CN104262245B - A kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor and its preparation method and application - Google Patents
A kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor and its preparation method and application, the structural formula of this inhibitor isWherein R1For acetyl groups, Mesylation group or pivaloyl group, R2For monosubstituted base or disubstituted, substituent group is alkyl, halogen.ABL1 kinases is had certain inhibitory action by this inhibitor series in vitro, and can suppress the propagation of tumor cell K562, can be used for the preparation of antitumor drug, especially CML (chronic myelocytic leukemia) medicine.The preparation method of 6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention, has raw material and is easy to get, and reaction condition is gentle, and course of reaction is simple to operate, the advantage that agents useful for same is cheap.
Description
Technical field
The invention belongs to biomedicine technical field, relate to a kind of antineoplastic compound, particularly to a kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor and its preparation method and application.
Background technology
Chronic granulocytic leukemia (CML) is a kind of blood system sex clone proliferative disease betiding hematopoietic stem cell, is also modal a kind of leukemia.In western countries, CML accounts for the 15-20% of adult leukemia, all can fall ill at each age group, with person in middle and old age's case for common.CML is by t (9；22) (q34；Q11) the Bcr-Abl albumen of breakaway poing Cu Ji district-abelson leukemia virus (BCR-ABL) fusion gene overexpression that chromosome translocation is formed causes.At present, occur in that multiple is the micromolecular inhibitor of target for BCR-ABL, but all there is drug resistance problems, therefore Bcr-Abl mutant drug-resistant is a great problem of this research field, therewith one of the research and development of novel B cr-Abl inhibitor just focus becoming pharmaceutical field.
Summary of the invention
Present invention solves the problem in that a kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor of offer and its preparation method and application, it is possible to be applied to the preparation of antitumor drug.
For reaching above-mentioned purpose, the present invention is achieved through the following technical solutions:
A kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor, is structured with formula:
Wherein, R1For acylate group, R2For monosubstituted base or disubstituted, substituent group is alkyl or halogen.
Described R1For acetyl groups, Mesylation group or pivaloyl group.
Described R2During for monosubstituted base, substituent group is positioned on phenyl ring between carboxyl position or para-position；
Described R2For time disubstituted, two substituent group differences, the position of substitution is adjacent.
The preparation method of a kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor, comprises the following steps:
5) 2-amino-6-bromopyridine and different acyl chloride reactions obtain different N-(6-bromopyridine-2-base) amide；
6) different N-(6-bromopyridine-2-base) amide and Carboxybenzeneboronic acid is obtained 4-[6-(amide) pyridine-2-base] benzoic acid by SUZUKI coupling reaction；
7) preparation of the benzoyl piperazine containing substituent group: benzoic acid and pivaloyl chloride reaction containing different substituents generate mixed acid anhydride intermediate, then are obtained by reacting the benzoyl piperazine containing different substituents with piperazine；
8) different 4-[6-(amide) pyridine-2-base] benzoic acid obtains 6-phenylpyridine-2-amine Bcr-Abl inhibitor with the benzoyl piperazine containing different substituents by mixed acid anhydride method.
Described step 1) concrete operations be: 2-amino-6-bromopyridine is dissolved in anhydrous methylene chloride, add anhydrous triethylamine, ice bath stir 30 minutes, then under condition of ice bath, drip the anhydrous methylene chloride solution of acyl chlorides, after dripping, remove ice bath, rise to room temperature reaction overnight；After reaction terminates, with dchloromethane, washing, then dry with anhydrous sodium sulfate, then through pillar layer separation, obtain N-(6-bromopyridine-2-base) amide；
Described step 2) concrete operations be: by N-(6-bromopyridine-2-base) amide, Carboxybenzeneboronic acid, tetra-triphenylphosphine palladium and cesium carbonate are dissolved in the mixed solution of acetonitrile and water, it is warming up to 90 DEG C to react 48 hours, react rear sucking filtration, filtrate be cooled to room temperature after with salt acid for adjusting pH value to precipitating out solid, filter, obtain 4-[6-(amide) pyridine-2-base] benzoic acid.
