Camptothecin compounds, their preparation and use

Abstract

The present invention relates to camptothecin compounds having antitumor activity, their preparation and their use. In particular, the present invention relates to camptothecin compounds having the following formula: JPEG2025515988000284.jpg37149 or a pharma- ceutically acceptable form thereof, a pharmaceutical composition thereof, a process for its preparation and its use. The compound can be used as a medicament for the treatment of diseases in abnormal cell proliferation.

Description

[Technical field]

[0001] This application is based on and claims priority from a Chinese patent application with patent application number 202210521215.2 and filing date May 13, 2022. The disclosure of the Chinese patent application is incorporated herein in its entirety.

[0002] [Technical field] The present invention relates to a class of camptothecin compounds and conjugates thereof having antitumor activity, as well as their preparation methods and medical applications. [Background technology]

[0003] Camptothecin (CPT, formula 1) is a compound with a five-ring quinoline core isolated from Camptotheca acuminata, a plant of the Davidia family. It consists of quinoline rings A and B, pyrrole ring C, pyridone ring D, and α-hydroxylactone ring E, with the 20-position in the S configuration (see the structure represented by the following formula). It was introduced into clinical practice in the early 1970s due to its excellent anticancer activity, but clinical trials were subsequently terminated due to severe side effects, such as diarrhea and hemorrhagic cystitis.

[0004] [ka]

[0005] Research data indicates that camptothecin forms a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, blocking DNA replication, and thus causing cell death (Cancer Res. 1989, 49, 6365). Camptothecin and its derivatives have potent antitumor activity in animal models of lung, breast, colorectal, and ovarian cancer (Nature Review Cancer, 2006, 6, 789).

[0006] Currently, several camptothecin drugs are approved for use in tumor therapy (Med. Res. Rev. 2015, 35, 753). Irinotecan is used for the treatment of colorectal cancer; Topotecan is used for the treatment of ovarian cancer; Belotecan is used for the treatment of ovarian cancer and small cell lung cancer. Camptothecan derivatives further include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, Elomotecan, etc.

[0007] Camptothecin drugs or their derivatives often have hematologic toxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, and gastrointestinal side effects, such as nausea, vomiting, and diarrhea. Clinical studies have found that the means for improving the safety and efficacy of camptothecin compounds include improving their pharmacokinetic properties, adjusting activity, reducing dosage, or using conjugates to form antibody-binding drugs with antibodies. Therefore, there is still a high clinical demand and application value for developing camptothecin compounds and their conjugates with novel structures, enhanced efficacy, and improved safety. Summary of the Invention

[0008] The present invention provides a novel camptothecin compound and its conjugate, the camptothecin compound has good antitumor activity, excellent permeability, low excretion rate, and good metabolic stability, and is promising for use in the treatment of tumor diseases, and the conjugate has the prospect of widespread application as an antibody binding drug.

[0009] A first aspect of the present invention provides a compound or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, and prodrugs thereof, which has the following structure: [ka] [In the formula, R 1 is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; or R 1 and R 2 are joined together with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle; R 3 is hydrogen, C 1~6 Alkyl, deuterated C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 cycloalkyl, and 3- to 6-membered heterocyclyl, or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; A is, [ka] selected from the group consisting of; Ring B is a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring; R 4 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 selected from the group consisting of cycloalkyl, and 3- to 6-membered heterocyclyl; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 3~6 Cycloalkyl and C 1~6haloalkyl, or R 5 Or R 6 together with the carbon atoms to which they are attached form a 3- to 6-membered ring; or R 4 and R 5 are connected to form a 4- to 6-membered ring; n=1 to 6, for example, 1, 2, 3, 4, 5 or 6.

[0010] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0011] In some embodiments, in formula (I), R 1 is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; or R 1 and R 2 are joined together with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle; R 3 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl and C 1~6 or, when joined to adjacent benzene ring carbon atoms, form a 6-membered carbocyclic ring; A is, [ka] selected from the group consisting of; Ring B is a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring; R 4 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6selected from the group consisting of cycloalkyl and 3- to 6-membered heterocyclyl; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 3~6 Cycloalkyl and C 1~6 haloalkyl; or R 5 Or R 6 together with the carbon atoms to which they are attached form a 3- to 6-membered ring; or R 4 and R 5 are connected to form a 4- to 6-membered ring; n=1 or 2.

[0012] In some embodiments, the compound has the structure of Formula (II): [ka] having In formula (II), R 1 ' is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 ' is selected from the group consisting of methyl, fluorine, chlorine, and hydroxyl; R 3 ' is hydrogen, C 1~6 Alkyl, deuterated C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 cycloalkyl, and 3- to 6-membered heterocyclyl; R 3 ' is preferably hydrogen, C 1~6 Alkyl, deuterated C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 is an alkoxyalkyl; R 4 ' is hydrogen, C 1~6 Alkyl and C 1~6 haloalkyl; R 4' is preferably hydrogen; R 1 ' is fluorine and R 2 If ' is methyl, then R 3 ' and R 4 ' is not hydrogen at the same time; R 1 ' is fluorine and R 2 If ' is methyl, then R 4 ' is C 1~6 Not alkyl. In some embodiments, in formula (II), R 1 ' is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 ' is selected from the group consisting of methyl, fluorine, chlorine, and hydroxyl; R 3 ' is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 R is selected from the group consisting of cycloalkyl and 3- to 6-membered heterocyclyl; 3 ' is preferably hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 is an alkoxyalkyl; R 4 ' is hydrogen, C 1~6 Alkyl and C 1~6 haloalkyl; R 4 ' is preferably hydrogen; R 1 ' is fluorine and R 2 If ' is methyl, then R 3 ' and R 4 ' is not hydrogen at the same time; R 1 ' is fluorine and R 2 If ' is methyl, then R 4 ' is C 1~6 Not alkyl. In some embodiments, in formula (II), R 1' is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 ' is chlorine or methyl; R 3 ' is hydrogen, methyl or deuteromethyl; R 4 ' is hydrogen. In some embodiments, in formula (II), R 1 ' is fluorine; R 2 ' is chlorine; R 3 ' is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 R is selected from the group consisting of cycloalkyl and 3- to 6-membered heterocyclyl; 3 ' is preferably hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl or C 1~6 Alkoxyalkyl; preferably hydrogen, C 1~6 Alkyl or C 1~6 haloalkyl; for example, hydrogen, methyl or deuterated methyl; R 4 ' is hydrogen, C 1~6 Alkyl and C 1~6 haloalkyl; R 4 ' is preferably hydrogen. In some embodiments, in formula (II), R 1 ' is chlorine; R 2 ' is methyl; R 3 ' is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 R is selected from the group consisting of cycloalkyl and 3- to 6-membered heterocyclyl; 3 ' is preferably hydrogen, C 1~6 Alkyl, C 1~6Haloalkyl or C 1~6 Alkoxyalkyl; preferably hydrogen, C 1~6 Alkyl or C 1~6 haloalkyl; for example, hydrogen, methyl or deuterated methyl; R 4 ' is hydrogen, C 1~6 Alkyl and C 1~6 haloalkyl; R 4 ' is preferably hydrogen.

[0013] In some embodiments, the compound has the structure of Formula (III): [ka] having In formula (III), R 1 is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; R 3 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 cycloalkyl, and 3- to 6-membered heterocyclyl, or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R 3 is preferably hydrogen; R 4 is hydrogen or C 1~6 is alkyl; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 1~6 Haloalkyl, C 3~6 cycloalkyl and 3- to 6-membered heterocyclyl, or R 5 and R 6form a 3- to 6-membered ring together with the carbon atom to which they are attached; or R 4 and R 5 are connected to form a 4- to 6-membered ring; n=1 or 2; R 1 is fluorine and R 2 is methyl and R 3 is hydrogen and R 4 If is hydrogen, R 5 and R 6 are not hydrogen and do not form a cyclopropyl group together with the carbon atom to which they are attached; R 1 is fluorine and R 2 is methyl and R 3 If is hydrogen, R 4 is not alkyl.

[0014] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0015] In some embodiments, the compound has the formula (III-1): [ka] having a structure represented by R 1 is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2 is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; R 3 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 R is selected from the group consisting of cycloalkyl and 3- to 6-membered heterocyclyl; 3 is preferably hydrogen; R 4 is hydrogen or C 1~6 is alkyl; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~6 cycloalkyl and 3- to 6-membered heterocyclyl, or R 5 and R 6 together with the carbon atoms to which they are attached form a 3- to 6-membered ring; R 1 is fluorine and R 2 is methyl and R 3 is hydrogen and R 4 If is hydrogen, R 5 and R 6 are not hydrogen and do not form a cyclopropyl group together with the carbon atom to which they are attached; R 1 is fluorine and R 2 is methyl and R 3 If is hydrogen, R 4 is not alkyl.

[0016] In some embodiments, R 2 is fluorine or chlorine.

[0017] In some embodiments, R 2 is selected from the group consisting of hydrogen, methyl, fluorine, and chlorine.

[0018] In some embodiments, R 2 is methyl or chlorine.

[0019] In some embodiments, R 3 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 1~6 Alkoxyalkyl, C 3~6 It is selected from the group consisting of cycloalkyl, and 3- to 6-membered heterocyclyl.

[0020] In some embodiments, R 4 is hydrogen.

[0021] In some embodiments, R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~6 Cycloalkyl and C 2~6 alkenyl, or R 5 and R 6 together with the carbon atom to which they are attached form a 3- to 6-membered ring.

[0022] In some embodiments, R 5 and R 6 are independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 3~6 cycloalkyl or R 5 and R 6 together with the carbon atom to which they are attached form a 3- to 6-membered carbocyclic ring.

[0023] In some embodiments, R 5 and R 6 are independently selected from the group consisting of hydrogen, methyl, vinyl, allyl, cyclopropyl, or R 5 and R 6 together with the carbon atom to which they are attached form a three-membered carbocyclic ring.

[0024] In some embodiments, R 5 and R 6 is hydrogen, C 1~6 Alkyl and C 3~6 cycloalkyl, or R 5 and R 6 together with the carbon atom to which they are attached form a 3- to 6-membered carbocyclic ring.

[0025] In some embodiments, R 5 and R 6 is independently selected from the group consisting of hydrogen, methyl, and cyclopropyl, or R 5 and R 6 together with the carbon atom to which they are attached form a three-membered carbocyclic ring.

[0026] In some embodiments, in Formula (III), when n=2, [ka] teeth, [ka] It is.

[0027] In some embodiments, R 4 and R 5 are connected to form a five-membered ring.

[0028] In some embodiments, R 4 and R 5 are connected to form a five-membered ring, and R 6 is hydrogen.

[0029] In some embodiments, in formula (III) or formula (III-1), R 1 is selected from the group consisting of fluorine and chlorine; R 2 is selected from the group consisting of chlorine and methyl; R 3 is hydrogen and C 1~6 alkyl, or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R 4 is hydrogen; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C3~6 Cycloalkyl and C 2~6 alkenyl, or R 5 and R 6 form a 3- to 6-membered ring together with the carbon atom to which they are attached; Or R 4 and R 5 are connected to form a 4- to 6-membered ring.

[0030] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0031] In some embodiments, in formula (III) or formula (III-1), R 1 is fluorine; R 2 is chlorine; R 3 is hydrogen or C 1~2 alkyl or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R 4 is hydrogen; R 5 and R 6 is hydrogen, C 1~2 Alkyl, C 1~2 Haloalkyl, C 3~6 Cycloalkyl and C 2~4 alkenyl, or R 5 and R 6 form a 3- to 6-membered ring together with the carbon atom to which they are attached; Or R 4 and R 5 are connected to form a 4- to 6-membered ring.

