CN114835640A - Fibroblast growth factor receptor inhibitor, preparation method and application - Google Patents

Fibroblast growth factor receptor inhibitor, preparation method and application Download PDF

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CN114835640A
CN114835640A CN202210569981.6A CN202210569981A CN114835640A CN 114835640 A CN114835640 A CN 114835640A CN 202210569981 A CN202210569981 A CN 202210569981A CN 114835640 A CN114835640 A CN 114835640A
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孙丽萍
王敏
王雨薇
张谨阳
兰丽
王晨艳
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Abstract

The invention discloses a fibroblast growth factor receptor inhibitor, a preparation method and application thereof. A compound of formula (i), stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein the substituents are as defined in the description and claims. The compound has the function of FGFR4 kinase inhibitor, and can be used for preparing medicaments for treating tumor diseases, in particular hepatocellular carcinoma, breast cancer, colon cancer, prostatic cancer, pancreatic cancer or rhabdomyosarcoma.

Description

Fibroblast growth factor receptor inhibitor, preparation method and application
Technical Field
The invention relates to the field of medicinal chemistry, in particular to a fibroblast growth factor receptor inhibitor, a preparation method and application thereof.
Background
Fibroblast Growth Factor Receptor 4 (FGFR 4) is a Receptor Tyrosine Kinase (RTK) and belongs to a subtype of the FGFR family. The human FGFR family includes four typical subtypes of FGFR1, FGFR2, FGFR3, and FGFR 4. Under physiological conditions, they can specifically bind to corresponding Fibroblast Growth Factors (FGFs) with high affinity, leading to dimerization and activation of two adjacent FGFR molecules, thereby mediating cell signaling and participating in many important physiological processes of embryonic development, organogenesis, angiogenesis, tissue homeostasis, tissue repair and inflammatory response (Haugsten E M et al. Molecular Cancer Research,2010,8(11): 1439-.
Under normal physiological conditions, FGF19 is an important metabolic regulator; under pathological conditions, FGF19 may be associated with the development of various cancers. FGFR4 is currently considered to be the only receptor for FGF19 that shows specificity, FGF19 can utilize β -Klotho as a cofactor to bind to FGFR4 and activate downstream signaling (Goetz R et al Nature Reviews Molecular Cell Biology,2013,14(3): 166-180). FGFR4 was found to be abnormally activated in a variety of tumors, including overexpression of FGFR4 in a variety of human cancers such as breast, liver, colon, prostate, rhabdomyosarcoma (Levine K M et al pharmacography & Therapeutics,2020,214: 107590); the N535K and V550E mutations found in the kinase domain of FGFR4 in rhabdomyosarcoma (Shern J F et al Cancer Discovery,2014,4(2): 216-340231; Taylor J G T et al Journal of Clinical Investigation,2009,119(11): 3395-3407); FGFR4-G388R is related to the occurrence, progression and prognosis of various cancers such as breast Cancer, colorectal Cancer, prostate Cancer, lung Cancer, soft tissue sarcoma and head and neck squamous cell carcinoma (Bange J et al Cancer Research,2002,62(3): 840-2257; Morimoto Y et al Cancer,2003,98(10): 2245-2250); overexpression of FGF19 was found in hepatocellular carcinomas (Miura S et al, BMC Cancer,2012,12: 56).
Disclosure of Invention
The invention designs a series of fibroblast growth factor receptor inhibitors with brand new structures by using a computer-aided drug design means.
The other purpose of the invention is to provide a preparation method of the fibroblast growth factor receptor inhibitor.
The invention also aims to provide application of the fibroblast growth factor receptor inhibitor.
