Camptothecin micromolecule, antibody drug conjugate thereof, preparation method and application

Abstract

The invention discloses a camptothecin micromolecule, an antibody drug conjugate thereof, a preparation method and an application. The invention provides a camptothecin micromolecule as shown in a formula I or a pharmaceutically acceptable salt thereof. The camptothecin micromolecule provided by the invention and the antibody drug conjugate thereof have good cytotoxic activity and can inhibit proliferation of tumor cells. # imgabs0 #

Description

[0001] This application claims the priority of Chinese patent application No. 2023109283984 filed on July 26, 2023. This application cites the entire text of the above 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] Camptothecin compounds are specific inhibitors of DNA topoisomerase I. Camptothecin compounds form a stable Topo I-DNA-CPT ternary complex by binding to the Topo I-DNA cleavable binary complex, which affects the reconnection of DNA, increases Topo I-mediated DNA damage, and ultimately leads to cell death.

[0004] The traditional camptothecin compound is a pentacyclic quinoline alkaloid, which contains a quinoline ring AB, a pyrrole ring C, a pyridone ring D and an α-hydroxy lactone 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 from the A ring, which also achieved good results in preclinical PDX models: WO2021148501. However, it is still necessary to further develop better ADC drugs to improve the efficacy of existing ADC drugs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the single structure of camptothecin derivatives and their antibody-drug conjugates in the prior art, and to provide camptothecin small molecules and their antibody-drug conjugates, preparation methods and applications. The camptothecin small molecules and their antibody-drug conjugates provided by the present invention have good cytotoxic activity and can inhibit the proliferation of tumor cells.

[0006] The present invention provides a camptothecin small molecule or a pharmaceutically acceptable salt thereof as shown in Formula I:

[0007]

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

[0009] L 1 For a single bond, -C1-6 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 (the content of this paragraph is hereinafter referred to as "in a preferred embodiment").

[0012] In a preferred embodiment, R 1 In the above, 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 The C in alkylene-NH- 1-6 The alkylene group is a straight chain alkylene group.

[0015] In a preferred embodiment, 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.

[0016] In a preferred embodiment, L 1 In the -C 1-6 The -C in alkylene-NH- 1-6 Alkylene- is -CH 2 -、-CH 2 -CH 2 -、-CH(CH 3 )-、-CH 2 -CH 2 -CH 2 -、-CH(CH 2 -CH 3 )-、-C(CH 3 ) 2 -、-CH 2 -CH(CH 3)- or butylene, such as -CH 2 -CH 2 -.

[0017] In a preferred embodiment, L 1 In the above, 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 above, the 3-10 membered heterocycloalkylene group is aziridinyl, aziridinyl or aziridinyl, for example For example Another example or Among them, the "1" position is connected to the hydroxyl group, and the "2" position is connected to L 2 connect.

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

[0021] In a preferred embodiment, L 2 In the C 1-6 Alkylene is methylene, -CH 2 -CH 2 -、-CH(CH 3 )-、-CH 2 -CH 2 -CH 2 -、-CH(CH 2 -CH 3 )-、-C(CH 3 ) 2 -、-CH 2 -CH(CH 3 )- or butylene, such as -CH 2 -or-CH 2 -CH 2 -.

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

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

[0024] In a preferred embodiment, L2 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] Scenario 1:

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

[0029] Scenario 2:

[0030] 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;

[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 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] Scenario 1:

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

[0041] Scenario 2:

[0042] 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;

[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, in the camptothecin small molecule as shown in Formula I or a pharmaceutically acceptable salt thereof, the camptothecin small molecule as shown in Formula I is selected from any of the following structures:

[0049]

[0050] The present invention also provides a linker-drug conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II:

[0051]

[0052] wherein Q is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0053] X is -(CR a R b )r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -、-(OCH 2 CH 2 ) 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 -;

[0054] 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;

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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;

[0059] Y is

[0060] R 1 , L 1 and L 2 The definition is as described in any one of the present invention;

[0061] m, p, q and r are independently integers of 0-14.

[0062] 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 (this paragraph is hereinafter referred to as "in a preferred embodiment").

[0063] In a preferred embodiment, each halogen is independently fluorine, chlorine, bromine or iodine.

[0064] In a preferred embodiment, each C 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0065] In a preferred embodiment, each C 1-6 Alkoxy is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

[0066] In a preferred embodiment, each 3-10 membered cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0067] 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.

[0068] In a preferred embodiment, each 6-14 membered aryl group is independently phenyl or naphthyl.

[0069] 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.

[0070] In a preferred embodiment, in Q, the single amino acid residue is -Phe-, -Lys-, -Val-, -Ala-, -Cit-, -Leu-, -Ile-, -Arg-, -Trp- or -Gly-.

[0071] 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 .

[0072] 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 .

[0073] 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 .

[0074] In a preferred embodiment, R a and R b For H.

[0075] In a preferred embodiment, m is 1, 2, 3, 4 or 5, for example 5.

[0076] In a preferred embodiment, p is 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0077] In a preferred embodiment, p is 8, 9, 10, 11, 12, 13 or 14, for example 8 or 12.

[0078] In a preferred embodiment, q is 1, 2, 3, 4 or 5, for example 2.

[0079] In a preferred embodiment, r is 1, 2, 3, 4 or 5, for example 2.

[0080] In a preferred embodiment, Q is a dipeptide residue or a tetrapeptide residue.

[0081] In a preferred embodiment, in the linker-drug conjugate as shown in Formula II, Q is a dipeptide residue, and the dipeptide residue is C＝O -Ala-Val- NH .

[0082] 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 .

[0083] In a preferred embodiment, in X, 1 -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p - 2 , 2 -(OCH 2 CH 2 ) m - 1 , 2 -O(CR a R b ) m - 1 , 2 -NH-(CR a Rb ) 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.

[0084] In a preferred embodiment, X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -or-(CR a R b ) m -; Preferably, X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -;For example Another example The "1" bit is connected to Y, and the "2" bit is connected to connect.

[0085] In the present invention, in Q, "C=O" in the dipeptide residue, tripeptide residue or tetrapeptide residue means that the left side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to NH connected to Q via a carbonyl group, and "NH" in the dipeptide residue, tripeptide residue or tetrapeptide residue means that the right side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the carbonyl connected to Q via an amino group. In a preferred embodiment, in the linker-drug conjugate shown in Formula II or a pharmaceutically acceptable salt thereof, Q is a dipeptide residue or a tetrapeptide residue;

[0086] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH2 ) 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]

[0097] The present invention also provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in Formula III:

[0098]

[0099] Among them, G L is an antibody; L It forms a covalent bond with Z through S on it;

[0100] t is the drug-ligand coupling ratio;

[0101] Z is The b end is connected to X, and the a end is connected to G L connect;

[0102] R 1 , L 1 , L 2 , Q and X are as defined in any one of the present invention.

[0103] 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 (this paragraph is hereinafter referred to as "in a preferred embodiment").