[0032] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R1 is the carbon atom at the meta-position of and R 2 and the ortho-position of the benzene ring.

[0033] In some embodiments, in formula (III) or formula (III-1), R 1 is chlorine; R 2 is methyl; R 3 is hydrogen or C 1~2 alkyl, or R 3 is connected to adjacent benzene ring carbon atoms to form a 6-membered carbon ring; R 4 is hydrogen; R 5 and R 6 are independently selected from the group consisting of hydrogen, C 1~2 alkyl, C 1~2 haloalkyl, C 3~6 cycloalkyl and C 2~4 alkenyl, or R 5 and R 6 together with the carbon atoms to which they are attached form a 3- to 6-membered ring; or, R 4 and R 5 are connected to form a 4- to 6-membered ring.

[0034] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a 6-membered carbon ring, and the carbon atoms are the carbon atoms at the meta-position of R 1 and the ortho-position of R 2 of the benzene ring.

[0035] In some embodiments, in formula (III) or formula (III-1), R 1 is fluorine; R 2 is methyl; R 3 is hydrogen or C 1~2 alkyl, or R 3 is connected to adjacent benzene ring carbon atoms to form a 6-membered carbon ring; R 4 is hydrogen; R 5 and R 6 is hydrogen, C 1~2 Alkyl, C 1~2 Haloalkyl, C 3~6 Cycloalkyl and C 2~4 alkenyl, or R 5 and R 6 form a 3- to 6-membered ring together with the carbon atom to which it is attached; preferably, R 5 and R 6 is not hydrogen at the same time; Or R 4 and R 5 are connected to form a 4- to 6-membered ring.

[0036] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0037] In some embodiments, in formula (III) or formula (III-1), R 1 is fluorine or chlorine; R 2 is methyl or chlorine; preferably chlorine; R 3 is hydrogen or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R 4 is hydrogen; R 5 and R 6 is hydrogen, C 1~6 Alkyl, C 1~6 Haloalkyl, C 3~6 Cycloalkyl and C 2~6 alkenyl, or R 5 and R 6 form a 3- to 6-membered ring together with the carbon atom to which they are attached; Or R 4 and R 5 are connected to form a 4- to 6-membered ring.

[0038] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0039] In some embodiments, in formula (III) or formula (III-1), R 1 is fluorine or chlorine; R 2 is chlorine; R 3 is hydrogen or R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R 4 is hydrogen; R 5 and R 6 is independently selected from the group consisting of hydrogen, methyl, trifluoromethyl, cyclopropyl, and vinyl, or R 5 and R 6 form a three-membered ring together with the carbon atom to which they are attached; Or R 4 and R 5 are connected to form a five-membered ring.

[0040] In some embodiments, R 3 is connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring, the carbon atoms being R 1 meta position and R 2 It is the ortho carbon atom of.

[0041] In some embodiments, the compound has formula (IV): [ka] having a structure represented by In formula (IV), R 1’ is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2’ is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; R 4’ is hydrogen, C 1~6 Alkyl, C 3~6 Cycloalkyl and C 1~6 haloalkyl; R 4’ is preferably hydrogen; B is C 1~6 selected from the group consisting of alkylene, a 3- to 6-membered carbocyclic ring, and a 3- to 6-membered heterocyclic ring; R 1’ is fluorine and R 2’ is methyl and a dashed carbocycle is present, R 4’ is hydrogen or C 1~6 Nor is it alkyl; R 2’ is methyl and R 1’ When is fluorine and the dashed carbocyclic ring is absent, then ring B is not a three-membered carbocyclic ring.

[0042] In some embodiments, in formula (IV), R 1’ is selected from the group consisting of hydrogen, fluorine, and chlorine; R 2’ is selected from the group consisting of hydrogen, methyl, fluorine, chlorine and hydroxyl; R 4’ is hydrogen, C 1~6 Alkyl, C 3~6 Cycloalkyl and C 1~6 haloalkyl; R 4’ is preferably hydrogen; B is selected from the group consisting of a 3- to 6-membered carbocyclic group or a 3- to 6-membered heterocyclic group; R 1’ is fluorine and R 2’ is methyl and a dashed carbocycle is present, R 4’ is neither hydrogen nor alkyl; R2’ is methyl and R 1’ When is fluorine and the dashed carbocyclic ring is absent, then ring B is not a three-membered carbocyclic ring.

[0043] In some embodiments, R 1’ is fluorine or chlorine.

[0044] In some embodiments, R 2’ is selected from the group consisting of hydrogen, methyl, fluorine, and chlorine.

[0045] In some embodiments, R 2’ is methyl or chlorine.

[0046] In some embodiments, R 4’ is hydrogen.

[0047] In some embodiments, B is C 1~6 alkylene; preferably, B is methylene, ethylene, propylene, isopropylene; more preferably, B is propylene or isopropylene.

[0048] In some embodiments, the B ring is a 3- to 6-membered carbocyclic ring; preferably, the B ring is a 3- to 6-membered saturated carbocyclic ring.

[0049] In some embodiments, the present invention provides the following compound: [ka] JPEG2025515988000013.jpg199149 JPEG2025515988000014.jpg186149 JPEG2025515988000015.jpg201149 JPEG2025515988000016.jpg206149 JPEG2025515988000017.jpg191149 JPEG2025515988000018.jpg200149 JPEG2025515988000019.jpg204149 JPEG2025515988000020.jpg202149 to provide.

[0050] On the one hand, the present invention relates to a compound of formula (V): MLED Formula (V) [In the formula, M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a linker connecting linking sites M and E; E represents a structural fragment connecting L and D; and D represents a structural fragment of a cytotoxic drug], or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, and prodrugs thereof.

[0051] In some embodiments, M has the structure: [ka] wherein X is selected from the group consisting of leaving groups such as chlorine, bromine, -OM, OT, and OTf.

[0052] In some embodiments, M has the structure: [ka] is selected from the group consisting of:

[0053] In some embodiments, L has the following structure: 1~6Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D- Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, [ka] [wherein R' is hydrogen, C 1~6 Alkyl or -(CH 2 CH 2 O) r - represents an alkyl group containing -; r is an integer selected from 1 to 10; and s is an integer selected from 1 to 20.

[0054] In some embodiments, L has the structure: [ka] [wherein s is an integer selected from 1 to 20].

[0055] In some embodiments, E is a single bond, -NH-CH 2 -, [ka] is selected from the group consisting of:

[0056] In some embodiments, E is -NH-CH 2 -It is.

[0057] In some embodiments, the cytotoxic agent is selected from the group consisting of the compounds described in any one of the clauses of the first aspect of the present invention.

[0058] In some embodiments, the cytotoxic agent is selected from the group consisting of compounds 1-1 through 1-2; 2-1 through 2-23; 3-1 through 3-4; and 4-1 through 4-12.

[0059] In some embodiments, D is selected from the group consisting of structures formed by removing a hydrogen atom from the compounds described in the first aspect of the invention.

[0060] In some embodiments, D is selected from the group consisting of structures formed by removing a hydrogen atom from compounds 1-1 to 1-2; 2-1 to 2-18; 3-1 to 3-18; 4-1 to 4-16; 5-1 to 5-10; 6-1 to 6-10; 7-1 to 7-2; 8-1 to 8-2, or 9-1 to 9-2.

[0061] In some embodiments, D has the structure: [ka] JPEG2025515988000027.jpg204149 JPEG2025515988000028.jpg195149 JPEG2025515988000029.jpg211149 JPEG2025515988000030.jpg194149 JPEG2025515988000031.jpg204149 JPEG2025515988000032.jpg207149 JPEG2025515988000033.jpg204149 JPEG2025515988000034.jpg166149 is selected from the group consisting of:

[0062] In some embodiments, the MLED is the following compound: A-1: [ka] A-2: [ka] A-3: [ka] A-4: [ka] A-5: [ka] A-6: [ka]

[0063] A-7: [ka] A-8: [ka] A-9: [ka] A-10: [ka] A-11: [ka] A-12:

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[0064] B-1:

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[0065] B-7:

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[0066] C-1:

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[0067] C-6:

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[0068] On the other hand, the present invention relates to a compound of formula (VI): Ab-(MLED) x Formula (VI) [In the formula, Ab stands for antibody or antigen-binding fragment thereof; M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a linker connecting linking sites M and E; E represents a structural fragment connecting L and D; D represents a structural fragment of a cytotoxic drug; and x is 1 to 10.

[0069] In some embodiments, M, L, E, and D in formula (VI) are as described above.

[0070] In some embodiments, the antibody-drug conjugate (ADC) has formula (VI-1): Ab-MLED Formula (VI-1) It is expressed by:

[0071] In some embodiments, the Ab-(MLED) x has the following structure: ADC A-1: [ka] ADC A-2:

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[0072] ADC A-7:

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[0073] ADC B-1:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0074] ADC B-7:

Chem.

Chem.

Chem.

Chem.

Chem.

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[0075] ADC C-1:

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[0076] ADC C-7:

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[0077] In some embodiments, x is 1 to 10, e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9.

[0078] In some embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0079] In some embodiments, the present invention provides the following antibody-drug conjugates (ADCs): Trastuzumab A-11: [ka] Trastuzumab B-1: [ka] Trastuzumab B-2: [ka] or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, and isotopically labeled compounds, metabolites, and prodrugs thereof are further provided.

[0080] In another aspect, the present application provides a composition of the antibody-drug conjugates (ADCs) described herein. Such a composition may include a plurality of ADCs described herein, each ADC includes a drug-linker described herein, and x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule of the present composition can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Accordingly, the present composition is characterized by a "drug-to-antibody" ratio (DAR) in the range of about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

[0081] For example, in any embodiment, the composition of the ADCs described herein has a DAR of about 1 to about 10, or any sub-range therebetween, such as: about 1-2, about 1-3, about 1-4, about 1-5, about 1-6, about 1-7, about 1-8, about 1-9, about 1-10, about 2-3, about 2-4, about 2-5, about 2-6, about 2-7, about 2-8, about 2-9, about 2-10, about 3-4, about 3-5, about 3-6, about 3-7, about 3-8, about 3-9, about 3-10, about 4-5, about 4-6, about 4-7, about 4-8, about 4-9, about 4-10, about 5-6, about 5-7, about 5-8, about 5-9, about 5-10, about 6-7, about 6-8, about 6-9, about 6-10, about 7-8, about 7-9, about 7-10, about 8-9, about 8-10, or about 9-10.

[0082] In certain embodiments, the ADC compositions described herein have a DAR of about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5 to 6. 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 5.0~8.0, 5.5~6.0, 5. 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5, about 7.5 to 8.0, about 7.5 to 8.5.

[0083] In some embodiments, the present invention further provides a composition comprising one or more antibody-drug conjugates as follows, the composition having a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 8.0. Trastuzumab A-11: [ka]

[0084] In some embodiments, the present invention further provides a composition comprising one or more antibody-drug conjugates as follows, the composition having a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 7.96. Trastuzumab B-1: [ka]

[0085] In some embodiments, the present invention also provides compositions comprising one or more of the following antibody-drug conjugates, wherein the compositions have a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 7.95: Trastuzumab B-2: [ka]

[0086] definition Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. The techniques used herein are intended to mean techniques commonly understood in the art, including obvious variations or equivalent substitutions of techniques that are apparent to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better describe the present invention.

[0087] As used herein, the terms "comprise," "include," "having," "containing," or "involving" and other variations thereof are inclusive or open-ended and do not exclude other, unrecited elements or method steps.

[0088] As used herein, the symbol "" in the structure of a compound * " indicates that the labeled carbon atom is an asymmetric carbon atom, and the present invention includes the pair of enantiomers formed by the asymmetric carbon atom. For example, if a compound contains two different asymmetric carbon atoms, the present invention includes the four optical isomers formed by the asymmetric carbon atoms.

