Anti-CDH6 antibody-drug conjugate and use thereof

Abstract

The present invention relates to a class of antibody-drug conjugates with a novel structure. Specifically, disclosed in the present invention is an antibody-drug conjugate targeting CDH6, which has good plasma stability in plasma of mammals (such as humans or monkeys), has a relatively strong anti-tumor effect, and has wide clinical application prospects in the treatment of diseases such as tumors.

Description

Anti-CDH6 antibody-drug conjugates and uses thereof

[0001] This disclosure claims priority to Chinese patent application No. 202310420429.5 filed with the Patent Office of China on April 14, 2023, entitled “Anti-CDH6 Antibody Drug Conjugates and Uses Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of biomedicine and relates to a class of antibody-drug conjugates with novel structures, a preparation method thereof, a pharmaceutical composition containing the conjugate, and use of the conjugate as an anti-tumor drug. Background Art

[0003] Antibody-drug conjugates (ADCs), as a new type of targeted drug, link monoclonal antibodies that specifically bind to tumor cell surface antigens with biologically active toxin molecules. These ADCs combine the tumor-targeting properties of the antibody with the highly effective killing properties of the toxin, while avoiding the drawbacks of the former, such as low efficacy, and the latter's excessive side effects and poor drugability. Compared to traditional chemotherapy drugs, ADCs precisely target tumor cells while minimizing their effects on normal cells, resulting in a safer and more effective anti-tumor effect.

[0004] ADCs generally consist of three parts: an antibody, a linker, and a toxin. Camptothecin derivatives are one type of toxin used in ADC development, which achieve anti-tumor effects by inhibiting topoisomerase I. Daiichi Sankyo used the camptothecin derivative Dxd as a toxin to target HER2 and developed the ADC drug Enhertu (Trastuzumab deruxtecan, DS-8201), which was approved for marketing by the US FDA in 2019. Clinical studies have shown that Enhertu has a good therapeutic effect on HER2-positive breast cancer, gastric cancer, and non-small cell lung cancer.

[0005] CDH6 is a type II classical cadherin, also known as K-cadherin. CDH6 is a single transmembrane protein consisting of 790 amino acids, with an extracellular domain divided into five regions (EC1-EC5). Studies have found that CDH6 is highly expressed in tumor tissues such as renal cancer, ovarian cancer, and thyroid cancer, while its expression in normal tissues is very low (Cancer Discov; 2017, 7(9): 1030-45). Like other members of the cadherin superfamily, CDH6 protein is localized to the basolateral membrane of epithelial cells and mediates calcium-dependent cell-cell adhesion, with the characteristic of rapid internalization. Therefore, CDH6 can be used as a potential target for ADC development for the treatment of cancers such as ovarian cancer and renal cancer.

[0006] Despite recent progress in the treatment of ovarian and renal cancers, significant unmet clinical needs remain. Currently, few ADCs targeting CDH6 are under development, and no such drugs have been approved for marketing. Therefore, developing ADCs targeting this target holds broad clinical and therapeutic potential.

[0007] Summary of the Invention

[0008] The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ,

[0009] in,

[0010] D is a cytotoxic drug;

[0011] L is a linker unit;

[0012] Pc is an antibody or antigen-binding fragment thereof that specifically binds to CDH6;

[0013] The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1-3 and / or the LCDR1-3 are selected from the following combinations:

[0014] (1) the HCDR1-3 are SEQ ID NOs: 11-13; or / and the LCDR1-3 are SEQ ID NOs: 14-16;

[0015] (2) the HCDR1-3 are SEQ ID NOs: 17-19; or / and the LCDR1-3 are SEQ ID NOs: 20-22;

[0016] (3) the HCDR1-3 are SEQ ID NOs: 23-25; or / and the LCDR1-3 are SEQ ID NOs: 26-28;

[0017] or,

[0018] The HCDR1-3 and / or the LCDR1-3 have a sequence that is at least 80% identical to each CDR in any of the HCDR1-3 and LCDR1-3 in groups (1) to (3), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;

[0019] Furthermore, n is a real number from 1 to 16.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following:

[0021] (1) the heavy chain variable region is the sequence shown in SEQ ID NO. 1, or / and the light chain variable region is the sequence shown in SEQ ID NO. 2;

[0022] (2) the heavy chain variable region is the sequence shown in SEQ ID NO. 3, or / and the light chain variable region is the sequence shown in SEQ ID NO. 4;

[0023] (3) the heavy chain variable region is the sequence shown in SEQ ID NO. 5, or / and the light chain variable region is the sequence shown in SEQ ID NO. 6;

[0024] or,

[0025] The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above-mentioned groups (1) to (3), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO:9, and the light chain is composed of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO:10.

[0027] In some embodiments, the antibody or antigen-binding fragment is:

[0028] (1) Chimeric antibodies or fragments thereof;

[0029] (2) humanized antibodies or fragments thereof; and / or,

[0030] (3) fully human antibodies or fragments thereof;

[0031] Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0032] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0033] In some embodiments, in the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, the cytotoxic drug D is selected from a chemotherapeutic drug or an antibiotic.

[0034] In some embodiments, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

[0035] In some embodiments, the cytotoxic drug D is selected from the compound represented by formula (DI),

[0036] in,

[0037] R 1 、R 2 Together with the atoms to which they are attached, they form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, which is optionally substituted by one or more D atoms;

[0038] R 4 Selected from H or C1-C3 alkyl;

[0039] R 5 is selected from H, halogen, CN, =O, OH, NH2 or C1-C3 alkyl;

[0040] R 6 Selected from H or C1-C3 alkyl;

[0041] R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by D, halogen, CN, ═O, OH, NH2 or C1-C3 alkyl.

[0042] In some embodiments, the R 1 、R 2 Together with the atoms they are connected to form

[0043] In some embodiments, R 4 Selected from H.

[0044] In some embodiments, R 5 is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl.

[0045] In some embodiments, R 5 Selected from H.

[0046] In some embodiments, R 6 Selected from H.

[0047] In some embodiments, R 7 Selected from cyclopropyl.

[0048] In some embodiments, the compound represented by formula (DI) is selected from one of the following compounds:

[0049] In some embodiments, the antibody-drug conjugate of the aforementioned general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, the linker unit L is selected from Its a end is covalently linked to the antibody unit Pc, and its b end is covalently linked to the cytotoxic drug D, wherein m1 is selected from integers 2 to 8, L 1A peptide residue selected from 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl.

[0050] In some embodiments, the L 1 It is a Gly-Gly-Phe-Gly tetrapeptide residue.

[0051] In some embodiments, m1 is 5.

[0052] In some embodiments, the linker unit L is Its a-terminal is covalently linked to the antibody unit Pc, and its b-terminal is covalently linked to the drug unit D.

[0053] In some embodiments, the aforementioned general formula is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of Pc-(LD)n, wherein n is selected from a real number of 1 to 16, for example, n is selected from a real number of 2 to 12, for example, n is selected from a real number of 4 to 10, for example, n is selected from a real number of 5 to 9, for example, n is selected from a real number of 6 to 8.

[0054] In some embodiments, n is a real number selected from 5 to 9, for example, n is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0055] In some embodiments, the antibody-drug conjugate of the present disclosure having the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0056] wherein Pc and n are as defined above.

[0057] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof.

[0058] In some embodiments, the present disclosure provides an expression vector comprising the nucleic acid molecule described above.

[0059] In some embodiments, the present disclosure provides an isolated host cell of the nucleic acid molecule described above, or the expression vector described above; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0060] On the other hand, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the aforementioned general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0061] On the other hand, the present disclosure provides a method for treating mammalian tumors, comprising administering a therapeutically effective amount of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

[0062] In some embodiments, the tumor is a CDH6-expressing tumor.

[0063] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and cholangiocarcinoma.

[0064] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a drug for treating tumors.

[0065] In some embodiments, the tumor is a CDH6-expressing tumor.

[0066] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and cholangiocarcinoma.

[0067] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating tumors.

[0068] In some embodiments, the tumor is a CDH6-expressing tumor.

[0069] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and cholangiocarcinoma.

[0070] In another aspect, the present disclosure provides the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating tumors.

[0071] Beneficial effects: The antibody-drug conjugate disclosed herein can specifically recognize the CDH6 target, has good plasma stability in mammalian (e.g., human or monkey) plasma, and / or has a strong tumor-suppressing effect, and has broad clinical application prospects in the treatment of diseases such as tumors.

[0072] Definitions and Explanations of Terms

[0073] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0074] The term "antibody-drug conjugate" (ADC) refers to an antibody or antigen-binding fragment thereof linked to a biologically active drug via a stable linker unit. The linker unit can be a covalent bond or a non-covalent interaction such as electrostatic forces. To form an immunoconjugate, various linkers known in the art can be used.

[0075] The term "DAR" or "drug-to-antibody ratio" refers to the average number of small molecule cytotoxic drugs attached to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0076] The term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004); the contents of which are incorporated herein by reference. Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0077] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0078] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0079] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, fragments of whole antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0080] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically existing in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.

[0081] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and the like.

[0082] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope on the same antigen.

[0083] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0084] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0085]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0086] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0087] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region. The "Fv fragment" is the smallest fragment produced by IgG and IgM that contains a complete antigen-binding site. Fv fragments have the same binding properties as Fab and similar three-dimensional binding characteristics. The VH and VL chains of an Fv fragment are bound together by non-covalent interactions.

