TWI624461B - Quinazoline compounds, method for preparing the same and use thereof - Google Patents

Abstract

A compound for treating a protein kinase-related disease having the structure of formula (I): Wherein G is heteroaryl, heterocyclyl or alkynyl; X is N or CH; L ¹ is -N(R ⁷ )-, -O-, -C(S)-, -C(O)- or - S-; L ² is -N(R ⁸ )- or -O-; R ¹ and R ^{2 are} each hydrogen, halogen, hydroxy, amine, cyano, nitro, carboxyl, C ₁ -C ₄ alkoxy , C ₁ -C ₄ alkoxy C ₁ -C ₄ alkoxy, N,N-(C ₁ -C ₄ dialkyl)amino C _1- C ₄ alkoxy, N-(C ₁ - C ₄ alkyl)amino C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkanoyl, C ₁ -C ₄ alkoxy, N-(C ₁ -C ₄ alkyl)amine, N An N-(C ₁ -C ₄ dialkyl)amino group, a C ₁ -C ₄ alkyl fluorenyl group or a heterocyclic group; wherein the C ₁ -C ₄ alkyl group may optionally be selected from one or more selected from the group consisting of fluorine and chlorine Substituted by a substituent; R ³ , R ⁴ and R ^{5 are} each hydrogen, fluoro or chloro; R ⁶ is C ₁ -C ₄ alkyl or aryl, which may optionally be selected from one or more selected from the group consisting of halogen, hydroxy, Substituted with a substituent of an amino group, a cyano group and a nitro group; or R ⁶ and R ⁸ are selected from a 5-6 membered ring group or a heterocyclic group.

Description

Quinazoline compound, preparation method and use thereof

This invention relates to a novel chemical compound and methods for its use in therapy and formulation. In particular, the present invention relates to certain substituted quinazoline compounds, and their use for inhibiting, regulating and/or modulating specific protein kinases and their associated signaling.

Protein kinases (PKs) play an important role in cellular signaling pathways that regulate a variety of cellular functions such as cell differentiation, proliferation, migration, survival, and apoptosis. These enzymes catalyze the transfer of the phosphate of ATP to the residues of tyrosine, serine or leucine on the receptor protein. Phosphorylation of kinases and dephosphorylation of dephosphatase are involved in numerous cellular processes, reflecting different intracellular signals, regulation of cellular functions, and activation or deactivation of cellular action.

Like metabolic, immune, and neurological diseases, abnormal PK activity has been shown to be associated with cancer. Therefore, protein kinases have become an attractive target for the treatment of human diseases. PK inhibitors, a compound that blocks PK activity, have been developed and widely used in clinical treatment. More than 30 PK inhibitors have been approved for the treatment of diseases, such as cancer treatment, but there is still a need for a new PK inhibitor to treat various diseases or overcome drug resistance. Thus, the identification of effective small molecule compounds that specifically inhibit message transmission and cell proliferation will be beneficial in modulating and regulating PK activity to modulate and regulate inappropriate cell proliferation, differentiation or metabolism leading to cancer.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the surprising discovery that certain quinazoline compounds can inhibit the activity of protein kinases (e.g., B-RAF, B-RAF (V600E), C-RAF). These properties make quinazoline compounds useful in the treatment of protein kinase related diseases, including cancer.

A specific embodiment of the invention is a quinazoline compound of formula (I): Wherein G is heteroaryl, heterocyclyl or alkynyl; X is N or CH; L ^{1 is} selected from -N(R ⁷ )-, -O-, -C(S)-, -C(O)- a group with -S-; L ^{2 is} selected from the group containing -N(R ⁸ )- and -O-; R ¹ and R ^{2 are} independently selected from hydrogen, halogen, hydroxy, amine, cyano, nitro , carboxy, alkoxy, alkoxyalkoxy, N,N-(dialkyl)aminoalkoxy, N-alkylaminoalkoxy, alkylamine, alkoxy, N- a group of an (alkyl)amino group, an N,N-(dialkyl)amino group, an alkylalkylamino group, a heterocyclic group, and a heteroalkenyl group, wherein the alkyl moiety is optionally one or more fluorine and Substituted by a substituent of chlorine; R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, fluorine and chlorine; R ^{6 is} selected from the group consisting of alkyl, alkenyl, alkynyl and aryl, wherein alkyl, alkenyl, alkynyl And the aromatic group is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amine, cyano and nitro; preferably, when L ² is -N(R ⁸ )-, R ⁶ and R ⁸ and the atom to which it is attached form a 5- to 6-membered cycloalkyl, cycloalkenyl or heterocycloalkyl group; R ⁷ and R ^{8 are} independently selected from the group consisting of hydrogen, alkyl and alkenyl.

A preferred embodiment of the invention is a compound selected from the group consisting of N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-yl) Amine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-morpholinpyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2 ,4-difluoro-3-(6-(6-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(3-(6-( 1H-pyrazol-4-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6) -(pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(4-fluoro-3-(6-(2-fluoropyridin-3-yl)) Quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(7-fluoro-6-(2-fluoropyridin-3-yl)quina Oxazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2-chloro-4-fluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4 -ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)benzene Methanesulfonamide; N-(2,4-difluoro-5-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonate Amine; N-(2,4-difluoro-3-(6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4 -difluoro-3-(6-(2-fluoropyridin-3-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3- (6-(pyridin-4-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N -(2,4-difluoro-3-(7-fluoro-6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2, 6-Difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,6-difluoro- 3-(6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2- Fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridine-3-) N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl) quinazoline) phenyl oxazol-4-ylamine) phenyl) ethane-1-sulfonamide Phenyl-4-ylamine)phenyl)propane-2-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-yl Amine)phenyl)benzenesulfonamide; N-(3-(7-(2-(dimethylamino)ethylamino)-6-(pyridin-4-yl)quinazolin-4-ylamine) -2,6-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-methoxypyridin-3-yl)quinolin-4-yl Amine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-4-yl)quinazolin-4-ylamine)phenyl) Propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-4-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonate amine; N-(2,4-Difluoro-3-(6-(2-oxo-1,2-dihydropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonate Guanidine; N-(2,4-difluoro-3-(6-(6-methoxypyridine-3-)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N-( 2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)-7-methoxyquinazolin-4-ylamine)phenyl)benzenesulfonamide; N-(2,4 -difluoro-3-(6-(2-fluoropyridin-3-yl)-7-(2-methoxyethoxy)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide N-(2,4-difluoro-3-(7-methoxy-6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N -(2,4-difluoro-3-(6-(2-methoxypyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2, 4-difluoro-3-(6-(6-oxo-1,6-dihydropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N- (2,4-Difluoro-3-(6-(6-(2-methoxyethoxy)pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-yloxy)phenyl)propane-1-sulfonamide; N-(3 -(6-(6-(2-(Dimethylamino)ethoxy)pyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonate Indoleamine; N-(2,4-difluoro-3-(6-(6-fluoropyridin-2-yl)quinazolin-4- Alkyl)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-methoxypyridin-2-yl)quinazolin-4-ylamine)benzene Propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-(2-methoxyethoxy)pyridin-2-yl)quinazolin-4-yl Amine)phenyl)propane-1-sulfonamide; N-(3-(6-(6-(2(dimethylamino)ethoxy)pyridin-2-yl)quinazolin-4-ylamine -2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4- Alkyl)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-morphopyridin-2-yl)quinazolin-4-ylamine)benzene Propane-1-sulfonamide; N-(2,4-difluoro-3-(6-morpholinium quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-( 3-(6-(1H-imidazol-1-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro -3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)-3-fluoropropane-1-sulfonamide; N-(3-(6-(2) -(2-dimethylamino)ethoxy)pyridin-4-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2 ,4-difluoro-3-(6-(5-fluoropyridin-3-yl)quinazolin-4-ylaminephenyl)propane-1-sulfonamide; N-(3-(6-(1H) -benzo[d]imidazol-1-yl)quinazolin-4- Base amine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazoline- 4-carbonyl)phenyl)propane-1-sulfonamide; N-(3-(6-ethynylquinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide N-(3-(6-(2-aminopyrimidin-5-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3 -(6-(2-(dimethylamino)pyrimidin-5-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3 -(6-(1H-1,2,3-triazol-4-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-( 2,difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-yl)(methyl)amino)phenyl)-N-methylpropane-1-sulfonamide N-(3-(6-(6-(dimethylamino)pyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane sulphonamide; N-(2, 4-difluoro-3-(5-fluoro-6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(3-( 6-(2-chloropyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3 -(6-(6-methoxy-5-nitropyridin-3-yl)quinazoline- 4-ylamine)phenyl)propane-1-sulfonamide; N-(3-(6-(3-aminophenyl)quinazolin-4-ylamine)-2,4-difluorophenyl) Propane-1-sulfonamide; N-(3-(6-(5-amino-6-methoxypyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl Propane-1-sulfonamide; N-(3-(6-(6-aminopyridin-2-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1 - sulfonamide; N-(2,4-difluoro-3-(6-(6-trifluoromethyl)pyridin-2-yl)quinazolin-4-ylamine)phenyl)propane-1- Sulfonamide; N-(3-(6-(6-cyanopyridin-2-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6-(2-cyanopyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2 4-difluoro-3-(6-(5-trifluoromethyl)pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2, 4-difluoro-3-(6-(4-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(3-(6-(6) -Amino-5-methylpyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide.

