Anti-b7-h3 antibody or fragment thereof, and antibody drug conjugate targeting b7-h3

Abstract

A series of novel anti-B7-H3 antibodies. The antibodies have high affinity to B7-H3 and have stable structures. Also provided is an antibody drug conjugate prepared by using said antibody. The antibody drug conjugate uses a camptothecin compound having a specific structure as a toxic molecule, has significant tumor inhibition activity, and has a bystander killing effect.

Description

Anti-B7-H3 antibodies or fragments thereof and antibody-drug conjugates targeting B7-H3

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the priority benefit of the Chinese invention patent application with application number CN202310655433.X filed on June 5, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology, and in particular, relates to an antibody-drug conjugate targeting B7-H3 and its application in tumor treatment. Background Art

[0004] B7-H3, also known as B7H3 or CD276, is a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. Encoded on chromosome 15q24, B7-H3 consists of an extracellular domain, a transmembrane domain, and a short intracellular domain. Immunohistochemical (IHC) analysis of a large number of normal tissues has shown that B7-H3 is only expressed at low levels, or even undetectable, in a few tissues. However, B7-H3 has been found to be overexpressed in various solid tumors, including bladder cancer, prostate cancer, and melanoma. Furthermore, with further research, increasing evidence indicates that B7-H3 primarily plays a co-inhibitory role in immune cells, facilitating tumor cell evasion of immune surveillance. Consequently, overexpression of B7-H3 is associated with poor prognosis in cancer patients and the invasive and metastatic potential of tumors in in vitro models.

[0005] Currently, over 20 clinical trials have been or are currently testing the safety and efficacy of monoclonal antibody immunotherapy strategies targeting B7-H3. Furthermore, several antibody-drug conjugates (ADCs) targeting B7-H3 have entered clinical trials both domestically and internationally.

[0006] Daiichi Sankyo developed DS-7300, an ADC targeting B7-H3, which is prepared by randomly conjugating the MC-GGFG-Dxd drug-containing linker to the hM30 antibody targeting B7H3 to obtain a B7-H3-targeting ADC with a DAR of 4. The structure is shown below:

[0007] MacroGenics developed MGC018, an ADC drug targeting B7-H3, which is composed of an anti-B7-H3 humanized IgG1 monoclonal antibody coupled to Duocarmycin (DUBA) via a cleavable linker. The average DAR is 2.7, and the structure is shown below:

[0008] BAT8009, an ADC drug targeting B7-H3 developed by BioAtla, is composed of a recombinant humanized anti-B7-H3 antibody and a DNA topoisomerase inhibitor coupled via a cleavable linker.

[0009] ABBV-155, an ADC drug targeting B7-H3 developed by AbbVie, is composed of an anti-B7-H3 antibody and a BCL-XL inhibitor that promotes cell apoptosis coupled via a linker. It is used to treat advanced solid tumors. The structure is shown below:

[0010] The above ADC drugs have shown certain therapeutic effects, but there are also certain problems. For example, MacroGenics announced a Phase I clinical trial (cohort expansion, NCT03729596) of MGC018 for the treatment of advanced solid tumors in 2021. In terms of safety, 43 patients experienced grade 3 or above treatment-related adverse events (TRAEs) (50%). Common serious TRAEs include neutropenia (22.1%) and thrombocytopenia (7%). The DS-7300 developed by Daiichi Sankyo uses a random conjugation method, which will cause the ADC product to have greater heterogeneity.

[0011] Currently, there are relatively few available ADCs containing camptothecin compounds that target B7-H3. In view of the above-mentioned deficiencies of existing ADCs of this type, there is still a need in the art to provide an ADC containing camptothecin compounds that has better targeting of B7-H3, lower product heterogeneity, fewer side effects, and thus better therapeutic effects.

[0012] Chinese patent application CN202111459992.0 (publication number CN114573695A), filed on December 2, 2021 and titled “An anti-human B7-H3 monoclonal antibody and its application,” discloses murine and humanized anti-human B7-H3 monoclonal antibodies. In this application, a murine anti-human B7-H3 monoclonal antibody capable of binding to the extracellular region of B7-H3 was prepared by hybridoma technology using the extracellular region of recombinant human B7-H3 as an immunogen; a human-mouse chimeric antibody capable of specifically binding to B7-H3 on the cell surface was constructed based on the murine antibody; further, a humanized antibody was constructed by CDRs transplantation and CDRs region mutation. The humanized antibody retains the ability to specifically bind to the extracellular region of human B7-H3 and B7-H3 on the cell membrane surface, and can be internalized by B7-H3 on the cell membrane surface. However, although the murine and humanized antibodies in this application have excellent effects, there is still room for further improvement.

[0013] Summary of the Invention

[0014] The inventors of the present invention discovered that a humanized anti-B7-H3 antibody disclosed in CN114573695A, while having a high affinity for human B7-H3, is susceptible to fragmentation. In practical applications, this susceptibility to fragmentation can lead to increased heterogeneity in products containing the antibody, thereby increasing the immunogenicity of the product, reducing its efficacy, and even causing toxic side effects. Therefore, whether used alone or in the development of other products, this humanized antibody has certain drawbacks.

[0015] Therefore, based on this known humanized anti-B7-H3 antibody, the inventors of the present invention used variable region site-directed mutagenesis to screen for novel anti-B7-H3 antibodies that can overcome the above-mentioned defects. Furthermore, they attempted to couple the mutated antibodies with camptothecin compounds of specific structures to obtain antibody-drug conjugates, in order to select antibody-drug conjugates with better B7-H3 targeting and B7-H3-expressing cell-killing effects.

[0016] Therefore, one object of the present invention is to provide an antibody or fragment thereof that specifically binds to B7-H3. Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof targeting B7-H3 prepared using the antibody or fragment thereof.

[0017] In the context of the present invention, halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0018] In the context of the present invention, "linker" is used interchangeably with "connector" and "linker compound".

[0019] In the context of the present invention, the term "drug-containing linker" refers to a compound in which a drug (eg, a small molecule drug such as a camptothecin compound) is directly or indirectly covalently bonded to a linker.

[0020] The technical solutions of the present invention are as follows.

[0021] First aspect

[0022] The present invention provides an anti-B7-H3 antibody or a fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain and the light chain comprise heavy chain complementary determining regions CDR-H1, CDR-H2, and CDR-H3 and light chain complementary determining regions CDR-L1, CDR-L2, and CDR-L3 derived from the following amino acid sequence combinations:

[0023] (i) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 3;

[0024] (ii) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 4; or

[0025] (iii) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 5.

[0026] In the context of the present invention, the term "fragment" encompasses various functional fragments of the anti-B7-H3 antibodies that retain the antigen binding ability and corresponding biological activity of the antibody. It is well known in the art that the antigen binding ability and corresponding biological activity of an antibody can be achieved by fragments of an intact antibody, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for intact antibodies. For example, antigen-binding fragments of the antibody can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of an intact antibody. For example, the fragment can be a Fab, F(ab')2, or scFv fragment, etc.

[0027] The amino acid sequences of the above items are respectively the amino acid sequences of the heavy chain variable region and the light chain variable region of the specific anti-B7-H3 antibody provided by the present invention; see the "Specific Embodiments" section of the present invention. Using the definition tools for the complementary determining regions in the heavy chain or light chain variable region of antibodies that are well known or conventional in the art (such as the Kabat scheme, the AbM scheme, the Chothia scheme, the Contact scheme, the IMGT scheme, the enhanced Chothia / Abm scheme), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained when the two are combined to form an antibody. For example, Example 1 of the present invention provides the position of the amino acid sequence of each CDR in the full length of the variable region when defining CDRs according to the Kabat scheme. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to definition tools that are well known or conventional in the art, and antibodies or fragments thereof that contain each of these heavy chain CDRs and light chain CDRs combinations are within the scope of protection of the present invention.

[0028] Furthermore, the anti-B7-H3 antibodies or fragments thereof provided herein comprise a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3; and, CDR-L1, CDR-L2, and CDR-L3) selected from the following:

[0029] (i) the CDR-H1, CDR-H2, and CDR-H3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, respectively;

[0030] (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 15, respectively; and

[0031] (iii) the CDR-H1, CDR-H2 and CDR-H3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0032] In the context of the present invention, the term "at least 75% identity" encompasses any percentage of identity between at least 75% identity and 100% identity, for example 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. Depending on the context in which the term is used, the up to 25% difference in amino acid sequence resulting from "at least 75% identity" may exist in the heavy or light chain CDRs, or in any region other than the heavy or light chain CDRs, such as the framework regions of the heavy and light chain variable regions of the antibodies or fragments thereof of the present invention, or the heavy and light chain constant regions of the antibodies or fragments thereof of the present invention. The differences may result from deletions, additions, or substitutions of amino acids at any position, wherein the substitutions may be conservative or non-conservative.

[0033] According to one embodiment of the present invention, the anti-B7-H3 antibody or fragment thereof provided by the present invention comprises a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3; and, CDR-L1, CDR-L2 and CDR-L3) selected from the following:

[0034] (i) the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, respectively;

[0035] (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 15, respectively; and

[0036] (iii) the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0037] The antibodies or fragments thereof provided by the present invention are antibodies or fragments thereof against B7-H3, particularly human B7-H3. The amino acid sequence of human B7-H3 is exemplarily provided in UniProtKB-Q5ZPR3.

[0038] The anti-B7-H3 antibodies or fragments thereof provided herein comprise at least a heavy chain variable region (VH) and a light chain variable region (VL), both of which include the aforementioned CDRs and a framework region (FR) therebetween, with the regions arranged in the pattern of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, in the anti-B7-H3 antibodies or fragments thereof provided herein, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 75% identity thereto; and / or the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3-5, or an amino acid sequence having at least 75% identity thereto.

[0039] Preferably, in the antibodies or fragments thereof provided by the present invention, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following:

[0040] (i) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 3;

[0041] (ii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 4; and

[0042] (iii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 5.

[0043] According to a specific embodiment of the present invention, the antibody or fragment thereof provided by the present invention comprises a combination of a heavy chain variable region and a light chain variable region selected from the following:

[0044] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3;

[0045] (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; and

[0046] (iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5.

[0047] The anti-B7-H3 antibodies or fragments thereof provided herein may be any form of antibodies or fragments thereof comprising the domains defined above, such as monoclonal antibodies, chimeric antibodies, partially or fully humanized antibodies, etc. The antibodies or fragments thereof provided herein may also be in the form of scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

[0048] In addition to the variable region, the anti-B7-H3 antibodies or fragments thereof provided herein may further comprise a constant region, such as a heavy chain constant region (CH) and / or a light chain constant region (CL), or any one or more domains in the constant region, such as any one or more of CH1, CH2, CH3, CH4, etc. Alternatively, the anti-B7-H3 antibodies or fragments thereof provided herein may comprise a heavy chain and / or a light chain, wherein the heavy chain may comprise, for example, a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and the light chain may comprise a light chain constant region of a κ or λ type.

[0049] Preferably, the present invention provides an anti-B7-H3 humanized antibody or a fragment thereof. More preferably, the present invention provides an anti-B7-H3 humanized monoclonal antibody or a fragment thereof.

[0050] The present invention provides humanized monoclonal antibodies that specifically bind to B7-H3 and have been isolated and structurally characterized. According to one embodiment of the present invention, the anti-B7-H3 antibodies provided by the present invention are hz10B4m1, hz10B4m2, or hz10B4m3. For details, see the "Detailed Description" section of the present invention.

[0051] Second aspect

[0052] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable region, heavy chain variable region, heavy chain and / or light chain contained in the anti-B7-H3 antibody or fragment thereof of the present invention.

[0053] The third aspect

[0054] The nucleic acid molecules of the present invention can be cloned into vectors and then used to transform or transfect host cells. Therefore, in a third aspect, the present invention also provides a vector comprising the nucleic acid molecules of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector.

[0055] The fourth aspect

[0056] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, in a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or transformed or transfected with the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant, or animal cell.

[0057] The anti-B7-H3 antibodies or fragments thereof provided by the present invention can be obtained using any method known in the art. For example, the host cells provided by the present invention are cultured while allowing them to express the heavy and light chains of the antibodies. Optionally, the method further comprises the step of recovering the produced antibodies. As described above, for example, antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact antibodies. The anti-B7-H3 antibodies or fragments thereof provided by the present invention can be obtained using any method known in the art. For example, the host cells provided by the present invention are cultured while allowing them to express the heavy and / or light chains of the antibodies to assemble into the antibodies. Optionally, the method further comprises the step of recovering the produced antibodies.

[0058] The fifth aspect

[0059] The anti-B7-H3 antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided herein can be included in pharmaceutical compositions, more particularly, in pharmaceutical preparations, and thus used for various purposes according to actual needs. Therefore, in a fifth aspect, the present invention also provides a composition comprising the anti-B7-H3 antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells described herein; preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient, or vehicle.

[0060] The sixth aspect

[0061] The present invention provides the use of the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating a disease associated with B7-H3 expression (including overexpression). Preferably, the disease is a tumor. More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms' tumor, and desmoplastic small round cell tumor.

[0062] Seventh aspect

[0063] The present invention also provides the use of the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a reagent for diagnosing a disease associated with B7-H3 expression (including overexpression). Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms' tumor and desmoplastic small round cell tumor.

[0064] The eighth aspect

[0065] The present invention also provides a method for treating a disease associated with B7-H3 expression (including overexpression), the method comprising administering an anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present invention to a subject in need thereof, and optionally other drugs or means. The optional other drugs or means refer to other drugs or means that can be administered in combination with the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present invention, such as small molecule drugs, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs and radiotherapy. The combined administration of the two can be carried out in any form, for example, simultaneously, continuously or at intervals of a certain time.

[0066] Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms tumor and desmoplastic small round cell tumor.

[0067] Preferably, the subject is a mammal, preferably a primate, more preferably a human.

[0068] The application of the above-mentioned disease treatment methods provided by the present invention depends on many factors, including the specific active ingredient administered, the patient's age, weight, gender or physical and medical condition, the severity of the condition to be treated, the route of administration, etc.

[0069] Ninth aspect

[0070] The present invention also provides a method for diagnosing a disease associated with B7-H3 expression (including overexpression), the method comprising contacting an anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition of the present invention with a sample from a subject. Preferably, the disease is a tumor or cancer. More preferably, the disease is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms' tumor, and desmoplastic small round cell tumor.

[0071] Preferably, the subject is a mammal; more preferably, the subject is a human.

[0072] Tenth Aspect

[0073] The present invention provides a kit comprising the anti-B7-H3 antibody or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition. The kit is used for the therapeutic or diagnostic purposes described above. Optionally, the kit may also include instructions for use.

[0074] Aspect 11

[0075] As described above, another object of the present invention is to provide an antibody-drug conjugate or salt thereof targeting B7-H3, wherein preferably, the antibody-drug conjugate or salt thereof is prepared using the anti-B7-H3 antibody or fragment thereof provided by the present invention. Therefore, in an eleventh aspect, the present invention provides an antibody-drug conjugate or salt thereof, wherein the antibody-drug conjugate or salt thereof has a structure as shown in Structural Formula Ia and / or Ib:

[0076] and / or

[0077] In formula Ia and / or Ib, Ab represents an anti-B7-H3 antibody or a fragment thereof. Preferably, the antibody or fragment thereof is an anti-B7-H3 antibody or a fragment thereof as defined above in the first aspect of the present invention.

[0078] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in a substituted phenylene group, the substituents are selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., halo-C1-6 alkyl, preferably halo-C1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester, amide and cyano groups; preferably, M is a halogen-substituted phenylene group.

[0079] In formula Ia and / or Ib, and unless otherwise specified in the context of the present invention, SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight-chain heteroalkylene, said C1-21 straight-chain heteroalkylene containing 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein said C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight-chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.

[0080] In formula Ia and / or Ib, and unless otherwise indicated in the context of the present invention, SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) a CO- or chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6.