Described step 3) concrete operations be: being dissolved in anhydrous methylene chloride by the benzoic acid containing different substituents, add triethylamine and pivaloyl chloride, at room temperature stir to clarify, then reaction 4 hours, obtain mixed acid anhydride intermediate；After having reacted, the ethanol solution of disposable addition piperazine, at room temperature reacts overnight, after reaction terminates, add concentrated hydrochloric acid, then with dichloromethane extraction, discarding dichloromethane phase, aqueous phase sodium hydroxide is adjusted to alkalescence, it is extracted with ethyl acetate again, merge organic facies, by the dry organic facies of anhydrous sodium sulfate, obtain benzoyl piperazine containing different substituents；
Described step 4) concrete operations be: 4-[6-(amide) pyridine-2-base] benzoic acid and 4-methyl morpholine are added sequentially in anhydrous methylene chloride, under condition of ice bath, the dichloromethane solution of dropping isobutyl chlorocarbonate, reacts 30～40min at 0 DEG C；After having reacted, under condition of ice bath, drip the dichloromethane solution of the benzoyl piperazine containing different substituents and 4-methyl morpholine, after dripping, remove ice bath, rise to room temperature reaction overnight, after reaction terminates, add dchloromethane, washing, then organic facies anhydrous slufuric acid ammonium dries, and then through column chromatography separating purification, obtains 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
The described preparation process to Carboxybenzeneboronic acid is: parabromotoluene prepares grignard reagent by grignard reaction, grignard reagent and methyl borate. are obtained by reacting methylphenylboronic acid ester, methylphenylboronic acid Ester hydrolysis is obtained methylphenylboronic acid, methylphenylboronic acid oxidation is obtained Carboxybenzeneboronic acid.
To the preparation process of Carboxybenzeneboronic acid particularly as follows: Mg bar and iodine are added in reaction bulb, with nitrogen protection, add the anhydrous THF solution to first bromobenzene in a heated condition, carry out back flow reaction, room temperature it is cooled to after having reacted, reaction unit is transferred in low-temp reaction instrument, add the anhydrous THF solution of methyl borate., room temperature reaction, add HCl solution afterwards to be hydrolyzed, after reacting completely, rotation is except THF, aqueous phase is extracted with ethyl acetate, merge organic facies, by the dry organic facies of anhydrous sodium sulfate, sucking filtration, filtrate rotation is except solvent, must to methylphenylboronic acid with dehydrated alcohol recrystallization；To methylphenylboronic acid be dissolved in the NaOH solution of 1mol/L under condition of ice bath, be added drop-wise to KMnO afterwards4, tetrabutyl ammonium bromide aqueous solution in, after dripping, remove ice bath, rise to room temperature reaction overnight, add a small amount of ethanol cancellation, then sucking filtration, filtrate regulates pH value to precipitating out solid with concentrated hydrochloric acid, obtains Carboxybenzeneboronic acid.
The application in preparing Bcr-Abl inhibitor medicaments of the described 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
The application in preparing antitumor drug of the described 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
Described antitumor drug is for treating leukemic medicine.
Compared with prior art, the method have the advantages that
6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention, is the novel compound with anti-Bcr-Abl kinase activity, in vitro Bcr-Abl kinases is had good inhibitory activity.Bcr-Abl kinases plays a significant role in cell signalling and conversion, and it is by phosphorylation and activates a series of stream substrates, promotes the CML unlimited hypertrophy of maturation granulocyte.Bcr-Abl does not express in normal cell, is the ideal targets for the treatment of CML.Bcr-Abl inhibitor can reach the purpose for the treatment of CML by suppressing Bcr-Abl kinase activity.Test result indicate that the 6-phenylpyridine-2-amine Bcr-Abl inhibitor prepared by the present invention can effectively suppress the activity of Bcr-Abl and suppress growth and the propagation of tumor cell, illustrate that prepared 6-phenylpyridine-2-amine Bcr-Abl inhibitor can be used for preparing Bcr-Abl inhibitor medicaments and antitumor drug.
