Ether-linked linker-drug molecules and antibody conjugate drugs, methods of making and use thereof

Abstract

The application discloses an ether bond connected linker-drug molecule and an antibody conjugated drug, a preparation method and application thereof, and particularly discloses a compound represented by formula (1) or (2), or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof, a composition containing the same or a use thereof. The compound of the application is connected with a drug molecule through an ether bond through a polypeptide (VC, VA, AAA)-PAB (self-elimination group) on a linker, greatly enhances the drugability (including hydrophilicity and stability) of the compound to an antibody drug conjugate, reduces the drug clearance in the body, and improves the tumor treatment effect.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to an ether-linked linker-drug molecule and antibody-drug conjugate, its preparation method and application. Background Technology

[0002] Antibody-drug conjugates (ADCs) significantly enhance the therapeutic window for tumors by targeting and delivering highly active toxins to lesions. However, their development is limited by the inherent limitations of linker-toxin conjugation technology. Traditional ADCs rely on acid-sensitive bonds (such as hydrazone bonds), enzyme-sensitive polypeptide bonds (such as Val-Cit), or non-cleavable bonds (such as thioether bonds) to link toxins, but these methods have significant shortcomings in terms of stability, release efficiency, and toxicity control. In particular, for camptothecin toxins (such as SN38 and DX-8951f), their high lipophilicity and rigid structure lead to problems such as antibody aggregation after conjugation, insufficient linker cleavage efficiency, and novel toxicities (such as interstitial lung disease), severely limiting their clinical application (e.g., Enhertu's objective response rate in HER2-low-expressing breast cancer was only 37%).

[0003] In existing technologies, acid-sensitive links (such as Mylotarg) are prone to off-target toxicity due to poor cyclic stability; enzyme-sensitive peptide links (such as Adcetris), while capable of releasing toxins via lysosomal enzymes, suffer from reduced cleavage efficiency due to steric hindrance when coupled with camptothecin (e.g., the mesmerization rate of WO2020233174A1 is >5%); and cleavable links (such as Kadcyla) cannot exert a side-effect killing effect because they rely on complete antibody degradation. Furthermore, while hydrophilic modifications (such as PEG) can improve hydrophobicity, they easily induce immunogenicity and functional masking. Therefore, there is an urgent need to develop a novel linker that combines high stability with precise release capability to adapt to the properties of camptothecin and overcome the efficacy-safety bottleneck. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a drug molecule with a stable ether-enzyme-triggered bifunctional linker, an antibody-drug conjugate, its preparation method, and its applications.

[0005] One object of the present invention is to provide a linker-drug molecule compound.

[0006] Another object of the present invention is to provide an antibody-drug conjugate.

[0007] Another object of the present invention is to provide a composition comprising the above-described compound or antibody-drug conjugate.

[0008] Another object of the present invention is to provide pharmaceutical use of the said compound, antibody-drug conjugate or composition.

[0009] According to one aspect of the invention, a compound represented by formula (1), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, is provided:

[0010]

[0011] Where Q represents the connector unit;

[0012] M is selected from absent, alkylene, or polyethylene glycol groups, wherein the alkylene or polyethylene glycol groups are optionally substituted with substituents;

[0013] L are each independently selected from amino acid residues or optionally substituted amino acid residues;

[0014] m can be 0, 1, 2, or 3;

[0015] D contains drug molecules.

[0016] In some implementations, Q is selected from N3-, BrCH2-, ICH2-,

[0017] in, This indicates that M is connected from this point.

[0018] In some embodiments, M is selected from one or more of C1-C6 straight-chain or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O). n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-, -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n - or -CH2O(CH2CH2O) n CH2-, where n is an integer from 2 to 12.

[0019] In some embodiments, L is independently selected from valine residues, D-valine residues, citrulline residues, phenylalanine residues, lysine residues, acetyllysine residues, leucine residues, glycine residues, alanine residues, asparagine residues, aspartic acid residues, arginine residues, etc. Where A is selected from j is an integer from 1 to 12, in In the diagram, * indicates a connection from that point to either M or another L. This indicates the connection from that point in equation (1) Or another L.

[0020] In some implementations, the D is selected from cytotoxins.

[0021] In some embodiments, the cytotoxic agent is selected from one or more of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, or RNA polymerase 2 inhibitors.

[0022] In some embodiments, the cytotoxin is selected from camptothecin derivatives, such as...

[0023] In some embodiments, the compound represented by formula (1) is selected from the following structures:

[0024]

[0025] According to another aspect of the invention, a compound represented by formula (2), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, is provided:

[0026]

[0027] Ab contains antibody or antigen-binding fragments;

[0028] Q stands for connector unit;

[0029] M is selected from absent, alkylene, or polyethylene glycol groups, wherein the alkylene or polyethylene glycol groups are optionally substituted with substituents;

[0030] L are each independently selected from amino acid residues or optionally substituted amino acid residues;

[0031] m can be 0, 1, 2, or 3;

[0032] D contains drug molecules;

[0033] i is an integer from 1 to 16.

[0034] In some implementations, Q is selected from one of the following structures:

[0035]

[0036] Where * indicates that Ab is connected from this point. This indicates that M is connected from this point.

[0037] In some embodiments, M is selected from one or more of C1-C6 straight-chain or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O). n -、-O(CH2CH2O)n -、-(CH2CH2O) n CH2-, -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n - or -CH2O(CH2CH2O) n CH2-, where n is an integer from 2 to 12.

[0038] In some embodiments, L is independently selected from valine residues, D-valine residues, citrulline residues, phenylalanine residues, lysine residues, acetyllysine residues, leucine residues, glycine residues, alanine residues, asparagine residues, aspartic acid residues, arginine residues, etc. Where A is selected from j is an integer from 1 to 12, in In the diagram, * indicates a connection from that point to either M or another L. This indicates the connection from that point in equation (2). Or another L.

[0039] In some implementations, the D is selected from cytotoxins.

[0040] In some embodiments, the cytotoxic agent is selected from one or more of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, or RNA polymerase 2 inhibitors.

[0041] In some embodiments, the cytotoxin is selected from camptothecin derivatives, such as...

[0042] In some embodiments, the compound is selected from the following structures:

[0043]

[0044]

[0045] The definitions of Ab and i are the same as those described above.

[0046] In some implementations, the antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0047] In some implementations, the antibody comprises a monoclonal antibody.

[0048] In some implementations, the antibody comprises a bispecific antibody.

[0049] In some embodiments, the Ab is selected from: anti-human folate receptor antibody, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-EGFR antibody, anti-Nectin-4 antibody, anti-c-MET antibody, anti-PD-L1 antibody, anti-Claudin18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-MSLN antibody, anti-FGFR antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, and antigen-binding fragments of the above antibodies.

