Camptothecin compounds and preparation methods and applications thereof

Abstract

Camptothecin compounds with anti-tumor activity, and preparation methods and uses thereof. Specifically, the present invention relates to the following compounds or their pharmaceutically acceptable forms, pharmaceutical compositions, preparation methods and uses thereof. The compounds can be used as drugs for treating diseases related to abnormal cell proliferation,

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202280008418.X, filed on January 27, 2022, and with the invention name “Camptothecin compounds, preparation methods and applications thereof”. Technical Field

[0002] The present invention relates to a class of camptothecin compounds with anti-tumor activity and conjugates thereof, as well as preparation methods and applications thereof in the medical field. Background Art

[0003] Camptothecin (CPT, Formula 1) is a pentacyclic quinoline nucleus compound isolated from Camptotheca acuminata, a plant of the Davidiaceae family. It consists of a quinoline ring AB, a pyrrole ring C, a pyridone ring D and an α-hydroxylactone ring E, wherein the 20-position is an S configuration (see the structural formula below). It was introduced into clinical practice in the early 1970s due to its excellent anticancer activity, but later clinical trials were terminated due to the occurrence of severe side effects such as diarrhea and hemorrhagic cystitis.

[0004]

[0005] Research data show that camptothecin can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, leading to DNA replication blockage and cell death (Cancer Res. 1989, 49, 6365). Camptothecin and its derivatives have strong anti-tumor activity in animal models of lung cancer, breast cancer, colorectal cancer, ovarian cancer, etc. (Nature Review Cancer. 2006, 6, 789).

[0006] Currently, several camptothecin drugs have been approved for marketing for tumor treatment (Med. Res. Rev. 2015, 35, 753). Irinotecan is a drug for colorectal cancer treatment; topotecan is used for the treatment of ovarian cancer; belotecan is used for the treatment of ovarian cancer and small cell lung cancer. Camptothecin derivatives include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, Elomotecan, etc.

[0007] Camptothecin drugs or derivatives often have blood toxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, etc., as well as gastrointestinal side effects, such as nausea, vomiting, diarrhea, etc. Clinical studies have found that measures to improve the safety and effectiveness of camptothecin compounds include improving their pharmacokinetic properties, regulating activity, reducing dosage, or using their conjugates to form antibody-coupled drugs with antibodies. Therefore, the research and development of camptothecin compounds and their conjugates with novel structures that can improve effectiveness and improve safety issues still have high clinical demand and application value. Summary of the invention

[0008] The present invention provides a camptothecin compound with a novel structure and a conjugate thereof. The camptothecin compound has good anti-tumor activity and is expected to be used in the treatment of tumor diseases; and the conjugate thereof has a wide application prospect in antibody-drug coupling.

[0009] In a first aspect, the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, wherein the compound has the structure shown below:

[0010]

[0011] in,

[0012] R 1 and R 2 are each independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, hydroxyl, cyano and C 3-6 Cycloalkyl; or, R 1 and R 2 Connected with adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

[0013] R 3 is hydrogen or with R 1 The adjacent carbon atoms are connected to form a six-membered carbon ring;

[0014] A is selected from and One of;

[0015] R 4 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0016] R 5 and R 6 are each independently selected from hydrogen, C 1-6Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylaminoalkyl, C 1-6 Alkoxyalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, aryl and heteroaryl; or R 5 and R 6 Connected with adjacent carbon atoms to form a 3-6 membered carbocyclic or heterocyclic ring;

[0017] m=1 or 2.

[0018] In some embodiments, the compound has the structure of Formula (I):

[0019]

[0020] In formula (I), R x Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0021] R y and R z are not hydrogen at the same time, and are independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkylaminoalkyl, C 1-6 Alkoxyalkyl, 3-6 membered heterocyclylalkyl and 3-6 membered heterocyclyl.

[0022] In some embodiments, R x Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0023] R y and R z are not hydrogen at the same time, and are independently selected from hydrogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkylaminoalkyl, C 1-6 Alkoxyalkyl, 3-6 membered heterocyclylalkyl and 3-6 membered heterocyclyl.

[0024] In some embodiments, in Formula (I), R x Selected from hydrogen, C1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 3- to 6-membered heterocyclic group;

[0025] R y and R z are not hydrogen at the same time, and are independently selected from hydrogen, C 2-6 Alkenyl, C 2-6 Alkynyl.

[0026] In some embodiments, R x Selected from hydrogen or C 1-6 alkyl.

[0027] In some embodiments, R x For hydrogen.

[0028] In some embodiments, R y and R z are not hydrogen at the same time, and are independently selected from hydrogen, dimethylaminomethylene, morpholinylmethylene and methoxymethylene.

[0029] In some embodiments, R y and R z are not hydrogen at the same time, and are independently selected from hydrogen, Dimethylaminomethylene and methoxymethylene.

[0030] In some embodiments, R y is hydrogen, R z Selected from dimethylaminomethylene, morpholinylmethylene and methoxymethylene.

[0031] In some embodiments, R y is hydrogen, R z Selected from Dimethylaminomethylene and methoxymethylene.

[0032] In some embodiments, R x is hydrogen, R y is hydrogen, R z Selected from dimethylaminomethylene, morpholinylmethylene and methoxymethylene.

[0033] In some embodiments, R x is hydrogen, R y is hydrogen, R z Selected from Dimethylaminomethylene and methoxymethylene.

[0034] In some embodiments, R xis hydrogen, R y is hydrogen, R z Selected from and dimethylaminomethylene.

[0035] In some embodiments, in formula (I) Chuwei Configuration.

[0036] In some embodiments, in formula (I) Chuwei Configuration.

[0037] In some embodiments, in formula (I) Chuwei Configuration.

[0038] In some embodiments, in formula (I) Chuwei Configuration.

[0039] In some embodiments, in formula (I) Chuwei Configuration.

[0040] In some embodiments, in formula (I) Chuwei Configuration.

[0041] In some embodiments, in formula (I) Chuwei Configuration.

[0042] In some embodiments, in formula (I) Chuwei Configuration.

[0043] In some embodiments, the compound has the structure of Formula (II):

[0044]

[0045] In formula (II), A' is selected from and One of;

[0046] R x’ Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0047] R y’ and R z’ independently selected from hydrogen, C 1-6 Alkyl, C 1-6Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, aryl and heteroaryl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0048] In some embodiments, the structure of formula (II) is shown in formula (II)-1:

[0049]

[0050] In some embodiments, R x’ Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 3- to 6-membered heterocyclic group;

[0051] R y’ and R z’ independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 2-6 Alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0052] In some embodiments, the 3-6 membered ring is selected from a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring.

[0053] In some embodiments, R x’ Selected from hydrogen and C 1-6 alkyl.

[0054] In some embodiments, R x’ is selected from hydrogen and methyl.

[0055] In some embodiments, R y’ and R z’ independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl and C 2-6 Alkenyl, or R y’ and R z’Connected to adjacent carbon atoms to form a 3-6 membered cycloalkyl group.

[0056] In some embodiments, R y’ Selected from hydrogen and C 1-6 Alkyl, R z’ Selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0057] In some embodiments, R y’ is selected from hydrogen and methyl, R z’ is selected from hydrogen, methyl and cyclopropyl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0058] In some embodiments, R x’ is selected from hydrogen and methyl, R y’ is selected from hydrogen and methyl, R z’ is selected from hydrogen, methyl and cyclopropyl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0059] In some embodiments, R x’ is hydrogen, R y’ is selected from hydrogen and methyl, R z’ is selected from hydrogen, methyl and cyclopropyl, or R y’ and R z’ Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0060] In some embodiments, A' in formula (II) is

[0061] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0062] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0063] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0064] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0065] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0066] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0067] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0068] In some embodiments, in formula (II)-1 Chuwei Configuration.

[0069] In some embodiments, the compound has the structure of Formula (III):

[0070]

[0071] In formula (III), A" is selected from and One of;

[0072] R x” Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0073] R y” and R z” independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, 4-6 membered heterocyclyl, C 2-6 Alkenyl, C 2-6 Alkynyl, aryl and heteroaryl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0074] In some embodiments, the compound of formula (III) has a structure as shown in formula (III)-1:

[0075]

[0076] In some embodiments, R x” Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 3- to 6-membered heterocyclic group;

[0077] R y” and R z” independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 4-6 membered heterocyclic group, C 2-6 Alkenyl, C 2-6 Alkynyl, aryl, heteroaryl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0078] In some embodiments, R x” Selected from hydrogen and C 1-6 alkyl.

[0079] In some embodiments, R x” For hydrogen.

[0080] In some embodiments, the 3-6 membered ring is selected from a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring.

[0081] In some embodiments, R y” and R z” are each independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl and vinyl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0082] In some embodiments, R y” is hydrogen, R z” Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl and vinyl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-6 membered carbon ring.

[0083] In some embodiments, R y” is hydrogen, R z” is selected from hydrogen, methyl, cyclopropyl and vinyl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0084] In some embodiments, R x”is hydrogen, R y” is hydrogen, R z” is selected from hydrogen, methyl, cyclopropyl and vinyl, or R y” and R z” Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0085] In some embodiments, A" in formula (III) is

[0086] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0087] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0088] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0089] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0090] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0091] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0092] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0093] In some embodiments, in formula (III)-1 Chuwei Configuration.

[0094] In some embodiments, the compound has the structure of Formula (IV):

[0095]

[0096] In formula (IV),

[0097] R a and R b independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxyalkyl, C1-6 Alkoxy, C 1-6 haloalkyl, hydroxyl and cyano; or R a and R b Connected with adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

[0098] R c and R d independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C 2-6 Alkenyl and C 2-6 Alkynyl, or R c and R d Connected to adjacent carbon atoms to form a 3-6 membered carbocyclic or heterocyclic ring;

[0099] R e Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl and C 2 -C 5 Heterocyclic group;

[0100] q = 0 or 1;

[0101] When q=0, R c and R d Not at the same time as hydrogen.

[0102] In some embodiments, R a and R b independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 haloalkyl, hydroxyl and cyano; or R a and R b Connected with adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

[0103] R c and R d independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C 2-6 Alkenyl and C 2-6 Alkynyl, or R cand R d Connected to adjacent carbon atoms to form a 3-6 membered carbocyclic or heterocyclic ring;

[0104] R e Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl and 4-6 membered heterocyclic group;

[0105] q = 0 or 1;

[0106] When q=0, R c and R d Not at the same time as hydrogen.

[0107] In some embodiments, in Formula (IV), R a and R b independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 haloalkyl, hydroxy, and cyano; or R a and R b Connected with adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;

[0108] R c and R d independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, 4-6 membered heterocyclic group, C 2-6 Alkenyl, C 2-6 Alkynyl, or R c and R d Connected to adjacent carbon atoms to form a 3-6 membered carbocyclic or heterocyclic ring;

[0109] R e Selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, or 4-6 membered heterocyclic group;

[0110] q = 0 or 1;

[0111] When q=0, R c and R d Not at the same time as hydrogen.

[0112] In some embodiments, R a and R b independently selected from hydrogen, halogen and C 1-6 Alkyl, or Ra and R b Connected with adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring.

[0113] In some embodiments, R a and R b are independently selected from hydrogen, fluorine, chlorine and methyl, or R a and R b Together with the benzene ring connected to it, it forms

[0114] Wherein Z is selected from -CH 2 -、-CD 2 -、-CH 2 CH 2 -and-CF 2 -.

[0115] In some embodiments, R a is methyl, R b is fluorine, or R a and R b Together with the benzene ring connected to it, it forms

[0116] In some embodiments, R c and R d independently selected from hydrogen, C 1-6 Alkoxyalkyl and C 1-6 Alkylaminoalkyl, or R c and R d Connected to adjacent carbon atoms to form a 3-6 membered carbon ring.

[0117] In some embodiments, R c is hydrogen, R d Selected from hydrogen, Methoxyethyl and cyclopropyl, or R c and R d Connected to adjacent carbon atoms to form a 3-6 membered carbon ring.

[0118] In some embodiments, R e Selected from hydrogen and C 1-6 alkyl.

[0119] In some embodiments, R e is selected from hydrogen and isopropyl.

[0120] In some embodiments, R a is methyl, R b is fluorine, or R a and R b Together with the benzene ring connected to it, it forms R eis selected from hydrogen and isopropyl, R c is hydrogen, R d Selected from hydrogen, Methoxyethyl and cyclopropyl, or R c and R d Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0121] In some embodiments, R a is methyl, R b is fluorine, or R a and R b Together with the benzene ring connected to it, it forms R e is selected from hydrogen and isopropyl, R c is hydrogen, R d is selected from hydrogen, methoxyethyl and cyclopropyl, or R c and R d Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0122] In some embodiments, in formula (IV) Chuwei Configuration.

[0123] In some embodiments, in formula (IV) Chuwei Configuration.

[0124] In some embodiments, the compound has the structure of Formula (V):

[0125]

[0126] In formula (V), R is selected from C 3-6 Cycloalkyl and C 1-6 Alkoxy;

[0127] A"' is selected from and One of;

[0128] R x”’ Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group;

[0129] R y”’ and R z”’ independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6Cycloalkyl, 3-6 membered heterocyclyl, 3-6 membered heterocyclylalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, aryl and heteroaryl, or R y”’ and R z”’ Connected to adjacent carbon atoms to form a 3-6 membered ring.

[0130] In some embodiments, R is selected from methoxy and cyclopropyl.

[0131] In some embodiments, the compound of formula (V) has a structure as shown in formula (V)-1:

[0132]

[0133] In some embodiments, the 3-6 membered ring is selected from a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring.

[0134] In some embodiments, R y”’ and R z”’ are each independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 3-6 Cycloalkyl and vinyl, or R y”’ and R z”’ Connected to adjacent carbon atoms to form a 3-6 membered carbon ring.

[0135] In some embodiments, R y”’ and R z”’ are hydrogen, or R y”’ and R z”’ Connected to adjacent carbon atoms to form a 3-6 membered carbon ring.

[0136] In some embodiments, R x”’ Selected from hydrogen and C 1-6 alkyl.

[0137] In some embodiments, R x”’ For hydrogen.

[0138] In some embodiments, A"' is

[0139] In some embodiments, R is selected from methoxy and cyclopropyl, R x”’ is hydrogen, R y”’ and R z”’ are hydrogen, or R y”’ and R z”’ Connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0140] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0141] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0142] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0143] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0144] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0145] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0146] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0147] In some embodiments, in Formula (V)-1 Chuwei Configuration.

[0148] In some embodiments, the present invention provides the following compounds:

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] In another aspect, the present invention also provides a compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof:

[0156] MLED

[0157] Formula (VI)

[0158] in,

[0159] M is a linker site with an antibody or antigen-binding fragment thereof;

[0160] L is a linker between the linkers M and E;

[0161] E is a structural fragment connecting L and D;

[0162] D is a structural fragment of a cytotoxic drug.

[0163] In some embodiments, M is selected from the following structures:

[0164]

[0165] In some embodiments, M is selected from the following structures:

[0166]

[0167] In some embodiments, L is selected from a divalent structure consisting of one or more of the following: 1-6 Alkylene, -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, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly,

[0168]

[0169] Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH 2 CH 2 O) r -alkyl; r is selected from an integer of 1-10; s is selected from an integer of 1-10.

[0170] In some embodiments, L is selected from the following structures:

[0171]

[0172] In some embodiments, L is selected from the following structures:

[0173]

[0174] In some embodiments, E is selected from a single bond, -NH-CH 2 -、 In some embodiments, E is -NH-CH 2 -.

[0175] In some embodiments, the cytotoxic drug is selected from the compound described in any one of the first aspects of the present invention.

[0176] In some embodiments, the cytotoxic drug is selected from compounds 1-1 to 1-15; 2-1 to 2-27; 3-1 to 3-26; 4-1 to 4-15; or 5-1 to 5-36 described herein.

[0177] In some embodiments, D is selected from the structure of a compound described herein after dehydrogenation.

[0178] In some embodiments, D is selected from the structures of compounds 1-1 to 1-15; 2-1 to 2-27; 3-1 to 3-26; 4-1 to 4-15; or 5-1 to 5-36 described herein after dehydrogenation.

[0179] In some embodiments, D is selected from the following structures:

[0180]

[0181] In some embodiments, D is selected from the following structures:

[0182]

[0183] In some embodiments, the MLED is selected from the following compounds:

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] definition

[0190] Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology generally understood in the art, including those changes in technology or replacement of equivalent technology that are obvious to those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0191] The terms "comprises," "comprising," "having," "containing," or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0192] As used herein, the "*" marked in the compound formula indicates that the marked carbon atom is a chiral carbon atom, and the present invention includes a pair of enantiomers formed by the chiral carbon atom. If a compound contains two different chiral carbon atoms, the present invention includes four optical isomers formed by the chiral carbon atom.

[0193] As used herein, represents a bond that can be stereospecific ((R) or (S)) or non-stereospecific.

[0194] The term "alkyl" is defined as a straight or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, such as 1 to 6 carbon atoms. For example, as used herein, the term "C 1-6 "Alkyl" refers to a linear or branched group of 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl), which is optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents.

[0195] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-6 Alkenyl", "C 2-4 Examples include, but are not limited to, ethenyl, 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.

[0196] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. 2-6 Alkynyl", "C 4-6"Alkynyl" and the like. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl and the like.

[0197] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocyclic alkyl and bicyclic alkyl (such as spirocyclic alkyl, paracyclic alkyl and bridged cycloalkyl). 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may be optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents, for example methyl substituted cyclopropyl.

[0198] The term "carbocycle" or "carbocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon group connected by a ring carbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0199] The term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., a monocyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a cyclononane ring, or a bicyclic ring, including a spirocyclic, fused or bridged system (such as 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 may be optionally substituted with 1 or more (such as 1, 2 or 3) suitable substituents. The term "3-6 membered carbocycle" refers to a carbocycle containing 3, 4, 5 or 6 ring-forming carbon atoms.

[0200] The term "heterocyclyl" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as a bicyclic) non-aromatic cyclic structure, whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatom selected from nitrogen, oxygen, and sulfur. If the valence bond requirements are met, the heterocyclyl can be connected to the rest of the molecule via any one of the ring atoms. The heterocyclyl in the present invention is preferably a 3-6 membered heterocyclyl. The term "3-6 membered heterocyclyl" used in the present invention 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, an oxygen-containing heterocyclyl, such as a 4-6 membered heterocyclyl, such as a 4-6 membered nitrogen-containing heterocyclyl, a 4-6 membered oxygen-containing heterocyclyl. Common heterocyclic groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups of the present invention may be optionally substituted with one or more substituents described herein. The heterocyclic groups of the present invention are optionally fused with one or more aromatic or non-aromatic rings.

[0201] The term "oxygen-containing heterocycle" refers to a heterocycle as described above in which one or more (e.g., 1, 2, or 3) ring atoms are oxygen atoms, such as a 5-6-membered oxygen-containing heterocycle, and specific examples include but are not limited to an oxirane ring, a tetrahydrofuran ring, a furan ring, a tetrahydropyran ring, a pyran ring, etc. The "nitrogen-containing heterocycle" described in the present invention refers to a heterocycle as described above in which one or more (e.g., 1, 2, or 3) ring atoms are nitrogen atoms.

[0202] The term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1, 2 or 3) the same or different halogen atoms, wherein alkyl is defined as above. 1-6 "Haloalkyl" refers to a haloalkyl group 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 、-CH2 Cl, etc. The haloalkyl group in the present invention is optionally substituted with one or more substituents described in the present invention.

[0203] The term "aryl" refers to a group obtained by removing a hydrogen atom from the aromatic carbon atom of an aromatic hydrocarbon molecule, such as a 6-14-membered aryl group, and specific examples include but are not limited to phenyl, naphthyl, anthracenyl, etc.

[0204] The term "heteroaryl" refers to an aromatic cyclic group containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered heteroaryl, 5-6 membered nitrogen-containing heteroaryl, 5-6 membered oxygen-containing heteroaryl, and the like, such as furanyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, and the like.

[0205] The term "alkoxy" refers to a group having the structure "alkyl-O-", wherein alkyl is as defined above. 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, etc. The alkoxy group in the present invention is optionally substituted with one or more substituents described in the present invention.

[0206] () () The term "alkoxyalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3 or 4) alkoxy groups, wherein alkoxy and alkyl are as defined above. For example, the term "C 1-6 "Alkoxyalkyl" refers to an alkyl group having 1 to 6 carbon atoms, substituted by one or more (e.g., 1, 2, 3 or 4) alkoxy groups. Common alkoxyalkyl groups include (but are not limited to) CH 3 O-CH 2 -,C 2 H 5 -O-CH 2 -、C 2 H 5 -O-CH 2 CH 2 -wait.