[0104] In a preferred embodiment, G L It is an anti-HER2 antibody, such as trastuzumab.

[0105] In a preferred embodiment, t is a natural number and / or a decimal of 1-8; for example, a natural number and / or a decimal of 7-8; for example, 7.8.

[0106] In a preferred embodiment, t is a natural number and / or a decimal number of 6-8, for example, 6.2, 7.8, 7.7 or 7.9.

[0107] In a preferred embodiment, t is a natural number of 1-8; for example, t is a natural number of 7-8.

[0108] In a preferred embodiment, the a end of Z and the G L are connected by thioether bonds. 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.

[0109] In a preferred embodiment, G L It is an anti-HER2 antibody;

[0110] t is a natural number and / or decimal between 6 and 8;

[0111] Z is The b end is connected to X, and the a end is connected to G L connect;

[0112] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -or-(CR a R b ) m -;

[0113] R a and R b independently H or D;

[0114] r is 1, 2, 3, 4 or 5;

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

[0116] p is 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0117] m is 1, 2, 3, 4 or 5;

[0118] Q is a dipeptide residue or a tetrapeptide residue;

[0119] R 1 is hydrogen or halogen;

[0120] -L 1 -L 2 - Any of the following options:

[0121] Scenario 1:

[0122] L 1 is a single bond; L 2 C 1-6 Alkylene;

[0123] Scenario 2:

[0124] 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;

[0125] L 2 for

[0126] In a preferred embodiment, G L for trastuzumab;

[0127] t is a natural number and / or decimal between 6 and 8;

[0128] Z is The b end is connected to X, and the a end is connected to G L connect;

[0129] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -or-(CR a R b ) m -;

[0130] R a and Rb independently H or D;

[0131] r is 1, 2, 3, 4 or 5;

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

[0133] p is 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0134] m is 1, 2, 3, 4 or 5;

[0135] Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly) 2 - NH ;

[0136] R 1 is a halogen;

[0137] L 1 is a single bond;

[0138] L 2 C 1-6 Alkylene.

[0139] In a preferred embodiment, G L for trastuzumab;

[0140] t is a natural number and / or decimal between 6 and 8;

[0141] Z is The b end is connected to X, and the a end is connected to G L connect;

[0142] X is -(CR a R b ) r -CO-NH-(CR a R b ) q -(OCH 2 CH 2 ) p -;

[0143] R a and R b independently H or D;

[0144] r is 1, 2, 3, 4 or 5;

[0145] q is 1, 2, 3, 4 or 5;

[0146] p is 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0147] Q is C＝O -Ala-Val- NH or C＝O -Gly-Phe-(Gly) 2 - NH ;

[0148] R 1 is a halogen;

[0149] L 1 is a single bond;

[0150] L 2 C 1-6 Alkylene.

[0151] In a preferred embodiment, the antibody-drug conjugate as shown in formula III is any of the following structures:

[0152]

[0153] The anti-HER2 antibody is trastuzumab; t is defined as described in any one of the present invention;

[0154] Preferably, the antibody-drug conjugate as shown in formula III is any of the following structures:

[0155]

[0156]

[0157] The anti-HER2 antibody is trastuzumab.

[0158] The present invention also provides a pharmaceutical composition, comprising (a therapeutically effective amount of) substance N and pharmaceutical excipients, wherein the 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 as described in any one of the present invention.

[0159] The present invention also provides a use of the substance N (therapeutically effective amount) or the pharmaceutical composition described above in the preparation of a drug for preventing and / or treating cancer.

[0160] 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 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, 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.

[0161] The present invention provides a method for preventing and / or treating the cancer, which comprises administering (a therapeutically effective amount of) the substance N or the pharmaceutical composition described above to a subject.

[0162] The present invention also provides a (intermediate) compound of formula 1c, 2h, 2i, 3c, 4h, 5e or 7e (for preparing the camptothecin small molecule as shown in formula I),

[0163]

[0164] 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:

[0165] 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 in the following formula to obtain a camptothecin small molecule represented by Formula I;

[0166]

[0167] Among them, R 1 , L 1 and L 2 The definition is as described in any one of the present invention;

[0168] Method 2: In a solvent (such as water), in the presence of a base (such as 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 small molecule represented by formula I;

[0169]

[0170] Among them, R 1 , L 1 and L 2 The definition is as described in any one of the present invention;

[0171] 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 as shown in Formula 1-s5 is subjected to a condensation reaction with compound H as shown in the following formula to obtain a camptothecin small molecule as shown in Formula I;

[0172]

[0173] Among them, L 2 for

[0174] The compound H is HO-L 1 -H or its salt, L 1 For -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;

[0175] R 1 The definition of is as described in any one of the present invention.

[0176] The present invention also provides a method for preparing the compound as shown in formula 1-s1, which comprises the following steps:

[0177] In a solvent (such as methanol), in the presence of hydrogen and a catalyst (such as 5% palladium 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,

[0178]

[0179] Among them, R 1 , L 1 and L 2 The definition of is as described in any one of the present invention.

[0180] The present invention also provides a method for preparing the compound as shown in formula 1-s3, which comprises the following steps:

[0181] 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 a compound represented by Formula 1-s3,

[0182]

[0183] Among them, R 1 , L 1 and L 2 The definition of is as described in any one of the present invention.

[0184] The present invention also provides a method for preparing the compound as shown in formula 1-s5, which comprises the following steps:

[0185] In a solvent (such as methanol), in the presence of a base (such as sodium hydroxide, such as 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;

[0186]

[0187] 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).

[0188] The preparation method of the camptothecin small molecule as shown in Formula I, the preparation method of the compound as shown in Formula 1-s1, the preparation method of the compound as shown in Formula 1-s3 and the preparation method of the compound as 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.

[0189] Definition of terms

[0190] The stereochemical definitions and conventions used in the present invention 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.

[0191] Depending on the choice of starting materials and process, the compounds of the invention may exist in the form of one of the possible isomers or 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.

[0192] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0193] 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.

[0194] 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.

[0195] In the present invention, a substituent group may be preceded by a single dash "-" to indicate that the substituent group is connected to the parent moiety by a single bond. 3 -C(=O)- means that the C(=O) in the acetyl group is attached to the parent moiety through a single bond.

[0196] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0197] The term "alkyl" refers to a group having a specified number of carbon atoms (e.g., C 1 ~C 6 ) is a straight or branched alkyl group. The alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0198] The term "alkylene" refers to a substituent formed by removing two hydrogen atoms from a saturated straight or branched alkane. The two hydrogen atoms removed can be on the same carbon atom or on different carbon atoms (for example, the two hydrogen atoms removed are on the carbon atoms at the two ends). 1 Alkylene (i.e. methylene) refers to -CH 2 -, C 2 Alkylene (i.e. ethylene) refers to -CH 2 -CH 2 - or -CH(CH 3 )-.

[0199] 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 single ring, and each ring is saturated. Heterocycloalkylene includes, but is not limited to, azetidinyl, piperidinyl or piperazinyl, etc., for example

[0200] In the present application, the term "alkoxy" as a group or part of another group refers to -O-alkyl, and alkyl is as defined above.

