Anti-CDH6 antibody-drug conjugate and its use

Abstract

This invention relates to a class of antibody-drug conjugates having a novel structure. Specifically, this invention discloses an antibody-drug conjugate that targets CDH6, which has good plasma stability in mammalian (human or monkey, etc.) plasma, has a relatively strong antitumor effect, and has the potential for broad clinical application in the treatment of diseases such as tumors.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310420429.5, titled "ANTI-CDH6 ANTIBODY-DRUG CONJUGATE AND USE THEREOF," filed with the China Intellectual Property Office on 14 April 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to a novel antibody-drug conjugate having a novel structure, a method for preparing the same, a pharmaceutical composition containing the conjugate, and its use as an antitumor agent, in the field of biomedicine. [Background technology]

[0003] Antibody-drug conjugates (ADCs), as novel targeted therapies, are formed by linking a monoclonal antibody that specifically binds to tumor cell surface antigens to a biologically active toxin molecule. This combines the tumor-targeting ability of the antibody with the efficient killing ability of the toxin molecule, overcoming the drawbacks of conventional chemotherapy, such as insufficient therapeutic efficacy and the latter, such as serious toxicity, side effects, and low drug-making potential. Compared to conventional chemotherapy agents, ADC drugs can precisely target tumor cells and reduce their impact on normal cells, achieving safer and more effective antitumor effects.

[0004] ADCs generally consist of three parts: antibody, linker, and toxin. Camptothecin derivatives are a class of toxins applied to the development of ADCs, achieving antitumor effects by inhibiting topoisomerase I. Daiichi Sankyo used the camptothecin derivative Dxd as the toxin to develop Enhertu (trastuzumab deruxtecan, DS-8201), an ADC drug targeting HER2. It was approved for manufacture and sale by the US FDA in 2019. Clinical trials have shown that Enhertu exhibits favorable therapeutic effects against HER2-positive breast cancer, gastric cancer, non-small cell lung cancer, and others.

[0005] CDH6, also known as K-cadherin, is a type II classical cadherin. It is a single-pass membrane protein composed of 790 amino acids, with its extracellular domain divided into five subregions (EC1-EC5). Studies have shown that CDH6 is primarily highly expressed in tumor tissues such as renal, ovarian, and thyroid cancers, but is rarely expressed in normal tissues (Cancer Discov;2017,7(9):1030-45). Like other members of the cadherin superfamily, the CDH6 protein localizes to the basolateral membrane of epithelial cells, mediates calcium-dependent intercellular adhesion, and exhibits rapid internalization properties. Therefore, CDH6 can be used as a potential target for ADC development for the treatment of cancers such as ovarian and renal cancer.

[0006] Despite several recent advances in the treatment of ovarian and renal cancer, substantial unmet clinical needs still exist. Currently, only a limited number of CDH6-targeted ADC drugs are under research and development, and no related drugs have been approved for manufacture or sale. Therefore, the development of ADC drugs targeted at this site has broad clinical applications and therapeutic potential. [Overview of the Initiative]

[0007] This disclosure provides an antibody-drug conjugate having the general structural formula Pc-(LD)n, or a pharmaceutically acceptable salt thereof. During the ceremony, D is a cytotoxic drug, L is the linker unit, Pc is an antibody or its antigen-binding fragment that specifically binds to CDH6. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, or / or the light chain variable region comprising LCDR1, LCDR2, and LCDR3, where HCDR1-3 or / or LCDR1-3 are selected from the following combinations: (1) HCDR1-3 are sequence numbers 11-13, and / or LCDR1-3 are sequence numbers 14-16. (2) HCDR1-3 are sequence numbers 17-19, and / or LCDR1-3 are sequence numbers 20-22. (3) HCDR1-3 are sequence numbers 23-25, and / or LCDR1-3 are sequence numbers 26-28. or HCDR1-3 and / or LCDR1-3 have at least 80% identity with each CDR in any one of the HCDR1-3 and LCDR1-3 of groups (1)-(3), or have sequences with up to three insertions, deletions, or substitution mutations, preferably, 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 between 1 and 16.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region and / or a light chain variable region selected from the following: (1) The heavy chain variable region is the sequence shown in Sequence ID No. 1, and / or the light chain variable region is the sequence shown in Sequence ID No. 2. (2) The heavy chain variable region is the sequence shown in Sequence ID No. 3, and / or the light chain variable region is the sequence shown in Sequence ID No. 4. (3) The heavy chain variable region is the sequence shown in Sequence ID No. 5, and / or the light chain variable region is the sequence shown in Sequence ID No. 6. or The heavy chain variable region and / or light chain variable region has at least 80% identity with any one of the heavy chain variable regions and / or light chain variable regions of the above groups (1) to (3), or has a sequence with up to three insertion, deletion or substitution mutations, preferably, at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0009] 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 an intact constant region sequence or a fragment thereof, the constant region fragment comprising CH1, a hinge region, CH2, CH3, or Fc; optionally, the heavy chain constant region is selected from human and mouse IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from human and mouse kappa and lambda constant regions; optionally, the antibody or antigen-binding fragment comprises an intact heavy chain and a light chain, where the heavy chain consists of VH and a heavy chain constant region, the heavy chain constant region having the sequence shown in SEQ ID NO: 9, and the light chain consists of VL and a light chain constant region, the light chain constant region having the sequence shown in SEQ ID NO: 10.

[0010] In some embodiments, the antibody or antigen-binding fragment is (1) Chimeric antibody or fragment thereof, (2) Humanized antibodies or fragments thereof, and / or (3) A fully human antibody or a fragment thereof, Preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, native antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, and single-domain antibodies.

[0011] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and the lowest recognition unit of an antibody.

[0012] In some embodiments, in an antibody-drug conjugate having the general formula Pc-(LD)n described above or a pharmaceutically acceptable salt thereof, the cytotoxic drug D is selected from chemotherapeutic agents and antibiotics.

[0013] In some embodiments, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

[0014] In some embodiments, the cytotoxic drug D is selected from compounds represented by formula (DI), [ka] During the ceremony, R 1 and R 2 These atoms, together with the atoms to which they are bonded, form a 5-6 member heterocycle, where the 5-6 member heterocycle contains one or two oxygen atoms as ring atoms, and the 5-6 member heterocycle is optionally substituted with one or more D atoms. R 4 It is selected from H and C1-C3 alkyl groups. R 5 These are selected from H, halogens, CN, =O, OH, NH2, and C1-C3 alkyl groups. R 6 It is selected from H and C1-C3 alkyl groups. R 7 The group is selected from H, C1-C3 alkyl, and C3-C6 cycloalkyl, where the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with D, halogen, CN, =O, OH, NH2, or C1-C3 alkyl.

[0015] In some embodiments, R 1 and R 2 They, together with the atoms to which they are bonded, [ka] It forms.

[0016] In some embodiments, R4 is selected from H.

[0017] In some embodiments, R 5 is selected from H, halogen, CN, OH, NH2, and C1-C3 alkyl.

[0018] In some embodiments, R 5 is selected from H.

[0019] In some embodiments, R 6 is selected from H.

[0020] In some embodiments, R 7 is selected from cyclopropyl.

[0021] In some embodiments, the compound represented by formula (D-I) is selected from one of the following compounds:

Chemical formula

[0022] In some embodiments, in the antibody-drug conjugate having the general formula of Pc-(L-D)n described above, or a pharmaceutically acceptable salt thereof, the linker unit L is

Chemical formula

[0023] In some embodiments, L 1 This is a Gly-Gly-Phe-Gly tetrapeptide residue.

[0024] In some embodiments, m1 is 5.

[0025] In some embodiments, the linker unit L is [ka] Its terminal a is covalently bonded to the antibody unit Pc, and its terminal b is covalently bonded to the drug unit D.

[0026] In some embodiments, in an antibody-drug conjugate having the general formula Pc-(LD)n described above, or a pharmaceutically acceptable salt thereof, n is a real number selected from 1 to 16, for example, a real number selected from 2 to 12, for example, a real number selected from 4 to 10, for example, a real number selected from 5 to 9, for example, a real number selected from 6 to 8.

[0027] 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.

[0028] In some embodiments, the antibody-drug conjugate having the general formula Pc-(LD)n of the present disclosure, or a pharmaceutically acceptable salt thereof, is selected from the following compounds or pharmaceutically acceptable salts thereof: [ka] In the formula, Pc and n are as defined in any one of the prior embodiments.

[0029] In another embodiment, the present disclosure provides isolated nucleic acid molecules encoding the aforementioned antibody or its antigen-binding fragment.

[0030] In some embodiments, the present disclosure provides an expression vector comprising the nucleic acid molecule described above.

[0031] In some embodiments, the disclosure provides isolated host cells of the nucleic acid molecules or expression vectors described above, preferably eukaryotic or prokaryotic, more preferably host cells derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis, and more preferably host cells selected from Expi293 cells and CHO cells.

[0032] In another embodiment, the Disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate having the general formula Pc-(LD)n as described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0033] In another embodiment, the present disclosure provides a method for treating tumors in mammals, the method comprising administering to a mammal in need of such treatment, preferably a human, an antibody-drug conjugate having the general formula Pc-(LD)n described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0034] In some embodiments, the tumor is a CDH6-expressing tumor.

[0035] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, kidney cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct cancer.

[0036] In another aspect, the present disclosure provides the use of an antibody-drug conjugate having the general formula Pc-(LD)n described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a drug for treating tumors.

[0037] In some embodiments, the tumor is a CDH6-expressing tumor.

[0038] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, kidney cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct cancer.

[0039] In another aspect, the present disclosure provides the use of an antibody-drug conjugate having the general formula Pc-(LD)n described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the treatment of tumors.

[0040] In some embodiments, the tumor is a CDH6-expressing tumor.

[0041] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, kidney cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct cancer.

[0042] In another aspect, the present disclosure provides an antibody-drug conjugate having the general formula Pc-(LD)n described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the treatment of tumors.

[0043] Beneficial Effects: The antibody-drug conjugates of this disclosure can specifically recognize the CDH6 target, exhibit good plasma stability in mammalian (e.g., human or monkey) plasma, and / or exhibit relatively strong antitumor effects, thereby having broad applicability in the treatment of diseases such as tumors.

[0044] Terms and Definitions Unless otherwise specified, the terms used in this disclosure have the following meanings, and the definitions of the groups and terms described in this disclosure, including their illustrative definitions, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in examples, may be combined and incorporated into each other as appropriate. Certain terms should not be considered uncertain or unclear unless otherwise specifically defined, and should be interpreted according to their general meaning in the art. Where a trade name is referred to herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0045] The term "antibody-drug conjugate" (ADC) means that an antibody or its antigen-binding fragment is linked to a bioactive drug via a stable linker unit. Linking can be achieved by covalent bonds or non-covalent interactions such as electrostatic forces. Various linkers known in the art can be used to form immune complexes.