According to a particular embodiment of the invention, the compound has the structure of the following formula (I): Where G is selected from one of the following: among them Representing a pyridyl group, optionally substituted by one or more R ⁹ Representing a pyrimidinyl group, optionally substituted by one or more R ¹⁰ Representing a furanyl group, optionally substituted by one or more R ¹¹ Representing a pyrazolyl group, optionally substituted independently with R ¹² and one or more R ¹³ , Representing a triazolyl group, optionally substituted independently with R ¹⁴ and one or more R ¹⁵ , It represents an imidazolyl, which may optionally be substituted with one or more R ^16, wherein R ⁹ is independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, haloalkyl, alkoxy, methoxy a group consisting of an alkoxy group, a dialkylamino alkoxy group, a dialkylamino group and a heterocyclic group, wherein R ¹⁰ is hydrogen, a hydroxyl group, an amine group or a dialkylamino group, wherein R ¹¹ and R ¹³ And R ¹⁵ and R ¹⁶ are independently hydrogen, halogen or alkyl, and wherein R ¹² and R ¹⁴ are independently hydrogen, alkyl or alkanoyl.

According to a particular embodiment of the invention, the compound has the structure of formula (I): Where G is selected from: among them Representing a morpholinyl group, optionally substituted by one or more R ¹⁷ Representing a pyridone group, optionally substituted independently by R ¹⁸ and one or more R ¹⁹ , Representing an alkynyl group, which may be substituted by one R ²⁰ ; wherein R ^{17 is} independently selected from the group consisting of hydrogen, halogen, hydroxy, amine, cyano, alkyl and alkoxy; wherein R ^{18 is} selected from the group consisting of hydrogen and alkyl a group; wherein R ^{19 is} independently selected from the group consisting of hydrogen, halogen, hydroxy, amine, cyano, alkyl, and alkoxy; and wherein R ²⁰ is selected from hydrogen, alkyl, aryl, methoxy a group consisting of a base and an alkoxy group.

Other features and advantages of the present invention will become apparent from the description and appended claims.

definition

The term "alkyl", unless otherwise indicated, refers to a straight or branched monovalent saturated hydrocarbon group containing from 1 to 20 carbon atoms. Range of values in this specification is intended to include any numerical value within the scope of the definition. For example: an alkyl group of 1 to 20 carbons, including C ₁ , C ₂ ... C ₂₀ , and C ₁ - C ₂₀ , C ₁ - C ₁₅ , C ₁ - C ₁₀ , C ₁ - C ₆ , C ₁ -C ₄ and so on. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl. The term "alkenyl" refers to a straight or branched chain monovalent hydrocarbon radical containing from 2 to 20 carbon atoms (eg, C ₂ -C ₁₀ ) and one or more double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, and 1,4-butadiene. The term "alkynyl" refers to a 2-20 carbon atoms (e.g., C ₂ -C ₁₀₎ monovalent hydrocarbon radicals and linear or branched one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1- and 2-butynyl, and 1-methyl-2-butynyl. The term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. The term "decyloxy" refers to a -OC(O)-R group wherein R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl , aryl or heteroaryl. The term "amino" refers to NH _2. The term "alkylamino" refers to a -N(R)-alkyl group, wherein R can be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycle Alkenyl, aryl or heteroaryl.

The term "cycloalkyl" refers to a 3 to 30 carbon atoms (e.g., C ₃ -C ₆ or C ₃ -C ₁₂₎ monovalent saturated hydrocarbon ring system. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and adamantyl. The term "cycloalkenyl" refers to a (e.g., C ₃ -C ₆ or C ₃ -C ₁₂₎ and one or a monovalent non-aromatic hydrocarbon ring system having 3 to 30 carbon atoms, more double bonds. Examples include cyclopentenyl, cyclohexenyl and cycloheptenyl. The term "heterocycloalkyl" refers to a monovalent non-aryl 5-8 membered monocyclic ring, 8-12 membered bicyclic ring, or 11-14 membered three having one or more heteroatoms (eg, oxygen, nitrogen, sulfur, or selenium). Ring ring system. Examples of heterocycloalkyl groups include, but are not limited to, piperazinyl, pyridyl, piperidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl. The term "heterocyclenyl" refers to a monovalent non-aryl 5-8 membered monocyclic, 8-12 membered bicyclic ring having one or more heteroatoms (such as oxygen, nitrogen, sulfur or selenium) and one or more double bonds. Or a ring system of 11-14 members.

The term "aryl" refers to a monovalent 6-carbon monocyclic, 10-carbon bicyclic or 14-carbon tricyclic aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. The term "aryloxy" refers to -O-aryl. The term "arylamino" refers to -N(R)-aryl, wherein R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl , aryl or heteroaryl. The term "heteroaryl" refers to a ring of one or more heteroatoms (eg, oxygen, nitrogen, sulfur, or selenium) monovalent aryl 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring. system. Examples of heteroaryl groups include pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolyl, indolyl, thiazolyl, pyrrolyl, isoquinolyl, indolyl, oxazole Base, pyrazolyl and carbazolyl.

