CN111094286B

CN111094286B - 1, 2-dihydro-1, 6-naphthyridine derivatives, preparation method and medical application thereof

Info

Publication number: CN111094286B
Application number: CN201880057084.9A
Authority: CN
Inventors: 别平彦; 陈磊; 白骅
Original assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Shanghai Aryl Pharmtech Co Ltd
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Shanghai Aryl Pharmtech Co Ltd
Priority date: 2017-09-20
Filing date: 2018-09-14
Publication date: 2022-07-29
Anticipated expiration: 2038-09-14
Also published as: TWI707853B; WO2019056990A1; CN111094286A; TW201914999A

Abstract

1, 2-dihydro-1, 6-naphthyridine derivatives, preparation method and medical application thereof. In particular to 1, 2-dihydro-1, 6-naphthyridine derivatives shown in a general formula (I), a preparation method thereof, pharmaceutically acceptable salts thereof and application thereof as a therapeutic agent, in particular to a c-KIT inhibitor, wherein the definition of each substituent in the general formula (I) is the same as that in the specification.

Description

1, 2-dihydro-1, 6-naphthyridine derivatives, preparation method and medical application thereof

Technical Field

The invention relates to a novel 1, 2-dihydro-1, 6-naphthyridine derivative, a preparation method thereof, a pharmaceutical composition containing the derivative and application thereof as a therapeutic agent, in particular as a c-KIT inhibitor.

Background

c-KIT (also known as KIT, CD117 and stem cell factor receptor) is a 145kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT proto-oncogene located on chromosome 4q11-21 encodes c-KIT, the ligand of which is a stem cell factor. The receptor has tyrosine protein kinase activity and binding to the ligand SCF results in autophosphorylation of c-KIT and its association with a substrate such as phosphatidylinositol 3-kinase (PI 3K). Phosphorylation of tyrosine by protein tyrosine kinases is particularly important in cell signaling and can mediate signals for major cellular processes such as proliferation, survival, differentiation, apoptosis, ligation, invasion and migration. The c-KIT mutation typically occurs in the DNA encoding the membrane proximal region domain (exon 11). They also occur less frequently in exons 7, 8, 9, 11, 13, 14, 17 and 18. Mutations render c-KIT function independent of activation by SCF, resulting in high cell division rates and possible genomic instability. Functionally increasing mutations and constitutively phosphorylated c-KIT gene expression are found in most gastrointestinal stromal tumors (GISTs), mastocytosis and acute myeloid leukemia. It has mutations at different positions at different exons, and the first generation of c-KIT mutations and related drugs are mainly imatinib, sunitinib, dasatinib and PKC 412.

c-KIT is found in tissues such as germ cell tumor, teratoma and mast cell leukemia of gastrointestinal stromal tumor, acute myelogenous leukemia, melanoma, breast tumor, cervical tumor, glioblastoma multiforme, ovarian tumor, seminoma or dysgerminoma, and the protein expression level of the c-KIT is closely related to the occurrence and development of tumor. Wherein, gastrointestinal stromal tumor (GIST) is the most common tumor of mesenchymal origin in gastrointestinal tract, and epidemiological research shows that the incidence rate is 0.66-2.20/10 ten thousand according to the current GIST diagnosis standard. GIST are not very sensitive to traditional chemotherapy, the effective rate of chemotherapeutic drugs is less than 5%, and the median survival rate in the advanced stage is only about 18 months. Even with complete tumor resection, GIST have a 5-year survival rate of only 35% -65%, a recurrence metastasis rate within 2 years of 40% -50%, and metastases occur in as many as 15% -50% of patients at the time of first diagnosis. Researches find that functional activation mutation of transmembrane tyrosine kinase receptor c-KIT and platelet-derived growth factor receptor PDGFR alpha gene on the surface of stem cell factor is the key for development of GIST. Platelet-derived growth factor receptors (PDGF-R) are cell surface tyrosine kinase receptors for members of the platelet-derived growth factor (PDGF) family. PDGF subunits PDGF α and PDGF β are important regulators that regulate cell proliferation, cell differentiation, cell growth, development and many diseases, including cancer.

With the clinical application of the first generation inhibitor Imatinib (Imatinib), the problem of acquired resistance to Imatinib is becoming a serious challenge for the clinical use of this class of inhibitors. Therefore, research and development of new c-KIT inhibitors to meet the market demand are urgently needed. A series of c-KIT inhibitor patents have been disclosed so far, including WO2014039714, WO2014100620, WO2015134536a1, and WO2013184119, etc., and the research and application of c-KIT inhibitors have made some progress, but the increased space is still huge, and there is still a need to continue research and development of new c-KIT inhibitors.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a novel 1, 2-dihydro-1, 6-naphthyridine derivative shown in a general formula (I), or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, the compound has larger structural difference with the specifically disclosed compound in the prior art, and can treat or prevent diseases such as gastrointestinal stromal tumor, acute myelogenous leukemia, systemic mastocyte proliferation and the like by regulating the activity of c-KIT and PDGF alpha.

The invention relates to a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof:

Wherein:

R ¹ selected from a hydrogen atom, an alkyl group or-C (O) R ⁵ Wherein said alkyl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, alkoxy, cycloalkyl, heterocyclyl, -C (O) R ⁵ 、-OC(O)R ⁵ 、-C(O)OR ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-S(O) _n NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Substituted with the substituent(s);

R ² selected from alkyl groups, wherein said alkyl groups are further substituted by one or more halogensSubstitution by an element;

R ³ identical or different, each independently selected from hydrogen atom, alkyl, alkoxy, hydroxyl, cyano, nitro, halogen, cycloalkyl, heterocyclyl, -C (O) R ⁵ 、-OC(O)R ⁵ 、-C(O)OR ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-S(O) _n NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Wherein said alkyl, alkoxy, cycloalkyl or heterocyclyl is optionally further substituted with one or more groups selected from hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ Substituted with the substituent(s);

R ⁴ selected from aryl or heteroaryl, wherein said aryl or heteroaryl is optionally further substituted with one or more groups selected from alkyl, alkoxy, hydroxy, cyano, nitro, halogen, cycloalkyl, heterocyclyl, -C (O) R ⁵ 、-OC(O)R ⁵ 、-C(O)OR ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-S(O) _n NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Substituted with the substituent(s); wherein said alkyl or alkoxy is optionally further substituted with one or more halogens;

R ⁵ 、R ⁶ and R ⁷ Each independently selected from the group consisting of hydrogen, hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ By substitution ofSubstituted by a group;

or, R ⁶ And R ⁷ Together with the N atom to which they are attached form a 4-8 membered heterocyclic group containing one or more of N, O or S (O) _n And optionally further substituted on the 4-to 8-membered heterocycle with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -C (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ Substituted with the substituent(s);

R ⁸ 、R ⁹ and R ¹⁰ Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally further substituted by one or more substituents selected from the group consisting of hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxy or carboxylate;

m is selected from 1, 2, 3 or 4; and is

n is selected from 0, 1 or 2.

In a preferred embodiment of the present invention, the compound of formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof is a compound of formula (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof,

wherein:

R ³ selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, cyano, nitro, halogen, cycloalkyl, heterocyclyl, -C (O) R ⁵ 、-OC(O)R ⁵ 、-C(O)OR ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-S(O) _n NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Wherein said alkyl and alkoxy areThe group, cycloalkyl or heterocyclyl is optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ Substituted with the substituent(s);

R ¹ 、R ² 、R ⁴ ～R ¹⁰ and n is as defined in formula (I).

In a preferred embodiment of the present invention, a compound of formula (I) or (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ selected from hydrogen atoms or C _1-4 Alkyl, wherein said C _1-4 Alkyl optionally further substituted by one or more C _1-6 Alkoxy substituted; wherein said C _1-6 The alkoxy group is preferably a methoxy group.

R ² is selected from C _1-4 Alkyl, wherein said C _1-4 Alkyl is further substituted with one or more halogens; wherein said halogen is preferably fluorine, chlorine or bromine; more preferably fluorine.

In a preferred embodiment of the present invention, the compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein R is ² Is trifluoroethyl.

R ³ selected from hydrogen atoms, C _1-4 Alkyl or halogen;

wherein said C _1-4 Alkyl is preferably methyl or ethyl; and is

Wherein said halogen is preferably fluorine, chlorine or bromine.

R ⁴ selected from phenyl or benzothienyl, wherein said phenyl or benzothienyl is optionally further substituted by one or more groups selected from halogen, C _1-4 Alkyl or C _1-6 Substituted by a substituent of alkoxy;

wherein said C _1-4 Alkyl or C _1-6 Alkoxy is optionally further substituted with one or more halo;

wherein said C _1-4 Alkyl is preferably methyl or ethyl; and is

Wherein said halogen is preferably fluorine, chlorine or bromine; more preferably fluorine.

R ¹ selected from hydrogen atoms or alkyl groups, preferably C _1-4 Alkyl, wherein said alkyl is optionally further substituted with one or more C _1-6 Alkoxy substituted; wherein said C _1-6 The alkoxy group is preferably a methoxy group.

R ² Selected from alkyl, wherein said alkyl is further substituted with one or more halogens; preferably, the alkyl group is C _1-4 Alkyl, more preferably ethyl; the halogen is preferably fluorine, chlorine or bromine, more preferably fluorine.

R ³ Selected from the group consisting of hydrogen, alkyl, and halogen, wherein said alkyl is optionally further substituted with one or more halogens; the alkyl group is preferably C _1-4 Alkyl, more preferably methyl or ethyl; and the halogen is preferably fluorine, chlorine or bromine.

R ⁴ Selected from phenyl or benzothienyl, wherein said phenyl or benzothienyl is optionally further substituted by one or more substituents selected from halogen, alkyl or alkoxy; wherein said alkyl or alkoxy is optionally further substituted with one or more halogens; the alkyl group is preferably C _1-4 Alkyl radical(ii) a The alkoxy group is preferably C _1-6 An alkoxy group; and the halogen is preferably fluorine, chlorine or bromine; more preferably fluorine.

