CN113527299B

CN113527299B - Nitrogen-containing condensed ring compound, preparation method and application

Info

Publication number: CN113527299B
Application number: CN202010747478.6A
Authority: CN
Inventors: 万惠新; 查传涛; 马金贵; 潘建峰
Original assignee: Shanghai Lingda Biomedical Co ltd
Current assignee: Shanghai Lingda Biomedical Co ltd
Priority date: 2020-04-18
Filing date: 2020-07-29
Publication date: 2023-12-29
Anticipated expiration: 2040-07-29
Also published as: CN113527299A

Abstract

The invention discloses a nitrogen-containing condensed ring compound, a preparation method and application thereof, in particular to a nitrogen-containing condensed ring compound shown in a general formula I, or pharmaceutically acceptable salt thereof, or enantiomer, diastereoisomer, tautomer, torsional isomer, solvate, polymorph or prodrug thereof, a preparation method thereof and application thereof in pharmacy, wherein the definition of each group is as described in the specification.

Description

Nitrogen-containing condensed ring compound, preparation method and application

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a nitrogen-containing condensed ring compound, a compound for inhibiting activity of Ras mutein, a preparation method and application.

Background

RAS is the first oncogene identified in human tumors, and was first discovered in two murine sarcoma viruses. The RAS gene family has three members, hras, kras, nras respectively. In human tumors, kras mutations are most common, accounting for about 85%. Previous studies have shown that Kras mutations are oncogenic because of missense mutation at codon 12, altering the structure of the Kras protein and allowing it to remain active. Ras plays a major role in signaling pathway in activating kinases that control gene transcription, thereby regulating cell differentiation and proliferation, and is intimately involved in tumor cell survival, proliferation, migration, metastasis, and angiogenesis. It is counted that there are Kras G12C mutations in 11% -16% of cases of lung adenocarcinoma, and that some of pancreatic cancer, colorectal cancer, ovarian cancer and cholangiocarcinoma are caused by Kras mutations. However, since the first discovery of Kras oncogenes has been over thirty years, targeting drugs for common proto-oncogenes such as EGFR, BCL, etc. have been used for several generations, and targeting drugs for Kras have not been successfully developed all the time. Targeting drugs against KRas pathway mutant tumors have been mainly focused on farnesyl transferase inhibitors and Raf-MEK pathway inhibitors, but have had little effect. In recent years, inhibitors against KRas specific gene mutation have been developed as hot spots, and part of the inhibitors gradually move from preclinical hatching to clinical researches, such as KRas G12C inhibitor AMG510, MRTX1257, and the like, and show a certain curative effect in early clinical experiments. The first clinical data of the global first-line KRASG12C inhibitor AMG510 was formally published by the american clinical oncology institute held at month 6 of 2019, in which the advanced drug AMG510 was shown to be able to prevent tumor growth in most non-small cell lung and colorectal cancer patients with KRas mutations. Therefore, finding and searching for targeted drugs with high specificity and excellent pharmaceutical properties for KRAS specific mutant genes becomes a great hotspot in the industry.

Disclosure of Invention

One of the technical problems to be solved by the invention is to provide a novel KRAS G12C inhibitor for preparing tumor therapeutic drugs.

The scheme for solving the technical problems is as follows:

a nitrogen-containing fused ring compound represented by the general formula I, or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, stereoisomer, solvate, polymorph or prodrug thereof,

wherein:

r1 is independently selected from hydrogen, halogen, cyano, nitro, C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-SO ₂ -、C ₁ -C ₆ alkyl-SO-, or C ₁ -C ₆ Haloalkyl；

R2, R3 are independently selected from hydrogen, halogen, cyano, nitro, C ₁ -C ₆ Alkyl, C ₁ -C ₆ alkyl-SO ₂ -、C ₁ -C ₆ alkyl-SO-, N (R) _2a )(R _2b )-(CH ₂ ) X-; alternatively, R ₉ And R is ₁₀ C which together form a 5-10 membered quilt ₁ -C ₆ Alkyl-substituted nitrogen-containing heterocycloalkyl; wherein R is _2a And R is _2b Each independently selected from hydrogen or C ₁ -C ₆ Alkyl, x is selected from any integer from 0 to 5;

w, W1, W2, M is independently selected from CR4 or N, R4 is independently selected from H, halogen, cyano, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Alkoxy, haloalkyl, haloalkoxy, alkenyl, alkynyl, alkyl ether, cycloalkyl ether, heterocycloalkyl ether, alkyl sulfide, cycloalkyl sulfide, heterocycloalkyl sulfide, 3-8 membered cycloalkyl or heterocycloalkyl, alkylamino substituted alkoxy, alkylamino substituted alkylamino, cycloalkylalkylene ether, heterocycloalkylalkylene ether, cycloalkylalkylene amino, heterocycloalkylalkylene amino, and the like;

Ra, rb, rc, rd, re, rf, rg are each independently selected from hydrogen, C1-C6 alkyl, alkoxy, haloalkyl, etc., or Ra, rb, rc, rd, re, rf, and Rg form a 3-8 membered saturated or partially unsaturated ring system therebetween;

ar is independently selected from 5-12 membered aromatic rings or aromatic condensed rings, 5-12 membered aromatic heterocyclic rings or aromatic condensed rings; and the Ar ring may be substituted with one or more of the following groups: hydrogen, halogen, C1-C6 alkyl, alkoxy, 3-8 membered cycloalkyl or heterocycloalkyl, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted amino, amido, sulfonamide, and the like;

cy is independently selected from 4-8 membered saturated or partially unsaturated ring systems or aromatic ring systems;

one or more hydrogen atoms on any of the above groups may be substituted with a substituent selected from the group consisting of: including but not limited to deuterium, halogen C1-C8 alkyl; wherein the heteroaryl group comprises 1 to 3 heteroatoms selected from the group consisting of: n, O, P or S, said heterocycloalkyl comprising 1 to 3 heteroatoms selected from the group consisting of: n, O, P or S, said ring system comprising a spiro, bridged, fused, or other saturated or partially unsaturated ring system.

In some embodiments, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, torsional isomer, solvate, polymorph or prodrug thereof, is preferably a compound of formula (IIA), (IIB), (IIC), (IID), (IIE), or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug thereof:

Wherein n=1-5, R5 is independently selected from hydrogen, halogen, C1-C6 alkyl, alkoxy, haloalkyl, haloalkoxy, amino, substituted amino, cyano, and the like; r1, R2, R3, ra, rb, rc, rd, re, rf, rg, W, W1, W2, M, ar are as defined above.

In other embodiments, a compound of formula (1), or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, torsional isomer, solvate, polymorph or prodrug thereof, is characterized by:

r1 is preferably selected from hydrogen, fluorine, methyl, cyano, etc.;

ra, rb, rc, rd, re, rf, rg are each independently selected from hydrogen, fluoro, methyl, cyanomethylene;

w, W1, W2 are each independently preferably from CR4, R4 is independently selected from hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, and the like;

m is independently preferably selected from N OR CH, C-CN, C-OR5, R5 is preferably selected from C1-C6 alkyl, alkoxyalkylene, alkylaminoalkylene, cycloalkylalkylene, heterocycloalkylalkylene, and the like;

ar is independently preferably selected from a monocyclic aromatic group such as a substituted or unsubstituted phenyl group and a pyridyl group, or a bicyclic aromatic group such as a substituted or unsubstituted naphthyl group, naphthyridinyl group, indazolyl group, benzimidazolyl group, etc.; the one or more substituents are preferably selected from the group consisting of: hydrogen, halogen, C1-C4 alkyl, hydroxy, amino, cyano, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, and the like;

Cy is as defined above.

A process for the preparation of a compound of formula I, comprising steps a-c:

a) Converting a compound of formula (a) with an arylboronic acid or arylboronic acid ester or arylmetal reagent (Ar-M) to an intermediate (B) by a transition metal catalyzed coupling reaction; and

b) Removing a protecting group PG from the compound of the general formula (B) through a conventional functional group to obtain a compound of the general formula (C);

c) The compound of the general formula (C) and acrylic acid or acrylic acid chloride are subjected to condensation reaction under proper conditions to generate the general formula (I).