[0088] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); the contents of which are incorporated herein by reference). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 30) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 31) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448, the contents of which are incorporated herein by reference). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. (the contents of which are incorporated herein by reference). In some cases, a disulfide bond may exist between the VH and VL of an scFv to form a disulfide-linked Fv (dsFv).

[0089] The term "diabody" refers to a diabody in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994), the contents of which are incorporated herein by reference).

[0090] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0091] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0092] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0093] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, naked antibodies are not glycosylated when expressed by host cells that do not have their own glycosylation machinery (e.g., glycosylase). In certain embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0094] The term "variable region" refers to the region of an antibody heavy or light chain that is involved in binding the antibody to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0095] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0096] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CHI domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CHI domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CHI domain. For example, a typical "heavy chain constant region fragment" can be selected from the CHI, Fc, or CH3 domains.

[0097] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0098] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can be, for example, removed during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and therefore, the Fc region may or may not include Lys447.

[0099] The term "single domain antibody" herein refers to a single domain antibody consisting only of the heavy chain variable region obtained by cloning the variable region of a natural heavy chain antibody lacking the light chain present in camels.

[0100] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0101] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0102] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the present invention contain polynucleotide sequences and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES) and polyadenylation signal sites to direct the efficient transcription of the genes carried on the expression vector. The expression vectors of the present invention may also contain the following polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0103] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0104] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0105] Herein, “n is a real number from 1 to 16” means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0106] In this article Indicates the attachment site.

[0107] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0108] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0109] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0110] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0111] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0112] In this article, C m -C n , means having an integer number of carbon atoms in the range of mn.

[0113] The term "alkyl" refers to a group of the formula CnH2n+1 The term "C1-C6 alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0114] The "C1-C6 alkyl group" described herein may further include a "C1-C3 alkyl group".

[0115] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0116] The term "heterocyclyl" refers to a fully saturated or partially saturated monocyclic, fused, spiro or bridged ring group, which contains 1-5 heteroatoms or heteroatom groups (i.e., heteroatom-containing atomic groups) in its ring atoms, wherein the "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6 or 7 ring atoms, and containing 1-3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. The term "5-6 membered heterocyclyl" refers to a heterocyclyl having 5 or 6 ring atoms, wherein the ring atoms contain 1-3 heteroatoms or heteroatom groups independently selected from the above. Examples of 4-membered heterocyclyls include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclyls include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclyls include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclyls include, but are not limited to, diazepanyl. "4-7 membered heterocyclyl" may include, but are not limited to, "4-7 membered heterocyclylalkyl," "5-6 membered heterocyclyl," "5-6 membered heterocyclylalkyl," and the like.

[0117] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0118] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0119] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0120] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0121] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0122] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0123] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0124] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0125] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0126] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0127] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0128] Certain isotopically labeled compounds of the present invention (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0129] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0130] In all administration methods of the compounds described herein, the daily dosage is 0.001 mg / kg to 600 mg / kg body weight, preferably 0.05 mg / kg to 200 mg / kg body weight, more preferably 0.1 mg / kg to 100 mg / kg body weight, in single or divided doses.

[0131] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0132] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0133] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0135] Figure 1 X-ray single crystal diffraction analysis results of compound 19-P1.

[0136] Fig. 2 Inhibitory effect of ADC on OVCAR3 cell proliferation.

[0137] Fig. 3 Inhibitory effect of ADC on PA-1 cell proliferation.

[0138] Fig. 4 Inhibitory effect of ADC on OVCAR3 cell proliferation.

[0139] Fig. 5 Inhibitory effect of ADC on PA-1 cell proliferation.

[0140] Fig. 6 Tumor growth curve of OVCAR3 subcutaneous tumor model.

[0141] Fig. 7 Tumor growth curve of OVCAR3 subcutaneous tumor model.

[0142] Figure 8 Tumor growth curve of PA-1 subcutaneous tumor model.

[0143] Figure 9 Tumor growth curve of PA-1 subcutaneous tumor model.

[0144] Figure 10 Tumor growth curve of 786-O subcutaneous tumor model. DETAILED DESCRIPTION

[0145] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0146] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0147] Example 1-1 Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14) and Isomers 14-P1 and 14-P2

[0148] Step 1: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethanone (Intermediate 14-2)

[0149] Intermediate 14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL). Nitric acid (35.43 g, 365.50 mmol, 65% purity) was slowly added, and the reaction mixture was stirred at 25°C for 2.5 h. After the reaction, saturated sodium bicarbonate solution was slowly added to the reaction mixture to adjust the pH to 7-8. Dichloromethane (100 mL) was then added, and the organic phase was washed with water (50 mL x 2) and dried over anhydrous sodium sulfate. The title compound (5 g) was obtained by purification by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 1:2).

[0150] MS m / z(ESI):210.0[M+H]+.

[0151] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)ethanone (Intermediate 14-3)

[0152] Intermediate 14-2 (2.37 g, 11.33 mmol) was dissolved in anhydrous ethanol (25 mL), and palladium on carbon (0.2 g, 10% purity) was added. The reaction mixture was stirred at 25°C under hydrogen protection for 16 h. After the reaction, the reaction mixture was filtered, and the filter cake was washed twice with ethyl acetate. The filtrate was concentrated to dryness under reduced pressure to obtain the title compound (1.6 g).

[0153] MS m / z(ESI):180.1[M+H]+.

[0154] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 14-4)

[0155] Intermediate 14-3 (1.0 g, 5.58 mmol) was dissolved in dichloromethane (10 mL). The reaction mixture was cooled to 0°C, and N,N-diisopropylethylamine (DIEA) (1.08 g, 8.37 mmol) and acetyl chloride (569.55 mg, 7.26 mmol) were added. The reaction mixture was stirred at 25°C for 1.5 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure to obtain the title compound (1.23 g).

[0156] MS m / z(ESI):222.1[M+H]+.

[0157] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl)acetamide (Intermediate 14-5)

[0158] Intermediate 14-4 (1.23 g, 5.00 mmol) was dissolved in acetic acid (12 mL). A solution of hydrogen bromide in acetic acid (1.84 g, 7.51 mmol, 33% purity) was added, followed by the slow addition of liquid bromine (959.69 mg, 6.01 mmol). The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was poured into ice water and stirred for 10 minutes. The mixture was filtered, and the filter cake was washed twice with water and concentrated to dryness under reduced pressure. Ethyl acetate (2 mL) and petroleum ether (10 mL) were added to the residue, and the reaction mixture was stirred at 25°C for 0.5 hour. The reaction mixture was filtered, and the filter cake was dried to obtain the title compound (500 mg).

[0159] MS m / z(ESI):300.0[M+H]+.

[0160] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)-2-chloroethanone (Intermediate 14-6)

[0161] Intermediate 14-5 (0.2 g, 666.43 μmol) was dissolved in anhydrous ethanol (1 mL) and concentrated hydrochloric acid (1 mL). The reaction mixture was stirred at 60°C for 16 h. After the reaction was completed and cooled to room temperature, ice water (10 mL) and saturated sodium bicarbonate (10 mL) were slowly added, followed by dichloromethane (50 mL). The organic phase was washed with water (20 mL x 2) and dried over anhydrous sodium sulfate. The title compound (160 mg) was obtained by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 6:1).

[0162] MS m / z(ESI):214.0[M+H]+.

[0163] Step 6: Synthesis of (S)-14-(bromomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 14-7)

[0164] Intermediate 14-6 (50 mg, 234.06 μmol) and intermediate 1-3 (61.62 mg, 234.06 μmol) were dissolved in toluene (1 mL), and pyridinium p-toluenesulfonate (5.88 mg, 23.41 μmol) was added. The reaction mixture was stirred at 90°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, ethanol (1 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (2 mL x 2) and dried to obtain the title compound (60 mg).

[0165] MS m / z(ESI):441.1[M+H]+.

[0166] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Intermediate 14-8)

[0167] Intermediate 14-7 (55.00 mg, 124.76 μmol) was dissolved in ethanol (1 mL), and hexamethylenetetramine (52.47 mg, 374.29 μmol) was added. The reaction mixture was stirred at 80°C for 1.5 h. After completion of the reaction, the reaction mixture was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by preparative HPLC (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: decreasingly polar mixtures of water (containing 0.225% formic acid) and methanol; methanol gradient: 0% to 27% over 12 minutes) to afford the title compound (10 mg).

[0168] MS m / z(ESI):422.1[M+H]+.

[0169] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14)

[0170] Intermediate 14-8 (10.00 mg, 20.17 μmol) and intermediate 11-1 (23.42 mg, 201.71 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (11.50 mg, 30.26 μmol) and N,N-diisopropylethylamine (7.82 mg, 60.51 μmol) were added thereto. The reaction solution was stirred at 25 ° C for 1.5 h. After completion of the reaction, the reaction solution was filtered and purified by preparative HPLC (YMC-Actus Triart C18 column 5 μm, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluent; acetonitrile gradient ratio 4%-44%, elution time 9 minutes) to obtain the title compound (3 mg).

[0171] MS m / z(ESI):520.1[M+H]+.

[0172] 1H NMR (400MHz, DMSO-d6) δ = 8.37 (t, J = 5.8Hz, 1H), 7.62 (s, 1H), 7.28 (s, 1H), 7.00 (s, 1H) ,6.25(s,1H),6.05(s,2H),5.27(d,J=5.0Hz,1H),5.23(s,2H),5.18(s,2H),4.48(d,J= 5.5Hz,2H),1.66-1.55(m,2H),0.76-0.40(m,1H),0.63(t,J=7.3Hz,3H),0.14-0.06(m,2H),0.05-0.04(m,2H).

