Camptothecin compound as well as preparation method and application thereof

Abstract

The invention relates to a camptothecin compound with anti-tumor activity as well as a preparation method and application of the camptothecin compound. Specifically, the invention relates to a compound shown in the specification or a pharmaceutically acceptable form, a pharmaceutical composition, a preparation method and application thereof. Use of said compound as a medicament for the treatment of diseases in the aspect of abnormal cell proliferation, # imgabs0

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280008418.X, titled "Camptothecin Compounds and Their Preparation Methods and Applications", filed on January 27, 2022. Technical Field

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

[0003] Camptothecin (CPT, Formula 1) is a pentacyclic quinoline nucleus compound isolated from the plant Camptotheca acuminata of the Nyssaceae family. It consists of quinoline rings AB, pyrrole ring C, pyridone ring D, and α-hydroxy lactone ring E, where the 20-position has an S configuration (see the structural formula below). It was introduced into clinical practice in the early 1970s due to its excellent anti-cancer activity. Later, clinical trials were terminated due to 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, resulting in blocked DNA replication, and ultimately causing 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, multiple camptothecin drugs have been approved for marketing for tumor treatment (Med. Res. Rev. 2015, 35, 753). Irinotecan is a drug for the treatment of colorectal cancer; Topotecan is used for the treatment of ovarian cancer; Belotecan is used for the treatment of ovarian cancer and small cell lung cancer. Camptothecin derivatives also include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, Elomotecan, etc.

[0007] Camptothecin drugs or derivatives often have hematotoxicity 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-drug conjugates with antibodies, etc. Therefore, there is still a high clinical need and application value in developing camptothecin compounds and their conjugates with novel structures that can improve effectiveness and safety issues. Summary of the Invention

[0008] The present invention provides camptothecin compounds with novel structures and their conjugates. The camptothecin compounds have good antitumor activity and are expected to be used in the treatment of tumor diseases; their conjugates have broad application prospects as antibody-drug conjugates.

[0009] In the first aspect of the present invention, there is provided a compound or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug, wherein the compound has the structure shown below:

[0010]

[0011] Wherein,

[0012] R 1 and R 2 each independently selected from hydrogen, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, hydroxy, cyano and C 3-6 cycloalkyl; or, R 1 and R 2 are connected to adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocycle;

[0013] R 3 is hydrogen or is connected to the adjacent carbon atom of R 1 to form a six-membered carbon ring;

[0014] A is selected from and -NH 2 one of them;

[0015] R 4 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3- to 6-membered heterocyclic group;

[0016] R 5 and R 6 each independently selected from hydrogen, C1-6 alkyl, 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- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl group, aryl, and heteroaryl; or R 5 and R 6 are joined to adjacent carbon atoms to form a 3- to 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 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, and 3- to 6-membered heterocyclic group;

[0021] R y and R z are not both hydrogen 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- to 6-membered heterocyclic alkyl group, and 3- to 6-membered heterocyclic group.

[0022] In some embodiments, R x is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, and 3- to 6-membered heterocyclic group;

[0023] R y and R z are not both hydrogen and are independently selected from hydrogen, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkylaminoalkyl, C 1-6 alkoxyalkyl, 3- to 6-membered heterocyclic alkyl group, and 3- to 6-membered heterocyclic group.

[0024] In some embodiments, in formula (I), R xSelected from hydrogen, C 1-6 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 both hydrogen, and are independently selected from hydrogen, C 2-6 alkenyl, C 2-6 alkynyl.

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

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

[0028] In some embodiments, R y and R z are not both hydrogen, and are independently selected from hydrogen, dimethylaminomethylene, morpholinomethylene and methoxymethylene.

[0029] In some embodiments, R y and R z are not both hydrogen, and are independently selected from hydrogen, dimethylaminomethylene and methoxymethylene.

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

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

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

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

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

[0035] In some embodiments, at the position of formula (I) is configuration.

[0036] In some embodiments, at the position of formula (I) is configuration.

[0037] In some embodiments, at the position of formula (I) is configuration.

[0038] In some embodiments, at the position of formula (I) is configuration.

[0039] In some embodiments, at the position of formula (I) is configuration.

[0040] In some embodiments, at the position of formula (I) is configuration.

[0041] In some embodiments, at the position of formula (I) is configuration.

[0042] In some embodiments, at the position of formula (I) is configuration.

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

[0044]

[0045] In formula (II), A’ is selected from and -NH 2 one of;

[0046] R x’ is 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’ are independently selected from hydrogen, C 1-6alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y’ and R z’ are linked to adjacent carbon atoms to form a 3- to 6-membered ring.

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

[0049]

[0050]

[0051] In some embodiments, R x’ is 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;

[0052] R y’ and R z’ are independently 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, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, or R y’ and R z’ are linked to adjacent carbon atoms to form a 3- to 6-membered ring.

[0053] In some embodiments, the 3- to 6-membered ring is selected from a 3- to 6-membered carbocyclic ring or a 3- to 6-membered heterocyclic ring.

[0054] In some embodiments, R x’ is selected from hydrogen and C 1-6 alkyl.

[0055] In some embodiments, R x’ is selected from hydrogen and methyl.

[0056] In some embodiments, R y’ and R z’ are independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C3-6 Naphthenyl and C 2-6 alkenyl, or R y’ and R z’ are connected to adjacent carbon atoms to form a 3- to 6-membered naphthenyl ring.

[0057] In some embodiments, R y’ is selected from hydrogen and C 1-6 alkyl, and R z’ is selected from hydrogen, C 1-6 alkyl, and C 3-6 naphthenyl, or R y’ and R z’ are connected to adjacent carbon atoms to form a 3- to 6-membered ring.

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

[0059] 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’ are connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0060] 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’ are connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0061] In some embodiments, A' in formula (II) is -NH 2 .

[0062] In some embodiments, at in formula (II)-1 is configuration.

[0063] In some embodiments, at in formula (II)-1 is configuration.

[0064] In some embodiments, at in formula (II)-1 is configuration.

[0065] In some embodiments, at in formula (II)-1 is configuration.

[0066] In some embodiments, at is configuration.

[0067] In some embodiments, at is configuration.

[0068] In some embodiments, at is configuration.

[0069] In some embodiments, at is configuration.

[0070] In some embodiments, the compound has the structure of formula (III):

[0071]

[0072] In formula (III), A” is selected from and -NH 2 one of;

[0073] R x” is 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;

[0074] R y” and R z” are 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 heterocyclic group, 3-6 membered heterocyclic alkyl, 4-6 membered heterocyclic group, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y” and R z” are connected to adjacent carbon atoms to form a 3-6 membered ring.

[0075] In some embodiments, the structure of the compound of formula (III) is shown as formula (III)-1 below:

[0076]

[0077] In some embodiments, R x” is 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;

[0078] R y” and R z” are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, 4- to 6-membered heterocyclic group, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, or R y” and R z” are joined to adjacent carbon atoms to form a 3- to 6-membered ring.

[0079] In some embodiments, R x” is selected from hydrogen and C 1-6 alkyl.

[0080] In some embodiments, R x” is hydrogen.

[0081] In some embodiments, the 3- to 6-membered ring is selected from 3- to 6-membered carbocyclic or 3- to 6-membered heterocyclic rings.

[0082] 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” are joined to adjacent carbon atoms to form a 3- to 6-membered ring.

[0083] In some embodiments, R y” is hydrogen, R z” is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, and vinyl, or R y” and R z” are joined to adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

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

[0085] 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” are connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0086] In some embodiments, A” in formula (III) is -NH 2 .

[0087] In some embodiments, at in formula (III)-1 is configuration.

[0088] In some embodiments, at in formula (III)-1 is configuration.

[0089] In some embodiments, at in formula (III)-1 is configuration.

[0090] In some embodiments, at in formula (III)-1 is configuration.

[0091] In some embodiments, at in formula (III)-1 is configuration.

[0092] In some embodiments, at in formula (III)-1 is configuration.

[0093] In some embodiments, at in formula (III)-1 is configuration.

[0094] In some embodiments, at in formula (III)-1 is configuration.

[0095] In some embodiments, the compound has the structure of formula (IV):

[0096]

[0097] In formula (IV),

[0098] R a and Rb independently selected from hydrogen, halogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkoxyalkyl, C 1-6 alkoxy, C 1-6 haloalkyl, hydroxy and cyano; or R a and R b are linked to adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocycle;

[0099] R c and R d are 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- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl group, C 2-6 alkenyl and C 2-6 alkynyl, or R c and R d are linked to adjacent carbon atoms to form a 3- to 6-membered carbocyclic or heterocyclic ring;

[0100] R e is 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;

[0101] q = 0 or 1;

[0102] When q = 0, R c and R d are not both hydrogen.

[0103] In some embodiments, R a and R b are 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 are linked to adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocycle;

[0104] R c and R d are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C3-6 cycloalkyl, 3- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl group, C 2-6 alkenyl and C 2-6 alkynyl, or R c and R d are linked to adjacent carbon atoms to form a 3- to 6-membered carbocyclic or heterocyclic ring;

[0105] R e is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl and 4- to 6-membered heterocyclic group;

[0106] q = 0 or 1;

[0107] When q = 0, R c and R d are not both hydrogen.

[0108] In some embodiments, in formula (IV), R a and R b are 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 are linked to adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocyclic ring;

[0109] R c and R d are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, 4- to 6-membered heterocyclic group, C 2-6 alkenyl, C 2-6 alkynyl, or R c and R d are linked to adjacent carbon atoms to form a 3- to 6-membered carbocyclic or heterocyclic ring;

[0110] R e is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, or 4- to 6-membered heterocyclic group;

[0111] q = 0 or 1;

[0112] When q = 0, R c and R d are not both hydrogen.

[0113] In some embodiments, R a and R b are independently selected from hydrogen, halogen, and C 1-6 alkyl, or R a and R b are joined to adjacent carbon atoms to form a 5- or 6-membered oxygen-containing heterocycle.

[0114] 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 to which they are attached form

[0115] wherein Z is selected from -CH 2 -, -CD 2 -, -CH 2 CH 2 -, and -CF 2 -.

[0116] In some embodiments, R a is methyl, R b is fluorine, or R a and R b together with the benzene ring to which they are attached form

[0117] In some embodiments, R c and R d are independently selected from hydrogen, C 1-6 alkoxyalkyl, and C 1-6 alkylaminoalkyl, or R c and R d are joined to adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

[0118] In some embodiments, R c is hydrogen, R d is selected from hydrogen, methoxyethyl, and cyclopropyl, or R c and R d are joined to adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

[0119] In some embodiments, R e is selected from hydrogen and C 1-6 alkyl.

[0120] In some embodiments, R e is selected from hydrogen and isopropyl.

[0121] In some embodiments, R a is methyl, Rb is fluorine, or R a and R b together with the benzene ring to which it is attached form 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 are connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0122] In some embodiments, R a is methyl, R b is fluorine, or R a and R b together with the benzene ring to which it is attached form 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 are connected to adjacent carbon atoms to form a 3-membered carbon ring.

[0123] In some embodiments, at the position of formula (IV) is configuration.

[0124] In some embodiments, at the position of formula (IV) is configuration.

[0125] In some embodiments, the compound has the structure of formula (V):

[0126]

[0127] In formula (V), R is selected from C 3-6 cycloalkyl and C 1-6 alkoxy;

[0128] A”’ is selected from and -NH 2 one of;

[0129] R x”’ is 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;

[0130] R y”’ and R z”’ are independently selected from hydrogen, C1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl group, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y”’ and R z”’ are joined to adjacent carbon atoms to form a 3- to 6-membered ring.

[0131] In some embodiments, R is selected from methoxy and cyclopropyl.

[0132] In some embodiments, the compound of formula (V) has the structure shown in formula (V)-1 below:

[0133]

[0134] In some embodiments, the 3- to 6-membered ring is selected from 3- to 6-membered carbocyclic rings or 3- to 6-membered heterocyclic rings.

[0135] 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”’ are joined to adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

[0136] In some embodiments, R y”’ and R z”’ are both hydrogen, or R y”’ and R z”’ are joined to adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

[0137] In some embodiments, R x”’ is selected from hydrogen and C 1-6 alkyl.

[0138] In some embodiments, R x”’ is hydrogen.

[0139] In some embodiments, A''' is -NH 2 .

[0140] In some embodiments, R is selected from methoxy and cyclopropyl, R x”’ is hydrogen, R y”’ and R z”’are all hydrogen, or R y”’ and R z”’ is connected to an adjacent carbon atom to form a 3-membered carbon ring.

[0141] In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration.

[0142] In some embodiments, in formula (V)-1 is configuration. In some embodiments, in formula (V)-1 is configuration. In some embodiments, the present invention provides the following compounds:

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] On the other hand, the present invention also provides a compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, and prodrug thereof:

[0150] M-L-E-D

[0151] Formula (VI)

[0152] wherein,

[0153] M is a linker portion to an antibody or an antigen-binding fragment thereof;

[0154] L is a linker connecting the linker M and E;

[0155] E is a structural fragment connecting L and D;

[0156] D is a structural fragment of a cytotoxic drug.

[0157] In some embodiments, M is selected from the following structures:

[0158]

[0159] In some embodiments, M is selected from the following structures:

[0160]

[0161] In some embodiments, L is selected from the divalent structures consisting of one or more of the following: C 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,

[0162] wherein R' represents hydrogen, C 1-6 alkyl or an alkyl containing -(CH 2 CH 2 O) r -; r is an integer selected from 1 to 10; s is an integer selected from 1 to 10.

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

[0164]

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

[0166]

[0167] In some embodiments, E is selected from a single bond, -NH-CH 2 -, In some embodiments, E is -NH-CH 2 -.

[0168] In some embodiments, the cytotoxic drug is selected from the compounds described in any one of the first aspect of the present invention.

[0169] 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 in the present invention.

[0170] In some embodiments, D is selected from the structures after dehydrogenation of the compounds described in the present invention.

[0171] In some embodiments, D is selected from the structures after dehydrogenation 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 in the present invention.

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

[0173]

[0174]

[0175] In some embodiments, D is selected from the following structures:

[0176]

[0177] In some embodiments, M-L-E-D is selected from the following compounds:

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] In some embodiments, M-L-E-D is selected from the following compounds:

[0184]

[0185] Definition

[0186] Unless otherwise defined herein, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that are obvious to one of ordinary skill in the art. Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the present invention.

[0187] The terms "comprising", "including", "having", "containing", or "involving" and other variant forms thereof used herein are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0188] As used herein, the "*" marked in the compound structural formula indicates that the marked carbon atom is a chiral carbon atom, and the present invention includes a pair of enantiomers formed by this chiral carbon atom. If a compound contains two different chiral carbon atoms, the present invention includes 4 optical isomers formed by these chiral carbon atoms.

[0189] As used herein, represents a bond that can be stereospecific ((R) or (S)) or non-stereospecific.

[0190] The term "alkyl" is defined as a straight-chain or branched-chain saturated aliphatic hydrocarbon group. In some embodiments, the alkyl has 1 to 12, for example 1 to 6 carbon atoms. For example, as used herein, the term "C 1-6 alkyl" refers to a linear or branched group having 1 to 6 carbon atoms (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl), which is optionally substituted with 1 or more (such as 1, 2, or 3) suitable substituents.

[0191] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-6 alkenyl", "C 2-4 alkenyl", etc. Examples thereof include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0192] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon triple bond. It includes, for example, "C 2-6 alkynyl", "C 4-6"Alkynyl", etc. 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, etc.

[0193] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocyclic alkyl and bicyclic alkyl (such as spirocycloalkyl, fused cycloalkyl and bridged cycloalkyl). The term "C 3-6 cycloalkyl" refers to a cycloalkyl having 3 to 6 ring-forming carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by 1 or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0194] The term "carbocyclic" or "carbocyclic group" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon group connected through ring carbon. Examples include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl.

[0195] The term "carbocyclic" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (for example, monocyclic, such as cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, cycloheptane ring, cyclooctane ring, cyclononane ring, or bicyclic, including spiro, fused or bridged systems (such as bicyclo[1.1.1]pentane ring, bicyclo[2.2.1]heptane ring, bicyclo[3.2.1]octane ring or bicyclo[5.2.0]nonane ring, decahydronaphthalene ring, etc.), which may optionally be substituted by 1 or more (such as 1, 2 or 3) suitable substituents. The term "3-6 membered carbocyclic ring" refers to a carbocyclic ring containing 3, 4, 5 or 6 ring-forming carbon atoms.

[0196] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure, the ring atoms of which 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 heterocyclic group can be connected to the rest of the molecule through any one of the ring atoms. The heterocyclic group in the present invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" used in the present invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered heterocyclic groups, 4-membered heterocyclic groups, 5-membered heterocyclic groups and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups, oxygen-containing heterocyclic groups, e.g., 4-6 membered heterocyclic groups, e.g., 4-6 membered nitrogen-containing heterocyclic groups, 4-6 membered oxygen-containing heterocyclic groups. Common heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl. The heterocyclic group in the present invention may optionally be substituted with one or more substituents described in the present invention. The heterocyclic group in the present invention is optionally fused with one or more aromatic or non-aromatic rings.

[0197] 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 5-6 membered oxygen-containing heterocycles, specific examples of which include (but are not limited to) ethylene oxide ring, tetrahydrofuran ring, furan ring, tetrahydropyran ring, 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.

[0198] The term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1, 2 or 3) identical or different halogen atoms, wherein the alkyl group is defined as described above. For example, the term "C 1-6 haloalkyl" used in the present invention 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 by one or more substituents described in the present invention.

[0199] The term "aryl" refers to a group obtained by removing a hydrogen atom from an aromatic nuclear carbon atom of an aromatic hydrocarbon molecule. For example, aryl groups having 6 to 14 carbon atoms, and specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, etc.

[0200] 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- to 6-membered heteroaryl groups, 5- to 6-membered nitrogen-containing heteroaryl groups, 5- to 6-membered oxygen-containing heteroaryl groups, etc., such as furyl, 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, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, etc.

[0201] The term "alkoxy" refers to a group having the structure "alkyl-O-", where the alkyl group is defined as above. For example, C 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 by one or more substituents described in the present invention.

[0202] ()() The term "alkoxyalkyl" refers to an alkyl group substituted by one or more (e.g., 1, 2, 3, or 4) alkoxy groups, where the definitions of the alkoxy group and the alkyl group are as described above. For example, the term "C 1-6 alkoxyalkyl" used in the present invention refers to an alkyl group having 1 to 6 carbon atoms and 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 -, etc.

[0203] The term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0204] The term "nitrogen oxides" refers to oxides of at least one nitrogen atom in the structure of the compounds of the present application (e.g., mono- or di-oxides). The mono-oxide of nitrogen can exist in the form of a single positional isomer or a mixture of positional isomers.

[0205] The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the specified atom are replaced by a selection from the indicated groups, provided that the normal atomic valence of the specified atom in the current case 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.

[0206] If a substituent is described as "optionally substituted", then the substituent may (1) be unsubstituted or (2) be substituted. If a carbon of a substituent is described as optionally substituted by one or more substituents from a list of substituents, then one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced by independently selected optional substituents, either individually and / or together. If a nitrogen of a substituent is described as optionally substituted by one or more from a list of substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced by an independently selected optional substituent.

[0207] If a substituent is described as "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.

[0208] As used herein, the term "one or more" means 1 or more than 1 under reasonable conditions, e.g., 2, 3, 4, 5, or 10.

[0209] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0210] The term "stereoisomer" refers to isomers formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, there can be produced racemic mixtures, single enantiomers, mixtures of diastereomers, and individual diastereomers. A particular individual molecule can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is understood that the scope of the present application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0211] In this text, a solid line (-), a solid wedge or a dashed wedge can be used to depict the carbon-carbon bonds of the compounds of the present invention. Using a solid line to depict a bond attached to an asymmetric carbon atom is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., a specific enantiomer, a racemic mixture, etc.). Using a solid or dashed wedge to depict a bond attached to an asymmetric carbon atom is intended to indicate that the depicted stereoisomer exists. When present in a racemic mixture, solid and dashed wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist in the form of stereoisomers (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 present invention can exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0212] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which can be a single polymorph or a mixture of any proportion of more than one polymorph.