The definition of each group is as above;

preferably, each of said steps a), b), c) is performed in a solvent, and said solvent is selected from the group consisting of: water, methanol, ethanol, isopropanol, butanol, ethylene glycol methyl ether, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran, toluene, methylene chloride, 1, 2-dichloroethane, acetonitrile, N-dimethylformamide, N-dimethylacetamide, dioxane, or a combination thereof.

Preferably, the transition metal catalyst is selected from the group consisting of: tris (dibenzylideneacetone) dipalladium (Pd) ₂ (dba) ₃ ) Tetrakis (triphenylphosphine) palladium (Pd (PPh) ₃ ) ₄ ) Palladium acetate, palladium chloride, dichloro-bis (triphenylphosphine) palladium, trifluoropalladium acetate, triphenylphosphine palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ]Palladium dichloride and bis (tri-o-phenyl methylphosphine) palladium dichloride1, 2-bis (diphenylphosphino) ethane palladium dichloride, or a combination thereof; the catalyst ligand is selected from the group consisting of: tri-tert-butylphosphine, tri-tert-butylphosphine tetrafluoroborate, tri-n-butylphosphine, triphenylphosphine, tri-p-benzylphosphine, tricyclohexylphosphine, tri-o-benzylphosphine, or a combination thereof.

Preferably, the condensing agent is selected from the group consisting of: DCC, DIC, CDI, EDCI, HOAt, HOBt, BOP, pyBOP, HATU, TBTU, etc., or a combination thereof.

Preferably, the inorganic base is selected from the group consisting of: sodium hydride, potassium hydroxide, sodium acetate, potassium t-butoxide, sodium t-butoxide, potassium fluoride, cesium fluoride, potassium phosphate, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, or a combination thereof; the organic base is selected from the group consisting of: pyridine, triethylamine, N, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU), lithium hexamethyldisilazide, sodium hexamethyldisilazide, lutidine, or combinations thereof.

Preferably, the acid is selected from the group consisting of: hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, formic acid, acetic acid, trifluoromethanesulfonic acid or combinations thereof.

Preferably, the reducing agent is selected from the group consisting of: iron powder, zinc powder, stannous chloride, sodium thiosulfate, sodium sulfite, hydrogen and the like.

The invention provides a class of preferred compounds of formula (I) including, but not limited to, the following structures:

it is another object of the present invention to provide a medicament for treating or preventing tumors and a composition thereof. The technical scheme for achieving the purpose is as follows:

a pharmaceutical composition for treating tumor comprises nitrogen-containing condensed ring compound shown in the general formula (I), or pharmaceutically acceptable salt, enantiomer, diastereomer, tautomer, torsion isomer, solvate, polymorph or prodrug thereof and pharmaceutically acceptable carrier.

It is a further object of the present invention to provide the use of the above compounds. The technical scheme for achieving the purpose is as follows:

the nitrogen-containing condensed ring compound shown in the general formula (I), or pharmaceutically acceptable salt, enantiomer, diastereomer, tautomer, torsional isomer, solvate, polymorph or prodrug thereof are used for preparing medicaments for treating diseases related to activity or expression quantity of Ras mutant protein, in particular to medicaments for treating tumors. The tumor is independently selected from non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, liver cancer, skin cancer, gastric cancer, intestinal cancer, bile duct cancer, brain cancer, leukemia, lymphoma, fibroma, sarcoma, basal cell carcinoma, glioma, renal cancer, melanoma, bone cancer, thyroid cancer, nasopharyngeal cancer, pancreatic cancer, etc.

The invention relates to a compound with the structural characteristics of a general formula (I), which can inhibit various tumor cells, especially can efficiently kill tumors related to abnormal KRAS G12C mutant protein signal paths, and is a therapeutic drug with a brand-new action mechanism.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. The limited space is not described in any more detail herein.

Detailed Description

The inventor has studied deeply for a long time to prepare a compound with a novel structure shown in a formula I, and found that the compound has better KRAS G12C protein inhibition activity, and the compound has specific inhibition effect on KRAS G12C protein at a very low concentration (which can be as low as less than 100 nM), and quite excellent cell proliferation inhibition activity related to KRAS G12C, and the compound has stronger killing effect on KRAS G12C positive tumor cells at a very low concentration (which can be as low as less than 100 nM), so that the compound can be used for treating related diseases such as tumors caused by KRAS G12C mutation or abnormal expression quantity. Based on the above findings, the inventors have completed the present invention.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety unless otherwise indicated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the inventive subject matter. In this application, the singular is used to include the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the use of "or" means "and/or" unless stated otherwise. Furthermore, the terms "include," as well as other forms, such as "comprising," "including," and "containing," are not limiting.

The definition of standard chemical terms can be found in references (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY TH ED." vols. A (2000) and B (2001), plenum Press, new York). Conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy, and pharmacological methods are employed unless otherwise indicated. Unless specifically defined otherwise, the terms used herein in the description of analytical chemistry, organic synthetic chemistry, and pharmaceutical chemistry are known in the art. Standard techniques may be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. For example, the reaction and purification can be carried out using the manufacturer's instructions for the kit, or in a manner well known in the art or in accordance with the teachings of the present invention. The techniques and methods described above may generally be practiced according to conventional methods well known in the art, based on a number of general and more specific descriptions in the literature cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by one skilled in the art to provide stable moieties and compounds.

When substituents are described by conventional formulas written from left to right, the substituents also include chemically equivalent substituents obtained when writing formulas from right to left. For example, -CH 2O-is equivalent to-OCH 2-.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals, and treatises, are hereby incorporated by reference in their entirety.

Certain chemical groups defined herein are preceded by a simplified symbol to indicate the total number of carbon atoms present in the group. For example, C1-6 alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the reduced notation does not include carbon that may be present in a substituent of the group.

In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings indicated below, unless specifically indicated otherwise.

In the present application, the term "halogen" refers to fluorine, chlorine, bromine or iodine; "hydroxy" refers to an-OH group; "hydroxyalkyl" refers to an alkyl group as defined below substituted with a hydroxy (-OH); "carbonyl" refers to a-C (=o) -group; "nitro" means-NO ₂ The method comprises the steps of carrying out a first treatment on the surface of the "cyano" refers to-CN; "amino" means-NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the "substituted amino" refers to an amino group substituted with one or two alkyl, alkylcarbonyl, aralkyl, heteroaralkyl groups as defined below, e.g., mono-, di-, alkylamido, aralkylamino, heteroaralkylamino; "carboxy" refers to-COOH.

In the present application, as part of a group or other group (e.g. as used in halogen substituted alkyl groups or the like), the term "alkyl" means a straight or branched hydrocarbon chain group consisting of only carbon atoms and hydrogen atoms, free of unsaturated bonds, having, for example, from 1 to 12 (preferably from 1 to 8, more preferably from 1 to 6) carbon atoms and being linked to the remainder of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, decyl and the like.

In the present application, the term "alkenyl" as part of a group or other group means a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, containing at least one double bond, having, for example, 2 to 14 (preferably 2 to 10, more preferably 2 to 6) carbon atoms and being linked to the rest of the molecule by a single bond, such as, but not limited to, ethenyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1, 4-dienyl, and the like.

In this application, the term "alkynyl" as part of a group or other group means a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, containing at least one triple bond and optionally one or more double bonds, having, for example, 2 to 14 (preferably 2 to 10, more preferably 2 to 6) carbon atoms and being attached to the remainder of the molecule by a single bond, such as, but not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-en-4-ynyl, and the like.

In this application, as part of a group or other group, the term "cycloalkyl" means a stable, non-aromatic, mono-or polycyclic hydrocarbon group consisting of only carbon and hydrogen atoms, which may include fused ring systems, bridged ring systems, or spiro ring systems, having from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms, more preferably from 3 to 8 carbon atoms, and which is saturated or unsaturated and may be attached to the remainder of the molecule by a single bond via any suitable carbon atom. Unless otherwise specifically indicated in the specification, carbon atoms in cycloalkyl groups may optionally be oxidized. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexanyl, cyclooctyl, 1H-indenyl, 2, 3-indanyl, 1,2,3, 4-tetrahydro-naphthyl, 5,6,7, 8-tetrahydro-naphthyl, 8, 9-dihydro-7H-benzocyclohepten-6-yl, 6,7,8, 9-tetrahydro-5H-benzocycloheptenyl, 5,6,7,8,9, 10-hexahydro-benzocyclooctenyl, fluorenyl, bicyclo [2.2.1] heptyl, 7-dimethyl-bicyclo [2.2.1] heptyl, bicyclo [2.2.1] heptenyl, bicyclo [ 2.2.2.2 ] octyl, bicyclo [3.1.1] heptyl, bicyclo [3.2.1] octyl, bicyclo [2.2.2] octenyl, bicyclo [ 2.1.1 ] octadienyl, adamantylene, and the like.