[0089] As used herein, [ka] refers to the site at which the structural fragment is connected to the rest of the molecule.

[0090] The term "alkyl" is defined as a straight or branched chain saturated aliphatic hydrocarbyl. In some embodiments, an alkyl has 1 to 12, e.g., 1 to 6, carbon atoms. For example, as used herein, the term "C 1~6 "Alkyl" refers to a straight or branched chain group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl) optionally substituted with one or more (e.g., 1, 2, or 3) suitable substituents.

[0091] The term "alkenyl" refers to, for example, "C 2~6 alkenyl", "C 2~4 "Alkenyl" refers to a straight or branched chain hydrocarbon containing at least one carbon-carbon double bond, including, but not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, and the like.

[0092] The term "alkynyl" refers to, for example, "C 2~6 "C4-6 alkynyl" refers to straight or branched chain hydrocarbons containing at least one carbon-carbon triple bond, including, but not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadinyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadinyl, and the like.

[0093] The term "cycloalkyl" refers to saturated cyclic hydrocarbonyls, including, but not limited to, monocycloalkyls and bicycloalkyls (e.g., spirocycloalkyls, fused cycloalkyls and bridged cycloalkyls). 3~6 "Cycloalkyl" refers to a cycloalkyl having 3 to 6 ring-forming carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, which can be optionally substituted by one or more (e.g., 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl, and the like.

[0094] The term "carbocycle" or "carbocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon connected through a ring carbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0095] The term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (e.g., bicyclic) hydrocarbon ring (e.g., a monocyclic ring, a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a cyclononane ring, etc., or a bicyclic ring, including a spiro, fused or bridged system (e.g., a bicyclo[1.1.1]pentane ring, a bicyclo[2.2.1]heptane ring, a bicyclo[3.2.1]octane ring or a bicyclo[5.2.0]nonane ring, a decalin ring, etc.), which can be optionally substituted with one or more (e.g., 1, 2 or 3) suitable substituents. The term "3- to 6-membered carbocycle" refers to a carbocycle containing 3, 4, 5 or 6 ring-forming carbon atoms.

[0096] The term "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (e.g., bicyclic) non-aromatic ring structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur. A heterocyclyl can be connected to the remainder of the molecule through any ring atom, provided that the valence bond requirements are met. A heterocyclyl in the present invention is preferably a 3- to 6-membered heterocyclyl. As used in the present invention, the term "3- to 6-membered heterocyclyl" refers to a heterocyclyl having 3 to 6 ring atoms, including a 3-membered heterocyclyl, a 4-membered heterocyclyl, a 5-membered heterocyclyl, and a 6-membered heterocyclyl, including a nitrogen-containing heterocyclyl and an oxygen-containing heterocyclyl, such as a 4- to 6-membered heterocyclyl, such as a 4- to 6-membered nitrogen-containing heterocyclyl and a 4- to 6-membered oxygen-containing heterocyclyl. Common heterocyclyl groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. Heterocyclyls of the present invention may be optionally substituted with one or more substituents as described herein. Heterocyclyls of the present invention may be optionally fused with one or more aromatic or non-aromatic rings.

[0097] The term "oxygen-containing heterocycle" refers to the above-mentioned heterocycle in which one or more (e.g., 1, 2 or 3) ring atoms are oxygen atoms, for example, 5- to 6-membered oxygen-containing heterocycles. Specific examples include, but are not limited to, oxirane ring, tetrahydrofuran ring, furan ring, tetrahydropyran ring, pyran ring, etc. As used herein, the term "nitrogen-containing heterocycle" refers to the above-described heterocycle in which one or more (e.g., 1, 2 or 3) ring atoms are nitrogen atoms.

[0098] The term "haloalkyl" refers to an alkyl substituted with one or more (e.g., 1, 2, or 3) of the same or different halogen atoms, where alkyl is as defined above. For example, the term "C 1~6 "Haloalkyl" refers to a haloalkyl having 1 to 6 carbon atoms. Common haloalkyl groups include, but are not limited to, -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CF 3 , -CF 2 CF 3 , -CH 2 CH 2 CF 3 , -CH 2 Cl, etc. Haloalkyl groups of the present invention are optionally substituted with one or more substituents described herein.

[0099] The term "alkoxy" refers to a group having the structure "alkyl-O-", where alkyl is as defined above, e.g., C 1~6 Alkoxy, C 1~4 Alkoxy, C 1~3 Alkoxy or C 1~2 and alkoxy. Common alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. Alkoxy groups in the present invention are optionally substituted with one or more substituents described herein.

[0100] The term "alkoxyalkyl" refers to an alkyl substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups, where alkoxy and alkyl are as defined above. For example, the term "C 1~6 "Alkoxyalkyl" refers to an alkyl having 1 to 6 carbon atoms and substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups. Common alkoxyalkyl groups include, but are not limited to, CH 3O-CH 2 -, C 2 H 5 -O-CH 2 -, C 2 H 5 -O-CH 2 CH 2 --etc.

[0101] The term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0102] The term "N-oxide" refers to an oxide (e.g., mono- or di-oxide) of at least one nitrogen atom in the compound structure of the present application. N-monoxides can exist as a single positional isomer or as a mixture of positional isomers.

[0103] The term "substitution" refers to the selective replacement of one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom with a specified group, provided that the specified atom still has normal valences in this situation and the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0104] When a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. When a carbon of a substituent is described as being optionally substituted with one or more of the substituents in the list of substituents, one or more of the carbon-bearing hydrogens (to the extent of any hydrogens present) may be individually and / or together replaced with an independently selected optional substituent. When a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more of the nitrogen-bearing hydrogens (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

[0105] When substituents are described as "independently selected from" a group, each substituent is selected independently from the other substituents. Thus, each substituent can be the same or different from another (other) substituent.

[0106] As used herein, the term "one or more" means, for example, one or more than one, such as, for example, 2, 3, 4, 5 or 10, under appropriate conditions.

[0107] As used herein, unless otherwise specified, the point of attachment of a substituent may be from any suitable position on the substituent.

[0108] The term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, this can result in racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Some individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally distinct forms (often called tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, and imine-enamine tautomers, and the like. It goes without saying that the scope of the present application encompasses all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0109] The carbon-carbon bonds in the compounds of the present invention are represented by solid lines. [ka] , solid wedge [ka] , or dashed wedge [ka] The use of a solid line to indicate a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers at that carbon atom (e.g., a specific enantiomer, a racemic mixture, etc.) are included. The use of a solid wedge or a dashed wedge to indicate a bond to an asymmetric carbon atom is intended to indicate that the stereoisomer exists as shown. When present in a racemic mixture, the solid wedge and dashed wedge are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist in the form of stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereoisomers, geometric isomers, rotamers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention can exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0110] The present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, which may be a single polymorph or a mixture of two or more polymorphs in any ratio.

[0111] It is also understood that some compounds of the present invention may exist in free form for use in therapy, or, where appropriate, as their pharma- ceutically acceptable derivatives. In the present invention, pharma-ceutically acceptable derivatives include, but are not limited to, pharma-ceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which can provide the compounds of the present invention or their metabolites or residues directly or indirectly after administration to a patient in need thereof. Thus, when referring to "compounds of the present invention" herein, it is also intended to include various derivative forms of the compounds described above.

[0112] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0113] Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, and the like.

[0114] Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, and the like.

[0115] For a general review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0116] The term "ester" refers to esters derived from the compounds of each general formula herein, including physiologically hydrolysable esters (which can hydrolyze under physiological conditions to release the compounds of the present invention in the form of the free acid or alcohol). The compounds of the present invention may themselves be esters.

[0117] The compounds of the present invention may exist in the form of solvates, preferably hydrates, including polar solvents, particularly water, methanol, ethanol, etc., as structural elements of the crystal lattice of the compound. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric ratios.

[0118] Also included within the scope of the invention are metabolic products of the compounds of the invention, i.e., substances formed in the body following administration of a compound of the invention. Such products may result, for example, from the oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic hydrolysis, and the like, of the administered compound. Thus, the invention includes metabolites of compounds of the invention, including compounds prepared by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolite thereof.

[0119] The present invention further includes within its scope the prodrugs of the compounds of the present invention. Typically, such prodrugs are functional derivatives of the compounds, which are easily converted in vivo into the desired therapeutically active compounds. Thus, in these examples, the term "administration" when used in the treatment methods of the present invention includes the treatment of various diseases or conditions with one or more prodrug forms of the compound to be protected, which are converted in vivo into the compounds described above after being administered to an individual. For example, "Design of Prodrug", edited by H. Bundgaard, Elsevier, 1985, describes the general methods for selecting and preparing suitable prodrug derivatives.

[0120] The present invention further includes within its scope isotopically labeled compounds of the compounds of the present invention which are identical to the compounds of the present invention except that one or more atoms have been replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number of the atom predominant in nature.

[0121] The present invention also encompasses the compounds of the present invention that contain protective groups. In any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups in any relevant molecule, thereby forming chemically protected forms of the compounds of the present invention. This can be achieved by using conventional protective groups, such as those described in Protective Groups in Organic Chemistry, edited by JFW MacOmie, Plenum Press, 1973; and TW Greene and PG M Huts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protective groups can be removed at a later appropriate stage by methods known in the art.

[0122] Pharmaceutical Compositions In a third aspect, the present invention provides a pharmaceutical composition comprising a compound according to the first or second aspect of the invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvent, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, and one or more pharma- ceutically acceptable carriers.

[0123] The term "pharmaceutical composition" refers to a composition that can be used as a medicine and contains an active pharmaceutical ingredient (API) (or therapeutic agent), and optionally one or more pharma- ceutically acceptable carriers. The term "pharmaceutical acceptable carrier" refers to an excipient with which a therapeutic agent is administered, and which is suitable, within the scope of sound medical judgment, for contact with human and / or other animal tissues without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0124] The pharmaceutical compositions can act systemically and / or locally, which can be achieved by suitable dosage forms, including but not limited to tablets, capsules, lozenges, hard lozenges, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, salves, aqueous suspensions, injections, elixirs, and syrups.

[0125] The pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one compound of the present invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

[0126] The present invention provides a method for preparing the aforementioned pharmaceutical composition or its corresponding dosage form, the method comprising combining at least one compound of the present invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof with one or more pharma- ceutically acceptable carriers.

[0127] kit In a fourth aspect, the present invention provides a kit, the kit comprising: a) at least one compound according to the first or second aspect of the invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof as a first therapeutic agent, or a pharmaceutical composition according to the third aspect as a first pharmaceutical composition; b) optionally, at least one additional therapeutic agent as a second therapeutic agent, or a pharmaceutical composition containing an additional therapeutic agent as a second pharmaceutical composition; and c) Optionally, providing packaging and / or instructions.

[0128] The aforementioned kit may contain 0.01 mg to 1000 mg of at least one of the compounds of the present invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

[0129] The present invention also provides a method for preparing the aforementioned kit, the method comprising combining at least one compound of the invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or a pharmaceutical composition as described above, optionally with at least one additional therapeutic agent, or a pharmaceutical composition containing the additional therapeutic agent, packaging, and / or instructions.

[0130] medical use The compounds of the present invention can exhibit potent effects in inhibiting abnormal cell proliferation.

[0131] Thus, the present application provides compounds of the present invention or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites and prodrugs thereof, or pharmaceutical compositions as described above, for use in treating diseases associated with abnormal cell proliferation.

[0132] Furthermore, the present application also provides the use of a compound of the present invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for treating a disease associated with abnormal cell proliferation.

[0133] In some embodiments, diseases involving abnormal cell proliferation include, but are not limited to, tumors, such as advanced solid tumors.