[0173] Step 9: Preparation of 2-cyclopropyl-2-hydroxybenzyl acetate (Intermediate 14-9-P1 / P2)

[0174] Intermediate 14-9 was subjected to separation to prepare isomers 14-9-P1 and 14-9-P2. Intermediate 14-9 (1.3 g) was subjected to supercritical fluid chromatography (DAICEL CHIRALPAK AD column, 10 μm silica, 30 mm diameter, 250 mm length; using ethanol (containing 0.1% ammonia) as eluent) to obtain intermediate 14-9-P1 (600 mg) and intermediate 14-9-P2 (600 mg).

[0175] The two isomers were further analyzed by chiral supercritical fluid chromatography (SCFC) under the following conditions.

[0176] Intermediate 14-9-P1:

[0177] Under the above chiral supercritical fluid chromatography conditions, its retention time was 2.990 minutes;

[0178] 1 H NMR (400MHz, METHANOL-d4) δ7.43-7.29(m,5H),5.29-5.16(m,2H),3.67(d,J=7.6Hz,1H),1.19-1.07(m,1H),0.58-0.38(m,4H).

[0179] Intermediate 14-9-P2:

[0180] Under the above chiral supercritical fluid chromatography conditions, its retention time was 2.661 minutes;

[0181] 1H NMR (400MHz, METHANOL-d4) δ7.46-7.28 (m, 5H), 5.30-5.16 (m, 2H), 3.67 (d, J = 7.6Hz, 1H), 1.21-1.03 (m, 1H), 0.60-0.36 (m, 4H).

[0182] Step 10: Synthesis of 2-cyclopropyl-2-hydroxyacetic acid (Intermediate 14-10-P1 / P2)

[0183] Under a hydrogen atmosphere, intermediate 14-9-P1 (500 mg) was added to methanol (15 mL). Wet palladium on carbon (10 mg, 10%) was added to the reaction solution, and the reaction solution was stirred at 25°C under a hydrogen atmosphere for 16 hours. After the reaction, the reactant was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P1 (273 mg).

[0184] 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J = 7.2Hz, 1H), 1.21-1.09 (m, 1H), 0.61-0.40 (m, 4H).

[0185] Under a hydrogen atmosphere, intermediate 14-9-P2 (500 mg) was added to methanol (15 mL). Wet palladium on carbon (10 mg, 10%) was added to the reaction solution, and the reaction solution was stirred at 25°C under a hydrogen atmosphere for 16 hours. After the reaction, the reactant was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P2 (279 mg).

[0186] 1 H NMR (400MHz, METHANOL-d4) δ3.63 (d, J = 7.2Hz, 1H), 1.19-1.08 (m, 1H), 0.60-0.39 (m, 4H).

[0187] Step 11: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14-P1 / P2)

[0188] Intermediate 14-8 (40.00 mg) and intermediate 14-10-P1 (28.11 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL). 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (46.02 mg) and N,N-diisopropylethylamine (31.28 mg) were added, and the reaction mixture was stirred at 25°C for 1.5 h. After completion of the reaction, the reaction mixture was purified by preparative HPLC (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: a decreasingly polar mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile gradient 16% to 46% over 12 minutes) to obtain compound 14-P1 (22.00 mg).

[0189] MS m / z(ESI):520.1[M+H] + .

[0190] 1 H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 5.7Hz, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 6.51 (s, 1H), 6.29 (s, 2H), 5.47 (s, 2H), 5.43 (s, 2H), 4. 73(d,J=5.9Hz,2H),3.54(d,J=5.9Hz,1H),1.93-1.78(m,2H),1.06-0. 96(m,1H),0.87(t,J=7.3Hz,3H),0.39-0.30(m,2H),0.29-0.21(m,2H).

[0191] Intermediate 14-8 (10.00 mg) and intermediate 14-10-P2 (8.27 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.05 mg) and N,N-diisopropylethylamine (6.13 mg) were added. The reaction mixture was stirred at 25°C for 1.5 h. After completion of the reaction, the reaction mixture was directly purified by preparative HPLC (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents (acetonitrile gradient 16% to 46% over 12 minutes) to obtain compound 14-P2 (8.00 mg).

[0192] MS m / z(ESI):520.1[M+H] + .

[0193] 1 H NMR (400MHz, DMSO-d6) δ = 8.63 (t, J = 5.9Hz, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.30 (s, 2H), 5.46 (s, 2H), 5.43 (s, 2H), 4.72 (d, J=6.0Hz,2H),3.55(d,J=6.0Hz,1H),1.92-1.81(m,2H),1.03-0.97(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.30(m,2H),0.28-0.22(m,2H).

[0194] The two isomers were further analyzed separately by the following chiral supercritical fluid chromatography analysis method.

[0195] Compound 14-P1:

[0196] Under the above chiral supercritical fluid chromatography conditions, its retention time was 3.673 minutes;

[0197] Compound 14-P2:

[0198] Under the above chiral supercritical fluid chromatography conditions, its retention time is 3.735 minutes.

[0199] Example 1-2: (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14-S)

[0200] Step 1: Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidin-2-one (Intermediate 3)

[0201] Starting material 1 (150.0 g), 4-dimethylaminopyridine (160.15 g), and starting material 2 (221.2 g) were weighed and dissolved in 1500 mL of dichloromethane and stirred at room temperature for 15 minutes. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 359.0 g) was weighed and added to the reaction mixture in batches. After addition, the mixture was stirred at room temperature for approximately 5 hours. After completion of the reaction, dilution was added to the reaction mixture with dichloromethane (1500 mL). The mixture was then washed with water (500 mL) twice, 2N HCl (500 mL) once, saturated sodium bicarbonate solution (500 mL) once, and saturated saline solution (500 mL) once. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (302 g).

[0202] 1 H NMR (400MHz, CDCl3) δ7.32-7.35(m,2H),7.26-7.29(m,1H),7.21-7.23(m,2H),4.68-4.71(m,1H),4.17-4.23(m,2H), 3.30-3.33(dd,1H),2.91-2.95(dd,1H),2.78-2.82(m,2H),1.14-1.18(m,1H),0.59-0.63(m,2H),0.21-0.26(m,2H).

[0203] Step 2: Synthesis of (S)-4-benzyl-3-((S)-2-cyclopropyl-2-hydroxyacetyl)oxazolidin-2-one (Intermediate 5)

[0204] Intermediate 3 (200 g) was weighed and dissolved in 2000 mL of anhydrous tetrahydrofuran. Under nitrogen, the mixture was stirred at -78°C for 15 minutes. Subsequently, sodium bis(trimethylsilyl)amide (443.5 mL, 2 M solution in tetrahydrofuran) was added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was stirred at -78°C for 30 minutes. Intermediate 4 (201.5 g) was dissolved in 700 mL of tetrahydrofuran until clear and then slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred at -78°C for 2 hours. Subsequently, 220 mL of glacial acetic acid was added to the reaction mixture to quench the reaction. After the addition was complete, the mixture was gradually warmed to room temperature and 600 mL of 2N HCl was added to the reaction mixture. The mixture was stirred at room temperature (20-25°C) for 10 hours. The reaction mixture was then concentrated under reduced pressure, and ethyl acetate (1000 mL) and water (200 mL) were added to the residue and stirred for 20 minutes. The aqueous phase was separated and extracted twice with ethyl acetate (500 mL x 2). The organic phases were combined and washed twice with 400 mL of saturated NaHCO3 solution, 400 mL of saturated Na2S2O3 solution and saturated brine, respectively. The resulting organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1 / 30) to give the title compound (128.7 g).

[0205] 1 H NMR (400MHz, CDCl3) δ7.37 (dd, J=8.1, 6.8Hz, 2H), 7.33-7.29 (m, 1H), 7.27-7.23 (m ,2H),4.81(dd,J=7.9,5.9Hz,1H),4.72(ddt,J=10.0,7.5,2.9Hz,1H),4.33(t,J=8 .3Hz,1H),4.28(dd,J=9.1,2.5Hz,1H),3.48(dd,J=8.2,3.9Hz,1H),3.35(dd,J=13 .5,3.4Hz,1H),2.88(dd,J=13.5,9.4Hz,1H),1.36-1.29(m,1H),0.62-0.44(m,4H).

[0206] Step 3: Synthesis of (S)-4-benzyl-3-((S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetyl)oxazolidin-2-one (Intermediate 6)

[0207] Intermediate 5 (128.7 g) was weighed and dissolved in 1300 mL of dichloromethane. Imidazole (56.17 g) was added and stirred in an ice bath for 15 min. TBSCl (107.3 g) was then added portionwise to the reaction mixture and stirred at room temperature for 3 h. 200 mL of 2N HCl was added to the reaction mixture and stirred for 20 min. The mixture was then separated. The organic phase was washed twice with 200 mL of saturated NaHCO₃ solution and twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 80:1) to obtain the title compound (160 g).

[0208] 1 H NMR (400MHz, CDCl3) δ7.35-7.37(m,2H),7.30-7.32(m,1H),7.26-7.29(m,2H),5.26-5.31(m,1H),4.67-4.71(m,1H),4.20-4.26(m ,2H),3.41-3.44(dd,1H),2.72-2.76(dd,1H),1.25-1.31(m,1H),0.94(s,9H),0.54-0.56(m,2H),0.46-0.48(m,2H),0.12(s,6H).