In the present application, the term "heterocyclyl" as part of a group or other group means a stable 3-to 20-membered non-aromatic cyclic group consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, phosphorus, oxygen and sulfur. Unless specifically indicated otherwise in the present specification, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic or more cyclic ring system, which may include fused, bridged or spiro ring systems; the nitrogen, carbon or sulfur atoms in the heterocyclyl may optionally be oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclyl may be partially or fully saturated. The heterocyclic group may be attached to the remainder of the molecule via a carbon atom or a heteroatom and by a single bond. In heterocyclyl groups containing fused rings, one or more of the rings may be aryl or heteroaryl as defined below, provided that the point of attachment to the remainder of the molecule is a non-aromatic ring atom. For the purposes of the present invention, heterocyclyl groups are preferably stable 4-to 11-membered non-aromatic monocyclic, bicyclic, bridged or spiro groups comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably stable 4-to 8-membered non-aromatic monocyclic, bicyclic, bridged or spiro groups comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of heterocyclyl groups include, but are not limited to: pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2, 7-diaza-spiro [3.5] nonan-7-yl, 2-oxa-6-aza-spiro [3.3] heptan-6-yl, 2, 5-diaza-bicyclo [2.2.1] heptan-2-yl, azetidinyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxacyclopentyl, tetrahydroisoquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, quinolizinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, indolinyl, octahydroindolyl, octahydroisoindolyl, pyrrolidinyl, pyrazolidinyl, phthalimidyl, and the like.

In this application, the term "aryl" as part of a group or other group means a conjugated hydrocarbon ring system group having 6 to 18 carbon atoms, preferably having 6 to 10 carbon atoms. For the purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic or more ring systems, and may also be fused to cycloalkyl or heterocyclyl groups as defined above, provided that the aryl groups are linked to the remainder of the molecule by single bonds via atoms on the aromatic ring. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, fluorenyl, 2, 3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) -one-7-yl, and the like.

In the present application, the term "arylalkyl" refers to an alkyl group as defined above substituted with an aryl group as defined above.

In this application, as part of a group or other group, the term "heteroaryl" means a 5-to 16-membered conjugated ring system group having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur within the ring. Unless otherwise specifically indicated in the present specification, heteroaryl groups may be monocyclic, bicyclic, tricyclic or more ring systems, and may also be fused to cycloalkyl or heterocyclyl groups as defined above, provided that heteroaryl groups are attached to the remainder of the molecule via an atom on an aromatic ring by a single bond. The nitrogen, carbon, or sulfur atoms in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. For the purposes of the present invention, heteroaryl groups are preferably stable 5-to 12-membered aromatic groups comprising 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably stable 5-to 10-membered aromatic groups comprising 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur or 5-to 6-membered aromatic groups comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothienyl, oxatriazolyl, cinnolinyl, quinazolinyl, thiophenyl, indolizinyl, phenanthroline, isoxazolyl, phenoxazinyl, phenothiazinyl, 4,5,6, 7-tetrahydrobenzo [ b ] thienyl, naphthyridinyl, [1,2,4] triazolo [4, 3-triazolo [1, 4] pyridazine, 3-1, 4-imidazo [1, 4] triazolo [1, 4, 3-triazolo [1, 4] pyridazine, 3-1, 4-imidazo [ 2,4] a ] 1, 4-imidazo [ 2, 4-a ] and the like.

In the present application, the term "heteroarylalkyl" refers to an alkyl group as defined above substituted with a heteroaryl group as defined above.

In this application, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted aryl groups and unsubstituted aryl groups.

The terms "moiety", "structural moiety", "chemical moiety", "group", "chemical group" as used herein refer to a particular fragment or functional group in a molecule. Chemical moieties are generally considered to be chemical entities that are embedded or attached to a molecule.

"stereoisomers" refer to compounds that consist of the same atoms, are bonded by the same bonds, but have different three-dimensional structures. The present invention is intended to cover various stereoisomers and mixtures thereof.

When an olefinic double bond is contained in the compounds of the present invention, the compounds of the present invention are intended to include both E-and Z-geometric isomers unless otherwise specified.

"tautomer" refers to an isomer formed by the transfer of a proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the invention are also intended to be included within the scope of the invention.

The compounds of the invention or pharmaceutically acceptable salts thereof may contain one or more chiral carbon atoms and thus may be produced in enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R) -or (S) -, based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the invention may be prepared by selecting racemates, diastereomers or enantiomers as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

Conventional techniques for preparing/separating individual isomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography, see, for example, gerald gabiz and Martin g.schmid (eds.), chiral Separations, methods and Protocols, methods in Molecular Biology, vol.243,2004; m.stalcup, chiral Separations, annu.rev.animal.chem.3:341-63, 2010; fumigs et al (EDs.), VOGEL' S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY.sup.TH ED, longman Scientific and Technical Ltd., essex,1991,809-816; heller, acc.chem.Res.1990,23,128.

In the present application, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formate, acetate, 2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

By "pharmaceutically acceptable base addition salt" is meant a salt formed with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including natural substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

"polymorphs" refer to the different solid crystalline phases of certain compounds of the present invention in the solid state due to the presence of two or more different molecular arrangements. Certain compounds of the present invention may exist in more than one crystal form, and the present invention is intended to include various crystal forms and mixtures thereof.

In general, crystallization will produce solvates of the compounds of the present invention. The term "solvate" as used herein refers to an aggregate comprising one or more molecules of a compound of the invention and one or more solvent molecules. The solvent may be water, in which case the solvate is a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like, as well as the corresponding solvated forms. The compounds of the invention may form true solvates, but in some cases may also retain only adventitious water or a mixture of water plus a portion of the adventitious solvent. The compounds of the invention may be reacted in a solvent or precipitated or crystallized from a solvent. Solvates of the compounds of the present invention are also included within the scope of the present invention.

The invention also includes prodrugs of the above compounds. In this application, the term "prodrug" means a compound that can be converted into a biologically active compound of the invention under physiological conditions or by solvolysis. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the invention. Prodrugs may not be active when administered to an individual in need thereof, but are converted in vivo to the active compounds of the present invention. Prodrugs are typically rapidly converted in vivo to the parent compounds of the present invention, for example, by hydrolysis in blood. Prodrug compounds generally provide solubility, histocompatibility, or sustained release advantages in mammalian organisms. Prodrugs include known amino protecting groups and carboxyl protecting groups. Specific methods of prodrug preparation can be found in saunnier, m.g., et al, biorg. Med. Chem. Lett.1994,4,1985-1990; greenwald, r.b., et al, j.med.chem.2000,43,475.

In this application, "pharmaceutical composition" refers to a formulation of a compound of the invention with a medium commonly accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote the administration of organisms, facilitate the absorption of active ingredients and further exert biological activity.

The term "pharmaceutically acceptable" as used herein refers to a material (e.g., carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention, and is relatively non-toxic, i.e., the material can be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition.

In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonizing agent, solvent, or emulsifying agent that is approved by the relevant government regulatory agency as acceptable for human or livestock use.

The "tumor", "cell proliferation abnormality related disease", and the like of the present invention include, but are not limited to, leukemia, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, pancreatic cancer, lung squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, cervical cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, brain cancer, bone cancer, esophageal cancer, melanoma, renal cancer, oral cancer, and the like.

The terms "prevent", "preventing" and "preventing" as used herein include reducing the likelihood of a patient from developing or worsening a disease or condition.

The term "treatment" and other similar synonyms as used herein include the following meanings:

(i) Preventing the occurrence of a disease or disorder in a mammal, particularly when such mammal is susceptible to the disease or disorder, but has not been diagnosed as having the disease or disorder;

(ii) Inhibiting the disease or disorder, i.e., inhibiting its progression;

(iii) Alleviating a disease or condition, i.e., causing the state of the disease or condition to subside; or alternatively

(iv) Alleviating symptoms caused by the disease or condition.