[0050] In some implementations, the Ab is an anti-HER2 antibody or its antigen-binding fragment.

[0051] In some embodiments, the Ab is an anti-B7-H3 antibody or its antigen-binding fragment.

[0052] In some embodiments, the Ab is an anti-MSLN antibody or its antigen-binding fragment.

[0053] According to another aspect of the invention, a composition is provided comprising the above-described compound, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof.

[0054] According to another aspect of the invention, the above-described compound, or its tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug, or solvate, is provided for use in the preparation of a medicament for the treatment and / or prevention of tumors.

[0055] In some embodiments, the tumor is selected from tumors associated with expression of targets selected from the group consisting of: human folate receptor, HER2, HER3, TROP2, B7-H3, EGFR, Nectin-4, c-MET, PD-L1, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD70, CD73, CD74, MSLN, FGFR, FGFR2, and FGFR3.

[0056] In some implementations, tumors associated with the expression of the target include tumors that highly express the target and / or tumors that are positive for the target.

[0057] In some implementations, the tumor is selected from solid tumors, hematologic malignancies, and metastatic, refractory, or recurrent lesions of cancer.

[0058] In some implementations, the tumor is selected from: gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, thymic cancer, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), and glioblastoma.

[0059] Beneficial effects

[0060] This application employs chemically inert ether bonds (COC) instead of traditional carbamate bonds, significantly enhancing the stability of the conjugate. A cathepsin B-sensitive short peptide (such as VC, VA, AAA, etc.) and a self-eliminating group variant (PAB) are embedded after the ether backbone to achieve site-specific release of the ADC drug molecule in lysosomes. Simultaneously, the hydrophilicity of the drug molecule is adjusted using polysarcosine (PSAR) or polyethylene glycol (PEG), making it less prone to aggregation of the antibody conjugate at higher drug loadings. This drug molecule exhibits better biological activity, safety, and other drug-related properties, enhancing antitumor activity and / or improving the overall therapeutic window by increasing the drug's half-life in vivo and its concentration in tumor tissue.

[0061] This approach overcomes the core contradictions of ADC hydrophobic collapse, insufficient release efficiency, and uncontrollable toxicity, providing a safer and more efficient treatment option for solid tumors and tumors with low HER2 expression. Attached Figure Description

[0062] Figure 1 Size exclusion chromatography (SEC) chromatogram of antibody-drug conjugate 1 (control ADC) provided in this application.

[0063] Figure 2 This is a molecular size exclusion chromatography (SEC) chromatogram of antibody-drug conjugate 2 provided in this application.

[0064] Figure 3 This is a molecular size exclusion chromatography (SEC) chromatogram of antibody-drug conjugate 3 provided in this application.

[0065] Figure 4 This is a molecular size exclusion chromatography (SEC) chromatogram of antibody-drug conjugate 4 provided in this application.

[0066] Figure 5 This is the deconvolution mass spectrum of the heavy and light chains of antibody-drug conjugate 4 provided in this application.

[0067] Figure 6 Stability curves of antibody-drug conjugates 2-4 provided for this application and antibody-drug conjugate 1 as a control sample in PBS at 55°C.

[0068] Figure 7-9 Tumor therapeutic curves of antibody-drug conjugates 2-4 provided in this application and antibody-drug conjugate 1 as a control sample in the SHP-77 mouse subcutaneous tumor model. Detailed Implementation

[0069] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0070] In this invention, the linker-drug molecule compound refers to a compound formed by the combination of one or more linkers with a drug molecule, and also includes free radicals and ions formed by the compound.

[0071] In this invention, when the drug molecule (or cytotoxin) is linked to the linker, it encompasses the free radical and ionic forms of the drug molecule (or cytotoxin).

[0072] In this invention, the amino acid residue (such as lysine residue, acetyllysine residue, asparagine residue, etc.) refers to a fragment derived from the above-mentioned amino acid. For example, the amino group of the amino acid forms an amide bond and removes the carboxyl group, the carboxyl group forms an amide bond and removes the amino group, or the amino group removes a hydrogen and the carboxyl group removes a hydroxyl group. For example, "glycine (NH2CH2COOH) residue" can refer to "-CONHCH2-", "-CH2CONH-", or "-NHCH2CO-".

[0073] In this invention, "integers from 1 to 12" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, as well as any combination of these integers, such as "integers from 2 to 6". Similarly, "integers from 2 to 12" includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, as well as any combination of these integers, such as "integers from 3 to 8".

[0074] Example

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0076] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0077] Example 1 (Preparation of XY-11)

[0078]

[0079]

[0080] Synthesis of 1-2

[0081]

[0082] Compound 1-1 (15.00 g, 88.82 mmol) was added to methanol (750 mL) and stirred at room temperature. Raney nickel (15 g, 355.28 mmol) was added to the reaction vessel. The reaction mixture was stirred under hydrogen atmosphere for 2 hours until TLC showed complete reaction of the starting material. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a brown solid (12.90 g, 99.2% yield). The crude product was used directly in the next step without further purification.

[0083] LC-MS (ESI) + )m / z 196.3(M+H) + .

[0084] Synthesis of 1-4

[0085]

[0086] Compounds 1-2 (0.86 g, 4.41 mmol) were added to toluene (9 mL) and acetic acid (9 mL) and stirred at room temperature for 10 minutes. (S)-ethyl-4-hydroxy-7,8-dihydro-pyranO[3,4-F]indolazine-3,6,10(4H)-one (compounds 1-3, 1.42 g, 5.73 mmol) and pyridinium p-toluenesulfonate (332.16 mg, 1.32 mmol) were added to a reaction vessel. The reaction mixture was heated to 110 °C under nitrogen and refluxed, stirred for 24 hours, until TLC showed complete reaction of the starting materials. The reaction mixture was cooled to room temperature, and the reaction solvent was concentrated to obtain a crude product. The crude product was dissolved in acetic acid (4.5 mL), added to water (45 mL), and stirred for 30 minutes. A solid precipitated, was filtered, and the filter cake was washed with water (15 mL) and dried to give a brown solid (1.39 g, yield 74.7%).

[0087] LC-MS (ESI) + )m / z 423.2(M+H) + .

[0088] 1H NMR (400MHz, DMSO-d6) δ10.23(s,1H),7.73(d,J=11.8Hz,1H),7.24(s,1H),6.50(s,1H),5.42(s,2H),5.17(s,2H),3.08( t,J=6.2Hz,2H), 3.01(t,J=6.2Hz,2H), 2.01(p,J=6.5Hz,2H), 1.87(td,J=14.4,13.1,6.1Hz,2H), 0.88(t,J=7.3Hz,3H).

