A triptolide drug conjugate

Abstract

The present application relates to the technical field of medicine, in particular to a triptolide ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a use thereof in medicine. The present application also relates to a triptolide linker-drug compound or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, and a use thereof in medicine.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to triptolide ligand-drug conjugates, triptolide linker-drug compounds, and their uses in medicine. Background Technology

[0002] Ligand-drug conjugates (ADCs) are a novel class of targeted therapeutic agents, consisting of three parts: a ligand (including antibodies, antigen-binding fragments, peptides, cyclic peptides, polycyclic peptides, or small molecules), a linker, and a cytotoxic drug (payload / warhead). Combining the high selectivity of ligands with the high activity of cytotoxic drugs, ADCs have become a research hotspot in targeted cancer therapy. Antibody-drug conjugates (ADCs) link biologically active small-molecule drugs to antibodies via linkers. The antibody then acts as a carrier, targeting and delivering the small-molecule drug to target cells. There are two main types of linkers: cleavable linkers and non-cleavable linkers. In recent years, ADCs have developed rapidly and are playing an increasingly important role in targeted cancer therapy.

[0003] As a key component of ligand-drug conjugates (ADCs), the warhead directly affects the therapeutic efficacy of ADCs. Most ADCs that have been successfully marketed or entered clinical trials use microtubule inhibitors and DNA damaging agents as warheads, such as calicutamines, maytansinoids, auristatins, pyrrolobenzodiazepine dimers, and camptothecins. These have a single mechanism of action, and long-term use easily leads to drug resistance. Therefore, developing highly effective, low-toxicity ligand-drug conjugates that overcome drug resistance using warheads with different mechanisms of action is a promising strategy for cancer treatment.

[0004] Tripterygium wilfordii (a Chinese medicinal plant) Tripterygium wilfordii Tripterygium ligands are a common research subject for anti-tumor drugs. Their main active ingredient is triptolide, also known as triptolide, which possesses anti-inflammatory, anti-tumor, and immunosuppressive activities. However, studies have found that triptolide has significant toxic side effects and a very narrow therapeutic window, which severely limits its clinical development and utilization. Therefore, the development of triptolide ligand-drug conjugates is of great clinical significance. Summary of the Invention

[0005] One of the technical problems to be solved by this invention is to explore and discover better antitumor triptolide drug conjugates, especially triptolide ADC drugs, so that they have higher safety and efficacy, overcome drug resistance, and better meet clinical needs.

[0006] In a first aspect, the present invention provides ligand-drug conjugates as shown in general formula I, or pharmaceutically acceptable salts or solvates thereof.

[0007] in: Tg is a ligand unit selected from antibodies, antigen-binding fragments, polypeptides, cyclic peptides, polycyclic peptides, or small molecules; the antibody is selected from murine antibodies, rabbit antibodies, phage-displayed antibodies, yeast-displayed antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0008] M4 is the connector unit between Tg and L4; L4 is the connection unit connecting M4 and A2; A2 is the spacing unit between L4 and L5; L5 is the connection part that connects A2 and D; D is Or its stereoisomers or derivatives; q is selected from integers or decimals from 1 to 10.

[0009] In some implementations, M4 is selected from the following structures: , , , , , , , , , , , , , , and ; M4's position 1 is connected to Tg, and its position 2 is connected to L4; Q is selected independently from the following structures: or

[0010] A is selected from the following structure: and ; Preferably, A is selected from the following structures: , , and ; X1 can be O, S, NH or CH2 independently; X2 can be independently O, S, NH, -C(O)- or CH2; The 1-position of Q is connected to the amino group of Tg, and the 3-position is connected to the N atom; a are integers independently selected from 0 to 24; b are each independently selected from integers between 0 and 6; c are each independently selected from integers between 0 and 24; d are each independently selected from integers between 0 and 10; e is independently selected from integers between 0 and 24; Each t is independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type; When the M4 structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, that is, each maleimide can be independently in an open-ring or closed-ring form. Specifically, each can be independently... or .

[0011] In some implementations, M4 is selected from the following structures: , , , , , , , , , , , and ; M4's position 1 is connected to Tg, and its position 2 is connected to L4; Q is selected independently from the following structures: or

[0012] A is selected from the following structure: , , and ; X1 can be O, S, NH or CH2 independently; X2 can be independently O, S, NH, -(CO)- or CH2; The 1-position of Q is connected to the amino group of Tg, and the 3-position is connected to the N atom; a are integers independently selected from 0 to 24; b are each independently selected from integers between 0 and 6; c are each independently selected from integers between 0 and 24; d are each independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type; When the M4 structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, that is, each maleimide can be independently in an open-ring or closed-ring form. Specifically, each can be independently... or .

[0013] In some implementations, M4 is selected from the following structures: , , , , and ; M4's position 1 is connected to Tg, and its position 2 is connected to L4; Q is selected independently from the following structures: or

[0014] A is selected from the following structure: , , and ; X1 can be O, S, NH or CH2 independently; X2 can be independently O, S, NH, -(CO)- or CH2; The 1-position of Q is connected to the amino group of Tg, and the 3-position is connected to the N atom; a are integers independently selected from 0 to 24; b are each independently selected from integers between 0 and 6; c are each independently selected from integers between 0 and 24; d are each independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type; When the M4 structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, that is, each maleimide can be independently in an open-ring or closed-ring form. Specifically, each can be independently... or .

[0015] In some implementations, 'a' is independently 0, 1, 2, 3, or 4.

[0016] In some implementations, b is independently 0, 1, 2, 3 or 4.

[0017] In some implementations, c is independently 0, 1, 2, 3 or 4.

[0018] In some implementations, d is independently 0, 1, 2, 3 or 4.

[0019] In some implementations, e is independently 0, 1, 2, 3 or 4.

[0020] In some implementations, t is independently 0, 1, 2, 3 or 4.

[0021] In some implementations, M4 is selected from the following structures: , , , and ; M4's position 1 is connected to Tg, and its position 2 is connected to L4; When the M4 structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, that is, each maleimide can be independently in an open-ring or closed-ring form. Specifically, each can be independently... or .

[0022] In some implementations, L4 is a covalent bond or ; L4's position 2 is connected to M4, and its position 4 is connected to A2; e is an integer selected from 0 to 24; t are each independently selected from integers between 0 and 10.

[0023] In some implementations, L4 is a covalent bond.

[0024] In some implementations, L4 is Position 2 of L4 is connected to M4, and position 4 is connected to A2.

[0025] In some embodiments, A2 is a spacer unit formed by a covalent bond or 1-6 amino acids or their derivatives, wherein A2 is optionally substituted with R6; R6 is OH or ; R7 is selected from C 1-6 Alkoxy, carboxyl, monosaccharide, or disaccharide; f is an integer selected from 1 to 30.

[0026] In some implementations, A2 is selected from the following structures: , , , , , , ,and ; Wherein, R6 is OH, COOH, or R7 is selected from C 1-6 Alkoxy, carboxyl, monosaccharide, or disaccharide; Position 4 is connected to L4, and position 7 is connected to L5; f is an integer selected from 1 to 24; s is an integer selected from 0 to 1.

[0027] In some implementations, f is selected from an integer between 11 and 24, such as 11, 12, 23, or 24.

[0028] In some implementations, A2 is selected from the following structures: , , , , , , ,and ; Wherein, R6 is OH or R7 is selected from C 1-6 Alkoxy, carboxyl, monosaccharide, or disaccharide; Position 4 is connected to L4, and position 7 is connected to L5; f is an integer selected from 1 to 24; s is an integer selected from 0 to 1.

[0029] In some implementations, A2 is Position 4 is connected to L4, and position 7 is connected to L5.

[0030] In some embodiments, L5 is selected from non-cleavable linkers and cleavable linkers, wherein the cleavable linker can be cleaved by acid or by an enzyme present in a pathological environment, the enzyme being selected from proteases, phosphatases, pyrophosphatases, β-glucuronidases, β-galactosidases, and sulfatases.

[0031] In some implementations, L5 is , in, Lh is a covalent bond. , , , , and ; in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v are each independently selected from 0-10; The chiral carbon at the position indicated by $ can be either R-type or S-type. Li is selected from covalent bonds, amino acids or their derivatives, and peptide fragments formed by two or more amino acids, wherein each amino acid is independently selected from valine (Val), citrulline (Cit), glutamic acid (Glu), lysine (Lys), arginine (Arg), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), asparagine (Asn), and glutamine (Gln); Preferably, Li is covalently bonded or selected from the following structures: , , , , , , , , and ; Among them, R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; Lj is a covalent bond or selected from the following structures: , , , and ; in, R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; R 10 Selected from H, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0032] In some implementations, L5 is , in, Lh is a covalent bond. , , , and ; in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v are each independently selected from 0-10; The chiral carbon at the position indicated by $ can be either R-type or S-type. Li is selected from covalent bonds, amino acids or their derivatives, and peptide fragments formed by two or more amino acids, wherein each amino acid is independently selected from valine (Val), citrulline (Cit), glutamic acid (Glu), lysine (Lys), arginine (Arg), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), asparagine (Asn), and glutamine (Gln); Preferably, Li is covalently bonded or selected from the following structures: , , , , , , , , and ; Among them, R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; Lj is a covalent bond or selected from the following structures: , , , and ; in, R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; R 10 Selected from H, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0033] In some implementations, L5 is , in, Lh is a covalent bond. and ; in, g is an integer selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6). h is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9). k is an integer selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6). Li is selected from covalent bonds, amino acids or their derivatives, and peptide fragments formed by two or more amino acids, wherein each amino acid is independently selected from valine (Val), citrulline (Cit), glutamic acid (Glu), lysine (Lys), arginine (Arg), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), asparagine (Asn), and glutamine (Gln); Preferably, Li is covalently bonded or selected from the following structures: , , , , , , , , and ; Among them, R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; Lj is a covalent bond or selected from the following structures: , , and ; in, R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; R 10 Selected from H and C 1-4 alkyl.

[0034] In some implementations, L5 is selected from the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and , in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v is an integer selected from 0 to 10; R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

[0035] In some implementations, L5 is selected from the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , and , in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v is an integer selected from 0 to 10; R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

[0036] In some implementations, L5 is selected from the following structures: , , , , , , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , and ; in, L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

[0037] In some implementations, L5 is selected from the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ; in, L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

[0038] In some implementations, L5 is

[0039] in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; R4 is selected from H, hydroxyl group, or ; L5's 7th position is connected to A2, and its 8th position is connected to D.

[0040] In some implementations, L5 is .

[0041] In some embodiments, D is selected from the structure shown in Formula A or its stereoisomers, deuterated derivatives, and tritated derivatives: (A) in: R8 and R9 are each independently selected from H, D, T, hydroxyl, halogen, or C. 1-6 Alkyl groups; or R8 and R9 forming carbonyl groups or C groups with the carbon atoms they are attached to. 3-6 cycloalkyl; R 11 Selected from H, D, T, hydroxyl, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl; R 12 Selected from H, D, T, hydroxyl, halogen or C 1-6 alkyl.

[0042] In some embodiments, D is selected from the following structures or their stereoisomers, deuterated derivatives, and tritated derivatives: , , , and .

[0043] In some implementations, D is .

[0044] In some implementations, D is .