The purpose of the invention can be realized by the following technical scheme:
a compound of formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof,
Figure BDA0003659888500000021
wherein:
a is C 6-8 Aryl, 5-to 8-membered heteroaryl, C 3-8 Cycloalkyl, 3-to 8-membered heterocycloalkyl or C 3-8 A cycloalkenyl group;
b is phenyl or heteroaryl;
x is selected from-O-, -NH-or none;
y is selected from one of-NHCONH-, -NHCO or-CONH-;
l is a moiety capable of forming a covalent bond with a nucleophile and is selected from the group having the following structure:
Figure BDA0003659888500000022
wherein
Figure BDA0003659888500000023
Represents a double bond or a single bond;
R 1 、R 2 and R 3 Each independently selected from hydrogen, halogen, optionally substituted C 1-4 Alkyl or optionally substituted heteroCyclylalkyl;
R 4 and R 5 Selected from hydrogen, halogen, cyano, mercapto, hydroxy, amino, amido, ester, amido, sulfonyl, sulfinyl, and optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 An alkoxy group;
R 6 selected from a chlorine atom, a cyano group or a group selected from the following structures:
Figure BDA0003659888500000024
R 7 selected from hydrogen, hydroxy, cyano, optionally substituted C 1-4 Alkyl or optionally substituted heterocyclylalkyl;
m is 1 or 2; n is 0,1, 2, 3 or 4; p is 0,1, 2, 3 or 4
As a preferred aspect of the present invention, the compound of formula (I), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof,
wherein:
a is a pyridine ring;
b is selected from phenyl, pyridyl or pyrazinyl;
x is one of-O-or none;
y is-NHCONH-;
l is selected from
Figure BDA0003659888500000031
Wherein R is 1 、R 2 Each independently selected from hydrogen, C1-3 alkyl, R 3 Selected from hydrogen, halogen;
R 4 selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, halogen;
R 5 is trifluoromethyl;
R 6 selected from chlorine atom, 4-methylpiperazino-1-methylene, 4-ethylpiperazino-1 methylene, morpholine-1-methylene, pyrrole-1-methylene;
n is 1 or 2.
As a further preferred aspect of the present invention, said compound of formula (I), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof,
wherein:
a is a pyridine ring;
b is selected from phenyl, pyridyl or pyrazinyl;
x is one of-O-or none;
y is-NHCONH-;
l is selected from acrylamide, chloroacetamide, crotonamide, butynamide, 2-fluoroacrylamide or propionamide;
R 4 selected from hydrogen, methyl, fluorine, chlorine, methoxy;
R 5 is trifluoromethyl;
R 6 selected from 4-methylpiperazino-1-methylene, 4-ethylpiperazino-1-methylene;
n is 1.
As a still further preferred aspect of the present invention, the compound of formula (i) is selected from any one of:
Figure BDA0003659888500000032
Figure BDA0003659888500000041
Figure BDA0003659888500000051
the invention relates to a preparation method of a compound shown in formula (I), wherein the reaction route is selected from any one of the following:
route one:
Figure BDA0003659888500000052
firstly, the raw material a and BTC react under the catalysis of pyridine to generate phenyl isocyanate, and then commercial raw materials or materials prepared in a laboratory are addedThe intermediate b is subjected to condensation reaction to obtain an aryl urea intermediate c, and the raw material d is Pd (dppf) Cl 2 Under the catalysis of the intermediate B, carrying out Miyaura boronation reaction with pinacol diboron to obtain pinacol phenylboronate intermediate e, and carrying out Pd (PPh) reaction on the intermediate c and the intermediate e 3 ) 4 Carrying out Suzuki coupling reaction under the catalysis of the intermediate F to obtain an intermediate f; finally, the intermediate f is condensed with corresponding carboxylic acid and acyl chloride to obtain the compound (Ia) with the general formula.
And a second route:
Figure BDA0003659888500000061
firstly, nucleophilic substitution is carried out on a raw material g and a raw material h under an alkaline condition to generate an intermediate i, the intermediate i and BTC react under the catalysis of pyridine to generate phenyl isocyanate, then a commercial raw material or a laboratory preparation raw material b is added, and an aryl urea intermediate j is obtained through condensation reaction; reducing the nitro group of the intermediate j into an amino intermediate k through palladium carbon and hydrogen; finally, the intermediate k is condensed with corresponding carboxylic acid and acyl chloride to obtain the compound (Ib).
Methods for enantiomeric and diastereomeric mixtures are familiar to those skilled in the art. The invention encompasses any isolated racemic or optically active form of formula (i) having FGFR4 kinase inhibitory activity.
A pharmaceutical composition comprising a compound of formula (i), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable receptor, adjuvant or diluent.
The invention also provides application of the compound shown in the formula (I), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof in preparing medicines for treating diseases related to abnormal activation of FGFR 4.