The preparation method of 6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention, has raw material and is easy to get, and reaction condition is gentle, and course of reaction is simple to operate, the advantage that agents useful for same is cheap.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of 6-phenylpyridine-2-amine Bcr-Abl inhibitor；
Wherein compound 1 is parabromotoluene, and compound 2 is the grignard reagent of parabromotoluene, and compound 3 is to methylphenylboronic acid；Compound is 4 is to Carboxybenzeneboronic acid, compound 5 is 2-amino-6-bromopyridine, compound 6 is different N-(6-bromopyridine-2-base) amide, compound 7 is 4-[6-(amide) pyridine-2-base] benzoic acid, compound 8 is the benzoic acid of different substituents, compound 9 is the benzoic acid pivalic acid acid anhydride intermediate of different substituents, compound 10 is the benzoyl piperazine of different substituents, and compound L P1-LP9 is target compound and 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
In figure mark particularly as follows:
(a).Mg,I2, THF, backflow, N2；(b) B (OMe)3, THF ,-20 DEG C；(c) .NaOH (1M), KMnO4,TBAB,H2O；E, triethylamine, DCM, 0 DEG C → r.t. of (d) acetylchlorideormethanesulfonylchlorideorpivaloylchlorid；(e) Pd (Pph3)4,Cs2CO3,CH3CN/H2O (V:V=3:2), 90 DEG C；(f) pivaloylchloride, TEA, DCM, r.t.30min；(g) ethanol, r.t.；(h) ClCOO-iBu, NMM, DCM, 0 DEG C；Mono-acylatedpiperazine, NMM, DCM, 0 DEG C → r.t.
Detailed description of the invention
The present invention provides a kind of 6-phenylpyridine-2-amine Bcr-Abl inhibitor having anti-tumor activity, and this kind of inhibitor embodies Bcr-Abl kinase inhibiting activity in vitro, it is possible to be applied to the preparation of antitumor drug.
Below in conjunction with drawings and Examples, the present invention being described in detail, the explanation of the invention is not limited.
6-phenylpyridine-2-amine Bcr-Abl the inhibitor with anti-tumor activity provided by the invention, its chemical structural formula is:
Wherein, R1For acylate group, R2For monosubstituted base or disubstituted, substituent group is alkyl or halogen.
Further, described R1For acetyl groups, Mesylation group or pivaloyl group.
Described R2During for monosubstituted base, substituent group is positioned on phenyl ring between carboxyl position or para-position；
Described R2For time disubstituted, two substituent group differences, the position of substitution is adjacent.
Preparation method and the screening active ingredients of 6-phenylpyridine-2-amine Bcr-Abl inhibitor is described in detail below in conjunction with the synthetic route shown in Fig. 1 and concrete synthesis example.
Embodiment 1
In the structural formula of this inhibitor, R1For acetyl groups, R2For m-trifluoromethyl, prepared by following steps:
1) preparation to Carboxybenzeneboronic acid (compound 4)
The Mg bar (2.15g, 90mmol) that addition processed in the two-neck bottle of 250ml, 2 iodine, nitrogen protection, evacuation 3 times.It is slowly added to compound to first bromobenzene (compound 1 with syringe under heating condition, anhydrous THF solution 60mmol), reaction is in reflux state after causing, continuously add surplus solution, add rear back flow reaction 5 hours, obtain the grignard reagent (compound 2) of parabromotoluene, after being cooled to room temperature, reaction unit is transferred in low-temp reaction instrument, temperature regulates to-20 DEG C, methyl borate. (9.36g is added with syringe after 5 minutes, anhydrous THF solution 90mmol), add rear room temperature reaction 3 hours, add the HCl solution of 100ml2mol/L afterwards to be hydrolyzed reaction, TLC detects reaction.After reacting completely, decompression rotation is except THF, and aqueous phase is extracted with ethyl acetate 3 times, merges organic facies, with water, saturated common salt is washed to neutrality, and anhydrous sodium sulfate dries, sucking filtration, filtrate decompression rotation is except solvent, and dehydrated alcohol recrystallization must to methylphenylboronic acid (compound 3) 4.66g, productivity 57%.
Methylphenylboronic acid (compound 3,4.66g, 34.26mmol) will be dissolved under condition of ice bath the NaOH solution (103ml) of 1mol/L.After to be dissolved, under condition of ice bath, above-mentioned solution is added drop-wise to KMnO4In water (340ml) solution of (16.24g, 102.79mmol), tetrabutyl ammonium bromide (0.34g).After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, adding a small amount of ethanol cancellation, sucking filtration, filtrate is adjusted to pH=2 with concentrated hydrochloric acid, precipitates out solid, obtains Carboxybenzeneboronic acid (compound 4) 5.69g.
2) preparation of N-(6-bromopyridine-2-base) acetamide (compound 6)
Being dissolved in 100ml anhydrous methylene chloride by 2-amino-6-bromopyridine (compound 5,5.19g, 30mmol), add 20ml anhydrous triethylamine, ice bath stirs 30 minutes.Anhydrous methylene chloride (40ml) solution of chloroacetic chloride (60mmol, 4.24ml) is dripped under condition of ice bath.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, with dchloromethane, washing (30ml × 3), sodium bicarbonate solution is washed (30ml × 3), and saturated nacl aqueous solution washes (30ml), then dries with anhydrous sodium sulfate.Then through pillar layer separation (petroleum ether: ethyl acetate=1:1), obtain product 5.64g, productivity 88%.