[0213] It should also be understood that certain compounds of the present invention can exist in free form for therapeutic use, or, when appropriate, in the form of their pharmaceutically acceptable derivatives. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which, after administration to a patient in need thereof, are capable of directly or indirectly providing the compounds of the present invention or their metabolites or residues. Thus, when referring to "the compounds of the present invention" herein, it is also intended to cover the above various derivative forms of the compounds.

[0214] The pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0215] Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartates, fumarates, glucoheptonates, gluconates, glucuronates, hexafluorophosphates, and the like.

[0216] 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.

[0217] A review of suitable salts can be found in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing the pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0218] The term "ester" means an ester derived from each of the general formula compounds in the present application, which includes physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in the form of free acids or alcohols). The compounds of the present invention themselves can also be esters.

[0219] The compounds of the present invention can exist in the form of solvates (preferably hydrates), in which the compounds of the present invention contain a polar solvent, particularly for example water, methanol, or ethanol, as a structural element of the crystal lattice of the compound. The amount of the polar solvent, particularly water, can be present in a stoichiometric or non-stoichiometric ratio.

[0220] Also included within the scope of the present invention are the metabolites of the compounds of the present invention, i.e., substances formed in the body upon administration of the compounds of the present invention. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic hydrolysis, etc. of the administered compounds. Accordingly, the present invention includes the metabolites of the compounds of the present invention, including compounds prepared by a method of contacting the compounds of the present invention with a mammal for a time sufficient to produce its metabolite.

[0221] The present invention further includes within its scope prodrugs of the compounds of the present invention. Generally, such prodrugs will be functional group derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Accordingly, in these instances, the term "administering" in the context of the therapeutic methods of the present invention shall include treating various diseases or disorders with a prodrug form of one or more of the claimed compounds, provided that the prodrug form is convertible in vivo into the aforementioned compounds upon administration to an individual. For example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985, conventional methods for the selection and preparation of suitable prodrug derivatives are described.

[0222] The present invention further includes within its scope isotopically labeled compounds of the compounds of the present invention, which are identical to the compounds of the present invention except that one or more atoms 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 predominating in nature.

[0223] The present invention also encompasses compounds of the present invention containing protecting groups. In any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any relevant molecules, thereby forming chemically protected forms of the compounds of the present invention. This can be accomplished by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. The protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0224] Drug composition

[0225] In a third aspect, the present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof according to the first or second aspect of the present invention, and one or more pharmaceutically acceptable carriers.

[0226] The term "pharmaceutical composition" refers to a composition that can be used as a medicine, which contains a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an excipient administered together with the 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.

[0227] The above-mentioned 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, troches, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0228] The above-mentioned pharmaceutical composition can contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled substance, metabolite or prodrug.

[0229] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition or its corresponding preparation form, which includes combining at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled substance, metabolite or prodrug with one or more pharmaceutically acceptable carriers.

[0230] Kit product

[0231] In a fourth aspect, the present invention provides a kit product, which contains:

[0232] a) at least one compound or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled substance, metabolite or prodrug described in the first aspect or the second aspect of the present invention as a first therapeutic agent, or a pharmaceutical composition described in the third aspect as a first pharmaceutical composition;

[0233] b) optionally at least one other therapeutic agent as a second therapeutic agent, or a pharmaceutical composition containing the other therapeutic agent as a second pharmaceutical composition; and

[0234] c) optionally packaging and / or instructions.

[0235] The above-mentioned kit product can contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled substance, metabolite or prodrug.

[0236] The present invention also provides a method for preparing the above-mentioned medicine box, which includes combining at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled substance, metabolite or prodrug, or the above-mentioned pharmaceutical composition with at least one other therapeutic agent optionally present or a pharmaceutical composition containing other therapeutic agents, packaging and / or instructions.

[0237] Medical use

[0238] The compounds of the present invention can exhibit a strong effect in inhibiting abnormal cell proliferation.

[0239] Therefore, the present application provides the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotope-labeled substances, metabolites and prodrugs, or the above-mentioned pharmaceutical compositions, which are used for treating diseases related to abnormal cell proliferation.

[0240] 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, N-oxides, isotope-labeled substances, metabolites and prodrugs, or the above-mentioned pharmaceutical compositions in the preparation of a drug for treating diseases related to abnormal cell proliferation.

[0241] In some embodiments, the diseases related to abnormal cell proliferation include (but are not limited to) tumors, such as advanced solid tumors.

[0242] The present application also provides the use of the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, N-oxides, isotope-labeled substances, metabolites and prodrugs, or the pharmaceutical compositions of the present invention in the preparation of a preparation for inhibiting the proliferation of tumor cells. In certain embodiments, the preparation is for in vivo or in vitro administration. For example, the preparation can be administered to a subject in vivo to inhibit the proliferation of tumor cells in the subject; or, the preparation can be administered to in vitro cells (such as cell lines or cells from a subject) to inhibit the proliferation of in vitro tumor cells.

[0243] The tumors of the present invention include (but are not limited to): brain tumors, lung cancers, squamous cell carcinomas, bladder cancers, gastric cancers, ovarian cancers, peritoneal cancers, pancreatic cancers, breast cancers, head and neck cancers, cervical cancers, endometrial cancers, colorectal cancers, liver cancers, kidney cancers, esophageal adenocarcinomas, esophageal squamous cell carcinomas, prostate cancers, female genital tract cancers, carcinoma in situ, lymphomas, neurofibromas, thyroid cancers, bone cancers, skin cancers, brain cancers, colon cancers, testicular cancers, gastrointestinal stromal tumors, prostate tumors, mast cell tumors, multiple myelomas, melanomas, gliomas or sarcomas.

[0244] Treatment method

[0245] In another aspect, the present invention provides a method for treating diseases related to abnormal cell proliferation, which comprises the following steps: administering a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite and prodrug thereof, or the above-mentioned pharmaceutical composition, to an individual in need thereof.

[0246] The term "effective amount" refers to a dose that is capable of inducing a biological or medical response in a cell, tissue, organ or organism (such as an individual), and is sufficient to achieve the desired prophylactic and / or therapeutic effect.

[0247] The dosing regimen can be adjusted to provide the optimal desired response. For example, a single dose can be administered, doses can be administered in divided amounts over time, or the dose can be administered at a reduced or increased rate depending on the circumstances. It is understood that for any particular individual, the specific dosing regimen should be adjusted according to need and the professional judgment of the person administering the dosing composition or supervising the composition.

[0248] The dosage of the compound of the present invention will depend on the individual circumstances, the severity of the disease or condition, the rate of administration, the disposition of the compound, and the judgment of the prescribing physician. Generally, the effective amount is about 0.001 - 10000 mg / kg of the subject's body weight per day. In suitable cases, the effective amount is about 0.01 - 1000 mg / kg of the subject's body weight per day. The dose of about 0.01 - 1000 mg / kg of the subject's body weight can be administered daily, every two days or every three days, usually about 0.1 - 500 mg / kg of the subject's body weight. Exemplary dosing regimens are once or multiple times a day, or once or multiple times a week, or once or multiple times a month. When multiple doses are administered, the interval between single doses can usually be daily, weekly, monthly or annually. Alternatively, it can be administered in the form of a sustained-release preparation, in which case a lower dosing frequency is required. The dosing dose and frequency can vary depending on the half-life of the drug in the subject, and can also vary depending on whether it is a prophylactic application or a therapeutic application. In prophylactic applications, relatively low doses are administered at relatively long intervals over a long period; in therapeutic applications, relatively high doses are sometimes required to be administered at shorter intervals until the progression of the disease is retarded or stopped, preferably until the individual shows partial or complete improvement of the disease symptoms, after which prophylactic application can be adopted.

[0249] The term "treatment" refers to alleviating or eliminating the targeted disease or disorder. If a subject has received a therapeutically effective 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 show observable and / or detectable remission and / or improvement, it indicates that the subject has been successfully "treated". It is understood that treatment includes not only complete treatment but also treatment that does not reach complete treatment but achieves some biologically or medically relevant results.

[0250] The term "administer (administrate / administrating / administration)" (or "drug administration") refers to the process of applying a pharmaceutically active ingredient (such as a compound of the present invention) or a pharmaceutical composition containing 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 that the pharmaceutically active ingredient or pharmaceutical composition comes into contact with the individual or its cells, tissues, organs, biological fluids, etc. Common administration methods 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.

[0251] The term "in need thereof" refers to a doctor's or other caregiver's judgment that an individual needs or will benefit from a prevention and / or treatment process, and this judgment is based on various factors within the doctor's or other caregiver's area of expertise.

[0252] The term "individual" (or "subject") refers to a human or non-human animal. The individuals of the present invention include 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, domestic animals and / or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).

[0253] Preparation method

[0254] The fourth aspect of the present invention provides a method for synthesizing the said compound.

[0255] The compound of formula (I) in the present invention can be synthesized and prepared by the following synthetic route.

[0256]

[0257] Wherein, R x 、R y and R z have the meanings as described above; LG is a leaving group, selected from mesyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0258] Step 1:

[0259] The substitution reaction of the compound of formula (I)-SM1 and the compound of formula (I)-SM2 gives the compound of formula (I)-IM1.

[0260] 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;

[0261] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may 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.

[0262] In some embodiments, this step is carried out in the presence of a suitable base, and the base includes an organic base or an inorganic base. The organic base may be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py). The inorganic base may 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 ;

[0263] Step 2:

[0264] The condensation reaction of the compound of formula (I)-IM1 and the compound of formula (I)-SM3 gives the compound of formula (I);

[0265] In some embodiments, this step is carried out with a suitable condensing reagent, and the condensing reagent may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0266] 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;

[0267] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0268] In some embodiments, this step is carried out in a suitable base, and the base includes an organic base or an inorganic base. The organic base may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base may be selected from K 3 PO 4 、NaH、K 2 CO 3 、Na 2 CO 3 、Cs 2 CO 3 and NaOH, preferably DIPEA.

[0269] The compound of formula (II)-1 in the present invention can be synthesized and prepared by the following synthetic route.

[0270]

[0271] Among them, R x’ 、R y’ and R z’ have the meanings as described above; LG is a leaving group, selected from mesyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine; PG is a protecting group, selected from

[0272] Step 1

[0273] The compound of formula (II)-IM1 is obtained by a substitution reaction of the compound of formula (II)-SM1.

[0274] 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;

[0275] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably n-heptane.

[0276] Step 2

[0277] The reduction reaction of the compound of formula (II)-IM1 gives the compound of formula (II)-IM2;

[0278] In some embodiments, this step is carried out in the presence of a suitable reducing agent, and the reducing agent may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

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

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

[0281] Step Three

[0282] The substitution reaction of the compound of formula (II)-IM2 gives the compound of formula (II)-IM3,

[0283] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0284] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate;

[0285] In some embodiments, this step is carried out under basic conditions, and the reagents providing basic conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilyl)amine lithium, bis(trimethylsilyl)amine sodium; 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.

[0286] Step Four

[0287] The coupling reaction of the compound of formula (II)-IM3 gives the compound of formula (II)-IM4;

[0288] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 50°C, 60°C, 70°C, 100°C, preferably 70°C;

[0289] In some embodiments, this step is carried out in a suitable organic solvent, which may 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;

[0290] In some embodiments, this step is carried out under basic conditions. The reagents providing the basic 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 bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide; 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.

[0291] Step Five

[0292] The compound of formula (II)-IM5 is obtained by the reduction reaction of the compound of formula (II)-IM4;

[0293] In some embodiments, this step is carried out with a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

[0294] 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;

[0295] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably tetrahydrofuran.

[0296] Step Six

[0297] The compound of formula (II)-IM6 is obtained by the ring-closing reaction of the compound of formula (II)-IM5;

[0298] 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 5°C;

[0299] In some embodiments, this step is carried out in a suitable organic solvent, which may 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.

[0300] Step Seven

[0301] The compound of formula (II)-IM7 is obtained by subjecting the compound of formula (II)-IM6 to a substitution reaction;

[0302] 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 5 °C;

[0303] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

[0304] In some embodiments, this step is carried out under basic conditions, and the reagents providing the basic 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 bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide; 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.

[0305] Step Eight

[0306] The compound of formula (II)-IM8 is obtained by subjecting the compound of formula (II)-IM7 to a reduction reaction;

[0307] In some embodiments, this step is carried out with a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

[0308] 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;

[0309] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0310] Step Nine

[0311] The compound of formula (II)-IM9 is obtained by subjecting the compound of formula (II)-IM8 to a substitution reaction;

[0312] 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;

[0313] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the 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 bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide; the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert - butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, and pyridine is preferred.

[0314] Step Ten

[0315] The compound of formula (II)-IM9 undergoes a hydrolysis reaction to obtain the compound of formula (II)-IM10;

[0316] 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, and 60°C is preferred;

[0317] 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 methanol is preferred.

[0318] In some embodiments, the reaction is carried out under acidic conditions. The reagents providing the acidic conditions include hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, and hydrochloric acid is preferred.

[0319] Step Eleven

[0320] 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)-trione undergo a cyclization reaction to obtain the compound of formula (II)-IM11;

[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, 140°C, and 140°C is preferred;

[0322] 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, and toluene is preferred.

[0323] In some embodiments, the reaction is carried out under acidic conditions. The reagents providing the acidic conditions include p - toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, and p - toluenesulfonic acid is preferred.

[0324] Step Twelve

[0325] The compound of formula (II)-IM11 undergoes a hydrolysis reaction to obtain the compound of formula (II)-IM12;

[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 100°C;

[0327] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing the acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0328] Step Thirteen

[0329] The compound of formula (II)-IM12 and the compound of formula (II)-SM2 undergo a substitution reaction to obtain the compound of formula (II)-IM13.

[0330] 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;

[0331] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent 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.

[0332] In some embodiments, this step is carried out in the presence of a suitable base, and the base includes organic bases or inorganic bases. The organic bases can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py). The inorganic bases 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 2 CO 3or NaHCO 3 ;

[0333] Step Fourteen

[0334] The condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM3 gives the compound of formula (II)-IM14;

[0335] In some embodiments, this step is carried out under a suitable condensing reagent, and the condensing reagent can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0336] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;

[0337] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0338] In some embodiments, this step is carried out in a suitable base, and the base 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.

[0339] Step Fifteen

[0340] The acidolysis reaction of the compound of formula (II)-IM14 gives the compound of formula (II)-1;

[0341] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 25°C;

[0342] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0343] In some embodiments, the reaction is carried out under acidic conditions. The reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably hydrochloric acid.

[0344] The synthesis method of formula (II)-SM3 is shown as follows:

[0345] When PG is :

[0346]

[0347] Step 1

[0348] Compound (II)-SM3-3 is obtained by the substitution reaction of compound (II)-SM3-1 and compound (II)-SM3-2;

[0349] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, 140 °C, preferably 0-25 °C;

[0350] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably tetrahydrofuran.

[0351] In some embodiments, this step is carried out in a suitable base, and the base includes organic bases or inorganic bases. The organic bases can be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic bases 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 .

[0352] Step 2

[0353] Compound (II)-SM3 is obtained by the hydrogenation reaction of compound (II)-SM3-3;

[0354] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, 140 °C, preferably 25 °C;

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

[0356] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

[0357] When PG is R y’ and R z’ are hydrogen:

[0358]

[0359] Step 1

[0360] The compound of formula (II)-SM3 is obtained by the condensation reaction of the compound of formula (II)-SM3-4 and the compound of formula (II)-SM3-5;

[0361] 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;

[0362] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, and their mixed solvents, preferably N,N-dimethylformamide.

[0363] Alternatively, the compound of formula (II)-1 can be synthesized by the following synthetic route:

[0364]

[0365] Step 1

[0366] The compound of formula (II)-IM15 is obtained by the condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM4;

[0367] In some embodiments, this step is carried out in the presence of a suitable condensing agent, which may be selected from HATU, HBTU, EDCI, DCC, and HOBT, preferably HATU;

[0368] 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;

[0369] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0370] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base may be selected from K 3 PO 4 、NaH, K 2 CO 3 、Na 2 CO 3 、Cs 2 CO 3 and NaOH, preferably DIPEA.

[0371] Step Two

[0372] The silicon protecting group of the compound of formula (II)-IM15 is removed to obtain the compound of formula (II)-1;

[0373] Alternatively, the compound of formula (II) can be synthesized by the following synthetic route:

[0374]

[0375] Step One

[0376] The compound of formula (II) is obtained by the condensation reaction of the compound of formula (II)-IM13 and the compound of formula (II)-SM5;

[0377] In some embodiments, this step is carried out with a suitable condensing reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0378] 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;

[0379] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0380] 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.

[0381] The compound of formula (III)-1 in the present invention can be synthesized and prepared by the following synthetic route.

[0382]

[0383] Wherein, R x” 、R y” and R z” are as defined above; LG is a leaving group, selected from mesyl, trifluoromethanesulfonyloxy, and halogen, preferably trifluoromethanesulfonyloxy or chlorine; PG is a protecting group, selected from

[0384] Step 1

[0385] The compound of formula (III)-IM1 is obtained by a substitution reaction of the compound of formula (III)-SM1.

[0386] 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;

[0387] 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.

[0388] Step 2

[0389] The compound of formula (III)-IM2 is obtained by a reduction reaction of the compound of formula (III)-IM1;

[0390] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from palladium catalyst, platinum catalyst, rhodium catalyst, preferably platinum catalyst;

[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 60°C;

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

[0393] Step Three

[0394] The compound of formula (III)-IM3 is obtained by a substitution reaction of the compound of formula (III)-IM2.

[0395] 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.

[0396] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0397] In some embodiments, this step is carried out under basic conditions. The reagents providing basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilyl)amine lithium, bis(trimethylsilyl)amine sodium; 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.

[0398] Step Four

[0399] The compound of formula (III)-IM4 is obtained by a coupling reaction of 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, 70°C, 100°C, preferably 70°C.

[0401] In some embodiments, this step is carried out in a suitable organic solvent, which may 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.

[0402] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the 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 bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide; the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert - butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, and preferably N,N - diisopropylethylamine.

[0403] Step Five

[0404] The compound of formula (III)-IM5 is obtained by the reduction reaction of the compound of formula (III)-IM4;

[0405] In some embodiments, this step is carried out with a suitable reducing agent. The reducing agent can be selected from palladium catalysts, platinum catalysts, rhodium catalysts, and preferably a platinum catalyst;

[0406] 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, and preferably 40°C;

[0407] In some embodiments, this step is carried out in a suitable organic solvent. The organic solvent can be selected from tetrahydrofuran, dichloromethane, N,N - dimethylformamide, N - methylpyrrolidone, dimethyl sulfoxide, n - heptane, n - hexane, ethyl acetate, and preferably tetrahydrofuran.

[0408] Step Six

[0409] The compound of formula (III)-IM6 is obtained by the ring - closing reaction of the compound of formula (III)-IM5;

[0410] 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, and preferably 5°C;

[0411] In some embodiments, this step is carried out in a suitable organic solvent. The organic solvent can be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N - dimethylformamide, N - methylpyrrolidone, dimethyl sulfoxide, n - heptane, n - hexane, ethyl acetate, tert - butanol, and preferably a mixed solvent of tetrahydrofuran and tert - butanol.

[0412] Step Seven

[0413] The compound of formula (III)-IM7 is obtained by the substitution reaction of the compound of formula (III)-IM6;

[0414] 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 5°C;

[0415] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may be selected from trifluoroacetic acid, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably trifluoroacetic acid;

[0416] In some embodiments, this step is carried out under basic conditions, and the reagents providing basic conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilyl)amine lithium, bis(trimethylsilyl)amine sodium; 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.