Typical compounds of the invention include, but are not limited to:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

The invention provides a preparation method of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps:

Reacting a compound of formula (IA) with a compound of formula (IB) under basic conditions to give a compound of formula (I);

wherein: r ¹ ～R ⁴ And m is as defined in formula (I).

In the preparation method, the alkaline condition is provided by organic base or inorganic base, the organic base is selected from diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine and 4-dimethylaminopyridine, and preferably diisopropylethylamine or triethylamine; the inorganic base is selected from sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, potassium hydride, preferably cesium carbonate or potassium carbonate.

The invention provides a preparation method of a compound shown in a general formula (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps:

reacting a compound of a general formula (IIA) with a compound of a general formula (IB) under an alkaline condition to obtain a compound of a general formula (II);

wherein: r ¹ ～R ⁴ Is as defined in formula (II).

In the preparation method, the alkaline condition is provided by organic base or inorganic base, the organic base is selected from diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine and 4-dimethylaminopyridine, and preferably diisopropylethylamine and/or triethylamine; the inorganic base is selected from sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, potassium hydride, preferably cesium carbonate and/or potassium carbonate.

Further, the present invention provides an intermediate compound of the general formula (IA) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof,

wherein: r ¹ ～R ³ And m is as defined in formula (I).

The invention provides an intermediate compound shown in a general formula (IA) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, which is an intermediate compound shown in a general formula (IIA) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof,

wherein: r ¹ ～R ³ Is as defined in formula (II).

Typical intermediate compounds of formula (IA) of the present invention include, but are not limited to:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

The present invention provides a process for the preparation of an intermediate compound of general formula (IA) or a stereoisomer, tautomer or a pharmaceutically acceptable salt thereof, which process comprises:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (Ib) under heating condition to obtain a compound of a general formula (Ic); reacting the compound of the general formula (Ic) with the compound of the general formula (Id) under heating to obtain an intermediate compound of the general formula (IA);

wherein:

x is halogen;

R ^a is an alkyl group; and is

R ¹ ～R ³ And m is as defined in formula (IA), and R ¹ Not a hydrogen atom.

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (Ib) under heating condition to obtain a compound of a general formula (Ic); carrying out condensation reaction on the compound of the general formula (Ic) and the compound of the general formula (Ie) under heating condition to obtain a compound of the general formula (If); reacting the compound of the general formula (If) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IA);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^a is an alkyl group; r ^b Is an alkyl group; and is

R ² 、R ³ And m is as defined in formula (IA).

The invention provides a preparation method of an intermediate compound shown in a general formula (IIA) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (IIa) under heating conditions to obtain a compound of a general formula (IIb); reacting the compound of the general formula (IIb) with the compound of the general formula (Id) under heating to obtain an intermediate compound of the general formula (IIA);

wherein:

x is halogen;

R ^a is an alkyl group; and is

R ¹ ～R ³ Is as defined in formula (IIA), and R ¹ Not a hydrogen atom.

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (IIa) under heating conditions to obtain a compound of a general formula (IIb); carrying out condensation reaction on the compound of the general formula (IIb) and the compound of the general formula (Ie) under heating condition to obtain a compound of the general formula (IIc); reacting the compound of the general formula (IIc) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IIA);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^a is an alkyl group; r ^b Is an alkyl group; and is

R ² 、R ³ The definition of (A) is as described in formula (IIA).

Further, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or a combination thereof.

The present invention provides a method of inhibiting c-KIT comprising contacting a c-KIT receptor with a compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

The invention provides a compound of general formula (I) or (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the preparation of a medicament for treating a disease mediated by c-KIT or mutated c-KIT, wherein the disease mediated by c-KIT or mutated c-KIT is preferably gastrointestinal stromal tumor, systemic mastocytosis, acute myelogenous leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia; more preferably gastrointestinal stromal tumors, systemic mastocytosis, glioblastoma multiforme and acute myeloid leukemia, most preferably gastrointestinal stromal tumors, glioblastoma multiforme and systemic mastocytosis; preferably the mutation of said mutated c-KIT is located at exon 9, 11, 13, 14, 17 and/or 18, or at amino acid residue 816, 670, 560 and/or 654, wherein said mutation at amino acid residue 816 is preferably D816V or D816H; wherein said mutation at amino acid residue 670 is preferably T670I; wherein said mutation at amino acid residue 560 is preferably V560G; wherein the mutation at amino acid residue 654 is preferably V654A.

The invention provides an application of a compound shown in a general formula (I) or (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a c-KIT inhibitor.

The invention provides an application of a compound shown in a general formula (I) or (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a PDFGR alpha inhibitor.

The invention provides a compound of general formula (I) or (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the preparation of a medicament for treating a disease mediated by a mutant or wild-type PDFGR alpha, wherein the disease mediated by the mutant or wild-type PDFGR alpha is preferably gastrointestinal stromal tumor, systemic mastocytosis, acute myelogenous leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia; more preferably gastrointestinal stromal tumors, systemic mastocytosis, glioblastoma multiforme and acute myeloid leukemia, most preferably gastrointestinal stromal tumors, glioblastoma multiforme and systemic mastocytosis; preferably, the mutation of the mutated PDFGR α is located at exon 18 and/or at amino acid residue 842, wherein the mutation at amino acid residue 842 is preferably a D842V mutation.

The present invention provides a method for treating a disease mediated by c-KIT or mutant c-KIT, comprising administering to a patient a therapeutically effective amount of a compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, wherein said disease mediated by c-KIT or mutant c-KIT is preferably gastrointestinal stromal tumor, systemic mastocytosis, acute myelogenous leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia; more preferably gastrointestinal stromal tumors, systemic mastocytosis, glioblastoma multiforme and acute myeloid leukemia, most preferably gastrointestinal stromal tumors, glioblastoma multiforme and systemic mastocytosis; wherein said mutation of the mutated c-KIT is located at exon 9, 11, 13, 14, 17 and/or 18, or at amino acid residue 816, 670, 560 and/or 654, wherein said mutation at amino acid residue 816 is preferably D816V or D816H; wherein said mutation at amino acid residue 670 is preferably T670I; wherein said mutation at amino acid residue 560 is preferably V560G; wherein the mutation at amino acid residue 654 is preferably V654A.

The present invention provides a method for treating a disease mediated by pdfgra or mutated pdfgra, comprising administering to a patient a therapeutically effective amount of a compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, wherein the pdfgra or mutated pdfgra mediated disease is preferably gastrointestinal stromal tumor, systemic mastocytosis, acute myelogenous leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia; more preferably gastrointestinal stromal tumors, systemic mastocytosis, glioblastoma multiforme and acute myeloid leukemia, most preferably gastrointestinal stromal tumors, glioblastoma multiforme and systemic mastocytosis; wherein the mutation of the mutated PDFGR alpha is located at exon 18 and/or at amino acid residue 842, wherein the mutation at amino acid residue 842 is preferably a D842V mutation.

Detailed description of the invention

Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:

"alkyl" when taken as a group or part of a group means including C ₁ -C ₂₀ Straight-chain or branched aliphatic hydrocarbon groups. Preferably C ₁ -C ₁₀ Alkyl, more preferably C ₁ -C ₆ An alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 11-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. Alkyl groups may be substituted or unsubstituted.

"alkenyl" means an aliphatic hydrocarbon group containing one carbon-carbon double bond, which may be straight or branched, and representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-or 3-butenyl, and the like. The alkenyl group may be optionally substituted or unsubstituted.

"alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, and can be straight or branched. Preferably selected is C ₂ -C ₁₀ Alkynyl of (2), more preferably C ₂ -C ₆ Alkynyl, most preferably C ₂ -C ₄ Alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like. Alkynyl groups may be substituted or unsubstituted.

"cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged, and spiro carbocyclic rings, but no ring has an aromatic system of completely conjugated pi electrons. Preferably C ₃ -C ₁₂ Cycloalkyl, more preferably C ₃ -C ₈ Cycloalkyl, most preferably C ₃ -C ₆ A cycloalkyl group. Examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopropyl, cyclohexenyl being preferred.

"spiro cyclic group" refers to a polycyclic group of 5 to 18 membered, two or more cyclic structures, and single rings share one carbon atom (called spiro atom) with each other, and one or more rings thereof may contain 1 or more double bonds, but none of the rings has an aromatic system of completely conjugated pi electrons. Preferably 6 to 14, more preferably 7 to 10. Spirocycloalkyl groups are classified according to the number of spiro atoms shared between rings into mono-spiro, di-spiro, or multi-spiro cycloalkyl groups, preferably mono-spiro and di-spiro cycloalkyl groups, preferably 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro [4.5] decyl, spiro [4.4] nonyl, spiro [3.5] nonyl, spiro [2.4] heptyl.

"fused cyclic group" means a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms with each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic fused alkyl groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicycloalkyl groups. Non-limiting examples of "fused ring alkyl" include, but are not limited to: bicyclo [3.1.0] hexyl, bicyclo [3.2.0] hept-1-enyl, bicyclo [3.2.0] heptyl, decalinyl or tetradecaphenanthryl.

"bridged cyclic group" means a 5 to 18 membered all carbon polycyclic group containing two or more cyclic structures sharing two non-directly attached carbon atoms with each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12, more preferably 7 to 10. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged cycloalkyl groups according to the number of constituent rings, and are preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" groups include, but are not limited to: (1s, 4s) -bicyclo [2.2.1] heptyl, bicyclo [3.2.1] octyl, (1s, 5s) -bicyclo [3.3.1] nonyl, bicyclo [2.2.2] octyl, and (1r, 5r) -bicyclo [3.3.2] decyl.

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring to which the parent structure is attached is cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted.

"Heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein and all refer to non-aromatic heterocyclic groups in which one or more of the ring-forming atoms is a heteroatom, such as oxygen, nitrogen, sulfur, and the like, including monocyclic, fused, bridged, and spiro rings. Preferably having a 5 to 7 membered monocyclic ring or a 7 to 10 membered bi-or tricyclic ring, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1, 1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclic group may be substituted or unsubstituted.

"spiroheterocyclyl" refers to a 5-to 18-membered polycyclic group having two or more cyclic structures wherein the individual rings share an atom with one another and which contains 1 or more double bonds within the ring, but none of the rings have a fully conjugated pi-electron aromatic system wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O) _n (wherein n is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. The spirocycloalkyl group is classified into a single spiroheterocyclic group, a double spiroheterocyclic group or a multiple spiroheterocyclic group, preferably a single spiroheterocyclic group and a double spiroheterocyclic group, according to the number of spiro atoms shared between rings. More preferred are 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclic groups. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to: 1, 7-dioxaspiro [4.5 ]]Decyl, 2-oxa-7-azaspiro [4.4]Nonyl, 7-oxaspiro [3.5 ]]Nonyl and 5-oxaspiro [2.4 ]]A heptyl group.

"fused heterocyclyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a pair of atoms with each other, one or more of which rings may contain one or more double bonds, but none of which rings has a fully conjugated pi-electron aromatic system, wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O) _n (wherein n is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. According to the number of constituent rings, they may be bicyclic, tricyclic, pyridone or polycyclic fusedThe heterocyclic group is preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: octahydropyrrolo [3, 4-c ]Pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo [3.1.0]Hexyl, octahydrobenzo [ b ]][1，4]Dioxins (dioxines).

"bridged heterocyclyl" means a 5-to 14-membered, 5-to 18-membered polycyclic group containing two or more cyclic structures sharing two atoms not directly attached to each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system in which one or more ring atoms are selected from nitrogen, oxygen, or S (O) _n (wherein n is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, pyridone or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or pyridone, more preferably bicyclic or tricyclic. Non-limiting examples of "fused heterocyclic groups" include, but are not limited to: 2-azabicyclo [2.2.1]Heptyl, 2-azabicyclo [2.2.2]Octyl and 2-azabicyclo [3.3.2]A decyl group. The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl. The heterocyclic group may be optionally substituted or unsubstituted.

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C ₆ -C ₁₀ Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. The aryl group may be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples include, but are not limited to:

"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 9-to 10-membered bicyclic ring containing 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1, 2, 3-thiadiazolyl, benzodioxolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, and benzisoxazolyl. Heteroaryl groups may be substituted or unsubstituted. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples include, but are not limited to:

"alkoxy" refers to a radical of (alkyl-O-). Wherein alkyl is as defined herein. C ₁ -C ₆ Alkoxy groups of (4) are preferred. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"hydroxy" refers to an-OH group.

"halogen" refers to fluorine, chlorine, bromine and iodine.

"amino" means-NH ₂ 。

"cyano" means-CN.

"nitro" means-NO ₂ 。

"benzyl" means-CH ₂ -phenyl.

"carboxy" refers to-C (O) OH.

"carboxylate" means a-C (O) O-alkyl group or a-C (O) O-cycloalkyl group, wherein alkyl and cycloalkyl are as defined above.

"DMSO" refers to dimethyl sulfoxide.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

As used herein, "substituted" or "substituted," unless otherwise specified, means that the group may be substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxy, carboxylate, ═ O, -c (O) R ⁵ 、-OC(O)R ⁵ 、-C(O)OR ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-S(O) _n NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ 。

R ⁵ 、R ⁶ And R ⁷ Each independently selected from the group consisting of hydrogen, hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ Substituted with the substituent(s);

or, R ⁶ And R ⁷ Together with the N atom to which they are attached form a 4-to 8-membered heterocyclic group containing one or more N, O, S (O) atoms in the 4-to 8-membered heterocyclic group _n Atomic, and 4 &The 8-membered heterocycle is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, ═ O, -c (O) R ⁸ 、-C(O)OR ⁸ 、-OC(O)R ⁸ 、-NR ⁹ R ¹⁰ 、-C(O)NR ⁹ R ¹⁰ 、-S(O) _n NR ⁹ R ¹⁰ or-NR ⁹ C(O)R ¹⁰ Substituted with the substituent(s).

R ⁸ 、R ⁹ And R ¹⁰ Each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is optionally further substituted by one or more substituents selected from the group consisting of hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxy or carboxylate; and is

n is selected from 0, 1 or 2.

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salts of the compounds of formula (I) may be metal salts, amine salts with suitable acids.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

The invention relates to a preparation method of a compound shown in a general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, which comprises the following steps:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (Ib) under heating condition to obtain a compound of a general formula (Ic); reacting the compound of the general formula (Ic) with the compound of the general formula (Id) under heating to obtain a compound of the general formula (IA); reacting a compound of formula (IA) with a compound of formula (IB) under basic conditions to give a compound of formula (I);

wherein:

x is halogen;

R ^a is an alkyl group; and is

R ¹ ～R ⁴ And m is as defined in formula (I), and R ¹ Not a hydrogen atom.

carrying out condensation reaction on the compound of the general formula (Ic) and the compound of the general formula (Ie) under heating condition to obtain a compound of the general formula (If); reacting the compound of the general formula (If) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IA); reacting a compound of formula (IA) with a compound of formula (IB) under basic conditions to give a compound of formula (I);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^b Is an alkyl group; and is

R ² ～R ⁴ The definition of (A) is described in the general formula (I).

The invention relates to a preparation method of a compound shown in a general formula (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, which comprises the following steps:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (IIa) under heating conditions to obtain a compound of a general formula (IIb); reacting a compound of a general formula (IIb) with a compound of a general formula (Id) under heating to obtain a compound of a general formula (IIA); reacting a compound of a general formula (IIA) with a compound of a general formula (IB) under an alkaline condition to obtain a compound of a general formula (II);

wherein:

x is halogen;

R ^a is an alkyl group; and is

R ¹ ～R ⁴ Is as defined in formula (II), and R ¹ Not a hydrogen atom.

reacting the compound of the general formula (IIb) with the compound of the general formula (Ie) under heating to obtain a compound of the general formula (IIc); reacting the compound of the general formula (IIc) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IIA); reacting a compound of a general formula (IIA) with a compound of a general formula (IB) under an alkaline condition to obtain a compound of a general formula (II);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^b is an alkyl group; and is

R ² ～R ⁴ Is as defined in formula (II).

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and are not intended to limit the invention. ¹ The H NMR spectrum was obtained using a Bruker instrument (400MHz) and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00ppm) was used. ¹ Method for H NMR expression: s is singlet, d is doublet, t is triplet, m is multiplet, br is broadened, dd is doublet of doublet, dt is doublet of triplet. If a coupling constant is provided, it is in Hz.

The mass spectrum is measured by an LC/MS instrument, and the ionization mode can be ESI or APCI.

Thin layer chromatography silica gel plate is prepared from HSGF254 of tobacco yellow sea or GF254 of Qingdao by Thin Layer Chromatography (TLC)

The silica gel plate is 0.15-0.2 mm in specification, and the thin layer chromatography separation and purification product is 0.4-0.5 mm in specification.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

In the following examples, all temperatures are in degrees Celsius unless otherwise indicated, and unless otherwise indicated, the various starting materials and reagents are commercially available or synthesized according to known methods, and none of the commercially available materials and reagents are used without further purification, and unless otherwise indicated, commercially available manufacturers include, but are not limited to, Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Prov Chemical science Inc. and Sci Chemical science Inc., and the like.

CD ₃ OD: deuterated methanol.

CDCl ₃ : deuterated chloroform.

DMSO-d ₆ : deuterated dimethyl sulfoxide.

The argon atmosphere means that the reaction flask is connected with an argon balloon having a volume of about 1L.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

Purifying the compound by silica gel column chromatography using an eluent system selected from the group consisting of: a: petroleum ether and ethyl acetate systems; b: dichloromethane and methanol systems; c: dichloromethane: ethyl acetate; the volume ratio of the solvent is different according to the polarity of the compound, and a small amount of acidic or basic reagent such as acetic acid or triethylamine can be added for adjustment.

Example 1

1- (4-chloro-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3-phenylurea

First step of

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinic acid ethyl ester

Ethyl 4, 6-dichloronicotinate 1a (10.1g, 45.9mmol), 2, 2, 2-trifluoroethylamine (15.5g, 157mmol) and N, N-diisopropylethylamine (17.8g, 138mmol) were dissolved in 200mL of dimethyl adipate and reacted at 100 ℃ for 8 hours. Concentration under reduced pressure and purification of the obtained residue by silica gel column chromatography (eluent: system a) gave 6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinic acid ethyl ester 1b (4g, white solid), yield: 31 percent.

MS m/z(ESI)：282.9[M+1]

Second step of

(6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) pyridin-3-yl) methanol

Ethyl 6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinate 1b (4g, 14.18mmol) was dissolved in 50mL of tetrahydrofuran and lithium aluminium hydride (1.89g, 51mmol) was added portionwise at-50 ℃ and the temperature was slowly raised to 0 ℃ for reaction at 0 ℃ for 1.5 hours. The reaction was quenched by slowly dropping 20mL of 10% sodium hydroxide solution at 0 ℃, filtered, the filtrate was extracted with ethyl acetate (30mL), the aqueous layer was separated, the organic phase was washed with saturated sodium chloride solution (30mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) pyridin-3-yl) methanol 1c (3.4g, yellow solid), yield: 100 percent.

MS m/z(ESI)：240.9[M+1]

The third step

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde

(6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) pyridin-3-yl) methanol 1c (3.4g, 14.18mmol) and activated manganese dioxide (18.5g, 212.7mmol) were dissolved in 52mL of dichloromethane and stirred at room temperature overnight. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to obtain 6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde 1d (2.4g, white solid), yield: 71 percent.