[0089] Synthesis of 11-2

[0090]

[0091] Compound 11-1 (10.00 g, 23.84 mmol) was added to tetrahydrofuran (200 mL) and stirred at room temperature. p-Aminobenzyl alcohol (5.20 g, 42.23 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (10.44 g, 42.23 mmol) were added to the reaction vessel, and the reaction mixture was stirred under nitrogen for 24 hours. The reaction proceeded until TLC showed complete reaction of the starting material. The reaction solvent was concentrated to obtain a crude product. Tetrahydrofuran (14 mL) and methyl tert-butyl ether (140 mL) were added to the crude product, and the mixture was stirred for 2 hours. A solid precipitated, which was filtered to obtain a yellow solid (8.80 g, yield 63.5%).

[0092] LC-MS (ESI+) m / z 378.2 (M+H) + .

[0093] 1 H NMR (400MHz, DMSO-d6) δ9.90 (s, 1H), 8.14 (d, J = 7.0Hz, 1H), 7.52 (d, J = 8.1Hz, 2H), 7. 24(t,J=9.9Hz,3H),5.90(ddt,J=16.5,10.8,5.5Hz,1H),5.29(d,J=17.2Hz,1H),5.1 6(d,J=10.6Hz,1H),5.09(t,J=5.6Hz,1H),4.44(dd,J=21.5,5.4Hz,5H),3.88(t,J=7 .9Hz, 1H), 1.96 (d, J = 6.8Hz, 1H), 1.29 (d, J = 7.0Hz, 3H), 0.85 (dd, J = 18.9, 6.8Hz, 6H).

[0094] Synthesis of 11-3

[0095]

[0096] Compound 11-2 (4.50 g, 11.92 mmol) was added to anhydrous tetrahydrofuran (90 mL) and stirred at 0 °C. Thionyl chloride (1.73 mL, 23.84 mmol) was slowly added dropwise to the reaction vessel, and the reaction mixture was stirred under nitrogen for 2 hours. The reaction proceeded until TLC showed complete reaction of the starting material. The mixture was concentrated to remove the reaction solvent and thionyl chloride, yielding a yellow solid (4.44 g, 94.1% yield). The crude product was used directly in the next step without further purification.

[0097] LC-MS (ESI) + )m / z 396.2(M+H) + .

[0098] Synthesis of 11-4

[0099]

[0100] Compounds 1-4 (4.00 g, 9.47 mmol) were added to N,N-dimethylformamide (80 mL) and stirred at room temperature. Compounds 11-3 (8.81 g, 22.25 mmol) and pentamethylpiperidine (8.05 mL) were added to the reaction vessel. The reaction mixture was heated to 50 °C under nitrogen and stirred for 24 hours until TLC showed complete reaction of the starting materials. The reaction mixture was cooled to room temperature and water (800 mL) was added dropwise. A brown solid gradually precipitated out. The mixture was filtered, the filter cake was washed with water, and dried to give a crude brown solid. Column chromatography of the crude product gave a white solid (2.80 g, yield 38.8%).

[0101] LC-MS (ESI) + )m / z 782.3(M+H) + .

[0102] 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.18(d,J=6.9Hz,1H),7.80(d,J=12.3Hz,1H),7.60(d,J=8.1Hz,2H),7.38(d ,J=8.1Hz,2H),7.32-7.24(m,2H),6.52(d,J=1.9Hz,1H),5.91(ddt,J=17.0,11.7,5.8Hz,1H),5.42(s,2H),5.30(d ,J=17.2Hz,1H),5.17(d,J=12.3Hz,3H),5.08(s,2H),4.45(dd,J=24.4,6.0Hz,3H),3.89(t,J=8.0Hz,1H),3.01(dt ,J=39.7,6.1Hz,4H),1.91(ddq,J=27.2,14.0,6.7Hz,5H),1.31(d,J=7.0Hz,3H),0.87(dq,J=16.0,6.7,5.0Hz,9H).

[0103] Synthesis of 1-5

[0104]

[0105] Compound 1-1 (2.28 g, 10.10 mmol) was added to N,N-dimethylformamide (40 mL) and stirred at room temperature. Compound 11-3 (4.00 g, 10.10 mmol) and pentamethylpiperidine (9.2 mL) were added to the reaction vessel. The reaction mixture was heated to 50 °C under nitrogen and stirred for 24 hours until TLC showed complete reaction of the starting materials. The reaction mixture was cooled to room temperature and water (800 mL) was added dropwise. A brown solid gradually precipitated out. The mixture was filtered, the filter cake was washed with water, and dried to give a crude brown solid. Column chromatography of the crude product gave a yellow solid (3.00 g, yield 50.8%).

[0106] LC-MS (ESI) + )m / z 585.2(M+H) + .

[0107] Synthesis of 1-6

[0108]

[0109] Compounds 1-5 (2.90 g, 4.96 mmol) were added to glacial acetic acid (30 mL) and stirred at room temperature. Iron powder (831 mg, 14.88 mmol) was added to the reaction vessel. The reaction mixture was stirred at room temperature (25 °C) for 24 hours under nitrogen atmosphere until TLC showed complete reaction of the starting material. The mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the crude product was concentrated. Column chromatography of the crude product gave a pale yellow solid (3.00 g, yield 50.8%).

[0110] LC-MS (ESI) + )m / z 555.2(M+H) + .

[0111] Another synthesis of 11-4

[0112]

[0113] Compounds 1-6 (2.0 g, 3.61 mmol) were added to N-methylpyrrolidone (9 mL) and stirred at room temperature for 10 minutes. (S)-ethyl-4-hydroxy-7,8-dihydro-pyranO[3,4-F]indolazine-3,6,10(4H)-one (compounds 1-3) (1.14 g, 4.33 mmol) and pyridinium p-toluenesulfonate (272 mg, 1.08 mmol) were added to a reaction vessel. The reaction mixture was heated to 110 °C under nitrogen and refluxed, and stirred for 6 hours until TLC showed complete reaction of the starting materials. The reaction mixture was cooled to room temperature, and the reaction solution was added to water (45 mL). The mixture was stirred for 30 minutes, and a solid precipitated. The solid was filtered, the filter cake was washed with water (15 mL), and dried to give a brown solid (2.0 g, yield 70.9%).

[0114] LC-MS (ESI) + )m / z 782.3(M+H) + .

[0115] Synthesis of 11-5

[0116]

[0117] Compound 11-4 (2.30 g, 9.47 mmol) was added to N,N-dimethylformamide (23 mL) and stirred at room temperature. Tetra(triphenylphosphine)palladium (33.99 mg, 29.42 μmol) and piperidine (2.32 mL, 23.53 mmol) were added to the reaction vessel, and the reaction mixture was stirred under nitrogen for 2 hours until TLC showed complete reaction of the starting material. The reaction solvent was concentrated to give a crude brown solid. Column chromatography of the crude product gave a yellow solid (1.60 g, yield 78.1%).