[0046] The term “DAR” or “drug-to-antibody ratio” refers to the average number of small molecule cytotoxic drugs conjugated to each antibody molecule. In the antibody-drug conjugates of this disclosure, DAR is defined by a variable “n” which may be either an integer or a decimal.

[0047] The term “antibody” is used in its broadest sense to refer to a polypeptide or combination of polypeptides containing a sufficient sequence from the immunoglobulin heavy chain variable region and / or a sufficient sequence from the immunoglobulin light chain variable region that can specifically bind to an antigen. “Antibody” as used herein encompasses a variety of forms and structures, insofar as they exhibit the desired antigen-binding activity. “Antibody” as used herein includes alternative protein scaffolds or artificial scaffolds having implanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include, for example, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antibody, and, for example, fully synthetic scaffolds containing biocompatible polymers. For example, see Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); and Roque et al., Biotechnol. Prog. 20:639-654 (2004), the contents of which are incorporated herein by reference. Such scaffolds may also include, but are not limited to, non-antibody-derived scaffold proteins such as tenascin, fibronectin, and peptide aptamers, which are well known in the art to be useful for transplanting CDRs.

[0048] In this specification, "antibody" includes a typical "quadrilateral antibody," which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting 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, from the N-terminus to the C-terminus. Furthermore, if the full-length antibody is an IgE isoform, the heavy chain optionally further includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL), from the N-terminus to the C-terminus. The heavy chains are linked to each other and to the light chains via disulfide bonds, forming a Y-shaped structure. The heavy chain constant regions of immunoglobulins differ in amino acid composition and arrangement, and therefore in antigenicity. Therefore, "immunoglobulin" as used herein can be classified into five classes, or isoforms, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same class can also be classified into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4, and IgA can be classified into IgA1 and IgA2. The light chains are classified into κ chains or λ chains depending on differences in the constant region. Each of the five classes of Ig may have either a κ chain or a λ chain.

[0049] In this specification, “antibodies” may be derived from any animal, including but not limited to humans and non-human animals, and non-human animals may be selected from primates, mammals, rodents, and vertebrates, for example, species of the camelid family, Lama glama, Lama guanicoe, Vicugna pacos, sheep, rabbits, mice, rats, or Chondrichthyes (e.g., sharks).

[0050] As used herein, "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0051] The term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any variants that may arise (e.g., spontaneous mutations or variants that occur during the production of the formulation, which are generally present in small amounts). In contrast to polyclonal antibody formulations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. The modifier “monoclonal” as used herein should not be interpreted as requiring that the antibody or antigen-binding molecule be produced by any particular method. For example, monoclonal antibodies can be prepared by a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and other methods well known in the art.

[0052] The term "natural antibody" refers to antibodies produced and counteracted by the immune system of multicellular organisms. The term "engineered antibody" as used herein refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. Exemplary examples of "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), and bispecific antibodies.

[0053] The term "single-specific" means that a substance has one or more binding sites, and each of these binding sites binds to the same epitope of the same antigen.

[0054] The term "multispecific antibody" means that it has at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. Therefore, terms such as "bispecific," "triplespecific," and "quadrispecific" refer to the number of different epitopes to which the antibody / antigen-binding molecule can bind.

[0055] The term "valency" indicates the presence of a specific number of binding sites in an antibody / antigen-binding molecule. Therefore, "monovalent," "divalent," "tetravalent," and "hexavalent" indicate the presence of one, two, four, and six binding sites, respectively, within the antibody / antigen-binding molecule.

[0056] In this specification, the terms "full-length antibody," "complete antibody," and "intact antibody" are used synonymously and refer to antibodies having a structure substantially similar to that of naturally occurring antibodies.

[0057] In this specification, “antigen-binding fragment” and “antibody fragment” are used synonymously and include only a portion of an intact antibody or a variant of that portion that retains the ability to bind to an antigen, although it does not have the complete structure of an intact antibody. In this specification, “antigen-binding fragment” or “antibody fragment” includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0058] An intact antibody is digested with papain to produce two identical antigen-binding fragments called "Fab" fragments, each containing a heavy chain variable domain and a light chain variable domain, as well as a light chain constant domain and a first heavy chain constant domain (CH1). Therefore, the term "Fab fragment" as used herein refers to a light chain fragment containing the VL domain and constant domain (CL) of the light chain, and an antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments are distinguished from Fab fragments by the addition of several 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 ​​residue in the constant domain possesses a free thiol group. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. "Fv fragments" are the smallest fragments produced by IgG and IgM and contain an intact antigen-binding site. The Fv fragment has the same binding properties as Fab and similar three-dimensional binding properties. The VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0059] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain containing VL and VH domains, where VL and VH are linked via a linker (see, for example, 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 Ed., Springer-Verlag, New York, pp. 269-315 (1994); the contents of these references are incorporated herein by reference). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Appropriate linkers in the prior art consist of repeats or variants of the GGGGS (SEQ ID NO: 30) amino acid sequence. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 31) may be used, or a variant thereof may be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448, which is incorporated herein by reference). Other linkers that may be used in this disclosure are described in 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 these references are incorporated herein by reference). In some cases, a disulfide bond may be present between the VH and VL of scFv, forming a disulfide linkage Fv (dsFv).

[0060] The term "diabody" refers to an antibody that has VH and VL domains expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing of the two domains on the same chain, thereby forcing these domains to pair with complementary domains on another chain and generating two antigen-binding sites (see, for example, 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).

[0061] The term "chimeric antibody" refers to an antibody in which a portion of the light chain and / or heavy chain originates from one antibody (which may originate from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain originates from another antibody (which may originate from the same or different species or belong to the same or different antibody class or subclass), but nevertheless retains binding activity to a target antigen (Cabilly et al. USP4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term "chimeric antibody" may include an antibody (e.g., a human-mouse chimeric antibody) in which the heavy chain and light chain variable regions of the antibody originate from a first antibody (e.g., a mouse antibody), and the heavy chain and light chain constant regions of the antibody originate from a second antibody (e.g., a human antibody).

[0062] The term "humanized antibody" refers to a genetically modified non-human antibody that has an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or part of the CDR of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain, or partially retain, desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response.

[0063] The term "fully human antibody" refers to an antibody in which both the FR and CDR have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also derives from a human germline immunoglobulin sequence. Fully human antibodies as used herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation). However, "fully human antibody" as used herein does not include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0064] In this specification, the term “naked antibody” refers to an antibody that is not linked, fused, or conjugated to another drug or molecule (e.g., a label or drug), peptide, or polypeptide. In certain embodiments, a naked antibody expressed by a mammalian host cell may be glycosylated by the host cell’s glycosylation mechanism (e.g., glycosylase). In certain embodiments, a naked antibody is not glycosylated when expressed by a host cell lacking its own glycosylation mechanism (e.g., glycosylase). In certain embodiments, the naked antibody is an intact antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of an intact antibody, such as a Fab antibody.

[0065] The term "variable region" refers to the region of the antibody's heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used synonymously with "VH" and "HCVR," while "light chain variable region" is used synonymously with "VL" and "LCVR." The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each containing 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 provide antigen-binding specificity. In this specification, the terms “complementarity-determining region” and “CDR” are used synonymously and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL), which is also known as the complementarity-determining region because it is precisely complementary to the antigenic epitope in spatial structure. The heavy chain variable region CDR may be abbreviated as HCDR, and the light chain variable region CDR may be abbreviated as LCDR. The terms “framework region” and “FR” are used synonymously and refer to amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. Generally, the variable region of a typical antibody consists of four FRs and three CDRs, in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0066] In this specification, “CDR” may be labeled and defined in a manner well known in the art, including but not limited to the Kabat numbering scheme, the Chothia numbering scheme, or the IMGT numbering scheme. Websites of tools used include, but are not limited to, the AbRSA site (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abysis site (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT site (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDR in this specification includes duplicates and subsets of amino acid residues, defined in different ways.

[0067] In this specification, the term “heavy chain constant region” refers to the carboxyl-terminal portion of an antibody heavy chain that does not directly participate in the binding of the antibody to the antigen but exhibits effector functions such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domain of the antibody. The “heavy chain constant region” includes at least the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The “heavy chain constant region” includes a “full-length heavy chain constant region” having a structure substantially similar to that of the constant region of a native antibody, while the “heavy chain constant region fragment” includes only a “part of the full-length heavy chain constant region.” Exemplarily, a typical “full-length antibody heavy chain constant region” consists of the CH1 domain-hinge region-CH2 domain-CH3 domain. If the antibody is IgE, it further includes the CH4 domain, and if the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical “heavy chain constant region fragment” may be selected from the CH1, Fc, and CH3 domains.

[0068] In this specification, the term “light chain constant region” refers to the carboxyl-terminal portion of the antibody light chain that is not directly involved in the binding of the antibody to the antigen. The light chain constant region may be selected from a constant κ domain and a constant λ domain.

[0069] In this specification, the term "Fc" refers to the carboxyl-terminal portion of an antibody formed by the hydrolysis of an intact antibody with papain. Typically, it includes the CH3 and CH2 domains of the antibody. The Fc region includes, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. While the boundaries of the Fc region of immunoglobulin heavy chains may vary slightly, the Fc region of human IgG heavy chains is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) may be removed, for example, during antibody production or purification, or by recombinant operation of the nucleic acid encoding the antibody heavy chain. Therefore, the Fc region may include or exclude Lys447.

[0070] In this specification, the term "single-domain antibody" refers to a single-domain antibody consisting solely of a heavy-chain variable region obtained by cloning the variable region of a heavy-chain antibody that naturally lacks a light chain in camels.

[0071] The term "identity" as used herein is calculated as follows: To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison). Next, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0072] As used herein, the term “nucleic acid” includes any compound and / or substance containing polymers of nucleotides. Each nucleotide consists 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. Generally, a nucleic acid molecule is described as a sequence of bases, thereby the bases representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is generally represented as 5' to 3'. As used herein, the term “nucleic acid molecule” includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term “nucleic acid molecule” includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbone links or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable for use as vectors for the direct expression of the antibodies of this disclosure in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may 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, thereby allowing the mRNA to be injected into a target to produce antibodies in vivo (e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0073] As used herein, the term “vector” includes nucleic acid vectors, e.g., DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins to prokaryotic or eukaryotic cells. Expression vectors in this disclosure include, in addition to polynucleotide sequences, additional sequence elements, for example, to express proteins and / or to integrate these polynucleotide sequences into the genome of mammalian cells. Certain vectors that may be used to express antibodies and antibody fragments in this disclosure include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) to direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that increase 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, intra-sequence ribosome entry sites (IRESs), and polyadenylation signaling sites, which direct the effective transcription of the genes supported in the expression vector. The expression vectors disclosed herein may also include polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics (e.g., ampicillin, chloramphenicol, kanamycin, or noseoslysin).