The term "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" as used herein refers to an amount of at least one agent or compound that is sufficient to alleviate one or more symptoms of the disease or disorder being treated to some extent after administration. The result may be a reduction and/or alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide clinically significant relief from a disorder. Effective amounts suitable in any individual case can be determined using techniques such as a dose escalation test.

The terms "administering," "administering," and the like as used herein refer to a method capable of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, duodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Application techniques useful in the compounds and methods described herein are well known to those skilled in the art, for example, at Goodman and Gilman, the Pharmacological Basis of Therapeutics, current ed.; pergamon; and Remington's, pharmaceutical Sciences (current edition), mack Publishing co., easton, pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

The terms "pharmaceutical combination", "co-administration", "administration of other treatments", "administration of other therapeutic agents" and the like as used herein refer to a pharmaceutical treatment obtained by mixing or combining more than one active ingredient, which includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient in the form of a single entity or single dosage form. The term "ambulatory combination" refers to the simultaneous administration, co-administration, or sequential administration of at least one compound described herein and at least one synergistic formulation as separate entities to a patient at variable intervals. These also apply to cocktail therapies, for example, administration of three or more active ingredients.

It will also be appreciated by those skilled in the art that in the methods described below, the intermediate compound functional groups may need to be protected by appropriate protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable hydroxy protecting groups include trialkylsilyl or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino groups include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable mercapto-protecting groups include-C (O) -R "(wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, and the like. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

Protecting groups may be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, t.w. and p.g.m. wuts, protective Groups in Organic Synthesis, (1999), 4th Ed. The protecting group may also be a polymeric resin.

The invention will be further illustrated with reference to specific examples. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are generally in accordance with conventional conditions, or in accordance with the conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

The preparation method of the intermediate comprises the following steps: synthesis of nitroquinoline compounds

Intermediate 1A: 7-bromo-4, 6-dichloro-8-fluoro-3-nitroquinoline

The intermediate compound 1A is prepared by taking commercial reagents as raw materials and referring to a synthetic route and a method of patent WO2019110751A 1.

Intermediate 1B: 7-bromo-4, 6-dichloro-3-nitro-8- (2, 2-trifluoroethoxy) quinoline

The first step: will 1Bromine-2-fluoro-3-nitrobenzene (10 g,45.7 mmol) and trifluoroethanol (13.7 g,137 mmol) were dissolved in N.N-Dimethylformamide (DMF) (100 mL), potassium carbonate (12.6 g,91.4 mmol) was added, and the pot was warmed to 90℃and reacted overnight. The reaction solution was cooled to room temperature, poured into water, and solid was precipitated, filtered, and after drying the cake, 1-bromo-3-nitro-2- (2, 2-trifluoroethoxy) benzene (6.5 g, yellow solid) was obtained by pulping and purifying with ethyl acetate. LC-MS ESI [ M+H ]] ⁺ ＝300.1。

And a second step of: 1-bromo-3-nitro-2- (2, 2-trifluoroethoxy) benzene (6.0 g,20.1 mmol) was dissolved in acetic acid (HOAc) (60 mL), and reduced iron powder (2.2 g,40.2 mmol) was added in portions and reacted overnight at room temperature. The reaction solution was filtered, concentrated under reduced pressure, saturated sodium bicarbonate solution was added, solids were precipitated, and the filter cake was dried to give 3-bromo-2- (2, 2-trifluoroethoxy) aniline (4.6 g, yellow solid). LC-MS ESI [ M+H ] ] ⁺ ＝270.1。

And a third step of: 3-bromo-2- (2, 2-trifluoroethoxy) aniline (4.0 g,14.9 mmol) was dissolved in ethanol (EtOH) (40 mL), N-chlorosuccinimide (NCS) (4.0 g,30.0 mmol) was added in portions, and the mixture was reacted overnight at room temperature. The reaction solution was filtered, concentrated under reduced pressure, extracted three times with saturated sodium bicarbonate solution, dried, concentrated under reduced pressure, and purified by column chromatography to give 3-bromo-4-chloro-2- (2, 2-trifluoroethoxy) aniline (2.2 g, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝304.3。

Fourth step: 3-bromo-4-chloro-2- (2, 2-trifluoroethoxy) aniline (2.0 g,6.6 mmol) was dissolved in EtOH (20 mL), diethyl 2- (ethoxyenyl) malonate (1.7 g,7.9 mmol) was added, and the temperature was raised to 80℃and reacted overnight. The reaction solution was cooled to room temperature, concentrated under reduced pressure, purified by beating with petroleum ether, filtered, and the filter cake was dried and dissolved in diphenyl ether (10 mL), and the temperature was raised to 245℃for 0.5 hour. Cooled to room temperature, poured into petroleum ether, and solid precipitated, filtered and dried to obtain 7-bromo-6-chloro-4-hydroxy-8- (2, 2-trifluoroethoxy) quinoline-3-carboxylic acid ethyl ester (2.0 g, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝428.2。

Fifth step: 7-bromo-6-chloro-4-hydroxy-8- (2, 2-trifluoroethoxy) quinoline-3-carboxylic acid ethyl ester (2.0 g,4.7 mmol) was dissolved in EtOH (20 mL), and 2M aqueous sodium hydroxide solution (11 mL, 22.0 mmol) and the reaction temperature was raised to 100℃for 0.5 hours. The reaction mixture was cooled to room temperature, diluted with water (70 mL), pH was adjusted to 3 with 2N diluted hydrochloric acid, solids were precipitated, filtered, the cake was dried and suspended in concentrated hydrochloric acid (20 mL), and the temperature was raised to 100℃for 8 hours. Cooled to room temperature, filtered and dried to give 7-bromo-6-chloro-8- (2, 2-trifluoroethoxy) quinolin-4-ol (1.4 g, light brown solid). LC-MS ESI [ M+H ]] ⁺ ＝356.2。

Sixth step: 7-bromo-6-chloro-8- (2, 2-trifluoroethoxy) quinolin-4-ol (1.2 g,3.4 mmol) was dissolved in glacial acetic acid (10 mL), 48% concentrated nitric acid (1 mL,10.7 mmol) was added, and the temperature was raised to 80℃for 2 hours. The reaction solution was cooled to room temperature, diluted with water (30 mL), and solid precipitated, filtered and dried to give 7-bromo-6-chloro-3-nitro-8- (2, 2-trifluoroethoxy) quinolin-4-ol (0.6 g, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝401.2。

Seventh step: 7-bromo-6-chloro-3-nitro-8- (2, 2-trifluoroethoxy) quinolin-4-ol (0.6 g,1.5 mmol) was dissolved in phosphorus oxychloride (8 mL), DMF (0.1 mL) was added, and the temperature was raised to 100℃for 2 hours. The reaction solution was cooled to room temperature, toluene (20 mL) was added, concentrated under reduced pressure, and saturated sodium bicarbonate NaHCO was added to the residue ₃ In aqueous solution (10 mL), dichloromethane DCM was extracted 3 times, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford intermediate 1B (0.55 g, yellow solid). LC-MS ESI [ M+H ] ] ⁺ ＝419.1。

Intermediate 1C: 7-bromo-4-chloro-8- (2, 2-difluoroethoxy) -3-nitro-6-vinylquinoline

The intermediate 1C is prepared by taking 2, 2-difluoroethanol as a raw material and referring to the synthesis method and conditions of the intermediate 1B. LC-MS ESI [ M+H ]] ⁺ ＝393.1。

Example general preparation method one

The first step: nitroquinoline/cinnoline intermediate (1 eq.) was dissolved in anhydrous tetrahydrofuran, and N, N-Diisopropylethylamine (DIPEA) (1.6 eq.) and piperazine intermediate (1.5 eq.) were added in this order, followed by heating and refluxing under nitrogen for 18 hours. TLC monitored the reaction was complete, cooled to room temperature, concentrated under reduced pressure, the residue was taken up in water and dichloromethane for phase separation, the aqueous phase was extracted three times with dichloromethane, the extract was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was used directly in the next reaction.

