Camptothecin compounds, their preparation methods and applications

Abstract

Camptothecin compounds with antitumor activity, their preparation methods, and applications. Specifically, this relates to compounds as shown below, or pharmaceutically acceptable forms thereof, their pharmaceutical compositions, preparation methods, and uses. These compounds can be used as drugs for treating diseases involving abnormal cell proliferation.

Description

[0001] This application is based on and claims priority to the following applications: CN application number 202110159956.6, filed on February 5, 2021; CN application number 202110533304.4, filed on May 17, 2021; CN application number 202110718245.8, filed on June 28, 2021; CN application number 202110936768.X, filed on August 16, 2021; and CN application number 202111355330.9, filed on November 16, 2021. The disclosures of the aforementioned CN applications are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to a class of camptothecin compounds with antitumor activity and their conjugates, as well as their preparation methods and applications in the pharmaceutical field. Background Technology

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

[0004]

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

[0006] Several camptothecin derivatives are currently approved for marketing in the treatment of cancer (Med. Res. Rev. 2015, 35, 753). Irinotecan is used to treat colorectal cancer; topotecan is used to treat ovarian cancer; and belotetcan is used to treat ovarian cancer and small cell lung cancer. Other camptothecin derivatives include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, and Elomotecan.

[0007] Camptothecin-based drugs and their derivatives often exhibit hematologic toxicity due to bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, and anemia, as well as gastrointestinal side effects like nausea, vomiting, and diarrhea. Clinical studies have found that improving the safety and efficacy of camptothecin compounds includes modifying their pharmacokinetic properties, regulating their activity, reducing dosage, or utilizing their conjugates to form antibody-drug conjugates. Therefore, the development of novel camptothecin compounds and their conjugates that can enhance efficacy and improve safety remains of great clinical demand and application value. Summary of the Invention

[0008] This invention provides novel camptothecin compounds 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] The first aspect of the present invention provides a compound or thereof, a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, wherein the compound has the following structure:

[0010]

[0011] in,

[0012] R1 and R2 are each independently selected from hydrogen, halogen, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, hydroxyl, cyano and C 3-6 Cycloalkyl; or, R1 and R2 are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocycle;

[0013] R3 is either hydrogen or connected to a carbon atom adjacent to R1 to form a six-membered carbon ring;

[0014] A is selected from One of them;

[0015] R4 is selected from hydrogen, C1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 cycloalkyl groups and 3-6 membered heterocyclic groups;

[0016] R5 and R6 are each independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylamine alkyl, C 1-6 Alkoxyalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, aryl and heteroaryl; or R5 and R6 are connected to adjacent carbon atoms to form a 3-6 membered carbon ring or heterocycle;

[0017] m = 1 or 2.

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

[0019]

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

[0021] R y and R z Not both of them are hydrogen, and each is independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkylamine alkyl, C 1-6 Alkoxyalkyl, 3-6 membered heterocyclic alkyl and 3-6 membered heterocyclic.

[0022] In some implementation schemes, R x Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 cycloalkyl groups and 3-6 membered heterocyclic groups;

[0023] R y and R z Not both of them are hydrogen, and each is independently selected from hydrogen and C. 2-6 alkenyl, C 2-6 alkynyl group, C 1-6Alkylamine alkyl, C 1-6 Alkoxyalkyl, 3-6 membered heterocyclic alkyl and 3-6 membered heterocyclic.

[0024] In some implementations, in formula (I), R x Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 cycloalkyl groups, 3- to 6-membered heterocyclic groups;

[0025] R y and R z Not both of them are hydrogen, and each is independently selected from hydrogen and C. 2-6 alkenyl, C 2-6 Alkyne group.

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

[0027] In some implementation schemes, R x It is hydrogen.

[0028] In some implementation schemes, R y and R z Not both of them are hydrogen, and each is independently selected from hydrogen. Dimethylaminomethylene, morpholinomethylene, and methoxymethylene.

[0029] In some implementation schemes, R y and R z Not both of them are hydrogen, and each is independently selected from hydrogen. Dimethylaminomethylene and methoxymethylene.

[0030] In some implementation schemes, R y For hydrogen, R z Selected from Dimethylaminomethylene, morpholinomethylene, and methoxymethylene.

[0031] In some implementation schemes, R y For hydrogen, R z Selected from Dimethylaminomethylene and methoxymethylene.

[0032] In some implementation schemes, R x For hydrogen, R y For hydrogen, R z Selected from Dimethylaminomethylene, morpholinomethylene, and methoxymethylene.

[0033] In some implementation schemes, R x For hydrogen, Ry For hydrogen, R z Selected from Dimethylaminomethylene and methoxymethylene.

[0034] In some implementation schemes, R x For hydrogen, R y For hydrogen, R z Selected from And dimethylaminomethylene.

[0035] In some implementations, in formula (I) place as Configuration.

[0036] In some implementations, in formula (I) place as Configuration.

[0037] In some implementations, in formula (I) place as Configuration.

[0038] In some implementations, in formula (I) place as Configuration.

[0039] In some implementations, in formula (I) place as Configuration.

[0040] In some implementations, in formula (I) place as Configuration.

[0041] In some implementations, in formula (I) place as Configuration.

[0042] In some implementations, in formula (I) place as Configuration.

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

[0044]

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

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

[0047] R y’ and R z’ Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y’ and R z’ It connects with adjacent carbon atoms to form 3-6 membered rings.

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

[0049]

[0050] In some implementation schemes, R x’ Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 cycloalkyl groups, 3- to 6-membered heterocyclic groups;

[0051] R y’ and R z’ Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, or R y’ and R z’ It connects with adjacent carbon atoms to form 3-6 membered rings.

[0052] In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbon rings or 3-6 membered heterocycles.

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

[0054] In some implementation schemes, R x’ Selected from hydrogen and methyl.

[0055] In some implementation schemes, R y’ and R z’ Independently selected from hydrogen and C 1-6Alkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 cycloalkyl and C 2-6 alkenyl, or R y’ and R z’ It can be linked with adjacent carbon atoms to form 3-6 membered cycloalkyl groups.

[0056] In some implementation schemes, 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’ It connects with adjacent carbon atoms to form 3-6 membered rings.

[0057] In some implementation schemes, R y’ Selected from hydrogen and methyl, R z’ Selected from hydrogen, methyl, and cyclopropyl, or R y’ and R z’ It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0058] In some implementation schemes, R x’ Selected from hydrogen and methyl, R y’ Selected from hydrogen and methyl, R z’ Selected from hydrogen, methyl, and cyclopropyl, or R y’ and R z’ It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0059] In some implementation schemes, R x’ For hydrogen, R y’ Selected from hydrogen and methyl, R z’ Selected from hydrogen, methyl, and cyclopropyl, or R y’ and R z’ It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0060] In some implementations, A' in equation (II) is

[0061] In some implementations, in formula (II)-1 place as Configuration.

[0062] In some implementations, in formula (II)-1 place as Configuration.

[0063] In some implementations, in formula (II)-1 place as Configuration.

[0064] In some implementations, in formula (II)-1 place as Configuration.

[0065] In some implementations, in formula (II)-1 place as Configuration.

[0066] In some implementations, in formula (II)-1 place as Configuration.

[0067] In some implementations, in formula (II)-1 place as Configuration.

[0068] In some implementations, in formula (II)-1 place as Configuration.

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

[0070]

[0071] In equation (III), A” is selected from One of them;

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

[0073] R y” and R z” Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, 4-6 membered heterocyclic, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y” and R z” It connects with adjacent carbon atoms to form 3-6 membered rings.

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

[0075]

[0076] In some implementation schemes, R x” Selected from hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 cycloalkyl groups, 3- to 6-membered heterocyclic groups;

[0077] R y” and R z” Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 3-6 Cycloalkyl, 4-6 membered heterocyclic groups, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, or R y” and R z” It connects with adjacent carbon atoms to form 3-6 membered rings.

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

[0079] In some implementation schemes, R x” It is hydrogen.

[0080] In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbon rings or 3-6 membered heterocycles.

[0081] In some implementation schemes, R y” and R z” Each is independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 cycloalkyl and vinyl, or R y” and R z” It connects with adjacent carbon atoms to form 3-6 membered rings.

[0082] In some implementation schemes, R y” For hydrogen, R z” Selected from hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl and vinyl, or R y” and R z” It connects with adjacent carbon atoms to form 3-6 membered carbon rings.

[0083] In some implementation schemes, R y” For hydrogen, R z” Selected from hydrogen, methyl, cyclopropyl and vinyl, or Ry” and R z” It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0084] In some implementation schemes, R x” For hydrogen, R y” For hydrogen, R z” Selected from hydrogen, methyl, cyclopropyl and vinyl, or R y” and R z” It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0085] In some implementations, A” in formula (III) is

[0086] In some implementations, in formula (III)-1 place as Configuration.

[0087] In some implementations, in formula (III)-1 place as Configuration.

[0088] In some implementations, in formula (III)-1 place as Configuration.

[0089] In some implementations, in formula (III)-1 place as Configuration.

[0090] In some implementations, in formula (III)-1 place as Configuration.

[0091] In some implementations, in formula (III)-1 place as Configuration.

[0092] In some implementations, in formula (III)-1 place as Configuration.

[0093] In some implementations, in formula (III)-1 place as Configuration.

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

[0095]

[0096] In equation (IV),

[0097] Ra and R b Independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, hydroxyl, and cyano groups; or R a and R b It connects with adjacent carbon atoms to form a 5-6 member oxygen-containing heterocycle;

[0098] R c and R d Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, C 2-6 alkenyl and C 2-6 alkynyl group, or R c and R d It can connect with adjacent carbon atoms to form 3-6 membered carbon rings or heterocycles;

[0099] R e Selected from hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 1-6 alkoxyalkyl and C2-C5 heterocyclic groups;

[0100] q = 0 or 1;

[0101] When q = 0, R c and R d They are not both hydrogen.

[0102] In some implementation schemes, R a and R b Independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, hydroxyl, and cyano groups; or R a and R b It connects with adjacent carbon atoms to form a 5-6 member oxygen-containing heterocycle;

[0103] R c and R d Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, C 2-6 alkenyl and C 2-6 alkynyl group, or R c and R d It can connect with adjacent carbon atoms to form 3-6 membered carbon rings or heterocycles;

[0104] R e Selected from hydrogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 1-6 alkoxyalkyl and 4-6 membered heterocyclic groups;

[0105] q = 0 or 1;

[0106] When q = 0, R c and R d They are not both hydrogen.

[0107] In some implementations, in formula (IV), R a and R b Independently selected from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, hydroxyl, and cyano groups; or R a and R b It connects with adjacent carbon atoms to form a 5-6 member oxygen-containing heterocycle;

[0108] R c and R d Independently selected from hydrogen and C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 1-6 alkoxyalkyl, 4-6 membered heterocyclic, C 2-6 alkenyl, C 2-6 alkynyl group, or R c and R d It can connect with adjacent carbon atoms to form 3-6 membered carbon rings or heterocycles;

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

[0110] q = 0 or 1;

[0111] When q = 0, R c and R d They are not both hydrogen.

[0112] In some implementation schemes, R a and R b Independently selected from hydrogen, halogens and C 1-6 Alkyl, or R a and R b It connects with adjacent carbon atoms to form a 5-6 member oxygen-containing heterocycle.

[0113] In some implementation schemes, R a and R b Independently selected from hydrogen, fluorine, chlorine, and methyl, or R a and R b Together with the benzene ring attached to it, they form

[0114] Z is selected from -CH2-, -CD2-, -CH2CH2- and -CF2-.

[0115] In some implementation schemes, R a For methyl, R b It is fluorine, or R a and R b Together with the benzene ring attached to it, they form

[0116] In some implementation schemes, R c and R d Independently selected from hydrogen, C 1-6 Alkoxyalkyl and C 1-6 alkylaminoalkyl, or R c and R d It connects with adjacent carbon atoms to form 3-6 membered carbon rings.

[0117] In some implementation schemes, R c For hydrogen, R d Selected from hydrogen, Methoxyethyl and cyclopropyl, or R c and R d It connects with adjacent carbon atoms to form 3-6 membered carbon rings.

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

[0119] In some implementation schemes, R e Selected from hydrogen and isopropyl.

[0120] In some implementation schemes, R a For methyl, R b It is fluorine, or R a and R b Together with the benzene ring attached to it, they form Re Selected from hydrogen and isopropyl, R c For hydrogen, R d Selected from hydrogen, Methoxyethyl and cyclopropyl, or R c and R d It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0121] In some implementation schemes, R a For methyl, R b It is fluorine, or R a and R b Together with the benzene ring attached to it, they form R e Selected from hydrogen and isopropyl, R c For hydrogen, R d Selected from hydrogen, methoxyethyl, and cyclopropyl, or R c and R d It connects with adjacent carbon atoms to form a 3-membered carbon ring.

[0122] In some implementations, in formula (IV) place as Configuration.

[0123] In some implementations, in formula (IV) place as Configuration.

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

[0125]

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

[0127] A”' is selected from One of them;

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

[0129] R y”’ and R z”’ Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6Cycloalkyl, 3-6 membered heterocyclic, 3-6 membered heterocyclic alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl and heteroaryl, or R y”’ and R z”’ It connects with adjacent carbon atoms to form 3-6 membered rings.

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

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

[0132]

[0133] In some embodiments, the 3-6 membered ring is selected from 3-6 membered carbon rings or 3-6 membered heterocycles.

[0134] In some implementation schemes, R y”’ and R z”’ Each is independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyalkyl, C 1-6 Alkylamine alkyl, C 3-6 cycloalkyl and vinyl, or R y”’ and R z”’ It connects with adjacent carbon atoms to form 3-6 membered carbon rings.

[0135] In some implementation schemes, R y”’ and R z”’ All are hydrogen, or R y”’ and R z”’ It connects with adjacent carbon atoms to form 3-6 membered carbon rings.

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

[0137] In some implementation schemes, R x”’ It is hydrogen.

[0138] In some implementations, A”' is

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

[0140] In some implementations, in equation (V)-1 place as Configuration.

[0141] In some implementations, in equation (V)-1 place as Configuration.

[0142] In some implementations, in equation (V)-1 place as Configuration.

[0143] In some implementations, in equation (V)-1 place as Configuration.

[0144] In some implementations, in equation (V)-1 place as Configuration.

[0145] In some implementations, in equation (V)-1 place as Configuration.

[0146] In some implementations, in equation (V)-1 place as Configuration.

[0147] In some implementations, in equation (V)-1 place as Configuration.

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

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] On the other hand, the present invention also provides compounds of formula (VI) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs thereof:

[0155] MLED

[0156] Formula (VI)

[0157] in,

[0158] M is the linker site for binding to antibodies or their antigen fragments;

[0159] L is the connector between connectors M and E;

[0160] E is a structural segment connecting L and D;

[0161] D is a structural fragment of a cytotoxic drug.

[0162] In some implementations, M is selected from the following structures:

[0163]

[0164] In some implementations, M is selected from the following structures:

[0165]

[0166] In some implementations, L is selected from one or more of the following bivalent structures: 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,

[0167] Where R' represents hydrogen, C 1-6 Alkyl groups or those containing -(CH2CH2O) r - alkyl; r is selected from 1 to 10 integers; s is selected from 1 to 10 integers.

[0168] In some implementations, L is selected from the following structures:

[0169]

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

[0171]

[0172] In some implementations, E is selected from single bonds, -NH-CH2-,

[0173] In some implementations, E is -NH-CH2-.

[0174] In some embodiments, the cytotoxic drug is selected from the compounds described in any of the first aspects of the present invention.

[0175] In some embodiments, the cytotoxic agent 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 this invention.

[0176] In some embodiments, D is selected from the structure of the compound after dehydrogenation.

[0177] In some embodiments, D is selected from the dehydrogenated structures of compounds 1-1 to 1-15, 2-1 to 2-27, 3-1 to 3-26, 4-1 to 4-15, or 5-1 to 5-36 described in this invention.

[0178] In some implementations, D is selected from the following structures:

[0179]

[0180]

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

[0182]

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

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] In some implementations, MLED is selected from the following compounds:

[0190]

[0191] definition

[0192] Unless otherwise defined below, 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 technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0193] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0194] As used herein, an asterisk (*) in a compound structural formula indicates that the labeled carbon atom is a chiral carbon atom, and the invention includes a pair of enantiomers formed from that chiral carbon atom. If a compound contains two different chiral carbon atoms, the invention includes four optical isomers formed from that chiral carbon atom.

[0195] As used in this article, This indicates that the bond can be stereo-oriented ((R) or (S)) or non-stereo-oriented.

[0196] The term "alkyl" is defined as a straight-chain or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group 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 of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl), which is optionally substituted by one or more (such as 1, 2, or 3) suitable substituents.

[0197] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C..." 2-6 "alkenyl", "C" 2-4 Examples of these 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.

[0198] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Alkyne", "C" 4-6Examples of "alkynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, etc.

[0199] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by one or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0200] The term "carbocyclic" or "carbocyclic group" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic hydrocarbon group linked by a ring carbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0201] The term "carbocyclic ring" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic rings such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, or bicyclic rings, including spirocyclic, fused, or bridged systems (such as bicyclic [1.1.1]pentane, bicyclic [2.2.1]heptane, bicyclic [3.2.1]octane, or bicyclic [5.2.0]nonane, decahydronaphthalene, etc.), which may optionally be substituted by one or more (such as one, two, or three) suitable substituents. The term "3-6 membered carbocyclic ring" refers to a carbocyclic ring containing 3, 4, 5, or 6 cyclic carbon atoms.

[0202] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic group in this invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, for example, 4-6 membered nitrogen-containing heterocyclic groups and 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, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0203] 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 include, but are not limited to, ethylene oxide rings, tetrahydrofuran rings, furan rings, tetrahydropyran rings, and pyran rings. The term "nitrogen-containing heterocycle" as used in this invention refers to a heterocycle as described above, in which one or more (e.g., 1, 2, or 3) ring atoms are nitrogen atoms.

[0204] The term "halogenated alkyl" 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" as used in this invention... 1-6 "Halogenated alkyl" refers to an alkyl halogroup having 1 to 6 carbon atoms. Common alkyl halogroups include (but are not limited to) -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, etc. The alkyl halogroups in this invention are optionally substituted by one or more substituents described in this invention.

[0205] The term "aryl" refers to a group obtained by removing a hydrogen atom from the aromatic carbon atom of an aromatic hydrocarbon molecule. Examples include 6-14 membered aryl groups, and specific examples include, but are not limited to, phenyl, naphthyl, and anthracene groups.

[0206] The term "heteroaryl" refers to an aromatic cyclic group containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 member heteroaryl, 5-6 member nitrogen-containing heteroaryl, and 5-6 member oxygen-containing heteroaryl, such as furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, and 1,2,4,5-tetraazinyl.