[0201] The term "cycloalkyl" refers to a cycloalkyl radical having a specified number of carbon atoms (e.g. 3-6 , C 3-10 ) is a saturated monocyclic ring group consisting of only carbon atoms. Cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0202] 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), a specified heteroatom type (one or more of N, O, and S), which is a monocyclic, bridged, or spirocyclic ring, and each ring is saturated. Heterocycloalkyl includes, but is not limited to, azetidinyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, piperidinyl, and the like.

[0203] The term "aryl" refers to a radical having a specified number of carbon atoms (e.g. 6-10 , C 6-14 ) and is composed only of carbon atoms, which is monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). The aryl group is connected to other fragments in the molecule through an aromatic ring or a non-aromatic ring. The aryl group includes, but is not limited to, phenyl, naphthyl, etc.

[0204] The term "heteroaryl" refers to a cyclic group with a specified number of ring atoms (e.g., 5 to 10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), a specified type of heteroatoms (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, furanyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, and the like.

[0205] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) 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.

[0206] The term "pharmaceutical excipients" refers to excipients and additives used in the production of drugs and the preparation of prescriptions. It is all substances contained in drug preparations except active ingredients. For details, please refer to the Pharmacopoeia of the People's Republic of China (2020 edition) or Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009).

[0207] The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing 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.

[0208] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0209] The term "therapeutically effective amount" refers to an amount of a compound that is sufficient to effectively treat a disease or condition described herein when administered to a patient."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.

[0210] The term "patient" refers to any animal that is about to or has been administered the compound or composition according to the embodiments of the present invention, preferably a mammal, and most preferably a human. 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, humans, etc., with humans being the most preferred.

[0211] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0212] The reagents and raw materials used in the present invention are commercially available.

[0213] The positive and progressive effect of the present invention is that the present invention provides a class of camptothecin small molecules and antibody-drug conjugates thereof with novel structures, and the camptothecin small molecules and antibody-drug conjugates thereof have excellent cytotoxic activity and can inhibit the proliferation of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0214] Figure 1 This is a trend chart of the inhibition rate of ADC-1 and ADC-2 on the proliferation ability of NCl-N87 tumor cells at different concentrations.

[0215] Figure 2 This is a graph showing the inhibitory effect of ADC-1 and ADC-2 on tumor volume in the NOG mouse subcutaneously transplanted NCI-N87 cell animal model.

[0216] Figure 3 This is a diagram showing the effects of ADC-1 and ADC-2 on body weight in the NOG mouse subcutaneously transplanted NCI-N87 cell animal model. DETAILED DESCRIPTION

[0217] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0218] In the following examples, DCC represents dicyclohexylcarbodiimide; DMAP represents dimethylaminopyridine; DIPEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide; and PBS represents phosphate buffered saline.

[0219] Example 1: Synthesis of I-1:

[0220]

[0221] Under nitrogen flow, add 350mL glacial acetic acid to the bottle, add 5-hydroxy-1-tetralone (25g, 154.1mmol) in batches, dissolve 65% nitric acid (12mL, 157mmol) in 50mL glacial acetic acid, add to the reaction bottle under ice bath, gradually return to room temperature and stir for 3h. After TLC monitoring, pour the reaction solution into 1.5L ice water, filter out the solid, rinse the filter cake with water, dry the solid under reduced pressure, and beat the solid with PE:EA=2:1 to obtain 16g of light yellow solid with a yield of 50%.

[0222] 1H 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 + ).

[0223]

[0224] Under nitrogen flow, 1a (5.0 g, 24.1 mmol), potassium carbonate (6.5 g, 36.2 mmol), 20 mL of anhydrous DMF were added to the bottle, stirred at room temperature for 0.5 h, benzyl-2-bromoethyl ether (5.8 mL, 36.2 mmol) was added, and stirred at 60° C. for 16 hours. After the reaction was completed as monitored by TLC, the solid was filtered, the filter cake was rinsed with ethyl acetate, the solvent was removed under reduced pressure, and the obtained solid was slurried with PE: EA = 5: 1 to obtain 4.4 g of a light pink solid with a yield of 53%.

[0225] 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 + ).

[0226]

[0227] Under nitrogen flow, 1b (100 mg, 0.3 mmol) was added to the bottle, 5 mL of methanol was added, 5% palladium carbon (180 mg, 0.088 mmol) was added, hydrogen was replaced, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed by TLC monitoring, the mixture was filtered through diatomaceous earth 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 + ).

[0228]

[0229] Under nitrogen flow, 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), p-toluenesulfonic acid pyridinium salt (7 mg, 27 μmol), 5 mL toluene, o-cresol (65 μL, 622 μmol) were added to the bottle, and the reaction was stirred at 100°C for 16 hours. After the reaction was completed by TLC monitoring, the precipitated solid was filtered, the filtrate was collected and the solvent was removed under reduced pressure, the solid was combined with the filtered cake, and slurried with ethyl acetate to obtain 35 mg of yellow solid with a yield of 86%.

[0230] 1 H 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).

[0231] 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 + ).

[0232] Example 2: Synthesis of I-2:

[0233]

[0234] Under nitrogen flow, activated zinc powder (20.1 g, 307.6 mmol), 150 mL of anhydrous DMF, and iodine (2.0 g, 7.68 mmol) were added to the bottle. After the solution was clarified, ethyl 4-bromobutyrate (30.0 g, 153.8 mmol) was added and reacted at 90°C for 4-5 hours. After the reaction was complete as monitored by TLC, the reaction solution was allowed to stand and cool to room temperature for use, and the supernatant was taken for use, with a concentration of about 1 mol / L.

[0235] Under nitrogen flow, add 150mL of anhydrous DMF to the bottle, add 2-bromo-6-fluoroanisole (25g, 121.9mmol), add palladium acetate (548mg, 2.4mmol) and stir until dissolved, add S-PHOS (2.0g, 4.9mmol) and stir until dissolved, add 160mL of the above supernatant dropwise under ice bath, and stir at 50°C for 16 hours. After TLC monitoring, add saturated ammonium chloride to quench the reaction, pour the reaction solution into 1.5L of water, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride twice each, dry with anhydrous sodium sulfate, remove the organic solvent under reduced pressure, and obtain a reddish brown crude product, which can be directly used in the next step without purification.

[0236] 1 H NMR (400 MHz, CDCl 3 )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 + ).

[0237]

[0238] Add 2a (24.4 g, 101.6 mmol) to the bottle, add 100 mL of methanol, add 50 mL of 4M sodium hydroxide solution, and react at room temperature for 4 h. After the reaction is complete as monitored by TLC, remove the organic solvent under reduced pressure, then add 20 mL of water, wash twice with dichloromethane, wash once with petroleum ether, separate the aqueous phase, add hydrochloric acid to adjust the pH to 2 under ice bath, precipitate a large amount of solid, filter and dry, and obtain 15.4 g of light yellow solid, with a yield of 71%.

