Bispecific antibody conjugate, preparation method therefor and use thereof

Abstract

A bispecific antibody conjugate, a preparation method therefor and a use thereof, in the field of biopharmaceuticals, specifically relating to a bispecific antibody molecule binding to EGFR and / or c-Met, a conjugate represented by formula I thereof, a method for preparing and using the conjugate, a drug conjugate comprising a bispecific antibody, and a related use thereof in treating cancer.

Description

A bispecific antibody conjugate, its preparation method and application

[0001] This application claims priority to Chinese Patent Application No. 2024115705757, filed November 5, 2024, and Chinese Patent Application No. 202511563862X, filed October 29, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biopharmaceuticals, and more specifically, to bispecific antibody molecules that bind EGFR and / or c-Met, and their conjugates with deuterated camptothecin compounds and their pharmaceutically acceptable salts, as well as methods for preparing and using these conjugates. This disclosure also relates to pharmaceutical conjugates comprising said bispecific antibodies and their related applications in the treatment of cancer. Background Technology

[0003] Epidermal growth factor receptor (EGFR) is a key factor controlling epithelial cell growth and survival. Its mediated signaling pathway regulates a range of normal cellular biological behaviors, such as growth, differentiation, and migration. EGFR is also a significant driver of tumorigenesis and development; most tumors exhibit overexpression or genetic alterations of EGFR and its ligands. Drugs that inhibit EGFR signaling pathway activation, including monoclonal antibodies and small molecule kinase inhibitors (EGFR-TKIs), have been widely used in the treatment of cancers such as colorectal cancer, pancreatic cancer, head and neck cancer, and non-small cell lung cancer (NSCLC). For most patients with EGFR-mutant NSCLC (excluding those with primary EGFR-TKI resistance), the current standard of care is EGFR-TKIs. However, EGFR-TKI treatment inevitably leads to resistance. Studies have shown that amplification or overexpression of cellular-mesenchymal epithelial transition factor (c-MET) has been well-established as a crucial mechanism for clinical resistance to EGFR-TKIs. Especially with the widespread use of the third-generation EGFR-TKI osimertinib, the number of c-MET-driven drug-resistant individuals is increasing.

[0004] c-MET can initiate downstream signaling pathways similar to EGFR; hepatocyte cytokine (HGF) is its only ligand, and the c-MET / HGF signaling axis plays an important role in regulating normal processes such as wound healing, tissue regeneration, and organ development. c-MET gene mutations or dysfunction are also driving factors in tumorigenesis and development. Numerous reports have shown that c-MET pathway abnormalities or dysregulation exist in various human cancers, including non-small cell lung cancer, colorectal cancer, gastric cancer, head and neck cancer, pancreatic cancer, renal cell carcinoma, and hepatocellular carcinoma, and c-MET overexpression is negatively correlated with prognosis and survival in these indications. Clinical practice indicates that c-MET pathway activation is associated with resistance to anti-EGFR targeted therapy. When EGFR signaling is inhibited, the c-MET signaling pathway can act as an alternative pathway to EGFR in drug resistance mechanisms, thereby affecting the efficacy of EGFR monoclonal antibodies and TKI drugs.

[0005] Simultaneous inhibition of the EGFR and c-MET signaling pathways may be an effective strategy to combat c-MET-driven resistance. In clinical practice, amivantamab / rybrevant, as a dual-targeting drug that simultaneously inhibits the EGFR and c-MET signaling pathways, has demonstrated its effectiveness in treating EGFR-mutant non-small cell lung cancer (NSCLC). Current approved use includes: patients with advanced NSCLC harboring EGFR exon 20 insertion mutations (patients with primary EGFR-TKI resistance); patients with EGFR-mutant NSCLC whose disease has progressed during or after osimertinib treatment, in combination with chemotherapy; and patients with EGFR-mutant NSCLC as first-line treatment in combination with the third-generation EGFR inhibitor lazertinib. However, for cancers where downstream gene alterations lead to persistent signal activation, such as Ras-mutant lung adenocarcinoma and colorectal cancer, the killing mechanism based on signal pathway blocking has limited effectiveness; the EGFR monoclonal antibody Cetuximab is ineffective in treating Ras-mutant colorectal cancer patients, and ervantuximab has only been clinically explored in Ras wild-type colorectal cancer patients. Antibody-drug conjugates (ADCs) exert their effects through the direct killing of cells by small molecule toxins, which may compensate for this deficiency.

[0006] ADCs combine the advantages of precise targeting by monoclonal antibodies and the efficient killing effect of a potent payload (cytotoxic drug), demonstrating enormous clinical therapeutic potential. Several ADC drugs have already been approved for marketing. In clinical practice, the EGFR and HER3 bispecific antibody ADC (BL-B01D1) has shown clear efficacy in non-small cell lung cancer, nasopharyngeal carcinoma, and esophageal cancer. The EGFR single-target ADC (MRG003) has also shown clear efficacy in nasopharyngeal carcinoma and head and neck cancer, and the c-MET single-target ADC (ABBV-400) has shown clear efficacy in non-small cell lung cancer and colorectal cancer. This indicates that EGFR and c-MET are two well-validated drug targets, and EGFR and c-MET-based bispecific antibody ADCs may provide more patients with more effective treatment options. Furthermore, due to the limitation of the maximum tolerated dose of small molecule cytotoxic drugs, the clinical dosage of ADCs is lower than that of monoclonal antibodies. At the conventionally lower dosage of ADCs, the target-related toxicities exhibited by EGFR and c-MET bispecific antibodies such as Amivantamab at higher doses may be mitigated. Summary of the Invention

[0007] In a first aspect, this disclosure provides compounds of Formula I or pharmaceutically acceptable salts thereof or stereoisomers thereof:

[0008] Wherein, Ab represents an anti-EGFR / c-MET bispecific antibody or antigen-binding fragment; L represents a linker connecting Ab and the warhead drug molecule; n is selected from 4-9;

[0009] R1 is selected from H, halogens, OH, SH, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy;

[0010] R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0011] Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, or 3;

[0012] R3 and R4 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl or C 1-4 Halogenated alkoxy groups;

[0013] Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3, 4;

[0014] X is selected from H, OH, HO-CH(R5)-(CH2). p -CO-NH- or -N(R6)(R7), where p is selected from 0, 1, or 2;

[0015] R5 is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl;

[0016] R6 is selected from H and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups;

[0017] R7 is selected from H or R8-S(O)2-;

[0018] R8 is selected from C 1-4 Alkyl groups; and

[0019] t is selected from 0, 1, 2, 3, 4, 5.

[0020] In this disclosure, it is stated that "R1 and R2 cyclize to -O-(CH2)". m "-O-" means that R1 and R2 are connected to form -O-(CH2). m -O-. This indicates that "R3 and R4 cyclize to -(CH2)". k -” refers to the connection of R3 and R4 to form -(CH2). k -

[0021] In any embodiment of the compound of formula I disclosed herein, R1 is C 1-4 Alkyl group, and R2 is a halogen.

[0022] In any embodiment of the compound of formula I disclosed herein, R1 is methyl and R2 is F.

[0023] In any embodiment of the compound of formula I disclosed herein, R1 is H and R2 is H.

[0024] In any embodiment of the compound of formula I disclosed herein, R1 and R2 are cyclized to -O-CH2-O-.

[0025] In any embodiment of the compound of formula I disclosed herein, R1 is NH2, and R2 is H or a halogen.

[0026] In any embodiment of the compound of formula I disclosed herein, R1 is NH2 and R2 is H or F.

[0027] In any embodiment of the compound of formula I disclosed herein, R3 is H and R4 is H.

[0028] In any embodiment of the compound of formula I disclosed herein, R3 is H and R4 is C. 1-4 alkyl.

[0029] In any embodiment of the compound of formula I disclosed herein, R3 is H and R4 is methyl.

[0030] In any embodiment of the compound of formula I disclosed herein, R3 and R4 are cyclized to -CH2-CH2-.

[0031] In any embodiment of the compound of formula I disclosed herein, X is HO-CH(R5)-(CH2). p -CO-NH-, where p is selected from 0, 1, and 2.

[0032] In any embodiment of the compound of formula I disclosed herein, X is selected from OH or NH. 2。

[0033] In any embodiment of the compound of formula I disclosed herein, X is H and t is 0.

[0034] In any embodiment of the compound of formula I disclosed herein, X is -N(R6)(R7).

[0035] In any embodiment of the compound of formula I disclosed herein, n is selected from 4.0-7.0 or 7.0-9.0. In one embodiment of the compound of formula I disclosed herein, n is selected from 5.0-7.0, or 5.0-6.0, or 5.3-5.9, or 5.5-5.8, or 5.6-5.8; in another embodiment of the compound of formula I disclosed herein, n is selected from 7.0-8.5, or 7.2-7.8, or 7.3-7.6, or 7.4-7.6.

[0036] In one embodiment of the compound of formula I disclosed herein, it is a compound of formula Ia or a pharmaceutically acceptable salt thereof or a stereoisomer thereof;

[0037] Wherein, R1, R2, R3, and R4 are as defined in any embodiment of the compound of formula I; X1 is selected from chemical bonds, -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; p is selected from 0, 1, 2; R5 is selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

[0038] In any embodiment of the compound of formula Ia disclosed herein, R1 is selected from methyl or methoxy; or R1 is methyl.

[0039] In any embodiment of the compound of formula Ia disclosed herein, R2 is selected from F or Cl; or R2 is F.

[0040] In any embodiment of the compound of formula Ia disclosed herein, R1 is methyl and R2 is F.

[0041] In any embodiment of the compound of formula Ia disclosed herein, R3 and R4 are cyclized to -(CH2). k - where k is 2.

[0042] In any embodiment of the compound of formula Ia disclosed herein, R3 is H; and R4 is H.

[0043] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; where p is selected from 0 or 1, and R5 is selected from H or C. 1-4 Alkyl or 3-6 membered cycloalkyl.

[0044] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; where p is selected from 0 or 1, and R5 is selected from H, methyl or cyclopropyl.

[0045] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-CO-, wherein the -CO- end is connected to -NH-; and R5 is selected from H or 3-6 membered cycloalkyl.

[0046] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-CO-, wherein the -CO- end is connected to -NH-; and R5 is selected from H or cyclopropyl.

[0047] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- end is connected to -NH-; and R5 is C 1-4 alkyl.

[0048] In any embodiment of the compound of formula Ia disclosed herein, X1 is -O-CH(R5)-CH2-CO-, wherein the -CO- end is connected to -NH-; and R5 is methyl.

[0049] In any embodiment of the compound of formula Ia disclosed herein, X1 is selected from the following groups: The -CO- end is connected to -NH.

[0050] In some embodiments of the compounds of formula Ia disclosed herein, the compounds of formula Ia are selected from compounds of formula Ia-1 or Ia-2 below, or pharmaceutically acceptable salts thereof or stereoisomers thereof;

[0051] Wherein, Ab, L, R1, R2, X1, and n are as defined in any embodiment of the compound of formula Ia.

[0052] In any embodiment of the compound of formula Ia disclosed herein, Selected from the following compound fragments:

[0053] in This indicates that the position is connected to the linker L via a chemical bond.

[0054] In one embodiment of the compound of formula I disclosed herein, the compound of formula I is a compound of formula Ib or a pharmaceutically acceptable salt thereof or a stereoisomer thereof;

[0055] Wherein, Ab, L, and n are as defined in any embodiment of the compound of formula I;

[0056] R1 is selected from H, OH, halogens, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy;

[0057] R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0058] Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, 3;

[0059] R3 and R4 are each independently selected from H and C. 1-4 Alkyl or C 1-4 Alkoxy;

[0060] Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3;

[0061] X2 is selected from O or -N(R6)-;

[0062] R6 is selected from H or C. 1-4 alkyl;

[0063] q is selected from 0, 1, 2, 3, and 4.

[0064] In any embodiment of the compound of formula Ib disclosed herein, R1 is selected from methyl and R2 is selected from H, Cl, and F.

[0065] In any embodiment of the compound of formula Ib disclosed herein, R1 and R2 are cyclized to -O-(CH2). m -O-, where m is selected from 1 or 2.

[0066] In any embodiment of the compound of formula Ib disclosed herein, both R3 and R4 are H.

[0067] In any embodiment of the compound of formula Ib disclosed herein, R3 is H and R4 is methyl.

[0068] In any embodiment of the compound of formula Ib disclosed herein, X2 is selected from -O- or -NH-.

[0069] In any embodiment of the compound of formula Ib disclosed herein, X2 is selected from -O- or -NH-, and q is 0, 1, 2, 3 or 4.

[0070] In any embodiment of the compound of formula Ib disclosed herein, X2 is selected from -O- or -NH-, q is 1, 2, 3 or 4; R3 and R4 are both H; R1 is selected from methyl and R2 is selected from H, Cl or F, or R1 and R2 are cyclized to -O-(CH2). m -O-, where m is selected from 1 or 2.

[0071] In some embodiments of the compound of formula Ib disclosed herein, the compound of formula Ib is a compound of formula Ib-1.

[0072] In compounds of formula Ib-1, Ab, L, n, X2, and q are as defined in any embodiment of compounds of formula Ib.

[0073] In any implementation of formula Ib, Selected from the following compound fragments;

[0074] in This indicates that the position is connected to the linker L via a chemical bond.

[0075] In one embodiment of the compound of formula I disclosed herein, the compound of formula I is a compound of formula Ic or a pharmaceutically acceptable salt thereof:

[0076] Wherein, X3 is selected from NH, O, S; Ab, L, n, R2, R3, R4 are as defined in any embodiment of the compound of Formula I of this disclosure.

[0077] In any embodiment of the compound of formula Ic disclosed herein, R2 is selected from H or a halogen, or R2 is selected from H or F.

[0078] In any embodiment of the compound of formula Ic disclosed herein, R3 is H and R4 is C. 1-4 Alkyl; or, R3 is selected from H and R4 is selected from methyl.

[0079] In any embodiment of the compound of formula Ic disclosed herein, R3 and R4 are cyclized to -(CH2). k -, k is selected from 1, 2, 3.

[0080] In any embodiment of the compound of formula Ic disclosed herein, R3 and R4 are cyclized to -(CH2)2-.

[0081] In any embodiment of the compound of formula Ic disclosed herein, X3 is selected from NH or O; or X3 is selected from NH, or X3 is selected from O;

[0082] In any embodiment of the compound of formula Ic disclosed herein, Selected from the following compound fragments:

[0083] In one embodiment of the compound of formula I disclosed herein, the compound of formula I is a compound of formula Id or a pharmaceutically acceptable salt thereof:

[0084] Wherein, R1, R2, R3, R4, R6, and R7 are as defined in any embodiment of the compound of formula I of this disclosure, and Ab, L, and n are as defined in any embodiment of the compound of formula I of this disclosure; r is selected from 0, 1, 2, and 3.

[0085] In any embodiment of the compound of formula Id disclosed herein, R1 and R2 are both H.

[0086] In any embodiment of the compound of formula Id disclosed herein, R3 and R4 are both H.

[0087] In any embodiment of the compound of formula Id disclosed herein, r is 1.

[0088] In any embodiment of the compound of formula Id disclosed herein, R6 is selected from methyl, ethyl, and isopropyl; or, R6 is isopropyl.