[0207] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy groups, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention are optionally substituted by one or more substituents described in this invention.

[0208] The term "alkoxyalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups, wherein the definitions of alkoxy and alkyl groups are as described above. For example, the term "C" as used in this invention... 1-6 "Alkoxyalkyl" refers to an alkyl group having 1-6 carbon atoms that is substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups. Common alkoxyalkyl groups include (but are not limited to) CH3O-CH2-, C2H5-O-CH2-, C2H5-O-CH2CH2-, etc.

[0209] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0210] The term "nitrogen oxide" refers to an oxide (e.g., a mono- or di-oxide) of at least one nitrogen atom in the structure of the compounds of this application. Mono-oxides of nitrogen may exist as a single positional isomer or a mixture of positional isomers.

[0211] The term "substitution" refers to the selective replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0212] If a substituent is described as “optionally substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together by independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted by independently selected optional substituents.

[0213] If a substituent is described as being “independently selected” from a group, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0214] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

[0215] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0216] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally 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 to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0217] Solid lines (-) and solid wedges can be used in this article. Or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0218] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0219] It should also be understood that certain compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to encompass the various derivative forms of the compounds described above.

[0220] The pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0221] Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucoheponicate, glucuronide, glucuronide, hexafluorophosphate, etc.

[0222] Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, etc.

[0223] For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of this invention are known to those skilled in the art.

[0224] The term "ester" refers to esters derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (compounds of the present invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the present invention may themselves be esters.

[0225] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0226] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0227] This invention further includes, within its scope, prodrugs of the compounds of the invention. Typically, such prodrugs are functional group derivatives of the compounds that readily convert in vivo into the desired therapeutically active compound. Therefore, in these cases, the term "administration" for the treatment methods of the invention should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but after administration to an individual, the prodrug form is converted in vivo into the aforementioned compound. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug," ed. H. Bundgaard, Elsevier, 1985.

[0228] The invention further includes, within its scope, isotopic labels of the compounds of the invention, which are identical to the compounds of the invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature.

[0229] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved 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.W. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0230] Pharmaceutical Composition

[0231] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first or second aspect of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, and one or more pharmaceutically acceptable carriers.

[0232] The term "pharmaceutical composition" refers to a composition that can be used as a medicine, comprising a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceuticalally acceptable carrier" refers to an excipient administered co-administered with the therapeutic agent, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0233] The above-described pharmaceutical compositions can act systemically and / or locally, which can be achieved through suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0234] The above-mentioned pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one of the compounds of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites or prodrugs.

[0235] The present invention also provides a method for preparing the above-described pharmaceutical composition or its corresponding formulation, comprising combining at least one of the compounds of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites or prodrugs with one or more pharmaceutically acceptable carriers.

[0236] Pillbox products

[0237] In a fourth aspect, the present invention provides a medicine box product comprising:

[0238] a) at least one compound of the first or second aspect of the present invention as a first therapeutic agent, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, or a pharmaceutical composition of the third aspect as a first pharmaceutical composition;

[0239] b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a pharmaceutical composition comprising another therapeutic agent as a second pharmaceutical composition; and

[0240] c) Optional packaging and / or instructions.

[0241] The aforementioned medicine box product may contain 0.01 mg to 1000 mg of at least one of the compounds of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites or prodrugs.

[0242] 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, nitrogen oxide, isotope label, metabolite or prodrug, or the above-mentioned pharmaceutical composition with at least one other therapeutic agent or a pharmaceutical composition containing other therapeutic agents, packaging and / or instructions.

[0243] Medical Use

[0244] The compounds of this invention exhibit a strong inhibitory effect on abnormal cell proliferation.

[0245] Therefore, this application provides the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites and prodrugs or the above-described pharmaceutical compositions for the treatment of diseases involving abnormal cell proliferation.

[0246] In addition, this application also provides the use of the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites and prodrugs or the above-described pharmaceutical compositions in the preparation of a medicament for treating diseases of abnormal cell proliferation.

[0247] In some implementations, the diseases involving abnormal cell proliferation include (but are not limited to) tumors, such as advanced solid tumors.

[0248] This application also provides the use of the compounds of the present invention, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs thereof, or pharmaceutical compositions of the present invention, in the preparation of formulations for inhibiting the proliferation of tumor cells. In some embodiments, the formulations are for in vivo or in vitro administration. For example, the formulations may be administered to a subject to inhibit the proliferation of tumor cells in the subject; or, the formulations may be administered to in vitro cells (e.g., cell lines or cells derived from the subject) to inhibit the proliferation of tumor cells in vitro.

[0249] The tumors described in this invention include (but are not limited to): brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumors, prostate tumors, mast cell tumors, multiple myeloma, melanoma, glioma, or sarcoma.

[0250] Treatment

[0251] In another aspect, the present invention provides a method for treating a disease involving abnormal cell proliferation, comprising the steps of administering a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, or a pharmaceutical composition thereof, to an individual in need of it.

[0252] The term "effective dose" refers to a dose that is sufficient to induce a biological or medical response in cells, tissues, organs, or organisms (e.g., individuals) and to achieve the desired preventive and / or therapeutic effects.

[0253] The dosing regimen can be adjusted to provide the optimal required response. For example, it can be administered as a single dose, divided into doses over time, or the dose can be reduced or increased proportionally as needed. It is understood that, for any given individual, the specific dosing regimen should be adjusted as required and with the professional judgment of the person administering the composition or supervising the administration of the composition.

[0254] The dosage of the compounds of this invention will depend on individual circumstances, the severity of the disease or condition, the rate of administration, the disposal of the compound, and the prescribing physician's judgment. Generally, the effective dose is about 0.001-10000 mg / kg body weight / day. Where appropriate, the effective dose is about 0.01-1000 mg / kg body weight / day. About 0.01-1000 mg / kg body weight can be administered daily, every two days, or every three days, typically about 0.1-500 mg / kg body weight. Exemplary dosing regimens are once or more daily, once or more weekly, or once or more monthly. With multiple administrations, the interval between single doses can typically be daily, weekly, monthly, or annually. Alternatively, it can be administered in the form of a sustained-release formulation, in which case a lower dosing frequency is required. The dosage and frequency of administration may vary depending on the half-life of the drug in the subject and may also vary depending on whether it is for prophylactic or therapeutic use. In prophylactic use, relatively low doses are administered at relatively low frequency intervals over a long period; in therapeutic use, relatively high doses are sometimes required at shorter intervals until disease progression is slowed or stopped, preferably until the individual shows partial or complete improvement in disease symptoms, after which prophylactic use can be adopted.

[0255] The term "treatment" refers to the reduction or elimination of a targeted disease or symptom. If a subject receives a therapeutic amount of a compound of the present invention or its pharmaceutically acceptable form, or a pharmaceutical composition of the present invention, and at least one indicator and symptom of the subject shows observable and / or detectable relief and / or improvement, the subject is considered to have been successfully "treated." It is understood that treatment includes not only complete cure but also the achievement of some biological or medically relevant outcome without achieving complete cure.

[0256] The term "administrate / administrating / administration" (or "drug administration") refers to the process of applying an active pharmaceutical ingredient (such as the compound of the present invention) or a pharmaceutical composition containing an active pharmaceutical ingredient (such as the pharmaceutical composition of the present invention) to an individual or its cells, tissues, organs, biological fluids, etc., so as to bring the active pharmaceutical ingredient or pharmaceutical composition into contact with the individual or its cells, tissues, organs, biological fluids, etc. Common methods of administration include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, and vaginal administration.

[0257] The term “needs” refers to the judgment of a physician or other caregiver regarding an individual’s need for or potential benefit from preventive and / or treatment processes, which is based on various factors within the physician’s or other caregiver’s area of ​​expertise.

[0258] The term "individual" (or subject) refers to a human or non-human animal. Individuals in this invention include individuals suffering from diseases and / or conditions (patients) and healthy individuals. Non-human animals in this invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0259] Preparation method

[0260] The fourth aspect of the present invention provides a method for synthesizing the compound.

[0261] The compound of formula (I) in this invention can be synthesized by the following synthetic route.

[0262]

[0263] Among them, R x R y and R z The meaning is as described above; LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0264] Step 1:

[0265] Compound (I)-IM1 is obtained by substitution reaction of compound (I)-SM1 and compound (I)-SM2.

[0266] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 50°C;

[0267] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0268] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base 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 (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably Na2CO3 or NaHCO3.

[0269] Step Two:

[0270] Compound (I) is obtained by condensation reaction of compound (I)-IM1 and compound (I)-SM3;

[0271] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0272] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0273] 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.

[0274] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0275] The compound of formula (II)-1 in this invention can be synthesized by the following synthetic route.

[0276]

[0277] Among them, R x’ R y’ and R z’ The meaning is as described above; LG is the leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy, and halogens, preferably trifluoromethanesulfonyloxy or iodine; PG is the protecting group, selected from...

[0278] Step 1

[0279] Compound (II)-IM1 is obtained by substitution reaction of compound (II)-SM1.

[0280] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 50°C;

[0281] 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 n-heptane.

[0282] Step Two

[0283] Compound (II)-IM2 is obtained by reducing compound (II)-IM1.

[0284] 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, or rhodium catalysts, with platinum catalysts being preferred.

[0285] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 60°C;

[0286] 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.

[0287] Step 3

[0288] Compound (II)-IM3 is obtained by substitution reaction of compound (II)-IM2.

[0289] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 20°C;

[0290] 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;

[0291] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably triethylamine.

[0292] Step Four

[0293] Compound (II)-IM4 is obtained by coupling reaction of compound (II)-IM3;

[0294] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, 70°C, or 100°C, preferably 70°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, water, preferably a mixture of tetrahydrofuran and water.

[0296] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably N,N-diisopropylethylamine.

[0297] Step 5

[0298] Compound (II)-IM5 is obtained by reducing compound (II)-IM4.

[0299] 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, or rhodium catalysts, with platinum catalysts being preferred.

[0300] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 40°C;

[0301] 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, with tetrahydrofuran being preferred.

[0302] Step Six

[0303] Compound (II)-IM6 is obtained by cyclization reaction of compound (II)-IM5;

[0304] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 5°C;

[0305] 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 mixture of tetrahydrofuran and tert-butanol.

[0306] Step Seven

[0307] Compound (II)-IM7 is obtained by substitution reaction of compound (II)-IM6;

[0308] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 5°C;

[0309] 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;

[0310] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably potassium tert-butoxide.

[0311] Step 8

[0312] Compound (II)-IM8 is obtained by reducing compound (II)-IM7;

[0313] In some implementations, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, or rhodium catalysts, with palladium catalysts being preferred.

[0314] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 20°C;

[0315] 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, with methanol being preferred.

[0316] Step Nine

[0317] Compound (II)-IM9 is obtained by substitution reaction of compound (II)-IM8;

[0318] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 20°C.

[0319] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably pyridine.

[0320] Step 10

[0321] Compound (II)-IM10 is obtained by hydrolysis of compound (II)-IM9.

[0322] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 60°C;

[0323] 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, with methanol being preferred.

[0324] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing the acidic conditions include hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, and methanesulfonic acid, preferably hydrochloric acid.

[0325] Step Eleven

[0326] Compound (II)-IM11 was obtained by cyclization of compound (II)-IM10 with (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranO[3,4-F]indoleazine-3,6,10(4H)-one.

[0327] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 140°C;

[0328] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, with toluene being preferred.

[0329] 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, and methanesulfonic acid, preferably p-toluenesulfonic acid.

[0330] Step Twelve

[0331] Compound (II)-IM12 is obtained by hydrolysis of compound (II)-IM11;

[0332] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 100°C;

[0333] 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, and methanesulfonic acid, preferably hydrochloric acid.

[0334] Step Thirteen

[0335] Compound (II)-IM13 is obtained by substitution reaction of compound (II)-IM12 and compound (II)-SM2.

[0336] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 50°C;

[0337] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Diox), dimethyl sulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0338] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base 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 (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably Na2CO3 or NaHCO3.

[0339] Step Fourteen

[0340] Compound (II)-IM14 is obtained by condensation reaction of compound (II)-IM13 and compound (II)-SM3;

[0341] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0342] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0343] 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.

[0344] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0345] Step Fifteen

[0346] Compound (II)-1 was obtained by acid hydrolysis of compound (II)-IM14;

[0347] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0348] 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 mixed solvents thereof, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0349] 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, and methanesulfonic acid, preferably hydrochloric acid.

[0350] The synthesis method of formula (II)-SM3 is as follows:

[0351] When PG is hour:

[0352]

[0353] Step 1

[0354] Compound (II)-SM3-3 is obtained by a substitution reaction between compound (II)-SM3-1 and compound (II)-SM3-2;

[0355] In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-25°C.

[0356] 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 mixtures thereof, preferably tetrahydrofuran.

[0357] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with K2CO3 being preferred.

[0358] Step Two

[0359] Compound (II)-SM3 is obtained by hydrogenation of compound (II)-SM3-3;

[0360] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0361] 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 mixtures thereof, with methanol being preferred.

[0362] In some implementations, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, or rhodium catalysts, with palladium catalysts being preferred.

[0363] When PG is At that time, R y’ and R z’ For hydrogen:

[0364]

[0365] Step 1

[0366] Compound (II)-SM3 is obtained by condensation reaction of compound (II)-SM3-4 and compound (II)-SM3-5;

[0367] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0368] 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 mixtures thereof, preferably N,N-dimethylformamide.

[0369] Alternatively, the compound of formula (II)-1 can be prepared by the following synthetic route:

[0370]

[0371] Step 1

[0372] Compound (II)-IM15 is obtained by condensation reaction of compound (II)-IM13 and compound (II)-SM4;

[0373] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0374] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0375] 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.

[0376] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0377] Step Two

[0378] Compound (II)-1 was obtained by removing the silicon protecting group from compound (II)-IM15;

[0379] Alternatively, the compound of formula (II) can be prepared by the following synthetic route:

[0380]

[0381] Step 1

[0382] Compound (II) is obtained by condensation reaction of compound (II)-IM13 and compound (II)-SM5;

[0383] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0384] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0385] 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.

[0386] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0387] The compound of formula (III)-1 in this invention can be synthesized by the following synthetic route.

[0388]

[0389] Among them, R x” R y” and R z” The meaning is as described above; LG is the leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy, and halogen, preferably trifluoromethanesulfonyloxy or chlorine; PG is the protecting group, selected from...

[0390] Step 1

[0391] Compound (III)-IM1 is obtained by substitution reaction of compound (III)-SM1.

[0392] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 50°C;

[0393] 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 n-heptane.

[0394] Step Two

[0395] Compound (III)-IM1 is reduced to give compound (III)-IM2;

[0396] 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, or rhodium catalysts, with platinum catalysts being preferred.

[0397] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 60°C;

[0398] 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.

[0399] Step 3

[0400] Compound (III)-IM2 is substituted to give compound (III)-IM3.

[0401] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 20°C;

[0402] 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;

[0403] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably triethylamine.

[0404] Step Four

[0405] Compound (III)-IM4 is obtained by coupling reaction of compound (III)-IM3;

[0406] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, 70°C, or 100°C, preferably 70°C;

[0407] 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 mixture of tetrahydrofuran and water.

[0408] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably N,N-diisopropylethylamine.

[0409] Step 5

[0410] Compound (III)-IM5 is obtained by reducing compound (III)-IM4.

[0411] 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, or rhodium catalysts, with platinum catalysts being preferred.

[0412] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 40°C;

[0413] 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, with tetrahydrofuran being preferred.

[0414] Step Six

[0415] Compound (III)-IM6 is obtained by cyclization reaction of compound (III)-IM5;

[0416] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 5°C;

[0417] 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 mixture of tetrahydrofuran and tert-butanol.

[0418] Step Seven

[0419] Compound (III)-IM7 is obtained by substitution reaction of compound (III)-IM6;

[0420] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 5°C;

[0421] 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;

[0422] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably potassium tert-butoxide.

[0423] Step 8

[0424] Compound (III)-IM7 is reduced to give compound (III)-IM8;

[0425] In some implementations, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, or rhodium catalysts, with palladium catalysts being preferred.

[0426] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 20°C;

[0427] 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, with methanol being preferred.

[0428] Step Nine

[0429] Compound (III)-IM9 is obtained by substitution reaction of compound (III)-IM8;

[0430] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 20°C.

[0431] In some embodiments, this step is carried out under alkaline conditions. The reagents providing the alkaline conditions include organic and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis(trimethylsilylaminolithium), and bis(trimethylsilylaminosodium); the inorganic bases include, but are not limited to, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium tert-butoxide, sodium hydride, sodium hydroxide, and potassium hydroxide, preferably pyridine.

[0432] Step 10

[0433] Compound (III)-IM10 is obtained by hydrolysis of compound (III)-IM9;

[0434] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 60°C;

[0435] 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, with methanol being preferred.

[0436] In some embodiments, the reaction is carried out under acidic conditions, and the reagents providing the acidic conditions include hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, and methanesulfonic acid, preferably hydrochloric acid.

[0437] Step Eleven

[0438] Compound (III)-IM11 was obtained by cyclization of compound (III)-IM10 with (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranO[3,4-F]indolinazine-3,6,10(4H)-one.

[0439] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 140°C;

[0440] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from toluene, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, with toluene being preferred.

[0441] 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, and methanesulfonic acid, preferably p-toluenesulfonic acid.

[0442] Step Twelve

[0443] Compound (III)-IM12 is obtained by hydrolysis of compound (III)-IM11;

[0444] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 100°C;

[0445] 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, and methanesulfonic acid, preferably hydrochloric acid.

[0446] Step Thirteen

[0447] Compound (III)-IM13 is obtained by substitution reaction of compound (III)-IM12 and compound (III)-SM2.

[0448] A substitution reaction occurs to give compound (II)-IM13.

[0449] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 50°C;

[0450] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0451] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base 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 (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably Na2CO3 or NaHCO3.

[0452] Step Fourteen

[0453] Compound (III)-IM14 is obtained by condensation reaction of compound (III)-IM13 and compound (III)-SM3;

[0454] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0455] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0456] 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.

[0457] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0458] Step Fifteen

[0459] Compound (III)-1 was obtained by acid hydrolysis of compound (III)-IM14;

[0460] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0461] 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 mixed solvents thereof, preferably a mixed solution of dichloromethane and methanol (volume ratio 2:1).

[0462] 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, and methanesulfonic acid, preferably hydrochloric acid.

[0463] The synthesis method of formula (III)-SM3 is as follows:

[0464] When PG is hour:

[0465]

[0466] Step 1:

[0467] Compound (III)-SM3-3 is obtained by a substitution reaction between compound (III)-SM3-1 and compound (II)-SM3-2;

[0468] In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-25°C.

[0469] 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 mixtures thereof, preferably tetrahydrofuran.