[0045] In some embodiments, the ligand-drug conjugates of the present invention are selected from the following structures: , , , , , , , , , , , , , , , , ; in, R4 is selected from H, or ; R8 is selected from H or D; R9 is selected from hydroxyl or F; c is selected from 1, 2, 3, 4, 5, 6, 7, or 8; e can be independently selected from 1, 2, 3, or 4; f is selected from 11, 12, 23, or 24; q is selected from integers or decimals from 1 to 10; When the ligand-drug conjugate structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, i.e., each maleimide can be independently in an open-ring or closed-ring form; specifically, each can be independently... or ; All chiral carbons can be configured as either R-type or S-type.

[0046] In some embodiments, the ligand-drug conjugate is selected from the following structures:

[0047] q is selected from integers or decimals from 1 to 10; When the ligand-drug conjugate structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, i.e., each maleimide can be independently in an open-ring or closed-ring form; specifically, each can be independently... or .

[0048] The S group attached to Tg originates from the thiol group of Tg, and the NH group attached to Tg originates from the amino group of Tg. Those skilled in the art will understand that... , Same meaning , They have the same meaning.

[0049] In some embodiments, the Tg of the present invention is not limited to being selected from: anti-EGFRvIII antibody, anti-PD 1 antibody, anti-PD L1 antibody, anti-DLL3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM 1 antibody, anti-LRRC15 antibody, anti-LIV 1 antibody, anti-CanAg / AFP antibody, anti-claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-cMET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7 antibody. H3(CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-Trop2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P cadherin antibody, anti-GD3 antibody, anti-Cadherin 6. Antibodies, including anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, anti-CD123 antibody, anti-GPC3 antibody, anti-EGFR×HER3 bispecific antibody, or any combination of antibodies targeting the above targets.

[0050] In a second aspect, the present invention provides a linker-drug compound of Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof.

[0051] in: M4' is a connector that can connect to ligand units; A2, L4, L5, and D are defined as in any embodiment of the present invention.

[0052] In some implementations, M4' is selected from: , , , , , , , , , , , , , , and ; Position 2 of M4' is connected to L4; a is an integer selected from 1 to 24; b is an integer selected from 1 to 6; e is an integer selected from 0 to 24; Each t is independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type.

[0053] In some implementations, M4' is selected from: , , , , , , , , , , , and ; Position 2 of M4' is connected to L4; a is an integer selected from 1 to 24; b is an integer selected from 1 to 6; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type.

[0054] In some implementations, M4' is selected from: , , , , and ; Position 2 of M4' is connected to L4; a is an integer selected from 1 to 24; b is an integer selected from 1 to 6; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type.

[0055] In some implementations, M4' is selected from: , , and ; where position 2 of M4' is connected to L4.

[0056] In some embodiments, the linker-drug compound of the present invention is selected from the following structures: , , , , , , , , , , , , , , , , ; in, R4 is selected from H, or ; e can be selected from 1, 2, 3, or 4; f is selected from 11, 12, 23, or 24; All chiral carbons can be configured as either R-type or S-type.

[0057] In some embodiments, the linker-drug compound is selected from the following structures: .

[0058] In a third aspect, the present invention provides a pharmaceutical composition comprising: (1) The ligand-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvate, or the linker-drug compound of the present invention or its stereoisomer, tautomer, pharmaceutically acceptable salt or solvate; (2) Pharmaceutically acceptable carriers, diluents or excipients; And optional (3) one or more other active ingredients.

[0059] In a fourth aspect, the present invention also provides the use of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvation thereof, or the linker-drug compound of the present invention or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvation thereof, or the use of the pharmaceutical composition of the present invention for the preparation of a medicament for the treatment or prevention of tumors.

[0060] In a fourth aspect, the present invention also provides the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvation thereof, or the linker-drug compound of the present invention or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvation thereof, or the pharmaceutical composition of the present invention for the treatment or prevention of tumors.

[0061] In some embodiments, the tumor is selected from solid tumors or non-solid tumors, such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

[0062] In a fifth aspect, the present invention also provides the use of the linker-drug compound of the present invention or its stereoisomers, tautomers, pharmaceutically acceptable salts or solvates for the preparation of ligand-drug conjugates or their pharmaceutically acceptable salts or solvates; in some embodiments, the ligand-drug conjugate is selected from the ligand-drug conjugates of the present invention or their pharmaceutically acceptable salts or solvates.

[0063] In some embodiments, M4 comprises maleimide. In these embodiments, the ligand-drug conjugate can undergo hydrolysis under readily hydrolyzable conditions, with the hydrolysis site being the maleimide portion of the linker unit. When the ligand-drug conjugate contains multiple linker-drug compounds, the following may occur depending on the degree of hydrolysis: Maleimides are completely non-hydrolyzed, meaning that all maleimides are in closed-ring form. ; Maleimide is not completely hydrolyzed, meaning that some maleimide exists in a closed-ring form. The other part of maleimide is in the ring-opening form. or ; Maleimide is completely hydrolyzed, meaning that all maleimide forms are in the open-ring form. or .

[0064] Therefore, when multiple maleimide-containing linker units M4 (i.e., Ab is connected to multiple maleimide-containing drug-linkers) exist simultaneously in an ADC, these maleimide groups can all be in closed-ring form, some in open-ring form, or all in open-ring form.

[0065] In the above structural formulas of maleimide, the wavy line on the left indicates the connection site to Ab, and the wavy line on the right indicates the connection to other structures in M4.

[0066] In a sixth aspect, this application also provides a method for treating or preventing tumors, comprising administering to a subject in need an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvation thereof, or the linker-drug compound of the present invention or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvation thereof, or a pharmaceutical composition of the present invention. In some embodiments, the subject is a mammal, such as a human.

[0067] When a trade name is used in this invention, the applicant intends to include the formulation of the product under that trade name, the generic drug under that trade name, and the active pharmaceutical ingredient.

[0068] The invention also includes compounds labeled with all pharmaceutically acceptable isotopes of the compound, which are identical to the compound except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compound include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds (e.g., those doped with radioactive isotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and detection. Using positron-emitting isotopes (e.g.) 11 C 18 F, 15 O and 13 Substitution of N) can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, by replacing the previously used unlabeled reagent with a suitable isotopically labeled reagent.

[0069] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0070] The term "ligand" is a targeting agent that specifically binds to a target moiety. The ligand is capable of specifically binding to cellular components or to other target molecules of interest. The target moiety or target is typically located on the cell surface. In some aspects, the role of the ligand is to deliver a drug unit to a specific target cell population with which the ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides, and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies and their antigen-binding fragments. In embodiments where the ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecules or substances. In some embodiments, a linker is covalently linked to a sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine ​​residue that forms an interchain disulfide bond in the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been incorporated into the ligand unit that forms an interchain disulfide bond in the antibody. In another aspect, the sulfur atom is introduced into the cysteine ​​residue of the ligand unit through, for example, site-directed mutagenesis or a chemical reaction.

[0071] The term "drug" refers to cytotoxic drugs, which are molecules that have a strong ability to disrupt the normal growth of tumor or cancer cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also cause apoptosis in normal cells while killing tumor or cancer cells, easily leading to serious side effects.

[0072] The term "ligand-drug conjugate" refers to a molecule formed by linking a ligand to a drug via a stable linker. In this invention, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody, a functional antibody fragment, or a targeting protein linked to a cytotoxic drug via a stable linker.

[0073] The term "antibody" or "functional antibody fragment" within its scope includes any part of an antibody structure. This unit can bind to, reactively associate with, or chelate a receptor, antigen, or other receptor units present in a target cell population. Antibodies can be any protein or protein-like molecule that can bind to, chelate with, or react with a portion of a population of cells to be treated or bioengineered.

[0074] The antibodies of the present invention include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0075] The amino acid three-letter codes and single-letter codes used in this invention are as follows: J. boil. Chem 1968, 243 As described in ,3558.

[0076] The term "natural amino acid" refers to amino acids that can be synthesized by biotechnology. Natural amino acids are generally... L -Type, but there are a few exceptions, such as glycine, including both natural and biologically synthesized forms.

[0077] The term "non-natural amino acid" refers to amino acids that can only be synthesized artificially.

[0078] In this document, the drug-to-antibody ratio (DAR) refers to the average amount of drug loaded onto each antibody (e.g., q in Formula I), which can be an integer or a decimal. In some embodiments of the invention, the DAR value is expressed as q, which can be, for example, the average of 1, 2, 3, 4, 5, 6, 7, or 8. The DAR value can be characterized using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC to determine the average amount of drug per molecule after the coupling reaction.

[0079] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 or 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2-dimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2, 2-Dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 (e.g., 1 to 4) carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be replaced at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo. The "alkyl" group may optionally be further substituted by one or more substituents.

[0080] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, wherein the ring of the cycloalkyl group contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. The term "cycloalkyl" may optionally be further substituted with one or more substituents.

[0081] The term "alkoxy" refers to an alkyl group in which at least one C atom is replaced by an O atom, or -O- (cycloalkyl); wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. The "alkoxy" may optionally be further substituted with one or more substituents.

[0082] When the "alkyl", "alkoxy", or "cycloalkyl" mentioned above is substituted, it can be further replaced by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 selected from F, Cl, Br, I, hydroxyl, mercapto, nitro, cyano, amino, C 1-6 Alkylamino, =O, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, -NR q4 R q5 =NR q6 -C(=O)OC 1-6 Alkyl group, -OC (=O)C 1-6 Alkyl, -C(=O)NR q4 R q5 C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl, C 6-10 Aryl, C 5-10 heteroaryl, -C(=O)OC 6-10 Aryl, -OC(=O)C 6-10 Aryl, -OC(=O)C 5-10 heteroaryl, -C(=O)OC 5-10 heteroaryl, -OC(=O)C 3-8 Heterocyclic alkyl, -C(=O)OC 3-8 Heterocyclic alkyl groups, -OC (=O)C 3-8 Cycloalkyl, -C(=O)OC 3-8 cycloalkyl, -NHC(=O)C 3-8 Heterocyclic alkyl groups, -NHC(=O)C6-10 Aryl, -NHC(=O)C 5-10 heteroaryl, -NHC(=O)C 3-8 cycloalkyl, -NHC(=O)C 3- 8 heterocyclic alkyl groups, -NHC(=O)C 2-6 alkenyl or -NHC(=O)C 2-6 The alkynyl group is replaced by a substituent, and the substituent C is described in the figure. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl, C 6-10 Aryl, C 5-10 heteroaryl, -NHC(=O)C 6-10 Aryl, -NHC(=O)C 5-10 heteroaryl, -NHC(=O)C 3-8 Heterocyclic alkyl groups or -NHC(=O)C 3-8 The cycloalkyl group may optionally be further surrounded by one to three elements selected from OH, F, Cl, Br, I, C. 1-6 Alkyl, C 1-6 Alkoxy, -NR q4 R q5 Or replaced by the =O substituent; R q1 Selected from C 1-6 Alkyl, C 1-6 Alkoxy or C 6-10 Aryl; R q2 R q3 Selected from H or C 1-6 Alkyl; wherein, R q4 R q5 Selected from H, C 1-6 Alkyl group, -NH (C=NR) q1 )NR q2 R q3 -S(=O)2NR q2 R q3 -C(=O)R q1 Or -C(=O)NR q2 R q3 The C mentioned therein 1-6 The alkyl group may optionally be further influenced by one or more elements selected from OH, F, Cl, Br, I, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 6-10 Aryl, C 5-10 heteroaryl, C 3-8 cycloalkyl or C 3-8 Substituents of heterocyclic alkyl groups; or Rq4 With R q5 The N atom forms a 3- to 8-membered heterocycle, which may contain one or more heteroatoms selected from N, O or S.

