Camptothecin small molecule and antibody drug conjugate thereof, preparation method therefor and use thereof

Abstract

A camptothecin small molecule and an antibody drug conjugate thereof, a preparation method therefor and the use thereof. Provided is a camptothecin small molecule as shown in formula I or a pharmaceutically acceptable salt thereof. The provided camptothecin small molecule and the antibody drug conjugate thereof have a good cytotoxic activity, and can inhibit the proliferation of tumor cells.

Description

Camptothecin small molecules and antibody-drug conjugates thereof, preparation methods and applications

[0001] This application claims priority to Chinese Patent Application No. 2023109283984, filed on July 26, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to camptothecin small molecules and antibody-drug conjugates thereof, as well as preparation methods and applications. Background Art

[0003] Camptothecins are specific inhibitors of DNA topoisomerase I. Camptothecins bind to the Topo I-DNA cleavable binary complex to form a stable Topo I-DNA-CPT ternary complex, which affects DNA religation, increases Topo I-mediated DNA damage, and ultimately leads to cell death.

[0004] The traditional camptothecin compound is a pentacyclic quinoline alkaloid, whose structure contains a quinoline ring AB, a pyrrole ring C, a pyridone ring D, and an α-hydroxylactone ring E, of which the 20-position is in S configuration. Recent studies have found that the camptothecin derivative exatecan with an F ring has good activity and can be used in antibody-drug conjugates (ADCs): WO2014057687; Clinical Cancer Research (2016) 2 2(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. In addition, AstraZeneca constructed a camptothecin ADC with an F ring based on the A ring, which also achieved good results in preclinical PDX models: WO2021148501. However, further development of better ADC drugs is still needed to improve the efficacy of existing ADC drugs.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the structural shortcomings of existing camptothecin derivatives and their antibody-drug conjugates, and to provide camptothecin small molecules and their antibody-drug conjugates, as well as preparation methods and applications. The camptothecin small molecules and their antibody-drug conjugates provided by the present invention have excellent cytotoxic activity and can inhibit the proliferation of tumor cells.

[0007] The present invention provides a camptothecin small molecule or a pharmaceutically acceptable salt thereof as shown in Formula I:

[0008] Among them, R 1 is hydrogen, halogen or C 1-6 alkyl;

[0009] L 1 For a single bond, -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0010] L 2 For a single key, or C 1-6 Alkylene.

[0011] In a preferred embodiment, in the camptothecin small molecule or a pharmaceutically acceptable salt thereof as shown in Formula I, certain groups have the following definitions, and the definitions of the unmentioned groups are as described in any embodiment of the present invention (this paragraph is hereinafter referred to as "in a preferred embodiment").

[0012] In a preferred embodiment, R 1 wherein the halogen is fluorine, chlorine, bromine or iodine, for example fluorine.

[0013] In a preferred embodiment, R 1 In the C 1-6 Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl.

[0014] In a preferred embodiment, L 1 In the -C 1-6 C in alkylene-NH- 1-6 Alkylene is a straight-chain alkylene.

[0015] In a preferred embodiment, L 1 In the -C 1-6 C in alkylene-NH- 1-6 Alkylene is connected to hydroxyl, NH is connected to L 2 connect.

[0016] In a preferred embodiment, L 1 In the -C 1-6 -C in alkylene-NH- 1-6 Alkylene- is -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene, for example -CH2-CH2-.

[0017] In a preferred embodiment, L 1 wherein the heteroatom of the 3-10 membered heterocycloalkylene group is N, and the number of the heteroatom is 1.

[0018] In a preferred embodiment, L 1 wherein the 3-10 membered heterocycloalkylene group is a 3-6 membered heterocycloalkylene group.

[0019] In a preferred embodiment, L 1 In the embodiment, the 3-10 membered heterocycloalkylene group is an aziridinyl group, an aziridinyl group or an aziridinyl group, for example For example For example Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to the L 2 connect.

[0020] In a preferred embodiment, L 2 In the C 1-6 Alkylene is a straight-chain alkylene.

[0021] In a preferred embodiment, L 2 In the C 1-6 Alkylene is methylene, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene, for example -CH2- or -CH2-CH2-.

[0022] In a preferred embodiment, L 1 is a single bond or L 1 -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene.

[0023] In a preferred embodiment, L 1 For a single bond.

[0024] In a preferred embodiment, L 2 C 1-6 Alkylene (preferably, L 2 For straight chain C 1-6 alkylene) or

[0025] In a preferred embodiment, L 2 C 1-6 Alkylene.

[0026] In a preferred embodiment, -L 1 -L 2 - Any of the following options:

[0027] Option 1:

[0028] L 1 is a single bond; L 2 C 1-6alkylene;

[0029] Option 2:

[0030] L 1 -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0031] L 2 for

[0032] In a preferred embodiment, R 1 is hydrogen or halogen, for example R 1 is hydrogen or fluorine.

[0033] In a preferred embodiment, R 1 It is a halogen.

[0034] In a preferred embodiment, -L 1 - is a single key, Another example is a single key, Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to the L 2 connect.

[0035] In a preferred embodiment, -L 2 -for

[0036] In a preferred embodiment, -L 1 -L 2 -for Preferably, -L 1 -L 2 -for Among them, the "1" position is connected to the hydroxyl group and the "2" position is connected to the methylene group.

[0037] In a preferred embodiment, R 1 is hydrogen or halogen;

[0038] -L 1 -L 2 - Any of the following options:

[0039] Option 1:

[0040] L 1 is a single bond; L 2 C 1-6 alkylene;

[0041] Option 2:

[0042] L1 -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0043] L 2 for

[0044] In a preferred embodiment,

[0045] R 1 is a halogen;

[0046] L 1 is a single bond;

[0047] L 2 C 1-6 Alkylene.

[0048] In a preferred embodiment, the camptothecin small molecule as represented by Formula I or a pharmaceutically acceptable salt thereof is selected from any of the following structures:

[0049] The present invention also provides a linker-drug conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II:

[0050] wherein Q is a single amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

[0051] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -、-(OCH2CH2) m -、-(CR a R b ) m -、-O(CR a R b ) m -、-NH-(CR a R b ) m -CR c R d -or-S(CR a R b ) m -CR c R d -;

[0052] R a and R b are independently H, D, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, cyano, nitro, 3-10 membered cycloalkyl or 3-10 membered heterocyclic group;

[0053] Or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group;

[0054] R c and R d Independently H, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, deuterated C 1-6 alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-14 membered aryl or 5-10 membered heteroaryl;

[0055] Or, R c and R d Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group;

[0056] The heteroatoms in each 3-10 membered heterocyclyl and 5-10 membered heteroaryl are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3;

[0057] Y is

[0058] R 1 , L 1 and L 2 The definition is as described in any one of the present invention;

[0059] m, p, q and r are independently integers of 0-14.

[0060] In a preferred embodiment, in the linker-drug conjugate as shown in Formula II, certain groups have the following definitions, and the definitions of the groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in a preferred embodiment" in this paragraph).

[0061] In a preferred embodiment, each halogen is independently fluorine, chlorine, bromine or iodine.

[0062] In a preferred embodiment, each C 1-6Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0063] In a preferred embodiment, each C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

[0064] In a preferred embodiment, each 3-10 membered cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0065] In a preferred embodiment, each 3-10 membered heterocyclic group is independently a 3-6 membered heterocyclic group, the heteroatoms of the 3-6 membered heterocyclic group are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3.

[0066] In a preferred embodiment, each 6-14 membered aryl group is independently phenyl or naphthyl.

[0067] In a preferred embodiment, each 5-10 membered heteroaryl group is independently a 5-6 membered heteroaryl group, the heteroatoms of the 5-6 membered heteroaryl group are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3.

[0068] In a preferred embodiment, in Q, the single amino acid residue is -Phe-, -Lys-, -Val-, -Ala-, -Cit-, -Leu-, -Ile-, -Arg-, -Trp- or -Gly-.

[0069] In a preferred embodiment, in Q, the dipeptide residue is C＝O -Lys-Phe- NH 、 C＝O -Ala-Val- NH 、 C＝O -Lys-Val- NH 、 C＝O -Lys-Ala- NH 、 C＝O -Cit-Val- NH 、 C＝O Cit-Phe- NH 、 C＝O -Cit-Leu- NH 、 C＝O Cit-Ile- NH 、 C＝O -Arg-Phe- NH 、 C＝O -Cit-Trp- NH or C＝O-Val-Gly- NH ;For example C＝O -Ala-Val- NH .

[0070] In a preferred embodiment, in Q, the tripeptide residue is C＝O -Ala-Val-Glu- NH 、 C＝O -Cit-Val-Glu- NH 、 C＝O -Ala-Val-αGlu- NH or C＝O -Cit-Val-αGlu- NH .

[0071] In a preferred embodiment, in Q, the tetrapeptide residue is C＝O -Gly-Phe-(Gly)2- NH or C＝O –(Gly)2-Phe-Gly- NH ,For example C＝O -Gly-Phe-(Gly)2- NH .

[0072] In a preferred embodiment, R a and R b For H.

[0073] In a preferred embodiment, m is 1, 2, 3, 4 or 5, for example 5.

[0074] In a preferred embodiment, p is 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0075] In a preferred embodiment, p is 8, 9, 10, 11, 12, 13 or 14, for example 8 or 12.

[0076] In a preferred embodiment, q is 1, 2, 3, 4 or 5, for example 2.

[0077] In a preferred embodiment, r is 1, 2, 3, 4 or 5, for example 2.

[0078] In a preferred embodiment, Q is a dipeptide residue or a tetrapeptide residue.

[0079] In a preferred embodiment, in the linker-drug conjugate shown in Formula II, Q is a dipeptide residue, and the dipeptide residue is C＝O -Ala-Val- NH .

[0080] In a preferred embodiment, in the linker-drug conjugate as shown in Formula II, Q is a tetrapeptide residue, and the tetrapeptide residue is C＝O -Gly-Phe-(Gly)2- NH .

[0081] In a preferred embodiment, in X, 1 -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p - 2 、 2 -(OCH2CH2) m - 1 、 2 -O(CR a R b ) m - 1 、 2 -NH-(CR a R b ) m -CR c R d-1 and 2 -S(CR a R b ) m -CR c R d-1 In the “1” position, the Y is connected, and the “2” position is connected. connect.

[0082] In a preferred embodiment, X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -; Preferably, X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -;For example For example The "1" bit is connected to Y, and the "2" bit is connected to connect.

[0083] In the present invention, in Q, the dipeptide residue, tripeptide residue or tetrapeptide residue " C＝O " indicates that the left side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to NH connected to Q through a carbonyl group, and the " NH " indicates that the right side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the carbonyl group connected to Q through the amino group.

[0084] In a preferred embodiment, in the linker-drug conjugate shown in Formula II or a pharmaceutically acceptable salt thereof,

[0085] Q is a dipeptide residue or a tetrapeptide residue;

[0086] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -;

[0087] Y is

[0088] R a and R b is H;

[0089] m is 1, 2, 3, 4 or 5;

[0090] p is an integer of 8, 9, 10, 11, 12, 13 or 14;

[0091] q is 1, 2, 3, 4, or 5;

[0092] r is 1, 2, 3, 4 or 5;

[0093] R 1 is hydrogen or halogen;

[0094] L 1 is a single bond; L 2 C 1-6 Alkylene.

[0095] In one embodiment, the linker-drug conjugate as shown in Formula II is any of the following structures:

[0096] The present invention also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in Formula III:

[0097] Among them, G L is an antibody; said G L Form a covalent bond with Z through S on it;

[0098] t is the drug-ligand coupling ratio;

[0099] Z is The b end is connected to X, and the a end is connected to G L connect;

[0100] R 1 , L 1 , L 2 , Q and X are as defined in any one of the present invention.

[0101] In a preferred embodiment, in the antibody drug conjugate as shown in Formula III, certain groups have the following definitions, and the definitions of the groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in a preferred embodiment" in this paragraph).

[0102] In a preferred embodiment, G L Anti-HER2 antibodies, such as trastuzumab.

[0103] In a preferred embodiment, t is a natural number and / or decimal from 1 to 8; for example, a natural number and / or decimal from 7 to 8; for example, 7.8.

