, hydroxy, or hydroxyalkyl groups; the atoms in the heterocyclic groups are each independently selected from C, N, O, or S atoms; and the heterocyclic group is substituted by R4; the value of m is an integer from 1 to 24. 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, hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; 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, hydroxy, or hydroxyalkyl groups; m is an integer from 1 to 24. R5 is selected from hydrogen atoms, alkyl groups, cycloalkyl groups, or heterocyclic groups; In R5, the alkyl, cycloalkyl, and heterocyclic groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxy, 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 takes the value of an integer from 1 to 10; j takes the value of an integer from 1 to 20; ** is the 4th bit; L3 is selected from amino acid residues or short peptides composed of 2 to 10 amino acid residues; In L3, the amino acid residues are either natural amino acid residues or non-natural amino acid residues. L4 is selected from , Or chemical bonds; In L4, The numbers 5 and 6 represent connection sites. The connection site marked with the number 5 is connected to L3, and the connection site marked with the number 6 is connected to D.

2. The antibody-drug conjugate as described in claim 1, characterized in that, D is a cytotoxic compound, immunomodulator, or enzyme that has an inhibitory effect on tumor cells.

3. The antibody-drug conjugate as described in claim 1, characterized in that, D is a kinase inhibitor that has an inhibitory effect on tumor cells.

4. The antibody-drug conjugate as described in claim 1, characterized in that, L1 is In L1, The numbers represent connection sites. Connection sites marked with number 1 are connected to Ab via S atoms, while connection sites marked with number 2 are connected to L2.

5. The antibody-drug conjugate as described in claim 1, characterized in that, The L2 is In L2, The numbers 3 and 4 represent connection sites. Connection sites marked with digit 3 are connected to L1, and connection sites marked with digit 4 are connected to L3.

6. The antibody-drug conjugate as described in claim 5, characterized in that, Z2 is selected from , , , , , , , or ; In Z2, n takes the value of an integer from 1 to 13.

7. The antibody-drug conjugate as described in claim 5, characterized in that, The Z3 is selected from -(CH2). i -C(=O)-** or -(CH2) i (OCH2CH2) j -C(=O)-**; In Z3, i takes the value of an integer from 1 to 10; j takes the value of an integer from 1 to 20; ** is the 4th bit.

8. The antibody-drug conjugate according to any one of claims 1 to 7, characterized in that, The tumor is a tumor that expresses anti-δ-like ligand 3.

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