[0417] Step Eight

[0418] The compound of formula (III)-IM8 is obtained by the reduction reaction of the compound of formula (III)-IM7;

[0419] In some embodiments, this step is carried out with a suitable reducing agent, and the reducing agent may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

[0420] 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;

[0421] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably methanol.

[0422] Step Nine

[0423] The compound of formula (III)-IM9 is obtained by the substitution reaction of the compound of formula (III)-IM8;

[0424] 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;

[0425] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the 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 bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide; the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, and pyridine is preferred.

[0426] Step Ten

[0427] The compound of formula (III)-IM10 is obtained by hydrolysis of the compound of formula (III)-IM9;

[0428] 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, and 60 °C is preferred;

[0429] 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 methanol is preferred.

[0430] In some embodiments, the reaction is carried out under acidic conditions. The reagents providing the acidic conditions include hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, and hydrochloric acid is preferred.

[0431] Step Eleven

[0432] The compound of formula (III)-IM11 is obtained by cyclization of 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)-trione;

[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, 140 °C, and 140 °C is preferred;

[0434] 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, and toluene is preferred.

[0435] In some embodiments, the reaction is carried out under acidic conditions. The reagents providing the acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, and p-toluenesulfonic acid is preferred.

[0436] Step Twelve

[0437] Compound (III)-IM12 is obtained by hydrolysis of compound (III)-IM11;

[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 100 °C;

[0439] 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.

[0440] Step Thirteen

[0441] Compound (III)-IM13 is obtained by substitution reaction of compound (III)-IM12 and compound (III)-SM2.

[0442] Compound (II)-IM13 is obtained by substitution reaction.

[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 50 °C;

[0444] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent 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.

[0445] In some embodiments, this step is carried out in the presence of a suitable base, and the base includes organic bases or inorganic bases. The organic bases can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py). The inorganic bases 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 ;

[0446] Step Fourteen

[0447] The condensation reaction of the compound of formula (III)-IM13 and the compound of formula (III)-SM3 gives the compound of formula (III)-IM14;

[0448] In some embodiments, this step is carried out under a suitable condensing reagent, and the condensing reagent can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0449] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, preferably 25 °C;

[0450] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0451] In some embodiments, this step is carried out in a suitable base, and the base 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.

[0452] Step Fifteen

[0453] The compound of formula (III)-IM14 undergoes acidolysis reaction to obtain the compound of formula (III)-1;

[0454] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, 140 °C, preferably 25 °C;

[0455] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, and their mixed solvents, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0456] 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.

[0457] The synthetic method of formula (III)-SM3 is as follows:

[0458] When PG is :

[0459]

[0460] Step 1:

[0461] Compound of formula (III)-SM3-3 is obtained by the substitution reaction of the compound of formula (III)-SM3-1 and the compound of formula (II)-SM3-2;

[0462] 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;

[0463] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, and their mixed solvents, preferably tetrahydrofuran.

[0464] In some embodiments, this step is carried out in a suitable base, which includes organic bases or inorganic bases. The organic bases may be selected from DIPEA, TEA, t-BuOK, and Py, and the inorganic bases may 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 .

[0465] Step 2:

[0466] The compound of formula (III)-SM3 is obtained by subjecting the compound of formula (III)-SM3-3 to a hydrogenation reaction;

[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 25 °C;

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

[0469] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

[0470] When PG is , R y’ and R z’ are hydrogen:

[0471]

[0472] Step 1:

[0473] The compound of formula (III)-SM3 is obtained by subjecting the compound of formula (III)-SM3-4 and the compound of formula (III)-SM3-5 to a condensation reaction;

[0474] 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;

[0475] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate and their mixed solvents, preferably N,N-dimethylformamide.

[0476] Alternatively, the compound of formula (III)-1 can be synthesized by the following synthetic route:

[0477]

[0478] Step 1:

[0479] The compound of formula (III)-IM15 is obtained by subjecting the compound of formula (III)-IM13 and the compound of formula (III)-SM4 to a condensation reaction;

[0480] In some embodiments, this step is carried out under a suitable condensing reagent, and the condensing reagent can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0481] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, preferably 25 °C;

[0482] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0483] In some embodiments, this step is carried out in a suitable base, and the base 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.

[0484] Step Two:

[0485] The silyl protecting group of the compound of formula (III)-IM15 is removed to obtain the compound of formula (III)-1;

[0486] Alternatively, the compound of formula (III)-1 can be synthesized by the following synthetic route:

[0487]

[0488] Step One:

[0489] The compound of formula (III)-1 is obtained by a condensation reaction of the compound of formula (III)-IM13 and the compound of formula (III)-SM5;

[0490] In some embodiments, this step is carried out under a suitable condensing reagent, and the condensing reagent can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HATU;

[0491] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, preferably 25 °C;

[0492] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0493] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic base may be selected from K 3 PO 4 、NaH、K 2 CO 3 、Na 2 CO 3 、Cs 2 CO 3 and NaOH, preferably DIPEA.

[0494] The compound of formula (IV) in the present invention can be synthesized and prepared by the following synthetic route:

[0495] Wherein, R a 、R b 、R c 、R d and R e have the meanings as described above;

[0496] When q = 1,

[0497]

[0498] Step 1

[0499] The compound of formula (IV)-IM1 is obtained by carrying out a nitration reaction on the compound of formula (IV)-SM1;

[0500] In some embodiments, this step is carried out at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

[0501] Step 2

[0502] The compound of formula (IV)-IM2 is obtained by carrying out a hydrogenation reaction on the compound of formula (IV)-IM1;

[0503] In some embodiments, this step is carried out with a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;

[0504] 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;

[0505] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0506] Step Three

[0507] The compound of formula (IV)-IM3 is obtained by acylating the compound of formula (IV)-IM2;

[0508] 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;

[0509] Step Four

[0510] The compound of formula (IV)-IM4 is obtained by reacting the compound of formula (IV)-IM3 with DMF-DMA;

[0511] 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;

[0512] Step Five

[0513] The compound of formula (IV)-IM5 is obtained by substituting the compound of formula (IV)-IM4 and the compound of formula (IV)-SM2;

[0514] 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;

[0515] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, ethanol, N,N-methylpyrrolidone, dimethyl sulfoxide, preferably ethanol;

[0516] Step Six

[0517] The compound of formula (IV)-IM6 is obtained by reducing the compound of formula (IV)-IM5;

[0518] In some embodiments, this step is carried out with a suitable reducing agent, and the reducing agent is preferably sodium borohydride;

[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, preferably 0 - 25 °C;

[0520] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from tetrahydrofuran, glacial acetic acid, methanol and their mixed solutions, preferably glacial acetic acid.

[0521] Step Seven

[0522] The amino group of the compound of formula (IV)-IM6 is protected with Fmoc to obtain the compound of formula (IV)-IM7;

[0523] Step Eight

[0524] The acetyl protecting group of the amino group of the compound of formula (IV)-IM7 is removed to obtain the compound of formula (IV)-IM8;

[0525] Step Nine

[0526] The compound of formula (IV)-IM8 undergoes a ring-closure reaction under acidic conditions to obtain the compound of formula (IV)-IM9;

[0527] 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;

[0528] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably toluene and xylene;

[0529] In some embodiments, this step is carried out under acidic conditions;

[0530] The reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0531] Step Ten

[0532] The Fmoc protecting group of the compound of formula (IV)-IM9 is removed to obtain the compound of formula (IV)-IM10;

[0533] Step Eleven

[0534] The compound of formula (IV)-IM10 and the compound of formula (IV)-SM4 undergo a condensation reaction to obtain the compound of formula (IV);

[0535] In some embodiments, this step is carried out in the presence of a suitable condensing agent, which may be selected from HATU, HBTU, EDCI, DCC, and HOBT, preferably HBTU;

[0536] 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;

[0537] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0538] In some embodiments, this step is carried out in a suitable base, which includes an organic base or an inorganic base. The organic base may be selected from DIPEA, TEA, t-BuOK, and Py, and the inorganic base may be selected from K 3 PO 4 、NaH, K 2 CO 3 、Na 2 CO 3 、Cs 2 CO 3 and NaOH, preferably DIPEA.

[0539] Alternatively, when q = 0,

[0540]

[0541] LG is a leaving group, selected from mesyl, trifluoromethanesulfonyloxy, and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0542] Step 1

[0543] The compound of formula (IV)-IM11 is obtained by a reduction reaction of the compound of formula (IV)-SM5,

[0544] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably platinum catalysts;

[0545] 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;

[0546] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate and tetrahydrofuran.

[0547] Step Two

[0548] The Friedel-Crafts acylation reaction of the compound of formula (IV)-IM11 gives the compound of formula (IV)-IM12;

[0549] In some embodiments, this step is carried out at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.

[0550] Step Three

[0551] The ring-closing reaction of the compound of formula (IV)-IM12 under acidic conditions gives the compound of formula (IV)-IM13;

[0552] 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;

[0553] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably toluene and xylene;

[0554] In some embodiments, this step is carried out under acidic conditions;

[0555] The reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.

[0556] Step Four

[0557] The substitution reaction of the compound of formula (IV)-IM13 gives the compound of formula (IV)-IM14;

[0558] 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;

[0559] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from methanol, ethanol, N,N-methylpyrrolidone, dimethyl sulfoxide, preferably dimethyl sulfoxide;

[0560] Step Five

[0561] The compound of formula (IV)-IM15 is obtained by the reduction reaction of the compound of formula (IV)-IM14.

[0562] In some embodiments, this step is carried out in the presence of a suitable reducing agent, and the reducing agent can be selected from palladium catalysts, platinum catalysts, rhodium catalysts, triphenylphosphine, triethyl phosphite, preferably triethyl phosphite;

[0563] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 50°C, 60°C, 80°C, 100°C, preferably 80°C;

[0564] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, toluene, and their mixed solutions, preferably the mixed solution of methanol and toluene.

[0565] Step Six

[0566] The compound of formula (IV)-IM16 is obtained by the substitution reaction of the compound of formula (IV)-IM15 and the compound of formula (IV)-SM6.

[0567] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;

[0568] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent 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.

[0569] In some embodiments, this step is carried out in the presence of a suitable base, and the base includes organic bases or inorganic bases. The organic bases can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py). The inorganic bases 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 ;

[0570] Step Seven

[0571] The compound of formula (IV) is obtained by the condensation reaction of the compound of formula (IV)-IM16 and the compound of formula (IV)-SM4;

[0572] In some embodiments, this step is carried out in the presence of a suitable condensation reagent, and the condensation reagent can be selected from HATU, HBTU, EDCI, DCC, and HOBT, preferably HBTU;

[0573] In some embodiments, this step is carried out at a suitable temperature, and the temperature is 20 °C, 25 °C, 40 °C, 50 °C, 60 °C, 100 °C, preferably 25 °C;

[0574] In some embodiments, this step is carried out in a suitable organic solvent, and the organic solvent can be selected from methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.

[0575] In some embodiments, this step is carried out in a suitable base, and the base 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.

[0576] The compound of formula (V)-1 in the present invention can be synthesized and prepared using the starting materials according to the same synthetic route as formula (III).

[0577] Advantages of the invention

[0578] The present invention provides camptothecin compounds represented by formula (I)-formula (IV), and their pharmaceutical compositions, preparation methods, and uses. These compounds have good anti-tumor activity, have the potential to overcome drug resistance, and can be used to treat cell abnormal proliferation diseases, including but not limited to advanced solid tumors. Detailed Embodiments

[0579] The present application will be further described below through the description of specific embodiments, but this is not a limitation to the present application. Those skilled in the art can make various modifications or improvements according to the teachings of the present application without departing from the basic idea and scope of the present application.

[0580] The abbreviations in the present invention have the following meanings:

[0581]

[0582]

[0583] In the following examples, the structures of the compounds described are determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0584] For nuclear magnetic resonance ( 1 H NMR), the measuring instrument used is a Bruker 400 MHz nuclear magnetic resonance spectrometer; hexadeuterodimethyl sulfoxide (DMSO-d 6 6); the internal standard substance is tetramethylsilane (TMS).

[0585] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0586] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad peak, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The δ value is expressed in ppm value.

[0587] For mass spectrometry (MS), the measuring instrument used is an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0588] 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

[0589]

[0590] 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. The reaction was carried out 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 3.24 mg of the title compound 1-1-A and 3.98 mg of 1-1-B.

[0591] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0592] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0593] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 30 70 28 18 90 10 28

[0594] Retention time: 1-1-A: 10.8 min; 1-1-B: 11.1 min.

[0595] The structural characterization data of 1-1-A are as follows:

[0596] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.58 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 6.17 (d, J = 6.0 Hz, 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 = 7.2 Hz, 3H).

[0597] ESI-MS (m / z): 532.2 [M + H] + .

[0598] The structural characterization data of 1-1-B are as follows:

[0599] 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,1H),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).

[0600] ESI-MS(m / z):532.2[M+H] + .

[0601] Example 2 (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-hydroxy-3-enamide and (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-hydroxy-3-enamide

[0602]

[0603] Dissolve compound vinyl glycolic acid (9.61 mg, 94.07 μmol) in DMF (2 mL), add HATU (44.70 mg, 117.58 μmol), compound SM1-1 (25.00 mg, 0.047 mmol) and DIPEA (24.30 mg, 188.13 μmol), and react 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 compound 1-7-A (4.00 mg) and 1-7-B (1.38 mg).

[0604] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0605] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0606]

[0607]

[0608] Retention time: 1-7-A: 8.7 min; 1-7-B: 9.1 min.

[0609] The structural characterization data of 1-7-A are as follows:

[0610] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.51 (s, J = 8.8 Hz, 1H), 7.76 (s, J = 10.8 Hz, 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 = 4.9 Hz, 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 = 7.2 Hz, 3H).

[0611] ESI-MS (m / z): 520.1 [M + H] + .

[0612] The structural characterization data of 1-7-B are as follows:

[0613] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.48 (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 = 1.7 Hz, 1H), 5.18 (s, 2H), 5.16 - 5.14 (m, 1H), 4.5 - 4.52 (m, 1H), 3.22 - 3.08 (m, 2H), 2.38 (s, 3H), 2.25 - 2.16 (m, 1H), 2.16 - 2.06 (m, 1H), 1.93 - 1.79 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0614] ESI-MS (m / z): 520.1 [M + H] + .

[0615] 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

[0616]

[0617] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene

[0618] At 25 °C, dissolve compound 2-1-01 (5.00 g, 29.14 mmol) in n-heptane (25 mL), add concentrated sulfuric acid (25 mL), heat to 50 °C, add NBS (6.22 g, 34.97 mmol) in batches at 50 °C, maintain the reaction at 50 °C for 2 hours, detect the reaction by thin-layer chromatography (ethyl acetate: petroleum ether = 1:10), drop the reaction solution cooled to room temperature into ice water, extract with toluene, combine the organic phases, wash with sodium sulfite solution, water, saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, purify the crude product by preparative high-performance liquid chromatography, and freeze-dry the preparation solution to obtain 4.88 g of the title compound.

[0619] Chromatographic column: C18 ODS 45 mm × 450 mm × 8.0 μm

[0620] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0621] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 60 40 60 10 60 40 60 40 100 0 60

[0622] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline

[0623] At 25 °C, dissolve compound 2-1-02 (4.88 g, 19.48 mmol) in ethyl acetate (100 mL), add platinum-carbon (2.00 g, 19.48 mmol, 5% content), replace with hydrogen and react at 60 °C for 4 hours under the protection of a hydrogen balloon, and monitor the reaction by high-performance liquid chromatography-mass spectrometry. Filter the reaction solution, concentrate the filtrate to obtain 3.68 g of the crude title compound, which is directly used in the next step without further purification.

[0624] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide

[0625] 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 carried out at 20 °C for 20 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. Water was added to the reaction solution, 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 a crude product. The crude product was slurried with a mixed solvent of ethyl acetate:petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0626] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid

[0627] At 20 °C, compound 2-1-04 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol), tris(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. After the reaction system was purged with nitrogen, it was heated to 70 °C and reacted for 5 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. 1N sodium hydroxide solution was added to the reaction solution to adjust the pH to 8, and then extracted with ethyl acetate. The remaining aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid and extracted with ethyl acetate. 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 of the reaction.

[0628] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butanoic acid

[0629] 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. After the system was purged with hydrogen, the reaction was carried out at 40 °C for 2 hours under the protection of a hydrogen balloon. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43 g of the title compound, which was used directly in the next step of the reaction without further purification.

[0630] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0631] Compound 2-1-06 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL). The temperature was lowered to 5 °C, and trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise. The reaction was carried out at 5 °C for 4 hours, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was added to water, and the pH was adjusted to 9 with 10 N sodium hydroxide. Ethyl acetate was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 0 - 20%), to obtain 1.53 g of the title compound.

[0632] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0633] At 5 °C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL). A THF solution (16 mL) of compound 2-1-07 (1.53 g, 6.08 mmol) was added dropwise. After 10 minutes, amyl nitrite (1.14 g, 9.73 mmol) was added dropwise. The reaction was carried out at 5 °C for 1 hour, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was adjusted to pH = 5 with 1 N hydrochloric acid, extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was triturated with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0634] Step 8: N-(7-Amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0635] At 20 °C, compound 2-1-08 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2 N hydrochloric acid (8 mL). Pd / C (0.15 g, 10% content) was added. After the system was replaced with hydrogen, the reaction was carried out at 5 °C under the protection of a hydrogen balloon for 2 hours, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.52 g of the hydrochloride salt of the title compound, which was used directly in the next step without further purification.

[0636] Step 9: Synthesis of N,N’-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide

[0637] 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 carried out at 20 °C for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was added to water, extracted with ethyl acetate, the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 30%) to obtain 0.22 g of the title compound.

[0638] Step Ten: Synthesis of N-(8-Amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

[0639] 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 reaction was heated to 60 °C for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. After cooling, saturated sodium bicarbonate solution was added to the reaction solution to adjust the pH to 8, 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 directly used in the next step without further purification.

[0640] Step Eleven: Synthesis of N-((9S)-5-Chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[f]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

[0641] 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), p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added, and the reaction was heated to 140 °C for 5 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated, and the crude product was purified by flash silica gel column (methanol: dichloromethane = 0 - 10%) to obtain 0.15 g of the title compound.

[0642] Step Twelve: Synthesis of (9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[f]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0643] Compound 2-1-12 (40.00 mg, 0.08 mmol) was added to concentrated hydrochloric acid (1 mL), and the mixture was heated to 100 °C and reacted for 5 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography. The preparation was freeze-dried to obtain 12.00 mg of the trifluoroacetate salt of the title compound 2-23.

[0644] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0645] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0646] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0647] The structure characterization data are as follows:

[0648] ESI-MS (m / z): 452.1 [M+H] + .

[0649] 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

[0650] At 25 °C, the trifluoroacetate of compound 2-23 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL). Then, 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 successively. The reaction was maintained at 25 °C for 0.5 h, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with dichloromethane / methanol (v / v = 10 / 1). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (dichloromethane:methanol = 20:1) to obtain two isomers, which were named 2-1-13-A (15.00 mg, Rf value of 0.3) and 2-1-13-B (12.00 mg, Rf value of 0.35) according to the Rf values.

[0651] 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

[0652] At 25 °C, 2-1-13-A (15.00 mg) and 2-1-13-B (12.00 mg) were separately dissolved in tetrahydrofuran (1 mL) in two reaction flasks. A mixture of tetrabutylammonium fluoride (1 M solution in tetrahydrofuran) / glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise, and the reaction was maintained at 25 °C for 0.5 h. The reaction was monitored by high-performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solutions were separately purified by preparative high-performance liquid chromatography, and the preparations were lyophilized to obtain the title compounds 2-1-A (6.94 mg) and 2-1-B (4.00 mg) respectively.