[0104] In a preferred embodiment, t is a natural number and / or decimal number from 6 to 8, such as 6.2, 7.8, 7.7 or 7.9.

[0105] In a preferred embodiment, t is a natural number of 1-8; for example, t is a natural number of 7-8.

[0106] In a preferred embodiment, the a end of Z and the G L Connected by thioether bond. For example, the G L It can be obtained by reducing the disulfide bonds of an antibody containing disulfide bonds (such as trastuzumab) to sulfhydryl groups under the action of a reducing agent.

[0107] In a preferred embodiment, G L It is an anti-HER2 antibody;

[0108] t is a natural number and / or decimal between 6 and 8;

[0109] Z is The b end is connected to X, and the a end is connected to GL connect;

[0110] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -;

[0111] R a and R b independently H or D;

[0112] r is 1, 2, 3, 4 or 5;

[0113] q is 1, 2, 3, 4, or 5;

[0114] p is 6, 7, 8, 9, 10, 11, 12, 13, or 14;

[0115] m is 1, 2, 3, 4 or 5;

[0116] Q is a dipeptide residue or a tetrapeptide residue;

[0117] R 1 is hydrogen or halogen;

[0118] -L 1 -L 2 - Any of the following options:

[0119] Option 1:

[0120] L 1 is a single bond; L 2 C 1-6 alkylene;

[0121] Option 2:

[0122] L 1 -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0123] L 2 for

[0124] In a preferred embodiment, G L for trastuzumab;

[0125] t is a natural number and / or decimal between 6 and 8;

[0126] Z is The b end is connected to X, and the a end is connected to G L connect;

[0127] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -;

[0128] R a and R b independently H or D;

[0129] r is 1, 2, 3, 4 or 5;

[0130] q is 1, 2, 3, 4, or 5;

[0131] p is 6, 7, 8, 9, 10, 11, 12, 13, or 14;

[0132] m is 1, 2, 3, 4 or 5;

[0133] Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly)2- NH ;

[0134] R 1 is a halogen;

[0135] L 1 is a single bond;

[0136] L 2 C 1-6 Alkylene.

[0137] In a preferred embodiment, G L for trastuzumab;

[0138] t is a natural number and / or decimal between 6 and 8;

[0139] Z is The b end is connected to X, and the a end is connected to G L connect;

[0140] X is -(CR a R b ) r -CO-NH-(CRa R b ) q -(OCH2CH2) p -;

[0141] R a and R b independently H or D;

[0142] r is 1, 2, 3, 4 or 5;

[0143] q is 1, 2, 3, 4, or 5;

[0144] p is 6, 7, 8, 9, 10, 11, 12, 13, or 14;

[0145] Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly)2- NH ;

[0146] R 1 is a halogen;

[0147] L 1 is a single bond;

[0148] L 2 C 1-6 Alkylene.

[0149] In a preferred embodiment, the antibody-drug conjugate as shown in Formula III is any of the following structures:

[0150] wherein the anti-HER2 antibody is trastuzumab; t is defined as described in any one of the present invention;

[0151] Preferably, the antibody-drug conjugate as shown in Formula III is any of the following structures:

[0152] The anti-HER2 antibody is trastuzumab.

[0153] The present invention also provides a pharmaceutical composition comprising (a therapeutically effective amount of) substance N and a pharmaceutical excipient, wherein substance N is a camptothecin small molecule as shown in Formula I or a pharmaceutically acceptable salt thereof, a linker-drug conjugate as shown in Formula II or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in Formula III or a pharmaceutically acceptable salt thereof according to any one of the present invention.

[0154] The present invention also provides a use of the substance N (in a therapeutically effective amount) or the pharmaceutical composition described above in the preparation of a drug for preventing and / or treating cancer.

[0155] The cancer is preferably breast cancer, lung cancer (such as lung adenocarcinoma), kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer or esophageal cancer, preferably breast cancer or preferably lung adenocarcinoma, gastric cancer or colon cancer.

[0156] The present invention provides a method for preventing and / or treating the cancer, comprising administering (a therapeutically effective amount of) the substance N or the pharmaceutical composition described above to a subject.

[0157] The present invention also provides a (intermediate) compound of formula 1c, 2h, 2i, 3c, 4h, 5e or 7e (for preparing the camptothecin small molecule shown in formula I),

[0158] The present invention also provides a method for preparing a camptothecin small molecule as shown in Formula I, which is the following method 1, method 2 or method 3:

[0159] Method 1: In a solvent (e.g., toluene and o-cresol, in a volume ratio of 77:1 or 5:1, or toluene and glacial acetic acid, in a volume ratio of 1:1), in the presence of a catalyst (e.g., pyridinium p-toluenesulfonate), a compound represented by Formula 1-s1 and a compound represented by Formula 1d are reacted as shown below to obtain a camptothecin-like small molecule represented by Formula I;

[0160] Among them, R 1 , L 1 and L 2 The definition as described in any one of the present invention;

[0161] Method 2: In a solvent (e.g., water), in the presence of a base (e.g., sodium hydroxide), the compound represented by Formula 1-s3 is subjected to a hydrolysis reaction as shown in the following formula to obtain a camptothecin-like small molecule represented by Formula I;

[0162] Among them, R 1 , L 1 and L 2 The definition as described in any one of the present invention;

[0163] Method 3: In a solvent (e.g., anhydrous dichloromethane), in the presence of a catalyst (e.g., 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HATU), a compound represented by Formula 1-s5 is subjected to a condensation reaction with Compound H as shown in the following formula to obtain a camptothecin-like small molecule represented by Formula I;

[0164] Among them, L 2 for

[0165] The compound H is HO-L 1 -H or its salt, L 1 -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene (e.g. 4-hydroxypiperidine, 3-hydroxyazetidine, 3-hydroxyazetidine hydrochloride or ethanolamine); for example: -L 1 -L 2 -for Among them, the "1" position is connected to the hydroxyl group and the "2" position is connected to the methylene group;

[0166] R 1 The definition of is as described in any one of the present invention.

[0167] The present invention also provides a method for preparing the compound shown in formula 1-s1, which comprises the following steps:

[0168] In a solvent (e.g., methanol), in the presence of hydrogen and a catalyst (e.g., 5% palladium on carbon or Raney nickel), the compound represented by Formula 1-s2 is subjected to a hydrogenation reaction as shown in the following formula to obtain a compound represented by Formula 1-s1:

[0169] Among them, R 1 , L 1 and L 2 The definition of is as described in any one of the present invention.

[0170] The present invention also provides a method for preparing the compound shown in formula 1-s3, which comprises the following steps:

[0171] In a solvent (e.g., toluene and o-cresol, in a volume ratio of 77:1 or toluene and glacial acetic acid, in a volume ratio of 1:1), in the presence of a catalyst (e.g., pyridinium p-toluenesulfonate), the compound represented by Formula 1-s4 and the compound represented by Formula 1d are reacted as shown in the following formula to obtain the compound represented by Formula 1-s3.

[0172] Among them, R 1 , L 1 and L2 The definition of is as described in any one of the present invention.

[0173] The present invention also provides a method for preparing the compound shown in formula 1-s5, which comprises the following steps:

[0174] In a solvent (e.g., methanol), in the presence of a base (e.g., sodium hydroxide, or an aqueous sodium hydroxide solution), the compound represented by Formula 1-s6 is subjected to a hydrolysis reaction as shown in the following formula to obtain a compound represented by Formula 1-s5;

[0175] Among them, R 1 The definition of R is as described in any one of the present invention; s C 1-6 Alkyl (eg, ethyl).

[0176] The preparation method of the camptothecin small molecule shown in Formula I, the preparation method of the compound shown in Formula 1-s1, the preparation method of the compound shown in Formula 1-s3, and the preparation method of the compound shown in Formula 1-s5 can all be conventional preparation methods in the art, and the preparation conditions and operations can all be conventional conditions and operations for this type of reaction in the art.

[0177] Definition of terms

[0178] Stereochemical definitions and conventions used herein generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0179] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or as a mixture thereof, such as a racemate and a diastereomeric mixture (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0180] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0181] In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds. When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents obtained when the structural formula is written from right to left.

[0182] It will be understood by those skilled in the art that the structural formulas used in the present invention to describe groups are based on the conventions used in the art. It means that the corresponding group is connected to other fragments and groups in the compound through this site.

[0183] In the present invention, a substituent may be preceded by a single dash "-" to indicate that the named substituent is connected to the parent moiety via a single bond. For example, CH3-C(=O)- means that the C(=O) in the acetyl group is connected to the parent moiety via a single bond.

[0184] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0185] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0186] The term "alkylene" refers to a substituent formed by removing two hydrogen atoms from a saturated straight-chain or branched alkane. The two hydrogen atoms removed can be on the same carbon atom or on different carbon atoms (e.g., the two hydrogen atoms removed are on carbon atoms at the ends). Thus, C1 alkylene (i.e., methylene) refers to -CH2-, and C2 alkylene (i.e., ethylene) refers to -CH2-CH2- or -CH(CH3)-.

[0187] The term "heterocycloalkylene" refers to a substituent formed by eliminating two hydrogen atoms from a heterocycloalkane. The two hydrogen atoms eliminated can be on the same ring atom or on different ring atoms. Among them, "heterocycloalkane" refers to a saturated cycloalkane parent structure in which the ring carbon atom at a certain position is replaced by a heteroatom, and has a specified number of ring atoms (e.g., 3-10 members, 3-6 members), a specified number of heteroatoms (e.g., 1, 2 or 3), and a specified type of heteroatom (one or more of N, O and S). "Heterocycloalkane" is a monocyclic ring, and each ring is saturated. Heterocycloalkylene includes but is not limited to azetidinyl, piperidinyl or piperazinyl, for example

[0188] In this application, the term "alkoxy" as a group or part of another group refers to -O-alkyl, and alkyl is as defined above.

[0189] The term "cycloalkyl" refers to a cycloalkyl group having the specified number of carbon atoms (e.g., C 3-6 、C 3-10 ) is a saturated monocyclic ring group consisting only of carbon atoms. Cycloalkyl includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0190] The term "heterocyclyl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 members, 3-10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which is monocyclic, bridged, or spirocyclic, and each ring is saturated. Heterocycloalkyl groups include, but are not limited to, azetidinyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, piperidinyl, and the like.

[0191] The term "aryl" refers to a radical having the specified number of carbon atoms (e.g., C 6-10 、C 6-14 ) cyclic groups consisting only of carbon atoms, which are monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). Aryl groups are connected to other fragments in the molecule through aromatic or non-aromatic rings. Aryl groups include but are not limited to phenyl, naphthyl, etc.

[0192] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which is monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). The heteroaryl group is connected to other fragments in the molecule through an aromatic ring or a non-aromatic ring. Heteroaryl includes, but is not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, and the like.

[0193] The term "pharmaceutically acceptable salt" refers to a salt formed by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for patient use) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent.

[0194] The term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and in the preparation of prescriptions. These excipients are all substances, other than the active ingredient, contained in a pharmaceutical preparation. For details, see the Pharmacopoeia of the People's Republic of China (2020 edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009).

[0195] The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0196] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0197] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a patient, is sufficient to effectively treat a disease or condition described herein. The "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as needed by those skilled in the art.

[0198] The term "patient" refers to any animal, preferably a mammal, and most preferably a human, that is about to receive or has received a compound or composition according to embodiments of the present invention. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0199] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0200] The reagents and raw materials used in the present invention are commercially available.

[0201] The positive progress of the present invention is that the present invention provides a class of novel camptothecin small molecules and antibody-drug conjugates thereof, which have good cytotoxic activity and can inhibit the proliferation of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0202] FIG1 is a trend diagram of the inhibition rate of ADC-1 and ADC-2 on the proliferation ability of NCl-N87 tumor cells at different concentrations.

[0203] Figure 2 shows the inhibitory effect of ADC-1 and ADC-2 on tumor volume in the NOG mouse subcutaneously transplanted NCI-N87 cell animal model.