And a second step of: the crude product of the above first step (1 eq.) was dissolved in anhydrous glacial acetic acid and reduced iron powder (3 eq.) was slowly added and heated to 80℃under nitrogen protection with stirring for 0.5 hour. After the reaction, celite was filtered, washed with ethyl acetate, and the filtrate was concentrated. The residue was diluted with dichloromethane, washed with saturated sodium bicarbonate solution and saturated saline in this order, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to give the target product, whose structure was confirmed by nuclear magnetism and mass spectrometry.

And a third step of: the second product (1 eq.) was dissolved in a mixed solvent of acetonitrile/triethylamine (3/1), phosphorus pentasulfide (1 eq.) was added in portions, and heated under reflux for 3 hours under nitrogen. After the reaction is finished, cooling to room temperature, precipitating solid, filtering, and vacuum drying a filter cake to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

Fourth step: the product of the third step (1 eq.) was dissolved in a mixed solvent of ethanol/diisopropylethylamine DIPEA (5/1), 2-dimethoxyethylamine (1 eq.) was added, and heated under reflux for 6 hours under nitrogen. After the reaction, cooling to room temperature, concentrating under reduced pressure, dissolving the residue in glacial acetic acid, heating to 100 ℃ and stirring for 2 hours. Cooling to room temperature, concentrating under reduced pressure, diluting the residue with dichloromethane, washing with saturated sodium bicarbonate solution and saturated saline solution sequentially, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying the residue with silica gel column chromatography to obtain the target product, and confirming the structure by nuclear magnetism and mass spectrum.

Fifth step: dissolving the product (1 eq.) of the fourth step in a mixed solvent of anhydrous dioxane/water (4/1), and sequentially adding boric acid or boric acid pinacol ester (2 eq.) and anhydrous carbonic acidPotassium powder (2.5 eq.) and [1,1' -bis (diphenylphosphino) ferrocene ]Palladium dichloride (Pd (dppf) Cl) ₂ ) (0.1 eq.) under nitrogen for 2 hours under reflux. Thin Layer Chromatography (TLC) is used for monitoring the reaction completion, cooling to room temperature, concentrating under reduced pressure, diluting the residue with dichloromethane, washing with saturated ammonium chloride solution and saturated saline water in sequence, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying the residue with silica gel column chromatography to obtain a target product, and confirming the structure by adopting nuclear magnetism and mass spectrum.

Sixth step: the product of the fifth step (1 eq.) was dissolved in methanol, 4M hydrogen chloride (HCl) in methanol (20 eq.) was added and stirred at room temperature for 3 hours. The reaction was monitored by TLC, concentrated under reduced pressure, the residue was dissolved in dichloromethane, DIPEA (3 eq.) and acryloyl chloride (1 eq.) were added sequentially at 0 ℃ and stirred for 0.5 hours, the reaction solution was washed with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to give the target compound, which was confirmed in structure by nuclear magnetism and mass spectrometry.

Example general preparation method two

The first step: the quinoline/cinnoline amide intermediate (1 eq.) was dissolved in anhydrous methylene chloride, and trimethyloxonium tetrafluoroborate (1.2 eq.) was added at 0℃and stirred at room temperature under nitrogen for 12 hours. TLC monitored completion of the reaction, the reaction was washed with saturated sodium bicarbonate solution and saturated brine in this order, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was directly used for the next reaction.

And a second step of: the crude product of the first step (1 eq.) was dissolved in a mixed solvent of methanol/concentrated ammonia (1/1), placed in a closed tank, added with catalytic amount of p-toluene sulfonic acid, heated to 100 ℃ and stirred for 6 hours. After the reaction, the reaction solution was concentrated under reduced pressure. The residue was diluted with methylene chloride, washed with a saturated sodium bicarbonate solution and then a saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was directly used for the next reaction.

And a third step of: the above second crude product (1 eq.) was dissolved in ethanol, phosphate buffer (10 ml, ph=6.7), sodium acetate (2 eq.) and 40% aqueous chloroacetaldehyde (10 eq.) were added, heated to 80 degrees and stirred for 24 hours. Cooled to room temperature, and the reaction solution was concentrated under reduced pressure. The residue was diluted with dichloromethane, washed with saturated ammonium chloride solution and saturated saline in this order, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography to give the objective product, whose structure was confirmed by nuclear magnetism and mass spectrometry.

Fourth step: dissolving the product (1 eq.) of the third step in a mixed solvent of anhydrous dioxane/water (4/1), and sequentially adding boric acid or boric acid pinacol ester (2 eq.), anhydrous potassium carbonate powder (2.5 eq.) and Pd (dppf) Cl ₂ (0.1 eq.) under nitrogen for 2 hours under reflux. TLC monitoring reaction is complete, cooling to room temperature, decompressing and concentrating, diluting residues with dichloromethane, washing with saturated ammonium chloride solution and saturated saline water in turn, drying with anhydrous sodium sulfate, filtering, decompressing and concentrating, separating and purifying residues with silica gel column chromatography to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

Fifth step: the product of the fourth step (1 eq.) was dissolved in methanol, 4M HCl in methanol (20 eq.) was added and stirred at room temperature for 3 hours. TLC monitoring reaction is complete, decompression concentration, residual is dissolved in dichloromethane, DIPEA (3 eq.) and acryloyl chloride (1 eq.) are added in sequence at 0 ℃, stirring is carried out for 0.5 hours, the reaction liquid is washed by saturated ammonium chloride solution and saturated saline water, anhydrous sodium sulfate is dried, filtration and decompression concentration are carried out, the residual is separated and purified by silica gel column chromatography to obtain the target compound, and nuclear magnetism and mass spectrum structure is adopted for confirmation.

Example general preparation method three

The first step: quinoline/cinnoline-based sulfamide intermediate (1 eq.) was dissolved in tetrahydrofuran, hydrazine hydrate (1.5 eq.) was added, and heated under reflux for 6 hours under nitrogen. After the reaction is finished, cooling to room temperature, concentrating under reduced pressure, precipitating solid, filtering, and vacuum drying a filter cake to obtain a target product, and adopting nuclear magnetism and mass spectrum to confirm a structure.

And a second step of: the product of the first step (1 eq.) was dissolved in dichloromethane, trimethyl orthoformate (4 eq.) was added, and after stirring for ten minutes trifluoroacetic acid (1 eq.) was added. After stirring for one hour at room temperature, concentrating under reduced pressure, separating and purifying the residue by silica gel column chromatography to obtain the target product, and confirming the structure by nuclear magnetism and mass spectrum.

And a third step of: the second product (1 eq.) was dissolved in a mixed solvent of anhydrous dioxane/water (4/1), boric acid or pinacol borate (2 eq.) was added sequentially, anhydrous potassium carbonate powder (2.5 eq.) and Pd (dppf) Cl ₂ (0.1 eq.) under nitrogen for 2 hours under reflux. TLC monitoring reaction is complete, cooling to room temperature, decompressing and concentrating, diluting residues with dichloromethane, washing with saturated ammonium chloride solution and saturated saline water in turn, drying with anhydrous sodium sulfate, filtering, decompressing and concentrating, separating and purifying residues with silica gel column chromatography to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

Fourth step: the product of the third step (1 eq.) was dissolved in methanol, 4m hci in methanol (20 eq.) was added and stirred at room temperature for 3 hours. TLC monitoring reaction is complete, decompression concentration, residual is dissolved in dichloromethane, DIPEA (3 eq.) and acryloyl chloride (1 eq.) are added in sequence at 0 ℃, stirring is carried out for 0.5 hours, the reaction liquid is washed by saturated ammonium chloride solution and saturated saline water, anhydrous sodium sulfate is dried, filtration and decompression concentration are carried out, the residual is separated and purified by silica gel column chromatography to obtain the target compound, and nuclear magnetism and mass spectrum structure is adopted for confirmation.

Example general preparation method four

The first step: the quinoline/cinnoline hydrazine intermediate (1 eq.) was dissolved in dry pyridine, and then acid chloride (1.5 eq.) was added dropwise under ice-bath cooling, and the mixture was heated to 100℃under nitrogen and stirred overnight. After the reaction is finished, the reaction solution is cooled to room temperature, reduced pressure concentration is carried out, the residue is diluted by saturated sodium carbonate solution, dichloromethane extraction is carried out, an organic phase is washed by water and saturated saline water sequentially, anhydrous sodium sulfate is dried, filtration and reduced pressure concentration are carried out, the residue is separated and purified by silica gel column chromatography to obtain a target product, and a nuclear magnetism and mass spectrum structure is adopted to confirm the structure.

