CN103304438B - N-substituted salicylamide compounds, preparation method and application - Google Patents

N-substituted salicylamide compounds, preparation method and application Download PDF

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CN103304438B
CN103304438B CN201310241976.3A CN201310241976A CN103304438B CN 103304438 B CN103304438 B CN 103304438B CN 201310241976 A CN201310241976 A CN 201310241976A CN 103304438 B CN103304438 B CN 103304438B
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hydroxyl
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benzamide
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CN103304438A (en
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赵桂森
李奕静
刘洋
刘建珍
王远游
张林娜
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Shandong University
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Abstract

The present invention relates to N-substituted salicylamide compounds, preparation method and application.N-substituted salicylamide compounds is the compound with general formula (I) structure, and wherein, X is hydrogen, halogen; Y is hydrogen, halogen; Z be hydrogen, methyl, methoxyl group, halogen or n is 0,1 or 2.The present invention also provides the preparation method of formula (I) compound and described compound as the application of EGFR tyrosine kinase inhibitor.

Description

N-substituted salicylamide compounds, preparation method and application
Technical field
The present invention relates to organic compound and medical applications field, particularly relate to N-substituted salicylamide compounds and preparation method thereof and application.
Background technology
Human epidermal growth factor receptor (humanepidermalgrowthfactorreceptor, HER) family is extensively present in organism, plays vital effect in cell signalling; HER and Growth of Cells, apoptosis, differentiation, migration and vasculogenesis etc. are closely bound up.HER is the single transmembrane albumen containing 1186 amino-acid residues, is divided into four hypotypes: EGFR, HER-2, HER-3 and HER-4; They have high homology; Its structure comprises three regions: (1) extracellular space, containing NH 2-end, with ligand binding; (2) hydrophobic transmembrane region is one section of αhelix containing 23 amino-acid residues, can on anchored receptor and cytolemma, and this region is relevant with Receptor dimerization; (3), for containing C-terminal, there are typical ATP-binding site and Tyrosylprotein kinase district in intracellular space.See: Liu Baoquan; Fan Sheng, EGFR(EGF-R ELISA) signal transduction mechanism and targeted therapy, Dalian Nationality College's journal 2008,10 (1), 13-16.Under normal operation, the form of HER family regulating cell and differentiation situation, and promote the normal formation of organ, when it expresses disorderly, oncogenic generation may be led.See: Woodburn, J.R., TheEpidermalGrowthFactorReceptorandItsInhibitioninCancer Therapy.Pharmacology & Therapeutics1999,82 (2 – 3), 241-250.HER family is divided into four hypotypes: EGFR, HER-2, HER-3 and HER-4.EGFR(epidermalgrowthfactorreceptor) be a member the most widely that distributes in HER family; its continuous activation and growth of tumour cell and survive closely related; in kinds of tumor cells, find EGFR process LAN at present, see: Herbst, R.S.; Langer, C.J., Epidermalgrowthfactorreceptorsasatargetforcancertreatmen t:TheemergingroleofIMC-C225inthetreatmentoflungandheadan dneckcancers.SeminarsinOncology2002,29 (1, Supplement4), 27-36.Therefore, one of the research and development study hotspot becoming neoplasm targeted therapy of EGFR inhibitor.
Be that the inhibitor of target is divided into two large classes: monoclonal antibody and micromolecular inhibitor with HER.The EGFR tyrosine kinase inhibitor gone on the market has Gefitinib, Tarceva, lapatinibditosylate and Conmana, and its constitutional features is all containing 4-anilinoquinazoline parent nucleus.
Summary of the invention
One of task of the present invention is to provide a kind of N-substituted salicylamide compounds with suppression EGFR tyrosine kinase activity.
Another task of the present invention is the preparation method and the application that provide this compound.
Summary of the invention
The present invention for parent nucleus with N-phenyl salicylic acid amides, introduces methoxyethoxy in the 4-position of salicylic amide phenyl ring, N-phenyl ring is introduced the substituting group of opposed polarity, has synthesized a series of N-substituted salicylamide compounds, had the structure of general formula (I); This compounds has EGFR tyrosine-kinase enzyme inhibition activity.
Detailed Description Of The Invention
There is the compound of general formula (I) structure:
Wherein, X is hydrogen, halogen; Y is hydrogen, halogen; Z be hydrogen, methyl, methoxyl group, halogen or wherein R is hydrogen, halogen, nitro; N is 0,1 or 2.
According to the present invention, preferably, in the compound of above-mentioned general formula (I), X is hydrogen, fluorine; Y is hydrogen, fluorine, chlorine; Z be hydrogen, chlorine, bromine, methyl, methoxyl group or wherein R is hydrogen, fluorine, chlorine, bromine, nitro; N is 0,1 or 2.
According to the present invention, preferred compound is one of following:
N-(4-(benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (7a),
N-(4-(3-fluorine benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (7b),
N-(4-(3-chlorine benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (7c),
N-(4-(3-bromo-benzyloxy-) phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (7d),
N-(4-(3-nitro benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (7e),
N-phenyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide (8a),
N-(3-chloro-phenyl-)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (8b),
N-(4-aminomethyl phenyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (8c),
N-benzyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9a),
N-(3-chlorobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9b),
N-(4-chlorobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9c),
N-(4-bromobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9d),
N-(4-methoxybenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9e),
N-(the chloro-2-luorobenzyl of 3-)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9f),
N-styroyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide (9g),
N-(the chloro-4-of 3-((3-luorobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide (12a),
N-(the chloro-4-of 3-((3-chlorobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide (12b),
N-(the chloro-4-of 3-((3-bromobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide (12c).
Be the code name of its correspondence in bracket after above-mentioned preferred 18 compound titles, in order to describe convenient and be concise in expression, the code name in above-mentioned bracket will be used directly in the following content of this specification sheets.
According to the present invention, preferred compound is compound 7a, 7b, 7c, 7d, 7e, 8b, 12a, 12b further.