[0089] In any embodiment of the compound of formula Id disclosed herein, R7 is selected from H.

[0090] In any embodiment of the compound of formula Id disclosed herein, R7 is selected from R8-S(O)2-, wherein R8 is selected from methyl or ethyl; or, R8 is selected from methyl.

[0091] In any embodiment of the compound of formula Id disclosed herein, Selected from the following compound fragments:

[0092] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, which comprises two domains that specifically bind to the EGFR protein and two domains that specifically bind to the c-MET protein.

[0093] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, having two homologous heavy chains and two homologous light chains, the amino acid sequence of the heavy chains being shown in SEQ ID NO:1, and the amino acid sequence of the light chains being shown in SEQ ID NO:2.

[0094] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, having two homologous heavy chains and two homologous light chains, the amino acid sequence of the heavy chains being as shown in SEQ ID NO:1, and the amino acid sequence of the light chains being as shown in SEQ ID NO:2; and n is selected from 7.0-8.5 or 7.2-7.8 or 7.3-7.6, or 7.4-7.6.

[0095] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, which includes a domain that specifically binds to the EGFR protein and a domain that specifically binds to c-MET.

[0096] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, having two heterologous heavy chains and one light chain, wherein the amino acid sequence of one heavy chain is as shown in SEQ ID NO:3, the amino acid sequence of the other heavy chain is as shown in SEQ ID NO:4, and the amino acid sequence of the light chain is as shown in SEQ ID NO:5.

[0097] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, the Ab is an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment, having two heterologous heavy chains and one light chain, wherein the amino acid sequence of one heavy chain is as shown in SEQ ID NO:3, the amino acid sequence of the other heavy chain is as shown in SEQ ID NO:4, the amino acid sequence of the light chain is as shown in SEQ ID NO:5, and n is selected from 5.0-7.0, or 5.0-6.0, or 5.3-5.9, or 5.5-5.8, or 5.6-5.8.

[0098] In one embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, L is L1-L2-L3-L4, wherein one end of L1 is connected to Ab and one end of L4 is connected to the warhead drug molecule D.

[0099] L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to Ab:

[0100] L2 is selected from chemical bonds, -N(R) 10 )-CH2-CO-、 Furthermore, one end of the CO pin of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1-4 Halogenated alkyl groups;

[0101] L3 is selected from polypeptide residues consisting of 2-6 amino acids, and the C-terminus of the polypeptide residue is connected to L4;

[0102] L4 is selected from chemical bonds, -NH-CH2-, One end of the CH2 group is connected to the warhead drug molecule, and the -NH- end is connected to L3.

[0103] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, L is L1-L2-L3-L4, and L2 is a chemical bond.

[0104] In any embodiment of the compound of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic or Id disclosed herein, L is L1-L2-L3-L4, and L2 is -N(CH3)-CH2-CO-.

[0105] In any embodiment of the compounds of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, L is L1-L2-L3-L4, and L3 is selected from the following polypeptide residues: GFG, GGFG, GGGFG, GGVA, V-Cit, VA.

[0106] In any embodiment of the compounds of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, L is L1-L2-L3-L4, and L1 is selected from... L2 is selected from chemical bonds or -N(CH3)-CH2-CO-, L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH-.

[0107] In any embodiment of the compounds of formula I, Ia, Ia-1, Ia-2, Ib, Ib-1, Ic, or Id disclosed herein, L is selected from the following fragments:

[0108] In this context, "*" indicates the position where the linker connects to the warhead drug molecule, while the other end indicates the position where the linker connects to Ab.

[0109] In any embodiment of the compound of formula I disclosed herein, Selected from the following structural fragment:

[0110] In one embodiment of the compound of formula I disclosed herein, the compound of formula I is a compound selected from the following compounds or pharmaceutically acceptable salts thereof, or their stereoisomers:

[0111] Wherein, n has the same meaning as in Equation I; for example, n is selected from 4.0-7.0, 7.0-9.0, or n is selected from 5.0-7.0, 5.0-6.0, 5.3-5.9, 5.5-5.8, 5.6-5.8; or n is selected from 7.0-8.5, 7.2-7.8, 7.3-7.6, 7.4-7.6.

[0112] A second aspect of this disclosure provides pharmaceutical compositions comprising compounds of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, their stereoisomers or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

[0113] The third aspect of this disclosure provides the use of the compounds of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions thereof in the preparation of a medicament for treating tumors.

[0114] The fourth aspect of this disclosure provides a bispecific antibody or an antigen-binding fragment thereof, characterized in that the antibody or the antigen-binding fragment thereof can bind to EGFR and c-MET.

[0115] In one embodiment, this disclosure provides a bispecific antibody or its antigen-binding fragment comprising two domains specifically binding to the EGFR protein, i.e., capable of binding to the EGFR protein in a bivalent manner, and further comprising two domains specifically binding to the c-MET protein, i.e., capable of binding to the c-MET protein in a bivalent manner. This bispecific antibody or its antigen-binding fragment is defined as a 2+2 bispecific antibody-drug conjugate, and the resulting drug conjugate (bispecific antibody-drug conjugate) is defined as a 2+2 bispecific antibody-drug conjugate (2+2 bispecific antibody ADC).

[0116] In another embodiment, this disclosure provides a bispecific antibody or its antigen-binding fragment comprising a domain specifically binding to the EGFR protein (i.e., capable of binding to the EGFR protein monovalently) and a domain specifically binding to the c-MET protein (i.e., capable of binding to the c-MET protein monovalently). This bispecific antibody or its antigen-binding fragment is defined as a 1+1 bispecific antibody, and the resulting drug conjugate (bispecific antibody-drug conjugate) is defined as a 1+1 bispecific antibody-drug conjugate (1+1 bispecific antibody ADC).

[0117] In one embodiment, this disclosure provides a 2+2 bispecific antibody or an antigen-binding fragment thereof, comprising: first and second antigen-binding domains for bivalent binding of EGFR; and third and fourth antigen-binding domains for bivalent binding of c-Met. The 2+2 bispecific antibody consists of two homologous heavy chains and two homologous light chains. The heavy chain of the 2+2 bispecific antibody has the heavy chain sequence shown in SEQ ID NO:1, and the light chain of the 2+2 bispecific antibody has the sequence shown in SEQ ID NO:2. In a more preferred embodiment, the structure of the 2+2 bispecific antibody of this disclosure is constructed as follows: a human c-MET binding domain is linked to the C-terminus of the heavy chain of the full-length anti-human EGFR antibody via a linker peptide shown in SEQ ID NO:10, and an amino acid peptide with the sequence shown in SEQ ID NO:8 is added to the end of the c-MET binding domain to eliminate the risk of binding to a pre-existing anti-drug antibody. Preferably, to reduce the antibody's immune-related activities such as ADCC, the Fc segment undergoes amino acid substitutions (TM mutations) of L234F, L235E, and P331S.

[0118] In one embodiment, this disclosure also provides a 1+1 bispecific antibody or its antigen-binding fragment thereof, comprising: a first antigen-binding domain that monovalently binds to EGFR, and a second antigen-binding domain that monovalently binds to c-Met. The antigen-binding domain binding to c-Met comprises a Fab fragment, while the antigen-binding domain binding to EGFR comprises a heavy chain single-domain antibody fragment (VHH). The 1+1 bispecific antibody or its antigen-binding fragment thereof consists of two heterologous heavy chains and one light chain, wherein one heavy chain comprises a heavy chain variable region sequence and a CH1 sequence of the heavy chain containing the antigen-binding domain binding to c-Met, the other heavy chain comprises a VHH sequence binding to EGFR, and the light chain comprises a light chain variable region sequence and a CL sequence of the light chain containing the antigen-binding domain binding to c-Met. The heavy chain of the antibody containing the heavy chain variable region sequence and the CH1 sequence of the antigen-binding domain binding to c-Met employs a "knob" structure design, including amino acid substitutions at the S354C and T366W sites. The heavy chain containing the VHH domain that binds to EGFR employs a "hole" structure design, including amino acid substitutions at four sites: Y349C, T366S, L368A, and Y407V. To facilitate the removal of the "hole" structure dimer during purification, the "hole" heavy chain also undergoes H435R substitution. Preferably, to reduce the antibody's ADCC and other immune-related activities, the Fc segments of both heavy chains undergo amino acid substitutions (TM mutations) at L234F, L235E, and P331S. Preferably, the sequence of the "hole" heavy chain is shown in SEQ ID NO:3, the sequence of the "knob" heavy chain is shown in SEQ ID NO:4, and the light chain sequence is shown in SEQ ID NO:5.

[0119] In a preferred embodiment, this disclosure provides a bispecific antibody or its antigen-binding fragment capable of binding to EGFR and c-MET. The bispecific antibody or its antigen-binding fragment has two homologous heavy chains and two homologous light chains, wherein the amino acid sequence of the heavy chains is shown in SEQ ID NO:1 and the amino acid sequence of the light chains is shown in SEQ ID NO:2; or, the bispecific antibody or its antigen-binding fragment has two heterologous heavy chains and one light chain, wherein the amino acid sequence of one heavy chain is shown in SEQ ID NO:3, the amino acid sequence of the other heavy chain is shown in SEQ ID NO:4, and the amino acid sequence of the light chain is shown in SEQ ID NO:5.

[0120] The fifth aspect of this disclosure is an antibody-drug conjugate comprising a bispecific antibody or its antigen-binding fragment as described in any of the technical solutions of the fourth aspect of this disclosure.

[0121] The sixth aspect of this disclosure provides a method for treating tumors, comprising administering to a patient in need an effective amount of the compound of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, its stereoisomers or pharmaceutically acceptable salts thereof, or administering to a patient in need an effective amount of the pharmaceutical composition of the second aspect of this disclosure; or administering to a patient in need an effective amount of the bispecific antibody or its antigen-binding fragment of any of the technical solutions of the fourth aspect of this disclosure, or administering to a patient in need an effective amount of the antibody-drug conjugate of the fifth aspect of this disclosure.

[0122] The seventh aspect of this disclosure provides compounds of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, stereoisomers thereof, or pharmaceutically acceptable salts thereof for treating tumors, or pharmaceutical compositions of the second aspect of this disclosure, or bispecific antibodies or antigen-binding fragments thereof of the fourth aspect of this disclosure, or antibody-drug conjugates of the fifth aspect of this disclosure.

[0123] The eighth aspect of this disclosure provides the use of compounds of formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, their stereoisomers or pharmaceutically acceptable salts thereof, pharmaceutical compositions of the second aspect of this disclosure, or bispecific antibodies or antigen-binding fragments thereof of the fourth aspect of this disclosure, or antibody-drug conjugates of the fifth aspect of this disclosure in the treatment of tumors.

[0124] The loaded compounds (i.e., the warhead drug molecules) in the compounds of formulas I, Ia, Ib, Ic, Id, Ia-1, Ia-2, and Ib-1 of this disclosure exhibit better stability in plasma, shorter half-lives in vivo, and lower in vivo exposure levels. Therefore, the loaded compounds of this disclosure are more easily eliminated after detachment in vivo, resulting in lower toxicity. Furthermore, toxicity tests of this disclosure have demonstrated that the compounds exhibit lower in vivo and hematologic toxicity, exhibiting better safety.

[0125] Terms and Explanations

[0126] Unless otherwise stated, the terms used herein have their general meanings within the technical field. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0127] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0128] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0129] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within those ranges, such as 1, 2, 3, 4, 5, 6. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range.

[0130] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0131] The three-letter and single-letter codes for amino acids used in this article are as described in J. BIOL. CHEM, 243, P3558 (1968).

[0132] As used herein, the term "anti-EGFR / C-MET bispecific antibody" or "anti-EGFR and C-MET bispecific antibody" refers to an antibody that can specifically bind to anti-EGFR and / or C-MET antibodies, or fragments thereof, with sufficient affinity.

[0133] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH).

[0134] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0135] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinctly different types based on the amino acid sequence of their constant domains, termed κ and λ.

[0136] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0137] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0138] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0139] As used herein, the broad category of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0140] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this document to refer to antibodies whose structure is substantially similar to that of natural antibodies or that contain the FC region.

[0141] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0142] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0143] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0144] It should be noted that the division of the CDR and FR in the variable region of the monoclonal antibody disclosed herein is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0145] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0146] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its epitope, or a fusion protein product further derived from such fragment, or a product conjugated with other compounds, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0147] The term "Fab" or "Fab fragment" includes the variable regions of the heavy chain and the light chain, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment. The term "F(ab')2 fragment" contains two Fab fragments and a hinge region, which is a bivalent antibody fragment.

[0148] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites.

[0149] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0150] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins together through genetic recombination or chemical methods. Linkers can be used for this linking, or not.

[0151] The term "multispecific antibody" refers to a novel antibody construct formed by functionally linking an antibody to one or more other binding molecules (e.g., chemical conjugation, gene fusion, non-covalent binding, or other methods) to bind to two or more different sites and / or targets. Among these, "bispecific antibody" is more commonly used, specifically referring to an antibody construct that is specific to two different antigens. Typically, bispecific or multispecific antibodies include at least two antigen-binding domains.

[0152] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, or intact extracellular domains (ECDs) or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0153] The term "epitope," also known as an "antigenic determinant," refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost after treatment with denaturing solvents. Epitopes typically consist of 3–15 amino acid residues. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0154] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by glycosylation, esterification, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0155] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "replacement" means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substitution of S32A means that the serine at position 32 is replaced by alanine.

[0156] The sequence identity or homology between the variable region of the humanized antibody and the variable region of the human receptor can be determined as discussed herein, and when such a determination is made, preferably at least 60% or 65% sequence identity will be shared, more preferably at least 70%, 75%, 80%, 85%, or 90% sequence identity, and even more preferably at least 93%, 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. A “conserved substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region or frame region of the disclosed antibody can be replaced with amino acid residues of other similar side chains. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution.

[0157] During monoclonal antibody production, various physicochemical factors can easily generate post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs can cause changes in the physicochemical properties of antibodies, alter their interaction with the antibody Fc receptor, and affect their binding activity to the target antigen. Some PTMs can even reduce antibody stability and induce immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTMs can be eliminated by modifying the amino acid sites, such as through conserved substitutions. Amino acid substitutions of antibody CDRs for the purpose of modifying PTMs are also explicitly kept within the scope of this disclosure.

[0158] The antibodies disclosed herein may also include substitutions or modifications to constant regions (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding to Fc receptors (FcRs), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0159] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0160] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0161] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a therapeutic active substance or active pharmaceutical ingredient (API), thereby enabling the therapeutic active substance or active pharmaceutical ingredient (API) to target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent linking of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC.

[0162] The term “pharmaceutical carrier” or “pharmaceuticalally acceptable carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or medium that is administered with a therapeutic agent.

[0163] The term "effective dose" refers to a dosage of a pharmaceutical formulation of the antibody or its antigen-binding fragment disclosed herein, which, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0164] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0165] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0166] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0167] The terms "isolated polynucleotide" or "isolated nucleic acid" refer to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain such polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location other than their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-stranded, negative-stranded, and double-stranded forms.