And a second step of: the product of the first step (1 eq.) was dissolved in a mixed solvent of anhydrous dioxane/water (4/1 volume ratio), boric acid or pinacol borate (2 eq.) was added sequentially, anhydrous potassium carbonate powder (2.5 eq.) and Pd (dppf) Cl ₂ (0.1 eq.) under nitrogen for 2 hours under reflux. TLC monitoring reaction is complete, cooling to room temperature, decompressing and concentrating, diluting residues with dichloromethane, washing with saturated ammonium chloride solution and saturated saline water in turn, drying with anhydrous sodium sulfate, filtering, decompressing and concentrating, separating and purifying residues with silica gel column chromatography to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

And a third step of: the above second-step product (1 eq.) was dissolved in methanol, and a 4M HCl methanol solution (20 eq.) was added and stirred at room temperature for 3 hours. TLC monitoring reaction is complete, decompression concentration, residual is dissolved in dichloromethane, DIPEA (3 eq.) and acryloyl chloride (1 eq.) are added in sequence at 0 ℃, stirring is carried out for 0.5 hours, the reaction liquid is washed by saturated ammonium chloride solution and saturated saline water, anhydrous sodium sulfate is dried, filtration and decompression concentration are carried out, the residual is separated and purified by silica gel column chromatography to obtain the target compound, and nuclear magnetism and mass spectrum structure is adopted for confirmation.

Example general preparation method five

The first step: the quinoline/cinnoline amine intermediate (1 eq.) was suspended in isopropanol and dimethylformamide-dimethylacetal (1.2 eq.) was added dropwise at room temperature. After the completion of the dropwise addition, the reaction solution was heated and refluxed for three hours, cooled to room temperature, and hydroxylamine hydrochloride (1.2 eq.) was added to the reaction solution, followed by stirring at 50℃overnight. The reaction solution was cooled to room temperature, concentrated under reduced pressure, dried, and the residue was suspended in anhydrous tetrahydrofuran, cooled in an ice bath, and then trifluoroacetic anhydride (1.5 eq.) was slowly added dropwise. After the completion of the dropwise addition, the ice bath was removed and stirred at room temperature overnight. And slowly dripping saturated sodium bicarbonate solution into the reaction liquid, extracting with dichloromethane, washing with water and saturated saline water in sequence, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying the residue by silica gel column chromatography to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

And a second step of: the product of the first step (1 eq.) was dissolved in a mixed solvent of anhydrous dioxane/water (4/1), boric acid or pinacol borate (2 eq.) was added sequentially, anhydrous potassium carbonate powder (2.5 eq.) and Pd (dppf) Cl ₂ (0.1 eq.) under nitrogen for 2 hours under reflux. TLC monitoring reaction is complete, cooling to room temperature, decompressing and concentrating, diluting residues with dichloromethane, washing with saturated ammonium chloride solution and saturated saline water in turn, drying with anhydrous sodium sulfate, filtering, decompressing and concentrating, separating and purifying residues with silica gel column chromatography to obtain a target product, and confirming a structure by adopting nuclear magnetism and mass spectrum.

And a third step of: the above second stage product (1 eq.) was dissolved in methanol, 4M HCl in methanol (20 eq.) was added and stirred at room temperature for 3 hours. TLC monitoring reaction is complete, decompression concentration, residual is dissolved in dichloromethane, DIPEA (3 eq.) and acryloyl chloride (1 eq.) are added in sequence at 0 ℃, stirring is carried out for 0.5 hours, the reaction liquid is washed by saturated ammonium chloride solution and saturated saline water, anhydrous sodium sulfate is dried, filtration and decompression concentration are carried out, the residual is separated and purified by silica gel column chromatography to obtain the target compound, and nuclear magnetism and mass spectrum structure is adopted for confirmation.

Example preparation

Example 1:1- ((15 aR) -9-chloro-7-fluoro-8- (2-fluoro-6-hydroxyphenyl) -12,13,15 a-tetrahydro-14H-pyrazin [1',2':4,5] [1,2,4] triazol [4',3':1,6] pyrazin [2,3-c ] quinolin-14-yl) prop-2-en-1-one

The first step: intermediate 7-bromoo-4, 6-dichlorio-8-fluoroo-3-nitroquinoline (4.1 g,12.1 mmol) was dissolved in 1, 4-dioxane (40 mL) and 1- (t-butoxycarbonyl) -piperazine-3- (R) -carboxylic acid was addedMethyl acid ester (3.3 g,13.5 mmol) and DIPEA (4.6 g,35.7 mmol) were reacted at 100℃for 2 days under nitrogen. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography to give methyl 1- (tert-butoxycarbonyl) - (R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) piperazine-3-carboxylate (2.5 g, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝546.8； ¹ H NMR(400MHz,DMSO_d6):δ9.16(s,1H),8.35(d,J＝1.2Hz,1H),4.37(s,1H),3.85-4.15(m,2H),3.62-3.75(m,2H),3.55(s,3H),3.22-3.32(m,2H),1.44(s,9H)。

And a second step of: methyl 1- (tert-butoxycarbonyl) - (R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) piperazine-3-carboxylate (2.2 g,4.0 mmol) was dissolved in glacial acetic acid (30 mL), reduced iron powder (790 mg,14.1 mmol) was added, and the mixture was reacted at 80℃under nitrogen for 40 minutes. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and saturated sodium bicarbonate (NaHCO) was added to the residue ₃ ) The pH value of the solution is regulated to 8, dichloromethane is extracted for three times, anhydrous sodium sulfate is dried, and reduced pressure concentration is carried out to obtain (R) -10-bromo-11-chloro-9-fluoro-5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazine [1',2':4,5 ]]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (1.91 g, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝485.2； ¹ H NMR(400MHz,DMSO_d6):δ11.06(s,1H),8.62(s,1H),8.08(s,1H),4.66-4.70(m,1H),3.84(brs,2H),3.19-3.34(m,3H),2.70(brs,1H),1.44(s,9H)。

And a third step of: (R) -10-bromo-11-chloro-9-fluoro-5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazin [1',2':4,5 ]Pyrazine [2,3-c ]]Tert-butyl quinoline-3-carboxylate (250 mg,0.52 mmol) was suspended in toluene (15 mL) and L-form (210 mg,0.52 mmol) was added under nitrogen and heated at reflux overnight. The reaction solution is cooled to room temperature, concentrated under reduced pressure, and the residue is purified by column chromatography to obtain (R) -10-bromo-11-chloro-9-fluoro-5-thio-1, 2, 4a,5, 6-hexahydro-3H-pyrazine [1',2':4,5 ]]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (204 mg, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝500.9/502.9。

Fourth step: (R) -10-bromo-11-chloro-9-fluoro-5-thio-1, 2, 4a,5, 6-hexahydro-3H-pyrazin [1',2':4,5]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (154 mg,0.31 mmol) was dissolved in Tetrahydrofuran (THF) (4 mL), hydrazine hydrate (154 mg,3.1 mmol) was added and the mixture was refluxed under nitrogen for 4 hours. The reaction solution is cooled toConcentrating under reduced pressure at room temperature to obtain (R, Z) -10-bromo-11-chloro-9-fluoro-5-hydrazinoalkenyl-1, 2, 4a,5, 6-hexahydro-3H-pyrazine [1',2':4,5 ]]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (159 mg, crude, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝498.8/500.8。

Fifth step: (R, Z) -10-bromo-11-chloro-9-fluoro-5-hydrazinoalkenyl-1, 2, 4a,5, 6-hexahydro-3H-pyrazin [1',2':4,5]Pyrazine [2,3-c ]]Tert-butyl quinoline-3-carboxylate (159 mg, crude) was suspended in trimethyl orthoformate (2 mL) and reacted at 80℃overnight under nitrogen. The reaction solution is cooled to room temperature, concentrated under reduced pressure, and the residue is purified by column chromatography to obtain (R) -8-bromo-9-chloro-7-fluoro-12, 13,15 a-tetrahydro-14H-pyrazine [1',2':4,5 ] ][1,2,4]Triazole [4',3':1,6]Pyrazine [2,3-c ]]Quinoline-14-carboxylic acid tert-butyl ester (136 mg, yellow solid). LC-MS ESI [ M+H ]] ⁺ ＝508.9/510.9； ¹ H NMR(400MHz,DMSO_d6):δ9.56(s,1H),9.49(s,1H),8.28(s,1H),4.84-4.88(m,1H),4.60-4.64(m,1H),3.67-3.83(m,2H),3.23-3.38(m,2H),2.56-2.67(m,1H),1.48(s,9H)。