[0470] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with K2CO3 being preferred.

[0471] Step Two:

[0472] Compound (III)-SM3 is obtained by hydrogenation of compound (III)-SM3-3;

[0473] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0474] 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 mixtures thereof, with methanol being preferred.

[0475] In some implementations, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, or rhodium catalysts, with palladium catalysts being preferred.

[0476] When PG is At that time, R y’ and R z’ For hydrogen:

[0477]

[0478] Step 1:

[0479] Compound (III)-SM3 is obtained by condensation reaction of compound (III)-SM3-4 and compound (III)-SM3-5;

[0480] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0481] 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 mixtures thereof, preferably N,N-dimethylformamide.

[0482] Alternatively, the compound of formula (III)-1 can be prepared by the following synthetic route:

[0483]

[0484] Step 1:

[0485] Compound (III)-IM15 is obtained by condensation reaction of compound (III)-IM13 and compound (III)-SM4;

[0486] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0487] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0488] 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.

[0489] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0490] Step Two:

[0491] Compound (III)-1 was obtained by removing the silicon protecting group from compound (III)-IM15;

[0492] Alternatively, the compound of formula (III)-1 can be prepared by the following synthetic route:

[0493]

[0494] Step 1:

[0495] Compound (III)-1 is obtained by condensation reaction of compound (III)-IM13 and compound (III)-SM5;

[0496] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HATU being preferred;

[0497] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0498] 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.

[0499] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0500] The compound of formula (IV) in this invention can be synthesized and prepared by the following synthetic route:

[0501] Among them, R a R b R c R d and R e The meaning is as described above;

[0502] When q = 1

[0503]

[0504] Step 1

[0505] Compound (IV)-IM1 is obtained by nitration of compound (IV)-SM1.

[0506] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C.

[0507] Step Two

[0508] Compound (IV)-IM2 is obtained by hydrogenation of compound (IV)-IM1.

[0509] In some implementations, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalysts, platinum catalysts, or rhodium catalysts, with palladium catalysts being preferred.

[0510] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0511] 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.

[0512] Step 3

[0513] Compound (IV)-IM3 is obtained by acylation of compound (IV)-IM2.

[0514] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0515] Step Four

[0516] Compound (IV)-IM4 was obtained by reacting compound (IV)-IM3 with DMF-DMA;

[0517] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 120°C, preferably 120°C;

[0518] Step 5

[0519] Compound (IV)-IM5 is obtained by substitution reaction of compounds (IV)-IM4 and (IV)-SM2;

[0520] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 50°C;

[0521] 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, with ethanol being preferred;

[0522] Step Six

[0523] Compound (IV)-IM6 is obtained by reducing compound (IV)-IM5.

[0524] In some embodiments, this step is carried out in the presence of a suitable reducing agent, preferably sodium borohydride;

[0525] In some embodiments, this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 0-25°C;

[0526] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from tetrahydrofuran, glacial acetic acid, methanol, and mixtures thereof, with glacial acetic acid being preferred.

[0527] Step Seven

[0528] Compound (IV)-IM7 was obtained by protecting the amino group of compound (IV)-IM6 with Fmoc.

[0529] Step 8

[0530] Compound (IV)-IM8 was obtained by removing the acetyl protecting group from the amino group of compound (IV)-IM7.

[0531] Step Nine

[0532] Compound (IV)-IM9 was obtained by cyclization reaction of compound (IV)-IM8 under acidic conditions;

[0533] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 120°C, preferably 120°C;

[0534] 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, with toluene and xylene being preferred;

[0535] In some implementations, this step is carried out under acidic conditions;

[0536] Reagents that provide acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, and methanesulfonic acid, with p-toluenesulfonic acid being preferred.

[0537] Step 10

[0538] Compound (IV)-IM10 was obtained by removing the Fmoc protecting group from compound (IV)-IM9;

[0539] Step Eleven

[0540] Compound (IV) is obtained by condensation reaction of compound (IV)-IM10 and compound (IV)-SM4;

[0541] In some embodiments, this step is performed with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HBTU being preferred;

[0542] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0543] 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.

[0544] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0545] Or, when q = 0,

[0546]

[0547] LG is a leaving group, selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyloxy or iodine;

[0548] Step 1

[0549] The reduction reaction of compound (IV)-SM5 yields compound (IV)-IM11.

[0550] 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, or rhodium catalysts, with platinum catalysts being preferred.

[0551] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0552] 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.

[0553] Step Two

[0554] Compound (IV)-IM12 was obtained by Friedel-Crafts acylation of compound (IV)-IM11.

[0555] In some embodiments, this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C.

[0556] Step 3

[0557] Compound (IV)-IM13 was obtained by a ring-closing reaction of (IV)-IM12 under acidic conditions;

[0558] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 120°C, preferably 120°C;

[0559] 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, with toluene and xylene being preferred;

[0560] In some implementations, this step is carried out under acidic conditions;

[0561] Reagents that provide acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, and methanesulfonic acid, with p-toluenesulfonic acid being preferred.

[0562] Step Four

[0563] The (IV)-IM14 compound was obtained by a substitution reaction of (IV)-IM13;

[0564] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 25°C;

[0565] 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;

[0566] Step 5

[0567] Compound (IV)-IM15 is obtained by reducing compound (IV)-IM14.

[0568] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from palladium catalyst, platinum catalyst, rhodium catalyst, triphenylphosphine, triethyl phosphite, preferably triethyl phosphite;

[0569] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 50°C, 60°C, 80°C, or 100°C, preferably 80°C;

[0570] 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, toluene, and mixtures thereof, preferably a mixture of methanol and toluene.

[0571] Step Six

[0572] Compound (IV)-IM16 is obtained by substitution reaction of compound (IV)-IM15 and compound (IV)-SM6.

[0573] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 50°C;

[0574] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably acetonitrile.

[0575] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base 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 (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably Na2CO3 or NaHCO3.

[0576] Step Seven

[0577] Compound (IV) is obtained by condensation reaction of compound (IV)-IM16 and compound (IV)-SM4;

[0578] In some embodiments, this step is carried out with a suitable condensation reagent, which may be selected from HATU, HBTU, EDCI, DCC and HOBT, with HBTU being preferred;

[0579] In some embodiments, this step is performed at a suitable temperature, said temperature being 20°C, 25°C, 40°C, 50°C, 60°C, or 100°C, preferably 25°C;

[0580] 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.

[0581] In some embodiments, this step is carried out in a suitable alkali, which includes an organic alkali or an inorganic alkali. The organic alkali may be selected from DIPEA, TEA, t-BuOK and Py, and the inorganic alkali may be selected from K3PO4, NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, with DIPEA being preferred.

[0582] The compound of formula (V)-1 in this invention can use starting materials. The synthesis was carried out following the same synthetic route as in formula (III).

[0583] Beneficial effects of the invention

[0584] This invention provides camptothecin compounds represented by formulas (I)-(IV), pharmaceutical compositions thereof, preparation methods and uses. These compounds possess good antitumor activity, have the potential to overcome drug resistance, and can be used to treat diseases of abnormal cell proliferation, including but not limited to advanced solid tumors. Detailed Implementation

[0585] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of the application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its fundamental ideas and scope.

[0586] The abbreviations used in this invention have the following meanings:

[0587]

[0588]

[0589] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0590] Nuclear magnetic resonance (NMR) 1 The H NMR (H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; hexadeuterated dimethyl sulfoxide (DMSO-d6) was used; and tetramethylsilane (TMS) was used as the internal standard.

[0591] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0592] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0593] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0594] 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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypentan-3-yneamide 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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypentan-3-yneamide

[0595]

[0596] 2-Hydroxypentyl-3-acetic 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 mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high-performance liquid chromatography (under the following conditions) to give the title compounds 1-1-A 3.24 mg and 1-1-B 3.98 mg.

[0597] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0598] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0599]

[0600]

[0601] Retention time: 1-1-A: 10.8 min; 1-1-B: 11.1 min.

[0602] The structural characterization data for 1-1-A are as follows:

[0603] 1 H NMR (400MHz, DMSO-d6) δ8.58(d,J=8.4Hz,1H),7.78(d,J=11.2Hz,1H),7.31(s,1H),6.54(s,1H),6.17(d,J=6.0Hz,1H),5.58-5.47(m,1H), 5.42(s,2H),5.23(s,2H),4.73-4.64(m,1H),3.25-3.06(m,2H),2.39(s,3H),2.27-2.05(m,2H),1.96-1.77(m,5H),0.87(t,J=7.2Hz,3H).

[0604] ESI-MS (m / z): 532.2 [M+H] + .

[0605] The structural characterization data for 1-1-B are as follows:

[0606] 1H NMR (400MHz, DMSO-d6) 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).

[0607] ESI-MS (m / z): 532.2 [M+H] + .

[0608] Example 2: (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxy-3-enamide and (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxy-3-enamide

[0609]

[0610] Ethylene glycolic acid (9.61 mg, 94.07 μmol) was dissolved in DMF (2 mL), and HATU (44.70 mg, 117.58 μmol), SM1-1 (25.00 mg, 0.047 mmol), and DIPEA (24.30 mg, 188.13 μmol) were added. The mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-7-A (4.00 mg) and 1-7-B (1.38 mg).

[0611] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0612] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0613]

[0614]

[0615] Retention time: 1-7-A: 8.7 min; 1-7-B: 9.1 min.

[0616] The structural characterization data for 1-7-A are as follows:

[0617] 1 H NMR (400MHz, DMSO-d6) δ8.51(s,J=8.8Hz,1H),7.76(s,J=10.8Hz,1H),7.29(s,1H),6.52(s,1H),6.15–6.04(m,1H),5.58–5.49(m,1H),5.42(s,2 H),5.39(s,1H),5.24–5.01(m,3H),4.54(s,J=4.9Hz,1H),3.24–3.05(m, 2H),2.37(s,3H),2.16(s,2H),1.91–1.79(m,2H),0.87(t,J=7.2Hz,3H).

[0618] ESI-MS (m / z): 520.1 [M+H] + .

[0619] The structural characterization data for 1-7-B are as follows:

[0620] 1 H NMR(400MHz,DMSO-d6)δ8.48(s,1H),7.78(s,1H),7.30(s,1H),6.12–5.92( m,1H),5.54–5.47(m,1H),5.42(s,2H),5.37(dt,J=1.7Hz,1H),5.18(s,2H) ,5.16–5.14(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=7.2Hz,3H).

[0621] ESI-MS (m / z): 520.1 [M+H] + .

[0622] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0623]

[0624] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene

[0625] Compound 2-1-01 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL) at 25 °C, and concentrated sulfuric acid (25 mL) was added. The mixture was heated to 50 °C, and NBS (6.22 g, 34.97 mmol) was added in batches at 50 °C. The reaction was maintained at 50 °C for 2 hours. The reaction was detected by thin-layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction mixture, cooled to room temperature, was added dropwise to ice water and extracted with toluene. The organic phases were combined, washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography. The preparative solution was freeze-dried to give 4.88 g of the title compound.

[0626] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0627] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0628] 0 60 40 60 10 60 40 60 40 100 0 60

[0629] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline

[0630] Compound 2-1-02 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL) at 25 °C. Platinum carbon (2.00 g, 19.48 mmol, 5%) was added, and the mixture was purged with hydrogen. The reaction was carried out at 60 °C for 4 hours under hydrogen balloon protection, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to give 3.68 g of the crude title compound, which was used directly in the next step of the reaction without further purification.

[0631] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide

[0632] Compound 2-1-03 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL) at 20 °C. Triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the reaction was maintained at 20 °C for 20 hours. 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 then slurried with a 1:5 mixture of ethyl acetate and petroleum ether to obtain 2.86 g of the title compound.

[0633] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid

[0634] Compound 2-1-04 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL) at 20 °C. Vinylacetic acid (708.31 mg, 8.23 ​​mmol), DIPEA (1.95 g, 15.08 mmol), and tris(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction mixture was purged with nitrogen and heated to 70 °C for 5 hours. The reaction was monitored by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pH of the reaction mixture was adjusted to 8 with 1 N sodium hydroxide solution, and the mixture was extracted with ethyl acetate. The remaining aqueous phase was adjusted to pH 3 with 1 N hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 0.82 g of the title compound, which was directly used in the next reaction.

[0635] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid

[0636] Compound 2-1-05 (2.60 g, 9.71 mmol) was dissolved in 50 mL of THF at 20 °C. Pd / C (0.52 g, 10%) was added, and the system was purged with hydrogen and reacted at 40 °C for 2 hours under hydrogen balloon protection. The reaction was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43 g of the title compound, which was used directly in the next reaction without further purification.

[0637] Step Six: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0638] Compound 2-1-06 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5 °C, and trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise. The reaction was maintained 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 solution. Ethyl acetate was added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-20%) to give 1.53 g of the title compound.

[0639] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0640] 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 maintained at 5°C for 1 hour, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. The pH of the reaction solution was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure, and the concentrate was slurried with methyl tert-butyl ether to give 1.20 g of the title compound.

[0641] Step 8: N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0642] Compound 2-1-08 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL) at 20 °C. Pd / C (0.15 g, 10%) was added, and the system was purged with hydrogen and reacted at 5 °C for 2 hours under hydrogen balloon protection. 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 of the reaction without further purification.

[0643] Step Nine: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide

[0644] Compound 2-1-09 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL) at 20 °C, and acetic anhydride (2 mL) was added. The reaction was maintained at 20 °C for 2 hours, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was added to water, extracted with ethyl acetate, and the organic phases were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-30%) to give 0.22 g of the title compound.

[0645] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide

[0646] Compound 2-1-10 (0.45 g, 1.46 mmol) was dissolved in methanol (16 mL) at 20 °C, and 2N hydrochloric acid (16 mL) was added. The mixture was heated to 60 °C and reacted for 2 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. The pH of the cooled reaction solution was adjusted to 8 by adding saturated sodium bicarbonate 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 give 0.23 g of the title compound, which was used directly in the next reaction without further purification.

[0647] Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide

[0648] Compound 2-1-11 (0.23 g, 0.78 mmol) was dissolved in toluene (10 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (0.23 g, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added. The mixture was heated to 140 °C and reacted 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 rapid silica gel column chromatography (methanol:dichloromethane = 0-10%) to give 0.15 g of the title compound.

[0649] Step Twelve: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione

[0650] 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 for 5 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was filtered, and the filtrate was purified by preparative HPLC. The preparative solution was freeze-dried to obtain 12.00 mg of trifluoroacetate of the title compound 2-23.

[0651] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0652] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0653] 0 5 95 28 2 5 95 28 18 50 50 28

[0654] The structural characterization data are as follows:

[0655] ESI-MS (m / z): 452.1 [M+H] + .

[0656] Step Thirteen: 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)acetamide

[0657] At 25°C, the trifluoroacetate of compound 2-23 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of 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). The reaction was maintained at 25°C for 0.5 hours, 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 separate two isomers. Based on their Rf values, the two isomers were named 2-1-13-A (15.00 mg, Rf value 0.3) and 2-1-13-B (12.00 mg, Rf value 0.35).

[0658] Step Fourteen: 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0659] At 25°C, 2-1-13-A (15.00 mg) and 2-1-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two separate reaction flasks. A mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and glacial acetic acid (v / v = 13 / 1) (50 μL) was then 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 (HPLC-MS). After the reaction was complete, the reaction solutions were purified by preparative HPLC, and the prepared solutions were lyophilized to obtain the title compounds 2-1-A (6.94 mg) and 2-1-B (4.00 mg), respectively.

[0660] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0661] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0662] 0 20 80 28 3 20 80 28 18 90 10 28

[0663] The structural characterization data for 2-1-A are as follows:

[0664] 1 H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.65-5.36(m,4H),5.21(q,J=19.0H z,2H),3.95(d,J=5.7Hz,2H),3.26-3.11(m,2H),2.53(s,3H),2.30-2.08(m,2H),1.94-1.79(m,2H),0.87(t,J=7.3Hz,3H).

[0665] ESI-MS (m / z): 510.1 [M+H] + .

[0666] The structural characterization data for 2-1-B are as follows:

[0667] 1 H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.64-5.35(m,4H),5.19(q,J=19.0H z,2H),3.97(d,J=5.2Hz,2H),3.27-3.10(m,2H),2.51(s,3H),2.27-2.10(m,2H),1.93-1.80(m,2H),0.88(t,J=7.3Hz,3H).

[0668] ESI-MS (m / z): 510.1 [M+H] + .

[0669] 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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[0670]

[0671] Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)propylamine Synthesis of (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]indolazino[1,2-b]quinoline-1-yl)propylamine

[0672] At 25°C, 30.00 mg (61.43 μmol) of 2-23 hydrochloride was dissolved in 1 mL of N,N-dimethylformamide, followed by the addition of (S)-2-((tert-butyldiphenylsilyl)oxy)propionic acid (24.21 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol), and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol). 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 separate two isomers. Based on their Rf values, the two isomers were named 2-7-01-A (6.00 mg, Rf value 0.35) and 2-7-01-B (6.00 mg, Rf value 0.40).

[0673] 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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine and (2S)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[0674] At 25°C, 2-7-01-A (6.00 mg, 7.87 μmol) and 2-7-01-B (6.00 mg, 7.87 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two separate reaction flasks. A mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and 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 (HPLC-MS). After the reaction was complete, the reaction solutions were purified by preparative HPLC, and the preparative solutions were lyophilized to obtain the title compounds 2-7-A (2.50 mg) and 2-7-B (3.00 mg), respectively.

[0675] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0676] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0677] 0 15 85 28 16 90 10 28

[0678] The structural characterization data for 2-7-A are as follows:

[0679] 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=8.8Hz,1H),8.15(s,1H),7.31(s,1H),6.55(s,1H),5.56-5.47(m,2H),5.42(s,2H),5.26-5.11(m,2H),4.1 7-4.06(m,1H),3.27-3.10(m,2H),2.52(s,3H),2.27-2.08(m,2H),1.8 6(tt,J=14.1,7.3Hz,2H), 1.30(d,J=6.7Hz,3H), 0.87(t,J=7.3Hz,3H).

[0680] ESI-MS (m / z): 524.2 [M+H] + .

[0681] The structural characterization data for 2-7-B are as follows:

[0682] 1H NMR (400MHz, DMSO-d6) δ8.48(d,J=9.1Hz,1H),8.12(s,1H),7.30(s,1H),6.54(s,1H),5.67(d,J=4.8Hz,1H),5. 55(dd,J=14.6,7.3Hz,1H),5.43(s,2H),5.24(d,J=19.0Hz,1H),5.03(d,J=19.0Hz,1H),4.21-4.08(m,1H),3.28 -3.08(m,2H),2.51(s,3H),2.16(d,J=6.2Hz,2H),1.94-1.82(m,2H),1.42(d,J=6.8Hz,3H),0.88(t,J=7.3Hz,3H).ESI-MS(m / z):524.2[M+H] + .