[0074] In this specification, the term “host cell” refers to a cell into which an exogenous nucleic acid has been introduced, and includes the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may contain mutations, although they may not be completely identical to the parent cells in terms of nucleic acid content. Mutant progeny having the same function or biological activity as those screened or selected in the originally transformed cells are included herein.

[0075] The percentage of identity between two sequences varies depending on the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap, as well as the identical positions shared by the sequences.

[0076] In this specification, "n is a real number between 1 and 16" means that n is any real number between 1 and 16, inclusive.

[0077] In this specification, JPEG2026514033000007.jpg9159 represents the connection point.

[0078] The exemplary methods for racemic or enantiomerically pure compounds described herein are from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, the absolute configuration of stereocenters is wedge and wedge-type dashed bonds. Represented by JPEG2026514033000008.jpg8159, the relative configuration of the stereocenters (e.g., cis or trans configuration of an alicyclic compound) is shown as solid black bonds and dashed dashed bonds. Represented by JPEG2026514033000009.jpg9159.

[0079] The term "stereoisomer" refers to isomers that arise from different spatial arrangements of atoms within a molecule, including cis-trans isomers, enantiomers, and diastereoisomers.

[0080] The compounds of this disclosure may have chiral atoms, such as carbon atoms, sulfur atoms, nitrogen atoms, and phosphorus atoms, or chiral double bonds, and therefore the compounds of this disclosure may exist in the form of specific geometric isomers or stereoisomers. These specific geometric isomers or stereoisomers include cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as enantiomer or diastereoisomer enriched mixtures. All of the above isomers and mixtures thereof are included within the definition of the compounds of this disclosure. Additional chiral carbon atoms, chiral sulfur atoms, chiral nitrogen atoms, or chiral phosphorus atoms may be present in substituents such as alkyl groups. All of these isomers and mixtures thereof involved in substituents are also included within the definition of the compounds of this disclosure. Compounds containing the asymmetric atoms of this disclosure can be separated in optically active pure form or racemic form. The optically active pure form can be obtained by separating a racemic mixture or by synthesis using chiral starting materials or chiral reagents.

[0081] The term "substituted" means that one or more hydrogen atoms on a particular atom are replaced by a substituent, provided that the valence of the atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo substituents do not occur on aromatic groups.

[0082] The terms “optional” or “optionally” mean that the events or situations described therein may, but do not necessarily, occur. The descriptions include cases where the events or situations occur and cases where they do not. For example, when ethyl is “optionally” substituted with a halogen, ethyl may be unsubstituted (CH2CH3), monosubstituted (e.g., CH2CH2F, CH2CH2Cl), polysubstituted (e.g., CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2), or completely substituted (e.g., CF2CF3, CF2CCl3, CCl2CCl3). It will be understood by those skilled in the art that no substitutions or substitution patterns are introduced for any group containing one or more substituents that are spatially impossible and / or cannot be synthesized.

[0083] C in this specification m ~C n This means that it has an integer number of carbon atoms within the range of m to n.

[0084] The term "alkyl" refers to CnH 2n+1 This refers to a hydrocarbon group having the general formula C1-C6 alkyl. Alkyl groups may be linear or branched. The term "C1-C6 alkyl" should be understood to represent a linear or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of alkyl groups include, but are 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, and 1,2-dimethylbutyl. The term "C1-C3 alkyl" refers to alkyl groups containing one to three carbon atoms, such as methyl, ethyl, n-propyl, or isopropyl.

[0085] The "C1-C6 alkyl" as described herein may further include "C1-C3 alkyl".

[0086] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic ring, fused ring, bridging ring, or spiro ring. The term "C3-C6 cycloalkyl" should be understood to represent a saturated monocyclic, fused, spiro, or bridging ring having 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0087] The term "heterocyclyl" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridging cyclic group, where the ring atoms of the group consist of 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatom-containing groups). Examples of "heteroatoms or heteroatomic groups," though not limited to them, include nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)²-, -S(=O)-, -P(=O)²-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, -NHC(=O)NH-, etc. The term "4- to 7-membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6, or 7 ring atoms, where 1 to 3 ring atoms are independently selected from the heteroatoms and heteroatomic groups described above. The term "5- to 6-membered heterocyclil" refers to a heterocyclil group having 5 or 6 ring atoms, where 1 to 3 ring atoms are independently selected from the heteroatoms and heteroatomic groups described above. Examples of 4-membered heterocyclils include, but are not limited to, azetidinyl or oxetanil; examples of 5-membered heterocyclils include, but are not limited to, tetrahydrofuranil, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinil, 4,5-dihydroxazolyl, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclils include, but are not limited to, tetrahydropyranil, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianil, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclils include, but are not limited to, diazepanil. The term "4-7 member heterocyclyl" may include ranges such as "4-7 member heterocycloalkyl," "5-6 member heterocyclyl," and "5-6 member heterocycloalkyl."

[0088] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0089] The term “treatment” refers to surgical treatment or therapeutic procedure aimed at preventing or slowing (reducing) the progression of undesirable physiological or pathological changes in the subject being treated, such as cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization of the condition (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the condition, and remission of the condition (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those already suffering from a disability or disease, those susceptible to developing a disability or disease, or those for whom prevention of disability or disease is intended. Where terms such as slowing, mitigation, reduction, relief, and remission are used, their meanings also include elimination, disappearance, and non-occurrence.

[0090] The term “effective dose” refers to the amount of a therapeutic agent, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, that is effective in preventing or mitigating the symptoms or progression of a disease. “Effective dose” also refers to the amount of a compound sufficient to alleviate symptoms, for example, to treat, cure, prevent, or reduce an associated medical disorder, or to increase the rate at which such disorder is treated, cured, prevented, or reduced. When an active ingredient is administered alone to an individual, the therapeutically effective dose simply refers to the amount of the ingredient. When a combination is used, the therapeutically effective dose refers to the combined amount of the active ingredients that produce the therapeutic effect, regardless of whether they are administered together, sequentially, or simultaneously.

[0091] The term “Subject” refers to an organism receiving treatment for a specific disease or disorder as described in this disclosure. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys), or non-primate mammals receiving treatment for a disease or disorder.

[0092] The amount of the compound in this disclosure that constitutes the "therapeutic dose" will vary depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal being treated, but a person skilled in the art can routinely determine it in accordance with their knowledge and this disclosure.

[0093] In this specification, the term “pharmaceutically acceptable” is used to describe compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of safe medical judgment, and that are commensurate with a reasonable benefit / risk ratio.

[0094] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed from a compound and an inorganic or organic acid, as well as salts formed from a compound and an inorganic or organic base.

[0095] The term "pharmaceutical composition" refers to a mixture comprising one or more compounds of the present disclosure or salts thereof and pharmaceutically acceptable excipients. Pharmaceutical compositions are intended to facilitate the administration of the compounds of the present disclosure to living organisms.

[0096] The term "pharmaceutically acceptable excipient" refers to an excipient that does not exhibit significant irritant effects on living 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 carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0097] The term "comprise" and its variations such as "comprises" or "comprising" can be understood in an open and non-exclusive sense, meaning "including but not limited to."

[0098] This disclosure also includes isotope-labeled compounds of this disclosure that are identical to the compounds described herein but in which one or more atoms are replaced with atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 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, 36 Cl, etc.

[0099] Certain isotope-labeled compounds of the present disclosure (e.g., 3 H or 14 Those labeled with C can be used for compound and / or substrate tissue distribution analysis. Tritiation (i.e., 3 H) and carbon-14 (i.e., 14 C) Isotopes are particularly preferred in terms of their ease of preparation and detectability. 15 O, 13 N, 11 C and 18 Positron-emitting isotopes such as fluorine may be used in positron emission tomography (PET) tests to determine substrate occupancy. The isotope-labeled compounds of this disclosure can generally be prepared by procedures similar to those disclosed in the following schemes and / or examples, using isotope-labeled reagents instead of non-isotope-labeled reagents.

[0100] The pharmaceutical compositions of this disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in a suitable unit dosage form. For example, the pharmaceutical compositions of this disclosure may be in the form of sterile aqueous solutions for injection for intramuscular or subcutaneous administration. When the pharmaceutical compositions of this disclosure are used, other vehicles or solvents, such as water, Ringer's solution, or isotonic sodium chloride solution, are acceptable.

[0101] In all methods of administering the compounds described herein, the daily dose administered is 0.001 mg / kg body weight to 600 mg / kg body weight, preferably 0.05 mg / kg body weight to 200 mg / kg body weight, more preferably 0.1 mg / kg body weight to 100 mg / kg body weight, and is administered as a single dose or in divided doses.

[0102] The compounds of this disclosure can be prepared by various synthesis methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthesis methods, and their equivalents well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of this disclosure.

[0103] The chemical reactions of the specific embodiments of this disclosure are carried out in a suitable solvent that must be compatible with the chemical changes and required reagents and materials described herein. To obtain the compounds of this disclosure, those skilled in the art may need to modify or select synthesis procedures or reaction processes based on existing embodiments.

[0104] An important consideration in synthetic route planning in the art is the selection of suitable protecting groups for reactive functional groups (e.g., amino and carboxyl in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed.), Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0105] Unless otherwise defined in this disclosure, scientific and technical terms used in connection with this application shall have the meanings generally understood by those skilled in the art. [Brief explanation of the drawing]

[0106] [Figure 1] The results of the X-ray single-crystal diffraction analysis of compound 19-P1 are shown. [Figure 2] This study demonstrates the inhibitory effect of ADCs on OVCAR3 cell proliferation. [Figure 3] This study demonstrates the inhibitory effect of ADCs on PA-1 cell proliferation. [Figure 4] This study demonstrates the inhibitory effect of ADCs on OVCAR3 cell proliferation. [Figure 5] This study demonstrates the inhibitory effect of ADCs on PA-1 cell proliferation. [Figure 6] The tumor growth curve for the OVCAR3 subcutaneous tumor model is shown. [Figure 7] The tumor growth curve for the OVCAR3 subcutaneous tumor model is shown. [Figure 8] The tumor growth curve for the PA-1 subcutaneous tumor model is shown. [Figure 9] The tumor growth curve for the PA-1 subcutaneous tumor model is shown. [Figure 10] The tumor growth curve for the 786-O subcutaneous tumor model is shown. [Modes for carrying out the invention]

[0107] This disclosure will be further described with reference to specific examples, and the advantages and features of this disclosure will become more apparent with the description. Experimental procedures where no conditions are specified in the examples will be carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or equipment other than those from specific manufacturers used herein are conventional products available on the market.