Preferably, the disease associated with abnormal activation of FGFR4 is selected from hepatocellular carcinoma, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, rhabdomyosarcoma, lung cancer, soft tissue sarcoma, head and neck squamous cell carcinoma, and cervical cancer.
The administration mode is as follows:
the compounds of the present invention may be administered to a mammal, preferably a human, alone or in a pharmaceutical composition in combination with a pharmaceutically acceptable recipient, adjuvant or diluent; the compounds can be administered orally or subcutaneously, intramuscularly, intraperitoneally, intravenously, rectally, and topically, ocularly, pulmonarily, nasally, parenterally.
Drug metabolism and prodrugs:
metabolites of the compounds and pharmaceutically acceptable salts thereof to which the present invention relates, and prodrugs which can be converted in vivo into structures of the compounds and pharmaceutically acceptable salts thereof to which the present invention relates, are also included in the claims of the present application.
The invention relates to application of a compound shown as a formula (I), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof in preparing a fibroblast growth factor receptor inhibitor.
The invention has the beneficial effects that: the invention designs a small molecular compound with the structural characteristics of a formula (I), the compound can effectively inhibit the activity of fibroblast growth factor receptor 4 and has obvious antiproliferative activity on a liver cancer cell line mediated by FGFR 4.
The compounds of the invention and the corresponding preparation processes are further illustrated and exemplified below by means of examples and preparations. It is to be understood that although typical or preferred reaction conditions (e.g., reaction temperature, time, molar ratios of reactants, reaction solvent, and pressure, etc.) are given in the specific examples, other reaction conditions may be used by one skilled in the art. The optimum reaction conditions may vary with the particular Sanskrit fifth or solvent used, but can be determined by routine experimentation by one skilled in the art.
The structures of the compounds of the following examples were characterized by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). The compounds were dissolved in appropriate deuterated reagents using a Bruker AV-300 type (300MHz) nuclear magnetic resonance apparatus and subjected to 1H-NMR analysis at ambient temperature using TMS as internal standard. NMR chemical shifts (δ) are in ppm and are abbreviated as follows: s, singlet; d, double peak; t, triplet; q, quartet; m, multiplet; brs, broad singlet. MS was determined by a Water Q-TOF MicroS (TM) mass spectrometer.
The starting materials, intermediates and compounds of the examples of the reaction can be isolated and purified by conventional techniques such as precipitation, filtration, recrystallization, evaporation, distillation and chromatography (e.g., column chromatography, TLC analysis, etc.).
The present invention will now be further described with reference to examples. It is to be understood that the invention is not limited to these examples, which are provided solely to provide methods of practicing the invention and are not intended to limit the scope of the invention in any way.
Detailed Description
Example 1 Synthesis of N- (5- (3-methyl-4- (3- (4- (4-methylpiperazine-1-methylene) -3- (trifluoromethyl) benzene Yl) ureido) phenyl) pyridin-2-yl) acrylamide (W-1)
Figure BDA0003659888500000081
Step 1, preparing intermediate 4-nitro-2-trifluoromethyl benzyl bromide
Figure BDA0003659888500000082
The starting material, 2-methyl-5-nitrotrifluorotoluene (3.20g,15.6mmol), was dissolved in 1, 2-dichloroethane (40mL), and N-bromosuccinimide (3.04g,17.1mmol) and azobisisobutyronitrile (256mg,1.56mmol) were added with stirring at room temperature, followed by warming to 80 ℃ for reaction. After the completion of the reaction was monitored by TLC, the reaction mixture was diluted with water (100mL), extracted with dichloromethane (100mL × 3), and the organic phase was washed with saturated aqueous sodium chloride (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent PE: EA ═ 400:1 to 50:1) to obtain a pale yellow liquid (3.32g, yield 75.8%).