Sixth step: (R) -8-bromo-9-chloro-7-fluoro-12, 13,15 a-tetrahydro-14H-pyrazine [1',2':4,5][1,2,4]Triazole [4',3':1,6]Pyrazine [2,3-c ]]Quinoline-14-carboxylic acid tert-butyl ester (136 mg,0.27 mmol) and (2-fluoro-6-hydroxyphenyl) boric acid (104 mg,0.67 mmol) were dissolved in dioxane 1.4-dioxane/water (6 mL/2 mL), and potassium carbonate (K) was added sequentially under nitrogen ₂ CO ₃ ) (186 mg,1.35 mmol), 2-dicyclohexylphosphorus-2 ',6' -diisopropyloxy-1, 1' -biphenyl Ruphos (13 mg,0.027 mmol) and a third generation palladium catalyst (Pd-Ruphos-G3) (23 mg,0.027 mmol) were then heated to 90 degrees for reaction overnight. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography to give (15 aR) -9-chloro-7-fluoro-8- (2-fluoro-6-hydroxyphenyl) -12,13,15 a-tetrahydro-14H-pyrazine [1',2':4,5 ]][1,2,4]Triazole [4',3':1,6]Pyrazine [2,3-c ]]Quinoline-14-carboxylic acid tert-butyl ester torsional isomers 1 (22 mg, yellow solid) and 2 (15 mg, yellow solid). Isomer 1: LC-MS ESI [ M+H ]] ⁺ ＝495.1，RT：6.369min； ¹ H NMR (400 MHz, CD3 OD): delta 9.48 (s, 1H), 9.36 (s, 1H), 8.30 (s, 1H), 7.23-7.37 (m, 2H), 6.79-6.81 (m, 2H), 6.72-6.76 (m, 1H), 6.25-6.29 (m, 1H), 5.78-5.87 (m, 1H), 5.11-5.14 (m, 1H), 4.71-4.82 (m, 1H), 4.13-4.17 (m, 1H), 3.42-3.47 (m, 2H), 2.67-2.71 (m, 1H); isomer 2: LC-MS: ESI[M+H] ⁺ ＝495.1,RT:6.564min； ¹ H NMR(400MHz,CD3OD):δ9.48(s,1H),9.36(s,1H),8.30(s,1H),7.22-7.37(m,2H),6.72-6.81(m,2H),6.25-6.29(m,1H),5.77-5.88(m,1H),5.11-5.15(m,1H),4.72-4.82(m,1H),4.47-4.51(m,1H),4.14-4.17(m,1H),3.79-3.85(m,1H),3.39-3.47(m,2H),2.67-2.71(m,1H)。

Isomer analysis chromatographic conditions: sunfire C184.6 x 150mm 5um 13min; mobile phase a 0.1% formic acid-water) FA-H2O, mobile phase B0.1% (formic acid-acetonitrile) FA-aCN.

Example 2:1- ((15 aR) -9-chloro-7-fluoro-8- (2-fluoro-6-hydroxyphenyl) -12,13,15 a-tetrahydro-14H-imidazo [1',2':1,6] pyrazin [1',2':4,5] pyrazin [2,3-c ] quinolin-14-yl) prop-2-en-1-one

The first step: (R) -10-bromo-11-chloro-9-fluoro-5-thio-1, 2, 4a,5, 6-hexahydro-3H-pyrazin [1',2':4,5]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (50 mg,0.10 mmol) was dissolved in tetrahydrofuran THF (1 mL), aminoacetaldehyde dimethyl acetal (52 mg,0.50 mmol) was added, and the mixture was heated under reflux under nitrogen for 3 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure to give (R, Z) -10-bromo-11-chloro-5- ((2, 2-dimethoxyethyl) imine) -9-fluoro-1, 2, 4a,5, 6-hexahydro-3H-pyrazine [1',2':4,5]]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (45 mg, crude, yellow solid) was used directly in the next reaction. LC-MS ESI [ M+H ]] ⁺ ＝572.0/574.0。

And a second step of: (R, Z) -10-bromo-11-chloro-5- ((2, 2-dimethoxyethyl) imine) -9-fluoro-1, 2, 4a,5, 6-hexahydro-3H-pyrazine [1',2':4,5]Pyrazine [2,3-c ]]Quinoline-3-carboxylic acid tert-butyl ester (400 mg) was dissolved in dioxane (10 mL), p-toluenesulfonic acid monohydrate (200 mg) was added, and the mixture was stirred overnight. Extracting with dichloromethane, washing the organic phase with saturated sodium bicarbonate aqueous solution and distilled water, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating and purifying by column chromatography to obtain tert-butyl (R) -8-bromo-9-chloro-7-fluoro-12, 13,15 a-tetrahydro-14H-imidazo [1',2':1,6 ]Pyrazine [1',2':4,5]Pyrazino [2,3-c ]]Quinoline-14-carboxylic acid ester (180 mg), LC-MS: ESI [ M+H ]] ⁺ ＝500.9/503.0。 ¹ H NMR(400MHz,CDCl3):δ9.02(s,1H),8.13(d,J＝2.0Hz,1H),7.58(d,J＝2.0Hz,1H),7.29(s,1H),5.12-5.15(m,1H),4.25(bs,1H),4.02-4.16(m,1H),3.65-3.68(m,1H),3.35-3.72(m,1H),3.09-3.12(m,1H),2.69-2.72(m,1H),1.46(s,9H)。

Third to fifth steps: the product obtained in the above step is used as raw material, and synthesized by the same operation method in the fifth to seventh steps of example 1, to prepare the compound 1- ((15 aR) -9-chloro-7-fluoro-8- (2-fluoro-6-hydroxyphenyl) -12,13,15 a-tetrahydro-14H-imidazole [1',2':1, 6) of example 2]Pyrazine [1',2':4,5]Pyrazine [2,3-c ]]Quinolin-14-yl) prop-2-en-1-one. LC-MS ESI [ M+H ]] ⁺ ＝494.1。 ¹ H NMR(400MHz,CDCl3):δ9.32(s,1H),8.24(d,J＝2.0Hz,1H),7.56(d,J＝2.0Hz,1H),7.32(s,1H),6.51(m,1H),6.23(m,1H),5.81(m,1H),5.16-5.18(m,1H),4.27(bs,1H),4.03-4.15(m,1H),3.62-3.66(m,1H),3.37-3.49(m,1H),3.06-3.15(m,1H),2.58-2.71(m,1H)。

The following compounds of examples were synthesized by the same method as in example 1 and example 2.

Test example 1 KrasG12C functional analysis

All enzyme and substrate solutions were formulated with reaction buffer (20 mM HEPES (pH 7.5), 5mM MgCl2,150mM NaCl and 0.01% tween 20). The experimental procedure was as follows: the biotinylated KRASG12C loaded with GDP and 37.5ng/ml of streptavidin europium cryptate were prepared using reaction buffer, 5. Mu.L of the above protein reaction solution was added per well in 384-well HiBase micro polystyrene microwell plates, while the test or control compound prepared from DMSO was added and incubated for 4 hours. In addition, 20nM GST-Raf Ras binding domain (GST-Raf RBD) and 4. Mu.g/ml anti-GST XL665 antibody (Cisbio) were mixed separately, formulated in reaction buffer (50 mM potassium fluoride and 0.05mg/ml BSA), and after equilibration for 4 hours 0.6. Mu.M GTPγS (Sigma) and 0.08. Mu.M SOS were added. Mu.l of GST-RAF RBD mix was added to each well of the microplate. The addition of this step of the mixture initiates a nucleotide exchange reaction which promotes the conversion of the KRASG12C of the non-activated GDP-loaded KRASG12C to activated GTPγS. The specific binding between activated GTPγS KRASG12C and GST-RAF RBD pulls in the europium and XL665 distance to enhance FRET signals detected using a Pherastar (BMG) reader equipped with an HTRF filter module. Any compound that inhibited nucleotide exchange or activated KRAS binding to RAF RBD resulted in a decrease in FRET signal was curve fitted to FRET dose response data using Genedata Screener and IC50 was calculated.