According to the present invention, the preparation method of the N-substituted salicylamide compounds of general formula (I), wherein, X is hydrogen, fluorine; Y is hydrogen, fluorine, chlorine; Z be hydrogen, chlorine, bromine, methyl, methoxyl group or wherein R is hydrogen, fluorine, chlorine, bromine, nitro; N is 0,1 or 2; The method comprises:
I. formula 3 compound 2-hydroxyl-4-(2-methoxy ethoxy) phenylformic acid and formula 6 compound 4-(substituted benzyloxy) aniline is made, at 4-lutidine and N, under N '-dicyclohexylcarbodiimide exists, in DMF, reaction forms formula 7 compound: N-(4-(substituted benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide;
Described 4-(substituted benzyloxy) aniline is 4-(benzyloxy) aniline, 4-(3-fluorine benzyloxy) aniline, 4-(3-chlorine benzyloxy) aniline, 4-(3-bromo-benzyloxy-) aniline, 4-(3-nitro benzyloxy) aniline; Or,
Ii. formula 3 compound 2-hydroxyl-4-(2-methoxy ethoxy) phenylformic acid and substituted aniline is made, at 4-lutidine and N, under N '-dicyclohexylcarbodiimide exists, in DMF, reaction forms formula 8 compound: 2-hydroxyl-4-(2-methoxyethoxy)-N-substituted-phenyl benzamide;
Described substituted aniline is aniline, 3-chloroaniline, 4-monomethylaniline; Or,
Iii. formula 3 compound 2-hydroxyl-4-(2-methoxy ethoxy) phenylformic acid and alpha substituted benzylamine or phenylethylamine is made, at N, under N-carbonyl dimidazoles exists, in anhydrous tetrahydro furan, reaction forms formula 9 compound: N-substituted benzyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide or N-styroyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide;
9n=1,2
Described alpha substituted benzylamine or phenylethylamine are benzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 4-bretylium, 4-methoxybenzylamine, the chloro-benzylamine of the fluoro-3-of 2-or β-phenylethylamine; Or,
Iv. make formula 3 compound 2-hydroxyl-4-(2-methoxy ethoxy) phenylformic acid under N-hydroxybenzotriazole and 1-ethyl-3 (3-dimethylamino-propyl) phosphinylidyne diimmonium salt hydrochlorate exist, form formula 12 compound with formula 11 compound 3-chlorin-4-((substituted benzyl) oxygen base) aniline reaction: N-(the chloro-4-of 3-((substituted benzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide;
The chloro-4-of described 3-((substituted benzyl) oxygen base) aniline is the chloro-4-of 3-(3-fluorine benzyloxy) aniline, the chloro-4-of 3-(3-chlorine benzyloxy) aniline, the chloro-4-of 3-(3-bromo-benzyloxy-) aniline.
According to the present invention, the N-substituted salicylamide compounds of above-mentioned general formula (I), synthetic route is as follows:
In above-mentioned synthesis type: X is hydrogen, fluorine; Y is hydrogen, fluorine, chlorine; Z be hydrogen, chlorine, bromine, methyl, methoxyl group or (wherein R is hydrogen, fluorine, chlorine, bromine, nitro); N is 0,1 or 2.
In above-mentioned synthesis type, reagent and reaction conditions as follows: (a) 2-chloroethyl methyl ether, salt of wormwood, DMF, 90 DEG C 24 hours; (b) potassium hydroxide, 95wt% ethanol, refluxes 4 hours; C () replaces benzyl halogen, salt of wormwood, DMF; (d) potassium hydroxide, 95wt% ethanol, refluxes 12 hours; (e) 4-lutidine, N, N '-dicyclohexylcarbodiimide DMF, 80 DEG C 24 hours; (f) substituted aniline, 4-lutidine, N, N '-dicyclohexylcarbodiimide, 80 ° of C24 hour; G () replaces amine, N, N '-carbonyl dimidazoles, tetrahydrofuran (THF), 50 DEG C are spent the night; H () replaces benzyl halogen, potassium tert.-butoxide, DMF, room temperature 2 hours; (i) N-hydroxybenzotriazole, 1-ethyl-3 (3-dimethylamino-propyl) phosphinylidyne diimmonium salt hydrochlorate, DMF, room temperature reaction spends the night.
Preparation in accordance with the present invention, preferably, the preparation method of intermediate formula 3 compound is as follows:
(1) by 2, 4-methyl dihydroxy benzoate 1 and salt of wormwood in molar ratio 1:2 join N, in dinethylformamide, be warming up to 90 ° of C, add the 2-chloroethyl methyl ether of 2 mol ratios, react 24 hours, be cooled to room temperature, by the N of reaction solution impouring 10 times of volumes, in the frozen water of dinethylformamide, be extracted with ethyl acetate three times, combined ethyl acetate, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, remove solvent under reduced pressure, column chromatography obtains intermediate formula 2 compound: (2-hydroxyl-4-(2-methoxy ethoxy) methyl benzoate),
(2) by above-mentioned intermediate formula 2 compound and potassium hydroxide solution in molar ratio 1:5 join in 95wt% ethanol, reflux 4 hours, remove solvent under reduced pressure, obtain yellow oil, drop in the hydrochloric acid soln of 1M, separate out white solid, filter, filter cake acetic acid ethyl dissolution, filter, filtrate decompression is steamed and is desolventized, and obtain yellow solid, dehydrated alcohol recrystallization obtains intermediate formula 3 compound: 2-hydroxyl-4-(2-methoxy ethoxy) phenylformic acid.
Preparation in accordance with the present invention, preferably, the preparation method of the salicylic amide compounds of formula 7, X is hydrogen; Y is hydrogen; R is hydrogen, fluorine, chlorine, bromine, nitro; When n is 0, step is as follows:
(1) by paracetamol 4 and salt of wormwood in molar ratio 1:1.5 join N, in dinethylformamide, after stirring, add the replacement benzyl chlorine of 1.2 mol ratios or replace benzyl bromine, 80 DEG C of reactions are reacted 5 hours for 12 hours or 30 DEG C, be cooled to room temperature, by reaction solution impouring 10 times of N, in the frozen water of dinethylformamide volume, separate out precipitation, filter, filter cake frozen water washs to obtain intermediate 5 crude product, without the need to purifying, with potassium hydroxide solution in molar ratio 1:10 join in 95wt% ethanol, backflow 10h, remove solvent under reduced pressure, obtain thick solid, dissolve with anhydrous diethyl ether, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography, obtain intermediate formula 6 compound: 4-(substituted benzyloxy) aniline,
(2) by above-mentioned intermediate formula 6 compound, 4-lutidine and N, N '-dicyclohexylcarbodiimide in molar ratio 1.2:1:1.2 is dissolved in N, in dinethylformamide, the intermediate 3 of 1 mol ratio is added under ice bath, nitrogen protection, 80 DEG C of reaction 24h, be cooled to room temperature, by reaction solution impouring 10 times of N, in the frozen water of dinethylformamide volume, be extracted with ethyl acetate three times, combined ethyl acetate, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, remove solvent under reduced pressure, column chromatography obtains target product 7:N-(4-(substituted benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide.