1 H NMR(300MHz,Chloroform-d)δ(ppm):8.54(d,J＝2.3Hz,1H),8.42(dd,J＝8.6,2.4Hz,1H),7.84(d,J＝8.6Hz,1H),4.67(s,2H).
Step 2, preparing an intermediate 4- (4-methyl)Piperazine-1-methylene) -3-trifluoromethyl-1-nitrobenzene
Figure BDA0003659888500000083
4-Nitro-2-trifluoromethylbromobenzyl (3.29g,11.7mmol) was dissolved in methylene chloride (50mL) and anhydrous potassium carbonate (1.62g,11.7mmol) was added with stirring at room temperature. Subsequently, a solution of N-methylpiperazine (2.6mL,23.4mmol) in methylene chloride (5mL) was added dropwise to the reaction solution while cooling on ice, and the reaction was stirred at room temperature after the addition of dropwise. After the completion of the reaction was monitored by TLC, the reaction solution was diluted with water (100mL), extracted with dichloromethane solution (100mL × 3), and the organic phase was washed with saturated aqueous sodium chloride solution (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluted with DCM: MeOH ═ 80:1, TEA 1%) to give a light yellow liquid (3.23g, yield 89.8%).
1 H NMR(300MHz,Chloroform-d)δ(ppm):8.51(d,J＝2.3Hz,1H),8.38(dd,J＝8.6,2.4Hz,1H),8.11(d,J＝8.6Hz,1H),3.75(s,2H),2.54(s,8H),2.32(s,3H).
Step 3, preparing intermediate 4- (4-methylpiperazine-1-methylene) -3-trifluoromethylaniline
Figure BDA0003659888500000091
4- (4-methylpiperazine-1-methylene) -3-trifluoromethyl-1-nitrobenzene (3.03g,10mmol) was dissolved in methanol (40mL), 10% palladium on carbon (300mg) and 85% hydrazine hydrate (4.9mL,100mmol) were added with stirring at room temperature, followed by warming to 65 ℃ for reaction. After completion of the reaction monitored by TLC, the reaction mixture was filtered with celite, the filtrate was concentrated under reduced pressure, then the reaction mixture was diluted with water (100mL), extracted with dichloromethane (100 mL. times.3), the organic phase was washed with a saturated aqueous sodium chloride solution (100mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent and then dried to give a pale yellow solid (2.14g, yield 78.5%).
1 H NMR(300MHz,Chloroform-d)δ(ppm):7.47(d,J＝8.3Hz,1H),6.92(d,J＝2.5Hz,1H),6.79(dd,J＝8.2,2.4Hz,1H),3.76(s,2H),3.53(s,2H),2.46(s,8H),2.29(s,3H).
Step 4, preparing an intermediate 1- (4-bromo-2-methylphenyl) -3- (4- (4-methylpiperazine-1-methylene) -3- (tris) Fluoromethyl) phenyl) urea
Figure BDA0003659888500000092
Triphosgene (302mg,1.02mmol) was dissolved in anhydrous tetrahydrofuran (10mL) under nitrogen protection. A solution of pyridine (943. mu.L, 18mmol) and 2-methyl-4-bromoaniline (558mg,3mmol) in tetrahydrofuran (3mL) was slowly added dropwise to the reaction system at-5 ℃ and the reaction was allowed to warm to room temperature for 30 minutes. Then, a solution of the compound 4- (4-methylpiperazin-1-methylene) -3-trifluoromethylaniline (727mg,2.7mmol) in tetrahydrofuran (2mL) was added dropwise, and the reaction was continued at room temperature. After TLC monitoring reaction, suction filtration, filter cake washing three times with tetrahydrofuran and drying. A pink solid was obtained (1.11g, 84.8% yield).
1 H NMR(300MHz,DMSO-d 6 )δ(ppm):9.51(s,1H),8.14(s,1H),7.97(s,1H),7.82(d,J＝8.7Hz,1H),7.63(d,J＝8.6Hz,1H),7.56(d,J＝8.4Hz,1H),7.40(s,1H),7.32(d,J＝8.7Hz,1H),3.53(s,2H),2.39(s,8H),2.25(s,3H),2.20(s,3H).
Step 5, preparing intermediate 2-aminopyridine-5-boric acid pinacol ester
Figure BDA0003659888500000101
2-amino-5-bromopyridine (2.60g,15mmol), pinacol diboron (4.57g,18mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (548mg,0.75mmol) and potassium acetate (4.42g,45mmol) were put into a dry two-necked flask in this order, anhydrous dioxane (30mL) was added thereto, nitrogen was replaced three times, and the mixture was heated to 100 ℃ and stirred for reaction. After completion of the TLC monitoring reaction, celite was filtered, the filtrate was spin dried under reduced pressure, slurried with petroleum ether, filtered and dried to give a light brown solid (3.25g, 98.5% yield).