[0108] The examples provided are illustrative only and do not in any way limit the scope of this disclosure. Various modifications or substitutions in form and detail may be made to the technical solutions of this disclosure without departing from the spirit and scope of this disclosure, and it will be understood by those skilled in the art that such modifications and substitutions are within the scope of protection of this disclosure.

[0109] 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]indolidino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14) and isomers 14-P1 and 14-P2 [ka]

[0110] Step 1: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)etanone (intermediate 14-2) Intermediate 14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL), and nitric acid (35.43 g, 365.50 mmol, 65% purity) was slowly added thereto. The reaction mixture was stirred at 25°C for 2.5 hours. After the reaction was complete, saturated sodium bicarbonate solution was slowly added to the reaction mixture to adjust the pH to 7-8, and then dichloromethane (100 mL) was added thereto. The organic phase was washed with water (50 mL x 2), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The residue was purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 1:2) to obtain the title compound (5 g).

[0111] MS m / z(ESI):210.0[M+H]+.

[0112] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)etanone (intermediate 14-3) Intermediate 14-2 (2.37 g, 11.33 mmol) was dissolved in anhydrous ethanol (25 mL), and palladium carbon (0.2 g, 10% purity) was added thereto. The reaction mixture was stirred at 25°C for 16 hours under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered, the filter cake was washed twice with ethyl acetate, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (1.6 g).

[0113] MS m / z(ESI):180.1[M+H]+.

[0114] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl)acetamide (intermediate 14-4) 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 thereto. The reaction mixture was stirred at 25°C for 1.5 hours. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure to obtain the title compound (1.23 g).

[0115] MS m / z(ESI):222.1[M+H]+.

[0116] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl)acetamide (intermediate 14-5) Intermediate 14-4 (1.23 g, 5.00 mmol) was dissolved in acetic acid (12 mL), and a solution of hydrogen bromide in acetic acid (1.84 g, 7.51 mmol, 33% purity) was added thereto, 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 the reaction was complete, the reaction mixture was poured into ice water and stirred for 10 minutes. The mixture was then filtered. The filtered 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 resulting reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filtered cake was dried to obtain the title compound (500 mg).

[0117] MS m / z(ESI):300.0[M+H]+.

[0118] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl)-2-chloroethanone (intermediate 14-6) Intermediate 14-5 (0.2 g, 666.43 μmol) was dissolved in anhydrous ethanol (1 mL) and concentrated hydrochloric acid (1 mL), and the reaction mixture was stirred at 60°C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature, and ice water (10 mL) and saturated sodium bicarbonate (10 mL) were slowly and continuously added, followed by the addition of dichloromethane (50 mL). The organic phase was washed with water (20 mL x 2), and the washed organic phase was dried over an appropriate amount of anhydrous sodium sulfate. The residue was subjected to preparative thin-layer chromatography (petroleum ether:ethyl acetate = 6:1) to obtain the title compound (160 mg).

[0119] MS m / z(ESI):214.0[M+H]+.

[0120] 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]indolidino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-7) 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 thereto. The reaction mixture was stirred at 90°C for 16 hours. After the reaction was complete, 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 hours. The reaction mixture was filtered, the filter cake was washed with ethanol (2 mL x 2), dried, and the title compound was obtained (60 mg).

[0121] MS m / z(ESI):441.1[M+H]+.

[0122] 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]indolidino[1,2-b]quinoline-8,11(7H)-dione (intermediate 14-8) Intermediate 14-7 (55.00 mg, 124.76 μmol) was dissolved in ethanol (1 mL), and urotropin (52.47 mg, 374.29 μmol) was added thereto. The reaction mixture was stirred at 80°C for 1.5 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 25 mm diameter, 100 mm length; mixture of reduced polar water (containing 0.225% formic acid) and methanol used as eluent; methanol gradient: 0%~27%, elution time: 12 min) to obtain the title compound (10 mg).

[0123] MS m / z(ESI):422.1[M+H]+.

[0124] 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]indolidino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14) 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 O-(7-azabenzotriazol-1-yl)-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 mixture was stirred at 25°C for 1.5 hours. After the reaction was complete, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm, 30 mm diameter, 150 mm length; mixture of reduced polar water (containing 0.225% formic acid) and acetonitrile was used as the eluent; acetonitrile gradient: 4%~44%, elution time: 9 min) to obtain the title compound (3 mg).

[0125] MS m / z(ESI): 520.1[M+H]+.

[0126] 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).

[0127] [ka] Step 9: Preparation of 2-cyclopropyl-2-hydroxybenzyl acetate (intermediate 14-9-P1 / P2) Intermediate 14-9 was divided to prepare isomers 14-9-P1 and 14-9-P2. Intermediate 14-9 (1.3 g) was subjected to preparative supercritical fluid chromatography (DAICEL CHIRALPAK AD column, 10 μm silica, 30 mm diameter, 250 mm length, ethanol (containing 0.1% aqueous ammonia) as the eluent) to obtain intermediate 14-9-P1 (600 mg) and intermediate 14-9-P2 (600 mg).

[0128] The two isomers described above were further analyzed under the following chiral supercritical fluid chromatography conditions. [Table 1]

[0129] Intermediate 14-9-P1 Under the chiral supercritical fluid chromatography conditions described above, the retention time was 2.990 minutes.

[0130] 1 ¹H NMR (400MHz, methanol-d4): δ 7.43-7.29 (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).

[0131] Intermediate 14-9-P2 Under the chiral supercritical fluid chromatography conditions described above, the retention time was 2.661 minutes.

[0132] 1 ¹H NMR (400MHz, methanol-d4): δ 7.46-7.28 (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).

[0133] Step 10: Synthesis of 2-cyclopropyl-2-hydroxyacetic acid (intermediate 14-10-P1 / P2) Intermediate 14-9-P1 (500 mg) was added to methanol (15 mL) under a hydrogen atmosphere, and wet palladium carbon (10 mg, 10%) was added to the reaction mixture. The reaction mixture was stirred under a hydrogen atmosphere at 25°C for 16 hours. After the reaction was complete, the reaction product was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-10-P1 (273 mg).

[0134] 1 ¹H NMR (400MHz, methanol-d4): δ 3.63 (d, J=7.2Hz, 1H), 1.21-1.09 (m, 1H), 0.61-0.40 (m, 4H).

[0135] Intermediate 14-9-P2 (500 mg) was added to methanol (15 mL) under a hydrogen atmosphere, and wet palladium carbon (10 mg, 10%) was added to the reaction mixture. The reaction mixture was stirred under a hydrogen atmosphere at 25°C for 16 hours. After the reaction was complete, the reaction product was filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 14-9-P2 (279 mg).

[0136] 1 ¹H NMR (400MHz, methanol-d4): δ 3.63 (d, J=7.2Hz, 1H), 1.19-1.08 (m, 1H), 0.60-0.39 (m, 4H).

[0137] 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]indolidino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-P1 / P2) Intermediate 14-8 (40.00 mg) and intermediate 14-10-P1 (28.11 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (46.02 mg) and N,N-diisopropylethylamine (31.28 mg) were added thereto. The reaction mixture was stirred at 25°C for 1.5 hours. After the reaction was complete, the reaction mixture was purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length; a mixture of reduced polar water (containing 0.225% formic acid) and acetonitrile was used as the eluent (acetonitrile gradient: 16%~46%, elution time: 12 min)) to obtain compound 14-P1 (22.00 mg).

[0138] MS m / z (ESI): 520.1 [M+H] + .

[0139] 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).

[0140] 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 O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.05 mg) and N,N-diisopropylethylamine (6.13 mg) were added thereto. The reaction mixture was stirred at 25°C for 1.5 hours. After the reaction was complete, the reaction mixture was directly purified by preparative high-performance liquid chromatography (Boston Green ODS C18 column, 5 μm silica, 30 mm diameter, 150 mm length; a mixture of reduced polar water (containing 0.225% formic acid) and acetonitrile was used as the eluent (acetonitrile gradient: 16%~46%, elution time: 12 min)) to obtain compound 14-P2 (8.00 mg).

[0141] MS m / z (ESI): 520.1 [M+H] + .

[0142] 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).

[0143] The two isomers were further analyzed separately using the following chiral supercritical fluid chromatography method. [Table 2]

[0144] Compound 14-P1: Under the chiral supercritical fluid chromatography conditions described above, the retention time was 3.673 minutes.

[0145] Compound 14-P2: Under the chiral supercritical fluid chromatography conditions described above, the retention time was 3.735 minutes.

[0146] Examples 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]indolidino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-S) [ka]

[0147] Step 1: Synthesis of (S)-4-benzyl-3-(2-cyclopropylacetyl)oxazolidine-2-one (intermediate 3) 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 the mixture was 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 the addition was complete, the resulting mixture was stirred at room temperature for 5 hours. After the reaction was complete, dichloromethane (1500 mL) was added to the reaction mixture for dilution. The mixture was then sequentially washed twice with water (500 mL), once with 2N HCl (500 mL), once with saturated sodium bicarbonate solution (500 mL), and once with saturated brine solution (500 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain the title compound (302 g).

[0148] 1H 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).

[0149] Step 2: Synthesis of (S)-4-benzyl-3-((S)-2-cyclopropyl-2-hydroxyacetyl)oxazolidine-2-one (intermediate 5) Intermediate 3 (200 g) was weighed and dissolved in 2000 mL of anhydrous tetrahydrofuran, and the mixture was stirred under a nitrogen atmosphere at -78°C for 15 minutes. Subsequently, sodium bis(trimethylsilyl)amide (443.5 mL, 2 M 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 completely dissolved in 700 mL of tetrahydrofuran, and the resulting solution was 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, 600 mL of 2N HCl was added to the reaction mixture, and the resulting mixture was stirred at room temperature (20-25°C) for 10 hours. Next, the reaction mixture was concentrated under reduced pressure, and ethyl acetate (1000 mL) and water (200 mL) were added to the residue. The mixture was stirred for 20 minutes. The aqueous phase was separated and then extracted twice with ethyl acetate (500 mL x 2). The organic phase was combined and washed sequentially twice with 400 mL of saturated NaHCO3 solution, twice with 400 mL of saturated Na2S2O3 solution, and twice with saturated brine. The resulting organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1 / 30) to obtain the title compound (128.7 g).

[0150] 1H 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).

[0151] Step 3: Synthesis of (S)-4-benzyl-3-((S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetyl)oxazolidine-2-one (intermediate 6) Intermediate 5 (128.7 g) was weighed and dissolved in 1300 mL of dichloromethane. Imidazole (56.17 g) was added, and the mixture was stirred in an ice bath for 15 minutes. Then, TBSCl (107.3 g) was added to the reaction mixture in batches, and the resulting mixture was stirred at room temperature for 3 hours. 200 mL of 2N HCl was added to the reaction mixture, and the resulting mixture was stirred for 20 minutes, followed by liquid separation. The organic phase was sequentially washed twice with 200 mL of saturated NaHCO3 solution and twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 80:1) to obtain the title compound (160 g).

