Camptothecin compound and use thereof

Abstract

Disclosed are a novel camptothecin compound, a linker-camptothecin compound, and a use thereof in a ligand-drug conjugate. Specifically, the involved camptothecin compound and an antibody-drug conjugate prepared therefrom can produce relatively good inhibitory activity on tumors in vivo and in vitro and exhibit significant toxicity and efficacy; in addition, a suitable metabolic rate also provides sufficient safety guarantee. These advantages are of great value for the development of antibody-drug conjugates in clinical treatment.

Description

Camptothecin compound and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a drug toxin, a toxin-linker conjugate, intermediates thereof, pharmaceutically acceptable salts thereof and the use thereof in the preparation of antibody drug conjugates, more specifically, the drug involved in the present application is camptothecin or a camptothecin compound. BACKGROUND

[0002] At present, cancer has become the second largest disease threatening human health. According to the investigation of the World Health Organization, there were 10 million cases of cancer deaths worldwide in 2020, and in 2022, the number of new cancer cases in China and the United States was about 4.82 million and 2.37 million, respectively, and the number of new deaths was about 3.21 million and 0.64 million, respectively. In the past few decades, chemotherapy based on cytotoxic drugs has become the main treatment for cancer, but most of the chemotherapy drugs show low therapeutic effect and serious side effects due to non-specific drug exposure to off-target tissues. In order to solve the above problems, scientists have developed a new type of cancer treatment method with higher targeting, i.e. monoclonal antibody (mAb). Monoclonal antibodies can change the mode of cancer treatment by precisely targeting tumor surface antigens, but compared with chemotherapy, the lethal effect of monoclonal antibodies on cancer cells is not ideal, and the use of monoclonal antibodies alone to treat cancer is far from enough. Antibody drug conjugates (ADC) combine the high specific targeting ability of monoclonal antibodies and the strong killing effect of cytotoxic drugs, and can achieve precise killing of cancer cells, and have become one of the focuses of anti-cancer drug research and development.

[0003] Antibody-Drug Conjugates (ADCs) are a new type of biopharmaceuticals coupled by monoclonal antibodies and potent cytotoxic payloads through bioactive linkers. The mechanism of action of ADCs is that the specific binding between the monoclonal antibody and the target antigen is realized, and then the ADCs are internalized into the target cancer cells. The cytotoxic payload exerts cytotoxicity to kill cancer cells after the ADCs are internalized into the cancer cells (Fu Z, Li S, Han S, et al. Antibody drug conjugate: the "biological missile" for targeted cancer therapy [J]. Signal Transduction and Targeted Therapy, (2022) 7:93.). Since the first ADC (Mylotarg, gemtuzumab ozogamicin) was launched in 2000, after decades of research and exploration, as of April 2025, only 16 ADC drugs (see Table 1 for basic information) have been approved for marketing and used for a small number of indications, which is difficult to meet the treatment needs of patients with various types of cancer. The main reason for the small number of ADC market products is that the development of ADC drugs is difficult, and the technical barriers are high. ADC drugs need to go through multiple steps to exert their effects after entering the human body, and each step has technical difficulties to overcome. From the perspective of toxins, the types of toxins that can be used to construct effective ADCs are very limited. The 16 marketed ADCs involve only 10 types of toxins, including calicheamicin, PE38 (a fragment of Pseudomonas aeruginosa exotoxin a), PBD (pyrrolobenzodiazepine), auristatin compounds (MMAE, MMAF), maytansinoid compounds (such as DM1, DM4), camptothecin compounds (such as DXD, SN38, KL6100023), etc. Table 1 Basic information of ADC drugs approved for marketing worldwide

[0004] Among them, the camptothecin compound is a group modification of one or more sites of the parent camptothecin (CPT), which is a topoisomerase I inhibitor and can be separated from the bark of the tree (a traditional Chinese medicine for treating cancer), and its specific molecular structure was first disclosed by Wall et al. in 1966 (Wall M. Plant antitumor agents I. The isolation and structure of camptothecin-A novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata [J]. J Am Chem Soc, 1966, 88.), as shown below.

[0005] Camptothecin (CPT) can bind to topoisomerase and DNA through hydrogen bonds, prevent DNA recombination and cause cell apoptosis by damaging DNA. However, due to the poor water solubility, low targeting, high toxicity to normal tissues, and instability in plasma, camptothecin has many limitations in application (Dancey J, Eisenhauer E A. Current perspectives on camptothecins in cancer treatment [J]. Br J Cancer, 1996; Proulx M E, Desormeaux A, Marquis J F, et al. Treatment of visceral leishmaniasis with sterically stabilized liposomes containing camptothecin [J]. Antimicrobial Agent and Chemotherapy, 2019.). Since the structure of camptothecin was first disclosed more than 50 years ago, although a large number of camptothecin compounds have been disclosed later, and some camptothecin compounds have been used as toxins to construct antibody drug conjugates. However, up to now, the number of antibody drug conjugates containing camptothecin compounds with significant drug properties is very small. As can be seen from Table 1, among the 16 currently marketed antibody drug conjugates, only four use camptothecin compounds, namely DXD in Enhertu (trastuzumab deruxtecan) and Datroway (datopotamab deruxtecan), SN38 in Trodelvy (Sacituzumab Govitecan), and KL6100023 (structure shown below) in Sacituzumab Tirumotecan. Because each load has different killing effects on different types of tumor diseases, there is a high difference, and the combination with different linkers also brings differences in treatment effect, so there is still a great demand and need for improvement for the development of new camptothecin loads.

[0006] From the perspective of drug composition and technical characteristics, ADC has undergone four iterations in the development process, namely: first-generation ADC drugs, second-generation ADC drugs, third-generation ADC drugs, and fourth-generation ADC drugs. Among them, ① the first-generation ADC drugs (Mylotarg, Besponsa) are mainly composed of conventional chemotherapeutic drugs connected with mouse antibodies through non-cleavable linkers. The efficacy of such ADC drugs is not superior to that of free cytotoxic drugs, and there are often problems such as immunogenicity, uncontrollable release of toxic load, antibody aggregation, metabolic instability, narrow safety window, and high challenges in drug development. ② The second-generation ADC drugs (Adcetris, Kadcyla) optimize monoclonal antibodies, cytotoxic payloads, and linkers. Compared with IgG4, IgG1 isotype monoclonal antibodies are more suitable for biological coupling with small molecule payloads and have higher cancer cell targeting ability. Another breakthrough is the use of more effective cytotoxic drugs, which improves their water solubility and coupling level. In addition, the second-generation ADC improves the linkers to achieve better plasma stability and drug uniformity. Through the improvement of the three components of antibodies, linkers, and toxins, the second-generation ADC has good clinical effects and safety, but still has shortcomings such as insufficient therapeutic window due to off-target toxicity, aggregation or rapid clearance of high DAR value ADC, etc. ③ The third-generation ADC drugs (such as Polivy, Padcev) introduce site-specific coupling technology to produce ADC with good characterization of DAR value (2 or 4) and expected cytotoxicity. Such ADCs exhibit lower off-target toxicity, lower immunogenicity, and higher pharmacokinetic efficiency, but still have problems such as insufficient effective payload release and high drug resistance. ④ The fourth-generation ADC drugs (Enhertu, Trodelvy) adopt a high DAR value (4-8) and a low-to-moderate activity cytotoxic payload strategy, and the payload has a high bystander effect on tumors with low and high expression of surface antigens. Among the currently marketed fourth-generation ADC drugs, Enhertu and Trodelvy both use camptothecin compounds as cytotoxic payloads, while the linker-toxin of Enhertu falls off in the body circulation, causing cytotoxicity. The payload used by Tropelvy is less active, and its stability in the body circulation is poor.

[0007] In summary, after decades of development, although antibody drug conjugates containing camptothecin compounds have significantly improved in terms of therapeutic effect, there are still many problems such as poor stability, insufficient efficacy, high toxicity, poor tolerance, and inability to meet the diverse needs of cancer treatment, which need further development of new camptothecin compounds to provide more and more cancer treatment-specific payload options for the development of antibody drug conjugates.

[0008] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss Prot accession numbers, are hereby incorporated by reference in their entirety, i.e., each individual reference is specifically and individually incorporated by reference herein. SUMMARY

[0009] The present application provides a new type of camptothecin compound, in particular to the camptothecin compound and its antibody drug conjugate can produce better inhibitory activity on tumor in vitro and in vivo, which shows significant toxicity and efficacy, and also shows excellent stability in the body circulation. The above advantages have great value for the development of antibody drug conjugate in clinical treatment.

[0010] In a specific aspect, the present application provides a compound represented by formula (I), 1) R 1 selected from hydrogen, methoxy, methyl, halogen, amino and R 2 selected from hydrogen, methyl, methoxy, amino, hydroxyl, hydroxyl alkyl amido, halogen; or R 1 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 2 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 3 selected from hydrogen, C1-C6 alkyl, R 4 selected from hydrogen, and R 5 is null; or R 3 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 4 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 5 is not null; 3) the selected from single bond, double bond or null; wherein, the R 6 , R 7 are each independently selected from hydrogen, C1-C6 alkyl, or R 6 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 7 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 6 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 7 and the carbon atom connected thereto forms a dioxo five-membered ring; and 2) R 8 selected from hydrogen, C2-C6 alkenyl or C1-C6 alkylene; the R 9 selected from C1-C6 alkyl, C2-C6 alkenyl, the R10 Selected from C1-C6 alkoxy groups, The R 11 Selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, methylene, The R 12 Selected from C1-C6 alkoxy, hydroxy, and C1-C6 alkylhydroxy groups; (2) One or more hydrogen atoms in the nitrogen heterocycle are optionally substituted with hydroxyl groups, C1-C6 hydroxyalkyl groups, or F; the six-membered carbon ring contains 0, 1, or more double bonds; the R 5 When not empty, it is selected from the following structures: hydrogen, carbonyl, fluorine, methylene, The R 5 When the hydrogen or fluorine is used, any one or more hydrogen atoms (including but not limited to 2, 3, or 4 hydrogen atoms) in the six-membered carbon ring are optionally replaced by fluorine atoms; The R 13 Selected from hydroxyl, The R 14 Selected from hydrogen or C1-C6 alkyl; The R 15 Selected from -O- or -NH-; the R 16 Selected from hydroxyl, amino, or C1-C6 aminoalkyl; The R 17 Selected from -O- or -NH-; the R 18 Selected from C1-C6 hydroxyalkyl The R 19 Selected from The R 20 Selected from The R 21 Selected from hydrogen and C1-C6 alkyl groups; The R 22 Selected from C1-C6 hydroxyalkyl or The numbers n1, n2, n2, n3, n4, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14, n15, n16, n17, n18, n19, n20, n21, n22, n23, n24, n25, and n26 are each independently selected from 0, 1, 2, 3, 4, 5, or 6.

[0011] In some embodiments, the compound has the following structure: Among them, 1)R 1 Selected from fluorine and R 2 Selected from methyl, hydroxy, amino, methoxy; or R 1 With R 2 The carbon atom bonded to it forms a dioxo five-membered ring; 2) R 5’Selected from hydrogen, fluorine, or empty; 3) R 23 Selected from hydrogen or fluorine, when R 23 When it is hydrogen, the R 5 Selected from the following structures: fluorine, When R 23 When it is fluorine, the R 5 It is hydrogen; 3) It can be a single or double bond; preferably a single bond; 4) It can be a single bond, a double bond, or empty; 5) as described It is a single key or empty; when the stated When it is a double bond, the empty and R 5’ Empty; when the stated When it is a single bond, the For single bonds and R 5’ Selected from hydrogen or fluorine.

[0012] In some embodiments, the compound has the following structure: Among them, 1)R 1 Selected from fluorine, hydrogen; and 2)R 2 For amino; and 3)R 12 Selected from hydroxyl, methoxy, and hydroxymethyl.

[0013] In some embodiments, the compound has the following structure: Among them, 1) the R 1 Selected from fluorine, hydrogen, methyl, amino, or methoxy; and 2) the R 2 Selected from hydrogen, amino, fluorine, methoxy, or methyl; or R 1 With R 2 The carbon atom attached to it forms a dioxopentanone ring; and R as described in 3) 6 Selected from hydrogen, isopropyl or and 4) R 7 Selected from Or R 6 With R 7 The nitrogen atom bonded to it forms the following structure: and 4) R 8 Selected from hydrogen, Where n2 is selected from 0, 1, 2, and 3.

[0014] In some embodiments, the compound has the following structure: Among them, 1) the R 1 R 2 Each is independently selected from hydrogen, amino, methyl, methoxy, or fluorine; or R1 with the carbon atom to which it is attached forming a dioxolane ring; and 2) said R 2 with the carbon atom to which it is attached forming a dioxolane ring; and 2) said R 9 is selected from the following structures:

[0015] In some embodiments, the compound is of the following structure: wherein, 1) said R 1 , R 2 are each independently selected from hydrogen, amino, methyl, methoxy, fluorine; or R 1 with the carbon atom to which it is attached forming a dioxolane ring; and 2) said R 2 with the carbon atom to which it is attached forming a dioxolane ring; and 2) said R 10 is selected from methoxy, and 3) said R 11 is selected from hydrogen,

[0016] In some embodiments, the compound is of the following structure: wherein, 1) said R 1 is selected from fluorine, chlorine; and 2) said R 2 is selected from chlorine, methyl, and 3) said R 3 is selected from hydrogen, ethyl, and 4) said R 4 is selected from hydrogen,

[0017] In some embodiments, the compound is selected from the following structures:

[0018] In some embodiments, at least one H in the compound is an isotope of H; the isotope of H is further preferably deuterium or tritium; in some embodiments, the compound is of the following structure:

[0019] In another aspect, the present application provides a compound, which is a stereoisomer of any one of the compounds in any of the embodiments described above, or a pharmaceutically acceptable salt or solvate thereof.

[0020] In another aspect, the present application provides a bis-molecular compound formed by linking any two identical or different compounds according to any of the embodiments described above.

[0021] In some embodiments, the two identical or different compounds are linked by a short chain segment, the longest chain of which contains 1-10 atoms and the short chain contains one or more of the following groups: heteroalkyl, heterocycloalkyl, aryl, heteroaryl, or combinations thereof; in some preferred embodiments, the short chain contains an oxaalkyl group.

[0022] In some specific embodiments, the bis-molecular compound is selected from the following structures:

[0023] In another aspect, the present application provides a linker-payload compound, which has a structure according to Formula (II-A): M-V1-V2-V3-Q (II-A), wherein, 1) the M is a linker; the V1 is a linking unit; the V2 is a polypeptide unit consisting of amino acids; the V3 is a structural segment linking V2 and Q; and 2) the Q is a therapeutic agent.

[0024] In some embodiments, the M- is selected from the following structures: wherein the X1 is selected from hydrogen, Cl, Br, or I.

[0025] In some embodiments, the V1 is selected from the structure consisting of one or more of the following: wherein the a1, a2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0026] In some embodiments, the M-V1- is selected from the following structures: wherein, m1 is selected from 1, 2, 3, 4, 5, 6; m2 is selected from 1, 2, 3, 4, 5, 6; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8; m4 is selected from 1, 2, 3, 4, 5, 6; m5 is selected from 1, 2, 3, 4, 5, 6; m6 is selected from 1, 2, 3, 4, 5, 6; m7 is selected from 1, 2, 3, 4, 5, 6; m8 is selected from 1, 2, 3, 4, 5, 6; m9 is selected from 1, 2, 3, 4, 5, 6.

[0027] In some embodiments, the M-V1- is selected from:

[0028] In some embodiments, said -V2- is selected from the group consisting of one or more of the amino acids phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, said -V2- is selected from the following structures: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; in some preferred embodiments, said -V2- is selected from -Gly-Gly-Phe-Gly-, -Val-Ala-, or -Val-Cit-.

[0029] In some embodiments, said -V3- is null or selected from the following structures:

[0030] In some embodiments, said linker-payload compound (II-A) is selected from:

[0031] In some embodiments, said therapeutic agent Q is selected from a cytotoxic molecule, an immune potentiator, and a radioisotope.

[0032] In some embodiments, said cytotoxic molecule is selected from a tubulin inhibitor or a DNA damaging agent.

[0033] In some embodiments, the tubulin inhibitor is selected from the group consisting of dolastatins, auristatin-based cytotoxic molecules, or maytansine-based cytotoxic molecules; the DNA damaging agent is selected from the group consisting of calicheamicins, duocarmycins, antrmycin-based derivatives PBDs, camptothecins, and camptothecin-based derivatives; in some preferred embodiments, the camptothecin-based derivative is selected from the group consisting of the compounds described in any of the above embodiments.

[0034] In another aspect, the present application provides a linker-payload compound, characterized in that the payload is selected from the group consisting of the compounds described in any of the above embodiments.

[0035] In some embodiments, the linker-payload compound has a structure as described in formula (II-B): W-A1-A2-A3-D (II-B); wherein, 1) the W is a linker; the -A1- is a connecting unit; the -A2- is a polypeptide unit consisting of amino acids; the -A3- is a spacer unit; and 2) the D is selected from the group consisting of the compounds described in any of the above embodiments after removal of hydrogen.

[0036] In some embodiments, the W- is selected from the group consisting of the following structures: wherein the X1is selected from hydrogen, Cl, Br, or I.

[0037] In some embodiments, the -A1- is selected from the group consisting of one or more of the following structures: wherein the a1, a2are independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0038] In some embodiments, the W-A1- is selected from the group consisting of the following structures: wherein, q1is selected from 1, 2, 3, 4, 5, 6; q2is selected from 1, 2, 3, 4, 5, 6; q3is selected from 1, 2, 3, 4, 5, 6, 7, 8; q4is selected from 1, 2, 3, 4, 5, 6; q5is selected from 1, 2, 3, 4, 5, 6; q6is selected from 1, 2, 3, 4, 5, 6; q7is selected from 1, 2, 3, 4, 5, 6; q8is selected from 1, 2, 3, 4, 5, 6; q9is selected from 1, 2, 3, 4, 5, 6.

[0039] In some embodiments, the W-A1- is selected from the group consisting of the following structures:

[0040] In some embodiments, said -A2- is selected from the group consisting of an amino acid in phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit) and a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0041] In some embodiments, said -A2- is selected from the group consisting of an amino acid in phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit) and a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0042] In some embodiments, said -A2- is selected from the group consisting of -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly- or -Ala-Ala-Ala-; in some preferred embodiments, said -A2- is selected from -Gly-Gly-Phe-Gly-, -Val-Ala- or -Val-Cit-.

[0043] In some embodiments, said -A3- is null or selected from the group consisting of:

[0044] In some embodiments, said W-A1-A2-A3- structure is as follows:

[0045] In some embodiments, the linker-load compound is selected from the following structures:

[0046] In another aspect, the present application provides a ligand-drug conjugate comprising a targeting unit, characterized in that the linker-load compound used in the ligand-drug conjugate is selected from the linker-load compounds described in any of the above embodiments.

[0047] In another aspect, the present application provides a ligand-drug conjugate comprising a targeting unit, characterized in that the linker-load compound used in the ligand-drug conjugate is selected from the linker-load compounds described in any of the above embodiments.

[0048] In some embodiments, the ligand-drug conjugate is obtained by reacting the targeting unit with the linker-load compound described in any of the above embodiments; in some embodiments, the reaction comprises a chemical coupling reaction, an enzymatic coupling reaction, a sugar chain remodeling, a glycosyl coupling reaction or a site-directed coupling reaction; in some preferred embodiments, the chemical coupling reaction comprises a chemical coupling reaction based on lysine in the targeting moiety and the linker, a chemical coupling reaction based on cysteine and the linker, or a chemical coupling reaction based on a non-natural amino acid and the linker.

[0049] In some embodiments, the ligand is selected from an antibody, an antigen binding fragment or a targeting peptide.

[0050] In some embodiments, the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody.

[0051] In some embodiments, the antibody heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0052] In some embodiments, the antibody is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody; in some embodiments, the antibody is a monoclonal antibody.

[0053] In some embodiments, the antibody is selected from a humanized monoclonal antibody or a fully human monoclonal antibody.

[0054] In some embodiments, the antigen binding fragment is selected from a scFv, a Fab, a Fab', a (Fab')2, a Fv fragment, a disulfide-linked Fv (dsFv).

[0055] In some embodiments, the ligand has at least one cysteine.

[0056] In some embodiments, the ligand-drug conjugate is selected from the following structures: wherein, A represents the ligand; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, representing the number of linker-load units connected on the ligand is 1, 2, 3, 4, 5, 6, 7, or 8.