[0118] LC-MS (ESI) + )m / z 698.6(M+H) + .

[0119] 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.17(d,J=7.2Hz,1H),7.80(d,J=12.3Hz, 1H),7.60(d,J=8.1Hz,2H),7.39(d,J=8.1Hz,2H),7.28(s,1H),6.51(s,1H),5.42 (s,2H),5.12(d,J=27.7Hz,4H),4.45(q,J=7.6,7.1Hz,1H),3.19-2.90(m,5H),1. 98-1.72(m,5H),1.30(d,J=7.0Hz,3H),0.98-0.84(m,6H),0.78(d,J=6.8Hz,3H).

[0120] XY-11 synthesis

[0121]

[0122] Compound 11-5 (0.30 g, 429.94 μmol) was added to acetonitrile (3 mL) and stirred at 0 °C. (444.81 mg, 644.92 μmol) and diethylamine (59.93 μL, 429.94 μmol) were added to a reaction vessel. The reaction mixture was stirred under nitrogen for 0.5 hours until HPLC showed complete reaction of the starting material. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was dissolved in dimethyl sulfoxide (5 mL) and purified by reverse preparative chromatography. Purification method: Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. The gradient range was 10–50% B. The solvent was concentrated under reduced pressure to give a white solid (240 mg, yield 43.9%).

[0123] LC-MS (ESI) + )m / z 1273.20(M+H) + .

[0124] 1H NMR (400MHz, DMSO-d6) δ10.04(s,1H),8.29(d,J=49.7Hz,2H),8.11-7.93(m,1H),7.82(h,J=7.6,7.1Hz,1H),7.62(dd,J=19.3,12.9Hz,3H),7 .49-7.17(m,3H),6.99(s,2H),5.56-4.83(m,6H),4.64-3.77(m,23H), 3.13-2.64(m,38H),2.18-1.08(m,25H),0.84(tt,J=20.0,9.9Hz,9H).

[0125] Example 2 (Preparation of XY-21)

[0126]

[0127] Synthesis of 21-2

[0128]

[0129] Compound 21-1 (5 g, 13.57 mmol) was added to methanol (50 mL), dichloromethane (10 mL), and water (25 mL) and stirred at room temperature. Potassium carbonate (3.75 g, 27.14 mmol), copper sulfate (217 mg, 1.36 mmol), and 1H-imidazolium-1-sulfonyl azidohydrochloride (3.00 g, 14.25 mmol) were added to the reaction vessel. The reaction mixture was stirred under nitrogen for 5 hours until TLC showed that starting material 21-1 had reacted completely. The system was diluted with water (13 mL), and the methanol was concentrated. The pH was adjusted to 3 with 4 mol / L hydrochloric acid under an ice-water bath. The aqueous phase was extracted with ethyl acetate (150 mL), and the organic phase was washed with saturated sodium chloride (100 mL). After standing and separation, the upper organic layer was collected. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and concentrated under reduced pressure to give a light green oil (3.00 g, yield 56.1%).

[0130] LC-MS (ESI) + )m / z 395.2(M+H) + .

[0131] 1H NMR (400MHz, DMSO-d6) δ13.31(s,1H),7.89(d,J=7.5Hz,2H),7.69(d,J=7.5Hz,2H),7.42(t,J=7.6Hz,2H),7.32(q,J=7.5Hz,3H ), 4.26 (dd, J = 30.9, 7.0Hz, 3H), 4.09 (t, J = 6.7Hz, 1H), 2.98 (q, J = 6.6Hz, 2H), 1.68 (dp, J = 39.8, 6.9Hz, 2H), 1.50-1.11 (m, 4H).

[0132] Synthesis of 21-3

[0133]

[0134] Compound 11-5 (1.50 g, 2.15 mmol) was added to N,N-dimethylformamide (30 mL) and stirred at room temperature. Compound 21-2 (1.02 g, 2.58 mmol), triethylamine (0.51 mL, 3.65 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.06 g, 2.79 mmol) were added to the reaction vessel. The reaction mixture was stirred under nitrogen for 1 hour until TLC showed complete reaction of the starting materials. The reaction mixture was added to water (300 mL), and a yellow solid gradually precipitated and was filtered. The filter cake was washed with water and dried to give a yellow solid (2.3 g, 99.6% yield).

[0135] LC-MS (ESI) + m / z 1074.9(M+H) + .

[0136] 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.31(d,J=6.7Hz,1H),8.19(d,J=8.7Hz,1H),7.84(dd,J=17.1,9.9Hz,3H),7.67(d, J=7.4Hz,2H),7.60(d,J=8.1Hz,2H),7.39(dd,J=8.4,5.2Hz,4H),7.36-7.25(m,4H),6.52(s,1H),5.42(s,2H),5.13(d,J=3 5.6Hz,4H),4.51-4.11(m,5H),3.86(t,J=7.1Hz,1H),3.02(dd,J=40.9,6.3Hz,6H),1.93(dddd,J=42.9,28.0,13.8,6.9Hz ,5H),1.66(q,J=7.9Hz,2H),1.42(p,J=7.2Hz,2H),1.31(d,J=6.8Hz,3H),1.27-1.03(m,2H),0.87(dd,J=18.5,7.0Hz,9H).

[0137] Synthesis of 21-4

[0138]

[0139] Compound 21-3 (0.62 g, 0.58 mmol) was added to N,N-dimethylformamide (12 mL) and stirred at room temperature. Diethylamine (89.21 μL, 0.87 mmol) was added to the reaction vessel. The reaction mixture was stirred under nitrogen for 5 h until TLC showed complete reaction of the starting material. 226.74 μL (0.58 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.26 g, 0.69 mmol), and N,N-diisopropylethylamine (0.15 mL, 0.87 mmol) were added to a reaction vessel. The reaction mixture was stirred under nitrogen for 1 hour until TLC showed complete reaction of the starting materials. The reaction mixture was then added to water (200 mL), and a yellow solid gradually precipitated. The mixture was filtered, the filter cake was washed with water, and dried to give a yellow solid (0.67 g, yield 57.8%).

[0140] LC-MS (ESI) + )m / z 1246.9(M+H) + .