[0683] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetamide Amides 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[0684]

[0685] Step 1: (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropylethyl Amides 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]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropylacetamide and (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropylacetamide and Synthesis of (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropylacetamide

[0686] At 25°C, 30.00 mg (61.43 μmol) of 2-23 hydrochloride was dissolved in 1 mL of N,N-dimethylformamide, followed by the sequential addition of 2-((tert-butyldiphenylsilyl)oxy)-2-cyclopropylacetic acid (26.13 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol), and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol). 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 separate two isomers. Based on the Rf value, the two isomers were named 2-12-01-A (8.00 mg, Rf value 0.35) and 2-12-01-B (10.00 mg, Rf value 0.40).

[0687] Step 2: (2S)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetyl) Synthesis of amines 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[0688] At 25°C, 2-12-01-A (8.00 mg, 10.15 μmol) and 2-12-01-B (10.00 mg, 12.68 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two separate reaction flasks. Then, a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was maintained at 25°C for 0.5 hours, 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 (HPLC). The reaction using 2-12-01-A as the starting material yielded two isomers, and the preparative solutions were lyophilized to obtain compounds 2-12-A (0.77 mg) and 2-12-B (1.03 mg), respectively. The reaction using 2-12-01-B as the starting material yielded two isomers, and the preparative solutions were lyophilized to obtain compounds 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:

[0689] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0690] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0691]

[0692]

[0693] Peak retention times were: 2-12-A: 10.0-11.0 min, 2-12-B: 11.0-12.5 min.

[0694] The purification conditions for 2-12-C / 2-12-D are as follows:

[0695] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0696] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0697] 0 20 80 28 2 20 80 28 18 80 20 28

[0698] Peak retention times were: 2-12-C: 10.6-11.4 min, 2-12-D: 11.4-12.5 min.

[0699] The structural characterization data for 2-12-A are as follows:

[0700] 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=8.9Hz,1H),8.15(s,1H),7.30(s,1H),6.54(s,1H),5. 63-5.53(m,1H),5.51(d,J=5.1Hz,1H),5.42(s,2H),5.28(d,J=19.2Hz,1H),5.16(d,J=19 .1Hz,1H),3.60(t,J=5.6Hz,1H),3.28-3.11(m,2H),2.52(s,3H),2.22-2.10(m,2H),1.86 (tt,J=14.1,7.3Hz,2H),1.23(d,J=4.9Hz,1H),0.87(t,J=7.2Hz,3H),0.56-0.36(m,4H).

[0701] ESI-MS (m / z): 550.2 [M+H] + .

[0702] The structural characterization data for 2-12-B are as follows:

[0703] 1H NMR(400MHz,DMSO-d6)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).

[0704] ESI-MS (m / z): 550.2 [M+H] + .

[0705] The structural characterization data for 2-12-C are as follows:

[0706] 1 H NMR (400MHz, DMSO-d6) 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).

[0707] ESI-MS (m / z): 550.1 [M+H] + .

[0708] The 2-12-D structure characterization data are as follows:

[0709] 1H NMR(400MHz,DMSO-d6)δ8.38(d,J=8.7Hz,1H),8.16(s,1H),7.31(s,1H),6.54(s,1H),5.59 -5.49(m,1H),5.43(d,J=5.3Hz,1H),5.43(s,2H),5.26(d,J=19.1Hz,1H),5.17(d,J=19.0Hz ,1H),3.64(t,J=5.8Hz,1H),3.17(dd,J=15.6,8.3Hz,2H),2.27-2.07(m,2H),1.94-1.79(m, 2H),1.19-1.06(m,1H),0.87(t,J=7.3Hz,3H),0.49-0.28(m,4H).ESI-MS(m / z):550.1[M+H] + .

[0710] 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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxy-2-methylpropylamine and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxy-2-methylpropylamine

[0711]

[0712] At 25°C, 30.00 mg (61.43 μmol) of 2-23 hydrochloride was dissolved in 1 mL of N,N-dimethylformamide. Then, 16.10 mg (73.72 μmol) of 2-((tert-butyldimethylsilyl)oxy)-2-methylpropionic acid, 35.01 mg (92.14 μmol) of HATU, and 23.82 mg (184.29 μmol) of N,N-diisopropylethylamine were added sequentially. The reaction was maintained at 25°C for 1 hour, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry (HPLC-MS). After the reaction was complete, the reaction solution was concentrated, and the crude product was purified by preparative HPLC to obtain two isomers. The preparative solutions were lyophilized, and the two isomers were named 2-17-A (2.65 mg) and 2-17-B (2.69 mg) based on their peak retention times.

[0713] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0714] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0715] 0 20 80 28 2 20 80 28 18 80 20 28

[0716] The peak retention times were: 2-17-A: 9.5-10.2 min and 2-17-B: 10.4-10.6 min.

[0717] The structural characterization data for 2-17-A are as follows:

[0718] 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=9.1Hz,1H),8.13(s,1H),7.30(s,1H),6 .54(s,1H),5.55-5.45(m,2H),5.42(s,2H),5.28(d,J=19.0Hz,1H),5.05( d,J=19.0Hz,1H),3.27-3.11(m,2H),2.51(s,1H),2.24-2.10(m,2H),1.86 (tt,J=14.0,7.2Hz,2H),1.46(s,3H),1.35(s,3H),0.87(t,J=7.3Hz,3H).

[0719] MS m / z (ESI): 538.2 [M+H] + .

[0720] The structural characterization data for 2-17-B are as follows:

[0721] 1 H NMR(400MHz,DMSO-d6)δ8.40(d,J=9.2Hz,1H),8.13(s,1H),7.30(s,1H),6 .53(s,1H),5.60-5.46(m,2H),5.42(s,2H),5.29(d,J=19.0Hz,1H),5.02( d,J=19.0Hz,1H),3.28-3.08(m,2H),2.50(s,3H),2.22-2.10(m,2H),1.87 (tt,J=14.2,7.2Hz,2H),1.47(s,3H),1.35(s,3H),0.88(t,J=7.3Hz,3H).

[0722] MS m / z (ESI): 538.2 [M+H] + .

[0723] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0724]

[0725] Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide Synthesis of 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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide

[0726] At 25°C, 30.00 mg (61.43 μmol) of 2-23 hydrochloride was dissolved in 1 mL of N,N-dimethylformamide, followed by the sequential addition of 2-(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). 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 separate two isomers. Based on their Rf values, the two isomers were named 2-20-01-A (4.00 mg, Rf value 0.30) and 2-20-01-B (4.00 mg, Rf value 0.35).

[0727] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0728] At 25°C, 2-20-01-A (4.00 mg, 5.17 μmol) and 2-20-01-B (4.00 mg, 5.17 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) in two separate reaction flasks. A mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and 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 (HPLC-MS). After the reaction was complete, the reaction solutions were purified by preparative HPLC, and the preparative solutions were lyophilized to obtain the title compounds 2-20-A (0.71 mg) and 2-20-B (1.05 mg), respectively.

[0729] The purification conditions for 2-20-A are as follows:

[0730] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0731] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0732] 0 15 85 28 16 90 10 28

[0733] The purification conditions for 2-20-B are as follows:

[0734] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0735] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0736] 0 20 80 28 2 20 20 28 18 80 20 28

[0737] The structural characterization data for 2-20-A are as follows:

[0738] 1H NMR(400MHz,DMSO-d6)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).

[0739] ESI-MS (m / z): 536.2 [M+H] + .

[0740] The structural characterization data for 2-20-B are as follows:

[0741] 1 H NMR(400MHz,DMSO-d6)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).

[0742] ESI-MS (m / z): 536.2 [M+H] + .

[0743] 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]indolazin[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]indolazin[1,2-b]quinoline-10,13-dione

[0744]

[0745] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline

[0746] Compound 3-1-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. After the addition was complete, the reaction was allowed to proceed at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified by fast silica gel column chromatography (ethyl acetate: petroleum ether = 0-25%) to give 0.95 g of the title compound.

[0747] The structural characterization data are as follows:

[0748] 1 H NMR (400MHz, DMSO-d6) δ6.77 (dd, J=2.5, 1.4Hz, 1H), 6.51 (dd, J=11.7, 2.5Hz, 1H), 5.84 (s, 2H).

[0749] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide

[0750] Compound 3-1-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL). Acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection. After the addition was complete, the mixture was heated to 50 °C and reacted for 15 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with methanol (5 mL) and then evaporated directly to dryness under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0751] The structural characterization data are as follows:

[0752] ESI-MS (m / z): 265.9 [M+H] + .

[0753] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid

[0754] 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-methylphenyl)phosphine (114.21 mg, 375.24 μmol), and palladium acetate (42.12 mg, 187.62 μmol) were added. After the addition was complete, the reaction system was purged three times with nitrogen, and the mixture was heated to 100 °C for 16 hours under a nitrogen atmosphere. The reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). After cooling the reaction solution to room temperature, 60 mL of 1 N sodium hydroxide aqueous solution and 50 mL of ethyl acetate were added, and the mixture was shaken to separate the layers. After separating the lower aqueous phase, the pH was adjusted to about 3 with 4 mol / L hydrochloric acid aqueous 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 under reduced pressure to obtain 1.00 g of crude product of the title compound.

[0755] The structural characterization data are as follows:

[0756] ESI-MS (m / z): 272.0 [M+H] + .

[0757] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid

[0758] The crude product of compound 3-1-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and then 10% palladium on carbon (0.10 g) was added. After the addition was complete, the reaction system was purged three times with hydrogen balloons, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

[0759] The structural characterization data are as follows:

[0760] ESI-MS (m / z): 274.0 [M+H] + .

[0761] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0762] The crude 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 the addition was complete, the reaction was maintained at 5 °C for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography to give 0.43 g of the title compound.

[0763] The structural characterization data are as follows:

[0764] ESI-MS (m / z): 256.1 [M+H] + .

[0765] Step Six: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0766] 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 flask. After 10 minutes, isoamyl nitrite (315.24 mg, 2.69 mmol) was added. After the addition was complete, the reaction was maintained at 5°C for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give 455.00 mg of the crude product of the title compound.

[0767] The structural characterization data are as follows:

[0768] ESI-MS (m / z): 285.0 [M+H] + .

[0769] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0770] The crude product of compound 3-1-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), followed by the addition of 3 mol / L hydrochloric acid aqueous solution (1 mL) and 10% palladium on carbon (40.00 mg). After the addition was complete, the reaction system was purged three times with a hydrogen balloon, 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 crude hydrochloride of the title compound.

[0771] The structural characterization data are as follows:

[0772] ESI-MS (m / z): 271.0 [M+H] + .

[0773] Step 8: Synthesis of (9H-fluorene-9-yl)methyl(8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[0774] The crude hydrochloride of compound 3-1-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), followed by the addition of sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL), and 9-fluorenemethyl-N-succinimide carbonate (481.81 mg, 1.43 mmol). After addition, the mixture was stirred at room temperature for 2 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was poured into water and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reversed-phase column chromatography (acetonitrile: 0.05% formic acid water = 20%-100%) to give 301.00 mg of the title compound.

[0775] The structural characterization data are as follows:

[0776] ESI-MS (m / z): 493.2 [M+H] + .

[0777] Step Nine: Synthesis of (9H-fluorene-9-yl)methyl (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[0778] Compound 3-1-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL), and 12 mol / L concentrated hydrochloric acid (1 mL) was added. After the addition was complete, the mixture was heated to 60 °C and reacted for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%) to give 198.00 mg of the title compound.

[0779] The structural characterization data are as follows:

[0780] ESI-MS (m / z): 451.1 [M+H] + .

[0781] Step 10: Synthesis of (9H-fluorene-9-yl)methyl((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)carbamate

[0782] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-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), followed by p-toluenesulfonic acid (75.53 mg, 439.13 μmol). After the addition was complete, the mixture was heated to 140 °C and reacted 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 rapid silica gel column chromatography (methanol:dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0783] The structural characterization data are as follows:

[0784] ESI-MS (m / z): 678.1 [M+H] + .

[0785] Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[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]indolazino[1,2-b]quinoline-10,13-dione

[0786] Compound 3-1-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), followed by the addition of diethylamine (108.49 mg, 1.48 mmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). After distilling off the ethylenediamine from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution. The reaction solution was then directly purified by preparative high-performance liquid chromatography (PPLC) to obtain the title compounds 3-1-A (44.00 mg) and 3-1-B (43.00 mg).

[0787] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0788] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0789] 0 10 90 28 3 10 90 28 18 70 30 28

[0790] 3-1-A (6 min LCMS peak early, retention time: 1.276 min)

[0791] The structural characterization data are as follows:

[0792] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H),5.62(d,J=19.3Hz,1H),5.44(s,2H),5.38(d,J=19.3Hz,1H),4.43 -4.38(m,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=7.3Hz,3H).ESI-MS(m / z):456.1[M+H] + .

[0793] The structural characterization data of 3-1-B (6 min LCMS peak late, retention time: 1.300 min) are as follows:

[0794] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=10.3Hz,1H),7.32(s,1H),5.61(d,J=19.4Hz,1H),5.44(s,2H),5.32(d,J=19 .4Hz,1H),4.44-4.36(m,1H),3.33-3.25(m,1H),3.22-3.11(m,1H),2.23-2.13(m,1H),2.11-2.03(m,1H),1.96 -1.82(m,2H),0.89(t,J=7.3Hz,3H).

[0795] ESI-MS (m / z): 456.1 [M+H] + .

[0796] 6-minute LCMS conditions:

[0797] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

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

[0799] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0800] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0801]

[0802] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxo-5,8,11,14,17-pentazaoctadecane-22-acetylic acid

[0803] Compound 3-4-01 (30.00 mg, 70.00 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonic acid)pyrimidin-5-yl)hexyl-5-acetylamide (28.00 mg, 77.00 μmol) was added. The reaction was carried out at room temperature for 1 hour, and 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 preparative solution was freeze-dried to give the title compound 3-4-03 (20.00 mg).

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

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

[0806] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0807] The structural characterization data are as follows:

[0808] ESI-MS (m / z): 691.0 [M+18] + .

[0809] Step 2: N-((S)-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazo[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-amide Synthesis of 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]indolazo[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide

[0810] Compound 3-1-A (36.00 mg, 79.70 μmol) and compound 3-4-03 (64.43 mg, 95.64 μmol) of the single configuration were dissolved in N,N-dimethylformamide (2 mL), followed by the addition of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 1 hour, and the reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was directly purified by HPLC to obtain the title compound 3-4-04-A (51.00 mg) of the single configuration.

[0811] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0812] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0813] 0 30 70 28 3 30 70 28 18 90 10 28

[0814] The structural characterization data are as follows:

[0815] ESI-MS (m / z): 1111.0 [M+H] + .

[0816] Compound 3-1-B (36.00 mg, 79.70 μmol) in its single configuration and compound 3-4-03 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). Then, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After the addition was complete, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was directly purified by HPLC to obtain the title compound 3-4-04-B (52.00 mg) in its single configuration.

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

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

[0819] 0 30 70 28 3 30 70 28 18 90 10 28

[0820] The structural characterization data are as follows:

[0821] ESI-MS (m / z): 1111.0 [M+H] + .

[0822] 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]indolazino[1,2-b]quinoline-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]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[0823] Compound 3-4-04-A (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). Then, 4 mol / L ethyl acetate hydrochloride (1 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. 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. The crude product was purified by high performance liquid chromatography to obtain the title compound 3-4-A (4.75 mg) with a single configuration.

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

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

[0826] 0 15 85 28 3 15 85 28 18 90 10 28

[0827] The structural characterization data are as follows:

[0828] 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=8.9Hz,1H),8.05(d,J=10.3Hz,1H),7.33(s,1H),6.55(s,1H),5.67-5.60(m,1H),5.49(t,J=5.8Hz, 1H),5.43(s,2H),5.21(s,2H),3.96(d,J=5.8Hz,2H),3.32-3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

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

[0830] 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, 4 mol / L ethyl acetate hydrochloride (1 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. 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. The crude product was purified by high performance liquid chromatography to obtain the title compound 3-4-B (8.24 mg) in a single configuration.

[0831] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0832] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0833] 0 15 85 28 3 15 85 28 18 90 10 28

[0834] The structural characterization data are as follows:

[0835] 1 H NMR (400MHz, DMSO-d6) δ8.52(d,J=9.0Hz,1H),8.05(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.68-5.58(m,1H),5.53(t,J=5.8Hz,1H),5.43 (d,J=2.9Hz,2H),5.20(d,J=7.3Hz,2H),3.97(d,J=5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J=7.3Hz,3H).

[0836] ESI-MS (m / z): 514.0 [M+H] + .

[0837] Example 10 (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indolazido[1,2-b]quinoline-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0838]

[0839] Step 1: Synthesis of 1-(4-fluoro-3-methylphenyl)-3-(isopropylamino)propane-1-one

[0840] Compound 4-12-01 (500.00 mg, 3.29 mmol), formaldehyde aqueous solution (2.5 mL, 37%), and isopropylamine (388.46 mg, 6.57 mmol) were added to isopropanol (5 mL) at 20 °C. Concentrated hydrochloric acid (2.5 mL) was added dropwise at 0 °C. The reaction mixture was stirred at 100 °C for 16 hours, and the reaction was detected 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 preparative solution was freeze-dried to obtain 200.00 mg of the title compound.

[0841] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0842] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0843] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0844] The structural characterization data are as follows:

[0845] ESI-MS (m / z): 224.1 [M+H] + .

[0846] Step 2: Synthesis of 1-(4-fluoro-5-methyl-2-nitrophenyl)-3-(isopropylamino)propane-1-one

[0847] Compound 4-12-02 (100.00 mg, 0.49 mmol) was added to concentrated sulfuric acid (0.5 mL) at 0 °C, along with potassium nitrate (54.34 mg, 0.54 mmol). The reaction mixture was maintained at 0 °C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction mixture was then poured into ice water and purified by reverse-phase column chromatography (acetonitrile: 0.05% formic acid water = 0-30%) to give 90.00 mg of the title compound.

[0848] The structural characterization data are as follows:

[0849] ESI-MS (m / z): 269.0 [M+H] + .

[0850] Step 3: Synthesis of 1-(2-amino-4-fluoro-5-methylphenyl)-3-(isopropylamino)propane-1-one

[0851] Compound 4-12-03 (200.00 mg, 0.75 mmol) was dissolved in methanol (20.0 mL) at 25 °C, and 10% palladium on carbon (10.00 mg) was added. The reaction solution was purged with hydrogen, and the reaction was maintained at 20 °C for 16 hours in a hydrogen atmosphere. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered and concentrated under reduced pressure to obtain 183.00 mg of the title compound.