[0204] Figure 3 shows the effects of ADC-1 and ADC-2 on body weight in the NOG mouse subcutaneously transplanted NCI-N87 cell animal model. DETAILED DESCRIPTION

[0205] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0206] In the following examples, DCC represents dicyclohexylcarbodiimide; DMAP represents dimethylaminopyridine; DIPEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide; and PBS represents phosphate buffered saline.

[0207] Example 1: Synthesis of I-1:

[0208] Under a nitrogen stream, 350 mL of glacial acetic acid was added to the flask. 5-Hydroxy-1-tetralone (25 g, 154.1 mmol) was added portionwise. 65% nitric acid (12 mL, 157 mmol) was dissolved in 50 mL of glacial acetic acid and added to the reaction flask under an ice bath. The mixture was gradually returned to room temperature and stirred for 3 h. After TLC monitoring of the reaction, the reaction solution was poured into 1.5 L of ice water, the solid was filtered, the filter cake was rinsed with water, and the solid was dried under reduced pressure. The solid was slurried with PE:EA = 2:1 to obtain 16 g of a light yellow solid, with a yield of 50%.

[0209] 1 H NMR (400MHz, DMSO) δ10.88(s,1H),7.48(d,J=8.6Hz,1H),7.07(d,J=8.6Hz,1H),2. 81(t,J=6.1Hz,2H),2.68–2.60(m,2H),2.09–2.01(m,2H).MS(ESI)m / z=208.1(M+H + ).

[0210] Under a nitrogen stream, 1a (5.0 g, 24.1 mmol) and potassium carbonate (6.5 g, 36.2 mmol) were added to a vial, along with 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h. Benzyl 2-bromoethyl ether (5.8 mL, 36.2 mmol) was then added, and the mixture was stirred at 60°C for 16 h. After completion of the reaction as monitored by TLC, the solid was filtered, the filter cake rinsed with ethyl acetate, and the solvent removed under reduced pressure. The resulting solid was slurried with PE:EA = 5:1 to give 4.4 g of a light pink solid (53% yield).

[0211] 1 H NMR (400MHz, DMSO) δ7.63 (d, J=8.7Hz, 1H), 7.38–7.26 (m, 6H), 4.58 (s, 2H), 4.34–4.27 (m, 2H), 3.85 –3.79(m,2H),2.87(t,J=6.0Hz,2H),2.68–2.63(m,2H),2.11–2.02(m,2H).MS(ESI)m / z=342.1(M+H + ).

[0212] Under nitrogen flow, 1b (100 mg, 0.3 mmol) was added to the bottle, followed by 5 mL of methanol and 5% palladium carbon (180 mg, 0.088 mmol), and the hydrogen atmosphere was replaced. The mixture was stirred at room temperature for 16 hours. After TLC monitoring, the reaction was completed, filtered through celite, and the solvent was removed under reduced pressure to obtain 53 mg of a light yellow solid with a yield of 80%. MS (ESI) m / z = 222.1 (M+H + ).

[0213] Under a nitrogen stream, 1c (20 mg, 90 μmol), (S)-4-ethyl-4-hydroxy-1,6-dihydro-10H-pyrano[3,4-f]indolizine-3,7,10(4H,8H)-trione (1d, 30 mg, 29 μmol), and pyridinium p-toluenesulfonate (7 mg, 27 μmol) were added to the vial. 5 mL of toluene and o-cresol (65 μL, 622 μmol) were added, and the mixture was stirred at 100°C for 16 hours. After completion of the reaction as monitored by TLC, the precipitated solid was filtered, and the filtrate was collected and the solvent removed under reduced pressure. The resulting solid was combined with the filter cake and slurried with ethyl acetate to obtain 35 mg of a yellow solid in an 86% yield.

[0214] 1H NMR (400MHz, DMSO) δ7.96(d,J=9.2Hz,1H),7.67(d,J=9.3Hz,1H),7.25(s,1H),6.50(s,1H),5.42(s,2H),5.13(s,2H),4.93(t,J=5.1Hz,1H),4. 18(t,J=4.3Hz,2H),3.78(d,J=4.5Hz,2H),3.07(s,2H),3.01(s,2H),2. 01(d,J=5.2Hz,2H),1.87(dt,J=13.7,6.7Hz,2H),0.89(t,J=7.1Hz,3H).

[0215] 13 C NMR (101MHz, DMSO) δ173.01,157.31,153.18,150.48,149.89,146.69,143.82,140.37,128.25,126.42,126.02,121.18 ,118.75,118.71,96.54,72.87,71.21,65.73,60.34,49.79,30.71,26.64,23.04,21.04,8.26.MS(ESI)m / z=449.2(M+H + ).

[0216] Example 2: Synthesis of I-2:

[0217] Under a nitrogen stream, activated zinc powder (20.1 g, 307.6 mmol) and 150 mL of anhydrous DMF were added to the flask. Iodine (2.0 g, 7.68 mmol) was then added. After the solution clarified, ethyl 4-bromobutyrate (30.0 g, 153.8 mmol) was added and the mixture was allowed to react at 90°C for 4-5 h. After the reaction was complete as monitored by TLC, the reaction mixture was allowed to cool to room temperature and the supernatant was collected for use. The concentration was approximately 1 mol / L.

[0218] Under a nitrogen stream, 150 mL of anhydrous DMF was added to the flask, followed by 2-bromo-6-fluoroanisole (25 g, 121.9 mmol). Palladium acetate (548 mg, 2.4 mmol) was added and stirred until dissolved. S-PHOS (2.0 g, 4.9 mmol) was added and stirred until dissolved. 160 mL of the supernatant was added dropwise under an ice bath, and the mixture was stirred at 50°C for 16 hours. After TLC monitoring of the reaction completion, saturated ammonium chloride was added to quench the reaction. The reaction solution was poured into 1.5 L of water and extracted with ethyl acetate. The organic phase was washed twice with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain a reddish-brown crude product, which was used directly in the next reaction without purification.

[0219] 1 H NMR (400MHz, CDCl3) δ6.97–6.89(m,3H),4.13(q,J=7.1Hz,2H),3.91(d,J=1.8Hz,3H),2.71–2.65 (m,2H),2.33(t,J=7.5Hz,2H),1.96–1.88(m,2H),1.26(t,J=7.1Hz,3H).MS(ESI)m / z=241.1(M+H + ).

[0220] To a flask, 2a (24.4 g, 101.6 mmol) was added, along with 100 mL of methanol and 50 mL of 4 M sodium hydroxide solution. The mixture was allowed to react at room temperature for 4 h. After completion of the reaction as monitored by TLC, the organic solvent was removed under reduced pressure. 20 mL of water was then added, and the mixture was washed twice with dichloromethane and once with petroleum ether. The aqueous phase was separated and the pH was adjusted to 2 with hydrochloric acid under ice. A large amount of solid precipitated, which was filtered and dried to yield 15.4 g of a pale yellow solid (71% yield).

[0221] 1 H NMR (400MHz, CDCl3) δ6.98–6.88(m,3H),3.92(d,J=1.8Hz,3H),2.74–2.65(m,2H),2.39(t,J=7.4Hz,2H),1.98–1.89(m,2H).MS(ESI)m / z=213.1(M+H + ).

[0222] Under a nitrogen stream, 2b (11.0 g, 51.8 mmol) was added to a flask, followed by 120 mL of anhydrous dichloromethane and dropwise addition of thionyl chloride (5.6 mL, 77.7 mmol). The reaction was allowed to react at room temperature for 3 h. After TLC monitoring of the formation of the acid chloride, anhydrous aluminum chloride (17.3 g, 129.5 mmol) was added under an ice bath. The reaction was returned to room temperature and allowed to react for 3 h. After TLC monitoring of the reaction completion, the reaction solution was quenched by pouring into 10% citric acid. The organic phase was separated, washed with saturated sodium bicarbonate three times and saturated sodium chloride once, dried over anhydrous sodium sulfate, and the organic solvent removed under reduced pressure to yield 8.2 g of a light yellow oily liquid in an 82% yield.

[0223] 1H NMR(400MHz,DMSO)δ7.68(dd,J=8.7,5.4Hz,1H),7.30–7.22(m,1H),3.87(s,3H),2 .92(t,J=6.0Hz,2H),2.60–2.55(m,2H),2.07–2.00(m,2H).MS(ESI)m / z=195.1(M+H + ).

[0224] Under a nitrogen stream, 2c (7 g, 36.0 mmol) was added to a flask, along with 80 mL of 40% aqueous hydrogen bromide solution. The mixture was stirred at 100°C for 16 hours. After completion of the reaction as monitored by TLC, the reaction solution was poured into cold water and the solid was filtered. The solid was dried under reduced pressure and slurried with a PE:EA ratio of 5:1 to yield 6.1 g of a pale yellow solid (93% yield).

[0225] 1 H NMR (400MHz, DMSO) δ9.88 (s, 1H), 7.40 (dd, J = 8.5, 5.4Hz, 1H), 7.14 (t, J = 9.6Hz, 1H), 2.84 (t,J=5.9Hz,2H),2.56–2.51(m,2H),2.01(dd,J=12.5,6.2Hz,2H).MS(ESI)m / z=181.1(M+H + ).

[0226] Under a nitrogen stream, 90 mL of glacial acetic acid was added to the flask, followed by portionwise addition of 2d (6.1 g, 33.8 mmol). 65% nitric acid (2.8 mL, 40.6 mmol) was dissolved in 10 mL of glacial acetic acid and added to the reaction flask under an ice bath. The mixture was gradually warmed to room temperature and stirred for 0.5 h. After completion of the reaction as monitored by TLC, the reaction solution was poured into 400 mL of ice water, the solid was filtered, the filter cake was rinsed with water, and the solid was dried under reduced pressure. The solid was then slurried with PE:EA = 2:1 to obtain 6.6 g of a pale yellow solid (87% yield).

[0227] 1 H NMR (400MHz, DMSO-d6) δ11.01 (s, 1H), 7.78 (d, J = 10.1Hz, 1H), 2.89 (t, J = 6.1Hz, 2H), 2.63 (t, J = 6.6Hz, 2H), 2.05 (p, J = 6.4Hz, 2H). MS (ESI) m / z = 226.0 (M + H) + ).

[0228] Under a nitrogen stream, 2f (2.0 g, 8.9 mmol) and potassium carbonate (2.5 g, 13.4 mmol) were added to a flask, followed by 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h. 2-Bromoethyl acetate (1.5 mL, 13.4 mmol) was then added, and the mixture was stirred at 60°C for 16 h. After completion of the reaction as monitored by TLC, the reaction solution was poured into 100 mL of water and extracted three times with ethyl acetate. The organic phases were separated and combined, washed twice with water and twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 4:1 to 1:1) to afford 1.6 g of a pale yellow solid (59% yield).

[0229] 1 H NMR(400MHz, CDCl3)δ7.12(d,J=10.3Hz,1H),4.37(s,4H),2.96(t,J=6.1Hz,2 H),2.64–2.60(m,2H),2.13–2.08(m,2H),2.05(s,3H).MS(ESI)m / z=312.1(M+H + ).

[0230] Under nitrogen flow, 2 g (900 mg, 2.9 mmol) of the product was added to the flask, followed by 10 mL of methanol and 1.0 g of Raney nickel. The hydrogen atmosphere was replaced and the mixture was stirred at room temperature. After TLC monitoring, the Raney nickel was filtered and the solvent was removed under reduced pressure to obtain 668 mg of a light yellow solid with a yield of 84%.

[0231] Under a nitrogen stream, 2h (650 mg, 2.3 mmol), 1d (730 mg, 2.8 mmol), and pyridinium p-toluenesulfonate (174 mg, 0.7 mmol) were added to the vial, along with 10 mL of toluene and 10 mL of glacial acetic acid. The reaction was stirred at 100°C for 16 hours. After TLC monitoring of the reaction completion, the organic solvent was removed under reduced pressure, followed by the addition of 10 mL of 2M sodium hydroxide solution and stirring for 2 hours. After TLC monitoring of the reaction completion, 2M hydrochloric acid was added to adjust the pH to 2-3, and the precipitated solid was filtered. The solid was slurried with ethyl acetate, filtered, and dried under reduced pressure to obtain 783 mg of a white solid with a yield of 73%.