[0239] 1 H NMR (400 MHz, CDCl 3)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 + ).

[0240]

[0241] Under nitrogen flow, add 2b (11.0 g, 51.8 mmol) to the bottle, add 120 mL of anhydrous dichloromethane, add dichlorothionyl (5.6 mL, 77.7 mmol) dropwise, and react at room temperature for 3 h. After the reaction generates acyl chloride by TLC monitoring, add anhydrous aluminum chloride (17.3 g, 129.5 mmol) under ice bath. Return to room temperature and react for 3 h. After the reaction is complete by TLC monitoring, pour the reaction solution into 10% citric acid to quench, separate the organic phase, wash with saturated sodium bicarbonate 3 times, wash with saturated sodium chloride once, dry with anhydrous sodium sulfate, remove the organic solvent under reduced pressure, and obtain 8.2 g of light yellow oily liquid with a yield of 82%.

[0242] 1 H 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 + ).

[0243]

[0244] In a nitrogen stream, 2c (7 g, 36.0 mmol) was added to the bottle, and 80 mL of 40% aqueous hydrogen bromide solution was added, and the mixture was stirred at 100°C for 16 hours. After the reaction was complete as monitored by TLC, the reaction solution was poured into cold water and the solid was filtered out. The solid was dried under reduced pressure and slurried with PE:EA=5:1 to obtain 6.1 g of a light yellow solid with a yield of 93%.

[0245] 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 + ).

[0246]

[0247] Under nitrogen flow, 90 mL of glacial acetic acid was added to the bottle, 2d (6.1 g, 33.8 mmol) was added in batches, 65% nitric acid (2.8 mL, 40.6 mmol) was dissolved in 10 mL of glacial acetic acid, added to the reaction bottle under ice bath, gradually returned to room temperature and stirred for 0.5 h. After TLC monitoring of the reaction was complete, the reaction solution was poured into 400 mL of ice water, the solid was filtered out, the filter cake was washed with water, the solid was dried under reduced pressure, and the solid was slurried with PE: EA = 2: 1 to obtain 6.6 g of light yellow solid with a yield of 87%.

[0248] 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) + ).

[0249]

[0250] Under nitrogen flow, 2f (2.0 g, 8.9 mmol), potassium carbonate (2.5 g, 13.4 mmol), 20 mL of anhydrous DMF were added to the bottle, stirred at room temperature for 0.5 h, 2-bromoethyl acetate (1.5 mL, 13.4 mmol) was added, and stirred at 60 ° C for 16 hours. After the reaction was completed by TLC monitoring, the reaction solution was poured into 100 mL of water, extracted with ethyl acetate 3 times, the organic phases were separated and combined, washed with water 2 times, washed with saturated sodium chloride 2 times, dried over anhydrous sodium sulfate, and column chromatography (PE: EA = 4: 1 to 1: 1) was performed to obtain 1.6 g of a light yellow solid with a yield of 59%.

[0251] 1 H NMR (400 MHz, CDCl 3 )δ7.12(d,J=10.3Hz,1H),4.37(s,4H),2.96(t,J=6.1Hz,2H),2.64–2.60(m,2H),2.13–2.08(m,2H),2.05(s,3H).MS(ESI)m / z=312.1(M+H + ).

[0252]

[0253] Under nitrogen flow, add 2g (900mg, 2.9mmol) to the bottle, add 10mL methanol, add 1.0g Raney nickel, replace hydrogen, and stir at room temperature. After TLC monitoring, filter the Raney nickel and remove the solvent under reduced pressure to obtain 668mg of light yellow solid with a yield of 84%.

[0254]

[0255] Under nitrogen flow, 2h (650 mg, 2.3 mmol), 1d (730 mg, 2.8 mmol), p-toluenesulfonic acid pyridinium salt (174 mg, 0.7 mmol) were added to the bottle, 10 mL of toluene, 10 mL of glacial acetic acid were added, and the reaction was stirred at 100 ° C for 16 hours. After the reaction was complete as monitored by TLC, the organic solvent was removed under reduced pressure, and then 10 mL of 2M sodium hydroxide solution was added and stirred for 2 hours. After the reaction was complete as monitored by TLC, 2M hydrochloric acid was added to adjust the pH to 2-3, and the precipitated solid was filtered out. The solid was slurried with ethyl acetate, the solid was filtered out, and it was dried under reduced pressure to obtain 783 mg of a white solid with a yield of 73%.

[0256] 1 H NMR (400 MHz, DMSO-d 6 )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),4.12(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).

[0257] 13 C NMR (100 MHz, DMSO-d 6 )δ172.93,157.26,156.24,151.77,150.48,146.32,145.97,143.12,141.79,129.85,126.50,123.09,119.31,112.13,111.9 4,97.02,76.21,72.85,65.72,60.79,60.23,49.87,30.72,26.66,23.74,21.24,21.16,14.56,8.26.MS(ESI)m / z=467.2(M+H + ).

[0258] Example 3: Synthesis of I-3:

[0259]

[0260] Under nitrogen flow, 1a (10 g, 44.4 mmol), potassium carbonate (9.2 g, 66.6 mmol) were added to the bottle, and 100 mL of anhydrous DMF was added, and the mixture was stirred at room temperature for 0.5 h. 3-bromo-1-propanol (5 mL, 66.6 mmol) was added, and the mixture was stirred at 60°C for 16 h. After the reaction was completed as monitored by TLC, the salt was filtered out, the DMF was removed under reduced pressure, and the mixture was redissolved in ethyl acetate, washed twice with water, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain solid PE: EA = 2: 1 slurry, and 9.5 g of light yellow solid was obtained, with a yield of 81%.

[0261] 1 H NMR (400 MHz, DMSO) δ 7.63 (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=3.9Hz,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 + ).

[0262]

[0263] Under nitrogen flow, 3b (1.0 g, 3.2 mmol) was added to the bottle, followed by 10 mL of methanol and 1.5 g of Raney nickel, and the hydrogen was replaced and stirred at room temperature for 3 h. After the reaction was completed as monitored by TLC, the mixture was filtered through diatomaceous earth, the solvent was removed under reduced pressure, and column chromatography (PE: EA = 4: 1 to 1: 1) was performed to obtain 785 mg of a light yellow solid with a yield of 89%.

[0264] 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 + ).

[0265]

[0266] Under nitrogen flow, 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), p-toluenesulfonic acid pyridinium salt (208 mg, 0.84 mmol), 10 mL toluene, o-cresol (2 mL, 19.6 mmol) were added to the bottle, and the reaction was stirred at 100°C for 16 hours. After the reaction was complete as monitored by TLC, the precipitated solid was filtered, the filtrate was collected and the solvent was removed under reduced pressure, the solid was combined with the filtered cake, and slurried with ethyl acetate to obtain 1.0 g of yellow solid with a yield of 78%.

[0267] 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).

[0268] 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 + ).