[0134] The present application also provides the use of the compounds of the present invention or their pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites and prodrugs, or pharmaceutical compositions of the present invention, in the manufacture of a preparation for inhibiting tumor cell proliferation. In some embodiments, the preparation is used for in vivo or in vitro administration. For example, the preparation can be administered to a subject to inhibit tumor cell proliferation in the subject; alternatively, the preparation can be administered to cells in vitro (e.g., cell lines or cells derived from a subject) to inhibit tumor cell proliferation in vitro.

[0135] Tumors described in the present invention include (but are not limited to): brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, squamous cell esophageal carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

[0136] Treatment method In another aspect, the present invention provides a method for treating a disease associated with abnormal cell proliferation, comprising the step of: administering to an individual in need thereof a therapeutically effective amount of a compound of the present invention or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition as described above.

[0137] The term "effective amount" refers to an amount sufficient to induce a biological or medical response in a cell, tissue, organ or organism (e.g., an individual) and to achieve a desired prophylactic and / or therapeutic effect.

[0138] Dosage regimen can be adjusted to provide the best desired response.For example, it can be administered in a single dose, in divided doses over time, or proportionally reduced or increased doses according to actual conditions.It goes without saying that for any particular individual, the specific dosage regimen should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the compound of the present invention.

[0139] The amount of the compound of the invention administered will depend on the individual circumstances, the severity of the disease or condition, the rate of administration, the nature of the compound, and the judgment of the prescribing physician. Generally speaking, an effective amount is about 0.001-10,000 mg per kg of subject body weight per day. In appropriate cases, an effective amount is about 0.01-1000 mg per kg of subject body weight per day. Administration can be performed daily, every other day, or every third day at a dose of about 0.01-1000 mg / kg of subject body weight, usually about 0.1-500 mg per kg of subject body weight. Exemplary dosage regimes are once or more daily, or once or more weekly, or once or more monthly. When multiple doses are administered, the interval between single doses can generally be daily, weekly, monthly, or yearly. Alternatively, it can be administered in the form of a sustained release formulation, in which case the frequency of administration needs to be reduced. The dosage and frequency of administration may vary based on the half-life of the drug in the subject and may vary based on whether the application is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered less frequently over a long period of time; in therapeutic applications, relatively high doses may need to be administered at shorter intervals until the progression of the disease is slowed or stopped, preferably until the individual shows partial or complete improvement in disease symptoms, after which prophylactic applications can be employed.

[0140] The term "treatment" refers to the alleviation or elimination of a targeted disease or condition. When a subject is administered a therapeutic amount of a compound of the present invention or a pharma- ceutically acceptable form thereof or a pharmaceutical composition of the present invention, and at least one indicator and symptom of the subject shows observable and / or detectable amelioration and / or improvement, the subject is shown to be successfully "treated". It goes without saying that treatment includes not only complete treatment, but also treatment that achieves some biologically or medically relevant results that are not complete.

[0141] The term "administer / administration" refers to the process of applying a pharmacologic active ingredient (e.g., a compound of the present invention) or a pharmaceutical composition containing a pharmacologic active ingredient (e.g., a pharmaceutical composition of the present invention) to an individual or its cells, tissues, organs, biological fluids, and other parts, such that the pharmacologic active ingredient or pharmaceutical composition comes into contact with the individual or its cells, tissues, organs, biological fluids, and other parts. Common methods of administration include (but are not limited to) oral, subcutaneous, intramuscular, subperitoneal, ocular, nasal, sublingual, rectal, intravaginal, etc.

[0142] The term "in need thereof" refers to the judgment of a physician or other caregiver that an individual needs or would benefit from prophylactic and / or therapeutic treatment, based on a variety of factors that the physician or other caregiver has in his or her area of ​​expertise.

[0143] The term "individual" (or subject) refers to a human or non-human animal. Individuals of the present invention include individuals (patients) suffering from a disease and / or disorder as well as normal individuals. Non-human animals of the present invention include all vertebrates, e.g., non-mammals such as birds, amphibians, reptiles, and mammals such as, e.g., non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0144] Preparation method A fourth aspect of the invention provides a method for synthesizing the compounds.

[0145] R 3 is hydrogen, the compound of formula (I) in the present invention can be prepared by the following synthesis route: [ka] It can be synthesized by R 1 , R 2 , R 3 , R 4 and A are as defined above, LG is a leaving group selected from the group consisting of methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine; Step 1 Compound (I)-SM1 and compound (I)-SM2 are subjected to Friedel-Crafts acylation to obtain compound (I)-IM1; In some embodiments, this step is carried out at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

[0146] Step 2 Compound (IV)-IM1 and compound (I)-SM3 are subjected to a ring-closing reaction under acidic conditions to obtain compound (I)-IM2; In some embodiments, this step is carried out at a suitable temperature, which is 20° C., 25° C., 40° C., 50° C., 60° C., 100° C., 120° C., preferably 120° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, preferably toluene and xylene; In some embodiments, this step is performed under acidic conditions; The acidic conditions are provided by an agent selected from the group consisting of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0147] Step 3: Compound (I)-IM2 undergoes a Derepin reaction to give compound (I)-IM3.

[0148] In some embodiments, this step is carried out at a suitable temperature, which may be 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 85°C, 100°C, preferably 85°C.

[0149] Step 4: Compound (I)-IM3 and compound (I)-SM4 were subjected to a substitution reaction to obtain compound (I)-IM4.

[0150] In some embodiments, this step is carried out at a suitable temperature, the temperature being 20° C., 25° C., 40° C., 50° C., 60° C., 100° C., 140° C., preferably 50° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (dioxane), dimethylsulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0151] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), and the inorganic base can be potassium phosphate (K 3 PO 4 ), sodium hydride (NaH), potassium carbonate (K 2 CO 3), Sodium Carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), Cesium carbonate (Cs 2 CO 3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 It could be; Step 5 A is [ka] In the case where R is a compound of formula (I)-IM4, the compound of formula (I)-IM4 is condensed with the compound of formula (I)-SM5-1 or the compound of formula (I)-SM5-2 to obtain the compound of formula (I), 5 , R 6 , n and ring B have the same meanings as above.

[0152] [ka]

[0153] In some embodiments, this step is carried out in the presence of a suitable condensing reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU and HATU; In some embodiments, this step is carried out at a suitable temperature, the temperature being 20° C., 25° C., 40° C., 50° C., 60° C., 100° C., preferably 25° C.; In some embodiments, this step is carried out in a suitable organic solvent, the organic solvent being selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0154] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, with DIPEA being preferred.

[0155] A is [ka] If R 4 and R 5 is hydrogen, and the compound of formula (I)-IM4 is reacted with trimethylsilyl cyanide to give the compound of formula (I).

[0156] [ka]

[0157] In some embodiments, this step is carried out at a suitable temperature, the temperature being 5° C., 20° C., 25° C., 40° C., 50° C., 60° C., 90° C., 100° C., preferably 90° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, n-methylpyrrolidone, dimethylsulfoxide and 1,4-dioxane, preferably 1,4-dioxane.

[0158] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, with TEA being preferred.

[0159] A, [ka] and R 5 and R 6 When is hydrogen, the compound of formula (I)-IM4 is reacted with chlorosulfonyl isocyanate, followed by removal of the amino protecting group to give the compound of formula (I).

[0160] [ka]

[0161] In some embodiments, this step is carried out at a suitable temperature, the temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 0-25°C; In some embodiments, this step is carried out in a suitable organic solvent, the organic solvent being selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0162] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO3 and NaOH, with TEA being preferred.

[0163] R 3 is connected to the adjacent benzene ring carbon atom to form a 6-membered carbocyclic ring, the compound of formula (I) in the present invention can be synthesized by the following synthesis route: [ka] It can be synthesized by R 1 , R 2 , R 3 , R 4 and A are as defined above, LG is a leaving group selected from the group consisting of methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine; Step 1: Compound (I)-SM6 and compound (I)-SM4 undergo a substitution reaction to give compound (I)-IM5.

[0164] In some embodiments, this step is carried out at a suitable temperature, the temperature being 20° C., 25° C., 40° C., 50° C., 60° C., 100° C., 140° C., preferably 50° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (dioxane), dimethylsulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0165] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), and the inorganic base can be potassium phosphate (K 3 PO 4 ), sodium hydride (NaH), potassium carbonate (K 2 CO 3 ), Sodium Carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), Cesium carbonate (Cs 2 CO 3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 It could be; Step 2 A [ka] In the case where R is a condensation reaction between the compound (I)-IM5 and the compound (I)-SM5-2, the compound (I)-IM5 is obtained. 5 , R 6 and the meaning of ring B is as described above.

[0166] [ka]

[0167] In some embodiments, this step is carried out under a suitable condensing reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC and HOBT, and may be preferably HBTU and HATU; In some embodiments, this step is carried out at a suitable temperature, the temperature being 20° C., 25° C., 40° C., 50° C., 60° C., 100° C., preferably 25° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, n-heptane, n-hexane, ethyl acetate, and preferably N,N-dimethylformamide.

[0168] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, with DIPEA being preferred.

[0169] A is [ka] If R 4 and R 5 is hydrogen, and the compound of formula (I)-IM5 is reacted with trimethylsilyl cyanide to give the compound of formula (I).

[0170] [ka]

[0171] In some embodiments, this step is carried out at a suitable temperature, the temperature being 5° C., 20° C., 25° C., 40° C., 50° C., 60° C., 90° C., 100° C., preferably 90° C.; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide and 1,4-dioxane, preferably 1,4-dioxane.

[0172] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, with TEA being preferred.

[0173] A, [ka] and R 5 and R 6 When is hydrogen, the compound of formula (I)-IM5 is reacted with chlorosulfonyl isocyanate, followed by removal of the amino protecting group to give the compound of formula (I).

[0174] [ka]

[0175] In some embodiments, this step is carried out at a suitable temperature, the temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 0-25°C; In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, n-heptane, n-hexane, ethyl acetate, and preferably N,N-dimethylformamide.

[0176] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from the group consisting of DIPEA, TEA, t-BuOK, and Py, and the inorganic base can be K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, with TEA being preferred.

[0177] Beneficial Effects of the Invention The present invention provides camptothecin compounds represented by formula (I) to formula (IV), their pharmaceutical compositions, preparation methods and uses. These compounds have good antitumor activity, may overcome drug resistance, and can be used to treat abnormal cell proliferation disorders, including but not limited to advanced solid tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0178] Detailed Model for Carrying Out the Invention The present application will be further described below through the description of detailed embodiments, which are not intended to limit the present application. Those skilled in the art can make various modifications or improvements based on the teachings of the present application without departing from the basic idea and scope of the present application.

[0179] The abbreviations in the present invention have the following meanings: [Table 1]

[0180] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 The molecular weight was determined by 1 H NMR or mass spectrometry (MS).

[0181] Nuclear magnetic resonance used (1 The H NMR measurement equipment was a Bruker 400 MHz nuclear magnetic resonance spectrometer; 6 ); the internal standard was tetramethylsilane (TMS).

[0182] The abbreviations for nuclear magnetic resonance (NMR) spectra used in the present examples are as follows: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d 6 : deuterated dimethyl sulfoxide. δ values ​​are expressed as ppm values.