[0083] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0084] The term "hydroxyl group" refers to -OH.

[0085] The term "amino" refers to -NH2.

[0086] The term "nitro" refers to -NO2.

[0087] The term "cyano" refers to -CN.

[0088] The term "carboxyl group" refers to -COOH.

[0089] The term "derivative" refers to a substance that has a similar chemical structure to a compound but also contains at least one chemical group not present in the compound and / or lacks at least one chemical group present in the compound. The compound to which the derivative is compared is called the "parent" compound. Typically, a "derivative" can be generated from a parent compound in one or more chemical reaction steps.

[0090] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including meso compounds, racemic compounds, enantiomers, diastereomers, cis-trans isomers, conformational isomers, or mixtures thereof.

[0091] The term "tautomer" or "tautomer form" refers to structural isomers with different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton transfer, such as keto-enol isomerization, imine-enamine isomerization, lactam-lactamimide isomerization, etc.

[0092] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug compound, or a ligand-drug conjugate). The compound or conjugate may contain at least one amino or carboxyl group and is therefore capable of forming an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to: sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid salts, salicylates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, etc.

[0093] The term "solvent" refers to the linker-drug compound or ligand-drug conjugate formed by one or more solvent molecules in this invention, including but not limited to water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.

[0094] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described in this invention or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components such as physiologically / pharmacologically acceptable carriers and / or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0095] The term "carrier" refers to a system that can alter the way drugs enter the body and their distribution within the body, control the rate of drug release, and deliver drugs to their target sites. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability.

[0096] The term "excipient" refers to any additive or excipient in a pharmaceutical preparation other than the active pharmaceutical ingredient. Examples include binders, fillers, disintegrants, and lubricants in tablets; matrix components in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, penetration enhancers, osmotic pressure regulators, and colorants in liquid preparations.

[0097] The terms "diluent" or "filler" are primarily used to increase the weight and / or volume of a formulation. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug.

[0098] The terms “optional,” “optionally,” “selectively,” or “selectively” mean that the event or condition described below may or may not occur, and the description includes both cases in which the event or condition occurs and cases in which it does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that the alkyl group may or may not be present, and the description includes cases in which the heterocyclic group is substituted with an alkyl group and cases in which the heterocyclic group is not substituted with an alkyl group.

[0099] The term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, narrowing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonging survival compared to expected survival (if no treatment was received).

[0100] The term "subject" refers to a mammal, such as a primate mammal, like a human. In some implementations, the subject (e.g., a human) has a tumor or cancer.

[0101] The term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for treating a disease is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc. Detailed Implementation

[0102] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, proportions, ratios, or parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0103] Compound A1

[0104] Step 1: Compound A1-2

[0105] In a 25 mL single-necked flask, A1-1 (1.0 g, 2.51 mmol) and DMF (10 mL) were added and dissolved. After dissolution, propargylamine (200 μL, 3.1 mmol) and EEDQ (1.25 g, 5.0 mmol) were added. The reaction was allowed to proceed overnight at room temperature. HPLC analysis showed that the reaction was complete. The mixture was purified by preparative HPLC and lyophilized to give a white solid compound A1-2 (645 mg, 59% yield).

[0106] Step 2: Compound A1

[0107] Compound A1-2 (500 mg, 1.15 mmol) and DCM (4 mL) were added to a 10 mL single-necked flask. After dissolution, TFA (4 mL) was added in an ice-water bath. The reaction was allowed to proceed at room temperature for 4 h. HPLC analysis showed the reaction was complete. The solution was directly concentrated and lyophilized with water to give compound A1 (349 mg, 109% yield, containing trifluoroacetic acid). LC-MS: 280.1 [M+H] + .

[0108] Compound A2

[0109] Step 1: Compound A2-2

[0110] Weigh serine benzyl ester hydrochloride A2-1 (43.16 mmol, 40 g) and dissolve it in DCM (200 mL). Cool the system to 0 °C and add TEA (90.46 mmol, 12.6 mL) dropwise. After the addition is complete, stir for 20 min. At this temperature, add o-nitrobenzenesulfonyl chloride (45.32 mmol, 10.04 g) in DCM (30 mL). After the reaction is complete as monitored by TLC, quench the reaction by adding saturated NaHCO3 aqueous solution (100 mL). Extract with DCM (100 mL × 2 times). Combine the organic phases, dry with anhydrous Na2SO4, filter the system, concentrate, and purify by column chromatography (PE / EtOAc = 1 / 1). After solvent concentration, obtain an oily liquid compound A2-2. Equivalent yield, LC-MS [M+H] + : m / z 381.1.

[0111] Step 2: Compound A2-3

[0112] At room temperature, PPh3 (27.6 mmol, 7.2 g) was weighed into a three-necked flask. The system was purged under a nitrogen atmosphere, and ultradry THF (80 mL) was added to dissolve it. The system was cooled to 0 °C, and an ultradry THF solution of compound A2-2 (26.3 mmol, 10.0 g) (24 mL) was added dropwise. After 10 min, the addition was completed, and then an ultradry THF solution of DEAD (27.6 mmol, 4.3 mmol) (30 mL) was added dropwise. The reaction was allowed to proceed at this temperature for 1 h, and TLC was used to monitor the reaction until it was complete. The reaction was quenched with saturated NaHCO3 aqueous solution (120 mL), and extracted with EtOAc (120 mL × 2 times). The organic phases were combined, dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE / EtOAc = 7 / 1). After solvent concentration, 8.6 g of yellow solid product compound A2-3 was obtained, with a yield of 91%. LC-MS [M+H] + : m / z 363.5.

[0113] Step 3: Compound A2-5

[0114] At room temperature, compound A2-3 (21.0 mmol, 7.6 g) was weighed and dissolved in THF (200 mL). The system was cooled to 0°C, and then (…) was added. S3-Amino-2-benzyloxycarbonylaminopropionate tert-butyl ester A2-4 (25.3 mmol, 7.44 g), Et3N (4.2 mmol, 0.584 mL). The mixture was allowed to warm to room temperature and reacted overnight. The reaction was monitored by TLC until complete. The reaction mixture was then concentrated and purified by column chromatography (PE / EtOAc = 10 / 1-2 / 1), yielding 9.8 g of a yellow oily liquid compound A2-5, in 71.1% yield. LC-MS [M+H] + : m / z 657.2.

[0115] Step 4: Compound A2-6

[0116] Compound A2-5 (14.8 mmol, 9.72 g) was weighed at room temperature and dissolved in DCM (330 mL). The mixture was cooled to 0 °C, and Et3N (44.7 mmol, 6.21 mL) was added dropwise. After the addition was complete, the mixture was stirred for 20 min, followed by the addition of (Boc)2O (61.0 mmol, 14 mL). The mixture was then heated to 35 °C and reacted for 5.5 h. The product compound A2-6 was purified by direct concentration and column chromatography (PE / EtOAc = 10 / 1-2 / 1) to give 6.4 g of product compound A2-6, with a yield of 57.2%. LC-MS [M+H] + : m / z 757.6.

[0117] Step 5: Compound A2-7

[0118] Compound A2-6 (8.5 mmol, 6.4 g) was weighed at room temperature and dissolved in DMF (120 mL). K2CO3 (170 mmol, 23.4 g) and 4-methoxythiophenol (85 mmol, 10.5 mL) were added sequentially. The reaction was allowed to proceed for 30 min at room temperature, and TLC was used to monitor the reaction until complete. The mixture was extracted three times with water (300 mL) and EtOAc (500 mL). The organic phase was dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE / EtOAc = 10 / 1-5 / 1-2 / 1) to give 4.0 g of oily compound A2-7, with a yield of 82%. LC-MS [M+H] + : m / z 572.0.

[0119] Step 6: Compound A2-8

[0120] Compound A2-7 (3 g, 5.2 mmol) was weighed at room temperature and dissolved in methanol (50 mL). 10% Pd / C (0.52 g) was added, and the system was replaced with an H2 atmosphere. The reaction was allowed to proceed for 15 h, and TLC was used to monitor the reaction until complete. Pd / C was removed by filtration, and the product was concentrated to obtain an oily product A2-8, which could be used directly in the next step without further purification.

[0121] Step 7: Compound A2-9

[0122] At room temperature, crude compound A2-8 (5.2 mmol) was dissolved in DCM (55 mL), followed by the addition of AcOH (26 mmol, 1.49 mL) and maleic anhydride (10.4 mmol, 1.02 g). After reacting at room temperature for 2 h, the reaction was monitored by HPLC to ensure complete reaction. The system was then directly concentrated, purified, and lyophilized to obtain 2.12 g of white solid product A2-9, with a yield of 75%. LC-MS [MH] - : m / z 542.3.

[0123] Step 8: Compound A2-10

[0124] In a 25 mL single-necked flask, add A2-9 (680 mg, 1.26 mmol), toluene (10 mL), and DIPEA (828 μL, 5.01 mmol). Reflux at 120 °C for 1 h, concentrate to remove solvent, and obtain crude compound A2-10. Use directly in the next step.

[0125] Step 9: Compound A2-11

[0126] Compound A2-10 was dissolved in DMF, followed by the addition of propargylamine (97 μL, 1.51 mmol) and EEDQ (932 mg, 3.77 mmol). The reaction was allowed to proceed overnight at room temperature. HPLC analysis confirmed the reaction was complete. The mixture was purified by preparative HPLC and lyophilized to give a white solid, compound A2-11 (200 mg, 39.9% yield). LC-MS: 445.2 [M-Boc+H] + .

[0127] Step 10: Compound A2

[0128] Compound A2-11 (500 mg, 0.919 mmol) and DCM (2 mL) were added to a 10 mL single-necked flask and dissolved. TFA (4 mL) was then added. The reaction was allowed to proceed at room temperature for 3 h. HPLC analysis showed the reaction was complete. After concentration, the mixture was purified by preparative HPLC and lyophilized to give compound A2 (320 mg, yield 89.9%). LC-MS: 389.2 [M+H] + .

[0129] Compound A3

[0130] Step 1: Compound A3-2

[0131] In a 250 mL single-necked flask, A3-1 (21.2 g, 49.8 mmol), propargylamine (3.3 g, 60 mmol), EDCI (14.3 g, 74.7 mmol), HOBt (10.1 g, 74.7 mmol), and THF (100 mL) were added. DIEPA (12.9 g, 99.6 mmol) was added dropwise under an ice-water bath. After the addition was complete, the mixture was allowed to rise to room temperature for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was extracted with EtOAc, dried, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain A3-2 (10.37 g), yield: 45.1%; LC-MS: [M+H] + =463.2.

[0132] Step 2: Compound A3-3

[0133] Add A3-2 (9.44 g, 20.4 mmol) and TFA / DCM (80 mL, 37.5%) to a 50 mL single-necked flask. React at room temperature for 2 h, monitored by TLC. Concentrate under reduced pressure to remove the solvent, and remove TFA three times with DCM. Dry under an oil pump. Pulverize with methyl ether for 2 h. Filter, wash the filter cake, and collect A3-3 (7.54 g), yield: 90.9%, LC-MS: [M+H]. + =407.2.