[0209] Step 4: Synthesis of (S)-benzyl 2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetate (Intermediate 7)

[0210] Weigh benzyl alcohol (62.18 g) and dissolve it in 500 mL of tetrahydrofuran. Stir at -25°C. Weigh n-butyl lithium (213.6 mL, 2.5 M in tetrahydrofuran) and slowly add it dropwise to the reaction mixture. Stir at -25°C for 1 hour. Weigh intermediate 6 (160 g) and dissolve it in 320 mL of tetrahydrofuran. Slowly add it dropwise to the reaction mixture at -25°C. Stir at -15°C for 3 hours. Add saturated NH4Cl (200 mL) to the reaction mixture to quench the reaction. The mixture was then concentrated under reduced pressure, and 400 mL of methyl tert-butyl ether and water (150 mL) were added to the reaction solution, stirred for 30 min, and separated. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL x 2), and the organic phases were combined, washed once with saturated brine (250 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 100: 1) to obtain the title compound (125 g).

[0211] 1H NMR (400MHz, CDCl3) δ7.28-7.40(m,5H),5.17-5.26(m,2H),3.86-3.89(m,1H),1.21-1.46(m,1H),0.90(s,9H),0.45-0.51(m,4H),0.05(s,6H).

[0212] Step 5: Synthesis of (S)-2-cyclopropyl-2-hydroxybenzyl acetate (Intermediate 8)

[0213] Intermediate 7 (125 g) was weighed and dissolved in 1200 mL of tetrahydrofuran. Glacial acetic acid (35.1 g) was added and stirred at room temperature for 5 min. Tetrabutylammonium fluoride (TBAF, 585 mL, 1 M solution in tetrahydrofuran) was then added to the reaction solution, and the reaction solution was placed at 45°C for 4 h. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran (600 mL). 300 mL of water and 400 mL of methyl tert-butyl ether were added to the residue and stirred for 20 min. The layers were separated, and the aqueous phase was extracted twice with methyl tert-butyl ether (200 mL). The organic phases were combined and washed twice with 200 mL of saturated NaHCO3 solution and saturated brine. The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 60:1) to obtain the title compound (68.9 g).

[0214] 1 H NMR (400MHz, CDCl3) δ7.27-7.39(m,5H),5.22-5.28(m,2H),3.82(d,1H),2.7(brs,1H),1.11-1.15(m,1H),0.41-0.56(m,4H).

[0215] Intermediate 8 was further analyzed by the following chiral supercritical fluid chromatography conditions.

[0216] The retention time of intermediate 8 under the above-mentioned chiral supercritical fluid chromatography conditions is 3.013 minutes, which is basically consistent with the retention time (2.990 minutes) of intermediate 14-9-P1 in Example 1-1 under the same chromatographic analysis conditions. Intermediate 8 and intermediate 14-9-P1 have the same configuration and are the same compound.

[0217] Step 6: Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid (Intermediate 9)

[0218] Intermediate 8 (5 g) was dissolved in methanol (80 mL). Wet palladium on carbon (10% by mass, 0.7 g) was added to the reaction mixture, and the mixture was stirred at 25°C for 16 h under a hydrogen atmosphere. After the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (2.4 g).

[0219] 1 H NMR (400MHz, METHANOL-d4) δ = 3.63 (d, J = 7.3Hz, 1H), 1.20-1.09 (m, 1H), 0.61-0.39 (m, 4H).

[0220] Step 7: Synthesis of (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)-2-hydroxyacetamide (Compound 14-S)

[0221] Intermediate 14-8 (90 mg) and Intermediate 9 (49.60 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (121.81 mg) and N,N-diisopropylethylamine (82.81 mg) were added. The reaction mixture was stirred at 25°C for 16 h. After completion of the reaction, the reaction mixture was purified by HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 20%-50%, 12 min) to obtain the title compound (21 mg).

[0222] MS m / z(ESI):520.0[M+H] + .

[0223] 1 H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 6.1 Hz, 1H), 7.87 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.30 (s, 2H), 5.48 (s, 2H), 5.43 (s, 2H), 4. 73(d,J=5.6Hz,2H),3.54(d,J=5.5Hz,1H),1.93-1.80(m,2H),1.05-0. 97(m,1H),0.88(t,J=7.3Hz,3H),0.39-0.30(m,2H),0.30-0.22(m,2H).

[0224] Compound 14-S was further analyzed by the following chiral supercritical fluid chromatography conditions.

[0225] Compound 14-S exhibited a retention time of 3.654 minutes under the aforementioned chiral supercritical fluid chromatography conditions, which is substantially consistent with the retention time (3.673 minutes) of compound 14-P1 prepared in Example 1-1 under the same chromatographic analysis conditions. Therefore, it was determined that compound 14-S and compound 14-P1 prepared in Example 1-1 have the same configuration and are identical compounds.

[0226] Example 2-1. Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19) and Isomers 19-P1 and 19-P2

[0227] Step 1: Synthesis of 1-(Benzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate 19-2)

[0228] Intermediate 19-1 (3 g) was dissolved in anhydrous DMF (25 mL), and deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added. After addition, the mixture was heated to 90°C and stirred for 16 h. The reaction solution was then added to water (100 mL) and extracted with ethyl acetate (200 mL x 2). The combined organic phases were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (2.4 g).

[0229] MS m / z(ESI):167.1[M+H] + .

[0230] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl-2,2-d2)ethan-1-one (Intermediate 19-3)

[0231] Intermediate 19-2 (2.4 g) was dissolved in anhydrous acetic acid (10 mL). Concentrated nitric acid (32.50 g, 70% content) was added dropwise at 0°C. Stirring was continued at 0°C for 10 minutes. The mixture was then warmed to room temperature and stirred for 1 hour. After completion of the reaction, the reaction mixture was added dropwise to ice water (200 mL). After filtration, the filter cake was dried to obtain the title compound (1.9 g).

[0232] MS m / z(ESI):212.0[M+H] + .

[0233] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0234] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate 19-4)

[0235] Intermediate 19-3 (1.8 g) was dissolved in acetic acid (25 mL), and acetic anhydride (1.84 g) and reduced iron powder (4.76 g) were added. The mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (1.5 g).

[0236] MS m / z(ESI):224.1[M+H] + .

[0237] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (Intermediate 19-5)

[0238] A solution of HBr in acetic acid (2.39 g, 33% content) was added dropwise to a solution of intermediate 19-4 (1.45 g) in anhydrous acetic acid (25 mL). Br2 (1.07 g) was then added dropwise. The mixture was stirred at room temperature for 1 h. After completion of the reaction, the reaction solution was concentrated to dryness under reduced pressure. The residue was added to water (50 mL) and extracted with ethyl acetate (50 mL x 2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to obtain the title compound (1.3 g).

[0239] MS m / z(ESI):302.1[M+H] + .

[0240] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl-2,2-d2)-2-chloroethane-1-one (Intermediate 19-6)

[0241] Intermediate 19-5 (1.2 g) and concentrated hydrochloric acid (144.82 mg) were dissolved in ethanol (15 mL), and the reaction mixture was stirred at 60°C for 16 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by HPLC (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% NH4HCO3) and acetonitrile as eluents (acetonitrile gradient ratio 40%-50%)) to obtain the title compound (577 mg).

[0242] MS m / z(ESI):216.0[M+H] + .

[0243] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate 19-7)

[0244] Intermediate 19-6 (100.0 mg) and Intermediate 1-3 (109.87 mg) were dissolved in toluene (1 mL) and acetic acid (1 mL), and pyridinium p-toluenesulfonate (5.24 mg) was added. The reaction mixture was stirred at 100°C for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 h. The reaction mixture was filtered, and the filter cake was washed with ethanol (5 mL x 2) to obtain the title compound (100.0 mg).

[0245] MS m / z(ESI):443.0[M+H] + .

[0246] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (Intermediate 19-8)

[0247] Intermediate 19-7 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and hexamethylenetetramine (94.97 mg) was added. The reaction mixture was stirred at 50°C for 6 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 0%-30%, 12 min) to obtain the title compound (25.0 mg).

[0248] MS m / z(ESI):424.0[M+H] + .

[0249] Step 8: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19)

[0250] Intermediate 19-8 (7 mg) and Intermediate 11-1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was filtered and purified by preparative HPLC (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: decreasingly polar mixtures of water (containing 0.05% formic acid) and acetonitrile (acetonitrile gradient 20%-50%, elution time 12 minutes) to obtain the title compound (2.60 mg).

[0251] MS m / z(ESI):522.1[M+H] + .

[0252] 1H NMR (400MHz, DMSO-d6) δ=8.62(t,J=5.9Hz,1H),7.84(s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),5.48-5.41(m,5H),4.72(d,J= 5.5Hz,2H),3.59-3.52(m,1H),2.00-1.76(m,2H),1.05-0.96(m,1H),0.88(t,J=7.4Hz,3H),0.37-0.30(m,2H),0.29-0.19(m,2H).

[0253] Step 9: Synthesis of 2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19-P1 / P2)

[0254] Intermediate 19-8 (7 mg) and intermediate 14-10-P1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: decreasingly polar mixtures of water (containing 0.05% formic acid) and acetonitrile; acetonitrile gradient: 15% to 45% over 12 minutes) to afford compound 19-P1 (3.30 mg).

[0255] MS m / z(ESI):522.1[M+H] + .