[0152] 1H 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).

[0153] Step 4: Synthesis of benzyl(S)-2-((tert-butyldimethylsilyl)oxy)-2-cyclopropylacetate (intermediate 7) Benzyl alcohol (62.18 g) was weighed and dissolved in 500 mL of tetrahydrofuran, and the mixture was stirred at -25°C. n-butyllithium (213.6 mL, 2.5 M in tetrahydrofuran) was weighed and slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred at -25°C for 1 hour. Intermediate 6 (160 g) was weighed and dissolved in 320 mL of tetrahydrofuran, and the resulting solution was slowly added dropwise to the reaction mixture at -25°C. After the addition was complete, the mixture was stirred at -15°C for 3 hours. Saturated NH4Cl solution (200 mL) was added to the reaction mixture to quench the reactants. The reaction mixture was then concentrated under reduced pressure, and 400 mL of methyl tert-butyl ether and water (150 mL) were added to the reaction mixture. The resulting mixture was stirred for 30 minutes, and then separated into liquids. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL x 2). The organic phases were combined, washed once with saturated brine (250 mL), dried on anhydrous sodium sulfate, and filtered. The filtrate was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 100:1) to obtain the title compound (125 g).

[0154] 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).

[0155] Step 5: Synthesis of benzyl(S)-2-cyclopropyl-2-hydroxyacetate (intermediate 8) Intermediate 7 (125 g) was weighed and dissolved in 1200 mL of tetrahydrofuran. Glacial acetic acid (35.1 g) was added, and the mixture was stirred at room temperature for 5 minutes. Next, tetrabutylammonium fluoride (TBAF, 585 mL, 1 M in tetrahydrofuran) was added to the reaction mixture, and the reaction mixture was reacted at 45°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran (600 mL), and 300 mL of water and 400 mL of methyl tert-butyl ether were added to the residue. The mixture was stirred for 20 minutes, and then separated into liquids. The aqueous phase was extracted twice with methyl tert-butyl ether (200 mL). The organic phases were combined and washed sequentially twice with 200 mL of saturated NaHCO3 solution and twice with saturated brine, dried on anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 60:1) to obtain the title compound (68.9 g).

[0156] 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).

[0157] Intermediate 8 was further analyzed under the following chiral supercritical fluid chromatography conditions. [Table 3]

[0158] Under the chiral supercritical fluid chromatography conditions described above, the retention time of intermediate 8 was 3.013 minutes, which was substantially consistent with the retention time of intermediate 14-9-P1 in Example 1-1 (2.990 minutes) under the same chromatographic analysis conditions. Intermediate 8 has the same structure as intermediate 14-9-P1, and both are the same compound.

[0159] Step 6: Synthesis of (S)-2-cyclopropyl-2-hydroxyacetic acid (intermediate 9) Intermediate 8 (5 g) was dissolved in methanol (80 mL), and wet palladium carbon (10% by mass, 0.7 g) was added to the reaction mixture. The resulting mixture was stirred at 25°C for 16 hours under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (2.4 g).

[0160] 1 ¹H NMR (400MHz, methanol-d4): δ=3.63 (d, J=7.3Hz, 1H), 1.20-1.09 (m, 1H), 0.61-0.39 (m, 4H).

[0161] 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]indolidino[1,2-b]quinoline-14-yl)methyl)-2-hydroxyacetamide (compound 14-S) Intermediate 14-8 (90 mg) and intermediate 9 (49.60 mg) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (121.81 mg) and N,N-diisopropylethylamine (82.81 mg) were added thereto. The reaction mixture was stirred at 25°C for 16 hours. After the reaction was complete, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 20%~50%, 12 min) to obtain the title compound (21 mg).

[0162] MS m / z (ESI): 520.0 [M+H] + .

[0163] 1 H NMR(400MHz,DMSO-d6)δ=8.62(t,J=6.1Hz,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).

[0164] Compound 14-S was further analyzed under the following chiral supercritical fluid chromatography conditions. [Table 4]

[0165] Under the chiral supercritical fluid chromatography conditions described above, the retention time of compound 14-S was 3.654 minutes, which was 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 has the same structure as compound 14-P1 prepared in Example 1-1, and that both are the same compound.

[0166] 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]indolidino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19) and isomers 19-P1 and 19-P2 [ka]

[0167] Step 1: Synthesis of 1-(benzo[d][1,3]dioxol-5-yl-2,2-d2)ethane-1-one (intermediate 19-2) Intermediate 19-1 (3 g) was dissolved in anhydrous DMF solution (25 mL), and deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added. After the addition was complete, the mixture was heated to 90°C and stirred for 16 hours. The reaction mixture was added to water (100 mL), and the resulting mixture was extracted with ethyl acetate (200 mL x 2). The organic phases were combined, washed with saturated brine (100 mL), dried on anhydrous sodium sulfate, and filtered. 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).

[0168] MS m / z (ESI): 167.1 [M+H] + .

[0169] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxol-5-yl-2,2-d2)ethane-1-one (intermediate 19-3) Intermediate 19-2 (2.4 g) was dissolved in acetic anhydride (10 mL), and concentrated nitric acid (32.50 g, 70% content) was added dropwise at 0°C. After the addition was complete, the mixture was stirred at 0°C for 10 minutes. Then, the mixture was warmed to room temperature and stirred for 1 hour. After the reaction was complete, the reaction mixture was added dropwise to ice water (200 mL). The resulting mixture was filtered, and the filter cake was dried to obtain the title compound (1.9 g).

[0170] MS m / z (ESI): 212.0 [M+H] + .

[0171] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0172] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (intermediate 19-4) 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 hour. After the reaction was complete, the mixture was filtered. 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 (1.5 g).

[0173] MS m / z (ESI): 224.1 [M+H] + .

[0174] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxol-5-yl-2,2-d2)acetamide (intermediate 19-5) 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 acetic anhydride (25 mL), followed by the dropwise addition of Br2 (1.07 g). After the addition was complete, the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was added to water (50 mL), and the mixture was 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).

[0175] MS m / z (ESI): 302.1 [M+H] + .

[0176] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxol-5-yl-2,2-d2)-2-chloroethane-1-one (intermediate 19-6) 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 hours. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by high-performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of reduced polar water (containing 0.05% NH4HCO3) and acetonitrile as the eluent (acetonitrile gradient: 40%~50%)) to obtain the title compound (577 mg).

[0177] MS m / z (ESI): 216.0 [M+H] + .

[0178] 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]indolidino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 19-7) 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 thereto. The reaction mixture was stirred at 100°C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and then concentrated to dryness under direct reduced pressure. Ethanol (5 mL) was added, and the reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was then filtered, and the filter cake was washed with ethanol (5 mL x 2) to obtain the title compound (100.0 mg).

[0179] MS m / z (ESI): 443.0 [M+H] + .

[0180] 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]indolidino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 19-8) Intermediate 19-7 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and urotropin (94.97 mg) was added thereto. The reaction mixture was stirred at 50°C for 6 hours. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by high-performance liquid chromatography (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).

[0181] MS m / z (ESI): 424.0 [M+H] + .

[0182] 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]indolidino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19) Intermediate 19-8 (7 mg) and intermediate 11-1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added thereto. The reaction mixture was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was filtered and purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; using a mixture of reduced polar water (containing 0.05% formic acid) and acetonitrile as the eluent (acetonitrile gradient: 20%~50%, elution time: 12 min)) to obtain the title compound (2.60 mg).

[0183] MS m / z (ESI): 522.1 [M+H] + .

[0184] 1 H 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).

[0185] 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]indolidino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19-P1 / P2) [ka]

[0186] Intermediate 19-8 (7 mg) and intermediate 14-10-P1 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added thereto. The reaction mixture was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; mixture of reduced polar water (containing 0.05% formic acid) and acetonitrile was used as the eluent; acetonitrile gradient: 15%~45%, elution time: 12 min) to obtain compound 19-P1 (3.30 mg).

[0187] MS m / z (ESI): 522.1 [M+H] + .

[0188] 1H NMR(400MHz,DMSO-d6)δ=8.62(t,J=6.0Hz,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).

[0189] Intermediate 19-8 (7 mg) and intermediate 14-10-P2 (5.76 mg) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.57 mg) and diisopropylethylamine (4.27 mg) were added thereto. The reaction mixture was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Waters Xbridge C18 column, 5 μm, 25 mm diameter, 100 mm length; a mixture of reduced polar water (containing 0.05% formic acid) and acetonitrile was used as the eluent (acetonitrile gradient: 15%~45%, elution time: 12 min)) to obtain compound 19-P2 (4.0 mg).

[0190] MS m / z (ESI): 522.1 [M+H] + .

[0191] 1H 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).

[0192] The two isomers were further analyzed separately using the following chiral supercritical fluid chromatography method. [Table 5]

[0193] Compound 19-P1: Under the chiral high-performance liquid chromatography conditions described above, the retention time was 2.877 minutes.

[0194] Compound 19-P2: Under the chiral high-performance liquid chromatography conditions described above, the retention time was 2.690 minutes.

[0195] Structure confirmation of compound 19-P1 (X-ray single crystal diffraction) Single crystal culture method: 10 mg of compound 19-P1 sample was weighed and placed in a 1.5 mL centrifuge tube, and 300 μL of pyridine was added. After complete dissolution by sonication, the tube was sealed with sealing film, three small holes were made in the film with a needle, and the mixture was slowly evaporated at 20-30°C for 48 hours to obtain needle-shaped crystals.

[0196] The obtained single-crystal samples were subjected to X-ray analysis. The test results are shown in Table 1 and Figure 1. [Table 6]

[0197] The chemical structure of compound 19-P1 was determined by the X-ray crystal diffraction experiment described above as follows: [ka]

[0198] 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]indolidino[1,2-b]quinoline-14-yl-2,2-d2)methyl)-2-hydroxyacetamide (compound 19-S) [ka]

[0199] Intermediate 19-8 (2.4 g) and intermediate 9 (1645.5 mg) were dissolved in anhydrous N,N-dimethylformamide (25 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (3.24 g) and N,N-diisopropylethylamine (1465.11 mg) were added thereto. The reaction mixture was stirred at 25°C for 3 hours. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. Ethyl acetate (100 mL) was added to the residue, and the mixture was stirred for 16 hours and filtered. Then methanol (50 mL) was added to the filter cake, and the mixture was stirred for 16 hours and filtered to obtain the title compound (1.6 g).

[0200] MS m / z (ESI): 522.1 [M+H] + .