[0057] In some embodiments, there are 1 to 8 loads connected in each ligand-drug conjugate.

[0058] In another aspect, the present application provides a ligand-drug conjugate comprising a linker structure as shown in (IV), characterized in that the linker structure is: -M-V1-V2-V3- (IV), wherein, -M- is a linker; -V1- is a connecting unit; -V2- is a polypeptide unit consisting of amino acids; -V3- is a connecting fragment; 1) -M- is selected from the following structures: 2) -V1- is selected from the following structures consisting of one or more of the following: wherein, a1, a2 are independently selected from 1, 2, 3, 4, 5, or 6; and 3) -V2- is a peptide consisting of one or more of phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); and 4) -V3- is null or selected from the following structures:

[0059] In some embodiments, -M-V1- is selected from the following structures: wherein, m1 is selected from 1, 2, 3, 4, 5, 6; m2 is selected from 1, 2, 3, 4, 5, 6; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8; m4 is selected from 1, 2, 3, 4, 5, 6; m5 is selected from 1, 2, 3, 4, 5, 6; m6 is selected from 1, 2, 3, 4, 5, 6; m7 is selected from 1, 2, 3, 4, 5, 6; m8 is selected from 1, 2, 3, 4, 5, 6; m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8.

[0060] In some embodiments, -M-V1- is selected from the following structures:

[0061] In some embodiments, said -V2- is selected from the group consisting of: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe- Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly- -Ala-Ala-Ala-.

[0062] In some embodiments, said -V2- is selected from the group consisting of: -Gly-Gly-Phe-Gly-, -Val-Ala- or -Val-Cit-.

[0063] In some embodiments, said -V3- is null or selected from the group consisting of:

[0064] In some embodiments, said formula (IV) is selected from the group consisting of: wherein, 1) said is linked to an antibody; and 2) said is linked to a payload.

[0065] In some embodiments, said ligand is selected from the group consisting of an antibody, an antigen binding fragment or a targeting peptide; in some preferred embodiments, said antibody is a targeting CDCP1 antibody.

[0066] In another aspect, the present application provides use of a compound or a linker-payload compound as described in any of the technical solutions above in the preparation of a ligand-drug conjugate; in some embodiments, said ligand is selected from the group consisting of an antibody, an antigen binding fragment or a targeting peptide; in some preferred embodiments, said antibody is a targeting CDCP1 antibody.

[0067] In another aspect, the present application provides a pharmaceutical composition, characterized in that said pharmaceutical composition comprises a ligand-drug conjugate as described in any of the embodiments above.

[0068] In some embodiments, the average number of linkages per 1 ligand of the payload is in the range of 1-8; in some preferred embodiments, the average number of linkages per 1 ligand of the payload is selected from the group consisting of 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, or 3-5.

[0069] In another aspect, the present application provides a use of the ligand-drug conjugate and the pharmaceutical composition of any of the above embodiments in the treatment of cancer; in some embodiments, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer; in some preferred embodiments, the lung cancer is selected from the group consisting of non-small cell lung cancer and lung squamous carcinoma.

[0070] In another aspect, the present application provides a method for preparing the ligand-drug conjugate of any of the above embodiments, comprising: coupling the ligand with the linker-payload compound of any of the above embodiments in a suitable solution and under suitable conditions.

[0071] In another aspect, the present application provides a method for preparing the ligand-drug conjugate of any of the above embodiments, comprising: 1) coupling the ligand with the linker M-V1-V2-V3 or W-A1-A2-A3 in the linker-payload compound of any of the above embodiments in a suitable solution and under suitable conditions to form a ligand-linker structure combination; 2) reacting the ligand-linker structure combination with the compound of any of the above embodiments in a suitable solution and under suitable conditions to obtain the ligand-drug conjugate.

[0072] In another aspect, the present application provides a method for preparing the ligand-drug conjugate of any of the above embodiments, comprising: 1) coupling the ligand with a part of the linker M-V1-V2-V3 or W-A1-A2-A3 in the linker-payload compound of any of the above embodiments in a suitable solution and under suitable conditions to form a partial linker structure combination; 2) reacting the part of M-V1-V2-V3 or W-A1-A2-A3 with the compound of any of the above embodiments in a suitable solution and under suitable conditions to form a payload-partial linker structure combination; 3) reacting the ligand-partial linker structure combination with the payload-partial linker structure combination in a suitable solution and under suitable conditions to obtain the ligand-drug conjugate.

[0073] In another aspect, the present application provides a linker-payload compound, which is a stereoisomer of any of the linker-payload compounds described in the above technical solutions, or a pharmaceutically acceptable salt or solvate thereof.

[0074]

DEFINITIONS

[0075] Unless otherwise indicated, the terms "a," "an," and "the" are inclusive of both singular and plural referents. denotes the point of attachment of a structural fragment to the rest of the molecule.

[0076] Unless otherwise indicated, the terms "may be substituted," "may contain," "contains," and other variations thereof herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0077] Unless otherwise indicated, the term "short chain" in the present invention refers to a structure that can link different or same compounds in the present invention as a bimolecular compound, such as heteroalkyl, heterocycloalkyl, aryl, heteroaryl, or any combination thereof.

[0078] Unless otherwise indicated, the term "substituted" in the present invention means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0079] Unless otherwise indicated, the term "alkyl" by itself or as part of another term in the present invention refers to a substituted or unsubstituted straight or branched chain, saturated or unsaturated hydrocarbon (e.g., -C1-C6alkyl refers to an alkyl group having from 1 to 6 carbon atoms), representative straight chain "-C1-C6alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl; while branched -C3-C6alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated -C2-C8alkyl groups include, but are not limited to, -ethenyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-iso-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl, the above alkyl groups can be unsubstituted, or can be substituted with one or more groups.

[0080] The term "alkenyl" refers to straight chain or branched chain hydrocarbon groups containing at least one carbon-carbon double bond, including, for example, "C 2-6 alkenyl," "C 2-4"Alkenyl" and the like. Examples include, but are not limited to, ethenyl, 1- propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4- hexadienyl, and the like.

[0081] Unless otherwise indicated, the term "heteroalkyl" by itself or in combination with another term, means a stable straight or branched chain, or combination thereof, having from 1 to 6, preferably 1 to 3, heteroatoms selected from O, N, P, or S, and wherein the nitrogen and sulfur atoms can optionally be oxidized and the nitrogen heteroatom can optionally be quaternized. The heteroatom O, N, S can be placed at any interior position of the heteroalkyl group or at the position of attachment thereof to the rest of the molecule. Typically, a Ci to C6heteroalkyl group has 1 to 6 carbon atoms and 1 to 3 heteroatoms.

[0082] Unless otherwise indicated, the term "cycloalkyl" by itself or as part of another term means a 3-, 4-, 5-, or 6-membered monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic, monocyclic or bicyclic carbon ring ring derived by removal of one hydrogen atom from a ring atom of a parent ring system. Representative C3-C6cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0083] Unless otherwise indicated, the term "aryl" by itself or as part of another term means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical derived by removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system, having the stated number of carbon atoms, typically 5-20 carbon atoms. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like, an exemplary aryl group is a phenyl group.

[0084] The term "heteroaryl" by itself or as part of another term means an aromatic C3-C8heterocycle, where the subscript indicates the total number of ring members of the heterocyclic ring system or the total number of aromatic carbons of the aromatic ring system of the heteroaryl group and does not imply the size of the ring system or the presence or absence of ring fusions, representative examples of aromatic C3-C8heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl. When explicitly given, the ring system size of a heterocycle or heteroaryl is indicated by the total number of atoms in the ring, typical heteroaryls include, but are not limited to, 5- or 6-membered heteroaryl indicates the total number of aromatic atoms in the heteroaromatic ring system of the heteroaryl (i.e., 5 or 6), but does not represent the number of aromatic heteroatoms or aromatic carbons in the ring system.

[0085] The term "heterocycloalkyl" by itself or as part of another term means a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having the number of carbon atoms (also referred to as ring members) stated and one to four ring members that are heteroatoms independently selected from N, O, P, or S and obtained by removing one hydrogen atom from a ring atom of the parent ring system, unless otherwise indicated. A heterocycle in which all ring atoms are involved in the structure of an aromatic compound is referred to as a heteroaryl, otherwise it is referred to as a heterocarbocycle.

[0086] The term "heterocycloalkyl" by itself or as part of another term means a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having the number of carbon atoms (also referred to as ring members) stated and one to four ring members that are heteroatoms independently selected from N, O, P, or S and obtained by removing one hydrogen atom from a ring atom of the parent ring system, unless otherwise indicated. A heterocycle in which all ring atoms are involved in the structure of an aromatic compound is referred to as a heteroaryl, otherwise it is referred to as a heterocarbocycle.

[0087] The term "hydroxyalkyl" means an alkyl group, where alkyl is as defined above, substituted with one or more (e.g., 1, 2, 3, or 4) hydroxyl groups, where the hydroxyl structure is: -OH. For example, the term "C 1-6 The term "hydroxyalkyl" means an alkyl group, where alkyl is as defined above, substituted with one or more (e.g., 1, 2, 3, or 4) hydroxyl groups, where the hydroxyl structure is: -OH. For example, the term "C

[0088] The term "aminoalkyl" means an alkyl group, where alkyl is as defined above, substituted with one or more (e.g., 1, 2, 3, or 4) amino groups, where amino includes, but is not limited to, a primary amine, a secondary amine, a tertiary amine, or an ammonium radical. For example, the term "C 1-6 The term "aminoalkyl" means an alkyl group, where alkyl is as defined above, substituted with one or more (e.g., 1, 2, 3, or 4) amino groups, where amino includes, but is not limited to, a primary amine, a secondary amine, a tertiary amine, or an ammonium radical. For example, the term "C

[0089] The term "carbonylalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) carbonyl groups, where alkyl is defined above and the carbonyl structure is: -(C=0)-. For example, the term "C 1-6 carbonylalkyl" refers to an alkyl group having 1 to 6 carbon atoms, substituted with one or more (e.g., 1, 2, 3, or 4) carbonyl groups.

[0090] The term "hydroxyalkylamido" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) hydroxyl and / or amido groups, where alkyl is defined above, the hydroxyl structure is: -OH, and the amido structure is: -(C=0)-amino-, which includes, but is not limited to, primary amines, secondary amines, tertiary amines, or ammonium radicals. Unless otherwise indicated, the term "fluoroalkyl" in the present application refers to an alkyl group substituted with one or more (such as 1, 2, or 3) fluorine atoms, where alkyl is defined above. For example, the term "C 1-6 fluoroalkyl" refers to a fluoroalkyl group having 1 to 6 carbon atoms. Common haloalkyl groups include, but are not limited to, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, and the like.

[0091] Unless otherwise indicated, the term "halogen" in the present application includes F, Cl, Br, or I.

[0092] Unless otherwise indicated, the term "alkoxy" in the present application refers to a group having the structure "alkyl-O-," where alkyl is defined above.

[0093] Unless otherwise indicated, the term "independently selected from" in the present application means that each substituent is selected independently of the other. Thus, each substituent can be the same as or different from the other substituent(s).

[0094] Unless otherwise indicated, the solid line "-" and solid wedge or dashed wedge Carbon-carbon bonds of the compounds of the present application are depicted. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate all possible stereoisomers (e.g., a particular enantiomer, a racemic mixture, etc.) including at that carbon atom. The use of a solid or dashed wedge to depict a bond to an asymmetric carbon atom is intended to indicate that the depicted stereoisomer is present. When present in a racemic mixture, the solid and dashed wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds of the present application are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational stereo-isomers, atropisomers, and mixtures thereof. The compounds of the present application can exhibit more than one type of isomerism, and consist of mixtures (e.g., racemic mixtures and diastereomeric pairs) thereof.

[0095] Unless otherwise indicated, all possible crystalline forms or polymorphs of the compounds encompassed by the present application are intended, which can be a single polymorph or a mixture of more than one polymorph in any ratio.

[0096] Unless otherwise indicated, the term "aryl" by itself or as part of another term in the present application refers to a substituted or unsubstituted, monovalent carbocyclic aromatic hydrocarbon radical obtained by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system, having the stated number of carbon atoms, typically 5 to 20 carbon atoms. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like, with an exemplary aryl group being a phenyl group.

[0097] Unless otherwise indicated, the term "amino acid" in the present application includes a natural amino acid, a molecule containing an N capable of forming an amide bond and a carboxylic acid, a molecule of the general formula NH2-CHR-COOH, or a residue within a peptide bearing the parent amino acid, wherein "R" is one of many different side chains. "R" can be a substituent found in a natural amino acid. "R" can also refer to a substituent that is not a natural amino acid.

[0098] Unless otherwise indicated, the term "stereoisomer" in the present application refers to a compound having the same chemical composition and connectivity but whose atoms or atom groups have different positioning in space, including but not limited to geometric isomer, cis-trans isomer, optical isomer, conformational stereo-isomer, rotamer, enantiomer, or diastereomer.

[0099] Unless otherwise indicated, the term "pharmaceutically acceptable salt" in the present application refers to a pharmaceutically acceptable organic or inorganic salt of a compound (linker-drug or ligand-drug conjugate thereof). Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, tartrate, oleate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate salts. Pharmaceutically acceptable salts can involve the incorporation of another molecule such as an acetate ion, a succinate ion, or other counter ion into the crystal lattice of the compound. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. In the case where multiple charged atoms are part of the pharmaceutically acceptable salt, multiple counter ions can be present, thus a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0100] Unless otherwise indicated, the term "ligand-drug conjugate" in the present application refers to a substance resulting from the attachment of a biologically active molecule (drug molecule) to a targeting unit, in some embodiments of the present application, the biologically active molecule is attached to the targeting unit via a linker, which is capable of being cleaved under a particular environment (e.g., hydrolytic enzymes and / or pH environment within a tumor) and a particular action (e.g., action of lysosomal proteases), thereby separating the biologically active molecule from the targeting unit. In some embodiments of the present application, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present application, the biologically active molecule is directly attached to the targeting unit via a covalent bond, which is capable of being cleaved under a particular environment or action, thereby separating the biologically active molecule from the targeting unit.

[0101] Unless otherwise indicated, the term "targeting unit" in the present application refers to a structure that binds or associates with a biological moiety or fragment thereof, including but not limited to an antibody fragment or functional fragment, a surrogate, a variant, a protein ligand, a protein scaffold, an RNA, a DNA, an RNA or DNA fragment, a small molecule ligand, or an antigen binding fragment.

[0102] The term "monoclonal antibody" in the present application includes, unless otherwise indicated, but is not limited to, a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric human-mouse (or other species) monoclonal antibody. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be produced by any of a variety of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

[0103] The term "antibody" in the present application, unless otherwise indicated, can be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen), or other antibody that binds to a tumor cell or stroma, "functionally active" meaning that the fragment, derivative, or analog is capable of immunospecifically binding to a target cell; can be a mono-specific antibody, a bi-specific antibody, or a multi-specific antibody; other useful antibodies include, but are not limited to, fragments of antibodies such as, without limitation, F(ab')2 fragments, Fab fragments, Fvs, single-chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as an antibody; antibodies include modified analogs and derivatives, i.e., analogs and derivatives modified by covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, and without limitation, derivatives and analogs of antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be introduced into an antibody by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Antibodies immunospecific for a cancer cell antigen are commercially available or produced by any method known to one of skill in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or similar database, from a literature publication, or by routine cloning and sequencing.

[0104] The term "antigen-binding fragment" in the present application, unless otherwise indicated, refers primarily to a functional fragment of an antibody that is capable of specifically binding to an antigen, such as a scFv (single-chain antibody), Fv, Fab, F(ab')2, Fab', scFv-Fc, a single-domain antibody, an antibody-like binding protein, a bi-specific antibody, a multi-specific antibody, or any antibody fragment modified by chemical modification.

[0105] Unless otherwise indicated, the term "therapeutic agent" in the present application means any substance or entity capable of exerting a therapeutic effect (e.g., treating, preventing, alleviating, or inhibiting any disease / disorder), including but not limited to: chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, thermal therapeutic agents, cytotoxic molecules, immunopotentiators, and radioisotopes, etc.

[0106] Unless otherwise indicated, the term "chemical coupling reaction" in the present application means the reaction that occurs between an amino acid residue on the surface of an antibody and a reactive functional group on a linker.

[0107] Unless otherwise indicated, the term "linker" in the present application has two reactive ends, one end for binding or associating with a biological moiety or fragment thereof, such as a targeting unit, and the other end for coupling, for example, a payload.

[0108] Unless otherwise indicated, "cancer" in the present application refers to or describes the physiological condition or disorder that is typically characterized by uncontrolled growth of cells in a mammal, and "tumor" comprises one or more cancerous cells.

[0109] Unless otherwise indicated, "treatment" in the present application means to alleviate or eliminate the disease or disorder in question. A subject is said to have been successfully "treated" if, after receiving a therapeutic amount of a compound of the present application or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present application, at least one indicia and symptom of the subject exhibits observable and / or detectable alleviation and / or improvement. It is understood that treatment includes not only complete treatment, but also treatment that fails to achieve complete treatment, but achieves some biologically or medically relevant result.

[0110] The structure of DX8951 used in the present application is as follows:

[0111] Unless otherwise indicated, the present application also includes compounds with various radioactive or non-radioactive isotopes of atoms. The atoms constituting the compounds of the present application can also contain atoms of isotopes in a non-natural proportion or be replaced with atomic isotopes. As atomic isotopes, there can be mentioned, for example, deuterium ( 2 H), tritium ( 3 H), or carbon-14 ( 14 C), etc. BRIEF DESCRIPTION OF DRAWINGS

[0112] Figure 1: Tumor volume change chart in the pharmacodynamic study of human lung squamous carcinoma cell EBC-1 subcutaneous transplantation tumor model;

[0113] Figure 2: Tumor volume change chart in the pharmacodynamic study of human lung squamous carcinoma cell EBC-1 subcutaneous transplantation tumor model;

[0114] Figure 3: Tumor volume change chart in the pharmacodynamic study of human pancreatic cancer cell BxPC-3 subcutaneous tumor model;

[0115] Figure 4: Tumor volume change chart in the pharmacodynamic study of human colon cancer cell RKO subcutaneous tumor model. DETAILED DESCRIPTION

[0116] Synthesis of the structure of the intermediate in Example 1

[0117] Synthesis of compound V-1 in Example 1-1

[0118] Preparation of compound V-1b: N-benzyloxycarbonyl-L-phenylalanine (150 g, 0.50 mol), N-hydroxysuccinimide (69.21 g, 0.60 mol) were dissolved with 750 mL of tetrahydrofuran, the reaction solution was cooled to 0°C, DCC (124.08 g, 0.60 mol) was weighed, and the DCC THF solution was ultrasonically dissolved with 310 mL of tetrahydrofuran, and the temperature was controlled at 0-5°C. The DCC THF solution was added dropwise. The reaction was carried out at room temperature for 4 h. The system was cooled to 5°C, filtered, the filter cake was washed with a small amount of tetrahydrofuran, the filtrate was replaced with EtOH twice, and the slurry was left overnight. Filtration gave 185.42 g of V-1b with a yield of 93%. LC-MS (ESI+) 397.27 [M+H] + .

[0119] Preparation of compound V-1a: Compound V-1b (185.42 g, 0.47 mol) was dissolved with 1390 mL of tetrahydrofuran, and bisglycine (92.70 g, 0.70 mol), sodium bicarbonate (47.16 g, 0.56 mol) were weighed, and the bisglycine sodium bicarbonate aqueous solution was ultrasonically dissolved with 705 mL of water, and the bisglycine sodium bicarbonate aqueous solution was added dropwise at room temperature. The reaction was carried out at room temperature for 2 h. The reaction solution was adjusted to PH≤2, extracted with DCM three times, washed with 1N HCl once, and dried. Filtration, washing with DCM, and rotary evaporation of the filtrate to a foaming solid were carried out. The system of EA:Hep.=2:1 was added to the slurry overnight. Filtration, washing with EA, Hep. three times, respectively, and drying of the filter cake were carried out. The target compound V-1a was obtained in a yield of 162.02 g, 93.78%. LC-MS (ESI+) 414.34 [M+H] + .

[0120] Preparation of compound V-1: Compound V-1a (20.00 g, 48.38 mmol) was dissolved with 240 mL of tetrahydrofuran, and lead tetraacetate (27.05 g, 128.68 mmol) was added at room temperature. The reaction was carried out at room temperature overnight. Filtration, washing of the filter cake with a small amount of tetrahydrofuran, rotary evaporation of the mother liquor, and column chromatography were carried out. The target compound V-1 was obtained in a yield of 14.55 g, 70.36%. LC-MS (ESI+) 450.34 [M+Na]+ .