[0256] 1H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 6.0 Hz, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 6.51 (s, 1H), 5.54-5.51 (m, 1H), 5.47 (s, 2H), 5.43 (s, 2H) ),4.72(d,J=6.0Hz,2H),3.55-3.53(m,1H),1.94-1.78(m,2H),1.05-0. 96(m,1H),0.88(t,J=7.3Hz,3H),0.40-0.30(m,2H),0.29-0.19(m,2H).

[0257] Intermediate 19-8 (7 mg) and intermediate 14-10-P2 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added. The reaction mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; eluent: decreasingly polar mixtures of water (containing 0.05% formic acid) and acetonitrile (acetonitrile gradient 15% to 45% over 12 minutes) to afford compound 19-P2 (4.0 mg).

[0258] MS m / z(ESI):522.1[M+H] + .

[0259] 1 H NMR (400MHz, DMSO-d6) δ = 8.62 (t, J = 6.1Hz, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 6.50 (s, 1H), 5.54-5.51 (m, 1H), 5.46 (s, 2H), 5 .43(s,2H),4.72(d,J=5.8Hz,2H),3.55-3.52(m,1H),1.94-1.80(m,2H),1.04-0.95(m,1H),0.88(t,J=7.4Hz,3H),0.39-0.30(m,2H), 0.29-0.21(m,2H).

[0260] The two isomers were further analyzed separately by the following chiral supercritical fluid chromatography analysis method.

[0261] Compound 19-P1:

[0262] Under the above chiral HPLC conditions, its retention time was 2.877 minutes;

[0263] Compound 19-P2:

[0264] Under the above chiral HPLC conditions, the retention time was 2.690 minutes.

[0265] Configuration confirmation of compound 19-P1 (X-ray single crystal diffraction method)

[0266] Single crystal cultivation method: Weigh 10 mg of compound 19-P1 sample and place it in a 1.5 ml centrifuge tube. Add 300 μl of pyridine, sonicate and then seal with parafilm. Use a needle to poke three small holes in the parafilm. Slowly evaporate at 20-30°C for 48 hours to obtain needle-shaped crystals.

[0267] The obtained single crystal sample was subjected to X-ray analysis, and the test results are shown in Table 1 and Figure 1.

[0268] Table 1 Single crystal sample and crystal data of compound 19-P1

[0269] Through the above X-ray crystal diffraction experiment, the chemical structure of compound 19-P1 was determined to be:

[0270] Example 2-2, Synthesis of (S)-2-cyclopropyl-N-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (Compound 19-S)

[0271] Intermediate 19-8 (2.4 g) and Intermediate 9 (1645.5 mg) were dissolved in anhydrous N,N-dimethylformamide (25 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (3.24 g) and N,N-diisopropylethylamine (1465.11 mg) were added. The reaction mixture was stirred at 25°C for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure. Ethyl acetate (100 mL) was added to the residue and stirred for 16 h. After filtration, methanol (50 mL) was added to the filter cake and stirred for 16 h. Filtration afforded the title compound (1.6 g).

[0272] MS m / z(ESI):522.1[M+H] + .

[0273] 1 H NMR (400MHz, DMSO-d6) δ = 8.60 (t, J = 5.9Hz, 1H), 7.84 (s, 1H), 7.50 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 5.49 (d, J = 5.1Hz, 1H), 5.47-5.37 (m, 4H), 4 .71(d,J=5.8Hz,2H),3.54(t,J=5.6Hz,1H),1.97-1.75(m,2H),1.07-0. 94(m,1H),0.87(t,J=7.3Hz,3H),0.40-0.29(m,2H),0.29-0.20(m,2H).

[0274] Compound 19-S was further analyzed by the following chiral supercritical fluid chromatography analysis method.

[0275] Compound 19-S prepared in this example exhibited a retention time of 2.853 minutes under the aforementioned chiral supercritical fluid chromatography conditions, which was substantially consistent with the retention time (2.877 minutes) of compound 19-P1 prepared in Example 2-1 under the same chromatographic analysis conditions. Therefore, it was determined that compound 19-S and compound 19-P1 had the same configuration and were identical compounds.

[0276] Example 3. Synthesis of N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-3,8,11,14,17-pentaoxo-5-oxyl-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-14) and Isomers L1-14-P1 and L1-14-P2

[0277] Step 1: Synthesis of intermediate L1-14-2

[0278] The starting material L1-14-1 (25 g), lead acetate (43.79 g) and pyridine (6.98 g) were dissolved in a mixed solvent of tetrahydrofuran (600 mL) and toluene (200 mL), heated to 85°C under a nitrogen atmosphere, and stirred for 18 hours. After the reaction was completed, it was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (chromatographic column: 330g Silica Flash Column, mobile phase gradient 0-75% ethyl acetate / petroleum ether, flow rate 100 mL / min) to obtain the title compound (18 g).

[0279] 1 H NMR (400MHz, METHANOL-d4) δ = 7.82 (d, J = 7.5Hz, 2H), 7.69 (d, J = 7.3Hz, 2H), 7.44-7.38 (m, 2H), 7. 36-7.31(m,2H),5.22(s,2H),4.39(d,J=6.8Hz,2H),4.28-4.22(m,1H),3.81(s,2H),2.03(s,3H).

[0280] MS m / z(ESI):391.1[M+Na] + .

[0281] Step 2: Synthesis of intermediate L1-14-3

[0282] The intermediate L1-14-2 (5g), 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (8.40g), and p-toluenesulfonic acid pyridinium salt (PPTS, 341.09mg) were dissolved in dichloromethane (150mL). The reaction solution was heated to 65°C under a nitrogen atmosphere and stirred for 48h. After the reaction was completed, it was cooled to room temperature, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (chromatographic column: 120g Silica Flash Column, mobile phase gradient 0-45% ethyl acetate / petroleum ether, flow rate 80 mL / min), followed by further purification by HPLC (chromatographic column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 42%-82%, 13 min) to obtain the title compound (1.4 g).

[0283] MS m / z(ESI):537.2[M+Na] + .

[0284] Step 3: Synthesis of intermediates L1-14-4-P1 and L1-14-4-P2

[0285] The intermediate L1-14-3 (1.4 g) was dissolved in a mixed solvent of methanol (15 mL) and water (15 mL), wet palladium carbon (10% mass content, 0.15 g) was added, and the reaction solution was stirred at 25° C. under a hydrogen atmosphere for 16 h. After the reaction, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by high performance liquid chromatography (chromatographic column: Boston Prime C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 24%-64%, 13 min), and then further purified by supercritical fluid chromatography (chromatographic column: DAICEL CHIRALPAK IC column, 10μm silica, 30mm diameter, 250mm length; using isopropanol (containing 0.1% ammonia water) as eluent) to obtain intermediate L1-14-4-P1 (130 mg) and intermediate L1-14-4-P2 (130 mg).

[0286] The two isomers were further analyzed separately by the following chiral HPLC analysis method.

[0287] Chiral HPLC conditions are as follows:

[0288] Intermediate L1-14-4-P1:

[0289] Under the above chiral HPLC conditions, its retention time was 4.471 minutes;

[0290] MS m / z(ESI):447.5[M+Na] + .

[0291] Intermediate L1-14-4-P2:

[0292] Under the above chiral HPLC conditions, its retention time was 5.692 minutes;

[0293] MS m / z(ESI):447.3[M+Na] + .

[0294] Step 4: Synthesis of intermediates L1-14-5-P1 and L1-14-5-P2

[0295] 2-Chlorotrityl chloride resin (2-CTC-resin) (specification: approximately 1.19 mmol / g) (257 mg) was added to dichloromethane (3 mL), followed by intermediate L1-14-4-P1 (130 mg) and diisopropylethylamine (59.37 mg). The reaction mixture was shaken on a shaker at 25°C under a nitrogen atmosphere for 16 hours. After completion of the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). Filter the mixture, and dry the filter cake to obtain intermediate L1-14-5-P1 (330 mg).

[0296] Intermediate L1-14-4-P2 (130 mg) was used as the starting material to prepare intermediate L1-14-5-P2 (350 mg) according to the above method.

[0297] Step 5: Synthesis of intermediates L1-14-6-P1 and L1-14-6-P2

[0298] Intermediate L1-14-5-P1 (330 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). The mixture was filtered and the filter cake was dried to obtain L1-14-6-P1 (220 mg).

[0299] Intermediate L1-14-5-P2 (350 mg) was used as the starting material to prepare intermediate L1-14-6-P2 (220 mg) according to the above method.

[0300] Step 6: Synthesis of intermediates L1-14-7-P1 and L1-14-7-P2

[0301] Intermediate L1-14-6-P1 (220 mg) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalanine (230.17 mg) were dissolved in N,N-dimethylformamide (5 mL). Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (225.31 mg) and diisopropylethylamine (99.96 mg) were added to the reaction solution. The reaction solution was shaken on a shaker at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times. Filter and dry the filter cake to obtain L1-14-7-P1 (377 mg).

[0302] Intermediate L1-14-7-P2 (361 mg) was prepared using intermediate L1-14-6-P2 (220 mg) as starting material according to the above method.

[0303] Step 7: Synthesis of intermediates L1-14-8-P1 and L1-14-8-P2

[0304] Intermediate L1-14-7-P1 (377 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (37.22 mg) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). Filter the mixture, and dry the filter cake to obtain L1-14-8-P1 (270 mg).

[0305] Intermediate L1-14-7-P2 (361 mg) was used as the starting material to prepare intermediate L1-14-8-P2 (260 mg) according to the above method.