[0183] The mass spectrometry (MS) measuring device used was an Agilent (ESI) mass spectrometer, the model of which was Agilent 6120B. EXAMPLES

[0184] Example 1: Preparation of (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (Compound 1-1)

[0185] [ka]

[0186] Step 1: Preparation of 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethan-1-one To an ice-water cooled solution of boron trichloride (1M, 36mL) in 1,2-dichloroethane (40mL), 4-chloro-3-fluoroaniline (4.36g, 29.95mmol) in 1,2-dichloroethane (20mL) solution was added dropwise, then 2-chloroacetonitrile (2.71g, 35.94mmol) and titanium tetrachloride (6.82g, 35.94mmol) were added successively at room temperature, and the temperature was raised to reflux and the reaction was carried out for 16 hours. After the reaction solution was cooled to room temperature, 2.5N hydrochloric acid (55.0mL) was added and the reaction was carried out again by heating to 85°C for 30 minutes. The reaction solution was cooled to room temperature and extracted with dichloromethane (3x25mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was filtered and concentrated to give the title compound (2g, 7.21mmol), which was used directly in the next reaction. ESI-MS(m / z):222[M+H] + .

[0187] Step 2: Preparation of (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethan-1-one (1-1-2, 0.5 g, 1.80 mmol) was added to toluene (10 mL), then (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (IM-1, 500.00 mg, 1.90 mmol) and p-toluenesulfonic acid monohydrate (68.53 mg, 360.29 μmol) were added successively, and the reaction solution was heated to 120° C. and reacted for 4 hours. After concentration, the reaction solution was slurried with ethyl acetate and the solid was collected by filtration to obtain the title compound (0.6 g, 1.34 mmol), which was used directly in the next reaction. ESI-MS(m / z):449[M+H] + .

[0188] Step 3: Preparation of (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1-3, 0.1 g, 222.59 μmol) was dissolved in ethanol (5 mL) and urotropine (94 mg, 670.54 μmol) was added. The reaction solution was heated to 85° C. and reacted for 5 hours. After the reaction solution was cooled to room temperature, 0.2 mL of concentrated hydrochloric acid was added. After stirring for 10 minutes, it was directly concentrated. The crude product was slurried with ethyl acetate. The solid was collected by filtration and dried to give the hydrochloride salt of the title compound (180 mg, 386.03 μmol). The hydrochloride salt of 1-1 (20 mg, 46.62 μmol) was added to a mixed solvent of DMF (1 mL) and water (1 mL), adjusted to pH=8 with sodium bicarbonate, and purified by preparative high performance liquid chromatography (purification conditions were as follows) to obtain (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1, 3 mg).

[0189] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0190] [Table 2]

[0191] The structural characterization data was as follows: 1 H NMR (400MHz, DMSO-d 6)δ8.62(d,J=8.1Hz,1H),8.16(d,J=10.3Hz,1H),7.33(s,1H),6.56(s,1H),5 .45(d,J=8.6Hz,4H),4.35(s,2H),1.97-1.74(m,2H),0.88(t,J=7.3Hz,3H). ESI-MS(m / z):430[M+H] + .

[0192] Example 2: Preparation of (S)-N-((9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxyacetamide (Compound 2-1)

[0193] [ka]

[0194] (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1, 60 mg, 128.68 μmol, hydrochloride salt) was dissolved in DMF (1 mL), glycolic acid (20 mg, 262.98 μmol), triethylamine (26.04 mg, 257.35 μmol) and DMTMM (71.21 mg, 257.35 μmol) were added successively, and the reaction system was reacted at 20° C. for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography to obtain the title compound (4 mg, 8.04 μmol).

[0195] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0196] [Table 3]

[0197] The structural characterization data was as follows: 1 H NMR (400MHz, DMSO-d 6 )δ8.87(d,J=4.1Hz,1H),8.85(s,1H),8.19(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.55(s,2H) ,5.45(s,2H),4.82(d,J=6.0Hz,2H),3.82(s,2H),1.87(dd,J=9.7,7.5Hz,2H),0.87(t,J=7.3Hz,3H). ESI-MS (m / z): 488[M+H] + .

[0198] Example 3: Preparation of (1S,9S)-1-sulfamoylamido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (Compound 7-1)

[0199] [ka]

[0200] Step 1: Preparation of tert-butyl (N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)carbamate tert-Butanol (20.92mg, 0.282mmol) was dissolved in DCM (0.5mL), cooled to 0℃ and stirred, chlorosulfonyl isocyanate (39.94mg, 0.282mol, 24.56μL) was added dropwise, maintained at that temperature, and reacted for 0.5h for further use. Exatecan mesylate (100mg, 0.188mol) was dissolved in DMF (2mL), TEA (57.11mg, 0.564mmol, 78.45μL) was added dropwise, cooled to 0℃ and stirred, then the above DCM reaction solution was added dropwise to the reaction system, allowed to warm to room temperature naturally, and reacted for 1h. The reaction was monitored by LCMS, and the product was evident. The reaction solution was used directly in the next reaction. ESI-MS(m / z):615.1[M+1] + .

[0201] Step 2: Preparation of (1S,9S)-1-sulfamoylamido-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione The tert-butyl (N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)sulfamoyl)carbamate reaction solution was diluted by adding dichloromethane (1 mL), and then trifluoroacetic acid (0.5 mL) was added dropwise, stirred and reacted for 1 hour. The product was monitored by LCMS. After concentration under reduced pressure, 52.26 mg of the title compound was obtained.

[0202] ESI-MS(m / z):515.2[M+1] + . 1H NMR (400MHz, DMSO): δ7.78(d,J=10.9Hz,1H),7.30(d,J=9.0Hz,2H),6.98(s,2H),6.53(s,1H),5.55-5.36(m,4H),4.92(d,J=4.2H) z,1H),3.27(dd,J=19.1,6.9Hz,1H),3.14-3.04(m,1H),2.38(s,3H),2.36-2.22(m,2H),1.93-1.79(m,2H),0.87(t,J=7.3Hz,3H).

[0203] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0204] [Table 4]

[0205] Example 4: Preparation of 1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)urea (Compound 7-2)

[0206] [ka]

[0207] Exatecan mesylate (50 mg, 0.082 mmol) was dissolved in 1,4-dioxane (1 mL), triethylamine (16.65 mg, 0.165 mmol, 22.87 μL) was added dropwise, trimethylsilyl cyanide (6.53 mg, 0.165 mmol, 8.24 μL) was added dropwise with stirring, and the mixture was heated to 90° C. for 2 h. The reaction was monitored by LCMS until the disappearance of the starting material. The reaction solution was cooled to room temperature, MeOH (1 mL) was added dropwise, stirred for 10 min, concentrated under reduced pressure, and purified to give the title compound 12.92 mg.

[0208] ESI-MS(m / z):479.1[M+1] + . 1 H NMR (400MHz, DMSO): δ7.78(d,J=10.9Hz,1H),7.30(s,1H),6.74(d,J=9.1Hz,1H),6.53(s,1H),5.69(s,2H),5.42(s,2H), 5.29(q,J=19.2Hz,3H),3.15(s,2H),2.39(s,3H),2.20-2.07(m,2H),1.86(dd,J=10.4,7.4Hz,2H),0.87(t,J=7.3Hz,3H).

[0209] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0210] [Table 5]

[0211] Example 5: Preparation of N-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-cyclopropyl-2-hydroxyacetamide (compound 2-4)

[0212] [ka]

[0213] (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1, 30 mg, 69.8 μmol) was dissolved in DMF (1 mL), and 2-cyclopropyl-2-hydroxyacetic acid (16.21 mg, 139.6 μmol), DIPEA (22.55 mg, 174.5 μmol) and HATU (31.83 mg, 83.75 μmol) were added successively. The reaction system was reacted at 20° C. for 1 hour. The reaction solution was directly purified by high-performance chromatography to obtain 11 mg of the title compound.

[0214] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0215] [Table 6]

[0216] The structural characterization data was as follows: 1 H NMR (400MHz, DMSO-d 6 )δ8.81(d,J=8Hz,1H),8.75(m,1H),8.19(d,J=8Hz,1H),7.34(s,1H),6.55(s,2H),5.53(s,2H),5.45(s, 2H),4.82(m,2H),3.57(m,1H),1.86(m,2H),0.97(m,1H),0.87(t,J=8Hz,3H),0.32(m,2H),0.23(m,2H). ESI-MS(m / z):529[M+H] + .

[0217] Example 6: Preparation of N-(((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-2-hydroxy-3-enamine (Compound 2-22)

[0218] [ka]

[0219] (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1, 30 mg, 73.28 μmol) was dissolved in DMF (1 mL), 2-hydroxy-3-enoic acid (7.48 mg, 73.28 μmol), DIPEA (23.68 mg, 183.19 μmol) and HATU (31.42 mg, 87.93 μmol) were added successively, and the reaction system was reacted at 20 ° C. for 1 hour, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 5.36 mg of the title compound.

[0220] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0221] [Table 7]

[0222] The structural characterization data was as follows: 1 H NMR (400MHz, DMSO-d 6)δ8.58(m,1H),7.97(m,1H),7.63(m,1H),5.59(m,1H),5.51(s,1H),5.40(m,2H),5.18( d,J=12Hz,1H),4.95(s,2H),4.61(s,2H),4.58(m,1H),1.95(m,2H),0.98(t,J=8Hz,3H). ESI-MS(m / z):514[M+H] + .

[0223] Example 7: Preparation of N-(((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-3,3,3-trifluoro-2-hydroxypropionamide (Compound 2-19)

[0224] [ka]

[0225] (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (1-1, 22 mg, 51.18 μmol) was dissolved in DMF (1 mL), 3,3,3-trifluoro-2-hydroxypropionic acid (8.85 mg, 61.42 μmol), DIPEA (19.84 mg, 153.55 μmol) and HATU (23.34 mg, 61.42 μmol) were added successively, and the reaction system was reacted at 20 ° C. for 1 hour, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 2 mg of the title compound.

[0226] Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0227] [Table 8]

[0228] The structural characterization data was as follows: 1 H NMR (400MHz, DMSO-d 6 )δ8.78(d,J=8Hz,1H),8.19(d,J=8Hz 1H),7.34(s,1H),6.55(s,1H),5.91(m,1H),5.85(m,1H),5.54(s,2H),5 .45(s,2H),4.86(m,2H),4.58(m,1H),1.86(m,2H),0.87(t,J=8Hz,3H). ESI-MS(m / z):556[M+H] + .

[0229] Example 8: Preparation of (2R,3R,4R,5S,6R)-6-(4-((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolo[1,2-b]quinolin-11-yl)methyl)carbamoyl)oxy)methyl)-2-(2-(2-(N-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethylamino)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (A-11)

[0230] [ka]

[0231] Step 1: Preparation of (2R,3S,4R,5R,6R)-2-(2-(8-(9-fluorenylmethoxycarbonylamino)-3,6-dioxa-octanamide)-4-(((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2b]quinolin-11-yl)methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triacetate (A-11-2) Compound 1-1 (30 mg, 69.80 μmol) and compound A-11-1 (75.85 mg, 76.77 μmol, prepared with reference to page 81 of WO 2022253035) were dissolved in N,N-dimethylformamide (1 mL), and then HOBT (11.32 mg, 83.75 μmol) and N,N-diisopropylethylamine (22.55 mg, 174.49 μmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was spin-dried to obtain crude product A-11-2, which was used directly in the next reaction.

[0232] Step 2: Preparation of (2R,3R,4R,5S,6R)-6-(2-(2-(2-(2-aminoethyl)ethoxy)acetamido)-4-(((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11yl)methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A-11-3) A-11-2 was dissolved in MeOH (1 mL) and water (0.5 mL) and added with LiOH H 2 O (26.28 mg, 625.67 μmol) was added and reacted at room temperature for 4 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to obtain the title compound A-11-3 (20 mg).