The aforementioned alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, amine, alkylamino, arylamino, alkoxy, aryloxy, naphthenic The base, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl and heteroaryl groups may be substituted or unsubstituted. Suitable substituents include, but are not limited to, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₃ -C ₂₀ cycloalkyl, C ₃ -C ₂₀ cycloalkenene , C ₁ -C ₂₀ heterocycloalkyl, C ₁ -C ₂₀ heterocycloalkenyl, C ₁ -C ₁₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amine, C ₁ -C ₁₀ alkylamino group, aromatic amine group, hydroxyl group, halogen, oxo group (O=), thio group (S=), thio group, C ₁ -C ₁₀ alkylthio group, arylthio group, C ₁ -C ₁₀ alkylsulfonyl, arylsulfonyl, decylamino, amidino, amine sulfhydryl, decyl, decyl, decyl, thioureido, thiocyanyl, sulfonamide Base, mercapto, ureido, cyano, nitro, decyl, thiodecyl, decyloxy, ureido, amine carbaryl (-C(O)NH ₂ ), carboxyl and carboxylic acid esters. On the other hand, the substituent of the alkyl group, the alkenyl group or the alkynyl group may include the above-exemplified substituents except for the C ₁ -C ₁₀ alkyl group. The cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl and heteroaryl groups may also be fused to each other.

In the following reaction formulas (Reaction Formula 1 to Reaction Formula 14), a representative scheme for preparing the compound of the formula (I) is provided. However, it will be understood by those skilled in the art that the invention may be modified or changed without departing from the spirit and scope of the invention. The quinazoline compound synthesized according to a specific embodiment of the present invention can be purified by flash column chromatography, high performance liquid chromatography, crystallization, or any other suitable method.

Intermediate product I

Concentrated nitric acid (1.1 eq.) was added dropwise to the fuming sulfuric acid (1.25 M) containing ethyl benzoate (1) (1.0 eq.) at 0 °C. Then, stir the mixture at room temperature for a small Then, the reaction mixture was poured into ice, and the aqueous phase was extracted with ethyl acetate. The organic layer was separated, washed with saturated sodium bicarbonate and concentrated to give product 2.

Activated carbon (0.05 equivalent) containing 10% (wt.) of palladium was added to a flask having methyl ethyl-3-nitrobenzoate (2) (1.0 equivalent) under nitrogen. Methanol (0.25 M) was added to the flask and stirred under 2 hydrogen balloon overnight. After the reaction was completed, the flask was flushed with nitrogen, and the reaction mixture was filtered through Celite. The volatiles were removed to give the crude ethyl 3-aminobenzoate (3).

Alkyl-1-sulfonium chloride (1.2 equivalents) was slowly added to a solution of ethyl 3-aminobenzoate (3) (1.1 equivalents) in pyridine (0.5 M) maintained in a cold water bath. The reaction mixture was stirred at room temperature for 1 hour and then poured into cold water. The aqueous layer was extracted with ethyl acetate. The organic layer was separated and washed with saturated aqueous ammonium chloride and brine then dried over magnesium sulfate, filtered and concentrated to give ethyl 3-(N-(alkylsulfonyl)sulfinylamino)benzoate (4) ).

Add 1 N aqueous sodium hydroxide (3.0 eq.) to a solution of ethyl 3-(N-(alkylsulfonyl)sulfonamide) (4) in 4:1 THF / MeOH (0.2M) (1.0 eq.). The reaction mixture was stirred at room temperature overnight then most of the organic solvent was removed in vacuo. A 1 N hydrochloric acid solution was slowly added to the mixture, and the resulting solid was filtered and washed with water. This material was washed with diethyl ether to give 3-alkylsulfonamide (5).

Intermediate II

Reaction formula 2.

Add N,N-diisopropylethylamine (2.0 equivalents) and chloromethyl methyl ether (2.0 equivalents) to phenol (6) (1.0 equivalents) dissolved in dry dichloromethane at 0 ° C under nitrogen. In solution. The resulting yellow mixture was stirred at 0 ° C for 30 minutes and left at room temperature overnight. The organic mixture was diluted with a 10% aqueous sodium hydroxide solution and extracted with dichloromethane. The organic layers were combined, dried over magnesium sulfate, filtered The crude product was purified on a silica gel column eluted with ethyl acetate in hexane to afford the desired product.

Add 1.4 M n-butyllithium in hexane (0.99 eq.) to a solution of methoxymethoxybenzene (7) (1.0 eq.) in tetrahydrofuran (0.35 M) at -70 ° C under nitrogen. The addition time is 10 minutes. The mixture was stirred at -70 ° C for 1.5 hours and then poured into pulverized dry ice. Once the bubbling has disappeared, the mixture can be warmed to room temperature and water added. The aqueous solution was extracted twice with diethyl ether and then acidified to pH 1 by adding concentrated hydrochloric acid. The resulting suspension was subjected to ultrasonication for 5 minutes and then extracted twice with dichloromethane. The combined dichloromethane extracts were dried over magnesium sulfate and concentrated under reduced pressure to afford product 9.

Alkanesulfonium chloride (1.2 equivalents) was slowly added to a pyridine solution (0.5 M) containing 3-hydroxybenzoic acid (9) (1.0 equivalent) maintained in a cold water bath. The reaction mixture was stirred at room temperature for 1 hour. Water was added, the organic layer was separated and washed with water and brine then dried over magnesium sulfate, filtered and concentrated to afford product 10.

Intermediate III

Triethylamine (2.3 equivalents) and diphenylphosphorizidine (1.15 equivalents) were added to a solution of benzoic acid (11) (1 eq.) in tetrahydrofuran (0.25M). The reaction mixture was stirred at room temperature for 3 hours and then warmed to 80 ° C for 2 hours. Water was added, and the mixture was stirred at 80 ° C for 15 hours. The reaction mixture was diluted with EtOAc. The solvent was removed under reduced pressure, and the residue was purified to silica gel column chromatography.

Intermediate IV

A small amount of lithium aluminum hydride (3.85 eq.) was added to an anhydrous tetrahydrofuran solution (1.1 M) containing 3-alkylsulfonylaminobenzoic acid (5) (1 eq.) at -20 ° C at -20 ° C. The mixture was stirred for 4 hours and then stirred at room temperature for 1 hour to carry out a reaction. The reaction was then quenched with saturated ammonium chloride solution. The crude product was obtained after usual workup, dried in vacuo and dissolved in anhydrous dichloromethane <RTIgt; The reaction mixture was stirred for 40 min. The combined organic layers were washed with water and brine over anhydrous magnesium sulfate Dry and concentrate under reduced pressure. The chromatographic column chromatography was carried out using a hexane stream wash containing ethyl acetate to give the product 13.

Intermediate product V

Pyridine (1.05 equivalent) was added to a stirred solution of phenol (14) (1.0 eq.) in dichloromethane (0.5M), and stirred for 10 min. Methyl chloroformate (1.05 eq.) diluted with dichloromethane was added dropwise over 15 min and the mixture was warmed to room temperature and stirring was continued for 17 hr. The reaction mixture was diluted with dichloromethane and washed sequentially with 0.5N aqueous sodium hydroxide and brine. The organic layer was filtered through a pad of anhydrous magnesium sulfate and concentrated to dryness.

Phenylmethyl carbonate (15) (1.0 eq.) was dissolved in concentrated sulfuric acid under nitrogen and cooled to 0 °C. Nitric acid (2.0 equivalents) was then carefully added while maintaining the temperature at 0 °C. The mixture was then stirred for 2 hours while warming to room temperature. It was then carefully poured into ice and extracted twice with ether. The combined organics were dried with MgSO4 andEtOAc

20% palladium on carbon (10% wt) was added to a solution of 3-nitrobenzyl carbonate (16) (1.0 eq.) in methanol (0.2 M). The reaction mixture was stirred at room temperature under hydrogen overnight. After replacing with nitrogen, the catalyst was filtered off. The solvent was evaporated from EtOAc (EtOAc m.