[0207] The term "halo" or "halogen" group is defined to include F, Cl, Br or I.

[0208] The term "nitrogen oxide" refers to an oxide (eg, mono- or di-oxide) of at least one nitrogen atom in the structure of the compounds of the present application. The nitrogen mono-oxide may exist in the form of a single positional isomer or a mixture of positional isomers.

[0209] The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds.

[0210] If a substituent is described as being "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the substituents listed in the substituents list, then one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or together, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed in the substituents list, then one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

[0211] If substituents are described as being "independently selected" from a group, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0212] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

[0213] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0214] The term "stereoisomer" means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of the present application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0215] In this article, solid lines (-), solid wedges Virtual wedge Carbon-carbon bonds of the compounds of the invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomer shown is present. When present in a racemic mixture, the solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the invention are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0216] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

[0217] It should also be understood that certain compounds of the present invention may exist in free form for treatment, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites or prodrugs, which, after being administered to a patient in need thereof, can directly or indirectly provide a compound of the present invention or a metabolite or residue thereof. Therefore, when referring to "compounds of the present invention" herein, the above-mentioned various derivative forms of the compounds are also intended to be covered.

[0218] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.

[0219] Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts and include aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate and the like.

[0220] 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.

[0221] For a review of suitable salts see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0222] The term "ester" refers to esters derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form). The compounds of the present invention themselves may also be esters.

[0223] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol. The amount of polar solvents, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0224] Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0225] The present invention further includes within its scope prodrugs of the compounds of the present invention. Such prodrugs are usually functional group derivatives of the compounds, which are easily converted into desired therapeutically active compounds in vivo. Therefore, in these cases, the term "administration" used in the therapeutic methods of the present invention should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but the prodrug forms are converted into the above-mentioned compounds in vivo after administration to individuals. For example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985, conventional methods for selecting and preparing suitable prodrug derivatives are described.

[0226] The present invention further includes within its scope isotopically labeled compounds of the present invention which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature.

[0227] The present invention also encompasses compounds of the present invention containing protecting groups. In any process of preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any related molecules, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Using methods known in the art, the protecting groups can be removed at an appropriate subsequent stage.

[0228] Pharmaceutical composition

[0229] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first or second aspect of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, and one or more pharmaceutically acceptable carriers.

[0230] The term "pharmaceutical composition" refers to a composition that can be used as a drug, which contains an active pharmaceutical ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an excipient that is administered with a therapeutic agent and is suitable for contact with human and / or other animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit / risk ratio.

[0231] The pharmaceutical composition can act systemically and / or locally, which can be achieved through suitable dosage forms. The dosage forms include but are not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0232] The above-mentioned pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof.

[0233] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition or its corresponding formulation form, which comprises combining at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug with one or more pharmaceutically acceptable carriers.

[0234] Medicine box products

[0235] In a fourth aspect, the present invention provides a kit product comprising:

[0236] a) at least one compound of the first or second aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof as a first therapeutic agent, or the pharmaceutical composition of the third aspect as a first pharmaceutical composition;

[0237] b) optionally at least one additional therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising an additional therapeutic agent as a second pharmaceutical composition; and

[0238] c) optional packaging and / or instructions.

[0239] The above-mentioned kit product may contain 0.01 mg to 1000 mg of at least one compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof.

[0240] The present invention also provides a method for preparing the above-mentioned medicine kit, which comprises combining at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug or the above-mentioned pharmaceutical composition with at least one other therapeutic agent or a pharmaceutical composition containing other therapeutic agents, packaging and / or instructions.

[0241] Medical Uses

[0242] The compound of the present invention can exhibit a strong effect of inhibiting abnormal cell proliferation.

[0243] Therefore, the present application provides the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites and prodrugs or the above-mentioned pharmaceutical compositions, which are used for treating diseases related to abnormal cell proliferation.

[0244] In addition, the present application also provides the use of the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites and prodrugs or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating diseases involving abnormal cell proliferation.

[0245] In some embodiments, the disease involving abnormal cell proliferation includes, but is not limited to, a tumor, such as an advanced solid tumor.

[0246] The present application also provides the use of the compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug or the pharmaceutical composition of the present invention in the preparation of a preparation, the preparation is used to inhibit the proliferation of tumor cells. In certain embodiments, the preparation is used for in vivo or in vitro administration. For example, the preparation can be administered to a subject to inhibit the proliferation of tumor cells in the subject; or, the preparation can be administered to in vitro cells (e.g., cell lines or cells from a subject) to inhibit the proliferation of tumor cells in vitro.

[0247] The tumors described in the present invention include (but are not limited to): brain tumors, 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, esophageal squamous cell 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.

[0248] Treatment

[0249] In another aspect, the present invention provides a method for treating diseases involving abnormal cell proliferation, comprising the following steps: administering a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, or the above-mentioned pharmaceutical composition to an individual in need thereof.

[0250] The term "effective amount" refers to a dose that can induce a biological or medical response in cells, tissues, organs or organisms (eg, individuals) and is sufficient to achieve the desired preventive and / or therapeutic effect.

[0251] The dosage regimen can be adjusted to provide the best desired response. For example, the dosage may be a single dose, divided doses may be administered over time, or the dosage may be proportionally reduced or increased according to actual conditions. It is understood that for any particular individual, the specific dosage regimen should be adjusted according to the needs and the professional judgment of the person administering the composition or supervising the administration of the composition.

[0252] The dosage of the compound of the present invention will depend on the individual situation, the severity of the disease or illness, the rate of administration, the disposal of the compound and the judgment of the prescribing physician. In general, the effective amount is about 0.001-10000mg / kg subject weight / day. In appropriate cases, the effective amount is about 0.01-1000mg / kg subject weight / day. About 0.01-1000mg / kg subject weight, usually about 0.1-500mg / kg subject weight, can be administered every day, every two days or every three days. An exemplary dosage regimen is once or more per day, or once or more per week, or once or more per month. During multiple administrations, the interval between single doses can generally be daily, weekly, monthly or annual. Alternatively, it can be administered in the form of a sustained-release preparation, in which case a lower frequency of administration is required. The dosage and frequency of administration can be different according to the half-life of the drug in the subject, and can also be different according to whether it is a preventive application or a therapeutic application. In preventive applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time; in therapeutic applications, it is sometimes necessary to administer a relatively high dosage at shorter intervals until the progression of the disease is slowed or stopped, preferably until the individual exhibits partial or complete improvement in disease symptoms, after which preventive applications can be employed.

[0253] The term "treatment" refers to the alleviation or elimination of the targeted disease or condition. If a subject receives a therapeutic amount of a compound of the present invention or a pharmaceutically 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 relief and / or improvement, it indicates that the subject has been successfully "treated". It is understood that treatment includes not only complete treatment, but also includes not achieving complete treatment, but achieving some biological or medically relevant results.

[0254] The term "administrate / administrating / administration" (or "dosing") refers to the process of applying a pharmaceutically active ingredient (such as a compound of the present invention) or a pharmaceutical composition comprising a pharmaceutically active ingredient (such as a pharmaceutical composition of the present invention) to an individual or its cells, tissues, organs, biological fluids, etc., so as to bring the pharmaceutically active ingredient or pharmaceutical composition into contact with the individual or its cells, tissues, organs, biological fluids, etc. Common modes of administration include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, etc.

[0255] The term "in need of" refers to the physician's or other caregiver's judgment that an individual needs or will benefit from a preventive and / or therapeutic procedure, which judgment is based on a variety of factors within the physician's or other caregiver's area of ​​expertise.

[0256] The term "individual" (or subject) refers to a human or non-human animal. The individual of the present invention includes individuals (patients) suffering from diseases and / or disorders and normal individuals. The non-human animals of the present invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0257] Preparation method

[0258] The fourth aspect of the present invention provides a method for synthesizing the compound.

[0259] The compound of formula (I) in the present invention can be synthesized by the following synthetic route.

[0260]

[0261] Among them, R x , R y and R z The meaning is as described above; LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0262] Step 1:

[0263] The compound of formula (I)-IM1 is obtained by a substitution reaction between the compound of formula (I)-SM1 and the compound of formula (I)-SM2.

[0264] 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, 140°C, preferably 50°C;

[0265] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

[0266] In some embodiments, this step is carried out in the presence of a suitable base, the base includes an organic base or an inorganic base, the organic base can be selected from N, N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K 3 PO4), 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 ;

[0267] Step 2:

[0268] The compound of formula (I) is obtained by condensing the compound of formula (I)-IM1 with the compound of formula (I)-SM3;

[0269] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0270] 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, preferably 25°C;

[0271] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0272] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0273] The compound of formula (II)-1 in the present invention can be synthesized by the following synthetic route.

[0274]

[0275] Among them, R x’ , R y’ and R z’ The meaning is as described above; LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine; PG is a protecting group selected from and

[0276]

[0277] Step 1

[0278] The compound of formula (II)-IM1 is obtained by a substitution reaction of the compound of formula (II)-SM1.

[0279] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 50°C;

[0280] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably n-heptane.

[0281] Step 2

[0282] The compound of formula (II)-IM2 is obtained by reducing the compound of formula (II)-IM1;

[0283] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a platinum catalyst;

[0284] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 60°C;

[0285] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0286] Step 3

[0287] The compound of formula (II)-IM2 undergoes a substitution reaction to obtain the compound of formula (II)-IM3,

[0288] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0289] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate;

[0290] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably triethylamine.

[0291] Step 4

[0292] The compound of formula (II)-IM4 is obtained by coupling reaction of the compound of formula (II)-IM3;

[0293] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 70°C, 100°C, preferably 70°C;

[0294] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, water, preferably a mixed solvent of tetrahydrofuran and water;

[0295] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably N,N-diisopropylethylamine.

[0296] Step 5

[0297] The compound of formula (II)-IM4 is subjected to a reduction reaction to obtain the compound of formula (II)-IM5;

[0298] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a platinum catalyst;

[0299] 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, preferably 40°C;

[0300] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably tetrahydrofuran.

[0301] Step 6

[0302] The compound of formula (II)-IM6 is obtained by ring-closing reaction of the compound of formula (II)-IM5;

[0303] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

[0304] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, tert-butanol, preferably a mixed solvent of tetrahydrofuran and tert-butanol.

[0305] Step 7

[0306] The compound of formula (II)-IM7 is obtained by a substitution reaction of the compound of formula (II)-IM6;

[0307] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

[0308] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

[0309] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably potassium tert-butoxide.

[0310] Step 8

[0311] The compound of formula (II)-IM8 is obtained by reducing the compound of formula (II)-IM7;

[0312] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a palladium catalyst;

[0313] 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, preferably 20°C;

[0314] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0315] Step 9

[0316] The compound of formula (II)-IM9 is obtained by a substitution reaction of the compound of formula (II)-IM8;

[0317] 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 20°C;

[0318] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably pyridine.

[0319] Step 10

[0320] The compound of formula (II)-IM9 is subjected to a hydrolysis reaction to obtain a compound of formula (II)-IM10;

[0321] 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, preferably 60°C;

[0322] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0323] In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0324] Step 11

[0325] The compound of formula (II)-IM11 is obtained by a cyclization reaction between the compound of formula (II)-IM10 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one;

[0326] 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, 140°C, preferably 140°C;

[0327] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably toluene.

[0328] In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0329] Step 12

[0330] The compound of formula (II)-IM12 is obtained by hydrolyzing the compound of formula (II)-IM11;

[0331] 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, 140°C, preferably 100°C;

[0332] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0333] Step 13

[0334] The compound of formula (II)-IM13 is obtained by a substitution reaction between the compound of formula (II)-IM12 and the compound of formula (II)-SM2.

[0335] 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, 140°C, preferably 50°C;

[0336] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Diox), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

[0337] In some embodiments, this step is carried out in the presence of a suitable base, the base includes an organic base or an inorganic base, the organic base can be selected from N, N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from 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 2CO 3 or NaHCO 3 ;

[0338] Step 14

[0339] The compound of formula (II)-IM14 is obtained by condensing the compound of formula (II)-IM13 with the compound of formula (II)-SM3;

[0340] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0341] 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, preferably 25°C;

[0342] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0343] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0344] Step 15

[0345] The compound of formula (II)-1 is obtained by acid hydrolysis of the compound of formula (II)-IM14;

[0346] 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, 140°C, preferably 25°C;

[0347] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and mixed solvents thereof, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0348] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0349] The synthesis method of formula (II)-SM3 is as follows:

[0350] When PG is hour:

[0351]

[0352] Step 1

[0353] The compound of formula (II)-SM3-3 is obtained by a substitution reaction between the compound of formula (II)-SM3-1 and the compound of formula (II)-SM3-2;

[0354] 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, 140°C, preferably 0-25°C;

[0355] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably tetrahydrofuran.

[0356] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably K 2 CO 3 .

[0357] Step 2

[0358] The compound of formula (II)-SM3-3 is subjected to hydrogenation reaction to obtain the compound of formula (II)-SM3;

[0359] 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, 140°C, preferably 25°C;

[0360] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably methanol.

[0361] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a palladium catalyst;

[0362] When PG is When R y’ and R z’ For hydrogen:

[0363]

[0364] Step 1

[0365] The compound of formula (II)-SM3-4 and the compound of formula (II)-SM3-5 undergo condensation reaction to obtain the compound of formula (II)-SM3;

[0366] 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, 140°C, preferably 25°C;

[0367] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably N,N-dimethylformamide.

[0368] Alternatively, the compound of formula (II)-1 can be synthesized by the following synthetic route:

[0369]

[0370] Step 1

[0371] The compound of formula (II)-IM13 and the compound of formula (II)-SM4 undergo a condensation reaction to obtain a compound of formula (II)-IM15;

[0372] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0373] 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, preferably 25°C;

[0374] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0375] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0376] Step 2

[0377] The compound of formula (II)-1 is obtained by removing the silicon protecting group from the compound of formula (II)-IM15;

[0378] Alternatively, the compound of formula (II) can be prepared by the following synthetic route:

[0379]

[0380] Step 1

[0381] The compound of formula (II) is obtained by condensing the compound of formula (II)-IM13 and the compound of formula (II)-SM5;

[0382] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0383] 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, preferably 25°C;

[0384] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0385] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0386] The compound of formula (III)-1 in the present invention can be synthesized by the following synthetic route.

[0387]

[0388] Among them, R x” , R y” and R z” The meaning is as described above; LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or chlorine; PG is a protecting group selected from

[0389] Step 1

[0390] The compound of formula (III)-SM1 undergoes a substitution reaction to obtain the compound of formula (III)-IM1.

[0391] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 50°C;

[0392] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably n-heptane.

[0393] Step 2

[0394] The compound of formula (III)-IM2 is obtained by reducing the compound of formula (III)-IM1;

[0395] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a platinum catalyst;

[0396] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 60°C;

[0397] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0398] Step 3

[0399] The compound of formula (III)-IM2 undergoes a substitution reaction to obtain the compound of formula (III)-IM3,

[0400] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0401] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate;

[0402] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably triethylamine.

[0403] Step 4

[0404] The compound of formula (III)-IM4 is obtained by coupling reaction of the compound of formula (III)-IM3;

[0405] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 70°C, 100°C, preferably 70°C;

[0406] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, water, preferably a mixed solvent of tetrahydrofuran and water;

[0407] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably N,N-diisopropylethylamine.

[0408] Step 5

[0409] The compound of formula (III)-IM4 is subjected to a reduction reaction to obtain the compound of formula (III)-IM5;

[0410] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a platinum catalyst;

[0411] 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, preferably 40°C;

[0412] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably tetrahydrofuran.

[0413] Step 6

[0414] The compound of formula (III)-IM6 is obtained by ring-closing reaction of the compound of formula (III)-IM5;

[0415] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

[0416] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, tert-butanol, preferably a mixed solvent of tetrahydrofuran and tert-butanol.

[0417] Step 7

[0418] The compound of formula (III)-IM7 is obtained by a substitution reaction of the compound of formula (III)-IM6;

[0419] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 5°C;

[0420] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

[0421] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably potassium tert-butoxide.

[0422] Step 8

[0423] The compound of formula (III)-IM8 is obtained by reducing the compound of formula (III)-IM7;

[0424] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a palladium catalyst;

[0425] 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, preferably 20°C;

[0426] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0427] Step 9

[0428] The compound of formula (III)-IM9 is obtained by a substitution reaction of the compound of formula (III)-IM8;

[0429] 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 20°C;

[0430] In some embodiments, this step is carried out under alkaline conditions, and the reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide; the inorganic bases include but are not limited to potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, preferably pyridine.

[0431] Step 10

[0432] The compound of formula (III)-IM9 is subjected to a hydrolysis reaction to obtain a compound of formula (III)-IM10;

[0433] 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, preferably 60°C;

[0434] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0435] In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0436] Step 11

[0437] The compound of formula (III)-IM11 is obtained by a cyclization reaction between the compound of formula (III)-IM10 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one;

[0438] 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, 140°C, preferably 140°C;

[0439] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably toluene.

[0440] In some embodiments, the reaction is carried out under acidic conditions, and the reagent providing acidic conditions includes p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0441] Step 12

[0442] The compound of formula (III)-IM12 is obtained by hydrolyzing the compound of formula (III)-IM11;

[0443] 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, 140°C, preferably 100°C;

[0444] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0445] Step 13

[0446] The compound of formula (III)-IM13 is obtained by a substitution reaction between the compound of formula (III)-IM12 and the compound of formula (III)-SM2.

[0447] Substitution reaction occurs to obtain a compound of formula (II)-IM13.

[0448] 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, 140°C, preferably 50°C;

[0449] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

[0450] In some embodiments, this step is carried out in the presence of a suitable base, the base includes an organic base or an inorganic base, the organic base can be selected from N, N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from 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 CO3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 ;

[0451] Step 14

[0452] The compound of formula (III)-IM14 is obtained by condensing the compound of formula (III)-IM13 with the compound of formula (III)-SM3;

[0453] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0454] 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, preferably 25°C;

[0455] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0456] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0457] Step 15

[0458] The compound of formula (III)-1 is obtained by acid hydrolysis of the compound of formula (III)-IM14;

[0459] 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, 140°C, preferably 25°C;

[0460] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and mixed solvents thereof, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0461] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0462] The synthesis method of formula (III)-SM3 is as follows:

[0463] When PG is hour:

[0464]

[0465] Step 1:

[0466] The compound of formula (III)-SM3-1 and the compound of formula (II)-SM3-2 undergo a substitution reaction to obtain a compound of formula (III)-SM3-3;

[0467] 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, 140°C, preferably 0-25°C;

[0468] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably tetrahydrofuran.

[0469] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably K 2 CO 3 .

[0470] Step 2:

[0471] The compound of formula (III)-SM3-3 is subjected to hydrogenation reaction to obtain the compound of formula (III)-SM3;

[0472] 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, 140°C, preferably 25°C;

[0473] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably methanol.

[0474] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a palladium catalyst;

[0475] When PG is When R y’ and R z’ For hydrogen:

[0476]

[0477] Step 1:

[0478] The compound of formula (III)-SM3-4 and the compound of formula (III)-SM3-5 undergo condensation reaction to obtain the compound of formula (III)-SM3;

[0479] 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, 140°C, preferably 25°C;

[0480] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and a mixed solvent thereof, preferably N,N-dimethylformamide.