[0201] 11H NMR (400 MHz, DMSO-d6) δ = 8.60 (t, J = 5.9 Hz, 1H), 7.84 (s, 1H), 7.50 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 5.49 (d, J = 5.1 Hz, 1H), 5.47 - 5.37 (m, 4H), 4.71 (d, J = 5.8 Hz, 2H), 3.54 (t, J = 5.6 Hz, 1H), 1.97 - 1.75 (m, 2H), 1.07 - 0.94 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.40 - 0.29 (m, 2H), 0.29 - 0.20 (m, 2H).

[0202] Compound 19-S was further analyzed by the following chiral supercritical fluid chromatography analysis method. [Table 7]

[0203] Under the above chiral supercritical fluid chromatography conditions, the retention time of Compound 19-S prepared in this example was 2.853 minutes, which was substantially consistent with the retention time (2.877 minutes) of Compound 19-P1 prepared in Example 2-1 under the same chromatography analysis conditions. Therefore, Compound 19-S and Compound 19-P1 were determined to have the same structure and be the same compound.

[0204] 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]quinoline-14-yl)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecane-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 [Chemical formula] JPEG2026514033000025.jpg104159

[0205] Step 1: Synthesis of intermediate L1-14-2 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). The mixture was heated to 85°C under a nitrogen atmosphere and reacted with stirring for 18 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (chromatographic column: ISCO®; 330 g SepaFlash® Silica Flash Column, mobile phase gradient: 0-75% ethyl acetate / petroleum ether, flow rate: 100 mL / min) to obtain the title compound (18 g).

[0206] 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).

[0207] MS m / z(ESI):391.1[M+Na] + .

[0208] Step 2: Synthesis of intermediate L1-14-3 Intermediate L1-14-2 (5 g), 2-cyclopropyl-2-hydroxybenzyl acetate (8.40 g), and pyridinium p-toluenesulfonate (PPTS, 341.09 mg) were dissolved in dichloromethane (150 mL). The reaction mixture was heated to 65°C under a nitrogen atmosphere and stirred for 48 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (chromatograph column: ISCO®; 120g Sepa Flash® Silica Flash Column, mobile phase gradient: 0-45% ethyl acetate / petroleum ether, flow rate: 80 mL / min). Subsequently, it was further purified by high-performance liquid chromatography (chromatograph 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).

[0209] MS m / z(ESI): 537.2[M+Na] + .

[0210] Step 3: Synthesis of intermediates L1-14-4-P1 and L1-14-4-P2 Intermediate L1-14-3 (1.4 g) was dissolved in a mixed solvent of methanol (15 mL) and water (15 mL), and wet palladium carbon (10% mass content, 0.15 g) was added. The reaction mixture was allowed to react under a nitrogen atmosphere at 25°C with stirring for 16 hours. After the reaction was complete, the reaction mixture was filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (chromatograph column: Boston Prime C18 150×30mm×5μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 24%~64%, 13 min). Subsequently, it was further purified by preparative supercritical fluid chromatography (chromatograph column: DAIEL CHIRALPAK IC column, 10μm silica, diameter 30mm, length 250mm, using isopropanol (containing 0.1% aqueous ammonia) as the eluent) to obtain intermediates L1-14-4-P1 (130 mg) and L1-14-4-P2 (130 mg).

[0211] The two isomers were further analyzed separately using the following chiral high-performance liquid chromatography method.

[0212] The conditions for chiral high-performance liquid chromatography were as follows: [Table 8]

[0213] Intermediate L1-14-4-P1: Under the chiral high-performance liquid chromatography conditions described above, the retention time was 4.471 minutes.

[0214] MS m / z(ESI):447.5[M+Na] + .

[0215] Intermediate L1-14-4-P2: Under the chiral high-performance liquid chromatography conditions described above, the retention time was 5.692 minutes.

[0216] MS m / z (ESI): 447.3 [M+Na] + 。

[0217] Step 4: Synthesis of intermediates L1-14-5-P1 and L1-14-5-P2 2-Chlorotrityl chloride resin (2-CTC-resin) (specification: about 1.19 mmol / g) (257 mg) was added to dichloromethane (3 mL), and then intermediate L1-14-4-P1 (130 mg) and diisopropylethylamine (59.37 mg) were added. The reaction mixture was reacted with shaking on a shaker at 25 °C for 16 hours under a nitrogen atmosphere. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated 3 times. After filtration, the filter cake was dried to obtain intermediate L1-14-5-P1 (330 mg).

[0218] Intermediate L1-14-5-P2 (350 mg) was prepared according to the above method using intermediate L1-14-4-P2 (130 mg) as the starting material.

[0219] Step 5: Synthesis of intermediates L1-14-6-P1 and L1-14-6-P2 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 on a shaker at 25 °C for 1 hour. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated 3 times. After filtration, the filter cake was dried to obtain L1-14-6-P1 (220 mg).

[0220] Intermediate L1-14-6-P2 (220 mg) was prepared according to the above method using intermediate L1-14-5-P2 (350 mg) as the starting material.

[0221] Step 6: Synthesis of intermediates L1-14-7-P1 and L1-14-7-P2 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), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (225.31 mg) and diisopropylethylamine (99.96 mg) were added to the reaction mixture. The reaction mixture was shaken on a shaker at 25°C for 1 hour. After the reaction was complete, the resin was sequentially washed with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated three times. After filtration, the filtration cake was dried to obtain L1-14-7-P1 (377 mg).

[0222] Intermediate L1-14-7-P2 (361 mg) was prepared using intermediate L1-14-6-P2 (220 mg) as the starting material, according to the method described above.

[0223] Step 7: Synthesis of intermediates L1-14-8-P1 and L1-14-8-P2 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 on a shaker at 25°C for 1 hour. After the reaction was complete, the resin was sequentially washed with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated three times. After filtration, the filter cake was dried to obtain L1-14-8-P1 (270 mg).

[0224] Intermediate L1-14-8-P2 (260 mg) was prepared using intermediate L1-14-7-P2 (361 mg) as the starting material, according to the method described above.

[0225] Step 8: Synthesis of intermediates L1-14-9-P1 and L1-14-9-P2 Intermediate L1-14-8-P1 (270 mg) was dissolved in N,N-dimethylformamide (5 mL), and N-((9H-fluoren-9-ylmethoxy)carbonyl)glycylglycine (209.58 mg), O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (224.29 mg), and diisopropylethylamine (99.51 mg) were added sequentially. The reaction mixture was shaken on a shaker at 25°C for 1 hour. After the reaction was complete, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated three times. After filtration, the filtration cake was dried to obtain intermediate L1-14-9-P1 (403 mg).

[0226] Intermediate L1-14-9-P2 (420 mg) was prepared using intermediate L1-14-8-P2 (260 mg) as the starting material, according to the method described above.

[0227] Step 9: Synthesis of intermediates L1-14-10-P1 and L1-14-10-P2 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 on a shaker at 25°C for 1 hour. After the reaction was complete, the resin was sequentially washed with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated three times. After filtration, the filter cake was dried to obtain L1-14-10-P1 (300 mg).

[0228] Intermediate L1-14-10-P2 (320 mg) was prepared using intermediate L1-14-9-P2 (420 mg) as the starting material, according to the method described above.

[0229] Step 10: Synthesis of intermediates L1-14-12-P1 and L1-14-12-P2 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 on a shaker at 25°C for 16 hours. After the reaction was complete, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL). This procedure was repeated three times. After filtration, the filter cake was dried to obtain L1-14-12-P1 (374 mg).

[0230] Intermediate L1-14-12-P2 (387 mg) was prepared according to the method described above, using intermediates L1-14-10-P2 (320 mg) and L1-14-11 (194.27 mg) as starting materials.

[0231] Step 11: Synthesis of intermediates L1-14-13-P1 and L1-14-13-P2 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 allowed to react at 25°C for 0.5 hours while shaking on a shaker. After the reaction was complete, the reaction mixture was filtered to remove the resin. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by high-performance liquid chromatography (chromatograph column: Boston Prime C18 150 × 30 mm × 5 μm, mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 15%~35%, 9 min) to obtain intermediate L1-14-13-P1 (74 mg).

[0232] 1H 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).

[0233] MS m / z(ESI):679.7[M+Na] + .

[0234] Intermediate L1-14-13-P2 (86 mg) was prepared using intermediate L1-14-12-P2 (387 mg) as the starting material, according to the method described above.

[0235] 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).

[0236] MS m / z(ESI):679.5[M+Na] + .

[0237] Step 12: Synthesis of compounds L1-14-P1 and L1-14-P2 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), and the reaction mixture was reacted under a nitrogen atmosphere at 25°C for 2 hours with stirring. After the reaction was complete, the reaction mixture was purified by high-performance liquid chromatography (chromatograph column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 26%~46%, 12 min) to obtain compound L1-14-P1 (12.3 mg).

[0238] 1 H 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),2.85-2.74(m,1 H),2.14-2.06(m,2H),1.94-1.80(m,2H),1.52-1.42(m,4H),1.27-1.1 3(m,2H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.41-0.28(m,4H).

[0239] MS m / z (ESI): 1060.3 [M+H] + .

[0240] Compound L1-14-P2 (11.4 mg) was prepared according to the method described above, using intermediates L1-14-12-P2 (31.17 mg) and 14-8 (20 mg) as starting materials.

[0241] 1 H 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)

[0242] MS m / z (ESI): 1060.3 [M+H] + .

[0243] The two isomers were further analyzed separately using the following chiral high-performance liquid chromatography method.

[0244] The conditions for chiral high-performance liquid chromatography were as follows: [Table 9]

[0245] Under the chiral high-performance liquid chromatography conditions described above, the retention time for compound L1-14-P1 was 3.735 minutes.

[0246] Under the conditions of the above chiral high performance liquid chromatography, the retention time of compound L1-14-P2 was 3.901 minutes.

[0247] 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-oxa-2,7,10,13,16-pentaazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Compound L1-19-P1)

Chemical Structure

[0248] 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), and the reaction mixture was reacted with stirring at 25 °C for 2 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 22% - 52%, 12 minutes) to obtain L1-19-P1 (6.0 mg).

[0249] MS m / z (ESI): 1062.3 [M+H] + .

[0250] 1H 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,2H),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).

[0251] L1-19-P1 was further analyzed using the following chiral high-performance liquid chromatography method.

[0252] The conditions for chiral high-performance liquid chromatography were as follows: [Table 10]

[0253] Under the chiral supercritical fluid chromatography conditions described above, the retention time for L1-19-P1 was 3.775 minutes.

[0254] 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]indolidino[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentazaoctadecane-18-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide (compound L1-19-S) [ka]

[0255] Step 1: Synthesis of L1-14-3-S Intermediate L1-14-2 (28 g) and intermediate 8 (23.5 g, prepared in Examples 1-2) were dissolved in dichloromethane (25 mL), and silver trifluoromethanesulfonate (139.50 mg) was added to the reaction mixture. The reaction mixture was carried out under a nitrogen atmosphere at 25°C for 108 hours with stirring. After the reaction was complete, the mixture was filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by flash silica gel column (mobile phase gradient: tetrahydrofuran / dichloromethanol: 0-7%, flow rate: 70 mL / min) to obtain the title compound (10.3 g).