[0269] Example 4: Synthesis of I-4:

[0270]

[0271] Under nitrogen flow, 2f (1.5 g, 6.6 mmol), potassium carbonate (1.4 g, 9.9 mmol), 10 mL of anhydrous DMF were added to the bottle, stirred at room temperature for 0.5 h, 3-bromo-1-propanol (900 μL, 9.9 mmol) was added, and stirred at 60°C for 16 hours. After TLC monitoring of the reaction was complete, the reaction solution was poured into 100 mL of water, extracted with ethyl acetate three times, the organic phases were separated and combined, washed with water twice, washed with saturated sodium chloride twice, dried over anhydrous sodium sulfate, and column chromatography (PE: EA = 4: 1 to 1: 1) was performed to obtain 909 mg of a light yellow solid with a yield of 48%.

[0272] 1 H NMR (400 MHz, CDCl 3 )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=6.1 Hz,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 + ).

[0273]

[0274] Under nitrogen flow, 4 g (900 mg, 3.2 mmol) was added to the bottle, 10 mL of methanol was added, 1.0 g of Raney nickel was added, hydrogen was replaced, and the mixture was stirred at room temperature. After TLC monitoring, the reaction was completed, diatomaceous earth was used for filtration, the solvent was removed under reduced pressure, and column chromatography (PE: EA = 4: 1 to 1: 1) was performed to obtain 668 mg of a light yellow solid with a yield of 82%.

[0275] 1 H NMR (400 MHz, CDCl 3 )δ6.26(d,J=12.6Hz,1H),4.01(t,J=5.9Hz,2H),3.91(t,J=5.8Hz,2H),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 + ).

[0276]

[0277] Under nitrogen flow, 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), p-toluenesulfonic acid pyridinium salt (148 mg, 0.6 mmol) were added to the bottle, 10 mL of toluene and 10 mL of glacial acetic acid were added, and the reaction was stirred at 100 ° C for 16 hours. After TLC monitoring, the reaction was complete, and the target molecule and hydroxypropyl acetylated compounds were present in the reaction system. The organic solvent was removed under reduced pressure, and then 10 mL of 2M sodium hydroxide solution was added and stirred for 2 hours. After TLC monitoring, the reaction was complete, 2M hydrochloric acid was added to adjust the pH to 2-3, and the precipitated solid was filtered out. The solid was slurried with ethyl acetate, the solid was filtered out, and the solid was dried under reduced pressure to obtain 488 mg of white solid, with a yield of 51%.

[0278] 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).

[0279] 13 C 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 + ).

[0280] Example 5: Synthesis of Compound I-5:

[0281]

[0282] Under nitrogen flow, 2f (2.0 g, 8.9 mmol), potassium carbonate (2.4 mg, 0.017 mmol) were added to the bottle, and 10 mL of anhydrous DMF was added, and stirred at room temperature for 0.5 hours, followed by the addition of ethyl bromoacetate (1.5 mL, 13.5 mmol), and stirred at room temperature for 6 hours. After the reaction was complete as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (PE: EA = 5: 1) was performed to obtain 2.2 g of a white solid, with a yield of 79%.

[0283] 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 + ).

[0284]

[0285] Under nitrogen flow, 5b (1.6 g, 5.1 mmol) was added to the bottle, 20 mL of methanol was added, and 1.5 g of Raney nickel was added to replace the hydrogen, and the mixture was stirred at room temperature for 2.5 hours. After the reaction was completed as monitored by TLC, the mixture was filtered through diatomaceous earth, and the solvent was removed under reduced pressure to obtain 1.5 g of a yellow solid with a yield of 105%.

[0286] 1 H NMR (400 MHz, CDCl 3 )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 + ).

[0287]

[0288] Under nitrogen flow, 5c (500 mg, 1.8 mmol), 1d (561 mg, 2.1 mmol), p-toluenesulfonic acid pyridinium salt (143 mg, 0.57 mmol) were added to the bottle, 10 mL of toluene and 1 mL of acetic acid were added, and the mixture was stirred at 110°C for 16 hours. After the reaction was completed by TLC monitoring, the solid was filtered out, the filter cake was rinsed with 2 mL of methanol, and the solid was dried under reduced pressure to obtain 763 mg of gray-green solid with a yield of 83%.

[0289] 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) + ).

[0290]

[0291] Under nitrogen flow, 5d (450 mg, 0.88 mmol), 1M sodium hydroxide (2.0 mL, 2 mmol), 4 mL methanol, 6 mL water were added to the bottle and stirred at room temperature for 3 hours. After TLC monitoring, the solvent was removed under reduced pressure, the pH was adjusted to 1-2, the precipitated solid was filtered, the filter cake was washed with 2 mL methanol, and the solid was dried under reduced pressure to obtain 403 mg of light yellow solid, with a yield of 95%. MS (ESI) m / z = 481.1 (M+H + ).

[0292]

[0293] Under nitrogen flow, 5e (45 mg, 0.094 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (71 mg, 0.19 mmol) were added to the bottle, and 5 mL of anhydrous dichloromethane was added, and stirred for 0.5 hours, followed by 4-hydroxypiperidine (18 mg, 0.18 mmol), and stirred at room temperature for 16 hours. After TLC monitoring, the reaction was complete, the solvent was removed under reduced pressure, water was added to precipitate the 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 with a yield of 83%.

[0294] 1 H NMR (400 MHz, DMSO-d 6)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=37.9,8.2 Hz,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 + ).

[0295] Example 6: Synthesis of Compound I-6:

[0296]

[0297] Under nitrogen flow, 5e (50 mg, 0.10 mmol), HATU (80 mg, 0.21 mmol) and 10 mL of anhydrous dichloromethane were added to the bottle, stirred for 0.5 hours, and then DIPEA (45 μL, 0.26 mmol) and 3-hydroxyazetidine hydrochloride (17 mg, 0.16 mmol) were added, and stirred at room temperature for 16 hours. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (DCM: MeOH = 50: 1) was performed to obtain 32 mg of a light yellow solid with a yield of 60%.

[0298] 1 H 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 + ).

[0299] Example 7: Synthesis of Compound I-7:

[0300]

[0301] Under nitrogen flow, 1a (2.0 g, 9.7 mmol), potassium carbonate (2.7 g, 19.5 mmol) and 20 mL of anhydrous DMF were added to the bottle, stirred at room temperature for 0.5 hours, ethyl bromoacetate (1.6 mL, 14.4 mmol) was added, and stirred at 40°C for 6 hours. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure, and 1.8 g of a light yellow solid was obtained by column chromatography (PE: EA = 5: 1), with a yield of 60%.

[0302] 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 + ).

[0303]

[0304] Under nitrogen flow, 7b (1.8 g, 6.1 mmol) was added to the bottle, 30 mL of methanol was added, 1.5 g of Raney nickel was added, hydrogen was replaced, and the mixture was stirred at room temperature for 2.5 hours. After the reaction was completed as monitored by TLC, the mixture was filtered through diatomaceous earth, and the solvent was removed under reduced pressure to obtain 1.5 g of a yellow solid with a yield of 93%.