[0481] Alternatively, the compound of formula (III)-1 can be prepared by the following synthetic route:

[0482]

[0483] Step 1:

[0484] The compound of formula (III)-IM13 and the compound of formula (III)-SM4 undergo a condensation reaction to obtain a compound of formula (III)-IM15;

[0485] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0486] 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, preferably 25°C;

[0487] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0488] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0489] Step 2:

[0490] The compound of formula (III)-1 is obtained by removing the silicon protecting group from the compound of formula (III)-IM15;

[0491] Alternatively, the compound of formula (III)-1 can be prepared by the following synthetic route:

[0492]

[0493] Step 1:

[0494] The compound of formula (III)-1 is obtained by condensing the compound of formula (III)-IM13 with the compound of formula (III)-SM5;

[0495] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0496] 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, preferably 25°C;

[0497] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0498] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0499] The compound of formula (IV) in the present invention can be synthesized by the following synthetic route:

[0500] Among them, R a , R b , R c , R d and R e The meaning is as stated above;

[0501] When q=1,

[0502]

[0503] Step 1

[0504] The compound of formula (IV)-SM1 is subjected to nitration reaction to obtain the compound of formula (IV)-IM1;

[0505] In some embodiments, this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

[0506] Step 2

[0507] The compound of formula (IV)-IM2 is obtained by hydrogenating the compound of formula (IV)-IM1;

[0508] In some embodiments, this step is carried out under a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a palladium catalyst;

[0509] 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, preferably 25°C;

[0510] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0511] Step 3

[0512] The compound of formula (IV)-IM2 undergoes an acylation reaction to obtain the compound of formula (IV)-IM3,

[0513] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 25°C;

[0514] Step 4

[0515] The compound of formula (IV)-IM4 is obtained by reacting the compound of formula (IV)-IM3 with DMF-DMA;

[0516] 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;

[0517] Step 5

[0518] The compound of formula (IV)-IM4 and formula (IV)-SM2 undergo a substitution reaction to obtain a compound of formula (IV)-IM5;

[0519] 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, 140°C, preferably 50°C;

[0520] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, ethanol, N, N-methylpyrrolidone, dimethyl sulfoxide, preferably ethanol;

[0521] Step 6

[0522] The compound of formula (IV)-IM6 is obtained by reducing the compound of formula (IV)-IM5;

[0523] In some embodiments, this step is carried out under a suitable reducing agent, preferably sodium borohydride;

[0524] 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, preferably 0-25°C;

[0525] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, glacial acetic acid, methanol and mixed solutions thereof, preferably glacial acetic acid.

[0526] Step 7

[0527] The amino group of the compound of formula (IV)-IM6 is protected with Fmoc to obtain the compound of formula (IV)-IM7;

[0528] Step 8

[0529] The compound of formula (IV)-IM8 is obtained by removing the acetyl protecting group from the amino group of the compound of formula (IV)-IM7;

[0530] Step 9

[0531] The compound of formula (IV)-IM9 is obtained by a ring-closing reaction of the compound of formula (IV)-IM8 under acidic conditions;

[0532] 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;

[0533] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably toluene and xylene;

[0534] In some embodiments, this step is performed under acidic conditions;

[0535] The reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0536] Step 10

[0537] The compound of formula (IV)-IM10 is obtained by removing the Fmoc protecting group from the compound of formula (IV)-IM9;

[0538] Step 11

[0539] A compound of formula (IV) is obtained by condensing a compound of formula (IV)-IM10 with a compound of formula (IV)-SM4;

[0540] In some embodiments, this step is carried out under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU;

[0541] 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, preferably 25°C;

[0542] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0543] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0544] Or, when q = 0,

[0545]

[0546] LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0547] Step 1

[0548] The compound of formula (IV)-SM5 is subjected to a reduction reaction to obtain the compound of formula (IV)-IM11,

[0549] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, preferably a platinum catalyst;

[0550] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 25°C;

[0551] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate and tetrahydrofuran.

[0552] Step 2

[0553] The compound of formula (IV)-IM12 is obtained by subjecting the compound of formula (IV)-IM11 to Friedel-Crafts acylation reaction;

[0554] In some embodiments, this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

[0555] Step 3

[0556] The compound of formula (IV)-IM13 is obtained by a ring-closing reaction of formula (IV)-IM12 under acidic conditions;

[0557] 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;

[0558] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably toluene and xylene;

[0559] In some embodiments, this step is performed under acidic conditions;

[0560] The reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0561] Step 4

[0562] A compound of formula (IV)-IM14 is obtained by a substitution reaction of formula (IV)-IM13;

[0563] 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, 140°C, preferably 25°C;

[0564] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, ethanol, N, N-methylpyrrolidone, dimethyl sulfoxide, preferably dimethyl sulfoxide;

[0565] Step 5

[0566] The compound of formula (IV)-IM14 is subjected to a reduction reaction to obtain the compound of formula (IV)-IM15,

[0567] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from palladium catalysts, platinum catalysts, rhodium catalysts, triphenylphosphine, triethyl phosphite, preferably triethyl phosphite;

[0568] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 80°C, 100°C, preferably 80°C;

[0569] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, toluene, and mixed solutions thereof, preferably a mixed solution of methanol and toluene.

[0570] Step 6

[0571] The compound of formula (IV)-IM16 is obtained by a substitution reaction between the compound of formula (IV)-IM15 and the compound of formula (IV)-SM6.

[0572] 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, 140°C, preferably 50°C;

[0573] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.

[0574] In some embodiments, this step is carried out in the presence of a suitable base, the base includes an organic base or an inorganic base, the organic base can be selected from N, N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from potassium phosphate (K 3 PO4), sodium hydride (NaH), potassium carbonate (K 2 CO 3 ), sodium carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO3 ), cesium carbonate (Cs 2 CO 3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 ;

[0575] Step 7

[0576] A compound of formula (IV) is obtained by condensing a compound of formula (IV)-IM16 with a compound of formula (IV)-SM4;

[0577] In some embodiments, this step is carried out in the presence of a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU;

[0578] 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, preferably 25°C;

[0579] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0580] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 、Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.

[0581] The compound of formula (V)-1 in the present invention can be used as starting material The synthesis is carried out according to the same synthetic route as formula (III).

[0582] Advantageous Effects of the Invention

[0583] The present invention provides camptothecin compounds represented by formula (I) to formula (IV), and pharmaceutical compositions, preparation methods and uses thereof. The compounds have good anti-tumor activity, have the potential to overcome drug resistance, and can be used to treat abnormal cell proliferation diseases, including but not limited to advanced solid tumors. DETAILED DESCRIPTION

[0584] The present application is further described below by describing specific implementation methods, but this is not intended to limit the present application. Those skilled in the art may make various modifications or improvements based on the teachings of the present application without departing from the basic concept and scope of the present application.

[0585] The abbreviations in the present invention have the following meanings:

[0586]

[0587]

[0588] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0589] Nuclear magnetic resonance (NMR) 1 H NMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; hexadeuterated dimethyl sulfoxide (DMSO-d 6 ); the internal standard substance was tetramethylsilane (TMS).

[0590] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the Examples are shown below.

[0591] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0592] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0593] Example 1 (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypent-3-ynamide and (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypent-3-ynamide

[0594]

[0595] The compound 2-hydroxypent-3-ynoic acid (4.29 mg, 37.63 μmol) was dissolved in DMF (1 mL), and HATU (21.46 mg, 56.44 μmol), SM1-1 (10.00 mg, 22.94 μmol) and DIPEA (7.29 mg, 56.44 μmol) were added, and the mixture was reacted at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high performance liquid chromatography (conditions as follows) to obtain the title compounds 1-1-A 3.24 mg and 1-1-B 3.98 mg.

[0596] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0597] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0598]

[0599]

[0600] Retention time: 1-1-A: 10.8min; 1-1-B: 11.1min.

[0601] The structural characterization data of 1-1-A are as follows:

[0602] 1 H NMR (400 MHz, DMSO-d 6 )δ8.58(d,J=8.4Hz,1H),7.78(d,J=11.2Hz,1H),7.31(s,1H),6.54(s,1H),6.17(d,J=6.0Hz,1H),5.58-5.47(m,1H),5.42(s,2H ),5.23(s,2H),4.73-4.64(m,1H),3.25-3.06(m,2H),2.39(s,3H),2.27-2.05(m,2H),1.96-1.77(m,5H),0.87(t,J=7.2Hz,3H).

[0603] ESI-MS (m / z): 532.2 [M+H] + .

[0604] The structural characterization data of 1-1-B are as follows:

[0605] 1 H NMR (400 MHz, DMSO-d 6)δ8.62(d,J=8.8Hz,1H),7.78(d,J=10.8Hz,1H),7.31(s,1H),6.54(s,1 H),6.19(d,J=6.0Hz,1H),5.58-5.47(m,1H),5.42(s,2H),5.21(d,J=4. 8Hz,2H),4.73-4.65(m,1H),3.27-3.06(m,2H),2.39(s,3H),2.27-2.05 (m,2H),1.93-1.80(m,2H),1.80(d,J=2.0Hz,3H),0.87(t,J=7.2Hz,3H).

[0606] ESI-MS (m / z): 532.2 [M+H] + .

[0607] Example 2 (S)-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)-2-hydroxy-3-eneamide and (R)-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)-2-hydroxy-3-eneamide

[0608]

[0609] Compound ethylene glycolic acid (9.61 mg, 94.07 μmol) was dissolved in DMF (2 mL), HATU (44.70 mg, 117.58 μmol), compound SM1-1 (25.00 mg, 0.047 mmol) and DIPEA (24.30 mg, 188.13 μmol) were added, and the mixture was reacted at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-7-A (4.00 mg) and 1-7-B (1.38 mg).

[0610] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0611] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0612]

[0613]

[0614] Retention time: 1-7-A: 8.7 min; 1-7-B: 9.1 min.

[0615] The structural characterization data of 1-7-A are as follows:

[0616] 1 H NMR (400 MHz, DMSO-d 6 )δ8.51(s,J=8.8Hz,1H),7.76(s,J=10.8Hz,1H),7.29(s,1H),6.52(s,1H),6.15-6.04(m,1H),5.58-5.49(m,1H),5.42(s,2H),5.39(s ,1H),5.24-5.01(m,3H),4.54(s,J=4.9Hz,1H),3.24-3.05(m,2H),2.37(s,3H),2.16(s,2H),1.91-1.79(m,2H),0.87(t,J=7.2Hz,3H).

[0617] ESI-MS (m / z): 520.1 [M+H] + .

[0618] The structural characterization data of 1-7-B are as follows:

[0619] 1 H NMR (400 MHz, DMSO-d 6 )δ8.48(s,1H),7.78(s,1H),7.30(s,1H),6.12-5.92(m,1H),5.54-5.47(m,1H),5.42(s,2H),5.37(dt,J=1.7Hz,1H),5.18(s,2H),5.16-5.1 4(m,1H),4.5-4.52(m,1H),3.22-3.08(m,2H),2.38(s,3H),2.25-2.1 6(m,1H),2.16-2.06(m,1H),1.93-1.79(m,2H),0.87(t,J=7.2Hz,3H).

[0620] ESI-MS (m / z): 520.1 [M+H] + .

[0621] Example 3 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0622]

[0623] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene

[0624] At 25°C, compound 2-1-01 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), concentrated sulfuric acid (25 mL) was added, and the mixture was heated to 50°C. NBS (6.22 g, 34.97 mmol) was added in batches at 50°C, and the reaction was maintained at 50°C for 2 hours. The reaction was detected by thin layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction solution cooled to room temperature was added dropwise to ice water, extracted with toluene, and the organic phases were combined, washed with sodium sulfite solution, water, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography, and the preparative solution was freeze-dried to obtain 4.88 g of the title compound.

[0625] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0626] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0627] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 60 40 60 10 60 40 60 40 100 0 60

[0628] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline

[0629] At 25°C, compound 2-1-02 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), and platinum carbon (2.00 g, 19.48 mmol, content 5%) was added. After hydrogen replacement, the mixture was reacted at 60°C for 4 hours under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered, and the filtrate was concentrated to obtain 3.68 g of the crude title compound, which was directly used in the next step without further purification.

[0630] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide

[0631] At 20°C, compound 2-1-03 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the reaction was maintained at 20°C for 20 hours, and the reaction was monitored by HPLC-MS. Water was added to the reaction solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried with a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0632] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid

[0633] At 20°C, compound 2-1-04 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), and vinylacetic acid (708.31 mg, 8.23 ​​mmol), DIPEA (1.95 g, 15.08 mmol), tri(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction system was replaced with nitrogen and heated to 70°C for 5 hours. The reaction was monitored by HPLC-MS. 1N sodium hydroxide solution was added to the reaction solution to adjust pH=8, and ethyl acetate was added for extraction. The remaining aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was directly used in the next step.

[0634] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid

[0635] At 20°C, compound 2-1-05 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), Pd / C (0.52 g, 10% content) was added, the system was replaced with hydrogen and reacted at 40°C for 2 hours under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43 g of the title compound, which was directly used in the next step without further purification.

[0636] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0637] Compound 2-1-06 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise, and the reaction was maintained at 5°C for 4 hours. The reaction was monitored by HPLC-MS. The reaction solution was added to water, dissolved in 10N sodium hydroxide to adjust pH = 9, extracted with ethyl acetate, combined organic phases, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

[0638] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0639] At 5°C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), and a THF solution (16 mL) of compound 2-1-07 (1.53 g, 6.08 mmol) was added dropwise, and amyl nitrite (1.14 g, 9.73 mmol) was added dropwise after 10 minutes, and the reaction was maintained at 5°C for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was adjusted to pH = 5 with 1N hydrochloric acid, extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was beaten with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0640] Step 8: N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0641] At 20°C, compound 2-1-08 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), Pd / C (0.15 g, 10% content) was added, and the system was replaced with hydrogen and kept at 5°C for 2 hours under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next step without further purification.

[0642] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diethylamide

[0643] At 20°C, compound 2-1-09 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), acetic anhydride (2 mL) was added, and the reaction was maintained at 20°C for 2 hours. The reaction was monitored by HPLC-MS. The reaction solution was added to water, extracted with ethyl acetate, the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrate was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 0.22 g of the title compound.

[0644] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

[0645] At 20°C, compound 2-1-10 (0.45 g, 1.46 mmol) was dissolved in methanol (16 mL), 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 hours, and the reaction was monitored by HPLC-MS. Saturated sodium bicarbonate solution was added to the cooled reaction solution to adjust the pH to 8, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.23 g of the title compound, which was used directly in the next step without further purification.

[0646] Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

[0647] Compound 2-1-11 (0.23 g, 0.78 mmol) was dissolved in toluene (10 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (0.23 g, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added, and the mixture was heated to 140°C for 5 hours, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated, and the crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-10%) to obtain 0.15 g of the title compound.

[0648] Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0649] Compound 2-1-12 (40.00 mg, 0.08 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100°C for 5 hours, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered, and the filtrate was purified by preparative HPLC. The preparative solution was freeze-dried to obtain 12.00 mg of trifluoroacetate salt of the title compound 2-23.

[0650] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0651] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0652] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0653] The structural characterization data are as follows:

[0654] ESI-MS (m / z): 452.1 [M+H] + .

[0655] Step 13: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

[0656] At 25°C, the trifluoroacetate of compound 2-23 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 μmol), HATU (62.25 mg, 163.81 μmol) and N,N-diisopropylethylamine (42.34 mg, 327.63 μmol) were added in sequence. The reaction was maintained at 25°C for 0.5 hour and the reaction was monitored by HPLC-MS / MS. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) to separate two isomers, which are named 2-1-13-A (15.00 mg, Rf value is 0.3) and 2-1-13-B (12.00 mg, Rf value is 0.35) according to their Rf values.

[0657] Step 14: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0658] At 25°C, 2-1-13-A (15.00 mg) and 2-1-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two reaction bottles, and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid (v / v=13 / 1) (50 uL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 2-1-A (6.94 mg) and 2-1-B (4.00 mg).

[0659] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0660] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0661] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 3 20 80 28 18 90 10 28

[0662] 2-1-A structural characterization data are as follows:

[0663] 1 H NMR (400 MHz, DMSO-d 6 )δ8.43(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.65-5.36(m,4H),5.21(q,J=19.0Hz,2H),3.9 5(d,J=5.7Hz,2H),3.26-3.11(m,2H),2.53(s,3H),2.30-2.08(m,2H),1.94-1.79(m,2H),0.87(t,J=7.3Hz,3H).

[0664] ESI-MS (m / z): 510.1 [M+H] + .

[0665] The structural characterization data of 2-1-B are as follows:

[0666] 1 H NMR (400 MHz, DMSO-d 6 )δ8.45(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.64-5.35(m,4H),5.19(q,J=19.0Hz,2H),3.9 7(d,J=5.2Hz,2H),3.27-3.10(m,2H),2.51(s,3H),2.27-2.10(m,2H),1.93-1.80(m,2H),0.88(t,J=7.3Hz,3H).

[0667] ESI-MS (m / z): 510.1 [M+H] + .

[0668] Example 4 (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropylamine

[0669]

[0670] Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propylamine and (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propylamine

[0671] At 25°C, the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and (S)-2-((tert-butyldiphenylsilyl)oxy)propionic acid (24.21 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence. The reaction was maintained at 25°C for 1 hour and the reaction was monitored by HPLC-MS / MS. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=15:1) to separate two isomers, which are named 2-7-01-A (6.00 mg, Rf value is 0.35) and 2-7-01-B (6.00 mg, Rf value is 0.40) according to their Rf values.

[0672] Step 2: Synthesis of (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropylamine

[0673] At 25°C, 2-7-01-A (6.00 mg, 7.87 μmol) and 2-7-01-B (6.00 mg, 7.87 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two reaction bottles, and tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid mixed solution (v / v=13 / 1) (50 μL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 2-7-A (2.50 mg) and 2-7-B (3.00 mg).

[0674] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0675] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0676] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 16 90 10 28

[0677] The structural characterization data of 2-7-A are as follows:

[0678] 1 H NMR (400 MHz, DMSO-d 6 )δ8.38(d,J=8.8Hz,1H),8.15(s,1H),7.31(s,1H),6.55(s,1H),5.56-5.47(m,2H),5.42(s,2H),5.26-5.11(m,2H),4.17-4.06(m, 1H),3.27-3.10(m,2H),2.52(s,3H),2.27-2.08(m,2H),1.86(tt,J=14.1,7.3Hz,2H),1.30(d,J=6.7Hz,3H),0.87(t,J=7.3Hz,3H).

[0679] ESI-MS (m / z): 524.2 [M+H] + .

[0680] The structural characterization data of 2-7-B are as follows:

[0681] 1 H NMR (400 MHz, DMSO-d 6)δ8.48(d,J=9.1Hz,1H),8.12(s,1H),7.30(s,1H),6.54(s,1H),5.67(d,J=4.8Hz,1H),5.55(dd,J=1 4.6,7.3Hz,1H),5.43(s,2H),5.24(d,J=19.0Hz,1H),5.03(d,J=19.0Hz,1H),4.21-4.08(m,1H),3.28 -3.08(m,2H),2.51(s,3H),2.16(d,J=6.2Hz,2H),1.94-1.82(m,2H),1.42(d,J=6.8Hz,3H),0.88(t,J=7.3Hz,3H).ESI-MS(m / z):524.2[M+H] + .

[0682] Example 5 (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide Amide and (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[0683]

[0684] Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylethyl Amide and (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylacetamide and (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylacetamide and Synthesis of (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropylacetamide

[0685] At 25°C, the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2-((tert-butyldiphenylsilyl)oxy)-2-cyclopropylacetic acid (26.13 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence. The reaction was maintained at 25°C for 1 hour and the reaction was monitored by HPLC-MS / MS. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=15:1) to separate and obtain two groups of isomers, which are named 2-12-01-A (8.00 mg, Rf value is 0.35) and 2-12-01-B (10.00 mg, Rf value is 0.40) according to the Rf value.

[0686] Step 2: (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide Synthesis of 2-hydroxy-1-(2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide and 2-hydroxy-1-(2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[0687] At 25°C, 2-12-01-A (8.00 mg, 10.15 μmol) and 2-12-01-B (10.00 mg, 12.68 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two reaction bottles, and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was maintained at 25°C for 0.5 h and the reaction was monitored by HPLC-MS / MS. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography. The reaction with 2-12-01-A as the raw material separated two isomeric products, and the prepared solution was lyophilized to obtain corresponding compounds 2-12-A (0.77 mg) and 2-12-B (1.03 mg); the reaction with 2-12-01-B as the raw material separated two isomeric products, and the prepared solution was lyophilized to obtain corresponding compounds 2-12-C (2.50 mg) and 2-12-D (1.00 mg). The purification conditions of 2-12-A / 2-12-B are as follows:

[0688] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0689] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0690]

[0691]

[0692] Peak retention time: 2-12-A: 10.0-11.0min, 2-12-B: 11.0-12.5min

[0693] 2-12-C / 2-12-D purification conditions are as follows:

[0694] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0695] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0696] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0697] Peak retention time: 2-12-C: 10.6-11.4min, 2-12-D: 11.4-12.5min

[0698] The structural characterization data of 2-12-A are as follows:

[0699] 1 H NMR (400 MHz, DMSO-d 6 )δ8.38(d,J=8.9Hz,1H),8.15(s,1H),7.30(s,1H),6.54(s,1H),5.63-5.53(m,1H), 5.51(d,J=5.1Hz,1H),5.42(s,2H),5.28(d,J=19.2Hz,1H),5.16(d,J=19.1Hz,1H), 3.60(t,J=5.6Hz,1H),3.28-3.11(m,2H),2.52(s,3H),2.22-2.10(m,2H),1.86(tt, J=14.1,7.3Hz,2H),1.23(d,J=4.9Hz,1H),0.87(t,J=7.2Hz,3H),0.56-0.36(m,4H).