[0232] 1H NMR (400MHz, DMSO-d6) δ7.77(d,J=12.5Hz,1H),7.27(s,1H),6.51(s,1H),5.42(s,2H),5.15(s,2H),4.91(s,1H),4.1 2(s,2H),3.73(s,2H),3.18–3.06(m,4H),2.01(d,J=5.3Hz,2H),1.87(dt,J=13.3,6.5Hz,2H),0.89(t,J=7.0Hz,3H).

[0233] 13 C NMR (100MHz, DMSO-d6) δ172.93,157.26,156.24,151.77,150.48,146.32,145.97,143.12,141.79,129.85,126.50,123.09,119.31,112 .13,111.94,97.02,76.21,72.85,65.72,60.79,60.23,49.87,30.72,26.66,23.74,21.24,21.16,14.56,8.26.MS(ESI)m / z=467.2(M+H + ).

[0234] Example 3: Synthesis of I-3:

[0235] Under a nitrogen stream, 1a (10 g, 44.4 mmol) and potassium carbonate (9.2 g, 66.6 mmol) were added to a flask, followed by 100 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h. 3-Bromo-1-propanol (5 mL, 66.6 mmol) was then added, and the mixture was stirred at 60°C for 16 h. After completion of the reaction as monitored by TLC, the salts were filtered, the DMF was removed under reduced pressure, and the mixture was redissolved in ethyl acetate, washed twice with water and twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure to obtain a solid PE:EA = 2:1 slurry. 9.5 g of a pale yellow solid was obtained, with a yield of 81%.

[0236] 1 H NMR (400MHz, DMSO) δ7.63(d,J=8.7Hz,1H),7.29(d,J=8.7Hz,1H),4.60(s,1H),4.18(t,J=6.1Hz,2H),3.59(d,J=3.9 Hz,2H),2.86(t,J=6.0Hz,2H),2.69–2.62(m,2H),2.10–2.03(m,2H),1.91(p,J=6.1Hz,2H).MS(ESI)m / z=266.1(M+H+ ).

[0237] Under a nitrogen stream, 3b (1.0 g, 3.2 mmol) was added to a flask, along with 10 mL of methanol and 1.5 g of Raney nickel. The hydrogen atmosphere was replaced, and the mixture was stirred at room temperature for 3 h. After completion of the reaction as monitored by TLC, the mixture was filtered through celite, the solvent was removed under reduced pressure, and column chromatography (PE:EA = 4:1 to 1:1) afforded 785 mg of a pale yellow solid (89% yield).

[0238] 1 H NMR (400MHz, DMSO) δ7.05(d,J=9.0Hz,1H),6.96(s,2H),6.56(d,J=9.0Hz,1H),4.50(t,J=5.1Hz,1H),3.90(t,J=6.2Hz,2H),3.56( dd,J=11.4,6.0Hz,2H),2.76(t,J=6.0Hz,2H),1.89(dt,J=12.6,6.3Hz,2H),1.80(dd,J=12.5,6.2Hz,2H).MS(ESI)m / z=236.1(M+H + ).

[0239] Under a nitrogen stream, 3c (650 mg, 2.8 mmol), (S)-4-ethyl-4-hydroxy-1,6-dihydro-10H-pyrano[3,4-F]indolizine-3,7,10(4H,8H)-trione (873 mg, 3.4 mmol), and pyridinium p-toluenesulfonate (208 mg, 0.84 mmol) were added to the vial. 10 mL of toluene and o-cresol (2 mL, 19.6 mmol) were added, and the mixture was stirred at 100°C for 16 hours. After completion of the reaction as monitored by TLC, the precipitated solid was filtered, and the filtrate was collected and the solvent removed under reduced pressure. The resulting solid was combined with the filter cake and slurried with ethyl acetate to obtain 1.0 g of a yellow solid with a yield of 78%.

[0240] 1 H NMR (400MHz, DMSO) δ7.93(d,J=9.2Hz,1H),7.63(d,J=9.3Hz,1H),7.24(s,1H),6.49(s,1H),5.41(s,2H),5.04(s,2H),4.60(s,1H),4. 19(t,J=5.8Hz,2H),3.62(s,2H),3.02(s,2H),2.93(s,2H),1.98(d,J=5.5Hz,2H),1.88(dt,J=13.6,6.4Hz,4H),0.90(t,J=7.1Hz,3H).

[0241] 13 C NMR (101MHz, DMSO) δ173.02,157.28,153.06,150.44,149.76,146.65,143.65,140.20,128.25,126.26,125.91,120.80,11 8.71,118.27,96.53,72.87,66.00,65.74,57.77,49.73,32.88,30.75,26.57,22.96,20.97,8.25.MS(ESI)m / z=463.2(M+H + ).

[0242] Example 4: Synthesis of I-4:

[0243] Under a nitrogen stream, 2f (1.5 g, 6.6 mmol) and potassium carbonate (1.4 g, 9.9 mmol) were added to the vial, followed by 10 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h, followed by 3-bromo-1-propanol (900 μL, 9.9 mmol). The mixture was stirred at 60°C for 16 h. After completion of the reaction as monitored by TLC, the reaction solution was poured into 100 mL of water and extracted three times with ethyl acetate. The organic phases were separated and combined, washed twice with water and twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 4:1 to 1:1) to afford 909 mg of a light yellow solid (48% yield).

[0244] 1 H NMR (400MHz, CDCl3) δ7.17(d,J=10.3Hz,1H),4.33(td,J=6.1,1.6Hz,2H),3.90(t,J=6.0Hz,2H),2.98 (t,J=6.1Hz,2H),2.71–2.65(m,2H),2.18–2.12(m,2H),2.06(p,J=6.0Hz,2H).MS(ESI)m / z=284.1(M+H + ).

[0245] Under a nitrogen stream, 4 g (900 mg, 3.2 mmol) of the compound was added to the flask, along with 10 mL of methanol and 1.0 g of Raney nickel. The hydrogen atmosphere was replaced with stirring at room temperature. After TLC monitoring of the reaction, the product was filtered through celite, the solvent was removed under reduced pressure, and column chromatography (PE:EA = 4:1 to 1:1) afforded 668 mg of a pale yellow solid (82% yield).

[0246] 1H NMR(400MHz, CDCl3) δ6.26(d,J=12.6Hz,1H),4.01(t,J=5.9Hz,2H),3.91(t,J=5.8Hz,2 H),2.91(t,J=6.1Hz,2H),2.61–2.56(m,2H),2.04–1.98(m,4H).MS(ESI)m / z=254.1(M+H + ).

[0247] Under a nitrogen stream, 4h (500 mg, 2.0 mmol), (S)-4-ethyl-4-hydroxy-1,6-dihydro-10H-pyrano[3,4-F]indolizine-3,7,10(4H,8H)-trione (1i, 623 mg, 2.4 mmol), and pyridinium p-toluenesulfonate (148 mg, 0.6 mmol) were added to the vial. 10 mL of toluene and 10 mL of glacial acetic acid were added, and the mixture was stirred at 100°C for 16 hours. After TLC monitoring of the reaction, the target molecule and the hydroxypropyl acetylated compound were present in the reaction system. The organic solvent was removed under reduced pressure, and 10 mL of 2M sodium hydroxide solution was added and stirred for 2 hours. After TLC monitoring of the reaction, 2M hydrochloric acid was added to adjust the pH to 2-3. The precipitated solid was filtered, slurried with ethyl acetate, filtered, and dried under reduced pressure to obtain 488 mg of a white solid (51% yield).

[0248] 1 H NMR (400MHz, DMSO) δ7.78(d,J=12.5Hz,1H),7.27(s,1H),6.51(s,1H),5.42(s,2H),5.16(s,2H),4.58(s,1H),4.16( d,J=5.2Hz,2H),3.63(d,J=4.9Hz,2H),3.10(d,J=4.4Hz,4H),2.03(s,2H),1.96–1.83(m,4H),0.90(d,J=6.4Hz,3H).

[0249] 13C NMR (101MHz, DMSO) δ172.93,158.72,157.25,156.21,151.76,150.46,146.29,145.93,145.80,143.12,142.98,141.72,129.63,129.59,126.4 5,123.06,119.32,112.20,112.01,97.02,72.84,71.67,65.72,57.81, 49.84,33.62,30.74,26.62,23.74,21.14,8.26.MS(ESI)m / z=481.2(M+H + ).

[0250] Example 5: Synthesis of Compound I-5:

[0251] Under a nitrogen stream, 2f (2.0 g, 8.9 mmol) and potassium carbonate (2.4 mg, 0.017 mmol) were added to the vial, followed by 10 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 hour, followed by the addition of ethyl bromoacetate (1.5 mL, 13.5 mmol), and stirred at room temperature for 6 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (PE:EA = 5:1) afforded 2.2 g of a white solid, with a yield of 79%.

[0252] 1 H NMR (400MHz, DMSO) δ7.91(d,J=11.3Hz,1H),4.95(s,2H),4.18(q,J=7.0Hz,2H),3.04(t,J=5.9H z,2H),2.65(t,J=6.5Hz,2H),2.10–2.01(m,2H),1.21(t,J=7.1Hz,3H).MS(ESI)m / z=312.1(M+H + ).

[0253] Under a nitrogen stream, 5b (1.6 g, 5.1 mmol) was added to 20 mL of methanol and 1.5 g of Raney nickel to displace the hydrogen. The mixture was stirred at room temperature for 2.5 hours. After completion of the reaction as monitored by TLC, the mixture was filtered through celite and the solvent removed under reduced pressure to yield 1.5 g of a yellow solid in a 105% yield.

[0254] 1H NMR (400MHz, CDCl3) δ6.46 (s, 2H), 6.21 (d, J = 13.0Hz, 1H), 4.52 (s, 2H), 4.26 (q, J = 7.1Hz, 2H), 3.00 (t, J=6.1Hz,2H),2.59(t,J=6.4Hz,2H),2.04–1.97(m,2H),1.30(t,J=7.1Hz,3H).MS(ESI)m / z=282.2(M+H + ).

[0255] Under a nitrogen stream, 5c (500 mg, 1.8 mmol), 1d (561 mg, 2.1 mmol), and pyridinium p-toluenesulfonate (143 mg, 0.57 mmol) were added to a vial, along with 10 mL of toluene and 1 mL of acetic acid. The mixture was stirred at 110°C for 16 hours. After completion of the reaction as monitored by TLC, the solid was filtered, the filter cake rinsed with 2 mL of methanol, and dried under reduced pressure to yield 763 mg of a gray-green solid in 83% yield.

[0256] 1 H NMR (400MHz, DMSO) δ7.82(d,J=12.9Hz,1H),7.29(s,1H),6.50(s,1H),5.43(s,2H),5.21(s,2H),4.88(s,2H),4.19(q,J=7.1Hz,2H),3.16(d t,J=22.2,5.4Hz,4H),2.08–1.99(m,2H),1.87(dt,J=13.8,6.9Hz,2H),1.22(t,J=7.1Hz,3H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z=509.2(M+H) + ).

[0257] Under nitrogen flow, 5d (450 mg, 0.88 mmol), 1M sodium hydroxide (2.0 mL, 2 mmol), 4 mL of methanol, and 6 mL of water were added to the bottle and stirred at room temperature for 3 hours. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure, the pH was adjusted to 1-2, and the precipitated solid was filtered. The filter cake was rinsed with 2 mL of methanol and the solid was dried under reduced pressure to obtain 403 mg of a light yellow solid with a yield of 95%. MS (ESI) m / z = 481.1 (M+H + ).

[0258] Under a nitrogen stream, 5e (45 mg, 0.094 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (71 mg, 0.19 mmol) were added to the vial, followed by 5 mL of anhydrous dichloromethane. The mixture was stirred for 0.5 hours, followed by 4-hydroxypiperidine (18 mg, 0.18 mmol) and stirring at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and water was added to precipitate a solid. The pH was adjusted to 2-3, the solid was filtered, and the filter cake was rinsed with 2 mL of methanol to obtain 44 mg of a light yellow solid (83% yield).