MS m/z(ESI)：238.9[M+1]

The fourth step

3- (5-amino-2-chloro-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde 1d (1g, 4.2mmol), ethyl 2- (5-amino-2-chloro-4-fluorophenyl) acetate 1e (970mg, 4.2mmol, prepared according to published patent application WO 2013184119), potassium carbonate (1.74g, 12.6mmol) were dissolved in 70mL of a mixed solvent of N, N-dimethylformamide and toluene (V: V ═ 2: 5) and reacted at 150 ℃ for 12 hours. 100mL of ethyl acetate was added, washed with water (100 mL. times.2) and saturated sodium chloride solution (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-2-chloro-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 1f (870mg, yellow solid) in yield: 48 percent.

MS m/z(ESI)：405.8[M+1]

The fifth step

3- (5-amino-2-chloro-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2-chloro-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 1f (300mg, 0.74mmol) and a solution of methylamine in tetrahydrofuran (3mL, 6mmol) were dissolved in 20mL of 1, 4-dioxane and reacted at 100 ℃ for 16 hours in a sealed tube. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developer: system a) to give 1g (150mg, yellow solid) of 3- (5-amino-2-chloro-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one: 50 percent.

MS m/z(ESI)：400.8[M+1]

The sixth step

1g (100mg, 0.25mmol) of 3- (5-amino-2-chloro-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one, 1H (33mg, 0.28mmol, prepared according to published patent application WO 2005113626) of phenyl isocyanate and triethylamine (126mg, 1.25mmol) were dissolved in 2mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developer: system a) to give 1- (4-chloro-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3-phenylurea 1(20mg, white solid) in yield: 18 percent.

MS m/z(ESI)：520.1[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.17(s，1H)，8.76(s，1H)，8.46(s，1H)，8.21(d，J＝8.8Hz，1H)，7.87(s，1H)，7.56(d，J＝10.8Hz，1H)，7.43(d，J＝8.4Hz，2H)，7.28(t，J＝7.2Hz，2H)，7.21-7.20(m，1H)，6.99(t，J＝7.6Hz，1H)，6.37(s，1H)，5.13-5.11(m，2H)，2.87(d，J＝4.8Hz，3H).

Example 2

1- (4-chloro-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

1g (100mg, 0.25mmol) of 3- (5-amino-2-chloro-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one, 1-fluoro-3-phenylisocyanate 2a (51mg, 0.375mmol) and triethylamine (75mg, 0.75mmol) were dissolved in 2mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developer: system a) to give 1- (4-chloro-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 2(13mg, white solid), yield: 10 percent.

MS m/z(ESI)：537.9[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.35(s，1H)，8.80(s，1H)，8.45(s，1H)，8.19(d，J＝6.8Hz，1H)，7.87(s，1H)，7.58(d，J＝11.2Hz，1H)，7.48(d，J＝10.8Hz，1H)，7.32-7.29(m，1H)，7.21(s，1H)，7.10-7.08(m，1H)，6.81-6.79(m，1H)，6.37(s，1H)，5.76(s，1H)，5.13-5.09(m，1H)，2.87(d，J＝4.8Hz，3H).

Example 3

1- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

3- (5-amino-4-fluoro-2-methylphenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde 1d (1g, 4.2mmol), ethyl 2- (5-amino-4-fluoro-2-methylphenyl) acetate 3a (890mg, 4.2mmol, prepared according to published patent application WO 2013184119), potassium carbonate (1.74g, 12.6mmol) were dissolved in 70mL of a mixed solvent of N, N-dimethylformamide and toluene (V: V ═ 2: 5) and reacted at 150 ℃ for 8 hours. 50mL of ethyl acetate was added, washed with water (50 mL. times.2) and saturated sodium chloride solution (50mL) in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-4-fluoro-2-methylphenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3b (620mg, yellow solid) in yield: 39 percent.

MS m/z(ESI)：385.8[M+1]

Second step of

3- (5-amino-4-fluoro-2-methylphenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-4-fluoro-2-methylphenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3b (620mg, 1.61mmol) and a solution of methylamine in tetrahydrofuran (6mL, 12mmol) were dissolved in 36mL of 1, 4-dioxane and reacted at 100 ℃ for 48 hours in a sealed tube. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 3- (5-amino-4-fluoro-2-methylphenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3c (550mg, yellow solid) in yield: 89 percent.

MS m/z(ESI)：380.9[M+1]

The third step

3- (5-amino-4-fluoro-2-methylphenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3c (183mg, 0.48mmol), 1-fluoro-3-phenylisocyanate 2a (198mg, 1.44mmol) and triethylamine (243mg, 2.41mmol) were dissolved in 20mL of tetrahydrofuran and reacted at room temperature overnight. Concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give 1- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 3(50mg, white solid), yield: 20 percent.

MS m/z(ESI)：517.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.25(s，1H)，8.57(s，1H)，8.45(s，1H)，7.95(d，J＝8.4Hz，1H)，7.79(s，1H)，7.51-7.47(m，1H)，7.33-7.27(m，1H)，7.18-7.13(m，2H)，7.07(d，J＝7.6Hz，1H)，6.79(dt，J＝8.8，2.4Hz，1H)，6.37(s，1H)，5.14-5.12(m，2H)，2.86(d，J＝4.8Hz，3H)，2.08(s，3H).

Example 4

1- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea

First step of

3- (5-amino-4-fluoro-2-methylphenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3c (183mg, 0.48mmol), 1-fluoro-4-phenylisocyanate 4a (198mg, 1.44mmol) and triethylamine (243mg, 2.41mmol) were dissolved in 20mL of tetrahydrofuran and reacted at room temperature overnight. Concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to give 1- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea 4(50mg, white solid) in yield: 20 percent.

MS m/z(ESI)：517.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.07(s，1H)，8.50(s，1H)，8.44(s，1H)，7.95(d，J＝8.4Hz，1H)，7.78(s，1H)，7.46-7.42(m，2H)，7.17-7.09(m，4H)，6.37(s，1H)，5.14-5.12(m，2H)，2.86(d，J＝4.8Hz，3H)，2.07(s，3H).

Example 5

1- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

3- (5-amino-2, 4-difluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde 1d (300mg, 1.26mmol), ethyl 2- (5-amino-2, 4-difluorophenyl) acetate 5a (271.7mg, 1.26mmol, prepared according to published patent application WO 2013184119), and potassium carbonate (522mg, 3.78mmol) were dissolved in 28mL of a mixed solvent of N, N-dimethylformamide and toluene (V: V ═ 2: 5) and reacted at 150 ℃ for 8 hours. 50mL of ethyl acetate was added, washed successively with water (50 mL. times.2) and saturated sodium chloride solution (50mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-2, 4-difluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5b (237mg, pale yellow solid), yield: 48 percent.

MS m/z(ESI)：389.8[M+1]

Second step of

3- (5-amino-2, 4-difluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2, 4-difluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5b (237mg, 0.61mmol) and a solution of methylamine in tetrahydrofuran (3mL, 6mmol) were dissolved in 20mL of 1, 4-dioxane and reacted at 100 ℃ for 12 hours in a sealed tube. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 3- (5-amino-2, 4-difluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5c (168mg, pale yellow solid) in yield: 72 percent.

MS m/z(ESI)：384.9[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ8.43(s，1H)，7.86(s，1H)，7.17(d，J＝4.0Hz，1H)，7.07(t，J＝8.0Hz，1H)，6.82(t，J＝8.0Hz，1H)，6.34(s，1H)，5.11-5.09(m，2H)，5.04(s，2H)，2.85(d，J＝4.0Hz，3H).

The third step

3- (5-amino-2, 4-difluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5c (168mg, 0.44mmol), 1-fluoro-3-phenylisocyanate 2a (90mg, 0.66mmol) and triethylamine (727mg, 7.2mmol) were dissolved in 10mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 1- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 5(4.5mg, light yellow solid), yield: 2 percent.

MS m/z(ESI)：521.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.32(s，1H)，8.66(s，1H)，8.46(s，1H)，8.13(t，J＝8.5Hz，1H)，7.93(s，1H)，7.49(d，J＝11.8Hz，1H)，7.41(t，J＝10.3Hz，1H)，7.31(q，J＝7.9Hz，1H)，7.23(d，J＝5.1Hz，1H)，7.10(d，J＝8.2Hz，1H)，6.80(t，J＝8.1Hz，1H)，6.37(s，1H)，5.14-5.11(m，2H)，2.87(d，J＝4.7Hz，3H).

Example 6

1- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea

First step of

3- (5-amino-2, 4-difluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5c (300mg, 0.78mmol), 1-fluoro-4-phenylisocyanate 4a (321mg, 2.34mmol) and triethylamine (394mg, 3.9mmol) were dissolved in 20mL of tetrahydrofuran and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 1- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea 6(160mg, white solid), yield: 39 percent.

MS m/z(ESI)：521.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.58(s，1H)，8.83(s，1H)，8.65(s，1H)，8.40(s，1H)，8.18(t，J＝8.4Hz，1H)，8.10(s，1H)，7.86-7.45(m，3H)，7.13(t，J＝8.4Hz，2H)，6.76(s，1H)，5.21(s，2H)，3.00(s，3H).

Example 7

1- (4-bromo-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3-phenylurea

First step of

3- (5-amino-2-bromo-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

6-chloro-4- ((2, 2, 2-trifluoroethyl) amino) nicotinaldehyde 1d (600mg, 2.52mmol), ethyl 2- (5-amino-2-bromo-4-fluorophenyl) acetate 7a (698mg, 2.52mmol, prepared according to published patent application WO 2013184119), and potassium carbonate (1.04g, 7.56mmol) were dissolved in 70mL of a mixed solvent of N, N-dimethylformamide and toluene (V: V ═ 2: 5) and reacted at 150 ℃ for 12 hours. 100mL of ethyl acetate was added, washed with water (100 mL. times.2) and a saturated sodium chloride solution (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-2-bromo-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 7b (660mg, pale yellow solid), yield: 58 percent.