[0700] ESI-MS (m / z): 550.2 [M+H] + .

[0701] The structural characterization data of 2-12-B are as follows:

[0702] 1 H NMR (400 MHz, DMSO-d 6)δ8.36(d,J=8.7Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.57-5.48(m,1H ),5.42(s,2H),5.40(d,J=5.4Hz,1H),5.26(d,J=19.3Hz,1H),5.18(d,J=19.0Hz,1 H),3.65-3.60(m,1H),3.26-3.12(m,2H),2.52(s,3H),2.26-2.09(m,2H),1.86(tt ,J=14.1,7.2Hz,2H),1.19-1.08(m,1H),0.87(t,J=7.3Hz,3H),0.51-0.27(m,4H).

[0703] ESI-MS (m / z): 550.2 [M+H] + .

[0704] The structural characterization data of 2-12-C are as follows:

[0705] 1 H NMR (400 MHz, DMSO-d 6 )δ8.42(d,J=9.0Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.56(dd,J=14.8,6.8Hz,1H),5.52( d,J=5.2Hz,1H),5.42(s,2H),5.29(d,J=19.2Hz,1H),5.16(d,J=19.1Hz,1H),3.62-3.58(m,1H),3.27 -3.08(m,2H),2.51(s,3H),2.27-2.08(m,2H),1.87(tt,J=14.0,7.2Hz,2H ),1.25(dd,J=13.2,6.8Hz,1H),0.87(t,J=7.3Hz,3H),0.64-0.28(m,4H).

[0706] ESI-MS (m / z): 550.1 [M+H] + .

[0707] The structural characterization data of 2-12-D are as follows:

[0708] 1 H NMR (400 MHz, DMSO-d 6)δ8.38(d,J=8.7Hz,1H),8.16(s,1H),7.31(s,1H),6.54(s,1H),5.59-5.49(m,1H),5. 43(d,J=5.3Hz,1H),5.43(s,2H),5.26(d,J=19.1Hz,1H),5.17(d,J=19.0Hz,1H),3.64 (t,J=5.8Hz,1H),3.17(dd,J=15.6,8.3Hz,2H),2.27-2.07(m,2H),1.94-1.79(m,2H), 1.19-1.06(m,1H),0.87(t,J=7.3Hz,3H),0.49-0.28(m,4H).ESI-MS(m / z):550.1[M+H] + .

[0709] Example 6 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxy-2-methylpropylamine and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxy-2-methylpropylamine

[0710]

[0711] At 25°C, the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) was dissolved in N, N-dimethylformamide (1 mL), and 2-((tert-butyldimethylsilyl)oxy)-2-methylpropionic acid (16.10 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N, N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence, and the reaction was maintained at 25°C for 1 hour, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified and separated by preparative HPLC to obtain two isomers, and the preparative solutions were lyophilized respectively, and the two isomers were named 2-17-A (2.65 mg) and 2-17-B (2.69 mg) according to the peak retention time.

[0712] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0713] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0714] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0715] The peak retention times are: 2-17-A: 9.5-10.2 min and 2-17-B: 10.4-10.6 min.

[0716] The structural characterization data of 2-17-A are as follows:

[0717] 1 H NMR (400 MHz, DMSO-d 6 )δ8.36(d,J=9.1Hz,1H),8.13(s,1H),7.30(s,1H),6.54(s,1H),5.55-5.45(m,2H),5.42(s,2H),5.28(d,J=19.0Hz,1H),5.05(d,J=19.0H z,1H),3.27-3.11(m,2H),2.51(s,1H),2.24-2.10(m,2H),1.86(tt,J=14.0,7.2Hz,2H),1.46(s,3H),1.35(s,3H),0.87(t,J=7.3Hz,3H).

[0718] MS m / z(ESI):538.2[M+H] + .

[0719] The structural characterization data of 2-17-B are as follows:

[0720] 1 H NMR (400 MHz, DMSO-d 6 )δ8.40(d,J=9.2Hz,1H),8.13(s,1H),7.30(s,1H),6.53(s,1H),5.60-5.46(m,2H),5.42(s,2H),5.29(d,J=19.0Hz,1H),5.02(d,J=19.0H z,1H),3.28-3.08(m,2H),2.50(s,3H),2.22-2.10(m,2H),1.87(tt,J=14.2,7.2Hz,2H),1.47(s,3H),1.35(s,3H),0.88(t,J=7.3Hz,3H).

[0721] MS m / z(ESI):538.2[M+H] + .

[0722] Example 7 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0723]

[0724] Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide and 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

[0725] At 25°C, the hydrochloride of 2-23 (30.00 mg, 61.43 umol) was dissolved in N,N-dimethylformamide (1 mL), and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-1-carboxylic acid (25.10 mg, 73.72 umol), HATU (35.01 mg, 92.14 umol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence. The reaction was maintained at 25°C for 1 hour and the reaction was monitored by HPLC-MS / MS. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=15:1) to separate two isomers, which are named 2-20-01-A (4.00 mg, Rf value is 0.30) and 2-20-01-B (4.00 mg, Rf value is 0.35) according to their Rf values.

[0726] Step 2: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0727] At 25°C, 2-20-01-A (4.00 mg, 5.17 μmol) and 2-20-01-B (4.00 mg, 5.17 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two reaction bottles, and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid (v / v=13 / 1) (50 μL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 2-20-A (0.71 mg) and 2-20-B (1.05 mg).

[0728] 2-20-A purification conditions are as follows:

[0729] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0730] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0731] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 16 90 10 28

[0732] 2-20-B purification conditions are as follows:

[0733] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0734] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0735] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 20 28 18 80 20 28

[0736] The structural characterization data of 2-20-A are as follows:

[0737] 1 H NMR (400 MHz, DMSO-d 6)δ8.58(d,J=9.0Hz,1H),8.15(s,1H),7.31(s,1H),6.55(s,1H),6.30(s,1 H),5.55(dd,J=13.2,8.2Hz,1H),5.43(s,2H),5.26(d,J=19.0Hz,1H),5.10 (d,J=19.0Hz,1H),3.29-3.09(m,2H),2.52(s,3H),2.31-2.15(m,2H),1.93 -1.80(m,2H),1.25-1.14(m,2H),0.98-0.90(m,2H),0.87(t,J=7.3Hz,3H).

[0738] ESI-MS (m / z): 536.2 [M+H] + .

[0739] The structural characterization data of 2-20-B are as follows:

[0740] 1 H NMR (400 MHz, DMSO-d 6 )δ8.63(d,J=9.0Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),6.35(s,1H ),5.55(dd,J=13.5,8.6Hz,1H),5.43(s,2H),5.29(d,J=19.1Hz,1H),5.08( d,J=19.1Hz,1H),3.28-3.10(m,2H),2.51(s,3H),2.30-2.14(m,2H),1.93- 1.81(m,2H),1.26-1.14(m,2H),1.02-0.90(m,2H),0.89(d,J=10.9Hz,3H).

[0741] ESI-MS (m / z): 536.2 [M+H] + .

[0742] Example 8 (1S, 9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R, 9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0743]

[0744] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline

[0745] Compound 3-1-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. After addition, the mixture was reacted at room temperature for 16 hours, and the reaction was detected by HPLC-MS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

[0746] The structural characterization data are as follows:

[0747] 1 H NMR (400 MHz, DMSO-d 6 )δ6.77(dd,J=2.5,1.4Hz,1H),6.51(dd,J=11.7,2.5Hz,1H),5.84(s,2H).

[0748] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide

[0749] Compound 3-1-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection. After the addition was completed, the temperature was raised to 50°C for reaction for 15 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with methanol (5 mL), it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0750] The structural characterization data are as follows:

[0751] ESI-MS (m / z): 265.9 [M+H] + .

[0752] Step 3: Synthesis of (E)-4-(5-acetylamino-2-chloro-3-fluorophenyl)-3-butenoic acid

[0753] Compound 3-1-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL), and then N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tri(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and palladium acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100 ° C under a nitrogen atmosphere for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added to shake and layer. After separating the lower aqueous phase, the pH was adjusted to about 3 with a 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

[0754] The structural characterization data are as follows:

[0755] ESI-MS (m / z): 272.0 [M+H] + .

[0756] Step 4: Synthesis of 4-(5-acetylamino-2-chloro-3-fluorophenyl)butyric acid

[0757] The crude product of compound 3-1-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and then 10% palladium carbon (0.10 g) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

[0758] The structural characterization data are as follows:

[0759] ESI-MS (m / z): 274.0 [M+H] + .

[0760] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0761] The crude product of compound 3-1-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL), cooled to 5 °C, and trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition was completed, the temperature was kept at 5 °C for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column to obtain 0.43 g of the title compound.

[0762] The structural characterization data are as follows:

[0763] ESI-MS (m / z): 256.1 [M+H] + .

[0764] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0765] Tetrahydrofuran (16mL) and tert-butanol (4mL) were added to the reaction solution bottle, cooled to 5°C in an ice bath, potassium tert-butoxide (415.18mg, 3.70mmol) was added, and compound 3-1-06 (0.43mg, 1.68mmol) was dissolved in tetrahydrofuran (1mL) and slowly added dropwise to the reaction solution. Isoamyl nitrite (315.24mg, 2.69mmol) was added 10 minutes later. After the addition, the reaction was maintained at 5°C for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00mg of the crude product of the title compound.

[0766] The structural characterization data are as follows:

[0767] ESI-MS (m / z): 285.0 [M+H] + .

[0768] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0769] The crude product of compound 3-1-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), and then 3 mol / L aqueous hydrochloric acid solution (1 mL) and 10% palladium carbon (40.00 mg) were added. After the addition, the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out at room temperature for 1 hour under a hydrogen atmosphere. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of the crude hydrochloride of the title compound.

[0770] The structural characterization data are as follows:

[0771] ESI-MS (m / z): 271.0 [M+H] + .

[0772] Step 8: Synthesis of (9H-fluoren-9-yl)methyl(8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0773] The crude hydrochloride of compound 3-1-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), and then sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol) were added. After the addition, the mixture was stirred at room temperature for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse phase column (acetonitrile: 0.05% formic acid water = 20%-100%) to obtain 301.00 mg of the title compound.

[0774] The structural characterization data are as follows:

[0775] ESI-MS (m / z): 493.2 [M+H] + .

[0776] Step 9: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0777] Compound 3-1-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL), and 12 mol / L concentrated hydrochloric acid (1 mL) was added. After the addition, the temperature was raised to 60°C for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

[0778] The structural characterization data are as follows:

[0779] ESI-MS (m / z): 451.1 [M+H] + .

[0780] Step 10: Synthesis of (9H-fluoren-9-yl)methyl((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)carbamate

[0781] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72 mg, 526.96 μmol) and compound 3-1-10 (198.00 mg, 439.13 μmol) were added to toluene (10 mL), and then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition was completed, the temperature was raised to 140 ° C and reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C to obtain a crude product. The crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0782] The structural characterization data are as follows:

[0783] ESI-MS (m / z): 678.1 [M+H] + .

[0784] Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0785] Compound 3-1-11 (201.18 mg, 296.67 μmol) was dissolved in N, N-dimethylformamide (4 mL), and then diethylamine (108.49 mg, 1.48 mmol) was added. After the addition was completed, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was evaporated under reduced pressure to remove ethylenediamine, the pH was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 3-1-A (44.00 mg) and 3-1-B (43.00 mg).

[0786] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0787] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0788] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 70 30 28

[0789] 3-1-A (6min LCMS peak at the front, retention time: 1.276min)

[0790] The structural characterization data are as follows:

[0791] 1 H NMR (400 MHz, DMSO-d 6 )δ8.00(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H),5.62(d,J=19.3Hz,1H),5.44(s,2H),5.38(d,J=19.3Hz,1H),4.43-4.38(m,1 H),3.28-3.10(m,2H),2.22-2.12(m,1H),2.12-2.02(m,1H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).ESI-MS(m / z):456.1[M+H] + .

[0792] The structural characterization data of 3-1-B (6min LCMS peak is later, retention time: 1.300min) are as follows:

[0793] 1 H NMR (400 MHz, DMSO-d 6 )δ7.98(d,J=10.3Hz,1H),7.32(s,1H),5.61(d,J=19.4Hz,1H),5.44(s,2H),5.32(d,J=19.4Hz,1H), 4.44-4.36(m,1H),3.33-3.25(m,1H),3.22-3.11(m,1H),2.23-2.13(m,1H),2.11-2.03(m,1H),1.96 -1.82(m,2H),0.89(t,J=7.3Hz,3H).

[0794] ESI-MS (m / z): 456.1 [M+H] + .

[0795] 6min LCMS conditions:

[0796] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0797] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0798] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0799] Example 9 N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0800]

[0801] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaazaoctadecane-22-ynecarboxylic acid

[0802] Compound 3-4-01 (30.00 mg, 70.00 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2,5-dioxopyrrolidin-1-yl 6-(2-(methanesulfonic acid)pyrimidin-5-yl)hexyl-5-ynamide (28.00 mg, 77.00 μmol) was added, and the mixture was reacted at room temperature for 1 hour, and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by preparative HPLC, and the preparative solution was freeze-dried to obtain the title compound 3-4-03 (20.00 mg).

[0803] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0804] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0805] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0806] The structural characterization data are as follows:

[0807] ESI-MS (m / z): 691.0 [M+18] + .

[0808] Step 2: N-((S)-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide and N-((S)-10-benzyl-1-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide

[0809] The single-configuration compound 3-1-A (36.00 mg, 79.70 μmol) and compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N, N-dimethylformamide (2 mL), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 3-4-04-A (51.00 mg).

[0810] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0811] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0812] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0813] The structural characterization data are as follows:

[0814] ESI-MS (m / z): 1111.0 [M+H] + .

[0815] The single-configuration compound 3-1-B (36.00 mg, 79.70 μmol) and compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N, N-dimethylformamide (2 mL), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After the addition was completed, the reaction was carried out at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 3-4-04-B (52.00 mg).

[0816] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0817] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0818] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0819] The structural characterization data are as follows:

[0820] ESI-MS (m / z): 1111.0 [M+H] + .

[0821] Step 3: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0822] Weigh compound 3-4-04-A (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 3-4-A (4.75 mg) of a single configuration.

[0823] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0824] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0825] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0826] The structural characterization data are as follows:

[0827] 1 H NMR (400 MHz, DMSO-d 6 )δ8.50(d,J=8.9Hz,1H),8.05(d,J=10.3Hz,1H),7.33(s,1H),6.55(s,1H),5.67-5.60(m,1H),5.49(t,J=5.8Hz,1H),5.43( s,2H),5.21(s,2H),3.96(d,J=5.8Hz,2H),3.32-3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0828] ESI-MS (m / z): 514.0 [M+H] + .

[0829] Weigh compound 3-4-04-B (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 3-4-B (8.24 mg) of a single configuration.

[0830] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0831] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0832] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0833] The structural characterization data are as follows:

[0834] 1 H NMR (400 MHz, DMSO-d 6 )δ8.52(d,J=9.0Hz,1H),8.05(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.68-5.58(m,1H),5.53(t,J=5.8Hz,1H),5.43(d,J=2.9H z,2H),5.20(d,J=7.3Hz,2H),3.97(d,J=5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J=7.3Hz,3H).

[0835] ESI-MS (m / z): 514.0 [M+H] + .

[0836] Example 10 (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0837]

[0838] Step 1: Synthesis of 1-(4-fluoro-3-methylphenyl)-3-(isopropylamino)propan-1-one

[0839] Compound 4-12-01 (500.00 mg, 3.29 mmol), formaldehyde aqueous solution (2.5 mL, 37%) and isopropylamine (388.46 mg, 6.57 mmol) were dissolved in isopropanol (5 mL) at 20°C, and concentrated hydrochloric acid (2.5 mL) was added dropwise at 0°C. The reaction solution was stirred at 100°C for 16 hours, and the reaction was detected by HPLC-MS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC, and the preparative solution was freeze-dried to obtain 200.00 mg of the title compound.

[0840] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0841] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0842] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0843] The structural characterization data are as follows:

[0844] ESI-MS (m / z): 224.1 [M+H] + .

[0845] Step 2: Synthesis of 1-(4-fluoro-5-methyl-2-nitrophenyl)-3-(isopropylamino)propan-1-one

[0846] Compound 4-12-02 (100.00 mg, 0.49 mmol) was dissolved in concentrated sulfuric acid (0.5 mL) at 0°C, potassium nitrate (54.34 mg, 0.54 mmol) was added, the reaction solution was kept at 0°C for 1 hour, and the reaction was detected by HPLC-MS / MS. The reaction solution was poured into ice water and purified by reverse phase column (acetonitrile: 0.05% formic acid water = 0-30%) to obtain 90.00 mg of the title compound.

[0847] The structural characterization data are as follows:

[0848] ESI-MS (m / z): 269.0 [M+H] + .

[0849] Step 3: Synthesis of 1-(2-amino-4-fluoro-5-methylphenyl)-3-(isopropylamino)propan-1-one

[0850] At 25°C, compound 4-12-03 (200.00 mg, 0.75 mmol) was dissolved in methanol (20.0 mL), and 10% palladium carbon (10.00 mg) was added. The reaction solution was replaced with hydrogen and kept at 20°C in a hydrogen environment for 16 hours. The reaction was detected by HPLC-MS / MS. The reaction solution was filtered and concentrated under reduced pressure to obtain 183.00 mg of the title compound.

[0851] The structural characterization data are as follows:

[0852] ESI-MS (m / z): 239.1 [M+H] + .

[0853] Step 4: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(2-(isopropylamino)ethyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione

[0854] At 25°C, compound 4-12-04 (50.00 mg, 0.21 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (55.23 mg, 0.21 mmol) were dissolved in toluene (3 mL), p-toluenesulfonic acid (3.61 mg, 0.02 mmol) was added, and the reaction solution was reacted at 130°C for 4 hours, and the reaction was detected by HPLC-MS. The reaction solution was concentrated under reduced pressure, the crude product was purified by preparative HPLC, and the preparative solution was freeze-dried to obtain 2.00 mg of trifluoroacetate of the title compound.

[0855] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0856] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0857] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 8 92 28 2.00 8 92 28 18.00 60 40 28

[0858] The structural characterization data are as follows:

[0859] 1 H NMR (400 MHz, DMSO-d 6 )δ8.58(s,2H),8.22(d,J=8.1Hz,1H),7.97(d,J=10.7Hz,1H),7.35(s,1H),6.59(s,1H),5.47(s,2H),5.41(s,2H), 3.58-3.45(m,3H),3.31-3.23(m,2H),2.56(s,3H),1.98-1.80(m,2H),1.26(d,J=6.3Hz,6H),0.89(t,J=7.3Hz,3H).

[0860] ESI-MS (m / z): 466.2 [M+H] +

[0861] Step 5: Synthesis of (S)-2-((tert-butyldiphenylsilyl)oxy)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

[0862] Compound 4-12-05 (22.00 mg, 47.26 μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (16.35 mg, 51.99 μmol) were dissolved in N,N-dimethylformamide (1 mL), and then HATU (21.55 mg, 56.71 μmol) and N,N-diisopropylethylamine (18.32 mg, 141.78 μmol) were added. After addition, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to obtain 18.00 mg of the title compound.

[0863] The structural characterization data are as follows:

[0864] ESI-MS (m / z): 762.3 [M+H] + .

[0865] Step 6: Synthesis of (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0866] Compound 4-12-06 (18.00 mg, 23.62 μmol) was dissolved in N,N-dimethylformamide (1 mL), and potassium fluoride (6.86 mg, 118.12 μmol) was added. The temperature was raised to 50°C for 1 hour, and the reaction was detected by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain 1.53 mg of the title compound.

[0867] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0868] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0869] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 18.00 90 10 28

[0870] The structural characterization data are as follows:

[0871] 1 H NMR (400 MHz, DMSO-d 6 )δ8.53(d,J=8.2Hz,1H),7.91(d,J=10.8Hz,1H),7.32(s,1H),6.55(s,1H),5.44(d,J=13.8Hz,4H),4.72(t,J=5.5Hz,1H),4.21(d,J= 5.5Hz,2H),3.99-3.90(m,1H),3.54-3.38(m,4H),2.54(s,3H),1.92-1.83(m,2H),1.17(dd,J=6.6,3.1Hz,6H),0.87(t,J=7.3Hz,3H).

[0872] ESI-MS (m / z): 524.2 [M+H] + .