[0121] Synthesis of Compound V-2 in Examples 1-2

[0122] Preparation of compound V-2b: Benzyloxycarbonyl-L-valine-L-alanine (160 g, 500 mmol) and N-hydroxysuccinimide (85.71 g, 750 mol) were dissolved in 2000 mL of dichloromethane. The solution was cooled to 0 °C and maintained at 0-10 °C. 2,4,6-Collidine (180.64 g, 1490 mmol) was added, and the solution was stirred for 1 h. Then, a solution of trifluoroacetic anhydride (156.54 g, 750 mmol) in 300 mL of dichloromethane was added, and the solution was stirred for 3 h. The mixture was filtered to obtain 199 g of the target compound V-2b, with a yield of 95.0%. LC-MS (ESI+) 420.4 [M+H] + .

[0123] Preparation of compound V-2a: Compound V-2b (199 g, 470 mmol) was dissolved in 1600 mL of tetrahydrofuran, and a 5% sodium bicarbonate aqueous solution (1000 mL) containing glycine (42.74 g, 570 mmol) was added, followed by 400 mL of ethylene glycol dimethyl ether. The reaction was carried out at 40 °C for 5 h. 350 mL of 2N hydrochloric acid was added to the reaction solution, followed by extraction with 2 L of dichloromethane. The organic phase was collected and evaporated to dryness to obtain 150.0 g of the target compound V-2a, with a yield of 84.0%. LC-MS (ESI+) 380.3 [M+H] + .

[0124] Preparation of compound V-2: Compound V-2a (20.0 g, 52.71 mmol) was dissolved in 800 mL of tetrahydrofuran. 5 mL of glacial acetic acid was added, followed by the rapid addition of lead tetraacetate (35.0 g, 118.13 mmol). The mixture was stirred at room temperature for 3 h. The reaction solution was centrifuged, and the supernatant was concentrated. 600 mL of dichloromethane was added, and the mixture was washed twice with 100 mL of 1N hydrochloric acid each time. The solution was then washed once with 100 mL of saturated brine. The solution was dried over anhydrous sodium sulfate for 1 h, filtered, and the filtrate was concentrated to give 14.3 g of a white solid, compound V-2, in 68.9% yield. LC-MS (ESI+) 395.3 [M+H] + .

[0125] Synthesis of Compound V-3 in Examples 1-3

[0126] Preparation of compound V-3b: Compound V-2 (7.0 g, 17.8 mmol) was dissolved in 200 mL of dichloromethane, and benzyl glycolate (29.57 g, 178 mmol), PPTS (0.45 g, 0.18 mmol) were added, and stirred at reflux overnight. The reaction solution was concentrated to obtain a yellowish oil, which was the target compound V-3b 7.1 g, with a yield of 80.2%. LC-MS (ESI+) 500.3 [M+H] + .

[0127] Preparation of compound V-3a: Compound V-3b (7.1 g, 14.2 mmol) was dissolved in 150 mL of methanol, and 10% palladium-carbon (2.5 g, 0.35 w) was added, and hydrogen was introduced, and stirred at room temperature for 3 h. The reaction solution was added with 70 mL of water, filtered, and the filtrate was concentrated to obtain white solid compound V-3a 3.7 g, with a yield of 95.3%. LC-MS (ESI+) 276.1 [M+H] + .

[0128] Preparation of compound V-3: N3-PEG2-NHS (3.0 g, 10 mmol) was dissolved in 50 mL of tetrahydrofuran, and V-3a (2.75 g, 10 mmol) was added, and triethylamine (2.02 g, 20 mmol) was added. Stirring was performed at room temperature for 2 h. The system was rotary evaporated, and the crude product was purified by preparation to obtain the target compound V-3 4.2 g, with a yield of 91%. LC-MS (ESI+) 461.23 [M+H] + .

[0129] Synthesis of compound W-1 in Example 1-4

[0130] Maleimide hexanoic acid (2112.1 mg, 10 mmol), N-hydroxysuccinimide (1.38 g, 12 mmol) were dissolved in 50 mL of tetrahydrofuran, and the reaction solution was cooled to 0°C, and DCC (2.48 g, 12 mmol) was weighed, and the THF solution of DCC was ultrasonically dissolved in 40 mL of tetrahydrofuran, and the THF solution of DCC was added dropwise at a temperature of 0-5°C. The reaction was performed at room temperature for 4 h. The system was cooled to 5°C, filtered, and the filter cake was washed with a small amount of tetrahydrofuran, and the filtrate was replaced with EtOH twice, and the slurry was left overnight, and filtered to obtain 2.86 g of W-1, with a yield of 93%. LC-MS (ESI+) 331.3 [M+Na] + .

[0131] Synthesis of compound W-3 in Example 1-5

[0132] Preparation of compound W-2: Compound W-1 (616.3 mg, 2.00 mmol), bisglycine (264.2 mg, 2.00 mmol) were dissolved in 10 mL of dichloromethane, TEA (415.9 μl, 3.00 mmol) was added, and stirring was performed at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, and the solid crude product was purified by preparative liquid chromatography to obtain 572.2 mg of W-2 at a yield of 88%. LC-MS (ESI+) 326.1 [M+H] + .

[0133] Preparation of compound W-3: Compound W-2 (572.2 mg, 1.75 mmol) was dissolved in 10 mL of tetrahydrofuran, and N-hydroxysuccinimide (402.2 mg, 3.50 mmol) and DCC (722.1 mg, 3.50 mmol) were added at room temperature. The reaction was performed overnight at room temperature. The system was cooled to 5°C, filtered, the filter cake was washed with a small amount of tetrahydrofuran, the mother liquor was evaporated, and ethanol was slurried to obtain the crude product of the target compound W-3. LC-MS (ESI+) 423.2 [M+H] + .

[0134] Synthesis of compound W-4 of Example 1-6

[0135] Preparation of compound W-4b: 1-(4-carboxyphenyl)-5-(methoxytetrazolyl)-disodium (530.2 mg, 2.0 mmol) was dissolved in 10 mL of dichloromethane, and iodomethane (312.2 mg, 2.2 mmol) and triethylamine (506.1 mg, 5.0 mmol) were added, and stirring was performed at room temperature overnight. Purification by preparative liquid chromatography and lyophilization yielded 413.5 mg of the target compound W-4b at a yield of 87.5%. LC-MS (ESI+) 237.3 [M+H] + .

[0136] Preparation of compound W-4a: W-4b (413.5 mg, 0.88 mmol), propargylglycine (55.1 mg, 1.0 mmol), HATU (380.2 mg, 1.0 mmol), and triethylamine (202.1 mg, 2.0 mmol) were dissolved in 10 mL of N,N-dimethylformamide, and stirring was performed at room temperature for 3 h. Purification by preparative liquid chromatography and lyophilization yielded 202.7 mg of the target compound W-4a at a yield of 84.3%. LC-MS (ESI+) 274.3 [M+H] + .

[0137] Preparation of compound W-4: W-4a (202.7 mg, 0.74 mmol), m-CPBA (638.6 mg, 3.7 mmol) were dissolved in 10 mL of tetrahydrofuran, stirred at 70 °C for 3 h, the reaction solution was reduced to room temperature, diluted with 5 mL of water, prepared liquid phase purification, freeze-drying to obtain the target compound W-4 149.6 mg, yield 66.2%. LC-MS (ESI+) 306.3 [M+H] + .

[0138] Synthesis of compound W-5 in Example 1-7

[0139] Preparation of compound W-5: Compound W-4 (100.5 mg, 0.32 mmol) was placed in a 25 mL three-necked bottle and dissolved in 8 mL of acetone, N3-PEG2-COOH (74 mg, 0.36 mmol), copper sulfate pentahydrate (62 mg, 0.48 mmol) and vitamin C sodium (96 mg, 0.48 mmol) were added, stirred at room temperature for 1 h. The reaction was quenched with acetonitrile (8.0 mL). The system was purified by preparative liquid chromatography to obtain the target compound W-5 131.3 mg, yield 80.6%. LC-MS (ESI+) 509.10 [M+H] + .

[0140] Synthesis of compound W-6 in Example 1-8

[0141] Preparation of compound W-6: 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1.0 mmol), propargyl glycine (55.1 mg, 1.0 mmol), HATU (380.2 mg, 1.0 mmol) and triethylamine (202.1 mg, 2.0 mmol) were dissolved in 10 mL of N,N-dimethylformamide, stirred at room temperature for 3 h, prepared liquid phase purification, freeze-drying to obtain the target compound W-6 250.7 mg, yield 82.0%. LC-MS (ESI+) 306.08 [M+H] + .

[0142] Synthesis of compound W-7 in Example 1-9

[0143] Preparation of compound W-7: Compound W-6 (100 mg, 0.32 mmol) was dissolved in 8 mL of acetone in a 25 mL three-necked flask, and N3-PEG2-COOH (72 mg, 0.36 mmol), copper sulfate pentahydrate (63 mg, 0.48 mmol) and sodium ascorbate (95 mg, 0.48 mmol) were added, and stirred at room temperature for 1 h. The reaction was quenched with acetonitrile (8.0 mL). The system was purified by preparative liquid chromatography to obtain the target compound W-7 128.3 mg, yield 77.2%. LC-MS (ESI+) 509.17 [M+H] + .

[0144] Synthesis of compound W-8 in Example 1-10

[0145] Preparation of compound W-8: 6-(maleimido)hexanoic acid succinimidyl ester (200 mg, 0.65 mmol) was stirred and dissolved in 5.0 mL of dichloromethane, and amino-PEG8-carboxylic acid (266.96 mg, 0.55 mmol) was added, and the reaction was carried out at room temperature for 1 h. The product was precipitated with methyl tert-butyl ether, filtered, and the solid was dried to obtain the target compound W-8 380.56 mg, yield 92.42%. LC-MS (ESI+) 635.3 [M+H] + .

[0146] Synthesis of compound W-9 in Example 1-11

[0147] Preparation of compound W-9: According to the preparation method of W-8, the raw material was replaced with amino-PEG2-carboxylic acid to obtain the target compound W-9 560.1 mg, yield 91.3%. LC-MS (ESI+) 371.17 [M+H] + .

[0148] Synthesis of specific compounds of compound I in Example 2

[0149] Synthesis of compound I-1 in Example 2-1

[0150] Preparation of compound I-1b: Methylsulfonic acid irinotecan (10.0 g, 0.02 mol) was weighed into 100 mL of H2O, and NaHCO3(1.6 g, 0.02 mol) was added, and stirred for 6 h. The mixture was filtered, and the filter cake was collected and dried to obtain 7.1 g of I-1b, yield 86.6%. LC-MS (ESI+) 435.16 [M+H] + .

[0151] Preparation of compound I-1a: Compound I-1b (7.0 g, 0.016 mol) was weighed, 80 mL MeOH was added, 3,5-di-tert-butyl-o-benzoquinone (7.1 g, 0.032 mol) was added, and the reaction was stirred for 6 h. 100 mL EA and 100 mL heptane were added to the reaction system, and the mixture was stirred for 1 h. The filter cake was dried to obtain 7.7 g of compound I-1a, with a yield of 75.5%. LC-MS (ESI+) 637.30 [M+H] + .

[0152] Preparation of compound I-1: Compound I-1a (7.0 g, 0.011 mol) was weighed, 140 mL DCM, 14 mL H2O, and 14 mL TFA were added, and the mixture was stirred for 1 h. The reaction system was concentrated by rotary evaporation, 100 mL H2O was added, and the mixture was stirred for 2 h. The mixture was filtered to obtain a black solid. The solid was purified by preparative liquid chromatography to obtain 1.8 g of I-1, with a yield of 37.7%. LC-MS (ESI+) 434.13 [M+H] + .

[0153] Synthesis of compound I-2 in Example 2-2

[0154] Compound I-1 (43.4 mg, 0.1 mmol), hydroxylamine (5.0 mg, 0.15 mmol), and pyridine p-toluenesulfonic acid (25.1 mg, 0.1 mmol) were weighed into a 10 mL vial, 2 mL DMF was added, and the mixture was stirred at 50°C for 6 h. After the reaction was completed, the mixture was purified by preparative liquid chromatography to obtain 23.5 mg of I-2 (which can be separated into I-3a and I-3b), with a yield of 26.3%. LC-MS (ESI+) 450.4 [M+H] + .

[0155] Synthesis of compound I-4 in Example 2-3

[0156]

[0157] Compound I-1 (43.4 mg, 0.1 mmol), 2-aminoxyethanol (11.6 mg, 0.15 mmol), and pyridine p-toluenesulfonic acid (25.1 mg, 0.1 mmol) were weighed into a 10 mL vial, 2 mL DMF was added, and the mixture was stirred at 50°C for 6 h. After the reaction was completed, the mixture was purified by preparative liquid chromatography to obtain 30.7 mg of I-4, with a yield of 62.3%. LC-MS (ESI+) 494.5 [M+H] + .

[0158] Synthesis of compound I-5 in Example 2-4

[0159] I-1 (86.9 mg, 0.2 mmol), 2-hydroxyacetohydrazide (18 mg, 0.2 mmol), p-toluenesulfonic acid pyridine salt (50 mg, 0.2 mmol) were dissolved in 10 mL DMF, 10 mL ethanol was added, and the reaction was refluxed for 4 h. After the reaction was completed, preparative liquid phase purification was performed to obtain 40 mg of I-5 at a yield of 18%. LC-MS (ESI+): 507.3 [M+H] + .

[0160] Synthesis of compound I-6 in Example 2-5

[0161] Preparation of compound I-6a: 2-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)acetic acid (54 mg, 0.24 mmol) was dissolved in 2 mL N,N-dimethylformamide, and irinotecan methanesulfonate (108 mg, 0.2 mmol), HATU (97 mg, 0.25 mmol), and N,N-diisopropylethylamine (51 mg, 0.4 mmol) were added at room temperature, and stirring was performed at room temperature for 1 h. The system was dried under reduced pressure, and preparative liquid phase purification was performed to obtain 104 mg of the target compound I-6a at a yield of 80%. LC-MS (ESI+) 651.5 [M+H] + .

[0162] Preparation of compound I-6: Compound I-6a (45.4 mg, 0.07 mmol) was dissolved in 3 mL dichloromethane, and trifluoroacetic acid 0.6 mL was added at room temperature. Stirring was performed at room temperature for 2 h. The system was dried by evaporation of the solvent, and preparative liquid phase purification was performed to obtain 31.9 mg of the target compound I-6 at a yield of 83%. LC-MS (ESI+) 551.5 [M+H] + .

[0163] Synthesis of compound I-7 in Example 2-6

[0164] Preparation of compound I-7a: 3-[2-(3-ethoxy-3-oxopropoxy)-ethoxy]-propanoic acid (80 mg, 0.34 mmol) was dissolved in 5 mL tetrahydrofuran, and sodium borohydride (25.8 mg, 0.68 mmol) was added, and stirring was performed at room temperature for 2 h. The system was quenched to neutral by adding 4N hydrochloric acid, and methanol and dichloromethane were added and washed three times, and rotary evaporation was performed to obtain 24.3 mg of crude I-7a. LC-MS (ESI+) 193.14 [M+H] + .

[0165] Preparation of compound I-7: Compound I-7a (24.3 mg, 0.12 mmol) was dissolved in 3 mL of N,N-dimethylformamide, HATU (48 mg, 0.12 mmol) and N,N-diisopropylethylamine (24.5 mg, 0.19 mmol) were added and stirred for 1 h. Exelixatuximab methanesulfonate (74 mg, 0.14 mmol) was weighed and stirred at room temperature for 2 h. The system was purified by preparative liquid phase to obtain the target compound I-7 18 mg, yield 22%. LC-MS (ESI+) 610.23 [M+H] + .

[0166] Example 2-7 Synthesis of compound I-9

[0167] Preparation of compound I-9a: Compound tert-butyl (3-methylene cyclobutyl)carbamate (100 mg, 0.55 mmol) was dissolved in 3 mL of dichloromethane, 0.6 mL of trifluoroacetic acid was added at room temperature. Stirring at room temperature for 3 h. The system was spin-dried to obtain the target compound I-9a 41 mg, yield 91%. LC-MS (ESI+) 84.0 [M+H] + .

[0168] Preparation of compound I-9c: Aluminum trichloride (1 g, 6.6 mmol) was dissolved in 14 mL of toluene, 3-fluoro-4-methoxyaniline (847 mg, 6.0 mmol) and boron trichloride in dichloromethane (6.9 mL, 6.9 mmol) were slowly added at 0°C, and bromopentanenitrile (2.98 g, 18 mmol) was added. Stirring at 110°C for 4 hours. The system was added to 50 mL of water to quench, extracted with ethyl acetate, washed with water twice, washed with brine once, the organic layer was spin-dried, and purified by silica gel column to obtain the target compound I-9c 1.15 g, yield 63%. LC-MS (ESI+) 305.7 [M+H] + .

[0169] Preparation of compound I-9b: Compound I-9c (757 mg, 2.50 mmol) was dissolved in 50 mL of toluene, (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (790 mg, 3.0 mmol) and 4-methylbenzenesulfonic acid pyridine (630 mg, 2.5 mmol) were added at room temperature. Stirring at 110°C for 5 hours. Cool down, spin-dry the system, the solid was slurried with ACN / H2O to obtain the target compound I-9b 930 mg, yield 70%. LC-MS (ESI+) 531.4 [M+H] + .

[0170] Preparation of compound I-9: Compound I-9a (13 mg, 0.15 mmol), compound I-9b (40 mg, 0.075 mmol) were dissolved in 1 mL of N,N-dimethylformamide, and N,N- diisopropylethylamine (19 mg, 0.15 mmol) was added at room temperature. Stirring was performed at room temperature overnight. Purification by preparative liquid chromatography gave the target compound I-9 16 mg, yield 20%. LC-MS (ESI+) 534.7 [M+H] + .

[0171] Example 2-8 Synthesis of compound I-10

[0172] Preparation of compound I-10: Compound (2-hydroxyethoxy)acetic acid (95.6 mg, 0.75 mmol), belotecan (206.7 mg, 0.5 mmol), HATU (230 mg, 0.6 mmol) were dissolved in 6 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine was added to adjust the reaction solution to pH > 7, and stirring was performed at room temperature until overnight. Purification by preparative liquid chromatography of the system gave the target compound I-10 4.6 mg, yield 2%. LC-MS (ESI+) 536.2 [M+H] + .

[0173] Example 2-9 Synthesis of compound I-11

[0174] Preparation of compound I-11a: CPT (100 mg, 0.287 mmol) was added to formic acid (3 mL) and stirred, and FeSO4·7H2O (82.3 mg, 0.287 mmol) was dissolved in purified water (1 mL) and added to the CPT solution, and 3-benzyloxy-1-propanal (0.86 g, 5.17 mmol), tert-butyl hydroperoxide (0.4 mL) were added at -10 °C, and stirring was performed for 30 min. After the reaction was completed, the reaction solution was extracted with dichloromethane, quenched with 10% sodium sulfite solution, 25% potassium bicarbonate solution to adjust pH 6-7, and the dichloromethane phase was concentrated and purified by preparative liquid chromatography to give I-11a 80 mg, yield 54%. LC-MS (ESI+): 511.2 [M+H]+.

[0175] Preparation of compound I-11: I-11a (56 mg, 0.11 mmol) was dissolved in 3 mL of tetrahydrofuran, 5 mL of methanol, 0.2 mL of formic acid, 0.2 mL of trifluoroacetic acid, and 10% w / w Pd / C (120 mg) were added, and the temperature was raised to 35 °C and stirring was performed for 12 h. After the reaction was completed, the reaction solution was filtered and concentrated, and purification by preparative liquid chromatography gave compound I-11 22 mg, yield 47%. LC-MS (ESI+): 421.2 [M+H]+ .

[0176] Synthesis of compound I-12 in Example 2-10

[0177] I-11 (16 mg, 0.0376 mmol) was dissolved in 10 mL of ethanol, hydroxylamine hydrochloride (40 mg, 0.576 mmol), pyridine p-toluenesulfonate (60 mg, 0.239 mmol) was added and the reaction was refluxed for 4 h. After the reaction was completed, I-12 was obtained by preparative liquid phase purification 10 mg, yield 51%. LC-MS (ESI+): 436.2 [M+H] + .