[0081] In formula Ia and / or Ib, and in the context of the present invention, unless otherwise indicated, A represents a short peptide structure formed by 2 to 4 amino acids. Among them, when A represents a short peptide structure formed by two amino acids, it can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg- or -Gly-Val-, preferably -Phe-Lys-, -Val-Ala- or -Val-Cit-; when A represents a short peptide structure formed by three amino acids, it can be -Glu-Val-Ala-, -Glu-Val-Cit- or -Ala-Ala-Ala-, preferably -Glu-Val-Ala- or -Ala-Ala-Ala-; when A represents a short peptide structure formed by four amino acids, it can be -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-, preferably -Gly-Gly-Phe-Gly-. Preferably, A is -Val-Ala-, -Gly-Gly-Phe-Gly- or -Ala-Ala-Ala-. In formula Ia and / or Ib, group A is connected to SP2 via the amino group at the amino terminus of its short peptide structure.

[0082] In formula Ia and / or Ib, M is preferably a halogen-substituted phenylene group, in particular a fluorine-substituted phenylene group.

[0083] In formula Ia and / or Ib, SP1 is preferably a C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene group containing 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S.

[0084] In formula Ia and / or Ib, SP2 is preferably a chemical bond.

[0085] In formula Ia and / or Ib, and unless otherwise stated in the context of the present invention, m is 1 to 10, preferably 1 to 8 (eg 1 to 5), more preferably 3 to 8. m may be an integer or a non-integer.

[0086] Furthermore, the antibody drug conjugate or salt thereof provided by the present invention has the structure shown in structural formula Ic and / or Id:

[0087] and / or

[0088] In formula Ic and / or Id, Ab, group A and m are as defined in formula Ia and / or Ib.

[0089] In any of Formulas Ia, Ib, Ic and Id, and unless otherwise indicated in the context of the present invention, CPT is a camptothecin compound.

[0090] Furthermore, in any of Structural Formulas Ia, Ib, Ic, and Id, the structure of CPT is as shown in Structural Formula I, wherein Structural Formula I is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond, preferably, the amino group adjacent to Group G in Structural Formula I is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond:

[0091] In Structural Formula I, and in the context of the present invention, unless otherwise specified, R1, R2, R3, and R4 are independently hydrogen, halogen, hydroxy, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4, together with the carbon atoms to which they are attached, form a C3-6 (preferably C3-5) cyclic alkyl group. When R1, R2, R3, and R4 are independently C1-6 alkoxy, the C1-6 alkoxy group includes a straight-chain or branched C1-6 alkoxy group, preferably a straight-chain or branched C1-3 alkoxy group, and more preferably a methoxy group. When R1, R2, R3, and R4 are independently substituted amino, the substituted amino group is an amino group substituted with one or more substituents selected from methyl and ethyl. When R1, R2, R3, and R4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 (preferably C3-5, more preferably C4) alkyl or substituted C1-7 (preferably C3-5, more preferably C4) alkyl, and the substituted C1-7 alkyl is a C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halomethyl (preferably trifluoromethyl).

[0092] In formula I, and in the context of the present invention, unless otherwise specified, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0093] In Structural Formula I, and in the context of the present invention, unless otherwise specified, Y represents oxygen, sulfur, sulfone, sulfoxide, methylene, or a substituted methylene group. A substituted methylene group may have one or both hydrogen atoms replaced, and the substituent may be a benzyl group or an alkyl group. When the substituent is an alkyl group, the alkyl group, R3 and / or R4, and the carbon atoms to which they are attached may form a C3-6 membered cyclic or spirocyclic structure. Alternatively, when the substituent is two alkyl groups, the two alkyl groups may form a C3-6 membered spirocyclic structure with the group Y. When Y is a substituted methylene group, the substituent is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group.

[0094] Preferably, Y is oxygen, sulfur, sulfone, sulfoxide or methylene; or, preferably, Y is oxygen, sulfur or methylene.

[0095] In formula I, and unless otherwise indicated in the context of the present invention, X is oxygen or sulfur.

[0096] In formula I, and in the context of the present invention, unless otherwise stated, n=0 or 1.

[0097] In structural formula I, when R1, R2, R3, and R4 are all hydrogen, X is oxygen, and n=0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is oxygen or sulfur, G cannot be hydrogen.

[0098] Preferably, R1, R2, R3, and R4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4, together with the carbon atoms to which they are attached, form a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl). Furthermore, R1 and R2 may be the same; and / or R3 and R4 may be the same.

[0099] Preferably, Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and / or R4, and the carbon atoms to which they are connected can form a C3-6 membered cyclic or spirocyclic structure.

[0100] Preferably, X may be oxygen.

[0101] Preferably, X is oxygen, G is hydrogen, halogen (such as fluorine or chlorine), methyl or methoxy, and Y and R1, R2, R3, R4 are as defined above.

[0102] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, and R1, R2, R3, and R4 are as defined above.

[0103] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0104] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0105] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0106] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0107] Preferably, X is oxygen, G is hydrogen, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0108] Preferably, X is oxygen, G is fluorine, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0109] Preferably, n=0.

[0110] According to a specific embodiment of the present invention, in structural formula I:

[0111] G is hydrogen, Y is methylene, R1 and R2 are methyl, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0112] G is hydrogen, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0113] G is hydrogen, Y is methylene, one of R1 and R2 and one of R3 and R4 together with the carbon atom to which they are attached form a C3 cyclic alkyl group, the other of R1 and R2 and the other of R3 and R4 are hydrogen, X is oxygen, and n=0;

[0114] G is hydrogen, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0115] G is hydrogen, Y is sulfoxide, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0116] G is hydrogen, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0117] G is hydrogen, Y is sulfone, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0118] G is hydrogen, Y is methylene, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=1;

[0119] G is fluorine, Y is oxygen, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0120] G is fluorine, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0121] G is fluorine, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0122] G is fluorine, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0;

[0123] G is hydrogen, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0; or

[0124] G is fluorine, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n=0.

[0125] Preferably, in any of Structural Formulas Ia, Ib, Ic, and Id, the structure of CPT is as shown in Structural Formula IA, wherein Structural Formula IA is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond, preferably, the amino group on the left phenyl ring in Structural Formula IA is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond:

[0126] In formula IA, the groups R1, R2, R3, and R4 are the same as those in formula I above, except that R1, R2, R3, and R4 are not all hydrogen.

[0127] Alternatively, in any of Structural Formulas Ia, Ib, Ic, and Id, the structure of CPT is as shown in Structural Formula II, wherein Structural Formula II is linked to the carboxyl group of Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond, preferably, the amino group adjacent to Group G in Structural Formula II is linked to the carboxyl group of Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond:

[0128] In Structural Formula II, and unless otherwise specified in the context of the present invention, R5 is C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted with one or more substituents, phenyl, or substituted phenyl. When R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight-chain or branched C1-5 alkyl. Further, R5 is C1-4 straight-chain alkyl. When R5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl, and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted with one or more substituents selected from methyl and ethyl. When R5 is substituted phenyl, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-3) or halogen.

[0129] In formula II, and unless otherwise specified in the context of the present invention, G is hydrogen, halogen (eg fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0130] In structural formula II, X is oxygen or sulfur.

[0131] In structural formula II, n=0 or 1.

[0132] In structural formula II, when X is oxygen, G is hydrogen, and n=0, R5 cannot be n-butyl.

[0133] Preferably, in any of Structural Formulas Ia, Ib, Ic, and Id, the structure of CPT is as shown in Structural Formula IIA, wherein Structural Formula IIA is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond, preferably, the amino group on the left phenyl ring in Structural Formula IIA is linked to the carboxyl group in Group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond:

[0134] In formula IIA, the group R5 is the same as the group R5 in formula II above, but R5 cannot be n-butyl.

[0135] According to a specific embodiment of the present invention, in any of Structural Formulas Ia, Ib, Ic, and Id, the structure of the CPT is as follows, wherein each of the Structural Formulas is linked to the carboxyl group in the group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond, preferably, the amino group on the left benzene ring in each of the Structural Formulas is linked to the carboxyl group in the group A in any of Structural Formulas Ia, Ib, Ic, and Id via an amide bond:

[0136] Furthermore, in any one of the structural formulas Ia, Ib, Ic and Id, the structure of CPT is as shown in structural formula IV, wherein the hydroxyl group connected to the same carbon as R8 in structural formula IV is connected to the carboxyl group of group A in any one of the structural formulas Ia, Ib, Ic and Id via a self-releasing structure, and the self-releasing structure is, for example The solid line represents the site of attachment to the carboxyl group of group A in any of formulas Ia, Ib, Ic, and Id, and the wavy line represents the site of attachment to the hydroxyl group in formula IV:

[0137] In formula IV, and unless otherwise specified in the context of the present invention, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.

[0138] When R8 is a substituted C1-5 alkyl group or a substituted C3-6 cyclic alkyl group, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.

[0139] Aspect 12

[0140] The present invention provides an antibody drug conjugate or a salt thereof, wherein the antibody drug conjugate or the salt thereof has the general formula The structure shown, wherein mAb represents an anti-B7-H3 antibody or a fragment thereof. Preferably, mAb is an anti-B7-H3 antibody or a fragment thereof as defined above in the first aspect of the present invention.

[0141] In this general formula, the groups M, SP1, SP2, A and CPT are the same as those defined in the eleventh aspect above; N is 1 to 10, preferably 1 to 8 (e.g., 1 to 5), and more preferably 3 to 8.

[0142] In this general formula, E L is selected from the following groups, wherein, Indicates that it is linked to the cysteine ​​in mAb. Indicates connection with M:

[0143] E L -1a and / or E L -1b: and / or E L -2: E L -3: E L -4: E L -5: E L -6:

[0144] Thirteenth Aspect

[0145] The present invention provides a drug-containing linker (linker-payload) having a structure represented by the general formula "LA-CPT", wherein L represents a linker for an antibody-drug conjugate (ADC), A represents one or more amino acids, and CPT is a camptothecin compound.

[0146] In the thirteenth aspect of the present invention, the drug-containing linker having the structure represented by the general formula LA-CPT is a compound having the structure represented by structural formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0147] In formula III, and unless otherwise indicated in the context of the present invention, E is selected from the group consisting of: Indicates the attachment site to M:

[0148] E-1: E-2: E-3: E-4: E-5: and E-6:

[0149] In formula III, and in the context of the present invention, unless otherwise specified, M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in a substituted phenylene group, the substituents are selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., halo-C1-6 alkyl, preferably halo-C1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester, amide and cyano groups; preferably, M is a halogen-substituted phenylene group.

[0150] In formula III, and unless otherwise specified in the context of the present invention, SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight-chain heteroalkylene, wherein the C1-21 straight-chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight-chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.

[0151] In formula III, and unless otherwise specified in the context of the present invention, SP2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer from 1 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6.

[0152] In formula III, and unless otherwise specified in the context of the present invention, A represents a short peptide structure formed by 2-4 amino acids. Among them, when A represents a short peptide structure formed by two amino acids, it can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg- or -Gly-Val-, preferably -Phe-Lys-, -Val-Ala- or -Val-Cit-; when A represents a short peptide structure formed by three amino acids, it can be -Glu-Val-Ala-, -Glu-Val-Cit- or -Ala-Ala-Ala-, preferably -Glu-Val-Ala- or -Ala-Ala-Ala-; when A represents a short peptide structure formed by four amino acids, it can be -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-, preferably -Gly-Gly-Phe-Gly-. Preferably, A is -Val-Ala-, -Gly-Gly-Phe-Gly- or -Ala-Ala-Ala-. In formula III, group A is connected to SP2 via the amino group at the amino terminus of its short peptide structure.

[0153] In formula III, M is preferably a halogen-substituted phenylene group, particularly a fluorine-substituted phenylene group.

[0154] In formula III, SP1 is preferably a C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene group containing 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S.

[0155] In formula III, SP2 is preferably a chemical bond.

[0156] In formula III, and unless otherwise indicated in the context of the present invention, CPT is a camptothecin compound.

[0157] Wherein, when the group E is E1, the structural formula III provided by the present invention is further structural formula IIIA:

[0158] In formula IIIA, and in the context of the present invention, unless otherwise specified, R6 and R7 are independently hydrogen, halogen or Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in substituted phenyl, the substituents are selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl

[0159] For example, in Structural Formula III and Structural Formula IIIA, CPT is the compound represented by Structural Formula I or Structural Formula IA provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, as well as the corresponding specific compound.

[0160] When CPT is the compound represented by structural formula I provided in the eleventh aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0161] In Structural Formula I, the groups G, X, Y, R1, R2, R3, R4, and n are the same as those defined above for Structural Formula I in Aspect 11. Furthermore, in Structural Formula III or Structural Formula IIIA, the carboxyl group in Structural Formula I is linked to the carboxyl group of group A via an amide bond. Preferably, the amino group adjacent to group G in Structural Formula I is linked to the carboxyl group of A via an amide bond, i.e., an amide bond is formed between the amino group of the compound represented by Structural Formula I and the carboxyl group of A in Structural Formula III or IIIA.

[0162] When CPT is the compound represented by structural formula IA provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0163] In Structural Formula IA, the groups R1, R2, R3, and R4 are the same as those defined above for Structural Formula IA in Aspect 11, except that R1, R2, R3, and R4 may all be hydrogen. Furthermore, in Structural Formula III or Structural Formula IIIA, the carboxyl group in Structural Formula IA is linked to the carboxyl group in Group A via an amide bond. Preferably, the amino group on the left phenyl ring in Structural Formula IA is linked to the carboxyl group in Group A via an amide bond, i.e., the amino group of the compound represented by Structural Formula IA forms an amide bond with the carboxyl group of Group A in Structural Formula III or IIIA.

[0164] In particular, when "CPT" in the compound represented by Structural Formula IIIA is a compound represented by Structural Formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, R6 and R7 may be hydrogen at the same time or at different times. Similarly, R1, R2, R3, and R4 in Structural Formula IA may be hydrogen at the same time or at different times. According to a specific embodiment of the present invention, when R6 and R7 are hydrogen at the same time, R1, R2, R3, and R4 in Structural Formula IA may be hydrogen at different times; when R6 and R7 are different times, R1, R2, R3, and R4 in Structural Formula IA may be hydrogen at different times or at different times.

[0165] For example, in Structural Formula III and Structural Formula IIIA, CPT is the compound represented by Structural Formula II or Structural Formula IIA provided in the eleventh aspect of the present invention above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, as well as the corresponding specific compound.

[0166] When CPT is the compound represented by structural formula II provided in the eleventh aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0167] In Structural Formula II, the groups G, R5, X, and n are the same as those defined above for Structural Formula II in the eleventh aspect. Furthermore, in Structural Formula III or Structural Formula IIIA, the carboxyl group in Structural Formula II is linked to the carboxyl group in Group A via an amide bond. Preferably, the amino group adjacent to Group G in Structural Formula II is linked to the carboxyl group in Group A via an amide bond, i.e., the amino group of the compound represented by Structural Formula II forms an amide bond with the carboxyl group in Group A in Structural Formula III or IIIA.

[0168] When CPT is the compound represented by structural formula IIA provided in the eleventh aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0169] In Structural Formula IIA, the group R5 is the same as defined above for Structural Formula IIA in Aspect 11, but may be an n-butyl group. Furthermore, in Structural Formula III or Structural Formula IIIA, Structural Formula IIA is linked to the carboxyl group of Group A via an amide bond. Preferably, the amino group on the left phenyl ring of Structural Formula IIA is linked to the carboxyl group of Group A via an amide bond. In other words, the amino group of the compound represented by Structural Formula IIA forms an amide bond with the carboxyl group of Group A in Structural Formula III or IIIA.

[0170] Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14-P, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs thereof set forth in the eleventh aspect above can be used as the "CPT" in structural formula III or IIIA, and are linked to the carboxyl group of group A in structural formula III or IIIA through an amide bond via their amino group.

[0171] For example, in structural formula III and structural formula IIIA, CPT can be an exteacan derivative shown in structural formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0172] In formula IV, and unless otherwise specified in the context of the present invention, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen. And in formula III and formula IIIA, formula IV is connected to the carboxyl group in group A via a self-releasing structure via the hydroxyl group connected to the same carbon as R8, and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.

[0173] When R8 is a substituted C1-5 alkyl group or a substituted C3-6 cyclic alkyl group, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.

[0174] In particular, when "CPT" in the compound of Formula IIIA is a compound of Formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, R6 and R7 may be both hydrogen or different hydrogens. Similarly, R8 in Formula IV may be hydrogen or different hydrogens. According to specific embodiments of the present invention, when R6 and R7 are both hydrogens, R8 may be hydrogen or different hydrogens.