[0852] The structural characterization data are as follows:

[0853] ESI-MS (m / z): 239.1 [M+H] + .

[0854] 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]indolazino[1,2-b]quinoline-3,14(4H)-dione

[0855] Compound 4-12-04 (50.00 mg, 0.21 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (55.23 mg, 0.21 mmol) were added to toluene (3 mL) at 25 °C, followed by the addition of p-toluenesulfonic acid (3.61 mg, 0.02 mmol). The reaction mixture was reacted at 130 °C for 4 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction mixture was concentrated under reduced pressure, and the crude product was purified by preparative HPLC. The preparative solution was lyophilized to obtain 2.00 mg of trifluoroacetate of the title compound.

[0856] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0857] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0858] 0.00 8 92 28 2.00 8 92 28 18.00 60 40 28

[0859] The structural characterization data are as follows:

[0860] 1 H NMR (400MHz, DMSO-d6) δ8.58(s,2H),8.22(d,J=8.1Hz,1H),7.97(d,J=10.7Hz,1H),7.35(s,1H),6.59(s,1H),5.47(s,2H),5. 41(s,2H),3.58-3.45(m,3H),3.31-3.23(m,2H),2.56(s,3H),1.98-1.80(m,2H),1.26(d,J=6.3Hz,6H),0.89(t,J=7.3Hz,3H).

[0861] ESI-MS (m / z): 466.2 [M+H] +

[0862] 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]indolazino[1,2-b]quinoline-11-yl)ethyl)-N-isopropylacetamide

[0863] Compound 4-12-05 (22.00 mg, 47.26 μmol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (16.35 mg, 51.99 μmol) were dissolved in N,N-dimethylformamide (1 mL), followed by the addition of HATU (21.55 mg, 56.71 μmol) and N,N-diisopropylethylamine (18.32 mg, 141.78 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by a C18 reversed-phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to give 18.00 mg of the title compound.

[0864] The structural characterization data are as follows:

[0865] ESI-MS (m / z): 762.3 [M+H] + .

[0866] Step Six: Synthesis of (S)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indolazido[1,2-b]quinoline-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0867] Compound 4-12-06 (18.00 mg, 23.62 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the addition of potassium fluoride (6.86 mg, 118.12 μmol). After the addition was complete, the mixture was heated to 50 °C and reacted for 1 hour. The reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The reaction solution was directly purified by HPLC to give 1.53 mg of the title compound.

[0868] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0869] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0870] 0.00 15 85 28 18.00 90 10 28

[0871] The structural characterization data are as follows:

[0872] 1H NMR (400MHz, DMSO-d6) δ8.53(d,J=8.2Hz,1H),7.91(d,J=10.8Hz,1H),7.32(s,1H),6.55(s,1H),5.44(d,J=13.8Hz,4H),4.72(t,J=5.5Hz,1H), 4.21(d,J=5.5Hz,2H),3.99-3.90(m,1H),3.54-3.38(m,4H),2.54(s,3H ),1.92-1.83(m,2H),1.17(dd,J=6.6,3.1Hz,6H),0.87(t,J=7.3Hz,3H).

[0873] ESI-MS (m / z): 524.2 [M+H] + .

[0874] Example 11 (S)-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxy-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indolazin[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropaneformamide

[0875]

[0876] The starting materials (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyran[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H,12H)-dione (4-10-01, prepared according to the synthesis method of patent WO2020219287, 30.00 mg, 67.00 μmol) and 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 with stirring, and the reaction was carried out at room temperature for 4 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and 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 purified by preparative high-performance liquid chromatography to obtain 1.20 mg of solid.

[0877] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0878] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0879]

[0880]

[0881] The structural characterization data are as follows:

[0882] 1 H NMR(400MHz, DMSO-d6)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).

[0883] ESI-MS (m / z): 494.1 [M+1] + .

[0884] 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]indolazin[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]indolazin[1,2-b]quinoline-10,13-dione

[0885]

[0886] Step 1: Synthesis of (E)-4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid

[0887] Weigh out N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (2.00 g, 8.13 mmol) and 2-methyl-3-butenoic acid (976.44 mg, 9.75 mmol) and dissolve them in a mixed solvent of 1,4-dioxane (15 mL) and water (5 mL). Then add tris(o-tolyl)phosphine (247.37 mg, 812.76 μmol), palladium acetate (91.24 mg, 406.38 μmol), and N,N-diisopropylethylamine (2.31 g, 17.88 mmol). After the addition is complete, purge the reaction system with nitrogen three times and heat to 80 °C for 3 hours under a nitrogen atmosphere. Detect the reaction by high performance liquid chromatography-mass spectrometry. After cooling the reaction solution to room temperature, add 1 mol / L sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) and shake to separate the layers. After separating the lower aqueous phase, the pH was adjusted to about 3 with 4 mol / L hydrochloric acid aqueous 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 concentrated under reduced pressure to obtain 1.90 g of the title compound.

[0888] The structural characterization data are as follows:

[0889] ESI-MS (m / z): 266.1 [M+H] + .

[0890] Step 2: Synthesis of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutyric acid

[0891] (E)-4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid (1.90 g, 7.16 mmol) was dissolved in methanol (40 mL). Under nitrogen protection, 10% palladium on carbon (0.15 g) was added. The reaction system was then purged three times with hydrogen balloons, and the reaction was carried out for 2 hours 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 1.51 g of the title compound.

[0892] The structural characterization data are as follows:

[0893] ESI-MS (m / z): 268.1 [M+H] + .

[0894] Step 3: Synthesis of N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0895] 1.50 g (5.61 mmol) of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutyric acid was dissolved in 20 mL of trifluoroacetic acid. After cooling to 5 °C, trifluoroacetic anhydride (2.36 g, 11.22 mmol) was added dropwise. After the addition was complete, the reaction was maintained at 5 °C for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into a saturated sodium bicarbonate aqueous solution, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-30%) to obtain 1.05 g of the title compound.

[0896] The structural characterization data are as follows:

[0897] ESI-MS (m / z): 250.1 [M+H] + .

[0898] Step 4: Synthesis of N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0899] N-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (0.55 g, 2.21 mmol) was dissolved in acetic acid (8 mL), followed by the addition of bromine (387.85 mg, 2.43 mmol). After the addition was complete, the mixture was heated to 50 °C and reacted for 15 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly evaporated to dryness under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-30%) to obtain 461.00 mg of the title compound.

[0900] The structural characterization data are as follows:

[0901] ESI-MS (m / z): 328.0 [M+H] + .

[0902] Step 5: Synthesis of N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0903] Weigh 460.00 mg (1.40 mmol) of N-(7-bromo-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-1-yl)acetamide and dissolve it in 10 mL of N,N-dimethylformamide. Then add sodium azide (273.37 mg, 4.21 mmol). After the addition is complete, react at room temperature for 1 hour. Detect the reaction by high performance liquid chromatography-mass spectrometry. Slowly pour the reaction solution into water and 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 to dryness under reduced pressure to obtain the crude product. Purify the crude product by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%) to obtain 347.00 mg of the title compound.

[0904] The structural characterization data are as follows:

[0905] ESI-MS (m / z): 291.1 [M+H] + .

[0906] Step Six: Synthesis of N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0907] 347.00 mg (1.20 mmol) of N-(7-azido-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-1-yl)acetamide was weighed and dissolved in 10 mL of tetrahydrofuran. 30.00 mg of 10% palladium on carbon was added under nitrogen protection. The reaction system was then purged three times with hydrogen balloons, and the reaction was carried out for 2 hours 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 a crude product. The crude product was purified by a C18 reversed-phase column (acetonitrile: 0.05% formic acid aqueous solution = 0%-30%) to obtain 205.00 mg of the title compound.

[0908] The structural characterization data are as follows:

[0909] ESI-MS (m / z): 265.1 [M+H] + .

[0910] Step 7: Synthesis of (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[0911] Weigh N-(7-amino-3-fluoro-4,7-dimethyl-8-oxo5,6,7,8-tetrahydronaphth-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-succinimide carbonate (650.55 mg, 1.14 mmol). After the addition is complete, stir the mixture at room temperature for 2 hours and detect the reaction 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, filtered, and the filtrate was evaporated under reduced pressure to obtain the crude product. The crude product was purified by a C18 reversed-phase column (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) to obtain 301.00 mg of the title compound.

[0912] The structural characterization data are as follows:

[0913] ESI-MS (m / z): 487.0 [M+H] + .

[0914] Step 8: Synthesis of (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[0915] (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate (101.00 mg, 207.59 μmol) was dissolved in 1,4-dioxane (5 mL), followed by the addition of 3 mol / L hydrochloric acid aqueous solution (5 mL). After the addition was complete, the mixture was heated to 50 °C and reacted for 15 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into a saturated sodium bicarbonate aqueous 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 the crude product. The crude product was purified by rapid silica gel column chromatography (methanol:dichloromethane = 0%-5%) to give 71.00 mg of the title compound.

[0916] The structural characterization data are as follows:

[0917] ESI-MS (m / z): 445.2 [M+H] + .

[0918] Step Nine: Synthesis of (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)carbamate

[0919] (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo1,2,3,4-tetrahydronaphth-2-yl)carbamate (35.00 mg, 132.96 μmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (49.25 mg, 110.80 μmol) were added to toluene (3 mL), followed by p-toluenesulfonic acid (19.08 mg, 110.80 μmol). After the addition was complete, the mixture was heated to 140 °C and reacted 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 C18 reverse-phase column chromatography (acetonitrile: 0.05% formic acid aqueous solution = 20%-80%) to give 21.00 mg of the title compound.

[0920] The structural characterization data are as follows:

[0921] ESI-MS (m / z): 672.2 [M+H] + .

[0922] 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]indolazin[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]indolazin[1,2-b]quinoline-10,13-dione

[0923] (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)carbamate (21.00 mg, 31.26 μmol) was dissolved in N,N-dimethylformamide (1 mL), and then diethylamine (0.2 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After distilling off ethylenediamine from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution. The reaction solution was then 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).

[0924] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0925] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0926] 0.00 10 90 28 3.00 10 90 28 18.00 90 10 28

[0927] The structural characterization data of 5-13-A (6-min LCMS peak early, retention time: 1.373 min) are as follows:

[0928] 1 H NMR (400MHz, DMSO-d6) δ7.88(d,J=10.6Hz,1H),7.36(s,1H),6.58(s,1H),5.60(d,J=3.5Hz,2H),5.46(d,J=2.5H z,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=7.3Hz,3H).

[0929] ESI-MS (m / z): 450.2 [M+H] + .

[0930] The structural characterization data of 5-13-B (6 min LCMS peak late, retention time: 1.523 min) are as follows:

[0931] 1H NMR (400MHz, DMSO-d6) δ7.76(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1H),5.73(d,J=19.8Hz,1H),5.50-5.40(m,3H),3.26-3.17(m,1H), 3.08-2.96(m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J=13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J=7.3Hz,3H).

[0932] ESI-MS (m / z): 450.2 [M+H] + .

[0933] 6-minute LCMS conditions:

[0934] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0935] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0936]

[0937]

[0938] Example Thirteen: (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]indolazin[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]indolazin[1,2-b]quinoline-10,13-dione

[0939]

[0940] Step 1: Synthesis of N-(7-((dimethylamino)methylene)-3-fluoro-4-methyl-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0941] Compound 5-13-04 (1.00 g, 4.25 mmol) was dissolved in N,N-dimethylformamide dimethyl acetal (10 mL), and the mixture was heated 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 rapid silica gel column chromatography (ethyl acetate: petroleum ether = 20%-100%) to obtain 891.00 mg of the title compound.

[0942] The structural characterization data are as follows:

[0943] ESI-MS (m / z): 291.1 [M+H] + .

[0944] Step 2: Synthesis of N-(7-(aminomethylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0945] Compound 5-7-01 (0.89 g, 3.07 mmol) was dissolved in ethanol (25 mL), followed by the addition of ammonium acetate (2.36 g, 30.65 mmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The solvent in the reaction solution was evaporated to dryness under reduced pressure, and then dichloromethane (30 mL) and water (20 mL) were added. The mixture was stirred and allowed to stand to separate the organic phase, which was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness under reduced pressure to give 785.00 mg of the title compound.

[0946] The structural characterization data are as follows:

[0947] ESI-MS (m / z): 263.1 [M+H] + .

[0948] Step 3: Synthesis of N-(7-(aminomethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[0949] Compound 5-7-02 (0.80 g, 3.05 mmol) was dissolved in ethanol (200 mL), followed by the addition of 10% palladium on carbon (0.40 mg) and concentrated hydrochloric acid (0.2 mL). After the addition was complete, the reaction system was purged three times with a hydrogen balloon, and 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.

[0950] The structural characterization data are as follows:

[0951] ESI-MS (m / z): 265.1 [M+H] + .

[0952] Step 4: Synthesis of (9H-fluorene-9-yl)methyl ((8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)methyl)carbamate

[0953] The hydrochloride salt of compound 5-7-03 (0.90 g, 2.99 mmol) was dissolved in 1,4-dioxane (20 mL), followed by the addition of sodium bicarbonate (1.01 g, 11.97 mmol), water (10 mL), and 9-fluorenylmethyl-N-succinimide carbonate (1.21 g, 3.59 mmol). After addition, the mixture was stirred at room temperature for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water and extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%) to give 1.30 g of the title compound.

[0954] The structural characterization data are as follows:

[0955] ESI-MS (m / z): 487.1 [M+H] + .

[0956] Step 5: Synthesis of (9H-fluorene-9-yl)methyl ((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)methyl)carbamate

[0957] Compound 5-7-04 (0.80 g, 1.64 mmol) was dissolved in 1,4-dioxane (20 mL), and 3 mol / L hydrochloric acid aqueous solution (20 mL) was added under nitrogen protection. After the addition was complete, the mixture was heated to 60 °C and reacted for 15 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, 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 the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-40%) to give 561.00 mg of the title compound.

[0958] The structural characterization data are as follows:

[0959] ESI-MS (m / z): 445.1 [M+H] + .

[0960] Step Six: Synthesis of (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)methyl)carbamate

[0961] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-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), followed by p-toluenesulfonic acid (325.00 mg, 1.89 mmol). After the addition was complete, the mixture was heated to 140 °C and reacted for 4 hours. The reaction solution was then directly evaporated to dryness under reduced pressure at 140 °C to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (methanol:dichloromethane = 0-5%) to give 563.00 mg of the title compound.

[0962] The structural characterization data are as follows:

[0963] ESI-MS (m / z): 672.2 [M+H] + .

[0964] 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]indolazin[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]indolazin[1,2-b]quinoline-10,13-dione

[0965] Compound 5-7-06 (454.00 mg, 675.89 μmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of diethylamine (1 mL). The reaction was allowed to proceed at room temperature for 0.5 hours, and the reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). After distilling off the ethylenediamine from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with formic acid. The reaction solution was then directly purified by preparative HPLC, and the preparative solutions were lyophilized to obtain the title compounds 5-7-A (32.00 mg) and 5-7-B (56.00 mg), respectively.

[0966] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0967] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0968] 0 10 90 28 3 10 90 28 18 90 10 28

[0969] The structural characterization data of 5-7-A (6-min LCMS peak early, retention time: 1.488 min) are as follows:

[0970] 1 H NMR (400MHz, DMSO-d6) δ7.88(d,J=10.6Hz,1H),7.36(s,1H),6.58(s,1H),5.60(d,J=3.5Hz,2H),5.46(d,J=2.5H z,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=7.3Hz,3H).

[0971] ESI-MS (m / z): 450.2 [M+H] + .

[0972] The structural characterization data of 5-7-B (6 min LCMS peak elution is late, retention time: 1.596 min) are as follows:

[0973] 1 H NMR (400MHz, DMSO-d6) δ7.76(d,J=10.8Hz,1H),7.30(s,1H),6.52(s,1H),5.73(d,J=19.8Hz,1H),5.50-5.40(m,3H),3.26-3.17(m,1H), 3.08-2.96(m,1H),2.38(s,3H),2.19-2.11(m,1H),2.04(td,J=13.0,5.1Hz,1H),1.91-1.79(m,2H),1.34(s,3H),0.87(t,J=7.3Hz,3H).

[0974] ESI-MS (m / z): 450.2 [M+H] + .

[0975] 6-minute LCMS conditions:

[0976] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0977] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0978] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0979] Example Fourteen: 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0980]

[0981] Step 1: 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide Synthesis of 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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide

[0982] The single configuration of compound 5-13-A (10.00 mg, 22.25 μmol) and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-1-carboxylic acid (11.36 mg, 33.37 μmol) were dissolved in N,N-dimethylformamide (1 mL), followed by the addition of HATU (12.68 mg, 33.37 μmol) and N,N-diisopropylethylamine (8.63 mg, 66.74 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was directly purified by C18 reversed-phase column (acetonitrile: 0.05% formic acid aqueous solution = 30%-100%) to give the single title compound 5-16-01-A (7 mg).

[0983] The structural characterization data are as follows:

[0984] ESI-MS (m / z): 772.3 [M+H] + .

[0985] 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]indolazin[1,2-b]quinoline-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]indolazin[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

[0986] Compound 5-16-01-A (7.00 mg, 9.07 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the addition of potassium fluoride (2.63 mg, 45.34 μmol). After the addition was complete, the mixture was heated to 50 °C and reacted for 1 hour. The reaction was detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was directly purified by HPLC to obtain the single title compound 5-16-A (1.73 mg).

[0987] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0988] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0989] 0.00 10 90 28 18.00 90 10 28

[0990] The structural characterization data are as follows:

[0991] 1 H NMR (400MHz, DMSO-d6) δ8.26(s,1H),7.78(d,J=10.8Hz,1H),7.30(s,1H),6.56(s,1H),6.52(s,1H),5.52(d,J=19.3Hz,1H),5.43(d,J=4.4Hz,2H),4 .94(d,J=19.2Hz,1H),3.30-3.24(m,1H),3.11-3.00(m,1H),2.95-2.84(m ,1H),2.39(s,3H),1.98-1.80(m,3H),1.62(s,3H),0.87(t,J=7.9Hz,3H).

[0992] ESI-MS (m / z): 534.2 [M+H] + .

[0993] 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecane Amide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecaneamide

[0994]

[0995] Step 1: Isolation and purification of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione

[0996] Compound 2-23 (16.00 mg) was purified by preparative high performance liquid chromatography. Under the following purification conditions, two diastereomers were separated to obtain 5.10 mg of trifluoroacetate of 2-23-A (retention time 9.85 min) and 7.12 mg of trifluoroacetate of 2-23-B (retention time 10.62 min).

[0997] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0998] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0999] 0 5 95 28 2 5 95 28 18 50 50 28

[1000] The structural characterization data are as follows:

[1001] 2-23-A:

[1002] 1H NMR(400MHz,DMSO-d6)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).