3) N-(6-bromopyridine-2-base) acetamide (compound 6) and Carboxybenzeneboronic acid (compound 4) is obtained 4-[6-(acetylamino) pyridine-2-base] benzoic acid (compound 7) by SUZUKI coupling reaction
In 250ml reaction bulb, add Carboxybenzeneboronic acid (compound 4,2.49g, 15mmol), N-(6-bromopyridine-2-base) acetamide (compound 6,3.22g, 15mmol), tetra-triphenylphosphine palladium (0.9g, 0.75mmol), Carbon Dioxide caesium (19.5g, 60mmol), adds acetonitrile/water (V:V=3:2) 160ml afterwards.N2Protection, oil bath rises to 90 DEG C of reaction 48h.After reaction terminates, sucking filtration while hot, filtrate is adjusted to pH=4 with 6mol/L hydrochloric acid, precipitates out solid, and sucking filtration, filter cake is 4-[6-(acetylamino) pyridine-2-base] benzoic acid (compound 7) 3.76g, productivity 78%.
4) m-trifluoromethylbenzoic acid (compound 8) and piperazine obtain 1-[3-(trifluoromethyl) benzoyl] piperazine (compound 10) by mixed anhydride method
By m-trifluoromethylbenzoic acid (20mmol, 3.80g) it is dissolved in 80ml anhydrous methylene chloride, add triethylamine (4.6ml, 30mmol) with pivaloyl chloride (2.42ml, 20mmol), it is stirred at room temperature to clarification, reacts 4 hours, obtain mixed acid anhydride intermediate (compound 9).
After having reacted, by the disposable addition of dehydrated alcohol (80ml) solution of piperazine (3.44g, 40mmol), room temperature reaction is overnight.Reaction adds concentrated hydrochloric acid 4ml after terminating, and extracts with dichloromethane (30ml × 2), discards dichloromethane phase.It is alkalescence that aqueous phase sodium hydroxide 8g is adjusted to pH; being extracted with ethyl acetate (50ml × 3), merge organic facies, organic facies anhydrous sodium sulfate dries; obtain 1-[3-(trifluoromethyl) benzoyl] piperazine (compound 10) 4.13g, productivity 80%.
5) 4-[6-(acetylamino) pyridine-2-base] benzoic acid (compound 7) and 1-[3-(trifluoromethyl) benzoyl] piperazine (compound 10) obtain target compound LP1 by mixed anhydride method
In 100ml reaction bulb, 4-[6-(acetylamino) pyridine-2-base] benzoic acid (compound 7,1.02g, 4.0mmol), 4-methyl morpholine (1.3ml, 12mmol) are added sequentially in 15ml anhydrous methylene chloride.Under condition of ice bath, dichloromethane (8ml) solution of isobutyl chlorocarbonate (0.8ml, 6mmol) is slowly added dropwise in above-mentioned solution, reacts 30 minutes at 0 DEG C.After having reacted; under condition of ice bath; the dichloromethane solution (10ml) of 1-[3-(trifluoromethyl) benzoyl] (compound 10,6mmol) and 4-methyl morpholine (1.3ml, 12mmol) is joined in above-mentioned reaction system.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, adding dichloromethane (20ml) dilution, wash (10mL × 2), saturated sodium carbonate solution is washed (10mL × 3), and saturated nacl aqueous solution washes (10mL).Organic facies anhydrous slufuric acid ammonium dries, and then through column chromatography separating purification (petroleum ether: ethyl acetate=1:3), obtains target compound LP10.53g, productivity 27%.
The structure of gained compound is as follows:
Its physicochemical property is: Mp214-216 DEG C, EI-MS (m/z): 496 ([M]+)。
Hydrogen spectrum nuclear magnetic resonance data:1HNMR(400MHz,DMSO-d6): δ=8.16 (d, J=8.1Hz, 2H), 8.06 (d, J=8.1Hz, 1H), 7.87 (dd, J=15.7,7.6Hz, 2H), 7.80 (s, 1H), 7.76 (d, J=7.4Hz, 1H), 7.71 (d, J=7.3Hz, 2H), 7.56 (d, J=7.1Hz, 2H), 3.37-3.71 (m, 8H), 2.14 (s, 3H).