[0178] Synthesis of compound I-15 in Example 2-11

[0179] CPT (503.7 mg, 1.44 mmol), FeSO4·7H2O (805.3 mg, 2.90 mmol) was dissolved in 15 mL of formic acid and 5 mL of purified water, 2.07 mL of n-propyl aldehyde was added at 0°C and stirred, 65% of tert-butyl alcohol peroxide (2 mL) was added dropwise. The reaction was carried out at room temperature for 10 min. After the reaction was completed, it was quenched with sodium sulfite and concentrated to dryness under reduced pressure. I-15 was obtained by reverse phase preparative purification 381.0 mg, yield 65%. LC-MS (ESI+): 405.1 [M+H] + .

[0180] Synthesis of compound I-16 in Example 2-12

[0181] I-15 (101.0 mg, 0.25 mmol), hydroxylamine hydrochloride (88.1 mg, 1.27 mmol) was dissolved in 1 mL of DMF and 10 mL of anhydrous ethanol, and the reaction was carried out at 100°C for 3 h. After the reaction was completed, the product I-16 was obtained by reverse phase preparative liquid phase purification and freeze-drying 79.1 mg, yield 75.5%. LC-MS (ESI+): 420.2 [M+H] + .

[0182] Synthesis of compound I-19 in Example 2-13

[0183] CPT (300 mg, 0.861 mmol) was added to acetic acid (0.9 mL), purified water (7 mL) was stirred, and FeS04·7H20 (0.12 g) was dissolved in purified water (2 mL) and added to the CPT solution. The temperature was lowered to 0°C, and sulfuric acid (2.4 mL) was added dropwise. Then, n-butyraldehyde (1.3 g, 17.2 mmol), and tert-butyl hydroperoxide (1.2 mL) were added in sequence, and the mixture was stirred at room temperature for 20 min. Acetic acid (8 mL) was added, and the mixture was stirred for 30 min. After the reaction was completed, the reaction solution was extracted with dichloromethane, quenched with 10% sodium sulfite solution, and adjusted to pH 6-7 with 25% potassium bicarbonate solution. The dichloromethane phase was concentrated and purified by preparative liquid chromatography to obtain 40 mg of I-191 with a yield of 38%. LC-MS (ESI+): 419.8 [M+H] + .

[0184] Synthesis of compound I-20 in Example 2-14

[0185] I-19 (70 mg, 0.17 mmol), hydroxylamine hydrochloride (13.9 mg, 0.2 mmol), and pyridine p-toluenesulfonate (140 mg, 0.5 mmol) were dissolved in 10 mL of ethanol, and the mixture was stirred at reflux for 4 h. After the reaction was completed, the reaction solution was dried by rotary evaporation, and purified by preparative liquid chromatography to obtain 30 mg of I-20 with a yield of 42%. LC-MS (ESI+): 434.2 [M+H] + .

[0186] Synthesis of compound I-24 in Example 2-15

[0187] CPT (100.00 mg, 0.29 mmol) was dissolved in 3 mL of formic acid, FeS04·7H20 (119.71 mg, 0.43 mmol) was dissolved in 0.5 mL of purified water and added to the CPT solution, 5-hydroxyvaleraldehyde (293.18 g, 2.9 mmol) was added to the CPT formic acid solution, the temperature was lowered to 0°C, and 100 microliters of hydrogen peroxide (30%) was added dropwise. After the dropwise addition was completed, the temperature was returned to room temperature, and the reaction was allowed to proceed for 1 h. The reaction solution was extracted with dichloromethane and purified water, the dichloromethane phase was concentrated under reduced pressure, and the target compound I-24 was obtained by preparative liquid chromatography with a yield of 42.65 mg and a yield of 33.15%. LC-MS (ESI+) 448.2 [M+H] + .

[0188] Synthesis of compound I-26 in Example 2-16

[0189] Preparation of compound I-26a: 2-(benzyloxy)acetamide (16.5 mg, 0.1 mmol), compound I-15 (43.6 mg, 0.1 mmol) were taken in a 10 mL vial, DMF solvent was added, 20 ul of concentrated H2SO4 was added, stirred at 100 °C for 5 h. After the reaction was completed by LC-MS monitoring, it was purified by preparative liquid phase to obtain I-26a 20.6 mg, yield 35.2% LC-MS (ESI+) 584.6 [M+H] + .

[0190] Preparation of compound I-26: I-26a (20.6 mg, 0.035 mmol), Pd / C (5 mg, 30%) were dissolved in 3 mL of MeOH, replaced with hydrogen, stirred at room temperature for 0.5 h, after the reaction was completed by LC-MS monitoring, filtered, and the filtrate was purified by preparative liquid phase to obtain I-26 15.4 mg, yield 88.9%. LC-MS (ESI+) 494.5 [M+H] + .

[0191] Example 2-17 Synthesis of compound I-27

[0192] I-1 (86.9 mg, 0.2 mmol) was dissolved in 10 mL of DMF, acetic acid (180 mg, 3 mmol) was added, then 2-hydrazinoethanol (152 mg, 2 mmol) was added, 10 mL of ethanol was added, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, it was purified by preparative liquid phase to obtain I-27 10 mg, yield 8.8%. LC-MS (ESI+): 493.4 [M+H] + .

[0193] Example 2-18 Synthesis of compound I-28

[0194] Preparation of compound I-28j: 5-bromo-1-tetralone (100 g, 0.44 mol) was dissolved in H2SO4 (800 mL), after stirring at 0 °C, KNO3 (51.65 g, 0.51 mol) was added. The reaction mixture was stirred at 0 °C for 2 h, poured into ice, extracted with ethyl acetate (300 mL x 3). The organic layer was washed with saturated sodium bicarbonate solution, dried over Na2SO4 and concentrated. The residue was purified by Flash column chromatography (petroleum ether / dichloromethane = 1 / 1 eluent) to obtain the target compound I-28j 73 g, yield 60.83%. LC-MS (ESI+) 270.0 [M+H] + .

[0195] Preparation of compound I-28h: To a solution of I-28j (46.12 g, 0.17 mol) in methanol (500 mL) was added KOH (47.91 g, 0.85 mol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, and (diacetoxyiodo)benzene (55 g, 0.34 mol) was added. The reaction mixture was stirred at 25 °C for 2.5 h. Then the mixture was poured into water, the pH was adjusted to 2 with HCl solution, and extracted with ethyl acetate (500 mL x 3). The organic layer was washed with saturated sodium bicarbonate solution and dried over Na2SO4. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 1 elution) to give the target compound I-28h 22 g in 45.08% yield. LC-MS (ESI+) 285.9 [M+H] + .

[0196] Preparation of compound I-28g: I-28h (22 g, 76.90 mmol) was dissolved in 500 mL of dichloromethane, and acetyl chloride (6.69 g, 92.28 mmol), triethylamine (11.65 g, 115.35 mmol) and DMAP (50 mg) were added. The reaction mixture was stirred at 25 °C for 2 h, quenched with NaHCO3 solution (100 mL), and extracted with dichloromethane (100 mL x 3). The organic layer was washed with saturated sodium bicarbonate solution, dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 1 elution) to give the target compound I-28g 13.2 g in 52.17% yield. LC-MS (ESI+) 327.9 [M+H] + .

[0197] Preparation of compound I-28f: I-28g (13.2 g, 40.20 mmol) and 4,4-bipyridine (6.28 g, 40.20 mmol) were stirred in ethanol (200 mL), and tetrahydroxyboron (10.81 g, 12.06 mmol) was added at room temperature. The reaction mixture was stirred at 50 °C for 2 h. After evaporation, the residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 3:1 elution) to give the target compound I-28f 9.8 g in 81.84% yield. LC-MS (ESI+) 300.0 [M+H] + .

[0198] Preparation of compound I-28e: I-28f (16 g, 54 mmol) and (S)-4-ethyl-4-hydroxy-7,8- dihydro-lH-pyrano O[3,4-F]indolizine-3,6,10(4H)-one (12.7 g, 48.3 mmol) were dissolved in acetic acid (200 mL) and stirred, trifluoroacetic acid (8 mL) was added at room temperature. The reaction mixture was stirred at 85 °C for 15 h. Evaporated under reduced pressure. The residue obtained was purified by Flash column (eluted with dichloromethane: ethyl acetate = 5: 1) to give the target compound I-28e 15.5 g, yield 54.93%. LC-MS (ESI+) 524.9 [M+H] + .

[0199] Preparation of compound I-28d: I-28e (15 g, 28.6 mmol) was dissolved in tetrahydrofuran (500 mL) and stirred, a solution of NaOH (2.3 g, 57.2 mmol) in methanol (50 mL) was added at room temperature. The reaction mixture was stirred for 1 h. The reaction mixture was quenched with trifluoroacetic acid (5 mL) and evaporated under reduced pressure. The residue obtained was purified by Flash column (eluted with dichloromethane: ethyl acetate = 3: 1) to give the target compound I-28d 11 g, yield 79.72%. LC-MS (ESI+) 482.9 [M+H] + .

[0200] Preparation of compound I-28c: I-28d (11 g, 22.8 mmol) was dissolved in dichloromethane (2000 mL) and stirred, Dess-Martin oxidizing agent (19.34 g, 45.6 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 2 h. After completion, concentrated and purified directly by Flash column (eluted with dichloromethane: ethyl acetate = 10: 1) to give the target compound I-28c 7 g, yield 63.60%. LC-MS (ESI+) 480.9 [M+H] + .

[0201] Preparation of compound I-28b: I-28c (7 g, 14.5 mmol) and ethoxyamine hydrochloride (5.6 g, 58 mmol) were dissolved in dichloromethane / methanol (v:v = 10 / 1, 550 mL) and stirred at room temperature, pyridine (4.6 g, 58 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction was evaporated under reduced pressure. The residue obtained was purified by Flash column (eluted with dichloromethane: methanol = 20: 1) to give the target compound I-28b 5.5 g, yield 72.41%. LC-MS (ESI+) 524.0 [M+H] + .

[0202] Preparation of compound I-28a: I-28b (1 g, 1.9 mmol), BocNH2 (670 mg, 5.7 mmol) and Cs2CO3 (1.24 g, 3.8 mmol) were dissolved in dioxane (100 mL) and stirred at room temperature under nitrogen protection, and xantphos Pd G3 (180 mg, 0.02 mmol) was added. The reaction mixture was stirred at 90 °C for 2 h. Filtration and evaporation under reduced pressure gave the crude product. The crude product was purified by flash chromatography column (dichloromethane:methanol = 15:1 elution) to give the target compound I-28a 500 mg, yield 47.37%. LC-MS (ESI+) 561.2 [M+H] + .

[0203] Preparation of compound I-28: A solution of I-28a (500 mg, 0.9 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5 mL). Stirring at room temperature for 2 h. Evaporation under reduced pressure. The residue was purified by pre-high performance liquid chromatography column to give the target compound I-28 120 mg, yield 27.76%. LC-MS (ESI+) 461.1 [M+H] + .

[0204] Example 2-19 Synthesis of compound I-29

[0205] Preparation of compound I-29b: FeSO4·7H2O (598.54 mg, 2.15 mmol) was dissolved in 2.5 mL purified water and added to a solution of camptothecin (500.00 mg, 1.44 mmol) in formic acid (5 mL), and 5-(benzyloxy)pentanal (1379.75 mg, 7.18 mmol) was added, and the temperature was lowered to 0-5 °C, and 500 μL hydrogen peroxide (30%) was added dropwise. After the addition was completed, the temperature was restored to room temperature, and the reaction was allowed to proceed for 1 h. The reaction solution was prepared. The target compound I-29b was obtained in 290.26 mg, with a yield of 37.55%. LC-MS (ESI+) 539.2 [M+H] + .

[0206] Preparation of compound I-29a: I-29b (70.00 mg, 0.13 mmol) was dissolved in N,N-dimethylformamide and anhydrous ethanol, and hydroxylamine hydrochloride (45.16 mg, 0.65 mmol) and p-toluenesulfonic acid pyridine salt (163.30 mg, 0.65 mmol) were added, and the temperature was raised to 100 °C, and the reaction was allowed to proceed for 1 h, and the temperature was lowered to room temperature, and the pH was adjusted to 6-7 with 20% aqueous potassium bicarbonate solution, and dichloromethane was added, and the organic phase was dried to give the target compound I-29a 45.21 mg, with a yield of 62.86%. LC-MS (ESI+): 554.2 [M+H] +.

[0207] Preparation of compound I-29A / B: I-29a (40.00 mg, 0.07 mmol) was dissolved in 5.5 mL of tetrahydrofuran and 5.5 mL of methanol, then 0.28 mL of formic acid, 0.28 mL of trifluoroacetic acid and 220.00 mg of Pd / C were added, and the mixture was reacted at 30-35 °C for 5 hours. After filtration, extraction with dichloromethane, adjustment of the pH of the organic phase to 7 with 20% aqueous potassium bicarbonate solution, extraction, separation, rotary evaporation of the organic phase, and preparative liquid phase purification, the target compounds I-29A 8.60 mg, yield 24.93%; I-29B: 11.06 mg, yield 32.07% were obtained. LC-MS (ESI+) 464.36 [M+H] + .

[0208] Synthesis of compound I-34 of example 2-20

[0209] Preparation of compound I-34c: Sodium hydride (813 mg, 20.4 mmol) was added to N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.19 g, 5.1 mmol) in toluene (40 mL) in batches, stirred for 30 minutes, then dimethyl carbonate (6.1 g, 5.1 mmol) was added, and the mixture was warmed to 90 °C and reacted for 3 hours. After the reaction was completed, the reaction solution was slowly added dropwise to 100 mL of water for quenching, extracted with ethyl acetate, washed with water twice, and saturated brine once, separated, and the organic layer was rotary evaporated to dryness. The residue was purified by silica gel column chromatography to obtain the target compound I-34c 900 mg, yield 57.9%. LC-MS (ESI+) 308.2 [M+H] + .

[0210] Preparation of compound I-34b: A sodium hydroxide (1.04 g, 27.5 mmol) aqueous solution containing I-34c (334 mg, 1.1 mmol) was warmed to 80 °C and reacted for 5 hours. After the reaction was completed, the pH was adjusted to about 1 with 4 mol / L dilute hydrochloric acid aqueous solution, and the mixture was extracted twice with ethyl acetate. The combined organic phase was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound I-34b crude product 150 mg, which was used directly in the next step without further purification. LC-MS (ESI+) 238.0 [M+H] + .

[0211] Preparation of compound I-34a: The crude of I-34b (150 mg), ethanolamine (161.9 mg, 2.2 mmol), HATU (621.6 mg, 1.6 mmol) and N, N-diisopropylethylamine (760 uL, 4.4 mmol) were dissolved in N, N-dimethylformamide (5 mL) and reacted at room temperature for 1 hour. After the reaction was completed, high-pressure preparation purification was performed to obtain 90 mg of the target compound I-34a. LC-MS (ESI+) 281.2 [M+H] + .

[0212] Preparation of compound I-34A / B: I-34a (50 mg, 0.18 mmol), (S)-4-ethyl-4-hydroxy-7,8- dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (49 mg, 0.19 mmol) and p-toluenesulfonic acid (46.6 mg, 0.19 mmol) were added to toluene (4 mL) and reacted at 125 °C for 7 hours. After the reaction was completed, high-pressure preparation purification was performed to obtain 11.3 mg of compound I-34A and 1.3 mg of compound I-34B. LC-MS (ESI+) 508.7 [M+H] + .

[0213] Example 2-21 Synthesis of compound I-35

[0214] Preparation of compound I-35c: CPT (500 mg, 1.44 mmol) was added to a 14 mL methanol and 11.5 mL purified water solution and stirred while being cooled to 0 °C. 5 mL of concentrated sulfuric acid was added dropwise, and FeSO4·7H2O (598.56 mg, 2.16 mmol) was weighed and added to the reaction solution. 1.5 mL of hydrogen peroxide (30%) was added dropwise, and the reaction was performed at room temperature. After the reaction was completed, it was filtered, and the solid was washed with purified water and then with methanol, and then vacuum dried. The target compound I-35c was obtained in a yield of 346.28 mg, 63.80%. LC-MS (ESI+) 379.3 [M+H] + .

[0215] Preparation of compound I-35b: I-35c (300.00 mg, 0.79 mmol) was dissolved in 12 mL of methanol and 2 mL of concentrated sulfuric acid and stirred at room temperature. MnO2 (137.37 mg, 1.58 mmol) was added, and the reaction was performed at 50 °C overnight. It was filtered, extracted with dichloromethane and purified water, and the dichloromethane was concentrated under reduced pressure to obtain I-35b in a yield of 190.53 mg, 56.89%. LC-MS (ESI+) 423.2 [M+H] + .

[0216] Preparation of compound I-35a: I-35b (160.00 mg, 0.38 mmol) was dissolved in 5 mL acetonitrile, 3 mL purified water and 1 mL hydrochloric acid, and refluxed at 70 °C for 1 h. The reaction solution was extracted with dichloromethane and purified water, and the dichloromethane phase was concentrated under reduced pressure and column chromatography. I-35a 100.14 mg, yield 70.20%. LC-MS (ESI+) 377.3 [M+H]+.

[0217] Preparation of compound I-35: Compound I-35a (80.00 mg, 0.21 mmol) was dissolved in 4 mL N,N-dimethylformamide, 2-aminoethanol (81.92 mg, 1.06 mmol) was added, stirred, p-toluenesulfonic acid pyridine salt (63.33 mg, 0.25 mmol) was added, and the temperature was raised to 100 °C for 1 h. The reaction solution was prepared and purified to obtain the target compound I-35 37.33 mg, yield 40.33%. LC-MS (ESI+) 436.14 [M+H] + .

[0218] Example 2-22 Synthesis of compound I-36

[0219] I-15 (50.6 mg, 0.13 mmol), 2-oxoaminoethanol (54.4 mg, 0.71 mmol) and p-toluenesulfonic acid pyridine salt (37.2 mg, 0.15 mmol) were dissolved in 0.5 mL DMF and 5 mL anhydrous ethanol, and reacted at 100 °C for 3 h. After the reaction was completed, the product I-36 was obtained by freeze-drying after reverse phase preparative liquid chromatography purification, with a yield of 26.8 mg, 46.2%. LC-MS (ESI+): 464.2 [M+H] + .

[0220] Example 2-23 Synthesis of compound I-37

[0221] I-15 (50.6 mg, 0.13 mmol), hydroxylamine hydrochloride (49.3 mg, 0.71 mmol) and p-toluenesulfonic acid were dissolved in 0.5 mL DMF and 5 mL anhydrous ethanol, and reacted at 100 °C for 8 h. After the reaction was completed, the product I-37 was obtained by freeze-drying after reverse phase preparative liquid chromatography purification, with a yield of 26.8 mg, 46.2%. LC-MS (ESI+): 464.2 [M+H] + .

[0222] Example 2-24 Synthesis of compound I-38

[0223] I-19 (85 mg, 0.20 mmol), hydroxylamine hydrochloride (16.8 mg, 0.24 mmol) and p-toluenesulfonic acid were dissolved in 10 mL of ethanol, heated to reflux and stirred for 8 h. After the reaction was completed, the reaction solution was rotary evaporated and purified by preparative liquid chromatography to obtain I-38 31 mg, yield 38%. LC-MS (ESI+): 434.2 [M+H]+.

[0224] Example 2-25 Synthesis of compound I-39

[0225] Preparation of compound I-39A / B: Compound I-15 (97.47 mg, 0.24 mmol) was dissolved in 4 mL of N,N-dimethylformamide, 2-hydrazinoethanol (91.70 mg, 1.21 mmol) was weighed into the reaction solution, 2.0 mL of acetic acid was added, heated to 75 °C, and reacted for 4 h. After the reaction was completed, LC-MS was monitored, and the target compound I-39A 15.25 mg, yield 13.91%; I-39B 10.06 mg, yield 9.02% was obtained by preparative liquid chromatography purification. LC-MS (ESI+) 463.2 [M+H] + .

[0226] Example 2-26 Synthesis of compound I-40

[0227] Preparation of compound I-40A / B: Compound I-15 (97.47 mg, 0.24 mmol) was dissolved in 4 mL of N,N-dimethylformamide, hydroxyacetic hydrazide (111.37 mg, 1.24 mmol) was weighed into the reaction solution, 2.0 mL of acetic acid was added, heated to 75 °C, and reacted for 4 h. After the reaction was completed, LC-MS was monitored, and the target compound I-40A 9.38 mg, yield 7.96%; I-40B 26.06 mg, yield 22.12% was obtained by preparative liquid chromatography purification. LC-MS (ESI+) 477.2 [M+H] + .