[0134] Step 3: Compound A3-4

[0135] A3-3 (100 mg, 0.25 mmol), aminododecayl glycol monomethyl ether (149 mg, 0.25 mmol), HATU (190 mg, 0.5 mmol), HOBt (67.5 mg, 0.5 mmol), and 5 mL of DMF were added to a 25 mL single-necked flask. DIPEA (81.4 mg, 0.63 mmol) was added under ice-water bath conditions. After the addition was complete, the mixture was allowed to rise to room temperature and reacted for 1 h. After the reaction was completed as monitored by HPLC, the reaction solution was purified by preparative HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain A3-4 (174 mg), yield: 70.1%; LC-MS: [M+H] + =948.5.

[0136] Step 4: Compound A3-5

[0137] Add A3-4 (150 mg, 0.15 mmol) and MeCN (3 mL) to a 25 mL single-necked flask, and add DBU (27 mg, 0.18 mmol) dropwise under an ice-water bath. After the addition is complete, raise the temperature to room temperature and react for 30 min. Monitor the reaction by TLC until it is complete. The reaction solution of compound A3-5 is ready for use.

[0138] Step 5: Compound A3-8

[0139] A3-6 (155 mg, 0.39 mmol) and MeCN (2.3 mL) were dissolved in a 25 mL single-necked flask. EEDQ (100 mg, 0.41 mmol) was added under an ice-water bath, and the mixture was allowed to react at room temperature for 15 min. This solution was then added dropwise to A3-7 (69 mg, 0.39 mmol) in DMF (2 mL) under an ice-water bath, and the mixture was allowed to react at room temperature for 10 min. The reaction was monitored by HPLC until completion. After the reaction, the reaction solution was directly purified by preparative HPLC. The product solution was collected and lyophilized to obtain A3-8 (150 mg), yield: 69.1%; LC-MS: [MH]. - =556.2.

[0140] Step 6: Compound A3-9

[0141] A3-8 (83 mg, 0.15 mmol), HATU (114 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol), and 5 mL of DMF were added to a 25 mL single-necked flask. The A3-5 reaction solution from step four was added under ice-water bath conditions. After the addition was complete, the mixture was allowed to rise to room temperature and reacted for 1 h. After the reaction was complete as monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution. The preparative solution was lyophilized to obtain A3-9 (140 mg), yield: 71.4%; LC-MS: [M-Boc+H] + =1165.8.

[0142] Step 7: Compound A3

[0143] A3-9 (100 mg, 76.3 μmol) was added to a 25 mL single-necked flask and dissolved in 7 mL of DCM. TFA (3 mL) was added under ice-water bath conditions. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain the preparative solution. The preparative solution was lyophilized to obtain compound A3 (64 mg), yield: 73%; LC-MS: [MH]. - =1107.6.

[0144] Compound A4

[0145] Following the synthetic route of compound A3, compound A4 (93 mg) was obtained; LC-MS: [MH] - =1636.0.

[0146] Compound A5

[0147] Following the synthetic route of compound A3, compound A5 (40 mg) was obtained; LC-MS: [MH] - =1165.8.

[0148] Compound A6

[0149] Following the synthetic route of compound A3, compound A6 (40 mg) was obtained; LC-MS: [MH] - =1694.1.

[0150] Compound A7

[0151] Step 1: Compound A7-2

[0152] In a 250 mL single-necked flask, A7-1 (2.0 g, 13.3 mmol), DCM (130 mL), NPC (di(p-nitrobenzene) carbonate, 4.5 g, 14.6 mmol), and DIPEA (2.3 mL, 13.3 mmol) were added. The reaction was allowed to proceed at room temperature for 1 h. TLC analysis showed the reaction was complete. The mixture was quenched with water, and the organic layer was separated. The aqueous layer was extracted twice with DCM. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by HPLC to give a white solid A7-2 (3.03 g, yield 84.5%).

[0153] Step 2: Compound A7-3

[0154] In a 100 mL single-necked flask, A7-2 (2.0 g, 7.43 mmol), A3-7 (1.32 g, 7.43 mmol), DCM (50 mL), and DIPEA (1.3 mL, 7.43 mmol) were added. The reaction was carried out at room temperature for 1 h. The reaction was monitored by TLC and found to be complete. The solution was directly concentrated under reduced pressure at low temperature, and then purified by HPLC to give a white solid A7-3 (2.02 g, yield 77%).

[0155] Step 3: Compound A7

[0156] In a 25 mL single-necked flask, A7-3 (2 g, 5.65 mmol), HOSu (716 mg, 6.22 mmol), DMF (11 mL), and DCC (1.40 g, 6.78 mmol) were added. The reaction was carried out at room temperature for 1 h. TLC monitoring showed the reaction was complete. The mixture was filtered and purified by HPLC to give a white solid A7 (2.06 g, 81% yield). LC-MS: [M+Na] + =473.3.

[0157] Compound A8

[0158] Step 1: Compound A8-3

[0159] In a 100 mL single-necked flask, add A8-1 (5 g, 10.8 mmol) and DCM (50 mL), then add DBU (1.62 mL, 10.8 mmol) under ice-water bath conditions. Incubate at room temperature for 30 min. TLC analysis showed that the reaction was complete. Reaction solution A was obtained.

[0160] In a 250 mL single-necked flask, A8-2 (5.3 g, 10.8 mmol) and DCM (50 mL) were added. Under ice-water bath conditions, HATU (4.93 g, 13.0 mmol) and HOBt (1.46 g, 10.8 mmol) were added, followed by reaction solution A and DIPEA (2.7 mL, 16.2 mmol). The reaction was allowed to proceed at room temperature for 1 h. TLC analysis showed the reaction was complete. The mixture was quenched with water, and the organic layer was separated. The aqueous layer was extracted twice with DCM. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (DCM:CH3OH = 200:1, 100:1) to obtain a colorless oily substance A8-3 (4.5 g, yield 63%).

[0161] Step 2: Compound A8-4

[0162] In a 100 mL single-necked flask, A8-3 (4.5 g, 6.34 mmol) and DCM (20 mL) were added, followed by TFA (10 mL) under ice-water bath conditions. The reaction was allowed to proceed at room temperature for 1 h, and TLC analysis showed that the reaction was complete. The solvent was removed by concentration, and the mixture was purified by column chromatography (DCM / CH3OH = 80:1, 40:1) to give a white, foamy solid, A8-4 (2.8 g, yield 67.5%).

[0163] Step 3: Compound A8-5

[0164] In a 250 mL solid-phase synthesizer, 7 g of 2-CTC resin (approximately 7 mmol) and 50 mL of DCM were added. The resin was swollen at room temperature for 10 min. After filtration, a solution of A8-4 (2 g, 3.27 mmol) in 50 mL of DCM was added, followed by DIPEA (3.5 mL, 21 mmol). After shaking and reacting at room temperature for 3 h, 10 mL of methanol was added, and the mixture was capped for 30 min. After filtration and washing twice with DMF, A8-5 (resin) was obtained.

[0165] Step 4: Compound A8-6

[0166] Add 50 mL of 20% piperidine DMF solution to the above resin. React at room temperature for 30 min. Filter, wash four times with DMF and twice with DCM to obtain A8-6 (resin).

[0167] Step 5: Compound A8-7

[0168] In a 500 mL single-necked flask, add A2-9 (20 g, 36.8 mmol), toluene (200 mL), and DIPEA (12.8 mL, 73.6 mmol). Reflux at 120 °C for 1 h, concentrate to remove solvent, purify by liquid chromatography, and lyophilize to obtain 15.3 g of A8-7 white solid.

[0169] Step 6: Compound A8-8

[0170] In a 250 mL solid-phase synthesizer, A8-6 (resin) (3.27 mmol), DCM (100 mL), A8-7 (3.44 g, 6.54 mmol), HOPO (1.82 g, 16.4 mmol), and DIC (2.53 mL, 32.7 mmol) were added. The mixture was allowed to react overnight at room temperature with shaking. The reaction was detected by bromophenol blue, indicating completion. The mixture was then filtered, washed three times with DMF, and three times with DCM to obtain A8-8 (resin).

[0171] Step 7: Compound A8

[0172] A 10% DCM solution of hexafluoroisopropanol (100 mL) was added to the above resin. After reacting at room temperature for 1 h, the mixture was filtered to obtain a filtrate. The filter cake was washed twice with DCM, and the filtrates were combined and concentrated to obtain 2.4 g of a colorless oily substance. This oil was purified and lyophilized to give a white solid compound A8 (530 mg, yield 17.4%). LCMS: 921.3 [M+H] + 943.2 [M+Na] +

[0173] Compound A9

[0174] Following the synthetic routes of compounds A3 and A8, compound A9 (167 mg) was obtained; LC-MS: [M+Na] + =1768.9.

[0175] Compound A10

[0176] Step 1: Compound A10-2

[0177] In a 100 mL single-necked flask, A10-1 (5.0 g, 12.43 mmol), A3-7 (2.20 g, 12.43 mmol), DCM (50 mL), and DIPEA (2.2 mL, 12.43 mmol) were added. The reaction was carried out at room temperature for 1 h. The reaction was monitored by TLC and found to be complete. The solution was directly concentrated under reduced pressure at low temperature, and then purified by HPLC to give a white solid A10-2 (5.14 g, yield 89%).

[0178] Step 3: Compound A10

[0179] In a 50 mL single-necked flask, A10-2 (5.0 g, 10.76 mmol), HOSu (1.36 g, 11.84 mmol), and DMF (25 mL) were added. The mixture was cooled in an ice-water bath, and DCC (2.44 g, 11.84 mmol) was slowly added. The reaction was allowed to proceed at room temperature for 1 h. TLC monitoring showed the reaction was complete. The mixture was filtered, diluted with 200 mL of water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give a white solid A10 (5.26 g, 87% yield). LC-MS: [M+Na] + =584.3.

[0180] Compound L1

[0181] Step 1: Synthesis of compound L1-2

[0182] In a 500 mL single-necked flask, L1-1 (12 g, 29 mmol) and methanol (200 mL) were added and dissolved. Then, 5% Pd / C (0.6 g, 5% w / w) was added, and the gas was purged with hydrogen. The reaction was allowed to proceed overnight at room temperature. TLC analysis showed the reaction was complete. Water (100 mL) was added, and the mixture was stirred for 10 min. The mixture was filtered through filter paper to remove palladium on carbon. The filter cake was washed with a small amount of water, and the filtrate was directly concentrated under reduced pressure to obtain a white solid compound L1-2 (7.7 g), which was used directly in the next reaction.

[0183] Step 2: Synthesize compound L1

[0184] In a 250 mL single-necked flask, L1-3 (5.5 g, 27 mmol, commercially available) and acetonitrile (100 mL) were added and dissolved. HOSu (4.66 g, 40.5 mmol) and DCC (8.36 g, 40.5 mmol) were then added. The reaction was allowed to proceed at room temperature for 4 h. The insoluble solids were removed by filtration, and the filter cake was washed with a small amount of acetonitrile. Compound L1-2 (7.5 g, 27 mmol) and DIPEA (6.7 mL, 40.5 mmol) were added to the filtrate. The reaction was allowed to proceed overnight at room temperature. HPLC analysis showed the reaction was complete. The compound was purified by preparative HPLC and lyophilized to give compound L1 (9.2 g, 73% yield); LC-MS: 487.3 [M+Na] + .