[0259] 1 H NMR(400MHz,DMSO-d6)δ7.80(d,J=9.2Hz,1H),7.28(s,1H),6.50(s,1H),5.41(s,2H),5.14(s,2H ),5.09–4.98(m,2H),4.89(d,J=82.6Hz,1H),3.89(d,J=12.6Hz,1H),3.72(d,J=3.8Hz,2H),3.24– 3.18(m,1H),3.12–2.98(m,5H),2.07–1.96(m,2H),1.86(dt,J=14.4,7.0Hz,2H),1.75(dd,J=37.9 ,8.2Hz,2H),1.47–1.36(m,1H),1.31–1.21(m,1H),0.88(t,J=7.1Hz,3H).MS(ESI)m / z=564.2(M+H + ).

[0260] Example 6: Synthesis of Compound I-6:

[0261] Under a nitrogen stream, 5e (50 mg, 0.10 mmol) and HATU (80 mg, 0.21 mmol) were added to the vial, followed by 10 mL of anhydrous dichloromethane. The mixture was stirred for 0.5 hours, followed by DIPEA (45 μL, 0.26 mmol) and 3-hydroxyazetidine hydrochloride (17 mg, 0.16 mmol). The mixture was stirred at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and the product was purified by column chromatography (DCM:MeOH = 50:1) to afford 32 mg of a pale yellow solid (60% yield).

[0262] 1H NMR (400MHz, DMSO) δ7.80(d,J=12.7Hz,1H),7.28(s,1H),6.51(s,1H),5.77(d,J=6.1Hz,1H),5.4 2(s,2H),5.18(s,2H),4.67(s,2H),4.50(dd,J=10.9,4.8Hz,1H),4.41(t,J=7.8Hz,1H),4.15–4. 09(m,1H),3.96(dd,J=8.9,4.0Hz,1H),3.66(dd,J=10.0,4.1Hz,1H),3.13(dt,J=11.1,5.3Hz,4H ),2.06–1.99(m,2H),1.87(dt,J=13.7,6.8Hz,2H),0.88(t,J=7.2Hz,3H).MS(ESI)m / z=536.2(M+H + ).

[0263] Example 7: Synthesis of Compound I-7:

[0264] Under a nitrogen stream, 1a (2.0 g, 9.7 mmol) and potassium carbonate (2.7 g, 19.5 mmol) were added to a vial, along with 20 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h. Ethyl bromoacetate (1.6 mL, 14.4 mmol) was then added, and the mixture was stirred at 40°C for 6 h. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure. Column chromatography (PE:EA = 5:1) afforded 1.8 g of a pale yellow solid in a 60% yield.

[0265] 1 H NMR (400MHz, DMSO) δ7.62(d,J=8.7Hz,1H),7.27(d,J=8.8Hz,1H),5.00(s,2H),4.18(q,J=7.1Hz,2H),2.92 (t,J=6.0Hz,2H),2.67(t,J=6.5Hz,2H),2.11–2.04(m,2H),1.22(t,J=7.1Hz,3H).MS(ESI)m / z=294.1(M+H + ).

[0266] Under a nitrogen stream, 7b (1.8 g, 6.1 mmol) was added to 30 mL of methanol and 1.5 g of Raney nickel to displace the hydrogen. The mixture was stirred at room temperature for 2.5 hours. After completion of the reaction as monitored by TLC, the mixture was filtered through celite and the solvent removed under reduced pressure to yield 1.5 g of a yellow solid (93% yield).

[0267] 1H NMR (400MHz, CDCl3) δ6.92(d,J=8.9Hz,1H),6.45(d,J=8.9Hz,1H),6.17(s,2H),4.53(s,2H),4.26(q,J=7.1Hz,2H ),2.96(t,J=6.1Hz,2H),2.62(t,J=6.4Hz,2H),2.07–2.00(m,2H),1.30(t,J=7.1Hz,3H).MS(ESI)m / z=264.1(M+H + ).

[0268] Under a nitrogen stream, 7c (100 mg, 0.38 mmol), (S)-4-ethyl-4-hydroxy-1,6-dihydro-10H-pyrano[3,4-f]indolizine-3,7,10(4H,8H)-trione (120 mg, 0.46 mmol), and pyridinium p-toluenesulfonate (30 mg, 0.12 mmol) were added to a vial. 10 mL of toluene and 10 mL of acetic acid were added, and the mixture was stirred at 110°C for 16 hours. After completion of the reaction as monitored by TLC, the solid was filtered, the filter cake was rinsed with 2 mL of methanol, and the solid was dried under reduced pressure to obtain 150 mg of a gray-green solid with an 80% yield.

[0269] 1 H NMR (400MHz, DMSO) δ7.97(d,J=9.3Hz,1H),7.60(d,J=9.4Hz,1H),7.27(s,1H),6.50(s,1H),5.42(s,2H),5.20(s,2H),5.01(s,2H),4.19(q,J=7.0 Hz,2H),3.09(dt,J=19.1,5.1Hz,4H),2.06–1.99(m,2H),1.92–1.81(m,2 H),1.23(t,J=7.1Hz,3H),0.89(t,J=7.1Hz,3H).MS(ESI)m / z=491.2(M+H + ).

[0270] Under a nitrogen stream, 7d (140 mg, 0.28 mmol) and potassium carbonate (60 mg, 0.43 mmol) were added to the vial, along with 5 mL of DMF and 5 mL of water. The mixture was stirred at room temperature for 3 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, the pH was adjusted to 1-2, and the precipitated solid was filtered. The filter cake was rinsed with 2 mL of methanol and dried under reduced pressure to yield 105 mg of a light yellow solid (81% yield).

[0271] 1H NMR (400MHz, DMSO) δ13.09(s,1H),7.97(d,J=9.2Hz,1H),7.58(d,J=9.3Hz,1H),7.26(s,1H),6.49(s,1H),5.42(s,2H),5.19 (s,2H),4.90(s,2H),3.08(d,J=21.8Hz,4H),2.02(s,2H),1.91–1.82(m,2H),0.88(t,J=7.0Hz,3H).MS(ESI)m / z=463.1(M+H + ).

[0272] Under a nitrogen stream, 7e (60 mg, 0.13 mmol) and HATU (100 mg, 0.26 mmol) were added to the vial, followed by 5 mL of anhydrous dichloromethane. The mixture was stirred for 0.5 s. 4-Hydroxypiperidine (27 mg, 0.27 mmol) was then added and stirred at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and water was added to precipitate a solid. The pH was adjusted to 2-3, the solid was filtered, and the filter cake was rinsed with 2 mL of methanol to obtain 55 mg of a light yellow solid (77% yield).

[0273] 1 H NMR (400MHz, DMSO) δ7.94(d,J=9.2Hz,1H),7.54(d,J=9.3Hz,1H),7.25(s,1H),6.49(s,1H),5.4 1(s,2H),5.14(s,2H),5.09–4.98(m,2H),4.89(d,J=82.6Hz,1H),3.89(d,J=12.6Hz,1H),3.72(d ,J=3.8Hz,2H),3.24–3.18(m,1H),3.12–2.98(m,5H),2.07–1.96(m,2H),1.86(dt,J=14.4,7.0Hz ,2H),1.75(dd,J=37.9,8.2Hz,2H),1.47–1.36(m,1H),1.31–1.21(m,1H),0.88(t,J=7.1Hz,3H).

[0274] 13C NMR (101MHz, DMSO) δ173.00,166.05,157.32,152.68,150.48,149.97,146.67,143.91,140.41,127.99,126.46,125.98,120.91,118. 78,118.35,96.58,72.87,67.12,65.89,65.74,49.81,42.18,34.96,34.30,30.73,26.61,23.12,21.02,8.27.MS(ESI)m / z=546.2(M+H + ).

[0275] Example 8: Synthesis of Compound I-8:

[0276] Under a nitrogen stream, 7e (100 mg, 0.22 mmol) and HATU (164 mg, 0.43 mmol) were added to the vial, followed by 10 mL of anhydrous dichloromethane. The mixture was stirred for 0.5 hours, followed by DIPEA (113 μL, 0.64 mmol) and 3-hydroxyazetidine hydrochloride (48 mg, 0.45 mmol). The mixture was stirred at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and the product was purified by column chromatography (DCM:MeOH = 50:1) to afford 65 mg of a light yellow solid (57% yield).

[0277] 1 H NMR (400MHz, DMSO) δ7.98(d,J=9.3Hz,1H),7.55(d,J=9.4Hz,1H),7.26(s,1H),6.50(s,1H) ,5.77(d,J=6.0Hz,1H),5.42(s,2H),5.20(s,2H),4.87–4.77(m,2H),4.54–4.42(m,2H),4. 16–4.10(m,1H),3.99(d,J=5.1Hz,1H),3.66(dd,J=10.0,3.6Hz,1H),3.10(d,J=5.1Hz,2H) ,3.07–3.01(m,2H),2.06–1.98(m,2H),1.86(td,J=13.8,6.8Hz,2H),0.88(t,J=7.2Hz,3H).

[0278] 13C NMR (101MHz, DMSO) δ189.28,173.01,167.91,157.34,152.41,150.51,150.17,146.67,144.04,140.56,128.19,126.62,126.06,12 1.12,118.85,118.15,96.63,72.87,66.53,65.73,61.20,60.26,58.55,49.86,30.70,26.62,21.04,8.27.MS(ESI)m / z=518.2(M+H + ).

[0279] Example 9: Synthesis of Compound I-9:

[0280] Under a nitrogen stream, 7e (50 mg, 0.11 mmol) and HATU (82 mg, 0.22 mmol) were added to the vial, along with 2 mL of anhydrous dichloromethane. The mixture was stirred for 0.5 hours, followed by the addition of ethanolamine (13 μL, 0.22 mmol). The mixture was stirred at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (DCM:MeOH = 50:1) afforded 44 mg of a pale yellow solid (79% yield).

[0281] 1 H NMR (400MHz, DMSO-d6) δ7.54(s,2H),7.26(s,2H),6.98(s,2H),5.78–5.67(m,6H),4.93(d,J=6.0Hz,4H),4.44(s,4H),3.74(s,2H),3.4 6(s,4H),3.19(s,4H),3.14(s,2H),2.96(s,2H),2.24(d,J=3.0Hz,4H),1.98(s,2H),1.32(s,2H),0.89(s,3H).MS(ESI)m / z=506.2(M+H + ).

[0282] Example 10: Synthesis of Compound II-1:

[0283] Under a nitrogen stream, I-4 (100 mg, 208 μmol) and II-1a (92 mg, 270 μmol, CAS No. 1599440-06-8, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) were added to a vial. 5 mL of anhydrous DMF was added and stirred at room temperature for dissolution. Boron trifluoride etherate (35 μL, 312 μmol) was added and stirred at 40°C for 16 hours. LC-MS monitoring of the reaction system showed 70% conversion of the starting material to product II-1b. The reaction was stirred for an additional 8 hours at 40°C. LC-MS monitoring of the reaction system also showed 70% conversion of the starting material to product II-1b. The solvent was removed under reduced pressure, and 4 mL of tetrahydrofuran and 1 mL of diethylamine were added. The mixture was stirred at room temperature for 3 hours. LC-MS monitoring of the reaction system showed complete deprotection of II-1b to II-1c. The solvent was removed under reduced pressure and the product was purified using a reverse phase column (mobile phase: 0.1% trifluoroacetic acid aqueous solution and acetonitrile; mobile phase acetonitrile concentration: 5% acetonitrile, 2CV - 5% - 30% acetonitrile, 10CV - 30% - 40% acetonitrile, 5CV - 40% - 95% acetonitrile, 5CV - 95% acetonitrile, 5CV). Lyophilization gave 55 mg of a yellow solid with a yield of 47%. MS (ESI) m / z = 567.2 (M+H + ).

[0284] Under a nitrogen stream, MC-GGF-OH (44 mg, 93 μmol, CAS No. 1599440-15-9, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) and DMTMM (27 mg, 93 μmol) were added to the vial, followed by 5 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 hours. II-1c (35 mg, 62 μmol) and DIPEA (15 μL, 93 μmol) were then added, and the mixture was stirred at 40°C for 8 hours. After completion of the reaction as monitored by LC-MS, the solvent was removed under reduced pressure, and column chromatography (DCM:MeOH = 25:1) afforded 42 mg of a white solid in a 67% yield.