[0653] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0654] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0655] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 20 80 28 3 20 80 28 18 90 10 28

[0656] The characterization data of the 2-1-A structure are as follows:

[0657] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.43 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65 - 5.36 (m, 4H), 5.21 (q, J = 19.0 Hz, 2H), 3.95 (d, J = 5.7 Hz, 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 = 7.3 Hz, 3H).

[0658] ESI-MS (m / z): 510.1 [M+H] + .

[0659] The characterization data of the 2-1-B structure are as follows:

[0660] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.45 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64 - 5.35 (m, 4H), 5.19 (q, J = 19.0 Hz, 2H), 3.97 (d, J = 5.2 Hz, 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 = 7.3 Hz, 3H).

[0661] ESI-MS (m / z): 510.1 [M+H] + .

[0662] 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-hydroxypropanamine 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-hydroxypropanamine

[0663]

[0664] Step 1: Synthesis of (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)propanamine 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)propanamine

[0665] At 25 °C, dissolve the hydrochloride salt of 2-23 (30.00 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL). Sequentially add (S)-2-((tert-butyldiphenylsilyl)oxy)propanoic acid (24.21 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol), and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol). Maintain the reaction at 25 °C for 1 hour and monitor the reaction by high-performance liquid chromatography-mass spectrometry. After the reaction is completed, add water to the reaction solution, extract with dichloromethane / methanol (v / v = 10 / 1). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the crude product by preparative thin-layer chromatography (dichloromethane:methanol = 15:1) to separate two isomers. Name the two isomers 2-7-01-A (6.00 mg, Rf value of 0.35) and 2-7-01-B (6.00 mg, Rf value of 0.40) according to the Rf values.

[0666] 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-hydroxypropanamine 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-hydroxypropanamine

[0667] 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 separately dissolved in anhydrous tetrahydrofuran (1 mL) in two reaction flasks, and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / 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 high performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solutions were purified by preparative high performance liquid chromatography respectively, and the preparations were freeze-dried to obtain the title compounds 2-7-A (2.50 mg) and 2-7-B (3.00 mg) respectively.

[0668] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0669] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0670] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 16 90 10 28

[0671] The structure characterization data of 2-7-A are as follows:

[0672] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.38 (d, J = 8.8 Hz, 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, 7.3 Hz, 2H), 1.30 (d, J = 6.7 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0673] ESI-MS (m / z): 524.2 [M + H] + .

[0674] The structure characterization data of 2-7-B are as follows:

[0675] 1 H NMR (400 MHz, DMSO-d 6)δ 8.48 (d, J = 9.1 Hz, 1H), 8.12 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.67 (d, J = 4.8 Hz, 1H), 5.55 (dd, J = 14.6, 7.3 Hz, 1H), 5.43 (s, 2H), 5.24 (d, J = 19.0 Hz, 1H), 5.03 (d, J = 19.0 Hz, 1H), 4.21 - 4.08 (m, 1H), 3.28 - 3.08 (m, 2H), 2.51 (s, 3H), 2.16 (d, J = 6.2 Hz, 2H), 1.94 - 1.82 (m, 2H), 1.42 (d, J = 6.8 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 524.2 [M+H] + .

[0676] 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 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

[0677]

[0678] Step 1: Synthesis of (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-cyclopropaneacetamide, (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-cyclopropaneacetamide, (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-cyclopropaneacetamide, and (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-cyclopropaneacetamide

[0679] At 25 °C, dissolve the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL), and successively add 2-((tert-butyldiphenylsilyl)oxy)-2-cyclopropaneacetic acid (26.13 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol), and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol). Keep the reaction at 25 °C for 1 hour and monitor the reaction by high performance liquid chromatography-mass spectrometry. After the reaction is completed, add water to the reaction solution, extract with dichloromethane / methanol (v / v = 10 / 1). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol = 15:1) to separate two groups of isomers. Name the two groups of isomers 2-12-01-A (8.00 mg, Rf value 0.35) and 2-12-01-B (10.00 mg, Rf value 0.40) according to the Rf values.

[0680] 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-cyclopropyl-2-hydroxyacetamide, (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, (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

[0681] At 25 °C, dissolve 2-12-01-A (8.00 mg, 10.15 μmol) and 2-12-01-B (10.00 mg, 12.68 μmol) in anhydrous tetrahydrofuran (1 mL) in two reaction flasks respectively, and dropwise add a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) / glacial acetic acid (v / v = 13 / 1) (50 μL). Keep the reaction at 25 °C for 0.5 h, and monitor the reaction by high performance liquid chromatography-mass spectrometry. After the reaction is completed, purify the reaction solutions by preparative high performance liquid chromatography. Two isomeric products are separated from the reaction with 2-12-01-A as the raw material, and the preparative solutions are lyophilized to obtain compound 2-12-A (0.77 mg) and 2-12-B (1.03 mg) respectively; two isomeric products are separated from the reaction with 2-12-01-B as the raw material, and the preparative solutions are lyophilized to obtain compound 2-12-C (2.50 mg) and 2-12-D (1.00 mg) respectively. The purification conditions for 2-12-A / 2-12-B are as follows:

[0682] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0683] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0684]

[0685]

[0686] The elution retention times are as follows: 2-12-A: 10.0 - 11.0 min, 2-12-B: 11.0 - 12.5 min

[0687] The purification conditions for 2-12-C / 2-12-D are as follows:

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

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

[0690] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0691] The elution retention times are as follows: 2-12-C: 10.6 - 11.4 min, 2-12-D: 11.4 - 12.5 min

[0692] The structure characterization data of 2-12-A are as follows:

[0693] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.38 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63 - 5.53 (m, 1H), 5.51 (d, J = 5.1 Hz, 1H), 5.42 (s, 2H), 5.28 (d, J = 19.2 Hz, 1H), 5.16 (d, J = 19.1 Hz, 1H), 3.60 (t, J = 5.6 Hz, 1H), 3.28 - 3.11 (m, 2H), 2.52 (s, 3H), 2.22 - 2.10 (m, 2H), 1.86 (tt, J = 14.1, 7.3 Hz, 2H), 1.23 (d, J = 4.9 Hz, 1H), 0.87 (t, J = 7.2 Hz, 3H), 0.56 - 0.36 (m, 4H).

[0694] ESI-MS (m / z): 550.2 [M+H] + .

[0695] The structure characterization data of 2-12-B are as follows:

[0696] 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, 1H), 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).

[0697] ESI-MS(m / z): 550.2[M + H] + .

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

[0699] 1 H NMR(400MHz, 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).

[0700] ESI-MS(m / z): 550.1[M + H] + .

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

[0702] 1 H NMR(400MHz, 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).

[0703] ESI-MS(m / z):550.1[M+H] + .

[0704] 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-methylpropanamine 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-methylpropanamine

[0705]

[0706] At 25 °C, the hydrochloride of 2-23 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL). 2-((tert-Butyldimethylsilyl)oxy)-2-methylpropanoic 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 successively. The reaction was maintained at 25 °C for 1 hour, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography to obtain two isomers. The preparation 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 retention time of the peaks.

[0707] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0708] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0709] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0710] The retention times of the peaks are: 2-17-A: 9.5 - 10.2 min and 2-17-B: 10.4 - 10.6 min.

[0711] The structural characterization data of 2-17-A are as follows:

[0712] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.36 (d, J = 9.1 Hz, 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 = 19.0 Hz, 1H), 5.05 (d, J = 19.0 Hz, 1H), 3.27 - 3.11 (m, 2H), 2.51 (s, 1H), 2.24 - 2.10 (m, 2H), 1.86 (tt, J = 14.0, 7.2 Hz, 2H), 1.46 (s, 3H), 1.35 (s, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0713] MS m / z (ESI): 538.2 [M+H] + .

[0714] The structural characterization data of 2-17-B are as follows:

[0715] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.40 (d, J = 9.2 Hz, 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 = 19.0 Hz, 1H), 5.02 (d, J = 19.0 Hz, 1H), 3.28 - 3.08 (m, 2H), 2.50 (s, 3H), 2.22 - 2.10 (m, 2H), 1.87 (tt, J = 14.2, 7.2 Hz, 2H), 1.47 (s, 3H), 1.35 (s, 3H), 0.88 (t, J = 7.3 Hz, 3H).

[0716] MS m / z (ESI): 538.2 [M+H] + .

[0717] 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

[0718]

[0719] Step 1: Synthesis of 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

[0720] At 25 °C, the hydrochloride salt of 2-23 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-1-carboxylic acid (25.10 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added successively. The reaction was maintained at 25 °C for 1 hour, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with dichloromethane / methanol (v / v = 10 / 1). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by preparative thin layer chromatography (dichloromethane:methanol = 15:1) to obtain two isomers, which were named 2-20-01-A (4.00 mg, Rf value of 0.30) and 2-20-01-B (4.00 mg, Rf value of 0.35) according to the Rf values.

[0721] 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

[0722] At 25 °C, in two reaction flasks, 2-20-01-A (4.00 mg, 5.17 μmol) and 2-20-01-B (4.00 mg, 5.17 μmol) were respectively dissolved in anhydrous tetrahydrofuran (1 mL), 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 high performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solutions were respectively purified by preparative high performance liquid chromatography, and the prepared solutions were respectively freeze-dried to obtain the title compounds 2-20-A (0.71 mg) and 2-20-B (1.05 mg).

[0723] The purification conditions of 2-20-A are as follows:

[0724] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0725] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0726] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 16 90 10 28

[0727] The purification conditions of 2-20-B are as follows:

[0728] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0729] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0730] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 20 80 28 2 20 20 28 18 80 20 28

[0731] The structure characterization data of 2-20-A are as follows:

[0732] 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,1H),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).

[0733] ESI-MS(m / z):536.2[M+H] + .

[0734] The structural characterization data of 2-20-B are as follows:

[0735] 1 1H NMR(400MHz,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).

[0736] ESI-MS(m / z):536.2[M+H] + .

[0737] 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

[0738]

[0739] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline

[0740] Dissolve compound 3-1-01 (2.00 g, 10.53 mmol) in N,N-dimethylformamide (30 mL), then slowly add N-chlorosuccinimide (1.69 g, 12.63 mmol). After addition, react at room temperature for 16 hours and detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is concentrated under reduced pressure to obtain a crude product, and the crude product is purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 25%) to obtain 0.95 g of the title compound.

[0741] The structure characterization data are as follows:

[0742] 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.77 (dd, J = 2.5, 1.4 Hz, 1H), 6.51 (dd, J = 11.7, 2.5 Hz, 1H), 5.84 (s, 2H).

[0743] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide

[0744] Dissolve compound 3-1-02 (0.95 g, 4.23 mmol) in ethyl acetate (20 mL), add acetic anhydride (648.13 mg, 6.35 mmol) under nitrogen protection. After addition, heat to 50 °C and react for 15 hours, and detect the reaction by high performance liquid chromatography-mass spectrometry. Quench the reaction solution with methanol (5 mL), and then directly evaporate to dryness under reduced pressure to obtain a crude product. The crude product is purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 40%) to obtain 1.01 g of the title compound.

[0745] The structure characterization data are as follows:

[0746] ESI-MS (m / z): 265.9 [M+H] + .

[0747] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid

[0748] 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). Then, N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-tolyl)phosphine (114.21 mg, 375.24 μmol), and palladium(II) acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was purged with nitrogen three times and heated to 100 °C under a nitrogen atmosphere for 16 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS). After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added, and the mixture was shaken and separated into layers. After separating the lower aqueous phase, the pH was adjusted to about 3 with 4 mol / L aqueous hydrochloric acid solution, and 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 the crude product of the title compound.

[0749] The structure characterization data are as follows:

[0750] ESI-MS (m / z): 272.0 [M+H] + .

[0751] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid

[0752] The crude product of compound 3-1-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL). Then, 10% palladium on carbon (0.10 g) was added. After the addition, the reaction system was purged with a hydrogen balloon three times and reacted under a hydrogen atmosphere for 4 hours. The reaction was monitored by HPLC-MS. 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.

[0753] The structure characterization data are as follows:

[0754] ESI-MS (m / z): 274.0 [M+H] + .

[0755] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0756] The crude product of compound 3-1-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL). After cooling to 5 °C, trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After addition, the reaction was carried out 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, then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by flash silica gel column to obtain 0.43 g of the title compound.

[0757] The structure characterization data are as follows:

[0758] ESI-MS (m / z): 256.1 [M+H] + .

[0759] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0760] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction flask. After cooling to 5 °C in an ice bath, potassium tert-butoxide (415.18 mg, 3.70 mmol) was added. Then compound 3-1-06 (0.43 mg, 1.68 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (315.24 mg, 2.69 mmol) was added. After addition, the reaction was carried out at 5 °C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated ammonium chloride aqueous solution, then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of the crude product of the title compound.

[0761] The structure characterization data are as follows:

[0762] ESI-MS (m / z): 285.0 [M+H] + .

[0763] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0764] The crude product of compound 3-1-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), then 3 mol / L hydrochloric acid aqueous solution (1 mL) and 10% palladium on carbon (40.00 mg) were added. After 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 salt of the title compound.

[0765] The structure characterization data are as follows:

[0766] ESI-MS (m / z): 271.0 [M+H] + .

[0767] Step Eight: Synthesis of (9H-Fluoren-9-yl)methyl (8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0768] The crude hydrochloride salt of compound 3-1-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), 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 addition, the reaction 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, 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 a C18 reversed-phase column (acetonitrile: 0.05% formic acid in water = 20%-100%) to obtain 301.00 mg of the title compound.

[0769] The structure characterization data are as follows:

[0770] ESI-MS (m / z): 493.2 [M+H] + .

[0771] Step Nine: Synthesis of (9H-Fluoren-9-yl)methyl (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0772] Compound 3-1-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL), concentrated hydrochloric acid (1 mL, 12 mol / L) was added. After addition, the temperature was raised to 60 °C and the reaction was carried out for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was 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 concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 0 - 50%) to obtain 198.00 mg of the title compound.

[0773] The structure characterization data are as follows:

[0774] ESI-MS (m / z): 451.1 [M + H] + .

[0775] Step 10: Synthesis of (9H-fluoren-9-yl)methyl ((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)carbamate

[0776] (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), then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After addition, the temperature was raised to 140 °C and the reaction was carried out for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 °C to obtain the crude product. The crude product was purified by flash silica gel column chromatography (methanol: dichloromethane = 0 - 5%) to obtain 256.00 mg of the title compound.

[0777] The structure characterization data are as follows:

[0778] ESI-MS (m / z): 678.1 [M + H] + .

[0779] Step XI: 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

[0780] Dissolve compound 3-1-11 (201.18 mg, 296.67 μmol) in N,N-dimethylformamide (4 mL), then add diethylamine (108.49 mg, 1.48 mmol). After addition, react at room temperature for 0.5 h, and detect the reaction by high performance liquid chromatography-mass spectrometry. After the reaction solution is evaporated to remove ethylenediamine under reduced pressure, adjust the pH to 2 - 3 with 1 mol / L hydrochloric acid aqueous solution, and directly purify the reaction solution by preparative high performance liquid chromatography to obtain the title compound 3-1-A (44.00 mg) and 3-1-B (43.00 mg).

[0781] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0782] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0783] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 70 30 28

[0784] 3-1-A (the peak appears earlier in the 6 min LCMS, retention time: 1.276 min)

[0785] The structure characterization data are as follows:

[0786] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.00 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 19.3 Hz, 1H), 5.44 (s, 2H), 5.38 (d, J = 19.3 Hz, 1H), 4.43 - 4.38 (m, 1H), 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 = 7.3 Hz, 3H). ESI-MS (m / z): 456.1 [M + H] + .

[0787] Structural characterization data of 3-1-B (peaking late in 6 min LCMS, retention time: 1.300 min) are as follows:

[0788] 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.98 (d, J = 10.3 Hz, 1H), 7.32 (s, 1H), 5.61 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.32 (d, J = 19.4 Hz, 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 = 7.3 Hz, 3H). ESI-MS (m / z): 456.1 [M+H] + .

[0789] 6 min LCMS conditions:

[0790] Chromatographic column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0791] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0792] 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

[0793] 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

[0794]

[0795] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaazaoctadec-22-ynoic acid

[0796] Compound 3-4-01 (30.00 mg, 70.00 μmol) was dissolved in N,N-dimethylformamide (1 mL), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (28.00 mg, 77.00 μmol) was added, and the reaction was carried out at room temperature for 1 hour. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by preparative high performance liquid chromatography, and the preparation was freeze-dried to obtain the title compound 3-4-03 (20.00 mg).

[0797] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0798] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0799] 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

[0800] The structure characterization data are as follows:

[0801] ESI-MS (m / z): 691.0 [M+18] + .

[0802] Step 2: Synthesis of 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-tetraazacyclohexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-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-tetraazacyclohexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-amide

[0803] The compound 3-1-A in a single configuration (36.00 mg, 79.70 μmol) and the compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), then 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the reaction was carried out at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-A in a single configuration (51.00 mg).

[0804] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0805] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0806] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0807] The structure characterization data are as follows:

[0808] ESI-MS (m / z): 1111.0 [M+H] + .

[0809] The compound 3-1-B in a single configuration (36.00 mg, 79.70 μmol) and the compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL), then 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the reaction was carried out at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-B in a single configuration (52.00 mg).

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

[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 structure characterization data are as follows:

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

[0815] 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

[0816] Weigh 40.00 mg (35.99 μmol) of compound 3-4-04-A and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then add ethyl acetate hydrochloride (1 mL, 4 mol / L). After addition, react at room temperature for 0.5 h, and detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product is purified by high performance liquid chromatography to obtain the title compound 3-4-A with a single configuration (4.75 mg).

[0817] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0818] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0819] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0820] The structure characterization data are as follows:

[0821] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.50 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.67 - 5.60 (m, 1H), 5.49 (t, J = 5.8 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.32 - 3.22 (m, 2H), 2.28 - 2.15 (m, 2H), 1.93 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0822] ESI-MS (m / z): 514.0 [M+H] + .

[0823] Compound 3-4-04-B (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then, ethyl acetate hydrochloride (1 mL, 4 mol / L) was added. After the addition, the reaction was carried out at room temperature for 0.5 h, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by high performance liquid chromatography to obtain the title compound 3-4-B with a single configuration (8.24 mg).

[0824] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0825] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0826] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0827] The structure characterization data are as follows:

[0828] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.52 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.68 - 5.58 (m, 1H), 5.53 (t, J = 5.8 Hz, 1H), 5.43 (d, J = 2.9 Hz, 2H), 5.20 (d, J = 7.3 Hz, 2H), 3.97 (d, J = 5.7 Hz, 2H), 3.31 - 3.21 (m, 2H), 2.26 - 2.15 (m, 2H), 1.92 - 1.82 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0829] ESI-MS (m / z): 514.0 [M+H] + .

[0830] Example X (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-isopropylethanamide

[0831]

[0832] Step 1: Synthesis of 1-(4-Fluoro-3-methylphenyl)-3-(isopropylamino)propan-1-one

[0833] At 20 °C, compound 4-12-01 (500.00 mg, 3.29 mmol), aqueous formaldehyde solution (2.5 mL, 37%), and isopropylamine (388.46 mg, 6.57 mmol) were placed in isopropanol (5 mL). Concentrated hydrochloric acid (2.5 mL) was added dropwise at 0 °C, and the reaction mixture was stirred at 100 °C for 16 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by preparative high performance liquid chromatography. The preparation was lyophilized to obtain 200.00 mg of the title compound.

[0834] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0835] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0836] 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

[0837] The structure characterization data are as follows:

[0838] ESI-MS (m / z): 224.1 [M+H] + .