[0306] Step 8: Synthesis of intermediates L1-14-9-P1 and L1-14-9-P2

[0307] Intermediate L1-14-8-P1 (270 mg) was dissolved in N,N-dimethylformamide (5 mL). N-((9H-fluoren-9-ylmethoxy)carbonyl)glycylglycine (209.58 mg), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (224.29 mg), and diisopropylethylamine (99.51 mg) were added sequentially. The reaction mixture was shaken at 25°C for 1 hour. After completion of the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). Filter the mixture, and dry the filter cake to obtain intermediate L1-14-9-P1 (403 mg).

[0308] Intermediate L1-14-8-P2 (260 mg) was used as the starting material and intermediate L1-14-9-P2 (420 mg) was prepared according to the above method.

[0309] Step 9: Synthesis of intermediates L1-14-10-P1 and L1-14-10-P2

[0310] Intermediate L1-14-9-P1 (403 mg) was dissolved in N,N-dimethylformamide (5 mL) and piperidine (1.08 g) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). The mixture was filtered and the filter cake was dried to obtain L1-14-10-P1 (300 mg).

[0311] Intermediate L1-14-9-P2 (420 mg) was used as the starting material and the intermediate L1-14-10-P2 (320 mg) was prepared according to the above method.

[0312] Step 10: Synthesis of intermediates L1-14-12-P1 and L1-14-12-P2

[0313] Intermediate L1-14-10-P1 (300 mg) was dissolved in N,N-dimethylformamide (5 mL), and compound L1-14-11 (182.13 mg) and diisopropylethylamine (99.41 mg) were added sequentially. The reaction mixture was shaken at 25°C for 16 hours. After completion of the reaction, the resin was washed three times with methanol (10 mL) and dichloromethane (10 mL). Filter the mixture, and dry the filter cake to obtain L1-14-12-P1 (374 mg).

[0314] Intermediate L1-14-10-P2 (320 mg) and intermediate L1-14-11 (194.27 mg) were used as raw materials to prepare intermediate L1-14-12-P2 (387 mg) according to the above method.

[0315] Step 11: Synthesis of intermediates L1-14-13-P1 and L1-14-13-P2

[0316] Intermediate L1-14-12-P1 (374 mg) was added to a mixed solvent of dichloromethane (8 mL) and hexafluoroisopropanol (HFIP, 2 mL), and the reaction mixture was shaken at 25°C for 0.5 h. After completion of the reaction, the resin was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by HPLC (column: Boston Prime C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 15%-35%, 9 min) to obtain intermediate L1-14-13-P1 (74 mg).

[0317] 1 H NMR (400MHz, METHANOL-d4) δ = 7.33-7.20 (m, 5H), 6.81 (s, 2H), 4.77-4.70 ( m,2H),4.60-4.52(m,1H),3.96-3.70(m,6H),3.61(d,J=7.6Hz,1H),3.55-3 .47(m,2H),3.27-3.23(m,1H),3.05-2.98(m,1H),2.30(t,J=7.4Hz,2H),1 .72-1.55(m,4H),1.38-1.29(m,2H),1.16-1.07(m,1H),0.60-0.47(m,4H).

[0318] MS m / z(ESI):679.7[M+Na] + .

[0319] Intermediate L1-14-12-P2 (387 mg) was used as the starting material and intermediate L1-14-13-P2 (86 mg) was prepared according to the above method.

[0320] 1 H NMR (400MHz, METHANOL-d4)δ=7.40-7.21(m,5H),6.82(s,2H),4.81-4.67(m,2H),4.60-4.50(m,1H),3.97-3.70(m,6H),3.66-3.57(m,1H),3.56-3 .47(m,2H),3.27-3.22(m,1H),3.08-2.97(m,1H),2.35-2.26(m,2H),1.7 5-1.55(m,4H),1.41-1.32(m,2H),1.16-1.06(m,1H),0.60-0.45(m,4H).

[0321] MS m / z(ESI):679.5[M+Na] + .

[0322] Step 12: Synthesis of compounds L1-14-P1 and L1-14-P2

[0323] Intermediate L1-14-13-P1 (31.17 mg), intermediate 14-8 (20 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (18.20 mg), pyridine (11.26 mg), and 1-hydroxybenzotriazole (12.83 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 2 h. After completion of the reaction, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 26%-46%, 12 min) to obtain compound L1-14-P1 (12.3 mg).

[0324] 1H NMR (400MHz, DMSO-d6)δ=8.65(t,J=5.9Hz,1H),8.58(t,J=6.6Hz,1H),8.27(t,J=5.6Hz,1H),8.17-8.02(m,2H),7.99(t,J=5.6Hz,1H),7. 79(s,1H),7.52(s,1H),7.28-7.19(m,5H),7.19-7.14(m,1H),6.98(s,2H),6.49(s,1H),6.29(d,J=3.7Hz,2H),5.48-5.37(m,4H),4.85-4. 70(m,2H),4.70-4.65(m,1H),4.52-4.45(m,1H),4.43-4.37(m,1H),3.79-3.54(m,7H),3.49(d,J=7.1Hz,1H),3.08-3.01(m,1H),2.85-2.7 4(m,1H),2.14-2.06(m,2H),1.94-1.80(m,2H),1.52-1.42(m,4H),1.27-1.13(m,2H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.41-0.28 (m,4H).

[0325] MS m / z(ESI):1060.3[M+H] + .

[0326] Using intermediate L1-14-12-P2 (31.17 mg) and intermediate 14-8 (20 mg) as raw materials, compound L1-14-P2 (11.4 mg) was prepared according to the above method.

[0327] 1H NMR (400MHz, DMSO-d6)δ=8.72-8.52(m,2H),8.33-8.25(m,1H),8.17-7.94(m,3H),7.80(s,1H),7.51(s,1H),7.32- 7.19(m,5H),7.18-7.12(m,1H),6.99(s,2H),6.49(s,1H),6.35-6.25(m,2H),5.48-5.36(m,4H),4.84-4.59(m,3H), 4.54-4.45(m,1H),4.44-4.35(m,1H),3.81-3.54(m,7H),3.49(d,J=6.7Hz,1H),3.08-3.01(m,1H),2.87-2.73(m,1H ),2.14-2.05(m,2H),1.95-1.77(m,2H),1.53-1.39(m,4H),1.25-1.13(m,2H),1.03-0.82(m,4H),0.43-0.25(m,4H)

[0328] MS m / z(ESI):1060.3[M+H] + .

[0329] The two isomers were further analyzed separately by the following chiral high performance liquid chromatography method.

[0330] Chiral HPLC conditions are as follows:

[0331] Under the above chiral HPLC conditions, the retention time of compound L1-14-P1 is 3.735 minutes.

[0332] Under the above chiral HPLC conditions, the retention time of compound L1-14-P2 is 3.901 minutes.

[0333] Example 4-1. Synthesis of N-((12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxyl-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-19-P1)

[0334] Intermediate L1-14-13-P1 (7.75 mg), intermediate 19-8 (5.0 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.53 mg), pyridine (2.80 mg), and 1-hydroxybenzotriazole (3.19 mg) were dissolved in N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 25°C under a nitrogen atmosphere for 2 h. After completion of the reaction, the reaction mixture was purified by HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 22%-52%, 12 min) to obtain L1-19-P1 (6.0 mg).

[0335] MS m / z(ESI):1062.3[M+H] + .

[0336] 1 H NMR (400MHz, DMSO-d6) δ=8.74-8.69(m,1H),8.59(t,J=6.7Hz,1H),8.32-8.26(t,J= 5.6Hz,1H),8.11(d,J=7.8Hz,1H),8.06(t,J=5.3Hz,1H),7.99-7.94(m,1H),7.80(s,1H),7.52(s,1H),7.27-7.20(m,5H) ,7.19-7.11(m,1H),6.99(s,2H),6.50(s,1H),5.49-5.41(m,4H),4.84-4.72(m,2H),4.71-4.63(m,1H),4.53-4.48(m,1H ),4.44-4.36(m,1H),3.77-3.56(m,6H),3.49(d,J=7.0Hz,1H),3.06-3.02(m,1H),2.83-2.74(m,1H),2.10(t,J=7.6Hz,2 H),1.91-1.82(m,2H),1.50-1.42(m,4H),1.22-1.13(m,2H),1.02-0.90(s,1H),0.88(t,J=7.3Hz,3H),0.38-0.29(m,4H).

[0337] L1-19-P1 was further analyzed by the following chiral high performance liquid chromatography analysis method.

[0338] Chiral HPLC conditions are as follows:

[0339] Under the above chiral supercritical fluid chromatography conditions, the retention time of L1-19-P1 is 3.775 minutes.

[0340] Example 4-2: N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxyl-2,7,10,13,16-pentaazaoctadec-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-19-S)

[0341] Step 1: Synthesis of L1-14-3-S

[0342] Intermediate L1-14-2 (28 g) and intermediate 8 (23.5 g, prepared in Example 1-2) were dissolved in dichloromethane (25 mL). Silver trifluoromethanesulfonate (139.50 mg) was added to the reaction solution. The reaction solution was stirred at 25°C under nitrogen for 108 h. After the reaction, the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified on a flash silica gel column (mobile phase gradient tetrahydrofuran / dichloromethanol: 0-7%, flow rate 70 mL / min) to obtain the title compound (10.3 g).

[0343] MS m / z(ESI):537.3[M+Na] + .