Test example 2 KRASG12C Mass Spectrometry addition analysis

All enzyme and substrate solutions were formulated with reaction buffer (20 mM HEPES (pH 7.5), 5mM MgCl2,150mM NaCl and 0.01% tween 20). 50. Mu.L of GDP-loaded biotinylated KRAS G12C, 0.5. Mu.L of 1mM test compound (final concentration 10. Mu.M) was added to each well of a 96-well polypropylene microplate, and after 4 hours of reaction, 50. Mu.L of 1% formic acid was added to terminate the reaction. Plates were read for Xex G2 QTOF (Waters) and Acquity LC system (Waters) after sealing of the plates. 10 μl of sample was injected into Xbridge BEH300; c4;3.5 μm; gradient analysis was performed for 3 minutes on a 2.1x 50mm column (Waters). A blank sample needs to be run between each test sample. Data analysis was performed using Mass Lynx Software (Waters) using total ion number (TIC) and combining the eluted protein peak data. Apo-proteins KRASG12C (Apo) and kras+ were tested for relative compound mass (addition), and the calculation of the percentage addition was performed using the following formula: percent sum = 100x (sum peak area/sum of APO and sum peak).

Test example 3: test of the Effect of the Compounds of the invention on NCI-H358, miaPaca-2 cell proliferation Capacity

1. NCI-H358 (lung cancer) and MiaPaca-2 cells (pancreatic cancer) cells (100 μl/well, 20000 cells/mL) were inoculated in 96-well culture plates, respectively, and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin sulfate. Cells were treated with a solution of test compound diluted three times in eight gradients at an initial concentration of 10. Mu.M using 0.5% dimethyl sulfoxide as a blank, And incubated in a 5% CO2 incubator for a period of time (3-7 days). At the end of incubation, 10. Mu.L of MTT stock solution (5 mg/mL) was added to each well. The plates were incubated at 37℃for 4 hours, after which the medium was removed. Dimethyl sulfoxide (100 μl) was added to each well, followed by shaking well. The absorbance of the formazan product was measured at 570nm on a thermoscientificariskanflash multimode reader. IC was obtained by fitting dose response data to a three-parameter nonlinear regression model using graphpadprism6.0 software ₅₀ Values.

2. As a result, the compounds of the present invention provided example have proliferation-inhibiting activity on NCI-H358 and MiaPaca-2 cells, IC ₅₀ Values of less than 5000nM, mostly less than 1000nM, and especially cell proliferation inhibitory activity of less than 100nM as in examples 5, 6.

Test example 4: in vivo pharmacokinetic parameter testing of example Compounds in rats, mice

The 6 male SPF-class SD rats (Shanghai Sipulel-BiKai experimental animals) were divided into two groups, and the test compounds were formulated as appropriate solutions or suspensions; one group is administered by intravenous injection and one group is administered orally. Blood was collected via jugular vein puncture, each sample was collected at about 0.2 mL/time point, heparin sodium was anticoagulated, and the blood collection time points were as follows: 5, 15 and 30min,1, 2, 4, 6, 8 and 24h before and after administration; blood samples were collected and placed on ice, and plasma was centrifuged (centrifugation conditions: 8000 rpm, 6 min, 2-8 ℃) and stored at-80℃prior to analysis of the collected plasma. Plasma samples were analyzed by LC-MS/MS.

According to the blood concentration data of the medicine, respectively calculating the pharmacokinetic parameters AUC of the test sample by using a pharmacokinetic calculation software WinNonlin5.2 non-atrioventricular model _0-t 、AUC _0-∞ 、MRT _0-∞ 、C _max 、T _max 、T _1/2 And V _d Isoparametric parameters, mean and standard deviation. In addition, the bioavailability (F) will be calculated by the following formula.

For samples with concentrations below the lower limit of quantification, the pharmacokinetic parameter calculations were performed before C was reached _max The previously sampled sample should be calculated as zero value, until C is reached _max The sample points should be calculated as unqualified (BLQ) later.

Test example 5: test of Compounds of examples for MiaPaca-2, NCI-H358 tumor cell nude mice transplantation tumor growth inhibition

Cutting tumor tissue in vigorous growth period into 1.5mm ³ Left and right, under aseptic conditions, the mice were inoculated subcutaneously in the right armpit. The diameter of the transplanted tumor is measured by a vernier caliper for subcutaneous transplantation of the nude mice, and the average tumor volume is up to 130mm ³ Animals were randomly grouped left and right. The compound of the examples (prepared with 1% tween80 in water for injection to the desired concentration for use) was orally administered daily at given doses for three consecutive weeks, and the solvent control group was given an equivalent amount of solvent. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week while weighing the mice. The calculation formula of Tumor Volume (TV) is: tv=1/2×a×b ² Wherein a and b respectively represent length and width. Based on the measured results, a Relative Tumor Volume (RTV) is calculated as: rtv=vt/V0. Where V0 is the tumor volume measured at the time of divided cage administration (i.e., d 0), and Vt is the tumor volume at each measurement. The evaluation index of the antitumor activity is 1) the relative tumor proliferation rate T/C (%), and the calculation formula is as follows:

T/C (%) = (TRTV/CRTV) ×100%, TRTV: treatment group RTV; CRTV: negative control RTV; 2) Tumor volume increase inhibition rate GI), the calculation formula is as follows: GI% = [1- (TVt-TV 0)/(CVt-CT 0) ]x100%, TVt is the tumor volume measured each time in the treatment group; TV0 is the tumor volume obtained when therapeutic component is administered in the cage; CVt is the tumor volume measured each time in the control group; CV0 is the tumor volume obtained when the control component is administered in a cage; 3) Tumor weight inhibition rate was calculated as follows: tumor weight inhibition% = (Wc-WT)/wc×100%, wc: tumor weight of control group, WT: treating the tumor weight of the group.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A nitrogen-containing fused ring compound represented by the general formula (IIB), (IIC), (IID), or a pharmaceutically acceptable salt thereof:

wherein,

r5 is independently selected from hydrogen, halogen, C1-C6 alkyl, amino;

r1 is independently selected from hydrogen, C ₁ -C ₆ An alkyl group;

r2 and R3 are independently selected from hydrogen, C ₁ -C ₆ An alkyl group;

w is independently selected from CR4 or N, R4 is independently selected from H, halogen, C ₁ -C ₆ Alkyl, C ₁ -C ₄ An alkoxy group;

w1 is independently selected from CR4 or N, R4 is independently selected from H and halogen;

w2 is independently selected from CR4, R4 is independently selected from H, C ₁ -C ₆ An alkyl group;

m is independently selected from CR4, R4 is independently selected from H, C ₁ -C ₆ An alkyl group;

ra, rb, rc, rd, re, rf, rg are each independently selected from hydrogen, C ₁ -C ₆ An alkyl group;

ar is independently selected from phenyl, naphthyl, benzimidazolyl, benzopyrazolyl or benzothiazolyl; and the Ar ring may be substituted with one or more of the following groups: halogen, C1-C6 alkyl, amino, hydroxy.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

r1 is selected from hydrogen;

ra, rb, rc, rd, re, rf, rg are each independently selected from hydrogen, methyl;

w is independently selected from CR4 or N, R4 is independently selected from H, halogen, C ₁ -C ₄ Alkyl, C ₁ -C ₄ An alkoxy group;

W2 is independently selected from CR4, R4 is independently selected from H, C ₁ -C ₄ An alkyl group;

m is independently selected from CH;

ar is independently selected from phenyl, benzimidazolyl or benzothiazolyl; and the Ar ring may be substituted with one or more of the following groups: halogen, C1-C4 alkyl, hydroxy, amino.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure:

4. use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with mutations in the Ras protein.

5. The use according to claim 4, wherein the disease is a tumor.

6. The use of claim 5, wherein the neoplasm is independently selected from lung cancer, pancreatic cancer, liver cancer, colorectal cancer, cholangiocarcinoma, brain cancer, leukemia, lymphoma, melanoma, thyroid cancer, and nasopharyngeal carcinoma.

7. A pharmaceutical composition, said pharmaceutical composition comprising:

(i) An effective amount of a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof; and

(ii) A pharmaceutically acceptable carrier.