Embodiment 2
In the structural formula of this inhibitor, R1For Mesylation group, R2For the chloro-3-trifluoromethyl of 4-, prepared by following steps:
Step 1) with embodiment 1 in step 1) identical, namely by parabromotoluene (compound 1) prepare to Carboxybenzeneboronic acid (compound 4).
2) preparation of N-(6-bromopyridine-2-base) Methanesulfomide (compound 6)
Being dissolved in 100ml anhydrous methylene chloride by 2-amino-6-bromopyridine (compound 5,5.19g, 30mmol), add 20ml anhydrous triethylamine, ice bath stirs 30 minutes.Anhydrous methylene chloride (40ml) solution of mesyl chloride (60mmol, 4.64ml) is dripped under condition of ice bath.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, with dchloromethane, washing (30ml × 3), sodium bicarbonate solution is washed (30ml × 3), and saturated nacl aqueous solution washes (30ml), and anhydrous sodium sulfate dries.Then through pillar layer separation (petroleum ether: ethyl acetate=1:7), obtain product 6.40g, productivity 85%.
3) N-(6-bromopyridine-2-base) Methanesulfomide (compound 6) and Carboxybenzeneboronic acid (compound 4) is obtained 4-{6-[(methyl sulphonyl) amino] pyridine-2-base by SUZUKI coupling reaction benzoic acid (compound 7)
In 250ml reaction bulb, add Carboxybenzeneboronic acid (compound 4,2.49g, 15mmol), N-(6-bromopyridine-2-base) Methanesulfomide (compound 6,3.76g, 15mmol), tetra-triphenylphosphine palladium (0.9g, 0.75mmol), Carbon Dioxide caesium (19.5g, 60mmol), adds acetonitrile/water (V:V=3:2) 160ml afterwards.N2Protection, oil bath rises to 90 DEG C of reaction 48h.After reaction terminates, sucking filtration while hot, filtrate is adjusted to pH=4 with 6mol/L hydrochloric acid, precipitates out solid, sucking filtration, and filter cake is 4-{6-[(methyl sulphonyl) amino] pyridine-2-base } and benzoic acid (compound 7) 2.76g, productivity 63%.
4) 4-chloro-3-(Trifluoromethyl)benzoic acid. (compound 8) and piperazine obtain 1-[4-chloro-3-(trifluoromethyl) benzoyl] piperazine (compound 10) by mixed anhydride method
By chloro-for 4-3-(Trifluoromethyl)benzoic acid. (20mmol, 4.49g) it is dissolved in 80ml anhydrous methylene chloride, add triethylamine (4.6ml, 30mmol) with pivaloyl chloride (2.42ml, 20mmol), it is stirred at room temperature to clarification, reacts 4 hours, obtain mixed acid anhydride intermediate (compound 9).
After having reacted, by the disposable addition of dehydrated alcohol (80ml) solution of piperazine (3.44g, 40mmol), room temperature reaction is overnight.Reaction adds concentrated hydrochloric acid 4ml after terminating, and extracts with dichloromethane (30ml × 2), discards dichloromethane phase.It is alkalescence that aqueous phase sodium hydroxide 8g is adjusted to pH; being extracted with ethyl acetate (50ml × 3), merge organic facies, organic facies anhydrous sodium sulfate dries; obtain 1-[4-chloro-3-(trifluoromethyl) benzoyl] piperazine (compound 10) 4.68g, productivity 80%.
5) 4-{6-[(methyl sulphonyl) amino] pyridine-2-base } benzoic acid (compound 7) and 1-[4-chloro-3-(trifluoromethyl) benzoyl] piperazine (compound 10) obtain target compound LP5 by mixed anhydride method
In 100ml reaction bulb; by 4-{6-[(methyl sulphonyl) amino] pyridine-2-base } benzoic acid (compound 7; 1.17g, 4.0mmol), 4-methyl morpholine (1.3ml, 12mmol) is added sequentially in 15ml anhydrous methylene chloride.Under condition of ice bath, dichloromethane (8ml) solution of isobutyl chlorocarbonate (0.8ml, 6mmol) is slowly added dropwise in above-mentioned solution, reacts 40 minutes at 0 DEG C.