Alkyl-1-sulfonyl chloride (1.2 equivalents) was slowly added to a pyridine solution (0.5 M) containing ethyl methyl-3-aminobenzoate (17) (1.0 eq.) maintained in a cooling water bath. The reaction mixture was stirred at room temperature for 1 hour. Water was added and the organic layer was separated, the organic layer was washed with water and brine then dried over magnesium sulfate, filtered and concentrated to give product 18.

A methanol solution (0.2 M) containing benzyl carbonate (18) (1.0 eq.) was heated with a 1N sodium hydroxide (5.0 eq.) solution at 75 ° C for 14 hours. After cooling to room temperature, the methanol was evaporated in vacuo. The resulting aqueous mixture was acidified with a 1N aqueous solution of hydrochloric acid, diluted with brine, and extracted with dichloromethane. The combined organics were dried with anhydrous magnesium sulfate, filtered and evaporated. The resulting solid was purified by EtOAc (EtOAc) elute

Intermediate VI

A suspension of the compound 20 (1.0 equivalents) of formamide (15.0 equivalents) was heated to 165 ° C for 6 hours. After cooling to room temperature, water was added to the reaction mixture. sink The precipitate was collected by filtration, washed with water and dried in an oven at 120 ° C to give the title compound 21.

A suspension of compound 21 (1.0 eq.) in phosphorus oxychloride (1.0 M) was heated to reflux for 6 hours. The resulting clear solution was then cooled to room temperature and concentrated in vacuo to afford 4-chloro-6-bromoquinazoline (22).

Intermediate VII

Compound 12 (1.0 eq.) in ethanol (0.5 M) was added to compound 12 (1.1 eq.) and a catalytic amount of hydrochloric acid. After refluxing for 2 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was diluted with EtOAc (EtOAc)EtOAc. Further purification by column chromatography gave compound 24.

Intermediate VIII

60% sodium hydride was added to a stirred solution of 3-alkylsulfonamide (18) (1.1 eq.) and 4-chloroquinazoline (25) (1.0 eq.) in DMF (0.3 M). An oil suspension of 1.25 equivalents was then allowed to warm to room temperature and stirred for 5 hours. Water was added slowly to terminate the reaction and was diluted with ethyl acetate. It was then washed with water and brine, dried over anhydrous magnesium Further purification by column chromatography gave compound 26.

Intermediate IX

Sodium hydride (50% oil, 1.3 equivalents) was added to a solution containing 4-chloroquinazoline (25) (1.0 eq.), benzaldehyde (8) (1.3 eq.) and 1,3-dimethylimidazolium iodide ( 0.33 equivalents in tetrahydrofuran solution. The mixture was refluxed in an oil bath for a suitable period of time. After cooling, the mixture was poured into an ice-water mixture and extracted with ethyl acetate. The extract was dried over magnesium sulfate and concentrated to dryness. Further purification by column chromatography gave compound 27.

Final product I

Compound 24 (1.0 eq.), heteroarylboronic acid (1.5 eq.) and sodium carbonate (3.0 eq.) in dioxane/water (4:1, 0.2 M) with Pd(Ph ₃ P) ₄ (0.05 eq. The reaction was catalyzed and stirred at 80 ° C for 2 hours. The crude reaction mixture was filtered, dried over magnesium sulfate Further purification by column chromatography gave compound 28.

Final product II

Compound 29 (1.0 eq.), potassium acetate (3.0 eq.), Pd(dppf)Cl ₂ (0.03 eq.), and dipentane diboron (1.2 eq.) were added to dioxane (0.3 M) and heated to 5 Hours to 80 ° C. The solution was extracted with ethyl acetate and water, and the organic phase was washed again with water, dried over magnesium sulfate and evaporated to a small volume. Further purification by column chromatography gave compound 30.

Dioxolane quinazoline (30) (1.0 eq.), aryl halide or heteroaryl halide (1.2 eq.), tetrakis(triphenylphosphine)palladium(0) (0.05 eq.) and 2.0 M An aqueous solution of sodium carbonate (2.5 equivalents) was added to dioxane (0.3 M) and heated for 5 hours to 80 °C. The solution was extracted with ethyl acetate and water, and the organic phase was washed again with water, dried over magnesium sulfate and evaporated to a small volume. Further purification by column chromatography gave compound 31.

Final product III

Add cuprous iodide (0.1 eq.), L-valine (0.2 eq.), K ₂ CO ₃ (2 eq.), compound 29 (1.0 eq.) and imidazole (1.5 eq.) to the flask, and vacuum the flask and use Nitrogen backfill. DMSO (0.2 M) was added to the mixture solution as a syringe at room temperature under nitrogen. The mixture was heated to 100 ° C before the mixture was distributed between water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with brine, dried over magnesium sulfate Further purification by column chromatography gave compound 32.

Final product IV

Tetrakis(triphenylphosphine)palladium (0.02 eq.) was added to a solution of compound 29 (1.0 eq.), propargyl alcohol (1.5 eq.) and triethylamine (1.5 eq.) in tetrahydrofuran (0.2 M). 16 hours. The mixture was cooled and then filtered through celite and washed with methanol. Further purification by column chromatography gave Compound 33.

Final product V

Reaction formula 14.

Morpholine (0.5 M) was added to a solution of 5-fluoro-2-nitrobenzoic acid (1.0 eq.), and the reaction solution was heated to 90 ° C for 5 hr. The reaction was then cooled and water was added. After stirring for 10 minutes, the mixture was filtered and the filter cake was rinsed with water. Citric acid was added to the filtrate to precipitate more of the product. The filter cake was rinsed with water and then dried under vacuum at 40 ° C overnight to give product 35.

Activated carbon (0.05 equivalent) containing 10% by weight of palladium was added to a flask containing 2-nitrobenzoic acid (35) (1.0 equivalent) under a nitrogen atmosphere. Methanol (0.25 M) was then added to the flask and stirred under 2 hydrogen balloon overnight. After the reaction was completed, the flask was flushed with nitrogen and the reaction mixture was filtered over Celite. After removal of the volatiles, crude 2-aminobenzoic acid (36) was obtained.

A suspension containing the compound 36 (1.0 eq.) of formamide (7.0 eq.) was continuously heated at 165 °C for 6 hours. After cooling to room temperature, ethyl acetate was added to the reaction mixture and stirred vigorously. The precipitate was collected by filtration, washed with ethyl acetate and evaporated to dry

The suspension of phosphorus oxychloride (1.0 M) containing compound 37 (1.0 eq.) was heated to reflux for 6 h then the cooled solution was cooled to room temperature and concentrated in vacuo to give crude product 37, which Purification can be used in the next step.

Compound 12 (1.1 equivalents) and a catalytic amount of hydrochloric acid were added to a solution of compound 38 (1.0 eq.) in ethanol (0.5 M). After refluxing for 4 hours, the reaction mixture was cooled to room EtOAc. The residue was diluted with EtOAc (EtOAc)EtOAc. Further purification by column chromatography gave compound 39.