[0839] Step 2: Synthesis of 1-(4-fluoro-5-methyl-2-nitrophenyl)-3-(isopropylamino)propan-1-one

[0840] At 0 °C, compound 4-12-02 (100.00 mg, 0.49 mmol) was placed in concentrated sulfuric acid (0.5 mL), and potassium nitrate (54.34 mg, 0.54 mmol) was added. The reaction mixture was maintained at 0 °C for 1 hour, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction mixture was poured into ice water and purified by a reverse phase column (acetonitrile: 0.05% formic acid in water = 0 - 30%) to obtain 90.00 mg of the title compound.

[0841] The structure characterization data are as follows:

[0842] ESI-MS (m / z): 269.0 [M+H] + .

[0843] Step 3: Synthesis of 1-(2-amino-4-fluoro-5-methylphenyl)-3-(isopropylamino)propan-1-one

[0844] At 25 °C, compound 4-12-03 (200.00 mg, 0.75 mmol) was placed in methanol (20.0 mL), and 10% palladium on carbon (10.00 mg) was added. The reaction mixture was purged with hydrogen and reacted at 20 °C in a hydrogen atmosphere for 16 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction mixture was filtered and concentrated under reduced pressure to obtain 183.00 mg of the title compound.

[0845] The structural characterization data are as follows:

[0846] ESI-MS (m / z): 239.1 [M+H] + .

[0847] 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

[0848] 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), and p-toluenesulfonic acid (3.61 mg, 0.02 mmol) was added. The reaction mixture was stirred at 130 °C for 4 hours, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography. The purified product was lyophilized to obtain 2.00 mg of the trifluoroacetate salt of the title compound.

[0849] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0850] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0851] 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

[0852] The structural characterization data are as follows:

[0853] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.58 (s, 2H), 8.22 (d, J = 8.1 Hz, 1H), 7.97 (d, J = 10.7 Hz, 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 = 6.3 Hz, 6H), 0.89 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 466.2 [M+H] +

[0854] 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-isopropylethanamide

[0855] Dissolve compound 4-12-05 (22.00 mg, 47.26 μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (16.35 mg, 51.99 μmol) in N,N-dimethylformamide (1 mL), then add HATU (21.55 mg, 56.71 μmol) and N,N-diisopropylethylamine (18.32 mg, 141.78 μmol). After addition, react at room temperature for 0.5 h, and detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is directly purified by a C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution = 30% - 100%) to obtain 18.00 mg of the title compound.

[0856] The structure characterization data are as follows:

[0857] ESI-MS (m / z): 762.3 [M+H] + .

[0858] 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-isopropylethanamide

[0859] Dissolve compound 4-12-06 (18.00 mg, 23.62 μmol) in N,N-dimethylformamide (1 mL), then add potassium fluoride (6.86 mg, 118.12 μmol). After addition, heat to 50 °C and react for 1 h, and detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is directly purified by high performance liquid chromatography to obtain 1.53 mg of the title compound.

[0860] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0861] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0862] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 15 85 28 18.00 90 10 28

[0863] The structure characterization data are as follows:

[0864] 11H NMR (400 MHz, DMSO-d 6 ) δ 8.53 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.55 (s, 1H), 5.44 (d, J = 13.8 Hz, 4H), 4.72 (t, J = 5.5 Hz, 1H), 4.21 (d, J = 5.5 Hz, 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, 3.1 Hz, 6H), 0.87 (t, J = 7.3 Hz, 3H).

[0865] ESI-MS (m / z): 524.2 [M + H] + .

[0866] Example XI (S)-N-((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)methyl)-1-hydroxycyclopropanecarboxamide

[0867]

[0868] The starting material (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (4-10-01, prepared according to the synthetic method of patent WO2020219287, 30.00 mg, 67.00 μmol), 1-hydroxycyclopropanecarboxylic acid (7.56 mg, 0.074 mmol) were 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 reaction was carried out at room temperature for 4 hours. Water and ethyl acetate were added and stirred, and after standing, the layers were separated. 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 further purified by preparative high-performance liquid chromatography to obtain 1.20 mg of solid.

[0869] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0870] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0871] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

[0872] The structural characterization data is as follows:

[0873] 1 H NMR(400MHz,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).

[0874] ESI-MS(m / z):494.1[M+1] + .

[0875] 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

[0876]

[0877] Step 1: Synthesis of (E)-4-(5-Acetamido-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid

[0878] 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), dissolve them in a mixed solvent of 1,4-dioxane (15 mL) and water (5 mL), then add tris(o-tolyl)phosphine (247.37 mg, 812.76 μmol), palladium(II) acetate (91.24 mg, 406.38 μmol) and N,N-diisopropylethylamine (2.31 g, 17.88 mmol). After adding, displace the reaction system with nitrogen three times, and heat to 80 °C under a 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), 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. Combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 1.90 g of the title compound.

[0879] The structure characterization data are as follows:

[0880] ESI-MS(m / z): 266.1[M+H] + .

[0881] Step 2: Synthesis of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutyric acid

[0882] Dissolve (E)-4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid (1.90 g, 7.16 mmol) in methanol (40 mL), add 10% palladium on carbon (0.15 g) under nitrogen protection, then displace the reaction system with a hydrogen balloon three times, and react under a hydrogen atmosphere for 2 hours. Detect the reaction by high performance liquid chromatography-mass spectrometry. Filter the reaction solution, and concentrate the filtrate to dryness under reduced pressure to obtain 1.51 g of the title compound.

[0883] The structure characterization data are as follows:

[0884] ESI-MS(m / z): 268.1[M+H] + .

[0885] Step 3: Synthesis of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0886] Weigh 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutyric 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 addition, keep the reaction at 5 °C for 2 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, and evaporate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 30%) to obtain 1.05 g of the title compound.

[0887] The structure characterization data are as follows:

[0888] ESI-MS (m / z): 250.1 [M+H] + .

[0889] Step 4: Synthesis of N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0890] 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 addition, heat the reaction to 50 °C for 15 hours and detect the reaction by high performance liquid chromatography-mass spectrometry. Directly evaporate the reaction solution under reduced pressure to obtain the crude product. The crude product is purified by flash silica gel column (ethyl acetate: petroleum ether = 0 - 30%) to obtain 461.00 mg of the title compound.

[0891] The structure characterization data are as follows:

[0892] ESI-MS (m / z): 328.0 [M+H] + .

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

[0894] 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). After adding, 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, and evaporate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by flash silica gel column chromatography (ethyl acetate:petroleum ether = 0 - 50%) to obtain 347.00 mg of the title compound.

[0895] The structure characterization data are as follows:

[0896] ESI-MS(m / z): 291.1[M+H] + .

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

[0898] 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). Under nitrogen protection, add 10% palladium on carbon (30.00 mg), then displace the reaction system with a hydrogen balloon three times, and react under a hydrogen atmosphere for 2 hours. Detect the reaction by high performance liquid chromatography-mass spectrometry. Filter the reaction solution, concentrate the filtrate under reduced pressure to dryness to obtain the crude product. The crude product is purified by C18 reversed-phase column chromatography (acetonitrile:0.05% formic acid aqueous solution = 0% - 30%) to obtain 205.00 mg of the title compound.

[0899] The structure characterization data are as follows:

[0900] ESI-MS(m / z): 265.1[M+H] + .

[0901] Step 7: Synthesis of (9H-fluoren-9-yl)methyl (8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0902] 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 adding, stir the reaction at room temperature for 2 hours 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 phases over anhydrous sodium sulfate, filter, and evaporate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 20% - 80%) to obtain 301.00 mg of the title compound.

[0903] The structure characterization data are as follows:

[0904] ESI-MS (m / z): 487.0 [M+H] + .

[0905] 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

[0906] Dissolve (9H-fluoren-9-yl)methyl (8-acetamido-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 adding, heat the reaction to 50 °C and react 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 and wash with saturated brine, dry the organic phases over anhydrous sodium sulfate, filter, and evaporate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by a flash silica gel column (methanol: dichloromethane = 0% - 5%) to obtain 71.00 mg of the title compound.

[0907] The structure characterization data are as follows:

[0908] ESI-MS (m / z): 445.2 [M+H] + .

[0909] 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

[0910] (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 complete, the temperature was raised to 140 °C and the reaction was carried out for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 °C to obtain the crude product. The crude product was purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution = 20% - 80%) to obtain 21.00 mg of the title compound.

[0911] The structure characterization data are as follows:

[0912] ESI-MS (m / z): 672.2 [M+H] + .

[0913] 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

[0914] (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), then diethylamine (0.2 mL) was added. After the addition, the reaction was carried out at room temperature for 0.5 h, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the ethylenediamine was distilled off under reduced pressure from the reaction solution, 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 two isomers 5-13-A (1.30 mg) and 5-13-B (1.68 mg).

[0915] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0916] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0917] 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

[0918] For 5-13-A (the peak appears earlier at 6 min in LCMS, retention time: 1.373 min), the structure characterization data are as follows:

[0919] 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.88 (d, J = 10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J = 3.5 Hz, 2H), 5.46 (d, J = 2.5 Hz, 2H), 3.25 - 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 = 7.3 Hz, 3H).

[0920] ESI-MS (m / z): 450.2 [M + H] + .

[0921] For 5-13-B (the peak appears later at 6 min in LCMS, retention time: 1.523 min), the structure characterization data are as follows:

[0922] 1 H NMR (400 MHz, DMSO-d 6)δ 7.76 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J = 19.8 Hz, 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, 5.1 Hz, 1H), 1.91 - 1.79 (m, 2H), 1.34 (s, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0923] ESI-MS (m / z): 450.2 [M+H] + .

[0924] 6 min LCMS conditions:

[0925] Column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0926] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0927]

[0928]

[0929] Example XIII (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

[0930]

[0931] Step 1: Synthesis of N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0932] 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 and reacted for 3 hours. 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 the crude product. The crude product was purified by flash silica gel column (ethyl acetate: petroleum ether = 20%-100%) to obtain 891.00 mg of the title compound.

[0933] The structure characterization data are as follows:

[0934] ESI-MS (m / z): 291.1 [M+H] + .

[0935] Step 2: Synthesis of N-(7-(aminomethylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0936] 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 the addition, the reaction was carried out at room temperature for 16 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The solvent of the reaction solution was evaporated to dryness under reduced pressure, and then dichloromethane (30 mL) and water (20 mL) were added. After stirring and standing, the organic phase was separated, 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.

[0937] The structure characterization data are as follows:

[0938] ESI-MS (m / z): 263.1 [M+H] + .

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

[0940] Compound 5-7-02 (0.80 g, 3.05 mmol) was dissolved in ethanol (200 mL), and then 10% palladium on 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 the reaction was carried out at room temperature for 3 hours under a hydrogen atmosphere. 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 salt of the title compound.

[0941] The structure characterization data are as follows:

[0942] ESI-MS (m / z): 265.1 [M+H] + .

[0943] 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

[0944] Dissolve the hydrochloride salt of compound 5-7-03 (0.90 g, 2.99 mmol) in 1,4-dioxane (20 mL), then add sodium bicarbonate (1.01 g, 11.97 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.21 g, 3.59 mmol). After addition, stir the reaction at room temperature for 1 hour and detect the reaction by high performance liquid chromatography-mass spectrometry. 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, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 0 - 50%) to obtain 1.30 g of the title compound.

[0945] The structure characterization data are as follows:

[0946] ESI-MS (m / z): 487.1 [M+H] + .

[0947] 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

[0948] Dissolve compound 5-7-04 (0.80 g, 1.64 mmol) in 1,4-dioxane (20 mL), add 3 mol / L hydrochloric acid aqueous solution (20 mL) under nitrogen protection. After addition, heat the reaction to 60 °C for 15 hours 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, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product is purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 0 - 40%) to obtain 561.00 mg of the title compound.

[0949] The structure characterization data are as follows:

[0950] ESI-MS (m / z): 445.1 [M+H] + .

[0951] 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

[0952] (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 addition, the temperature was raised to 140 °C and the reaction was carried out for 4 hours. Then the reaction solution was directly evaporated to dryness under reduced pressure at 140 °C to obtain the crude product. The crude product was purified by flash silica gel column (methanol:dichloromethane = 0-5%) to obtain 563.00 mg of the title compound.

[0953] The structure characterization data are as follows:

[0954] ESI-MS (m / z): 672.2 [M+H] + .

[0955] 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

[0956] 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 addition, the reaction was carried out at room temperature for 0.5 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was evaporated to remove ethylenediamine under reduced pressure, the pH was adjusted to 2-3 with formic acid, and then the reaction solution was directly purified by preparative high performance liquid chromatography. The prepared solutions were freeze-dried respectively to obtain 32.00 mg of the title compound 5-7-A and 56.00 mg of 5-7-B.

[0957] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0958] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0959] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 90 10 28

[0960] Structural characterization data of 5-7-A (eluting earlier in 6 min LCMS, retention time: 1.488 min) are as follows:

[0961] 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.88 (d, J = 10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J = 3.5 Hz, 2H), 5.46 (d, J = 2.5 Hz, 2H), 3.25 - 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 = 7.3 Hz, 3H).

[0962] ESI-MS (m / z): 450.2 [M+H] + .

[0963] Structural characterization data of 5-7-B (eluting later in 6 min LCMS, retention time: 1.596 min) are as follows:

[0964] 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.76 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J = 19.8 Hz, 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, 5.1 Hz, 1H), 1.91 - 1.79 (m, 2H), 1.34 (s, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0965] ESI-MS (m / z): 450.2 [M+H] + .

[0966] 6 min LCMS conditions:

[0967] Column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0968] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0969] 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

[0970] 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

[0971]

[0972] Step 1: Synthesis of 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

[0973] Dissolve 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) in N,N-dimethylformamide (1 mL), then add HATU (12.68 mg, 33.37 μmol) and N,N-diisopropylethylamine (8.63 mg, 66.74 μmol). After addition, react at room temperature for 0.5 h, and detect the reaction by high-performance liquid chromatography-mass spectrometry. The reaction solution is directly purified by a 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).

[0974] The structure characterization data are as follows:

[0975] ESI-MS (m / z): 772.3 [M+H] + .

[0976] 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

[0977] Dissolve compound 5-16-01-A (7.00 mg, 9.07 μmol) in N,N-dimethylformamide (1 mL), then add potassium fluoride (2.63 mg, 45.34 μmol). After addition, heat the mixture to 50 °C and react for 1 hour. Detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is directly purified by high performance liquid chromatography to obtain the single title compound 5-16-A (1.73 mg).

[0978] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0979] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0980] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 10 90 28 18.00 90 10 28

[0981] The structure characterization data are as follows:

[0982] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.26 (s, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 6.56 (s, 1H), 6.52 (s, 1H), 5.52 (d, J = 19.3 Hz, 1H), 5.43 (d, J = 4.4 Hz, 2H), 4.94 (d, J = 19.2 Hz, 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 = 7.9 Hz, 3H).

[0983] ESI-MS (m / z): 534.2 [M + H] + .

[0984] 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-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide or 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-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide

[0985]

[0986] 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-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

[0987] Compound 2-23 (16.00 mg) was purified by preparative high performance liquid chromatography. Two diastereoisomers were separated under the following purification conditions to obtain 5.10 mg of the trifluoroacetate of 2-23-A (retention time 9.85 min) and 7.12 mg of the trifluoroacetate of 2-23-B (retention time 10.62 min).

[0988] Column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[0989] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0990] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0991] The structure characterization data are as follows:

[0992] 2-23-A:

[0993] 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),5.10-5.06(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).

[0994] ESI-MS(m / z):452.1[M+H] + .

[0995] 2-23-B:

[0996] 1 H NMR(400MHz,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),5.10-5.06(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).

[0997] ESI-MS(m / z):452.0[M+H] + .

[0998] Step 2: Synthesis of 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-tetraazapentadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide or 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-tetraazapentadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide

[0999] At 25 °C, dissolve the trifluoroacetate of 2-23-A (34.71 mg, 61.43 μmol) in N,N-dimethylformamide (1 mL). Then, sequentially add 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). Keep the reaction at 25 °C for 0.5 h and monitor the reaction by high-performance liquid chromatography-mass spectrometry. After the reaction is completed, purify the reaction solution by preparative high-performance liquid chromatography (conditions are as follows). Freeze-dry the preparation solution to obtain 11.04 mg of the title compound D-L-15, with a retention time of 7.5 min.

[1000] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1001] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[1002] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[1003] The structure characterization data are as follows:

[1004] D-L-15:

[1005] ESI-MS (m / z): 1107.3 [M + H] + .

[1006] Example XVI N-((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-cyclopropyl-2-hydroxyacetamide

[1007]

[1008] Step 1: Synthesis of N-((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-cyclopropyl-2-hydroxyacetamide

[1009] The formate of compound 3-1-A in a single configuration (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), then HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol) were added. After the addition, the reaction was carried out at room temperature for 0.5 h; the reaction was detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (3-12-A: 12.96 mg, 3-12-B: 13.56 mg).

[1010] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1011] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1012] 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

[1013] The structure characterization data of 3-12-A (the peak appears earlier in the 6 min LCMS) are as follows:

[1014] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.44 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.2 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (q, J = 6.7 Hz, 1H), 5.52 (d, J = 5.1 Hz, 1H), 5.42 (s, 2H), 5.24 (q, J = 19.2 Hz, 2H), 3.61 (dd, J = 6.2, 5.1 Hz, 1H), 3.32 - 3.21 (m, 2H), 2.19 (q, J = 6.5 Hz, 2H), 1.92 - 1.80 (m, 2H), 1.26 - 1.20 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.57 - 0.34 (m, 4H).

[1015] ESI-MS (m / z): 554.0 [M + H]+.

[1016] The structure characterization data of 3-12-B (the peak appears later in the 6 min LCMS) are as follows:

[1017] 11H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 5.57 (q, J = 6.7 Hz, 1H), 5.43 (s, 2H), 5.30 - 5.17 (m, 2H), 3.64 (d, J = 6.2 Hz, 1H), 3.29 (q, J = 6.7 Hz, 2H), 2.28 - 2.13 (m, 2H), 1.93 - 1.78 (m, 2H), 1.18 - 1.08 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.50 - 0.29 (m, 4H).

[1018] ESI-MS (m / z): 554.0 [M + H]+.

[1019] The formate of compound 3-1-B with another single configuration (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), then HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol) were added. After the addition, the reaction was carried out at room temperature for 0.5 h; the reaction was detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (3-12-C: 20.19 mg, 3-12-D: 18.33 mg).

[1020] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1021] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1022] 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

[1023] The structure characterization data of 3-12-C (with a shorter retention time in LCMS at 6 min) are as follows:

[1024] 1 1H NMR (400 MHz, DMSO-d 6) δ 8.47 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (q, J = 6.5 Hz, 1H), 5.53 (d, J = 5.1 Hz, 1H), 5.43 (s, 2H), 5.32 - 5.16 (m, 2H), 3.61 (dd, J = 6.3, 5.1 Hz, 1H), 3.32 - 3.22 (m, 2H), 2.19 (q, J = 6.5 Hz, 2H), 1.92 - 1.80 (m, 2H), 1.28 - 1.20 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.54 - 0.35 (m, 4H).

[1025] ESI-MS (m / z): 554.0 [M + H]+.

[1026] Structural characterization data of 3 - 12 - D (the peak appears at the back in 6 min LCMS) are as follows:

[1027] 1 1H NMR (400 MHz, DMSO - d 6 ) δ 8.44 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 10.2 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.58 (q, J = 6.7 Hz, 1H), 5.45 (d, J = 5.2 Hz, 1H), 5.43 (s, 2H), 5.31 - 5.14 (m, 2H), 3.65 (t, J = 5.7 Hz, 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 = 7.3 Hz, 3H), 0.46 - 0.31 (m, 4H).

[1028] ESI-MS (m / z): 554.0 [M + H] + .

[1029] Example XVII: Preparation of (S)-N - ((1S,9S)-4 - chloro - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benz[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 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benz[de]pyrano[3',4':6,7]indolizino[1,2 - b]quinolin - 1 - yl)-2 - hydroxypropylamine

[1030]

[1031] 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]indolizino[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), then HATU (121.1 mg, 351.0 μmol) and DIPEA (68.0 mg, 526.5 μmol) were added, and the reaction was carried out at room temperature for 2 h; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain compound 3-7-A (6.1 mg, yield 12%) and compound 3-7-B (9.6 mg, yield 20%).