[0344] Step 2: Synthesis of L1-14-4-S

[0345] Intermediate L1-14-3-S (10 g) was dissolved in tetrahydrofuran (100 mL), and wet palladium on carbon (10% by mass, 1 g) was added. The reaction mixture was stirred at 0°C under a hydrogen atmosphere for 16 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. Petroleum ether / ethyl acetate (10 mL / 0.5 mL) was added to the residue, stirred for 2 h, and filtered to obtain the title compound (6 g).

[0346] MS m / z(ESI):447.2[M+Na] + .

[0347] L1-14-4-S was further analyzed by the following chiral high performance liquid chromatography analysis method.

[0348] Chiral HPLC conditions are as follows:

[0349] Under the aforementioned chiral supercritical fluid chromatography conditions, the retention time of L1-14-4-S was 4.385 minutes, which is essentially consistent with the retention time of compound L1-14-4-P1 (4.471 minutes) under the same chromatographic analysis conditions. Therefore, L1-14-4-S and L1-14-4-P1 have the same configuration and are the same compound.

[0350] Step 3: Synthesis of L1-19-S

[0351] Intermediate L1-14-13-S (600 mg, prepared from L1-14-4-S according to the synthetic method of L1-14-13-P1), intermediate 19-8 (502.93 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (350.31 mg), pyridine (216.82 mg), and 1-hydroxybenzotriazole (246.91 mg) were dissolved in N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25°C under nitrogen for 2 h. After completion of the reaction, the reaction mixture was purified by HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [A: water (0.05% formic acid), B: acetonitrile]; B%: 24%-54%, 12 min) to obtain the title compound (286 mg).

[0352] 1 H NMR (400MHz, DMSO-d6)δ=8.72-8.63(m,1H),8.62-8.52(m,1H),8.34-8.26(m,1H),8.16-7.94(m,3H),7.78(s,1H),7.5 1(s,1H),7.26-7.13(m,6H),6.99(s,1H),6.56-6.42(m,1H),5.52-5.40(m,4H),4.85-4.60(m,3H),4.59-4.46(m,1H), 4.44-4.38(m,1H),3.79-3.53(m,6H),3.52-3.44(m,2H),3.08-3.02(m,1H),2.85-2.75(m,1H),2.09(t,J=7.8Hz,2H), 1.95-1.75(m,2H),1.58-1.38(m,4H),1.26-1.09(m,2H),1.04-0.96(m,1H),0.88(t,J=7.1Hz,3H),0.42-0.26(m,4H).

[0353] MS m / z(ESI):1062.5[M+H] +.

[0354] L1-19-S was further analyzed by the following chiral high performance liquid chromatography method.

[0355] Chiral HPLC conditions are as follows:

[0356] Under the above-mentioned chiral supercritical fluid chromatography conditions, the retention time of L1-19-S was 3.748 minutes, which is essentially consistent with the retention time (3.775 minutes) of compound L1-19-P1 prepared in Example 4-1 under the same chromatographic analysis conditions. Therefore, L1-19-S and L1-19-P1 are considered to have the same configuration and are the same compound.

[0357] Example 5. Construction and production of anti-human CDH6 antibodies

[0358] The heavy and light chain variable region sequences of the anti-human CDH6 monoclonal antibodies (CDH6-Ab-4, CDH6-Ab-3, CDH6-Ab-2, and CDH6-Ab) are shown in Table 2 below, and the CDR sequences classified according to Kabat notation are shown in Table 3 below. The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5 containing a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 29) and heavy chain constant region / light chain constant region sequences to generate recombinant plasmids expressing VH-CH / VL-CL. The plasmid and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and cultured in a shaker at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and 6 g / L glucose (Sigma, Catalog No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected and purified using Protein A (GE, Catalog No.: 28985254). The eluted sample was dialyzed into PBS (pH 7.4) to obtain an anti-human CDH6 monoclonal antibody. CDH6-Ab is a positive control human CDH6 antibody, the sequence of which is derived from patent WO2018212136A1.

[0359] Table 2 Sequences of heavy and light chain variable regions of anti-human CDH6 monoclonal antibodies

[0360] Table 3 Anti-human CDH6 monoclonal antibody CDR sequences (Kabat classification)

[0361] Example 6: Preparation of Antibody-Drug Conjugates

[0362] Conjugation: The antibody prepared in Example 5 was dialyzed into a 20 mM PB, 150 mM NaCl, 1 mM EDTA solution (pH 6.5). An 8-fold volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) was added to the antibody solution. The mixed solution was reduced on a thermostatic metal shaker at 37°C for 2.5 hours. A 15-fold volume of the linker-payload compound (prepared in Examples 3 and 4) was dissolved in DMSO and added to the reaction system. The reaction solution was conjugated at 25°C for 6 hours. The reaction product was desalted on a G25 column and exchanged into phosphate-buffered saline (PBS) buffer to remove unreacted free small molecule toxins. The ADC product was analyzed for purity and DAR value using SEC and LC-MS.

[0363] SEC Purity Analysis: The protein samples were analyzed using SEC-HPLC to characterize the size uniformity of the recombinant protein and determine its purity. The HPLC used in this method was an Agilent 1260, the chromatographic column was a TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropanol (Merck, 1.01040.4008) (v / v 9:1), the detection temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0364] DAR value determination: The DAR value of the ADC molecule was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-glycan modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37°C for 1 hour to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected onto a Waters ACQUITY Protein BEH molecular exclusion chromatography column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were as follows: spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, parent ion scan range 800-3000, etc. Finally, the mass spectrometry data analysis software Biopharma Finder was used. 4.1. Deconvolution processing is performed using the Respect algorithm to calculate the molecular weight information of the light and heavy chain mass spectrum peaks and the mass spectrum response signals of each component, respectively, and thus calculate the DAR value of the ADC sample to be tested.

[0365] Table 4 DAR values ​​and SEC purity of CDH6-ADC

[0366] The same method as above was used to prepare the isotype control (ISO) anti-FITC-hIgG1 antibody and conjugate it with the Linker-Payload compound to obtain the isotype control of the ADC. Its DAR value and SEC results are shown below.

[0367] Example 7: In vitro proliferation inhibition test of tumor cells by ADC

[0368] Cells and materials: Human ovarian cancer cell line OVCAR3 (CDH6 high-expressing cell line) was purchased from ATCC (#HTB-161), human ovarian teratoma cell line PA-1 (CDH6 medium-expressing cell line) was purchased from Nanjing Kebai Biotechnology Co., Ltd. (#CBP60800), bovine serum, 1640 culture medium, MEM medium (Gibco#11095-080), MEM NEAA (Gibco#11140-050), sodium pyruvate (Gibco#11360-070), penicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco, bovine insulin was purchased from Solarbio, 96-well plates were purchased from Corning, and Cell-Titer Glo reagent was purchased from Promega.

[0369] Cell Culture: OVCAR3 cells were cultured in 1640 medium supplemented with 20% fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C and 5% CO2. PA-1 cells were cultured in MEM supplemented with 10% fetal bovine serum, 1% MEM NEAA, 1% sodium pyruvate, and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used in experiments.

[0370] Cell proliferation activity assay: The inhibitory activity of ADCs against OVCAR3 and PA-1 cell lines was assessed using Cell-Titer Glo reagent. OVCAR3 cells (5000 cells per well) and PA-1 cells (800 cells per well) were seeded in 96-well plates and incubated at 37°C, 5% CO₂ for 24 hours. The ADC was diluted with culture medium from the corresponding cells to a concentration of 100 nM. A three-fold serial dilution was then performed in culture medium for a total of eight concentrations. 10 μL of the prepared ADC solution was transferred to a 96-well plate to a final concentration of 0-10 nM. After the test ADC solution was added, the plates were incubated at 37°C, 5% CO₂. OVCAR3 and PA-1 cells were cultured for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[0371] Data analysis: Calculate %inhibition and fit IC 50 % inhibition = 1-100% × (Signal-Bottom) / (Top-Bottom). Signal refers to the signal value of the ADC sample group, Bottom refers to the signal value of the same volume of culture medium without cells, and Top refers to the signal value of cells without ADC sample.

[0372] Experimental results: Under the experimental conditions of this study, the tested ADCs exhibited strong proliferation inhibitory activity against both OVCAR3 and PA-1 cells, as shown in Tables 5-6 and Figure 2-5.

[0373] Table 5 Inhibitory effect of ADC on OVCAR3 and PA-1 cell proliferation Note: “ / ” indicates IC cannot be fitted 50 .

[0374] Table 6 Inhibitory effect of ADC on OVCAR3 and PA-1 cell proliferation Note: “ / ” indicates IC cannot be fitted 50 .

[0375] Example 8: Evaluation of drug efficacy in OVCAR3 subcutaneous tumor model

[0376] Experimental reagents: Human ovarian cancer OVCAR3 cells were purchased from ATCC, RPMI-1640 medium was purchased from Gibco (catalog number A104910), fetal bovine serum was purchased from Excell (catalog number FND500), penicillin-streptomycin was purchased from Gibco (catalog number 15140122), bovine insulin was purchased from Yeasen (catalog number 40107ES60), 0.25% trypsin-EDTA was purchased from Gibco (catalog number 25200-072), D-PBS (phosphate buffered saline without calcium and magnesium ions) was purchased from Hyclone (catalog number SH30256.01), and Matrigel was purchased from Corning (catalog number 356237).

[0377] Experimental methods:

[0378] Animal information: Balb / c nude female mice, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to a standard certified commercial laboratory diet and free drinking water.

[0379] Cell Culture: Human ovarian cancer OVCAR3 cells were cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, and 10 μg / ml bovine insulin in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the required number of cells was reached.