The quinazoline compound mentioned in the specification of the present invention may contain a non-aromatic double bond and one or more asymmetric centers, for example, a substituent attached to a central aromatic ring. Thus, these compounds can be used as racemic mixtures and racemic mixtures, single palmier isomers, single diastereomeric stereoisomers, diastereomeric mixtures, and cis or trans isomer forms. All such isomeric forms are included within the scope of the invention. The quinazoline compounds of the present invention may have acidic or basic functional groups (e.g., on a substituent) to form salts, particularly pharmaceutically acceptable salts, which are conventional in the pharmaceutical industry. It is also within the scope of the invention to practice such quinazoline salts. Likewise, its acidic or basic groups can be functionalized, for example to form an ester. Such functionalized derivatives are hydrolyzed in vivo, and thus such quinazoline compound derivatives described in the present invention can be used as a prodrug. While the formation of prodrugs involves only conventional skills, one skilled in the art can understand how to prepare and use such prodrugs without undue experimentation.

The scope of the present invention also encompasses: (1) a pharmaceutical composition comprising an effective amount of at least one quinazoline compound of the present invention and a pharmaceutically acceptable carrier thereof; (2) a method for treating a protein kinase-related disease a method (eg, cancer) achieved by administering a desired effective amount of such a quinazoline compound to a body; and (3) reducing at least A method of protein kinase activity by contacting at least one protein kinase with at least one quinazoline compound of the invention.

The term "protein kinase-associated disease" as used herein refers to a disease or condition having an abnormal property of PK activity, or a disease or condition treatable by altering at least one PK activity. Abnormal PK activity is caused by an increase in the expression of the PK gene or in the absence of normal occurrence of PK. PK-related diseases described herein include, but are not limited to, cancer, diabetes, hyperproliferative diseases, renal hyperproliferative diseases, kidney diseases, von Hippel-Lindau disease, regenerative stenosis, fibers Degeneration, psoriasis, osteoarthritis, rheumatoid arthritis, inflammatory diseases, immune diseases such as autoimmune diseases (such as AIDS, lupus erythematosus, etc.), cardiovascular diseases (such as atherosclerosis) and vascular proliferative diseases Such as abnormal angiogenesis.

The term "treating" means administering a quinazoline compound to an individual suffering from or associated with a protein kinase-related disease for the purpose of curing, restoring, alleviating, ameliorating, altering, correcting, ameliorating, ameliorating, affecting or reducing the disease. The quality of the risk, symptoms or rickets. For example, cancer therapy refers to treatment that results in inhibition of tumor growth or tumor cell growth, degradation of tumor growth (ie, it reduces the size of a detectable tumor), or the disappearance of cancer.

The term "effective amount" refers to the amount of active agent required to achieve the desired therapeutic effect in an individual. Effective dosages can vary depending on the techniques known in the art, the route of administration, the use of excipients, and possibly other drugs. For the determination of an effective dose, an effective dose for a predetermined use can be determined by those skilled in the art without undue experimentation requiring only conventional skill. The individual in need of treatment can be a mammal. The term "mammal" refers to a human or non-human mammal such as a dog, cat, pig, cow, sheep, goat, horse, rat, or mouse.

Cancers treatable by the methods of the invention include the growth of any abnormal cells or tissues, such as tumors, whether malignant, pre-malignant or non-malignant. Cancer is characterized by uncontrolled proliferation of cells that may or may not invade surrounding tissues and, therefore, may or may not metastasize to new body parts. Cancer includes epithelial cell carcinoma, while epithelial cell carcinoma includes squamous cell carcinoma, adenocarcinoma, melanoma, and liver cancer. Cancer also includes sarcomas, which are tumors of stromal cell origin; sarcomas include osteosarcoma, leukemia, and lymphoma. Cancer can include one or more tumor cell types. The term "cancer" is exemplified by, but not limited to, lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer. , oral cancer, skin cancer, brain cancer, lymphoma and leukemia. It also refers to drug-resistant cancers (including but not limited to multi-drug resistant cancers).

The compounds of the invention may be administered to a mammal in combination with radiation therapy, immunotherapy, monoclonal antibody therapy, hormonal therapy, chemotherapy and/or surgery with other drugs. The combined treatments need not occur at the same time, and may be continuous, or alternate with each other and/or with rest and recovery periods.

In a specific embodiment of the invention, a method of treating a protein kinase-related disease, such as cancer, comprising administering to a mammal an effective amount of at least one quinazoline compound of the invention and at least one chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, PK inhibitors as described herein (eg, imatinib mesylate, gefitinib, dasatinib, erro) Erlotinib, lapatinib, sunitinib, nilotinib, sorafenib, etc.; antibodies, including eg: trastuzumab , rituximab, cetuximab; and bevacizumab; mitoxantrone; dexamethasone; Prednisone and temozolomide, alkylating agents (eg melphalan, chlorambucil, busulfan, saudipa) Thiotepa), ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, ring Cyclomelamide, mitotic inhibitor, antimetabolite (eg capecitibine, gemcitabine, 5-fluorouracil or 5-fluorouracil/leucovorin, fludarabine) , cytarabine, mercaptopurine, thioguanine, pentostatin, and methotrexate, cell cycle inhibitors, enzymes, hormones , anti-hormone, growth factor inhibitors, plant alkaloids and terpenoids, topoisomerase inhibitors (eg, etoposide, teniposide, camptothecin, topography) Topotecan, irinotecan, and mildew (doxorubicin), daunorubicin, anti-tumor antibiotics (eg actinomycin D, bleomycin, mitomycin C, adriamycin) , daunorubicin, idarubicin, doxorubicin, and pegylated liposomal doxorubicin, vinca alkaloids (eg, vincristine) (vincristine) and vinblastine (vinblastin), taxanes (eg paclitaxel and docetaxel), platinum reagents (eg cisplatin, carboplatin and ileoplatin) Oxaliplatin)), thalidomide and related analogs (eg CC-5013 and CC-4047), monoclonal antibodies and anti-angiogenic agents.

The term "contacting" as used herein refers to the use of a method for introducing a compound of the invention with at least one PK band which directly reduces the activity of PK by acting with PK itself. Or indirectly reduce the activity of PK by acting with another molecule involved in PK activity. "Contacting" can occur in vitro or in vivo, for example, by administering a compound of the invention to a test tube containing at least one PK, a petri dish having whole cells grown, or a mammal. Examples of targets targeting PK include, but are not limited to, EGFR, CDK1, Aurora A & B kinase, MAP, CDK2, Raf, NEK (including NEK 4a, NEK 4b, NEK 5 and NEK 6), BUB1, VEGFR, C-MET , HER2, HER3, HER4, IR, IGF-IR, IRR, PDGFRct, PDGFRO, CSFIR, C-Kit, C-fms, Flk-1 R, Flk4, KDRlFlk-1, FLT-1, FLT3, FGFR-1, FGFR-2, FGFR-3, FGFR4, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, Aur2 and Yrk.

For practicing the methods of the invention, the above pharmaceutical compositions may be administered by oral, parenterally spray inhalation, topical topical application, rectal, nasal, buccal, vaginal or implanted reservoirs. . The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraspinal, intralesional, and intracranial injection or infusion techniques. In one embodiment, a quinazoline compound of the invention is administered by intravenous injection, and suitable carriers may include, but are not limited to, physiological saline or phosphate buffered saline (PBS), as well as thickening and solubilizing agents. Solutions such as glucose, polyethylene glycol solutions and polypropylene glycol and mixtures thereof.

Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Mannitol, water, Ringer's solution, and isotonic sodium chloride solution can be employed between acceptable excipients and solvents. In addition, sterile Non-volatile oils are commonly used as solvents or suspending media (eg, synthetic mono- or diglycerides). Fatty acids such as oleic acid and its glyceride derivatives can be used in the preparation of injectables, and natural medicinal oils such as olive oil or castor oil, especially in their polyoxyethylated form, are also useful in the preparation of injectables. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethylcellulose or a similar dispersing agent. Other commonly used surfactants (such as Tweens, or Spans) or other similar emulsifiers or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms can also be used for the preparation purposes.

Compositions for oral administration can be any orally acceptable dosage forms including, but not limited to, capsules, troches, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, conventional carriers include lactose and corn starch. A lubricant such as magnesium stearate can also usually be added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When the aqueous suspension or emulsion is administered orally, the active ingredient can be suspended or dissolved in the oil phase in association with an emulsifier or suspension. Some sweeteners, flavoring or coloring agents may be added if desired. Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. Compositions containing a quinazoline compound can also be administered in the form of an embolic agent for rectal administration.

The carrier in the pharmaceutical composition must be "acceptable", i.e., compatible with the active ingredient in the composition (if it can be stabilized) and not deleterious to the individual to be treated. One or more solubilizing agents (e.g., cyclodextrin) that form a more soluble complex with the active quinazoline compound can serve as a pharmaceutical carrier for delivery of the active compound. Examples of other carriers include colloidal cerium oxide, magnesium stearate, and sodium lauryl sulfate.

Suitable in vitro assays can be used to initially assess the efficacy of the quinazoline compounds of the invention in inhibiting the anti-cancer activity of tumor cell growth. The efficacy of these compounds in the treatment of cancer can be further examined. For example, the compound can be administered to an animal having cancer (for example, a mouse model), and then its therapeutic effect is evaluated. The appropriate dosage range and route of administration can also be determined based on these results.

The above description has fully realized the present invention without further elaboration. Therefore, the following examples are to be construed as illustrative only and not limiting the scope of the invention.

Instance

Exemplary quinazoline compounds are listed in Table 1, and the calculated mass and ESI-MS data series are listed in Table 2.

Biological activity

A variety of different compounds of formula (I) were used to test their ability to inhibit a variety of protein kinases, and the different assays are outlined below.

1.B-Raf kinase activity analysis

The ability of the test compound to inhibit kinase activity can be estimated by quantifying the amount of [ ^33P ] in the presence of the compound to be tested. Standard assay conditions were 5 ng of recombinant B-Raf kinase (Upstate Biotechnology) and 500 ng of MEK1 (K97R) in assay buffer (8 μM ATP, 0.5 μCi [ ³³ P] ATP (specific activity 3000 Ci/mmol, PerkinElmer), 50 mM Tris/HCl (pH 7.5) and 1 mM EGTA, 1 mM Na ₃ VO ₄ , 1% 2-mercaptoethanol, 0.1% Brij 35, and 0.2 mg/ml BSA) were brought to a final volume of 25 μL. The reaction solution was allowed to act at 30 ° C for 30 minutes, and the reaction was stopped by adding 3% phosphoric acid, and the product was collected into a 96-well GF/B microporous disk (UniFilter, PerkinElmer) using a cell harvester (PerkinElmer), and used. A microplate scintillation counter (PerkinElmer) was counted. Inhibitor IC ₅₀ values obtained via line serially diluted 3-fold concentration of each compound tested, and the test is repeated two do. The results obtained were analyzed by linear regression software (GraphPad Prism 4; GraphPad Software Inc.).

The inhibition activity data for the compounds listed in Table 1 are summarized in Table 3. IC ₅₀ value is defined as the maximum kinase activity was inhibited half the concentration of compound tested. + indicates that the concentration is 10,000 to 1,000 nM; ++ indicates the concentration is 1,000 to 300 nM; and +++ indicates that the concentration is less than 300 nM.

2.B-Raf (V600E) kinase activity analysis

The inhibition of kinase activity by the compounds disclosed herein can be calculated by quantifying the amount of [ ³³ P] in the presence of the compound to be tested. Standard assay conditions were 5 ng of recombinant B-Raf (V600E) kinase (Upstate Biotechnology) and 500 ng of MEK1 (K97R) dissolved in buffer (8 μM ATP, 0.5 μCi [ ³³ P] ATP (activity 3000 Ci/mmol, PerkinElmer), 50 mM Tris/HCl (pH 7.5), with 1 mM EGTA, 1 mM Na ₃ VO ₄ , 1% 2-mercaptoethanol, 0.1% Brij 35, and 0.2 mg/ml BSA), and test compound (diluted in 4% DMSO) or Separate DMSO (as a control) with a final reaction volume of 25 μL. The reaction was carried out at 30 ° C for 30 minutes, and 3% phosphoric acid was added to terminate the reaction. The product was collected in a 96-well GF/B UniFilter (PerkinElmer) using a cell harvester (PerkinElmer) and the radioactivity was counted in a TopCount microplate scintillation counter (PerkinElmer). IC ₅₀ values via the test-based inhibitor 3-fold serial dilutions to give a concentration of each compound, and the repeated linear regression The results obtained software (GraphPad Prism 4; GraphPad Software Inc. ) analysis.

The inhibition activity data for the selected compounds listed in Table 1 are compiled in Table 4. IC ₅₀ value is defined as half of the maximum inhibition of kinase activity, the concentration of the test compound. + indicates that the concentration is 10,000 to 1,000 nM; ++ indicates the concentration is 1,000 to 300 nM; and +++ indicates that the concentration is less than 300 nM.

3.C-Raf kinase activity analysis

The inhibition of kinase activity by the compounds disclosed herein can be calculated by quantifying the amount of [ ³³ P] in the presence of the compound to be tested. Standard assay conditions 2ng recombinant C-Raf kinase (Upstate Biotechnology) and 500ng MEK1 (K97R) dissolved in a buffer ^{(8μM ATP, 0.5μCi [33 P} ] ATP ( activity 3000Ci / mmol, PerkinElmer), 50mM Tris / HCl (pH 7.5), with 1 mM EGTA, 1 mM Na ₃ VO ₄ , 1% 2-mercaptoethanol, 0.1% Brij 35, and 0.2 mg/ml BSA), and test compound (diluted in 4% DMSO) or DMSO alone (as a control), the final reaction volume was 25 μL. The reaction was allowed to act at 30 ° C for 30 minutes, the reaction was stopped by the addition of 3% phosphoric acid, and the product was collected in a 96-well GF/B UniFilter (PerkinElmer) using a cell harvester (PerkinElmer). The radioactivity was counted using a TopCount microplate scintillation counter (PerkinElmer). IC ₅₀ values each based inhibitor by 3-fold serial dilutions of the compound obtained, and repeats, results obtained using linear regression software (GraphPad Prism 4; GraphPad Software Inc. ) analysis.

Selected compounds for inhibition of the kinase activity of IC ₅₀ data lines of the C-Raf finishing in Table 5 below. IC ₅₀ value is defined as half of the maximum inhibition of kinase activity, the concentration of the test compound. + indicates that the concentration is 10,000 to 1,000 nM; ++ indicates that the concentration is 1,000 to 300 nM; and +++ indicates that the concentration is less than 300 nM.

4. Analysis of VEGFR2 and EGFR kinase activity

Quinazoline is a backbone structure well known to be useful in the development of ATP competitive kinase inhibitors, particularly against EGFR and VEGFR2 kinases. However, the novel quinazoline derivatives of the present invention unexpectedly show selective inhibitory activity against Raf kinase without inhibiting VEGFR2 or EGFR kinase, as shown below.