[1032] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1033] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1034] 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

[1035] Compound 3-7-A (the peak appears earlier in 6 min LC-MS, retention time 2.49 min) has the following structure characterization data:

[1036] MS m / z (ESI): 528.2 [M+H] +

[1037] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 8.55 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 6.67 (d, J = 4.8 Hz, 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 - 1.81 (m, 2H), 1.41 (d, J = 6.8 Hz, 3H), 1.30 - 1.23 (m, 1H), 0.89 - 0.85 (t, J = 7.2 Hz, 3H).

[1038] The structural characterization data of Compound 3-7-B (eluting at the end in 6 min LC-MS, retention time 2.50 min) are as follows:

[1039] MS m / z (ESI): 528.2 [M+H] +

[1040] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.46 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H), 5.60 - 5.53 (ms, 1H), 5.51 (d, J = 5.2 Hz, 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 = 6.8 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[1041] Example XVIII 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]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropanecarboxamide Compound 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]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropanecarboxamide Compound

[1042]

[1043] At 25 °C, (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenz[de]pyrano[3',4':6,7]indolizino[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), then HATU (121.14 mg, 350.98 μmol) and DIPEA (68.04 mg, 526.47 μmol) were added. After addition, the reaction was carried out at room temperature for 0.5 h; the reaction solution was concentrated to dryness and directly detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 5.3 mg of the title compound 3-17-A and 3.5 mg of 3-17-B.

[1044] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1045] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1046] 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

[1047] The structure characterization data are as follows:

[1048] For 3-17-A (eluting earlier at 6 min in LCMS, retention time: 2.657 min), the structure characterization data are as follows:

[1049] ESI-MS (m / z): 540.0 [M+H]+.

[1050] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.69 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.2 Hz, 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).

[1051] For 3-17-A (eluting later at 6 min in LCMS, retention time: 2.724 min), the structure characterization data are as follows:

[1052] ESI-MS (m / z): 540.0 [M+H]+.

[1053] 1 H NMR(400MHz,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).

[1054] 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 - octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2 - b]quinolin - 1 - yl)-2 - hydroxyacetamide Compound

[1055]

[1056] Step 1: Synthesis of N-(4 - chloro - 3 - fluoro - 7 - (hydroxyimino)-8 - oxo - 5,6,7,8 - tetrahydronaphthalen - 1 - yl)acetamide

[1057] Dissolve N-(4 - chloro - 3 - fluoro - 8 - oxo - 5,6,7,8 - tetrahydronaphthalen - 1 - yl)acetamide (570 mg, 2.23 mmol) and cyclopropylboronic acid (574.56 mg, 6.69 mmol) in 1,4 - dioxane, add dichlorobis(tert - butyl)-(4 - dimethylaminophenyl)phosphine palladium(II) (480 mg, 677.97 μmol) and cesium carbonate (2.17 g, 6.69 mmol). After protecting with nitrogen, react at 115 °C by microwave for 2 h; detect the reaction by high - performance liquid chromatography - mass spectrometry; dilute the reaction solution with ethyl acetate and filter, extract the filtrate with ethyl acetate (30 ml * 3), combine the organic phases, wash with saturated brine (50 mL), dry the organic phase with anhydrous sodium sulfate, then filter, evaporate the filtrate under reduced pressure, and purify the crude product by column chromatography on silica gel column (PE:EA = 1:4) to obtain 550 mg of the title compound.

[1058] The structure characterization data are as follows:

[1059] ESI - MS(m / z):262.1[M + H] + .

[1060] Step 2: Synthesis of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1061] Add tetrahydrofuran (30 mL) and tert-butanol (10 mL) into the reaction flask. After cooling the reaction solution to 5 °C in an ice bath, add potassium tert-butoxide (945 mg, 8.42 mmol). Then dissolve N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.0 g, 3.83 mmol) in tetrahydrofuran (1 mL), and slowly add it dropwise to the reaction solution. After 10 minutes, add isoamyl nitrite (718 mg, 6.12 mmol). After addition, keep the reaction at 5 °C for 1 hour; detect the reaction by high performance liquid chromatography-mass spectrometry; quench the reaction solution with saturated ammonium chloride aqueous solution (50 mL), extract with ethyl acetate (40 mL×3), combine the organic phases, wash with saturated brine (50 mL), dry the organic phase with anhydrous sodium sulfate, then filter, and evaporate the filtrate under reduced pressure to dryness to obtain 1.2 g of the crude product of the title compound.

[1062] The structure characterization data are as follows:

[1063] ESI-MS (m / z): 291.1 [M+H] + .

[1064] Step 3: Synthesis of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide hydrochloride

[1065] Dissolve the crude product of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.2 g, 1.41 mmol) in methanol (7.5 mL) and tetrahydrofuran (7.5 mL), then add 1 mol / L hydrochloric acid aqueous solution (7.5 mL) and 10% palladium on carbon (450 mg). After addition, displace the reaction system with a hydrogen balloon three times, and react at room temperature under a hydrogen atmosphere for 1 hour; detect the reaction by high performance liquid chromatography-mass spectrometry; filter the reaction solution, and concentrate the filtrate under reduced pressure to dryness to obtain 1.05 g of the crude product.

[1066] The structure characterization data are as follows:

[1067] ESI-MS (m / z): 277.1 [M+H] + .

[1068] 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

[1069] 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), 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 addition, the reaction mixture was stirred at room temperature for 2 hours; the reaction was monitored by high performance liquid chromatography-mass spectrometry; the reaction solution was poured into water (50 mL), then extracted with ethyl acetate (40 mL×3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by a C18 reversed-phase column to obtain 2.0 g of the title compound.

[1070] The structure characterization data are as follows:

[1071] ESI-MS (m / z): 499.2 [M+H] + .

[1072] 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

[1073] (9H-fluoren-9-yl)methyl (8-acetamido-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (2.0 g, 3.21 mmol, 80%) was dissolved in dioxane (20 mL), 12 mol / L concentrated hydrochloric acid (5 mL) was added. After addition, the temperature was raised to 70 °C and the reaction was carried out for 2 hours; the reaction was monitored by high performance liquid chromatography-mass spectrometry; the reaction solution was poured into water (40 mL), then extracted with ethyl acetate (30 mL×3). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by a silica gel column chromatography (PE:EA = 2:1) to obtain 740 mg of the title compound.

[1074] The structure characterization data are as follows:

[1075] ESI-MS (m / z): 457.3 [M+H] + .

[1076] Step 6: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-4-cyclopropyl-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)carbamate

[1077] (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 complete, the temperature was raised to 135 °C and the reaction was carried out for 2 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 °C to obtain the 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.

[1078] The structure characterization data are as follows:

[1079] ESI-MS (m / z): 684.1 [M+H] + .

[1080] 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]indolizino[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]indolizino[1,2-b]quinoline-10,13-dione

[1081] (9H-Fluoren-9-yl)methyl ((9S)-4-cyclopropyl-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)carbamate (1.02 g, 1.49 mmol) was dissolved in N,N-dimethylformamide (15 mL), and then diethylamine (5 mL) was added. After the addition was complete, the reaction was carried out at room temperature for 0.5 hour; the reaction was detected by high performance liquid chromatography-mass spectrometry; after ethylenediamine was evaporated under reduced pressure from the reaction solution, 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 two isomers of the title compound (5-22-7-A: 60 mg; 5-22-7-B: 55 mg).

[1082] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1083] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1084] 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

[1085] For 5-22-7-A (eluting earlier at 6 min in LCMS, retention time: 2.28 min), the structure characterization data are as follows:

[1086] ESI-MS (m / z): 462.2 [M+H] + .

[1087] For 5-22-7-B (eluting later at 6 min in LCMS, retention time: 2.35 min), the structure characterization data are as follows:

[1088] ESI-MS (m / z): 462.2 [M+H] + .

[1089] Step Eight: Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[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-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[1090] 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), then HATU (40 mg, 104 μmol) and DIPEA (36 mg, 258 μmol) were added. After addition, the reaction was carried out at room temperature for 0.5 h; the reaction solution was concentrated to dryness and directly detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 12.5 mg of compound 5-22-A.

[1091] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1092] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1093] 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

[1094] The structure characterization data are as follows:

[1095] The structural characterization data of 5-22-A (which elutes earlier in the 6-minute LCMS, retention time: 2.540 min) are as follows:

[1096] ESI-MS (m / z): 520.0 [M+H] + .

[1097] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.46 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 11.9 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.64 - 5.56 (m, 1H), 5.49 (t, J = 5.8 Hz, 1H), 5.42 (s, 2H), 5.19 (s, 2H), 3.96 (d, J = 5.7 Hz, 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 = 7.2 Hz, 3H), 0.80 - 0.70 (m, 2H).

[1098] 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), then HATU (40 mg, 104 μmol) and DIPEA (17 mg, 130 μmol) were added. After addition, the reaction was carried out at room temperature for 0.5 h; the reaction solution was concentrated to dryness and directly detected by high-performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain 13.83 mg of compound 5-22-B.

[1099] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[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-B (which elutes earlier in the 6-minute LCMS, retention time: 2.612 min) are as follows:

[1104] ESI-MS (m / z): 520.0 [M+H] + .

[1105] 11H NMR (400 MHz, DMSO-d 6 ) δ 8.49 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 11.9 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.60 (s, 1H), 5.51 (t, J = 5.9 Hz, 1H), 5.43 (s, 2H), 5.25 - 5.13 (m, 2H), 3.97 (d, J = 5.8 Hz, 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 = 7.2 Hz, 4H), 0.80 - 0.70 (m, 2H).

[1106] Synthesis of Example 20 (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]indolizino[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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1107]

[1108] Step 1: Synthesis of (9H-Fluoren-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]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate and (9H-Fluoren-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]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate

[1109] At 25 °C, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-benz[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione mesylate (72 mg, 166 μmol) and 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxypropanoic acid (65 mg, 199 μmol) were dissolved in DMF (2 mL), then HATU (95 mg, 250 μmol) and DIPEA (65 mg, 498 μmol) were added. After addition, the reaction was carried out at room temperature for 0.5 h; the reaction solution was concentrated to dryness and directly detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 24 mg of the title compound (Compound 1-10-1-A) and 28 mg of (Compound 1-10-1-B).

[1110] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1111] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1112] 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

[1113] Compound 1-10-1-A (the peak in the 6 min LCMS appears earlier, retention time: 3.283 min), the structure characterization data are as follows:

[1114] ESI-MS (m / z): 745.4 [M+H] + .

[1115] Compound 1-10-1-B (the peak in the 6 min LCMS appears later, retention time: 3.465 min), the structure characterization data are as follows:

[1116] ESI-MS (m / z): 745.4 [M+H] + .

[1117] 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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide and Synthesis of (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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1118] At 25 °C, dissolve compound 1-10-1-B (28 mg, 37 μmol) in DMF (2 mL), then add diethylamine (1 mL). After addition, react at room temperature for 1.0 h; concentrate the reaction solution to dryness to obtain 28 mg of the crude product (compound 1-10-2-B), which is directly used in the next step of the reaction.

[1119] At 25 °C, dissolve compound 1-10-1-A (24 mg, 33 μmol) in DMF (2 mL), then add diethylamine (1 mL). After addition, react at room temperature for 1.0 h; concentrate the reaction solution to dryness to obtain 24 mg of the crude product (compound 1-10-2-A), which is directly used in the next step of the reaction.

[1120] The structure characterization data is as follows:

[1121] ESI-MS (m / z): 523.2 [M+H] + .

[1122] 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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide and Synthesis of (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]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

[1123] At 25 °C, compound 1-10-2-B (28 mg, 37 μmol, 70%) was dissolved in methanol (2 mL), and then aqueous formaldehyde solution (1 mL) was added. After addition, the reaction was carried out at room temperature for 16.0 h, and then sodium cyanoborohydride (7.07 mg, 96.45 μmol) was added, and the reaction was carried out at room temperature for 1.0 h. The reaction solution was concentrated to dryness and directly detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by preparative high performance liquid chromatography to obtain 1.3 mg of the title compound (compound 1-10B).

[1124] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1125] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1126] 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

[1127] The structure characterization data of compound 1-10-B (with a relatively early peak in 6 min LCMS, retention time: 1.937 min) are as follows:

[1128] ESI-MS (m / z): 551.2 [M+H] + .

[1129] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.48 (d, J = 8.6 Hz, 1H), 8.31 (s, 2H), 7.80 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.54 (s, 1H), 5.43 (s, 2H), 5.34 (d, J = 19.2 Hz, 1H), 5.19 (d, J = 19.1 Hz, 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 = 7.0 Hz, 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 = 7.2 Hz, 3H).

[1130] At 25 °C, 24 mg (33 μmol, 70%) of Compound 1-10-2-A was dissolved in methanol (2 mL), and then aqueous formaldehyde solution (1 mL) was added. After addition, the reaction was carried out at room temperature for 16.0 h, and then sodium cyanoborohydride (6.06 mg, 96.45 μmol) was added, and the reaction was carried out at room temperature for 1.0 h; the reaction solution was concentrated to dryness and directly detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain 4.44 mg of the title compound (Compound 1-10-A).

[1131] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1132] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1133] 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

[1134] Compound 1-10-A (eluting earlier at 6 min in LCMS, retention time: 1.920 min) had the following structure characterization data:

[1135] ESI-MS (m / z): 551.2 [M+H] + .

[1136] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.58 (d, J = 9.0 Hz, 1H), 8.28 (s, 1H), 7.84 (d, J = 10.9 Hz, 1H), 7.37 (s, 1H), 6.61 (s, 1H), 5.66 - 5.59 (m, 1H), 5.49 (s, 2H), 5.35 (d, J = 19.1 Hz, 1H), 5.17 (d, J = 18.9 Hz, 1H), 4.23 - 4.16 (m, 1H), 3.23 (d, J = 7.8 Hz, 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 = 7.3 Hz, 3H).

[1137] Example 21 (S)-14-(2-(Cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione (Compound 4-14)

[1138]

[1139] Step 1: Synthesis of 2-nitro-4,5-methylenedioxyacetophenone

[1140] Dissolve compound 4-14-1 (10.0 g, 60.92 mmol) in nitromethane (100 mL), slowly add concentrated nitric acid (26 mL) dropwise with stirring, and react at room temperature for 2 hours. Monitor the reaction by TLC. There is a small amount of residual raw material and obvious product. Slowly add saturated sodium bicarbonate aqueous solution to neutralize the reaction, extract with dichloromethane 3 times, combine the organic phases, wash with saturated brine 3 times and then dry, and concentrate to obtain the crude product. Purify by silica gel column (eluent: 0 - 20% ethyl acetate / petroleum ether) to obtain 9.8 g of the title compound.

[1141] Step 2: Synthesis of 6-amino-3,4-methylenedioxyacetophenone

[1142] Dissolve compound 4-14-2 (2.0 g, 9.56 mmol) in ethyl acetate (20 mL), add 10% palladium on carbon (0.2 g), and stir the reaction for 4 hours under hydrogen replacement and protection. Filter, and concentrate the filtrate under reduced pressure to obtain 1.7 g of the crude product of the title compound.

[1143] Step 3: Synthesis of 6-acetamido-3,4-methylenedioxyacetophenone

[1144] Dissolve compound 4-14-3 (1.7 g, 9.49 mmol) in acetic anhydride (17 mL), and stir the reaction for 1 hour. Evaporate the solvent under reduced pressure, add water and stir, filter, wash the solid with water and then dry it under vacuum to obtain 2.08 g of the crude product of the title compound.

[1145] Step 4: Synthesis of (E)-N-(6-(3-(dimethylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

[1146] Dissolve compound 4-14-4 (1.88 g, 8.50 mmol) in DMF-DMA (30 mL), heat to 120 °C and react for 2 hours. Evaporate the solvent under reduced pressure to obtain 2.33 g of the crude product of the title compound.

[1147] The structure characterization data are as follows:

[1148] ESI-MS (m / z): 277.2 [M + 1] + .

[1149] Step 5: Synthesis of (E)-N-(6-(3-(cyclopropylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

[1150] Dissolve compound 4-14-5 (200 mg, 0.72 mmol) in ethanol (5 mL), add cyclopropylamine (413.3 mg, 7.24 mmol) dropwise, and react at 50 °C for 16 hours. Evaporate the solvent under reduced pressure to obtain 208 mg of the crude title compound.

[1151] The structure characterization data are as follows:

[1152] ESI-MS (m / z): 289.2 [M+1] + .

[1153] Step 6: Synthesis of N-(6-(3-(cyclopropylamino)propanoyl)benzo[d][1,3]dioxol-5-yl)acetamide

[1154] 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 stirring in an ice-water bath, and stir at room temperature for 3 hours. Evaporate the solvent under reduced pressure to obtain 209 mg of the crude title compound.

[1155] The structure characterization data are as follows:

[1156] ESI-MS (m / z): 291.1 [M+1] + .

[1157] Step 7: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-acetamidobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1158] Dissolve compound 4-14-7 (200 mg, 0.69 mmol) in 1,4-dioxane (20 mL) and water (20 mL), add 9-fluorenylmethyl N-succinimidyl carbonate (395 mg, 0.68 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) under stirring, and react at room temperature for 2 hours. Add water and ethyl acetate, stir, let stand for liquid separation, wash the organic phase with saturated brine, dry and concentrate, and purify by silica gel column (eluent: 30% ethyl acetate / petroleum ether) to obtain 350 mg of the title compound.

[1159] Step 8: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-aminobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1160] 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. The temperature was raised to 60 °C and stirred for reaction for 16 hours. Water and ethyl acetate were added and stirred, and after standing for liquid separation, the organic phase was washed with water, dried and concentrated, and purified by silica gel column (eluent: 33% ethyl acetate / petroleum ether) to obtain 218 mg of the title compound.

[1161] Step 9: Synthesis of (S)-(9H-Fluoren-9-yl)methyl cyclopropyl (2-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)carbamate

[1162] 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 and reacted for 4 hours. The crude product of the title compound, 59 mg, was obtained by concentration under reduced pressure.

[1163] The structure characterization data are as follows:

[1164] ESI-MS (m / z): 698.1 [M+1] + .

[1165] Step 10: Synthesis of (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

[1166] Compound 4-14-10 (59 mg, 0.085 mmol) was dissolved in DMF (1 mL), diethylamine (0.5 mL) was added dropwise, and the mixture was stirred for reaction for 1 hour. Diethylamine was removed by evaporation under reduced pressure, and after acidification with 3N hydrochloric acid, it was purified by high performance liquid preparation (the purification conditions are as follows), and freeze-dried to obtain 12.66 mg of the trifluoroacetate salt of the title compound.

[1167] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1168] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1169] Time [min] Mobile Phase A [%] Mobile Phase B [%] Flow Rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[1170] The structural characterization data are as follows:

[1171] 1 H NMR(400MHz,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).

[1172] ESI-MS(m / z):476.1[M + 1] + .

[1173] Example 22 (S)-7-Ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione (Compound 4-15)

[1174]

[1175] Step 1: Synthesis of (E)-N-(6-(3-((2-methoxyethyl)amino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

[1176] 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 react at 50 °C for 16 hours. Monitor the reaction by LCMS. There is a small amount of residual raw material and obvious product. Evaporate the solvent under reduced pressure to obtain 221 mg of the title compound, which is directly used for the next step of the reaction.