[0873] Example 11 (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropanecarboxamide

[0874]

[0875] The raw material (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinoline-3,14(4H,12H)-dione (4-10-01, prepared according to the synthesis method of patent WO2020219287, 30.00 mg, 67.00 μmol), 1-hydroxycyclopropanecarboxylic acid (7.56 mg, 0.074 mmol) was dissolved in DMF (1 mL), HBTU (34.30 mg, 0.14 mmol) and diisopropylethylamine (26.09 mg, 0.20 mmol) were added under stirring, and the mixture was reacted at room temperature for 4 hours. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with saturated brine and concentrated under reduced pressure. The concentrate was purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) and then by preparative high performance liquid chromatography to obtain 1.20 mg of a solid.

[0876] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0877] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0878]

[0879]

[0880] The structural characterization data are as follows:

[0881] 1 H NMR (400 MHz, DMSO-d 6 )δ8.96(t,J=6.0Hz,1H),8.51(d,J=8.0Hz,1H),7.90(d,J=10.8Hz,1H),7.31(s,1H),6.53(s,1H),6.30(s,2H),6.30(s,1H),5.52(s,2H),5. 44(s,2H),4.84(d,J=6.0Hz,2H),2.51(s,3H),1.91-1.81(m,2H),1.01(dd,J=7.2,4.1Hz,2H),0.87(t,J=7.3Hz,3H),0.83(t,J=3.6Hz,2H).

[0882] ESI-MS (m / z): 494.1 [M+1] + .

[0883] Example 12 (1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo [de] pyrano [3',4':6,7] indolizino [1,2-b] quinoline-10,13-dione and (1R, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo [de] pyrano [3',4':6,7] indolizino [1,2-b] quinoline-10,13-dione

[0884]

[0885] Step 1: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid

[0886] Weigh N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (2.00 g, 8.13 mmol) and 2-methyl-3-butenoic acid (976.44 mg, 9.75 mmol) and dissolve them in a mixed solvent of 1,4-dioxane (15 mL) and water (5 mL), then add tri(o-tolyl)phosphine (247.37 mg, 812.76 μmol), palladium acetate (91.24 mg, 406.38 μmol) and N,N-diisopropylethylamine (2.31 g, 17.88 mmol). After the addition, replace the reaction system with nitrogen three times, and heat to 80 ° C under nitrogen atmosphere for 3 hours. Detect the reaction by high performance liquid chromatography-mass spectrometry. After the reaction solution is cooled to room temperature, add 1 mol / L sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) to shake and separate. After separating the lower aqueous phase, the pH was adjusted to about 3 with a 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.90 g of the title compound.

[0887] The structural characterization data are as follows:

[0888] ESI-MS (m / z): 266.1 [M+H] + .

[0889] Step 2: Synthesis of 4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methylbutyric acid

[0890] Dissolve (E)-4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid (1.90 g, 7.16 mmol) in methanol (40 mL), add 10% palladium carbon (0.15 g) under nitrogen protection, then replace the reaction system with a hydrogen balloon three times, and react for 2 hours under a hydrogen atmosphere, and detect the reaction by high performance liquid chromatography-mass spectrometry. Filter the reaction solution, and concentrate the filtrate under reduced pressure to obtain 1.51 g of the title compound.

[0891] The structural characterization data are as follows:

[0892] ESI-MS (m / z): 268.1 [M+H] + .

[0893] Step 3: Synthesis of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0894] Weigh 4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (1.50 g, 5.61 mmol) and dissolve it in trifluoroacetic acid (20 mL). After cooling to 5°C, add trifluoroacetic anhydride (2.36 g, 11.22 mmol) dropwise. After the addition is completed, keep 5°C for 2 hours and detect the reaction by high performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into a saturated sodium bicarbonate aqueous solution, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness under reduced pressure to obtain a crude product, which is purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 1.05 g of the title compound.

[0895] The structural characterization data are as follows:

[0896] ESI-MS (m / z): 250.1 [M+H] + .

[0897] Step 4: Synthesis of N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0898] Weigh N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (0.55 g, 2.21 mmol) and dissolve it in acetic acid (8 mL), then add bromine (387.85 mg, 2.43 mmol), after the addition, heat to 50 ° C for 15 hours, and detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is directly evaporated to dryness under reduced pressure to obtain a crude product, which is purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 461.00 mg of the title compound.

[0899] The structural characterization data are as follows:

[0900] ESI-MS (m / z): 328.0 [M+H] + .

[0901] Step 5: Synthesis of N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0902] Weigh N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (460.00 mg, 1.40 mmol) and dissolve it in N,N-dimethylformamide (10 mL), then add sodium azide (273.37 mg, 4.21 mmol), react at room temperature for 1 hour, and detect the reaction by high performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into water, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness under reduced pressure to obtain a crude product, which is purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 347.00 mg of the title compound.

[0903] The structural characterization data are as follows:

[0904] ESI-MS (m / z): 291.1 [M+H] + .

[0905] Step 6: Synthesis of N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0906] Weigh N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (347.00 mg, 1.20 mmol) and dissolve it in tetrahydrofuran (10 mL). Add 10% palladium carbon (30.00 mg) under nitrogen protection, then replace the reaction system with a hydrogen balloon three times, and react for 2 hours under a hydrogen atmosphere. Detect the reaction by HPLC-MS / MS. Filter the reaction solution, concentrate the filtrate under reduced pressure to obtain a crude product, and purify the crude product by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 0%-30%) to obtain 205.00 mg of the title compound.

[0907] The structural characterization data are as follows:

[0908] ESI-MS (m / z): 265.1 [M+H] + .

[0909] Step 7: Synthesis of (9H-fluoren-9-yl)methyl(8-acetylamino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0910] Weigh N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (200.00 mg, 756.73 μmol) and dissolve it in a mixed solvent of 1,4-dioxane (6 mL) and water (3 mL), then add sodium bicarbonate (254.28 mg, 3.03 mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (650.55 mg, 1.14 mmol). After the addition is complete, stir the reaction at room temperature for 2 hours, and detect the reaction by HPLC-MS / MS. The reaction solution was slowly poured into water, then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) to obtain 301.00 mg of the title compound.

[0911] The structural characterization data are as follows:

[0912] ESI-MS (m / z): 487.0 [M+H] + .

[0913] Step 8: Synthesis of (9H-fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0914] Dissolve (9H-fluoren-9-yl)methyl(8-acetylamino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (101.00 mg, 207.59 μmol) in 1,4-dioxane (5 mL), then add 3 mol / L hydrochloric acid aqueous solution (5 mL), after addition, heat to 50°C for 15 hours, and detect the reaction by high performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into saturated sodium bicarbonate aqueous solution, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness under reduced pressure to obtain a crude product, which is purified by rapid silica gel column (methanol: dichloromethane = 0%-5%) to obtain 71.00 mg of the title compound.

[0915] The structural characterization data are as follows:

[0916] ESI-MS (m / z): 445.2 [M+H] + .

[0917] Step 9: Synthesis of (9H-fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0918] (9H-fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (35.00 mg, 132.96 μmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (49.25 mg, 110.80 μmol) were added to toluene (3 mL), and then p-toluenesulfonic acid (19.08 mg, 110.80 μmol) was added. After the addition was completed, the temperature was raised to 140° C. for 4 hours, and the reaction solution was directly evaporated to dryness under reduced pressure at 140° C. to obtain a crude product. The crude product was purified by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) to obtain 21.00 mg of the title compound.

[0919] The structural characterization data are as follows:

[0920] ESI-MS (m / z): 672.2 [M+H] + .

[0921] Step 10: Synthesis of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0922] (9H-fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (21.00 mg, 31.26 μmol) was dissolved in N,N-dimethylformamide (1 mL), and then diethylamine (0.2 mL) was added. After the addition was complete, the mixture was reacted at room temperature for 0.5 hour, and the reaction was detected by HPLC-MS / MS chromatography. After the reaction solution was evaporated under reduced pressure to remove ethylenediamine, the pH was adjusted to 2-3 with 1 mol / L aqueous hydrochloric acid solution, and the reaction solution was directly purified by preparative HPLC to obtain two isomers 5-13-A (1.30 mg) and 5-13-B (1.68 mg).

[0923] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0924] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0925] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 3.00 10 90 28 18.00 90 10 28

[0926] The structural characterization data of 5-13-A (6min LCMS peak is near the front, retention time: 1.373min) are as follows:

[0927] 1 H NMR (400 MHz, DMSO-d 6 )δ7.88(d,J=10.6Hz,1H),7.36(s,1H),6.58(s,1H),5.60(d,J=3.5Hz,2H),5.46(d,J=2.5Hz,2H),3.2 5-3.17(m,2H),2.41(s,3H),2.38-2.28(m,2H),1.91-1.84(m,2H),1.79(s,3H),0.88(t,J=7.3Hz,3H).

[0928] ESI-MS (m / z): 450.2 [M+H] + .

[0929] The structural characterization data of 5-13-B (6min LCMS peak later, retention time: 1.523min) are as follows:

[0930] 1 H NMR (400 MHz, DMSO-d 6)δ7.76(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1H),5.73(d,J=19.8Hz,1H),5.50-5.40(m,3H),3.26-3.17(m,1H),3.08-2.96 (m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J=13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J=7.3Hz,3H).

[0931] ESI-MS (m / z): 450.2 [M+H] + .

[0932] 6min LCMS conditions:

[0933] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0934] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0935]

[0936]

[0937] Example 13 (1S, 9S)-1-(aminomethyl)-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 and (1R, 9S)-1-(aminomethyl)-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

[0938]

[0939] Step 1: Synthesis of N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0940] Compound 5-13-04 (1.00 g, 4.25 mmol) was dissolved in N,N-dimethylformamide dimethyl acetal (10 mL), and then the temperature was raised to 120°C for 3 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 20%-100%) to obtain 891.00 mg of the title compound.

[0941] The structural characterization data are as follows:

[0942] ESI-MS (m / z): 291.1 [M+H] + .

[0943] Step 2: Synthesis of N-(7-(aminomethylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0944] Compound 5-7-01 (0.89 g, 3.07 mmol) was dissolved in ethanol (25 mL), and then ammonium acetate (2.36 g, 30.65 mmol) was added. After addition, the mixture was reacted at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution solvent was evaporated to dryness under reduced pressure, and then dichloromethane (30 mL) and water (20 mL) were added, stirred and allowed to stand to separate the organic phase, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 785.00 mg of the title compound.

[0945] The structural characterization data are as follows:

[0946] ESI-MS (m / z): 263.1 [M+H] + .

[0947] Step 3: Synthesis of N-(7-(aminomethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0948] Compound 5-7-02 (0.80 g, 3.05 mmol) was dissolved in ethanol (200 mL), and then 10% palladium carbon (0.40 mg) and concentrated hydrochloric acid (0.2 mL) were added. After the addition, the reaction system was replaced with a hydrogen balloon three times, and then reacted at room temperature for 3 hours under a hydrogen atmosphere, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 905.00 mg of the hydrochloride of the title compound.

[0949] The structural characterization data are as follows:

[0950] ESI-MS (m / z): 265.1 [M+H] + .

[0951] Step 4: Synthesis of (9H-fluoren-9-yl)methyl((8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)carbamate

[0952] The hydrochloride of compound 5-7-03 (0.90 g, 2.99 mmol) was dissolved in 1,4-dioxane (20 mL), and then sodium bicarbonate (1.01 g, 11.97 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.21 g, 3.59 mmol) were added. After the addition was completed, the mixture was stirred at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 1.30 g of the title compound.

[0953] The structural characterization data are as follows:

[0954] ESI-MS (m / z): 487.1 [M+H] + .

[0955] Step 5: Synthesis of (9H-fluoren-9-yl)methyl((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)carbamate

[0956] Compound 5-7-04 (0.80 g, 1.64 mmol) was dissolved in 1,4-dioxane (20 mL), and 3 mol / L hydrochloric acid aqueous solution (20 mL) was added under nitrogen protection. After the addition, the temperature was raised to 60 ° C for 15 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, the organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 561.00 mg of the title compound.

[0957] The structural characterization data are as follows:

[0958] ESI-MS (m / z): 445.1 [M+H] + .

[0959] Step 6: Synthesis of (9H-fluoren-9-yl)methyl(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methyl)carbamate

[0960] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (597.00 mg, 2.27 mmol) and compound 5-7-05 (840.00 mg, 1.89 mmol) were added to toluene (60 mL), and then p-toluenesulfonic acid (325.00 mg, 1.89 mmol) was added. After the addition was completed, the temperature was raised to 140°C for reaction for 4 hours, and then the reaction solution was directly evaporated to dryness under reduced pressure at 140°C to obtain a crude product. The crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-5%) to obtain 563.00 mg of the title compound.

[0961] The structural characterization data are as follows:

[0962] ESI-MS (m / z): 672.2 [M+H] + .

[0963] Step 7: Synthesis of (1S,9S)-1-(aminomethyl)-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 and (1R,9S)-1-(aminomethyl)-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

[0964] Compound 5-7-06 (454.00 mg, 675.89 μmol) was dissolved in N, N-dimethylformamide (5 mL), and then diethylamine (1 mL) was added. After the addition, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was decompressed to remove ethylenediamine, the pH was adjusted to 2-3 with formic acid, and the reaction solution was directly purified by preparative high performance liquid chromatography. The preparative solution was lyophilized to obtain the corresponding title compounds 5-7-A (32.00 mg) and 5-7-B (56.00 mg).

[0965] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0966] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0967] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 90 10 28

[0968] The structural characterization data of 5-7-A (6min LCMS peak is near the front, retention time: 1.488min) are as follows:

[0969] 1 H NMR (400 MHz, DMSO-d 6 )δ7.88(d,J=10.6Hz,1H),7.36(s,1H),6.58(s,1H),5.60(d,J=3.5Hz,2H),5.46(d,J=2.5Hz,2H),3.2 5-3.17(m,2H),2.41(s,3H),2.38-2.28(m,2H),1.91-1.84(m,2H),1.79(s,3H),0.88(t,J=7.3Hz,3H).

[0970] ESI-MS (m / z): 450.2 [M+H] + .

[0971] The structural characterization data of 5-7-B (6min LCMS peak is later, retention time: 1.596min) are as follows:

[0972] 1 H NMR (400 MHz, DMSO-d 6 )δ7.76(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1H),5.73(d,J=19.8Hz,1H),5.50-5.40(m,3H),3.26-3.17(m,1H),3.08-2.96 (m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J=13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J=7.3Hz,3H).

[0973] ESI-MS (m / z): 450.2 [M+H] + .

[0974] 6min LCMS conditions:

[0975] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0976] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0977] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0978] Example 14 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0979]

[0980] Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide or 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide

[0981] The single configuration compound 5-13-A (10.00 mg, 22.25 μmol) and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-1-carboxylic acid (11.36 mg, 33.37 μmol) were dissolved in N,N-dimethylformamide (1 mL), and then HATU (12.68 mg, 33.37 μmol) and N,N-diisopropylethylamine (8.63 mg, 66.74 μmol) were added. After the addition was completed, the reaction was allowed to react at room temperature for 0.5 hours, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to obtain the single title compound 5-16-01-A (7 mg).

[0982] The structural characterization data are as follows:

[0983] ESI-MS (m / z): 772.3 [M+H] + .

[0984] Step 2: Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0985] Compound 5-16-01-A (7.00 mg, 9.07 μmol) was dissolved in N,N-dimethylformamide (1 mL), and potassium fluoride (2.63 mg, 45.34 μmol) was added. After addition, the temperature was raised to 50°C for 1 hour, and the reaction was detected by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain the single title compound 5-16-A (1.73 mg).

[0986] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0987] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0988] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 18.00 90 10 28

[0989] The structural characterization data are as follows:

[0990] 1 H NMR (400 MHz, DMSO-d 6 )δ8.26(s,1H),7.78(d,J=10.8Hz,1H),7.30(s,1H),6.56(s,1H),6.52(s,1H),5.52(d,J=19.3Hz,1H),5.43(d,J=4.4Hz,2H),4.94(d,J=1 9.2Hz,1H),3.30-3.24(m,1H),3.11-3.00(m,1H),2.95-2.84(m,1H),2.39(s,3H),1.98-1.80(m,3H),1.62(s,3H),0.87(t,J=7.9Hz,3H).

[0991] ESI-MS (m / z): 534.2 [M+H] + .

[0992] Example 15 N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecane N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecaneamide

[0993]

[0994] Step 1: Isolation and purification of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0995] Compound 2-23 (16.00 mg) was purified by preparative HPLC and two diastereomers were separated under the following purification conditions to obtain 5.10 mg of trifluoroacetate salt of 2-23-A (retention time 9.85 min) and 7.12 mg of trifluoroacetate salt of 2-23-B (retention time 10.62 min).

[0996] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0997] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0998] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0999] The structural characterization data are as follows:

[1000] 2-23-A:

[1001] 1 H NMR (400 MHz, DMSO-d 6)δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.59(s,1H),5.78-5.63(m,1H),5.50-5.36(m,3H),5.10-5.0 6(m,1H),3.20-3.04(m,2H),2.56(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H),0.88(t,J=7.2Hz,3H).

[1002] ESI-MS (m / z): 452.1 [M+H] + .

[1003] 2-23-B:

[1004] 1 H NMR (400 MHz, DMSO-d 6 )δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.58(s,1H),5.78-5.63(m,1H),5.50-5.36(m,3H),5.10-5.0 6(m,1H),3.20-3.04(m,2H),2.55(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H),0.88(t,J=7.2Hz,3H).

[1005] ESI-MS (m / z): 452.0 [M+H] + .

[1006] Step 2: N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecaneamide or the synthesis of N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecaneamide

[1007] At 25°C, the trifluoroacetate of 2-23-A (34.71 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 3-4-03 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence. The reaction was maintained at 25°C for 0.5 hours. The reaction was monitored by HPLC-MS / MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions are as follows). The preparative solution was freeze-dried to obtain 11.04 mg of the title compound DL-15 with a retention time of 7.5 min.

[1008] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1009] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1010] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[1011] The structural characterization data are as follows:

[1012] DL-15:

[1013] ESI-MS (m / z): 1107.3 [M+H] + .

[1014] Example 16 N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-cyclopropyl-2-hydroxyacetamide

[1015]

[1016] Step 1: Synthesis of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-cyclopropyl-2-hydroxyacetamide

[1017] The formate salt of the single-configuration compound 3-1-A (50 mg, 109.68 μmol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol) were added. After the addition was completed, the reaction was allowed to react at room temperature for 0.5 hour; the reaction was detected by HPLC-MS / MS chromatography; the reaction solution was directly purified by preparative HPLC to obtain two isomers of the title compound (3-12-A: 12.96 mg, 3-12-B: 13.56 mg).

[1018] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1019] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1020] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[1021] The structural characterization data of 3-12-A (6min LCMS peak is near the front) are as follows:

[1022] 1 H NMR (400 MHz, DMSO-d 6 )δ8.44(d,J=9.0Hz,1H),8.05(d,J=10.2Hz,1H),7.33(s,1H),6.54(s,1H),5 .62(q,J=6.7Hz,1H),5.52(d,J=5.1Hz,1H),5.42(s,2H),5.24(q,J=19.2Hz,2 H),3.61(dd,J=6.2,5.1Hz,1H),3.32-3.21(m,2H),2.19(q,J=6.5Hz,2H),1. 92-1.80(m,2H),1.26-1.20(m,1H),0.87(t,J=7.3Hz,3H),0.57-0.34(m,4H).

[1023] ESI-MS (m / z): 554.0 [M+H]+.

[1024] The structural characterization data of 3-12-B (6min LCMS peak is later) are as follows:

[1025] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.7Hz,1H),8.06(d,J=10.3Hz,1H),7.33(s,1H),5.57(q,J=6.7Hz,1H),5.43(s,2H),5.30-5.17(m,2H),3. 64(d,J=6.2Hz,1H),3.29(q,J=6.7Hz,2H),2.28-2.13(m,2H),1.93-1. 78(m,2H),1.18-1.08(m,1H),0.87(t,J=7.3Hz,3H),0.50-0.29(m,4H).

[1026] ESI-MS (m / z): 554.0 [M+H]+.

[1027] The formate salt of another single-configuration compound 3-1-B (50 mg, 109.68 μmol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) were dissolved in N,N-dimethylformamide (2 mL), and then HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol) were added. After the addition was completed, the mixture was reacted at room temperature for 0.5 hour; the reaction was detected by HPLC-MS / MS chromatography; the reaction solution was directly purified by preparative HPLC to obtain two isomers of the title compound (3-12-C: 20.19 mg, 3-12-D: 18.33 mg).