[0305] 1 H NMR (400 MHz, CDCl 3 )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 + ).

[0306]

[0307] Under nitrogen flow, 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), p-toluenesulfonic acid pyridinium salt (30 mg, 0.12 mmol) were added to the bottle, 10 mL of toluene and 10 mL of acetic acid were added, and the mixture was stirred at 110°C for 16 hours. After the reaction was complete as monitored by TLC, the solid was filtered out, the filter cake was rinsed with 2 mL of methanol, and the solid was dried under reduced pressure to obtain 150 mg of gray-green solid with a yield of 80%.

[0308] 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 + ).

[0309]

[0310] Under nitrogen flow, 7d (140 mg, 0.28 mmol), potassium carbonate (60 mg, 0.43 mmol), 5 mL DMF, 5 mL water were added to the bottle, and stirred at room temperature for 3 hours. After the reaction was complete as monitored by TLC, the solvent was removed under reduced pressure, the pH was adjusted to 1-2, the precipitated solid was filtered, the filter cake was washed with 2 mL of methanol, and the solid was dried under reduced pressure to obtain 105 mg of a light yellow solid with a yield of 81%.

[0311] 1 H 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),1.91–1.8 2 (m,2H),0.88(t,J=7.0Hz,3H).MS(ESI)m / z=463.1(M+H + ).

[0312]

[0313] Under nitrogen flow, 7e (60 mg, 0.13 mmol), HATU (100 mg, 0.26 mmol) and 5 mL of anhydrous dichloromethane were added to the bottle, stirred for 0.5 minutes, and then 4-hydroxypiperidine (27 mg, 0.27 mmol) was added and stirred at room temperature for 16 hours. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure, water was added to precipitate the solid, the pH was adjusted to 2-3, the solid was filtered, and the filter cake was washed with 2 mL of methanol to obtain 55 mg of a light yellow solid with a yield of 77%.

[0314] 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).

[0315] 13 C 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 + ).

[0316] Example 8: Synthesis of Compound I-8:

[0317]

[0318] Under nitrogen flow, 7e (100 mg, 0.22 mmol) HATU (164 mg, 0.43 mmol) was added to the bottle, and 10 mL of anhydrous dichloromethane was added, and stirred for 0.5 hours, followed by DIPEA (113 μL, 0.64 mmol) and 3-hydroxyazetidine hydrochloride (48 mg, 0.45 mmol), and stirred at room temperature for 16 hours. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (DCM: MeOH = 50: 1) was performed to obtain 65 mg of a light yellow solid, with a yield of 57%.

[0319] 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).

[0320] 13 C 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 + ).

[0321] Example 9: Synthesis of Compound I-9:

[0322]

[0323] Under nitrogen flow, 7e (50 mg, 0.11 mmol) and HATU (82 mg, 0.22 mmol) were added to the bottle, and 2 mL of anhydrous dichloromethane was added, and stirred for 0.5 hours, followed by the addition of ethanolamine (13 μL, 0.22 mmol), and stirred at room temperature for 16 hours. After the reaction was complete as monitored by TLC, the solvent was removed under reduced pressure, and column chromatography (DCM: MeOH = 50: 1) was performed to obtain 44 mg of a light yellow solid, with a yield of 79%.

[0324] 1 H NMR (400 MHz, DMSO-d 6 )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),3.46(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 + ).

[0325] Example 10: Synthesis of Compound II-1:

[0326]

[0327] Under nitrogen flow, 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 the bottle, 5 mL of anhydrous DMF was added and stirred at room temperature to dissolve, and boron trifluoride etherate (35 μL, 312 μmol) was added and stirred at 40°C for 16 hours. LC-MS monitored the reaction system, and 70% of the raw material was converted into product II-1b, and then stirred at 40°C for 8 hours. LC-Ms monitored the reaction system, and 70% of the raw material was still converted into product II-1b. The solvent was removed under reduced pressure, 4 mL of tetrahydrofuran and 1 mL of diethylamine were added, and stirred at room temperature for 3 hours. LC-Ms monitored the reaction system, and II-1b was completely deprotected to II-1c. The solvent was removed under reduced pressure and purified by reverse phase column (mobile phase: 0.1 trifluoroacetic acid aqueous solution and acetonitrile; mobile phase method 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 yellow solid with a yield of 47%. MS (ESI) m / z = 567.2 (M+H + ).

[0328]

[0329] Under nitrogen flow, MC-GGF-OH (44 mg, 93 μmol, CAS No. 1599440-15-9, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) DMTMM (27 mg, 93 μmol) was added to 5 mL of anhydrous DMF, stirred at room temperature for 0.5 hours, II-1c (35 mg, 62 μmol) was added, DIPEA (15 μL, 93 μmol) was added, and stirred at 40°C for 8 hours. After the reaction was complete as monitored by LC-Ms, the solvent was removed under reduced pressure, and column chromatography (DCM: MeOH = 25: 1) was performed to obtain 42 mg of a white solid with a yield of 67%.

[0330] 1 H 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 + ).

[0331] Example 11: Synthesis of Compound II-2:

[0332]

[0333] Under nitrogen flow, 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), HOBT (8.4 g, 62.0 mmol), 30 mL of anhydrous dichloromethane, glycine tert-butyl ester (370 μL, 2.6 mmol), DIPEA (10.8 mL, 62.0 mmol) were added to the bottle, and stirred at room temperature for 16 hours. After the reaction was completed by TLC monitoring, the organic solvent was removed under pressure, and column chromatography (PE: EA = 5: 1 to 1: 1) was performed to obtain 24.5 g of a white solid with a yield of 91%.

[0334] 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 + ).

[0335]

[0336] Under nitrogen flow, II-2b (20.0 g, 45.9 mmol), 100 mL of dichloromethane, and 20 mL of trifluoroacetic acid were added to the bottle, and stirred at room temperature for 5 h. After the reaction was completed 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 white solid, with a yield of 94%.

[0337] 1H NMR (500MHz, DMSO-d 6 )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 + ).

[0338]

[0339] Under nitrogen flow, add Ⅱ-2c (10.0 g, 26.4 mmol), lead tetraacetate (23.4 g, 52.8 mmol), copper acetate (472 mg, 2.6 mmol), 40 mL of anhydrous toluene, 160 mL of anhydrous tetrahydrofuran, pyridine (4.3 mL, 52.8 mmol) into the bottle, and heat and stir at 60°C for 6 hours. After TLC monitoring, the inorganic salts were filtered out, the filter cake was washed with 20 mL of ethyl acetate, the solvent was removed under reduced pressure, the mixture was slurried with water, and then slurried with PE:EA=1:1 to obtain 8.2 g of white solid, with a yield of 79%.

[0340] 1 H NMR (400 MHz, DMSO-d 6)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=14.2,7.1Hz,1H),3.9 0–3.85(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 + ).