[0168] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0169] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and available in the prior art, such as in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. The antibodies or antigen-binding fragments described in this invention are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the journal Immunoglobulins, (2001) ISBN: 012441351.

[0170] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0171] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0172] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0173] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0174] As used herein, the term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. These therapies can be physical, such as radiation therapy, or chemical, such as administering a drug to the subject, including combination drugs. "Combination drugs" refers to a combination of two or more pharmaceutical preparations, each containing an active ingredient, that are administered to a subject in combination. The active ingredients may be mixed together to form a single dosing unit or may be administered separately as independent dosing units; during administration, the different pharmaceutical preparations may be administered substantially synchronously, simultaneously, or sequentially.

[0175] In any embodiment of the compound of formula I disclosed herein, when R1 and R2 are cyclized to -O-(CH2) m When -O-, the compound of formula I is the compound of formula I-1 as follows. In the compound of formula I disclosed herein, or in any embodiment thereof, when R3 and R4 are cyclized to -(CH2) k - When, it is presented in the form of I-2 as follows.

[0176] In the compound of formula I disclosed herein, This means that the linker L can be chemically bonded to any linkable site of the warhead drug molecule D in Formula I (in which case a hydrogen atom is removed from the linking site of the warhead drug molecule D), as long as it is chemically feasible and can yield a robust drug.

[0177] Those skilled in the art will understand that in Formula I, Ia, Ib, Ic, Id, Ia-1, Ia-2, Ib-1, or specific compounds thereof disclosed herein, the linker L or its corresponding specific structure is connected to Ab via a sulfur atom from Ab. This sulfur atom S may be embodied in the structural or chemical formula of L or L1, or it may be omitted, hidden, or not embodied. For example It can also be written as Both represent the same structure and meaning; It can also be written as It can be written as It can also be written as Other L1 segments can be understood in the same way.

[0178] The term "warhead drug molecule," also known as "load," "loaded drug," or simply "payload," refers to a substance with preventative or therapeutic effects against diseases. In antibody-drug conjugates, the drug typically refers to a cytotoxic drug, a chemical molecule that can strongly disrupt the normal growth of tumor cells.

[0179] The term "linker" refers to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being linked to a drug.

[0180] The term "drug-linker conjugate" is also called a load-linker conjugate or a linker-load conjugate, and has the same meaning in this disclosure.

[0181] The terms “optional” or “optionally” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, the term “optionally” means that the group may be unsubstituted or substituted with a specified substituent; whether substituted or not, it falls within the scope of “optionally”.

[0182] The terms "independently selected" or "each independently selected" mean that each substituent in the preceding term can choose different optional variables from a specified range of choices, unaffected by the selection results of other substituents. When a substituent that can be "independently selected" exists as multiple identical substituents in the general chemical formula, it can still choose the same or different optional variables.

[0183] The term "halogen" or "halogen atom" refers to fluorine, chlorine, bromine, and iodine.

[0184] The term "halogenated" refers to a group formed when one or more hydrogen atoms in a substituent are replaced by halogen atoms.

[0185] The term "alkyl" refers to a straight-chain or branched hydrocarbon group in which carbon atoms are linked by single bonds. Alkyl groups are preferably C14-245 ... 1-4 Or C 1-6 Alkyl group. C 1-4 "Alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3, or 4 carbon atoms. C 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. C 1-6 Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl.

[0186] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen atoms. 1-4 Examples of alkyl halogens include, but are not limited to, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, and 2,2,2-trichloroethyl.

[0187] The term "alkoxy" refers to an alkyl-O- group, wherein the alkyl group, as defined above, includes C0. 1-4 Alkoxy or C 1-6 Alkyl group. C 1-4 Alkoxy can be understood as "C 1-4 Alkyl-O-", examples of which include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and tert-butoxy. C 1-6 Alkoxy can be understood as "C 1-6 Alkyl-O-”, examples of which include the aforementioned C 1-4 In addition to specific examples of alkoxy groups, other examples include, but are not limited to, n-pentoxy, neopentoxy, and n-hexyloxy.

[0188] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by halogen atoms. 1-4 Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, and 2,2,2-trichloroethoxy.

[0189] The term "cycloalkyl" refers to a cyclic saturated monocyclic or polycyclic hydrocarbon group formed by single bonds between carbon atoms. The hydrogen atom of the ring carbon atom is optionally oxidized, i.e., the "-CH2-" in the ring is optionally oxidized to form "-C(O)-". 3- to 6-membered cycloalkyl refers to 3, 4, 5, or 6-membered cycloalkyl groups, specific examples of which include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0190] The term "heterocyclic alkyl" refers to a cycloalkyl group in which one or more (e.g., 2, 3, or 4) cyclic carbon atoms are replaced by heteroatoms or heteroatom groups (i.e., groups containing heteroatoms). The cyclic carbon atoms are the carbon atoms that form the cyclic framework; the heteroatoms are atoms in an organic compound other than C and H, such as nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), or boron (B), and the heteroatoms, such as nitrogen and sulfur atoms, can be oxidized, and the nitrogen atom can be quaternized; examples of heteroatom groups include, but are not limited to, -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, or -NHC(=O)NH-, etc. 3-6 membered heterocyclic alkyl refers to 3, 4, 5 or 6 membered heterocyclic alkyl, and specific examples include, but are not limited to, azirropropane, oxacyclopropane, thiohexanepropane, azirrobutyl, oxacyclobutyl, thiohexanebutyl, azirropentane, oxacyclopentane, thiohexanepentane, piperazinyl, piperidinyl, oxacyclohexyl, morpholinyl, and 1,4-dioxanecycloyl.

[0191] In this disclosure, the letters G, F, V, A, and Cit used to represent amino acids or amino acid residues represent glycine, phenylalanine, valine, alanine, and citrulline, respectively.

[0192] The term "composition" means a product comprising specified amounts of each of the specified ingredients, and any product derived directly or indirectly from a combination of the specified amounts of the specified ingredients. Those skilled in the art can vary the actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure so that the resulting amount of active compound is effectively targeted to a specific patient, composition, and route of administration to obtain the desired therapeutic response.

[0193] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0194] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0195] The term "excipient" generally refers to the carrier, diluent, and / or medium required to formulate an effective pharmaceutical composition.

[0196] In this disclosure, the substituents in This indicates the connection point of the substituent to the parent structure or other segments. A dash "-" in the substituent structure indicates the connection point of the substituent; for example, -CH3 indicates that the group is connected to the parent structure or other segments through a carbon atom. The absolute configuration representing the center of a solid, i.e., the R or S configuration.

[0197] In this disclosure, chemical bonds are depicted by solid and dashed lines. Whether a chemical bond is a single or double bond can be determined based on the valence of the atoms at both ends of the bond, within the range known to those skilled in the art.

[0198] In this disclosure, the term "isomer" includes geometric isomers and stereoisomers, such as blocked trans isomers, cis-trans isomers, enantiomers, diastereomers, tautomers, racemic mixtures thereof, and other mixtures, all of which are within the scope of this disclosure. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a functional group isomer that has different hydrogen bonding sites through one or more double bond shifts; for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers of molecules having two or more chiral centers and being non-mirror images of each other. The term "cis-trans isomer" refers to different spatial configurations of a molecule where double bonds or single bonds of cyclic carbon atoms cannot rotate freely. The term "blocked trans isomer" refers to a stereoisomer that can be separated due to impeded or very slow rotation of single bonds.

[0199] The stereoisomers of the compounds disclosed herein can be prepared by chiral synthesis or using chiral reagents or other conventional techniques. For example, an enantiomer of a compound disclosed herein can be prepared by asymmetric catalysis or chiral derivative derivatization. Alternatively, a single stereoisomer can be obtained from a mixture using chiral resolution techniques. Alternatively, it can be prepared directly from chiral starting materials. The separation of optically pure compounds in this disclosure is typically accomplished using preparative chromatography, employing chiral chromatographic columns to achieve the separation of chiral compounds.

[0200] In this disclosure, the unit of solution concentration M represents mol / L, mM represents mmol / L, and nM represents nmol / L. The unit of solution concentration N represents equivalent concentration, which is expressed as the number of gram equivalents of solute contained in 1 liter of solution, denoted by the symbol N. For example, if 1 liter of concentrated hydrochloric acid contains 12.0 gram equivalents of hydrochloric acid (HCl), then the concentration is 12.0N. Equivalent concentration = number of gram equivalents of solute / solution volume (liters). In in vivo drug activity experiments, the unit of administered dose mpk refers to mg / kg.

[0201] The chemical abbreviations used in this disclosure and the chemical names they refer to are as follows:

[0202] Table 1 Attached Figure Description

[0203] Figure 1. Schematic diagram of the structure of the 2+2 dual-antibody ADC;

[0204] Figure 2. Schematic diagram of the 1+1 dual-antibody ADC structure;

[0205] Figure 3. Inhibitory effect of bispecific antibody ADC on HCC-827 tumor cells;

[0206] Figure 4. Inhibitory effect of bispecific antibody ADC on EBC-1 tumor cells;

[0207] Figure 5. Inhibitory effect of bispecific antibody ADC on KYSE30 tumor cells;

[0208] Figure 6. Inhibitory effect of bispecific antibody ADC on normal immortalized skin cells HACAT cells;

[0209] Figure 7. IHC staining images of the four CDX models;

[0210] Figure 8. In vivo efficacy of bispecific antibody ADC 1 at 3mpk in the HCC827 CDX model;

[0211] Figure 9. In vivo efficacy of bispecific antibody ADC 1 at 3mpk in the HCC827 CDX model;

[0212] Figure 10. In vivo efficacy of bispecific antibody ADC at 2 mpk in HCC827 CDX model.

[0213] Figure 11. In vivo efficacy of bispecific antibody ADC in H1975 CDX;

[0214] Figure 12. In vivo efficacy of bispecific antibody ADC at 1 mpk in the FaDu CDX model;

[0215] Figure 13. In vivo efficacy of bispecific antibody ADC-1 at 3 mpk in the FaDu CDX model;

[0216] Figure 14. In vivo efficacy of bispecific antibody ADC at 1 mpk in the EBC-1CDX model. Detailed Implementation

[0217] The present disclosure is further described in detail below through specific preparation examples and biological experiments. However, it should be understood that these examples and biological experiments are for illustrative purposes only and should not be construed as limiting the present disclosure in any way. Those skilled in the art will understand that, unless otherwise specified, the materials used below are well-known in the art and can be obtained commercially or by those skilled in the art based on published literature or conventional methods. Unless otherwise stated, all reactions in this disclosure are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, using a dry solvent, wherein: (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature, generally 15-35°C, preferably 20-30°C, more preferably 20-25°C; (ii) solvent removal is performed using a rotary evaporator under reduced pressure, with a bath temperature not exceeding 60°C; (iii) the reaction process is monitored by thin-layer chromatography (TLC); (iv) the final product has a satisfactory proton nuclear magnetic resonance spectrum (NMR). 1 H-NMR and / or mass spectrometry (MS) data.

[0218] Example 1: Preparation of Compound 1

[0219] Step 1:

[0220] 86.4 mL of 2,2,6,6-tetramethylpiperidine was added to a 3 L three-necked flask, followed by 1 L of anhydrous tetrahydrofuran. The system was cooled to -78 °C and reacted for 15 min. Then, 272 mL of a 2.5 M n-butyllithium solution in petroleum ether was added, and the reaction was allowed to proceed for 30 min. 32 g of compound L-1 was dissolved in 200 mL of anhydrous tetrahydrofuran and added to the system, reacting for 1 h. 50 mL of 1-penten-3-one was dissolved in 200 mL of anhydrous tetrahydrofuran and added to the system, reacting for 1 h. After the reaction was complete as monitored by TLC, 640 mL of 4N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated sodium bicarbonate aqueous solution, then washed with saturated brine, and dried over anhydrous sodium sulfate. The system was concentrated under vacuum and purified by column chromatography to give 21 g of compound L-2 (yield: 49%).

[0221] ESI-MS m / z: 254.1, [M+H] + .

[0222] 1H NMR (400MHz, CDCl3) δ6.93 (s, 1H), 5.96 (dd, J = 17.2, 10.7Hz, 1H), 5.44 (d, J = 17.2Hz, 1H), 5.28 (d, J = 10. 8Hz, 1H), 4.15 (s, 3H), 2.10 (dq, J=14.7, 7.4Hz, 1H), 1.93 (dq, J=14.6, 7.3Hz, 1H), 0.85 (t, J=7.3Hz, 3H).

[0223] Step 2

[0224] 21 g of compound L-2 was added to a 1 L three-necked flask, followed by 400 mL of anhydrous ethanol. After cooling to 0 °C, 14.21 g of sodium borodeuteride was added, and the mixture was stirred for 15 minutes. The reaction was then brought to room temperature and allowed to proceed for 12 hours. The reaction was monitored by TLC until complete. 30 mL of 1N hydrochloric acid was added, and the mixture was extracted with dichloromethane. The combined organic phases were washed successively with saturated sodium bicarbonate solution, saturated brine, and dried over anhydrous sodium sulfate. After vacuum concentration, the mixture was purified by column chromatography to obtain 17 g of compound L-3 as a clear, viscous liquid (yield: 80%). ESI-MS m / z: 260.1, [M+H] + . 1 H NMR(400MHz, CDCl3) δ6.99(s,1H),6.13(dd,J=17.3,10.6Hz,1H),5.29(d,J=7.0Hz,1H),5.26(s,1H ),3.99(s,3H),2.73(s,2H),2.04(dt,J=14.8,7.3Hz,1H),1.98–1.87(m,1H),0.91(t,J=7.3Hz,3H).

[0225] Step 3:

[0226] 2 g of compound L-3 was added to a 250 mL round-bottom flask, followed by 100 mL of dichloromethane. The system was cooled to -78 °C, and ozone was bubbled through it. After the reaction was complete as monitored by TLC, 0.5 mL of dimethyl sulfide was added, and the mixture was stirred at room temperature for 30 minutes. The system was concentrated under vacuum and purified by column chromatography to obtain 1.9 g of compound L-4, yield: 95%. The product was used directly in the next reaction without further purification. ESI-MS m / z: 262.1, [M+H] + .

[0227] 1H NMR (400MHz, CDCl3) δ7.15 (s, 1H), 5.19 (d, J = 4.8Hz, 1H), 3.96 (s, 3H), 3.21 (d, J = 4.8Hz, 1H), 2.64 (s, 1H), 1.80 (q, J = 7.5Hz, 2H), 0.92 (t, J = 7.5Hz, 3H).

[0228] Step 4:

[0229] 12 g of compound L-4 was added to a 1 L single-necked flask, followed by 240 mL of dichloromethane. The mixture was stirred for 30 minutes, and then cooled to 0 °C. 286 mg of 2,2,6,6-tetramethylpiperidine oxide, 616 mg of sodium bicarbonate, 654 mg of potassium bromide, and 18 mL of water were added, and the mixture was stirred for 15 minutes. 120 mL of sodium hypochlorite aqueous solution (>7.5 wt%) was added, and the reaction was allowed to proceed for 30 minutes. The reaction was monitored by TLC until complete. 12 g of sodium bisulfite was added, and the mixture was extracted with dichloromethane. The organic phases were combined, washed with water and saturated brine, and dried over anhydrous sodium sulfate. After vacuum concentration, the mixture was purified by column chromatography to obtain 6.6 g of compound L-5, yield: 55%. ESI-MS m / z: 260.1, [M+H] + .