MS m/z(ESI)：449.7[M+1]

Second step of

3- (5-amino-2-bromo-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2-bromo-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 7b (160mg, 0.35mmol) and a solution of methylamine in tetrahydrofuran (1.5mL, 3mmol) were dissolved in 5mL of 1, 4-dioxane and reacted at 100 ℃ for 8 hours in a sealed tube. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 3- (5-amino-2-bromo-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 7c (123mg, yellow solid) in yield: 78 percent.

MS m/z(ESI)：444.8[M+1]

The third step

3- (5-amino-2-bromo-4-fluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 7c (114mg, 0.26mmol), phenyl isocyanate 1H (34mg, 0.28mmol), and triethylamine (130mg, 1.28mmol) were dissolved in a mixed solution of 20mL of toluene and dichloromethane (V: V ═ 1: 1) and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 1- (4-bromo-2-fluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3-phenylurea 7(20mg, white solid) in yield: 12 percent.

MS m/z(ESI)：563.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.16(s，1H)，8.76(s，1H)，8.45(s，1H)，8.22(d，J＝8.8Hz，1H)，7.84(s，1H)，7.69(d，J＝10.8Hz，1H)，7.43(d，J＝7.6Hz，2H)，7.28(t，J＝7.6Hz，2H)，7.22-7.20(m，1H)，6.99(t，J＝7.2Hz，1H)，6.37(s，1H)，5.14-5.11(m，2H)，2.86(d，J＝4.8Hz，3H).

Example 8

1- (benzo [ b ] thiophen-3-yl) -3- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) urea

First step of

3-Isocyanatobenzo [ b ] thiophen esters

Benzo [ b ] thiophene-3-carboxylic acid 8a (500mg, 2.8mmol) and triethylamine (319mg, 3.2mmol) were dissolved in 10mL of toluene and stirred at room temperature until 8a was completely dissolved. Diphenyl phosphorazidate (847mg, 3.1mmol) was added and reacted at 50 ℃ for 1 hour, and the reaction was continued at 100 ℃ for 0.5 hour. Concentration under reduced pressure gave crude 3-isocyanatobenzo [ b ] thiophenyl ester 8b (490mg, colorless oil), yield: 100 percent.

Second step of

3- (5-amino-4-fluoro-2-methylphenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 3c (183mg, 0.48mmol), 3-isocyanatobenzo [ b ] thiophene 8b (252mg, 1.44mmol) and triethylamine (243mg, 2.41mmol) were dissolved in 20mL of tetrahydrofuran and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 1- (benzo [ b ] thiophen-3-yl) -3- (2-fluoro-4-methyl-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) urea 8(60mg, yellow solid), yield: 22 percent.

MS m/z(ESI)：555.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.42(s，1H)，8.86(s，1H)，8.46(s，1H)，8.10(d，J＝8.8Hz，1H)，7.97(d，J＝8.0Hz，1H)，7.89(d，J＝8.4Hz，1H)，7.81(s，1H)，7.70(s，1H)，7.51(t，J＝6.4Hz，1H)，7.43(t，J＝8.0Hz，1H)，7.20(d，J＝12Hz，1H)，7.15-7.13(m，1H)，6.38(s，1H)，5.15-5.10(m，2H)，2.87(d，J＝4.8Hz，3H)，2.09(s，3H).

Example 9

1- (benzo [ b ] thiophen-3-yl) -3- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) urea

First step of

3- (5-amino-2, 4-difluorophenyl) -7- (methylamino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5c (200mg, 0.52mmol), 3-isocyanatobenzo [ b ] thiophene 8b (91mg, 0.52mmol) and triethylamine (157mg, 1.56mmol) were dissolved in 5mL of tetrahydrofuran and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: system a) to give 1- (benzo [ b ] thiophen-3-yl) -3- (2, 4-difluoro-5- (7- (methylamino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) urea 9(50mg, white solid), yield: 17 percent.

MS m/z(ESI)：559.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.41(s，1H)，8.89(s，1H)，8.48(s，1H)，8.28(t，J＝8.4Hz，1H)，7.99-7.96(m，2H)，7.89(d，J＝8.0Hz，1H)，7.71(s，1H)，7.51(t，J＝7.6Hz，1H)，7.46-7.41(m，2H)，7.23-7.20(m，1H)，6.37(s，1H)，5.14-5.09(m，2H)，2.87(d，J＝4.8Hz，3H).

Example 10

1- (4-bromo-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

3- (5-amino-2-bromo-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2-bromo-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 7b (340mg, 0.76mmol), 2-methoxyethylamine (568mg, 7.57mmol) and 1, 8-diazabicycloundecen-7-ene (DBU) (230mg, 1.51mmol) were dissolved in 7mL of N-methylpyrrolidone and reacted with a microwave at 180 ℃ for 1.5 hours. 70mL of water was added, extraction was performed with dichloromethane (100 mL. times.2), the organic phases were combined, washed with a saturated sodium chloride solution (100mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-2-bromo-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 10a (370mg, yellow oil), yield: 100 percent.

MS m/z(ESI)：488.8[M+1]

Second step of

3- (5-amino-2-bromo-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 10a (100mg, 0.21mmol), 1-fluoro-3-phenylisocyanate 2a (42mg, 0.31mmol) and triethylamine (62mg, 0.62mmol) were dissolved in 3mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developing solvent: system a) to give 1- (4-bromo-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 10(30mg, white solid), yield: 23 percent.

MS m/z(ESI)：625.7[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.36(s，1H)，8.81(s，1H)，8.45(s，1H)，8.18(d，J＝8.8Hz，1H)，7.83(s，1H)，7.71(d，J＝10.4Hz，1H)，7.48(d，J＝12.4Hz，1H)，7.31(q，J＝8.4Hz，1H)，7.27-7.23(m，1H)，7.08(d，J＝8.0Hz，1H)，6.81(t，J＝8.8Hz，1H)，6.51(s，1H)，5.06-5.04(m，2H)，3.53-3.50(m，4H)，3.29(s，3H).

Example 11

1- (4-bromo-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea

First step of

3- (5-amino-2-bromo-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 10a (100mg, 0.21mmol), 1-fluoro-4-phenylisocyanate 4a (42mg, 0.31mmol) and triethylamine (62mg, 0.62mmol) were dissolved in 3mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developing solvent: system a) to give 1- (4-bromo-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea 11(10mg, white solid), yield: 8 percent.

MS m/z(ESI)：625.7[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.21(s，1H)，8.76(s，1H)，8.44(s，1H)，8.19(d，J＝8.4Hz，1H)，7.83(s，1H)，7.69(d，J＝10.8Hz，1H)，7.46-7.43(m，2H)，7.25-7.24(m，1H)，7.13(t，J＝8.8Hz，2H)，6.50(s，1H)，5.07-5.02(m，2H)，3.53-3.50(m，4H)，3.29(s，3H).

Example 12

1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

3- (5-amino-2-chloro-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2-chloro-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 1f (400mg, 1mmol), 2-methoxyethylamine (750mg, 10mmol) and 1, 8-diazabicycloundec-7-ene (DBU) (304mg, 2mmol) were dissolved in 8mL of N-methylpyrrolidone and reacted with a microwave at 180 ℃ for 1 hour. 100mL of water was added, extraction was performed with dichloromethane (50 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: System A) to give 3- (5-amino-2-chloro-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 12a (400mg, yellow oil) in yield: 90 percent.

MS m/z(ESI)：444.9[M+1]

Second step of

3- (5-amino-2-chloro-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 12a (70mg, 0.16mmol), 1-fluoro-3-phenylisocyanate 2a (26mg, 0.19mmol) and triethylamine (48mg, 0.47mmol) were dissolved in 3mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developing solvent: system a) to give 1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 12(9mg, white solid), yield: 10 percent.

MS m/z(ESI)：581.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.36(s，1H)，8.80(s，1H)，8.45(s，1H)，8.18(d，J＝8.8Hz，1H)，7.86(s，1H)，7.58(d，J＝10.8Hz，1H)，7.50-7.43(m，2H)，7.31(q，J＝6.8Hz，1H)，7.08(d，J＝8.4Hz，1H)，6.83-6.79(m，1H)，6.51(s，1H)，5.06-5.03(m，2H)，3.51-3.50(m，4H)，3.29(s，3H).

Example 13

1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea

First step of

3- (5-amino-2-chloro-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 12a (100mg, 0.225mmol), 1-fluoro-4-phenylisocyanate 4a (37mg, 0.27mmol) and triethylamine (68mg, 0.675mmol) were dissolved in 3mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developing solvent: system a) to give 1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4-fluorophenyl) urea 13(9mg, white solid), yield: 10 percent.

MS m/z(ESI)：581.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.15(s，1H)，8.71(s，1H)，8.45(s，1H)，8.19(d，J＝8.8Hz，1H)，7.86(s，1H)，7.57(d，J＝11.2Hz，1H)，7.46-7.43(m，2H)，7.25-7.23(m，1H)，7.13(d，J＝8.8Hz，2H)，6.51(s，1H)，5.06-5.04(m，2H)，3.53-3.48(m，4H)，3.29(s，3H).

Example 14

1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4- (trifluoromethyl) phenyl) urea

First step of

3- (5-amino-2-chloro-4-fluorophenyl) -7- ((2-methoxyethyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 12a (100mg, 0.225mmol), 1-4- (trifluoromethyl) phenyl 1-isocyanate 14a (50mg, 0.27mmol) and triethylamine (68mg, 0.675mmol) were dissolved in 3mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography (developer: system a) to give 1- (4-chloro-2-fluoro-5- (7- ((2-methoxyethyl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (4- (trifluoromethyl) phenyl) urea 14(16mg, white solid), yield: 11 percent.