[0285] 1H NMR (400MHz, DMSO) δ8.51 (s, 1H), 8.31 (s, 1H), 7.82 (d, J = 12.4Hz, 1H), 7.39–7.09 (m, 6H), 7. 00(d,J=13.6Hz,1H),6.53(d,J=16.7Hz,1H),5.34(d,J=73.4Hz,3H),4.64–4.39(m,2H),4.17 (ddd,J=36.5,30.8,18.2Hz,4H),3.76–3.50(m,7H),3.24–3.03(m,5H),2.15–1.83(m,7H),1. 54(d,J=53.4Hz,3H),1.26(t,J=6.4Hz,12H),0.88(t,J=7.1Hz,3H).MS(ESI)m / z=1021.4(M+H + ).

[0286] Example 11: Synthesis of Compound II-2:

[0287] Under a nitrogen stream, Cbz-VA-OH (20.0 g, 62.0 mmol, CAS No. 24787-89-1, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.), DECI (11.8 g, 62.0 mmol), and HOBT (8.4 g, 62.0 mmol) were added to a vial. 30 mL of anhydrous dichloromethane was then added, along with glycine tert-butyl ester (370 μL, 2.6 mmol) and DIPEA (10.8 mL, 62.0 mmol). The mixture was stirred at room temperature for 16 hours. After completion of the reaction as monitored by TLC, the organic solvent was removed under pressure. The mixture was purified by column chromatography (PE:EA = 5:1 to 1:1) to afford 24.5 g of a white solid in a 91% yield.

[0288] 1H NMR (400MHz, DMSO-d6) δ7.32(d,J=5.0Hz,5H),6.61(s,1H),5.76(s,1H),5.06(d,J=13.4Hz,3H),4.93(s,1 H),4.69(s,1H),4.35(s,1H),4.10(s,1H),2.73(s,1H),1.42(s,12H),0.96(s,6H).MS(ESI)m / z=436.3(M+H + ).

[0289] Under a nitrogen stream, II-2b (20.0 g, 45.9 mmol), 100 mL of dichloromethane, and 20 mL of trifluoroacetic acid were added to the vial, and the mixture was stirred at room temperature for 5 h. After the reaction was complete as monitored by TLC, the solvent was removed under reduced pressure, and the solid was slurried with PE:EA = 1:1 to obtain 16.3 g of a white solid, with a yield of 94%.

[0290] 1H NMR(500MHz,DMSO-d6)δ7.30(d,J=5.0Hz,5H),5.49(s,1H),5.37(s,1H),5.03(t,J=3.9Hz,4H),4 .70(s,1H),4.13(d,J=6.3Hz,2H),2.72(s,1H),1.41(s,3H),0.96(s,6H).MS(ESI)m / z=380.2(M+H + ).

[0291] Under a nitrogen stream, II-2c (10.0 g, 26.4 mmol), lead tetraacetate (23.4 g, 52.8 mmol), and copper acetate (472 mg, 2.6 mmol) were added to a flask. 40 mL of anhydrous toluene and 160 mL of anhydrous tetrahydrofuran were also added, along with pyridine (4.3 mL, 52.8 mmol). The mixture was heated and stirred at 60°C for 6 hours. After completion of the reaction as monitored by TLC, the inorganic salts were filtered out, and the filter cake was rinsed with 20 mL of ethyl acetate. The solvent was removed under reduced pressure, and the mixture was slurried with water and then with PE:EA = 1:1 to obtain 8.2 g of a white solid (79% yield).

[0292] 1 H NMR (400MHz, DMSO-d6) δ8.88(t,J=6.8Hz,1H),8.06(d,J=7.1Hz,1H),7.39–7.28(m,7H),5.12–4.99(m,4H),4.30(td,J=14.2,7.1H z,1H),3.90–3.85(m,1H),1.98(s,3H),1.20(d,J=6.9Hz,3H),0.86(d,J=6.7Hz,3H),0.82(d,J=6.6Hz,3H).MS(ESI)m / z=394.2(M+H + ).

[0293] Under a nitrogen flow, II-2d (164 mg, 416 μmol), I-4 (100 mg, 208 μmol), and PPTS (78 mg, 312 μmol) were added to a vial, along with 5 mL of anhydrous tetrahydrofuran, and stirred at 45°C for 16 hours. LC-MS monitoring of the reaction system revealed 60% conversion of the starting material to product II-2e, and the solvent was removed under reduced pressure. Under a nitrogen flow, 1 mL of DMA and 9 mL of methanol were added, along with 10 mg of Pd / C, and the atmosphere was replaced with hydrogen. The product was stirred at room temperature for 2 hours. After completion of the reaction, as monitored by LC-MS, the Pd / C was removed by filtration, the solvent was removed under reduced pressure, and the product was purified using a reverse-phase column (mobile phase: 0.1% trifluoroacetic acid in water and acetonitrile; mobile phase acetonitrile concentration: 5% acetonitrile in 2 CV, 5% to 30% acetonitrile in 10 CV, 40% to 95% acetonitrile in 5 CV, 95% to 95% acetonitrile in 5 CV, and 5 CV). The solid was lyophilized to give 71 mg with a yield of 50%. MS (ESI) m / z = 680.3 (M+H + ).

[0294] 1 H NMR(400MHz,DMSO)δ8.82(s,1H),8.63(d,J=6.9Hz,1H),8.10(s,2H),7.77(d,J =11.6Hz,1H),7.27(s,1H),5.41(s,2H),5.15(s,2H),4.58(dd,J=13.0,6.6Hz,2 H),4.37–4.29(m,1H),4.10(s,2H),3.08(s,4H),2.03(d,J=3.4Hz,3H),1.98–1 .93(m,2H),1.91–1.82(m,2H),1.30–1.19(m,6H),0.89(dd,J=17.3,6.9Hz,9H).

[0295] Under a nitrogen flow, II-2f (20 mg, 29 μmol) was added to the vial, along with 2 mL of anhydrous DMF, DIPEA (5.0 μL, 29 μmol), and Mc-PEG12-OSu (25 mg, 29 μmol, CAS No. 756525-92-5, purchased from Bomei Biotechnology Co., Ltd.), and stirred at room temperature for 6 hours. After completion of the reaction, monitored by LC-MS, the product was purified using a reverse-phase column (mobile phase: 0.05% formic acid in water and acetonitrile; acetonitrile concentration in the mobile phase ranged from 5% acetonitrile in 2 CV to 5% to 50% acetonitrile in 20 CV, then from 50% to 95% acetonitrile in 5 CV to 95% acetonitrile in 5 CV). Lyophilization afforded 23 mg of a solid in a 55% yield. MS (ESI) m / z = 1430.7 (M+H + ).

[0296] 1 H NMR (400MHz, DMSO) δ8.65–8.59(m,1H),8.30(t,J=24.6Hz,1H),8.06(d,J=6.9Hz,1H),7.98(dd,J=15.9,6 .4Hz,1H),7.86(d,J=8.8Hz,1H),7.79(d,J=12.4Hz,1H),7.28(s,1H),6.98(s,1H),5.42(s,2H),5.19(s, 2H),4.62–4.50(m,3H),4.28–4.21(m,2H),4.15(dd,J=19.3,11.9Hz,6H),3.58–3.57(m,3H),3.49(s,44H ),3.16–3.06(m,6H),2.48–2.24(m,6H),2.03(s,2H),1.96(s,2H),1.94–1.81(m,4H),0.88–0.80(m,9H).

[0297] Example 12: Synthesis of Compound II-3:

[0298] Under a nitrogen stream, Cbz-GG-OH (25.0 g, 93.9 mmol, CAS No. 2566-19-0, Shanghai Bid Pharmaceutical Technology Co., Ltd.), lead tetraacetate (83.3 g, 187.8 mmol), and copper acetate (1.7 g, 9.4 mmol) were added to the flask. 40 mL of anhydrous toluene and 160 mL of anhydrous tetrahydrofuran were also added, along with pyridine (15 mL, 187.8 mmol). The mixture was heated and stirred at 60°C for 6 hours. After TLC monitoring of the reaction, the inorganic salts were filtered out, and the filter cake was rinsed with 20 mL of ethyl acetate. The solvent was removed under reduced pressure, and the mixture was slurried with water and then with PE:EA = 1:1 to obtain 14.5 g of a white solid (yield: 55%).

[0299] 1H NMR (400MHz, DMSO) δ8.94(t,J=6.8Hz,1H),7.49(t,J=6.0Hz,1H),7.38–7.32(m,5H),5.10(d, J=6.9Hz,2H),5.04(s,2H),3.66(d,J=6.2Hz,2H),1.99(s,3H).MS(ESI)m / z=281.2(M+H + ).

[0300] Under a nitrogen flow, II-3a (88 mg, 312 μL), I-4 (50 mg, 104 μL), and pyridinium p-toluenesulfonate (40 mg, 156 μL) were added to a vial, along with 5 mL of anhydrous tetrahydrofuran. The mixture was stirred at 45°C for 16 hours. LC-MS monitoring of the reaction system revealed 70% conversion of the starting material to product 12c. The solvent was then removed under reduced pressure. Under a nitrogen flow, 5 mL of 0.1% aqueous trifluoroacetic acid, 10 mL of methanol, and 1 mL of DMF were added to the vial. 13 mg of Pd / C was added to displace the hydrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction as monitored by LC-MS, the Pd / C was filtered, the solvent was removed under reduced pressure, and the product was purified using a reverse-phase column (mobile phase: 0.1% trifluoroacetic acid aqueous solution and acetonitrile; mobile phase acetonitrile concentration: 5% acetonitrile, 2CV to 5%-30% acetonitrile, 10CV to 30%-40% acetonitrile, 5CV to 40%-95% acetonitrile, 5CV to 95% acetonitrile, 5CV). Lyophilization afforded 32 mg of a yellow solid in a 54% yield.

[0301] 1 H NMR (400MHz, DMSO) δ9.11 (s, 1H), 8.03 (s, 2H), 7.84 (d, J = 12.3Hz, 1H), 7.29 (s,1H),6.52(s,1H),5.43(s,2H),5.25(s,2H),4.64(d,J=6.2Hz,2H),4.14 (t,J=5.7Hz,2H),3.67–3.60(m,4H),3.18–3.09(m,4H),2.03(dd,J=11.3,5 .5Hz,4H),1.91–1.82(m,2H),0.88(t,J=6.9Hz,3H).MS(ESI)m / z=567.2(M+H + ).

[0302] Under a nitrogen flow, Cbz-GGF-OH (76 mg, 185 μmol, CAS No. 13171-93-2, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) and DMTMM (36 mg, 185 μmol) were added to a vial, followed by 5 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 hours. II-3c (70 mg, 124 μmol) and DIPEA (21 μL, 124 μmol) were then added, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete as monitored by LC-MS, the solvent was removed under reduced pressure. Under a nitrogen flow, 13 mL of methanol and 2 mL of DMA were added, along with 5 mg of Pd / C and 5 mg of PdOH. The hydrogen atmosphere was replaced, and the mixture was allowed to react at room temperature for 16 hours. After the reaction was complete as monitored by LC-MS, the Pd / C and PdOH were removed by filtration, and the solvent was removed under reduced pressure. Purify using a reverse phase column (mobile phase: 0.1% trifluoroacetic acid aqueous solution and acetonitrile; mobile phase acetonitrile concentration: 5% acetonitrile, 2CV - 5% - 50% acetonitrile, 20CV - 50% - 95% acetonitrile, 5CV - 95% acetonitrile, 5CV). Lyophilize to obtain 84 mg of solid, yield 82%.

[0303] 1 H NMR (400MHz, DMSO) δ8.73(s,1H),8.62(t,J=6.1Hz,1H),8.51(t,J=5.2Hz,1H),8.41–8.30(m,2 H),8.00(s,2H),7.80(d,J=12.4Hz,1H),7.28(s,1H),7.24(d,J=11.2Hz,4H),7.17(d,J=5.7Hz, 1H),5.43(s,2H),5.18(s,2H),4.59(d,J=6.3Hz,2H),4.12(d,J=5.9Hz,2H),3.93–3.78( m,2H),3.78–3.70(m,3H),3.69–3.64(m,1H),3.63–3.56(m,3H),3.11(d,J=4.8Hz,2H),3 .05(dd,J=13.9,4.2Hz,1H),2.75(dd,J=13.7,10.4Hz,1H),2.54(d,J=5.4Hz,2H),2.07– 1.97(m,4H),1.87(dt,J=13.5,6.6Hz,2H),0.89(t,J=7.2Hz,3H).MS(ESI)m / z=828.3(M+H + ).