[0185] Compound L2

[0186] Step 1: Synthesis of compound L2-3

[0187] L2-1 (548 mg, 1.17 mmol), L2-2 (methyl-docosahexaethylene glycol-amino, 1.28 g, 1.17 mmol), EDCI (336 mg, 1.75 mmol), HOBt (237 mg, 1.75 mmol), and THF (15 mL) were added to a 50 mL single-necked flask. DIEPA (611 μL, 3.51 mmol) was added dropwise under an ice-water bath. After the addition was complete, the mixture was allowed to rise to room temperature for 2 h, monitored by TLC. After the reaction was complete, the mixture was extracted with EtOAc, dried, and concentrated to obtain the crude product. The crude product was purified by HPLC and lyophilized to obtain L2-3 (1.6 g), yield: 88.9%; LC-MS: [M+H] + =1539.0.

[0188] Step 2: Synthesize compound L2-5

[0189] In a 25 mL single-necked flask, L2-3 (991 mg, 0.644 mmol) and acetonitrile (10 mL) were added and dissolved. Then, hexahydropyridine (2.5 mL) was added, and the mixture was reacted at room temperature for 30 minutes. TLC monitoring showed complete disappearance of the starting material. The reaction solution was concentrated under reduced pressure, and then DMF (10 mL), L2-4 (211 mg, 0.644 mmol), and DIEPA (168 μL, 0.966 mmol) were added to the crude product. The mixture was reacted at room temperature for 2 hours, and HPLC analysis showed complete reaction. After HPLC purification, compound L2-5 (505 mg, yield 51.3%) was obtained by lyophilization. LC-MS: [M+H] + =1529.0.

[0190] Step 3: Synthesize compound L2

[0191] In a 25 mL single-necked flask, L2-5 (505 mg, 0.330 mmol) and dichloromethane (5 mL) were added. Trifluoroacetic acid (1 mL) was added under ice bath conditions, and the reaction was carried out at room temperature for 30 minutes. The reaction was monitored by HPLC until complete. The reaction solution was concentrated under reduced pressure and purified by HPLC. The resulting compound, L2 (437 mg, 92.6% yield), was lyophilized. LC-MS: [M+H] + =1428.9.

[0192] Compound L3

[0193] Step 1: Synthesis of compound L3-1

[0194] In a 25 mL single-necked flask, L2-3 (567 mg, 0.368 mmol) and dichloromethane (5 mL) were added, followed by the addition of TFA (1 mL) under ice bath conditions. The reaction was carried out at room temperature for 30 minutes, and HPLC monitoring showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by HPLC, then lyophilized to give compound L3-1 (489 mg, yield 92.3%). LC-MS: [M+H] + =1438.9.

[0195] Step 2: Synthesize compound L3-2

[0196] Succinic anhydride (37 mg, 0.374 mmol) was dissolved in dioxane (2 mL), and a solution of L3-1 (489 mg, 0.340 mmol) in dioxane (4 mL) was slowly added dropwise. The mixture was heated to 80°C and reacted for 30 minutes. The reaction solution was then concentrated under reduced pressure, purified by HPLC, and lyophilized to give compound L3-2 (325 mg, yield 62.2%). LC-MS: [M+H] +=1538.9.

[0197] Step 3: Synthesize compound L3

[0198] In a 25 mL single-necked flask, L3-2 (325 mg, 0.211 mmol) and acetonitrile (4 mL) were added and dissolved. Hexahydropyridine (1 mL) was then added, and the mixture was reacted at room temperature for 30 minutes. TLC monitoring showed complete disappearance of the starting material. The reaction solution was concentrated under reduced pressure and purified by HPLC, then lyophilized to give compound L3 (259 mg, yield 93.3%). LC-MS: [M+H] + =1316.9.

[0199] Compound L4

[0200] Step 1: Compound L4-1

[0201] A3-1 (2.0 g, 4.7 mmol), L2-2 (methyl-docosahexaethylene glycol-amino, 5.12 g, 4.7 mmol), EDCI (992 mg, 5.17 mmol), HOBt (701 mg, 5.17 mmol), and THF (40 mL) were added to a 100 mL single-necked flask. DIEPA (1.65 mL, 9.4 mmol) was added dropwise under an ice-water bath. After the addition was complete, the mixture was allowed to rise to room temperature for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was extracted with EtOAc, dried, and concentrated to obtain the crude product. The crude product was purified by HPLC and lyophilized to obtain L4-1 (6.33 g), yield: 90%; LC-MS: [M+Na] + =1518.0.

[0202] Step 2: Compound L4

[0203] In a 25 mL single-necked flask, L4-1 (5.0 g, 3.34 mmol) and DMF (10 mL) were added. DBU (0.5 mL, 3.34 mmol) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 30 minutes, and TLC was used to monitor the reaction until complete. The reaction solution was purified by HPLC and lyophilized to give compound L4 (3.53 g, 83% yield). LC-MS: [M+H] + =1273.8.

[0204] Compound L5

[0205] Step 1: Synthesize compound L5-2

[0206] L5-1 (glycyl-glycine, 10 g, 75.68 mmol, 1.00 eq.), NaHCO3 (12.72 g, 151.37 mmol, 2.00 eq.), and H2O (70 mL) were added to a 250 mL single-necked flask. A THF solution of AllocCl (10.95 g, 90.82 mmol, 1.20 eq.) in 35 mL was added at 0 °C. After the addition was complete, the reaction mixture was brought to room temperature and reacted for 5 h. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the mixture was acidified to pH 5 with 1 M HCl. The precipitate was collected by filtration and treated with 1 M HCl (2... Wash with 50 mL of water. Dry the solid to give L5-2 (9 g), yield: 55%; LC-MS: [M+H] + =217.2.

[0207] Step 2: Synthesize compound L5-3

[0208] In a 500 mL single-necked flask, L5-2 (9 g, 41.62 mmol, 1.00 eq.), Cu(OAc)2 (0.76 g, 4.16 mmol, 0.1 eq.), and THF (220 mL) were added. The mixture was heated to 60 °C for 1 h under N2 protection. The reaction solution was then cooled to room temperature, and Pd(OAc)4 (22.15 g, 49.95 mmol, 1.2 eq.) was added. The mixture was stirred at room temperature for 1 h. After the reaction was confirmed to be complete by LCMS, the reaction solution was filtered, and the filter cake was treated with MeOH (3... Wash with 50 mL of solution, collect all filtrate, and concentrate under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound L5-3 (5 g, yield 52%); LC-MS: [M+Na] + =253.3.

[0209] Step 3: Synthesize compound L5

[0210] In a 25 mL single-necked flask, L5-3 (100 mg, 0.43 mmol, 1.00 eq.) and DCM (1 mL) were added, followed by TMSCl (220 μL, 1.72 mmol, 4.00 eq.) at 0 °C. The reaction mixture was reacted at 0 °C for 2 h under N2 protection. The reaction solution was quenched with methanol and analyzed by LC-MS. The complete conversion of the derivatives to methanol confirmed the complete reaction. The reaction solution was then evaporated to dryness under reduced pressure and used directly in the next step. LC-MS: [M+H] + =203.3 (methanol derivative).

[0211] compound LD2

[0212] Step 1: Compound LD2-2

[0213] Weigh 3.0 g (8.32 mmol) of triptolide LD2-1 and place it in a 250 ml single-necked reaction flask. Dissolve the product in ultra-dry dichloromethane (85 ml), cool to 0 °C, and add dropwise 2,6-dimethylpyridine (1.45 ml, 12.5 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (TBSOTf, 2.3 ml, 10.0 mmol). After reacting for 1 h, monitor the reaction by TLC. The reaction is complete. Concentrate the system and purify by rapid column chromatography (PE:EtOAc = 2:1) to obtain 2.1 g of product LD2-2, yield: 53%.

[0214] Step 2: Compound LD2-3

[0215] LD2-2 (2.0 g, 4.2 mmol) was placed in a 100 ml single-necked reaction flask and dissolved in ultra-dry tetrahydrofuran (42 ml). Under nitrogen protection, the mixture was cooled to -78 °C. After 15 min, lithium diisopropylaminolithium (LDA, 1.26 ml, 5.06 mmol) was added. After reacting for 40 min, LD1-1 ([[2-(Fmoc-amino)acetamido]methyl]acetate, 2.32 g, 6.3 mmol) was added. After reacting for 1 h, the reaction was spotted onto a TLC plate. The reaction was complete. The reaction was quenched with saturated ammonium chloride, extracted with dichloromethane, and purified by column chromatography (PE:EtOAc = 1:1) to obtain 1.22 g of product LD2-3, yield: 37%.

[0216] Step 3: Compound LD2-4

[0217] LD2-3 (1.22 g, 1.56 mmol) was placed in a 25 ml single-necked reaction flask, dissolved in DCM (9 ml), and trifluoroacetic acid (1 mL) was added dropwise. After reacting for 3 h, the reaction was spotted onto a TLC plate. The reaction was then quenched in an aqueous sodium bicarbonate solution under an ice-water bath. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, concentrated directly, and purified by column chromatography to give 948 mg of product LD2-4, yield: 91%.

[0218] Step 4: Compound LD2

[0219] Compound LD2-4 (500 mg, 0.75 mmol) and DMF (4 mL) were added to a 25 mL single-necked flask. DBU (112 μL, 0.75 mmol) was added under an ice-water bath. After the addition was complete, the mixture was brought to room temperature and reacted for 30 min. The reaction was monitored by TLC and recorded as reaction solution 1.

[0220] Compound L1 (350 mg, 0.75 mmol) and DMF (4 mL) were cooled in an ice-water bath. PyBOP (780 mg, 1.50 mmol), HOBt (203 mg, 1.50 mmol), reaction solution 1, and DIPEA (0.26 mL, 1.50 mmol) were added sequentially to the reaction flask. After addition, the mixture was brought to room temperature and reacted for 2 h. The reaction was monitored for completeness by HPLC. The mixture was purified by preparative liquid chromatography and lyophilized to give 482 mg of the white compound LD2 (72% yield). LC-MS [M+Na] was also analyzed. + : m / z 915.5.

[0221] compound LD2

[0222] Step 1: Compound LD2-2

[0223] Weigh 3.0 g (8.32 mmol) of triptolide LD2-1 and place it in a 250 ml single-necked reaction flask. Dissolve the product in ultra-dry dichloromethane (85 ml), cool to 0 °C, and add dropwise 2,6-dimethylpyridine (1.45 ml, 12.5 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (TBSOTf, 2.3 ml, 10.0 mmol). After reacting for 1 h, monitor the reaction by TLC. The reaction is complete. Concentrate the system and purify by rapid column chromatography (PE:EtOAc = 2:1) to obtain 2.1 g of product LD2-2, yield: 53%.

[0224] Step 2: Compound LD2-3A

[0225] LD2-2 (2.0 g, 4.2 mmol, 1.00 eq.) and 20 mL of ultra-dry THF were added to the reaction flask to replace N2. The reaction solution was then cooled to -78 °C. At this temperature, LDA (4 mL, 4.2 mmol, 1.00 eq.) was added, and the reaction was continued at -78 °C for 1 h. Subsequently, a 20 mL solution of L5 THF was added to the reaction solution, and the reaction was continued for another 1 h. After the reaction was confirmed to be complete by LC-MS, H2O (50 mL) was added directly to the reaction solution to quench the reaction. The solution was then extracted twice with EtOAc (50 mL). The organic phases were combined and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase preparative column chromatography and lyophilized to obtain 680 mg of white solid compound LD2-3A, with a yield of 25.13%. LC-MS: [M+H] + =645.5.