After having reacted; under condition of ice bath; the dichloromethane solution (10ml) of 1-[4-chloro-3-(trifluoromethyl) benzoyl] piperazine (compound 10,6mmol) and 4-methyl morpholine (1.3ml, 12mmol) is joined in above-mentioned reaction system.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, adding dichloromethane (20ml) dilution, wash (10mL × 2), saturated sodium carbonate solution is washed (10mL × 3), and saturated nacl aqueous solution washes (10mL).Organic facies anhydrous slufuric acid ammonium dries, and then through column chromatography separating purification (petroleum ether: ethyl acetate=1:7), obtains target compound LP50.36g, productivity 16%.
The structure of gained compound is as follows:
Its physicochemical property is: Mp120-122 DEG C, EI-MS (m/z): 566 ([M]+)。
Hydrogen spectrum nuclear magnetic resonance data:1HNMR(400MHz,DMSO-d6): δ=8.13 (d, J=8.0Hz, 2H), 7.91 (s, 1H), 7.84 (d, J=7.9Hz, 2H), 7.78 (s, 1H), 7.68 (d, J=7.6Hz, 1H), 7.56 (d, J=7.6Hz, 2H), 6.92 (d, J=8.1Hz, 1H), 3.44 (s, 3H), 3.79 3.39 (m, 8H).
Embodiment 3
In the structural formula of this inhibitor, R1For pivaloyl group, R2For to the tert-butyl group, being prepared by following steps:
Step 1) with embodiment 1 in step 1) identical, namely by parabromotoluene (compound 1) prepare to Carboxybenzeneboronic acid (compound 4).
2) preparation of N-(6-bromopyridine-2-base)-2,2-dimethylpropionamide (compound 6)
Being dissolved in 100ml anhydrous methylene chloride by 2-amino-6-bromopyridine (compound 5,5.19g, 30mmol), add 20ml anhydrous triethylamine, ice bath stirs 30 minutes.Anhydrous methylene chloride (40ml) solution of pivaloyl chloride (60mmol, 7.39ml) is dripped under condition of ice bath.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, with dchloromethane, washing (30ml × 3), sodium bicarbonate solution is washed (30ml × 3), and saturated nacl aqueous solution washes (30ml), then dries with anhydrous sodium sulfate.Then through pillar layer separation (petroleum ether), obtain product 6.93g, productivity 90%.
3) N-(6-bromopyridine-2-base)-2; 2-dimethylpropionamide (compound 6) and Carboxybenzeneboronic acid (compound 4) is obtained 4-{6-[(2,2-Dimethylpropanoyl) amino] pyridine-2-base by SUZUKI coupling reaction benzoic acid (compound 7)
In 250ml reaction bulb, add Carboxybenzeneboronic acid (compound 4,2.49g, 15mmol), N-(6-bromopyridine-2-base)-2,2-dimethylpropionamide (compounds 6,3.85g, 15mmol), tetra-triphenylphosphine palladium (0.9g, 0.75mmol), Carbon Dioxide caesium (19.5g, 60mmol), acetonitrile/water (V:V=3:2) 160ml is added afterwards.N2Protection, oil bath rises to 90 DEG C of reaction 48h.After reaction terminates, sucking filtration while hot, filtrate is adjusted to pH=4 with 6mol/L hydrochloric acid, precipitates out solid, sucking filtration, and filter cake is 4-{6-[(2,2-Dimethylpropanoyl) amino] pyridine-2-base } and benzoic acid (compound 7) 3.49g, productivity 78%.
4) p-tert-butyl benzoic acid (compound 8) and piperazine obtain 1-(4-tert-butyl-benzoyl) piperazine (compound 10) by mixed anhydride method
By p-tert-butyl benzoic acid (20mmol, 3.56g) it is dissolved in 80ml anhydrous methylene chloride, add triethylamine (4.6ml, 30mmol) with pivaloyl chloride (2.42ml, 20mmol), it is stirred at room temperature to clarification, reacts 4 hours, obtain mixed acid anhydride intermediate (compound 9).
After having reacted, by the disposable addition of dehydrated alcohol (80ml) solution of piperazine (3.44g, 40mmol), room temperature reaction is overnight.Reaction adds concentrated hydrochloric acid 4ml after terminating, and extracts with dichloromethane (30ml × 2), discards dichloromethane phase.It is alkalescence that aqueous phase sodium hydroxide 8g is adjusted to pH; being extracted with ethyl acetate (50ml × 3), merge organic facies, organic facies anhydrous sodium sulfate dries; obtain 1-(4-tert-butyl-benzoyl) piperazine (compound 10) 4.09g, productivity 83%.