[1003] ESI-MS (m / z): 452.1 [M+H] + .

[1004] 2-23-B:

[1005] 1 H NMR(400MHz,DMSO-d6)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).

[1006] ESI-MS (m / z): 452.0 [M+H] + .

[1007] Step 2: N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecaneamide Synthesis of N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolazo[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexadecanoamide

[1008] At 25°C, trifluoroacetate of 2-23-A (34.71 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of 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). The reaction was maintained at 25°C for 0.5 hours. The reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions). The preparative solution was freeze-dried to obtain 11.04 mg of the title compound DL-15 with a retention time of 7.5 min.

[1009] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1010] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1011] 0 30 70 28 3 30 70 28 18 90 10 28

[1012] The structural characterization data are as follows:

[1013] DL-15:

[1014] ESI-MS (m / z): 1107.3 [M+H] + .

[1015] Example 16 N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[1016]

[1017] Step 1: Synthesis of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-cyclopropyl-2-hydroxyacetamide

[1018] The formate (50 mg, 109.68 μmol) of the single configuration of compound 3-1-A and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) were dissolved in N,N-dimethylformamide (2 mL), followed by the addition of HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. 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).

[1019] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1020] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1021] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[1022] The structural characterization data of 3-12-A (with a prominent 6-min LCMS peak) are as follows:

[1023] 1 H NMR (400MHz, DMSO-d6) δ8.44(d,J=9.0Hz,1H),8.05(d,J=10.2Hz,1H),7.33(s,1H) ,6.54(s,1H),5.62(q,J=6.7Hz,1H),5.52(d,J=5.1Hz,1H),5.42(s,2H),5.24(q,J =19.2Hz,2H),3.61(dd,J=6.2,5.1Hz,1H),3.32–3.21(m,2H),2.19(q,J=6.5Hz,2H ),1.92–1.80(m,2H),1.26–1.20(m,1H),0.87(t,J=7.3Hz,3H),0.57–0.34(m,4H).

[1024] ESI-MS (m / z): 554.0 [M+H]+.

[1025] The structural characterization data of 3-12-B (6-min LCMS peak elution is late) are as follows:

[1026] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.7Hz,1H),8.06(d,J=10.3Hz,1H),7.33(s,1H),5.57(q,J=6.7Hz,1H),5.43(s,2H),5.30–5.17(m,2H),3. 64(d,J=6.2Hz,1H),3.29(q,J=6.7Hz,2H),2.28–2.13(m,2H),1.93–1. 78(m,2H),1.18–1.08(m,1H),0.87(t,J=7.3Hz,3H),0.50–0.29(m,4H).

[1027] ESI-MS (m / z): 554.0 [M+H]+.

[1028] The formate (50 mg, 109.68 μmol) of another single configuration of compound 3-1-B and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 μmol) were dissolved in N,N-dimethylformamide (2 mL), followed by the addition of HATU (7.57 mg, 219.36 μmol) and N,N-diisopropylethylamine (42.53 mg, 329.04 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. 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 the two isomers of the title compound (3-12-C: 20.19 mg, 3-12-D: 18.33 mg).

[1029] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1030] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1031] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[1032] The structural characterization data of 3-12-C (with a prominent peak elution at 6 min LCMS) are as follows:

[1033] 1H NMR(400MHz,DMSO-d6)δ8.47(d,J=9.0Hz,1H),8.06(d,J=10.3Hz,1H),7.33(s,1H ),6.54(s,1H),5.62(q,J=6.5Hz,1H),5.53(d,J=5.1Hz,1H),5.43(s,2H),5.32–5 .16(m,2H),3.61(dd,J=6.3,5.1Hz,1H),3.32–3.22(m,2H),2.19(q,J=6.5Hz,2H) ,1.92–1.80(m,2H),1.28–1.20(m,1H),0.87(t,J=7.3Hz,3H),0.54–0.35(m,4H).

[1034] ESI-MS (m / z): 554.0 [M+H]+.

[1035] The structural characterization data of 3-12-D (6-min LCMS peak elution is late) are as follows:

[1036] 1 H NMR (400MHz, DMSO-d6) δ8.44(d,J=8.8Hz,1H),8.05(d,J=10.2Hz,1H),7.34(s, 1H),6.55(s,1H),5.58(q,J=6.7Hz,1H),5.45(d,J=5.2Hz,1H),5.43(s,2H),5. 31–5.14(m,2H),3.65(t,J=5.7Hz,1H),3.33–3.21(m,2H),2.28–2.13(m,2H),1 .95–1.80(m,2H),1.16–1.09(m,1H),0.88(t,J=7.3Hz,3H),0.46–0.31(m,4H).

[1037] ESI-MS (m / z): 554.0 [M+H] + .

[1038] Example 17: Preparation of (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine and (S)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazin[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[1039]

[1040] 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]indoleazine[1,2-b]quinoline-10,13(1H,9H)-dione (80.0 mg, 175.5 μmol) and L-lactic acid (31.6 mg, 351.0 μmol) were dissolved in DMF (3 mL), and then HATU (121.1 mg, 351.0 μmol) and DIPEA (68.0 mg, 526.5 μmol) were added. The reaction was carried out at room temperature for 2 hours. 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%).

[1041] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1042] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1043] 0.00 30 70 24 2.00 30 70 24 18.00 90 10 24

[1044] The structural characterization data of compound 3-7-A (high elution rate at 6 min LC-MS, retention time 2.49 min) are as follows:

[1045] MS m / z (ESI): 528.2 [M+H] +

[1046] 1H NMR(400MHz,DMSO-d6)δ8.55(d,J=9.2Hz,1H),8.06(d,J=10.4Hz,1H),7.33(s,1H), 6.55(s,1H),6.67(d,J=4.8Hz,1H),5.65–5.59(m,1H),5.43(s,2H),5.29–5.21(m,1H ),5.14–5.10(m,1H),4.15–4.10(m,,1H),3.27–3.20(m,1H),2.22–2.15(m,2H),1.92 –1.81(m,2H),1.41(d,J=6.8Hz,3H),1.30–1.23(m,1H),0.89–0.85(t,J=7.2Hz,3H).

[1047] The structural characterization data of compound 3-7-B (later elution peak on LC-MS at 6 min, retention time 2.50 min) are as follows:

[1048] MS m / z (ESI): 528.2 [M+H] +

[1049] 1 H NMR(400MHz,DMSO-d6)δ8.46(d,J=8.8Hz,1H),8.06(d,J=10.4Hz,1H),7.33(s ,1H),6.56(s,1H),5.60–5.53(ms,1H),5.51(d,J=5.2Hz,1H),5.43(s,2H),5.2 7–5.14(m,2H),4.16–4.08(m,1H),3.28–3.22(m,1H),2.22–2.19(m,2H),1.92 –1.81(m,2H),1.49–1.39(m,1H),1.29(d,J=6.8Hz,3H),0.87(t,J=7.2Hz,3H).

[1050] Example 18: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl-1-hydroxycyclopropylformamide 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]indoleazine[1,2-b]quinoline-1-yl-1-hydroxycyclopropylformamide

[1051]

[1052] 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]indoleazine[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), followed by the addition of HATU (121.14 mg, 350.98 μmol) and DIPEA (68.04 mg, 526.47 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. The reaction solution was concentrated to dryness and the reaction was 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 3-17-B. 3.5mg.

[1053] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1054] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1055] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1056] The structural characterization data are as follows:

[1057] The structural characterization data of 3-17-A (6-min LCMS peak early, retention time: 2.657 min) are as follows:

[1058] ESI-MS (m / z): 540.0 [M+H]+.

[1059] 1 H NMR (400MHz, DMSO-d6) 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).

[1060] The structural characterization data of 3-17-A (6-min LCMS peak late, retention time: 2.724 min) are as follows:

[1061] ESI-MS (m / z): 540.0 [M+H]+.

[1062] 1 H NMR (400MHz, DMSO-d6) 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).

[1063] Example 19 Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide compound

[1064]

[1065] Step 1: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[1066] N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1-group)acetamide (570 mg, 2.23 mmol) and cyclopropylboronic acid (574.56 mg, 6.69 mmol) were dissolved in 1,4-dioxane. Dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (480 mg, 677.97 μmol) and cesium carbonate (2.17 g, 6.69 mmol) were added. After nitrogen protection, the mixture was reacted in a microwave oven at 115 °C for 2 h. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was diluted with ethyl acetate and filtered. The filtrate was extracted with ethyl acetate (30 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel column (PE:EA = 1:4) to give 550 mg of the title compound.

[1067] The structural characterization data are as follows:

[1068] ESI-MS (m / z): 262.1 [M+H] + .

[1069] Step 2: Synthesis of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[1070] Tetrahydrofuran (30 mL) and tert-butanol (10 mL) were added to the reaction solution flask. After cooling to 5 °C in an ice bath, potassium tert-butoxide (945 mg, 8.42 mmol) was added. Then, N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.0 g, 3.83 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (718 mg, 6.12 mmol) was added. After the addition was complete, the reaction was maintained at 5 °C for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated ammonium chloride aqueous solution (50 mL) and extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain 1.2 g of crude product of the title compound.

[1071] The structural characterization data are as follows:

[1072] ESI-MS (m / z): 291.1 [M+H] + .

[1073] Step 3: Synthesis of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide hydrochloride

[1074] Crude N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.2 g, 1.41 mmol) was dissolved in methanol (7.5 mL) and tetrahydrofuran (7.5 mL), followed by the addition of 1 mol / L hydrochloric acid aqueous solution (7.5 mL) and 10% palladium on carbon (450 mg). After the addition was complete, the reaction system was purged three times with a hydrogen balloon, 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 1.05 g of crude product.

[1075] The structural characterization data are as follows:

[1076] ESI-MS (m / z): 277.1 [M+H] + .

[1077] Step 4: Synthesis of (9H-fluorene-9-yl)methyl(8-acetamido-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[1078] Crude N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide hydrochloride (1.05 g, 3.80 mmol) was dissolved in 1,4-dioxane (10 mL), followed by the addition of sodium bicarbonate (1.3 g, 15.20 mmol), water (10 mL), and 9-fluorenylmethyl-N-succinimide carbonate (1.54 g, 4.56 mmol). After addition, the mixture was stirred at room temperature for 2 hours. The reaction was detected by high-performance liquid chromatography-mass spectrometry. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain the crude product. The crude product was purified by C18 reversed-phase column chromatography to give 2.0 g of the title compound.

[1079] The structural characterization data are as follows:

[1080] ESI-MS (m / z): 499.2 [M+H] + .

[1081] Step 5: Synthesis of (9H-fluorene-9-yl)methyl(8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[1082] (9H-fluorene-9-yl)methyl(8-acetamido-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate (2.0 g, 3.21 mmol, 80%) was dissolved in dioxane (20 mL), and 12 mol / L concentrated hydrochloric acid (5 mL) was added. After the addition was complete, the mixture was heated to 70 °C and reacted for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water (40 mL) and then extracted with ethyl acetate (30 mL * 3). The organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain the crude product. The crude product was purified by column chromatography on silica gel column (PE:EA = 2:1) to give 740 mg of the title compound.

[1083] The structural characterization data are as follows:

[1084] ESI-MS (m / z): 457.3 [M+H] + .

[1085] Step Six: Synthesis of (9H-fluorene-9-yl)methyl((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indoleazine[1,2-b]quinoline-1-yl)carbamate

[1086] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (442 mg, 1.68 mmol) and (9H-fluorene-9-yl)methyl(8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)carbamate (640 mg, 1.40 mmol) were added to toluene (30 mL), followed by p-toluenesulfonic acid (242 mg, 1.40 mmol). After the addition was complete, the mixture was heated to 135 °C and reacted 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 using silica gel column chromatography (DCM:MeOH = 33:1) to obtain 1.02 g of the title compound.

[1087] The structural characterization data are as follows:

[1088] ESI-MS (m / z): 684.1 [M+H] + .

[1089] 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]indoleazine[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]indoleazine[1,2-b]quinoline-10,13-dione

[1090] (9H-fluorene-9-yl)methyl((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indoleazine[1,2-b]quinoline-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 allowed to proceed at room temperature for 0.5 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. After distilling off the ethylenediamine from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution. The reaction solution was then 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).

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

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

[1093] 0.00 10 90 28 3.00 10 90 28 18.00 70 30 28

[1094] The structural characterization data of 5-22-7-A (6-min LCMS peak early, retention time: 2.28 min) are as follows:

[1095] ESI-MS (m / z): 462.2 [M+H] + .

[1096] The structural characterization data of 5-22-7-B (6 min LCMS peak late, retention time: 2.35 min) are as follows:

[1097] ESI-MS (m / z): 462.2 [M+H] + .

[1098] Step 8: Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[1099] At 25°C, a single configuration of compound 5-28-7-A (40 mg, 86 μmol) and glycolic acid (8 mg, 104 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (40 mg, 104 μmol) and DIPEA (36 mg, 258 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hr. The reaction solution was concentrated to dryness and the reaction was 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.

[1100] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1101] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1102] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1103] The structural characterization data are as follows:

[1104] The structural characterization data of 5-22-A (6-min LCMS peak early, retention time: 2.540 min) are as follows:

[1105] ESI-MS (m / z): 520.0 [M+H] + .

[1106] 1 H NMR(400MHz,DMSO-d6)δ8.46(d,J=8.9Hz,1H),7.74(d,J=11.9Hz,1H),7.3 0(s,1H),6.53(s,1H),5.64–5.56(m,1H),5.49(t,J=5.8Hz,1H),5.42(s,2H ),5.19(s,2H),3.96(d,J=5.7Hz,2H),2.25–2.10(m,2H),2.04–1.79(m,4H) ,1.23(s,2H),1.15–1.05(m,2H),0.87(t,J=7.2Hz,3H),0.80–0.70(m,2H).

[1107] At 25°C, 30 mg (65 μmol) of the single configuration of compound 5-28-7-B and glycolic acid (6 mg, 78 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (40 mg, 104 μmol) and DIPEA (17 mg, 130 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. The reaction solution was concentrated to dryness and the reaction was 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.

[1108] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1109] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1110] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1111] The structural characterization data are as follows:

[1112] The structural characterization data of 5-22-B (6 min LCMS peak late, retention time: 2.612 min) are as follows:

[1113] ESI-MS (m / z): 520.0 [M+H] + .

[1114] 1H NMR(400MHz,DMSO-d6)δ8.49(d,J=8.9Hz,1H),7.74(d,J=11.9Hz,1H),7.31(s ,1H),6.53(s,1H),5.60(s,1H),5.51(t,J=5.9Hz,1H),5.43(s,2H),5.25–5.13 (m,2H),3.97(d,J=5.8Hz,2H),2.18(s,2H),2.04–1.91(m,4H),1.90–1.80(m, 1H), 1.23 (s, 6H), 1.15–1.05 (m, 2H), 0.87 (t, J = 7.2Hz, 4H), 0.80–0.70 (m, 2H).

[1115] Example 20 Synthesis of (R)-3-(dimethylamine)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropamide 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]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropamide

[1116]

[1117] Step 1: (9H-fluoro-9-yl)methyl((S)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate Synthesis of (9H-fluoro-9-yl)methyl((R)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate

[1118] At 25°C, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-benzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (72 mg, 166 μmol) and 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxypropionic acid (65 mg, 199 μmol) were mixed. Dissolve 1 mol in DMF (2 mL), then add HATU (95 mg, 250 μmol) and DIPEA (65 mg, 498 μmol). After the addition is complete, react at room temperature for 0.5 hr. Concentrate the reaction solution to dryness and directly detect the reaction by high performance liquid chromatography-mass spectrometry. The reaction solution is 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).

[1119] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1120] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1121] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1122] The structural characterization data of compound 1-10-1-A (elevation rate high at 6 min LCMS, retention time: 3.283 min) are as follows:

[1123] ESI-MS (m / z): 745.4 [M+H] + .

[1124] The structural characterization data of compound 1-10-1-B (later elution peak at 6 min LCMS, retention time: 3.465 min) are as follows:

[1125] ESI-MS (m / z): 745.4 [M+H] + .

[1126] 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]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxypropamide and (S)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxypropamide

[1127] At 25°C, compound 1-10-1-B (28 mg, 37 μmol) was dissolved in DMF (2 mL), and then diethylamine (1 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 1.0 hr. The reaction solution was concentrated to dryness to obtain 28 mg of crude product (compound 1-10-2-B), which was directly used in the next reaction.

[1128] At 25°C, compound 1-10-1-A (24 mg, 33 μmol) was dissolved in DMF (2 mL), and then diethylamine (1 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 1.0 hr. The reaction solution was concentrated to dryness to obtain 24 mg of crude product (compound 1-10-2-A), which was directly used in the next step of the reaction.

[1129] The structural characterization data are as follows:

[1130] ESI-MS (m / z): 523.2 [M+H] + .

[1131] 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]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxypropamide 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]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxypropamide

[1132] At 25°C, compound 1-10-2-B (28 mg, 37 μmol, 70%) was dissolved in methanol (2 mL), followed by the addition of formaldehyde aqueous solution (1 mL). After the addition was complete, the reaction was allowed to proceed at room temperature for 16.0 hr. Then, sodium cyanoborohydride (7.07 mg, 96.45 μmol) was added, and the reaction was allowed to proceed at room temperature for 1.0 hr. The reaction solution was concentrated to dryness and the reaction was 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).

[1133] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1134] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1135] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1136] The structural characterization data of compound 1-10-B (later elution peak at 6 min LCMS, retention time: 1.937 min) are as follows:

[1137] ESI-MS (m / z): 551.2 [M+H] + .

[1138] 1 H NMR (400MHz, DMSO-d6) δ8.48(d,J=8.6Hz,1H),8.31(s,2H),7.80(d,J=11.0Hz,1H),7.31(s,1H),6.55( s,1H),5.54(s,1H),5.43(s,2H),5.34(d,J=19.2Hz,1H),5.19(d,J=19.1Hz,1H),4.09–4.06(m,1H),3.2 0–3.15(m,2H),2.59–2.53(m,1H),2.45–2.42(m,1H),2.42–2.38(s,3H),2.23–2.19(d,J=7.0Hz,1H),2. 13(s,6H),2.12–2.08(m,1H),2.02–1.95(m,1H),1.90–1.85(m,2H),1.23(s,2H),0.88(d,J=7.2Hz,3H).

[1139] At 25°C, compound 1-10-2-A (24 mg, 33 μmol, 70%) was dissolved in methanol (2 mL), followed by the addition of formaldehyde aqueous solution (1 mL). After the addition was complete, the reaction was allowed to proceed at room temperature for 16.0 hr. Then, sodium cyanoborohydride (6.06 mg, 96.45 μmol) was added, and the reaction was allowed to proceed at room temperature for 1.0 hr. The reaction solution was concentrated to dryness and the reaction was 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).