[0141] 1H NMR (400MHz, CD3OD) δ7.51(d,J=7.7Hz,2H),7.37-7.10(m,4H),5.45(d,J=29.2Hz,1H),5.38(s,1H) ,5.23(s,1H),5.19(s,1H),4.77(s,1H),4.53(dd,J=24.2,15.1Hz,2H),4.29(d,J=7.2Hz,1H),3.92( t,J=6.8Hz,1H),3.61(s,30H),3.34(s,3H),3.17(t,J=6.7Hz,2H),2.79(d,J=27.1Hz,4H),2.43(t, J=6.1Hz,2H),2.25-1.67(m,7H),1.48(dq,J=20.7,13.6,10.4Hz,7H),0.97(dt,J=14.6,7.1Hz,9H).

[0142] Synthesis of 21-5

[0143]

[0144] Compound 21-4 (0.6 g, 0.48 mmol) was added to a phosphate buffer solution (pH = 6) (12 mL) and stirred in an ice bath. Trimethylphosphine (1 mol / L tetrahydrofuran solution) (2.41 mL) was added to the reaction vessel. The reaction mixture was stirred in an ice bath for 2 hours until TLC showed complete reaction of the starting material. The reaction mixture was dissolved in methanol (20 mL) and purified by reverse preparative purification. Purification method: Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. The gradient range was 20%–95% of mobile phase B. The solvent was concentrated under reduced pressure to give a white solid (0.28 g, yield 47.7%).

[0145] LC-MS (ESI) + )m / z 1221.2(M+H) + .

[0146] 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.32(d,J=25.7Hz,2H),8.14(d,J=8.6Hz,1H),7.83( d,J=12.8Hz,2H),7.60(d,J=8.1Hz,2H),7.47-7.10(m,3H),5.42(s,2H),5.15(d,J=39.1Hz ,4H),4.38(q,J=7.2Hz,1H),4.25(d,J=6.8Hz,1H),3.69-3.30(m,31H),3.23(s,3H),3.04( dt,J=34.9,6.2Hz,6H),2.30(q,J=8.4,6.6Hz,2H),2.12-1.16(m,14H),1.05-0.68(m,9H).

[0147] XY-21 synthesis

[0148]

[0149] Compound 21-5 (0.10 g, 81.94 μmol) was added to N,N-dimethylformamide (1 mL) and acetonitrile (2 mL) and stirred at room temperature. 6-(maleimide)hexanoic acid succinimide ester (75.78 mg, 0.25 mmol) and triethylamine (34.17 μL, 0.25 mmol) were added to the reaction vessel. The reaction mixture was stirred at 0 °C for 0.5 h until HPLC showed complete reaction of the starting material. The mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse column chromatography. Purification method: Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. The gradient range was 10–50% B. Concentration of the solvent under reduced pressure gave a white solid (62 mg, yield 53.5%).

[0150] LC-MS (ESI) + )m / z 1414.1(M+H) + .

[0151] 1H NMR (400MHz, DMSO-d6) δ10.02(s,1H),8.18(d,J=6.6Hz,1H),7.96(d,J=7.9Hz,1H), 7.80(h,J=4.2Hz,2H),7.63(dd,J=21.5,8.4Hz,3H),7.45-7.22(m,3H),7.00(s,2H), 6.52(s,1H),5.57-4.83(m,6H),4.44-4.13(m,3H),3.61-3.34(m,32H),3.23(d,J=3 .0Hz,3H),3.14-2.86(m,6H),2.37-1.00(m,24H),0.85(dq,J=24.3,9.0,8.1Hz,9H).

[0152] Example 3 (Preparation of XY-22)

[0153]

[0154]

[0155] Synthesis of 22-1

[0156]

[0157] Compound 21-3 (1.00 g, 0.93 mmol) was added to N,N-dimethylformamide (20 mL) and stirred at room temperature. Diethylamine (145.90 μL) was added to the reaction vessel. The reaction mixture was stirred under nitrogen for 5 hours until TLC showed complete reaction of the starting material. Acetylated-10 polysarcosine (Ac-PSar10-OH, 0.72 g, 0.93 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.71 g, 1.86 mmol), and N,N-diisopropylethylamine (0.32 mL, 1.86 mmol) were added to the reaction vessel. The reaction mixture was stirred under nitrogen for 1 hour until TLC showed complete reaction of the starting material. The reaction mixture was added dropwise to water (200 mL), and a yellow solid gradually precipitated out. The mixture was filtered, the filter cake was washed with water, and dried to obtain a yellow solid (1.28 g, yield 85.9%).

[0158] LC-MS (ESI) + )m / z 1605.5(M+H) + .

[0159] 1H NMR (400MHz, DMSO-d6) δ10.06(s,1H),8.34(d,J=6.5Hz,1H),8.19(d,J=8.4Hz,1 H),7.82(t,J=15.1Hz,2H),7.60(d,J=8.0Hz,2H),7.39(d,J=8.1Hz,2H),7.28(s ,1H),6.53(s,1H),5.42(s,2H),5.12(d,J=28.5Hz,4H),4.52-3.79(m,23H),3.2 5-2.67(m,36H),2.51(s,6H),2.19-1.16(m,17H),0.87(dd,J=19.1,7.4Hz,9H).

[0160] Synthesis of 22-2

[0161]

[0162] Compound 22-1 (0.5 g, 0.31 mmol) was added to a phosphate buffer solution (pH = 6) (10 mL) and stirred in an ice bath. Trimethylphosphine (1 mol / L tetrahydrofuran solution) (1.56 mL) was added to the reaction vessel, and the reaction mixture was stirred in an ice bath for 2 hours until TLC showed complete reaction of the starting material. The reaction mixture was dissolved in 20 mL of methanol. Purification was performed using a reverse preparative method. The purification method was as follows: mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. The gradient range was 10–80% B. The solvent was concentrated under reduced pressure to give a white solid (0.15 g, yield 30.5%).

[0163] LC-MS(ESI+).m / z 1579.2(M+H)+.

[0164] 1 H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.42-8.25(m,2H),8.10(s,1H),7.84(d,J=12.2Hz,1H),7.60(d,J=8.0Hz,2H),7.39(d, J=8.1Hz,2H),7.29(s,1H),5.59-4.90(m,6H),4.51-3.81(m,22H),3.29-2.57(m,37H),2.07-1.18(m,17H),0.99-0.71(m,9H).

[0165] XY-22 synthesis

[0166]

[0167] Compound 22-2 (0.10 g, 63.34 μmol) was added to N,N-dimethylformamide (1 mL) and acetonitrile (2 mL) and stirred at room temperature. 6-(maleimide)hexanoic acid succinimide ester (58.58 mg, 0.19 mmol) and triethylamine (26.41 μL, 0.19 mmol) were added to the reaction vessel at 0 °C. The reaction mixture was stirred under nitrogen atmosphere for 30 minutes until HPLC showed complete reaction of the starting material. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse preparative method. Purification method: Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. The gradient range was 10–50% B. Concentration of the solvent under reduced pressure gave a white solid (52 mg, yield 46.3%).