[1028] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1029] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1030] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[1031] The structural characterization data of 3-12-C (6min LCMS peak is near the front) are as follows:

[1032] 1 H NMR (400 MHz, DMSO-d 6)δ8.47(d,J=9.0Hz,1H),8.06(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H), 5.62(q,J=6.5Hz,1H),5.53(d,J=5.1Hz,1H),5.43(s,2H),5.32-5.16(m,2H) ,3.61(dd,J=6.3,5.1Hz,1H),3.32-3.22(m,2H),2.19(q,J=6.5Hz,2H),1.9 2-1.80(m,2H),1.28-1.20(m,1H),0.87(t,J=7.3Hz,3H),0.54-0.35(m,4H).

[1033] ESI-MS (m / z): 554.0 [M+H]+.

[1034] The structural characterization data of 3-12-D (6min LCMS peak is later) are as follows:

[1035] 1 H NMR (400 MHz, DMSO-d 6 )δ8.44(d,J=8.8Hz,1H),8.05(d,J=10.2Hz,1H),7.34(s,1H),6.55(s,1H ),5.58(q,J=6.7Hz,1H),5.45(d,J=5.2Hz,1H),5.43(s,2H),5.31-5.14(m ,2H),3.65(t,J=5.7Hz,1H),3.33-3.21(m,2H),2.28-2.13(m,2H),1.95- 1.80(m,2H),1.16-1.09(m,1H),0.88(t,J=7.3Hz,3H),0.46-0.31(m,4H).

[1036] ESI-MS (m / z): 554.0 [M+H] + .

[1037] Example 17: Preparation of (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxypropylamine and (S)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxypropylamine

[1038]

[1039] At 25°C, (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13(1H,9H)-dione (80.0 mg, 175.5 μmol) and L-lactic acid (31.6 mg, 351.0 μmol) were dissolved in DMF (3 mL), and then HATU (121.1 mg, 351.0 μmol) and DIPEA (68.0 mg, 526.5 μmol) were added and reacted at room temperature for 2 hr; the reaction solution was directly purified by preparative HPLC to obtain compound 3-7-A (6.1 mg, yield 12%) and compound 3-7-B (9.6 mg, yield 20%).

[1040] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1041] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1042] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 24 2.00 30 70 24 18.00 90 10 24

[1043] The structural characterization data of compound 3-7-A (6min LC-MS peak at the front, retention time 2.49min) are as follows:

[1044] MS m / z(ESI):528.2[M+H] +

[1045] 1 H NMR (400 MHz, DMSO-d 6 )δ8.55(d,J=9.2Hz,1H),8.06(d,J=10.4Hz,1H),7.33(s,1H),6.55(s,1H),6.6 7(d,J=4.8Hz,1H),5.65-5.59(m,1H),5.43(s,2H),5.29-5.21(m,1H),5.14-5. 10(m,1H),4.15-4.10(m,,1H),3.27-3.20(m,1H),2.22-2.15(m,2H),1.92-1.8 1(m,2H),1.41(d,J=6.8Hz,3H),1.30-1.23(m,1H),0.89-0.85(t,J=7.2Hz,3H).

[1046] The structural characterization data of compound 3-7-B (6min LC-MS peak is later, retention time is 2.50min) are as follows:

[1047] MS m / z(ESI):528.2[M+H] +

[1048] 1 H NMR (400 MHz, DMSO-d 6 )δ8.46(d,J=8.8Hz,1H),8.06(d,J=10.4Hz,1H),7.33(s,1H),6.56(s,1H ),5.60-5.53(ms,1H),5.51(d,J=5.2Hz,1H),5.43(s,2H),5.27-5.14(m, 2H),4.16-4.08(m,1H),3.28-3.22(m,1H),2.22-2.19(m,2H),1.92-1.81 (m,2H),1.49-1.39(m,1H),1.29(d,J=6.8Hz,3H),0.87(t,J=7.2Hz,3H).

[1049] Example 18 Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide

[1050]

[1051] At 25°C, (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13(1H,9H)-dione (80 mg, 175.49 μmol) and 1-hydroxycyclopropanecarboxylic acid (35.83 mg, 350.98 μmol) were dissolved in DMF (2 mL), and then HATU (121.14 mg, 350.98 μmol) and DIPEA (68.04 mg, 526.47 μmol) were added. After the addition, the mixture was reacted at room temperature for 0.5 hr; the reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS; the reaction solution was directly purified by preparative HPLC to obtain 5.3 mg of the title compounds 3-17-A and 3-17-B 3.5mg.

[1052] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1053] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1054] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1055] The structural characterization data are as follows:

[1056] The structural characterization data of 3-17-A (6min LCMS peak is near the front, retention time: 2.657min) are as follows:

[1057] ESI-MS (m / z): 540.0 [M+H]+.

[1058] 1 H NMR (400 MHz, DMSO-d 6 )δ8.69(d,J=8.8Hz,1H),8.06(d,J=10.2Hz,1H),7.35(s,1H),6.57(s,1H),6.32(s,1H),5.62(s,1H),5.45(s,2H), 5.34-5.24(m,1H),5.20-5.10(m,1H),2.26(s,2H),2.00(s,1H),1.88(s,2H),1.47-1.12(m,8H),1.01-0.80(m,6H).

[1059] The structural characterization data of 3-17-A (6min LCMS peak is later, retention time: 2.724min) are as follows:

[1060] ESI-MS (m / z): 540.0 [M+H]+.

[1061] 1 H NMR (400 MHz, DMSO-d 6 )δ8.69(d,J=8.8Hz,1H),8.06(d,J=10.2Hz,1H),7.35(s,1H),6.57(s,1H),6.32(s,1H),5.62(s,1H),5.45(s,2H), 5.34-5.24(m,1H),5.20-5.10(m,1H),2.26(s,2H),2.00(s,1H),1.88(s,2H),1.47-1.12(m,8H),1.01-0.80(m,6H).

[1062] Example 19 Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[1063]

[1064] Step 1: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1065] N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (570 mg, 2.23 mmol) and cyclopropylboronic acid (574.56 mg, 6.69 mmol) were dissolved in 1,4-dioxane, and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (II) (480 mg, 677.97 μmol) and cesium carbonate (2.17 g, 6.69 mmol) were added. After nitrogen protection, the reaction was carried out at 115°C for 2 h in a microwave oven. The reaction was detected by HPLC-MS / MS chromatography. The reaction solution was diluted with ethyl acetate and then filtered. The filtrate was extracted with ethyl acetate (30 ml*3). The organic phases were combined and washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and then filtered. The filtrate was evaporated to dryness under reduced pressure. The crude product was purified by column chromatography on a silica gel column (PE:EA=1:4) to obtain 550 mg of the title compound.

[1066] The structural characterization data are as follows:

[1067] ESI-MS (m / z): 262.1 [M+H] + .

[1068] Step 2: Synthesis of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1069] Tetrahydrofuran (30 mL) and tert-butanol (10 mL) were added to the reaction solution bottle, cooled to 5°C in an ice bath, and potassium tert-butoxide (945 mg, 8.42 mmol) was added. Then, N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.0 g, 3.83 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (718 mg, 6.12 mmol) was added. After the addition was completed, the reaction was maintained at 5°C for 1 hour. The reaction was detected by HPLC-MS / MS. The reaction solution was quenched with saturated aqueous ammonium chloride solution (50 mL), extracted with ethyl acetate (40 ml*3), the organic phases were combined, washed with saturated brine (50 mL), the organic phases were dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain 1.2 g of a crude product of the title compound.

[1070] The structural characterization data are as follows:

[1071] ESI-MS (m / z): 291.1 [M+H] + .

[1072] Step 3: Synthesis of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide hydrochloride

[1073] The crude product (1.2 g, 1.41 mmol) of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide was dissolved in methanol (7.5 mL) and tetrahydrofuran (7.5 mL), and then 1 mol / L aqueous hydrochloric acid solution (7.5 mL) and 10% palladium carbon (450 mg) were added. After the addition, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out at room temperature under a hydrogen atmosphere for 1 hour; the reaction was detected by HPLC-MS / MS; the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 1.05 g of a crude product.

[1074] The structural characterization data are as follows:

[1075] ESI-MS (m / z): 277.1 [M+H] + .

[1076] Step 4: Synthesis of (9H-fluoren-9-yl)methyl(8-acetamido-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1077] The crude hydrochloride of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.05 g, 3.80 mmol) was dissolved in 1,4-dioxane (10 mL), and then sodium bicarbonate (1.3 g, 15.20 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.54 g, 4.56 mmol) were added. After the addition, the mixture was stirred at room temperature for 2 hours. The reaction was detected by HPLC-MS / MS. The reaction solution was poured into water (50 mL), and then extracted with ethyl acetate (40 mL*3). The organic phases were combined and washed with saturated brine (50 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse phase column to obtain 2.0 g of the title compound.

[1078] The structural characterization data are as follows:

[1079] ESI-MS (m / z): 499.2 [M+H] + .

[1080] Step 5: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1081] Dissolve (9H-fluoren-9-yl)methyl(8-acetamide-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (2.0 g, 3.21 mmol, 80%) in dioxane (20 mL), add 12 mol / L concentrated hydrochloric acid (5 mL), after addition, heat to 70°C and react for 2 hours; detect the reaction by HPLC-MS / MS; pour the reaction solution into water (40 mL), then extract with ethyl acetate (30 mL*3), combine the organic phases, wash with saturated brine (40 mL), dry the organic phase with anhydrous sodium sulfate, filter, and dry the filtrate under reduced pressure to obtain a crude product. The crude product was purified by column chromatography on silica gel (PE:EA=2:1) ​​to obtain 740 mg of the title compound.

[1082] The structural characterization data are as follows:

[1083] ESI-MS (m / z): 457.3 [M+H] + .

[1084] Step 6: Synthesis of (9H-fluoren-9-yl)methyl((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)carbamate

[1085] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (442 mg, 1.68 mmol) and (9H-fluoren-9-yl)methyl(8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (640 mg, 1.40 mmol) were added to toluene (30 mL), and then p-toluenesulfonic acid (242 mg, 1.40 mmol) was added. After the addition was completed, the temperature was raised to 135°C for reaction for 2 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140°C to obtain a crude product; the crude product was purified by column chromatography on a silica gel column (DCM:MeOH=33:1) to obtain 1.02 g of the title compound.

[1086] The structural characterization data are as follows:

[1087] ESI-MS (m / z): 684.1 [M+H] + .

[1088] Step 7: Synthesis of (1S,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (Compound 5-29-1) & (1R,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione

[1089] Dissolve (9H-fluoren-9-yl)methyl((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)carbamate (1.02 g, 1.49 mmol) in N,N-dimethylformamide (15 mL), then add diethylamine (5 ml), and react at room temperature for 0.5 hour after the addition is complete; detect the reaction by HPLC-MS / MS chromatography; after the reaction solution is decompressed to remove ethylenediamine, adjust the pH to 2-3 with 1 mol / L hydrochloric acid aqueous solution, and directly purify the reaction solution by preparative HPLC to obtain two isomers of the title compound (5-22-7-A: 60 mg; 5-22-7-B: 55 mg).

[1090] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1091] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1092] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 3.00 10 90 28 18.00 70 30 28

[1093] The structural characterization data of 5-22-7-A (6min LCMS peak at the front, retention time: 2.28min) are as follows:

[1094] ESI-MS (m / z): 462.2 [M+H] + .

[1095] The structural characterization data of 5-22-7-B (6min LCMS peak later, retention time: 2.35min) are as follows:

[1096] ESI-MS (m / z): 462.2 [M+H] + .

[1097] Step 8 Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[1098] At 25°C, the single-configuration compound 5-28-7-A (40 mg, 86 μmol) and glycolic acid (8 mg, 104 μmol) were dissolved in DMF (2 mL), and then HATU (40 mg, 104 μmol) and DIPEA (36 mg, 258 μmol) were added. After the addition, the mixture was reacted at room temperature for 0.5 hr; the reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS; the reaction solution was directly purified by preparative HPLC to obtain 12.5 mg of compound 5-22-A.

[1099] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1100] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1101] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1102] The structural characterization data are as follows:

[1103] The structural characterization data of 5-22-A (6min LCMS peak is near the front, retention time: 2.540min) are as follows:

[1104] ESI-MS (m / z): 520.0 [M+H] + .

[1105] 1 H NMR (400 MHz, DMSO-d 6 )δ8.46(d,J=8.9Hz,1H),7.74(d,J=11.9Hz,1H),7.30(s,1H),6.53(s,1H),5.64-5.56(m,1H),5.49(t,J=5.8Hz,1H),5.42(s,2H),5.19(s, 2H),3.96(d,J=5.7Hz,2H),2.25-2.10(m,2H),2.04-1.79(m,4H),1.23(s,2H),1.15-1.05(m,2H),0.87(t,J=7.2Hz,3H),0.80-0.70(m,2H).

[1106] At 25°C, the single-configuration compound 5-28-7-B (30 mg, 65 μmol) and glycolic acid (6 mg, 78 μmol) were dissolved in DMF (2 mL), and then HATU (40 mg, 104 μmol) and DIPEA (17 mg, 130 μmol) were added. After the addition, the mixture was reacted at room temperature for 0.5 hr. The reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS chromatography. The reaction solution was directly purified by preparative HPLC to obtain 13.83 mg of compound 5-22-B.

[1107] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1108] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1109] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1110] The structural characterization data are as follows:

[1111] The structural characterization data of 5-22-B (6min LCMS peak later, retention time: 2.612min) are as follows:

[1112] ESI-MS (m / z): 520.0 [M+H] + .

[1113] 1H NMR (400 MHz, DMSO-d 6 )δ8.49(d,J=8.9Hz,1H),7.74(d,J=11.9Hz,1H),7.31(s,1H),6.53(s,1H ),5.60(s,1H),5.51(t,J=5.9Hz,1H),5.43(s,2H),5.25-5.13(m,2H),3. 97(d,J=5.8Hz,2H),2.18(s,2H),2.04-1.91(m,4H),1.90-1.80(m,1H),1 .23(s,6H),1.15-1.05(m,2H),0.87(t,J=7.2Hz,4H),0.80-0.70(m,2H).

[1114] Example 20 Synthesis of (R)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide and (S)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1115]

[1116] Step 1: (9H-fluoro-9-yl)methyl((S)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate and (9H-fluoro-9-yl)methyl((R)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate

[1117] At 25°C, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (72 mg, 166 μmol) and 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxypropionic acid (65 mg, 199 μmol) were added. mol,) was dissolved in DMF (2mL), and then HATU (95mg, 250μmol) and DIPEA (65mg, 498μmol) were added. After the addition, the reaction was allowed to react at room temperature for 0.5hr; the reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS; the reaction solution was directly purified by preparative HPLC to obtain 24mg of the title compound (Compound 1-10-1-A) and 28mg of (Compound 1-10-1-B).

[1118] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1119] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1120] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1121] The structural characterization data of compound 1-10-1-A (6min LCMS peak at the front, retention time: 3.283min) are as follows:

[1122] ESI-MS (m / z): 745.4 [M+H] + .

[1123] The structural characterization data of compound 1-10-1-B (6min LCMS peak is later, retention time: 3.465min) are as follows:

[1124] ESI-MS (m / z): 745.4 [M+H] + .

[1125] Step 2: Synthesis of (R)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide and (S)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1126] At 25°C, compound 1-10-1-B (28 mg, 37 μmol) was dissolved in DMF (2 mL), and then diethylamine (1 mL) was added. After the addition, the mixture was reacted at room temperature for 1.0 hr. The reaction solution was concentrated to dryness to obtain 28 mg of the crude product (compound 1-10-2-B), which was directly used in the next step.

[1127] At 25°C, compound 1-10-1-A (24 mg, 33 μmol) was dissolved in DMF (2 mL), and then diethylamine (1 mL) was added. After the addition, the mixture was reacted at room temperature for 1.0 hr. The reaction solution was concentrated to dryness to obtain 24 mg of the crude product (compound 1-10-2-A), which was directly used in the next step.

[1128] The structural characterization data are as follows:

[1129] ESI-MS (m / z): 523.2 [M+H] + .

[1130] Step 3: Synthesis of (R)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide and (S)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1131] At 25°C, compound 1-10-2-B (28 mg, 37 μmol, 70%) was dissolved in methanol (2 mL), and then formaldehyde aqueous solution (1 mL) was added. After the addition was complete, the mixture was reacted at room temperature for 16.0 hr, and then sodium cyanoborohydride (7.07 mg, 96.45 μmol) was added and reacted at room temperature for 1.0 hr; the reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS; the reaction solution was directly purified by preparative HPLC to obtain 1.3 mg of the title compound (compound 1-10B).

[1132] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1133] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1134] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1135] The structural characterization data of compound 1-10-B (6min LCMS peak is later, retention time: 1.937min) are as follows:

[1136] ESI-MS (m / z): 551.2 [M+H] + .

[1137] 1 H NMR (400 MHz, DMSO-d 6 )δ8.48(d,J=8.6Hz,1H),8.31(s,2H),7.80(d,J=11.0Hz,1H),7.31(s,1H),6.55(s,1H),5.54(s,1 H),5.43(s,2H),5.34(d,J=19.2Hz,1H),5.19(d,J=19.1Hz,1H),4.09-4.06(m,1H),3.20-3.15(m, 2H),2.59-2.53(m,1H),2.45-2.42(m,1H),2.42-2.38(s,3H),2.23-2.19(d,J=7.0Hz,1H),2.13(s ,6H),2.12-2.08(m,1H),2.02-1.95(m,1H),1.90-1.85(m,2H),1.23(s,2H),0.88(d,J=7.2Hz,3H).

[1138] At 25°C, compound 1-10-2-A (24 mg, 33 μmol, 70%) was dissolved in methanol (2 mL), and then formaldehyde aqueous solution (1 mL) was added. After the addition was complete, the reaction was allowed to react at room temperature for 16.0 hr, and then sodium cyanoborohydride (6.06 mg, 96.45 μmol) was added, and the reaction was allowed to react at room temperature for 1.0 hr; the reaction solution was concentrated to dryness and the reaction was directly detected by HPLC-MS / MS; the reaction solution was directly purified by preparative HPLC to obtain 4.44 mg of the title compound (compound 1-10-A).

[1139] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1140] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1141] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1142] The structural characterization data of compound 1-10-A (6min LCMS peak is near the front, retention time: 1.920min) are as follows:

[1143] ESI-MS (m / z): 551.2 [M+H] + .

[1144] 1 H NMR (400 MHz, DMSO-d 6 )δ8.58(d,J=9.0Hz,1H),8.28(s,1H),7.84(d,J=10.9Hz,1H),7.37(s,1H),6.61(s,1H) ,5.66-5.59(m,1H),5.49(s,2H),5.35(d,J=19.1Hz,1H),5.17(d,J=18.9Hz,1H),4.23- 4.16(m,1H),3.23(d,J=7.8Hz,2H),2.75-2.67(m,2H),2.45(s,3H),2.30(s,6H),2.27- 2.17(m,2H),2.14-1.99(m,1H),1.98-1.87(m,2H),1.30(s,2H),0.93(t,J=7.3Hz,3H).

[1145] Example 21 (S)-14-(2-(Cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolan-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione (Compound 4-14)

[1146]

[1147] Step 1: Synthesis of 2-nitro-4,5-methylenedioxyacetophenone

[1148] Compound 4-14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL), and concentrated nitric acid (26 mL) was slowly added dropwise under stirring. The reaction was allowed to react at room temperature for 2 hours. TLC was used to monitor the reaction. A small amount of raw material remained and the product was obvious. Slowly dropwise saturated sodium bicarbonate aqueous solution was added to neutralize the reaction. Dichloromethane was added to extract 3 times. The organic phases were combined, washed 3 times with saturated brine, dried, and concentrated to obtain a crude product. Purification by silica gel column (eluent: 0-20% ethyl acetate / petroleum ether) gave 9.8 g of the title compound.

[1149] Step 2: Synthesis of 6-amino-3,4-methylenedioxyacetophenone

[1150] Dissolve compound 4-14-2 (2.0 g, 9.56 mmol) in ethyl acetate (20 mL), add 10% palladium carbon (0.2 g), replace with hydrogen and stir under protection for 4 hours, filter, and concentrate the filtrate under reduced pressure to obtain 1.7 g of crude title compound.

[1151] Step 3: Synthesis of 6-acetylamino-3,4-methylenedioxyacetophenone

[1152] Dissolve compound 4-14-3 (1.7 g, 9.49 mmol) in acetic anhydride (17 mL), stir and react for 1 hour, evaporate the solvent under reduced pressure, add water and stir, filter, wash the solid with water and dry in vacuo to obtain 2.08 g of crude title compound.