[1140] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1141] Mobile phase A: methanol; Mobile phase B: water (0.05% formic acid)

[1142] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[1143] The structural characterization data of compound 1-10-A (elevation early on LCMS at 6 min, retention time: 1.920 min) are as follows:

[1144] ESI-MS (m / z): 551.2 [M+H] + .

[1145] 1 H NMR (400MHz, DMSO-d6) δ8.58(d,J=9.0Hz,1H),8.28(s,1H),7.84(d,J=10.9Hz,1H),7.37(s, 1H),6.61(s,1H),5.66–5.59(m,1H),5.49(s,2H),5.35(d,J=19.1Hz,1H),5.17(d,J=18.9Hz, 1H),4.23–4.16(m,1H),3.23(d,J=7.8Hz,2H),2.75–2.67(m,2H),2.45(s,3H),2.30(s,6H), 2.27–2.17(m,2H),2.14–1.99(m,1H),1.98–1.87(m,2H),1.30(s,2H),0.93(t,J=7.3Hz,3H).

[1146] Example 21 (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxapentano[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(10H,13H)-dione (compound 4-14)

[1147]

[1148] Step 1: Synthesis of 2-nitro-4,5-methylenedioxyacetophenone

[1149] Compound 4-14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL), and concentrated nitric acid (26 mL) was slowly added dropwise with stirring. The reaction was carried out at room temperature for 2 hours. The reaction was monitored by TLC; a small amount of the starting material remained, and the product was obvious. The reaction was neutralized by slow dropwise addition of saturated sodium bicarbonate aqueous solution. The mixture was extracted three times with dichloromethane, and the organic phases were combined, washed three times with saturated brine, dried, and concentrated to give the crude product. Purification by silica gel column chromatography (eluent: 0-20% ethyl acetate / petroleum ether) yielded 9.8 g of the title compound.

[1150] Step 2: Synthesis of 6-amino-3,4-methylenedioxyacetophenone

[1151] Compound 4-14-2 (2.0 g, 9.56 mmol) was dissolved in ethyl acetate (20 mL), and 10% palladium on carbon (0.2 g) was added. The mixture was stirred for 4 hours under hydrogen purging and protection. After filtration, the filtrate was concentrated under reduced pressure to give 1.7 g of the crude title compound.

[1152] Step 3: Synthesis of 6-acetamido-3,4-methylenedioxyacetophenone

[1153] Compound 4-14-3 (1.7 g, 9.49 mmol) was dissolved in acetic anhydride (17 mL), and the reaction was stirred for 1 hour. The solvent was removed by vacuum evaporation, water was added and stirred, the mixture was filtered, and the solid was washed with water and dried under vacuum to give 2.08 g of the crude title compound.

[1154] Step 4: Synthesis of (E)-N-(6-(3-(dimethylamino)acryloyl)benzo[d][1,3]dioxapentane-5-yl)acetamide

[1155] Compound 4-14-4 (1.88 g, 8.50 mmol) was dissolved in DMF-DMA (30 mL) and reacted at 120 °C for 2 hours. After removing the solvent under reduced pressure, 2.33 g of the crude title compound was obtained.

[1156] The structural characterization data are as follows:

[1157] ESI-MS (m / z): 277.2 [M+1] + .

[1158] Step 5: Synthesis of (E)-N-(6-(3-(cyclopropylamino)acryloyl)benzo[d][1,3]dioxapentane-5-yl)acetamide

[1159] Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), and cyclopropylamine (413.3 mg, 7.24 mmol) was added dropwise. The mixture was heated to 50 °C and reacted for 16 hours. The solvent was removed by vacuum evaporation to give 208 mg of the crude title compound.

[1160] The structural characterization data are as follows:

[1161] ESI-MS (m / z): 289.2 [M+1] + .

[1162] Step Six: Synthesis of N-(6-(3-(cyclopropylamino)propionyl)benzo[d][1,3]dioxapentane-5-yl)acetamide

[1163] Compound 4-14-6 (208 mg, 0.72 mmol) was dissolved in glacial acetic acid (4 mL), and sodium borohydride (13.65 mg, 0.36 mmol) was added while stirring in an ice-water bath. The mixture was then stirred for 3 hours at room temperature. The solvent was removed by vacuum distillation to give 209 mg of the crude title compound.

[1164] The structural characterization data are as follows:

[1165] ESI-MS (m / z): 291.1 [M+1] + .

[1166] Step 7: Synthesis of (9H-fluorene-9-yl)methyl(3-(6-acetamidobenzo[d][1,3]dioxapentane-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1167] Compound 4-14-7 (200 mg, 0.69 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL). 9-fluorenemethyl-N-succinimide carbonate (395 mg, 0.68 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were added with stirring, and the reaction was carried out at room temperature for 2 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (eluent: 30% ethyl acetate / petroleum ether) to give 350 mg of the title compound.

[1168] Step 8: Synthesis of (9H-fluorene-9-yl)methyl(3-(6-aminobenzo[d][1,3]dioxapentane-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[1169] 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 mixture was heated to 60 °C and stirred for 16 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water, dried, and concentrated. The solution was purified by silica gel column chromatography (eluent: 33% ethyl acetate / petroleum ether) to give 218 mg of the title compound.

[1170] Step Nine: Synthesis of (S)-(9H-fluorene-9-yl)methylcyclopropyl (2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxapentano[4,5-g]pyrano[3,4]:6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)carbamate

[1171] Compound 4-14-9 (40 mg, 0.085 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indoleazine-3,6,10(4H)-trione (24.62 mg, 0.094 mmol) were dissolved in toluene (1 mL), and p-toluenesulfonic acid (2.93 mg, 0.017 mmol) was added. The mixture was heated to 120 °C and reacted for 4 hours. The solution was concentrated under reduced pressure to give 59 mg of the crude title compound.

[1172] The structural characterization data are as follows:

[1173] ESI-MS (m / z): 698.1 [M+1] + .

[1174] Step 10: Synthesis of (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxapentano[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(10H,13H)-dione

[1175] Compound 4-14-10 (59 mg, 0.085 mmol) was dissolved in DMF (1 mL), and diethylamine (0.5 mL) was added dropwise. The mixture was stirred for 1 hour. The diethylamine was removed by vacuum distillation, and the mixture was acidified with 3N hydrochloric acid. The solution was then purified by high performance liquid chromatography (purification conditions as shown below), and lyophilized to obtain 12.66 mg of the trifluoroacetate of the title compound.

[1176] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1177] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1178] 0 20 80 28 2 20 80 28 18 80 20 28

[1179] The structural characterization data are as follows:

[1180] 1 H NMR(400MHz,DMSO-d6)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).

[1181] ESI-MS (m / z): 476.1 [M+1] + .

[1182] Example 22: (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxane[4,5-g]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-8,11(10H,13H)-dione (compounds 4-15)

[1183]

[1184] Step 1: Synthesis of (E)-N-(6-(3-((2-methoxyethyl)amino)acryloyl)benzo[d][1,3]dioxane-5-yl)acetamide

[1185] Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), and 2-methoxyethylamine (543.7 mg, 7.24 mmol) was added dropwise. The reaction was carried out at 50 °C for 16 hours. The reaction was monitored by LCMS. A small amount of starting material remained, and the product was obvious. The solvent was removed by vacuum distillation to obtain 221 mg of the title compound, which was then directly proceeded to the next step of the reaction.

[1186] The structural characterization data are as follows:

[1187] ESI-MS (m / z): 307.1 [M+1] +

[1188] Step 2: Synthesis of N-(6-(3-((2-methoxyethyl)amino)propionyl)benzo[d][1,3]dioxane-5-yl)acetamide

[1189] 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 in an ice-water bath, and the mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS; the starting material disappeared, and the product was clearly visible. The solvent was removed by vacuum distillation to obtain 200 mg of the title compound, which was then directly used for the next reaction.

[1190] ESI-MS (m / z): 309.1 [M+1] +

[1191] Step 3: Synthesis of (9H-fluorene-9-yl)methyl(3-(6-acetamidobenzo[d][1,3]dioxo-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[1192] 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-succinimide carbonate (372 mg, 0.65 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) were added with stirring, and the reaction was carried out at room temperature for 2 hours. The starting material disappeared under TLC monitoring, and the product was clearly visible. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with saturated brine, dried, and concentrated. Purification was performed by silica gel column chromatography (eluent: 50% ethyl acetate / petroleum ether) to give 180 mg of the title compound.

[1193] Step 4: Synthesis of ((9H-fluorene-9-yl)methyl(3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[1194] Compound 4-15-3 (180 mg, 0.68 mmol) was dissolved in 1,4-dioxane (5 mL), and 3N hydrochloric acid aqueous solution (5 mL) was added dropwise. The mixture was heated to 60 °C and stirred for 16 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water, dried, and concentrated. The solution was purified by silica gel column chromatography (eluent: 45% ethyl acetate / petroleum ether) to give 132 mg of the title compound.

[1195] Step 5: Synthesis of (S)-(9H-fluorene-9-yl)methyl(2-(7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxane[4,5-g]pyrano[3',4]:6,7]indolazino[1,2-b]quinoline-14-yl)ethyl)(2-methoxyethyl)carbamate

[1196] Compound 4-15-4 (130 mg, 0.266 mmol) and rac-(4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10-trione (70.05 mg, 0.266 mmol) were dissolved in toluene (4 mL), and p-toluenesulfonic acid (9.16 mg, 0.053 mmol) was added. The reaction was heated to 120 °C for 4 hours. The reaction was monitored by LC-MS; the starting material disappeared, and the product was clearly visible. The product was concentrated under reduced pressure to give 190 mg of the crude product of the title compound.

[1197] The structural characterization data are as follows:

[1198] ESI-MS (m / z): 716.1 [M+1] +

[1199] Step Six: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxane[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(10H,13H)-dione

[1200] Compound 4-15-5 (190 mg, 0.265 mmol) was dissolved in DMF (3 mL), and diethylamine (2 mL) was added dropwise. The mixture was stirred for 1 hour. The reaction was monitored by LCMS. The starting material disappeared, and the product was clearly visible. Diethylamine was removed by vacuum distillation, and the mixture was purified by acidification with 3N hydrochloric acid. The purified product was then lyophilized to give 99.28 mg of the title compound.

[1201] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1202] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1203] 0 20 80 28 2 20 80 28 18 80 20 28

[1204] The structural characterization data are as follows:

[1205] 1H NMR (400MHz, DMSO-d6): δ8.68(s,2H),7.68(s,1H),7.56(s,1H),7.26(s,1H),6.52(s,1H),6.32(s,2H),5.44(s,2H),5.31(s,2H),3.65–3. 58(m,2H),3.42(d,J=10.2Hz,2H),3.36(s,3H),3.22(d,J=4.0Hz,4H),1.94–1.80(m,2H),0.87(t,J=7.3Hz,3H).ESI-MS(m / z):494.2[M+1] +

[1206] Example 23 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide & N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[1207]

[1208] Step 1: Synthesis of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide

[1209] 3-Bromo-5-fluoro-4-methoxyaniline (1.7 g, 7.73 mmol) was dissolved in tetrahydrofuran (30 mL), followed by the addition of triethylamine (2.35 g, 23.18 mmol) and acetic anhydride (1.18 g, 11.59 mmol). After the addition was complete, the mixture was heated to 50 °C and stirred for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, it was diluted with ethyl acetate (50 mL), and then washed once each with distilled water (30 mL) and saturated brine (30 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain the crude product. The crude product was purified by pulping with petroleum ether:ethyl acetate = 5:1 to obtain 1.1 g of the title compound.

[1210] The structural characterization data are as follows:

[1211] ESI-MS (m / z): 262.0 [M+H] + .

[1212] Step 2: Synthesis of (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)-3-butenoic acid

[1213] N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (1.1 g, 4.20 mmol) and 3-butenoic acid (397.47 mg, 4.62 mmol) were dissolved in a mixed solvent of 1,4-dioxane (20 mL) and water (5 mL). Then, triethylamine (1.27 g, 12.59 mmol), tris(o-methylphenyl)phosphine (127.75 mg, 419.73 μmol), and palladium acetate (47.12 mg, 209.89 μmol) were added. After the addition was complete, the reaction system was purged with nitrogen three times and heated to 100 °C for 4 hours under a nitrogen atmosphere. The reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1 mol / L sodium hydroxide aqueous solution (50 mL) and ethyl acetate (50 mL) were added and the mixture was shaken to separate the layers. After separating the lower aqueous phase, the pH was adjusted to about 3 with 4 mol / L hydrochloric acid aqueous solution, and then extracted with ethyl acetate (40 mL * 2). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to obtain 1.1 g of crude product of the title compound.

[1214] The structural characterization data are as follows:

[1215] ESI-MS (m / z): 268.1 [M+H] + .

[1216] Step 3: Synthesis of 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid

[1217] The crude product of (E)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)-3-butenoic acid (1.1 g, 4.12 mmol) was dissolved in methanol (20 mL), and then 10% palladium on carbon (100 mg) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 1.05 g of the crude product of the title compound.

[1218] The structural characterization data are as follows:

[1219] ESI-MS (m / z): 270.1 [M+H] + .

[1220] Step 4: Synthesis of N-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[1221] Crude 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 was complete, the mixture was allowed to rise naturally to room temperature for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water (60 mL) and then extracted with ethyl acetate (40 mL * 3). The organic phases were combined and washed with saturated sodium bicarbonate aqueous solution until neutral, and then washed with saturated brine (40 mL). The organic phase was dried over anhydrous sodium sulfate and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain the crude product, which was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0-40%) to obtain 503 mg of the title compound.

[1222] The structural characterization data are as follows:

[1223] ESI-MS (m / z): 252.1 [M+H] + .

[1224] Step 5: Synthesis of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[1225] Tetrahydrofuran (15 mL) and tert-butanol (4 mL) were added to the reaction solution 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-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide (500 mg, 1.99 mmol) was dissolved in tetrahydrofuran (5 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (373.01 mg, 3.18 mmol) was added. After the addition was complete, the reaction was maintained at 5 °C for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated ammonium chloride aqueous solution (50 mL) and extracted with ethyl acetate (40 x 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 crude product of the title compound.

[1226] The structural characterization data are as follows:

[1227] ESI-MS (m / z): 281.1 [M+H] + .

[1228] Step Six: Synthesis of N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide

[1229] Crude N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide (520 mg, 1.86 mmol) was dissolved in a mixed solution of methanol (10 mL) and tetrahydrofuran (10 mL), followed by the addition of 1 mol / L hydrochloric acid aqueous solution (3.71 mL) and 10% palladium on carbon (50 mg). After the addition was complete, the reaction system was purged three times with a hydrogen balloon, 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 obtain 551 mg of crude hydrochloride of the title compound.

[1230] The structural characterization data are as follows:

[1231] ESI-MS (m / z): 267.1 [M+H] + .

[1232] Step 7: Synthesis of (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[1233] Crude N-(7-amino-3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide hydrochloride (550 mg, 1.64 mmol) was dissolved in 1,4-dioxane (15 mL), followed by the addition of sodium bicarbonate (549.45 mg, 6.54 mmol), water (5 mL), and 9-fluorenylmethyl-N-succinimide carbonate (1.12 g, 1.96 mmol). After the addition was complete, the mixture 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) and 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 dried under reduced pressure to obtain the crude product. The crude product was purified by a C18 reverse-phase column (acetonitrile / 0.05% formic acid aqueous solution, 20% acetonitrile to 100% acetonitrile) to give 410 mg of the title compound.

[1234] The structural characterization data are as follows:

[1235] ESI-MS (m / z): 489.1 [M+H] + .

[1236] Step 8: Synthesis of (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphth-2-yl)carbamate

[1237] (9H-fluorene-9-yl)methyl(8-acetamido-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphth-2-yl)carbamate (410 mg, 839.29 μmol) was dissolved in dioxane (10 mL), and 12 mol / L concentrated hydrochloric acid (2 mL) was added. After the addition was complete, the mixture was heated to 70 °C and reacted for 2 hours. The reaction was detected 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 dried under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel (ethyl acetate: petroleum ether = 0-60%) to obtain 351 mg of the title compound.

[1238] The structural characterization data are as follows:

[1239] ESI-MS (m / z): 447.1 [M+H] + .

[1240] Step Nine: Synthesis of (9H-fluorene-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indoleazine[1,2-b]quinoline-1-yl)carbamate

[1241] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (247.64 mg, 940.71 μmol) and (9H-fluorene-9-yl)methyl(8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphthyl-2-yl)carbamate (350 mg, 783.93 μmol) were added to toluene (15 mL), followed by p-toluenesulfonic acid (134.84 mg, 783.93 μmol). After the addition was complete, the mixture was heated to 135 °C and reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 135 °C to obtain the crude product. The crude product was purified by rapid silica gel (methanol:dichloromethane = 0-6%) column chromatography to obtain 358 mg of the title compound.

[1242] The structural characterization data are as follows:

[1243] ESI-MS (m / z): 674.2 [M+H] + .

[1244] 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]indoleazine[1,2-b]quinoline-10,13-dione

[1245] (9H-fluorene-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinoline-1-yl)carbamate (358 mg, 531.41 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then diethylamine (0.4 mL) was added. After the addition was complete, the reaction was carried out at room temperature for 0.5 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by slurrying with ethyl acetate to obtain 220 mg of the title compound.

[1246] The structural characterization data are as follows:

[1247] ESI-MS (m / z): 452.1 [M+H] + .

[1248] Step 11: Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide & N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indoleazine[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

[1249] (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]indoleazine[1,2-b]quinoline-10,13-dione (50 mg, 110.76 μmol) and 2-hydroxyacetic acid (16.85 mg, 221.51 μmol) were dissolved in N,N-dimethylformamide (2 The solution was added to a solution containing HATU (84.17 mg, 221.51 μmol) and N,N-diisopropylethylamine (42.94 mg, 332.27 μmol). After addition, the mixture was allowed to react at room temperature for 0.5 hours. 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 with a single configuration (5-34-A: 6.22 mg, 5-34-B: 9.81 mg).

[1250] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1251] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1252] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1253] The structural characterization data for 5-34-A are as follows:

[1254] 1 H NMR (400MHz, DMSO-d6) δ8.53(d,J=9.0Hz,1H),7.89(d,J=12.3Hz,1H),7.33(s,1H),5.58(q,J=7.5,7.0Hz,1H),5.42(d,J=2.1Hz,2H),5. 25–5.09(m,2H),3.98(s,2H),3.96(d,J=1.1Hz,3H),3.30–3.10(m,2H),2.15(q,J=7.4Hz,2H),1.93–1.80(m,2H),0.87(t,J=7.3Hz,3H).

[1255] ESI-MS (m / z): 510.2 [M+H] + .