MS m/z(ESI)：631.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.67(s，1H)，8.91(s，1H)，8.45(s，1H)，8.18(d，J＝8.4Hz，1H)，7.87(s，1H)，7.64(s，4H)，7.59(d，J＝11.2Hz，1H)，7.25-7.24(m，1H)，6.51(s，1H)，5.06-5.04(m，2H)，3.53-3.50(m，4H)，3.29(s，3H).

Example 15

(R) -1- (4-chloro-2-fluoro-5- (7- ((1-methoxypropan-2-yl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea

First step of

(R) -3- (5-amino-2-chloro-4-fluorophenyl) -7- ((1-methoxypropan-2-yl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2-chloro-4-fluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 1f (202mg, 0.4mmol), (R) -1-methoxypropan-2-amine (223mg, 2.5mmol) and 1, 8-diazabicycloundec-7-ene (DBU) (152mg, 1mmol) were dissolved in 4mL of N-methylpyrrolidone and reacted with a microwave at 180 ℃ for 1.5 hours. 40mL of water was added, extraction was performed with dichloromethane (30 mL. times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: System A) to give (R) -3- (5-amino-2-chloro-4-fluorophenyl) -7- ((1-methoxyprop-2-yl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 15a (229mg, yellow solid), yield: 100 percent.

MS m/z(ESI)：458.9[M+1]

Second step of

(R) -3- (5-amino-2-chloro-4-fluorophenyl) -7- ((1-methoxypropan-2-yl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 15a (100mg, 0.22mmol), 1-fluoro-3-phenylisocyanate 2a (45mg, 0.33mmol) and triethylamine (66mg, 0.65mmol) were dissolved in 2mL of dichloromethane and reacted at room temperature overnight. Concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography (developer: system a) to give (R) -1- (4-chloro-2-fluoro-5- (7- ((1-methoxypropan-2-yl) amino) -2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-naphthyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea 15(12mg, white solid), yield: 10 percent.

MS m/z(ESI)：595.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.38(s，1H)，8.81(s，1H)，8.44(s，1H)，8.18(d，J＝8.4Hz，1H)，7.85(s，1H)，7.58(d，J＝10.8Hz，1H)，7.48(d，J＝11.6Hz，1H)，7.32-7.29(m，1H)，7.11-7.08(m，2H)，6.83-6.79(m，1H)，6.48(m，1H)，5.06-5.034(m，2H)，4.25-4.22(m，1H)，3.53-3.50(m，2H)，3.30(s，3H)，1.16(d，J＝7.2Hz，3H).

Example 16

1- (5- (7-amino-2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-diazonaphthyridin-3-yl) -2, 4-difluorophenyl) -3- (4-fluorophenyl) urea

First step of

3- (5-amino-2, 4-difluorophenyl) -7- ((4-methoxybenzyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2, 4-difluorophenyl) -7-chloro-1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 5b (500mg, 1.28mmol) was dissolved in 5mL of 4-methoxybenzylamine 16a and reacted at 130 ℃ overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, and 10mL of a mixed solvent of ethyl acetate and water (V: 1) was added thereto, followed by stirring at room temperature for 30 minutes. Extraction with ethyl acetate (30mL × 2), combination of the organic phases, washing with saturated sodium chloride solution (30mL), drying over anhydrous sodium sulfate, filtration and concentration under reduced pressure gave a residue which was purified by silica gel column chromatography (eluent: system a) to give 3- (5-amino-2, 4-difluorophenyl) -7- ((4-methoxybenzyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 16b (375mg, light yellow solid) in yield: 60 percent.

MS m/z(ESI)：490.9[M+1]

Second step of

7-amino-3- (5-amino-2, 4-difluorophenyl) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one

3- (5-amino-2, 4-difluorophenyl) -7- ((4-methoxybenzyl) amino) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 16b (375mg, 0.76mmol) was dissolved in 4mL of trifluoroacetic acid and reacted at 60 ℃ overnight. After the reaction was completed, it was cooled to room temperature, concentrated under reduced pressure, and a saturated sodium bicarbonate solution (20mL) was added at 0 ℃, extracted with ethyl acetate (30mL × 2), the organic phases were combined, washed with a saturated sodium chloride solution (30mL), dried over anhydrous sodium sulfate, filtered, and concentrated under pressure, and the obtained residue was purified by silica gel column chromatography (eluent: system a) to obtain 7-amino-3- (5-amino-2, 4-difluorophenyl) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 16c (200mg, pale yellow solid), yield: 71 percent.

MS m/z(ESI)：370.9[M+1]

The third step

Triethylamine (40.4mg, 0.4mmol) was dissolved in 1mL of tetrahydrofuran, and 7-amino-3- (5-amino-2, 4-difluorophenyl) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 16c (50mg, 0.1mmol) was added at 0 ℃ followed by dropwise addition of 1-fluoro-4-phenylisocyanate 4a (13.7mg, 0.1mmol) and reaction at room temperature overnight. After completion of the reaction, concentration was performed under reduced pressure, and the obtained residue was purified by preparative chromatography to give 1- (5- (7-amino-2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-diazonaphthyridin-3-yl) -2, 4-difluorophenyl) -3- (4-fluorophenyl) urea 16(9mg, light yellow solid), yield: 15 percent.

MS m/z(ESI)：507.8[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.10(s，1H)，8.58(s，1H)，8.42(s，1H)，8.10(t，J＝12.0Hz，1H)，7.94(s，1H)，7.47-7.37(m，3H)，7.12(t，J＝8.0Hz，2H)，6.78(s，2H)，6.42(s，1H)，5.01(d，J＝8.0Hz，2H).

Example 17

1- (5- (7-amino-2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-diazonaphthyridin-3-yl) -2, 4-difluorophenyl) -3- (3-fluorophenyl) urea

First step of

Triethylamine (54.6mg, 0.54mmol) was dissolved in 3mL of tetrahydrofuran, and 7-amino-3- (5-amino-2, 4-difluorophenyl) -1- (2, 2, 2-trifluoroethyl) -1, 6-naphthyridin-2 (1H) -one 16c (100mg, 0.27mmol) was added thereto at 0 ℃ followed by dropwise addition of 1-fluoro-3-phenylisocyanate 2a (40.76mg, 0.29mmol) and reaction at room temperature overnight. After completion of the reaction, concentration was performed under reduced pressure, and the obtained residue was purified by preparative chromatography to give 1- (5- (7-amino-2-oxo-1- (2, 2, 2-trifluoroethyl) -1, 2-dihydro-1, 6-diazonaphthyridin-3-yl) -2, 4-difluorophenyl) -3- (3-fluorophenyl) urea 17(54mg, white solid), yield: 41 percent.

MS m/z(ESI)：507.9[M+1]

¹ H NMR(400MHz，DMSO-d ₆ )δ9.31(s，1H)，8.68(s，1H)，8.48(s，1H)，8.10(t，J＝8.0Hz，1H)，7.99(s，1H)，7.51-7.40(m，2H)，7.34-7.28(q，J＝8.0Hz，1H)，7.10(d，J＝8.0Hz，3H)，6.82-6.78(m，1H)，6.53(s，1H)，5.04(d，J＝8.0Hz，2H).

Biological evaluation

Test example 1 determination of Activity of Compound of the present invention on recombinant human c-KIT [ D816V ] kinase

The following method was used to determine the degree of inhibition of the kinase activity of recombinant human c-KIT [ D816V ] (D816V mutation) by the compounds of the invention under in vitro conditions.

The method uses Cisbio

The tyrosine kinase kit (cat # 62TK0PEB) is based on time-resolved fluorescence energy resonance transfer (TF-FRET) and reflects the inhibition of protein kinase activity by compounds by measuring the degree of protein-mediated phosphorylation of biotinylated polypeptide substrates. The detailed experimental procedures can be referred to the kit instructions. Recombinant human c-KIT [ D816V ]]Protein kinases were purchased from Carna bioscience (Japan, cat # c-KIT [ D816V ]]#08-505)。

The experimental procedure is briefly described as follows: test compounds (compounds listed in table 1) were first dissolved in DMSO to prepare stock solutions, and then diluted in a gradient of buffer provided in the kit to give a final concentration of test compounds in the reaction system ranging from 10 μ M to 0.1 nM. The ATP solution (Biotechnology (Shanghai) Ltd., # A600311) used in the assay had a concentration of ATP Km value determined in advance for each kinase, where c-KIT [ D816V)]The ATP Km value concentration of (B) was 30. mu.M. The reaction is carried out in a 384-well microplate, firstly, a compound to be tested and 0.66ng of a protein to be tested are added into empty wells, the incubation is carried out for 5 minutes at room temperature, then, an ATP solution and a biotinylated polypeptide substrate solution are added into a reaction solution, the reaction solution is subjected to shaking incubation for 50 minutes at room temperature, then, an anti-phosphotyrosine antibody coupled with a europium series element compound and streptavidin coupled with modified allophycocyanin XL665 are added into the reaction solution, and the shaking incubation is continued for 1 hour at room temperature. After the incubation, the fluorescence intensity values of each well at an excitation wavelength of 304nM, an emission wavelength of 620nM and 665nM are measured in a microplate reader in TF-FRET mode. Percent inhibition of compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control (0.1% DMSO) and the compound IC was obtained by nonlinear regression analysis of the compound concentration log-inhibition by GraphPad Prism 5 software ₅₀ Values, see table 1.

TABLE 1 Compound of the invention vs c-KIT [ D816V]Inhibited IC ₅₀ Value of

And (4) conclusion: the compound has better inhibition effect on c-KIT D816V.