Replacement benzyl chlorine described in above-mentioned steps (1) or replacement benzyl bromine are benzyl chloride, 3-fluorobenzyl chloride, 3-chlorine bromobenzyl, a bromobenzyl bromine, a nitrobenzyl bromine.
Preparation in accordance with the present invention, preferably, the preparation method of the salicylic amide compounds of formula 8, X is hydrogen; Y is hydrogen, chlorine; Z is hydrogen, methyl; N is 0; Step is as follows:
By substituted aniline, 4-lutidine and N, N '-dicyclohexylcarbodiimide in molar ratio 1.2:1:1.2 be dissolved in DMF, add the intermediate 3 of 1 mol ratio under ice bath, nitrogen protection, 80 DEG C reaction 24h.Be cooled to room temperature, by system impouring 10 times of N, in the frozen water of dinethylformamide volume, be extracted with ethyl acetate three times, combined ethyl acetate, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filters, remove solvent under reduced pressure, column chromatography obtains target product 8:2-hydroxyl-4-(2-methoxyethoxy)-N-substituted-phenyl benzamide.Wherein, described substituted aniline is aniline, 3-chloroaniline or 4-monomethylaniline.
Preparation in accordance with the present invention, preferably, the preparation method of the salicylic amide compounds of formula 9, X is hydrogen, fluorine; Y is hydrogen, chlorine; Z is hydrogen, chlorine, bromine, methoxyl group; N is 1 or 2; Step is as follows:
By intermediate 3 and N, N '-carbonyl dimidazoles 1:1.2 is dissolved in anhydrous tetrahydro furan, nitrogen protection, add alpha substituted benzylamine or the phenylethylamine of 1.2 mol ratios, be warming up to 50 DEG C of reactions to spend the night, filter, filtrate decompression is steamed and is desolventized to obtain oily matter or colored solid, with acetic acid ethyl dissolution, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography purification obtains target product 9:N-substituted benzyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide or N-styroyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide.Wherein, described alpha substituted benzylamine or phenylethylamine are benzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 4-bretylium, 4-methoxybenzylamine, the chloro-benzylamine of the fluoro-3-of 2-or β-phenylethylamine.
Preparation in accordance with the present invention, preferably, the preparation method of the salicylic amide compounds of formula 12, when X is hydrogen; Y is chlorine; R is fluorine, chlorine, bromine; When n is 0, step is as follows:
(1) 4-amino-2-chlorophenol 10 is dissolved in N, dinethylformamide, the potassium tert.-butoxide of 1.2 mol ratios is added under ice bath, stir, slowly add the replacement benzyl halogen of 1 mol ratio, room temperature reaction 2h, by the frozen water of system impouring 10 times of DMF volumes, be extracted with ethyl acetate three times, combined ethyl acetate, uses water successively, and saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, remove solvent under reduced pressure, column chromatography obtains the chloro-4-of intermediate 11:3-((substituted benzyl) oxygen base) aniline;
(2) under ice bath, intermediate 3 is dissolved in N, dinethylformamide, stir 5, add the 1-hydroxyl-benzo-triazole of 2 mol ratios and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate of 2 mol ratios, stir after 30 minutes, add the intermediate 11 of 1 mol ratio, reaction is spent the night, by system impouring 10 times of N, in the frozen water of dinethylformamide volume, be extracted with ethyl acetate three times, combined ethyl acetate, use water successively, saturated nacl aqueous solution washs, anhydrous sodium sulfate drying, filter, remove solvent under reduced pressure, column chromatography obtains target product formula 12 compound: N-(the chloro-4-of 3-((substituted benzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide.
Replacement benzyl halogen described in above-mentioned steps (1) is 3-fluorobenzyl chloride, 3-chlorine bromobenzyl or a bromobenzyl bromine.
N-substituted salicylamide compounds of the present invention is as the application of EGFR tyrosine kinase inhibitor.
N-substituted salicylamide compounds of the present invention has EGFR kinase inhibiting activity, for the preparation of anti-nonsmall-cell lung cancer, breast cancer medicines.Wherein, compound 7a, 7b, 7c, 7d, 7e, 8b, 12a, 12b, 12c has good EGFR kinase inhibiting activity (Detailed Experimental will be illustrated in example 2).Most preferred compound 8b.
Compared with prior art excellent results of the present invention:
The design and synthesis of the present invention N-substituted salicylamide compounds of novel structure, its innovative point is on salicylic amide parent nucleus, by introducing hydrophobic group and changing the novel salicylic amide derivative of amino substituent size design and synthesis.And measuring its inhibit activities to EGFR Tyrosylprotein kinase, N-substituted salicylamide compounds of the present invention has EGFR tyrosine-kinase enzyme inhibition activity.Wherein, the EGFR tyrosine-kinase enzyme inhibition activity IC of compound 8b 50be less than 0.3 μM.
Embodiment
Further describe the present invention below in conjunction with embodiment, understand the present invention and advantage thereof and effect in order to more deep, but described embodiment is only for illustration of the present invention instead of restriction the present invention.