[0228] Example 2-27 Synthesis of compound I-41

[0229] Preparation of compound I-41a: I-29b (100.00 mg, 0.19 mmol) was dissolved in tetrahydrofuran and anhydrous ethanol, and methoxyammonium chloride (77.53 mg, 0.93 mmol) and p-toluenesulfonic acid pyridine salt (233.21 mg, 0.93 mmol) were added. The reaction was heated to 100 °C for 1 h, and then cooled to room temperature. The pH was adjusted to 6-7 with 20% aqueous potassium bicarbonate solution, and the organic phase was extracted with dichloromethane. The organic phase was dried and concentrated to give the target compound I-41a 52.48 mg, with a yield of 49.80%. LC-MS (ESI+) 567.2 [M+H] + .

[0230] Preparation of compound I-41A / B: I-41a (50.00 mg, 0.09 mmol) was dissolved in 5.5 mL of tetrahydrofuran and 5.5 mL of methanol, and 0.30 mL of formic acid, 0.30 mL of trifluoroacetic acid and 200.00 mg of Pd / C were added. The reaction was carried out at 30-35 °C for 5 h, and then filtered. The organic phase was extracted with dichloromethane, and the pH was adjusted to 7 with 20% aqueous potassium bicarbonate solution. The target compound I-41A was obtained by extraction, separation, drying and concentrating of the organic phase, and purification by preparative liquid chromatography, with a yield of 9.46 mg, 24.49%; and I-41B was obtained with a yield of 15.23 mg, 36.21%. LC-MS (ESI+) 464.36 [M+H] + .

[0231] Synthesis of compound I-44 of example 2-28

[0232] Preparation of compound I-44b: Compound I-1 (1000 mg, 2.3 mmol) was dispersed in 40 mL of tetrahydrofuran, and cooled to 0 °C. Sodium triacetylborohydride (244 mg, 4.6 mmol) was added to the reaction system, and the temperature was controlled at 0-5 °C for 0.33 h. Ice acetic acid (552 mg, 9.2 mmol) was added, and 80 mL of n-heptane was added and stirred for 2 h. The target compound I-44b was obtained by filtration, with a yield of 812 mg, 81%. LC-MS (ESI+) 436.2 [M+H] + .

[0233] Preparation of compound I-44aA / B: Compound I-44b (800 mg, 1.8 mmol) was dissolved with 8 mL of N,N-dimethylformamide, and di(p-nitrophenyl) carbonate (2189 mg, 7.2 mmol), N,N-diisopropyl ethylamine (464 mg, 3.6 mmol) were added under nitrogen protection, and the reaction was carried out at room temperature for 2 h. The system was added with glacial acetic acid (432 mg, 7.2 mmol), diluted with 40 mL of ethyl acetate, and the organic phase was washed with 20 mL of saturated brine for 3 times, and the organic phase was dried with anhydrous sodium sulfate, concentrated to solid by rotary evaporation, and the solid was dissolved with 10 mL of tetrahydrofuran, and 20 mL of n-heptane was added for crystallization, and the crude product was obtained by filtration, and the obtained crude product was purified by preparative liquid chromatography to obtain the target compound I-44aA 395 mg, yield 19.5%; I-44aB 309 mg, yield 15.3%. LC-MS (ESI+) 601.1 [M+H] + .

[0234] Preparation of compound I-44A / B: I-44aA (20 mg, 0.033 mmol) was placed in a single-neck flask, dissolved with 1 mL of dichloromethane, and azetidin-3-ol (5.0 mg, 0.05 mmol) was added, and stirred at room temperature for 4 h. The system was rotary evaporated, and purified by preparative liquid chromatography to obtain the target compound I-44A 12 mg, yield 64%; I-44B 12 mg, yield 64% was obtained by replacing I-44aA with I-44aB using the same method as above. LC-MS (ESI+) 564.1 [M+H] + .

[0235] Example 2-29 Synthesis of compound I-45

[0236] Preparation of compound I-45: I-1a (100.00 mg, 0.16 mmol), propyl hydrazine (116.22 mg, 1.57 mmol) and p-toluenesulfonic acid pyridine salt (39.40 mg, 0.16 mmol) were dissolved in 10 mL of N,N-dimethylformamide, 10 mL of ethanol was added, and the temperature was raised to 80-90°C, and the reaction was carried out for 4 h. Purification by preparative liquid chromatography to obtain the target compound I-45 36.26 mg, yield 47.15%. LC-MS (ESI+) 491.2 [M+H] + .

[0237] Example 2-30 Synthesis of compound I-49

[0238] Preparation of compound I-49a: To 3.0 mL dimethylsulfoxide was added 305 mg ethanolamine and 0.5 mL hydrochloric acid, the reaction was heated to 110 °C in an oil bath, after 30 minutes at this temperature, 7,10-dimethyl-11-fluoro camptothecin (395 mg, 1.0 mmol) was added, the temperature was raised to 130 °C, and the reaction was allowed to proceed for 5 hours. Upon completion, the reaction was directly purified by high pressure preparative purification to give the target compound I-49a 165 mg, yield 35.2%. LC-MS (ESI+) 468.2 [M+H] + .

[0239] Preparation of compound I-49: Dimethylphosphoryl chloride (112 mg, 1.0 mmol) was slowly added dropwise to a solution of I-49a (100 mg, 0.21 mmol) and N,N diisopropylethylamine (129 mg, 1.0 mmol) in dichloromethane (5 mL) at 0 °C, and the reaction was allowed to proceed for 1 hour. Upon completion, the reaction was purified by high pressure preparative purification to give the target compound I-49 45 mg, yield 38.7%. LC-MS (ESI+) 544.3 [M+H] + .

[0240] Example 2-31 Synthesis of compound I-50

[0241] Preparation of compound I-50c: To a solution of DX8951 (430 mg, 0.99 mmol), 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid (202.1 mg, 1.0 mmol), HATU (570.3 mg, 1.5 mmol), and N,N diisopropylethylamine (516 mg, 4.0 mmol) in N,N-dimethylformamide 5 mL was stirred at room temperature for 3 hours. Upon completion, the reaction was purified by high pressure preparative purification to give the target compound I-50c 520 mg, yield 85.1%. LC-MS (ESI+) 619.4 [M+H] + .

[0242] Preparation of compound I-50b: To a solution of compound I-50c (502 mg, 0.81 mmol) in N,N-dimethylformamide (5 mL) was added trifluoroacetic acid (1 mL), and the reaction was stirred at room temperature for 2 hours. Upon completion, the reaction was purified by high pressure preparative purification to give the target compound I-50b 410 mg, yield 97.4%. LC-MS (ESI+) 519.2 [M+H] + .

[0243] Preparation of compound I-50a: To a solution of compound I-50b (400 mg, 0.77 mmol) in acetonitrile (5 mL) was added N, N-diisopropylethylamine (0.65 mL, 3.7 mmol) and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, high pressure preparative purification was performed to obtain the target compound I-50a 135 mg, yield 28.6%. LC-MS (ESI+) 572.6 [M+H] + .

[0244] Preparation of compound I-50: To a solution of compound I-50a (126 mg, 0.22 mmol) in anhydrous methanol (3 mL) was added m-CPBA (meta-chloroperoxybenzoic acid) (77%, 80 mg, 0.36 mmol) and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was completed, high pressure preparative purification was performed to obtain the target compound I-50 23 mg, yield 19.5%. LC-MS (ESI+) 535.2 [M+H] + .

[0245] Example 2-32 Synthesis of compound I-59

[0246] Preparation of compound I-59i: In a 500 mL three-necked flask was added 250 mL of concentrated sulfuric acid, and the temperature was lowered to 0-5 °C in an ice bath. Compound I-59j (31.25 g, 0.20 mol) was added, and NIS (67.13 g, 0.30 mol) was added while controlling the temperature below 10 °C. The reaction was stirred overnight at room temperature. The reaction solution was added dropwise to 1 L of ice water, filtered, and the solid was dissolved in 500 mL of ethyl acetate. The ethyl acetate phase (300 mL x 3) was washed with an aqueous solution of sodium thiosulfate, and then with brine (500 mL) once. The solid was filtered and dried under reduced pressure, and then slurried with n-heptane. The solid was filtered and dried under reduced pressure to obtain I-59i 40.59 g, yield 72%. LC-MS (ESI+) 284.0 [M+H] + .

[0247] Preparation of compound I-59h: Compound I-59i (37.78 g, 0.1335 mol) was stirred in a 1000 mL three-necked flask with 450 mL of ethanol and 20 mL of water. Iron powder (26.17 g, 0.4672 mol) was added, and the temperature was raised to 85 °C for 5 h. After the reaction was completed, celite was added to assist filtration, and then rotary evaporation was performed. The solid was dissolved in ethyl acetate, and the ethyl acetate phase was washed with saturated sodium chloride. The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure. Column chromatography (dichloromethane:methanol = 10:1) was performed to obtain I-59h 16.3 g, yield 48%. LC-MS (ESI+) 254.01 [M+H] + .

[0248] Preparation of compound I-59g: Compound I-59h (16.3 g, 0.0644 mol) was stirred in a 250 mL single-neck flask with 163 mL of dichloromethane, and triethylamine (21.5 g, 0.2126 mol) was added. Acetic anhydride (17.1 g, 0.1675 mol) was added dropwise while controlling the temperature at 0-5°C, and the reaction was allowed to proceed overnight. Water (100 mL) was added to the reaction solution, followed by the addition of 200 mL of ethyl acetate. The organic phase was washed with water three times (100 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in 75 mL of ethyl acetate and 15 mL of n-heptane to obtain the target compound I-59g (20.3 g) with a yield of 94%. LC-MS (ESI+) 338.09 [M+H] + .

[0249] Preparation of compound I-59f: Compound I-59g (20.3 g, 0.06 mol) was stirred in a 250 mL three-neck flask with 162 mL of N,N-dimethylformamide, and triethylamine (24.35 g, 0.24 mol) was added. 3-Butyn-1-ol (8.44 g, 0.12 mol) was added, followed by the addition of Pd(PPh3)Cl2 (1.06 g, 1.5 mmol) and CuI (1.15 g, 0.6 mmol). The reaction was allowed to proceed for 3 h under nitrogen. The reaction solution was added dropwise to 500 mL of 1N HCl solution, followed by the addition of 200 mL of ethyl acetate. The organic phase was washed with water three times (200 mL x 3), washed with lithium chloride (200 mL x 3), and washed once with saturated sodium chloride. The organic phase was passed through a silica gel pad, and concentrated under reduced pressure to obtain the target compound I-59f (15.1 g) with a yield of 90%. LC-MS (ESI+) 280.27 [M+H] + .

[0250] Preparation of compound I-59e: Compound I-59f (14.0 g, 0.05 mol) was stirred in a 500 mL single-neck flask with 250 mL of tetrahydrofuran and 250 mL of methanol. Platinum dioxide (1.4 g) was added, and the reaction was allowed to proceed overnight under hydrogen. The filtrate was dried under reduced pressure, and the target compound I-59e (13.5 g) was obtained with a yield of 95%. LC-MS (ESI+) 284.4 [M+H] + .

[0251] Preparation of compound I-59d: Compound I-59e (13.0 g, 0.0463 mol) was stirred in a 500 mL three-neck flask with 260 mL of acetone, and the temperature was controlled at 0-5°C. Jones reagent (26 mL) was added dropwise, and the reaction was allowed to proceed for 1 h. Isopropyl alcohol (26 mL) was added dropwise, and the reaction was filtered, concentrated under reduced pressure, dissolved in ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The target compound I-59d (7.8 g) was obtained by further passing through a silica gel column (dichloromethane:methanol = 10:1). LC-MS (ESI+) 298.28 [M+H]+ .

[0252] Preparation of compound I-59c: Compound I-59d (7.0 g, 0.0236 mol) was added with 12.81 mL trifluoroacetic acid in a 50 mL flask, cooled to 0-5 °C, added 11.26 mL trifluoroacetic anhydride dropwise, after dropwise addition, warmed to 50 °C, reacted for 3 h. The reaction solution was added dropwise into ice water, extracted with ethyl acetate, and dried to obtain an oil, which was purified by column chromatography (n-heptane: ethyl acetate = 15: 1) to obtain 4.1 g of the target compound I-59c with a yield of 62%. LC-MS (ESI+) 280.27 [M+H] + .

[0253] Preparation of compound I-59b: Compound I-59c (2.0 g, 7.2 mmol) was added with 50 mL of tetrahydrofuran in a 100 mL flask, cooled to -70 °C, added 28.7 mL of LiHMDS (1 N) dropwise, added a solution of N-phenylsulfonylimine (6.7 g, 21.5 mmol) in tetrahydrofuran (20 mL) dropwise, moved to room temperature overnight after dropwise addition. 2N HCl methanol solution (100 mL) was added to the reaction solution, warmed to 45 °C, and reacted for 3 h. The aqueous phase was extracted with 100 mL of dichloromethane, and the crude product I-59b 1.5 g was obtained after drying. LC-MS (ESI+) 232.24 [M+H] + .

[0254] Preparation of compound I-59: Compound I-59b crude (1.0 g, 4.3 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranoO[3,4-F]indolizine-3,6,10(4H)-one (1.4 g, 5.2 mmol), and p-toluenesulfonic acid (0.74 g, 4.3 mmol) were added in a 250 mL flask, stirred with 100 mL of toluene, and warmed to 130 °C for 8 h. The toluene was removed by rotary evaporation, and the target compound I-59 310 mg was obtained by further silica gel column chromatography (dichloromethane:methanol = 10:1). LC-MS (ESI+) 459.4 [M+H] + .

[0255] Example 2-33 Synthesis of compound I-61

[0256] Preparation of compound I-61A / B: The synthesis of compound I-61A / B was performed according to the method of I-44A / B, with azetidin-3-ylmethanol as the starting material to obtain 11.4 mg of target compound I-61A and 10.5 mg of target compound I-61B. LC-MS (ESI+) 550.2 [M+H] + .

[0257] Example 2-34 Synthesis of compound I-62A

[0258] Preparation of compound I-62A: The synthesis of compound I-62A was according to the method of I-44A, the starting material was replaced by (R)-pyrrolidine-3-methanol, and the target compound I-62A 12.2 mg was obtained. LC-MS (ESI+) 564.3 [M+H] + .

[0259] Example 2-35 Synthesis of compound I-65

[0260] Preparation of compound I-65A / B: The synthesis of compound I-65A / B was according to the method of I-44A / B, the starting material was replaced by 4-hydroxypiperidine, and the target compound I-65A 10.1 mg, I-65B 9.8 mg was obtained. LC-MS (ESI+) 564.1 [M+H] + .

[0261] Example 2-36 Synthesis of compound I-76

[0262] Preparation of compound I-76b: Ethanol acid (0.2 g, 2.63 mmol) was weighed into a single-necked flask, 2 mL of acetonitrile was added to dissolve it completely, and 1.6 mL of N,N-diisopropylethylamine was added under ice bath stirring. TBS-Cl (872.01 mg, 2.63 mmol) was dissolved in 2 mL of acetonitrile and added dropwise to the above flask, and the reaction was carried out at 0°C for 2 h. After the reaction was completed, it was directly fed into the next step. LC-MS (ESI+) 191.3 [M+H] + .

[0263] Preparation of compound I-76a: 1 mL of I-76b reaction solution was spin-dried, 4 mL of N,N-dimethylformamide was added to dissolve it, and HATU (109.89 mg, 289 μmol) was weighed into the flask and ultrasonically dissolved. N,N-diisopropylethylamine 69 μL was added dropwise to the reaction system, and stirred at room temperature for 2 h. 10-amino-11-F camptothecin (100 mg, 262.7 μmol) was weighed into the flask, and stirred at room temperature for 4 h. After the reaction was completed, high-pressure preparation purification was carried out, and I-76a 23.3 mg was obtained with a yield of 15.8%. LC-MS (ESI+) 554.6 [M+H] + .

[0264] Preparation of compound I-76: I-76a (15 mg, 27.09 μmol) was weighed into 1 mL of tetrahydrofuran, ultrasonically dissolved, 0.5 mL of HCOOH and 0.5 mL of purified water were added, and stirred at room temperature for 2 h. The system was purified by high pressure preparation, to obtain 3.7 mg of I-76, with a yield of 31.08%. LC-MS (ESI+) 440.4 [M+H] + .

[0265] Example 2-37 Synthesis of compound I-77

[0266] Preparation of compound I-77e: I-77f (850 mg, 2.5 mmol) was weighed into a 50 mL three-necked flask, 6 mL of acetonitrile was added under nitrogen protection. The temperature was lowered to 0-5 °C. NBS (445 mg, 2.5 mmol) was added in batches, and stirred for 30 min. It was removed to room temperature and reacted for 1 h. The reaction was completed. The reaction solution was rotary evaporated. Methyl tert-butyl ether (15 mL) was added to the slurry for 1 h. It was filtered, and the filtrate was washed with saturated brine (15 mL). The organic phase was concentrated to obtain compound I-77e 1045 mg, with a yield of 100%. LC-MS (ESI+) 341.2 [M+H] + .

[0267] Preparation of compound I-77d: I-77e (1045 mg, 2.5 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano O[3,4-F]indolizine-3,6,10(4H)-one (799 mg, 3.0 mmol), PTSA (397 mg, 2.25 mmol), toluene (50 mL) were added to a 250 mL single-necked flask, and refluxed for 4 h. The reaction was completed. It was cooled and rotary evaporated, and the crude product was purified by column chromatography, with methanol:dichloromethane=10%~25% as the eluent. Concentration obtained 1100 mg of compound I-77d, with a yield of 68%. LC-MS (ESI+) 646.2 [M+H] + .

[0268] Preparation of compound I-77c: I-77d (1100 mg, 1.7 mmol), 10% Pd / C (110 mg) were weighed into a 100 mL single-necked flask, 20 mL of methanol was added under nitrogen protection, and hydrogenated for 1 h. The sample was taken to control the completion of the reaction. It was filtered, and the filtrate was concentrated to obtain 703 mg of compound I-77c, with a yield of 75%. LC-MS (ESI+) 512.1 [M+H] + .

[0269] Preparation of compound I-77b: I-77c (700 mg, 1.3 mmol) was weighed into a 50 mL single-neck flask, and 7 mL of N,N-dimethylformamide was added under nitrogen protection, and N,N-diisopropylethylamine (2120 mg, 15 mmol) was stirred and dissolved. The solution was cooled in an ice bath. Methylsulfonyl chloride (628 mg, 5.2 mmol) was added to the reaction solution. The reaction was carried out at room temperature for 1 h. The reaction was sampled and controlled. The reaction was diluted with 50 mL of dichloromethane, washed with 2*20 mL of water, and dried with anhydrous sodium sulfate. The organic phase was concentrated to obtain 700 mg of compound I-77b with a yield of 93%. LC-MS (ESI+) 590.1 [M+H] + .

[0270] Preparation of compound I-77a: I-77b (640 mg, 1.1 mmol), Cbz-NH2 (197 mg, 1.3 mmol), X-phose (104 mg, 0.22 mmol), Pd(dba)3 (50 mg, 0.055 mmol), cesium carbonate (710 mg, 2.2 mmol), and toluene (10 mL) were added to a 50 mL single-neck flask, heated to 100°C, and reacted for 48 h. The reaction was sampled and controlled. The reaction solution was concentrated and prepared. Compound I-77a was obtained in a yield of 344 mg, 6.2%. LC-MS (ESI+) 661.2 [M+H] + .

[0271] Preparation of compound I-77: I-77a (44 mg) and 10% Pd / C (10 mg) were added to a 10 mL single-neck flask, 10 mL of methanol was added under nitrogen protection, and hydrogenation was carried out for 1 h. The reaction was sampled and controlled. The filtrate was collected, concentrated, and prepared and purified to obtain 15 mg of compound I-77 with a yield of 35%. LC-MS (ESI+) 527.2 [M+H] + .

[0272] Example 2-38 Synthesis of compound I-81

[0273] Preparation of compound I-81c: Compound camptothecin (1.00 g, 2.87 mmol) and FeSO4·7H2O (1.22 g, 4.32 mmol) were added to a reaction flask, stirred to completely dissolve in 28 mL of methanol, 24 mL of purified water, and 10 mL of concentrated sulfuric acid, and stirred at a temperature of 0-5°C. 3 mL of hydrogen peroxide (30%) was added dropwise. The reaction was carried out at room temperature for 2 h. 100 mL of purified water was added to the reaction system, filtered, and the solid was dried to obtain 927.71 mg of target compound I-81c with a yield of 85.26%. LC-MS (ESI+) 379.12 [M+H] + .