Step 6, preparing the intermediate 1- (4- (6-aminopyridin-3-yl) -2-methylphenyl) -3- (4- (4-methylpiperazine- 1-methylene) -3- (trifluoromethyl) phenyl) urea
Figure BDA0003659888500000102
2-aminopyridine-5-boronic acid pinacol ester (660mg,3mmol), 1- (4-bromo-2-methylphenyl) -3- (4- (4-methylpiperazin-1-methylene) -3- (trifluoromethyl) phenyl) urea (970mg,2mmol) (970mg,2mmol), tetrakis (triphenylphosphine) palladium (115mg,0.01mmol) and cesium carbonate (1.30g,4mmol) were sequentially put into a Schlenk's tube, toluene/ethanol/water (9/6/3mL) was added thereto, nitrogen was replaced three times, and the reaction was stirred while warming to 100 ℃. After TLC monitoring of the completion of the reaction, celite was filtered off with suction, concentrated under reduced pressure, and purified by column chromatography (eluent DCM: MeOH: 100:1 ~ 40:1, 1% TEA) to give a pale yellow solid (511mg, 51.3% yield).
1 H NMR(300MHz,DMSO-d 6 )δ(ppm):9.32(s,1H),8.22(d,J＝2.5Hz,1H),8.01(s,1H),7.98(d,J＝2.1Hz,1H),7.83(d,J＝8.4Hz,1H),7.69–7.64(m,1H),7.61(s,1H),7.59–7.54(m,1H),7.41(d,J＝2.2Hz,1H),7.35(dd,J＝8.4,2.3Hz,1H),6.51(d,J＝8.6Hz,1H),5.99(s,2H),3.53(s,2H),2.38(s,8H),2.29(s,3H),2.18(s,3H).
Step 7, preparing N- (5- (3-methyl-4- (3- (4- (4-methylpiperazine-1-methylene) -3- (trifluoromethyl) benzene Yl) ureido) phenyl) pyridin-2-yl) acrylamide (W-1)
Figure BDA0003659888500000103
1- (4- (6-aminopyridin-3-yl) -2-methylphenyl) -3- (4- (4-methylpiperazin-1-methylene) -3- (trifluoromethyl) phenyl) urea (99.7mg,0.2mmol) was dissolved in anhydrous dichloromethane (3mL) and triethylamine (83. mu.L, 0.6mmol) was added with stirring at room temperature. Then, a solution of acryloyl chloride (20 μ L,0.25mmol) in dichloromethane (1mL) was slowly added dropwise while cooling on ice, and the mixture was stirred overnight at room temperature after dropping. After completion of the TLC detection reaction, the reaction was quenched by addition of water (20mL), extracted with dichloromethane (20 mL. times.3), and the organic phase was washed with saturated aqueous ammonium chloride (20mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM: MeOH ═ 60:1, elution with 1% TEA solution). The mixed solvent of dichloromethane and n-hexane was slurried, suction-filtered and dried to obtain a white solid (46.7mg, yield 42.3%).
1 H NMR(300MHz,DMSO-d6)δ(ppm):10.82(s,1H),9.43(s,1H),8.66(d,J＝2.4Hz,1H),8.26(d,J＝8.6Hz,1H),8.10(d,J＝8.5Hz,2H),7.98(d,J＝8.8Hz,2H),7.64(d,J＝8.6Hz,1H),7.59(s,2H),7.53(d,J＝9.7Hz,1H),6.64(dd,J＝17.0,10.1Hz,1H),6.33(d,J＝17.1Hz,1H),5.84–5.77(m,1H),3.53(s,2H),2.38(s,8H),2.33(s,3H),2.17(s,3H).
HRMS(ESI)m/z calcd for C 29 H 31 F 3 N 6 O 2 [M+H] + ,553.2533；found,553.2531.