Subsequent results show that VEGFR2, EGFR or Raf kinase inhibition. The inhibitory activity against VEGFR2 or EGFR was determined by radiation kinase assay. The experimental conditions for VEGFR2 kinase assay were 6.25 ng of recombinant VEGFR2 kinase (only VEGFR2 kinase domain, Millipore), 5 μg of poly(glutamate-tyrosine) substrate (4:1, Sigma), kinase reaction buffer. (20 mM MOPS pH 7.0, 1 mM EDTA, 5% glycerol, 0.01% Brij-35, 0.1-mercaptoethanol, 1 mg/mL BSA, 100 μM ATP and 0.1 μCi/well of [ ³³ P]ATP (specific activity 3000 Ci/mmol, PerkinElmer)), as well as test compounds (diluted with 4% DMSO) or DMSO alone (as a control), with a final volume of 25 [mu]L. The reaction was allowed to proceed at 30 ° C for 30 minutes, and 3% phosphoric acid was added to terminate the reaction, and collected in a 96-well GF/B UniFilter (PerkinElmer) using a cell harvester (PerkinElmer), and counted using a TopCount microplate scintillation counter (PerkinElmer).

The experimental conditions for EGFR kinase activity assay were 25 ng of EGFR kinase (only EGFR kinase domain, Millipore), 3 μg of receptor poly(Glu-Tyr) (Sigma), kinase reaction buffer (10 mM MOPS pH 7.0, 0.3 mM EDTA). , 0.5 glycerol, 0.001% Brij-35, 10 mM manganese chloride, 0.1 mg/mL BSA, 100 μM ATP and 0.1 μCi/well of [ ³³ P]ATP (specific activity 3000 Ci/mmol, PerkinElmer) and test compound (4%) DMSO dilution) or DMSO (as a control), the final volume was 25 μL. The reaction was carried out at 30 ° C for 30 minutes, and the reaction was stopped by the addition of 3% phosphoric acid, and 96-well GF/B UniFilter (PerkinElmer) was collected using a cell harvester (PerkinElmer). The radioactivity was counted using a TopCount microplate scintillation counter (PerkinElmer).

VEGFR2 and selected compounds for inhibition of EGFR kinase activity results in Table 6 finishing train, wherein the selected compound B-Raf kinase inhibition concentration (IC ₅₀₎ less than the Department of 300nM. IC ₅₀ value is defined as a half-maximal inhibition of the kinase activity values, the concentration of the test compound. + indicates that the concentration is 3000 to 1000 nM; ++ indicates the concentration is 1,000 to 300 nM; +++ indicates that the concentration is less than 300 nM.

As shown in Table 6, the novel quinazoline derivatives showed selective inhibitory activity against Raf kinase compared to VEGFR2 or EGFR kinase. These compounds IC Raf kinase _IC50 values are less than 300 nM, however, for the inhibition of EGFR kinase VEGFR2, or weak, the IC ₅₀ value higher (e.g., typically greater than 3μM). The unexpected selectivity of these novel quinazoline compounds has been shown to be useful in therapeutic methods that require selective control of Raf kinase activity.

5. Cell proliferation assay

As indicated above, the compounds of the invention are useful in the treatment of diseases or conditions associated with protein kinases. The protein kinase-related disease may be cancer, diabetes, kidney disease, von Hip-pel-Lindau disease, fibrosis, osteoarthritis autoimmune disease, or vascular proliferative disorder. The cancer may be lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer, oral cancer. , skin cancer, brain tumors, lymphoma, or leukemia.

Using CellTiter ^TM -96 Assay Kit (Promega) in accordance with the operating manual, the compounds can be measured of formula (I) for the B-Raf mutations in human cancer cell line (the V600E), inhibition of A375 melanoma cell proliferation of colon cancer cells and COLO205 Activity. Briefly, cells were cultured in DMEM medium containing 10% FCS at 37 ° C, 5% CO ₂ , and cells were plated in 96-well plates at a density of 2000 cells in one well and cultured for 24 hours. These cells were then treated with different concentrations of compound and incubated for an additional 72 hours. At the end time added ^{CellTiter TM -96 Aqueous One Solution Reagent (} Promega) and then incubated for 4 hours. Sample readings using a microplate machine ^{(EMax ® microplate reader, Molecular Devices} ) by measuring absorbance at 490nm of cell survival rate was measured, and to GraphPad Prism for analysis and data processing. The test compound had an antiproliferative activity with an IC ₅₀ < 2 μM, and the results of the correlation analysis are summarized in Table 7.

The data in Table 7 clearly shows that the compounds of the invention can inhibit cancer cell growth. Therefore, these compounds are useful for treating cancer.

While the invention has been described with respect to the embodiments of the invention, it will be understood that Therefore, the scope of the invention should be limited only by the scope of the patent application in this application.

Claims (11)