The preparation of embodiment 1. formula 3 compound (key intermediate)
(i) the preparation of formula 2 compound 2-hydroxyl-4-(2-methoxy ethoxy) methyl benzoate
By 2,4-methyl dihydroxy benzoate (0.02mol) and K 2cO 3(0.04mol) be dissolved in 4mLN, in dinethylformamide, be warming up to 90 ° of C, stir 10 minutes, at this temperature, slowly drip the DMF solution of 2mL2-chloroethyl methyl ether (0.04mol), stir 12h.Be cooled to room temperature, by reaction solution impouring 60mL frozen water, extraction into ethyl acetate three times, each ethyl acetate consumption are 60mL (being designated as 60mL × 3), combined ethyl acetate, uses water (30mL × 3) successively, and saturated nacl aqueous solution (30mL × 3) washs, anhydrous sodium sulfate drying, filter, steam and desolventize to obtain oily matter, column chromatography, elution system is petrol ether/ethyl acetate=10:1, obtain white solid, yield 91%, mp:43.5 ~ 46.4 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):10.328(s,1H),7.850(m,2H),7.750(d,J=7.8Hz,2H),7.451(s,1H),7.380(t,J=7.8Hz,2H),7.293(m,2H),7.138(t,J=7.8Hz,1H),4.129(s,3H)。MS(ESI)m/zcalcdforC 10H 12O 5:212.1993,found:213.0753[M+H] +。
(ii) formula 3 compound 2-hydroxyl-4-(2-methoxy ethoxy) benzoic preparation
Formula 2 compound 2-hydroxyl-4-(2-methoxy ethyl by above-mentioned (i) obtained) methyl benzoate (0.02mol) is suspended in 80mL ethanol, adds the aqueous solution of 10mL containing KOH (0.1mol), backflow 4h.Remove solvent under reduced pressure, obtain yellow oil.Gained oily matter drops in the hydrochloric acid soln of 100mL1M under stirring by ice bath, separates out white solid, filters.Filter cake acetic acid ethyl dissolution, filters, and filtrate decompression is steamed and desolventized, and obtain yellow solid, dehydrated alcohol recrystallization obtains white solid, yield 98%, mp:136 ~ 138 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):10.763(s,1H),7.706(dd,J 1=8.4Hz,J 2=1.2Hz,1H),6.536(m,2H),4.138(t,J=6.0Hz,2H),3.863(s,3H),3.570(t,J=4.8Hz,4H),2.683(t,J=6.0Hz,2H),2.484(m,4H)。MS(ESI)m/zcalcdforC 11H 14O 5:226.0841,found:227.0913[M+H] +。
The preparation of embodiment 2. target product 7a ~ 7e
(i) by paracetamol (2mmol) and salt of wormwood (3mmol), be dissolved in 2mLN, dinethylformamide, stirs 10 minutes, and gradation adds and replaces benzyl chlorine (2.4mmol), be warming up to 80 DEG C, reaction is spent the night, and is cooled to room temperature, by reaction system impouring 20mL frozen water, separate out white solid, filter to obtain the crude product of intermediate 5.Transfer them in reaction flask, add 20mL95wt% ethanol and substantially dissolve to solid, add 2mL containing KOH(20mmol) the aqueous solution, be warming up to 95 DEG C, reflux 10 hours.Be cooled to room temperature, remove solvent under reduced pressure, obtain thick solid, anhydrous diethyl ether extraction (30mL × 3), merge ether, use water (30mL × 3), saturated nacl aqueous solution (30mL × 3) to wash successively, anhydrous sodium sulfate drying, filter, steaming desolventizes to obtain intermediate 6 crude product, column chromatography, elution system is petrol ether/ethyl acetate, obtains intermediate formula 6 compound.
Above-mentioned replacement benzyl chlorine selects benzyl chloride, 3-fluorobenzyl chloride respectively, obtains following intermediate formula 6 compound respectively.
6a:4-(benzyloxy) aniline, gold solid, yield 82%, 1h-NMR (DMSO-d 6) δ (ppm): 7.404 (d, J=7.2Hz, 2H), 7.367 (t, J=7.8Hz, 2H), 7.301 (t, J=7.8Hz, 1H), 6.713 (d, J=4.8Hz, 2H), 6.496 (d, J=4.8Hz, 2H), 4.935 (s, 2H), 4.614 (s, 2H).MS(ESI)m/zcalcdforC 13H 13ON:199.1,found:200.3[M+H] +。
6b:4-((3-luorobenzyl) oxygen base) aniline, dark brown solid, yield 68%, 1H-NMR data (DMSO-d 6) δ (ppm): 7.405 (m, 1H), 7.233 (m, 2H), 7.126 (dd, J 1=12Hz, J 2=3.6Hz, 1H), 6.724 (dd, J 1=12Hz, J 2=3.6Hz, 2H), 6.406 (dd, J 1=9.6Hz, J 2=2.4Hz, 2H), 4.953 (s, 2H), 4.633 (s, 2H).MS(ESI)m/zcalcdforC 13H1 2FNO:217.1,found:218.5[M+H] +。
(ii) by paracetamol (2mmol) and salt of wormwood (3mmol), be dissolved in 2mLN, dinethylformamide, stir 10 minutes, gradation adds and replaces benzyl bromine (2.4mmol), and room temperature reaction 5 hours, by reaction system impouring 10 times amount frozen water, separate out white solid, filter to obtain the crude product of intermediate 5.Transfer them in reaction flask, add 20mL95wt% ethanol and substantially dissolve to solid, add 2mL containing KOH(20mmol) the aqueous solution, be warming up to 95 DEG C, reflux 10 hours.Be cooled to room temperature, remove solvent under reduced pressure, obtain thick solid, anhydrous diethyl ether extraction (30mL × 3), merge ether, use water (30mL × 3) successively, saturated nacl aqueous solution (30mL × 3) washs, anhydrous sodium sulfate drying, filter, steam and desolventize to obtain intermediate 6 crude product, column chromatography, elution system is petrol ether/ethyl acetate, obtains intermediate formula 6 compound.