Example 2 Synthesis of N- (5- (3-methyl-4- (3- (4- (4-methylpiperazine-1-methylene) -3- (trifluoromethyl) benzene Yl) ureido) phenoxy) pyridin-2-yl) acrylamide (W-23)
Figure BDA0003659888500000111
Step 1, preparing an intermediate 2-methyl-4- ((6-nitropyridin-3-yl) oxy) aniline
Figure BDA0003659888500000112
The starting material, 3-methyl-4-aminophenol (1.60g,13mmol), was dissolved in anhydrous N, N-dimethylformamide (20mL) under nitrogen protection, cesium carbonate (9.45g,29mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Then, a solution of 2-nitro-5-chloropyridine (2.30g,14.5mmol) in DMF (5mL) was slowly added dropwise to the reaction solution, and the temperature was raised to 90 ℃ after completion of the dropwise addition. After the completion of the reaction was monitored by TLC, water (50mL) was added to dilute, extracted with ethyl acetate (50mL × 3), washed with saturated aqueous sodium chloride (50mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent PE: EA ═ 8:1) to obtain a yellow solid (1.71g, yield 53.7%).
1 H NMR(300MHz,Chloroform-d)δ(ppm):8.28(d,J＝2.9Hz,1H),8.19(d,J＝8.9Hz,1H),7.33(dd,J＝9.0,2.9Hz,1H),6.84–6.77(m,2H),6.71(d,J＝8.3Hz,1H),3.67(s,2H),2.19(s,3H).
Step 2, preparing an intermediate 1- (2-methyl-4- ((6-nitropyridin-3-yl) oxy) phenyl) -3- (4- (4-methyl) Piperazine-1-methylene) -3- (trifluoromethyl) phenyl) urea
Figure BDA0003659888500000121
Triphosgene (164mg,0.55mmol) was dissolved in anhydrous tetrahydrofuran (5mL) under nitrogen. A solution of pyridine (785. mu.L, 9.72mmol) and 2-methyl-4- ((6-nitropyridin-3-yl) oxy) aniline (412mg,1.62mmol) in tetrahydrofuran (2mL) was slowly added dropwise to the reaction system under ice-cooling, and the reaction was allowed to warm to room temperature for 30 minutes after completion of the addition. Subsequently, a solution of 4- (4-methylpiperazin-1-methylene) -3-trifluoromethylaniline (398mg,1.46mmol) in tetrahydrofuran (2mL) was added dropwise, and the reaction was continued at room temperature. After TLC monitoring completion of the reaction, it was directly concentrated under reduced pressure and purified by column chromatography (eluent DCM: MeOH ═ 100:1, 1% TEA) to give a yellow solid (625mg, 78.7% yield).
Step 3, preparation of intermediate 1- (4- ((6-aminopyridin-3-yl) oxy) -2-methylphenyl) -3- (4- (4-methyl) Piperazine-1-methylene) -3- (trifluoromethyl) phenyl) urea
Figure BDA0003659888500000122
1- (2-methyl-4- ((6-nitropyridin-3-yl) oxy) phenyl) -3- (4- (4-methylpiperazin-1-methylene) -3- (trifluoromethyl) phenyl) urea (1.50g,2.76mmol) was dissolved in ethanol water (10:5mL), and iron powder (1.21g,22mmol), ammonium chloride (1.47g,27.6mmol) were added with stirring at room temperature, followed by warming to 85 ℃ for reaction. After completion of the TLC detection reaction, the reaction solution was filtered with celite, the filtrate was dried under reduced pressure, then diluted with water (30mL), extracted with dichloromethane (50 mL. times.3), and the organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure and dried to obtain a white solid (1.26g, yield 88.7%).
Step 4, preparing N- (5- (3-methyl-4- (3- (4- (4-methylpiperazine-1-methylene) -3- (trifluoromethyl) benzene Yl) ureido) phenoxy) pyridin-2-yl) acrylamide (W-23)
Figure BDA0003659888500000123
1- (4- ((6-aminopyridin-3-yl) oxy) -2-methylphenyl) -3- (4- (4-methylpiperazin-1-methylene) -3- (trifluoromethyl) phenyl) urea (98mg, 0.2mmol) was dissolved in anhydrous dichloromethane and triethylamine (83.4ul, 0.6mmol) was added with stirring at room temperature, followed by slowly adding a solution of acryloyl chloride (25ul, 0.25mmol) in dichloromethane dropwise while cooling on ice, and stirring overnight at room temperature after rising dropwise. After completion of the TLC detection reaction, the reaction was quenched by addition of water (20mL), extracted with dichloromethane (20 mL. times.3), and the organic phase was washed with saturated aqueous ammonium chloride (20mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM: MeOH ═ 60:1, elution with 1% TEA solution). A mixed solvent of dichloromethane and n-hexane was slurried, suction-filtered and dried to obtain a white solid (41.6mg, yield 38.5%).