[0226] Step 3: Compound LD2-4A

[0227] LD2-3A (600 mg, 0.93 mmol) was placed in a 25 mL single-necked reaction flask, dissolved in DCM (5 mL), and then trifluoroacetic acid (0.4 mL) was added dropwise. After reacting for 3 h, the reaction was spotted onto a TLC plate. The reaction was complete, and the reaction mixture was quenched in an aqueous sodium bicarbonate solution under ice-water bath. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, concentrated directly, and purified by column chromatography to give 460 mg of product LD2-4A, yield: 93.22%. LC-MS: [M+H] + =531.4.

[0228] Step 4: Compound LD2-5A

[0229] Compound LD2-4A (400 mg, 0.75 mmol, 1.00 eq.) and THF (4 mL) were added to a 25 mL single-necked flask. Pd(PPh3)4 (86.7 mg, 0.075 mmol, 0.1 eq.) and phenylsilane (163.4 mg, 1.51 mmol, 2.00 eq.) were added under ice-water bath conditions. After the addition was complete, N2 was introduced, and the mixture was stirred at room temperature for 3 h. After the reaction was confirmed to be complete by LC-MS, the reaction solution was directly purified by reverse-phase preparative column chromatography. The preparative solution was lyophilized to give 256 mg of white solid compound LD2-5A, yield 76.45%. LC-MS: [M+H] + =447.3.

[0230] Step 5: Compound LD2

[0231] Compound L1 (209 mg, 0.45 mmol, 1.00 eq.) and DMF (2 mL) were cooled in an ice-water bath. PyBOP (468.4 mg, 0.90 mmol, 2.00 eq.), HOBt (121.6 mg, 0.90 mmol, 2.00 eq.), a DMF solution of compound LD2-5 (200 mg, 0.45 mmol, 1.00 eq.), and DIPEA (157 μL, 0.90 mmol, 2.00 eq.) were added sequentially to the reaction flask. After addition, the mixture was brought to room temperature and reacted for 2 h. The reaction was monitored for completeness by HPLC. The mixture was purified by preparative liquid chromatography and lyophilized to obtain 182 mg of the white compound LD2, with a yield of 45.29%. LC-MS [M+H] + = 893.8.

[0232] compound LD3

[0233] Step 1: Compound LD3-1

[0234] LD2-1 (triptolide, 1 g, 2.77 mmol, 1.00 eq.) and 80 mL of ultra-dry DCM were added to a 250 mL reaction flask. Under ice bath stirring, Dysmart reagent (1.65 g, 3.89 mmol, 1.40 eq.) was added. After replacing N2 three times, the mixture was stirred at room temperature for 2 h (a white solid precipitated, forming a suspension). TLC monitoring showed that the starting material reacted completely. 50 mL of saturated NaHCO3 solution and 50 mL of saturated Na2S2O3 solution were added to the reaction system under ice bath stirring for 10 min (the reaction solution became clear). After extraction three times with DCM, the organic phases were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness to obtain 1.00 g of white solid compound LD3-1, yield 80.1%. LC-MS: [2M+Na] + =739.1.

[0235] Step 2: Compound LD3-2

[0236] LD3-1 (900 mg, 2.51 mmol, 1.00 eq.) and 15 mL of ultra-dry MeOH were added to a reaction flask and stirred to form a suspension. NaBH4 (190 mg, 5.00 mmol, 2.00 eq.) was added under ice bath conditions. After the addition was complete, N2 was substituted, and the reaction mixture was brought to room temperature and stirred for 4 h. TLC monitoring showed that the starting material reacted completely. A saturated NH4Cl solution was added to the reaction system under ice bath conditions. The mixture was then concentrated under reduced pressure and extracted twice with EtOAc. The combined organic phases were dried over anhydrous Na2SO4, filtered, and evaporated to dryness to obtain 1.15 g of crude product. The crude product was purified by column chromatography to obtain 460 mg of white solid compound LD3-2, with a yield of 47.0%. LC-MS: [M+H] + =361.3.

[0237] Step 3: Compound LD3-3

[0238] LD3-2 (200 mg, 0.55 mmol, 1.00 eq.) and 10 mL of ultra-dry DCM were added to the reaction flask. After replacing the nitrogen with nitrogen, DAST (367 μL, 2.77 mmol, 5.00 eq.) was added with stirring in an ice bath. The reaction mixture was allowed to return to room temperature and reacted for 4 h. TLC monitoring showed that the starting material had reacted completely. Stirring was stopped, and the reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by directional preparative column chromatography. The preparative solution was extracted twice with DCM, and the organic phase was collected and concentrated under reduced pressure to obtain 12 mg of white solid compound LD3-3, with a yield of 5.97%. LC-MS: [M+H] + =363.3.

[0239] Step 4: Compound LD3-4

[0240] Following the synthetic method of LD2-3A, compound LD3-4 was synthesized from compounds LD3-3 and L5. LC-MS m / z (ES+): [M+H] + =533.3.

[0241] Step 5: Compound LD3-5

[0242] Following the synthetic method for LD2-5A, compound LD3-5 was synthesized from compound LD3-4. LC-MS m / z (ES+): [M+H] + =449.3.

[0243] Step 6: Compound LD3

[0244] Following the synthetic method for LD2, compound LD3 was synthesized from compounds LD3-5 and L1. LC-MS m / z (ES+): [M+H] + =895.5.

[0245] Compound LP5

[0246] LD2 (50 mg, 0.056 mmol), A4 (92 mg, 0.056 mmol), copper sulfate pentahydrate (42 mg, 0.168 mmol), sodium ascorbate (33 mg, 0.168 mmol), and DMF (2 mL) were added to a 10 mL reaction tube. Water (0.5 mL) was then added under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature. HPLC analysis showed that the reaction was complete after 30 minutes. The reaction solution was purified by HPLC and lyophilized to obtain LP5 (126 mg), yield: 89%; LC-MS: [M+H] + =2531.5.

[0247] Compound LP6

[0248] Step 1: Compound LP6-1

[0249] LD2 (50 mg, 0.056 mmol), A4-5 (70 mg, 0.056 mmol), copper sulfate pentahydrate (42 mg, 0.168 mmol), sodium ascorbate (33 mg, 0.168 mmol), and DMF (2 mL) were added to a 10 mL reaction tube. Water (0.5 mL) was then added under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature. HPLC showed that the reaction was complete after 30 minutes. The reaction solution was purified by HPLC and lyophilized to obtain LP6-1 (109 mg), yield: 91%; LC-MS: [M+H] + =2148.2.

[0250] Step 2: Compound LP6

[0251] LP6-1 (59 mg, 0.027 mmol), A7 (13 mg, 0.027 mmol), and DMF (2 mL) were added to a 10 mL reaction flask. DIPEA (5 μL, 0.027 mmol) was then added under ice bath conditions. The reaction was allowed to proceed at room temperature after the addition was complete. HPLC analysis showed that the reaction was complete after 30 minutes. The reaction solution was purified by HPLC and lyophilized to obtain LP6 (55 mg), yield: 82%; LC-MS: [M+H] + =2483.5.

[0252] Compound LP7

[0253] LD2 (50 mg, 0.056 mmol), A9 (96 mg, 0.056 mmol), copper sulfate pentahydrate (42 mg, 0.168 mmol), sodium ascorbate (33 mg, 0.168 mmol), and DMF (2 mL) were added to a 10 mL reaction tube. Water (0.5 mL) was then added under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature. HPLC showed that the reaction was complete after 30 minutes. The reaction solution was purified by HPLC and lyophilized to obtain LP7 (126 mg), yield: 86%; LC-MS: [M+H] + =2639.3.

[0254] Compound LP8

[0255] LP6-1 (40 mg, 0.018 mmol), A10 (10.5 mg, 0.018 mmol), and DMF (2 mL) were added to a 10 mL reaction flask. DIPEA (5 μL, 0.027 mmol) was then added under ice bath conditions. The reaction was allowed to proceed at room temperature after 30 minutes. HPLC analysis showed the reaction was complete. The reaction solution was purified by HPLC and lyophilized to obtain LP8 (40 mg), yield: 83%; LC-MS: [M+2H] 2+ / 2=1297.7.

[0256] Compound LP9

[0257] Following the synthetic method for LP5, compound LP9 was synthesized using compounds LD3 and A4 as starting materials. LC-MS m / z (ES+): [M+H] + =2532.4.

[0258] Compound LP10

[0259] Following the synthetic method for LP6, compound LP10 was synthesized using compounds LD3, A4-5, and A7 as starting materials. LC-MS m / z (ES+): [M+H] + =2485.5.

[0260] Compound LP11

[0261] Following the synthetic method for LP7, compound LP11 was synthesized using compounds LD3 and A9 as starting materials. LC-MS m / z (ES+): [M+H] + =2641.4.

[0262] Compound LP12

[0263] Following the synthetic method for LP8, compound LP12 was synthesized using compounds LP10-1 and A10 as starting materials. LC-MS m / z (ES+): [M+H] + =2595.4.

[0264] Antibody T000

[0265] Antibody T000 is an antibody targeting HER2, and its preparation method is as follows: The day before transfection, Expi293 cells were loaded with 0.9 × 10⁻⁶ cells. 6The plasmid was inoculated into the culture medium at a density of 1 / mL and cultured overnight in a CO2 shaking incubator at 37 °C, 5% CO2, and 120 rpm. On the day of transfection, the expression plasmid was transfected using PEI-MAX. On the first day after transfection, 5% (v / v) feed was added. On the third day after transfection, 5% (v / v) feed was added again. On the sixth day after transfection, expression was terminated, and the supernatant was collected by centrifugation.

[0266] The harvested supernatant was purified using Protein A affinity chromatography. After elution with 0.05 M sodium acetate (pH 3.6), the pH was adjusted to 7.0 with 1 M Tris-HCl (pH 8.8), and then impurities such as polymers were removed by gel filtration chromatography.

[0267] The sequence of antibody T000 is as follows:

[0268] Note: CDRs are numbered according to Kabat.

[0269] Antibody H000

[0270] Antibody H000 is an antibody that targets TROP2, and its preparation method is the same as that of antibody T000.

[0271] The sequence of antibody H000 is as follows:

[0272] Note: CDRs are numbered according to IMGT.

[0273] General Method A for Coupling ADC Preparation

[0274] After cell expression and purification via Protein A affinity chromatography and molecular sieve chromatography, the antibody was replaced with 20 mM acetate, pH 6.0 buffer and concentrated or diluted to a protein concentration of 3 mg / mL. The linker-payload was dissolved in DMA to a concentration of 20 mg / mL for later use. To break the interchain disulfide bonds of the antibody, 20 times the molecular weight of TECP was added, and the reaction was carried out at room temperature for 3 hours. Then, 20 times the molecular weight of the linker-payload solution was added, and the reaction was carried out at room temperature for 1 hour. After the reaction, ultrafiltration was performed using 30 kDa ultrafiltration centrifuge tubes to remove the linker-payload that was not conjugated with the antibody, thus obtaining the corresponding antibody-drug conjugate (ADC) sample.

[0275] General Method B for Coupling ADC Preparation

[0276] A certain volume of antibody containing an azide group was taken and diluted with 20 mM sodium acetate buffer (5 mg / mL). Then, 20 times the amount of DMA solution of the linker-drug compound (20 mg / mL) was added according to the molecular ratio, and the reaction was carried out overnight at room temperature. After the reaction was completed, the sample was transferred to 20 mM sodium acetate buffer at pH 6.5 to obtain the corresponding antibody-drug conjugate (ADC) sample.