Above-mentioned replacement benzyl bromine selects 3-chlorine bromobenzyl, a bromobenzyl bromine, a nitrobenzyl bromine respectively, obtains following intermediate formula 6 compound respectively:
6c:4-((3-chlorobenzyl) oxygen base) aniline, brown solid, yield 74%, 1h-NMR (DMSO-d 6) δ (ppm): 7.456 (s, 1H), 7.387 (m, 3H), 6.716 (dd, J 1=8.4Hz, J 2=1.2Hz, 2H), 6.503 (dd, J 1=8.4Hz, J 2=1.2Hz, 2H), 4.962 (s, 2H), 4.634 (s, 2H).MS(ESI)m/zcalcdforC 13H 12ClNO:233.1,found:234.3[M+H] +。
6d:4-((3-bromobenzyl) oxygen base) aniline, brown solid, yield 78%, 1h-NMR (DMSO-d 6) δ (ppm): 7.605 (s, 1H), 7.390 (m, 3H), 6.729 (dd, J 1=9.6Hz, J 2=3.6Hz, 2H), 6.526 (dd, J 1=9.6Hz, J 2=3.6Hz, 2H), 4.960 (s, 2H), 4.650 (s, 2H).MS(ESI)m/zcalcdforC 13H 12BrNO:277.0,found:278.3[M+H] +。
6e:4-((3-nitrobenzyl) oxygen base) aniline, red brown solid, yield 76%, 1h-NMR (DMSO-d 6) δ (ppm): 7.705 (s, 1H), 7.500 (m, 3H), 6.742 (dd, J 1=12Hz, J 2=3.6Hz, 2H), 6.553 (dd, J 1=12Hz, J 2=3.6Hz, 2H), 4.960 (s, 2H), 4.670 (s, 2H).MS(ESI)m/zcalcdforC 13H 12N 2O 3:244.1,found:245.3[M+H] +。
(iii) under ice bath, by intermediate 6(1.2mmol), 4-lutidine (0.1mmol) N, N '-dicyclohexylcarbodiimide (1.2mmol) is dissolved in the anhydrous N of 40mL, dinethylformamide, stir 5 minutes, gradation adds intermediate formula 3 compound (1mmol), nitrogen protection, be warming up to 80 DEG C of reaction 24h, by in reaction system impouring 10 times amount frozen water, extraction into ethyl acetate (20mL × 3), combined ethyl acetate, use water (20mL × 3) successively, saturated nacl aqueous solution (20mL × 3) washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography purification obtains compound 7, elution system is petrol ether/ethyl acetate=6:1.
7a:N-(4-(benzyloxy) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide, white solid, yield 56%, mp:173 ~ 175 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.589(s,1H),10.134(s,1H),7.967(d,J=9.0Hz,1H),7.563(d,J=8.4Hz,2H),7.456(d,J=7.8Hz,2H),7.398(t,J=7.8Hz,2H),7.339(d,J=7.2Hz,1H),7.020(d,J=8.4Hz,2H),6.554(d,J=7.8Hz,1H),6.482(s,1H),7.138(t,J=7.8Hz,1H),5.103(s,2H),4.136(s,2H),3.659(s,2H),3.129(s,3H)。MS(ESI)m/zcalcdforC 23H 23NO 5:393.1576,found:394.1643[M+H] +。
7b:N-(4-((3-luorobenzyl) oxygen base) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide, white solid, yield 57%, mp:161.1 ~ 161.3 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.413(s,1H),7.679(s,1H),7.442(d,J=9.0Hz,2H),7.355(d,J=8.4Hz,2H),7.349(m,1H),7.195(d,J=7.8Hz,1H),7.158(d,J=9.6Hz,1H),6.997(m,3H),6.511(m,2H),5.067(s,2H),4.143(t,J=4.2Hz,2H),3.765(t,J=4.2Hz,2H),3.458(s,3H)。MS(ESI)m/zcalcdforC 23H 22FNO 5:411.1482,found:412.1551[M+H] +。
7c:N-(4-((3-chlorobenzyl) oxygen base) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide, white solid, yield 48%, mp:159 ~ 160.4 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.570(s,1H),10.158(s,1H),7.961(d,J=9.0Hz,1H),7.568(d,J=9.0Hz,2H),7.521(s,1H),7.415(m,3H),7.023(d,J=9.0Hz,2H),6.550(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.477(d,J=2.4Hz,1H),5.128(s,2H),4.135(t,J=4.2Hz,2H),3.662(t,J=4.2Hz,2H),3.309(s,3H)。MS(ESI)m/zcalcdforC 23H 22ClNO 5:427.1187,found:428.1249[M+H] +。
7d:N-(4-((3-bromobenzyl) oxygen base) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide, white solid, yield 52%, mp:173.4 ~ 174.6 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.563(s,1H),10.137(s,1H),7.962(d,J=9.0Hz,1H),7.660(s,1H),7.550(m,3H),7.463(d,J=7.8Hz,1H),7.366(t,J=7.8Hz,1H),7.022(d,J=9.6Hz,2H),6.552(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.479(d,J=2.4Hz,1H),5.122(s,2H),4.136(t,J=4.2Hz,2H),3.323(t,J=4.2Hz,2H),3.310(s,3H)。MS(ESI)m/zcalcdforC 23H 22BrNO 5:471.0681,found:472.0740[M+H] +。
7e:N-(4-((3-nitrobenzyl) oxygen base) phenyl)-2-hydroxyl-4-(2-methoxy ethoxy) benzamide, white solid, yield 43%, mp:154.9 ~ 156.0 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):8.333(s,1H),8.202(d,J=7.8Hz,1H),7.778(d,J=7.8Hz,1H),7.663(s,1H),7.581(t,J=8.4Hz,1H)7.476(d,J=9.0Hz,2H),7.400(d,J=8.4Hz,1H),7.003(d,J=9.0Hz,2H),6.522(m,2H),5.173(s,2H),4.151(t,J=4.8Hz,2H),3.769(t,J=4.8Hz,2H),3.461(s,3H)。MS(ESI)m/zcalcdforC 23H 22N 2O 7:438.1427,found:439.1491[M+H] +。
The preparation of embodiment 3. target product 8a ~ 8c
Under ice bath, by substituted aniline (1.2mmol), 4-lutidine (0.1mmol), N, N '-dicyclohexylcarbodiimide (1.2mmol) is dissolved in the anhydrous N of 4mL, dinethylformamide, stir 5 minutes, gradation adds intermediate formula 3 compound (1mmol), nitrogen protection, be warming up to 80 DEG C of reaction 24h, by in reaction system impouring 40mL frozen water, extraction into ethyl acetate (20mL × 3), combined ethyl acetate, use water (20mL × 3) successively, saturated nacl aqueous solution (20mL × 3) washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography purification obtains compound 7, elution system is petrol ether/ethyl acetate=10:1.