[0168] LC-MS (ESI) + m / z 1772.60(M+H) + .

[0169] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),8.29(d,J=49.7Hz,2H),8.09-7.91(m,1H),7.82(h,J=7.6,7.1Hz,1H),7.62(dd,J=19.3,12.9Hz,3H),7 .50-7.18(m,3H),6.99(s,2H),5.66-4.85(m,6H),4.50-3.85(m,23H), 3.18-2.66(m,38H),2.18-1.08(m,25H),0.84(tt,J=20.0,9.9Hz,9H).

[0170] Experimental Example (Preparation of Compound (2))

[0171] A general method for ADC preparation (total reduction, drug-to-antibody ratio of 8):

[0172] The antibody was transferred to pH 7.4 10mM phosphate buffer using an ultrafiltration centrifuge tube. 10-15 times the molar amount of tris(2-hydroxyethyl)phosphonic acid hydrochloride (TCEP) was added. TCEP was prepared as a 10mM stock solution using 10mM sodium phosphate buffer at pH 7.4 (TCEP manufacturer: Sigma, catalog number C4706-2G, CAS. 51805-45-9). The reaction was carried out at 37°C for 1 hour to open the interchain disulfide bonds of the antibody. After reduction, the antibody was transferred to pH 7.4 10mM phosphate buffer using a 3KD ultrafiltration centrifuge tube to remove excess TCEP. 12-20 times the molar amount of Linker-Payload (the compounds of formula (1) prepared in Examples 1-3 and compound MC-GGFG-Dxd) was added. The Linker-Payload was dissolved in dimethyl sulfoxide (DMSO) to 5mM and reacted at room temperature for 2 hours. After the reaction was complete, cysteine ​​was added in quantities twice the molar number of Linker-Drug to quench the reaction. The mixture was then centrifuged at 8000g at 2–8°C using 30KD ultrafiltration centrifuge tubes (Merck, catalog number UFC9030) and the solution was changed to pH 5.5 acetate buffer to reduce residual small molecules to 0.1% of the reaction concentration. The antibody-drug conjugate sample after the buffer change was filtered through a 0.22-micron sterile filter, the concentration was determined, and the sample was aliquoted and frozen.

[0173] Comparative Example 1: Preparation of Antibody-Conjugate 1 (Control ADC)

[0174] Following the general method described above, a 12-fold excess of the reference standard Deruxtecan (i.e., the linker drug compound MC-GGFG-Dxd, synthesized using the method described in WO2019044947A1) was conjugated with reduced ifinatamab monoclonal antibody (refer to US9808537) to obtain the corresponding antibody-conjugate 1. The content of the polymer was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical chromatogram is shown in [reference missing]. Figure 1 The proportion of aggregates was approximately 3.03%. The drug loading (DAR) obtained by mass spectrometry analysis was approximately 8.

[0175] Experimental Example 1: Preparation of Antibody Conjugate 2

[0176] Following the general method described above, a 12-fold excess of compound (XY-11) was conjugated with the reduced ifinatamab monoclonal antibody to obtain the corresponding antibody-conjugate 2. The content of the polymer was analyzed by size exclusion chromatography-HPLC, and the analytical chromatogram is shown below. Figure 2 The proportion of aggregates was approximately 1.4%. The drug loading (DAR) obtained by mass spectrometry analysis was approximately 8.

[0177] Experimental Example 2: Preparation of Antibody Conjugate 3

[0178] Following the general method described above, a 12-fold excess of compound (XY-21) was conjugated with reduced ifinatamab monoclonal antibody to obtain the corresponding antibody conjugate 3. The content of the polymer was analyzed by size exclusion chromatography-HPLC, and the analytical chromatogram is shown in the figure. Figure 3 The proportion of aggregates was approximately 2.80%. The drug loading (DAR) obtained by mass spectrometry analysis was approximately 8.

[0179] Experimental Example 3: Preparation of Antibody Conjugate 4

[0180] Following the general method described above, a 12-fold excess of compound (XY-22) was conjugated with the reduced ifinatamab monoclonal antibody to obtain the corresponding antibody conjugate 4. The content of the polymer was analyzed by size exclusion chromatography-HPLC, and the analytical chromatogram is shown in the figure. Figure 4 The proportion of aggregates was approximately 1.77%. The drug loading (DAR) obtained by mass spectrometry analysis was approximately 8.

[0181] Conjugated antibody sequence

[0182] The heavy chain amino acid sequence of Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER2), is as follows: SEQ ID NO:1

[0183] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0184] The light chain amino acid sequence of Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER2), is as follows: SEQ ID NO:2

[0185] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0186] The heavy chain amino acid sequence of the humanized monoclonal antibody ifinatamab against the immunomodulatory protein B7-CD28 (B7H3), SEQ ID NO:3, is as follows:

[0187] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLFPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0188] The light chain amino acid sequence of the humanized monoclonal antibody ifinatamab against the immunomodulatory protein B7-CD28 (B7H3), SEQ ID NO:4, is as follows:

[0189] SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0190] The heavy chain amino acid sequence of the humanized monoclonal antibody Anetumab against human cortisol (MSLN), SEQ ID NO:5, is as follows:

[0191] QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0192] The light chain amino acid sequence of the humanized monoclonal antibody Anetumab against human cortisol (MSLN), SEQ ID NO:6, is as follows:

[0193] DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0194] Chromatographic analysis of antibody-drug conjugates

[0195] The antibody-drug conjugate was detected by SEC-HPLC molecular sieve method using a TSKgel G3000SWXL (TOSOH, catalog number 0008541) column. Approximately 0.3 mg of the sample was centrifuged at 8000g for 5 minutes in a 300 μL volume, and the supernatant was collected. The mobile phase was 50 mM sodium phosphate + 0.1 M sodium chloride, pH 6.8. The column was connected to a Shimadzu LC-20-AT HPLC system at a flow rate of 0.6 mL / min and a column temperature of 30 °C. After rinsing with the mobile phase for at least 30 minutes until the UV baseline at 280 nm stabilized, 100 μg of sample was injected, followed by rinsing with the mobile phase for 30 minutes. Detection wavelengths were 280 nm and 365 nm. The percentage of each peak (polymer, monomer, and low molecular weight) was calculated based on peak area. The monomer concentration was used as the sample purity value. The results are shown in Table 1. Figures 1-4 As shown.