[0341]

[0342] Under nitrogen flow, II-2d (164 mg, 416 μmol), I-4 (100 mg, 208 μmol), PPTS (78 mg, 312 μmol) were added to the bottle, and 5 mL of anhydrous tetrahydrofuran was added, and the mixture was stirred at 45 °C for 16 hours. The reaction system was monitored by LC-Ms, and 60% of the raw material was converted into product II-2e, and the solvent was removed under reduced pressure. Under nitrogen flow, 1 mL of DMA and 9 mL of methanol were added to the bottle, and 10 mg of Pd / C was added to replace hydrogen, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by LC-Ms, Pd / C was removed by filtration, the solvent was removed under reduced pressure, and the mixture was purified by reverse phase column (mobile phase: 0.1 trifluoroacetic acid aqueous solution and acetonitrile; mobile phase method acetonitrile concentration: 5% acetonitrile, 2CV—5%-30% acetonitrile, 10CV—30%-40% acetonitrile, 5CV—40%-95% acetonitrile, 5CV—95% acetonitrile, 5CV). The solid was freeze-dried to give 71 mg, with a yield of 50%. MS (ESI) m / z = 680.3 (M+H + ).

[0343] 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).

[0344]

[0345] Under nitrogen flow, II-2f (20 mg, 29 μmol) was added to the bottle, followed by 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 the reaction was completed by LC-Ms monitoring, it was purified by reverse phase column (mobile phase: 0.05% formic acid aqueous solution and acetonitrile; mobile phase method acetonitrile concentration: 5% acetonitrile, 2CV-5%-50% acetonitrile, 20CV-50%-95% acetonitrile, 5CV-95% acetonitrile, 5CV). Lyophilization gave 23 mg of solid with a yield of 55%. MS (ESI) m / z=1430.7 (M+H + ).

[0346] 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).

[0347] Example 12: Synthesis of Compound II-3:

[0348]

[0349] Under nitrogen flow, 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), copper acetate (1.7 g, 9.4 mmol), 40 mL of anhydrous toluene, 160 mL of anhydrous tetrahydrofuran, pyridine (15 mL, 187.8 mmol) were added to the bottle, and heated and stirred at 60 ° C for 6 hours. After TLC monitoring, the reaction was complete, the inorganic salt was filtered out, the filter cake was washed with 20 mL of ethyl acetate, the solvent was removed under reduced pressure, and the mixture was slurried with water, and then slurried with PE:EA=1:1 to obtain 14.5 g of white solid, with a yield of 55%.

[0350] 1HNMR(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 + ).

[0351]

[0352] Under nitrogen flow, II-3a (88 mg, 312 μL), I-4 (50 mg, 104 μL), p-toluenesulfonic acid pyridinium salt (40 mg, 156 μL) were added to the bottle, and 5 mL of anhydrous tetrahydrofuran was added, and stirred at 45 ° C for 16 hours. The reaction system was monitored by LC-Ms, and 70% of the raw material was converted into product 12c. The solvent was removed under reduced pressure. Under nitrogen flow, 5 mL of 0.1% trifluoroacetic acid aqueous solution, 10 mL of methanol, 1 mL of DMF were added to the bottle, 13 mg of Pd / C was added, hydrogen was replaced, and stirred at room temperature for 2 hours. After the reaction was completed as monitored by LC-Ms, Pd / C was filtered, the solvent was removed under reduced pressure, and the product was purified by reverse phase column (mobile phase: 0.1 trifluoroacetic acid aqueous solution and acetonitrile; mobile phase method acetonitrile concentration: 5% acetonitrile, 2CV—5%-30% acetonitrile, 10CV—30%-40% acetonitrile, 5CV—40%-95% acetonitrile, 5CV—95% acetonitrile, 5CV). Lyophilization gave 32 mg of a yellow solid with a yield of 54%.

[0353] 1H 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 + ).

[0354]

[0355] Under nitrogen flow, Cbz-GGF-OH (76 mg, 185 μmol, CAS No. 13171-93-2, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.), DMTMM (36 mg, 185 μmol), 5 mL of anhydrous DMF were added to the bottle, and the mixture was stirred at room temperature for 0.5 hours. Ⅱ-3c (70 mg, 124 μmol) was added, and DIPEA (21 μL, 124 μmol) was 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 nitrogen flow, 13 mL of methanol, 2 mL of DMA, 5 mg of Pd / C, and 5 mg of palladium hydroxide were added to the bottle, hydrogen was replaced, and the mixture was reacted at room temperature for 16 hours. After the reaction was complete as monitored by LC-Ms, Pd / C and palladium hydroxide were filtered out, and the solvent was removed under reduced pressure. Purify with reverse phase column (mobile phase: 0.1 trifluoroacetic acid aqueous solution and acetonitrile; mobile phase method acetonitrile concentration: 5% acetonitrile, 2CV—5%-50% acetonitrile, 20CV—50%-95% acetonitrile, 5CV—95% acetonitrile, 5CV). Freeze-dry to obtain 84 mg of solid, yield 82%.

[0356] 1H 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,2H),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=1 3.7,10.4Hz,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 + ).

[0357]

[0358] Under nitrogen flow, II-3e (30 mg, 36.μmol) was added to the bottle, followed by 2 mL of anhydrous DMF, DIPEA (4.6 μL, 36.2 μmol), and Mc-PEG12-OSu (31 mg, 36.2 μmol), and stirred at room temperature for 6 hours. After the reaction was completed by LC-Ms monitoring, it was purified by reverse phase column (mobile phase: 0.05 formic acid aqueous solution and acetonitrile; mobile phase method acetonitrile concentration: 5% acetonitrile, 2CV-5%-50% acetonitrile, 20CV-50%-95% acetonitrile, 5CV-95% acetonitrile, 5CV). Lyophilization gave 35 mg of solid with a yield of 61%. MS (ESI) m / z=1578.7 (M+H + ).

[0359] 1H 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).

[0360] Embodiment 13:

[0361]

[0362] 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 + ).

[0363] Embodiment 14:

[0364]

[0365] 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 + ).

[0366] Synthesis of compounds ADC-1 and ADC-2 in Examples 15 and 16:

[0367] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 37.2μL, 372nmol) to the PBS buffer solution (pH=6.5 0.05M PBS buffer solution; 10.0mg / mL, 1.0mL, 67.6nmol) of antibody Trastuzumab (HER2 antibody, CAS: 180288-69-1) purchased from Hangzhou Haoyang Biotechnology Co., Ltd.), place in a water bath oscillator, oscillate at 37°C for 3 hours, and then stop the reaction. Cool the reaction solution to 25°C with a water bath. Dissolve compound II-2 or II-3 (1014nmol) in 50μL DMSO, add to the above reaction solution, place in a water bath oscillator, oscillate at 25°C for 3 hours, and then stop 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 PBS buffer solution (1.1 mg / mL, 7.5 mL) of the conjugates ADC-1 and ADC-2, which were stored at 4°C.

[0368]

[0369] Average drug loading calculated by RP-HPLC: ADC-1, t = 7.8; ADC-2, t = 7.8

[0370] Embodiment 17

[0371] Referring to the synthesis examples of compounds ADC-1 and ADC-2, compound ADC-3 was prepared.