Remarking: imatinib was purchased from Seikagaku industries (CAS: 152459-95-5, purity: 98.87%, batch No. 1604105, off-white solid);

reference compounds 1, 2 and 3 are the compounds prepared in example 31, example 59 and example 15, respectively, disclosed in WO 2013184119; the concrete structure is as follows:

see, respectively, example 31, example 59 and example 15 of WO2013184119 for preparation method and structural identification thereof;

preparation of reference compound 4 was prepared with reference to example 15 of WO2013184119, with the following structural identification:

MS m/z(ESI)：468.8[M+1]；

1H NMR(400MHz，DMSO-d6)δ9.07(s，1H)，8.55(s，1H)，8.45(s，1H)，8.12(t，J＝12.0Hz，1H)，7.84(s，1H)，7.45-7.35(m，3H)，7.15-7.08(m，3H)，6.25(s，1H)，4.16(q，J＝4.0Hz，2H)，2.88(d，J＝8.0Hz，3H)，1.23(t，J＝4.0Hz，3H)。

test example 2 determination of the Activity of the Compounds of the invention on recombinant human c-KIT [ T670I ] and c-KIT [ V560G/D816V ] kinases

The following method was used to determine the degree of inhibition of the kinase activity of recombinant human c-KIT [ T670I ] and c-KIT [ V560G/D816V ] protein kinases by the compounds of the invention under in vitro conditions.

The method uses Cisbio

Tyrosine kinase kit (cat # 62TK0PEB) whose principle is based on time resolutionFluorescence resonance energy transfer (TF-FRET) is a measure of the extent of protein-mediated phosphorylation of biotinylated polypeptide substrates to reflect the strong or weak inhibition of protein kinase activity by compounds. The detailed experimental procedures can be referred to the kit instructions. Recombinant human c-KIT [ T670I ] ]Protein kinase and c-KIT [ V560G/D816V]Purchased from Carna bioscience (Japan, cat # c-KIT [ T670I)]#08-195 and c-KIT [ V560G/D816V # 08-535).

The experimental procedure is briefly described as follows: test compounds (compounds described in Table 2) were first dissolved in DMSO to prepare stock solutions, and then diluted in a gradient of buffer provided in the kit to give a final concentration of test compounds in the reaction system ranging from 10. mu.M to 0.1 nM. The ATP solution (Biotechnology (Shanghai) Ltd., # A600311) used in the test had a concentration of ATP Km value determined in advance for each kinase, where c-KIT [ T670I]The ATP Km value of (E) was 30. mu.M, c-KIT [ V560G/D816V]The ATP Km value concentration of (B) was 10. mu.M. The reaction was carried out in 384-well microplates by first adding the test compound and the test protein (c-KIT [ T670I ] to empty wells]0.66ng or c-KIT [ V560G/D816V]0.03ng) was added to the reaction solution, and incubated at room temperature for 5 minutes, then an ATP solution and a biotinylated polypeptide substrate solution were added to the reaction solution, and after incubation at room temperature for 50 minutes with shaking, an anti-phosphotyrosine antibody conjugated with a europium-based element compound and streptavidin conjugated with a modified allophycocyanin XL665 were added to the reaction, and incubation with shaking was continued for 1 hour at room temperature. After the incubation, the fluorescence intensity values of each well at an excitation wavelength of 304nM, an emission wavelength of 620nM and 665nM are measured in a microplate reader in TF-FRET mode. Percent inhibition of compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control (0.1% DMSO) and the compound IC was obtained by nonlinear regression analysis of the compound concentration log-inhibition by GraphPad Prism 5 software ₅₀ Values, see table 2.

TABLE 2 Compound pairs of the invention c-KIT [ T670I]And c-KIT [ V560G/D816V]Inhibited IC ₅₀ Value of

And (4) conclusion: the compound has better inhibition effect on c-KIT 760I and c-KIT V560G/D816V.

Test example 3 determination of Activity of the Compound of the present invention on recombinant human PDFGR alpha [ D842V ] kinase

The following methods were used to determine the inhibition of the kinase activity of recombinant human PDGFR α [ D842V ] (D842V mutation) by compounds represented herein under in vitro conditions.

The method uses Cisbio

The tyrosine kinase kit (cat # 62TK0PEB) is based on time-resolved fluorescence energy resonance transfer (TF-FRET) and reflects the inhibition of protein kinase activity by compounds by measuring the degree of protein-mediated phosphorylation of biotinylated polypeptide substrates. The detailed experimental procedures can be referred to the kit instructions. Recombinant humanized PDGFR alpha [ D842V]Protein kinases were purchased from Carna bioscience (Japan, Cat. No. PDFGR alpha [ D842V ]]#08-506)。

The experimental procedure is briefly described as follows: test compounds were first dissolved in DMSO to prepare a stock solution, followed by gradient dilution with the buffer provided in the kit, and the final concentration of the test compound in the reaction system ranged from 10 μ M to 0.1 nM. The ATP solution used for the test (Biotechnology engineering (Shanghai); A600311) was measured at a concentration of 30. mu.M for the previously determined ATP Km value. The reaction is carried out in a 384-well microplate, firstly, a compound to be tested and 0.66ng of a protein to be tested are added into empty wells, the incubation is carried out for 5 minutes at room temperature, then, an ATP solution and a biotinylated polypeptide substrate solution are added into a reaction solution, the reaction solution is subjected to shaking incubation for 50 minutes at room temperature, then, an anti-phosphotyrosine antibody coupled with a europium series element compound and streptavidin coupled with modified allophycocyanin XL665 are added into the reaction solution, and the shaking incubation is continued for 1 hour at room temperature. After the incubation, the fluorescence intensity values of each well at an excitation wavelength of 304nM, an emission wavelength of 620nM and 665nM are measured in a microplate reader in TF-FRET mode. Chemical combination was calculated by comparison with the fluorescence intensity ratio of the control group (0.1% DMSO) Percent inhibition of the compound at each concentration, and nonlinear regression analysis of the inhibition by GraphPad Prism 5 software using the logarithm of the compound concentration to obtain the IC of the compound ₅₀ Values, see table 3.

Table 3 compound pairs PDGFR α [ D842V are represented by this application]Inhibited IC ₅₀ Value of

Example numbering	IC ₅₀ (nM)/PDGFRα[D842V]
		16	61
17	18

And (4) conclusion: representative compounds 16 and 17 of the present application have a superior inhibitory effect on PDGFR α [ D842V ].

Test example 4 determination of Activity of Compound of the present invention on mouse mast cell tumor P815

The following methods were used to determine the effect of the compounds of the present invention on tumor Cell proliferation by using Cell Counting Kit-8 Kit (Dojindo, Donor's chemical technology) according to the instructions. For c-KIT [ D816V ], mouse mast cell tumor P815 (purchased from cell resource center of Shanghai Life sciences institute of Chinese academy of sciences) was used for culture.

The experimental method is briefly described as follows: test compounds (compounds described in Table 3) were first dissolved in DMSO to prepare stock solutions, and then diluted in a gradient with the culture medium of the corresponding cells to prepare test samples at final concentrations ranging from 30. mu.M to 0.01 nM. Tumor cells in logarithmic growth phase were added at 1000/well Was seeded into 96-well cell culture plates at 37 ℃ in 5% CO ₂ After overnight in the incubator, cells were incubated for an additional 48 hours after addition of the test compound sample. After incubation, 10. mu.L of CCK-8 assay solution was added to each well and incubated at 37 ℃ for 1-2 hours, followed by reading the absorbance values at 450nM for each well of the sample on a microplate reader. Percent inhibition of compound at each concentration point was calculated by comparison with absorbance values of control (0.3% DMSO), followed by non-linear regression analysis in GraphPad Prism 5 software at compound concentration log-inhibition to obtain IC of compound inhibition of cell proliferation ₅₀ Values, see table 4.

TABLE 4 IC inhibition of mouse mast cell tumor Activity by Compounds of the invention ₅₀ Value of

Compound numbering	IC ₅₀ (nM)/P815
		Imatinib	3252
Reference Compound 1	44
		5	25
6	19
		9	20

And (4) conclusion: the compound of the invention has obvious inhibition effect on proliferation of mouse mast cell tumor P815.

Test example 5 pharmacokinetic testing of the Compound of the invention

1. Abstract

SD rats are used as test animals, and the drug concentrations in blood plasma of the rats at different times after the rats are gavaged with the reference compound 1 and the compound 6 are measured by an LC/MS/MS method, so that the pharmacokinetic characteristics of the compound in the rats are researched.

2. Experimental protocol

2.1 Experimental drugs and animals

Reference compound 1 and compound 6 of the present invention;

healthy adult SD male rats were divided into 3 groups and purchased from sipelo bikeka laboratory animals ltd.

2.2 drug formulation and administration

Weighing appropriate amount of experimental medicine, adding 0.5% sodium carboxymethylcellulose (CMC-Na), ultrasonically treating to dissolve, and sucking 100 μ L with pipette for concentration determination to obtain solution with concentration of 0.3 mg/mL.

Healthy adult SD male rats, 6, were divided into 3 groups and were individually gavaged after overnight fasting at a dose of 3mg/kg and a volume of 10 mL/kg.

2.3 operation

0.25mL of cervical vein blood was collected before and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, placed in heparinized tubes, centrifuged at 8000 rpm at 2-8 ℃ for 6 minutes, stored at-70 ℃, and fed after 2 hours of administration. The LC-MS/MS method is used for determining the content of the compound to be tested in the plasma of SD male rats after the intragastric administration of different compounds. Pharmacokinetic parameters were calculated from blood concentration data at different time points using WinNonlin.

3. Pharmacokinetic parameter results

The pharmacokinetic parameters of the compounds of the invention are shown in table 5.

TABLE 5 pharmacokinetic data table for the compounds of the invention

And (4) conclusion: compared with the reference compound 1, the compound 6 has higher blood concentration and area under the curve of the drug time, simultaneously prolongs the half-life period and has better pharmacokinetic property.

Test example 6 ICR mouse pharmacokinetic test of preferred Compound of the invention

1. Abstract (abstract)

ICR mice are used as test animals, and the drug concentrations in blood plasma of the mice at different times after the mice are injected by tail vein and administered by gastric lavage with the compounds of reference compound 1, example 5 and example 6 are measured by an LC/MS/MS method, so that the pharmacokinetic characteristics of the compounds in the mice are researched.