[0196] Table 1: Size Exclusion Chromatography (SEC) of Each Experimental Example and Comparative Example

[0197]

[0198] Summary: From Table 1 and Figures 1-4 It is known that ifinatamab antibody (refer to US9808537), a humanized monoclonal antibody against the immunomodulatory protein B7-CD28 (B7H3), was used as a model antibody to conjugate with the designed linker drug compound and the control linker drug compound to prepare antibody-drug conjugates.

[0199] Experimental Example 5 (Drug-Antibody Ratio (DAR))

[0200] Sample processing:

[0201] The antibody conjugate 4 prepared in Example 4 was diluted to 1 mg / mL with ultrapure water. 2 μL of PNGase and 2 μL of CpB were added to every 100 μg of antibody conjugate 4, and the mixture was incubated in a water bath at 37°C for 2 h.

[0202] LC-MS parameters:

[0203] Mobile phase A consisted of 0.01% FA and 100% aqueous solution, while mobile phase B consisted of 0.01% FA and 100% acetonitrile solution. The separation gradient is shown in Table 2 below.

[0204] Table 2:

[0205]

[0206]

[0207] Flow rate: 0.25 mL / min. Column: BEH C4 Column (1.7μm, 2.1×150mm), column temperature 70℃.

[0208] The mass spectrometer was Themo Q Exactive Plus, using positive ion mode Full Scan, with a spray voltage of 3.8 kV and a scan range of 1600-3600 m / z.

[0209] The results are shown in Table 3 and Figure 5 As shown.

[0210] Table 3: Theoretical and detectable molecular weights of the main components of antibody conjugates at 4LC / MS

[0211] Components Theoretical molecular weight (Da) Measured molecular weight (Da) LC+1*XY-22 25185.85 25187.81 HC+3*XY-22(G0F) 54028.52 53903.52 HC+3*XY-22(G1F) 55799.36 55675.31

[0212] Summary: From Table 3 and Figure 5 It is known that because antibody conjugate 4 uses cysteine ​​coupling, it underwent a pretreatment of reduced-state glycosylation. The results showed that the light chain (LC) and heavy chain (HC) each had a major component. Mass spectrometry molecular weight analysis identified the main coupling form of the light chain as one XY-22 molecule and the heavy chain as three XY-22 molecules. The deconvolution results are shown below. Figure 5 As shown, the results are consistent with the theoretical molecular weight. Furthermore, partial ring-opening of the XY-22 molecule and heteromorphs with varying numbers of heavy chain couplings were also discovered. The DAR value of antibody-drug conjugate 4 was calculated to be 8.04 using the relative intensity normalization method of the liquid chromatography spectrum, which is comparable to the theoretical drug-antibody ratio of 8.

[0213] Experiment Example 6 (Accelerated Stability Experiment: Stability Test in PBS at 55°C)

[0214] The molecular samples to be tested were diluted with PBS 7.2 to a concentration of 0.5 mg / mL. 35 μL of the sample was injected directly or incubated at 55°C for 1 h, 2.5 h, 5 h, 8 h, 12 h, 24 h, and 48 h, respectively. The samples were then analyzed by SEC-HPLC molecular sieve method. The results are shown in Table 4. Figure 6 As shown in Table 4, the data represent the purity of the remaining compounds as determined by SEC liquid chromatography.

[0215] Table 4: Stability test of antibody conjugates 1-4 in PBS at 55°C

[0216] Time Antibody-conjugate 1 Antibody-conjugate 2 Antibody-conjugate 3 Antibody-conjugate 4 h ％ ％ ％ ％ 0 0 0 0 0 1 8.745 13.226 9.033 1.773 2.5 16.611 20.468 11.476 6.467 5 26.693 30.909 20.746 11.38 8 37.473 38.805 28.499 14.171 12 44.865 48.446 36.461 18.781 24 63.05 63.558 58.112 32.484 48 71.325 77.139 73.652 45.244

[0217] Summary: Table 4 shows that the stability of antibody-drug conjugates in PBS at 55 degrees Celsius is: antibody-drug conjugate 4 > 1 > 3 > 2. The results show that antibody-drug conjugate 4 of the candidate compounds has better stability.

[0218] Experimental Example 7 (In vitro tumor cell killing activity)

[0219] SHP-77 cell line, a human small cell lung cancer cell line

[0220] Human small cell lung cancer cell line SHP-77 cells were cultured in RPMI 1640 medium containing 10% FBS in a 37℃, 5% CO2 incubator in a semi-adherent, semi-suspension manner. When the cell confluence was greater than 80%, the cells were passaged at a ratio of 1:3 to 1:5, and passaged every 3 to 4 days.

[0221] Adherent SHP-77 cells were digested with 0.25% trypsin-EDTA digestion solution and the cells were resuspended. After counting, an appropriate amount of cells was resuspended in RPMI 1640 + 10% FBS and the cell density was adjusted to 1.875 × 10⁻⁶ cells / year. 4 Cells / mL were seeded at 80 μL / well into 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. The next day, ADC and small molecule toxin were serially diluted 3-fold starting from 5 μM (final concentration 1 μM), for a total of 10 concentration points; a negative control with a sample concentration of 0 and a blank control with only culture medium and no cells were also set up. The diluted sample was added at 100 μL / well (double replicates) to 96-well cell culture plates containing cells and incubated at 37°C with 5% CO2 for 120 hours. 25 μL of CCL assay reagent (VKEY-Bio) was added to each well, centrifuged at 200g for 1 minute, and incubated at 37°C for 10 minutes. Readings were performed using a multi-plate reader with the Lum program. The cell viability of the negative control was set at 100%, and the cell viability (%) of the experimental wells was calculated using the following formula.

[0222] Cell viability (%) = (Lum value of experimental wells - Lum value of blank wells) / (Lum value of negative control wells - Lum value of blank control wells) × 100%

[0223] The sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) was used for nonlinear regression to calculate the corresponding IC50 (Half-Maximal Inhibitory Concentration). The results are shown in Table 5.

[0224] Table 5: Half-maximal effective concentrations (IC50) of 3101 antibody-drug conjugate and control antibody-drug conjugate in SHP-77 cells for in vitro cell killing.

[0225] Test sample IC50 value (nM) Antibody-drug conjugate 1 (control ADC) 124.5 Antibody-conjugate 2 186.6 Antibody-conjugate 3 32.17 Antibody-conjugate 4 39.74

[0226] In summary, Table 5 shows that all ifinatamab antibody-drug conjugates exhibited specific killing of SHP-77, with IC50 values ​​ranging from 32.17 to 186.6 nM. The IC50 values ​​(32.17–39.74 nM) of antibody-drug conjugates 3 and 4, containing branched polysarcosine (PSAR) and polyethylene glycol (PEG) hydrophilic side chains, were superior to those of branched antibody-drug conjugates 1 and 2 (124.5–186.6 nM).