[0230] 1 H NMR (400MHz, CDCl3) δ7.20 (s, 1H), 4.00 (s, 3H), 3.64 (s, 1H), 1.79 (q, J = 7.4Hz, 2H), 0.97 (t, J = 7.4Hz, 3H).

[0231] Step 5:

[0232] Under a carbon monoxide atmosphere, 3 g of compound L-5 was added to a 100 mL three-necked flask, along with 286 mg of 1,3-bis(diphenylphosphine)propane, 2.4 g of potassium carbonate, 129 mg of palladium acetate, 15 mL of N,N-dimethylformamide, and 30 mL of deuterated methanol. The mixture was heated to 65 °C and reacted for 12 hours. The reaction was monitored by TLC until complete. 20 mL of 1N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The combined organic phases were washed successively with saturated sodium bicarbonate aqueous solution and saturated brine, and dried over anhydrous sodium sulfate. After vacuum concentration, the mixture was purified by column chromatography to give 1.33 g of compound L-6 (yield: 40%). ESI-MS m / z: 287.1, [M+H] + .

[0233] 1H NMR (400MHz, CDCl3) δ7.98 (d, J = 2.0 Hz, 1H), 4.09 (d, J = 2.0 Hz, 3H), 3.71 (d, J = 2.6 Hz, 1H), 1.88–1.75 (m, 2H), 0.96 (td, J = 7.3, 1.9 Hz, 3H).

[0234] Step 6:

[0235] 1 g of compound L-6 was added to a 50 mL double-necked flask, along with 1.05 g of sodium iodide and 10 mL of acetonitrile. The mixture was cooled to 0 °C and stirred for 30 minutes. Then, 0.89 mL of trimethylchlorosilane was added, and the mixture was heated to room temperature and reacted for 12 hours. The reaction was monitored by TLC until complete. 40 mL of water and 1 mL of saturated sodium bisulfite aqueous solution were added, and the mixture was stirred for 1 hour. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. After vacuum concentration, the mixture was purified by column chromatography to obtain 800 mg of compound L-7, yield: 84%. ESI-MS m / z: 273.1, [M+H] + .

[0236] 1 H NMR (400MHz, CDCl3) δ10.03 (s, 1H), 7.32 (s, 1H), 3.90 (s, 1H), 1.81 (dd, J = 14.5, 7.2Hz, 2H), 0.99 (t, J = 7.4Hz, 3H).

[0237] Step 7:

[0238] 620 mg of compound L-7 was added to a 25 mL double-necked flask, along with 1.48 g of cesium carbonate, 8 mL of dimethyl sulfoxide, and 1.67 mL of tert-butyl acrylate. The mixture was heated to 50 °C and reacted for 12 hours. The reaction was monitored by TLC until complete. 1 mL of concentrated hydrochloric acid was added, followed by 40 mL of water. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. After vacuum concentration, the mixture was subjected to preparative SPC chiral separation (instrument model: SFC-350 (Waters), column: AS25*250 mm, 10 μm, mobile phase: CO2 / MEOH = 65 / 35, flow rate: 200 mL / min) to obtain 468 mg of compound L-8, yield: 55%. ESI-MS m / z: 366.1, [M+H] + .

[0239] Step 8:

[0240] 200 mg of compound L-8 was added to a 10 mL single-necked flask, along with 5 mL of toluene and 0.5 mL of trifluoroacetic acid. The mixture was heated to 110 °C and reacted for 2 hours. The reaction was monitored by TLC until complete. After vacuum concentration, the mixture was purified by column chromatography to obtain 140 mg of compound L-9, yield: 98%. ESI-MS m / z: 266.1, [M+H] + .

[0241] Step 9:

[0242] 300 mg of compound L-9 was added to a 25 mL single-necked flask, along with 266 mg of N-(5-methyl-6-fluoro-8-amino-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide, 9 mL of toluene, 770 mL of o-cresol, and 133 mg of pyridine p-toluenesulfonic acid. The system was heated to 110 °C and reacted for 18 hours. The reaction was monitored by TLC until complete. After vacuum concentration, the system was purified by column chromatography (dichloromethane:methanol = 10:1) to give 443 mg of compound L-10, yield: 81%.

[0243] Step 10:

[0244] 443 mg of intermediate compound L-10 was added to a 25 mL single-necked flask, followed by 7 mL of purified water and 2.2 mL of methanesulfonic acid. The system was then purged under a nitrogen atmosphere. The mixture was heated to 85 °C and reacted for 10 hours. The reaction was monitored by TLC until complete. After vacuum concentration, the mixture was purified by column chromatography (dichloromethane:methanol) to give 141 mg of compound 1 or its formate sulfonate, yield: 35%. ESI-MS m / z: 438.2, [M+H] + .

[0245] 1 H NMR(400MHz,D2O)δ7.20–7.06(m,2H),5.42–5.14(m,5H),3.30(dd,J=18.1,4.0Hz,1H),3.06–2.91 (m,1H),2.69(s,2H),2.62–2.47(m,1H),2.18(s,3H),1.80(q,J=7.3Hz,2H),0.79(t,J=7.4Hz,3H).

[0246] Example 2 Preparation of Compound 2

[0247] To a mixture of anhydrous sodium sulfate (2.25 eq), ethyl 2-oxime cyanoacetate (2.25 eq), 2-hydroxyacetic acid (1.45 eq), and methanesulfonate of compound 1 (100 mg), 2.4 mL of water and 3.0 mL of tetrahydrofuran were added. After stirring at room temperature for 15 minutes, N-methylmorpholine (1.10 eq) was added, and the mixture was stirred at room temperature for 15 minutes. Then, EDCI (2 eq) was added, and the mixture was stirred at room temperature for 3 hours. The reaction of compound 1 was monitored by HPLC until complete. The mixture was filtered, the filter cake was dried, and then purified by silica gel column chromatography (DCM:MeOH) to obtain compound 2.

[0248] 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.9Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.52(s,1H),5.58(d,J=5.4Hz,1H),5.49(t,J=5.7Hz,1H),5.20(t ,J=13.9Hz,2H),3.96(d,J=5.5Hz,2H),3.28–3.05(m,2H),2.39(s,3H),2.2 9–2.11(m,2H),1.96–1.78(m,2H),0.87(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :496.1.

[0249] Example 3 Preparation of Compound 3

[0250] Following the preparation method of Example 2, 52 mg of compound 3 was prepared from (R)-2-cyclopropyl-2-hydroxyacetic acid, with a yield of 52%.

[0251] 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=8.4Hz,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H),6.5 2(s,1H),5.58–5.48(m,1H),5.38(d,J=5.3Hz,1H),5.21(q,J=19.0Hz,2H),3.62(d,J=5. 2Hz,1H),3.16(dd,J=11.2,5.7Hz,2H),2.39(s,3H),2.22–2.12(m,2H),1.86(dt,J=14.3 ,7.1Hz,2H),1.14(q,J=7.3Hz,1H),0.87(t,J=7.2Hz,3H),0.38(dd,J=20.9,5.6Hz,4H).

[0252] MS(ESI)m / z[M+H] + :536.2.

[0253] Example 4: Preparation of Compound 4

[0254] Following the preparation method of Example 2, 54 mg of compound 4 was prepared from (S)-2-cyclopropyl-2-hydroxyacetic acid, with a yield of 54%.

[0255] 1 H NMR (400MHz, DMSO-d6) δ8.37(d,J=9.0Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.51 (s,1H),5.58(q,J=6.7Hz,1H),5.49(d,J=5.2Hz,1H),5.35–5.06(m,2H),3.60(t,J=5.7Hz ,1H),3.17(q,J=17.2Hz,2H),2.39(s,3H),2.16(d,J=6.1Hz,2H),1.85(dq,J=14.2,7.1H z,2H),1.24(q,J=8.1Hz,1H),0.87(t,J=7.3Hz,3H),0.59–0.32(m,4H).MS(ESI)m / z[M+H] + :536.2.

[0256] Example 5: Preparation of Compound 5

[0257] Following the preparation method of Example 2, 55 mg of compound 5 was prepared from (R)-3-hydroxybutyric acid, with a yield of 56%.

[0258] 1 H NMR (400MHz, DMSO-d6) δ8.42(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.52(s, 1H),5.55(dt,J=9.3,5.0Hz,1H),5.29–5.12(m,2H),4.66(d,J=4.7Hz,1H),4.05(dt,J=12.1 ,6.0Hz,1H),3.25–3.09(m,2H),2.39(s,3H),2.33–2.17(m,2H),2.13(dd,J=10.7,5.4Hz,2H ),1.86(hept,J=7.1Hz,2H),1.09(d,J=6.2Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H]+ :524.2.

[0259] Example 6 Preparation of Compound 6

[0260] Following the preparation method of Example 2, 51 mg of compound 6 was prepared from (S)-3-hydroxybutyric acid, with a yield of 52%.

[0261] 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.7Hz,1H),7.78(d,J=11.0Hz,1H),7.30(s,1H),6.53(s ,1H),5.57(dt,J=8.8,4.5Hz,1H),5.22(d,J=2.7Hz,2H),4.64(d,J=4.5Hz,1H),4.05(dt,J =11.9,6.1Hz,1H),3.16(t,J=5.7Hz,2H),2.39(s,3H),2.32–2.17(m,2H),2.17–2.04(m,2H ),1.86(hept,J=7.1Hz,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :524.2.

[0262] Example 7 Preparation of Compound 7

[0263] Compound 7-1 (87 mg, 1 eq), compound 7-2 (144 mg, 1.1 eq), p-toluenesulfonic acid pyridinium salt (50 mg, 0.4 eq), acetic acid (2.6 mL, 30 V), and toluene (2.6 mL, 30 V) were added to a reaction flask and reacted at 110 °C for 18 hours. The reaction was monitored by TLC until complete. The solvent was removed by rotation, and the mixture was purified by silica gel column chromatography (dichloromethane: methanol) to give 123 mg of compound 7, with a yield of 61%.

[0264] 1 H NMR (400MHz, DMSO-d6) δ7.72(d,J=9.0Hz,1H),7.29(d,J=9.0Hz,1H),7.17(s,1H),6.45(s,1H),5.65(s,2H),5.16(s,2H),3.04 (t,J=6.1Hz,2H),2.75(t,J=6.1Hz,2H),2.08–1.95(m,2H),1.85(hept,J=7.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H]+ :406.1.

[0265] Example 8: Preparation of Compound 8

[0266] Following the preparation method of Example 7, 127 mg of compound 8 was prepared from compounds 8-1 and 7-2 as starting materials, with a yield of 60%.

[0267] 1 H NMR(400MHz,DMSO-d6)δ7.56(d,J=12.4Hz,1H),7.14(s,1H),5.10(s,2H),2.99( t,J=6.1Hz,2H),2.78(t,J=6.1Hz,2H),1.95(t,J=5.8Hz,2H),1.79(m,2H),1.40 1.00(m,3H),0.81(t,J=7.4Hz,3H).MS(ESI)m / z[M+H] + :424.1.

[0268] Example 9: Preparation of Compound 9

[0269] Step 1:

[0270] 250 mg of compound 9-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of p-toluenesulfonic acid pyridine salt were added to a 50 mL single-necked flask and reacted at 110 °C for 18 hours. The reaction was monitored by TLC until complete. The solvent was evaporated under reduced pressure, and the mixture was purified by silica gel column chromatography (DCM:MeOH) to give 386 mg of compound 9-3, yield: 81%. MS (ESI) m / z [M+H] + :480.2.

[0271] Step 2:

[0272] 380 mg of compound 9-3 was added to a 100 mL single-necked flask, along with 7.6 mL of purified water and 3.8 mL of methanesulfonic acid. The reaction was carried out at 85 °C for 10 hours under nitrogen protection. The reaction was monitored by TLC until complete. The mixture was then cooled to room temperature, and 22.8 mL of methanol was added. The mixture was stirred at room temperature for 2 hours, filtered, and the crude product was obtained. Purification was performed by silica gel column chromatography (dichloromethane:methanol) to yield 148 mg of compound 9, with a yield of 35%.

[0273] 1H NMR(400MHz,D2O)δ7.20–7.06(m,2H),5.42–5.14(m,5H),3.30(dd,J=18.1,4.0Hz,1H),3.06–2.91 (m,1H),2.69(s,2H),2.62–2.47(m,1H),2.18(s,3H),1.80(q,J=7.3Hz,2H),0.79(t,J=7.4Hz,3H). MS(ESI)m / z[M+H]+:438.2.

[0274] Example 10: Preparation of Compound 10

[0275] The preparation method described in Example 9 was used to prepare 70 mg of compound 10 using compound 10-1 and compound 7-2 as raw materials, with a yield of 34%.

[0276] 1 H NMR(400MHz,DMSO-d6)δ8.32(d,J=8.4Hz,1H),8.20(dd,J=8.4,1.2Hz,1H),7.93 -7.84(m,1H),7.79(t,J=7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.42(s,2H),3.98 (p,J=6.7Hz,1H),3.50(t,J=8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.88(he pt,J=7.3Hz,2H),1.15(d,J=6.7Hz,6H),0.88(t,J=7.3Hz,3H).MS(ESI)m / z[M+H] + :514.2.

[0277] Example 11 Preparation of Compound 11

[0278] The preparation method described in Example 9 was used to prepare 80 mg of compound 11 using compound 11-1 and compound 7-2 as raw materials, with a yield of 37%.

[0279] 1H NMR (500MHz, DMSO-d6) δ0.88(t,J=7.25Hz,3H),1.32(t,J=7.5Hz,3H),1.85(m,2H),3.11(q,J=7.5Hz,2 H), 5.26 (s, 2H), 6.48 (s, 1H), 7.23 (s, 1H), 7.41 (d, J = 10.0Hz, 2H), 8.01 (d, J = 10.0Hz, 1H), 10.3 (s, 1H). MS(ESI)m / z[M+H] + :395.1.

[0280] Example 12 Preparation of Compound 12

[0281] 255 mg of compound 12-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of p-toluenesulfonic acid pyridinium salt were added to a 50 mL single-necked flask and reacted at 110 °C for 18 hours. The reaction was monitored by TLC until complete. The solvent was evaporated under reduced pressure, and the mixture was purified by silica gel column chromatography (DCM: MeOH) to give 330 mg of compound 12-3, yield: 70%.

[0282] 1 H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.26(s,2H ),3.81(d,J=5.9Hz,2H),3.22(s,2H),1.98(d,J=6.7Hz,4H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :471.1.

[0283] 300 mg of intermediate compound 12-3 was dissolved in 15 mL of 10% sulfuric acid and reacted at 110 °C for 48 hours. The reaction was monitored by TLC until complete. The solution was adjusted to neutral by adding saturated sodium carbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was then purified by silica gel column chromatography (DCM:MeOH) to give 89 mg of compound 12, yield: 31%.

[0284] 1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.32-5.19(m ,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.92–1.76(m,4H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :453.2.