a compound of formula (I), Or a stereoisomer, tautomer thereof or a pharmacologically acceptable salt thereof, wherein: G is a substituent selected from the group consisting of: among them Representing a pyridyl group, optionally substituted by one or more R ⁹ , wherein Representing a pyrimidinyl group, optionally substituted by one or more R ¹⁰ , wherein Representative pyrazolyl, optionally substituted independently with R ¹² and one or more R ¹³ , wherein Representing a triazolyl group, optionally substituted independently with R ¹⁴ and one or more R ¹⁵ , wherein Generation represents an imidazolyl group, optionally substituted by one or more R ¹⁶ ; Representing morpholinyl, optionally substituted by one or more R ¹⁷ , wherein Representing a pyridone group, optionally independently substituted by R ¹⁸ and one or more R ¹⁹ , wherein Representing an alkynyl group, optionally substituted by R ²⁰ , wherein R ⁹ is independently selected from the group consisting of hydrogen, halogen, hydroxy, amine, C ₁ -C ₄ alkoxy, methoxy C ₁ -C ₄ alkoxy a group of a C ₁ -C ₄ dialkylamino group and a heterocyclic group; R ^{10 is} independently hydrogen, hydroxy or C ₁ -C ₄ dialkylamino; R ¹¹ , R ¹³ , R ¹⁵ and R ^{16 are} independently Is hydrogen, halogen or C ₁ -C ₄ alkyl; R ¹² and R ^{14 are} independently hydrogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkylindenyl; R ¹⁷ is independently selected from hydrogen, a group consisting of a halogen, a hydroxyl group, an amine group, a nitro group, a hydrogen group, a C ₁ -C ₄ amine group and a C ₁ -C ₄ alkoxy group; and R ¹⁸ is selected from the group consisting of hydrogen containing a C ₁ -C ₄ alkyl group. a group; R ¹⁹ is a group independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, cyano, C ₁ -C ₄ alkyl, and C ₁ -C ₄ alkoxy; and R ²⁰ is selected from hydrogen, a group consisting of C ₁ -C ₄ alkyl, methoxy C ₁ -C ₄ alkoxy and aryl; X is N or CH; L ¹ is selected from -N(R ⁷ )-, -O-, a group consisting of -C(S)-, -C(O)-, and -S-, wherein R ⁷ is hydrogen or C ₁ -C ₄ alkyl; L ² is selected from -N(R ⁸ )- and -O-, wherein R ⁸ is hydrogen or C ₁ -C ₄ alkyl; R ¹ and R ² are independently selected Hydrogen, halogen, hydroxy, amino, cyano, nitro, carboxy, C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkoxy C ₁ -C ₄ alkoxy, N, N- (C _{1 -} C ₄ dialkyl) amino C ₁ -C ₄ alkoxy, N- (C _1- C ₄ alkyl) amino C ₁ -C ₄ alkoxy, C ₁ -C ₄ -alkyl, C ₁ -C ₄ alkyl alkoxy, N-(C ₁ -C ₄ alkyl)amino, N,N-(C ₁ -C ₄ dialkyl)amine, C ₁ -C ₄ alkanoyl and hetero a group consisting of a cyclic group, wherein the C ₁ -C ₄ alkyl group may be optionally substituted by one or more substituents selected from the group containing fluorine or chlorine; R ³ , R ⁴ and R ⁵ are independently a group selected from the group consisting of hydrogen, fluorine, and chlorine; and R ⁶ is selected from the group consisting of a C ₁ -C ₄ alkyl group and an aryl group, wherein the C ₁ -C ₄ alkyl group and the aryl group may be optionally independently Substituted by one or more substituents selected from the group consisting of a halogen, a hydroxyl group, an amine group, a cyano group and a nitro group; when L ² is -N(R ⁸ )-, R ⁶ and R ⁸ are bonded thereto An atom forms a 5- to 6-membered ring or heterocyclic group. A compound of claim 1, wherein L ² is NH. The compound of claim 1, wherein L ¹ is NH. The compound of claim 1, wherein R ⁵ is hydrogen. The compound of claim 1, wherein R ³ and R ⁴ are fluorine. A pharmaceutical composition comprising a compound as described in claim 1 of the patent application, a salt thereof or a stereoisomer thereof; and a pharmaceutically acceptable carrier. The compound of claim 1, wherein the compound is: N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine Phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-morpholinopyridin-3-yl)quinazolin-4-ylamine)phenyl) Propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonate Indoleamine; N-(3-(6-(1H-pyrazol-4-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N- (2,4-difluoro-3-(6-(pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(4-fluoro-3-() 6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(7-fluoro-6) -(2-fluoropyridin-3-yl)quinazolin-4-ylamine)propane-1-sulfonamide; N-(2-chloro-4-fluoro-3-(6-(2-fluoropyridine)- 3-yl)quinazolin-4-ylamine)phenyl)propan-1-aminesulfonate Amine; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)methanesulfonamide; N-(2,4 -difluoro-5-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3 -(6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoro) Pyridin-3-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(pyridin-4-yl)quinoline- 4-ylamine)phenyl)propane-1-indolesulfonamide; N-(2,4-difluoro-3-(7-fluoro-6-(pyridin-4-yl)quinazolin-4-yl) Amine)phenyl)propane-1-sulfonamide; N-(2,6-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl) Propane-1-sulfonamide; N-(2,6-difluoro-3-(6-(pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)butane-1-sulfonamide; N-(2 ,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)ethanesulfonamide; N-(2,4-difluoro-3 -(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-2-sulfonamide; N-(2,4-difluoro-3-(6-( 2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)benzenesulfonamide; N-( 3-(7-(2-(Dimethylamino)ethylamine)-6-(pyridin-4-yl)quinazolin-4-ylamine)-2,6-difluorophenyl)propane-1- Sulfonamide; N-(2,4-difluoro-3-(6-(2-methoxypyridin-3-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N -(2,4-difluoro-3-(6-(2-fluoropyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4 -difluoro-3-(6-(2-fluoropyridin-4-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3- (6-(2-oxo-1,2-dihydropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro -3-(6-(6-methoxypyridin-3-yl)quinolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6) -(2-fluoropyridin-3-yl)-7-methoxyquinazolin-4-ylamine)phenyl)benzenesulfonamide; N-(2,4-Difluoro-3-(6-(2-fluoropyridin-3-yl)-7-(2-methoxyethoxy)quinazolin-4-ylamine)phenyl)propane 1-sulfonamide; N-(2,4-difluoro-3-(7-methoxy-6-pyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonate Indoleamine; N-(2,4-difluoro-3-(6-(2-methoxypyridin-4-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N- (2,4-Difluoro-3-(6-(6-oxo-1,6-dihydropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide N-(2,4-difluoro-3-(6-(6-(2-methoxyethoxy)pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1- Sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-yloxy)phenyl)propane-1-sulfonamide; N -(3-(6-(6-(2-(dimethylamino)ethoxy)pyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1 - sulfonamide; N-(2,4-difluoro-3-(6-(6-fluoropyridin-2-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-methoxypyridin-2-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2 4-difluoro-3-(6-(6-(2-methoxyethoxy)pyridin-2-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N -(3-(6-(6-(2-(dimethylamino)ethoxy)pyridin-2-yl)quina Phenyl-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonylamine; N-(2,4-difluoro-3-(6-(2-(fluoropyridin-3-yl)) a quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(6-morpholinpyridin-2-yl)quinazoline) 4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-morpholinoquinazolin-4-ylamine)phenyl)propane-1- Sulfonamide; N-(3-(6-(1H-imidazol-1-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2 4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)-3-fluoropropane-1-sulfonamide; N-(3-(6-(2-(2-(Dimethylamino)ethoxy)pyridin-4-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane- 1-sulfonamide; N-(2,4-difluoro-3-(6-(5-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide N-(3-(6-(1H-benzo[d]imidazol-1-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2-fluoropyridin-3-yl)quinazolin-4-carbonyl)phenyl)propane-1-sulfonamide; N-(3-( 6-ethynylquinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6-(2-aminopyrimidin-5-yl)quinaquine Oxazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6-(2-(dimethylamino)pyrimidin-5-yl)quina Oxazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6-(1H-1,2,3-triazol-4-yl)) Quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-((6-(2-fluoropyridine-3) -yl)quinazolin-4-yl)(methyl)amine)phenyl)-N-methylpropan-1-sulfonamide; N-(3-(6-(6-(dimethylamino)) Pyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(2) -fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane sulphonamide; N- (2,4-difluoro-3-(5-fluoro-6-(2-fluoropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-( 3-(6-(2-chloropyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-di Fluor-3-(6-(6-methoxy-5-nitropyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(3-(6-(3-Aminophenyl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6- (5-Amino-6-methoxypyridin-3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(3-(6 -(6-Aminopyridin-2-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonylamine; N-(2,4-difluoro-3 -(6-(6-(trifluoromethyl)pyridin-2-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(3-(6-(6-) Cyanopyridin-2-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonylamine; N-(3-(6-(2-cyanopyridine)- 3-yl)quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(5-() Trifluoromethyl)pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; N-(2,4-difluoro-3-(6-(4-fluoro) Pyridin-3-yl)quinazolin-4-ylamine)phenyl)propane-1-sulfonamide; or N-(3-(6-(6-amino-5-methylpyridin-3-yl) Quinazolin-4-ylamine)-2,4-difluorophenyl)propane-1-sulfonamide. The pharmaceutical composition according to claim 6, which is for use in the treatment of a protein kinase-related disease. The pharmaceutical composition according to claim 8, wherein the protein kinase-related diseases are cancer, diabetes, kidney disease, von Hip-pel-Lindau disease, fibrosis, osteoarthritis , autoimmune disease, or vascular proliferative disorder. The pharmaceutical composition according to claim 8, wherein the protein kinase-related disease is cancer. The pharmaceutical composition according to claim 10, wherein the cancer is lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney Cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma, or leukemia.