[0175] In particular, for the compound represented by structural formula IIIA, when R6 and R7 are Ar'S, M is preferably a phenylene or a substituted phenylene, and SP1 is a C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight-chain heteroalkylene group, wherein the straight-chain heteroalkylene group contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S.

[0176] Preferably, the compounds represented by structural formula III and structural formula IIIA provided by the present invention are further represented by structural formula V:

[0177] In formula V, R6 and R7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in which the substituents are selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide and cyano. Preferably, Ar' is phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl

[0178] In formula V, and in the context of the present invention, unless otherwise specified, Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g., haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

[0179] In formula V, and in the context of the present invention, unless otherwise specified, m is an integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.

[0180] In structural formula V, A represents a short peptide structure formed by 2-4 amino acids, as defined above.

[0181] In Formula V, the definition of CPT and its linkage in Formula V are the same as those of CPT in Formulas III and IIIA, as defined above.

[0182] Preferably, the compound represented by structural formula V provided by the present invention is further represented by structural formula VA:

[0183] In structural formula VA, the groups A, G, Y, R1, R2, R3, R4, X, and n are the same as the definitions of the groups A, G, Y, R1, R2, R3, R4, X, and n in structural formula III or structural formula IIIA above.

[0184] Preferably, in formula VA, G is hydrogen, fluorine or chlorine.

[0185] Preferably, in formula VA, Y is methylene, sulfur or oxygen.

[0186] In formula VA, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0187] Preferably, the compound represented by structural formula VA provided by the present invention is further represented by structural formula VA-1:

[0188] Alternatively, the compound represented by structural formula V provided by the present invention is further represented by structural formula VB:

[0189] In structural formula VB, the groups A, G, R5, X, and n are the same as those defined above in structural formula III or structural formula IIIA.

[0190] In formula VB, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0191] Preferably, the compound represented by structural formula VB provided by the present invention is further represented by structural formula VB-1:

[0192] Alternatively, the compound represented by structural formula V provided by the present invention is further represented by structural formula VC:

[0193] In structural formula VC, the groups A and R8 are the same as those defined above in structural formula III or structural formula IIIV.

[0194] In the structural formula VC, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0195] In the structural formula VC, and in the context of the present invention, unless otherwise specified, The self-releasing structure is the same as that when the CPT in the above structural formula III or structural formula IIIV is structural formula IV.

[0196] According to a specific embodiment of the present invention, the compound represented by structural formula V provided by the present invention is further represented by structural formula VI:

[0197] In formula VI, A represents a short peptide structure formed by 2-4 amino acids, as defined above.

[0198] In formula VI, CPT is a camptothecin compound. The definition of CPT and its connection relationship in formula VI are the same as those of CPT in formulas III and IIIA, as defined above.

[0199] Preferably, the compound represented by structural formula VI provided by the present invention is further represented by structural formula VI-A:

[0200] In structural formula VI-A, the groups A, G, Y, R1, R2, R3, R4, X, and n have the same definitions as those of the groups A, G, Y, R1, R2, R3, R4, X, and n in structural formula III or structural formula IIIA above.

[0201] Preferably, in structural formula VI-A, G is hydrogen, fluorine or chlorine.

[0202] Preferably, in formula VI-A, Y is methylene, sulfur or oxygen.

[0203] In formula VI-A, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0204] Preferably, the compound represented by structural formula VI-A provided by the present invention is further represented by structural formula VI-A-1:

[0205] Alternatively, the compound represented by structural formula VI is further represented by structural formula VI-B:

[0206] In structural formula VI-B, the groups A, G, R5, X, and n are the same as those defined in structural formula III or structural formula IIIA above.

[0207] In formula VI-B, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group in the CPT shown forms an amide bond with the carboxyl group in A.

[0208] More preferably, the compound represented by structural formula VI-B is further represented by structural formula VI-B-1:

[0209] Alternatively, the compound represented by structural formula VI provided by the present invention is further represented by structural formula VI-C:

[0210] In structural formula VI-C, the definitions of group A and R8 are the same as those of group A and R8 in structural formula III or structural formula IIIA above.

[0211] In formula VI-C, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0212] When the self-releasing structure in the structural formula VC is When , structural formula VC can be further structured as shown in structural formula VI-C.

[0213] According to a specific embodiment of the present invention, in the thirteenth aspect of the present invention, the compound has the following structure:

[0214] Fourteenth Aspect

[0215] The present invention provides a method for preparing an antibody-drug conjugate or a salt thereof, the method comprising the following steps:

[0216] a. Antibody reduction: adding ≥5.5 times the molar equivalent of the antibody to a phosphate buffer containing 5-30 mg / mL antibody, and reacting for 1.5-2 h, wherein the reducing agent is selected from one or more of TCEP, DTT, 2-MEA and DTBA;

[0217] b. Antibody conjugation and hydrolysis: The reduced antibody from step a is replaced with a phosphate buffer at pH 6.5-7.8, and the antibody is diluted to a concentration of 3.5-15 mg / mL. 4.5-6.5 times the molar equivalent of the antibody, dissolved in an organic cosolvent, is added to the antibody dilution solution and stirred at 15-35°C for ≥0.5 h, wherein the organic cosolvent is one or more selected from DMA, DMSO, DMF, and ACN. The antibody conjugated product is replaced with a phosphate buffer at pH 7.4-9.0 and heated at 35±10°C for 2-24 h to obtain a hydrolyzed product.

[0218] c. Hydrophobic chromatography: The antibody-coupled product was purified by hydrophobic chromatography using a hydrophobic filler.

[0219] Wherein, in step a, the antibody is an antibody against tumor-associated antigens, such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, etc. C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2, etc.

[0220] Preferably, the antibody is an anti-B7-H3 antibody. More preferably, the antibody is the anti-B7-H3 antibody provided in the first aspect of the present invention. For example, the anti-B7-H3 antibody may be the anti-B7-H3 antibody hz10B4, hz10B4m1, hz10B4m2, or hz10B4m3 provided in the Examples of the present invention.

[0221] Preferably, in step b, the drug-containing linker is the compound defined in the thirteenth aspect of the present invention.

[0222] Aspect 15

[0223] The antibody drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Therefore, in a fifteenth aspect, the present invention also provides a composition comprising the antibody drug conjugate or its salt according to the present invention; preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient or vehicle. The pharmaceutical composition provided by the present invention can be prepared into various dosage forms known in the medical or pharmaceutical fields and administered in an appropriate manner.

[0224] Aspect 16

[0225] The present invention also provides use of the antibody-drug conjugate or its salt and / or a composition containing the same in preparing a drug for treating tumors.

[0226] Preferably, the tumor is expressed in a tumor-associated antigen such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, or a combination thereof. C), mesothelin (Mesothelin), SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, and Claudin 18.2 positive or high expression.

[0227] Preferably, the tumor is associated with B7-H3 expression. More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms tumor and desmoplastic small round cell tumor.

[0228] Seventeenth Aspect

[0229] Accordingly, the present invention also provides a method for treating tumors, comprising administering the antibody-drug conjugate or its salt and / or a composition comprising the same provided by the present invention to a subject in need thereof.

[0230] Preferably, the tumor is expressed in a tumor-associated antigen such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, or a combination thereof. C), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein 3, and Claudin 18.2 positive or high expression.

[0231] Preferably, the tumor is associated with B7-H3 expression (including overexpression). More preferably, the tumor is a solid tumor, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, Wilms tumor and desmoplastic small round cell tumor.

[0232] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0233] The application of the above-mentioned disease treatment methods provided by the present invention depends on many factors, including the specific active ingredient administered, the patient's age, weight, gender or physical and medical condition, the severity of the condition to be treated, the route of administration, etc.

[0234] Based on a known humanized anti-B7-H3 antibody, the inventors of the present invention used site-directed mutagenesis of the variable regions to screen and obtain a series of novel humanized anti-B7-H3 antibodies. Compared to the parent antibody, these antibodies retain the parent antibody's high affinity for B7-H3 while also possessing significantly higher purity, thereby overcoming the parent antibody's vulnerability to breakage and reducing toxic side effects. Furthermore, the inventors of the present invention conjugated this series of novel humanized antibodies with a camptothecin compound of a specific structure to produce antibody-drug conjugates. By leveraging the stable, robust structure and high targeting of these antibodies to B7-H3-expressing cells, combined with the potent cytotoxicity of the camptothecin compound of this specific structure, they achieved excellent tumor-killing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0235] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0236] FIG1 shows the binding activity of the anti-B7H3 antibody of the present invention to B7H3 expressed on the surface of cancer cells.

[0237] FIG2 shows the efficacy of the B7H3-targeting antibody-drug conjugate of the present invention in a lung cancer mouse model.

[0238] FIG3 shows the efficacy of the B7H3-targeting antibody-drug conjugate and anti-B7H3 antibody of the present invention in a pancreatic cancer mouse model.

[0239] FIG4 shows the results of inducing cancer cell apoptosis by the antibody-drug conjugate targeting B7H3 and the anti-B7H3 antibody of the present invention.

[0240] FIG5 shows the bystander effect detection results of the antibody drug conjugate targeting B7H3 of the present invention (mean±SD, n=2).

[0241] FIG6 shows the test results of the ability of the B7H3-targeting antibody drug conjugate of the present invention to induce B7H3-positive cell cycle arrest (mean±SD, n=2); the results of G0 / G1, G2 / M, and S phases are arranged from top to bottom.

[0242] Best Mode for Carrying Out the Invention

[0243] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0244] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0245] Human B7H3: UniProtKB-Q5ZPR3;

[0246] Control antibody hum30: an antibody targeting B7H3, prepared according to the patent method of US20160368990A1.

[0247] (I) Screening and characterization of novel anti-B7-H3 humanized antibodies

[0248] Example 1 Preparation of anti-B7H3 antibodies

[0249] The antibody hz10B4 was prepared according to the method provided in patent application CN202111459992.0 (CN114573695A). The amino acid sequences of the heavy and light chain variable regions of the antibody hz10B4 are shown below (i.e., SEQ ID NO: 20 and SEQ ID NO: 22 in CN114573695A), with the CDRs (based on the Kabat scheme) underlined.

[0250] >hz10B4-VH(VH / CDR-H1 / CDR-H2 / CDR-H3:SEQ ID NOs:1 / 8 / 9 / 10)

[0251] >hz10B4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:2 / 11 / 12 / 13)

[0252] Five point mutations were designed for hz10B4 LCDR3, and the VLs obtained after the point mutations were combined with hz10B4-VH, respectively, according to the method described in Example 9 of CN114573695A, to obtain a series of anti-B7-H3 antibodies (see Table 1).

[0253] Table 1. Point mutations in hz10B4 LCDR3

[0254] >hz10B4-Lm1-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:3 / 11 / 12 / 14)

[0255] >hz10B4-Lm2-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:4 / 11 / 12 / 15)

[0256] >hz10B4-Lm3-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:5 / 11 / 12 / 16)

[0257] >hz10B4-Lm4-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:6 / 11 / 12 / 17)

[0258] >hz10B4-Lm5-VL(VL / CDR-L1 / CDR-L2 / CDR-L3:SEQ ID NOs:7 / 11 / 12 / 18)

[0259] Example 2 Functional evaluation of anti-B7H3 antibodies

[0260] 2.1 Antibody Binding Affinity Study for Human B7H3 ECD-His

[0261] The binding affinity of hz10B4, hz10B4m1-m5 to human B7H3 was determined using Fortebio Octet QKe.

[0262] hz10B4, hz10B4m1-m5, and the control antibody hum30 were diluted to 4 μg / ml in PBS buffer and flowed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 seconds. A recombinant human B7-H3 extracellular domain protein (SEQ ID NO: UniProtKB-Q5ZPR3, 1aa-461aa, C-His tag) (60 nM) was used as the mobile phase with an association time of 300 seconds and a dissociation time of 300 seconds. After the experiment, a 1:1 Langmuir binding model was fitted using software to calculate the kinetic constants for antigen-antibody binding.

[0263] The results of affinity testing of hz10B4 (SEQ ID NOs: 1+2), hz10B4m1 (SEQ ID NOs: 1+3), hz10B4m2 (SEQ ID NOs: 1+4), hz10B4m3 (SEQ ID NOs: 1+5), hz10B4m4 (SEQ ID NOs: 1+6), hz10B4m5 (SEQ ID NOs: 1+7), and hum30 for the recombinant human B7-H3 extracellular domain protein are shown in Table 2. The results showed that the affinities of hz10B4m1, hz10B4m2, and hz10B4m3 for the recombinant human B7-H3 extracellular domain protein were similar to those of the parental hz10B4, with a slightly lower Koff. The affinity of hz10B4m1 was slightly superior to that of hz10B4m2 and hz10B4m3.

[0264] Table 2. Affinity test results of antibodies and human B7-H3 extracellular domain recombinant protein

[0265] 2.2 Study on the binding activity of antibodies to SKOV3 cell surface antigens

[0266] Using SKOV3 cells naturally expressing human B7H3 as target cells, the binding activity of hz10B4, hz10B4m1-m5 to SKOV3 cell surface antigens was detected by FACS.

[0267] SKOV3 cells naturally expressing human B7H3 that had grown to the logarithmic phase were collected by centrifugation and the cell density was adjusted to 2×10 5 Pipette 1 ml of cell suspension into EP tubes, i.e. 2×10 5 The cells were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. 200 μL each of antibodies hz10B4 and hz10B4m1-m5 (at a concentration of 198 nM, 3-fold serial dilution, 12 concentrations) were added to the cells and incubated at 37°C for 30 min. In addition, the following controls were set: (1) positive control (PC): control antibody hum30; (2) negative control (NC-huIgG1): isotype control antibody. After incubation, the cells were washed three times with PBS, and 1:200 diluted goat anti-human IgG-FITC (Cat: F9512, Sigma) was added and incubated for 30 min. After washing the cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of the test antibodies to B7-H3 on the surface of SKOV3 cells.

[0268] The results of the binding activity assays of hz10B4, hz10B4m1, hz10B4m2, hz10B4m3, hz10B4m4, hz10B4m5, and hum30 to human SKOV3 cell surface antigens are shown in Table 3 and Figure 1. The results showed that the binding activities of hz10B4m1, hz10B4m2, and hz10B4m3 to SKOV3 cell surface antigens were similar to those of the parental hz10B4.

[0269] Table 3. Binding activity of antibodies to SKOV3 cell surface antigens

[0270] Example 3 Purity Analysis of Anti-B7H3 Antibodies

[0271] The purity of the anti-B7H3 antibody was determined using capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) under reducing and non-reducing conditions. The purity of the antibody product was determined according to General Chapter 3127 of the Chinese Pharmacopoeia, "Determination of Monoclonal Antibody Size Variants (CE-SDS Method)." The results are shown in Table 4.

[0272] Table 4. Anti-B7H3 antibody purity analysis results

[0273] NR-CE-SDS and R-CE-SDS analysis results showed that the purity of hz10B4m1, hz10B4m2, and hz10B4m3 were all above 98%, and were all superior to the parent antibody hz10B4.

[0274] (II) Synthesis of Camptothecin Compounds

[0275] General method for the synthesis of camptothecin compounds:

[0276] The camptothecin compounds of the present invention can be obtained by reacting the compound represented by formula A with a tricyclic compound via a Friedlander reaction. The general reaction formula is as follows:

[0277] Among them, CDE tricyclic compound was purchased from MCE:

[0278] The HCDE tricyclic compound was prepared according to the method of Bioorganic and Medicinal Chemistry, 2010, vol. 18, #9, p. 3140–3146:

[0279] 1. Synthesis of the compound represented by formula A

[0280] Example 4 Synthesis of Compound A1

[0281] Method 1: Compound A1 was synthesized according to the method of patent publication document WO2020200880A1. The synthetic route is as follows:

[0282] Method 2: Compound A1 was synthesized by zinc reagent palladium catalyzed coupling method. The synthetic route is as follows:

[0283] (1) Acetylation

[0284] 3-Bromo-4-nitroaniline (25.00 g, 0.12 mol) and 200 mL of acetic acid were added to a 500 mL flask, followed by the slow dropwise addition of 50 mL of acetic anhydride. The mixture was allowed to react at room temperature for 16 hours. After TLC analysis of the reaction, the reaction mixture was filtered and concentrated under reduced pressure to remove the acetic acid. The combined filter cake was slurried with 200 mL of MTBE, and 27.6 g of a yellow solid (A1-a) was obtained by filtration and dried. Yield: 92%. LC-MS (ESI): m / z 259 (M+H).