Above-mentioned substituted aniline selects aniline, 3-chloroaniline, 4-anisidine respectively, obtains following target product formula 8 compound respectively:
8a:2-hydroxyl-4-(2-methoxyethoxy)-N-phenylbenzamaide, white solid, yield 66%, mp:138.3 ~ 140.0 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.189(s,1H),7.753(s,1H),7.702(s,1H),7.398(d,J=8.4Hz,2H),7.302(t,J=8.4Hz,1H),7.157(m,1H),6.519(m,2H),4.148(t,J=4.2Hz,2H),3.770(t,J=4.2Hz,2H),3.461(s,3H)。MS(ESI)m/zcalcdforC 16H 17NO 4:287.1158,found:288.1226[M+H] +。
8b:2-hydroxyl-4-(2-methoxyethoxy)-N-(3-chloro-phenyl-)-benzamide, white solid, yield 76%, mp:146.0 ~ 147.5 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.369(s,1H),7.739(s,1H),7.556(d,J=8.4Hz,2H),7.404(m,3H),7.196(t,J=7.8Hz,1H),4.152(t,J=4.8Hz,2H),3.770(t,J=4.8Hz,2H),3.461(s,3H)。MS(ESI)m/zcalcdforC 16H 16ClNO 4:321.0768,found:322.0836[M+H] +。
8c:2-hydroxyl-4-(2-methoxyethoxy)-N-(p-methylphenyl) benzamide, white solid, yield 74%, mp:160.1 ~ 161.8 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.498(s,1H),10.153(s,1H),7.977(d,J=9.0Hz,1H),7.551(d,J=8.4Hz,2H),7.170(d,J=8.4Hz,2H),6.558(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.485(d,J=3.0Hz,1H),4.138(t,J=4.8Hz,2H),3.661(t,J=4.8Hz,2H),3.310(s,3H)。MS(ESI)m/zcalcdforC 16H 17NO 4:301.1314,found:302.1385[M+H] +。
The preparation of embodiment 4. target product 9a ~ 9g
By intermediate formula 3 compound (1mmol) and N, N '-carbonyl dimidazoles (1.2mmol) is dissolved in 4mL anhydrous tetrahydro furan, nitrogen protection, add alpha substituted benzylamine or phenylethylamine (1.2mmol), be warming up to 50 DEG C of reactions to spend the night, filter, filtrate decompression is steamed and is desolventized to obtain oily matter or colored solid, extraction into ethyl acetate (30mL × 3), combined ethyl acetate, use water (30mL × 3) successively, saturated nacl aqueous solution (30mL × 3) washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography purification must obtain target product 9, elution system is petrol ether/ethyl acetate=5:1.
Alpha substituted benzylamine described in above-mentioned steps or phenylethylamine are benzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 4-bretylium, 4-methoxybenzylamine, the chloro-benzylamine of the fluoro-3-of 2-, β-phenylethylamine, obtain following target product formula 9 compound respectively.
9a:N-benzyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 88%, mp:72.1 ~ 73.4 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):13.024(s,1H),9.295(s,1H),7.821(d,J=9.0Hz,1H),7.327(m,4H),7.249(t,J=6.6Hz,1H),6.461(d,J=8.4Hz,1H),6.410(s,1H),4.485(d,J=8.4Hz,2H),4.103(t,J=4.2Hz,2H),3.641(t,J=4.2Hz,2H),3.296(s,3H)。MS(ESI)m/zcalcdforC 17H 19NO 4:301.1314,found:301.1386[M+H] +。
9b:N-(3-chlorobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 52%, mp:90.0 ~ 90.1 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.848(s,1H),9.206(s,1H),7.818(d,J=9.0Hz,1H),7.372(m,2H),7.323(m,1H),7.285(d,J=7.8Hz,1H),6.502(dd,J 1=9.0Hz,J 2=3.0Hz,1H),6.437(d,J=2.4Hz,1H),4.485(d,J=6.6Hz,2H),4.116(m,2H),3.644(m,2H),3.328(s,3H)。MS(ESI)m/zcalcdforC 17H 18ClNO 4:335.0924,found:336.0988[M+H] +。
9c:N-(4-chlorobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 77%, mp:107.1 ~ 108.9 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.894(s,1H),9.203(s,1H),7.811(d,J=9.0Hz,1H),7.396(d,J=8.4Hz,2H),7.336(d,J=8.4Hz,2H),6.493(dd,J 1=10.8Hz,J 2=2.4Hz,1H),6.429(d,J=2.4Hz,1H),4.465(d,J=6.0Hz,2H),4.113(t,J=4.2Hz,2H),3.642(t,J=4.2Hz,2H),3.298(s,3H)。MS(ESI)m/zcalcdforC 17H 18ClNO 4:335.0924,found:336.1005[M+H] +。
9d:N-(4-bromobenzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 82%, mp:109.0 ~ 128.2 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.889(s,1H),9.202(s,1H),7.810(d,J=9.0Hz,1H),7.527(m,2H),7.277(d,J=8.4Hz,2H),6.493(dd,J 1=8.4Hz,J 2=2.4Hz,1H),6.428(d,J=3.0Hz,1H),4.447(d,J=6.0Hz,2H),4.112(m,2H),3.645(m,2H),3.298(s,3H)。MS(ESI)m/zcalcdforC 17H 18BrNO 4:379.0419,found:380.0482[M+H] +。
9e:N-(4-methoxy-benzyl)-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 68%, mp:85.2 ~ 86.7 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):13.025(s,1H),9.122(s,1H),7.806(d,J=9.0Hz,1H),7.243(d,J=8.4Hz,2H),6.892(dd,J 1=6.6Hz,J 2=2.4Hz,2H),6.474(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.417(d,J=3.0Hz,1H),4.404(d,J=6.0Hz,2H),4.107(m,2H),3.724(s,3H),3.639(m,2H),3.296(s,3H)。MS(ESI)m/zcalcdforC 18H 21NO 5:331.1420,found:332.1452[M+H] +。
9f:N-(the chloro-2-luorobenzyl of 3-)-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 76%, mp:133.4 ~ 134.9 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):9.186(s,1H),7.827(d,J=9.0Hz,1H),7.502(t,J=7.2Hz,1H),7.340(t,J=7.2Hz,1H),7.210(t,J=7.2Hz,1H),6.501(dd,J 1=9.0Hz,J 2=1.8Hz,1H),6.438(s,1H),4.550(d,J=5.4Hz,2H),4.115(s,3H),3.647(t,J=3.6Hz,2H)。MS(ESI)m/zcalcdforC 17H 17ClFNO 4:353.0830,found:354.0910[M+H] +。