[0285] Example 13 Preparation of Compound 13

[0286] Following the preparation method of Example 12, 74 mg of compound 13 was prepared from compound 13-1 and compound 7-2, with a two-step yield of 16%.

[0287] 1 H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.32-5.19(m ,2H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.94–1.76(m,6H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :467.2.

[0288] Example 14 Preparation of Compound 14

[0289] Step 1:

[0290] 213 mg of compound 14-1, 300 mg of compound 7-2, 7.5 mL of acetic acid, 7.5 mL of toluene, and 68 mg of p-toluenesulfonic acid pyridine salt were added to a 50 mL single-necked flask and reacted at 110 °C for 18 hours. The reaction was monitored by TLC until complete. The solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography (DCM:MeOH) to give 300 mg of compound 14-3, yield: 68%. MS (ESI) m / z [M+H]+: 443.1.

[0291] Step 2:

[0292] 300 mg of compound 14-3 was dissolved in 3 mL of N,N-dimethylformamide, and sodium azide (48 mg, 1.1 eq) was added. The reaction was carried out at 80 °C for 16 hours. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, 18 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give 186 mg of crude compound 14-4, with a yield of 63%. MS (ESI) m / z [M+H]+: 450.1.

[0293] Step 3:

[0294] 186 mg of compound 14-4 was dissolved in 4 mL of tetrahydrofuran, and triphenylphosphine (134 mg, 1.2 eq) was added. The mixture was stirred at room temperature for 4 hours, and the reaction was monitored by TLC until complete. Hydrochloric acid (4 M, 1 mL) was added to the reaction system, and the reaction was carried out at 55 °C for 16 hours. The mixture was then concentrated under reduced pressure using a reversed-phase column (SEPA FLASH SW025, Spherical C18, 20-45 μm). Mobile phase A: 0.05% formic acid / water, mobile phase B: acetonitrile; mobile phase A: mobile phase B = 40:60, flow rate 20 mL / min) was used for separation and purification to obtain 63 mg of compound 14, yield: 35%.

[0295] 1 H NMR(400MHz,DMSO-d6)δ8.62(s,2H),8.45(s,1H),7.50(s,1H),7.25(s,1H),6.48(s,1H),6.28(s,2H),5 .20(s,2H),4.70(d,J=6.0Hz,2H),1.86(tt,J=14.2,6.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :424.1.

[0296] Example 15 Preparation of Compound 15

[0297] Following the preparation method of Example 14, 50 mg of compound 15 was prepared using compounds 15-1 and 7-2 as starting materials, with a three-step yield of 11%.

[0298] 1H NMR(400MHz,DMSO-d6)δ8.62(s,2H),8.45(s,1H),7.50(s,1H),7.25(s,1H),6.48(s,1H),6.28(s,2H),5.20(s,2 H),3.51-3.46(m,2H),3.17-3.13(m,2H),1.86(tt,J=14.2,6.1Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :438.2.

[0299] Example 16 Preparation of Compound 16

[0300] 1 g of compound 16-1, 1.5 g of compound 7-2, 30 mL of acetic acid, 30 mL of toluene, and 340 mg of p-toluenesulfonic acid pyridine salt were added to a 250 mL single-necked flask and reacted at 110 °C for 18 hours. The reaction was monitored by TLC until complete. The solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography (dichloromethane:methanol) to give 1.6 g of the target intermediate compound 16-3, yield: 75%. MS (ESI) m / z [M+H]+: 431.1.

[0301] 1.6 g of compound 16-3 was dissolved in 16 mL of N,N-dimethylformamide, and sodium azide (256 mg, 1.1 eq) was added. The reaction was carried out at 80 °C for 16 hours. The reaction was monitored by TLC until complete. The mixture was cooled to room temperature, 96 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with water, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give 1.1 g of crude compound 16-4, with a yield of 68%. MS (ESI) m / z [M+H]+: 438.2.

[0302] 1.1 g of compound 16-4 was dissolved in 22 mL of tetrahydrofuran, and 0.79 g (1.2 eq) of triphenylphosphine was added. The mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. Hydrochloric acid (4 M, 5 mL) was added to the reaction system, and the reaction was carried out at 55 °C for 16 hours. The mixture was concentrated under reduced pressure and purified by reversed-phase column chromatography (instrument model: SEPAFLASH SW025, column: Spherical C18, 20-45 μm). Mobile phase A: 0.05% formic acid / water, mobile phase B: acetonitrile; mobile phase A: mobile phase B = 45:55, flow rate 20 mL / min), yielded 0.58 g of compound 16, yield: 56%.

[0303] 1H NMR(400MHz, DMSO-d6)δ8.62(s,3H),8.43(d,J=8.0Hz,1H),7.99(d,J=10.7Hz,1H),7.35(s,1H),5.59(s,2H),4 .70(d,J=6.0Hz,2H),2.55(s,3H),1.88(hept,J=7.1Hz,2H),0.88(t,J=7.3Hz,3H).MS(ESI)m / z[M+H]+:412.2.

[0304] Example 17 Preparation of Compound 17

[0305] To a mixture of anhydrous sodium sulfate (2.25 eq), 2-hydroxyacetic acid (1.45 eq), and compound 16 (100 mg), 2.4 mL of water and 3.0 mL of tetrahydrofuran were added. After stirring at room temperature for 15 minutes, N-methylmorpholine (1.10 eq) was added, and the mixture was stirred at room temperature for 15 minutes. Then, EDCI (2 eq) was added, and the mixture was stirred at room temperature for 3 hours. HPLC monitoring showed that the reaction of compound 16 was complete. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (DCM:MeOH) to give 82 mg of compound 17, with a yield of 72%.

[0306] 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.9Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.52(s,1H),5.58(d,J=5.4Hz,1H),5.49(t,J=5.7Hz, 1H),5.39(s,2H),5.20(t,J=13.9Hz,2H),3.96(d,J=5.5Hz,2H),2.55(s,3H),1.96–1.78(m,2H),0.87(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :470.2.

[0307] Example 18 Preparation of Compound 18

[0308] Following the preparation method of Example 17, 78 mg of compound 18 was prepared using compound 3-1 and compound 16 as starting materials, with a yield of 63%.

[0309] 1H NMR (400MHz, DMSO-d6) δ8.76(t,J=5.8Hz,1H),8.41(d,J=8.9Hz,1H),7.78(d,J=1 1.0Hz,1H),7.31(s,1H),6.52(s,1H),5.58–5.48(m,1H),5.38(d,J=5.7Hz,2H),4 .85(t,J=13.9Hz,2H),3.96(d,J=5.5Hz,1H),2.39(s,3H),1.86(dt,J=14.3,7.1H z, 2H), 1.14 (q, J = 7.3Hz, 1H), 0.87 (t, J = 7.2Hz, 3H), 0.38 (dd, J = 20.9, 5.6Hz, 4H).

[0310] MS(ESI)m / z[M+H] + :510.2.

[0311] Example 19 Preparation of Compound 19

[0312] Following the preparation method of Example 17, 74 mg of compound 19 was prepared using compound 4-1 and compound 16 as starting materials, with a yield of 60%.

[0313] 1 H NMR (400MHz, DMSO-d6) δ8.76(t,J=6.0Hz,1H),8.37(d,J=8.9Hz,1H),7.77(d,J=1 1.0Hz,1H),7.30(s,1H),6.51(s,1H),5.58(d,J=5.4Hz,1H),5.49(d,J=5.7Hz,2H ),4.85(t,J=13.9Hz,2H),3.96(d,J=5.5Hz,1H),2.40(s,3H),1.96–1.78(m,2H), 1.24(q,J=8.1Hz,1H),0.87(t,J=7.2Hz,3H),0.59–0.32(m,4H).MS(ESI)m / z[M+H] + :510.2.

[0314] Example 20 Preparation of Compound 20

[0315] Following the preparation method of Example 17, 70 mg of compound 20 was prepared using compound 5-1 and compound 16 as starting materials, with a yield of 58%.

[0316] 1H NMR (400MHz, DMSO-d6) δ8.76(t,J=6.0Hz,1H),8.42(d,J=8.9Hz,1H),7.78(d,J=11.0 Hz,1H),7.30(s,1H),6.52(s,1H),5.55(d,J=6.0Hz,1H),5.38(d,J=5.7Hz,2H),5.29 –5.12(m,2H),4.05(dt,J=12.1,6.0Hz,1H),2.39(s,3H),2.13(dd,J=10.7,5.4Hz,2H ),1.96–1.78(m,2H),1.09(d,J=6.2Hz,3H),0.87(t,J=7.2Hz,3H).MS(ESI)m / z[M+H] + :498.2.

[0317] Example 21 Preparation of Compound 21

[0318] Following the preparation method of Example 17, 75 mg of compound 21 was prepared using compound 6-1 and compound 16 as starting materials, with a yield of 62%.

[0319] 1 H NMR (400MHz, DMSO-d6) δ8.76(t,J=6.0Hz,1H),8.41(d,J=8.7Hz,1H),7.78(d,J =11.0Hz,1H),7.30(s,1H),6.53(s,1H),5.57(d,J=6.0Hz,1H),5.38(d,J=5.7Hz ,2H),5.29–5.12(m,2H),4.05(dt,J=12.1,6.0Hz,1H),2.39(s,3H),2.17–2.04 (m,2H),1.86(hept,J=7.1Hz,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).

[0320] MS(ESI)m / z[M+H] + :498.2.

[0321] Example 22 Preparation of compound LK1-1

[0322] Step 1:

[0323] Compound 22-2 (160 g, 1.0 eq) was added to a 10 L four-necked flask, followed by 2.4 L of ethylene glycol dimethyl ether. The mixture was mechanically stirred, then compound 22-1 (123.37 mL, 2.0 eq) was added. The mixture was stirred at 0 °C, and 10 mol / L sodium hydroxide (43.43 mL, 1.0 eq) was added dropwise using a constant pressure dropping funnel. The mixture was stirred for 1 h. TLC monitoring was performed. After the reaction was complete, acetic acid (12.42 mL) was added, and the mixture was stirred for 1 h. Water (660 mL) was added, and the mixture was stirred for 1 h. Distilled water (1.54 L) was added, and the mixture was stirred for 1 h. The mixture was filtered, and the filter cake was washed with 50% (v / v) ethylene glycol dimethyl ether aqueous solution (640 mL). The filter cake was dried in an oven at 40 °C to obtain 204.3 g of compound 22-3, with a yield of 99%. MS (ESI) m / z: 475.2 [M+H] + .

[0324] Step 2:

[0325] Compound 22-3 (180 g, 90%, 1.0 eq) was added to a 10 L four-necked flask, followed by 5.4 L of acetonitrile. The mixture was mechanically stirred, and DBU (25.5 mL, 0.5 eq) was added. The mixture was stirred at room temperature for 4 h, and then HOBT (101.5 g, 2.2 eq) was added. The mixture was stirred at room temperature for 0.5 h, cooled to 0 °C, and stirred overnight. The mixture was filtered, and the filter cake was washed with acetonitrile (800 mL) and dried in an oven at 40 °C to give 119.0 g of compound 22-4, with a yield of 81%. 1 H NMR (400MHz, MeOD) δ7.69(dd,J=12.9,8.6Hz,2H),7.40–7.24(m,7H),5.19(s,2H),4.80(s,2H),4.21(s,2H),3.66(s,2H).

[0326] Step 3:

[0327] Compound 22-5 (55 g, 1.0 eq) was added to a 5 L three-necked flask, dissolved in 600 mL acetonitrile and 100 mL water. Compound 22-4 (50 g, 1.0 eq) was then added, and EDCI (24 g, 1.0 eq) was added under stirring at 0 °C for 4 h. After the reaction was complete as monitored by TLC, 500 mL ethanol and 750 mL water were added, and the mixture was stirred overnight at room temperature. Then, 1450 mL water was added, and the mixture was stirred for 2 h. The mixture was filtered, and the filter cake was dried in an oven at 40 °C to give 80.0 g of compound 22-6, with a yield of 96%. MS (ESI) m / z: 648.3 [M+H] + .

[0328] Step 4:

[0329] Under nitrogen protection, 840 mL of tetrahydrofuran and 540 mL of water were added to compound 22-6 (40.0 g, 1.0 eq), followed by the addition of Pd (5%) / C (8.8 g, 0.07 eq) to displace hydrogen. The mixture was stirred overnight at room temperature. After the reaction was complete as monitored by HPLC, Pd / C was filtered off using diatomaceous earth. The Pd / C was washed with 300 mL of water, and the filtrates were combined. The filtrate was concentrated under reduced pressure, and then concentrated again under reduced pressure with 400 mL of ethanol. This process was repeated three times. Subsequently, 800 mL of ethanol was added, and the mixture was magnetically stirred and filtered. The filter cake was dried in an oven at 40 °C to give 19.6 g of compound 22-7, with a yield of 75%. MS (ESI) m / z: 424.2 [M+H] + .

[0330] 1 H NMR (400MHz, D2O) δ7.31(ddd,J=22.4,14.6,7.3Hz,5H),4.72–4.56(m,3H),3.94(s,2H),3.93–3.73(m,6H),3.08(ddd,J=22.1,13.7,7.7Hz,2H).

[0331] Step 5:

[0332] 5.4 g (1.5 eq) of 6-(maleimide)hexanoic acid succinimide ester was added to a 250 mL single-necked flask and dissolved in 45 mL of acetonitrile. Compound 22-7 (5.0 g, 1.0 eq), 105 mL of water, and DIPEA (1.56 mL, 0.8 eq) were then added, and the mixture was stirred overnight at room temperature. After the reaction was monitored by HPLC to be complete, 50 mL of isopropyl acetate, 10 g of anhydrous sodium dihydrogen phosphate, and 0.65 g of disodium hydrogen phosphate were added to the reaction mixture, and the mixture was stirred for 0.5 h. The organic phase was removed by separation. Another 50 mL of isopropyl acetate was added, and the organic phase was removed by separation. 25 mL of ethylene glycol dimethyl ether, 25 mL of ethyl acetate, 2.5 mL of acetonitrile, and 40 g of anhydrous sodium dihydrogen phosphate were added to the aqueous phase, and the mixture was stirred for 1 h before separating the aqueous phase. The organic phase was added twice, with 75 mL of acetonitrile, 11.3 mL of water, 3 g of sodium chloride, and 750 mg of anhydrous sodium dihydrogen phosphate. After stirring, the aqueous phase was separated. The organic phase was concentrated to 50 mL under reduced pressure, and 75 mL of ethylene glycol dimethyl ether was added to concentrate it to 50 mL. Then, 1 mL of water and 100 mL of ethylene glycol dimethyl ether were added, and the mixture was stirred overnight at room temperature. The mixture was then filtered, and the filter cake was added to 200 mL of ethylene glycol dimethyl ether and 6.5 mL of water. The mixture was stirred at 45 °C for 0.5 h, washed with 15 mL of a mixture of ethylene glycol dimethyl ether and water (v / v: 97 / 3), and the combined filtrates were concentrated to 100 mL. 25 mL of ethylene glycol dimethyl ether was added, and the mixture was stirred overnight at room temperature. The precipitated solid was washed with 25 mL of ethylene glycol dimethyl ether and dried under reduced pressure at 25 °C to give the 1,2-dimethoxyethane adduct of compound 22-9, in 58% yield. MS (ESI) m / z: 617.3 [M+H] + .