[0285] (2) Preparation of 4-ethoxy-4-oxobutylzinc bromide

[0286] Activated zinc powder (19.0 g, 0.29 mol, 2.00 eq) was added to a 250 mL three-necked flask (thermometer, reflux condenser, rubber stopper). After nitrogen displacement, anhydrous DMF (145 mL) was added, and nitrogen was replaced again. Iodine (1.86 g, 0.015 mol, 0.1 eq) was added at room temperature. The solution was observed to change from colorless to brown-red, then gradually to light yellow and finally to colorless (2-3 min). Then, ethyl 4-bromobutyrate (28.6 g, 0.15 mol, 1.00 eq) was added, and the temperature was raised to 80°C (internal temperature) for 4 to 5 hours. After the reaction was complete by TLC, the reaction solution was allowed to stand and cooled to room temperature for use. The supernatant was light yellow with a concentration of 1 mol / L.

[0287] (3) Zinc reagent coupling

[0288] A 500 mL reaction flask was charged with anhydrous DMF (120 mL) and intermediate A1-a (25.0 g, 1.00 eq). After purging the atmosphere with nitrogen, palladium acetate (433 mg, 0.02 eq) was added. The mixture was stirred at room temperature for 10 min, followed by the addition of S-PHOS (1.6 g, 0.04 eq). The atmosphere was purged with nitrogen again, and the mixture was stirred for 20 min. After that, 4-ethoxy-4-oxobutylzinc bromide (145 mL, 1.50 eq) prepared in the previous step was added dropwise at room temperature (25-30°C). The reaction was maintained at 25-30°C for 16 hours. After complete reaction, TLC (DCM:EA = 5:1) confirmed that the starting materials had reacted completely. The reaction was cooled to room temperature and quenched by the addition of ammonium chloride solution (15 mL). The reaction solution was poured into 1 L of water, and 400 mL of ethyl acetate was added. The mixture was separated and filtered using a Buchner funnel. The aqueous phase was extracted with ethyl acetate (400 mL x 2). The organic phase was washed twice with water (500 mL) and once with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 37.0 g of a reddish-brown oily liquid (A1-b), with a yield of 130%. The crude product was used directly in the next reaction without further purification. LC-MS (ESI): [M+1]+ = 295.

[0289] (4) Synthesis of Intermediate A1-c

[0290] Intermediate A1-b (37.0 g, 1.0 eq) and ethanol (1.5 L) were added to a 3 L three-necked reaction flask, and the starting materials dissolved completely. The reaction system was cooled to 5°C, and 5% Pd / C (5.7 g, 1.0 eq) was added. The temperature was raised to 25°C under a hydrogen atmosphere (normal pressure) and the reaction was allowed to proceed for 16 hours. TLC (DCM:EA = 5:1) confirmed the completion of the reaction. The reaction system was filtered through Celite, and the organic phase was concentrated to yield 33.0 g of the crude product (A1-c), in a 130% yield. The crude product was used directly in the next step without further purification. LC-MS (ESI): [M+1]+ = 265.

[0291] (5) Synthesis of Intermediate A1-d

[0292] To a 1L flask, intermediate A1-c (33.0g, 1.00eq) and 260mL of acetic acid were added, followed by the slow dropwise addition of 46mL of acetic anhydride, and the reaction was allowed to proceed at room temperature for 2 hours. After the reaction was complete, as determined by TLC (DCM:MeOH = 10:1), the reaction solution was filtered and concentrated under reduced pressure to remove the acetic acid. 250mL of water was added, and the aqueous phase was extracted with ethyl acetate (400mL x 2). The organic phase was washed twice with water (500mL) and once with saturated sodium chloride solution (500mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 32g of crude product. The crude product was slurried with 400mL of MTBE, and the dried product (A1-d) was filtered to yield 27.0g of a pale yellow solid (91%). The total yield of the four-step reaction was 83.7%.

[0293] LC-MS (ESI): [M+1]+=307.

[0294] 1H NMR (400MHz, DMSO) δ9.87(s,1H),9.19(s,1H),7.41(s,1H),7.37(d,J=8.7Hz,1H),7.20(d,J=8.5Hz,1H),4.06(q, J=7.0Hz,2H),2.52(d,J=8.5Hz,2H),2.29(t,J=7.3Hz,2H),2.02(s,6H),1.79–1.64(m,2H),1.18(t,J=7.1Hz,3H).

[0295] (6) Synthesis of Intermediate A1-e

[0296] To a 500 mL three-necked reaction flask, intermediate A1-d (27.0 g, 88 mmol, 1.0 eq), water (100 mL), and tetrahydrofuran (200 mL) were added, and the starting materials dissolved completely. Lithium hydroxide monohydrate (18.5 g, 441 mmol, 5.0 eq) was added at room temperature, and the reaction system was maintained at room temperature for 3 hours. TLC confirmed the reaction was complete, and most of the tetrahydrofuran was evaporated under reduced pressure. 300 mL of water was added to the residue, and the aqueous phase was extracted with EA (2 × 100 mL). The organic phase was discarded. The aqueous phase was adjusted to pH 4 with 6N hydrochloric acid in an ice bath. A solid precipitated, which was filtered to obtain a white solid (Intermediate A1-e), 19.1 g, in a 78% yield.

[0297] 1H NMR (400MHz, DMSO) δ12.09(s,1H),9.86(s,1H),9.18(s,1H),7.38(d,J=15.5Hz,2H),7.23(d ,J=8.5Hz,1H),2.51(d,J=8.1Hz,2H),2.23(t,J=7.1Hz,2H),2.02(s,6H),1.76–1.61(m,2H)

[0298] (7) Synthesis of Compound A1

[0299] To a 250mL reaction flask, add 40mL of polyphosphoric acid and heat to 90°C. Intermediate A1-5 (5.0g, 17.97mmol) was added portionwise and the internal temperature maintained at 95-100°C for 5 hours. Upon completion of the reaction by TLC, remove the heat, cool the system to 50-60°C, and quench the reaction by dropwise addition of 15mL of 4M HCl (aq) (the temperature will rise to 100°C). After completion of the dropwise addition, adjust the pH to 10 by dropwise addition of 600mL of 4M NaOH in an ice bath. The aqueous phase was extracted with ethyl acetate (50mL x 3). The combined organic phases were washed once with saturated sodium chloride solution (50mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 3.75g of a yellow solid (Intermediate A1-f) in an 80.5% yield. The product was used directly in the next step without purification. LC-MS (ESI): [M+1]+ = 261.

[0300] In a 250 mL three-necked reaction flask, the raw intermediate A1-f (3.75 g) was suspended in 19% hydrochloric acid (30 mL). The reaction system was heated to 90°C (internal temperature) for 3 hours. TLC confirmed the completion of the reaction. The mixture was cooled to below 5°C in an ice-salt bath, and 4M NaOH solution (45 mL, 30 eq) was added dropwise to adjust the pH to 10. The aqueous phase was extracted with ethyl acetate (80 mL x 5). The combined organic phases were washed once with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 2.45 g of crude product. The crude product was purified by column chromatography using DCM as the eluent to afford 1.93 g of a yellow solid (Compound A1), in a 76% yield. The total yield for the two-step reaction was 61.2%.

[0301] LC-MS (ESI): [M+1]+=177.

[0302] 1H NMR(400MHz,DMSO)δ6.76(d,J=8.7Hz,1H),6.68(s,2H),6.42(d,J=8.7Hz,1H) ,4.17(s,2H),2.55(t,J=5.9Hz,2H),2.46(t,J=6.2Hz,2H),2.00–1.80(m,2H)

[0303] Method 3: Compound A1 was prepared by lactam hydrolysis. The synthetic route is as follows:

[0304] The aminolactam compound was prepared according to the method of patent publication document CN106349233A.

[0305] In a 500ml three-necked flask, the aminolactam compound (17.6g, 0.1moml), ethanol (250ml), and 98% sulfuric acid (5ml) were mixed and heated under reflux for 24 hours. Sampling was performed and after the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. The residue was added with dichloromethane (200ml) and water (100ml), cooled to below 10°C in an ice bath, and the pH was adjusted to 7-8 with 1N sodium hydroxide aqueous solution. The mixture was stirred and separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to remove the organic solvent to obtain a crude diaminoethyl ester intermediate (25g), which was directly used for the next step.

[0306] The diaminoethyl ester intermediate was dissolved in dichloromethane (200 mL), and triethylamine (20.2 g, 0.2 mol, 2 eq) was added. The mixture was cooled to below 10°C in an ice bath, and acetic anhydride (25.5 g, 0.25 mol, 2.5 eq) was added dropwise. The mixture was incubated for 1 hour, sampled, and tested. Upon completion of the reaction, the reaction solution was poured into icy 1N hydrochloric acid and stirred for 15 minutes. The layers were separated, and the aqueous phase was extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to remove the organic solvent. The crude product was slurried with 400 mL of MTBE and filtered to dryness to yield 26.9 g of intermediate A1-e as a pale yellow solid. The total yield of the two-step reaction was 87.8%. LC-MS (ESI): m / z 307 (M+H)+.

[0307] According to method 2, compound A1 was prepared from intermediate A1-e.

[0308] Example 5 Synthesis of Compound A2

[0309] According to the method of patent publication WO2021148501, compound A2 was synthesized using the following synthesis route:

[0310] Example 6 Synthesis of Compound A3

[0311] Compound A3 was synthesized according to the method of patent publication document US2004266803A.

[0312] Example 7 Synthesis of Compound A4

[0313] Compound A4 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0314] Step 1:

[0315] Intermediate A4-2 was prepared from compound A4-1 according to the literature method.

[0316] Step 2:

[0317] Intermediate A4-2 (3.4 g) was dissolved in dichloromethane at -5°C to 5°C. Triethylamine (3.0 g) was added, and AllocCl (2.8 g) was slowly added dropwise. After the addition was complete, the mixture was stirred for 1-2 hours. The mixture was quenched with water, washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 5.5 g of crude intermediate A4-3. LC-MS (ESI): [M+1]+ = 255.7.

[0318] Step 3:

[0319] At room temperature, intermediate A4-3 (5.5 g) was mixed with TBAF (55 ml) and acrylic acid (110 ml), heated to 50-55°C, and stirred for 24 hours. The reaction mixture was then concentrated to dryness and eluted by column chromatography with petroleum ether followed by methanol:dichloromethane (1:50) to afford approximately 5.2 g of crude intermediate A4-4. LC-MS (ESI): [M+1]+ = 327.4.

[0320] Step 4:

[0321] At room temperature, intermediate A4-4 (5.0 g) was mixed in 100 ml of a mixed solution of ethanol:water = 3:1, and ammonium chloride (5.5 g) and iron powder (5.5 g) were added. The mixture was heated to reflux for 1 to 2 hours. The reaction mixture was also cooled to room temperature, filtered, and the solid was washed with ethanol and concentrated to dryness to obtain approximately 3.8 g of crude intermediate A4-5. LC-MS (ESI): [M+1]+ = 297.4.

[0322] Step 5:

[0323] At 20-30°C, intermediate A4-5 (3.8 g) was mixed with trifluoroacetic acid (38 ml), and trifluoroacetic anhydride (38 ml) was added. The mixture was stirred for 18-24 hours. Post-treatment: the mixture was concentrated to dryness and purified by column chromatography using petroleum ether and eluted with ethyl acetate:petroleum ether (1:4) to afford intermediate A4-6 (1.0 g). LC-MS (ESI): [M+1]+ = 375.3.

[0324] Step 6:

[0325] At room temperature, intermediate A4-6 (1.0 g) was mixed in methanol (20 ml), and potassium carbonate (2.0 g) and water (5 ml) were added, and the mixture was stirred for 1 to 2 hours. The reaction solution was diluted with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a crude product. The crude product was eluted by column chromatography with petroleum ether until ethyl acetate:petroleum ether = 1:2 to obtain intermediate A4-7: 0.65 g, LC-MS (ESI): [M+1]+ = 279.3.

[0326] Step 7:

[0327] At room temperature, intermediate A4-7 (500 mg) was dissolved in tetrahydrofuran (20 ml). Under nitrogen, pyrrole (120 mg) and tetrakistriphenylphosphine palladium (160 mg) were added, and the mixture was stirred for 1 to 2 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography using dichloromethane:methanol = 30:1 to obtain A4 as a brown solid, 30 mg; LC-MS (ESI): [M+1]+ = 195.4.

[0328] Example 8 Synthesis of Compound A5

[0329] Compound A5 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0330] Step 1:

[0331] At 20-30°C, compound A5-1 (25 g) was mixed with acetic acid (100 ml), and acetic anhydride (24.9 g) was slowly added. After the addition was complete, the mixture was stirred for 3-4 hours. The reaction solution was slowly transferred into ice water and stirred to precipitate a solid. The solid was collected by filtration, washed with water, and dried in vacuo to obtain intermediate A5-2 as a yellow solid, 31.0 g, LC-MS (ESI): [M+1]+=197.2.

[0332] Step 2:

[0333] At 20-30°C, intermediate A5-2 (29 g), potassium carbonate (40 g), potassium iodide (5 g) and bromopropyl alcohol (25 g) were mixed in DMF (300 ml), heated to 100-110°C, and stirred for 3-4 hours. The reaction solution was cooled to room temperature, quenched by adding ice water, extracted three times with 2 L of ethyl acetate, combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to dryness, and slurried with petroleum ether to obtain a solid, which was dried in vacuo to obtain intermediate A5-3 as a yellow solid: 33.0 g, LC-MS (ESI): [M+1]+=255.3.

[0334] Step 3:

[0335] At 20-25° C., intermediate A5-3 (23 g) was dissolved in acetonitrile (1 L), and sodium dihydrogen phosphate solution (0.67 mol, pH 6.7, 900 ml), TEMPO (5 g), sodium chlorite solution (52.0 g dissolved in 60 ml of water), and sodium hypochlorite solution (38 ml mixed with 38 ml of water) were added sequentially. After the addition was completed, the reaction solution turned dark brown and was stirred for 30 minutes. The reaction solution was cooled to room temperature, the pH was adjusted to 2-3 with 2N hydrochloric acid, and extracted with ethyl acetate (1000 ml×3). The ethyl acetate layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and slurried with petroleum ether to obtain 24.0 g of crude intermediate A5-4, LC-MS (ESI): [M+1]+=269.2.

[0336] Step 4:

[0337] At room temperature, intermediate A5-4 (25 g) and 5% palladium on carbon (5 g) were mixed in methanol (500 ml) and subjected to pressure hydrogenation for 3 to 4 hours. The palladium on carbon was filtered off, the solid was washed with methanol, the filtrate was concentrated, and the residue was slurried with petroleum ether to obtain a crude product, which was then dried in vacuo to obtain intermediate A5-5 as a yellow solid: 18.0 g, LC-MS (ESI): [M+1]+=239.5.

[0338] According to a method similar to steps 5 and 6 in the synthesis of compound A4, intermediate A5-7 was synthesized from intermediate A5-5. Finally, the acetyl protection was removed with 6N hydrochloric acid to prepare compound A5 as a yellow solid. LC-MS (ESI): [M+1]+=179.2.

[0339] Example 9 Synthesis of Compound A6

[0340] Compound A6 was synthesized according to the method of Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0341] Compound A6 is a yellow solid, LC-MS (ESI): [M+1]+=248.9.