[1153] Step 4: Synthesis of (E)-N-(6-(3-(dimethylamino)acryloyl)benzo[d][1,3]dioxolan-5-yl)acetamide

[1154] Compound 4-14-4 (1.88 g, 8.50 mmol) was dissolved in DMF-DMA (30 mL), heated to 120°C for 2 hours, and the solvent was evaporated under reduced pressure to obtain 2.33 g of a crude product of the title compound.

[1155] The structural characterization data are as follows:

[1156] ESI-MS (m / z): 277.2 [M+1] + .

[1157] Step 5: Synthesis of (E)-N-(6-(3-(cyclopropylamino)acryloyl)benzo[d][1,3]dioxolan-5-yl)acetamide

[1158] Dissolve compound 4-14-5 (200 mg, 0.72 mmol) in ethanol (5 mL), drop cyclopropylamine (413.3 mg, 7.24 mmol), raise the temperature to 50°C and react for 16 hours. Evaporate the solvent under reduced pressure to obtain 208 mg of a crude product of the title compound.

[1159] The structural characterization data are as follows:

[1160] ESI-MS (m / z): 289.2 [M+1] + .

[1161] Step 6: Synthesis of N-(6-(3-(cyclopropylamino)propionyl)benzo[d][1,3]dioxolan-5-yl)acetamide

[1162] Dissolve compound 4-14-6 (208 mg, 0.72 mmol) in glacial acetic acid (4 mL), add sodium borohydride (13.65 mg, 0.36 mmol) under ice-water cooling and stirring, return to room temperature and stir for 3 hours. Evaporate the solvent under reduced pressure to obtain 209 mg of the crude title compound.

[1163] The structural characterization data are as follows:

[1164] ESI-MS (m / z): 291.1 [M+1] + .

[1165] Step 7: Synthesis of (9H-fluoren-9-yl)methyl(3-(6-acetylaminobenzo[d][1,3]dioxolan-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1166] Compound 4-14-7 (200 mg, 0.69 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (395 mg, 0.68 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were added under stirring, and the mixture was reacted at room temperature for 2 hours. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with saturated brine, dried, concentrated, and purified by silica gel column (eluent: 30% ethyl acetate / petroleum ether) to obtain 350 mg of the title compound.

[1167] Step 8: Synthesis of (9H-fluoren-9-yl)methyl(3-(6-aminobenzo[d][1,3]dioxolan-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1168] Compound 4-14-8 (350 mg, 0.68 mmol) was dissolved in 1,4-dioxane (10 mL), and 3N hydrochloric acid aqueous solution (10 mL) was added dropwise, and the mixture was heated to 60°C and stirred for 16 hours. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with water, dried, concentrated, and purified by silica gel column (eluent: 33% ethyl acetate / petroleum ether) to obtain 218 mg of the title compound.

[1169] Step 9: Synthesis of (S)-(9H-fluoren-9-yl)methylcyclopropyl(2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolan[4,5-g]pyrano[3,4]:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)carbamate

[1170] Compound 4-14-9 (40 mg, 0.085 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (24.62 mg, 0.094 mmol) were dissolved in toluene (1 mL), p-toluenesulfonic acid (2.93 mg, 0.017 mmol) was added, and the temperature was raised to 120°C for 4 hours. The mixture was concentrated under reduced pressure to obtain 59 mg of a crude product of the title compound.

[1171] The structural characterization data are as follows:

[1172] ESI-MS (m / z): 698.1 [M+1] + .

[1173] Step 10: Synthesis of (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolan-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

[1174] Compound 4-14-10 (59 mg, 0.085 mmol) was dissolved in DMF (1 mL), and diethylamine (0.5 mL) was added dropwise, and the mixture was stirred for 1 hour. Diethylamine was evaporated under reduced pressure, and 3N hydrochloric acid was added dropwise to acidify the mixture, and then purified by high performance liquid chromatography (purification conditions are as follows), and lyophilized to obtain 12.66 mg of the trifluoroacetate salt of the title compound.

[1175] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1176] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1177] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[1178] The structural characterization data are as follows:

[1179] 1 H NMR (400 MHz, DMSO-d 6 )δ8.73(s,2H),7.67(s,1H),7.57(s,1H),7.26(s,1H),6.54(s,1H),6.33(s,2H),5.44(s,2H),5.34 (s,2H),3.40(s,4H),2.82(s,1H),1.91-1.81(m,2H),0.87(t,J=7.2Hz,5H),0.79(d,J=7.4Hz,2H).

[1180] ESI-MS (m / z): 476.1 [M+1] + .

[1181] Example 22 (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-8,11(10H,13H)-dione (Compound 4-15)

[1182]

[1183] Step 1: Synthesis of (E)-N-(6-(3-((2-methoxyethyl)amino)acryloyl)benzo[d][1,3]dioxetane-5-yl)acetamide

[1184] Dissolve compound 4-14-5 (200 mg, 0.72 mmol) in ethanol (5 mL), add 2-methoxyethylamine (543.7 mg, 7.24 mmol) dropwise, and heat to 50°C for 16 hours. Monitor the reaction by LCMS, and a small amount of raw material remains, and the product is obvious. Evaporate the solvent under reduced pressure to obtain 221 mg of the title compound, which is directly used for the next step.

[1185] The structural characterization data are as follows:

[1186] ESI-MS (m / z): 307.1 [M+1] +

[1187] Step 2: Synthesis of N-(6-(3-((2-methoxyethyl)amino)propionyl)benzo[d][1,3]dioxetane-5-yl)acetamide

[1188] Dissolve compound 4-15-1 (200 mg, 0.65 mmol) in glacial acetic acid (4 mL), add sodium borohydride (12.35 mg, 0.33 mmol) under ice-water cooling and stirring, return to room temperature and stir for 3 hours. Monitor the reaction by LCMS, the starting material disappears, and the product is obvious. Evaporate the solvent under reduced pressure to obtain 200 mg of the title compound, which is directly used for the next step.

[1189] ESI-MS (m / z): 309.1 [M+1] +

[1190] Step 3: Synthesis of (9H-fluoren-9-yl)methyl(3-(6-acetylaminobenzo[d][1,3]dioxol-5-yl)-3-oxypropyl)(2-methoxyethyl)carbamate

[1191] The crude product of compound 4-15-2 (200 mg, 0.65 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (372 mg, 0.65 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were added under stirring, and the reaction was carried out at room temperature for 2 hours. The raw material disappeared under TLC monitoring, and the product was obvious. Water and ethyl acetate were added and stirred, and the liquid was separated by standing. The organic phase was washed with saturated brine, dried and concentrated, and purified by silica gel column (eluent: 50% ethyl acetate / petroleum ether) to obtain 180 mg of the title compound.

[1192] Step 4: Synthesis of ((9H-fluoren-9-yl)methyl(3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(2-methoxyethyl)carbamate

[1193] Compound 4-15-3 (180 mg, 0.68 mmol) was dissolved in 1,4-dioxane (5 mL), and 3N hydrochloric acid aqueous solution (5 mL) was added dropwise, and the mixture was heated to 60°C and stirred for 16 hours. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with water, dried, concentrated, and purified by silica gel column (eluent: 45% ethyl acetate / petroleum ether) to obtain 132 mg of the title compound.

[1194] Step 5: Synthesis of (S)-(9H-fluoren-9-yl)methyl(2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxacyclo[4,5-g]pyrano[3',4]:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(2-methoxyethyl)carbamate

[1195] Compound 4-15-4 (130 mg, 0.266 mmol) and rac-(4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione (70.05 mg, 0.266 mmol) were dissolved in toluene (4 mL), p-toluenesulfonic acid (9.16 mg, 0.053 mmol) was added, and the temperature was raised to 120°C for 4 hours. The reaction was monitored by LCMS, the starting material disappeared, and the product was obvious. Concentrated under reduced pressure to obtain 190 mg of a crude product of the title compound.

[1196] The structural characterization data are as follows:

[1197] ESI-MS (m / z): 716.1 [M+1] +

[1198] Step 6: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

[1199] Compound 4-15-5 (190 mg, 0.265 mmol) was dissolved in DMF (3 mL), and diethylamine (2 mL) was added dropwise. The mixture was stirred and reacted for 1 hour. The reaction was monitored by LCMS. The starting material disappeared and the product was obvious. Diethylamine was evaporated under reduced pressure, and 3N hydrochloric acid was added dropwise to acidify the mixture. The mixture was prepared and purified, and lyophilized to obtain 99.28 mg of the title compound.

[1200] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1201] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1202] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[1203] The structural characterization data are as follows:

[1204] 1 H NMR (400 MHz, DMSO-d 6): δ8.68(s,2H),7.68(s,1H),7.56(s,1H),7.26(s,1H),6.52(s,1H),6.32(s,2H),5.44(s,2H),5.31(s,2H),3.65-3.58(m,2H), 3.42(d,J=10.2Hz,2H),3.36(s,3H),3.22(d,J=4.0Hz,4H),1.94-1.80(m,2H),0.87(t,J=7.3Hz,3H).ESI-MS(m / z):494.2[M+1] +

[1205] Example 23 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide & N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[1206]

[1207] Step 1: Synthesis of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide

[1208] 3-Bromo-5-fluoro-4-methoxy-aniline (1.7 g, 7.73 mmol) was dissolved in tetrahydrofuran (30 mL), and then triethylamine (2.35 g, 23.18 mmol) and acetic anhydride (1.18 g, 11.59 mmol) were added. After the addition was completed, the temperature was raised to 50°C and stirred for reaction for 4 hours; the reaction was detected by HPLC-MS / MS chromatography; after the reaction solution was cooled to room temperature, it was diluted with ethyl acetate (50 mL), and then washed once with clean water (30 mL) and saturated brine (30 mL), the organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by beating with (petroleum ether: ethyl acetate = 5:1) to obtain 1.1 g of the title compound.

[1209] The structural characterization data are as follows:

[1210] ESI-MS (m / z): 262.0 [M+H] + .

[1211] Step 2: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methoxyphenyl)-3-butenoic acid

[1212] N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (1.1 g, 4.20 mmol) and 3-butenoic acid (397.47 mg, 4.62 mmol) were dissolved in a mixed solvent of 1,4-dioxane (20 mL) and water (5 mL), and then triethylamine (1.27 g, 12.59 mmol), tri(o-methylphenyl)phosphine (127.75 mg, 419.73 μmol) and palladium acetate (47.12 mg, 209.89 μmol) were added. After the addition was completed, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100°C under a nitrogen atmosphere for 4 hours; the reaction was detected by high performance liquid chromatography-mass spectrometry; after the reaction solution was cooled to room temperature, 1 mol / L sodium hydroxide aqueous solution (50 mL) and ethyl acetate (50 mL) were added to shake and separate the layers. After separating the lower aqueous phase, adjust the pH to about 3 with 4 mol / L hydrochloric acid aqueous solution, then extract with ethyl acetate (40 mL*2), combine the organic phases, wash with saturated brine (40 mL), dry over anhydrous sodium sulfate, filter, and evaporate the filtrate under reduced pressure to obtain 1.1 g of a crude product of the title compound.

[1213] The structural characterization data are as follows:

[1214] ESI-MS (m / z): 268.1 [M+H] + .

[1215] Step 3: Synthesis of 4-(5-acetylamino-3-fluoro-2-methoxyphenyl)butyric acid

[1216] The crude product of (E)-4-(5-acetylamino-3-fluoro-2-methoxyphenyl)-3-butenoic acid (1.1 g, 4.12 mmol) was dissolved in methanol (20 mL), and then 10% palladium on carbon (100 mg) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon and reacted for 4 hours under a hydrogen atmosphere; the reaction was detected by HPLC-MS / MS; the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 1.05 g of a crude product of the title compound.

[1217] The structural characterization data are as follows:

[1218] ESI-MS (m / z): 270.1 [M+H] + .

[1219] Step 4: Synthesis of N-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1220] The crude product of 4-(5-acetylamino-3-fluoro-2-methoxyphenyl)butyric acid (1.1 g, 4.09 mmol) was dissolved in trifluoroacetic acid (10 mL). After cooling to 5°C, trifluoroacetic anhydride (4.29 g, 20.43 mmol) was slowly added. After the addition was completed, the temperature was naturally raised to room temperature for reaction for 2 hours. The reaction was detected by HPLC-MS / MS chromatography. The reaction solution was slowly poured into water (60 mL), and then extracted with ethyl acetate (40 mL*3). The organic phases were combined, washed with saturated sodium bicarbonate aqueous solution until neutral, and then washed with saturated brine (40 mL). The organic phase was dried over anhydrous sodium sulfate and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 503 mg of the title compound.

[1221] The structural characterization data are as follows:

[1222] ESI-MS (m / z): 252.1 [M+H] + .

[1223] Step 5: Synthesis of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1224] Tetrahydrofuran (15 mL) and tert-butanol (4 mL) were added to the reaction solution bottle, cooled to 5°C in an ice bath, and potassium tert-butoxide (491.26 mg, 4.38 mmol) was added. Then, N-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (500 mg, 1.99 mmol) was dissolved in tetrahydrofuran (5 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (373.01 mg, 3.18 mmol) was added. After the addition was completed, the reaction was maintained at 5°C for 1 hour. The reaction was detected by HPLC-MS / MS. The reaction solution was quenched with saturated aqueous ammonium chloride solution (50 mL), extracted with ethyl acetate (40*2), the organic phases were combined, washed with saturated brine (40 mL), the organic phase was dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain 550 mg of a crude product of the title compound.

[1225] The structural characterization data are as follows:

[1226] ESI-MS (m / z): 281.1 [M+H] + .

[1227] Step 6: Synthesis of N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1228] The crude product of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (520 mg, 1.86 mmol) was dissolved in a mixed solution of methanol (10 mL) and tetrahydrofuran (10 mL), and then 1 mol / L aqueous hydrochloric acid solution (3.71 mL) and 10% palladium on carbon (50 mg) were added. After the addition was completed, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out at room temperature under a hydrogen atmosphere for 1 hour; the reaction was detected by HPLC-MS / MS; the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain 551 mg of a crude hydrochloride of the title compound.

[1229] The structural characterization data are as follows:

[1230] ESI-MS (m / z): 267.1 [M+H] + .

[1231] Step 7: Synthesis of (9H-fluoren-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1232] The crude hydrochloride of N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (550 mg, 1.64 mmol) was dissolved in 1,4-dioxane (15 mL), and then sodium bicarbonate (549.45 mg, 6.54 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.12 g, 1.96 mmol) were added. After the addition was completed, the mixture was stirred at room temperature for 2 hours; the reaction was detected by HPLC-MS / MS chromatography; the reaction solution was poured into water (50 mL), and then extracted with ethyl acetate (40 mL*2), the organic phases were combined, washed with saturated brine (40 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by C18 (acetonitrile / 0.05% formic acid aqueous solution, 20% acetonitrile to 100% acetonitrile) reverse phase column to obtain 410 mg of the title compound.

[1233] The structural characterization data are as follows:

[1234] ESI-MS (m / z): 489.1 [M+H] + .

[1235] Step 8: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1236] Dissolve (9H-fluoren-9-yl)methyl(8-acetamide-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphthalene-2-yl)carbamate (410 mg, 839.29 μmol) in dioxane (10 mL), add 12 mol / L concentrated hydrochloric acid (2 mL), after addition, heat to 70°C and react for 2 hours; detect the reaction by HPLC-MS / MS; pour the reaction solution into water (30 mL), then extract with ethyl acetate (30 mL*2), combine the organic phases, wash with saturated brine (30 mL), dry the organic phase with anhydrous sodium sulfate, filter, and dry the filtrate under reduced pressure to obtain a crude product. Purify by rapid silica gel (ethyl acetate: petroleum ether = 0-60%) to obtain 351 mg of the title compound.

[1237] The structural characterization data are as follows:

[1238] ESI-MS (m / z): 447.1 [M+H] + .

[1239] Step 9: Synthesis of (9H-fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)carbamate

[1240] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (247.64 mg, 940.71 μmol) and (9H-fluoren-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (350 mg, 783.93 μmol) were added to toluene (15 mL), and then p-toluenesulfonic acid (134.84 mg, 783.93 μmol) was added. After the addition was completed, the temperature was raised to 135° C. and the reaction was carried out for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 135° C. to obtain a crude product; the crude product was purified by a rapid silica gel (methanol: dichloromethane = 0-6%) column to obtain 358 mg of the title compound.

[1241] The structural characterization data are as follows:

[1242] ESI-MS (m / z): 674.2 [M+H] + .

[1243] Step 10: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione

[1244] (9H-fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)carbamate (358 mg, 531.41 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then diethylamine (0.4 mL) was added. After the addition was complete, the reaction was allowed to react at room temperature for 0.5 hour. The reaction was detected by HPLC-MS / MS chromatography. The reaction solution was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by beating with ethyl acetate to obtain 220 mg of the title compound.

[1245] The structural characterization data are as follows:

[1246] ESI-MS (m / z): 452.1 [M+H] + .

[1247] Step 11: Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide & N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[1248] (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (50 mg, 110.76 μmol) and 2-hydroxyacetic acid (16.85 mg, 221.51 μmol) were dissolved in N,N-dimethylformamide (2 mL), then add HATU (84.17 mg, 221.51 μmol) and N,N-diisopropylethylamine (42.94 mg, 332.27 μmol), after addition, react at room temperature for 0.5 hour; detect the reaction by HPLC-MS / MS; the reaction solution is directly purified by preparative HPLC to obtain two isomers of the title compound with a single configuration (5-34-A: 6.22 mg, 5-34-B: 9.81 mg)

[1249] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1250] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1251] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1252] The structural characterization data of 5-34-A are as follows:

[1253] 1 H NMR (400 MHz, DMSO-d 6 )δ8.53(d,J=9.0Hz,1H),7.89(d,J=12.3Hz,1H),7.33(s,1H),5.58(q,J=7.5,7.0Hz,1H),5.42(d,J=2.1Hz,2H),5.25-5.09(m ,2H),3.98(s,2H),3.96(d,J=1.1Hz,3H),3.30-3.10(m,2H),2.15(q,J=7.4Hz,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[1254] ESI-MS (m / z): 510.2 [M+H] + .

[1255] The structural characterization data of 5-34-B are as follows:

[1256] 1 H NMR (400 MHz, DMSO-d 6)δ8.55(d,J=9.0Hz,1H),7.90(d,J=12.3Hz,1H),7.34(s,1H),5.59(q,J=7.4,6.8Hz,1H),5.43(s,2H),5.25-5.11(m,2H) ,3.99(s,2H),3.96(d,J=1.1Hz,3H),3.30-3.13(m,2H),2.15(q,J=6.4Hz,2H),1.93-1.82(m,2H),0.88(t,J=7.3Hz,3H).

[1257] ESI-MS (m / z): 510.2 [M+H] + .

[1258] Example 24 (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropylamine & (2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropylamine

[1259]

[1260] Step 1: Synthesis of (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizin[1,2-b]quinolin-1-yl)-2-hydroxypropylamine & (2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizin[1,2-b]quinolin-1-yl)-2-hydroxypropylamine

[1261] (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (50 mg, 110.64 μmol) and (2R)-2-hydroxypropionic acid (19.93 mg, 221.29 μmol) were dissolved in N,N-dimethylformamide. amide (2mL), then add HATU (84.09mg, 221.29μmol) and N,N-diisopropylethylamine (42.90mg, 331.93μmol), after addition, react at room temperature for 0.5 hours; detect the reaction by HPLC-MS / MS; the reaction solution is directly purified by preparative HPLC to obtain two isomers of the title compound (2-27-A: 5.73mg, 2-27-B: 7.59mg)

[1262] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1263] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1264] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 90 28 2.00 30 90 28 18.00 90 10 28

[1265] The structural characterization data of 2-27-A are as follows:

[1266] 1 H NMR (400 MHz, DMSO-d 6 )δ8.45(d,J=9.1Hz,1H),8.14(s,1H),7.30(s,1H),6.54(s,1H),5.63(s,1 H),5.56(q,J=8.0Hz,1H),5.42(s,2H),5.25(d,J=19.0Hz,1H),5.08(d,J=1 9.0Hz,1H),4.13(q,J=6.7Hz,1H),3.27-3.12(m,2H),2.51(s,3H),2.23-2. 13(m,2H),1.92-1.80(m,2H),1.41(d,J=6.8Hz,3H),0.87(t,J=7.3Hz,3H).

[1267] ESI-MS (m / z): 524.2 [M+H] + .