1 H NMR(300MHz,DMSO-d 6 )δ(ppm):10.77(s,1H),9.39(s,1H),8.22(d,J＝9.0Hz,1H),8.12(d,J＝2.9Hz,1H),8.08(s,1H),7.97(d,J＝2.2Hz,1H),7.73(d,J＝8.8Hz,1H),7.62(d,J＝8.6Hz,1H),7.56(dd,J＝8.6,2.2Hz,1H),7.50(dd,J＝9.0,3.0Hz,1H),6.94(d,J＝2.9Hz,1H),6.87(dd,J＝8.7,2.9Hz,1H),6.60(dd,J＝17.0,10.1Hz,1H),6.30(dd,J＝17.0,2.1Hz,1H),5.78(dd,J＝10.2,2.0Hz,1H),3.53(s,2H),2.41(s,8H),2.23(d,J＝2.9Hz,6H).
HRMS(ESI)m/z calcd for C 29 H 31 F 3 N 6 O 3 [M+H] + ,569.2482；found,569.2494.
Examples and structural characterization information summaries are shown in table 1:
the synthesis was as in example 1 or example 2:
table 1, examples and their structural characterization information:
Figure BDA0003659888500000131
Figure BDA0003659888500000141
Figure BDA0003659888500000151
Figure BDA0003659888500000161
Figure BDA0003659888500000171
Figure BDA0003659888500000181
Figure BDA0003659888500000191
Figure BDA0003659888500000201
Figure BDA0003659888500000211
Figure BDA0003659888500000221
Figure BDA0003659888500000231
Figure BDA0003659888500000241
biological assay
1. In vitro kinase Activity assay
All compounds were completely dissolved in DMSO and prepared as a stock solution at a concentration of 10 mM. Then, it was diluted to 50 times the highest concentration required. 100 μ L of dilution was added to a 96-well plate, while 100 μ L of DMSO was added to two empty wells as a no-compound control and a no-enzyme control, and the plate was labeled as a source plate. mu.L of compound was transferred from the source plate to a new 96-well plate as an intermediate plate, 90. mu.L of 1 Xkinase base buffer was added to each well of the intermediate plate, and mixed by shaking for 10 minutes. From each well of the intermediate plate, 5. mu.L of the liquid was transferred to a 384-well plate in duplicate. A kinase solution was prepared at 2.5x final concentration using 1x kinase base buffer, and FAM-labeled peptide and ATP were added to 1x kinase base buffer to prepare a 2.5x polypeptide solution. mu.L of 2.5 Xkinase solution was added to each well of 384-well assay plates and incubated at room temperature for 10 minutes, then 10. mu.L of 2.5 Xpolypeptide solution was added to each well and incubated at 28 ℃ for a period of time, and the kinase reaction was stopped by adding 30. mu.L of stop buffer. The conversion was read with a microplate reader. Data analysis, inhibition rate (%) (max-conversion)/(max-min): 100
Wherein conversion is the conversion reading for the compound; max is the positive control well, representing the conversion reading for the no compound well; min is a negative control well representing a conversion reading for wells without enzyme. The results are shown in Table 2.
TABLE 2 inhibition of FGFR4 kinase by compounds at 1. mu.M test concentration
Figure BDA0003659888500000251
The data show that the compound has a remarkable inhibitory effect on FGFR4, and is worthy of further study.
2. In vitro tumor cell antiproliferation assay
Firstly, cells are digested and counted to prepare the cell with the density of 4.5 multiplied by 10 4 one/mL of cell suspension, 100. mu.L of cell suspension per well in a 96-well cell culture plate, and then placing the 96-well cell culture plate at 37 ℃ in 5% CO 2 The culture was carried out in an incubator for 24 hours. Diluting the drug with culture medium to desired concentration, adding 100 μ L of corresponding drug-containing culture medium into each well, setting up negative control group, placing 96-well cell culture plate at 37 deg.C and 5% CO 2 The incubator was continued for 72 hours. Then, 10. mu.L of CCK-8 was added to each well, and the culture was continued in an incubator for 2 to 3 hours. Mixing the mixture gently by a shaking table for 10 minutes to remove bubbles in the 96-well plate; the absorbance of each well at a wavelength of 450nm was measured on a microplate reader (BioTek) to calculate the inhibition rate. Inhibition (%) - (negative control OD value-experimental OD value)/negative control OD value 100%. The results are shown in Table 3.