[0342] 1 H NMR (400MHz, d6-DMSO): δ12.289 (1H, s), 8.666-8.648 (1H, d), 8.198-8.1 (1H, d ), 3.138-3.108(2H, t), 2.768-2.734(2H, t), 2.219(3H, s), 2.050-1.991(2H, m)

[0343] Step 1:

[0344] In a 250ml three-necked flask under nitrogen, compound A1-4 (1.25g, 5mmol, 1eq) was dissolved in 150ml of tetrahydrofuran, cooled to -60°C, and LDA (2M in THF, 7.6ml, 15.2mmol, 3.04eq) was added. After the addition, the mixture was stirred at -60°C for 30 minutes. MeI (1.45g, 10.21mmol, 2.04eq) was added. After the addition, the dry ice bath was removed, the mixture was allowed to warm naturally, and the reaction was allowed to proceed overnight at room temperature. The reaction was quenched by the addition of 50ml of aqueous ammonium chloride solution and extracted with ethyl acetate (150ml x 3). The organic phases were combined, washed once with water, and concentrated to dryness to obtain the crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate in a ratio of 1:0 to 5:1 to afford compound A6-1 as a yellow solid, 350mg, in a 28% yield.

[0345] 1 H NMR (CDCl3): δ12.49 (1H, s), 8.77 (1H, d), 8.03 (1H, d), 3.21 (2H, m), 2.22 (3H, s), 1.90 (2H, t), 1.20 (6H, s)

[0346] Step 2:

[0347] In a 100ml single-necked flask, combine compound A6-1 (280mg) with 20ml of 6N hydrochloric acid and heat under reflux with stirring for 2 hours. After cooling to room temperature, adjust the pH of the reaction mixture to 7-8 with sodium bicarbonate. Extract with dichloromethane (30ml x 3). Combine the organic phases and concentrate to dryness to obtain compound A6-2 as a brown-gray solid (275mg). The crude product was used directly in the next step without purification.

[0348] Step 3:

[0349] In a 50ml three-necked flask, compound A6-2 (220mg) was dissolved in acetic acid (25ml). 1.3g of iron powder was added and the mixture was stirred at 80-85°C for 1-2 hours. After the reaction was complete, the acetic acid was distilled off under reduced pressure. Water (30ml) was added to the residue and the pH was adjusted to 7-8 with sodium bicarbonate. The mixture was extracted with dichloromethane (30ml*3) and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 1:0-2:1 as eluent to obtain compound A6 as a brown solid (122mg). The two-step reaction yield was 75%. LC-MS (ESI): [M+1]+ = 205.0

[0350] Example 10 Synthesis of Compound A7

[0351] The synthetic route is as follows:

[0352] In a 250 ml three-necked flask, compound A1 (2.18 g, 124 mmol, 1 eq) was dissolved in tetrahydrofuran (100 ml), and Boc anhydride (8.2 g, 376 mmol, 3 eq) was added. The mixture was stirred at 40-45°C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound A7-1 as a yellow solid (3.0 g, 90% yield).

[0353] 1H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J=6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

[0354] In a 250 ml three-necked flask, compound A7-1 (3.0 g, 10.8 mmol, 1 eq) and DIPEA (3.5 g, 27.1 mmol, 1.5 eq) were mixed in dichloromethane (125 ml). The reaction solution was cooled to -5 to 0°C with an ice-salt bath. Acetyl chloride (1.3 g, 16.5 mmol, 2 eq) was added dropwise. After the addition was complete, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours. The reaction solution was concentrated to dryness, and the residue was purified by column chromatography with petroleum ether:ethyl acetate = 1:0 to 1:3 as the eluent to obtain compound A7-2 as a light yellow solid, 3.48 g, in a yield of 100%.

[0355] 1H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J=9.1Hz,1H),7.57(t,J=36.2Hz,1H),6.09(s, 1H),2.80(t,J=6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

[0356] In a 500 ml three-necked flask, under nitrogen protection, compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C in a dry ice-acetone bath. KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition was complete, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. q) was dissolved in tetrahydrofuran (70 ml), added dropwise, and stirred at -70 to -60°C for 10 minutes, then naturally warmed to 20 to 30°C and stirred for 3 hours. Saturated aqueous ammonium chloride (80 ml) was added to quench the reaction. The reaction solution was extracted with ethyl acetate (150 ml * 3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound A7-3 as a yellow solid, 0.85 g, in a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0357] 1H NMR (400MHz, CDCl3) δ11.49 (s, 1H), 8.64 (d, J = 9.2Hz, 1H), 7.74 (d, J = 8.5Hz, 1H),6.06(s,1H),3.01(t,J=6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

[0358] In a 100ml single-necked flask, compound A7-3 (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. Heating was stopped, and after the reaction solution cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate. The mixture was extracted with dichloromethane (30ml*3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain the crude product. Purification by column chromatography with petroleum ether:ethyl acetate = 1:0 to 1:3 as eluent yielded compound A7 as a brown solid (0.25g, 49.1% yield). LC-MS (ESI): [M+1]+ = 213.9

[0359] Example 11 Synthesis of Compound A8

[0360] The synthetic route is as follows:

[0361] Compound A8-1 was synthesized according to the method described in Journal of Medicinal Chemistry, 1989, vol. 32, #6, p. 1217-1230. LC-MS: (ESI): [M+1]+ = 146.3.

[0362] Compound A8-1 (1.8 g) and DIEA (4.8 g) were added to DCM (50 ml). The mixture was cooled to 0°C. Trifluoroacetic anhydride (4.8 g) was added, the temperature was raised to room temperature, and the reaction was allowed to react for 1 hour. Water (20 ml) was added, the organic layer was separated, and the mixture was dried by spin drying. Purification by column chromatography (EA:PE = 0-30%) afforded intermediate A8-2 as a white solid (2.7 g). LC-MS: (ESI): [M+1]+ = 242.2.

[0363] Zn(Et)2 (33.6 ml) was added to DCM (70 ml) and cooled to 0°C. TFA (3.8 g) and CH2I2 (9.0 g) were added. The mixture was allowed to react for 15 minutes. A solution of compound A8-2 (2.7 g) in DCM (30 ml) was added dropwise. After completion of the addition, the mixture was stirred at room temperature overnight. Water (30 ml) was added, the organic layer was separated, dried, and spin-dried to afford intermediate A8-3 as an off-white solid (2.7 g). LC-MS (ESI): [M+1]+ = 256.1.

[0364] Intermediate A8-3 (1.5 g) was added to DCM (100 ml) and acetic anhydride (30 ml), cooled to 0°C, and 65% nitric acid (2.8 g) was added. The mixture was allowed to react at room temperature overnight. Water (50 ml) and DCM (100 ml) were added. The organic layer was separated and spin-dried. The crude intermediate A8-4 (1.6 g) was obtained by oil pump drying and used directly in the next step. LC-MS (ESI): [M+1]+ = 301.2.

[0365] Intermediate A8-4 (1.6 g crude product), iron powder (3.2 g), and ammonium chloride (6.4 g) were added to anhydrous ethanol (100 ml) and refluxed overnight. The mixture was cooled, filtered, dried, and purified by column chromatography (EA:PE = 0-50%) to afford intermediate A8-5 as a brown oil (0.25 g). LC-MS (ESI): [M+1]+ = 271.3.

[0366] Intermediate A8-5 (250 mg) and DIEA (400 mg) were added to DCM (10 ml), cooled to 0°C, and trifluoroacetic anhydride (320 mg) was added. The mixture was reacted at room temperature for 1 hour. Water (5 ml) was added for washing. The organic layer was separated, dried, filtered, and spin-dried to afford intermediate A8-6 as a yellow solid (340 mg). LC-MS (ESI): [M+1]+ = 367.3.

[0367] Intermediate A8-6 (150 mg) and CrO₃ (400 mg) were added to acetic acid (8 ml) and acetic anhydride (4 ml) and reacted at 30-40°C for 3 hours. The solvent was evaporated. Water (5 ml) was added and the mixture was extracted with DCM (20 ml). The organic layer was separated, dried, filtered, and evaporated to dryness to obtain 160 mg of crude intermediate A8-7, which was used directly in the next step. LC-MS (ESI): [M+1]⁺ = 381.3.

[0368] The intermediate A8-7 was deprotected from the bis-trifluoroacetyl group by 6N hydrochloric acid and purified by column chromatography to obtain compound A8. LC-MS (ESI): [M+1]+=189.3.

[0369] The following compounds can be synthesized according to the similar method described above

[0370] 2. Synthesis of Camptothecin Compounds (Friedlander Reaction)

[0371] Synthesis of control camptothecin compound MWC-1:

[0372] At room temperature, compound A1 (176 mg, 1 eq), CDE ring compound (315 mg, 1.2 eq), and PPTS (301 mg, 1.2 eq) were mixed in toluene (50 ml) and refluxed to separate water for 3 to 4 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether as eluent to methanol / dichloromethane = 1:20 to obtain MWC-1 as a yellow solid, 260 mg, LC-MS (ESI): [M+1]+ = 404.

[0373] Example 12 Synthesis of Camptothecin Compound 1

[0374] The synthetic route is as follows:

[0375] In a 50 ml single-necked reaction flask, under nitrogen protection, compound A6 (120 mg, 0.59 mmol, 1 eq), CDE tricyclic compound (180 mg, 0.68 mmol, 1.2 eq) and PPTS (35 mg, 0.14 mmol, 0.25 eq) were mixed in 15 ml and heated under reflux for 3 to 4 hours. Most of the acetic acid was removed by distillation under reduced pressure, and the residue was crudely separated by column chromatography with an eluent of dichloromethane:methanol = 15:1 to obtain 80 mg of a crude product, which was then purified by preparative liquid phase separation to obtain 3 mg of camptothecin compound 1 as a yellow solid.

[0376] LC-MS (ESI): [M+1]+=432.4.

[0377] Example 13 Synthesis of Camptothecin Compounds 2 (2A and 2B)

[0378] The synthetic route is as follows:

[0379] The products were synthesized by a method similar to Example 12, using A9 instead of A6. The products were separated and purified by preparative liquid phase to obtain compound 2A (early retention time) as a yellow solid, LC-MS (ESI): [M+1]+=418.3; compound 2B (late retention time) as a yellow solid, LC-MS (ESI): [M+1]+=418.4.

[0380] Example 14 Synthesis of Camptothecin Compound 3

[0381] The synthetic route is as follows:

[0382] The camptothecin compound 3 was synthesized in a similar manner to Example 12, using A7 instead of A6 and toluene instead of acetic acid as solvent to obtain a yellowish brown solid. LC-MS (ESI): [M+1]+=440.8

[0383] 1H NMR (400MHz, Acetone) δ7.89(d,J=9.1Hz,1H),7.48(d,J=9.1Hz,1H),7.37(s,1H),7.31(d,J=8.9Hz,0H), 5.59–5.26(m,5H),3.11–3.05(m,4H),2.73–2.59(m,3H),1.98(dt,J=14.0,7.0Hz,1H),1.05–0.98(m,3H)

[0384] 1H NMR (400MHz, DMSO) δ7.87(d,J=9.1Hz,0H),7.41(d,J=9.1Hz,0H),7.21(s,0H),6.50(s,0H),6.15(s,1H),5.36(d ,J=46.6Hz,1H),2.93(t,J=6.5Hz,1H),2.66–2.54(m,1H),1.97–1.68(m,3H),1.24(s,1H),0.88(t,J=7.3Hz,3H)

[0385] Example 15 Synthesis of Camptothecin Compounds 4 (4A and 4B)

[0386] According to a similar synthetic method as Example 12, A10 was used instead of A6 to prepare the product. After preparative liquid separation and purification, compound 4A (early retention time) was obtained as a khaki solid. LC-MS (ESI): [M+1]+=422.3; compound 4B (late retention time) was obtained as a khaki solid. LC-MS (ESI): [M+1]+=422.3.

[0387] Example 16 Synthesis of Camptothecin Compound 5

[0388] The synthetic route is as follows:

[0389] A camptothecin compound 5 was prepared as a yellow solid using a similar method to Example 12, using A12 instead of A6. LC-MS (ESI): [M+1]+=444.3.

[0390] Example 17 Synthesis of Camptothecin Compound 6

[0391] The synthetic route is as follows:

[0392] Camptothecin compound 6 was synthesized by a similar method to Example 12, using A11 instead of A6, as a yellow solid. LC-MS (ESI): [M+1]+=430.5.

[0393] Example 18 Synthesis of Camptothecin Compound 7

[0394] The synthetic route is as follows:

[0395] Compound 7 was synthesized by a similar method to Example 12, using A8 instead of A6, to obtain a yellow solid (0.7 mg). LC-MS (ESI): [M+1]+ = 416.3.

[0396] Example 19 Synthesis of Camptothecin Compound 9

[0397] The synthetic route is as follows:

[0398] According to a similar synthesis method as Example 12, A3 was used instead of A6 to obtain camptothecin compound 9 as a brown-red solid. LC-MS (ESI): [M+1]+=420.3.

[0399] Example 20 Synthesis of Camptothecin Compound 12

[0400] Compound 12 was synthesized according to a method similar to that described in Journal of Medicinal Chemistry, 1998, 41(13), 2308-2318. The synthetic route is as follows:

[0401] According to the similar synthesis method of Example 12, using A4 instead of A6 and using toluene instead of acetic acid as solvent, compound 12 was synthesized as a brown solid, 30 mg; LC-MS (ESI): [M+1]+=422.5.

[0402] Example 21 Synthesis of Camptothecin Compound 13

[0403] The synthetic route is as follows:

[0404] According to the synthetic method similar to Example 12, using A15 instead of A6 and using toluene as solvent, compound 13 was synthesized as a brown solid. LC-MS (ESI): [M+1]+=458.4.

[0405] Example 22 Synthesis of Camptothecin Compound 14

[0406] The synthetic route is as follows:

[0407] According to a synthetic method similar to Example 12, using A5 instead of A6 and using toluene as solvent, compound 14 was prepared as a yellow solid. LC-MS (ESI): [M+1]+=406.1.

[0408] Example 23 Synthesis of Camptothecin Compound 14-P

[0409] Compound 12 (200 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml) at room temperature. Under nitrogen, the temperature was lowered to 5°C, 30% H₂O₂ (580 mg) was added, and the mixture was stirred for 2 hours. The reaction solution was concentrated to dryness and separated by preparative HPLC to give compounds 14-P1 and 14-P2. Freeze-dried to give 42 mg of compound 14-P1 and 25 mg of compound 14-P2 as a brown solid; LC-MS (ESI): [M+1]⁺ = 438.5.

[0410] Example 24 Synthesis of Camptothecin Compound 15

[0411] Compound 12 (100 mg) was dissolved in a mixed solvent of acetic acid (24 ml) and water (3 ml) at room temperature. Under nitrogen, the temperature was lowered to 5°C, 30% H₂O₂ (750 mg) was added, and the mixture was stirred for 2 hours. The mixture was then returned to room temperature (25°C) and stirred overnight. LCMS analysis indicated the reaction was complete. The reaction solution was concentrated to dryness, separated by preparative HPLC, and the solid was collected and lyophilized to afford 8 mg of compound 15 as a brown solid. LC-MS (ESI): [M+1]⁺ = 454.4.

[0412] Example 25 Synthesis of Camptothecin Compound 16

[0413] Intermediate 16a was synthesized according to the patent method of WO2020200880A1, and then 16a-thiocamptothecin structure intermediate 16c was prepared according to the method of the literature (J.Med.Chem.2008, 51, 3040-3044). Finally, the acetyl protection was removed to obtain compound 16, LC-MS (ESI): [M+1]+=420.3.

[0414] Example 26 Synthesis of Camptothecin Compound 17

[0415] The synthetic route is as follows:

[0416] According to the synthesis method similar to Example 12, using A1 instead of A6, HCDE instead of CDE, and toluene as solvent, homocamptothecin compound 17 (S, R mixture) was obtained, LC-MS (ESI): [M+1]+=418.5.

[0417] Example 27 Synthesis of Camptothecin Compound 18

[0418] The synthetic route is as follows:

[0419] Compound 18 was synthesized according to a similar method to Example 12, except that A16 was substituted for A6 and toluene was used as solvent. The compound 18 was a light yellow solid. LC-MS (ESI): [M+1]+=424.2.

[0420] Example 28 Synthesis of Camptothecin Compound 19

[0421] The synthetic route is as follows:

[0422] According to the synthetic method similar to Example 12, using A17 instead of A6 and toluene as solvent, compound 19 was synthesized as a yellow solid. LC-MS (ESI): [M+1]+=460.5.