[1268] The structural characterization data of 2-27-B are as follows:

[1269] 1 H NMR (400 MHz, DMSO-d6 )δ8.40(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.55-5.49(m,1H),5.43(d,J=2.2Hz,2H),5.18(q,J=19.0Hz,2H),4.1 3(q,J=6.6Hz,1H),3.24-3.12(m,2H),2.51(s,3H),2.22-2.10(m,2H),1.92-1.82(m,2H),1.30(d,J=6.7Hz,3H),0.88(t,J=7.3Hz,3H).

[1270] ESI-MS (m / z): 524.2 [M+H] + .

[1271] Example 25: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropylamine and (R)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropylamine

[1272]

[1273] At 25°C, 3-1-A (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added and reacted at room temperature for 2 hours; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative HPLC to obtain compound 3-26-A (15.4 mg, yield 64%).

[1274] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1275] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1276] Retention time: 5.3~6.2min

[1277] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

[1278] The structural characterization data are as follows:

[1279] MS m / z(ESI):528.2[M+H] +

[1280] 1 H NMR (400 MHz, DMSO-d 6 )δ8.52(d,J=9.2Hz,1H),8.05(d,J=10.0Hz,1H),7.33(s,1H),6.55(s,1H),5.62-5.59(m,2H),5.43(s,2H),5.28-5.10(m,2H),4.13-4 .11(m,1H),3.41-3.38(m,1H),3.28-3.22(m,1H),2.20-2.18(m,2H),1.92-1.80(m,2H),1.40(d,J=6.8Hz,3H),0.87(t,J=7.2Hz,3H).

[1281] At 25°C, 3-1-B (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added and reacted at room temperature for 2 hours; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative HPLC to obtain compound 3-26-B (4.8 mg, yield 20%).

[1282] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1283] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1284] Retention time: 7.5~8.5min

[1285] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 28 4.00 30 70 28 20.00 90 10 28

[1286] The structural characterization data are as follows:

[1287] MS m / z(ESI):528.2[M+H] +

[1288] 1 H NMR (400 MHz, DMSO-d 6)δ8.48(d,J=8.8Hz,1H),8.06(d,J=10.4Hz,1H),7.34(s,1H),6.57(br,1H),5.61-5.55(m,1H),5.48-5.39(m,2H),5.27-5.16(m ,2H),4.15-4.10(m,1H),3.32-3.21(m,3H),2.23-2.16(m,2H),1.92-1.81(m,2H),1.30(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[1289] Biological evaluation

[1290] 1. Tumor cell proliferation inhibition test

[1291] 1. Inhibitory effect of compounds on HT29 cell proliferation

[1292] (1) Cell plating: First, culture tumor cells HT29 in the corresponding culture medium, digest the cells with trypsin, resuspend the cells after centrifugation and count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 1.

[1293] Table 1. Tumor cell sources

[1294] Cell Name Tumor type source HT29 Human colon cancer cells Chinese Academy of Sciences Cell Bank

[1295] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1296] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50 μL, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 2.

[1297] (2) Data results

[1298] Table 2. HT29 cell proliferation inhibitory activity

[1299]

[1300]

[1301] The test results show that the compounds of the present invention in Table 2 have a strong proliferation inhibitory effect on the proliferation of HT29 colon cancer cells.

[1302] 2. Inhibitory effect of compounds on A549 cell proliferation

[1303] (1) Cell plating: First, tumor cells A549 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 3.

[1304] Table 3. Origin of tumor cells

[1305] Cell Name Tumor type source A549 Human lung cancer cells Chinese Academy of Sciences Cell Bank

[1306] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1307] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 500L, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 4.

[1308] (2) Data results

[1309] Table 4. A549 cell proliferation inhibitory activity

[1310] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 94.18 Embodiment 1 (1-1-A) 66.56 Embodiment 4 (2-7-A) 6.97 Embodiment 4 (2-7-B) 16.39 Embodiment 5 (2-12-A) 6.96 Embodiment 5 (2-12-B) 5.52 Embodiment 5 (2-12-C) 6.64 Embodiment 7 (2-20-A) 8.14 Embodiment 7 (2-20-B) 23.06 Embodiment 11 (4-10) 68.48

[1311] The test results show that the compounds of the present invention in Table 4 have a significant inhibitory effect on the proliferation of A549 human lung cancer cells.

[1312] 3. Inhibitory effect of compounds on HCC1806 cell proliferation

[1313] (1) Cell plating: First, tumor cells HCC1806 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 5.

[1314] Table 5. Tumor cell sources

[1315] Cell Name Tumor type source HCC1806 Human breast squamous cell carcinoma ATCC

[1316] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1317] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50 μL, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 6.

[1318] (2) Data results

[1319] Table 6. HCC1806 cell proliferation inhibitory activity

[1320] name <![CDATA[IC 50 (nM) <!-- 106 -->]]> Comparative Compound 1 3.03 Embodiment 3 (2-1-A) 1.43 Embodiment 4 (2-7-A) 1.25 Embodiment 5 (2-12-A) 1.40 Embodiment 5 (2-12-B) 1.07 Embodiment 5 (2-12-C) 2.08 Embodiment 12 (5-13-A) 0.96 Embodiment 16 (3-12-A) 0.16 Embodiment 16 (3-12-B) 1.76 Embodiment 16 (3-12-C) 1.91 Embodiment 16 (3-12-D) 1.53 Embodiment 17 (3-7-B) 1.94 Embodiment 18 (3-17-A) 1.70 Embodiment 18 (3-17-B) 2.26 Embodiment 19 (5-22-A) 2.57 Embodiment 19 (5-22-B) 3.71 Example 24 (2-27-A) 4.55 Example 24 (2-27-B) 3.93 Example 25 (3-26-A) 1.27 Example 25 (3-26-B) 4.93

[1321] The test results show that the compounds of the present invention in Table 6 have a significant inhibitory effect on the proliferation of HCC1806 human breast squamous cell carcinoma cells.

[1322] 4. Inhibitory effect of compounds on SKOV-3 cell proliferation

[1323] (1) Cell plating: First, the tumor cells SKOV-3 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 7.

[1324] Table 7. Tumor cell sources

[1325] Cell Name Tumor type source SKOV-3 Human ovarian cancer cells Nanjing Kebai Biological

[1326] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 h.

[1327] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μ0, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 8.

[1328] (2) Data results

[1329] Table 8. SKOV-3 cell proliferation inhibitory activity

[1330] name <![CDATA[IC 50 (nM) <!-- 107 -->]]> Comparative Compound 1 17.14 Embodiment 1 (1-1-A) 7.81 Embodiment 3 (2-1-A) 7.12 Embodiment 4 (2-7-A) 8.64 Embodiment 4 (2-7-B) 9.47 Embodiment 5 (2-12-A) 6.85 Embodiment 5 (2-12-B) 4.99 Embodiment 5 (2-12-C) 6.52 Embodiment 7 (2-20-A) 6.79 Embodiment 12 (5-13-A) 2.09 Example 21 (4-14) 2.16

[1331] The test results show that the compounds of the present invention in Table 8 have a significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

[1332] 5. Inhibitory effect of compounds on NCI-H358 cell proliferation

[1333] (1) Cell plating: First, tumor cells NCI-H358 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 9.

[1334] Table 9. Tumor cell sources

[1335] Cell Name Tumor type source NCI-H358 Human non-small cell lung cancer cells Nanjing Kebai Biological

[1336] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1337] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μ0, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 10.

[1338] (2) Data results

[1339] Table 10. NCI-H358 cell proliferation inhibitory activity

[1340] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 58.84 Embodiment 1 (1-1-A) 65.22 Embodiment 2 (1-7-A) 11.38 Embodiment 2 (1-7-B) 51.52 Embodiment 3 (2-1-A) 68.62 Embodiment 4 (2-7-A) 35.42 Embodiment 4 (2-7-B) 51.06 Embodiment 5 (2-12-A) 17.65 Embodiment 5 (2-12-B) 15.76 Embodiment 5 (2-12-C) 23.08 Embodiment 7 (2-20-A) 18.56 Embodiment 12 (5-13-A) 7.01 Embodiment 16 (3-12-A) 15.45 Embodiment 16 (3-12-B) 19.77 Embodiment 16 (3-12-C) 28.29 Embodiment 16 (3-12-D) 20.75 Embodiment 17 (3-7-A) 49.38 Embodiment 17 (3-7-B) 22.22 Embodiment 18 (3-17-A) 13.34 Embodiment 18 (3-17-B) 37.34 Embodiment 19 (5-22-A) 29.94 Embodiment 19 (5-22-B) 61.36 Example 21 (4-14) 11.98 Example 22 (4-15) 17.76 Example 24 (2-27-A) 56.46 Example 24 (2-27-B) 69.12 Example 25 (3-26-A) 31.64

[1341] The test results show that the compounds of the present invention in Table 10 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[1342] 6. Inhibitory effect of compounds on NCI-N87 cell proliferation

[1343] (1) Cell plating: First, culture the tumor cells NCI-N87 in the corresponding culture medium, digest the cells with trypsin, resuspend the cells after centrifugation and count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 11.

[1344] Table 11. Tumor cell sources

[1345] Cell Name Tumor type source NCI-N87 Human gastric cancer cells ATCC

[1346] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1347] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS).TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 12.

[1348] (2) Data results

[1349] Table 12. NCI-N87 cell proliferation inhibitory activity

[1350]

[1351]

[1352] The test results show that the compounds of the present invention in Table 12 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

[1353] 7. Inhibitory effect of compounds on Hela cell proliferation

[1354] (1) Cell plating: First, culture Hela tumor cells in the corresponding culture medium, digest the cells with trypsin, resuspend the cells after centrifugation and count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 13.

[1355] Table 13. Tumor cell sources

[1356] Cell Name Tumor type source Hela Human cervical cancer cells Nanjing Kebai Biological

[1357] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1358] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 14.

[1359] (2) Data results

[1360] Table 14. Hela cell proliferation inhibitory activity

[1361]

[1362]

[1363] The test results show that the compounds of the present invention in Table 14 have a significant inhibitory effect on the proliferation of Hela human cervical cancer cells.

[1364] 8. Inhibitory effect of compounds on HCC70 cell proliferation

[1365] (1) Cell plating: First, culture HCC70 tumor cells in the corresponding culture medium, digest the cells with trypsin, resuspend the cells after centrifugation and count them, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 15.

[1366] Table 15. Tumor cell sources

[1367] Cell Name Tumor type source HCC70 Human breast cancer cells Nanjing Kebai Biological

[1368] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1369] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μ0, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 16.

[1370] (2) Data results

[1371] Table 16. HCC70 cell proliferation inhibitory activity

[1372] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 226.10 Embodiment 1 (1-1-A) 224.85 Embodiment 4 (2-7-A) 150.70 Embodiment 4 (2-7-B) 192.20 Embodiment 5 (2-12-A) 170.93 Embodiment 5 (2-12-B) 176.16 Embodiment 5 (2-12-C) 210.68 Embodiment 7 (2-20-A) 104.99 Embodiment 12 (5-13-A) 36.65

[1373] The test results show that the compounds of the present invention in Table 16 have a significant inhibitory effect on the proliferation of HCC70 human breast cancer cells.

[1374] 9. Inhibitory effect of compounds on MDA-MB-231 cell proliferation

[1375] (1) Cell plating: First, the tumor cells MDA-MB-231 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 17.

[1376] Table 17. Tumor cell sources

[1377] Cell Name Tumor type source MDA-MB-231 Human breast cancer cells Nanjing Kebai Biological

[1378] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1379] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50) m, shake and mix in the dark, react for 10 min before testing, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 18.

[1380] (2) Data results

[1381] Table 18. MDA-MB-231 cell proliferation inhibitory activity

[1382] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 381.70 Embodiment 1 (1-1-A) 363.60 Embodiment 3 (2-1-A) 299.70 Embodiment 4 (2-7-A) 123.50 Embodiment 5 (2-12-A) 123.50 Embodiment 9 (3-4-A) 284.80 Embodiment 12 (5-13-A) 332.60 Embodiment 13 (5-7-B) 172.40 Embodiment 20 (1-10-A) 97.57 Embodiment 20 (1-10-B) 27.24 Example 22 (4-15) 175.40

[1383] The test results show that the compounds of the present invention in Table 18 have a significant inhibitory effect on the proliferation of MDA-MB-231 human breast cancer cells.

[1384] 10. Inhibitory effect of compounds on Jeko-1 cell proliferation

[1385] (1) Cell plating: First, the tumor cells Jeko-1 were cultured in the corresponding culture medium, the cells were centrifuged and resuspended, and the cells were counted, and finally the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 19.

[1386] Table 19. Tumor cell sources

[1387]

[1388]

[1389] Co-incubation of the compound of the present invention and tumor cells: After the cells are plated, the diluted bioactive molecules (compound of the present invention) are added to the above plate wells according to a certain dilution ratio and incubated for 72 h.

[1390] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 500 μg, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, according to the four-parameter model fitting curve, calculate the half inhibition concentration (IC50) of the compound, the test results are shown in Table 20.

[1391] (2) Data results

[1392] Table 20. Jeko-1 cell proliferation inhibitory activity

[1393] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 1.25 Embodiment 1 (1-1-A) 1.33 Embodiment 2 (1-7-A) 0.15 Embodiment 2 (1-7-B) 0.66 Embodiment 4 (2-7-A) 0.28 Embodiment 9 (3-4-A) 0.27 Embodiment 9 (3-4-B) 0.15 Embodiment 11 (4-10) 0.45 Embodiment 13 (5-7-A) 0.16 Embodiment 13 (5-7-B) 0.18 Example 21 (4-14) 0.34 Example 22 (4-15) 0.11

[1394] The test results show that the compounds of the present invention in Table 20 have a significant inhibitory effect on the proliferation of Jeko-1 human mantle cell lymphoma cells.

[1395] 11. Inhibitory effect of compounds on MDA-MB-453 cell proliferation

[1396] (1) Cell plating: First, the tumor cells MDA-MB-453 were cultured in the corresponding culture medium, the cells were digested with trypsin, the cells were resuspended after centrifugation and counted, and the cells were adjusted to an appropriate concentration for plating. The sources of tumor cells are shown in Table 21.

[1397] Table 21. Tumor cell sources

[1398] Cell Name Tumor type source MDA-MB-453 Human breast cancer cells Connaughty

[1399] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecules (compound of the present invention) are added to the above plate wells and incubated for 72 hours.

[1400] In vitro cell activity detection: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 500 μg, shake and mix in the dark, react for 10 minutes before detection, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM ) to obtain background RLU, the culture medium containing cells (containing Cell Counting-Lite TM ) to obtain solvent RLU. Cell inhibition rate = 1-(sample RLU-sample RLU) / (solvent RLU-background RLU)×100%, and the half inhibitory concentration (IC) of the compound was calculated according to the four-parameter model fitting curve. 50 ), the test results are shown in Table 22.

[1401] (2) Data results

[1402] Table 22. MDA-MB-453 cell proliferation inhibitory activity

[1403] name <![CDATA[IC 50 (nM)]]> Comparative Compound 1 6.99 Embodiment 16 (3-12-A) 0.47 Embodiment 16 (3-12-B) 4.32 Embodiment 16 (3-12-C) 5.52 Embodiment 16 (3-12-D) 3.98 Embodiment 17 (3-7-A) 6.23 Embodiment 17 (3-7-B) 4.87 Embodiment 18 (3-17-A) 3.32 Embodiment 18 (3-17-B) 6.16 Embodiment 19 (5-22-A) 7.34 Embodiment 19 (5-22-B) 5.67 Example 24 (2-27-A) 11.83 Example 25 (3-26-A) 3.95

[1404] The test results show that the compounds of the present invention in Table 22 have a significant inhibitory effect on the proliferation of MDA-MB-453 human breast cancer cells.

[1405] The structures of comparative compound 1 and comparative compound 2 are shown below:

[1406]

[1407] 2. Antibody Coupling Test

[1408] The coupling preparation of ADC DL-15 sample is as follows:

[1409] Take 1.036mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg / mL), dilute with 0.1M disodium edetate solution (pH 7.6), and then use 1M Na 2 HPO 4 The solution was adjusted to pH 7.6, and 2.4 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) was added and mixed, and allowed to stand at room temperature for 90 min. 5 times the amount of compound DL-15 dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain antibody drug conjugate ADC DL-15 (1.77 mL, 8.60 mg / mL).

[1410]

[1411] The molecular weight of ADC DL-15 was determined by LC-MS, and the drug / antibody ratio DAR value was calculated to be 4.11, as shown in Tables 23 and 24.

[1412] Table 23: Measured molecular weight of ADC DL-15

[1413]

[1414] Table 24: DAR values ​​for ADC DL-15

[1415]

[1416] Chromatographic conditions:

[1417] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[1418] Mobile phase A: 0.1% FA / H 2 O; mobile phase B: 0.1% FA / ACN;

[1419] Flow rate: 0.25 mL / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μL;

[1420]

[1421]

[1422] Mass spectrometry conditions:

[1423] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[1424] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10;

[1425] Accumulation time 0.5s; m / z 600-4000; Time bins to sum 40.

[1426] The coupling preparation of ADC 3-4-04-A sample is as follows:

[1427] Take 0.518mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg / mL), dilute with 0.1M disodium edetate solution (pH 7.6), and then use 1M Na 2 HPO 4 The solution was adjusted to pH 7.6, and 5.5 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) was added and mixed, and allowed to stand at room temperature for 90 min. 10 times the amount of compound 3-4-04-A dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain antibody drug conjugate ADC 3-4-04-A (1.50 mL, 5.60 mg / mL).

[1428]

[1429] The molecular weight of ADC 3-4-04-A was determined by LC-MS, and the drug / antibody ratio DAR value was calculated to be 7.47, as shown in Tables 25 and 26. The chromatographic determination conditions were the same as those for ADC DL-15.

[1430] Table 25: ADC 3-4-04-A measured molecular weight

[1431]

[1432] Table 26: DAR values ​​for ADC 3-4-04-A

[1433]

[1434]

[1435] The coupling preparation of ADC 3-4-04-B sample is as follows:

[1436] Take 0.518mL hIgG antibody (anti-chicken lysozyme antibody, 19.3mg / mL), dilute with 0.1M disodium edetate solution (pH 7.6), and then use 1M Na 2 HPO 4 The solution was adjusted to pH 7.6, and 5.5 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) was added and mixed, and allowed to stand at room temperature for 90 min. 10 times the amount of compound 3-4-04-B dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain antibody drug conjugate ADC 3-4-04-B (1.50 mL, 5.60 mg / mL).

[1437]

[1438] The molecular weight of ADC 3-4-04-B was determined by LC-MS, and the drug / antibody ratio DAR value was calculated to be 8.04, as shown in Tables 27 and 28. The chromatographic determination conditions were the same as those for ADC DL-15.

[1439] Table 27: ADC 3-4-04-B measured molecular weight

[1440]

[1441] Table 28: DAR values ​​for ADC 3-4-04-B

[1442]

[1443] The above experiments prove that the cytotoxic drug-linker compounds of the present invention can be smoothly coupled with antibodies to obtain antibody-drug conjugates.

[1444] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (I): In the above formula (I), R x is hydrogen; R y is hydrogen, R z Selected from Dimethylaminomethylene.

2. A compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is as follows:

3. A compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is as follows:

4. A compound represented by formula (VI) or a pharmaceutically acceptable salt thereof, wherein the compound structure is as follows: MLED Formula (VI) in, M is a linker site with an antibody or antigen-binding fragment thereof; L is a linker between the linkers M and E; E is a structural fragment connecting L and D; D is selected from the structure of the compound after dehydrogenation according to any one of claims 1 to 3; Where M has the following structure: L is selected from the following structures: and E is selected from a single bond, -NH-CH2-, 5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein L has the following structure:

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein E is -NH-CH2-.

7. A compound represented by formula (VI) or a pharmaceutically acceptable salt thereof, wherein the compound structure is as follows: MLED Formula (VI) in, M is a linker site with an antibody or antigen-binding fragment thereof; L is a linker between the linkers M and E; E is a structural fragment connecting L and D; D is selected from the following structures: M has the following structure: L is selected from the following structures: and E is selected from a single bond, -NH-CH2-, 8. A compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is as follows:

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

10. A medicine kit product, comprising: a) at least one compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9 as a first therapeutic agent; and b) optional packaging and / or instructions.

11. Use of the compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 9 or the kit product of claim 10 in the preparation of a medicament for treating a disease involving abnormal cell proliferation; the disease involving abnormal cell proliferation is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, cervical cancer, and lymphoma.

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