[0256] MS m / z(ESI): 537.3[M+Na] + .

[0257] Step 2: Synthesis of L1-14-4-S Intermediate L1-14-3-S (10 g) was dissolved in tetrahydrofuran (100 mL), and wet palladium carbon (10% by mass, 1 g) was added. The reaction mixture was allowed to react under a nitrogen atmosphere at 0°C for 16 hours with stirring. After the reaction was complete, the mixture was filtered. The filtrate was concentrated to dryness under reduced pressure, and petroleum ether / ethyl acetate (10 mL / 0.5 mL) was added to the residue. The mixture was stirred for 2 hours and filtered to obtain the title compound (6 g).

[0258] MS m / z(ESI):447.2[M+Na] + .

[0259] L1-14-4-S was further analyzed by the following chiral high-performance liquid chromatography method.

[0260] The conditions for chiral high-performance liquid chromatography were as follows: [Table 11]

[0261] Under the chiral supercritical fluid chromatography conditions described above, the retention time of L1-14-4-S was 4.385 minutes, which was substantially 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 structure and are the same compound.

[0262] Step 3: Synthesis of L1-19-S Intermediate L1-14-13-S (600 mg, prepared using L1-14-4-S as a starting material, referring to the synthesis method for 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), and the reaction mixture was reacted under a nitrogen atmosphere at 25°C for 2 hours with stirring. After the reaction was complete, the reaction mixture was purified by high-performance liquid chromatography (column: Boston Green ODS 150×30mm×5μm; mobile phase: [A: water (0.05% formic acid), B: acetonitrile]; B%: 24%~54%, 12 min) to obtain the title compound (286 mg).

[0263] 1H 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).

[0264] MS m / z (ESI): 1062.5 [M+H] + .

[0265] L1-19-S was further analyzed by the following chiral high-performance liquid chromatography method.

[0266] The conditions for chiral high-performance liquid chromatography were as follows: [Table 12]

[0267] Under the chiral supercritical fluid chromatography conditions described above, the retention time of L1-19-S was 3.748 minutes, which was substantially 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, it was determined that L1-19-S and L1-19-P1 have the same structure and belong to the same compound.

[0268] Example 5: Construction and production of anti-human CDH6 antibody Table 2 below shows the heavy chain variable region sequences and light chain variable region sequences of anti-human CDH6 monoclonal antibodies (CDH6-Ab-4, CDH6-Ab-3, CDH6-Ab-2, and CDH6-Ab), and Table 3 below shows the CDR sequences of the antibodies defined according to the Kabat numbering scheme. The nucleic acid sequences encoding VH and VL of the antibodies were recombinant into the expression vector pTT5, which has a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 29) and heavy chain constant region / light chain constant region sequences, to obtain recombinant plasmids for VH-CH / VL-CL expression. The plasmids and transfection reagent PEI (Polysciences, catalog number 24765-1) were added to OPTI-MEM (Gibco, catalog number 11058021). The mixtures were thoroughly mixed and allowed to stand for 15 minutes. Expi293F cells (Thermofisher, catalog number A14527) were added, and the system was incubated on a shaker at 37°C, 5% CO2, and 120 rpm. On day 2 of transfection, OPM-293 ProFeed (Shanghai OPM Biosciences Co.,Ltd., catalog number F081918-001) and 6 g / L glucose (Sigma, catalog number G7528) were added. On day 6 of transfection, the cell supernatant was collected. The sample was purified with protein A (GE, catalog number 28985254), and the eluted sample was dialyzed against PBS (pH 7.4) to obtain an anti-human CDH6 monoclonal antibody. CDH6-Ab was used as the positive control human CDH6 antibody, and its sequence is derived from patent WO2018212136A1. [Table 13] JPEG2026514033000034.jpg118159

[0269] [Table 14]

[0270] Example 6: Method for preparing antibody-drug conjugates Conjugation: The antibody prepared in Example 5 was buffer-exchanged by dialysis into a solution containing 20 mM PB, 150 mM NaCl, and 1 mM EDTA (pH 6.5). Eight times the volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) was added to the antibody solution, and the mixture was incubated in a metal shaker at a constant temperature at 37°C for 2.5 hours to reduce the antibody. Fifteen times the volume of the linker payload compound (prepared in Examples 3 and 4) was dissolved in DMSO, and the resulting solution was added to the reaction system. The reaction mixture was incubated at 25°C for 6 hours for conjugation. The reaction product was desalted and buffer-exchanged with phosphate-buffered saline (PBS) through a G25 column to remove free unreacted small molecule toxins. The ADC product was analyzed for purity and DAR value using SEC and LC-MS.

[0271] SEC Purity Analysis: Test protein samples were analyzed using the SEC-HPLC method to characterize the uniformity of the molecular size of recombinant proteins and determine their purity. In this method, the HPLC instrument used 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 / v9:1), the detection temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein load was 50 μg, and the analysis time was 40 minutes.

[0272] Determination of DAR values: The DAR values ​​of ADC molecules were determined using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). First, test ADC molecules were treated with PNGase F (NEB#P0705L) to remove N-glycosylation modifications, then treated with dithiothreitol (DTT, Sigma#646563), incubated at 37°C for 1 hour to reduce to light and heavy chains, and then analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometer. Next, 2 μg of protein was injected into 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 minutes. The mass spectrometer used was a Thermo Q Exactive Plus. The main parameters of the mass spectrometry were a spray voltage of 3.8 kV, a capillary heating temperature of 300 °C, a sheath gas flow rate of 35 arb, and a precursor ion scan range of 800-3000. Finally, using the mass spectral data analysis software Biopharma Finder 4.1, deconvolution was performed through the Respect algorithm to obtain molecular weight information for the light and heavy chain mass spectral peaks. The DAR value of the test ADC sample was calculated by separately calculating the mass spectral response signal of each component. [Table 15] JPEG2026514033000037.jpg44159

[0273] Using the same method as described above, an isotype control (ISO), which is an anti-FITC-hIgG1 antibody, was prepared and conjugated with a linker payload compound to obtain an isotype control ADC. The DAR value and SEC results for the ADC are shown below. [Table 16]

[0274] Example 7. Assay on the inhibition of in vitro proliferation of tumor cells by ADC. Cells and materials: Human ovarian cancer cell line OVCAR3 (high CDH6 expression cell line) was purchased from ATCC (#HTB-161). Human ovarian teratoma cell line PA-1 (moderate CDH6 expression cell line) was purchased from Nanjing Cobioer Biosciences Co.,Ltd. (#CBP60800). Bovine serum, 1640 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 Cornin. Cell-Titer Glo reagent was purchased from Promega.

[0275] Cell culture: OVCAR3 cells were cultured at 37°C under 5% CO2 in 1640 medium containing 20% ​​fetal bovine serum + 2 μg / mL bovine insulin + 1% penicillin-streptomycin. PA-1 cells were cultured at 37°C under 5% CO2 in MEM medium containing 10% fetal bovine serum + 1% MEM NEAA + 1% sodium pyruvate + 1% penicillin-streptomycin. Cells in the logarithmic growth phase were available for the experiment.

[0276] Cell proliferation activity assay: The inhibitory activity of ADCs on the proliferation of both OVCAR3 and PA-1 cell lines was assayed using Cell-Titer Glo reagent. OVCAR3 cells (5000 cells per well) and PA-1 cells (800 cells per well) were seeded in a 96-well plate and cultured at 5% CO2 and 37°C for 24 hours. Each ADC molecule was diluted in the medium for the corresponding cells as described above to achieve an ADC concentration of 100 nM. Then, a 3-fold gradient dilution was performed in the medium to obtain a total of eight concentrations, and 10 μL of each prepared ADC solution was transferred to the 96-well plate to obtain final concentrations ranging from 0 to 10 nM. After adding the test ADC solution, the plate was incubated at 5% CO2 and 37°C. OVCAR3 and PA-1 cells were cultured for 5 days. Cell activity was measured after adding Cell-Titer Glo reagent.

[0277] Data analysis: Calculate inhibition % (inhibition rate) and fit to IC 50 The following was determined: Inhibition % = 1 - 100% × (Signal - Bottom) / (Top - Bottom). Signal refers to the signal value of the ADC sample group, Bottom refers to the signal value obtained when only an equal volume of medium is added without cells, and Top refers to the signal value obtained when only cells are added without the ADC sample.

[0278] Experimental results: Under the conditions of this experiment, all ADCs tested showed strong inhibitory activity against the proliferation of OVCAR3 and PA-1 cells, as detailed in Tables 5-6 and Figures 2-5. [Table 17] [Table 18]

[0279] Example 8: Evaluation of efficacy in an OVCAR3 subcutaneous tumor model 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). Matrigel was purchased from Corning (catalog number 356237).

[0280] Experimental method Animal Information: Balb / c nude mice (female, 5-6 weeks old, weighing approximately 14-20g) were purchased from Beijing Vital River Biotechnology Co., Ltd. The mice were housed in an SPF-level environment in independently ventilated cages. All animals were given free access to standard, certified, commercially available laboratory feed and drinking water.

[0281] Cell Culture: Human ovarian cancer OVCAR3 cell lines were cultured in vitro under RPMI-1640 conditions, supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, and 10 μg / mL bovine insulin, in an incubator at 37°C and 5% CO2. Following conventional procedures, cells were digested weekly with 0.25% trypsin-EDTA digestion solution and subcultured. Cells were harvested and counted when they reached 80%–90% confluence and met the density requirements.

[0282] Cell inoculation: 0.1 mL of OVCAR3 cell suspension (RPMI-1640: 1 × 10⁶ cells in Matrigel (v / v=1:1)) 7 A solution containing cells was subcutaneously inoculated into the axilla of each mouse. On day 26 after cell inoculation, the mice were randomly divided into groups and administered based on tumor volume. The day of group division was designated as day 0.

[0283] Tumor measurement and experimental indices: The tumor diameter was measured twice a week using calipers. The tumor volume was calculated using the following formula: V = 0.5 a × b 2 (where a and b represent the major and minor axes of the tumor, respectively). The body weight of the mice was measured twice a week.

[0284] The anti-tumor therapeutic effect of the test drug was evaluated by the tumor growth inhibition rate TGI (%). TGI (%) = [(1 - (mean tumor volume at the end of dosing in the treatment group - mean tumor volume at the start of dosing in the treatment group)) / (mean tumor volume at the end of treatment in the vehicle control group - mean tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0285] Experimental results: In the mouse subcutaneous xenograft tumor OVCAR3 model, both ADC-L1-14-P1-12 and ADC-L1-14-P1-5 showed a significant inhibitory effect on tumor growth (P < 0.0001) after a single intravenous administration at a dose of 3 mg / kg. The results are shown in Table 7 and Figure 6.