[0423] Example 29 Synthesis of Camptothecin Compound 20

[0424] The synthetic route is as follows:

[0425] According to the synthetic method similar to Example 12, using A13 instead of A6 and using toluene as solvent, compound 20 was synthesized as a yellow solid. LC-MS (ESI): [M+1]+=458.2.

[0426] Example 30 Synthesis of Camptothecin Compound 21

[0427] The synthetic route is as follows:

[0428] According to the synthetic method similar to Example 12, using A24 instead of A6 and using toluene as solvent, compound 21 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=418.4.

[0429] Example 31 Synthesis of Camptothecin Compound 22

[0430] The synthetic route is as follows:

[0431] According to the synthetic method similar to Example 12, using A25 instead of A6 and using toluene as solvent, compound 22 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=434.4.

[0432] Example 32 Synthesis of Camptothecin Compound 23

[0433] The synthetic route is as follows:

[0434] According to the synthetic method similar to Example 12, using A21 instead of A6 and using toluene as solvent, compound 23 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=432.4.

[0435] Example 33 Synthesis of Camptothecin Compound 24

[0436] The synthetic route is as follows:

[0437] According to the synthetic method similar to Example 12, using A20 instead of A6 and using toluene as solvent, compound 24 (a pair of isomers) was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=418.4.

[0438] Example 34 Synthesis of Camptothecin Compound 25

[0439] The synthetic route is as follows:

[0440] According to the synthetic method similar to Example 12, using A32 instead of A6 and using toluene as solvent, compound 25 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=432.4.

[0441] Example 35 Synthesis of Camptothecin Compound 26

[0442] According to the synthetic method similar to Example 12, using A18 instead of A6 and using toluene as solvent, compound 26 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=440.4.

[0443] Example 36 Synthesis of Camptothecin Compound 27

[0444] According to a synthetic method similar to Example 12, using A19 instead of A6 and using toluene as solvent, compound 27 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=476.4.

[0445] Example 37 Synthesis of Camptothecin Compound 31

[0446] According to the synthetic method similar to Example 12, using A14 instead of A6 and using toluene as solvent, compound 31 was obtained as a yellow solid. LC-MS (ESI): [M+1]+=442.4.

[0447] Example 38 Synthesis of Camptothecin Compound 32

[0448] According to the synthetic method similar to Example 12, using A2 instead of A6, HCDE instead of CDE, and toluene as solvent, compound 32 was synthesized as a yellow solid. LC-MS (ESI): [M+1]+=436.4.

[0449] Example 39 Synthesis of Camptothecin Compound 33

[0450] The synthetic route is as follows:

[0451] According to the synthesis method similar to Example 12, using A23 instead of A6 and using toluene as solvent, compound 33 was obtained as a yellow solid. LC-MS (ESI): [M+1]+=458.5.

[0452] Example 40 Synthesis of Camptothecin Compound 34

[0453] The synthetic route is as follows:

[0454] According to a synthetic method similar to that of Example 12, using A22 instead of A6 and using toluene as solvent, compound 34 was synthesized as a yellow solid, LC-MS (ESI): [M+1]+=458.5.

[0455] Example 41 Synthesis of Camptothecin Compounds 35 and 38

[0456] The synthetic route is as follows:

[0457] According to a similar synthetic method as in Example 12, using A26 instead of A6 and using toluene as solvent, compound 35 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=438.7.

[0458] According to the synthetic method similar to Example 12, using A27 instead of A6 and using toluene as solvent, compound 38 was obtained as a brown-red solid. LC-MS (ESI): [M+1]+=474.7.

[0459] Example 42 Synthesis of Camptothecin Compounds 36 and 39

[0460] The synthetic route is as follows:

[0461] According to the synthetic method similar to Example 12, using A30 instead of A6 and using toluene as solvent, compound 36 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=456.7.

[0462] According to the synthetic method similar to that of Example 12, A31 was used instead of A6, and toluene was used as solvent to synthesize compound 39 as a brownish red solid. LC-MS (ESI): [M+1]+=492.7

[0463] Example 43 Synthesis of Camptothecin Compounds 37 and 40

[0464] The synthetic route is as follows:

[0465] According to the synthetic method similar to Example 12, using A28 instead of A6 and using toluene as solvent, compound 37 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=440.8.

[0466] According to a synthetic method similar to that of Example 12, using A29 instead of A6 and using toluene as solvent, compound 40 was synthesized as a brown-red solid. LC-MS (ESI): [M+1]+=476.7.

[0467] (III) Synthesis of drug-containing linkers

[0468] Example 44 Synthesis of drug-containing linker MWD-L1

[0469] The synthetic route is as follows:

[0470] GGFG-Dxd was synthesized according to the method described in patent publication (US20190151328A1):

[0471] GGFG-Dxd is a light yellow solid, LC-MS (ESI): M+1=841;

[0472] The BL linker compound was synthesized according to the method described in patent publication WO2018 / 095422A1:

[0473] The BL linker compound is a yellow solid, LC-MS (ESI): M+1=858

[0474] The BL linker compound (857 mg, 1 mmol), GGFG-Dxd (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), and HATU (570 mg, 1.5 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred for 2 h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20 ml) was added, and the mixture was stirred for 0.5 h. The layers were separated, and the aqueous phase was extracted with DCM (30 ml x 2). The organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. Purification by column chromatography using DCM:MeOH = 50:1 to 10:1 as eluent yielded 500 mg of the drug-containing linker MWD-L1 as a yellow solid in a yield of 29.8%. LC-MS (ESI) results showed M+1 = 1681, (M+1) / 2 = 840.9.

[0475] Example 45 Synthesis of drug-containing linker MWC-L2

[0476] Step 1

[0477] Boc-Val-Ala-OH (288 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml of DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography using an eluent of PE:EA (10:1 to 5:1) to quantitatively obtain 450 mg of intermediate 2-1 as a gray-green solid.

[0478] Step 2

[0479] Intermediate 2-1 (450 mg, 1 mmol), the CDE tricyclic compound (263 mg, 1 mmol, 1 eq), and pyridinium p-toluenesulfonate (PPTS) (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and the reaction mixture was allowed to cool. The solid precipitate was collected as the crude product of Intermediate 2-2, a brown solid (750 mg). LC-MS (ESI): M+1 = 574. The product was used in the next step without purification.

[0480] Step 3

[0481] The BL linker compound (857 mg, 1 mmol), HoSu (138 mg, 1.2 mmol, 1.2 eq), and DCC (310 mg, 1.5 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was then filtered, and the filtrate was the DCM solution of A-Osu. The filtrate was added to a mixed solution of the crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The layers were separated, the aqueous phase was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1 to 10:1, to obtain linker MWC-L2 as an orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1 = 1414, (M+1) / 2 = 707

[0482] Example 46 Synthesis of drug-containing linker MWC-L3

[0483] Step 1

[0484] Boc-Gly-OH (175 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 mL of DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography using an eluent of PE:EA (10:1 to 5:1) to quantitatively obtain 335 mg of intermediate 3-1 as a light green solid.

[0485] Step 2

[0486] Intermediate 3-1 (335 mg, 1 mmol), the CDE tricyclic compound (263 mg, 1 mmol, 1 eq), and PPTS (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and the reaction solution was allowed to cool. The solid precipitate was collected as the crude product of Intermediate 3-2, a brown solid (560 mg). LC-MS (ESI): M+1 = 461. The product was used in the next step without purification.

[0487] Step 3

[0488] Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 20 ml of DMF and stirred for 3 h. 1N hydrochloric acid (30 ml) was added and stirred for 0.5 h. The mixture was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification was performed by column chromatography using an eluent of DCM:MeOH = 50:1 to 10:1 to obtain intermediate 3-3 as a brown solid (350 mg). LC-MS (ESI): M+1 = 945

[0489] Step 4

[0490] Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), and diethylamine (2 ml) was added. The mixture was stirred for 3 h. The reaction solution was dried to give the crude intermediate 3-4 as a brown viscous product. LC-MS (ESI): M+1=722. The crude product was directly used for the next step without purification.

[0491] Step 5

[0492] The BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), and DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was then filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The layers were separated, the aqueous phase was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1 to 10:1, to obtain 120 mg of linker MWC-L3 as an orange-red solid, yield 20.8%, LC-MS (ESI): M+1 = 1562, (M+1) / 2 = 781

[0493] Example 47 Synthesis of Drug-Containing Linked MWE-L4

[0494] MWE-L4 was synthesized by a similar method to Example 45 using A3 instead of A1 as an orange solid. LC-MS (ESI): M+1=1430

[0495] Example 48 Synthesis of drug-containing linker MWG-L5

[0496] MWG-L5 was synthesized by a similar method to Example 45 using A6 instead of A1 as an orange solid. LC-MS (ESI): M+1=1442

[0497] Example 49 Synthesis of drug-containing linker MWF-L6

[0498] MWF-L6 was synthesized by a similar method to Example 45 using A7 instead of A1 as an orange solid. LC-MS (ESI): M+1=1450

[0499] Example 50 Synthesis of drug-containing linker MWF-L7

[0500] MWF-L7 was synthesized by a similar method to Example 45, using A7 instead of A1 and Alloc-Ala-Ala-Ala-OH instead of Alloc-Val-Ala-OH. The product was an orange-red solid. LC-MS (ESI): M+1=1493.5

[0501] Example 51 Synthesis of drug-containing linker MWF-L8

[0502] MWF-L8 was synthesized according to the method of Example 46 by substituting A7 for A1 as an orange solid. LC-MS (ESI): M+1=1598.4.

[0503] Example 52 Synthesis of drug-containing linker MWF-L9

[0504] MWF-L9 was synthesized according to the method of Example 45 by substituting A5 for A1 as a yellow solid. LC-MS (ESI): M+1=1416.4.

[0505] Example 53 Synthesis of drug-containing linker MWF-L10

[0506] MWF-L10 was synthesized according to the method of Example 45 by replacing A1 with A13 as a yellow solid. LC-MS (ESI): M+1=1468.4.

[0507] Example 54 Synthesis of drug-containing linker MWF-L11

[0508] MWF-L11 was synthesized according to the method of Example 45 by replacing A1 with A18 as a yellow solid. LC-MS (ESI): M+1=1450.4.

[0509] Example 55 Synthesis of drug-containing linker MWF-L12

[0510] MWF-L12 was synthesized according to the method of Example 45 by replacing A1 with A16 as a yellow solid. LC-MS (ESI): M+1=1434.4.

[0511] Example 56 Synthesis of drug-containing linker MWF-L13

[0512] MWF-L13 was synthesized according to the method of Example 45 using A19 instead of A1 as a yellow solid. LC-MS (ESI): M+1=1486.4.

[0513] Example 57 Synthesis of drug-containing linker MWF-L14

[0514] MWF-L14 was synthesized according to the method of Example 45 by replacing A1 with A17 as a yellow solid. LC-MS (ESI): M+1=1470.4.

[0515] Example 58 Synthesis of drug-containing linker MWF-L15

[0516] MWF-L15 was synthesized according to the method of Example 45 by substituting A2 for A1 as a yellow solid. LC-MS (ESI): M+1=1432.4.

[0517] Example 59 Synthesis of drug-containing linker MWD-L7

[0518] The synthetic route is as follows:

[0519] Step 1

[0520] Fmoc-Val-Cit-PAB-PNP (153 mg, 0.2 mmol), exatecan mesylate (106 mg, 0.2 mmol, 1 eq), and DIPEA (65 mg, 0.5 mmol, 2.5 eq) were dissolved in 30 ml of DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography using a DCM:MeOH ratio of 50:1 to 10:1 eluent to afford 180 mg of intermediate 5-1 as a pale yellow solid. LC-MS (ESI): M+1 = 1064.

[0521] Step 2

[0522] Intermediate 5-1 (180 mg, 0.17 mmol) was dissolved in methanol (20 ml), and diethylamine (2 ml) was added. The mixture was stirred for 3 h. The reaction solution was dried to give the crude intermediate 5-2 as a pale yellow viscous product. LC-MS (ESI): M+1=842. The crude product was directly used for the next step without purification.

[0523] Step 3

[0524] The BL linker compound (146 mg, 0.17 mmol), HoSu (25 mg, 0.21 mmol, 1.2 eq), and DCC (54 mg, 0.26 mmol, 1.5 eq) were dissolved in 20 ml of DCM and stirred at room temperature for 3 h. The reaction solution was then filtered. The filtrate was added to a mixed solution of the crude intermediate 5-1, DIPEA (56 mg, 0.43 mmol, 2.5 eq), and DCM (20 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The layers were separated, the aqueous phase was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1-10:1, to obtain 67 mg of the drug-containing linker MWD-L7 as a yellow solid, with a yield of 23.1%. LC-MS (ESI): M+1 = 1682, (M+1) / 2 = 841.

[0525] Example 60 Synthesis of drug-containing linker MWD-L8

[0526] According to the similar synthesis method of Example 44, MWD-L8 was synthesized instead of BL compound as a light yellow solid, LC-MS (ESI): M+1=1645.7.

[0527] Example 61 Synthesis of drug-containing linker MWD-L9

[0528] According to the similar synthesis method of Example 44, MWD-L9 was synthesized instead of BL compound as a light yellow solid, LC-MS (ESI): M+1=1713.7.

[0529] Example 62 Synthesis of drug-containing linker LD

[0530] According to the similar synthesis method of Example 48, MC-OSU was used instead of BL-OSU to synthesize LD as a light yellow solid. LC-MS (ESI): M+1=795.9.

[0531] Example 63 Synthesis of drug-containing linker LE

[0532] According to the similar synthesis method of Example 45, MC-OSU was used instead of BL-OSU to synthesize LE as a light yellow solid. LC-MS (ESI): M+1=803.8.

[0533] Example 64 Synthesis of drug-containing linker LF

[0534] LF was prepared according to a similar method to Example 45, except that A7 was used instead of A1 and MaL-PEG8-COOH was used instead of BL. The product was a yellow solid. LC-MS (ESI): M+1=1185.

[0535] Example 65 Synthesis of drug-containing linker LG

[0536] According to Example 46, MC-OSu was used instead of BL-OSu to prepare LG as a yellow solid. MS (ESI): M+1=965.98.

[0537] Example 66 Synthesis of drug-containing linker LH

[0538] According to the similar synthesis method of Example 47, MaL-PEG8-COOH was used instead of BL compound to synthesize LH as a light yellow solid. LC-MS (ESI): M+1=1165.3.

[0539] Example 67 Synthesis of Drug-Containing Linker LI

[0540] A similar method to Example 45 was used, using A13 instead of A1, to prepare LI as a yellow solid. LC-MS (ESI): M+1=1203.4.

[0541] Example 68 Synthesis of drug-containing linker LJ

[0542] A similar method to Example 45 was used, using A18 instead of A1, to prepare LJ as a yellow solid. LC-MS (ESI): M+1=1185.4.

[0543] Example 69 Synthesis of drug-containing linker LK

[0544] A similar method to Example 45 was used, but A16 was used instead of A1, to prepare LK as a yellow solid. LC-MS (ESI): M+1=1169.3.

[0545] Example 70 Synthesis of Drug-Containing Linker LL

[0546] A similar method to Example 45 was used, using A19 instead of A1, to prepare LL as a yellow solid. LC-MS (ESI): M+1=1221.4.

[0547] Example 71 Synthesis of drug-containing linker LM

[0548] LM was prepared by the method of Example 45, using A17 instead of A1, as a yellow solid. LC-MS (ESI): M+1=1205.4.

[0549] Example 72 Synthesis of drug-containing linker LN

[0550] Synthesized according to a method similar to Example 45, using HCDE intermediate instead of CDE intermediate; using MaL-PEG8-COOH instead of BL compound to prepare LN, a yellow solid, LC-MS (ESI): M+1=1163.2 (a mixture of a pair of diastereomers).

[0551] Example 73 Synthesis of drug-containing linker MWS-L1

[0552] According to the similar method of Example 45, A13 was used instead of A1.

[0553] MWS-L1 was prepared instead of the BL linker compound as a yellow solid, LC-MS (ESI): M+1=1236.3.

[0554] Example 74 Synthesis of drug-containing linker MWS-L2

[0555] A similar method to Example 45 was used, except that A18 was used instead of A1, to prepare MWS-L2 as a yellow solid. LC-MS (ESI): M+1=1218.4.