[0277] The general procedure for preparing antibodies with azido groups is as follows: Take a certain volume of antibody, dilute it with 20 mM sodium acetate buffer (5 mg / ml), add 4-15 eq of DMA solution of the affinity peptide, and incubate at 25°C for 1-2 h. Subsequently, add 4-15 eq of DMA solution of the azido compound, incubate at 25°C for 1-2 h, then add 0.1 M NaHCO3 solution to adjust the pH to 9.0 ± 0.5, and incubate the reaction mixture at 25°C for 2-4 h. After the reaction is complete, terminate the reaction with 0.1 M glycine at pH 2.5.

[0278] General Method for Coupling ADC Preparation C

[0279] Antibody molecules with a monomer content greater than 95% after preliminary purification were transferred to phosphate buffer containing EDTA (10 mg / mL) using an ultrafiltration centrifuge tube. Eight times the molar number of antibody molecules of TCEP were added, and the reaction was carried out at room temperature for 3 hours. The mixture was then transferred to phosphate buffer at pH 6.5 using an ultrafiltration centrifuge tube, and eight times the molar number of antibody molecules of DHA (dehydroascorbic acid) were added, and the reaction was carried out at room temperature for 3 hours. Then, five times the molar number of antibody molecules of the linker-drug compound were added, and the reaction was carried out at room temperature for 3 hours. After the reaction, the mixture was transferred to PBS using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and the unconjugated linker-drug compound was removed to obtain the site-conjugated ADC.

[0280] Preparation of ADC-1

[0281] ADC-1 was prepared by conjugating LP5 with anti-HER2 trastuzumab (purchased from Genentech, batch number: SH0418, hereinafter the same) according to general method A.

[0282]

[0283] Preparation of ADC-2

[0284] ADC-2 was prepared by coupling LP6 with trastuzumab containing an azide group according to general method B.

[0285]

[0286] Preparation of ADC-3

[0287] ADC-3 was prepared by coupling LP6 with trastuzumab containing an azide group according to general method B.

[0288]

[0289] Preparation of ADC-4

[0290] ADC-4 was prepared by conjugation of LP7 and trastuzumab according to general method A.

[0291]

[0292] Preparation of ADC-5

[0293] ADC-5 is prepared by conjugation of LP5 with the anti-TROP2 antibody hu4D3 (the sequence of antibody hu4D3 and its preparation method are described in Example 91 of patent WO2023098889, the contents of which are incorporated herein by reference in their entirety, hereinafter the same) according to general method A.

[0294]

[0295] Preparation of ADC-6

[0296] ADC-6 was prepared by conjugating LP6 with an anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0297]

[0298] Preparation of ADC-7

[0299] ADC-7 was prepared by conjugating LP6 with an anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0300]

[0301] Preparation of ADC-8

[0302] ADC-8 was prepared by conjugating LP7 with the anti-TROP2 antibody hu4D3 according to general method A.

[0303]

[0304] Preparation of ADC-9

[0305] ADC-9 is prepared by conjugating LP5 and antibody T000 according to general method C.

[0306]

[0307] Preparation of ADC-10

[0308] ADC-10 is prepared by conjugation of LP7 and antibody T000 according to general method C.

[0309]

[0310] Preparation of ADC-11

[0311] ADC-11 is prepared by conjugation of LP5 and antibody H000 according to general method C.

[0312]

[0313] Preparation of ADC-12

[0314] ADC-12 is prepared by conjugation of LP7 and antibody H000 according to general method C.

[0315]

[0316] ADC-13

[0317] ADC-13 was prepared by coupling LP8 with trastuzumab containing an azide group according to general method B.

[0318]

[0319] ADC-14

[0320] ADC-14 was prepared by conjugating LP8 with the anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0321]

[0322] Preparation of ADC-15

[0323] ADC-15 is prepared by conjugating LP9 with anti-HER2 trastuzumab using general method A.

[0324]

[0325] Preparation of ADC-16

[0326] ADC-16 is prepared by coupling LP10 with trastuzumab containing an azide group according to general method B.

[0327]

[0328] Preparation of ADC-17

[0329] ADC-17 is prepared by coupling LP10 with trastuzumab containing an azide group according to general method B.

[0330]

[0331] Preparation of ADC-18

[0332] ADC-18 is prepared by conjugation of LP11 and trastuzumab according to general method A.

[0333]

[0334] Preparation of ADC-19

[0335] ADC-19 is prepared by conjugating LP9 with the anti-TROP2 antibody hu4D3 according to general method A.

[0336]

[0337] Preparation of ADC-20

[0338] ADC-20 was prepared by conjugating LP10 with the anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0339]

[0340] Preparation of ADC-21

[0341] ADC-21 was prepared by conjugating LP10 with the anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0342]

[0343] Preparation of ADC-22

[0344] ADC-22 was prepared by conjugating LP11 with the anti-TROP2 antibody hu4D3 according to general method A.

[0345]

[0346] Preparation of ADC-23

[0347] ADC-23 is prepared by conjugating LP9 with antibody T000 according to general method C.

[0348]

[0349] Preparation of ADC-24

[0350] ADC-24 was prepared by conjugating LP11 with antibody T000 according to general method C.

[0351]

[0352] Preparation of ADC-25

[0353] ADC-25 is prepared by conjugation of LP9 and antibody H000 according to general method C.

[0354]

[0355] Preparation of ADC-26

[0356] ADC-12 is prepared by conjugation of LP11 and antibody H000 according to general method C.

[0357]

[0358] Preparation of ADC-27

[0359] ADC-27 is prepared by coupling LP12 with trastuzumab containing an azide group according to general method B.

[0360]

[0361] Preparation of ADC-28

[0362] ADC-28 was prepared by conjugating LP12 with an anti-TROP2 antibody hu4D3 containing an azide group according to general method B.

[0363]

[0364] ADC Evaluation

[0365] 1. Detection of DAR by reversed-phase high-performance liquid chromatography (RP-HPLC)

[0366] Place the sample vials containing the samples onto the sample plate, and set the corresponding position, injection volume, number of injection needles, and injection method for each sample according to the "UPLC Standard Operating Procedure". The DAR value (corresponding to the q value in the ADC example) detection results are shown in Table 1.

[0367] Table 1. Detection results of DAR value of ADC

[0368] The results show that the DAR values ​​of the ADCs disclosed in this invention are stable and close to the theoretical values, indicating that the ADCs of this invention have good uniformity.

[0369] 2. SEC detection of antibody or ADC monomer rate

[0370] Place the sample vials on the sample plate and set the corresponding position, injection volume, number of injection needles and injection method for each sample according to the "UPLC Standard Operating Procedure". The results are shown in Table 2.

[0371] Table 2. Monomer content detection results of ADC

[0372] The results show that the ADCs disclosed in this invention have excellent properties such as low degradation rate, low aggregation rate and high monomer rate, indicating that the ADCs of this invention have high chemical purity and good stability.

[0373] 3. Plasma stability study

[0374] A mixture of ADC and IgG-free plasma was prepared to achieve a final ADC concentration of 0.6 mg / mL. The mixture was incubated in a water bath at 37 ℃ for 0, 3, and 7 days. A control group without plasma incubation was also included. After incubation, the drug-antibody ratio (DAR) was measured after purification and extraction to reflect the stability of the ADC in plasma. The results are shown in Table 3.

[0375] Table 3 Results of in vitro plasma stability test of ADC

[0376] Note: Incubation for 0 days refers to the DAR value measured after the purification and extraction steps begin immediately after the ADC is added to plasma; incubation for 3 days refers to the DAR value measured after the purification and extraction steps begin immediately after the ADC is added to plasma and placed at 37°C for 3 days; incubation for 7 days refers to the DAR value measured after the purification and extraction steps begin immediately after the ADC is added to plasma and placed at 37°C for 7 days. The results show that the ADCs disclosed in this invention have excellent plasma stability, indicating that the ADCs of this invention have good stability and low toxicity.

[0377] 4. ADC anti-tumor cell activity test

[0378] This embodiment utilizes human tumor cell lines from different cancer types and mixed tumor cells (JIMT-1 + MDA-MB-468) as experimental models (cell lines are shown in Table 4) to evaluate the in vitro tumor cell killing activity of antibody-drug conjugates. A certain number of tumor cells were seeded in 96-well plates, and serially diluted test antibodies or ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay, and the half-maximal inhibitory concentration (IC50) was calculated. 50 To evaluate the killing effect of the tested antibodies and ADC drugs on tumor cell lines.

[0379] The initial dose of antibody or ADC drug was 100 nM, diluted 10-fold, with a total of 8 detection concentration points, and the treatment lasted for 5 days. Cell viability = (experimental group - blank) / (control group - blank) × 100%.

[0380] Table 4. Cell line information

[0381] The JIMT-1 + MDA-MB-468 in vitro mixed tumor cell model was constructed according to the following protocol: Collect test cells in the logarithmic growth phase, discard the culture medium, and wash the cells with sterile 1×PBS. Add an appropriate amount of 0.25% Trpsin-EDTA to digest the cells, depending on the size of the culture flask. After digestion, resuspending, and counting the cells, first seed the HER2-moderately expressing breast cancer cell line JIMT-1 into a 96-well plate, then seed the HER2-negative triple-negative breast cancer cell line MDA-MB-468 into the same 96-well plate. Finally, add 300 μL of sterile 1×PBS or sterile ddH2O to the edges of the 96-well plates (excluding those containing cell suspension) for sealing, and incubate overnight in a CO2 incubator until ready for use.

[0382] Table 5. Inhibitory effects of HER2-ADC on the proliferation of different tumor cells.

[0383] Table 6. Inhibitory effects of TROP2-ADC on the proliferation of different tumor cells

[0384] The experimental results are shown in Tables 5 and 6. The results show that the ADCs disclosed in this invention have significant in vitro antitumor activity in a variety of antigen-positive tumor cell lines.

[0385] 5. In vivo efficacy evaluation of ADC in monosomal tumors (JIMT-1)

[0386] This embodiment established a HER2+ human breast cancer cell line JIMT-1 BALB / c-nu mouse subcutaneous xenograft model to evaluate the in vivo efficacy of HER2-ADCs. JIMT-1 (5×10⁻⁶ cells / year) was used as the target cell line. 6 Cell suspension (0.1 mL / mouse) was injected subcutaneously into the right scapula of 6-7 week old BALB / c-nu mice. When the average tumor volume of the mice grew to a suitable size, they were randomly divided into groups and the tested ADC drugs were administered to observe the inhibitory effect on tumor growth and the effect on the body weight of the mice.

[0387] The results showed that the ADCs disclosed in this invention have significant in vivo antitumor activity and safety in the monosomal tumor (JIMT-1) model.

[0388] 6. In vivo efficacy evaluation of ADC in mixed tumors (JIMT-1 + MDA-MB-468)

[0389] This embodiment established a subcutaneous mixed xenograft model of HER2+ human breast cancer cells JIMT-1 and HER2- human breast cancer cells MDA-MB-468 in BALB / c-nu mice to evaluate the in vivo efficacy of HER2-ADCs. A mixture of JIMT-1 and MDA-MB-468 cells (0.1 mL / mouse) was subcutaneously injected into the right scapula of 6-7 week old BALB / c-nu mice. Once the average tumor volume reached a suitable size, the mice were randomly divided into groups, and the inhibitory effect of the tested ADC on tumor growth and its effect on mouse body weight were observed.