[1177] The structural characterization data are as follows:

[1178] ESI-MS(m / z):307.1[M + 1] +

[1179] Step 2: Synthesis of N-(6-(3-((2-methoxyethyl)amino)propanoyl)benzo[d][1,3]dioxol-5-yl)acetamide

[1180] Compound 4-15-1 (200 mg, 0.65 mmol) was dissolved in glacial acetic acid (4 mL). Sodium borohydride (12.35 mg, 0.33 mmol) was added under stirring while cooling in an ice-water bath. The mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The raw material disappeared and the product was obvious. The solvent was removed by distillation under reduced pressure to obtain 200 mg of the title compound, which was directly used for the next reaction.

[1181] ESI-MS (m / z): 309.1 [M+1] +

[1182] Step 3: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-acetamidobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[1183] 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). 9-Fluorenylmethyl N-succinimidyl carbonate (372 mg, 0.65 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were added under stirring. The reaction was carried out at room temperature for 2 hours. The reaction was monitored by TLC. The raw material disappeared and the product was obvious. Water and ethyl acetate were added and stirred. After standing for liquid separation, the organic phase was washed with saturated brine, dried and concentrated, and purified by silica gel column chromatography (eluent: 50% ethyl acetate / petroleum ether) to obtain 180 mg of the title compound.

[1184] Step 4: Synthesis of ((9H-fluoren-9-yl)methyl (3-(6-aminobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[1185] Compound 4-15-3 (180 mg, 0.68 mmol) was dissolved in 1,4-dioxane (5 mL). 3N hydrochloric acid aqueous solution (5 mL) was added dropwise, and the mixture was stirred at 60 °C for 16 hours. Water and ethyl acetate were added and stirred. After standing for liquid separation, the organic phase was washed with water, dried and concentrated, and purified by silica gel column chromatography (eluent: 45% ethyl acetate / petroleum ether) to obtain 132 mg of the title compound.

[1186] Step 5: Synthesis of (S)-(9H-fluoren-9-yl)methyl (2-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(2-methoxyethyl)carbamate

[1187] 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 mixture was heated to 120 °C and reacted for 4 hours. The reaction was monitored by LCMS, the raw materials disappeared, and the product was obvious. The mixture was concentrated under reduced pressure to obtain 190 mg of the crude product of the title compound.

[1188] The structure characterization data are as follows:

[1189] ESI-MS (m / z): 716.1 [M+1] +

[1190] Step 6: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(10H,13H)-dione

[1191] Compound 4-15-5 (190 mg, 0.265 mmol) was dissolved in DMF (3 mL), diethylamine (2 mL) was added dropwise, and the mixture was stirred and reacted for 1 hour. The reaction was monitored by LCMS, the raw materials disappeared, and the product was obvious. Diethylamine was removed by evaporation under reduced pressure, and the mixture was acidified with 3N hydrochloric acid, then prepared for purification and freeze-dried to obtain 99.28 mg of the title compound.

[1192] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1193] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1194] Time [min] Mobile Phase A [%] Mobile Phase B [%] Flow Rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[1195] The structure characterization data are as follows:

[1196] 1 H NMR (400 MHz, DMSO-d 6): δ 8.68 (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 = 10.2 Hz, 2H), 3.36 (s, 3H), 3.22 (d, J = 4.0 Hz, 4H), 1.94 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[1197] ESI-MS (m / z): 494.2 [M + 1] +

[1198] Example 23 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methoxy-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 & N-((1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methoxy-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

[1199]

[1200] Step 1: Synthesis of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide

[1201] Dissolve 3-bromo-5-fluoro-4-methoxyaniline (1.7 g, 7.73 mmol) in tetrahydrofuran (30 mL), then add triethylamine (2.35 g, 23.18 mmol) and acetic anhydride (1.18 g, 11.59 mmol). After addition, heat to 50 °C and stir for 4 hours; detect the reaction by high-performance liquid chromatography-mass spectrometry; after cooling the reaction solution to room temperature, dilute it with ethyl acetate (50 mL), then wash it once with water (30 mL) and saturated brine (30 mL) respectively. Separate the organic phase, dry it with anhydrous sodium sulfate, filter, and evaporate the filtrate under reduced pressure to obtain the crude product. Purify it by slurrying with (petroleum ether:ethyl acetate = 5:1) to obtain 1.1 g of the title compound.

[1202] Structural characterization data are as follows:

[1203] ESI-MS (m / z): 262.0 [M + H] + .

[1204] Step 2: Synthesis of (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)-3-butenoic acid

[1205] Dissolve N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (1.1 g, 4.20 mmol) and 3-butenoic acid (397.47 mg, 4.62 mmol) in a mixed solvent of 1,4-dioxane (20 mL) and water (5 mL), then add triethylamine (1.27 g, 12.59 mmol), tris(o-tolyl)phosphine (127.75 mg, 419.73 μmol) and palladium acetate (47.12 mg, 209.89 μmol). After addition, the reaction system is purged with nitrogen three times and heated to 100 °C under a nitrogen atmosphere for 4 hours; the reaction is monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS); after the reaction solution is cooled to room temperature, 1 mol / L aqueous sodium hydroxide solution (50 mL) and ethyl acetate (50 mL) are added and shaken to separate the layers. After separating the lower aqueous phase, the pH is adjusted to about 3 with 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate (40 mL × 2), the combined organic phases are washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness under reduced pressure to obtain 1.1 g of the crude product of the title compound.

[1206] The structure characterization data are as follows:

[1207] ESI-MS (m / z): 268.1 [M+H] + .

[1208] Step 3: Synthesis of 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butanoic acid

[1209] Dissolve the crude product of (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)-3-butenoic acid (1.1 g, 4.12 mmol) in methanol (20 mL), then add 10% palladium on carbon (100 mg). After addition, the reaction system is purged with a hydrogen balloon three times and reacted under a hydrogen atmosphere for 4 hours; the reaction is monitored by HPLC-MS; the reaction solution is filtered, and the filtrate is concentrated to dryness under reduced pressure to obtain 1.05 g of the crude product of the title compound.

[1210] The structure characterization data are as follows:

[1211] ESI-MS (m / z): 270.1 [M+H] + .

[1212] Step 4: Synthesis of N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1213] The crude product of 4-(5-acetamido-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, the mixture was allowed to warm to room temperature and react for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS). 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 aqueous sodium bicarbonate until neutral, and then washed with saturated brine (40 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column chromatography (ethyl acetate:petroleum ether = 0 - 40%) to obtain 503 mg of the title compound.

[1214] The structure characterization data are as follows:

[1215] ESI-MS (m / z): 252.1 [M+H] + .

[1216] Step 5: Synthesis of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1217] Tetrahydrofuran (15 mL) and tert-butanol (4 mL) were added to a reaction flask. After cooling to 5 °C in an ice bath, potassium tert-butoxide (491.26 mg, 4.38 mmol) was added. Then, N-(3-fluoro-4-methoxy-8-oxo-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 mixture. After 10 minutes, isoamyl nitrite (373.01 mg, 3.18 mmol) was added. After the addition, the mixture was maintained at 5 °C and reacted for 1 hour. The reaction was monitored by HPLC-MS. The reaction solution was quenched with saturated aqueous ammonium chloride (50 mL), extracted with ethyl acetate (40 × 2), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain 550 mg of the crude product of the title compound.

[1218] The structure characterization data are as follows:

[1219] ESI-MS (m / z): 281.1 [M+H] + .

[1220] Step 6: Synthesis of N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[1221] The crude product of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-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), then 1 mol / L hydrochloric acid aqueous solution (3.71 mL) and 10% palladium on carbon (50 mg) were added. After 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 under reduced pressure to dryness to obtain 551 mg of the crude hydrochloride of the title compound.

[1222] The structure characterization data are as follows:

[1223] ESI-MS (m / z): 267.1 [M+H] + .

[1224] Step 7: Synthesis of (9H-fluoren-9-yl)methyl (8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1225] The crude hydrochloride of N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (550 mg, 1.64 mmol) was dissolved in 1,4-dioxane (15 mL), 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 addition, the reaction was stirred at room temperature for 2 hours; the reaction was detected by high performance liquid chromatography-mass spectrometry; the reaction solution was poured into water (50 mL), then extracted with ethyl acetate (40 mL×2), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain the crude product. The crude product was purified by a C18 (acetonitrile / 0.05% formic acid aqueous solution, 20% acetonitrile to 100% acetonitrile) reverse phase column to obtain 410 mg of the title compound.

[1226] The structure characterization data are as follows:

[1227] ESI-MS (m / z): 489.1 [M+H] + .

[1228] Step 8: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1229] (9H-Fluoren-9-yl)methyl (8-acetamido-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (410 mg, 839.29 μmol) was dissolved in dioxane (10 mL), and concentrated hydrochloric acid (2 mL, 12 mol / L) was added. After addition, the temperature was raised to 70 °C and the reaction was carried out for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water (30 mL), and then extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel (ethyl acetate:petroleum ether = 0 - 60%) to obtain 351 mg of the title compound.

[1230] The structure characterization data are as follows:

[1231] ESI-MS (m / z): 447.1 [M+H] + .

[1232] Step 9: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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

[1233] (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-oxo-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 addition, the temperature was raised to 135 °C and the reaction was carried out for 4 hours. The reaction solution was directly concentrated under reduced pressure at 135 °C to obtain the crude product. The crude product was purified by flash silica gel column (methanol:dichloromethane = 0 - 6%) to obtain 358 mg of the title compound.

[1234] The structure characterization data are as follows:

[1235] ESI-MS (m / z): 674.2 [M+H] + .

[1236] 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]indolizino[1,2-b]quinoline-10,13-dione

[1237] Dissolve (9H-Fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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 (358 mg, 531.41 μmol) in N,N-dimethylformamide (4 mL), then add diethylamine (0.4 mL). After addition, react at room temperature for 0.5 h; detect the reaction by high performance liquid chromatography-mass spectrometry; evaporate the reaction solution to dryness under reduced pressure to obtain the crude product, and purify the crude product by pulping with ethyl acetate to obtain 220 mg of the title compound.

[1238] The structure characterization data are as follows:

[1239] ESI-MS (m / z): 452.1 [M+H] + .

[1240] Step 11: Synthesis of N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methoxy-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 & N-((1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methoxy-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

[1241] (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]indolizino[1,2-b]quinoline-10,13-dione (50 mg, 110.76 μmol) and glycolic acid (16.85 mg, 221.51 μmol) were dissolved in N,N-dimethylformamide (2 mL), then HATU (84.17 mg, 221.51 μmol) and N,N-diisopropylethylamine (42.94 mg, 332.27 μmol) were added. After the addition, the reaction was carried out at room temperature for 0.5 h; the reaction was detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the single configuration title compound (5-34-A: 6.22 mg, 5-34-B: 9.81 mg)

[1242] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1243] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1244] 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

[1245] The structural characterization data of 5-34-A are as follows:

[1246] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.53 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 12.3 Hz, 1H), 7.33 (s, 1H), 5.58 (q, J = 7.5, 7.0 Hz, 1H), 5.42 (d, J = 2.1 Hz, 2H), 5.25 - 5.09 (m, 2H), 3.98 (s, 2H), 3.96 (d, J = 1.1 Hz, 3H), 3.30 - 3.10 (m, 2H), 2.15 (q, J = 7.4 Hz, 2H), 1.93 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[1247] ESI-MS (m / z): 510.2 [M + H] + .

[1248] The structural characterization data of 5-34-B are as follows:

[1249] 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).

[1250] ESI-MS(m / z):510.2[M+H] + .

[1251] Example 24 (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-hydroxypropanamine & (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-hydroxypropanamine

[1252]

[1253] Step 1: Synthesis of (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-hydroxypropanamine & (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-hydroxypropanamine

[1254] (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]indolizino[1,2-b]quinoline-10,13-dione (50 mg, 110.64 μmol) and (2R)-2-hydroxypropanoic acid (19.93 mg, 221.29 μmol) were dissolved in N,N-dimethylformamide (2 mL), then HATU (84.09 mg, 221.29 μmol) and N,N-diisopropylethylamine (42.90 mg, 331.93 μmol) were added. After the addition, the reaction was carried out at room temperature for 0.5 h; the reaction was detected by high performance liquid chromatography-mass spectrometry; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (2-27-A: 5.73 mg, 2-27-B: 7.59 mg)

[1255] Chromatographic column: SunFire Prep C18 OBD 19 mm×150 mm×5.0 μm

[1256] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1257] 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

[1258] The structure characterization data of 2-27-A are as follows:

[1259] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.45 (d, J = 9.1 Hz, 1H), 8.14 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63 (s, 1H), 5.56 (q, J = 8.0 Hz, 1H), 5.42 (s, 2H), 5.25 (d, J = 19.0 Hz, 1H), 5.08 (d, J = 19.0 Hz, 1H), 4.13 (q, J = 6.7 Hz, 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 = 6.8 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[1260] ESI-MS (m / z): 524.2 [M+H] + .

[1261] The structure characterization data of 2-27-B are as follows:

[1262] 1 H NMR (400 MHz, DMSO-d6 ) δ 8.40 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.55 - 5.49 (m, 1H), 5.43 (d, J = 2.2 Hz, 2H), 5.18 (q, J = 19.0 Hz, 2H), 4.13 (q, J = 6.6 Hz, 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 = 6.7 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H).

[1263] ESI-MS (m / z): 524.2 [M + H] + .

[1264] Example 25: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropylamine and (R)-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-hydroxypropylamine

[1265]

[1266] 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), then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added, and the reaction was carried out at room temperature for 2 h; most of the DMF in the reaction solution was concentrated, and the residue was purified by preparative high-performance liquid chromatography to obtain compound 3-26-A (15.4 mg, yield 64%).

[1267] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1268] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1269] Retention time: 5.3 - 6.2 min

[1270] 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

[1271] The structural characterization data are as follows:

[1272] MS m / z(ESI): 528.2 [M+H] +

[1273] 1 H NMR(400 MHz, DMSO-d 6 ) δ 8.52 (d, J = 9.2 Hz, 1H), 8.05 (d, J = 10.0 Hz, 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 = 6.8 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[1274] 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), then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added, and the reaction was carried out at room temperature for 2 h; most of the DMF in the reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography to obtain compound 3-26-B (4.8 mg, yield 20%).

[1275] Chromatographic column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[1276] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1277] Retention time: 7.5 - 8.5 min

[1278] 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

[1279] The structural characterization data are as follows:

[1280] MS m / z(ESI): 528.2 [M+H] +

[1281] 1 H NMR(400 MHz, DMSO-d 6)δ8.48 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 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 -

[1282] 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 = 6.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[1283] Biological Evaluation

[1284] I. Tumor Cell Proliferation Inhibition Assay

[1285] 1. Inhibitory effect of the compound on the proliferation of HT29 cells

[1286] (1) Cell seeding: First, culture the tumor cells HT29 in the corresponding medium, digest the cells with trypsin, centrifuge and resuspend the cells for counting, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 1.

[1287] Table 1. Sources of tumor cells

[1288] Cell Name Tumor Type Source HT29 Human Colon Cancer Cells Cell Bank of the Chinese Academy of Sciences

[1289] Co - incubation of the compound of the present invention with tumor cells: After the cells adhered, remove the medium in the cells, add the diluted bioactive molecule (the compound of the present invention) into the above - mentioned wells, and incubate for 72 h.

[1290] In vitro cell activity detection: After the incubation, add 50 μL of Cell Counting - Lite TM 2.0 reagent (Vazyme / Nowizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read with an enzyme - linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar - FS). Obtain the background RLU using the medium without cells (containing Cell Counting - Lite TM ), and obtain the vehicle RLU using the medium containing cells (containing Cell Counting - Lite TM ). Cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU)×100%, fit the curve according to the four - parameter model, and calculate the half - maximal inhibitory concentration (IC50) of the compound. The detection results are shown in Table 2.

[1291] (2) Data results

[1292] Table 2. Proliferation inhibitory activity of HT29 cells

[1293]

[1294]

[1295] The test results show that the compounds of the present invention in Table 2 have a strong inhibitory effect on the proliferation of HT29 colon cancer cells.

[1296] 2. Inhibitory effect of the compound on the proliferation of A549 cells

[1297] (1) Cell seeding: First, culture the tumor cells A549 using the corresponding medium, digest the cells with trypsin, centrifuge and resuspend the cells for counting, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 3.

[1298] Table 3. Sources of tumor cells

[1299] Cell Name Tumor Type Source A549 Human Lung Cancer Cells Cell Bank of the Chinese Academy of Sciences

[1300] Co-incubation of the compound of the present invention with tumor cells: After the cells adhered, remove the medium in the cells, add the diluted bioactive molecule (the compound of the present invention) to the above wells, and incubate for 72 h.

[1301] In vitro cell activity detection: After the incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read the values using an enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar-FS). Obtain the background RLU using the medium without cells (containing Cell Counting-Lite TM ), and obtain the solvent RLU using the medium containing cells (containing Cell Counting-Lite TM ). The cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%, fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC50) of the compound. The detection results are shown in Table 4.

[1302] (2) Data results

[1303] Table 4. Proliferation inhibitory activity of A549 cells

[1304] Name <![CDATA[IC 50 (nM)]]> Reference Compound I 94.18 Example 1 (1-1-A) 66.56 Example 4 (2-7-A) 6.97 Example 4 (2-7-B) 16.39 Example 5 (2-12-A) 6.96 Example 5 (2-12-B) 5.52 Example 5 (2-12-C) 6.64 Example 7 (2-20-A) 8.14 Example 7 (2-20-B) 23.06 Example 11 (4-10) 68.48

[1305] 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.

[1306] 3. Inhibitory effect of the compound on the proliferation of NCI-H1806 cells

[1307] (1) Cell seeding: First, culture the tumor cells HCC1806 with the corresponding medium, digest the cells with trypsin, centrifuge and resuspend the cells for counting, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 5.

[1308] Table 5. Sources of tumor cells

[1309] Cell Name Tumor Type Source HCC1806 Human Breast Squamous Carcinoma Cells ATCC

[1310] Co-incubation of the compound of the present invention with tumor cells: After the cells adhered to the wall, remove the medium in the cells, add the diluted bioactive molecule (the compound of the present invention) to the above well, and incubate for 72 h.

[1311] In vitro cell activity detection: After the incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read with an enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar-FS). Obtain the background RLU using the medium without cells (containing Cell Counting-Lite TM ), and obtain the vehicle RLU using the medium containing cells (containing Cell Counting-Lite TM ). Cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU) × 100%, fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC50) of the compound. The detection results are shown in Table 6.

[1312] (2) Data results

[1313] Table 6. Proliferation inhibitory activity of HCC1806 cells

[1314] Name <![CDATA[IC 50 (nM)]]> Reference Compound I 3.03 Example 3 (2-1-A) 1.43 Example 4 (2-7-A) 1.25 Example 5 (2-12-A) 1.40 Example 5 (2-12-B) 1.07 Example 12 (5-13-A) 0.96 Example 16 (3-12-A) 0.16 Example 16 (3-12-B) 1.76 Example 16 (3-12-C) 1.91 Example 16 (3-12-D) 1.53 Example 17 (3-7-B) 1.94 Example 18 (3-17-A) 1.70 Example 18 (3-17-B) 2.26 Example 19 (5-22-A) 2.57 Example 19 (5-22-B) 3.71 Example 25 (3-26-A) 1.27 Example 25 (3-26-B) 4.93

[1315] 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 mammary squamous carcinoma cells.

[1316] 4. Inhibitory effect of the compound on the proliferation of SKOV-3 cells

[1317] (1) Cell seeding: First, culture the tumor cells SKOV-3 in the corresponding medium, digest the cells with trypsin, resuspend the cells after centrifugation, count the cells, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 7.

[1318] Table 7. Sources of tumor cells

[1319] Cell Name Tumor Type Source SKOV-3 Human Ovarian Cancer Cells Nanjing Kebai Biotechnology

[1320] Co-incubation of the compound of the present invention with tumor cells: After the cells adhered to the wall, remove the medium in the cells, add the diluted bioactive molecule (the compound of the present invention) to the above wells, and incubate for 72 h.