Table 3, results of anti-proliferative activity of compound on tumor cells in vitro:
Figure BDA0003659888500000261
the data show that the compound has obvious inhibition effect on hepatocellular carcinoma strains Hep3B and HuH 7.

Claims (9)

1. A compound of formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof,
Figure FDA0003659888490000011
wherein:
a is C 6-8 Aryl, 5-to 8-membered heteroaryl, C 3-8 Cycloalkyl, 3-to 8-membered heterocycloalkyl or C 3-8 A cycloalkenyl group;
b is phenyl or heteroaryl;
x is selected from-O-, -NH-or none;
y is selected from one of-NHCONH-, -NHCO or-CONH-;
l is a moiety capable of forming a covalent bond with a nucleophile and is selected from the group having the following structure:
Figure FDA0003659888490000012
wherein
Figure FDA0003659888490000013
Represents a double bond or a single bond;
R 1 、R 2 and R 3 Each independently selected from hydrogen, halogen, optionally substituted C 1-4 Alkyl or optionally substituted heterocyclylalkyl;
R 4 and R 5 Selected from hydrogen, halogen, cyano, mercapto, hydroxy, amino, amido, ester, amido, sulfonyl, sulfinyl, and optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 An alkoxy group;
R 6 selected from a chlorine atom, a cyano group or a group selected from the following structures:
Figure FDA0003659888490000014
R 7 selected from hydrogen, hydroxy, cyano, optionally substituted C 1-4 Alkyl or optionally substituted heterocyclylalkyl;
m is 1 or 2; n is 0,1, 2, 3 or 4; p is 0,1, 2, 3 or 4.
2. A compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein:
a is a pyridine ring;
b is selected from phenyl, pyridyl or pyrazinyl;
x is one of-O-or none;
y is-NHCONH-;
l is selected from
Figure FDA0003659888490000021
T-shirt
Figure FDA0003659888490000022
Wherein R is 1 、R 2 Each independently selected from hydrogen, C1-3 alkyl, R 3 Selected from hydrogen, halogen;
R 4 selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, halogen;
R 5 is trifluoromethyl;
R 6 selected from chlorine atom, 4-methylpiperazino-1-methylene, 4-ethylpiperazino-1 methylene, morpholine-1-methylene, pyrrole-1-methylene;
n is 1 or 2.
3. A compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, according to claim 2, wherein:
a is a pyridine ring;
b is selected from phenyl, pyridyl or pyrazinyl;
x is one of-O-or none;
y is-NHCONH-;
l is selected from acrylamide, chloroacetamide, crotonamide, butynamide, 2-fluoroacrylamide or propionamide;
R 4 selected from hydrogen, methyl, fluorine, chlorine, methoxy;
R 5 is trifluoromethyl;
R 6 selected from 4-methylpiperazino-1-methylene, 4-ethylpiperazino-1-methylene;
n is 1.
4. A compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, according to claim 2, wherein said compound of formula (i) is selected from any one of the following:
Figure FDA0003659888490000023
Figure FDA0003659888490000031
5. a process for the preparation of a compound of formula (I) as claimed in any one of claims 1 to 4, wherein the reaction scheme is selected from any one of the following:
route one:
Figure FDA0003659888490000041
and a second route:
Figure FDA0003659888490000042
6. a pharmaceutical composition comprising a compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable adjuvant.
7. The use of a compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the treatment of a disease state associated with abnormal activation of FGFR 4.
8. The use according to claim 7, characterized in that the disease associated with abnormal activation of FGFR4 is selected from hepatocellular carcinoma, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, rhabdomyosarcoma, lung cancer, soft tissue sarcoma, head and neck squamous cell carcinoma or cervical cancer.
9. The use of a compound of formula (i), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, according to claim 1, for the preparation of an inhibitor of fibroblast growth factor receptors.
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