[0274] Preparation of compound I-81b: To I-81c (460.00 g, 1.23 mmol) was added 22 mL of 40% aqueous hydrogen bromide solution and 0.8 mL of concentrated sulfuric acid, warmed to 100°C, reacted for 2h, extracted with dichloromethane, concentrated, purified by column chromatography, and the target compound I-81b was obtained in a yield of 106.79 mg, 19.91%. LC-MS (ESI+) 441.0 [M+H] + .

[0275] Preparation of compound I-81a: (3-methylene cyclobutyl) tert-butyl carbamate (100.00 mg, 0.55 mmol) was stirred and dissolved in 5 mL of dichloromethane, 0.6 mL of hydrogen chloride ethanol solution was added, and the reaction was allowed to proceed at room temperature for 1h, and the reaction solution was concentrated to obtain the target compound I-81a in a yield of 53.69 mg, 82.27%. LC-MS (ESI+) 84.1 [M+H] + .

[0276] Preparation of compound I-81: I-81b (100.00 mg, 0.23 mmol) and I-81a (29.81 mg, 0.25 mmol) were added to a reaction bottle, 1 mL of N,N-dimethylformamide was added, stirred and dissolved, sodium carbonate (48.04 mg, 0.45 mmol) was added, and the reaction was allowed to proceed at room temperature for 0.5h, and the preparation liquid was purified by liquid chromatography to obtain the target compound I-81 in a yield of 31.72 mg, 31.56%. LC-MS (ESI+) 444.2 [M+H] + .

[0277] Example 2-39 Synthesis of compound I-100

[0278] Preparation of compound I-100a: I-1 (43.4 mg, 0.1 mmol), tert-butyl (2- (aminooxy)ethyl)carbamate (26.4 mg, 0.15 mmol), and p-toluenesulfonic acid pyridine (25.1 mg, 0.1 mmol) were weighed into a 10 mL vial, 2 mL of DMF was added, and the mixture was stirred at 50°C for 6h, LC-MS was used to monitor the completion of the reaction, and the preparation liquid was purified by liquid chromatography to obtain I-100a in a yield of 42.9 mg, 72.4%. LC-MS (ESI+) 593.6 [M+H] + .

[0279] Preparation of compound I-100: Compound I-100a (42.9 mg, 0.072 mmol) was dissolved in 2 mL of DCM, stirred at room temperature, 0.4 mL of TFA was added dropwise, stirred at room temperature for 2 h, after the reaction was completed by LC-MS monitoring, preparative liquid phase purification, I-100 33.0 mg, yield 93.1% LC-MS (ESI+) 493.5 [M+H] + .

[0280] Example 2-40 Synthesis of compound I-103

[0281] I-100 (49.3 mg, 0.1 mmol), paraformaldehyde (3.0 mg, 0.1 mmol) were placed in a 10 mL single-neck flask, 2 mL of MeOH, 27.8 ul of TEA were added, stirred at room temperature overnight, after the reaction was completed by LC-MS monitoring, preparative liquid phase purification, I-103 21.6 mg, yield 42.7% LC-MS (ESI+) 507.6 [M+H] + .

[0282] Example 2-41 Synthesis of compound I-115

[0283] Preparation of compound I-115b: To compound I-81c (927.71 mg, 2.45 mmol) was added 18 mL of methanol, 4.5 mL of concentrated sulfuric acid was stirred to dissolve, 463.12 mg of MnO2 was added. The temperature was raised to 50°C, and the reaction was carried out for 2 h. The reaction solution was filtered, dichloromethane was added to the filtrate, and the organic phase was concentrated under reduced pressure to obtain the target compound I-115b 832.24 mg, yield 80.03%. LC-MS (ESI+) 423.15 [M+H] + .

[0284] Preparation of compound I-115a: To compound I-115b (832.24 mg, 1.97 mmol) was added 24 mL of acetonitrile, 10 mL of purified water and 6 mL of concentrated hydrochloric acid was stirred to dissolve. The temperature was raised to 70°C, and the reaction was carried out for 1 h. It was lowered to 0-5°C, 75 mL of purified water was added to the reaction system, stirred for 10 min, then filtered, and the solid was dried under vacuum to obtain the target compound I-115a 563.24 mg, yield 73.40%. LC-MS (ESI+) 391.13 [M+H] + .

[0285] Preparation of compound I-115: Into a reaction flask was added compound I-115a (563.24 mg, 1.44 mmol), paraformaldehyde (259.85 mg, 2.88 mmol) and isopropylamine hydrochloride (276.01 mg, 2.88 mmol), which was stirred in 25 mL of N,N-dimethylformamide and 0.8 mL of purified water. After 5 h of reaction at room temperature, the preparation liquid was purified to obtain 140.05 mg of the target compound I-115 with a yield of 24.05%. LC-MS (ESI+) 403.12 [M+H] + .

[0286] Example 2-42 Synthesis of compound I-143 Preparation of compound I-43: I-29b (200 mg, 0.47 mol) was dissolved in 10 mL of methanol, 40.00 mg of Pd / C was added, and hydrogen was replaced for 3 times. After 1 h of reaction at room temperature, the reaction liquid was purified by preparation liquid phase to obtain 56.98 mg of the target compound I-143 with a yield of 34.22%. LC-MS (ESI+) 449.2 [M+H] + .

[0287] Example 3 Synthesis of linker-toxin compound

[0288] Example 3-1 Synthesis of compound II-1

[0289] Synthesis of compound II-1b: I-2 (50.0 mg, 0.11 mmol) was weighed into a single-neck flask and dissolved in 3 mL of tetrahydrofuran and stirred at room temperature. V-1 (274.4 mg, 0.56 mmol) and cesium carbonate (36.2 mg, 0.11 mmol) were sequentially weighed into the reaction flask and reacted at room temperature for 3 h. After the reaction was completed, the preparation liquid phase was purified to obtain 43.4 mg of the target product compound II-1b with a yield of 48%. LC-MS (ESI+) 817.2 [M+H] + .

[0290] Synthesis of compound II-1a: II-1b (43.4 mg, 0.05 mmol) was weighed into a single-neck flask, 2 mL of methanol and 1 mL of tetrahydrofuran were added to the flask to completely dissolve it, and palladium on carbon (9.2 mg, 0.08 mmol) was weighed into the flask, hydrogen was replaced, and it was stirred at room temperature for 2 h. After the reaction was completed, the palladium on carbon was filtered off, the solvent was rotary evaporated, and 21.4 mg of the target compound II-1a was obtained, which was directly subjected to the next step. LC-MS (ESI+) 683.8 [M+H] + .

[0291] Synthesis of compound II-1 : dissolve II-1a (21.4 mg, 0.03 mmol) in 2 mL of N,N- dimethylformamide, add W-3 (15.9 mg, 0.04 mmol) at room temperature, add N,N- diisopropylethylamine (6.1 mg, 0.05 mmol) at room temperature, stir for 4 h at room temperature. Purify by preparative liquid phase to obtain the target compound II-1 5.0 mg, yield 17%. LC-MS (ESI+) 990.7 [M+H] + .

[0292] Synthesis of compound II-2

[0293] Preparation of compound II-2b: weigh I-4 (60 mg, 0.12 mmol) into a single-mouth bottle, dissolve using 3 mL of tetrahydrofuran, stir at room temperature. Weigh V-1 (256 mg, 0.6 mmol), p-toluenesulfonic acid hydrate (34 mg, 0.18 mmol) into the reaction bottle in turn, react at room temperature for 3 h. After the reaction is completed, purify by preparative liquid phase. Obtain the target product II-2b 63 mg, yield 61%. LC-MS (ESI+) 861.5 [M+H] + .

[0294] Preparation of compound II-2a: weigh II-2b (63 mg, 0.073 mmol) into a single-mouth bottle, add 5 mL of methanol to the bottle to completely dissolve, weigh palladium on carbon (19 mg, 0.18 mmol) into the bottle, replace with hydrogen, stir for 2 h at room temperature. After the reaction is completed, filter out the palladium on carbon, spin dry the solvent system to obtain the target compound II-2a, which is directly subjected to the next step. LC-MS (ESI+) 727.5 [M+H] + .

[0295] Preparation of compound II-2: dissolve II-2a (21.4 mg, 0.03 mmol) in 2 mL of N,N- dimethylformamide, add W-3 (15.9 mg, 0.04 mmol) at room temperature, add N,N- diisopropylethylamine (6.1 mg, 0.05 mmol) at room temperature, stir for 4 h at room temperature. Purify by preparative liquid phase to obtain the target compound II-1 5.0 mg, yield 17%. LC-MS (ESI+) 990.7 [M+H] + .

[0296] Synthesis of compound II-4

[0297] Preparation of compound II-4b: Compound V-2 (1376.6 mg, 3.5 mmol), I-4 (345 mg, 0.7 mmol) were dissolved in 15 mL of N,N-dimethylformamide, 1.5 mL of trifluoroacetic acid was added, and stirred at room temperature for 3 h. The reaction system was purified by preparative liquid phase to obtain 372 mg of the target compound II-4b with a yield of 64%. LC-MS (ESI+) 827.7 [M+H] + .

[0298] Preparation of compound II-4a: Compound II-4b (372 mg, 0.45 mmol) was dissolved in 6 mL of methanol. Palladium on carbon (111.6 mg, 30%) was added at room temperature. Hydrogen was replaced three times and stirred at room temperature for 1 h. The system was filtered, and the palladium on carbon was washed with 10 mL of methanol three times, and the filtrate was rotary evaporated to obtain the crude product II-4a which was directly used in the next step. LC-MS (ESI+) 693.4 [M+H] + .

[0299] Preparation of compound II-4: Compound II-4a (89 mg, 0.12 mmol) was dissolved in 5 mL of N,N-dimethylformamide, W-5 (65 mg, 0.12 mmol), EDCI (49.3 mg, 0.26 mmol) and HOBT (35 mg, 0.26 mmol) were added at room temperature, followed by the addition of N,N-diisopropyl ethylamine (33.6 μL, 0.2 mmol) and stirred at room temperature for 2 h. The system was purified by preparative liquid phase to obtain 42.6 mg of the target compound II-4 with a yield of 30%. LC-MS (ESI+) 1183.76 [M+H] + .

[0300] Synthesis of compound II-5 in Example 3-4

[0301] Preparation of compound II-5: According to the preparation method of the last step of compound II-4, the raw material was changed to W-7 to obtain 51.5 mg of the target compound II-5 with a yield of 34%. LC-MS (ESI+) 1183.45 [M+H] + .

[0302] Synthesis of compound II-15 in Example 3-5

[0303] Preparation of compound II-15: According to the complete preparation method of compound II-4, the raw material was changed to I-27 to obtain 10.38 mg of the target compound II-15 with a yield of 58%. LC-MS (ESI+) 1182.45 [M+H] + .

[0304] Synthesis of compound II-16 in Example 3-6

[0305] Preparation of compound II-16: according to the preparation method of the last step of compound II-15, the raw material was changed to W-7, and the target compound II-16 9.83 mg was obtained with a yield of 68%. LC-MS (ESI+) 1182.48 [M+H] + .

[0306] Synthesis of compound II-25 of example 3-7

[0307] Preparation of compound II-25b: I-24 (45.12 mg, 0.10 mmol) and DMAP (14.52 mg, 0.30 mmol) were dissolved in 2 mL of dichloromethane, and the temperature was lowered to 0°C. Triflic anhydride (12.03 mg, 0.04 mmol) was weighed and dissolved in 0.5 mL of dichloromethane, and slowly added to the reaction solution. After 0.5 hours of reaction, Fmoc-VC-PAB-OH (63.25 mg, 0.10 mmol) was weighed and dissolved in 0.5 mL of dimethyl sulfoxide, and added dropwise to the reaction solution at 0°C. The temperature was restored to room temperature, and the reaction was stirred for 3 hours. The system was purified by preparative liquid chromatography to obtain the target compound II-25b 71.25 mg with a yield of 65.81%. LC-MS (ESI+) 1076.4 [M+H] + .

[0308] Preparation of compound II-25a: II-25b (53.81 mg, 0.05 mmol) was stirred and dissolved in 2 mL of N,N-dimethylformamide, diethylamine (18.28 mg, 0.25 mmol) was added, and the reaction was carried out at room temperature for 1 hour. The system was purified by preparative liquid chromatography to obtain the target compound II-25a 35.95 mg with a yield of 84.19%. LC-MS (ESI+) 854.4 [M+H] + .

[0309] Preparation of compound II-25: II-25a (26.83 mg, 0.03 mmol) and W-8 (19.22 mg, 0.03 mmol) were stirred and dissolved in 2.0 mL of N,N-dimethylformamide, HATU (13.82 mg, 0.04 mmol) and N,N diisopropyl ethylamine (11.74 mg, 0.10 mmol) were added, and the reaction was carried out at room temperature for 1 hour. The system was purified by preparative liquid chromatography to obtain II-25 29.43 mg with a yield of 64.67%, LC-MS (ESI+) 1052.7 [M+H] + .

[0310] Synthesis of compound II-26 of example 3-8

[0311] Preparation of compound II-26b: Compound I-1 (200 mg, 0.44 mmol), 80% hydrazine hydrate (35.2 mg, 0.88 mmol) were mixed with 8 mL dichloromethane and 2 mL methanol, stirred at room temperature for 24 h. The system was added with 20 mL n-heptane and stirred for 2 h, and the target compound II-26b was obtained by filtration, 165 mg, yield 80%. LC-MS (ESI+) 448.15 [M+H] + .

[0312] Preparation of compound II-26a: II-26b (250.11 mg, 0.56 mmol), V-3 (308.19 mg, 0.67 mmol) and HATU (253.88 mg, 0.67 mmol) were weighed and dissolved in 20 mL N,N-dimethylformamide, and N,N-diisopropylethylamine (216.26 mg, 1.68 mmol) was added, stirred at room temperature for 3 h, and the system was purified by preparative liquid chromatography to obtain the target compound II-26a 393.55 mg, yield 79.20%. LC-MS (ESI+) 891.38 [M+H] + .

[0313] Preparation of compound II-26: II-26a (90.05 mg, 0.10 mmol) and W-6 (34.22 mg, 0.10 mmol) were weighed and added to acetone (6 mL) and purified water (2 mL) and stirred to dissolve, and protected by nitrogen; copper sulfate pentahydrate (25.19 mg, 0.10 mmol) and sodium ascorbate (21.04 mg, 0.10 mmol) were weighed and dissolved in 0.6 mL purified water respectively, and added to the reaction liquid in turn, and stirred at room temperature for 0.5 h. The system was purified by preparative liquid chromatography to obtain II-26a 87.37 mg, yield 72.26%. LC-MS (ESI+) 1196.46 [M+H] + .

[0314] Synthesis of compound II-27 in Example 3-9

[0315] Preparation of compound II-27: According to the preparation method of the last step of compound II-26, the raw material was changed to W-4 to obtain the target compound II-27 91.89 mg, yield 76.89%. LC-MS (ESI+) 1196.43 [M+H] + .

[0316] Synthesis of compound II-30 in Example 3-10

[0317] Preparation of compound II-30d: V-2 (393.4 mg, 1.0 mmol), 3-hydroxymethylazetidine-1-carboxylic acid tert-butyl ester (936.2 mg, 5 mmol), p-toluenesulfonic acid monohydrate (190.2 mg, 1.0 mmol) were dissolved in a 50 mL three-necked flask, replaced with N2 for three times, added tetrahydrofuran 20 mL, stirred at room temperature overnight, the reaction solution was centrifuged, the filter cake was washed with tetrahydrofuran (5 mL*3), the combined filtrate was concentrated under reduced pressure, dissolved in N,N-dimethylformamide and purified by preparative liquid chromatography, and then freeze-dried to obtain the target compound II-30d 324.9 mg with a yield of 62.4%. LC-MS (ESI+) 521.6 [M+H] + .

[0318] Preparation of compound II-30c: III-30d (324.9 mg, 0.62 mmol) was dissolved in 5 mL dichloromethane, 1 mL trifluoroacetic acid was added, stirred at room temperature for 1 h, and the reaction solution was concentrated under reduced pressure to obtain the crude product II-30c 280.1 mg. LC-MS (ESI+) 421.6 [M+H] + .

[0319] Preparation of compound II-30b: II-30c crude product 280.1 mg, I-44aA (372.3 mg, 0.62 mmol) were dissolved in 5 mL N,N-dimethylformamide, N,N-diisopropylethylamine (250 mg, 1.93 mmol) was added to adjust the pH to weak alkaline, stirred at room temperature for 2 h, purified by preparative liquid chromatography, and then freeze-dried to obtain the target compound II-30b 499.8 mg with a yield of 91.3%. LC-MS (ESI+) 883.9 [M+H] + .

[0320] Preparation of compound II-30a: II-30b (441.5 mg, 0.5 mmol), 10% palladium-carbon 88.5 mg were placed in a 25 mL single-necked flask, replaced with H2, added 8 mL tetrahydrofuran, stirred at room temperature for 2 h, the reaction solution was filtered, 10 mL H2O was added, and then freeze-dried to obtain the target compound II-30a 349.5 mg with a yield of 93.3%. LC-MS (ESI+) 749.8 [M+H] + .

[0321] Preparation of compound II-30: II-30a (37.4 mg, 0.05 mmol), W-9 (22.3 mg, 0.06 mmol), HATU (22.8 mg, 0.06 mmol) in 10 mL vial, 3 mL N,N-dimethylformamide, N,N diisopropyl ethylamine (12.93 mg, 0.1 mmol), stirring at room temperature for 3 h, preparation of liquid phase purification, freeze-drying, target compound II-30 40.3 mg, yield 73.2%. LC-MS (ESI+) 1102.2 [M+H] + .

[0322] Example 3-11 Synthesis of compound II-28

[0323] Preparation of compound II-28: according to the preparation method of compound II-30, the raw material of the first step was changed to N-Boc-3-hydroxyazetidine, and finally the target compound II-28 30.5 mg was obtained. LC-MS (ESI+) 1087.47 [M+H] + .

[0324] Example 4 Preparation of antibody drug conjugate

[0325] The preparation of antibody drug conjugate adopts a general conjugation method: the reducing agent and the protecting agent are prepared with purified water as follows: 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine), 1-20 mM DTPA (Diethylene triamine pentacetate acid) stock solution, the amount of reducing agent can be added within a certain concentration range according to the different conjugation rates required, mixed with a certain concentration of monoclonal antibody (such as: 5-30 mg / mL), the final molar ratio of TCEP to antibody is 10-14:1, and the reaction is stirred at 25°C for 1 h. The antibody reduced by TCEP can be directly conjugated.

[0326] A certain concentration (5 mM) of linker-active drug unit compound is dissolved in DMSO (dimethyl sulfoxide), and the drug is slowly added according to the molar ratio of drug to thiol of 10-15:1, and the reaction is stirred at 25°C for 1-4 h. After the reaction is completed, centrifugal ultrafiltration is carried out with His.Hcl buffer, and free small molecules such as residual unreacted drugs and DMSO are removed by purification. The conjugation is detected by hydrophobic high performance liquid chromatography (HIC-HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods, and the purity of the conjugated sample is detected by size exclusion chromatography (SEC). Using the preparation method, the following antibody drug conjugates are exemplarily synthesized:

[0327] wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, representing the number of linker-load moieties attached on the antibody is 1, 2, 3, 4, 5, 6, 7, or 8. The RA1and RA6are two antibodies targeting CDCP1 prepared by the inventors themselves.

[0328] Example 5 Biological research test

[0329] Example 5-1 In vitro small molecule toxicity test

[0330] 1. Test method

[0331] Cells PC-3 (human prostate cancer cell line), EBC-1 (human lung cancer squamous cell line), HCC827 (human non-small cell lung cancer cell line), BXPC-3 (human pancreatic adenocarcinoma cell line in situ), MDA-MB-231 (human breast cancer cell line), BJ (human skin fibroblast cell line) in the exponential growth phase were inoculated in 96-well plates at a density of 5 x 10 4 ~ 1 x 10 5 / mL, 100 uL per well, and incubated overnight at 37°C in a 5% CO2incubator. The serial camptothecin compounds were prepared in complete culture medium at concentrations of 0-100 ug / mL, 100 uL was added to the wells, 3 replicates were set for each concentration, and a blank control group was also set. After 72±2h of drug action, CCK-8 reagent was added, and incubated at 37°C in a 5% CO2incubator for 1-4h, and the OD value of each well was detected at 450nm on a microplate reader. The inhibition rate IR% = (OD blank-OD drug) x 100 / OD blank was calculated. The inhibition rate and drug concentration were fitted with four parameters to calculate the IC50, and the specific IC50values are shown in Tables 2-1~2-8.