[0304] Under a nitrogen stream, II-3e (30 mg, 36 μmol) was added to the vial, followed by 2 mL of anhydrous DMF, DIPEA (4.6 μL, 36.2 μmol), and Mc-PEG12-OSu (31 mg, 36.2 μmol). The mixture was stirred at room temperature for 6 hours. After completion of the reaction, monitored by LC-MS, the product was purified using a reverse-phase column (mobile phase: 0.05% formic acid in water and acetonitrile; acetonitrile concentration in the mobile phase was: 5% acetonitrile in 2 CVs to 5% to 50% acetonitrile, 20 CVs to 50% to 95% acetonitrile, 5 CVs to 95% acetonitrile, 5 CVs). Lyophilization afforded 35 mg of a solid with a yield of 61%. MS (ESI) m / z = 1578.7 (M+H + ).

[0305] 1 H NMR (400MHz, DMSO) δ8.53(t,J=6.6Hz,1H),8.30(t,J=5.7Hz,1H),8.19(t,J=5.5Hz,1H),8.12(d,J=8.1Hz,1H),8.02(d,J=3.4Hz,2H),7.76(d,J=12.4 Hz,1H),7.32–7.10(m,7H),6.98(s,1H),6.52(s,1H),5.41(s,2H),5.13(s, 2H),4.57(d,J=6.5Hz,2H),4.47(dd,J=10.8,6.7Hz,1H),4.11(t,J=6.1Hz, 2H),3.77–3.67(m,6H),3.60(s,2H),3.58(s,2H),3.57(d,J=2.5Hz,2H),3. 48(d,J=6.7Hz,44H),3.35(t,J=5.8Hz,2H),3.14(d,J=5.7Hz,2H),3.08(d, J=3.9Hz,2H),3.06–2.99(m,2H),2.38(t,J=6.4Hz,2H),2.32(t,J=7.2Hz,2 H),2.04–1.95(m,4H),1.86(dt,J=13.4,6.8Hz,2H),0.88(t,J=7.3Hz,3H).

[0306] Example 13:

[0307] Referring to Example II-2, Mc-PEG8-Osu (CAS: 756525-93-6, purchased from Bomei Biotechnology Co., Ltd.) was used to replace Mc-PEG12-Osu to prepare compound II-4. MS (ESI) m / z = 1254.6 (M+H + ).

[0308] Example 14:

[0309] Referring to Example II-3, Mc-PEG8-Osu was used to replace Mc-PEG12-Osu to prepare compound II-5. MS (ESI) m / z = 1402.6 (M+H + ).

[0310] Synthesis of compounds ADC-1 and ADC-2 in Examples 15 and 16:

[0311] At 37°C, the antibody Trastuzumab (HER2 antibody, CAS: 180288-69-1) was purchased from Hangzhou Haoyang Biotechnology Co., Ltd.) in a PBS buffered aqueous solution (pH = 6.5 0.05M PBS buffered aqueous solution; 10.0 mg / mL, 1.0 mL, 67.6 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 37.2 μL, 372 nmol), placed in a water bath shaker, and oscillated at 37°C for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath. Compound II-2 or II-3 (1014 nmol) was dissolved in 50 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and oscillated at 25°C for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (mobile phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain conjugates ADC-1 and ADC-2 in PBS buffer (1.1 mg / mL, 7.5 mL), which were stored at 4°C.

[0312] Average drug loading calculated by RP-HPLC: ADC-1, t = 7.8; ADC-2, t = 7.8

[0313] Example 17

[0314] Compound ADC-3 was prepared by referring to the synthesis examples of compounds ADC-1 and ADC-2.

[0315] The average drug loading calculated by RP-HPLC was: ADC-3, t=6.2.

[0316] Example 18

[0317] Compound ADC-4 was prepared by referring to the synthesis examples of compounds ADC-1 and ADC-2.

[0318] The average drug loading calculated by RP-HPLC was: ADC-4, t=7.7.

[0319] Example 19

[0320] Compound ADC-5 was prepared by referring to the synthesis examples of compounds ADC-1 and ADC-2.

[0321] The average drug loading calculated by RP-HPLC was: ADC-5, t=7.9.

[0322] Biological evaluation

[0323] Camptothecin's ability to test tumor cell proliferation:

[0324] Effect Example 1: The CTG assay was used to test the effects of camptothecin derivatives I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, and I-9 on the proliferation ability of tumor cells (tumor cell types are shown in Table 1).

[0325] After digestion, resuspend the test cells, count the cells, centrifuge, remove the supernatant, and adjust the cell density. Count the cells using a cell counter. Pipette 20 μl of the cell suspension, mix with 20 μl of trypan blue, and then remove 20 μl of the suspension for cell counting. Plate the cells into a 96-well cell culture plate, adding 180 μl of culture medium to each well, seeding 1000 cells per well. Gently tap to mix, and incubate the cell culture plate at 37°C for 16 hours. Dilute the compound with McCoy's 5A medium and add 20 μl of culture medium to each well, ensuring a maximum concentration of 100 nM, as indicated in the experimental design. Three-fold dilutions are performed, with 10 points plated. DMSO is used as a vehicle control; 20 μl of McCoy's 5A medium is added to the blank control. Gently tap to mix, and incubate the cell culture plate at 37°C for 7 days. After 7 days of incubation, remove the cell plate from the incubator, aspirate the culture medium, add 20 μl of culture medium, and then add 20 μl of CTG to each well. The reaction mixture was shaken on a shaker for 5 minutes, allowed to stand at room temperature for 10 minutes, and 30 μl of the reaction solution was transferred to a Viewplate-96white, white, transparent-bottom ELISA plate. Data were analyzed using an EnVision plate reader. The effects of the compounds on tumor cell proliferation are shown in Table 2.

[0326] Calculation formula: background value = control group - blank group; inhibition rate = 1 - (experimental group - blank group) / (background value) * 100%.

[0327] Table 1. Overview of tumor cell lines used in this screening

[0328] Table 2. Effects of compounds on tumor cell proliferation

[0329] As shown in Table 2, the compounds of the present invention have good inhibitory activity against human gastric cancer cells, human lung adenocarcinoma cells and human colon cancer cells.

[0330] Effect Example 2: Test method for the tumor cell proliferation ability of ADC-1 and ADC-2:

[0331] After digestion, resuspend NCI-N87 cells and count them. Centrifuge and remove the supernatant, then adjust the cell density. Count cells using a cell counter. Aspirate 20 μL of the cell suspension and mix with 20 μL of VisStain AOPI staining solution. Count the cells using a 20 μL aliquot. Plate the cells in a 96-well cell culture plate, adding 180 μL per well to seed 1000 or 2000 cells per well. Gently tap to mix, and incubate the cell culture plate at 37°C for 16 hours. Dilute the compound in RPMI1640 medium / 10% FBS, adding 20 μL to each well to a maximum concentration of 100 nM. Plate 10 points with 3-fold dilutions. For a vehicle control using DMSO, add 20 μL of RPMI1640 medium / 10% FBS to the blank. Gently tap to mix, and incubate the cell culture plate at 37°C for 7 days. After 7 days of culture, remove the cell plate from the incubator, aspirate the culture medium, add 20 μL of culture medium, and then add 20 μL of CTG to each well. Oscillate on a shaker for 5 minutes, let it stand at room temperature for 10 minutes, and transfer 30 μL of the reaction solution to a Viewplate-96white, white, clear-bottomed microtiter plate for data analysis using an EnVision plate reader.

[0332] Calculation formula: The inhibition rate (IR) of the test antibody was calculated using the following formula: IR (%) = (1 – (antibody – blank control) / (vehicle control – blank control)) * 100%. The inhibition rates at different antibody concentrations were calculated in Excel, and inhibition curves were plotted and relevant parameters were calculated using GraphPad Prism software. The results are shown in Table 3 and Figure 1.

[0333] Table 3 Effects of ADC-1 and ADC-2 on the proliferation of NCl-N87 tumor cells

[0334] Effect Example 3. Antitumor efficacy test of ADC-1 and ADC-2 on the NOG mouse subcutaneously transplanted NCI-N87 cell animal model:

[0335] In this study, NCI-N87 cells were subcutaneously inoculated into NOG mice to determine the anti-tumor effects of ADC-1 and ADC-2.

[0336] NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

[0337] Cells: Human gastric cancer NCI-N87 cells (ATCC source, catalog number CRL-5822) were routinely subcultured according to the instructions; cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension (inoculation size was 1×10 7 ) were subcutaneously inoculated into the right axilla of female NOG mice to establish the NCI-N87 tumor-bearing mouse model.

[0338] Dosage:

[0339] On the 10th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with a tumor volume of 110 mm were selected. 3 ~250mm 3 Mice within the range were grouped according to tumor volume (5 mice per group) and administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), ADC-1 and ADC-2 on the 11th day after inoculation. The changes in tumor volume and body weight of mice in each group were monitored during the administration period. The monitoring frequency was 2 times / week for 4 consecutive weeks. Body weight and tumor volume were measured before each administration. The tumor volume inhibition rate (TGI%) was calculated on the 28th day after administration. The calculation formula is as follows: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100; where Ti: mean tumor volume of the administration group, T0: mean tumor volume of the administration group on D0 day, Vi: mean tumor volume of the isotype control group, V0: mean tumor volume of the isotype control group on D0 day. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: TV = 1 / 2 × a × b 2 Body weight was measured using an electronic balance.

[0340] The dosage and method of administration are shown in Table 4.

[0341] Table 4 Dosage study design

[0342] As shown in Table 5 and Figure 2, on day 28 after administration, ADC-1 and ADC-2 at a dose of 4 mg / kg significantly inhibited tumor growth, with tumor volume inhibition rates of 102.83% and 108.86%, respectively.

[0343] Table 5. Inhibitory effect of ADC on tumor growth in vivo at day 28 Note: P value is compared with negative control antibody

[0344] The above experiments showed that ADC-1 and ADC-2 significantly inhibited the growth of tumors in the NCI-N87 cell animal model transplanted subcutaneously in NOG mice, and had no significant effect on the body weight of NOG mice (as shown in Figure 3).

Claims

1. A camptothecin small molecule or a pharmaceutically acceptable salt thereof as shown in Formula I: in, R 1 is hydrogen, halogen or C 1-6 alkyl; L 1 For a single bond, -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3; L 2 For a single key, or C 1-6 Alkylene.

2. The camptothecin small molecule as shown in formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 1 wherein the halogen is fluorine, chlorine, bromine or iodine; for example, fluorine; (2)R 1 In the C 1-6 Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; for example, methyl; (3)L 1 In the -C 1-6 The -C in alkylene-NH- 1-6 Alkylene- is -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene; preferably, the -C 1-6 The C in alkylene-NH- 1-6 Alkylene is a straight chain alkylene group, for example -CH2-CH2-; (4)L 1 In the -C 1-6 The C in alkylene-NH- 1-6 Alkylene is connected to hydroxyl group, NH is connected to L 2 connect; (5)L 1 In the above, the 3-10 membered heterocycloalkylene group is a 3-6 membered heterocycloalkylene group; the heteroatom of the 3-6 membered heterocycloalkylene group is N, and the number of heteroatoms is 1; preferably, the 3-10 membered heterocycloalkylene group is an aziridinyl group, an aziridinyl group or an aziridinyl group, for example Another example More example Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to L 2 Connect; and (6)L 2 In the C 1-6 The alkylene group is methylene, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene. Preferably, the C 1-6 Alkylene is a straight chain alkylene; for example -CH2- or -CH2-CH2-.