[0390] The results showed that the ADCs disclosed in this invention have significant in vivo antitumor activity and safety in the mixed tumor (JIMT-1 + MDA-MB-468) model.

[0391] 7. In vivo efficacy evaluation of ADC in monosomal tumors (NCI-N87)

[0392] This embodiment uses BALB / c-Nude subcutaneously inoculated with a human gastric cancer cell line (NCI-N87) as an experimental model to evaluate the in vivo efficacy of ADCs. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude. When the tumor volume reached a suitable size, the cells were randomly divided into groups, and the test ADC drugs were administered to observe their inhibitory effect on tumor growth and their effect on mouse body weight.

[0393] The results showed that the ADCs disclosed in this invention have significant in vivo antitumor activity and safety in the monosomal tumor (NCI-N87) model.

[0394] 8. In vivo efficacy evaluation of ADC in monosomal tumors (NCI-H1975)

[0395] This embodiment uses BALB / c-Nude subcutaneously inoculated human non-small cell lung cancer cell line (NCI-H1975) as an experimental model to evaluate the in vivo efficacy of ADCs. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude cells. When the tumor volume reached a suitable size, the cells were randomly divided into groups, and the test ADC drugs were administered to observe their inhibitory effect on tumor growth and their effect on mouse body weight.

[0396] The results showed that the ADCs disclosed in this invention have significant in vivo antitumor activity and safety in the monosomal tumor (NCI-H1975) model.

[0397] 9. In vivo efficacy evaluation of ADC in monosomal tumors (A431)

[0398] This embodiment uses BALB / c-Nude subcutaneously inoculated human squamous cell carcinoma cell line (A431) as an experimental model to evaluate the in vivo efficacy of ADCs. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude. When the tumor volume reached a suitable size, the cells were randomly divided into groups, and the test ADC drugs were administered to observe their inhibitory effect on tumor growth and their effect on mouse body weight.

[0399] The results showed that the ADCs disclosed in this invention have significant in vivo antitumor activity and safety in a monotumor (A431) model.

[0400] 10. Pharmacokinetic (PK) evaluation of ADC in mice

[0401] This embodiment evaluates the in vivo pharmacokinetic characteristics of an ADC administered via a single intravenous (IV) injection to BALB / c mice. Pharmacokinetic parameters were calculated by measuring the concentration-time curves of total antibody (tAb), conjugated antibody (cAb, i.e., intact ADC), and free payload in the blood to assess the systemic exposure, clearance, and in vivo stability of the linker of the tested ADC. Healthy BALB / c mice were randomly assigned to groups based on body weight. Blood samples were collected at predetermined time points after a single tail vein administration of the tested ADC, processed, and used for PK parameter determination and calculation.

[0402] The results show that the ADCs disclosed in this invention have good pharmacokinetic characteristics.

[0403] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; the above modifications or equivalent substitutions are all within the protection scope of the present invention.

Claims

1. A ligand-drug conjugate of Formula I or a pharmaceutically acceptable salt or solvate thereof, in: Tg is a ligand unit selected from antibodies, antigen-binding fragments, polypeptides, cyclic peptides, polycyclic peptides, or small molecules; the antibody is selected from murine antibodies, rabbit antibodies, phage-display-derived antibodies, yeast-display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, monospecific antibodies, bispecific antibodies, or multispecific antibodies. M4 is the connector unit between Tg and L4; L4 is the connection unit connecting M4 and A2; A2 is the spacing unit between L4 and L5; L5 is the connection part that connects A2 and D; D is Or its stereoisomers or derivatives; q is selected from integers or decimals from 1 to 10.

2. The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, M4 is selected from the following structure: , , , , , , , , , , , , , , and ; M4's position 1 is connected to Tg, and its position 2 is connected to L4; Q is selected independently from the following structures: or A is selected from the following structure: and ; Preferably, A is selected from the following structures: , , and ; X1 can be O, S, NH or CH2 independently; X2 can be independently O, S, NH, -C(O)- or CH2; The 1-position of Q is connected to the amino group of Tg, and the 3-position is connected to the N atom; a are integers independently selected from 0 to 24; b are each independently selected from integers between 0 and 6; c are each independently selected from integers between 0 and 24; d are each independently selected from integers between 0 and 10; e is independently selected from integers between 0 and 24; Each t is independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type; When the M4 structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, that is, each maleimide can be independently in an open-ring or closed-ring form. Specifically, each can be independently... or .

3. The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, L4 is a covalent bond or ; L4's position 2 is connected to M4, and its position 4 is connected to A2; e is an integer selected from 0 to 24; t are each independently selected from integers between 0 and 10.

4. The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, A2 is a spacer unit formed by a covalent bond or 1-6 amino acids or their derivatives, wherein A2 is optionally substituted with R6; R6 is OH or ; R7 is selected from C 1-6 Alkoxy, carboxyl, monosaccharide, or disaccharide; f is an integer selected from 1 to 30.

5. The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, A2 is selected from the following structure: , , , , , , ,and ; Wherein, R6 is OH, COOH, or R7 is selected from C 1-6 Alkoxy, carboxyl, monosaccharide, or disaccharide; Position 4 is connected to L4, and position 7 is connected to L5; f is an integer selected from 1 to 24; s is an integer selected from 0 to 1.

6. The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, L5 is selected from non-cleavable linkers and cleavable linkers, wherein the cleavable linker can be cleaved by acid or by enzymes present in a pathological environment, and the enzymes are selected from proteases, phosphatases, pyrophosphatases, β-glucuronidase, β-galactosidase, and sulfatases.

7. The ligand-drug conjugate according to claim 6, or a pharmaceutically acceptable salt or solvate thereof, wherein, L5 is , in, Lh is a covalent bond. , , , , and ; in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v are each independently selected from 0-10; The chiral carbon at the position indicated by $ can be either R-type or S-type. Li is selected from covalent bonds, amino acids or their derivatives, and peptide fragments formed by two or more amino acids, wherein each amino acid is independently selected from valine (Val), citrulline (Cit), glutamic acid (Glu), lysine (Lys), arginine (Arg), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), asparagine (Asn), and glutamine (Gln); Preferably, Li is covalently bonded or selected from the following structures: , , , , , , , , and ; Among them, R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; Lj is a covalent bond or selected from the following structures: , , , and ; in, R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; R 10 Selected from H, C 1-6 Alkyl or C 3-6 Cycloalkyl.

8. The ligand-drug conjugate according to claim 7, or a pharmaceutically acceptable salt or solvate thereof, wherein, L5 is selected from the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and ， in, g is an integer selected from 1 to 6; h is an integer selected from 1 to 10; k is an integer selected from 1 to 6; v is an integer selected from 0 to 10; R3 is independently selected from H or C. 1-6 alkyl; R4 is selected from H, hydroxyl group, or ; R5 are each independently selected from C 1-6 Alkyl or C 3-6 cycloalkyl; L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

9. The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 8, characterized in that, L5 is selected from the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and ； in, L5 is connected to A2 at position 7 and to D at position 8; The chiral carbon at the position indicated by $ can have either an R-type or an S-type configuration.

10. The ligand-drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein, D is selected from the structure shown in formula A or its stereoisomers, deuterated products, and tritated products: （A） in: R8 and R9 are each independently selected from H, D, T, hydroxyl, halogen, or C. 1-6 Alkyl groups; or R8 and R9 forming carbonyl groups or C groups with the carbon atoms they are attached to. 3-6 cycloalkyl; R 11 Selected from H, D, T, hydroxyl, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl; R 12 Selected from H, D, T, hydroxyl, halogen or C 1-6 alkyl.

11. The ligand-drug conjugate according to claim 10, or a pharmaceutically acceptable salt or solvate thereof, characterized in that, D is selected from the following structures or their stereoisomers, deuterated derivatives, and tritium derivatives: , , , and .

12. The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1-11, characterized in that, The ligand-drug conjugate is selected from the following structures: , , , , , , 、 、 、 、 、 、 、 、 、 、 ； in, R4 is selected from H, or ; R8 is selected from H or D; R9 is selected from hydroxyl or F; c is selected from 1, 2, 3, 4, 5, 6, 7, or 8; e can be independently selected from 1, 2, 3, or 4; f is selected from 11, 12, 23, or 24; q is selected from integers or decimals from 1 to 10; When the ligand-drug conjugate structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, i.e., each maleimide can be independently in an open-ring or closed-ring form; specifically, each can be independently... or . Among them, the configuration of all chiral carbons can be either R-type or S-type; Preferably, the ligand-drug conjugate is selected from the following structures: q is selected from integers or decimals from 1 to 10; When the ligand-drug conjugate structure includes maleimide, the maleimide can optionally be hydrolyzed to open the ring, i.e., each maleimide can be independently in an open-ring or closed-ring form; specifically, each can be independently... or .

13. The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1-12, characterized in that: The Tg is not limited to selection from: anti-EGFRvIII antibody, anti-PD 1 antibody, anti-PD L1 antibody, anti-DLL3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM 1 antibody, anti-LRRC15 antibody, anti-LIV 1 antibody, anti-CanAg / AFP antibody, anti-claudin18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-cMET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7 antibody. H3(CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-Trop2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P Anti-cadherin antibody, anti-GD3 antibody, anti-Cadherin6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, anti-CD123 antibody, anti-GPC3 antibody, anti-EGFR×HER3 bispecific antibody, or any combination of antibodies targeting the above targets.

14. A linker-drug compound of Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, in: M4' is a connector that can connect to ligand units; A2, L4, L5, and D are as defined in any one of claims 1-11.

15. The linker-drug compound or its stereoisomers, tautomers, pharmaceutically acceptable salts or solvates according to claim 14, characterized in that, The M4' is selected from: , , , , , , , , , , , , , , and ; Position 2 of M4' is connected to L4; a is an integer selected from 1 to 24; b is an integer selected from 1 to 6; e is an integer selected from 0 to 24; Each t is independently selected from integers between 0 and 10; The configuration of the chiral carbon at the indicated position can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by # can be either R-type or S-type; The configuration of the chiral carbon at the position indicated by & can be either R-type or S-type.

16. The linker-drug compound or its stereoisomers, tautomers, pharmaceutically acceptable salts or solvates according to claim 14 or 15, wherein the linker-drug compound is selected from the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； in, R4 is selected from H, or ; e can be selected from 1, 2, 3, or 4; f is selected from 11, 12, 23, or 24; Among them, the configuration of all chiral carbons can be either R-type or S-type; Preferably, the linker-drug compound is selected from the following structures: 。 17. A pharmaceutical composition comprising: (1) The ligand-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1-13, or the linker-drug compound or its stereoisomer, tautomer, pharmaceutically acceptable salt or solvate according to any one of claims 14-16; (2) Pharmaceutically acceptable carriers, diluents or excipients; And optional (3) one or more other active ingredients.

18. The use of the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-13, or the linker-drug compound or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 14-16, or the use of the pharmaceutical composition of claim 17 for the preparation of a medicament for the treatment or prevention of tumors; Preferably, the tumor is selected from solid tumors or non-solid tumors, such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.

19. Use of the linker-drug compound or its stereoisomers, tautomers, pharmaceutically acceptable salts or solvates according to any one of claims 14-16 for the preparation of ligand-drug conjugates or their pharmaceutically acceptable salts or solvates; Preferably, the ligand-drug conjugate is selected from the ligand-drug conjugate of any one of claims 1-13 or its pharmaceutically acceptable salt or solvate.

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