[0332] Table 2-1 Inhibition activity IC50values of camptothecin compounds on various cancer cell lines

[0333] The experimental results of Table 2-1 show that 1) the inhibition activity of compounds I-1, I-3b, I-3a and I-4 on PC-3, EBC-1, HCC827 and BXPC-3 4 cancer cell lines is excellent and better than that of Dxd. 2) The inhibition activity of compounds I-1, I-3b, I-3a and I-4 on PC-3, EBC-1, HCC827 and BXPC-3 4 cancer cell lines is better than that on MDA-MB-231. 3) The inhibition activity of compounds I-3b, I-3a and I-4 on MDA-MB-231 cell line is significantly better than that of Dxd on the cell line.

[0334] Table 2-2 IC50 values of camptothecin compounds against various cancer cell lines

[0335] The experimental results of Table 2-2 show that 1) the inhibitory activity of compound I-27 is excellent and superior to that of Dxd in the 5 cancer cell lines tested; 2) the inhibitory activity of compound I-24 against EBC-1 and BXPC-3 is comparable to that of Dxd; 3) the inhibitory activity of compound I-19 against MDA-MB-231 is comparable to that of Dxd; 4) the inhibitory activity of compounds I-15, I-19, I-24 and I-27 against PC-3, EBC-1, HCC827 and BXPC-3 is significantly superior to that against MDA-MB-231.

[0336] Table 2-3 IC50 values of camptothecin compounds against various cancer cell lines

[0337] The experimental results of Table 2-3 show that the inhibitory activity of compound I-35 against PC-3, HCC827, BXPC-3 and MDA-MB-231 is comparable to that of Dxd.

[0338] Table 2-4 IC50 values of camptothecin compounds against various cancer cell lines

[0339] The experimental results of Table 2-4 show that the inhibitory activity of compounds I-44A and I-44B against PC-3, EBC-1, HCC827 and BXPC-3 is excellent and significantly superior to that of Dxd; and the inhibitory activity against MDA-MB-231 is also significantly superior to that of Dxd; the inhibitory activity of compounds I-44A and I-44B against PC-3, EBC-1, HCC827 and BXPC-3 is superior to that against MDA-MB-231.

[0340] Table 2-5 IC50 values of camptothecin compounds against various cancer cell lines

[0341] The experimental results of Table 2-5 show that the inhibitory activity of compounds I-61A and I-61B against the five cancer cell lines tested is excellent and superior to that of Dxd.

[0342] Table 2-6 IC50 values of camptothecin compounds against various cancer cell lines

[0343] The experimental results of Table 2-6 show that the inhibitory activity of compound I-65A on the five cancer cell lines verified is excellent and is superior to the inhibitory activity of Dxd.

[0344] Table 2-7 IC50 values of camptothecin compounds on various cancer cell lines

[0345] The experimental results of Table 2-7 show that the inhibitory activity of compound I-5 on the five cancer cell lines verified is excellent and is superior to the inhibitory activity of Dxd.

[0346] Table 2-8 IC50 values of camptothecin compounds on various cancer cell lines

[0347] The experimental results of Table 2-8 show that the inhibitory activity of compound I-28 on the five cancer cell lines verified is excellent and is superior to the inhibitory activity of Dxd.

[0348] Based on the above analysis, the camptothecin compounds in the application not only have broad-spectrum inhibitory activity on various cancer cell lines, but also have a differential advantage in MDA-MB-231 breast cancer cell lines compared with Dxd.

[0349] Example 5-2 SD rat in vivo pharmacokinetic study

[0350] The quarantine qualified SD rats were taken, 6 in each group, half male and half female. The compound was prepared on the day of administration in the example, and the solvent was 5% DMSO+10% solutol+85% saline. Before administration, the weight was weighed, and the administration amount was calculated according to the weight, the administration dose was 1 mg / kg, the administration volume was 5 mL / kg, and the administration was performed by intravenous injection. Before administration and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration, blood was collected from the jugular vein, about 0.2 mL of each sample was collected, heparin sodium was anticoagulated, and the plasma was separated by centrifugation (centrifugation conditions: 6800g, 6 minutes, 2-8℃) within 1 hour. 40uL of plasma sample was added with 160uL of acetonitrile for precipitation, mixed and centrifuged at 3500xg for 5-20 minutes, and 100uL of supernatant after treatment was used for LC / MS / MS analysis of the concentration of the test compound. By using the blood drug concentration data at different time points, the pharmacokinetic parameters were calculated by Phoenix WinNonlin7.0 to provide AUClast (reflecting the total exposure in vivo), MRTlast, Cmax (maximum blood drug concentration), Tmax (time to reach maximum blood drug concentration), and T1 / 2 (drug half-life), Cl_obs (clearance rate) and other parameters and their mean and standard deviation as shown in Tables 3-1 to 3-3, wherein,

[0351] 1) The formula for calculating the percentage of decrease in in vivo exposure of the camptothecin compound group relative to Dxd is as follows: a. Calculate the amount of decrease in in vivo exposure: Decrease = (AUG (Dxd) - AUG (喜树碱化合物) ) b. Calculate the percentage of decrease in in vivo exposure: Decrease percentage = (Decrease / AUG (Dxd) ) x 100%

[0352] 2) The formula for calculating the percentage of acceleration in in vivo clearance of the camptothecin compound group relative to Dxd is as follows: Acceleration percentage = ((Cl_obs (喜树碱化合物) - Cl_obs (Dxd) ) / Cl_obs (Dxd) ) x 100%

[0353] Table 3-1 Camptothecin compound pharmacokinetic (PK) data

[0354] The experimental results in Table 3-1 show that, compared with Dxd, compound I-76 has a shorter half-life, lower in vivo exposure (decrease percentage: 24.21%) and faster in vivo clearance rate (acceleration percentage: 43.32%) in rats, indicating that the in vivo safety of I-76 is superior to that of Dxd.

[0355] Table 3-2 Camptothecin compound pharmacokinetic (PK) data

[0356] The experimental results in Table 3-2 show that, compared with Dxd, compound I-5 has a longer half-life in rats, but compound I-5 has lower in vivo exposure (decrease percentage: 24.92%) and faster in vivo clearance rate (acceleration percentage: 52.69%).

[0357] Table 3-3 Camptothecin compound pharmacokinetic (PK) data

[0358] The experimental results in Table 3-3 show that, compared with Dxd, compound I-49 has a longer half-life in rats, but compound I-49 has lower in vivo exposure (decrease percentage: 45.48%) and faster in vivo clearance rate (acceleration percentage: 74.73%).

[0359] Example 5-3 Inhibition experiment of topoisomerase I activity

[0360] According to the experimental requirements, the reaction system is prepared, which contains 10x Reaction buffer, TOPO 1 and H2O, then 1 μL of camptothecin compound is added, and the reaction is carried out at room temperature for 15 min, 0.5 μL of pBR322 is added after the reaction is completed, and then vortexed, separated and placed in a 37℃ incubator for 30 min, the sample hole of the cooled agarose gel is close to the negative electrode, 2.5 μL of 10x DNA loading buffer is mixed with 25 μL of sample, and 10 μL is taken in turn; the voltage is set to 130V, and electrophoresis is carried out for 30 min; after electrophoresis, 0.05% Sparkgreen (60 mL purified water + 30 μL Sparkgreen) is prepared and dyed for 30 min, and then rinsed with water for 1 min, and photographed by using a gel imaging instrument, and two parallel experiments (experiments 1 and 2) are carried out for each compound, and the inhibitory activity of camptothecin compounds on topoisomerase I is shown in Table 4.

[0361] Table 4 Inhibitory activity of camptothecin compounds on topoisomerase I

[0362] Summary: The experimental results show that the camptothecin compounds in the application have significant inhibitory activity on topoisomerase I, especially compounds I-2, I-28, I-6, I-5, I-44B, I-41, I-29, I-27, I-65A, I-4, I-147, I-44A, which are equivalent to or better than DX8951 and Dxd in vitro on topoisomerase I activity, and the results are basically consistent with the results of cell activity evaluation.

[0363] Example 5-4 In vitro anti-proliferation effect test of antibody drug conjugate (ADC)

[0364] The cells PC-3, EBC-1, HCC827, BXPC-3, MDA-MB-231 and BJ in the exponential growth phase are inoculated in a 96-well plate at a density of 5x10 4 ~1x10 5 / mL, 100ul per well. Incubate in a 37℃, 5% CO2 incubator overnight. The ADC is prepared in a gradient of 0-3333.3nM with complete culture medium, 100ul is added to the well, and 3 replicate wells are set up for each concentration, and a blank control group is also set up. After 72-144h of drug action, add CCK-8 reagent, incubate in a 37℃, 5% CO2 incubator for 1-4h, and detect the OD value of each well at 450nm of the enzyme label instrument. Calculate the inhibition rate IR%=(OD blank-OD drug) x 100 / OD blank. The inhibition rate and drug concentration are fitted by four parameters, and the IC50 is calculated, and the specific IC50 values are shown in Tables 5-1 to 5-10.

[0365] Table 5-1 IC50 values of antibody drug conjugates against the anti-proliferation effect on various cancer cell lines

[0366] The test results of Table 5-1 show that the ADCs (RA6III-4 and RA6III-5) constructed with compound I-4 as the payload have a significantly superior anti-proliferation effect on the three cancer cell lines of BXPC-3, PC-3 and EBC-1 than the ADC (RA6-DL01-Dxd) constructed with Dxd as the payload.

[0367] Table 5-2 IC50 values of antibody drug conjugates against the anti-proliferation effect on various cancer cell lines

[0368] The test results of Table 5-2 show that the ADC (RA6III-27) constructed with compound I-5 as the payload has a superior anti-proliferation effect on the five cancer cell lines tested and is superior to the ADC (RA6-DL01-Dxd) constructed with Dxd as the payload, especially the anti-proliferation effect on EBC-1 is significantly superior to that of RA6-DL01-Dxd.

[0369] Table 5-3 IC50 values of antibody drug conjugates against the anti-proliferation effect on various cancer cell lines

[0370] The test results of Table 5-3 show that the ADC (RA1III-27) constructed with compound I-5 as the payload and having a DAR of 8 has a superior anti-proliferation effect on the five cancer cell lines tested and is superior to the ADC (RA1-DL01-Dxd) constructed with Dxd as the payload, especially the anti-proliferation effect on EBC-1 and MDA-MB-231 is significantly superior to that of RA1-DL01-Dxd.

[0371] Table 5-4 IC50 values of antibody drug conjugates against the anti-proliferation effect on various cancer cell lines

[0372] The test results of Table 5-4 show that the ADCs (RA1III-26 and RA6III-26) constructed with compound I-5 as the payload and having a DAR of 6 have a superior anti-proliferation effect on the two cancer cell lines of BXPC-3 and PC-3 and are comparable to the effect of RA6-DL01-Dxd; the anti-proliferation effect of RA1III-26 and RA6III-26 on EBC-1 is superior and significantly superior to that of RA6-DL01-Dxd.

[0373] Table 5-5 IC50 values of antibody drug conjugates against the anti-proliferation effect on various cancer cell lines

[0374] Table 5-5 shows that the ADC (RA6III-25) constructed by using compound I-24 as the payload has excellent anti-proliferative effects on four cancer cell lines, i.e., BXPC-3, PC-3, HCC827, and MDA-MB-231, and the anti-proliferative effects on BXPC-3 and HCC827 are significantly superior to those of the ADC (RA6-DL01-Dxd) constructed by using Dxd as the payload.

[0375] Table 5-6 shows the IC50 values of the anti-proliferative effects of the antibody drug conjugates on various cancer cell lines.

[0376] Table 5-6 shows that the ADC (RA6III-25) constructed by using compound I-24 as the payload has excellent anti-proliferative effects on four cancer cell lines, i.e., BXPC-3, PC-3, HCC827, and MDA-MB-231, and the anti-proliferative effects on BXPC-3 and HCC827 are significantly superior to those of the ADC (RA6-DL01-Dxd) constructed by using Dxd as the payload.

[0377] Table 5-7 shows the IC50 values of the anti-proliferative effects of the antibody drug conjugates on various cancer cell lines.

[0378] Table 5-7 shows that the ADC (RA1III-28) constructed by using compound I-44A as the payload has excellent anti-proliferative effects on five cancer cell lines, i.e., BXPC-3, PC-3, EBC-1, HCC827, and MDA-MB-231, and the anti-proliferative effect on EBC-1 is significantly superior to that of the ADC (RA1-DL01-Dxd), and the anti-proliferative effects on BXPC-3, HCC827, and MDA-MB-231 are superior to those of the ADC (RA1-DL01-Dxd).

[0379] Table 5-8 shows the IC50 values of the anti-proliferative effects of the antibody drug conjugates on various cancer cell lines.

[0380] Table 5-8 shows that the ADC (RA1III-30) constructed by using compound I-61A as the payload has excellent anti-proliferative effects on four cancer cell lines, i.e., BXPC-3, PC-3, EBC-1, and HCC827, and the anti-proliferative effects on BXPC-3, EBC-1, and BJ are superior to those of the ADC (RA1-DL01-Dxd) constructed by using Dxd as the payload, and RA1III-30 has a significant anti-proliferative advantage over the ADC (RA1-DL01-Dxd) on EBC-1.

[0381] Table 5-9 shows the IC50 values of the anti-proliferative effects of the antibody drug conjugates on various cancer cell lines.

[0382] The test results in Table 5-9 show that the anti-proliferation effect of the ADC (RA1 III-31) with DAR of 8 constructed by using compound I-65A as the payload on four cancer cell lines, including BXPC-3, PC-3, EBC-1, HCC827, and MDA-MB-231, is superior to that of RA1-DL01-Dxd.

[0383] Table 5-10 IC50 values of the anti-proliferation effect of the antibody drug conjugate on various cancer cell lines

[0384] The test results in Table 5-10 show that the anti-proliferation effect of the ADC (RA6 III-29) constructed by using compound I-76 as the payload on three cancer cell lines, including BXPC-3, PC-3, and EBC-1, is excellent, and the ADC constructed by using compound I-76 as the payload has a significant advantage on the MDA-MB-231 cell line compared with the ADC (RA6-DL01-Dxd) constructed by using Dxd as the payload.

[0385] Based on the above analysis, the antibody drug conjugate constructed by using the camptothecin compound in the application and different types of linkers on various cancer cell lines shows a significant anti-proliferation effect or selective advantage. Compared with the antibody drug conjugate (control group: RA1-DL01-Dxd and RA6-DL01-Dxd) constructed by using the camptothecin derivative Dxd, the antibody drug conjugate has a more excellent or equivalent anti-proliferation effect on the cancer cell line.

[0386] Example 6 Biological in vivo study test

[0387] Example 6-1 Pharmacodynamic study of the antibody drug conjugate in a human lung squamous carcinoma cell EBC-1 subcutaneous tumor model

[0388] 1. Research method: After the human lung squamous carcinoma cell EBC-1 (source: Nanjing Kebai) was cultured by amplification, it was inoculated subcutaneously on the back of the right front limb of BALB / c-nu mice (Jisui Pharmaceutical, female). The growth of the tumor was observed. When the tumor volume reached about 100-300 mm 3 , the tumor was randomly grouped according to the tumor volume and dosed. The day of dosing was recorded as D0. During the test period (D0-D21), the tumor long diameter, short diameter, and body weight were measured twice a week, and the clinical symptoms were observed. At the end of the test, the experimental mice were euthanized.

[0389] 2. Results: The dosing and test results are shown in Table 6 and FIG. 1.

[0390] Table 6 Anti-tumor results of the antibody drug conjugate in the EBC-1 tumor model

[0391] 3. Summary: According to the test results shown in Table 6 and Figure 1, the antibody drug conjugate constructed by the camptothecin compound in the application shows significant anti-tumor effect in the EBC-1 tumor model, and its tumor inhibition effect is significantly better than that of RA1-DL01-Dxd constructed by Dxd.

[0392] Example 6-2 Pharmacodynamic study of antibody drug conjugate in human lung squamous carcinoma cell EBC-1 subcutaneous tumor model

[0393] 1. Research method: After the human lung squamous carcinoma cell EBC-1 (source: Nanjing Kebai) was cultured by amplification, it was inoculated subcutaneously on the back of the right forelimb of BALB / c-nu mice (Jisui Pharmaceutical, female). The growth of the tumor was observed. When the tumor volume reached about 100-300mm 3 , the mice were randomly grouped according to the tumor volume and administered. The day of administration was recorded as D0. During the test period (D0-D21), the tumor long diameter, short diameter and body weight were measured twice a week, accompanied by clinical symptom observation. At the end of the test, the experimental mice were euthanized.

[0394] 2. Results: The administration dose and test results are shown in Table 7 and Figure 2.

[0395] Table 7 Anti-tumor results of antibody drug conjugate in EBC-1 tumor model

[0396] Summary: According to the test results shown in Table 7 and Figure 2, the antibody drug conjugate constructed by the camptothecin compound in the application with DAR of 4 shows significant anti-tumor effect in the EBC-1 tumor model and the effect is close to that of RA1-DL01-Dxd with DAR of 8.

[0397] Example 6-3 Pharmacodynamic study of antibody drug conjugate in human pancreatic cancer cell BxPC-3 subcutaneous tumor model

[0398] 1. Research method: After the human pancreatic cancer cell BxPC-3 was cultured by amplification, it was inoculated subcutaneously on the back of the right forelimb of CB17-SCID mice (Vitron Life Science, female). The growth of the tumor was observed. When the tumor volume reached 100-300mm 3 , the mice were randomly grouped according to the tumor volume and administered.

[0399] The day of administration was recorded as D0. During the test period (D0-D18), the tumor long diameter, short diameter and body weight were measured twice a week, accompanied by clinical symptom observation. At the end of the test, the experimental mice were euthanized.

[0400] 2. Results: The administration dose and test results are shown in Table 8 and Figure 3.

[0401] Table 8 Anti-tumor results of antibody drug conjugates in BxPC-3 tumor model

[0402] 3. Summary: According to the test results shown in Table 8 and Figure 3, the antibody drug conjugates of camptothecin compounds in the present application (RA1 III-27, RA1 III-31, RA1 III-4) showed significantly better anti-tumor effects than RA1-DL01-Dxd in the BxPC-3 tumor model.

[0403] Example 6-4 Pharmacodynamic study of antibody drug conjugates in human colon cancer cell RKO subcutaneous transplantation tumor model

[0404] 1. Research method: After the human colon cancer cell RKO was cultured, it was inoculated subcutaneously on the back of the right front limb of BALB / c-nu mice (Jisui Yakang, female). The growth of the tumor was observed. When the tumor volume reached 100-300 mm 3 3, the mice were randomly grouped according to the tumor volume and administered. The day of administration was recorded as D0. During the test period (D0-D21), the tumor long diameter, short diameter and body weight were measured twice a week, and the clinical symptoms were observed. At the end of the test, the experimental mice were euthanized.

[0405] 2. Results: The administration dose and test results are shown in Table 9 and Figure 4.

[0406] Table 9 Anti-tumor results of antibody drug conjugates in RKO tumor model

[0407] 3. Summary: According to the test results shown in Table 9 and Figure 4, the antibody drug conjugates of camptothecin compounds in the present application showed significant anti-tumor effects in the RKO tumor model. In particular, RA1 III-4 reached an 84.4% tumor inhibition effect at a dose of 3 mg / kg.

[0408] Example 7 In vivo toxicity study of antibody drug conjugates

[0409] Example 7-1 Toxicity study of RA1 III-4 (DAR8) and RA1 III-5 (DAR8)

[0410] 1. Purpose of test

[0411] ICR mice were administered RA1 III-4 (DAR8) and RA1 III-5 (DAR8) intravenously. The nature and degree of toxicity reactions that the antibody-drug conjugate can cause and the maximum tolerance of ICR mice were observed.

[0412] 2. Experimental method

[0413] 28 ICR mice, female, body weight at grouping 26-29 g, 2 molecules were divided into low dose group (150 mg / kg), medium dose group (450 mg / kg), high dose group (600 mg / kg), among which 450 mg / kg and 600 mg / kg, through clinical observation, body weight, etc. to investigate the tolerance of animals, body weight change and drug related toxicity, the test lasted for 14 days.

[0414] 3. Test results

[0415] The results of clinical performance of mice after administration are shown in Table 10.