2. Experimental protocol

2.1 Experimental drugs and animals

Reference compound 1, compound 5 of the present invention and compound 6;

54 healthy adult ICR male mice, 31.3-35.6 g, were divided into 6 groups, A-F, and purchased from Beijing Wintolite laboratory animal technologies, Inc. Animal quality certification number: 11400700310546.

2.2 drug formulation and administration

2.2.1 formulation of intravenous (IV-A) administration

Weighing a proper amount of experimental medicine, adding DMSO, Solutol HS-15, Saline 5: 10: 85(v/v/v), uniformly mixing by vortex, filtering by using a Nylon filter membrane (0.45 mu m), sucking 100 mu L multiplied by 2 by a pipette for concentration determination, configuring the concentration of the solution to be 0.2mg/mL, and storing at 2-8 ℃.

2.2.2 preparation of intragastric (IG-B) administration preparation

Weighing a proper amount of experimental medicine, adding 0.5% sodium carboxymethylcellulose (CMC-Na), performing ultrasonic treatment until the mixture is dissolved, sucking 100 mu L multiplied by 2 by a pipette for concentration determination, preparing a solution with the concentration of 0.3mg/mL, and storing at 2-8 ℃.

2.2.3 administration

54 healthy adult ICR male mice, 31.3-35.6 g, are divided into 6 groups and A-F groups, after fasting overnight, the A group, the C group and the E group are respectively administrated by tail vein injection, the administration dose is 1mg/kg, and the administration volume is 5 mL/kg; the group B, the group D and the group F are respectively administered by gastric lavage, the administration dose is 3mg/kg, and the administration volume is 10 mL/kg.

2.3 operation

80 μ L of blood was collected via orbital vein before intravenous administration and at 0.083 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after administration or before intragastric administration and at 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after administration, placed in EDTA-K2 anticoagulation tube, centrifuged at 1500g for 10 minutes, stored at-40 to-20 ℃, and fed after 4 hours after administration. The LC-MS/MS method is used for determining the content of the compound to be tested in the plasma of ICR male mice after different compounds are injected into the tail vein and are subjected to gastric lavage. Pharmacokinetic parameters were calculated from blood concentration data at different time points using WinNonlin.

3. Pharmacokinetic parameter results

The pharmacokinetic parameters of the compounds of the invention are shown in tables 6 and 7.

TABLE 6 pharmacokinetic data table for gavage administration of the compounds of the invention in mice

And (4) conclusion: compared with the reference compound 1, the compound 5 and the compound 6 have higher maximum blood concentration and area under the curve when the compound is administrated by intragastric administration to mice, and have better pharmacokinetic property.

TABLE 7 pharmacokinetic data table for intravenous administration of the compounds of the invention to mice

And (4) conclusion: compared with the reference compound 1, the compound 5 and the compound 6 have higher area under the curve when the mice are administrated by intravenous injection, and have better pharmacokinetic property.

Claims

1. A compound of the general formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R ¹ selected from hydrogen atoms or C _1-4 Alkyl, wherein said alkyl is optionally further substituted with one or more C _1-6 Alkoxy substituted;

R ² is trifluoroethyl;

R ³ same or different, each independently selected from hydrogen atom, C _1-4 Alkyl or halogen;

R ⁴ selected from phenyl or benzothienyl, wherein said phenyl or benzothienyl is optionally further substituted by one or more groups selected from halogen, C _1-4 Alkyl or C _1-6 Substituted by a substituent of alkoxy; wherein said C _1-4 Alkyl or C _1-6 Alkoxy is optionally further substituted with one or more halo;

m is selected from 1, 2, 3 or 4.

2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is ¹ Is selected from C _1-4 Alkyl radical, wherein said C _1-4 Alkyl is optionally further substituted with one or more methoxy groups.

3. The compound according to claim 1, which is a compound of the general formula (II) or a pharmaceutically acceptable salt thereof,

wherein:

R ³ selected from hydrogen atoms, C _1-4 Alkyl or halogen;

R ¹ 、R ² 、R ⁴ is as defined in claim 1.

4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:

R ³ selected from hydrogen, methyl, ethyl, fluorine, chlorine or bromine.

5. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:

R ⁴ selected from phenyl or benzothienyl, wherein said phenyl or benzothienyl is optionally further substituted by one or more groups selected from fluoro, chloro, bromo, methyl, ethyl or C _1-6 Substituted by a substituent of alkoxy.

6. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:

R ⁴ selected from phenyl or benzothienyl, wherein said phenyl or benzothienyl is optionally further substituted by one or more fluorine.

7. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is:

8. a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, which process comprises:

reacting a compound of formula (IA) with a compound of formula (IB) to give a compound of formula (I);

wherein: r ¹ ～R ⁴ And m is as defined in claim 1 or 2.

9. A process for the preparation of a compound of formula (II) or a pharmaceutically acceptable salt thereof according to claim 3, which process comprises:

reacting a compound of the general formula (IIA) with a compound of the general formula (IB) to obtain a compound of the general formula (II);

wherein: r ¹ ～R ⁴ Is as defined in claim 3.

10. An intermediate compound of formula (IA) or a pharmaceutically acceptable salt thereof,

wherein: r ¹ ～R ³ And m is as defined in claim 1 or 2.

11. The compound according to claim 10, which is a compound of formula (IIA), or a pharmaceutically acceptable salt thereof,

wherein: r ¹ ～R ³ Is as defined in claim 10.

12. The intermediate compound according to claim 10, wherein said compound is:

13. a process for the preparation of an intermediate compound of general formula (IA) or a pharmaceutically acceptable salt thereof, according to claim 10, which process comprises:

1) When R is ¹ When not a hydrogen atom:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (Ib) under heating condition to obtain a compound of a general formula (Ic); reacting the compound of formula (Ic) with a compound of formula (Id) to give an intermediate compound of formula (IA);

wherein:

x is halogen;

R ^a is C _1-4 An alkyl group; and is

R ¹ ～R ³ And m is as defined in claim 10, and R ¹ Is not H;

2) when R is ¹ When the hydrogen atom is:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (Ib) under heating condition to obtain a compound of a general formula (Ic); reacting the compound of formula (Ic) with a compound of formula (Ie) to give a compound of formula (If); reacting the compound of the general formula (If) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IA);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^a is C _1-4 An alkyl group;

R ^b is C _1-4 An alkyl group; and is

R ² 、R ³ And m is as defined in claim 10.

14. A process for the preparation of an intermediate compound of formula (IIA) as claimed in claim 11, which comprises:

1) when R is ¹ When not a hydrogen atom:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (IIa) under heating conditions to obtain a compound of a general formula (IIb); reacting a compound of formula (IIb) with a compound of formula (Id) to give an intermediate compound of formula (IIA);

Wherein:

x is halogen;

R ^a is C _1-4 An alkyl group; and is

R ¹ ～R ³ Is as defined in claim 11, and R ¹ Is not H;

2) when R is ¹ When the hydrogen atom is:

carrying out condensation reaction on a compound of a general formula (Ia) and a compound of a general formula (IIa) under heating conditions to obtain a compound of a general formula (IIb); reacting the compound of the general formula (IIb) with a compound of the general formula (Ie) to obtain a compound of the general formula (IIc); reacting the compound of the general formula (IIc) in the presence of trifluoroacetic acid to obtain an intermediate compound of the general formula (IIA);

wherein:

x is halogen;

R ¹ is a hydrogen atom;

R ^a is C _1-4 An alkyl group;

R ^b is C _1-4 An alkyl group; and is

R ² 、R ³ Is as defined in claim 11.

15. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

16. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, in the manufacture of a medicament for the treatment of a disease mediated by c-KIT or a mutated c-KIT, wherein the mutation of the mutated c-KIT is at amino acid residue exon 9, 11, 13, 14, 17 and/or 18, or position 816, 670, 560 and/or 654.

17. The use of claim 16, wherein the disease mediated by c-KIT or mutated c-KIT is gastrointestinal stromal tumor, systemic mastocytosis, acute myeloid leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia.

18. The use of claim 16, wherein the disease mediated by c-KIT or mutant c-KIT is gastrointestinal stromal tumor, systemic mastocytosis, glioblastoma multiforme, and acute myeloid leukemia.

19. The use of claim 16, wherein the disease mediated by c-KIT or mutated c-KIT is gastrointestinal stromal tumor, glioblastoma multiforme, and systemic mastocytosis.

20. The use of claim 16, wherein the mutation at amino acid residue 816 is D816V or D816H; wherein the 670 th amino acid residue is mutated into T670I; wherein the 560 th amino acid residue is mutated to V560G; wherein the 654 th amino acid residue is mutated into V654A.

21. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, in the manufacture of a c-KIT inhibitor.

22. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, for the manufacture of a medicament for the treatment of a disease mediated by mutant or wild-type pdfgra, wherein the mutation of the mutant pdfgra is at amino acid residue position 842 and/or exon 18.

23. The use according to claim 22, wherein the disease mediated by mutated or wild-type pdfgra is gastrointestinal stromal tumor, systemic mastocytosis, acute myeloid leukemia, ovarian cancer, melanoma, cervical cancer, seminoma, dysgerminoma, glioblastoma multiforme, teratoma, mast cell leukemia.

24. The use according to claim 22, wherein the disease mediated by mutated or wild-type pdfgra is gastrointestinal stromal tumor, systemic mastocytosis, glioblastoma multiforme and acute myeloid leukemia.

25. The use according to claim 22, wherein the disease mediated by mutant or wild-type pdfgra is gastrointestinal stromal tumor, glioblastoma multiforme, and systemic mastocytosis.

26. The use of claim 22, wherein the mutation at amino acid residue 842 is a D842V mutation.

27. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, in the preparation of a pdfgra inhibitor.