9g:N-styroyl-2-hydroxyl-4-(2-methoxyethoxy) benzamide, white solid, yield 67%, mp:81.8 ~ 83.3 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):7.341(t,J=7.2Hz,2H),7.251(m,4H),7.070(d,J=9.0Hz,1H),6.461(d,J=1.8Hz,1H),6.399(dd,J 1=3.0Hz,J 2=1.8Hz,1H),6.090(m,1H),4.113(t,J=8.4Hz,2H),3.743(t,J=8.4Hz,2H),3.688(q,J=6.6Hz,2H),3.443(s,3H),2.924(t,J=6.6Hz,2H)。MS(ESI)m/zcalcdforC 18H 21NO 4:315.1471,found:316.1540[M+H] +。
The preparation of embodiment 5. target product 12a ~ 12c
(i) 4-amino-2-chlorophenol (5mmol) is dissolved in 5mLN, dinethylformamide, potassium tert.-butoxide (6mmol) is added under ice bath, stir, slow dropping 5mL replaces the N of benzyl halogen (5mmol), dinethylformamide solution, room temperature reaction 2 hours, by in system impouring 100mL frozen water, extraction into ethyl acetate (50mL × 3), combined ethyl acetate, use water (30mL × 3) successively, saturated nacl aqueous solution (30mL × 3) washs, anhydrous sodium sulfate drying, filter, steaming desolventizes, column chromatography, elution system is petrol ether/ethyl acetate, obtain intermediate formula 11 compound: the chloro-4-of 3-((substituted benzyl) oxygen base) aniline,
Replacement benzyl halogen described in above-mentioned steps is 3-fluorobenzyl chloride, 3-chlorine bromobenzyl, a bromobenzyl bromine.
The chloro-4-of 11a:3-((3-luorobenzyl) oxygen base) aniline: yellow solid, yield 55.6%, mp:82.9 ~ 84.5 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):8.320(s,1H),7.431(dd,J 1=13.8Hz,J 2=7.2Hz,1H),7.262(t,J=8.4Hz,2H),7.149(t,J=8.4Hz,1H),6.910(d,J=9.0Hz,1H),6.648(s,1H),6.464(d,J=8.4Hz,1H),5.031(s,2H),4.966(s,2H)。MS(ESI)m/zcalcdforC 13H 11ClFNO:251.1,found:252.3[M+H] +。
The chloro-4-of 11b:3-((3-chlorobenzyl) oxygen base) aniline: yellow solid, yield 58.2%, mp:73.8 ~ 74.8 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):7.492(s,1H),7.405(m,3H),6.910(d,J=9.0Hz,1H),6.647(d,J=2.4Hz,1H),6.464(dd,J 1=8.4Hz,J 2=3.0Hz,1H),5.019(s,2H),4.963(s,2H)。MS(ESI)m/zcalcdforC 13H 11Cl 2NO:267.0,found:268.3[M+H] +。
The chloro-4-of 11c:3-((3-bromobenzyl) oxygen base)-3-aniline: yellow solid, yield 35.9%, mp:61 ~ 62.5 DEG C.1H-NMR(DMSO-d6)δ(ppm):7.635(s,1H),7.520(d,J=7.8Hz,1H),7.360(d,J=7.8Hz,1H),7.354(t,J=7.8Hz,1H),6.909(d,J=9.0Hz,1H),6.646(d,J=3.0Hz,1H),6.464(dd,J1=8.4Hz,J2=3.0Hz,1H),5.019(s,2H),4.963(s,2H)。MS(ESI)m/zcalcdforC13H11BrClNO:311.0,found:312.2[M+H]+。
(ii) under ice bath, intermediate formula 3 compound (1mmol) is dissolved in N, dinethylformamide, stir 5 minutes, add N-hydroxybenzotriazole (2mmol) and 1-ethyl-3 (3-dimethylamino-propyl) phosphinylidyne diimmonium salt hydrochlorate (2mmol), stir after 30 minutes, add intermediate 11(1mmol), reaction is spent the night, by system impouring 10 times of N, in the frozen water of dinethylformamide volume, be extracted with ethyl acetate (30mL × 3), combined ethyl acetate, use water (30mL × 3) successively, saturated nacl aqueous solution (30mL × 3) washs, anhydrous sodium sulfate drying, filter, remove solvent under reduced pressure, column chromatography obtains target product 12:N-(the chloro-4-of 3-((substituted benzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide.
12a:N-(the chloro-4-of 3-((3-luorobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide, white solid, yield 35%, mp:157.6 ~ 158.9 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.345(s,1H),10.209(s,1H),7.939(d,J=8.4Hz,1H),7.866(d,J=2.4Hz,1H),7.561(dd,J 1=9Hz,J 2=2.4Hz,1H),7.465(m,1H),7.307(m,2H),7.240(d,J=9.6Hz,1H),7.180(td,J 1=9.0Hz,J 2=3.0Hz,1H),6.570(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.494(d,J=2.4Hz,1H),5.238(s,2H),4.140(t,J=3.0Hz,2H),3.663(t,J=3.0Hz,2H),3.310(s,3H)。HRMS(ESI)m/zcalcdforC 23H 21ClFNO 5:445.1092,found:446.1166[M+H] +。
12b:N-(the chloro-4-of 3-((3-chlorobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide, white solid, yield 37%, mp:139.2 ~ 140.6 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.340(s,1H),10.210(s,1H),7.937(d,J=9.0Hz,1H),7.865(d,J=2.4Hz,1H),7.561(dd,J 1=9Hz,J 2=2.4Hz,1H),7.542(s,1H),7.432(m,3H),7.239(d,J=9.0Hz,1H),6.570(dd,J 1=9.0Hz,J 2=2.4Hz,1H),6.492(d,J=3.0Hz,1H),5.231(s,2H),4.138(t,J=4.2Hz,2H),3.663(t,J=2.4Hz,2H),3.309(s,3H)。HRMS(ESI)m/zcalcdforC 23H 21Cl 2NO 5:461.0797,found:462.0868[M+H] +。
12c:N-(the chloro-4-of 3-((3-bromobenzyl) oxygen base) phenyl)-2-hydroxyl-4 (2-methoxyethoxy)-benzamide, white solid, yield 46%, mp:148.8 ~ 150.5 DEG C. 1H-NMR(DMSO-d 6)δ(ppm):12.341(s,1H),10.210(s,1H),7.937(d,J=9.0Hz,1H),7.865(d,J=2.4Hz,1H),7.683(s,1H),7.556(m,2H),7.480(d,J=2.4Hz,1H),7.387(t,J=9.0Hz,J2=7.8Hz,1H),7.237(d,J=9.0Hz,1H),6.570(dd,J 1=8.4Hz,J 2=2.4Hz,1H),6.493(d,J=3.0Hz,1H),5.226(s,2H),4.140(t,J=3.0Hz,2H),3.663(t,J=2.4Hz,2H),3.309(s,3H)。MS(ESI)m/zcalcdforC 23H 21BrClNO 5:505.0292,found:506.0366[M+H] +。
Embodiment 6. compound is to the mensuration of EGFR kinase inhibiting activity.