3. The camptothecin small molecule as shown in formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or both of the following conditions: (1)R 1 is hydrogen or halogen, for example R 1 is hydrogen or fluorine; and (2)-L 1 -L 2 - Any of the following options: Option 1: L 1 is a single bond; L 2 C 1-6 Alkylene; Option 2: L 1 For -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3; L 2 for Preferably, -L 1 -L 2 -for More preferably, -L 1 -L 2 -for Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to the methylene group.

4. The camptothecin small molecule as shown in formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 1 is a halogen; (2)L 1 is a single bond or L 1 For -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; preferably, L 1 is a single bond; (3)L 2 C 1-6 Alkylene or Preferably, L 2 C 1-6 Alkylene; more preferably, L 2 For straight chain C 1-6 Alkylene; Preferably, in the camptothecin small molecule as shown in Formula I: R 1 is halogen; L 1 is a single bond; L 2 C 1-6 Alkylene; More preferably, the camptothecin small molecule as shown in Formula I satisfies one or two of the following conditions: (1)-L 1 - is a single key, Another example is a single key, Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to L 2 connect; (2)-L 2 -for 5. The camptothecin small molecule as shown in formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The camptothecin small molecule as shown in formula I is selected from any of the following structures:

6. A linker-drug conjugate as shown in Formula II or a pharmaceutically acceptable salt thereof: in, Q is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -、-(OCH2CH2) m -、-(CR a R b ) m -、-O(CR a R b ) m -、-NH-(CR a R b ) m -CR c R d - or -S(CR a R b ) m -CR c R d -; R a and R b are independently H, D, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, cyano, nitro, 3-10 membered cycloalkyl or 3-10 membered heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group; R c and R d Independently H, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-14 membered aryl or 5-10 membered heteroaryl; Or, R c and R d Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group; The heteroatoms in each 3-10 membered heterocyclyl and 5-10 membered heteroaryl are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3; Y is m, p, q and r are independently integers from 0 to 14; R 1 , L 1 and L 2 The definition as described in any one of claims 1 to 5.

7. The linker-drug conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: It meets one or more of the following conditions: (1) Each halogen is independently fluorine, chlorine, bromine or iodine; (2) Each C 1-6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; (3) Each C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy; (4) each 3-10 membered cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; (5) Each 3-10 membered heterocyclic group is independently a 3-6 membered heterocyclic group, the heteroatoms of the 3-6 membered heterocyclic group are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3; (6) Each 6-14 membered aryl group is independently phenyl or naphthyl; (7) each 5-10 membered heteroaryl group is independently a 5-6 membered heteroaryl group, the heteroatoms of the 5-6 membered heteroaryl group are independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3; (8) In Q, the single amino acid residue is -Phe-, -Lys-, -Val-, -Ala-, -Cit-, -Leu-, -Ile-, -Arg-, -Trp- or -Gly-; (9) In Q, the dipeptide residue is C＝O -Lys-Phe- NH , C＝O -Ala-Val- NH , C＝O -Lys-Val- NH , C＝O -Lys-Ala- NH , C＝O -Cit-Val- NH , C＝O Cit-Phe- NH , C＝O -Cit-Leu- NH , C＝O Cit-Ile- NH , C＝O -Arg-Phe- NH , C＝O -Cit-Trp- NH or C＝O -Val-Gly- NH ;For example C＝O -Ala-Val- NH ; (10) In Q, the tripeptide residue is C＝O -Ala-Val-Glu- NH , C＝O -Cit-Val-Glu- NH , C＝O -Ala-Val-αGlu- NH or C＝O -Cit-Val-αGlu- NH ;and (11) In Q, the tetrapeptide residue is C＝O -Gly-Phe-(Gly)2- NH or C＝O –(Gly)2-Phe-Gly- NH ,For example C＝O -Gly-Phe-(Gly)2- NH ; (12) In X, 1 -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p - 2 , 2 -(OCH2CH2) m - 1 , 2 -O(CR a R b ) m - 1 , 2 -NH-(CR a R b ) m -CR c R d_1 and 2 -S(CR a R b ) m -CR c R d_1 In the example, the "1" bit is connected to Y, and the "2" bit is connected to connect.

8. The linker-drug conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: It meets one or more of the following conditions: (1)R a and R b is H; (2) m is 1, 2, 3, 4 or 5; (3) p is 6, 7, 8, 9, 10, 11, 12, 13 or 14; preferably, p is 8, 9, 10, 11, 12, 13 or 14; (4) q is 1, 2, 3, 4 or 5; (5) r is 1, 2, 3, 4 or 5; (6) Q is a dipeptide residue or a tetrapeptide residue; for example C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly)2- NH ;and (7)X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -; Preferably, X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -;For example Another example The "1" bit is connected to Y, and the "2" bit is connected to connect.

9. The linker-drug conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: Q is a dipeptide residue or a tetrapeptide residue; X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -; Y is R a and R b is H; m is 1, 2, 3, 4 or 5; p is an integer of 8, 9, 10, 11, 12, 13 or 14; q is 1, 2, 3, 4 or 5; r is 1, 2, 3, 4 or 5; R 1 is hydrogen or halogen; L 1 is a single bond; L 2 C 1-6 Alkylene.

10. The linker-drug conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: The linker-drug conjugate shown in Formula II is any of the following structures:

11. An antibody-drug conjugate of formula III or a pharmaceutically acceptable salt thereof: in, G L For antibodies; t is the drug-ligand coupling ratio; Z is The b end is connected to X, and the a end is connected to G L connect; R 1 , L 1 and L 2 The definition as in any one of claims 1 to 5; Q and X are as defined in any one of claims 6-10.

12. The antibody-drug conjugate of formula III or a pharmaceutically acceptable salt thereof according to claim 11, characterized in that: It meets one or more of the following conditions: (1) G L Anti-HER2 antibodies, such as trastuzumab; (2) t is a natural number and / or decimal between 1 and 8, such as a natural number and / or decimal between 7 and 8, and another example is 7.8; or t is a natural number between 1 and 8, such as a natural number between 7 and 8, or t is a natural number and / or decimal between 6 and 8, such as 6.2, 7.8, 7.7 or 7.9; and (3) the a-end of the Z and the G L Connected by thioether bonds; Preferably, in the antibody-drug conjugate as shown in Formula III, G L It is an anti-HER2 antibody; t is a natural number and / or decimal between 6 and 8; Z is The b end is connected to X, and the a end is connected to G L connect; X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -; R a and R b independently H or D; r is 1, 2, 3, 4 or 5; q is 1, 2, 3, 4 or 5; p is 6, 7, 8, 9, 10, 11, 12, 13 or 14; m is 1, 2, 3, 4 or 5; Q is a dipeptide residue or a tetrapeptide residue; R 1 is hydrogen or halogen; -L 1 -L 2 - Any of the following options: Option 1: L 1 is a single bond; L 2 C 1-6 Alkylene; Option 2: L 1 For -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; the heteroatoms of the 3-10 membered heterocycloalkylene are selected from one, two or three of N, O and S, and the number of heteroatoms is 1, 2 or 3; L 2 for More preferably, in the antibody-drug conjugate as shown in Formula III, G L for trastuzumab; t is a natural number and / or decimal between 6 and 8; Z is The b end is connected to X, and the a end is connected to G L connect; X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -or-(CR a R b ) m -; R a and R b independently H or D; r is 1, 2, 3, 4 or 5; q is 1, 2, 3, 4 or 5; p is 6, 7, 8, 9, 10, 11, 12, 13 or 14; m is 1, 2, 3, 4 or 5; Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly)2- NH ; R 1 is a halogen; L 1 is a single bond; L 2 C 1-6 Alkylene; Optimally, in the antibody-drug conjugate of formula III, G L for trastuzumab; t is a natural number and / or decimal between 6 and 8; Z is The b end is connected to X, and the a end is connected to G L connect; X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH2CH2) p -; R a and R b independently H or D; r is 1, 2, 3, 4 or 5; q is 1, 2, 3, 4 or 5; p is 6, 7, 8, 9, 10, 11, 12, 13 or 14; Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly)2- NH ; R 1 is a halogen; L 1 is a single bond; L 2 C 1-6 Alkylene.

13. The antibody-drug conjugate of formula III or a pharmaceutically acceptable salt thereof according to claim 11, characterized in that: The antibody-drug conjugate shown in formula III is any of the following structures: The anti-HER2 antibody is trastuzumab; t is defined as in claim 11 or 12; Preferably, the antibody-drug conjugate as shown in formula III is any of the following structures: The anti-HER2 antibody is trastuzumab.

14. A pharmaceutical composition comprising a substance N and a pharmaceutical excipient, wherein the substance N is a camptothecin small molecule as shown in formula I as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, a linker-drug conjugate as shown in formula II as described in any one of claims 6 to 10 or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in formula III as described in any one of claims 11 to 13 or a pharmaceutically acceptable salt thereof.

15. Use of a substance N or a pharmaceutical composition as claimed in claim 14 in the preparation of a drug for preventing and / or treating cancer; the substance N is a camptothecin small molecule as shown in formula I as claimed in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, a linker-drug conjugate as shown in formula II as claimed in any one of claims 6 to 10 or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in formula III as claimed in any one of claims 11 to 13 or a pharmaceutically acceptable salt thereof; The cancer is preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penis cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, sarcoma, melanoma Preferably, the patient is a tumour, bladder cancer, stomach cancer or esophageal cancer, preferably a breast cancer or preferably a lung adenocarcinoma, stomach cancer or colon cancer.

16. A compound of formula 1c, 2h, 2i, 3c, 4h, 5e or 7e, 17. A method for preparing a camptothecin small molecule as shown in formula I, which is the following method 1, method 2 or method 3: Method 1: In a solvent, in the presence of a catalyst, a compound as shown in Formula 1-s1 and a compound as shown in Formula 1d are reacted as shown below to obtain a camptothecin small molecule as shown in Formula I; in, R 1 , L 1 and L 2 The definition as in any one of claims 1 to 5; Method 2: In a solvent, in the presence of a base, the compound represented by formula 1-s3 is subjected to a hydrolysis reaction as shown below to obtain a camptothecin small molecule represented by formula I; Among them, R 1 , L 1 and L 2 The definition as in any one of claims 1 to 5; Method 3: In a solvent, in the presence of a catalyst, the compound represented by formula 1-s5 is subjected to a condensation reaction with compound H as shown below to obtain a camptothecin small molecule represented by formula I; Among them, L 2 for The compound H is HO-L 1 -H or its salt, L 1 For -C 1-6 Alkylene-NH- or 3-10 membered heterocycloalkylene; R 1 The definition as described in any one of claims 1 to 5.

18. A method for preparing a compound as shown in Formula 1-s1, Formula 1-s3 or 1-s5, characterized in that: The method for preparing the compound as shown in formula 1-s1 comprises the following steps: In a solvent, in the presence of hydrogen and a catalyst, the compound represented by formula 1-s2 is subjected to a hydrogenation reaction represented by the following formula to obtain a compound represented by formula 1-s1, Among them, R 1 , L 1 and L 2 The definition as in any one of claims 1 to 5; The method for preparing the compound as shown in formula 1-s3 comprises the following steps: In a solvent, in the presence of a catalyst, the compound represented by Formula 1-s4 and the compound represented by Formula 1d are reacted as shown in the following formula to obtain a compound represented by Formula 1-s3; Among them, R 1 , L 1 and L 2 The definition as in any one of claims 1 to 5; The method for preparing the compound shown in formula 1-s5 comprises the following steps: In a solvent, in the presence of a base, the compound represented by Formula 1-s6 is subjected to a hydrolysis reaction represented by the following formula to obtain a compound represented by Formula 1-s5; Among them, R 1 The definition as in any one of claims 1 to 5; R s C 1-6 alkyl.

19. A method for preventing and / or treating cancer, comprising administering a substance N or a pharmaceutical composition as claimed in claim 14 to a subject; the substance N is a camptothecin small molecule as shown in formula I as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, a linker-drug conjugate as shown in formula II as described in any one of claims 6 to 10 or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate as shown in formula III as described in any one of claims 11 to 13 or a pharmaceutically acceptable salt thereof; The cancer is preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumors, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer or esophageal cancer, preferably breast cancer or preferably lung adenocarcinoma, gastric cancer or colon cancer.

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