[0556] Example 75 Synthesis of drug-containing linker MWS-L3

[0557] A similar method to Example 45 was used, except that A16 was used instead of A1, to prepare MWS-L3 as a yellow solid. LC-MS (ESI): M+1=1202.4.

[0558] Example 76 Synthesis of drug-containing linker MWS-L4

[0559] A similar method to Example 45 was used, except that A19 was used instead of A1, to prepare MWS-L4 as a yellow solid. LC-MS (ESI): M+1=1254.4.

[0560] Example 77 Synthesis of drug-containing linker MWS-L5

[0561] According to a similar method to Example 45, using A17 instead of A1, MWS-L5 was prepared as a yellow solid. LC-MS (ESI): M+1=1238.4.

[0562] Example 78 Synthesis of drug-containing linker MWS-L6

[0563] According to the similar method of Example 54,

[0564] MWS-L6 was prepared by replacing Mal-PEG8-COOH as a yellow solid. LC-MS (ESI): M+1=1302.4

[0565] Example 79 Synthesis of drug-containing linker MWS-L7

[0566] A similar method to Example 45 was used, except that A18 was used instead of A1, to prepare MWS-L7 as a yellow solid. LC-MS (ESI): M+1=1284.4.

[0567] Example 80 Synthesis of drug-containing linker MWS-L8

[0568] A similar method to Example 45 was used, except that A16 was used instead of A1, to prepare MWS-L8 as a yellow solid. LC-MS (ESI): M+1=1268.4.

[0569] Example 81 Synthesis of drug-containing linker MWS-L9

[0570] A similar method to Example 45 was used, except that A19 was used instead of A1, to prepare MWS-L9 as a yellow solid. LC-MS (ESI): M+1=1320.4.

[0571] Example 82 Synthesis of drug-containing linker MWS-L10

[0572] A similar method to Example 45 was used, except that A17 was used instead of A1, to prepare MWS-L10 as a yellow solid. LC-MS (ESI): M+1=1304.4.

[0573] (IV) Comparison of the synthesis of drug-containing linkers

[0574] Example 83 Synthesis of drug-containing linkers LA, LB and LC

[0575] 1. Synthesis of Randomly Conjugated Drug-Containing Linkers

[0576] The drug-containing linker MC-GGFG-Dxd was synthesized according to the method described in the patent publication (US20190151328A1) to obtain MC-GGFG-Dxd as a light yellow solid. LC-MS (ESI): M+1=1034, 1 H NMR (CDCl3).

[0577] The drug-containing linkers LA and LB were synthesized according to the method described in the patent disclosure document (WO2020200880), and LA was obtained as a yellow solid, LC-MS (ESI): M+1=767; LB was a brown viscous substance, LC-MS (ESI): M+1=1149.

[0578] 2. Synthesis of other ring-bridged drug-containing linkers LC

[0579] The synthetic route is as follows:

[0580] Linker LC was synthesized in a similar manner to drug-containing linker L-1. LC was a light yellow solid. LC-MS (ESI): M+1=1982, (M+1) / 2=991.

[0581] (V) Preparation and physicochemical characterization of antibody-drug conjugates

[0582] 1. General preparation method of antibody drug conjugates

[0583] a. General preparation method for site-directed coupling

[0584] The antibody is reduced to open the disulfide bond, then coupled with the linker to form a bridged antibody-drug conjugate, which is then hydrolyzed to open the maleimide ring and finally purified to obtain an antibody-drug conjugate with a DAR of 4. The details are as follows:

[0585] Antibody reduction: 120 mg of antibody sample was diluted to 10 mg / ml using a Sephadex G25-supported NAP-25 column and purged into a pH 7.0 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate / disodium hydrogen phosphate. 100 mg of antibody sample (10 ml) was diluted to 2.1 ml of a 10 mg / ml aqueous solution of TCEP (Sigma-Aldrich) at an antibody-TCEP molar ratio of 1 to 10. After incubation for 2 hours, the reaction solution was purged into a pH 6.5 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate / disodium hydrogen phosphate using a Sephadex G25 column.

[0586] Conjugation and hydrolysis of the antibody and drug-containing linker: Dilute the reduced antibody to 5 mg / mL. Add 0.38 mL of N,N-dimethylacetamide (DMA) (2% of the total reaction volume) as a presolvent. Add 0.67 mL of a DMA-drug-containing linker mixture containing 10 mg / mL of drug-containing linker at a molar ratio of 1:5.5 for antibody to small molecule drug. Stir at room temperature for 30 minutes. Replace the reaction solution with sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (pH 8.0) using a Sephadex G25-supported NAP-25 column. Remove excess drug-containing linker and heat in a 37°C water bath for 3 hours.

[0587] Purification of Antibody Drug Conjugates: The above sample was concentrated using an AMICOM ultrafiltration centrifuge tube to approximately 15 mg / mL. 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution was added until the conductivity reached 100 ms / cm. The sample was loaded onto a TOYOPEAL Butyl-650M packing (purchased from TOSOH) hydrophobic column. Phase A consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6 M ammonium sulfate, and phase B consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Phase B was eluted with a gradient of 0-100% over 8 column volumes, and the main peak was collected.

[0588] The final sample was replaced with 50 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 μm filter membrane (Sartorius stedim Ministart).

[0589] b. General preparation method for random coupling

[0590] The antibody is reduced to open the disulfide bonds and then coupled with the linker to form a bridged antibody drug conjugate.

[0591] Specifically, it is prepared according to the preparation method disclosed in CN104755494B patent.

[0592] 2. General Analytical Methods for Antibody Drug Conjugates

[0593] a. Determination of drug-antibody coupling ratio and concentration by UV spectrophotometry (UV-DAR method)

[0594] The concentration of the antibody drug conjugate can be determined by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of the small molecule, and then performing the following calculation.

[0595] a1. Determination of Drug-Antibody Ratio (DAR) of Antibody-Drug Conjugates

[0596] According to the literature [Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70], DAR in, is the molar absorption coefficient of the antibody at 280 nm, A 280 is the UV absorption value of the antibody drug conjugate at 280 nm, A z is the UV absorption value of the antibody drug conjugate at the characteristic absorption wavelength Znm of the drug-containing linker, is the molar absorption coefficient of the antibody at the characteristic absorption wavelength Z nm of the drug-containing linker, is the molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm, is the molar absorption coefficient of the drug-containing linker at 280 nm.

[0597] in:

[0598] a2. Drug concentration determination

[0599] Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additivity of absorbance), assuming that the molar absorptivity of the antibody and drug-containing linker does not change before and after conjugation, the concentration of the antibody-drug conjugate has the following relationship:

[0600] Therefore, the molar concentration of antibody drug conjugate (mol / L) The antibody drug conjugate concentration (g / L) was obtained. Substituting the DAR value into the formula gives the protein concentration.

[0601] Among them, MW ADC is the molecular weight of the antibody drug conjugate, MW Ab is the molecular weight of the antibody, MW D is the molecular weight of the drug-containing linker.

[0602] b. Hydrophobic chromatography

[0603] b1. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0604] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0605] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm x 35 mm;

[0606] Mobile phase A: 1.5 M (NH4)2SO4 + 25 mM PB, pH 7.0

[0607] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0608] Flow rate: 0.6 mL / min;

[0609] Detection wavelength: 280nm;

[0610] Column temperature: 30°C;

[0611] Sample volume: 10 μL;

[0612] HIC chromatographic gradient

[0613] DAR calculation formula:

[0614] DAR = ∑(weighted peak area) / 100, that is, DAR = (D0 peak area ratio*0+D1 peak area ratio*1+D2 peak area ratio*2+D3 peak area ratio*3+D4 peak area ratio*4+D5 peak area ratio*5+D6 peak area ratio*6+D7 peak area ratio*7+D8 peak area ratio*8) / 100.

[0615] b2. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0616] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0617] Chromatographic column: TSKgel Butyl-NPR, 2.5 μm, 4.6 mm x 100 mm;

[0618] Mobile phase A: 1.2 M (NH4)2SO4 + 25 mM PB, pH 7.0

[0619] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0620] Flow rate: 0.6 mL / min;

[0621] Detection wavelength: 280nm;

[0622] Column temperature: 30°C;

[0623] Sample volume: 10 μL;

[0624] HIC chromatographic gradient

[0625] DAR calculation formula: same as b1.

[0626] b3. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0627] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0628] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm x 35 mm;

[0629] Mobile phase A: 2.5 M (NH4)2SO4 + 125 mM PB, pH 7.0

[0630] Mobile phase B: 125 mM PB, pH 7.0

[0631] Mobile phase C: IPA

[0632] Mobile phase D: H2O

[0633] Flow rate: 0.5 mL / min;

[0634] Detection wavelength: 280nm;

[0635] Column temperature: 30°C;

[0636] Sample volume: 10 μL;

[0637] HIC chromatographic gradient

[0638] DAR calculation formula: same as b1.

[0639] c. Determination of DAR value by mass spectrometry LC-MS

[0640] Sample Preparation: Place an appropriate amount of sample in an ultrafiltration tube and replace with 50mM NH4HCO3 replacement buffer (pH 7.1). Refill the buffer and ultrafiltration centrifuge (13000g*5min). Add 8μl of PNGase F enzyme to the replaced sample and incubate at 37℃ for 5h to desugar. After incubation, centrifuge at 12000rpm for 5min. The supernatant is collected and placed in a vial as the test sample.

[0641] Chromatographic column: PolyHYDROXYETHYLA Column, 5um, 2.1mm×200mm;

[0642] Mobile phase: 50 mM ammonium acetate, pH 7.0;

[0643] Run time: 10 minutes;

[0644] Flow rate: 0.1 mL / min;

[0645] Injection volume: 2 μL;

[0646] Column temperature: 25°C;

[0647] Detection wavelength: 280nm;

[0648] Ionization method: ESI positive

[0649] Drying gas temperature: 325℃

[0650] Drying gas flow rate: 8L / min

[0651] Atomizer pressure: 20 psig

[0652] Sheath gas temperature: 325°C;

[0653] Sheath gas flow rate: 12L / min;

[0654] Scan settings: 900-8000 m / z.

[0655] d. Size exclusion chromatography SEC-HPLC determination of molecular size heterogeneity

[0656] Sample treatment: The sample was diluted to about 1.0 mg / ml with mobile phase, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for analysis.

[0657] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5 μm, 7.8 mm*300 mm;

[0658] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0659] Flow rate: 0.6 mL / min;

[0660] Detection wavelength: 280nm;

[0661] Column temperature: 30°C;

[0662] Sample volume: 20uL;

[0663] Elution time: 20 min;

[0664] Elution gradient: isocratic elution.

[0665] e. Purity determination by non-reducing capillary electrophoresis NR-CE-SDS

[0666] The determination was based on the monoclonal antibody size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.

[0667] f. Determination of small molecule / antibody coupling ratio (DAR value) of antibody drug conjugates by reversed-phase chromatography (RP-HPLC)

[0668] The analysis was performed according to the method described in patent publication US10227417 B2.

[0669] The DAR value was calculated according to the formula: DAR = 2 × (∑ light chain weighted peak area + ∑ heavy chain weighted peak area) / 100. That is, DAR = 2 × (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100.

[0670] Example 84 Preparation and Characterization of Antibody Drug Conjugates Targeting B7H3

[0671] According to the coupling method in this section, 100 mg each of the B7H3-targeting antibodies hz10B4m1, hz10B4m2, and hz10B4 were taken and coupled with the drug-containing linkers MWD-L1, MWC-L2, MWC-L3, MWF-L6, MWF-L8, and MWF-L11 to prepare bridged ADCs.

[0672] According to the method of patent CN104755494B, 100 mg each of the B7H3-targeting antibodies hum30 and hz10B4m1 were conjugated with MC-GGFG-Dxd, vcMMAE, MWS-L1, MWS-L7, LJ, and LI to prepare a randomly conjugated ADC.

[0673] The concentration, DAR value and purity of the bridged ADC were determined using 2.a UV spectrophotometry, 2.b hydrophobic chromatography and 2.d size exclusion chromatography in this section, respectively.

[0674] The DAR values ​​of ADC samples coupled with MC-GGFG-Dxd drug-containing linkers were determined using the reverse-action chromatography method described in section 2.f of this article. The DAR values ​​of ADC samples coupled with vcMMAE drug-containing linkers were determined using the hydrophobic chromatography method described in section 2.b of this article. The concentration and purity of non-bridged ADC drugs were determined using the ultraviolet spectrophotometry method described in section 2.a and the size exclusion chromatography method described in section 2.d of this article, respectively.

[0675] The results are shown in Table 5.

[0676] Table 5. Characterization results of antibody drug conjugates

[0677] Results showed that the UV-DAR values ​​for the prepared low-drug-loading ADCs ranged from 3.6 to 4.8, while the UV-DAR for the high-drug-loading ADC (B7H3-ADC-5) was 6.9. SEC results revealed that, with the exception of B7H3-ADC-3 (95.05% purity), the monomer content of all other ADC samples exceeded 98%, demonstrating the high purity of the prepared ADC samples.

[0678] (6) Activity evaluation

[0679] Example 85 Comparative Study on the Activity of Camptothecin Compounds

[0680] Oral epithelial cancer cells KB (purchased from ATCC) were cultured using DMEM medium supplemented with 10% FBS; human gastric cancer cells NCI-N87 (purchased from ATCC) were cultured using RPMI1640 medium supplemented with 10% FBS; human colon cancer cells HT29 (purchased from ATCC) were cultured using RPMI1640 medium supplemented with 10% FBS; and human pancreatic adenocarcinoma cells BxPC-3 (purchased from ATCC) were cultured using RPMI1640 medium supplemented with 10% FBS.

[0681] The cell density of KB cells was adjusted to 2 × 10 4 NCI-N87 cells were adjusted to a cell density of 5×10 4 HT29 cells were adjusted to a cell density of 5×10 4 Cells were plated in a 96-well transparent flat-bottom plate at 100 μL / well and cultured overnight. The cell density of BxPC-3 cells was adjusted to 5×10 4 pieces / mL.

[0682] Dilute the sample to be tested to 20 μM using culture medium, then dilute 5-fold, and set up 9 gradients (set the well without sample, which is the well with maximum cell growth, and the well without cells, which is the culture medium base). Add 100 μL / well to the cell culture plate and incubate in a carbon dioxide incubator for 96 hours. After incubation, add the prepared CCK-8 (purchased from Tongren Chemical) at 20 μL / well. After incubation in a carbon dioxide incubator for 3 hours, read the OD450 value with a microplate reader.

[0683] The data were fitted with a four-parameter curve using Prism7 software. The formula for calculating maximum killing was: 1-(OD450 value of the well with maximum killing - OD450 value of the culture medium) / (OD450 value of the well with maximum cell growth - OD450 value of the culture medium) × 100%. The results are shown in Table 6.

[0684] Table 6. Comparative study results of the activity of camptothecin compounds

[0685] As shown in Table 6, the cell-killing activity of the novel camptothecin compounds provided by the present invention is significantly better than that of the control molecule DXD.

[0686] Example 86 Comparative Study of In Vitro Activity of Antibody Drug Conjugates Targeting B7H3

[0687] Calu-6-B7H3 (homemade), a human non-small cell lung cancer cell line that highly expresses B7H3, was taken out of liquid nitrogen and revived. The cell density was adjusted to 5×10 4 After the cells were concentrated to 100 μl / well, 100 μl / well was added to the cell culture plate and cultured overnight.

[0688] The B7H3-targeting ADC prepared above was diluted to 5 μg / ml using complete medium, followed by 4-fold or 2-fold serial dilutions for a total of 9 gradients, with two replicates for all samples. A negative control (cells + medium) was also included. The cells were then incubated in a cell culture incubator for 168 hours (7 days). The cell culture plate was removed, and MTS was added at 40 μl / well. The cells were incubated at 37°C for 2-4 hours. The plate was removed and the OD value was read at 490 nm.

[0689] The results are shown in Table 7.