[1256] The structural characterization data for 5-34-B are as follows:

[1257] 1H NMR (400MHz, DMSO-d6) 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).

[1258] ESI-MS (m / z): 510.2 [M+H] + .

[1259] Example 24 (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine & (2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[1260]

[1261] Step 1: Synthesis of (2R)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine & (2R)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[1262] (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]indolezine[1,2-b]quinoline-10,13-dione (50 mg, 110.64 μmol) and (2R)-2-hydroxypropionic acid (19.93 mg, 221.29 μmol) were dissolved in N,N-dimethylformamide. The amide (2 mL) was then added, followed by the addition of HATU (84.09 mg, 221.29 μmol) and N,N-diisopropylethylamine (42.90 mg, 331.93 μmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The reaction solution was directly purified by preparative HPLC to obtain two isomers of the title compound (2-27-A: 5.73 mg, 2-27-B: 7.59 mg).

[1263] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1264] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1265] 0.00 30 90 28 2.00 30 90 28 18.00 90 10 28

[1266] The structural characterization data for 2-27-A are as follows:

[1267] 1 H NMR(400MHz,DMSO-d6)δ8.45(d,J=9.1Hz,1H),8.14(s,1H),7.30(s,1H),6.54(s ,1H),5.63(s,1H),5.56(q,J=8.0Hz,1H),5.42(s,2H),5.25(d,J=19.0Hz,1H),5 .08(d,J=19.0Hz,1H),4.13(q,J=6.7Hz,1H),3.27–3.12(m,2H),2.51(s,3H),2. 23–2.13(m,2H),1.92–1.80(m,2H),1.41(d,J=6.8Hz,3H),0.87(t,J=7.3Hz,3H).

[1268] ESI-MS (m / z): 524.2 [M+H] + .

[1269] The structural characterization data for 2-27-B are as follows:

[1270] 1H NMR (400MHz, DMSO-d6) δ8.40(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.55–5.49(m,1H),5.43(d,J=2.2Hz,2H),5.18(q,J=19.0H z,2H),4.13(q,J=6.6Hz,1H),3.24–3.12(m,2H),2.51(s,3H),2.22–2.10 (m,2H),1.92–1.82(m,2H),1.30(d,J=6.7Hz,3H),0.88(t,J=7.3Hz,3H).

[1271] ESI-MS (m / z): 524.2 [M+H] + .

[1272] Example 25: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine and (R)-N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolezine[1,2-b]quinoline-1-yl)-2-hydroxypropylamine

[1273]

[1274] At 25°C, 3-1-A (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added. The mixture was reacted at room temperature for 2 hours. Most of the DMF was concentrated from the reaction solution, and the residue was purified by preparative high performance liquid chromatography to obtain compound 3-26-A (15.4 mg, yield 64%).

[1275] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1276] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1277] Retention time: 5.3–6.2 min

[1278] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

[1279] The structural characterization data are as follows:

[1280] MS m / z (ESI): 528.2 [M+H] +

[1281] 1 H NMR (400MHz, DMSO-d6) δ8.52(d,J=9.2Hz,1H),8.05(d,J=10.0Hz,1H),7.33(s,1H),6.55(s,1H),5.62–5.59(m,2H),5.43(s,2H),5.28–5.10(m,2 H),4.13–4.11(m,1H),3.41–3.38(m,1H),3.28–3.22(m,1H),2.20–2.18 (m,2H),1.92–1.80(m,2H),1.40(d,J=6.8Hz,3H),0.87(t,J=7.2Hz,3H).

[1282] At 25°C, 3-1-B (20.0 mg, 43.9 μmol) and D-lactic acid (7.90 mg, 87.8 μmol) were dissolved in DMF (1.0 mL), and then HATU (33.4 mg, 87.8 μmol) and DIPEA (17.0 mg, 131.6 μmol) were added. The mixture was reacted at room temperature for 2 hours. Most of the DMF was concentrated from the reaction solution, and the residue was purified by preparative high performance liquid chromatography to obtain compound 3-26-B (4.8 mg, yield 20%).

[1283] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[1284] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1285] Retention time: 7.5–8.5 min

[1286] 0.00 30 70 28 4.00 30 70 28 20.00 90 10 28

[1287] The structural characterization data are as follows:

[1288] MS m / z (ESI): 528.2 [M+H] +

[1289] 1H NMR(400MHz,DMSO-d6)δ8.48(d,J=8.8Hz,1H),8.06(d,J=10.4Hz,1H),7.34(s,1 H),6.57(br,1H),5.61–5.55(m,1H),5.48–5.39(m,2H),5.27–5.16(m,2H),4.15–

[1290] 4.10(m,1H),3.32–3.21(m,3H),2.23–2.16(m,2H),1.92–1.81(m,2H),1.30(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[1291] Biological evaluation

[1292] I. Tumor Cell Proliferation Inhibition Test

[1293] 1. Inhibitory effect of the compound on the proliferation of HT29 cells

[1294] (1) Cell plating: First, tumor cells HT29 were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 1.

[1295] Table 1. Tumor cell origin

[1296] HT29 Human colon cancer cells Cell Bank of Chinese Academy of Sciences

[1297] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1298] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50 μL, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 2.

[1299] (2) Data Results

[1300] Table 2. Inhibitory activity of HT29 cell proliferation

[1301]

[1302]

[1303] 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.

[1304] 2. Inhibitory effect of the compound on the proliferation of A549 cells

[1305] (1) Cell plating: First, tumor cells A549 were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 3.

[1306] Table 3. Tumor cell origin

[1307] A549 Human lung cancer cells Cell Bank of Chinese Academy of Sciences

[1308] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1309] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50 μL, mix thoroughly by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting the curve according to the four-parameter model. The detection results are shown in Table 4.

[1310] (2) Data Results

[1311] Table 4. Inhibitory activity of A549 cell proliferation

[1312] Comparative compound 1 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

[1313] The test results show that the compounds of the present invention listed in Table 4 have a significant inhibitory effect on the proliferation of A549 human lung cancer cells.

[1314] 3. Inhibitory effect of the compound on the proliferation of HCC1806 cells

[1315] (1) Cell plating: First, HCC1806 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 5.

[1316] Table 5. Tumor cell origin

[1317] HCC1806 Human breast squamous cell carcinoma ATCC

[1318] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1319] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50 μL, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 6.

[1320] (2) Data Results

[1321] Table 6. Inhibitory activity against HCC1806 cell proliferation

[1322] Comparative compound 1 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 5 (2-12-C) 2.08 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 24 (2-27-A) 4.55 Example 24 (2-27-B) 3.93 Example 25 (3-26-A) 1.27 Example 25 (3-26-B) 4.93

[1323] The test results show that the compounds of the present invention listed in Table 6 have a significant inhibitory effect on the proliferation of HCC1806 human breast squamous cell carcinoma cells.

[1324] 4. Inhibitory effect of the compound on SKOV-3 cell proliferation

[1325] (1) Cell plating: First, SKOV-3 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 7.

[1326] Table 7. Tumor cell origin

[1327] SKOV-3 Human ovarian cancer cells Nanjing Kebai Biotechnology

[1328] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1329] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50μl, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting the curve according to the four-parameter model. The detection results are shown in Table 8.

[1330] (2) Data Results

[1331] Table 8. Inhibitory activity of SKOV-3 cell proliferation

[1332] Comparative compound 1 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

[1333] The test results show that the compounds of the present invention listed in Table 8 have a significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

[1334] 5. Inhibitory effect of the compound on the proliferation of NCI-H358 cells

[1335] (1) Cell plating: First, NCI-H358 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 9.

[1336] Table 9. Tumor cell origin

[1337] NCI-H358 Human non-small cell lung cancer cells Nanjing Kebai Biotechnology

[1338] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1339] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50μl, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 10.

[1340] (2) Data Results

[1341] Table 10. Inhibitory activity against NCI-H358 cell proliferation

[1342] Comparative compound 1 58.84 Example 1 (1-1-A) 65.22 Example 2 (1-7-A) 11.38 Example 2 (1-7-B) 51.52 Example 3 (2-1-A) 68.62 Example 4 (2-7-A) 35.42 Example 4 (2-7-B) 51.06 Example 5 (2-12-A) 17.65 Example 5 (2-12-B) 15.76 Example 5 (2-12-C) 23.08 Example 7 (2-20-A) 18.56 Example 12 (5-13-A) 7.01 Example 16 (3-12-A) 15.45 Example 16 (3-12-B) 19.77 Example 16 (3-12-C) 28.29 Example 16 (3-12-D) 20.75 Example 17 (3-7-A) 49.38 Example 17 (3-7-B) 22.22 Example 18 (3-17-A) 13.34 Example 18 (3-17-B) 37.34 Example 19 (5-22-A) 29.94 Example 19 (5-22-B) 61.36 Example 21 (4-14) 11.98 Example 22 (4-15) 17.76 Example 24 (2-27-A) 56.46 Example 24 (2-27-B) 69.12 Example 25 (3-26-A) 31.64

[1343] The test results show that the compounds of the present invention listed in Table 10 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[1344] 6. Inhibitory effect of the compound on the proliferation of NCI-N87 cells

[1345] (1) Cell plating: First, NCI-N87 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 11.

[1346] Table 11. Tumor cell origin

[1347] NCI-N87 Human stomach cancer cells ATCC

[1348] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1349] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM2.0 Reagent (Vazyme / Novozyme) 50μL, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting the curve according to the four-parameter model. The detection results are shown in Table 12.

[1350] (2) Data Results

[1351] Table 12. Inhibitory activity of NCI-N87 cell proliferation

[1352]

[1353]

[1354] The test results show that the compounds of the present invention listed in Table 12 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

[1355] 7. Inhibitory effect of the compound on HeLa cell proliferation

[1356] (1) Cell plating: First, HeLa tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The sources of tumor cells are shown in Table 13.

[1357] Table 13. Tumor cell origin

[1358] Hela Human cervical cancer cells Nanjing Kebai Biotechnology

[1359] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1360] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50μL, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TMBackground RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 14.

[1361] (2) Data Results

[1362] Table 14. HeLa cell proliferation inhibitory activity

[1363]

[1364]

[1365] The test results show that the compounds of the present invention listed in Table 14 have a significant inhibitory effect on the proliferation of HeLa human cervical cancer cells.

[1366] 8. Inhibitory effect of the compound on the proliferation of HCC70 cells

[1367] (1) Cell plating: First, HCC70 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 15.

[1368] Table 15. Tumor cell origin

[1369] HCC70 Human breast cancer cells Nanjing Kebai Biotechnology

[1370] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1371] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50μl, mix well by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TMThe solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 16.

[1372] (2) Data Results

[1373] Table 16. Inhibitory activity against HCC70 cell proliferation

[1374] Comparative compound 1 226.10 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

[1375] The test results show that the compounds of the present invention listed in Table 16 have a significant inhibitory effect on the proliferation of HCC70 human breast cancer cells.

[1376] 9. Inhibitory effect of the compound on the proliferation of MDA-MB-231 cells

[1377] (1) Cell plating: First, tumor cells MDA-MB-231 were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 17.

[1378] Table 17. Tumor cell origin

[1379] MDA-MB-231 Human breast cancer cells Nanjing Kebai Biotechnology

[1380] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1381] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50ml, mix well by shaking in the dark, react for 10 minutes before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 18.

[1382] (2) Data Results

[1383] Table 18. Inhibitory activity of MDA-MB-231 cell proliferation

[1384] Comparative compound 1 381.70 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

[1385] The test results show that the compounds of the present invention listed in Table 18 have a significant inhibitory effect on the proliferation of MDA-MB-231 human breast cancer cells.

[1386] 10. Inhibitory effect of the compound on Jeko-1 cell proliferation

[1387] (1) Cell plating: First, Jeko-1 tumor cells were cultured in the appropriate culture medium. After centrifugation, the cells were resuspended and counted. Finally, the cell concentration was adjusted to a suitable level for plating. The source of tumor cells is shown in Table 19.

[1388] Table 19. Tumor cell origin

[1389]

[1390]

[1391] Co-incubation of the compound of the present invention with tumor cells: After the cells are plated, the diluted bioactive molecule (the compound of the present invention) is added to the wells of the plate according to a certain dilution ratio and incubated for 72 hours.

[1392] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50 μL, mix thoroughly by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated according to the curve fitted by the four-parameter model. The detection results are shown in Table 20.

[1393] (2) Data Results

[1394] Table 20. Jeko-1 cell proliferation inhibitory activity

[1395] Comparative compound 1 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

[1396] The test results show that the compounds of the present invention listed in Table 20 have a significant inhibitory effect on the proliferation of Jeko-1 mantle cell lymphoma cells.

[1397] 11. Inhibitory effect of the compound on the proliferation of MDA-MB-453 cells

[1398] (1) Cell plating: First, tumor cells MDA-MB-453 were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cells were adjusted to a suitable concentration for plating. The source of tumor cells is shown in Table 21.

[1399] Table 21. Tumor cell origin

[1400] MDA-MB-453 Human breast cancer cells Conotech

[1401] Co-incubation of the compound of the present invention with tumor cells: After the cells adhere, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells above and incubated for 72 hours.

[1402] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent (Vazyme / Novozyme) 50 μL, mix thoroughly by shaking in the dark, react for 10 min before detection, and read the value using a microplate reader (manufacturer: BMG, model: PheraStar-FS). Use cell-free culture medium (containing Cell Counting-Lite). TM Background RLU was obtained using cell-containing culture medium (containing Cell Counting-Lite). TM The solvent RLU was obtained. Cell inhibition rate = 1 - (sample RLU - sample RLU) / (solvent RLU - background RLU) × 100%. The half-maximal inhibitory concentration (IC50) of the compound was calculated by fitting a curve using a four-parameter model. 50 The test results are shown in Table 22.

[1403] (2) Data Results

[1404] Table 22. Inhibitory activity of MDA-MB-453 cell proliferation

[1405] Comparative compound 1 6.99 Example 16 (3-12-A) 0.47 Example 16 (3-12-B) 4.32 Example 16 (3-12-C) 5.52 Example 16 (3-12-D) 3.98 Example 17 (3-7-A) 6.23 Example 17 (3-7-B) 4.87 Example 18 (3-17-A) 3.32 Example 18 (3-17-B) 6.16 Example 19 (5-22-A) 7.34 Example 19 (5-22-B) 5.67 Example 24 (2-27-A) 11.83 Example 25 (3-26-A) 3.95

[1406] The test results show that the compounds of the present invention listed in Table 22 have a significant inhibitory effect on the proliferation of MDA-MB-453 human breast cancer cells.

[1407] The structures of comparative compound 1 and comparative compound 2 are shown below:

[1408]

[1409] II. Antibody Conjugation Assay

[1410] The coupling preparation of the ADC DL-15 sample is as follows:

[1411] Take 1.036 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg / mL), dilute with 0.1 M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 2.4 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6), mix well, and incubate at room temperature for 90 min. Add 5 times the amount of compound DL-15 dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. After that, 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, and obtain the antibody-drug conjugate ADC DL-15 (1.77 mL, 8.60 mg / mL).

[1412]

[1413] The molecular weight of ADC DL-15 was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated to be 4.11, as shown in Tables 23 and 24.

[1414] Table 23: Measured molecular weight of ADC DL-15

[1415]

[1416] Table 24: DAR values ​​of ADC DL-15

[1417]

[1418] Chromatographic determination conditions:

[1419] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[1420] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[1421] Flow rate: 0.25 mL / min; Sample chamber temperature: 8℃; Column temperature: 60℃; Injection volume: 2 μL;

[1422]

[1423]

[1424] Mass spectrometry determination conditions:

[1425] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[1426] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time0.5s; m / z 600-4000; Time bins to sum 40.

[1427] The coupling preparation of the ADC 3-4-04-A sample is as follows:

[1428] Take 0.518 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg / mL), dilute with 0.1 M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 5.5 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) to the above solution system, mix well, and let stand at room temperature for 90 min. Add 10 times the amount of compound 3-4-04-A 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 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), then add sucrose and Tween 20, mix well, and obtain the antibody-drug conjugate ADC 3-4-04-A (1.50 mL, 5.60 mg / mL).

[1429]

[1430] The molecular weight of ADC 3-4-04-A was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated to be 7.47, as shown in Tables 25 and 26. The chromatographic conditions were the same as those for ADC DL-15.

[1431] Table 25: Measured molecular weight of ADC 3-4-04-A

[1432]

[1433] Table 26: DAR values ​​of ADC 3-4-04-A

[1434]

[1435]

[1436] The coupling preparation of the ADC 3-4-04-B sample is as follows:

[1437] Take 0.518 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg / mL), dilute with 0.1 M disodium edetate solution (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 5.5 times the amount of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) to the above solution system, mix well, and let stand at room temperature for 90 min. Add 10 times the amount of compound 3-4-04-B 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 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), then add sucrose and Tween 20, mix well, and obtain the antibody-drug conjugate ADC 3-4-04-B (1.50 mL, 5.60 mg / mL).

[1438]

[1439] The molecular weight of ADC 3-4-04-B was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated to be 8.04, as shown in Tables 27 and 28. The chromatographic conditions were the same as those for ADC DL-15.

[1440] Table 27: Measured molecular weight of ADC 3-4-04-B

[1441]

[1442] Table 28: DAR values ​​of ADC 3-4-04-B

[1443]

[1444] The above experiments demonstrate that the cytotoxic drug-linker compounds of the present invention can be successfully conjugated with antibodies to obtain antibody-drug conjugates.

[1445] Although specific embodiments of the 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 disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound having the following structure: 。 2. A compound or a pharmaceutically acceptable salt thereof, said compound having the following structure: 。 3. A pharmaceutical composition comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

4. A medicine box product comprising: a) at least one compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof as a first therapeutic agent, or a pharmaceutical composition of claim 3; b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a pharmaceutical composition comprising another therapeutic agent as a second pharmaceutical composition; and c) Optional packaging and / or instructions.

5. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 3, or the kit product of claim 4 in the preparation of a medicament for treating diseases of abnormal cell proliferation.

6. The use as described in claim 5, wherein, The disease described as involving abnormal cell proliferation is tumor.

7. The use as described in claim 5, wherein, The disease involving abnormal cell proliferation is an advanced solid tumor.

8. The use as described in claim 5, wherein, The diseases involving abnormal cell proliferation include brain tumors, lung cancer, bladder cancer, gastric cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, lymphoma, neurofibroma, bone cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, or sarcoma.

9. The use as described in claim 5, wherein, The diseases involving abnormal cell proliferation are squamous cell carcinoma, skin cancer, thyroid cancer, colon cancer, or glioma.

10. The use as described in claim 5, wherein, The diseases involving abnormal cell proliferation are ovarian cancer, cervical cancer, or endometrial cancer.

11. The use as described in claim 5, wherein, The disease involving abnormal cell proliferation is carcinoma in situ.

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