[0227] Experimental Example 8 (Therapeutic Effects on Tumors in Vivo)

[0228] SHP-77 cell line, a human small cell lung cancer cell line, is used to create a mouse xenograft tumor model.

[0229] M-NSG mice, female, 7-8 weeks old, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. All experimental animals were housed in individually ventilated, temperature- and humidity-controlled chambers, three mice per chamber, with alternating light and shadow for 12 hours. Animals had free access to water and food. SHP-77 cells were purchased from Zhejiang Meisen and cultured in RPMI-1640 medium containing 10% fetal bovine serum. SHP-77 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. The SHP-77 cells were identified by STR testing. 3 × 10⁶ cells were subcutaneously inoculated into the right back of each mouse. 6 SHP-77 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.10 ml / cell). Tumor growth was observed regularly until the tumor reached an average volume of approximately 141 mm². 3 Mice were randomly divided into groups of 5 mice each, based on tumor size and body weight.

[0230] Following tumor cell inoculation, routine monitoring included monitoring tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the experimental animals' activity levels, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight was measured 2-3 times per week, and tumor volume (TV, mm) was calculated. 3 ), and plot the tumor growth curve.

[0231] Formula for calculating tumor size: Tumor volume (mm) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0232] Tumor inhibition rate (%) = (TV) 对照组 -TV 给药组 )×100 / TV 对照组

[0233] All experimental results are expressed as MEAN (arithmetic mean) ± SEM (mean standard error). Statistical analysis between different groups was performed at the optimal treatment point (usually after the last dose). Independent samples t-tests were used to compare the relative tumor volume between the treatment and control groups to determine if there was a significant difference. All data were analyzed using SPSS 28.0. p < 0.05 was considered statistically significant.

[0234] The treatment regimens of antibody-drug conjugates in the SHP-77 mouse subcutaneous tumor model are shown in Table 6, and the tumor growth curves corresponding to the tumor treatment effects are shown in Table 6. Figure 7-9 .

[0235] Table 6: Grouping, administration regimens, and tumor inhibition rates of SHP-77 mouse subcutaneous tumor models after drug treatment

[0236]

[0237] Notes: ip: intraperitoneal administration; NA: not applicable

[0238] Summary: From Table 6 and Figure 7-9 It can be seen that, under a single dose of 5 mg / kg, the tumor growth of the control antibody-drug conjugate 1 was slowed ( Figure 7 At the end of the experiment, the tumor inhibition rate (TGI) was 103.95% ( Figure 8 However, antibody-drug conjugates 3 and 4 showed superior efficacy in cancer treatment. Figure 9 The TGI values ​​were 104.90% and 106.09%, respectively. In the SHP-77 model, the tumor volume from smallest to largest was antibody-drug conjugate 4 > 3 > 1 > 2, indicating that antibody-drug conjugate 4 among the candidate compounds showed the best activity.

Claims

1. A compound represented by formula (1), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof: in, Q is selected from N3-, BrCH2-, ICH2-, in, This indicates that M is connected from this point; M is selected from one or more of C1-C6 straight-chain or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O). n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-, -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n - or -CH2O(CH2CH2O) n CH2-, where n is an integer from 2 to 12; L is independently selected from valine residues, D-valine residues, citrulline residues, phenylalanine residues, lysine residues, acetyllysine residues, leucine residues, glycine residues, alanine residues, asparagine residues, aspartic acid residues, and arginine residues. Where A is selected from j is an integer from 1 to 12, in In the diagram, * indicates a connection from that point to either M or another L. This indicates the connection from that point in equation (1) Or another L; m can be 0, 1, 2, or 3; D contains drug molecules.

2. The compound according to claim 1, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The D is selected from cytotoxins. The cytotoxin is selected from one or more of the following: microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, or RNA polymerase 2 inhibitors.

3. The compound according to claim 2, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The cytotoxin is selected from...

4. The compound according to claim 1, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The compound represented by formula (1) is selected from the following structures:

5. A compound represented by formula (2), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof: Ab contains antibody or antigen-binding fragments; Q is selected from one of the following structures: in, * indicates that Ab is connected from this point. This indicates that M is connected from this point; M is selected from one or more of C1-C6 straight-chain or branched alkylene groups, or polyethylene glycol groups, wherein the polyethylene glycol group is -(CH2CH2O). n -、-O(CH2CH2O) n -、-(CH2CH2O) n CH2-, -(CH2CH2O) n CH2CH2-、-CH2O(CH2CH2O) n - or -CH2O(CH2CH2O) n CH2-, where n is an integer from 2 to 12; L is independently selected from valine residues, D-valine residues, citrulline residues, phenylalanine residues, lysine residues, acetyllysine residues, leucine residues, glycine residues, alanine residues, asparagine residues, aspartic acid residues, and arginine residues. Where A is selected from j is an integer from 1 to 12, in In the diagram, * indicates a connection from that point to either M or another L. This indicates the connection from that point in equation (2). Or another L; m can be 0, 1, 2, or 3; D contains drug molecules; i is an integer from 1 to 16.

6. The compound according to claim 5, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The D is selected from cytotoxins. The cytotoxin is selected from one or more of the following: microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, or RNA polymerase 2 inhibitors.

7. The compound according to claim 6, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The cytotoxin is selected from...

8. The compound according to claim 5, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The compound is selected from the following structures: in, The definitions of Ab and i are the same as in claim 5.

9. The compound according to claim 5 or 8, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein... The Ab is selected from: anti-human folate receptor antibody, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-EGFR antibody, anti-Nectin-4 antibody, anti-c-MET antibody, anti-PD-L1 antibody, anti-Claudin18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-MSLN antibody, anti-FGFR antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, and antigen-binding fragments of the above antibodies.

10. A composition comprising the compound according to claim 1 or 5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof.

11. The use of the compound according to claim 1 or 5, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, in the preparation of a medicament for the treatment and / or prevention of tumors.

12. The use according to claim 11, wherein, The tumors were selected from those associated with the expression of targets selected from the following groups: human folate receptor, HER2, HER3, TROP2, B7-H3, EGFR, Nectin-4, c-MET, PD-L1, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD70, CD73, CD74, MSLN, FGFR, FGFR2, and FGFR3.

13. The use according to claim 12, wherein, Tumors associated with the expression of the target include tumors with high expression of the target and / or tumors that are positive for the target.

14. The use according to claim 11, wherein, The tumor is selected from metastatic, refractory, or recurrent lesions of solid tumors, hematologic malignancies, and cancers.

15. The use according to claim 11, wherein, The tumors are selected from: gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, head and neck cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, thymic cancer, mesothelioma, lymphoma, myeloma, and glioblastoma.

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