Table 19

[0286] In the mouse subcutaneous xenograft tumor OVCAR3 model, both ADC-L1-19-P1-12 and ADC-L1-19-P1-5 showed a significant inhibitory effect on tumor growth (P < 0.0001) after a single intravenous administration at a dose of 3 mg / kg. The results are shown in Table 8 and Figure 7.

Table 20

[0287] Example 9: Evaluation of efficacy in the PA-1 subcutaneous tumor model Experimental reagents: Human ovarian cancer PA-1 cells were purchased from Nanjing Cobioer Biosciences 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 (phosphate-buffered saline without calcium and magnesium ions) was purchased from Hyclone (catalog number SH30256.01). Matrigel was purchased from Corning (catalog number 356237).

[0288] Experimental method Animal Information: Balb / c nude mice (female, 5-6 weeks old, weighing approximately 14-20g) were purchased from Beijing Vital River Biotechnology Co., Ltd. The mice were housed in an SPF-level environment in independently ventilated cages. All animals were given free access to standard, certified, commercially available laboratory feed and drinking water.

[0289] Cell Culture: Human ovarian cancer PA-1 cell lines were cultured in vitro in a 37°C, 5% CO2 incubator under MEM conditions supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% NEAA, and 1 mM sodium pyruvate. Following conventional procedures, cells were digested weekly with Versene digestion solution and subcultured. When the cells reached 80%–90% confluence and met the required density, they were harvested and counted.

[0290] Cell inoculation: 0.1 mL of PA-1 cell suspension (MEM: Matrigel (v / v=1:1) containing 1 × 10⁶ cells) 7A solution containing cells was subcutaneously inoculated into the axilla of each mouse. On day 17 after cell inoculation, the mice were randomly divided into groups and administered based on tumor volume. The day of group division was designated as day 0.

[0291] Tumor measurement and experimental index: Tumor diameter was measured twice a week using calipers. Tumor volume was calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively). The body weight of the mice was measured twice a week.

[0292] The antitumor therapeutic effect of the study drug was evaluated by the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment in the treatment group - mean tumor volume at the start of treatment in the treatment group)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0293] Experimental results: In a mouse subcutaneous xenograft tumor PA-1 model, both ADC-L1-14-P1-12 and ADC-L1-14-P1-5 showed a significant inhibitory effect on tumor growth (P<0.0001) after a single intravenous administration at a dose of 3 mg / kg. The results are shown in Table 9 and Figure 8. [Table 21]

[0294] In a mouse subcutaneous xenograft tumor PA-1 model, both ADC-L1-19-P1-12 and ADC-L1-19-P1-5 showed a significant inhibitory effect on tumor growth (P<0.0001) after a single intravenous administration at doses of 1 mg / kg and 3 mg / kg, and the inhibitory effect was dose-dependent. The results are shown in Table 10 and Figure 9. [Table 22]

[0295] Example 10: Evaluation of efficacy in a 786-O subcutaneous tumor model Experimental reagents: Human renal cell carcinoma 786-O cells (a cell line with low CDH6 expression) 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). Matrigel was purchased from Corning (catalog number 356237).

[0296] Experimental method Animal Information: NOD-SCID mice (female, 8-9 weeks old, weighing approximately 18-25g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were housed in an SPF-level environment in independently ventilated cages. All animals were given free access to standard, certified, commercially available laboratory feed and drinking water.

[0297] Cell Culture: Human renal cell carcinoma 786-O cell line was cultured in vitro under RPMI-1640 conditions, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, in an incubator at 37°C and 5% CO2. Following conventional practices, cells were digested weekly with 0.25% trypsin-EDTA digestant and subcultured. Cells were harvested and counted when they reached 80%–90% confluence and met the density requirements.

[0298] Cell inoculation: 5 × 10 cells in 0.1 mL of 786-O cell suspension (RPMI-1640: Matrigel (v / v=1:1)). 6 A solution containing cells was subcutaneously inoculated into the axilla of each mouse. Thirteen days after cell inoculation, the mice were randomly divided into groups and administered based on tumor volume. The day of group division was designated as day 0.

[0299] Tumor measurement and experimental index: Tumor diameter was measured twice a week using calipers. Tumor volume was calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively). The body weight of the mice was measured twice a week.

[0300] The antitumor therapeutic effect of the study drug was evaluated by the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment in the treatment group - mean tumor volume at the start of treatment in the treatment group)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0301] Experimental results: In the mouse subcutaneous xenograft tumor 786-O model, both ADC-L1-19-P1-12 and ADC-L1-19-P1-5 showed a significant inhibitory effect on tumor growth (P<0.0001) after a single intravenous administration at a dose of 3 mg / kg. The results are shown in Table 11 and Figure 10. [Table 23]

[0302] Example 11: Assay on the plasma stability of ADCs ADC molecules (final concentration: 100 μg / mL) were incubated separately with human plasma (Oribiotech, PB021-C) and monkey plasma (Shinunoda Bio, SND-X0107) in a 37°C incubator. The first day of incubation was designated as day 0. Samples were then collected on days 7, 14, and 28 and assayed for free small molecules.

[0303] An internal standard working solution (300 μL, prepared in acetonitrile) was added to 20 μL of the sample, and the mixture was thoroughly mixed by vortexing for 5 minutes and then centrifuged for 5 minutes (14000 rpm). Next, 4 μL of the supernatant was injected into an LC-MS / MS analyzer (API6500+). The results are shown in Table 12. The results indicate that the tested ADC molecule was stable in both human plasma and monkey plasma. [Table 24]

Claims

1. An antibody-drug conjugate having the general structural formula Pc-(L-D)n, or a pharmaceutically acceptable salt thereof, During the ceremony, D is a cytotoxic drug, L is the linker unit, Pc is an antibody or its antigen-binding fragment that specifically binds to CDH6. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and / or the light chain variable region comprising LCDR1, LCDR2, and LCDR3, and the HCDR1-3 and / or the LCDR1-3 are defined as follows: (1) HCDR1-3 are sequence numbers 11-13, and / or LCDR1-3 are sequence numbers 14-16. (2) The HCDR1-3 are sequence numbers 17-19, and / or the LCDR1-3 are sequence numbers 20-22. (3) The HCDR1-3 are sequence numbers 23-25, and / or the LCDR1-3 are sequence numbers 26-28. or The HCDR1-3 and / or the LCDR1-3 have at least 80% identity with any one of the HCDR1-3 and LCDR1-3 in groups (1)-(3), or have sequences with up to three insertions, deletions, or substitutions, and 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 from 1 to 16, the antibody-drug conjugate, or a pharmaceutically acceptable salt thereof.

2. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region and / or the light chain variable region is selected from the following: (1) The heavy chain variable region is the sequence shown in Sequence ID No. 1, and / or the light chain variable region is the sequence shown in Sequence ID No.

2. (2) The heavy chain variable region is the sequence shown in Sequence ID No. 3, and / or the light chain variable region is the sequence shown in Sequence ID No.

4. (3) The heavy chain variable region is the sequence shown in Sequence ID No. 5, and / or the light chain variable region is the sequence shown in Sequence ID No.

6. or The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the heavy chain variable region and / or the light chain variable region has at least 80% identity with any one of the heavy chain variable regions and / or the light chain variable region of the above-described group (1) to (3), or has a sequence having up to three insertion, deletion or substitution mutations, preferably the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

3. 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 is selected from intact constant region sequences or fragments thereof, the constant region fragment comprising CH1, a hinge region, CH2, CH3, or Fc; optionally, the heavy chain constant region is selected from human and mouse IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is human and mouse kappa constant region An antibody-drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, comprising an antibody selected from a normal region and a lambda constant region; optionally, the antibody or antigen-binding fragment comprises an intact heavy chain and a light chain, wherein the heavy chain consists of the VH and the heavy chain constant region having the sequence shown in SEQ ID NO: 9, and the light chain consists of the VL and the light chain constant region having the sequence shown in SEQ ID NO:

10.

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

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

6. The cytotoxic drug D is selected from the compounds represented by formula (D-I), 【Chemistry 1】 During the ceremony, R １ and R ２ These atoms, together with the atoms to which they are bonded, form a 5-6 member heterocycle, where the 5-6 member heterocycle contains one or two oxygen atoms as ring atoms, and the 5-6 member heterocycle is optionally substituted with one or more D atoms. R ４ H and C １ ~C ３ Selected from alkyl groups, R ５ is selected from H, halogen, CN, OH, NH ２ , and C １ ~C ３ alkyl, R ６ H and C １ ~C ３ Selected from alkyl groups, R ７ H, C １ ~C ３ Alkyl and C ３ ~C ６ Selected from cycloalkyl, where C １ ~C ３ Alkyl or the C ３ ~C ６ Cycloalkyls can be any of D, halogen, CN, =O, OH, or NH. ２ , or C １ ~C ３ An antibody-drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, substituted with an alkyl group.

7. R １ and R ２ However, together with the atoms to which they are bonded, 【Chemistry 2】 An antibody-drug conjugate according to claim 6, or a pharmaceutically acceptable salt thereof, which forms a .

8. R ４ , R ５ , and R ６ However, all are selected from H, the antibody-drug conjugate according to claim 6 or 7 or a pharmaceutically acceptable salt thereof.

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

10. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 6 to 9, wherein the compound represented by formula (D-I) is selected from one of the following compounds. 【Transformation 3】

11. The linker unit L is 【Chemistry 4】 A is selected from the above, its terminal a is covalently bound to the antibody unit Pc, and its terminal b is covalently bound to the cytotoxic drug D, where m1 is an integer selected from 2 to 8, and L １ The peptide residue is selected from 1 to 8 amino acids, and the peptide residue is halogen, CN, =O, C １ ~C ６ Alkyl, OH, O(C) １ ~C ６ Alkyl), NH ２ NH(C １ ~C ６ Alkyl), N (C １ ~C ６ Alkyl) ２ , C ３ ~C ６ An antibody-drug conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, optionally further substituted with one or more substituents selected from cycloalkyl and 4- to 7-membered heterocyclyl groups.

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

13. The linker unit L is 【Transformation 5】 The antibody-drug conjugate according to any one of claims 1 to 12, wherein terminal a is covalently bonded to the antibody unit Pc and terminal b is covalently bonded to the drug unit D, or a pharmaceutically acceptable salt thereof.

14. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody-drug conjugate or the pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugates or pharmaceutically acceptable salt thereof: 【Transformation 6】

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

16. The use of an antibody-drug conjugate according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, in the manufacture of a drug for treating a tumor, wherein the tumor is a CDH6-expressing tumor, and optionally the tumor is selected from ovarian cancer, kidney cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and cholangiocarcinoma.

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

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