[0233] The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0234] [Table 9]

[0235] Step 3: Preparation of (2R,3R,4R,5S,6R)-6-(4-(((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolo[1,2-b]quinolin-11-yl)methyl)carbamoyl)oxy)methyl)-2-(2-(2-(N-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethylamino)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (A-11) A-11-3 (20 mg, 21.83 μmol), IM-1 (7.98 mg, 21.83 μmol) and N,N-diisopropylethylamine (8.46 mg, 65.49 μmol) were dissolved in DMF (1 mL), and the reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to obtain 3.42 mg of the title compound.

[0236] Its structural characterization data is as follows: MS m / z(ESI): 1168.2 [M+H] + . The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0237] [Table 10]

[0238] Example 9: Preparation of N-((S)-12-benzyl-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13-tetraazapentadecan-15-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexa-5-ynamide (B-1)

[0239] [ka]

[0240] Compound 1-1 (30 mg, 69.80 μmol) and compound IM-2 (51.72 mg, 76.77 μmol, prepared with reference to page 92 of International Publication No. 2022253035) were dissolved in N,N-dimethylformamide (1 mL), and then HATU (29.17 mg, 76.77 μmol) and N,N-diisopropylethylamine (22.55 mg, 174.49 μmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to obtain 10 mg of the title compound.

[0241] Its structural characterization data is as follows: MS m / z(ESI): 1087.3[M+H] + .

[0242] The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm * 19mm * 150mm) Mobile phase A: Acetonitrile; Mobile phase B: water (0.05% formic acid)

[0243]

Table 11

[0244] Example 10: Preparation of N-((9S,12S,15S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-9,12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazapentadecane-15-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide (B-2)

[0245]

Chemical formula

[0246] Step 1: Preparation of (9H-fluoren-9-yl)methyl ((9S,12S,15S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-9,12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazapentadecane-15-yl)carbamate (B-2-1) Compound 1-1 (50 mg, 116.33 μmol) and Compound IM-3 (66.03 mg, 122.14 μmol) were dissolved in N,N-dimethylformamide (1 mL), then HATU (48.62 mg, 127.96 μmol) and N,N-diisopropylethylamine (37.58 mg, 290.81 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by high performance liquid chromatography and freeze-dried to obtain 100 mg of the title compound.

[0247] The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0248] [Table 12]

[0249] Step 2: Preparation of (S)-2-amino-N-((9S,12S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-9-methyl-3,8,11-trioxo-5-oxa-2,7,10-triazatridecan-12-yl)propionamide (B-2-2) Compound B-2-1 (100 mg, 105.00 μmol) was dissolved in N,N-dimethylformamide (1 mL), diethylamine (0.5 mL) was added, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product, which was used directly in the next step reaction.

[0250] Step 3: Preparation of N-((9S,12S,15S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-9,12-dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecan-15-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexa-5-ynamide (B-2) Compound IM-1 (17.66 mg, 48.39 μmol) and N,N-diisopropylethylamine (12.48 mg, 0.226 mmol) were dissolved in N,N-dimethylformamide (1 mL) containing the crude product of B-2-2, and the reaction system was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography and then lyophilized to obtain 5 mg of the title compound.

[0251] Its structural characterization data is as follows: MS m / z(ESI):981.7[M+H] + .

[0252] The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0253] [Table 13]

[0254] Example 11: Preparation of (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutyamide (2-25)

[0255] [ka]

[0256] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (prepared with reference to WO2022166762A1) (30 mg, 0.053 mmol), (S)-3- Hydroxybutyric acid (16.5 mg, 0.158 mmol), HATU (40.0 mg, 0.105 mmol) and DIPEA (20.4 mg, 0.158 mmol) were added to the reaction system in DMF (0.5 mL), and the reaction was carried out by stirring at 25°C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography, and then freeze-dried to obtain the title compound (13.5 mg, 0.024 mmol).

[0257] The isolation and purification method is as follows: Chromatography column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0258] [Table 14]

[0259] Its structural characterization data is as follows: ESI-MS(m / z):542.1[M+H] + 1 H NMR (400MHz, DMSO-d 6)δ8.44(d,J=8.8Hz,1H),8.09(d,J=10.4Hz,1H),7.34(s,1H),6.56(m,1 H),5.64-5.60(m,1H),5.44(s,2H),5.32-5.25(m,2H),4.63(d,J=4.8Hz, 1H),4.06-4.01(m,1H),3.31-3.26(m,2H),2.28-2.23(m,1H),2.20-2.13 (m,3H),1.90-1.82(m,2H),1.08(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[0260] Example 12: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (2-26)

[0261] [ka]

[0262] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (prepared with reference to WO2022166762A1) (30 mg, 52.64 μmol), (R)- 3-Hydroxybutyric acid (10.96 mg, 105.28 μmol), HATU (30.02 mg, 78.96 μmol) and DIPEA (20.41 mg, 157.93 μmol) were added to the reaction system in DMF (2 mL), the reaction was stirred at 25 °C for 2 h, and the reaction solution was directly purified by high performance liquid chromatography and lyophilized to give the title compound (17 mg, 31.42 μmol).

[0263] The isolation and purification method is as follows: Column: Waters XBridge Prep C18OBD (5μm * 19mm * 150mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0264] [Table 15]

[0265] Its structural characterization data is as follows: ESI-MS(m / z):542.1[M+H] + 1 H NMR(400MHz,DMSO)δ8.43(d,J=8.8Hz,1H),8.06(d,J=10.4Hz,1H),7.33(s,1H),6.5 4(s,1H),5.63-5.55(m,1H),5.43(s,2H),5.27(d,J=18.8Hz,1H),5.21(d,J=18.8Hz, 1H),4.66(d,J=4.8Hz,1H),4.09-3.98(m,1H),3.32-3.26(m,2H),2.32-2.25(m,1H) ,2.24-2.12(m,3H),1.93-1.78(m,2H),1.08(d,J=6.0Hz,3H),0.87(t,J=7.2Hz,3H).

[0266] Preparation of antibody-drug conjugates Preparation of Trastuzumab-A-11(DAR8) 1.781 mL of trastuzumab antibody (16 mg / mL) was taken and diluted with 89 μL of 20 mM phosphate buffer + 0.1 M EDTA (pH 7.60), and then diluted with 1 M Na 2 HPO 4The pH was adjusted to 7.60 with a solution, and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 108 μL, pH 7.60) solution was added, mixed thoroughly, and left at room temperature for 1.5 hours. Then, 10 times the amount of material (218 μL, 10 mM) A-11 dissolved in dimethyl sulfoxide solution was slowly added, mixed thoroughly, and left at room temperature for 2 hours. After that, the buffer was replaced with 20 mM histidine buffer (pH 6.0) using a NAP-5 gel column (Cytiva), thereby obtaining the antibody-drug conjugate trastuzumab-A-11. The DAR value was 8.0 as measured by mass spectrometry.

[0267] Preparation of Trastuzumab-B-1(DAR8) 1.973 mL of trastuzumab antibody (15.2 mg / mL) was taken and diluted with 98.7 μL of 20 mM phosphate buffer + 0.1 M EDTA (pH 7.60), and then diluted with 1 M Na 2 HPO 4 The pH was adjusted to 7.60 with a solution, and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.92 μL, pH 7.60) solution was added, mixed thoroughly, and left at room temperature for 1.5 hours. Then, 12 times the amount of material (258.16 μL, 10 mM) of B-1 dissolved in dimethyl sulfoxide solution was slowly added, mixed thoroughly, and left at room temperature for 2 hours. After that, the buffer was replaced with 20 mM histidine buffer (pH 6.0) using a NAP-5 gel column (Cytiva), thereby obtaining the antibody-drug conjugate trastuzumab-B-1. Its DAR value was 7.96 as determined by mass spectrometry.

[0268] Preparation of Trastuzumab-B-2 (DAR8) 2.16 mL of trastuzumab antibody (16.2 mg / mL) was taken and diluted with 108 μL of 20 mM phosphate buffer + 0.1 M EDTA (pH 7.60), and then diluted with 1 M Na 2 HPO 4The pH was adjusted to 7.60 with a solution, 10 mM TCEP (tris(2-carboxyethyl)phosphine, 88.42 μL, pH 7.60) solution was added, mixed thoroughly, and left at room temperature for 1.5 hours. Then, 20 times the amount of material (502.38 μL, 10 mM) of B-2 dissolved in dimethyl sulfoxide solution was slowly added, mixed thoroughly, and left at room temperature for 2 hours. After that, the buffer was replaced with 20 mM histidine buffer (pH 6.0) using a NAP-5 gel column (Cytiva), thereby obtaining the antibody-drug conjugate trastuzumab-B-2. Its DAR value was 7.95 as determined by mass spectrometry.

[0269] Biological evaluation I. Tumor cell proliferation inhibition test 1. Inhibitory effect of compounds on HT29 cell proliferation (1) Cell plating: First, tumor cells HT29 were cultured in the corresponding medium, the cells were digested with trypsin and centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The sources of tumor cells were shown in Table 1.

[0270] [Table 16]

[0271] Co-culture of the compound of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell adhesion, 10 μL of the bioactive molecule (compound of the present invention) diluted in medium was added to each well of the plate and cultured for 72 hours.

[0272] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate=1-(sample RLU-background RLU) / (cell control RLU-background RLU)×100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 2.

[0273] (2) Data results [Table 17]

[0274] The test results show that the compounds of the present invention in Table 2 have strong inhibitory effects on the proliferation of HT29 colon cancer cells.

[0275] 2. Inhibitory effect of compounds on HCC1806 cell proliferation (1) Cell plating: First, tumor cells HCC1806 were cultured in the corresponding medium, the cells were digested with trypsin, centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The sources of tumor cells were shown in Table 3.

[0276] [Table 18]

[0277] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0278] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 4.

[0279] (2) Data results [Table 19]

[0280] The test results show that the compounds of the present invention in Table 4 have significant inhibitory effects on the proliferation of HCC1806 human breast squamous cell carcinoma cells.

[0281] 3. Inhibitory effect of compounds on NCI-H358 cell proliferation (1) Cell plating: First, tumor cells NCI-H358 were cultured in the corresponding medium, the cells were digested with trypsin, centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 5.

[0282] [Table 20]

[0283] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0284] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 6.

[0285] (2) Data results [Table 21]

[0286] The test results show that the compounds of the present invention in Table 6 have significant inhibitory effects on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[0287] 4. Inhibitory effect of compounds on NCI-N87 cell proliferation (1) Cell plating: First, tumor cells NCI-N87 were cultured in the corresponding medium, the cells were digested with trypsin, centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 7.

[0288] [Table 22]

[0289] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0290] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 8.

[0291] (2) Data results [Table 23]

[0292] The test results show that the compounds of the present invention in Table 8 have significant inhibitory effects on the proliferation of NCI-N87 human gastric cancer cells.

[0293] 5. Inhibitory effect of compounds on MDA-MB-231 cell proliferation (1) Cell plating: First, tumor cells MDA-MB-231 were cultured in the corresponding medium, the cells were digested with trypsin, centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 9.

[0294] [Table 24]

[0295] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0296] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 10.

[0297] (2) Data results [Table 25]

[0298] The test results show that the compounds of the present invention in Table 10 have significant inhibitory effects on the proliferation of MDA-MB-231 human breast cancer cells.

[0299] 6. Inhibitory effect of compounds on Jeko-1 cell proliferation (1) Cell plating: First, the tumor cells Jeko-1 were cultured in the corresponding medium, the cells were resuspended and counted, and finally, the cells were adjusted to the appropriate concentration for plating. The sources of the tumor cells are shown in Table 11.

[0300] [Table 26]

[0301] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0302] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 12.

[0303] (2) Data results [Table 27] The test results show that the compounds of the present invention in Table 12 have significant inhibitory effects on the proliferation of Jeko-1 human mantle cell lymphoma cells.