[0690] Table 7. In vitro cell killing effect of ADC targeting B7H3

[0691] In vitro cell killing results of ADCs targeting B7H3 showed that the 7-day in vitro killing activity of B7H3-ADC-7 on Calu-6-B7H3 cells was equivalent to that of the control ADC (B7H3-ADC-9, i.e., DS-7300), and the 7-day in vitro killing activity of B7H3-ADC-6, B7H3-ADC-11, B7H3-ADC-13, and B7H3-ADC-14 on Calu-6-B7H3 cells was superior to that of the control ADC (DS-7300). In addition, the high-drug-loaded ADC (B7H3-ADC-5) had better in vitro killing activity than the low-drug-loaded ADC (B7H3-ADC-4).

[0692] Example 87 Comparative Study on the Efficacy of Camptothecin-Antibody Drug Conjugates Targeting B7H3 in Vivo

[0693] Calu-6 human lung cancer cells (purchased from ATCC) were used. Calu-6 cells were cultured adherently in 10-cm culture dishes in MEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged 2-3 times weekly. When cells reached the exponential growth phase, they were trypsinized, harvested, counted, and plated.

[0694] The antibody-drug conjugate B7H3-ADC-6 was compared across different dose groups. Nude mice implanted subcutaneously with human lung cancer Calu-6 cells were administered intravenously (IV) twice weekly. A vehicle group received the same volume of saline. Tumor volume and mouse body weight were measured twice weekly, and the data were recorded.

[0695] The experiment ends, the experimental endpoint (D21) is reached, or the tumor volume reaches 2000mm 3 The animals were sacrificed under CO2 anesthesia, and the tumors were removed and photographed. The results are shown in Table 8 and Figure 2.

[0696] Table 8. Efficacy of B7H3-ADC-6 on human lung cancer Calu-6 subcutaneous xenograft tumors in nude mice (TGI % calculated based on tumor volume).

[0697] The above results show that B7H3-ADC-6 has significant tumor inhibitory activity compared with the solvent group in the Calu-6 human lung cancer xenograft tumor model; at the same dose, B7H3-ADC-6 has better tumor inhibitory activity than DS-7300, and the tumor inhibitory activity of B7H3-ADC-6 at a dose of 3 mg / kg is equivalent to that of DS-7300 at a dose of 10 mg / kg.

[0698] Example 88 Comparative Study on the Efficacy of Camptothecin-Antibody Drug Conjugates Targeting B7H3 in Vivo

[0699] Human pancreatic cancer cells, Pan 05.04 (purchased from ATCC), were used. Pan 05.04 cells were cultured adherently in 10-cm dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged 2-3 times weekly. When cells reached the exponential growth phase, they were trypsinized, harvested, counted, and plated.

[0700] The antibody-drug conjugate B7H3-ADC-6 and its anti-B7-H3 antibody were compared at different doses. Nude mice implanted subcutaneously with human pancreatic cancer Pan 05.04 cells were administered intravenously (IV) twice weekly. A vehicle group received the same volume of saline. Tumor volume and mouse body weight were measured twice weekly, and the data were recorded.

[0701] The experiment ends, the experimental endpoint (D18) is reached, or the tumor volume reaches 2000mm 3 The animals were sacrificed under CO2 anesthesia, and the tumors were removed and photographed. The results are shown in Table 9 and Figure 3.

[0702] Table 9. Efficacy of B7H3-ADC-6 on subcutaneous transplanted tumors in mice (TGI% calculated based on tumor volume).

[0703] The above results show that in the Pan05.04 human pancreatic cancer xenograft model, B7H3-ADC-6 has significant tumor inhibitory activity compared with the solvent group; at the same dose, B7H3-ADC-6 has better tumor inhibitory activity than DS-7300; in three different dose groups (1 mg / kg, 3 mg / kg and 10 mg / kg), the tumor inhibitory activity of B7H3-ADC-6 alone is better than that of the combined administration group of anti-B7H3 antibody hz10B4m1 + compound 3 (B7H3 antibody 10 mg / kg + compound 3 0.11 mg / kg).

[0704] Example 89 Study on ADC Targeting B7H3 Inducing Cellular DNA Damage

[0705] Calu-6 cells were seeded in 6-well plates and treated with drugs B7H3-ADC-6 (0.6572, 6.572, 65.72, 657.2, 1971.7 nM), DS-7300 (0.6649, 6.649, 66.49, 664.9 nM), B7H3-ADC-6 antibody hz10B4m1 (675.4, 2026.3 nM), and compound 3 (0.1, 1 nM) for 72 h. The cells were then collected and lysed with lysis buffer (0.1 M MES, pH 6.6, 1 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 10% glycerol) and centrifuged at 12,000 rpm for 5 min. The supernatant was removed and 1× SDS sample buffer (50 mM Tris, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and denatured in a boiling water bath for 10 minutes, and then the γ-H2AX level in the cells was detected by Western blot.

[0706] The experimental results showed that B7H3-ADC-6, DS-7300, and compound 3 all induced DNA damage in Calu-6 cells, while hz10B4m1 had no significant DNA damage-inducing effect. The results are shown in Figure 4.

[0707] Example 90 Study on the Inhibition of Proliferation of Different Tumor Cells by ADC Drugs Targeting B7H3

[0708] The SRB assay was used to assess the effects of drugs on the proliferation of tumor cells cultured in vitro. A specific number of cells in logarithmic growth phase were seeded into a 96-well culture plate and allowed to adhere overnight. Various concentrations of drug were then added. After 144 hours of drug exposure, the cells were fixed with trichloroacetic acid. After staining with SRB (prepared with 1% glacial acetic acid, 4 mg / mL), 10 mM Tris solution was added to each well for dissolution. OD values ​​were read at 510 nm using a microplate reader.

[0709] Inhibition rate (%) = (OD value of control well - OD value of drug treatment well) / OD value of control well × 100%

[0710] The MTT assay was used to assess the effects of drugs on in vitro tumor cell proliferation in suspension-growing cells. A defined number of cells in logarithmic growth phase were seeded into 96-well culture plates and allowed to adhere overnight. Various concentrations of drugs were then added. After 144 hours of drug exposure, MTT was added to each well and the cells were incubated for another 4 hours at 37°C in a 5% CO2 saturated humidity incubator. 100 μL of the triplicate solution was added to each well, and the OD values ​​were measured at 570 nm and 690 nm on a microplate reader.

[0711] According to the inhibition rate of each concentration, the half-maximal inhibitory concentration (IC) was calculated using Graphpad Prism 8.0 software. 50 The experiment was repeated once, and the data are expressed as mean ± SD. A concentration (logarithm)-inhibition rate graph was generated. The results are shown in Table 10.

[0712] Table 10. Inhibitory effects of ADC drugs targeting B7H3 on proliferation of different tumor cells

[0713] The above studies show that B7H3-ADC-6 has a significant proliferation inhibitory effect on BxPC-3, ES-2, PANC05.04, Calu-6, Calu-3, HCT-116, MDA-MB-468, and PANC-1 tumor cells, which are all superior to the B7H3 antibody hz10B4m1.

[0714] Example 91 Study on the bystander killing effect of ADC targeting B7H3

[0715] B7H3-positive Calu-6 cells (purchased from ATCC) and B7H3-negative MV-4-11 / luc (MV-4-11 transformed with luciferase, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were plated in a 96-well plate at a ratio of 1:1, or B7H3-negative MV-4-11 / luc cells were seeded alone in a 96-well plate (control). The drug B7H3-ADC-6 (1000, 3000, 10000, 30000, 100000 ng / mL) was then added to each well for 144 hours, followed by centrifugation, removal of the supernatant, addition of lysis buffer to lyse the cells, centrifugation, removal of the supernatant, addition of the substrate luciferin, and detection of fluorescence intensity by microplate reader.

[0716] Inhibition rate (%) = (OD value of control well - OD value of drug treatment well) / OD value of control well × 100%

[0717] These studies indicate that B7H3-ADC-6 exhibits a strong bystander effect: while killing B7H3-positive Calu-6 cells, it also kills B7H3-negative MV-4-11 / luc cells. However, it has no significant effect on the proliferation of MV-4-11 / luc cells cultured alone. The results are shown in Figure 5.

[0718] Example 92 Study on cell cycle arrest induced by ADC targeting B7H3

[0719] Calu-6 cells (purchased from ATCC) were seeded in 6-well plates and treated with the drug B7H3-ADC-6 (0.6752, 6.752, 67.52, 675.2, 1971.7 nM), the B7H3-ADC-6 antibody hz10B4m1 (675.4, 2026.3 nM), and compound 3 (0.01, 0.1, 1 nM) for 48 hours. The cells were then collected and fixed with ethanol overnight. RNase and PI were then added, mixed, and stained at 37°C in the dark for 30 minutes. Finally, the DNA content in the cells was detected by flow cytometry (BD FACS CALIBUR 4). 1×10 cells were sampled for each group. 4 Calu-6 cells cultured alone were used as controls. The experimental results were analyzed using ModFit Lt Mac V3.0 software.

[0720] The above studies show that both B7H3-ADC-6 and compound 3 significantly induced G2 / M arrest in Calu-6 cells; the anti-B7H3 antibody hz10B4m1 had no significant cell cycle arrest. Furthermore, the results showed that B7H3-ADC-6 induced cell cycle arrest at the G2 / M phase, consistent with the activity of compound 3. The results are shown in Figure 6.

[0721] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

Claims

1. An antibody-drug conjugate or a salt thereof, wherein the antibody-drug conjugate or a salt thereof has a structure as shown in structural formula Ia and / or Ib: and / or in, Ab represents an anti-B7-H3 antibody or a fragment thereof; M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituents are selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., haloC1-6 alkyl, preferably haloC1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester group, amide group and cyano group; SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight chain heteroalkylene, wherein the C1-21 straight chain heteroalkylene contains 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano; SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-、-NH(CH2CH2O) a CH2CO-, -S(CH2) a CO- or chemical bond, wherein a is an integer from 1 to 20; A represents a short peptide structure formed by 2-4 amino acids; m is 1 to 10, preferably 1 to 8 (e.g. 1 to 5), more preferably 3 to 8; CPT is a camptothecin compound.

2. The antibody-drug conjugate or a salt thereof according to claim 1, characterized in that: The anti-B7-H3 antibody or fragment thereof comprises a combination of heavy chain CDRs and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3; and, CDR-L1, CDR-L2 and CDR-L3) selected from the following: (i) the CDR-H1, CDR-H2 and CDR-H3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise amino acid sequences that are at least 75% identical to the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:14, respectively; (ii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:15, respectively; and (iii) the CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:16, respectively; Preferably, The anti-B7-H3 antibody or fragment thereof comprises at least a heavy chain variable region (VH) and a light chain variable region (VL); Preferably, the heavy chain variable region and the light chain variable region comprise a sequence combination selected from the following: (i) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 3; (ii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO:4; and (iii) the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO:

5.

3. The antibody-drug conjugate or a salt thereof according to claim 1 or 2, characterized in that: The anti-B7-H3 antibody or fragment thereof further comprises a constant region, such as a heavy chain constant region (CH) and / or a light chain constant region (CL), or any one or more domains in the constant region; Alternatively, the anti-B7-H3 antibody or fragment thereof comprises a heavy chain and / or a light chain; for example, the heavy chain comprises IgG, IgA, The heavy chain constant region of IgM, IgD or IgE, and the light chain comprises a light chain constant region of type kappa or lambda.

4. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 3, characterized in that: In formula Ia and / or Ib, M is a halogen-substituted phenylene group, preferably a fluorine-substituted phenylene group; Preferably, SP1 is C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene, which contains 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S; Preferably, SP2 is a chemical bond.

5. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in Structural Formula I, wherein Structural Formula I is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond, preferably the amino group adjacent to Group G in Structural Formula I is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond: in, R1, R2, R3, and R4 are independently hydrogen, halogen, hydroxy, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms to which they are attached form a C3-6 (preferably C3-5) cyclic alkyl; G is hydrogen, halogen, methyl or methoxy; Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene; X is oxygen or sulfur; n=0 or 1.

6. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 5, characterized in that: The structure of CPT is shown in Structural Formula IA, wherein Structural Formula IA is connected to the carboxyl group in Group A in Structural Formulas Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in Structural Formula IA is connected to the carboxyl group in Group A in Structural Formulas Ia and / or Ib via an amide bond: Wherein, the groups R1, R2, R3, and R4 have the same definitions as the groups R1, R2, R3, and R4 in the structural formula I in claim 5, but R1, R2, R3, and R4 are not hydrogen at the same time.

7. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in structural formula II, wherein structural formula II is connected to the carboxyl group in group A in structural formula Ia and / or Ib via an amide bond, preferably the amino group adjacent to group G in structural formula II is connected to the carboxyl group in group A in structural formula Ia and / or Ib via an amide bond: Wherein, R5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl; G is hydrogen, halogen (e.g. fluorine), methyl or methoxy; X is oxygen or sulfur; n=0 or 1.

8. The antibody-drug conjugate or a salt thereof according to claim 7, characterized in that: The structure of CPT is shown in Structural Formula IIA, wherein Structural Formula IIA is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in Structural Formula IIA is connected to the carboxyl group in Group A in Structural Formula Ia and / or Ib via an amide bond: The group R5 has the same definition as the group R5 in the structural formula II in claim 7, but R5 cannot be n-butyl.

9. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 8, characterized in that: The structure of the CPT is shown below, wherein each structural formula is connected to the carboxyl group in the group A in the structural formula Ia and / or Ib via an amide bond, preferably the amino group on the left benzene ring in each structural formula is connected to the carboxyl group in the group A in the structural formula Ia and / or Ib via an amide bond:

10. The antibody-drug conjugate or a salt thereof according to any one of claims 1 to 4, characterized in that: The structure of CPT is shown in formula IV, wherein the hydroxyl group connected to the same carbon as R8 in formula IV is connected to the carboxyl group of group A in formula Ia and / or Ib via a self-releasing structure, such as The solid line indicates the site of attachment to the carboxyl group of group A in formula Ia and / or Ib, and the wavy line indicates the site of attachment to the hydroxyl group in formula IV: Wherein, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.

11. An antibody drug conjugate or a salt thereof, wherein the antibody drug conjugate or the salt thereof has the general formula The structure shown, In the general formula, mAb represents an anti-B7-H3 antibody or a fragment thereof; The groups M, SP1, SP2, A and CPT are the same as those defined in any one of claims 1 to 10; N is 1 to 10, preferably 1 to 8 (e.g. 1 to 5), more preferably 3 to 8; E L is selected from the following groups, wherein, Indicates that it is linked to the cysteine ​​in mAb. Indicates connection with M: E L -1a and / or E L -1b: and / or E L -2： E L -3： E L -4： E L -5： E L -6： 12. A method for preparing an antibody drug conjugate or a salt thereof according to any one of claims 1 to 11, comprising the following steps: a. Antibody reduction: adding ≥5.5 times the molar equivalent of the antibody to a phosphate buffer containing 5-30 mg / mL antibody, and reacting for 1.5-2 h, wherein the reducing agent is selected from TCEP, DTT, 2-MEA and DTBA; b. Antibody coupling and hydrolysis: replace the reduced antibody in step a into a phosphate buffer at pH 6.5-7.8, and dilute the antibody to a concentration of 3.5-15 mg / mL, add 4.5-6.5 times the molar equivalent of the antibody-containing linker dissolved in an organic co-solvent to the antibody dilution, and stir the reaction at 15-35° C. for ≥0.5 h, wherein the organic co-solvent is one or more selected from DMA, DMSO, DMF and ACN; replace the antibody coupling product into a phosphate buffer at pH 7.4-9.0, and heat at 35±10° C. for 2-24 h to obtain a hydrolyzate; c. Hydrophobic chromatography: Use hydrophobic filler to purify the antibody-coupled product by hydrophobic chromatography.

13. The preparation method according to claim 12, characterized in that: In step a, the antibody is an antibody against tumor-associated antigens, such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.

2. 14 . A composition comprising the antibody drug conjugate or a salt thereof according to any one of claims 1 to 11 .

15. Use of the antibody-drug conjugate or a salt thereof according to any one of claims 1 to 11 and / or the composition according to claim 14 in the preparation of a medicament for treating tumors.

16. The use according to claim 15, characterized in that The tumor and tumor-associated antigens such as HER2, B7-H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

17. A method for treating a tumor, comprising administering the antibody-drug conjugate or a salt thereof according to any one of claims 1 to 11 and / or the composition according to claim 14 to a subject in need thereof.

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