[1321] In vitro cell activity detection: After the incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read the results with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). Obtain the background RLU using the medium without cells (containing Cell Counting-Lite TM ), and obtain the vehicle RLU using the medium containing cells (containing Cell Counting-Lite TM ). Cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU) × 100%, fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC50) of the compound. The detection results are shown in Table 8.

[1322] (2) Data results

[1323] Table 8. Inhibitory activity of SKOV-3 cell proliferation

[1324] Name <![CDATA[IC 50 (nM)]]> Reference Compound I 17.14 Example 1 (1-1-A) 7.81 Example 3 (2-1-A) 7.12 Example 4 (2-7-A) 8.64 Example 4 (2-7-B) 9.47 Example 5 (2-12-A) 6.85 Example 5 (2-12-B) 4.99 Example 5 (2-12-C) 6.52 Example 7 (2-20-A) 6.79 Example 12 (5-13-A) 2.09 Example 21 (4-14) 2.16

[1325] 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.

[1326] 5. Inhibitory effect of the compound on the proliferation of NCI-H358 cells

[1327] (1) Cell seeding: First, culture the tumor cells NCI-H358 in the corresponding medium, digest the cells with trypsin, resuspend the cells after centrifugation, count the cells, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 9.

[1328] Table 9. Sources of tumor cells

[1329] Cell Name Tumor Type Source NCI-H358 Human Non-Small Cell Lung Cancer Cells Nanjing Kebai Biotechnology

[1330] Co-incubation of the compound of the present invention with tumor cells: After the cells adhered to the wall, the culture medium in the cells was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the above-mentioned wells and incubated for 72 h.

[1331] In vitro cell activity detection: After the incubation, 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) was added to each well, mixed well in the dark with shaking, and detection could be carried out after reacting for 10 min. The enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar-FS) was used for reading. The background RLU was obtained using the cell-free culture medium (containing Cell Counting-Lite TM ), and the vehicle RLU was obtained using the culture medium containing cells (containing Cell Counting-Lite TM ). The cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU) × 100%. According to the four-parameter model, the curve was fitted to calculate the half-maximal inhibitory concentration (IC50) of the compound. The detection results are shown in Table 10.

[1332] (2) Data results

[1333] Table 10. Proliferation inhibitory activity of NCI-H358 cells

[1334]

[1335]

[1336] The test results showed that the compounds of the present invention in Table 10 had a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[1337] 6. Inhibitory effect of the compound on the proliferation of NCI-N87 cells

[1338] (1) Cell seeding: First, the tumor cells NCI-N87 were cultured using the corresponding culture medium. The cells were digested with trypsin, centrifuged, resuspended, counted, and adjusted to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 11.

[1339] Table 11. Sources of tumor cells

[1340] Cell Name Tumor Type Source NCI-N87 Human Gastric Cancer Cells ATCC

[1341] Co-incubation of the compound of the present invention with tumor cells: After the cells adhered to the wall, the culture medium in the cells was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the above-mentioned wells and incubated for 72 h.

[1342] In vitro cell activity detection: After the incubation is completed, add 50 μL of Cell Counting-Lite TM Reagent 2.0 (Vazyme / Novizan) to each well, mix thoroughly by shaking in the dark. After reacting for 10 min, the detection can be carried out, and the readings are taken using an ELISA reader (manufacturer: BMG, model: PHERAStar-FS). The background RLU is obtained using a cell-free culture medium (containing Cell Counting-Lite TM ), and the vehicle RLU is obtained using a culture medium containing cells (containing Cell Counting-Lite TM ). The cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU) × 100%. Fit the curve according to the four-parameter model to calculate the half-maximal inhibitory concentration (IC50) of the compound. The test results are shown in Table 12.

[1343] (2) Data results

[1344] Table 12. Proliferation inhibitory activity of NCI-N87 cells

[1345]

[1346]

[1347] 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.

[1348] 7. Inhibitory effect of the compound on the proliferation of Hela cells

[1349] (1) Cell seeding: First, culture the tumor cells Hela using the corresponding culture medium. Digest the cells with trypsin, centrifuge, resuspend the cells and count them, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 13.

[1350] Table 13. Sources of tumor cells

[1351] Cell Name Tumor Type Source Hela Human Cervical Cancer Cells Nanjing Kebai Biotechnology

[1352] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, remove the culture medium in the cells, and add the diluted bioactive molecule (the compound of the present invention) to the above wells, and incubate for 72 h.

[1353] In vitro cell activity detection: After the incubation is completed, add Cell Counting-Lite TM50 μL of 2.0 reagent (Vazyme / Novizan), mix well by shaking in the dark. After reacting for 10 min, detection can be carried out, and read with an enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar-FS). Use cell-free medium (containing Cell Counting-Lite TM ) to obtain the background RLU, and cell-containing medium (containing Cell Counting-Lite TM ) to obtain the vehicle RLU. Cell inhibition rate = 1 - (sample RLU - sample background RLU) / (vehicle RLU - background RLU) × 100%. Fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC50) of the compound. The test results are shown in Table 14.

[1354] (2) Data results

[1355] Table 14. Proliferation inhibitory activity of Hela cells

[1356] Name <![CDATA[IC 50 (nM)]]> Reference Compound I 17.57 Example 1 (1-1-A) 4.57 Example 1 (1-1-B) 3.65 Example 3 (2-1-A) 16.98 Example 4 (2-7-A) 13.77 Example 5 (2-12-A) 13.25 Example 5 (2-12-B) 16.68 Example 7 (2-20-A) 11.17 Example 11 (4-10) 3.04

[1357] 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.

[1358] 8. Inhibitory effect of the compound on the proliferation of HCC70 cells

[1359] (1) Cell seeding: First, culture the tumor cells HCC70 with the corresponding medium, digest the cells with trypsin, centrifuge and resuspend the cells for counting, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 15.

[1360] Table 15. Sources of tumor cells

[1361] Cell Name Tumor Type Source HCC70 Human breast cancer cells Nanjing Kebai Biotechnology

[1362] Co-incubation of the compound of the present invention and tumor cells: After the cells adhere to the wall, remove the medium in the cells, and add the diluted bioactive molecule (the compound of the present invention) to the above wells, and incubate for 72 h.

[1363] In vitro cell activity detection: After the incubation is completed, add 50 μL of 2.0 reagent (Vazyme / Novizan) containing Cell Counting-Lite TM to each well, mix well by shaking in the dark. After reacting for 10 min, detection can be carried out, and read with an enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: BMG, model: PHERAStar-FS). Use cell-free medium (containing Cell Counting-Lite TM ) to obtain the background RLU, and cell-containing medium (containing Cell Counting-Lite TM)Obtain the solvent RLU. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%, fit the curve according to the four-parameter model, calculate the half-maximal inhibitory concentration (IC50) of the compound, and the test results are shown in Table 16.

[1364] (2) Data results

[1365] Table 16. Proliferation inhibitory activity of HCC70 cells

[1366] Name <![CDATA[IC 50 (nM)]]> Comparative Compound I 226.10 Example 1 (1-1-A) 224.85 Example 4 (2-7-A) 150.70 Example 4 (2-7-B) 192.20 Example 5 (2-12-A) 170.93 Example 5 (2-12-B) 176.16 Example 5 (2-12-C) 210.68 Example 7 (2-20-A) 104.99 Example 12 (5-13-A) 36.65

[1367] 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.

[1368] 9. Inhibitory effect of the compound on the proliferation of MDA-MB-231 cells

[1369] (1) Cell seeding: First, culture the tumor cells MDA-MB-231 with the corresponding medium, digest the cells with trypsin, centrifuge and resuspend the cells for counting, and adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 17.

[1370] Table 17. Sources of tumor cells

[1371] Cell Name Tumor Type Source MDA-MB-231 Human breast cancer cells Nanjing Kebai Biotechnology

[1372] Co-incubate the compound of the present invention with the tumor cells: After the cells adhere to the wall, remove the medium in the cells, add the diluted bioactive molecule (the compound of the present invention) to the above wells, and incubate for 72 h.

[1373] In vitro cell activity detection: After the incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). Use the medium without cells (containing Cell Counting-Lite TM ) to obtain the background RLU, and use the medium containing cells (containing Cell Counting-Lite TM ) to obtain the solvent RLU. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%, fit the curve according to the four-parameter model, calculate the half-maximal inhibitory concentration (IC50) of the compound, and the test results are shown in Table 18.

[1374] (2) Data results

[1375] Table 18. Inhibitory activity on the proliferation of MDA-MB-231 cells

[1376] Name <![CDATA[IC 50 (nM) <!-- 112 -->]]> Comparative Compound I 381.70 Example 1 (1-1-A) 363.60 Example 3 (2-1-A) 299.70 Example 4 (2-7-A) 123.50 Example 5 (2-12-A) 123.50 Example 9 (3-4-A) 284.80 Example 12 (5-13-A) 332.60 Example 13 (5-7-B) 172.40 Example 20 (1-10-A) 97.57 Example 20 (1-10-B) 27.24 Example 22 (4-15) 175.40

[1377] 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.

[1378] 10. Inhibitory effect of the compound on the proliferation of Jeko-1 cells

[1379] (1) Cell seeding: First, culture the tumor cells Jeko-1 with the corresponding medium. After centrifuging the cells, resuspend them and count the cells. Finally, adjust the cells to an appropriate concentration for seeding. The sources of the tumor cells are shown in Table 19.

[1380] Table 19. Sources of tumor cells

[1381] Cell Name Tumor Type Source Jeko-1 Human mantle cell lymphoma cells ATCC

[1382] Co-incubation of the compound of the present invention with tumor cells: After cell seeding, add the diluted bioactive molecule (the compound of the present invention) to the above wells according to a certain dilution ratio and incubate for 72 h.

[1383] In vitro cell activity detection: After incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark with shaking, and perform the detection after reacting for 10 min. Read the absorbance with an ELISA reader (manufacturer: BMG, model: PHERAStar-FS). Obtain the background RLU using the cell-free medium (containing Cell Counting-Lite TM ), and obtain the vehicle RLU using the cell-containing medium (containing Cell Counting-Lite TM ). The cell inhibition rate = 1 - (sample RLU - sample RLU) / (vehicle RLU - background RLU) × 100%, fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC50) of the compound. The test results are shown in Table 20.

[1384] (2) Data results

[1385] Table 20. Inhibitory activity on the proliferation of Jeko-1 cells

[1386] Name <![CDATA[IC 50 (nM)]]> Comparative Compound I 1.25 Example 1 (1-1-A) 1.33 Example 2 (1-7-A) 0.15 Example 2 (1-7-B) 0.66 Example 4 (2-7-A) 0.28 Example 9 (3-4-A) 0.27 Example 9 (3-4-B) 0.15 Example 11 (4-10) 0.45 Example 13 (5-7-A) 0.16 Example 13 (5-7-B) 0.18 Example 21 (4-14) 0.34 Example 22 (4-15) 0.11

[1387] 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.

[1388] 11. Inhibitory Effect of Compounds on the Proliferation of MDA-MB-453 Cells

[1389] (1) Cell Seeding: First, culture the tumor cells MDA-MB-453 using the corresponding medium. Digest the cells with trypsin, centrifuge, resuspend the cells, count them, and adjust the cell concentration to an appropriate level for seeding. The sources of the tumor cells are shown in Table 21.

[1390] Table 21. Sources of Tumor Cells

[1391] Cell Name Tumor Type Source MDA-MB-453 Human breast cancer cells Conote

[1392] Co-incubation of the Compounds of the Present Invention with Tumor Cells: After the cells adhered to the plate, remove the medium from the cells, and add the diluted bioactive molecules (compounds of the present invention) to the above wells and incubate for 72 h.

[1393] In Vitro Cell Activity Detection: After the incubation, add 50 μL of Cell Counting-Lite TM 2.0 reagent (Vazyme / Novizan) to each well, mix well in the dark by shaking, and perform the detection after reacting for 10 min. Read the values using a microplate reader (manufacturer: BMG, model: PHERAStar-FS). Obtain the background RLU using the medium without cells (containing Cell Counting-Lite TM ), and obtain the vehicle RLU using the medium containing cells (containing Cell Counting-Lite TM ). Cell Inhibition Rate = 1 - (Sample RLU - Sample RLU) / (Vehicle RLU - Background RLU) × 100%, fit the curve according to the four-parameter model, and calculate the half-maximal inhibitory concentration (IC 50 ) of the compound. The detection results are shown in Table 22.

[1394] (2) Data Results

[1395] Table 22. Inhibitory Activity of MDA-MB-453 Cell Proliferation

[1396]

[1397]

[1398] 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.

[1399] The structures of Comparative Compound I and Comparative Compound II are shown below:

[1400]

[1401] II. Antibody Conjugation Assay

[1402] The conjugation preparation of ADC D-L-15 is as follows:

[1403] Take 1.036 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg / mL), dilute it with a solution of 0.1 M disodium edetate (pH 7.6), and then adjust the pH to 7.6 with 1 M Na 2 HPO 4 solution. Add 2.4 times the amount of a 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6), mix well, and let stand at room temperature for 90 min. Add 5 times the amount of compound D-L-15 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let stand at room temperature for 2 h. After completion, replace the buffer with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), and then add sucrose and Tween 20, mix well, to obtain the antibody-drug conjugate ADC D-L-15 (1.77 mL, 8.60 mg / mL).

[1404]

[1405] The molecular weight of ADC D-L-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.

[1406] Table 23: Measured molecular weight of ADC D-L-15

[1407]

[1408] Table 24: DAR value of ADC D-L-15

[1409]

[1410] Chromatographic determination conditions:

[1411] Liquid chromatography column: Thermo MAbPac RP 3.0*100 mm;

[1412] Mobile phase A: 0.1% FA / H 2 O; Mobile phase B: 0.1% FA / ACN;

[1413] Flow rate: 0.25 mL / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μL;

[1414] Time (minutes) 2 20 22 25 26 30 Mobile Phase A (volume%) 75 60 5 5 75 75 Mobile Phase B (volume%) 25 40 95 95 25 25

[1415] Mass spectrometry determination conditions:

[1416] Mass spectrometry model: AB Sciex Triple TOF 5600+;

[1417] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10;

[1418] Accumulation time 0.5s; m / z 600 - 4000; Time bins to sum 40.

[1419] The conjugation preparation of ADC 3 - 4 - 04 - A sample is as follows:

[1420] Take 0.518 mL of hIgG antibody (anti - chicken lysozyme antibody, 19.3 mg / mL), dilute it with 0.1 M disodium edetate solution (pH 7.6), and then adjust the pH to 7.6 with 1 M Na 2 HPO 4 solution. Add 5.5 - fold molar amount of 10 mM TCEP (tris(2 - carboxyethyl)phosphine) solution (pH 7.6), mix well, and let it stand at room temperature for 90 min. Add 10 - fold molar amount of compound 3 - 4 - 04 - A dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. After completion, use a NAP - 5 gel column (Cytiva) to replace the buffer with 10 mM histidine buffer solution at pH 6.0, and then add sucrose and Tween 20, mix well to obtain the antibody - drug conjugate ADC 3 - 4 - 04 - A (1.50 mL, 5.60 mg / mL).

[1421]

[1422] 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 Table 25 and Table 26. The chromatographic determination conditions are the same as those of ADC D - L - 15.

[1423] Table 25: Measured molecular weight of ADC 3 - 4 - 04 - A

[1424]

[1425] Table 26: DAR value of ADC 3 - 4 - 04 - A

[1426]

[1427] The conjugation preparation of ADC 3 - 4 - 04 - B sample is as follows:

[1428] Take 0.518 mL of hIgG antibody (anti - chicken lysozyme antibody, 19.3 mg / mL), dilute it with a solution of 0.1 M disodium edetate (pH 7.6), and then adjust the pH to 7.6 with 1 M Na 2 HPO 4 solution. Add 5.5 - fold molar amount of 10 mM TCEP (tris(2 - carboxyethyl)phosphine) solution (pH 7.6), mix well, and leave it at room temperature for 90 min. Add 10 - fold molar amount of compound 3 - 4 - 04 - B dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. After completion, replace the buffer with 10 mM histidine buffer solution at pH 6.0 using a NAP - 5 gel column (Cytiva), then add sucrose and Tween 20, mix well, to obtain the antibody - drug conjugate ADC 3 - 4 - 04 - B (1.50 mL, 5.60 mg / mL).

[1429]

[1430] Determine the molecular weight of ADC 3 - 4 - 04 - B by LC - MS, and calculate the drug / antibody ratio DAR value to be 8.04. As shown in Table 27 and Table 28, the chromatographic determination conditions are the same as those of ADC D - L - 15.

[1431] Table 27: Measured molecular weight of ADC 3 - 4 - 04 - B

[1432]

[1433] Table 28: DAR value of ADC 3 - 4 - 04 - B

[1434]

[1435] The above experiments prove that the cytotoxic drug - linker - like compounds of the present invention can be successfully conjugated with antibodies to obtain antibody - drug conjugates.

[1436] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings that have been disclosed, and these changes are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein the compound has the structure of formula (II): In the above formula (II), A' is R x’ selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3- to 6-membered heterocyclic group; R y’ and R z’ are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, 3- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic group alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y’ and R z’ are linked to adjacent carbon atoms to form a 3- to 6-membered ring.

2. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 1, wherein: R x’ selected from hydrogen and C 1-6 alkyl group.

3. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 1 or 2, wherein: R y’ and R z’ are 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’ are connected to adjacent carbon atoms to form a 3- to 6-membered cycloalkyl ring.

4. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 3, wherein: 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’ are linked to adjacent carbon atoms to form a 3- to 6-membered cycloalkyl group.

5. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein the compound has the structure of formula (III): In the above formula (III), A” is R x” selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, and 3- to 6-membered heterocyclic group; R y” and R z” are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, C 1-6 alkoxyalkyl, 3- to 6-membered heterocyclic group, 3- to 6-membered heterocyclic alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl.

6. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 5, wherein: R x” is hydrogen.

7. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 5 or 6, wherein: R y” and R z” each independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 1-6 alkylaminoalkyl, and vinyl.

8. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 7, wherein: R y” is hydrogen, R z” is selected from hydrogen, C 1-6 alkyl and vinyl.

9. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, the structure of the compound is shown as follows:

10. A compound of formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein the structure of the compound is shown as follows: M-L-E-D Formula (VI) wherein, M is a linker moiety to an antibody or an antigen-binding fragment thereof; L is a linker connecting the linker M and E; E is a structural fragment connecting L and D; D is a structural fragment of a cytotoxic drug; the cytotoxic drug is selected from the compounds described in any one of claims 1-9; Preferably, M is selected from the following structures: Preferably, M is selected from the following structures: Preferably, L is a divalent structure selected from one or more of the following: C 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- wherein R’ represents hydrogen, C 1-6 alkyl or an alkyl group containing -(CH 2 CH 2 O) r -; r is an integer selected from 1 to 10; s is an integer selected from 1 to 10; Preferably, L is selected from the following structures: Preferably, L is selected from the following structures: Preferably, E is selected from a single bond, -NH-CH 2 -, Preferably, E is -NH-CH 2 -; Preferably, D is the structure after dehydrogenation of the compound described in any one of claims 1-5; Preferably, D is selected from the following structures: Preferably, D is preferably selected from the following structures:

11. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof according to claim 10, the structure of the compound is shown as follows: Preferably, the compound is selected from:

12. A pharmaceutical composition comprising a compound according to any one of claims 1-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, and one or more pharmaceutically acceptable carriers.

13. A kit product comprising: a) at least one compound according to any one of claims 1-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof as a first therapeutic agent, or the pharmaceutical composition of claim 12; b) optionally present, at least one other therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising the other therapeutic agent as a second pharmaceutical composition; and c) optionally present, a package and / or instructions.

14. Use of a compound according to any one of claims 1-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite and prodrug thereof, the pharmaceutical composition of claim 12 or the kit product of claim 13 in the manufacture of a medicament for the treatment of diseases associated with abnormal cell proliferation.

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