[0304] 7. Inhibitory effect of compounds on MDA-MB-453 cell proliferation (1) Cell plating: First, tumor cells MDA-MB-453 were cultured in the corresponding medium, the cells were digested with trypsin, centrifuged, then the cells were resuspended and counted, and the cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 13.

[0305] [Table 28]

[0306] Co-culture of the compounds of the present invention and tumor cells: 90 μL of cell suspension was added to each well of a 96-well plate. After cell attachment, 10 μL of bioactive molecules (compounds of the present invention) diluted in culture medium was added to each well of the plate and incubated for 72 hours.

[0307] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Light™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Culture wells without cells were used as background RLU, and culture wells with cells but no compound were used as cell control RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (cell control RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. The results of these studies are shown in Table 14.

[0308] (2) Data results [Table 29]

[0309] The test results show that the compounds of the present invention in Table 14 have significant inhibitory effects on the proliferation of MDA-MB-453 human breast cancer cells.

[0310] 8. Inhibitory effect of compounds on HCC827 cell proliferation (1) Cell plating: First, tumor cells HCC827 were cultured in corresponding medium, cells were digested with trypsin, centrifuged, then cells were resuspended and counted, and cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 15.

[0311] [Table 30]

[0312] Co-culture of the compound of the present invention and tumor cells: After cell attachment, the medium in the cells was removed, and the diluted bioactive molecule (compound of the present invention) was added to the wells of the plate and incubated for 72 hours.

[0313] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Cell Counting-Lite™ without cells was used to obtain background RLU, and Cell Counting-Lite™ with cells was used to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (solvent RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. RLU: Relative Luminescence Units. The results of these studies are shown in Table 16.

[0314] (2) Data results [Table 31]

[0315] The test results show that the compounds of the present invention in Table 16 have an inhibitory effect on the proliferation of HCC827 human non-small cell lung cancer cells.

[0316] 9. Inhibitory effect of compounds on HCC1954 cell proliferation (1) Cell plating: First, tumor cells HCC1954 were cultured in corresponding medium, cells were digested with trypsin, centrifuged, then cells were resuspended and counted, and cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 17.

[0317] [Table 32]

[0318] Co-culture of the compound of the present invention and tumor cells: After cell attachment, the medium in the cells was removed, and the diluted bioactive molecule (compound of the present invention) was added to the wells of the plate and incubated for 72 hours.

[0319] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Cell Counting-Lite™ without cells was used to obtain background RLU, and Cell Counting-Lite™ with cells was used to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (solvent RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50 ) was calculated by fitting the curve according to a four-parameter model. RLU: Relative Luminescence Units. The results of these studies are shown in Table 18.

[0320] (2) Data results [Table 33]

[0321] The test results show that the compounds of the present invention in Table 18 have an inhibitory effect on the proliferation of HCC1954 human breast cancer cells.

[0322] 10.Inhibitory effect of compounds on NCI-H1975 cell proliferation (1) Cell plating: First, tumor cells NCI-H1975 were cultured in corresponding medium, cells were digested with trypsin, centrifuged, then cells were resuspended and counted, and cells were adjusted to the appropriate concentration for plating. The source of tumor cells was shown in Table 19.

[0323] [Table 34]

[0324] Co-culture of the compound of the present invention and tumor cells: After cell attachment, the medium in the cells was removed, and the diluted bioactive molecule (compound of the present invention) was added to the wells of the plate and incubated for 72 hours.

[0325] In vitro cell viability detection: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme) was added to each well, shaken well in the dark, mixed thoroughly, and reacted for 10 minutes before examination. A microplate reader (manufacturer: BMG, model: PHERA Star-FS) was used for reading. Cell Counting-Lite™ without cells was used to obtain background RLU, and Cell Counting-Lite™ with cells was used to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (solvent RLU-background RLU) x 100%. The half inhibitory concentration of compound (IC 50) was calculated by fitting the curve according to a four-parameter model. RLU: Relative Luminescence Units. The results of these studies are shown in Table 20.

[0326] (2) Data results [Table 35]

[0327] The test results show that the compounds of the present invention in Table 20 have an inhibitory effect on the proliferation of NCI-H1975 human non-small cell lung cancer cells.

[0328] II. Permeability test The MDCK cell model is used to evaluate the permeability of compounds, and the LC-MS / MS method is used to determine the concentration of the substance to be tested and the apparent permeability coefficient (P app ) and the outflow ratio was calculated [outflow ratio = P app (B→A) / P app (A→B)].

[0329] 1. Preparation of Reagents Complete medium: MEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Digestion solution: 0.05% trypsin-EDTA. HBSS buffer: Ca 2+ , Mg 2+ HBSS containing Permeabilization solution: HBSS containing 10 mM Hepes, 4% BSA, pH 7.4. Compound stock solution: A certain amount of the compound to be tested was weighed out and mixed with DMSO to prepare a 10 mM stock solution.

[0330] 2.Cell culture MDCK cells (generation 26) in logarithmic growth phase were digested with 0.05% trypsin-EDTA, uniformly dispersed, and then inoculated into a transwell chamber at 3 × 10 5 cells / cm 2Then, 200 μL was inoculated into side A, and 1000 μL into side B. After inoculation, the medium was changed once a day and cultured for 3 to 4 days.

[0331] 3. Permeability test The cell medium was discarded by pipetting and then washed three times with permeate. 200 μL of permeate was added to side A and 1000 μL of permeate was added to side B. A voltage-ohm meter was used to measure the cell transmembrane resistance (TEER). After measuring the TEER value of the cells, the permeate on both sides was discarded by pipetting and solution was added. After 120 min, 150 μL samples were taken from side A and side B, respectively, and frozen at -80°C before testing. Then, 10 μg / ml of yellow fluorescence was added to side A. After 30 min of incubation, 100 μL of the solution from side B was pipetted and added to a 96-well white plate. The fluorescence value was measured using a microplate reader at wavelengths of Ex=485 nm and Em=530 nm, and the penetration was calculated (which was less than 1%).

[0332] 4. Sample Processing 80 μL of the compound to be tested was taken and added to 320 μL of acetonitrile solution containing the internal standard (tolbutamide 30 ng / mL), vortexed, centrifuged (4000 rpm, 4° C.) for 10 min, and the supernatant was taken for LC-MS / MS analysis.

[0333] 5. Data Processing Using Excel 2013, app Value and P of B side → A side vs. A side → B side app Ratios were calculated and the percent recovery was calculated for each control compound and compound being tested.

[0334] (1) Calculation of apparent permeability The apparent permeability coefficient of this compound (P app ) was calculated according to the following formula: P app =(dC r / dt) × V r / (A×C 0 ) C 0is the initial concentration of the drug to be tested in the donor (μM), and (dC r / dt) × V r is the rate of appearance of the drug to be tested at the receiver (μM / s), and V r is the volume of solution in the receiver (mL) and A is the surface area of ​​the polycarbonate membrane (cm 2 ) was. Evaluation criteria → Low permeability: P app ≦0.5(×10 -6 cm / s) Medium transparency: 0.5 <P app <2.5(×10 -6 cm / s) High permeability:P app ≧2.5(×10 -6 cm / s)

[0335] (2) Transporter The discharge ratio (ER) is P app Calculated according to: ER=P app (B→A) / P app (A → B) In the formula, P app (B→A) is the apparent permeability coefficient of the drug to be tested from the BL (basolateral) terminal to the AP (apical) terminal, P app (A→B) was the apparent permeability coefficient of the drug to be tested from the AP terminus to the BL terminus.

[0336] Evaluation criteria Possible efflux transporter substrate (high certainty): ERa ≥ 2 and ERa / ERi > 2 Likely (probably) a substrate for efflux transporters: ERa ≥ 2 Low or no possibility of being an efflux transporter substrate (poor or absent): ERa<2 where ERi and ERa were the efflux ratios of the compound to be tested in the presence and absence of a P-gp inhibitor, respectively.

[0337] The recovery rate was calculated according to the following formula: Recovery rate (%) = 100 × [(V r×C r )+(V d ×C d )] / (V d ×C 0 ) In the formula, V d and V r are the solution volumes on the donor and receiver sides, respectively, and C d and C r were the compound concentrations in the donor and receiver sides, respectively.

[0338] 6.Results The detailed data was as follows: [Table 36]

[0339] P app According to the results (A→B), control compound 2, control compound 3, control compound 4, and compound 2-19 are all low permeability compounds, especially control compound 2 has a relatively low permeability; control compound 1, compound 2-1, compound 2-22, and compound 2-25 are intermediate permeability compounds; compound 1-1 is a high permeability compound; the efflux ratio data showed that control compound 1, control compound 2, control compound 3, control compound 4, compound 2-22, and compound 2-25 may be efflux transporter substrates, especially control compound 2, control compound 3, and control compound 4 have relatively high efflux ratios; and compound 1-1, compound 2-1, and compound 2-19 are not efflux transporter substrates or are weak efflux transporter substrates.

[0340] III. Liver microsome stability test Liver microsomes were used as an in vitro model to assess the metabolic stability of compounds in human and monkey liver microsomes.

[0341] 1. Materials and Methods 1.1 Main test materials Control compounds and compounds of the invention: Testosterone: Dr. Ehrenstorfer, Germany Mixed human liver microsomes: Xenotech, USA Mixed male cynomolgus monkey liver microsomes: RILD, China

[0342] 1.2 Liver microsome incubation system The positive compound testosterone or test substance (liver microsome solution, 50 μL) was mixed with PBS (25 μL). After 5 min of pre-incubation (37°C), NADPH (25 μL) was added to make the final concentration of the positive compound or test substance 1 μM, the final concentration of human liver microsome protein was 0.5 mg / ml, and the final concentration of monkey liver microsome protein was 1 mg / ml. The test substance group and the positive compound group were incubated for 0 min and 15 min. After reacting for the corresponding time, the reaction was terminated by adding 300 μL of ice-cold acetonitrile containing the internal standard, vortexed, and stored at -80°C for later analysis. All incubated samples were duplicate samples.

[0343] 1.3 Sample preparation The test compounds were vortexed for 1 min and centrifuged (4° C., 4000 rpm) for 10 min, and 300 μL of the supernatant was taken for LC-MS / MS analysis.

[0344] 2. Data Processing The percentage of drug remaining in the primary form in the incubation system was calculated using Excel software: Primary form survival rate (%)=100×(A インキュベートしたサンプル / A 0h ) Note: A インキュベートしたサンプル represents the peak area ratio of the compound to the internal standard after incubation for the corresponding time, and A 0h represents the peak area ratio of compound to internal standard when unreacted.

[0345] 3. Results and Conclusions The percent remaining primary form of the compound after 15 minutes of incubation in human and monkey liver microsomes is shown in Table 22.

[0346] [Table 37]

[0347] The data in Table 22 showed that, compared with the control compound 4, compounds 2-25 had clearly superior stability in liver microsomes.

[0348] Structure of the control compound: [ka]

[0349] Although the detailed embodiments of the present invention have been described in detail, it goes without saying that those skilled in the art can make various modifications and substitutions to those details based on all the teachings disclosed, and these modifications are within the scope of the present invention. The full scope of the present invention is indicated by the appended claims and any equivalents thereof.

Claims

1. A compound or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, and prodrugs thereof, said compound having the following structure: 【Chemistry 1】 [In the formula, R １ is selected from the group consisting of hydrogen, fluorine, and chlorine; R ２ is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; or R １ and R ２ are joined together with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle; R ３ is hydrogen, C １～６ Alkyl, deuterated C １～６ Alkyl, C １～６ Haloalkyl, C １～６ Alkoxyalkyl, C ３～６ cycloalkyl, and 3- to 6-membered heterocyclyl, or R ３ are connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; A is, 【Chemistry 2】 selected from the group consisting of: Ring B is a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring; R ４ is hydrogen, C １～６ Alkyl, C １～６ Haloalkyl, C １～６ Alkoxyalkyl, C ３～６ selected from the group consisting of cycloalkyl, and 3- to 6-membered heterocyclyl; R ５ and R ６ is hydrogen, C １～６ Alkyl, C ２～６ Alkenyl, C ３～６ Cycloalkyl, and C １～６ haloalkyl, or R ５ Or R ６ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Or R ４ and R ５ are connected to form a 4- to 6-membered ring; n is 1 to 6; or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, and prodrugs thereof.