[0333] 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),8.57(s,1H),8.32(s,1H),8.14(d,J=7.1Hz,1H) ,8.08(s,1H),8.02(s,1H),7.21(d,J=22.0Hz,5H),7.00(s,2H),4.61(s,2H),4.50(s, 1H),3.98(s,2H),3.80–3.59(m,6H),3.43(s,4H),3.37(s,2H),3.24(s,6H),3.06(d,J =13.3Hz,1H),2.81(t,J=11.5Hz,1H),2.11(s,2H),1.47(d,J=5.8Hz,4H),1.19(s,2H).

[0334] Step 6:

[0335] In a 10 mL single-necked flask, anhydrous sodium sulfate (60 mg, 2.25 eq), ethyl 2-oxime cyanoacetate (60 mg, 2.25 eq), compound 22-9 (20 mg, 1.45 eq), 0.8 mL water, and 0.6 mL tetrahydrofuran were added. The mixture was stirred at room temperature for 30 min. Then, compound 22-10 (100 mg, 1.0 eq), 0.3 mL water, and 0.5 mL tetrahydrofuran were added. After stirring for 15 min, N-methylmorpholine (23 μL, 1.1 eq) and 0.3 mL tetrahydrofuran were added. After stirring for 15 min, EDCI (72 mg, 2.0 eq), 0.5 mL water, and 0.5 mL tetrahydrofuran were added. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by HPLC until complete. Extracted with dichloromethane, dried, and the combined organic phases were evaporated to dryness. Then, reversed-phase preparative separation was performed (Sante C18 column (20 g), 35-40% acetonitrile / water) to give 99 mg of compound LK1-1, yield 51%. MS (ESI) m / z: 1036.4 [M+H] + .

[0336] 1 H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J= 5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7 .75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2 H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz, 2H),4.53–4.42(m,1H),4.02(s,2H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3 .17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H) ,2.37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1 Hz,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0337] Example 23 Preparation of compound LK1-2

[0338] Following the preparation method of Example 22, compound 22-1 (i.e., benzyl glycolate) was replaced with (R)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester to prepare compound LK1-2. MS (ESI) m / z: 1076.4 [M+H] + .

[0339] 1 H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5. 7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75 (d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6. 51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.5 3–4.42(m,1H),3.79–3.53(m,7H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz,2H),3 .02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.18(dt,J= 12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),1.42(tt,J=14.3,7 .4Hz,4H),1.17(m,3H),0.85(dt,J=14.7,7.3Hz,3H),0.38(dd,J=20.9,5.6Hz,4H).

[0340] Example 24 Preparation of compound LK1-3

[0341] Following the preparation method of Example 22, compound 22-1 (i.e., benzyl glycolate) was replaced with (S)-2-cyclopropyl-2-hydroxyacetic acid benzyl ester to prepare compound LK1-3. MS (ESI) m / z: 1076.4 [M+H] + .

[0342] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7 Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d, J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s ,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.4 2(m,1H),3.79–3.53(m,7H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz,2H),3.02(dd ,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.18(dt,J=12.0,6. 9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),1.42(tt,J=14.3,7.4Hz,4H), 1.24(q,J=8.1Hz,1H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H),0.59–0.32(m,4H).

[0343] Example 25 Preparation of compound LK1-4

[0344] Following the preparation method of Example 22, compound LK1-4 was prepared by replacing compound 22-1 (i.e., benzyl glycolate) with (R)-3-hydroxybutyrate benzyl ester. MS (ESI) m / z: 1064.4 [M+H] + .

[0345] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7H z,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d,J= 10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H) ,5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1 H),4.05(dt,J=12.1,6.0Hz,1H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J =17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H) ,2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,4H),1.85(hept,J=7.1Hz,2H),1.42(tt, J=14.3,7.4Hz,4H),1.17(m,2H),1.09(d,J=6.2Hz,3H),0.85(dt,J=14.7,7.3Hz,3H).

[0346] Example 26 Preparation of compound LK1-5

[0347] Following the preparation method of Example 22, compound LK1-5 was prepared by replacing compound 22-1 (i.e., benzyl glycolate) with (S)-3-hydroxybutyrate benzyl ester. MS (ESI) m / z: 1064.4 [M+H] + .

[0348] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7H z,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.75(d,J= 10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H) ,5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1 H),4.05(dt,J=11.9,6.0Hz,1H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J =17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.37(s,3H) ,2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,4H),1.85(hept,J=7.1Hz,2H),1.42(tt, J=14.3,7.4Hz,4H),1.17(m,2H),1.08(d,J=6.1Hz,3H),0.85(dt,J=14.7,7.3Hz,3H).

[0349] Example 27 Preparation of compound LK1a-1

[0350] Step 1:

[0351] Glycine (3 g, 1.00 eq) was added to a 250 mL four-necked flask, followed by 90 mL of tetrahydrofuran and 30 mL of purified water. Sodium bicarbonate (3.70 g, 1.10 eq) was then added, and the mixture was mechanically stirred until the solution became clear. Compound 27-1 (12.8 g, 1.00 eq) was dissolved in 30 mL of DME and slowly added using a constant-pressure dropping funnel, followed by stirring overnight at room temperature. 100 mL of saturated sodium bicarbonate and 100 mL of purified water were added, and the mixture was washed three times with 100 mL of ethyl acetate. The pH was adjusted to 4 by slowly adding 12 M hydrochloric acid. The mixture was filtered, the filter cake was rinsed with water, and dried to give 10.1 g of compound 27-2, with a yield of 98%. MS (ESI) m / z: 281.1 [M+H] + .

[0352] Step 2:

[0353] In a 500 mL four-necked flask, 6.5 g of L-phenylalanine tert-butyl hydrochloride (1.00 eq) and 7.8 g of compound 27-2 (1.1 eq) were added to 200 mL of N,N-dimethylformamide, followed by triethylamine (3.5 mL, 1.00 eq) and DMTMM (8.9 g, 1.20 eq). The mixture was stirred at room temperature for 2 hours. The reaction solution was then added to 1 L of purified water, filtered, and the filter cake was washed with water to give 11 g of compound 27-3, with a yield of 90%. MS (ESI) m / z: 484.2 [M+H] + .

[0354] Step 3:

[0355] Compound 27-3 (10 g, 1.0 eq), 100 mL of dichloromethane, and 50 mL of trifluoroacetic acid (30.00 eq) were added to a 250 mL four-necked flask and stirred at room temperature for 2 hours. The reaction solution was concentrated, slurried with MTBE, filtered, and the filter cake was washed with water to give 8.5 g of compound 27-4, with a yield of 96%. MS (ESI) m / z: 428.2 [M+H] + .

[0356] Step 4:

[0357] Following the preparation method of Example 22, N-(benzyloxy)carbonylglycylglycyl-L-phenylalanine (i.e., compound 22-5) was replaced with N-((benzyloxy)carbonyl)-N-methylglycylglycyl-L-phenylalanine (i.e., compound 27-4) to prepare LK1a-1. MS (ESI) m / z: 1050.4 [M+H] + .

[0358] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J =5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),7.75(d,J=10.9Hz,1H ),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5H),6.99(s,2H),6.51(s,1H),5. 59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m ,1H),4.02(s,2H),3.79–3.53(m,6H),3.35(q,J=6.3Hz,2H),3.17(q,J=17.5Hz ,2H),3.02(dd,J=13.7,4.3Hz,1H),2.97(s,3H),2.77(dd,J=13.7,9.7Hz,1H),2 .37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1H z,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0359] Example 28 Preparation of compound LK1c-1

[0360] Following the preparation method of Example 22, compound 22-8 was replaced with 2,5-dioxopyrrolidone-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl)hexanoyl)glycine ester (CAS No.: 1956326-21-8) to prepare LK1c-1. MS (ESI) m / z: 1093.4 [M+H] + .

[0361] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7 Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t,J=5.5Hz,1H),7.96(t ,J=5.5Hz,1H),7.75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7.2,6.7Hz,5 H),6.99(s,2H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27–5.08(m,2H),4.64(d ,J=6.5Hz,2H),4.53–4.42(m,1H),4.02(s,2H),3.80–3.53(m,8H),3.35(q,J=6.3Hz ,2H),3.17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2.77(dd,J=13.7,9.7Hz ,1H),2.37(s,3H),2.18(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7 .1Hz,2H),1.42(tt,J=14.3,7.4Hz,4H),1.17(m,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0362] Example 29 Preparation of compound LK1d-1

[0363] Step 1;

[0364] Step 2:

[0365] Following the preparation method of Example 22, LK1d-1 was prepared by replacing compound 22-8 with 2,5-dioxo-1-pyrrolidinyl 3-[2-[2-(2,5-dihydro-2,5-dioxo-1H-pyrrolidinyl)ethoxy]ethoxy]propionate (CAS: 1433997-01-3). MS (ESI) m / z: 1082.4 [M+H] + .

[0366] 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.50(d,J=8.8Hz,1H),8.29(t,J=5.7Hz,1H),8.12(d,J=7.9Hz,1H),8.06(t,J=5.6Hz,1H),8.00(t ,J=5.5Hz,1H),7.75(d,J=10.9Hz,1H),7.30(s,1H),7.20(dp,J=20.5,7. 2,6.7Hz,5H),6.99(s,2H),6.51(s,1H),5.59(q,J=6.6,6.1Hz,1H),5.27 –5.08(m,2H),4.64(d,J=6.5Hz,2H),4.53–4.42(m,1H),4.02(s,2H),3.7 9–3.53(m,16H),3.17(q,J=17.5Hz,2H),3.02(dd,J=13.7,4.3Hz,1H),2. 77(dd,J=13.7,9.7Hz,1H),2.37(s,3H),2.28(dt,J=12.0,6.9Hz,2H),2.13–2.02(m,2H),1.85(hept,J=7.1Hz,2H),0.85(dt,J=14.7,7.3Hz,3H).

[0367] Example 30 Preparation of compound LK1b-1

[0368] Step 1:

[0369] In a 250 mL single-necked flask, 10.0 g (1.0 eq) of 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynyl acid and 5.15 g (1.2 eq) of N-hydroxysuccinimide were added and dissolved in 100 mL of dichloromethane. Then, 10.0 g (1.3 eq) of N,N'-dicyclohexylcarbodiimide was added, and the mixture was stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered, and the filtrate was concentrated under reduced pressure to give 14.0 g of compound 31-2. No further purification was required; it was used directly in the next reaction. MS (ESI) m / z: 366.4 [M+H] + .

[0370] In a 250 mL single-necked flask, 13.6 g (1.0 eq) of compound 31-2 was added and dissolved in 135 mL of acetonitrile. Then, 18.9 g (1.2 eq) of compound 31-3, 270 mL of water, and 5.3 mL (0.8 eq) of DIPEA were added, and the mixture was stirred overnight at room temperature. After the reaction was monitored by HPLC to be complete, the reaction solution was extracted three times with 300 mL of ethyl acetate. 10 g of anhydrous sodium dihydrogen phosphate was added to the aqueous phase, and the aqueous phase was extracted three times with 300 mL of ethylene glycol dimethyl ether. The ethylene glycol dimethyl ethers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the primary product. The crude product was separated using reverse-phase preparative chromatography (acetonitrile / water) to give 18.6 g of compound 31-4, with a yield of 74%.

[0371] In a 250 mL single-necked flask, compound 31-4 (8.3 g, 1.4 eq), compound 31-5 (4.9 g, 1.0 eq), and 175 mL of DMF were added. The mixture was stirred at room temperature, and then triethylamine (1.9 mL, 1.6 eq) and DMTMM (3.87 g, 1.5 eq) were added. The mixture was stirred at room temperature for 1 hour, and the reaction was monitored by HPLC until complete. The reaction mixture was then added to 500 mL of water, filtered, and dried to obtain 12 g of crude product. The crude product was purified by column chromatography to give 8.9 g of compound LK1b-1, yield: 85%.

[0372] MS(ESI)m / z:1093.4[M+H]+.

[0373] 1H NMR(400MHz, DMSO-d6)δ9.10(s,2H),8.64(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.31(t,J=5.7Hz,1H),8.26–8.00(m,3H),7.77(d,J=10.9Hz, 1H),7.36–7.10(m,6H),6.52(s,1H),5.67–5.52(m,1H),5.19(s,2H),4. 64(d,J=6.5Hz,2H),4.47(dt,J=12.9,6.7Hz,1H),4.02(s,2H),3.77–3.6 8(m,4H),3.63–3.57(m,1H),3.40(s,3H),3.17(td,J=12.8,11.5,6.7Hz,2H),3.02(dd,J=13.7,4.2Hz,1H),2.77(dd,J=13.7,9.7Hz,1H),2.38( s,3H),2.32(t,J=7.3Hz,2H),2.17(dd,J=13.4,6.8Hz,2H),2.08(s,1H) ,1.84(dq,J=18.9,7.0Hz,4H),1.28–1.22(m,2H),0.87(t,J=7.2Hz,3H).

[0374] Example 31 Construction and expression of anti-EGFR / c-MET bispecific antibody

[0375] The structure of the 2+2 bispecific antibody disclosed herein is constructed as follows: A human c-MET binding domain is linked to the C-terminus of the heavy chain of the full-length anti-human EGFR antibody via a linker peptide shown in SEQ ID NO:10. An amino acid peptide sequence as shown in SEQ ID NO:8 is added to the end of the c-MET binding domain to eliminate the risk of binding to pre-existing anti-drug antibodies. To reduce the antibody's ADCC and other immune-related activities, amino acid substitutions (TM mutations) of L234F, L235E, and P331S are performed in the Fc segment. The full-length heavy chain sequence of the 2+2 bispecific antibody (M-06C12-PTM) is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2.

[0376] The disclosed 1+1 bispecific antibody (1+1 bispecific antibody) consists of two heterologous heavy chains and one light chain, with no light chain mismatch. The heavy chain containing the c-MET binding domain employs a "knob" structure design, including amino acid substitutions at the S354C and T366W sites. The heavy chain containing the EGFR-binding VHH domain employs a "hole" structure design, including amino acid substitutions at the Y349C, T366S, L368A, and Y407V sites. To facilitate purification and removal of the "hole" structure dimer, the "hole" heavy chain also undergoes H435R substitution. To reduce the antibody's ADCC and other immune-related activities, the Fc segments of both heavy chains undergo amino acid substitutions at L234F, L235E, and P331S (TM mutation). The sequence of the "hole" heavy chain of the 1+1 bispecific antibody (T-E6-H6-TM) is shown in SEQ ID NO:3, the sequence of the "knob" heavy chain is shown in SEQ ID NO:4, and the light chain sequence is shown in SEQ ID NO:5. The bispecific antibody structure and related molecular sequences disclosed herein are summarized in Tables 2 and 3, respectively.

[0377] Table 2. Structure of bispecific antibodies

[0378] Table 3. Amino acid sequences of bispecific antibodies

[0379] The heavy and light chain target gene fragments of the aforementioned bispecific antibody molecules were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids, which were then transfected into Expi293 or ExpiCHO cells. Next, the transfected Expi293 or ExpiCHO cells were cultured in serum-free medium on a shaker at 37°C and 8% CO2 for 6–12 days. The supernatant was then collected for purification, and the final purified bispecific antibody was analyzed for purity by SEC-HPLC and A280 concentration was determined.