[0372]

[0373] The average drug loading value calculated by RP-HPLC was: ADC-3, t=6.2.

[0374] Embodiment 18

[0375] Referring to the synthesis examples of compounds ADC-1 and ADC-2, compound ADC-4 was prepared.

[0376]

[0377] Average drug loading calculated by RP-HPLC: ADC-4, t=7.7.

[0378] Embodiment 19

[0379] Referring to the synthesis examples of compounds ADC-1 and ADC-2, compound ADC-5 was prepared.

[0380]

[0381] The average drug loading value calculated by RP-HPLC was: ADC-5, t=7.9.

[0382] Biological evaluation

[0383] Camptothecin's ability to test tumor cell proliferation:

[0384] Effect Example 1: The CTG detection method 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 (the types of tumor cells are shown in Table 1).

[0385] After digestion, resuspend the test cells for cell counting, centrifuge to remove the supernatant, and adjust the cell density. Count with a cell counter, aspirate 20μl of cell suspension, mix with 20μl of trypan blue, take 20μl of liquid, and count the cells. Spread the cells in a 96-well cell culture plate, add 180ul to each well, and seed 1000 cells per well. Gently tap to mix, and place the cell culture plate in a 37℃ cell culture incubator for 16 hours. Dilute the compound with McCoy's5A medium, add 20ul to each well, so that the sample concentration is as shown in the experimental design, so that the maximum concentration is 100nM, 3-fold dilution, 10 points. The solvent control is DMSO, and 20μl McCoy's 5A medium is added to the blank control. Gently tap to mix, and place the cell culture plate in a 37℃ cell culture incubator for 7days. After 7 days of culture, remove the cell plate from the incubator, aspirate the culture medium, add 20ul culture medium, and then add 20ul CTG to each well. The mixture was shaken on an oscillator for 5 minutes, and then allowed to stand at room temperature for 10 minutes. 30 ul of the reaction solution was transferred to a Viewplate-96white white transparent bottom ELISA plate, and data was analyzed on an EnVision plate reader. The effects of the compounds on the proliferation ability of tumor cells are shown in Table 2.

[0386] Calculation formula: background value = control group - blank group; inhibition rate = 1 - (experimental group - blank group) / (background value) * 100%.

[0387] Table 1. Overview of tumor cell lines used in this screening

[0388] Cell code Cell line name Culture medium source NCI-N87 Human gastric cancer cells 1640+10%FBS ATCC Calu-3 Human lung adenocarcinoma cells MEM + 10% FBS ATCC HCT-116 Human colon cancer cells McCoy's 5A + 10% FBS ATCC

[0389] Table 2. Effects of compounds on tumor cell proliferation

[0390]

[0391] 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.

[0392] Effect Example 2: Test method for the proliferation ability of ADC-1 and ADC-2 on tumor cells:

[0393] After digestion, resuspend the NCI-N87 cells and count the cells. Centrifuge to remove the supernatant and adjust the cell density. Count with a cell counter, aspirate 20uL of cell suspension, mix with 20uL VisStain AOPI staining solution, take 20uL of liquid, and count the cells. Spread the cells in a 96-well cell culture plate, add 180uL to each well, and plant 1000 cells or 2000 cells per well. Gently tap to mix, and place the cell culture plate in a 37°C cell culture incubator for 16 hours. Dilute the compound with RPMI1640 medium / 10% FBS, add 20uL to each well, so that the maximum concentration is 100nM, 3-fold dilution, 10 points. The solvent control is DMSO, and 20uL RPMI1640 medium / 10% FBS is added to the blank control. Gently tap to mix, and place the cell culture plate in a 37°C cell culture incubator for 7days. After 7 days of culture, the cell plate was taken out of the incubator, the culture medium was removed, 20uL of culture medium was added, and then 20uL of CTG was added to each well. The plate was shaken for 5 minutes, and then allowed to stand at room temperature for 10 minutes. 30uL of the reaction solution was transferred to the Viewplate-96white white transparent bottom ELISA plate, and data was analyzed on the EnVision plate reader.

[0394] Calculation formula: The inhibition rate (IR) of the test antibody was calculated using the following formula: IR (%) = (1-(antibody-blank control) / (solvent control-blank control))*100%. The inhibition rates of different antibody concentrations were calculated in Excel, and then the inhibition curves were plotted and related parameters were calculated using GraphPad Prism software. The results are shown in Table 3 and Figure 1 shown.

[0395] Table 3 Effects of ADC-1 and ADC-2 on the proliferation ability of NCl-N87 tumor cells

[0396] Antibody Drug Conjugates <![CDATA[IC 50 (μg / mL)]]> ADC-1 0.0182 ADC-2 0.0153

[0397] Effect Example 3. Antitumor efficacy test of ADC-1 and ADC-2 on NOG mouse subcutaneous transplantation of NCI-N87 cells animal model:

[0398] In this study, NCI-N87 cells were subcutaneously inoculated into NOG mice to determine the antitumor effects of ADC-1 and ADC-2.

[0399] NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were adaptively housed for 7 days after arrival, and then the study began.

[0400] 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 volume was 1×10 7 ) were subcutaneously inoculated into the right axilla of female NOG mice to establish the NCI-N87 tumor-bearing mouse model.

[0401] Dosage:

[0402] On the 10th day after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of 110 mm 3 ~250mm 3 Mice within the range were grouped according to tumor volume (5 mice per group) and were given drugs on the 11th day after inoculation, negative control antibodies (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), ADC-1 and ADC-2. The tumor volume and body weight changes of mice in each group were monitored during the drug administration. The monitoring frequency was 2 times / week and the monitoring was continuous for 4 weeks. Body weight and tumor volume were measured before each drug administration. The tumor volume inhibition rate (TGI%) was calculated on the 28th day after drug administration. The calculation formula is as follows: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100; where Ti: mean tumor volume of the drug administration group, T0: mean tumor volume of the drug 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 with a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 × a × b 2 Body weight was measured using an electronic balance.

[0403] The dosage and method of administration are shown in Table 4.

[0404] Table 4 Drug administration experiment design

[0405] serial number Dosage Dosing frequency Dosage Negative control antibody 4mg / kg Single dose Intravenous injection ADC-1 4mg / kg Single dose Intravenous injection ADC-2 4mg / kg Single dose Intravenous injection

[0406] As shown in Table 5 and Figure 2 As shown, on the 28th day 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.

[0407] Table 5. Inhibitory effect of ADC on tumor growth in vivo on day 28

[0408]

[0409] Note: P value is compared with negative control antibody

[0410] The above experiments showed that ADC-1 and ADC-2 significantly inhibited the growth of tumors in NOG mice subcutaneously transplanted with NCI-N87 cells, and had no significant effect on the body weight of NOG mice (e.g. Figure 3 shown).

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 group; 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: Wherein the anti-HER2 antibody is trastuzumab; the definition of t is as described 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 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.

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.

Images