[0380] Cell inoculation: 0.1 ml of OVCAR3 cell suspension (containing 1×10 7Each mouse was subcutaneously inoculated in the axilla with 100 cells (RPMI-1640:Matrigel volume ratio of 1:1). On day 26 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping being designated Day 0.

[0381] Tumor measurements and experimental parameters:

[0382] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0383] The anti-tumor efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0384] Experimental results:

[0385] In the mouse subcutaneous xenograft OVCAR3 tumor model, ADC-L1-14-P1-12 and ADC-L1-14-P1-5 significantly inhibited tumor growth after a single intravenous administration at a dose of 3 mg / kg (P < 0.0001). The results are shown in Table 7 and Figure 6.

[0386] Table 7 Tumor volume of OVCAR3 subcutaneous tumor model

[0387] In the mouse subcutaneous xenograft OVCAR3 tumor model, ADC-L1-19-P1-12 and ADC-L1-19-P1-5 significantly inhibited tumor growth after a single intravenous administration at a dose of 3 mg / kg (P < 0.0001). The results are shown in Table 8 and Figure 7.

[0388] Table 8 Tumor volume of OVCAR3 subcutaneous tumor model

[0389] Example 9: Evaluation of drug efficacy in PA-1 subcutaneous tumor model

[0390] Experimental reagents: Human ovarian cancer PA-1 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd., MEM medium was purchased from Gibco (catalog number 32561-037), fetal bovine serum was purchased from Excell (catalog number FND500), penicillin-streptomycin was purchased from Gibco (catalog number 15140122), NEAA was purchased from Gibco (catalog number 11140-050), sodium pyruvate was purchased from Gibco (catalog number 11360-070), Versene was purchased from Gibco (catalog number 15040-066), D-PBS (calcium-free and magnesium-free phosphate buffer) was purchased from Hyclone (catalog number SH30256.01), and Matrigel was purchased from Corning (catalog number 356237).

[0391] Experimental methods:

[0392] Animal information: Balb / c nude female mice, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to a standard certified commercial laboratory diet and free drinking water.

[0393] Cell Culture: Human ovarian cancer PA-1 cells were cultured in MEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% NEAA, and 1 mM sodium pyruvate in an incubator at 37°C and 5% CO2. Cells were routinely digested and passaged weekly using Versene digestion solution. Cells were harvested and counted when cell saturation reached 80%-90% and the desired number of cells was reached.

[0394] Cell inoculation: 0.1 ml PA-1 cell suspension (containing 1×10 7 Each mouse was subcutaneously inoculated in the axilla with 100 cells (MEM:Matrigel volume ratio of 1:1). On day 17 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping as Day 0.

[0395] Tumor measurements and experimental parameters:

[0396] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0397] The anti-tumor efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0398] Experimental results:

[0399] In the PA-1 subcutaneous xenograft mouse model, ADC-L1-14-P1-12 and ADC-L1-14-P1-5 significantly inhibited tumor growth at a single intravenous dose of 3 mg / kg (P < 0.0001). The results are shown in Table 9 and Figure 8.

[0400] Table 9 Tumor volume of PA-1 subcutaneous tumor model

[0401] In the PA-1 subcutaneous xenograft mouse model, ADC-L1-19-P1-12 and ADC-L1-19-P1-5 significantly inhibited tumor growth at doses of 1 mg / kg and 3 mg / kg, respectively, after a single intravenous administration (P<0.0001), in a dose-dependent manner. The results are shown in Table 10 and Figure 9.

[0402] Table 10 Tumor volume of PA-1 subcutaneous tumor model

[0403] Example 10: Evaluation of drug efficacy in 786-O subcutaneous tumor model

[0404] Experimental reagents: Human renal cancer 786-O cells (CDH6 low-expressing cell line) were purchased from ATCC, RPMI-1640 medium was purchased from Gibco (catalog number A104910), fetal bovine serum was purchased from Excell (catalog number FND500), penicillin-streptomycin was purchased from Gibco (catalog number 15140122), 0.25% trypsin-EDTA was purchased from Gibco (catalog number 25200-072), D-PBS (phosphate buffered saline without calcium and magnesium ions) was purchased from Hyclone (catalog number SH30256.01), and Matrigel was purchased from Corning (catalog number 356237).

[0405] Experimental methods:

[0406] Animal information: NOD-SCID mice, female, 8-9 weeks old, weighing approximately 18-25 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with separate ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0407] Cell Culture: Human renal carcinoma 786-O cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the required number of cells was reached.

[0408] Cell inoculation: 0.1 ml of 786-O cell suspension (containing 5×10 6 Each mouse was subcutaneously inoculated in the axilla with 100 cells (RPMI-1640:Matrigel volume ratio of 1:1). On day 13 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping being designated Day 0.

[0409] Tumor measurements and experimental parameters:

[0410] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0411] The anti-tumor efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0412] Experimental results:

[0413] In the mouse subcutaneous xenograft 786-O tumor model, ADC-L1-19-P1-12 and ADC-L1-19-P1-5 significantly inhibited tumor growth after a single intravenous administration at a dose of 3 mg / kg (P < 0.0001). The results are shown in Table 11 and Figure 10.

[0414] Table 11 Tumor volume of 786-O subcutaneous tumor model

[0415] Example 11, ADC plasma stability test

[0416] ADC molecules (final concentration of 100 μg / ml) were incubated with human plasma (Aoneng Bio, PB021-C) and monkey plasma (Sinoda Bio, SND-X0107) in a 37°C incubator. The day of incubation was designated as day 0, and samples were subsequently removed on days 7, 14, and 28 for free small molecule detection.

[0417] A 20 μL sample was added to 300 μL of internal standard working solution (prepared in acetonitrile), vortexed for 5 minutes, centrifuged for 5 minutes (14,000 rpm), and 4 μL of the supernatant was injected for LC-MS / MS analysis (API 6500+). The results are shown in Table 12. The results show that the tested ADC molecules are relatively stable in both human and monkey plasma.

[0418] Table 12 Plasma stability of CDH6-ADC N / A means no free small molecules were detected and the % free small molecules could not be calculated.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD) n , in, D is a cytotoxic drug; L is a linker unit; Pc is an antibody or an antigen-binding fragment thereof that specifically binds to CDH6; The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 or / and the LCDR1-3 are: (1) the HCDR1-3 are SEQ ID NOs: 11-13; or / and the LCDR1-3 are SEQ ID NOs: 14-16; (2) the HCDR1-3 are SEQ ID NOs: 17-19; or / and the LCDR1-3 are SEQ ID NOs: 20-22; (3) the HCDR1-3 are SEQ ID NOs: 23-25; or / and the LCDR1-3 are SEQ ID NOs: 26-28; or, The HCDR1-3 and / or the LCDR1-3 have a sequence with at least 80% identity, or a sequence with at most 3 insertion, deletion or substitution mutations compared to each CDR in any group of HCDR1-3 and LCDR1-3 in groups (1) to (3); optionally, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; Furthermore, n is a real number ranging from 1 to 16.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following: (1) the heavy chain variable region is the sequence shown in SEQ ID NO.1, or / and the light chain variable region is the sequence shown in SEQ ID NO.2; (2) the heavy chain variable region is the sequence shown in SEQ ID NO.3, or / and the light chain variable region is the sequence shown in SEQ ID NO.4; (3) the heavy chain variable region is the sequence shown in SEQ ID NO.5, or / and the light chain variable region is the sequence shown in SEQ ID NO.6; or, The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above groups (1) to (3), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein: The antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, the constant region fragment comprising CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, the heavy chain constant region has the sequence shown in SEQ ID NO:9, and the light chain is composed of the VL and the light chain constant region, the light chain constant region has the sequence shown in SEQ ID NO:

10.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment is: (1) Chimeric antibodies or fragments thereof; (2) humanized antibodies or fragments thereof; and / or, (3) fully human antibodies or fragments thereof; Optionally, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics. Optionally, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: The cytotoxic drug D is selected from the compound represented by formula (DI), in, R 1 , R 2 Together with the atoms to which they are attached, they form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, which is optionally substituted by one or more D atoms; R 4 Selected from H or C1-C3 alkyl; R 5 is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl; R 6 Selected from H or C1-C3 alkyl; R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, wherein: The R 1 , R 2 Together with the atoms they are connected to form 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6 or 7, wherein: R 4 , R 5 , R 6 All are selected from H.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 8, wherein: R 7 Selected from cyclopropyl.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 9, wherein: The compound represented by formula (DI) is selected from one of the following compounds:

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein: The linker unit L is selected from Its a end is covalently linked to the antibody unit Pc, and its b end is covalently linked to the cytotoxic drug D, wherein m1 is selected from an integer of 2 to 8, L 1 A peptide residue selected from 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 11, wherein: The L 1 It is a Gly-Gly-Phe-Gly tetrapeptide residue.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein: The linker unit L is Its a-terminal is covalently linked to the antibody unit Pc, and its b-terminal is covalently linked to the drug unit D.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugate or a pharmaceutically acceptable salt thereof:

15. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

16. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15, in the preparation of a drug for treating tumors; optionally, the tumor is a tumor expressing CDH6; optionally, the tumor is selected from ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer and bile duct epithelial carcinoma.

17. A method for treating mammalian tumors, the method comprising administering a therapeutically effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15, to a mammal in need of such treatment, preferably a human; optionally, the tumor is a tumor expressing CDH6; optionally, the tumor is selected from ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer and bile duct epithelial carcinoma.