[0380] Example 32 Preparation of anti-EGFR / c-MET bispecific antibody ADC

[0381] Example 32-1 Analysis Method of ADC

[0382] 1. Determination of ADC DAR value using reversed-phase chromatography (RP-UPLC). Sample preparation: Sample concentration 0.1–0.5 mg / mL, completely reduced with excess DTT; Instrumentation: Waters Acquity H-Class UPLC; Column: Waters BioResolve™ RP mAb polyphenyl, 2.7 μm, 4.6 × 100 mm; Mobile phase A: 0.1% TFA water; Mobile phase B: 0.1% TFA acetonitrile; Flow rate: 0.3 mL / min; Detection wavelength: 280 nm & 360 nm; Column temperature: 60–90 °C; Sample loading volume: 5 μL; RP elution method: Under equilibrium conditions, phase B increases from ~30% to ~50% within 30 minutes.

[0383] RP-UPLC data analysis: By comparing the spectra of the sample with those of the bare antibody, the positions of the light and heavy chains are distinguished. Then, the spectrum of the tested sample is integrated to calculate the DAR value.

[0384] The calculation formula is as follows:

[0385] Table 4

[0386] Total LC peak area = LC peak area + LC+1 peak area

[0387] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0388] LC DAR = Σ(number of linked drugs * percentage of peak area) / total LC peak area

[0389] HC DAR = Σ(number of linked drugs * percentage of peak area) / total HC peak area

[0390] DAR = LC DAR + HC DAR

[0391] Where LC represents a light chain; LC+1 represents a light chain with one connector-load; HC represents a heavy chain; HC+1 represents a heavy chain with one connector-load; HC+2 represents a heavy chain with two connector-loads; HC+3 represents a heavy chain with three connector-loads.

[0392] 2. Determination of ADC purity by size exclusion chromatography-HPLC: Sample preparation: Sample concentration 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane; Instrumentation: Agilent 1260 Infinity II Bio-Inert LC System; Column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm; Mobile phase: 0.2 M PB, 5-15% isopropanol, pH 7.0; Flow rate: 0.5-1 mL / min; Detection wavelength: 280 nm & 360 nm; Column temperature: room temperature; Sample loading: 30 μg; SEC elution method: isocratic elution.

[0393] In this disclosure, PB refers to sodium phosphate buffer solution with disodium hydrogen phosphate (DHP) as the main component. DHP-DHP buffer solutions with different pH values ​​are typically prepared using sodium dihydrogen phosphate and disodium hydrogen phosphate solutions of the same concentration.

[0394] Example 32-2 Preparation of 2+2 Bispecific Antidote Drug Conjugate ADC-1

[0395] The anti-EGFR / c-MET bispecific antibody M-06C12-PTM was reduced in 20 mM PBS (pH 6.0-7.4) with 6-20 equivalents of TCEP reducing agent at 4-40°C for 0.5-6 hours. After reduction, 5-30% DMSO (DMF or DMA can also be used as organic solvents) was added, and 6-30 equivalents of the loading-linker (e.g., LK1b-1) were added at 4-40°C for 0.5-24 hours. After the coupling reaction was completed, 100 mM N-acetylcysteine ​​solution was added at a quenching ratio of 20:1 (quencher:antibody), and the mixture was shaken well and quenched at room temperature for 20 minutes to terminate the coupling reaction. The ADC buffer was changed to 20 mM His-HAc buffer at pH 5.5 using desalting chromatography or ultrafiltration, and the antibody-drug conjugate concentration was adjusted to approximately 3-10 mg / mL. The ADC stock solution was then filtered through a 0.22 μm PES filter and aliquoted according to experimental requirements, and stored at -20°C or -40°C. The absorbance was measured at 280 nm and 370 nm using UV chromatography to calculate the antibody concentration. The average DAR value was calculated using RP-HPLC, and the ADC purity was determined by SEC-HPLC. The obtained products are shown in Table 5 below, and their overall structure is shown in Figure 1.

[0396] Example 32-3 Preparation of 1+1 Bispecific Antidote Drug Conjugate ADC-2

[0397] The anti-EGFR / c-MET bispecific antibody T-E6-H6-TM was reduced in 20 mM PBS (pH 6.0-7.4) with 6-20 equivalents of TCEP reducing agent at 4-40°C for 0.5-6 hours. After reduction, 5-30% DMSO (DMF or DMA can also be used as organic solvents) was added, and 6-30 equivalents of the loading-linker (e.g., LK1b-1) were added at 4-40°C for 0.5-24 hours. After the coupling reaction was completed, 100 mM N-acetylcysteine ​​solution was added at a quenching ratio of 20:1 (quencher:antibody), and the mixture was shaken well and quenched at room temperature for 20 minutes to terminate the coupling reaction. The ADC buffer was changed to 20 mM His-HAc buffer at pH 5.5 using desalting chromatography or ultrafiltration to achieve an antibody-drug conjugate concentration of approximately 3-10 mg / mL. The ADC stock solution was then filtered through a 0.22 μm PES filter and aliquoted according to experimental requirements, and stored at -20°C or -40°C. The absorbance was measured at 280 nm and 370 nm using UV chromatography to calculate the antibody concentration. The average DAR value was calculated using RP-HPLC, and the ADC purity was determined using SEC-HPLC. The obtained products are shown in Table 5 below, and their overall structure is shown in Figure 2.

[0398] Table 5. ADC molecules and their DAR values ​​obtained in this publication.

[0399] The antibody-drug conjugates and load-linker conjugates provided in this disclosure can also be prepared by referring to the relevant preparation methods described in documents such as WO2014057687A, WO2020063676A, and WO2022068878, based on the preparation methods of the relevant compounds described in this disclosure.

[0400] Example 32-4 Preparation of control molecule AZD9592

[0401] The positive control molecule AZD9592 was prepared according to Example 11 of US20230183358, with a DAR value of 5.88. Its EGFR antibody heavy chain amino acid sequence (RAA22-H) is as follows:

[0402] The light chain amino acid sequence (RAA22-L) of its EGFR antibody is as follows:

[0403] Its c-MET antibody heavy chain amino acid sequence (B09-H) is as follows:

[0404] The light chain amino acid sequence (B09-H) of its c-MET antibody is as follows:

[0405] Its connector-load structure is as follows:

[0406] Example 32-5 Preparation of control molecule BL-B01D1

[0407] The positive control molecule BL-B01D1 was prepared according to Example 93 of CN116120460A, with a DAR value of 7.86. Its antibody heavy chain amino acid sequence is as follows.

[0408] The amino acid sequence of its antibody light chain is as follows:

[0409] Its connector-load structure is as follows:

[0410] Examples 32-6 Preparation of control molecule NC-LK1b-1

[0411] The positive control molecule NC-LK1b-1 can be prepared by methods known in the art, and its antibody heavy chain amino acid sequence is as follows:

[0412] The amino acid sequence of its antibody heavy chain is as follows:

[0413] Bioactivity test

[0414] 1. Detection of the in vitro antitumor activity of the loading compounds used in the ADC of this disclosure

[0415] Experimental objective: To detect the in vitro inhibitory activity of the loaded compounds in this disclosure against NCI-N87 (human gastric cancer cells), Calu-3 (human lung adenocarcinoma cells), MDA-MB-453 (human breast cancer cells), KPL-4 (human breast cancer cells), or MDA-MB-468 (human breast cancer cells).

[0416] Tumor cells in logarithmic growth phase, including NCI-N87 (ATCC, catalog number: CRL-5822), Calu-3 (ATCC, catalog number: HTB-55), MDA-MB-453 (ATCC, catalog number: HTB-131), KPL-4 (Nanjing Kebai Biotechnology, catalog number: CBP60379), or MDA-MB-468 (ATCC, catalog number: HTB-132), were added to cell culture plates at a density of 5000 cells / well. The plates were incubated at 37°C in a 5% CO2 cell culture incubator for 12-16 hours. 100 μL of sample (starting at 10 μM, 4-fold dilution, 10 concentrations) was added to each well, gently mixed, and incubated for 48 hours. After incubation, 70 μL of CellTiter-Glo was added. TM (Promega, catalog number: G7572) Working solution, gently shake to lyse cells, and read the plate on a microplate reader. Cell proliferation inhibition rate was calculated as: Cell proliferation inhibition rate = (1 - Sample well / Control well) × 100%. Using GraphPad Prism 8.0 software, a graph was plotted with the Log value of sample concentration on the x-axis and Cytotoxicity% on the y-axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC50 for each sample. 50 The values ​​are shown in Tables 6 and 7.

[0417] Table 6. In vitro activity test results of the loaded compounds disclosed herein.

[0418] Table 7. In vitro activity test results of the loaded compounds disclosed herein.

[0419] Experimental results show that the loading compounds disclosed herein have significant inhibitory activity against the proliferation of NCI-N87, Calu-3, MDA-MB-453, KPL-4, or MDA-MB-468.

[0420] 2. Pharmacokinetics of the loading compounds used in the ADC of this disclosure in mice.

[0421] Experimental objective: To evaluate the metabolic kinetics of the loaded compounds in this disclosure in mice.

[0422] Balb / c mice were randomly divided into groups of 12 (half male and half female) and injected with samples once (Formulation A: 5% DMSO + 95% (15% sulfobutyl-β-cyclodextrin - physiological saline); Formulation B: 5% DMSO - 10% Solutol HS15 - 85% physiological saline, diluted according to dosage). Plasma samples were collected at cross-collection points before administration and at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after administration. The concentration of the samples in the plasma was detected by LC-MS, and the pharmacokinetic parameters were calculated and are shown in Table 8.

[0423] Table 8. PK parameters of the disclosed loaded compounds in mice.

[0424] Experimental results show that the loading compound disclosed herein has a short in vivo half-life in mice, and when used as a loading compound for an ADC, the ADC can be cleared more quickly in vivo when load shedding occurs, thus exhibiting better in vivo safety.

[0425] 3. Pharmacokinetics of the loading compounds used in the ADC of this disclosure in rats.

[0426] Experimental objective: To evaluate the metabolic kinetics of the loaded compounds in this disclosure in rats.

[0427] SD rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into groups of 6 rats each, with half males and half females. Each group received a single injection of a sample (Formulation A: 5% DMSO + 95% (15% sulfobutyl-β-cyclodextrin-physiological saline), diluted according to the dosage). Plasma samples were collected at cross-collection points before administration and at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after administration. The concentration of the sample in the plasma was detected by LC-MS, and the pharmacokinetic parameters were calculated. The results are shown in Table 9.

[0428] Table 9. PK parameters of the load compounds disclosed herein in rats.

[0429] Experimental results show that the loaded compound disclosed herein also has a short in vivo half-life and a rapid in vivo clearance rate in rats.

[0430] 4. In vitro plasma stability of the loading compound used in the ADC of this disclosure

[0431] Take 398 μL of plasma and incubate at 37°C for 15 minutes. Add 2 μL of sample solution to the plasma to prepare a final sample concentration of 5 μM. Incubate the above solution at 37°C, and at 0, 15, 30, 60, and 120 minutes, take 50 μL of the solution and add it to 450 μL of ice-cold acetonitrile (containing internal standard). Vortex the solution for 10 minutes, centrifuge, and determine the residual content of the original substrate by LC-MS / MS. See Table 10 for specific results.

[0432] Table 10 Plasma stability of the disclosed loading compounds

[0433] Experimental results show that the loaded compound disclosed herein has good plasma stability.

[0434] 5. Repeated-dose toxicity test of the loading compound used in the ADC of this disclosure in rats.

[0435] Preparation of test sample: Weigh the test sample into a glass bottle according to the predetermined weight; first add 1 mL DMSO and mix well. After the test sample dissolves, add 1 mL Solutol HS-15 and mix well. Then add 8 mL sodium chloride injection and vortex mix well to obtain a clear and transparent solution.

[0436] SD rats (purchased from Vital River Pharmaceuticals, Beijing) were used as test animals. Compound 2 and the positive control Dxd were administered via tail vein injection for 7 consecutive days. Two doses of compound 2 and the positive control Dxd were set at 0.3 mg / kg and 1.0 mg / kg respectively. During the acclimatization period, cage-side observation was conducted once daily; during the drug administration period, all animals were clinically observed twice daily, once in the morning and once in the afternoon. Responses were recorded to various sites including skin, coat, eyes (sclera), ears, nose, mouth, chest, abdomen, urogenital tract, and limbs, as well as to changes in respiration, movement, urination, defecation (urine and fecal color, etc.) and behavior. Daily weight data for the main experimental group was recorded; for animals scheduled for dissection, their final weight was measured before dissection (after fasting). Food intake was also recorded. Specific results are shown in Table 11.

[0437] Table 11 Results of repeated-dose toxicity studies of the loaded compounds disclosed herein in rats. In this context, ♂ represents male and ♀ represents female;

[0438] At the end of administration, gross autopsy was performed on day 8. It was found that some of the dead rats in the Dxd control group had focal / punctate dark red discoloration in their lungs, while no such symptom was found in the compound 2 loading group.

[0439] The above experimental data demonstrate that the loading compound 2 used in the ADC of this disclosure has lower in vivo toxicity and better in vivo safety compared to the positive control Dxd.

[0440] 6. Affinity analysis of anti-EGFR / c-MET bispecific antibodies with FcγRs (FcγRIa, FcγRIIIa(F), FcγRIIIa(V))

[0441] Deactivating the immune-related function of the Fc receptor (Fc silencing) is an effective way to reduce the uptake of ADC drugs by immune cells, and is expected to improve load-induced hematologic toxicity and gastrointestinal reactions. Therefore, the Fc fragment of the EGFR / c-MET bispecific antibody disclosed in this paper has undergone amino acid substitutions (TM mutations) of L234F, L235E, and P331S to remove the binding of Fc to Fcγ receptors. To evaluate the effect of Fc silencing, the binding activity of TM-mutated naked antibodies and non-TM-mutated naked antibodies to different Fcγ receptors was detected using the SPR method.

[0442] FcγRIa was captured on the experimental channels of the chip using His capture. The bispecific antibody samples were diluted to their highest concentrations with running buffer (due to significant differences in affinity for FcγRIa among different samples, M-06C12-PTM and T-E6-H6 were diluted to a maximum concentration of 20 nM, and M-06C12-PTM and T-E6-H6-TM samples were diluted to a maximum concentration of 500 nM). Six serial dilutions were then performed, and samples of different concentrations were sequentially passed through the reference and experimental channels for binding and dissociation. The binding time was 180 s, and the dissociation time was 300 s. Finally, regeneration was achieved with 50 mM HCl. The resulting multi-concentration kinetic curves were analyzed using a 1:1 binding model.

[0443] The binding of FcγRIIIa(F) was carried out in a similar manner. FcγRIIIa(F) (0.6 μg / ml) was first captured in the experimental channel using the His tag capture method. Then, serially diluted antibody (concentration 3200-100 nM) was injected into the reference channel and the experimental channel. Binding was performed for 90 s, dissociation for 100 s, and regeneration was carried out with 50 mM HCl.