Compound 7a ~ 7e embodiment 1 to embodiment 5 prepared, 8a ~ 8c, 9a ~ 9g and 12a ~ 12c carries out biological activity determination by the following method, and acquired results is listed in table 1.
Compound adopts the method for time resolved fluorescence Resonance energy transfer (TR-FRET) to measure to the inhibit activities of EGFR Tyrosylprotein kinase.
The preparation of working fluid: precision takes N-(2-hydroxyethyl) piperazine-N'-ethyl sulfonic acid (Hepes, purchased from Sigma-Aldrich company) 2.9789g, ethylene glycol bis (2-amino-ethyl ether) tetraacethyl (EGTA, purchased from Sigma-Aldrich company) 0.0951g, MgCl 22.5mL and 0.05wt% tween 20 solution 25 μ L, in 30mL distilled water, adjusts pH to be 7.5 with 1MNaOH, adds distilled water and be settled to 50mL, obtain 5 × kinase buffer liquid; Get this damping fluid 1000 μ L, dithiothreitol (DTT) (DTT, purchased from Sigma-Aldrich company) the solution 10 μ L of distilled water 3990 μ L, 2 μm of ol/L, shakes up mixing and is made into 1 × kinase buffer liquid; Ice chest is got EGFR solution (purchased from CarnaBiosciences, Inc., concentration is 200ng/ μ L) 1.32 μ L, 1 × kinase buffer liquid 998.7 μ L, mixing, as kinases working fluid, preserves on ice; Ice chest is got kinase substrate (Μ Light-TKPeptide, purchased from PerkinElmer company) 40 μ L, get 1 × kinase buffer liquid 960 μ L and dilute, mix to obtain substrate working fluid; Get Triphosaden (ATP, purchased from Sigma-Aldrich company) solution (concentration is 10000 μm of ol/L) 18 μ L, add 1 × kinase buffer liquid 982 μ L and dilute, mix to obtain ATP working fluid.Get 10 × detection damping fluid (LANCE tMdetectionBuffer (10 × Conc.), purchased from PerkinElmer company) 600 μ L, distilled water 5400 μ L dilutes, mixing, and obtain 1 × detect damping fluid; Get ethylenediamine tetraacetic acid (EDTA) (EDTA likes (TCI, Shanghai) company purchased from ladder is uncommon) solution (concentration is 500 μm of ol/L) 160 μ L, 1 × detecting damping fluid 1840 μ L dilutes to obtain stop bath; Ice chest is got europium rubidium marking antibody (PT66, purchased from PerkinElmer company, concentration is 0.625 μm of ol/L) solution 8.29 μ L 1 × detection damping fluid 199.71 μ L dilution, mix to obtain antibody working fluid.
The preparation of solution to be measured: get testing compound respectively, precision weighing, dissolving with biochemical level dimethyl sulfoxide (DMSO) (DMSO) and be mixed with concentration is 2 × 10 4the storing solution of μm ol/L ,-20 DEG C of preservations; First 1 × kinase buffer liquid the dilution of this storing solution is become the solution that concentration is 600,180,60,20,6.7,2.2,0.74,0.25,0.082,0.027,0 μMs, then with kinase buffer liquid be diluted to concentration be 3000,900,300,100,33.3,11.1,3.70,1.23,0.40,0.10, the solution to be measured (final concentration of note: DMSO is not higher than 0.5%) of 0nM.
Solution to be measured is added respectively in 384 orifice plates, the each 2.5 μ l/ holes of EGFR kinases working fluid, centrifugal, add substrate working fluid 2.5 μ l/ hole, centrifugal, add ATP working fluid 2.5 μ l/ hole, centrifugal, 120min is hatched under being placed in 23 DEG C of conditions, add stop bath 5 μ l/ hole, hatch 5min for 23 DEG C, add antibody (PT66) working fluid 5 μ L/ hole, centrifugal, 1h is hatched under 23 DEG C of conditions, record the numerical value of every hole at 615nm and 665nm fluorescence (excitation wavelength 330nm/340nm), and both ratio (ratio value), curve is made by ratio value and concentration, calculate IC 50value.
Table 1. compound is to EGFR tyrosine-kinase enzyme inhibition activity determination data
Experimental result shows, the part of compounds in table 1 has certain inhibit activities to EGFR Tyrosylprotein kinase.Wherein, the EGFR tyrosine-kinase enzyme inhibition activity of 7a, 7b, 7c, 7e, 8b, 12a and 12b is lower than 3 μMs.Experiment shows that N-substituted salicylamide compounds of the present invention has EGFR kinase inhibiting activity.

Claims (2)

1.N-substituted salicylamide compounds, is characterized in that described compound is n-(3-chloro-phenyl-)-2-hydroxyl-4-(2-methoxyethoxy) benzamide (8b).
2. compound described in claim 1 is for the preparation of the purposes of EGFR tyrosine kinase inhibitor medicine.
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