2. The compound has the structure of formula (II): 【Chemistry 3】 having In formula (II), R １ ' is selected from the group consisting of hydrogen, fluorine, and chlorine; R ２ ' is selected from the group consisting of methyl, fluorine, chlorine, and hydroxyl; R ３ ' is hydrogen, C １～６ Alkyl, deuterated C １～６ Alkyl, C １～６ Haloalkyl, C １～６ Alkoxyalkyl, C ３～６ cycloalkyl, and 3- to 6-membered heterocyclyl; R ３ ' is preferably hydrogen, C １～６ Alkyl, deuterated C １～６ Alkyl, C １～６ Haloalkyl, or C １～６ is alkoxyalkyl; R ４ ' is hydrogen, C １～６ Alkyl and C １～６ haloalkyl; R ４ ' is preferably hydrogen; R １ ' is fluorine, R ２ When R′ is methyl, ３ ' and R ４ ' is not hydrogen at the same time; R １ ' is fluorine, R ２ When R′ is methyl, ４ ' is C １～６ 10. The compound of claim 1, or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, which is not alkyl.

3. The compound has the structure of formula (III): 【Chemistry 4】 having In formula (III), R １ is selected from the group consisting of hydrogen, fluorine, and chlorine; R ２ is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; R ３ is hydrogen, C １～６ Alkyl, C １～６ Haloalkyl, C １～６ Alkoxyalkyl, C ３～６ cycloalkyl, and 3- to 6-membered heterocyclyl, or R ３ are connected to adjacent benzene ring carbon atoms to form a six-membered carbocyclic ring; R ３ is preferably hydrogen; R ４ is hydrogen or C １～６ is alkyl; R ５ and R ６ is hydrogen, C １～６ Alkyl, C １～６ Haloalkyl, C ３～６ Cycloalkyl, C ２～６ alkenyl, and 3- to 6-membered heterocyclyl, or R ５ and R ６ together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Or R ４ and R ５ are connected to form a 4- to 6-membered ring; n=1 or 2; R １ is fluorine, R ２ is methyl, R ３ is hydrogen, R ４ is hydrogen, R ５ and R ６ are not hydrogen and do not form a cyclopropyl group together with the carbon atom to which they are attached; R １ is fluorine, R ２ is methyl, R ３ is hydrogen, R ４ The compound of claim 1, or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein is not alkyl.

4. The compound has the structure of formula (IV): 【Chemistry 5】 having In formula (IV), R １’ is selected from the group consisting of hydrogen, fluorine, and chlorine; R ２’ is selected from the group consisting of hydrogen, methyl, fluorine, chlorine, and hydroxyl; R ４’ is hydrogen, C １～６ Alkyl, C ３～６ Cycloalkyl, and C １～６ haloalkyl; R ４’ is preferably hydrogen; B is C １～６ selected from the group consisting of alkylene, a 3- to 6-membered carbocycle, and a 3- to 6-membered heterocycle; R １’ is fluorine, R ２’ is methyl and when the dashed carbocycle is present, R ４’ is hydrogen or C １～６ Not alkyl; R ２’ is methyl, R １’ is fluorine and when the dashed carbocyclic ring is not present, ring B is not a 3-membered carbocyclic ring; or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

5. The compound has the following structure: 【Chemistry 6】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

6. A compound represented by formula (V) or pharma- ceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, and prodrugs thereof, said compound having the following structure: M-L-E-D Formula (V) [In the formula, M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a linker connecting linking moieties M and E; E represents a structural fragment connecting L and D; D represents a structural fragment of a cytotoxic drug; Preferably, M has the following structure: 【Chemistry 7】 is selected from the group consisting of X is selected from leaving groups such as chlorine, bromine, -OM, OT, and OTf; Preferably, M has the following structure: 【Chemistry 8】 selected from the group consisting of: Preferably、Lは、:C １～６ アルキレン、-N(R')-、carbonyl、-O-、V------------------------------- Zal-Leu-Lys、Gly-Gly-Ars、Ala-Ala-Asn、Al-Ala-Als、Al--Ala-Ala、 The 【Chemistry 9】 R′ is a structure consisting of one or more selected from the group consisting of hydrogen, C １～６ Alkyl or -(CH ２ CH ２ O) ｒ represents an alkyl group containing -; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20; Preferably, L has the following structure: 【Chemistry 10】 wherein s is an integer selected from 1 to 20; Preferably, E is a single bond, -NH-CH ２ --, 【Chemistry 11】 selected from the group consisting of: Preferably, E is -NH-CH ２ - and Preferably, the cytotoxic agent is selected from the compounds according to any one of claims 1 to 5; Preferably, the cytotoxic drug is selected from the compounds according to claim 5; preferably, D is selected from the structure formed by removing a hydrogen atom from a compound according to any one of claims 1 to 5; Preferably, D is selected from the structure formed by removing a hydrogen atom from the compound of claim 5; Preferably, D has the following structure: 【Chemistry 12】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 selected from the group consisting of: Preferably, D has the following structure: 【Chemistry 13】 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound has formula (V):

7. The compound has the following structure: A-1: 【Chemistry 14】 A-2: 【Chemistry 15】 A-3: 【Chemistry 16】 A-4: 【Chemistry 17】 A-5: 【Chemistry 18】 A-6: 【Chemistry 19】 A-7: 【Chemistry 20】 A-8: 【Chemistry 21】 A-9: 【Chemical 22】 A-10: 【Chemistry 23】 A-11: 【Chemistry 24】 A-12: 【Chemistry 25】 B-1: 【Chemistry 26】 B-2: 【Chemistry 27】 B-3: 【Chemistry 28】 B-4: 【Chemical 29】 B-5: 【Chemistry 30】 B-6: 【Chemistry 31】 B-7: 【Chemistry 32】 B-8: 【Chemical 33】 B-9: 【Chemical Formula 34】 B-10: 【Chemistry 35】 B-11: 【Chemical 36】 B-12: 【Chemical 37】 C-1: 【Chemical Formula 38】 C-2: 【Chemical 39】 C-3: 【Chemistry 40】 C-4: 【Chemistry 41】 C-5: 【Chemistry 42】 C-6: 【Chemistry 43】 C-7: 【Chemistry 44】 C-8: 【Chemistry 45】 C-9: 【Chemistry 46】 C-10: 【Chemistry 47】 C-11: 【Chemistry 48】 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof.

8. Formula (VI): Ab-(M-L-E-D) ｘ Formula (VI) [In the formula, Ab stands for antibody; M represents a linking site connecting the antibody or antigen-binding fragment thereof; L represents a linker connecting linking moieties M and E; E represents a structural fragment connecting L and D; D represents a structural fragment of a cytotoxic drug; x is 1 to 10; M, L, E and D are as defined in any one of claims 1 to 7; Preferably, Ab-(ML-E-D) ｘ has the following structure: ADC A-1: 【Chemistry 49】 ADC A-2: 【Chemistry 50】 ADC A-3: 【Chemistry 51】 ADC A-4: 【Chemistry 52】 ADC A-5: 【Chemistry 53】 ADC A-6: 【Chemical 54】 ADC A-7: 【Chemistry 55】 ADC A-8: 【Chemistry 56】 ADC A-9: 【Chemistry 57】 ADC A-10: 【Chemistry 58】 ADC A-11: 【Chemistry 59】 ADC A-12: 【Chemistry 60】 ADC B-1: 【Chemistry 61】 ADC B-2: 【Chemistry 62】 ADC B-3: 【Chemistry 63】 ADC B-4: 【Chemistry 64】 ADC B-5: 【Chemistry 65】 ADC B-6: 【Chemistry 66】 ADC B-7: 【Chemistry 67】 ADC B-8: 【Chemistry 68】 ADC B-9: 【Chemistry 69】 ADC B-10: 【Chemistry 70】 ADC B-11: 【Chemistry 71】 ADC B-12: 【Chemical 72】 ADC C-1: 【Chemical Formula 73】 ADC C-2: 【Chemical 74】 ADC C-3: 【Chemistry 75】 ADC C-4: 【Chemical Formula 76】 ADC C-5: 【Chemical 77】 ADC C-6: 【Chemical 78】 ADC C-7: 【Chemical Formula 79】 ADC C-8: 【Chemistry 80】 ADC C-9: 【Chemistry 81】 ADC C-10: 【Chemistry 82】 ADC C-11: 【Chemistry 83】 The antibody-drug conjugate (ADC) is represented by the following formula:

9. The following structure: Trastuzumab A-11: 【Chemistry 84】 Trastuzumab B-1: 【Chemistry 85】 Trastuzumab B-2: 【Chemistry 86】 An antibody-drug conjugate (ADC) having the formula:

10. An antibody-drug conjugate of: Trastuzumab A-11: 【Chemistry 87】 wherein the DAR value (drug to antibody ratio) is 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 8.

0.

11. An antibody-drug conjugate of: Trastuzumab B-1: 【Chemistry 88】 wherein the DAR value (drug to antibody ratio) is 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 7.

96.

12. An antibody-drug conjugate of: Trastuzumab B-2: 【Chemistry 89】 wherein the DAR value (drug to antibody ratio) is 7.5 to 8.5, preferably 7.5 to 8.0, and more preferably 7.

95.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or an antibody-drug conjugate according to claim 8 or 9, or a composition according to any one of claims 10 to 12 and one or more pharma- ceutically acceptable carriers.

14. a) a first therapeutic agent which is at least one of the compound of any one of claims 1 to 7 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, the antibody-drug conjugate of claim 8 or 9, the composition of any one of claims 10 to 12, or the pharmaceutical composition of claim 13; b) an optional second therapeutic agent, which is at least one additional therapeutic agent, or an optional second pharmaceutical composition, which is a pharmaceutical composition containing an additional therapeutic agent; and c) A kit, optionally with packaging and / or instructions.

15. Preferably, the disease is a tumor, e.g., an advanced solid tumor; Use of the compound according to any one of claims 1 to 9 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite and prodrug thereof, the antibody-drug conjugate according to claim 8 or 9, the composition according to any one of claims 10 to 12, the pharmaceutical composition according to claim 13, or the kit according to claim 14, in the manufacture of a medicament for treating a disease associated with abnormal cell proliferation, preferably wherein the tumor is selected from the group consisting of lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, squamous cell esophageal carcinoma, prostate cancer, female genital tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

16. A method for treating a disease associated with abnormal cell proliferation, comprising the steps of: administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 9 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite and prodrug thereof, or an antibody-drug conjugate according to any one of claims 8 or 9, or a composition according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, or a kit according to claim 14; Preferably, the disease is a tumor, e.g., an advanced solid tumor; Preferably, the method wherein the tumor is selected from the group consisting of lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, squamous cell esophageal carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

17. Preferably, the disease is a tumor, e.g., an advanced solid tumor; The compound according to any one of claims 1 to 8 or a pharma- ceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite and prodrug thereof, or the antibody-drug conjugate according to claim 8 or 9, or the composition according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13, or the kit according to claim 14, for use in the treatment of a disease associated with abnormal cell proliferation, preferably wherein the tumor is selected from the group consisting of lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, squamous cell esophageal carcinoma, prostate cancer, female genital tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

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