[0444] Similarly, FcγRIIIa(V) (0.15 μg / ml) was captured in the experimental channel using the His capture method. Then, serially diluted antibody (concentration 3200–100 nM) was injected into the reference and experimental channels. Binding was performed for 90 s, dissociation for 100 s, and regeneration was carried out with 50 mM HCl.

[0445] The experimental results are shown in Table 12: The naked antibody with TM mutation did not bind significantly to FcγRIa, FcγRIIIa(F), and FcγRIIIa(V), while the naked antibody without TM mutation bound to the above three receptors.

[0446] Table 12. Naked antibodies against TM mutations showed no significant binding to FcγRs (FcγRIa, FcγRIIIa(F), FcγRIIIa(V)).

[0447] 7. Bispecific antibody ADC in vitro cell binding activity

[0448] Materials sourced from: HCC-827, Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; H1975, Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; KYSE30, Nanjing Kebai Biotechnology Co., Ltd.; EBC-1, Nanjing Kebai Biotechnology Co., Ltd.

[0449] FACS was used to detect the binding activity of bispecific antibody-drug conjugate (ADC) and naked antibody to cells. Four different expression types of tumor cells were selected for binding activity detection: HCC827 (EGFR c-MET co-high expression, EGFR>c-MET), H1975 (EGFR c-MET co-low expression), KYSE30 (EGFR mono-high expression, EGFR>>c-MET), and EBC-1 (c-MET high expression, c-MET>EGFR).

[0450] Cells were collected and resuspended in FACS buffer (PBS + 1% BSA + 0.5mM EDTA). After adjusting the cell concentration, cells were added to 96-well plates at a density of 1E5 cells per well. Then, serially diluted antibodies were added at pre-set concentrations. After incubation at 4°C for 1 hour on a shaker, the cells were washed twice by centrifugation with FACS buffer. Then, 100 μL of fluorescently labeled anti-human IgG secondary antibody was added to each well. After incubation at 4°C for 1 hour on a shaker, the cells were washed twice by centrifugation with FACS buffer and filtered into new 96-well plates. The prepared samples were then analyzed using flow cytometry. The mean fluorescence intensity (MFI) for each concentration was calculated using software, and the half-maximal binding concentration (EC50) was calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 13.

[0451] Table 13. Binding activity of bispecific antibody ADCs to cells.

[0452] Experimental results showed that both types of bispecific antibody ADCs had cell-binding activity that was essentially equivalent to that of naked antibodies.

[0453] 8. Bispecific antibody ADC in vitro cell-killing activity

[0454] In this experiment, the aim was to evaluate the in vitro potency of EGFR / c-MET bispecific ADCs by measuring IC50. 50IMAX was used to evaluate its in vitro activity. Four cell types were selected for cell killing activity assay: HCC827 (EGFR / c-MET co-high expression, EGFR>c-MET), KYSE30 (EGFR single high expression, EGFR>>c-MET), EBC-1 (c-MET high expression, c-MET>EGFR), and HACAT (immortalized normal skin cells).

[0455] Cells were digested with trypsin, neutralized with fresh culture medium, centrifuged at 1000 rpm, resuspended in culture medium, and counted. For HCC827 and EBC-1 cells, 1000 cells / well were added to each 96-well cell culture plate; for KYSE30 and HACAT cells, 800 cells / well were added. In all 96-well plates, column 11 was left uncoated with culture medium only, and cultured at 37°C for 16 h in 5% CO2. The highest ADC concentration was 200 nM, serially diluted 5-fold (9 concentrations total), incubated at 37°C for 5 days in 5% CO2. 70 μL CTG was added to each well, and the plates were incubated at room temperature in the dark for 10 minutes. A white backing membrane was attached to the bottom of the cell culture plate, chemiluminescence was read, and data were processed. The results are shown in Table 14 and Figures 3-6.

[0456] Table 14. Cell-killing activity of bispecific antibody ADCs Note: " / " indicates that no inhibitory effect was detected;

[0457] The above results indicate that the two bispecific antibody ADCs disclosed herein exhibit superior cytotoxic activity against cells expressing different cell types compared to the control molecule AZD9592. Their cytotoxicity against normal cells is weaker, and their in vitro therapeutic window is superior to that of AZD9592 and BL-B01D1.

[0458] 9. In vivo efficacy study of anti-EGFR / c-MET bispecific antibody ADC

[0459] This disclosure selects four models for in vivo drug efficacy evaluation: HCC827 co-high expression CDX model, FaDu EGFR single high expression CDX model, EBC-1 high expression CDX model, and H1975 (L858R / T790M / C797S) co-low expression - third-generation EGFR-TKI osimertinib resistance CDX model (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.); Figure 7 shows the IHC staining results performed according to the standard procedure.

[0460] The above-mentioned cells were seeded subcutaneously in the right rib area of ​​Balb / c nude mice, and the average tumor volume was approximately 200 mm. 3The mice were then divided into groups of 6-8 model mice. Each group received a single intravenous dose of ADC (0.1 mL / 10 g) per mouse. Daily behavioral monitoring was conducted for 24 days. Throughout the experiment, the long and wide diameters of the tumor were measured twice weekly using calipers, and the tumor volume (mm²) was also measured. 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 The relative tumor inhibition rate (TGI%) was calculated as follows: TGI% = (1 - T / C) × 100%. T / C% represents the relative tumor growth rate, which is the percentage of tumor volume or weight in the treatment group and the PBS control group at a certain time point. T and C represent the tumor volume (TV) or tumor weight (TW) in the treatment group and the PBS control group at a specific time point, respectively. All data are expressed as Mean ± SEM. The student's t-test was used to compare whether there was a significant difference in tumor volume and weight between the treatment group and the control group, with p < 0.05 indicating a significant difference. Specific measurement results are shown in Table 15 and Figures 8-14.

[0461] Table 15. Coupled products obtained in this disclosure and their properties

[0462] The results are shown in Figures 8, 9, and 10. In the HCC827 co-expression of CDX model, all groups showed significant antitumor effects. As shown in Figure 8, at a single dose of 3 mg / kg, ADC-1 was more effective than AZD9592 in vivo. As shown in Figure 9, at a single dose of 3 mg / kg, ADC-1 was more effective than BL-B01D1 in vivo. As shown in Figure 10, at a single dose of 2 mg / kg, ADC-1 and ADC-2 were comparable in efficacy.

[0463] As shown in Figure 11, in the CDX model of H1975 co-lower expression and osimertinib resistance, the third-generation EGFR-TKI osimertinib was ineffective at a daily dose of 10 mg / kg. Other ADC drugs all showed significant antitumor effects; at a single dose of 3 mg / kg molar concentration, the in vivo efficacy of ADC-1 was comparable to that of ADC-2, and the in vivo efficacy of the two bispecific antibody ADCs disclosed in this paper was superior to that of the positive controls AZD9592 and BL-B01D1.

[0464] As shown in Figure 12, in the FaDu EGFR-overexpressing CDX model, AZD9592 showed weak antitumor activity at a single dose of 1 mg / kg, while ADC-1 and ADC-2 exhibited significant antitumor activity, with ADC-1 showing better in vivo efficacy than ADC-2. As shown in Figure 13, at a single dose of 3 mg / kg, ADC-1 showed better in vivo efficacy than BL-B01D1.

[0465] As shown in Figure 14, in the CDX model with EBC-1 / c-MET predominant expression, at a single dose of 1 mg / kg, the in vivo efficacy of the two bispecific antibody ADCs disclosed herein was superior to the positive control AZD9592, and also superior to the higher dose of BL-B01D1.

[0466] The above results indicate that the two bispecific antibody ADCs disclosed herein have superior antitumor effects compared to the positive control molecules AZD9592 and BL-B01D1.

[0467] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this disclosure and are covered by the claims.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof, or its stereoisomers: in, Ab represents an antibody or antigen-binding fragment, and includes an anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment; L represents a linker connecting Ab and the warhead drug molecule; n is selected from 4-9; R1 is selected from H, halogens, OH, SH, NH2, and C. 1-4 Alkyl, C 1-4 Halogenated alkyl or C 1-4 Alkoxy; R2 is selected from H, halogens, and C. 1-4 Alkyl or C 1-4 Alkoxy; Alternatively, R1 and R2 can cyclize to form -O-(CH2). m -O-, where m is selected from 1, 2, or 3; R3 and R4 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl or C 1-4 Halogenated alkoxy groups; Alternatively, R3 and R4 can cyclize to form -(CH2). k - where k is selected from 1, 2, 3, 4; X is selected from H, OH, HO-CH(R5)-(CH2). p -CO-NH- or -N(R6)(R7), where p is selected from 0, 1, or 2; R5 is selected from H and C. 1-4 Alkyl, C 1-4 Halogenated alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl; R6 is selected from H and C. 1-4 Alkyl or C 1-4 Halogenated alkyl groups; R7 is selected from H or R8-S(O)2-; R8 is selected from C 1-4 alkyl; And t is selected from 0, 1, 2, 3, 4, 5.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein R1 is C 1-4 Alkyl group, and R2 is a halogen; or R1 is methyl group, and R2 is F; or R1 is H and R2 is H; or R1 and R2 are cyclized to form -O-CH2-O-; or R1 is NH2 and R2 is H or a halogen; or R1 is NH2 and R2 is H or F.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein R3 is H and R4 is H; or R3 is H and R4 is C. 1-4 Alkyl; R3 is H, R4 is methyl; or R3 and R4 are cyclized to form -CH2-CH2-.

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein X is HO-CH(R5)-(CH2). p -CO-NH-, where p is selected from 0, 1, 2; or X is selected from OH or NH2; or X is H and t is 0; or X is -N(R6)(R7).

5. The compound or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-4, wherein n is selected from 4.0-7.0 or 7.0-9.0, or n is selected from 5.0-7.0, or 5.0-6.0, or 5.3-5.9, or 5.5-5.8, or 5.6-5.8; or n is selected from 7.0-8.5, or 7.2-7.8, or 7.3-7.6, or 7.4-7.

6.

6. The compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of claims 1-5, wherein the compound is a compound of formula Ia, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, R1, R2, R3, and R4 are as defined in any one of claims 1-3; X1 is selected from chemical bonds, -O-CH(R5)-(CH2). p -CO-, where the -CO- end is connected to -NH-; p is selected from 0, 1, 2; R5 is selected from H, C 1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, or its stereoisomers, wherein R1 is selected from methyl or methoxy; or R2 is selected from F or Cl; or R1 is methyl and R2 is F; or R3 and R4 are cyclized to -(CH2). k - where k is 2; or, R3 is H; and R4 is H.

8. The compound according to any one of claims 6-7, or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein X1 is -O-CH(R5)-(CH2). p -CO-, p is selected from 0 or 1, R5 is selected from H, C 1-4 Alkyl or 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or 3-6 membered cycloalkyl; or, X1 is -O-CH(R5)-CO-, R5 is selected from H or cyclopropyl; or, X1 is -O-CH(R5)-CH2-CO-; R5 is C 1-4 Alkyl group, or X1 is -O-CH(R5)-CH2-CO-, where R5 is methyl; or X1 is selected from the following groups: in, The -CO- end of X1 is connected to NH.

9. The compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of claims 1-8, wherein the compound is a compound of formula Ia-1 or formula Ia-2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, Ab, L, R1, R2, X1, n are as defined in any one of claims 1-8.

10. The compound according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein... Selected from the following compound fragments: in This indicates the position where the linker L is connected via a chemical bond.

11. The compound or its pharmaceutically acceptable salt, or its stereoisomer, according to any one of claims 1-10, characterized in that, The anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment has two homologous heavy chains and two homologous light chains, the amino acid sequence of the heavy chains being shown in SEQ ID NO:1 and the amino acid sequence of the light chains being shown in SEQ ID NO:2; or the anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment has two heterologous heavy chains and one light chain, wherein the amino acid sequence of one heavy chain is shown in SEQ ID NO:3, the amino acid sequence of the other heavy chain is shown in SEQ ID NO:4, and the amino acid sequence of the light chain is shown in SEQ ID NO:

5.

12. The compound or a pharmaceutically acceptable salt thereof, or its stereoisomer, according to any one of claims 1-11, wherein L is -L1-L2-L3-L4-, wherein, One end of L1 is connected to the Ab, and one end of L4 is connected to the warhead drug molecule; L1 is selected from the following groups, and the end of L1 marked with an asterisk * is connected to the Ab: L2 is selected from chemical bonds, -N(R) 10 )-CH2-CO-、 Furthermore, one end of the CO pin of L2 is connected to L3, and the other end is connected to L1; R 10 Selected from C 1-4 Alkyl, C 1-4 Haloalkyl; L3 is selected from polypeptide residues consisting of 2-6 amino acids, wherein the C-terminus of the polypeptide residue is connected to L4; and L4 is selected from chemical bonds, -NH-CH2-, One end of the CH2 component is connected to the warhead drug molecule, and the -NH- end is connected to L3.

13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein L2 is a chemical bond; or, L2 is -N(CH3)-CH2-CO-; or / and L3 is selected from the following polypeptide residues: GFG, GGFG, GGGFG, GGVA, V-Cit, VA; or, L1 is selected from L2 is selected from chemical bonds or -N(CH3)-CH2-CO-, and L3 is selected from GFG, GGFG, GGGFG, and L4 is -NH-CH2-.

14. The compound according to any one of claims 1-13, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein L is selected from the following fragments: in, The asterisk (*) indicates the position where the connector is connected to the warhead drug molecule, while the other end indicates the position where the connector is connected to the Ab.

15. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, or its stereoisomer, wherein, Selected from the following structural fragment:

16. The compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of claims 1-15, wherein the compound is selected from the group consisting of a compound or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: in, n is selected from 4.0-7.0, 7.0-9.0, or n is selected from 5.0-7.0, 5.0-6.0, 5.3-5.9, 5.5-5.8, 5.6-5.8; or n is selected from 7.0-8.5, 7.2-7.8, 7.3-7.6, 7.4-7.

6.

17. A pharmaceutical composition comprising the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a pharmaceutically acceptable excipient.

18. Use of the compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof, its stereoisomers, and the pharmaceutical composition of claim 17 in the preparation of a medicament for treating tumors.

19. A method of treating tumors, comprising administering to a patient in need an effective amount of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a pharmaceutical composition of claim 17.

20. The compound of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or the pharmaceutical composition of claim 17, for the treatment of tumors.

21. An anti-EGFR / c-MET bispecific antibody or its antigen-binding fragment thereof, having two homologous heavy chains and two homologous light chains, wherein the amino acid sequence of the heavy chains is shown in SEQ ID NO:1 and the amino acid sequence of the light chains is shown in SEQ ID NO:2; or the anti-EGFR / c-MET bispecific antibody or its antigen fragment thereof has two heterologous heavy chains and one light chain, wherein the amino acid sequence of one heavy chain is shown in SEQ ID NO:3, the amino acid sequence of the other heavy chain is shown in SEQ ID NO:4, and the amino acid sequence of the light chain is shown in SEQ ID NO:

5.

22. An antibody-drug conjugate comprising the bispecific antibody of claim 21 or an antigen-binding fragment thereof.

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