[0416] Table 10 Results of clinical performance of mice after administration

[0417] Table 10 shows that RA1III-4 and RA1III-5 have 100% (4 / 4) mortality at 450 mg / kg and 600 mg / kg. No death was observed at 150 mg / kg dose, and the body weight changes of the two molecules were comparable. The RA1III-4 molecule showed transient piloerection, and the RA1III-5 molecule showed no abnormalities.

[0418] 4. Test conclusion

[0419] ICR mice were given RA1III-4 (DAR8) and RA1III-5 (DAR8) at 150 mg / kg, 450 mg / kg and 600 mg / kg. The tolerance of ICR mice to RA1III-4 (DAR8) and RA1III-5 (DAR8) was 150 mg / kg-450 mg / kg, and the toxicity was basically comparable.

[0420] Example 7-2 Toxicity study of RA1III-5 (DAR4) and RA1III-5 (DAR8)

[0421] 1. Purpose of test

[0422] ICR mice were given RA1III-5 (DAR4) and RA1III-5 (DAR8). The toxicity of the two molecules was compared in terms of toxicity reaction properties and degree.

[0423] 2. Experimental method

[0424] 12 ICR mice, female, body weight at grouping 25-28 g, 2 molecules were given 300 mg / kg. Through clinical observation, body weight, etc. to investigate the tolerance of animals, body weight change and drug related toxicity, the test lasted for 14 days.

[0425] 3. Test results

[0426] The results of the clinical manifestations of the mice after administration are shown in Table 11.

[0427] Table 11 Results of the clinical manifestations of the mice after administration

[0428] At a dose of 300 mg / kg, RA1 III-5 (DAR 8) caused 50% (2 / 4) of the mice to die, and the maximum weight loss was 26% on day 7 (D7); no death was observed for RA1 III-5 (DAR 4), and the maximum weight loss was 9% on day 4 (D4), and the clinical observation showed that 25% (1 / 4) of the mice had piloerection.

[0429] 4. Test Conclusion

[0430] The test results show that the toxicity of RA1 III-5 (DAR 8) is greater than that of RA1 III-5 (DAR 4), and the tolerance amount of RA1 III-5 (DAR 8) is less than 300 mg / kg, and the tolerance amount of RA1 III-5 (DAR 4) is greater than 300 mg / kg.

[0431] Example 7-3 Toxicity study of RA1 III-31 (DAR 8)

[0432] Test Purpose

[0433] The toxicity reaction and toxicity degree of BALB / c mice after intravenous injection of RA1 III-31 (DAR 8) were evaluated.

[0434] Experimental Method

[0435] Twelve BALB / c mice, female, weighing 17-22 g when grouped, were given 200 mg / kg and 300 mg / kg, respectively. The toxicity reaction of BALB / c to RA1 III-31 (DAR 8), body weight change, and the test lasted for 14 days were investigated by clinical observation, body weight, etc.

[0436] Test Results

[0437] At a dose of 200 mg / kg-300 mg / kg, RA1 III-31 (DAR 8) showed no obvious abnormalities in clinical observation, and the maximum weight loss was 1.81%, 7.1%, respectively.

[0438] Test Conclusion

[0439] BALB / c mice tolerated 200 mg / kg and 300 mg / kg of RA1 III-31 (DAR 8), and the maximum tolerated dose was greater than 300 mg / kg.

[0440] Therefore, the camptothecin compound in the application has a significant effect of inhibiting tumors or treating cancer, and the antibody drug conjugate developed thereby will also produce a significant therapeutic effect in various tumors and has a safer drug property, which provides a possibility for developing an antibody drug conjugate with a larger therapeutic window, and has great value and significance for the development of antibody drug conjugates.

[0441] The application is described by specific preferred embodiments, but it should be understood that the claimed application should not be unduly limited to these specific embodiments. Indeed, various modifications of the described modes of carrying out the application that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

1. A compound of formula (I) ###0001### (I) wherein, 1)R 1 Selected from hydrogen, methoxy, methyl, halogen, amino and R 2 Select hydrogen, methyl, methoxy, amino, hydroxy, hydroxyalkylamide, or halogen; or R 1 With R 2 It forms a dioxopentanone ring with the carbon atom it is attached to; and 2) R 3 selected from hydrogen, Ci-C6alkyl, R 4 selected from hydrogen, and R 5 is null; or R 3 and R 4 and the carbon atom to which it is attached form a six-membered carbocyclic ring and R 5 is not null; 3) said is selected from a single bond, a double bond, or null; wherein R is selected from the group consisting of hydrogen, C1-C6alkyl, 6 , R is independently selected from the group consisting of hydrogen, C1-C6alkyl, 7 ​ Or R 6 With R 7 The nitrogen atom bonded thereto forms a cyclosulfonyl group, preferably, one or more hydrogen atoms in the cyclosulfonyl group are optionally substituted with a C1-C6 hydroxyalkyl group; or R 6 With R 7 The nitrogen atom attached thereto forms a nitrogen heterocyclic alkyl group, preferably in which one or more hydrogen atoms are optionally substituted with methylene groups; said R 8 selected from hydrogen, C2-C6alkenyl or Ci-C6alkylene; said R 9 selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, said R 10 selected from C1-C6alkoxy, said R 11 selected from hydrogen, Ci-C6alkyl, C2-C6alkenyl, methylene, said R 12 selected from C1-C6alkoxy, hydroxy, C1-C6alkylhydroxy; in said 2) any one or more hydrogens in the nitrogen heterocycle is optionally substituted with hydroxyl, C1-C6 hydroxyalkyl, or F; the six-membered carbocycle contains 0, 1, or more double bonds internally; said R 5 when not null, is selected from the group consisting of hydrogen, carbonyl, fluorine, methylene, R is hydrogen or fluoro; and 5 when R is hydrogen or fluoro, then one or more hydrogens in said six-membered carbocyclic ring are optionally substituted with a fluoro atom; said R 13 selected from the group consisting of hydroxyl, said R 14 selected from hydrogen or Ci-C6alkyl; said R 15 is selected from -O- or -NH-; said R 16 selected from hydroxy, amino or Ci-C6aminoalkyl; said R 17 is selected from -O- or -NH-; said R 18 selected from C1-C6 hydroxyalkyl The R 19 selected from The R 20 selected from said R 21 selected from hydrogen, Ci-C6alkyl; said R 22 selected from C1-C6 hydroxyalkyl or each of n1, n2, n2, n3, n4, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14, n15, n16, n17, n18, n19, n20, n21, n22, n23, n24, n25, n26 is independently selected from 0, 1, 2, 3, 4, 5, or 6.

2. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) R 1 selected from the group consisting of fluorine and R 2 selected from the group consisting of methyl, hydroxy, amino, methoxy; or R 1 with the carbon atom to which it is attached forms a dioxolane ring; 2 with the carbon atom to which it is attached forms a dioxolane ring; 2) R 5’ selected from hydrogen, fluorine or is null; 3) R 23 selected from hydrogen or fluoro, when R 23 is hydrogen, said R 5 is selected from the following structures: fluorine, when R 23 is fluoro, said R 5 is hydrogen; 3) is a single bond or a double bond; preferably a single bond; 4) is a single bond, a double bond, or null; 5) said is selected from a single bond or is null; when said when the double bond is present, said For empty and R 5’ For empty; when the when is a single bond, said R is selected from the group consisting of hydrogen, halogen, and C1-C3alkyl; 5’ R is selected from the group consisting of hydrogen, halogen, and 3. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) R 1 selected from fluoro, hydrogen; and 2) R 2 is amino; and 3) R 12 selected from hydroxy, methoxy, hydroxymethyl.

4. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) said R 1 selected from fluoro, hydrogen, methyl, amino or methoxy; and 2) said R 2 selected from hydrogen, amino, fluoro, methoxy or methyl; or R 1 with R 2 form a dioxolane ring with the carbon atom to which they are attached; and 3) said R 6 selected from hydrogen, isopropyl or and 4) said R 7 selected from or R 6 with the nitrogen atom to which they are attached forming the following structure: 7 and the nitrogen atom to which they are attached forming the following structure: and 5) said R 8 selected from hydrogen, wherein, n2 is selected from 0, 1, 2, 3.

5. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) said R 1 , R 2 are each independently selected from hydrogen, amino, methyl, methoxy or fluorine; or R 1 and R 2 form together with the carbon atom to which they are attached a dioxolane ring; and 2) said R 9 selected from the following structures:

6. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) said R 1 , R 2 are each independently selected from the group consisting of hydrogen, amino, methyl, methoxy, fluorine; or R 1 and R 2 form together with the carbon atom to which they are attached a dioxolane ring; and 2) said R 10 selected from methoxy, and 3) said R 11 selected from hydrogen, 7. The compound of claim 1, wherein The compound is of the following structure: wherein, 1) said R 1 selected from fluoro, chloro; and 2) said R 2 selected from chloro, methyl, and 3) said R 3 selected from hydrogen, ethyl, and 4) said R 4 selected from hydrogen, 8. The compound of claim 1, wherein The compound is selected from the following structures:

9. The compound of any one of claims 1-8, wherein, at least one H in the compound is an isotope of H; the isotope of H is further preferably deuterium or tritium; preferably, the compound is of the following structure:

10. A compound, characterized in that, the compound is a stereoisomer of any one of the compounds of claims 1-9, or a pharmaceutically acceptable salt or solvate thereof.

11. A bis-molecular compound comprising any two identical or different compounds of any one of claims 1-10.

12. The bis-molecular compound of claim 11, wherein, the two identical or different compounds are connected by a short chain fragment, the longest chain in the short chain fragment comprising 1-10 atoms and the short chain comprising one or more of the following groups: heteroalkyl, heterocycloalkyl, aryl, heteroaryl, or a combination thereof; preferably, the short chain comprises an oxaalkyl group.

13. The bispecific compound of claim 12, wherein: The bis-molecular compound is selected from the following structures:

14. A linker-payload compound, characterized in that, the structure of the linker-payload compound is described by Formula (II-A): M-V1-V2-V3-Q (II-A), wherein, 1) the M is a linker; the -V1- is a connecting unit; the -V2- is a polypeptide unit consisting of amino acids; the -V3- is a structural fragment connecting V2 and Q; and 2) the Q is a therapeutic agent; Preferably, said M- is selected from the following structures: wherein, the X1 is selected from hydrogen, Cl, Br, or I; Preferably, said -V1- is selected from the structure consisting of one or more of the following: wherein, each of a1, a2 is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; Preferably, said M-V1- is selected from the following structures: wherein, m1 is selected from 1, 2, 3, 4, 5, 6; m2 is selected from 1, 2, 3, 4, 5, 6; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8; m4 is selected from 1, 2, 3, 4, 5, 6; m5 is selected from 1, 2, 3, 4, 5, 6; m6 is selected from 1, 2, 3, 4, 5, 6; m7 is selected from 1, 2, 3, 4, 5, 6; m8 is selected from 1, 2, 3, 4, 5, 6; m9 is selected from 1, 2, 3, 4, 5, 6; Preferably, said M-V1- is selected from the group consisting of: preferably, the -V2- is selected from a peptide consisting of one or more of the following amino acids: phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, the -V2- is selected from the following structures: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, said -V2- is selected from -Gly-Gly-Phe-Gly-, -Val-Ala-, or -Val-Cit-; Preferably, -V3- is null or selected from the group consisting of the following structures: Preferably, the linker-payload compound (II-A) is selected from: Preferably, said therapeutic agent Q is selected from a cytotoxic molecule, an immunopotentiator, and a radioisotope; preferably, said cytotoxic molecule is selected from a tubulin inhibitor or a DNA damaging agent; Preferably, said tubulin inhibitor is selected from a dolastatin, an auristatin cytotoxic molecule, or a maytansine cytotoxic molecule; said DNA damaging agent is selected from a calicheamicin, a duocarmycin, an anthramycin derivative PBD, a camptothecin, and a camptothecin derivative; further preferably, said camptothecin derivative is selected from a compound of any one of claims 1-13.

15. A linker-payload compound, characterized in that, said payload is selected from a compound of any one of claims 1-13.

16. The linker-payload compound of claim 15, wherein, said linker-payload compound has a structure according to formula (II-B): W-A1-A2-A3-D (II-B); wherein, 1) said W is a linker; said -A1- is a connecting unit; said -A2- is a polypeptide unit consisting of amino acids; said -A3- is a spacer unit; and 2) said D is selected from a compound of any one of claims 1-13 after removal of the hydrogen.

17. The linker-payload compound of claim 16, wherein, W - is selected from the following structures: wherein, said X1is selected from hydrogen, Cl, Br, or I.

18. The linker-payload compound of claim 16, wherein, said -A1- is selected from the structure consisting of one or more of the following: wherein, said a1, a2are each independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

19. The linker-payload compound of claim 16, wherein, W-A1- is selected from the following structures: wherein, q1is selected from 1, 2, 3, 4, 5, 6; q2is selected from 1, 2, 3, 4, 5, 6; q3is selected from 1, 2, 3, 4, 5, 6, 7, 8; q4is selected from 1, 2, 3, 4, 5, 6; q5is selected from 1, 2, 3, 4, 5, 6; q6is selected from 1, 2, 3, 4, 5, 6; q7 is selected from 1, 2, 3, 4, 5, 6; q8 is selected from 1, 2, 3, 4, 5, 6; q9 is selected from 1, 2, 3, 4, 5, 6.

20. The linker-payload compound of claim 16, wherein, W-A1- is selected from the following structures:

21. The linker-payload compound of any one of claim 16, wherein, said -A2- is selected from a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

22. The linker-payload compound of claim 16, wherein, said -A2- is selected from a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from the group consisting of phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

23. The linker-payload compound of claim 16, wherein, said -A2- is selected from the group consisting of -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, said -A2- is selected from -Gly-Gly-Phe-Gly-, -Val-Ala-, or -Val-Cit-.

24. The linker-payload compound of any one of claims 16-23, wherein, said -A3- is null or selected from the group consisting of the following structures:

25. The linker-payload compound of claim 16, wherein, The W-A1-A2-A3-structure is shown below:

26. The linker-payload compound of claim 14 or 15, wherein, The linker-payload compound is selected from the following structures:

27. A ligand-drug conjugate comprising a targeting unit, characterized in that, said ligand-drug conjugate uses a payload selected from the group consisting of the compounds of any one of claims 1-13.

28. A ligand-drug conjugate comprising a targeting unit, characterized in that, said ligand-drug conjugate uses a linker-payload compound selected from the group consisting of the linker-payload compounds of any one of claims 14-26.

29. The ligand-drug conjugate of claim 27 or 28, wherein The ligand-drug conjugate is reacted with the linker-payload compound of any one of claims 14-26 via the targeting unit; preferably, the reaction comprises a chemical coupling reaction, an enzymatic coupling reaction, a sugar chain remodeling, a glycosyl coupling reaction, or a site-directed coupling reaction; preferably, the chemical coupling reaction comprises a chemical coupling reaction based on lysine in the targeting moiety and the linker, a chemical coupling reaction based on cysteine and the linker, or a chemical coupling reaction based on a non-natural amino acid and the linker.

30. The ligand-drug conjugate of any one of claims 27-29, wherein The ligand is selected from an antibody, an antigen-binding fragment, or a targeting peptide.

31. The ligand-drug conjugate of claim 30, wherein The antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

32. The ligand-drug conjugate of claim 30, wherein The antibody heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.

33. The ligand-drug conjugate of claim 30, wherein The antibody is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a nanobody; preferably, the antibody is a monoclonal antibody.

34. The ligand-drug conjugate of claim 30, wherein The antibody is selected from a humanized monoclonal antibody or a fully human monoclonal antibody.

35. The ligand-drug conjugate of claim 30, wherein The antigen-binding fragment is selected from an scFv, a Fab, a Fab', a (Fab')2, an Fv fragment, a disulfide-linked Fv (dsFv).

36. The ligand-drug conjugate of any one of claims 27-35, wherein The ligand has at least one cysteine.

37. The ligand-drug conjugate of any one of claims 27 or 28, selected from the following structures: wherein, A represents the ligand; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, and represents the number of linker-payload units attached to the ligand, which is 1, 2, 3, 4, 5, 6, 7, or 8.

38. The ligand-drug conjugate of any one of claims 27-37, wherein Each ligand-drug conjugate is linked to 1 to 8 payloads.

39. A ligand-drug conjugate comprising a linker structure as shown in (Ⅳ), characterized in that, The linker structure is -M-V1-V2-V3- (IV), wherein -M- is a linker; -V1- is a connecting unit; -V2- is a polypeptide unit composed of amino acids; and -V3- is a connecting fragment. 1) -M- is selected from the following structures: and wherein a1 and a2 are each independently selected from 1, 2, 3, 4, 5, or 6; and 2) said -V1- is selected from the structure consisting of one or more of the following: 3) -V2- is a peptide composed of one or more amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), and citrulline (Cit); and wherein 4) said -V3- is null or selected from the group consisting of the structures:

40. The ligand-drug conjugate of claim 39, wherein: said -M-V1- is selected from the following structures: m1 is selected from 1, 2, 3, 4, 5, 6; m2 is selected from 1, 2, 3, 4, 5, 6; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8; m4 is selected from 1, 2, 3, 4, 5, 6; m5 is selected from 1, 2, 3, 4, 5, 6; m6 is selected from 1, 2, 3, 4, 5, 6; m7 is selected from 1, 2, 3, 4, 5, 6; m8 is selected from 1, 2, 3, 4, 5, 6; m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8. ​ 41. The ligand-drug conjugate of claim 40, wherein: said -M-V1- is selected from the following structures:

42. The ligand-drug conjugate of any one of claims 39-41, characterized in that: said -V2- is selected from the group consisting of -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-.

43. The ligand-drug conjugate of any one of claims 42, characterized in that: said -V2- is selected from the group consisting of -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-.

44. The ligand-drug conjugate of any one of claims 39-43, characterized in that: said -V3- is either null or selected from the group consisting of the following structures:

45. The ligand-drug conjugate of claim 44, wherein: said formula (IV) is selected from the group consisting of: wherein 1) said linked to an antibody; and 2) said linked to a payload.

46. The ligand-drug conjugate of any one of claims 27-45, characterized in that: said ligand is selected from the group consisting of an antibody, an antigen binding fragment, or a targeting peptide; preferably, said ligand is a targeting CDCP1 antibody.

47. Use of a compound of any one of claims 1-13 or a linker-payload compound of any one of claims 14-26 for the manufacture of a ligand-drug conjugate, said ligand is selected from the group consisting of an antibody, an antigen binding fragment, or a targeting peptide; preferably, said antibody is a targeting CDCP1 antibody.

48. A pharmaceutical composition comprising said pharmaceutical composition comprises a ligand-drug conjugate of any one of claims 27-46.

49. The pharmaceutical composition of claim 48, wherein, said average number of linkages of the payload per ligand is in the range of 1-8; further preferred is 2-8, 3-8, 4-8, 5-8, 6-8, 7-8, or 3-5.

50. Use of a ligand-drug conjugate of any one of claims 27-46 or a pharmaceutical composition of claim 48 or 49 for the treatment of a cancer; preferably, said cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer; more preferably, said lung cancer is selected from the group consisting of non-small cell lung cancer and lung squamous carcinoma.

51. A method of making a ligand-drug conjugate of any one of claims 27-46, comprising: coupling a ligand to a linker-payload compound of any one of claims 14-26 in a suitable solution and under suitable conditions.

52. A method of making a ligand-drug conjugate of any one of claims 27-46, comprising: 1) coupling a ligand to a linker M-V1-V2-V3 or W-A1-A2-A3 of a linker-payload compound of any one of claims 14-26 in a suitable solution and under suitable conditions, to form a ligand-linker structure combination; 2) reacting the ligand with the linker moiety in a suitable solution and conditions to form a ligand-linker conjugate; 53. A method of making a ligand-drug conjugate of any one of claims 27-46, comprising: 1) coupling the ligand to a portion of the linker-load compound of any one of claims 14-26, M-V1-V2-V3 or W-A1-A2-A3, in a suitable solution and conditions to form a partial linker moiety conjugate; 2) reacting the portion of M-V1-V2-V3 or W-A1-A2-A3 with the compound of any one of claims 1-13 in a suitable solution and conditions to form a load-partial linker moiety conjugate; 3) reacting the ligand-partial linker moiety conjugate with the load-partial linker moiety conjugate in a suitable solution and conditions to form a ligand-drug conjugate.

54. A linker-payload compound, characterized in that, the linker-load compound is a stereoisomer of any one of the linker-load compounds of claims 14-26, or a pharmaceutically acceptable salt or solvate thereof. the linker-load compound is a stereoisomer of any one of the linker-load compounds of claims 14-26, or a pharmaceutically acceptable salt or solvate thereof.

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