5) 4-{6-[(2,2-Dimethylpropanoyl) amino] pyridine-2-base } benzoic acid (compound 7) and 1-(4-tert-butyl-benzoyl) piperazine (compound 10) obtain target compound LP9 by mixed anhydride method
In 100ml reaction bulb; by 4-{6-[(2,2-Dimethylpropanoyl) amino] pyridine-2-base } benzoic acid (compound 7,1.19g; 4.0mmol), 4-methyl morpholine (1.3ml, 12mmol) is added sequentially in 15ml anhydrous methylene chloride.Under condition of ice bath, dichloromethane (8ml) solution of isobutyl chlorocarbonate (0.8ml, 6mmol) is slowly added dropwise in above-mentioned solution, reacts 35 minutes at 0 DEG C.
After having reacted; under condition of ice bath; the dichloromethane solution (10ml) of 1-(4-tert-butyl-benzoyl) piperazine (compound 10,6mmol) and 4-methyl morpholine (1.3ml, 12mmol) is joined in above-mentioned reaction system.After dripping, remove ice bath, rise to room temperature reaction overnight.After reaction terminates, adding dichloromethane (20ml) dilution, wash (10mL × 2), saturated sodium carbonate solution is washed (10mL × 3), and saturated nacl aqueous solution washes (10mL).Organic facies anhydrous slufuric acid ammonium dries, and then through column chromatography separating purification (petroleum ether: ethyl acetate=1:1), obtains target compound LP90.59g, productivity 28%.
The structure of gained compound is as follows:
Its physicochemical property is: Mp237-240 DEG C, EI-MS (m/z): 526 ([M]+)。
Hydrogen spectrum nuclear magnetic resonance data:1HNMR(400MHz,DMSO-d6): δ=8.20 (d, J=7.9Hz, 2H), 8.03 (d, J=8.2Hz, 1H), 7.89 (t, J=8.0Hz, 1H), 7.73 (d, J=7.6Hz, 1H), 7.55 (d, J=8.1Hz, 2H), 7.48 (d, J=7.8Hz, 2H), 7.38 (d, J=8.2Hz, 2H), 3.78 3.42 (m, 8H), 1.30 (s, 9H), 1.28 (s, 9H).
Measure the 6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention inhibitory activity to Bcr-Abl tyrosine kinase below.
Assay method is specific as follows:
Kinases ABL1 and substrate A bltide, purchased from Signal-Chem company, selects the ADP-Glob of Promega companyTMThe Inhibiting enzyme activity of KinaseAssays detection kit detection target compound, operational approach illustrates to carry out according to test kit.
By ATP (1mM) buffer (2 ×) (Tris80mM, MgCl220mM, BSA0.2mg/mL, DTT2mM) dilute 80 times of buffer (2 ×) solution being configured to ATP (125 μMs)；The mixed solution that the ATP solution of 125 μMs and Abltide liquor capacity 1:1 are hybridly prepared into ATP (62.5 μMs)-Abltide (0.5 μ g/ μ l) is standby；ABL1 kinase solution buffer (1 ×) (Tris40mM, MgCl210mM, BSA0.1mg/mL, DTT1mM) dilute 100 times of buffer (1 ×) solution for standby being configured to ABL1 (10ng/ μ l)；
Target compound (LP1～LP9) and positive control drug (Imatinib) buffer (1 ×) are configured to 1.5 × 10 respectively-5Mol/L, 1.5 × 10-6Mol/L, 1.5 × 10-7Mol/L, 1.5 × 10-8, 1.5 × 10-9, 1.5 × 10-10The sample solution of mol/L Concentraton gradient, on 384 orifice plates, every hole is sequentially added into the mixed solution of 2 μ LATP-Abltide, 1 μ L sample solution, 2 μ L enzymatic solution；Blank well adds the mixed solution of 3 μ L buffer and 2 μ LATP-Abltide；Control wells adds the mixed solution of 2 μ LATP-Abltide, 1 μ L buffer, and 2 μ L enzymatic solution finish, hatch 60min at 30 DEG C；Add ADP-Glo reagent 5 μ L, at 25 DEG C, hatch 40min；Add Kinasedetection reagent, then hatch 30min at 25 DEG C.The chemiluminescence module adopting the multi-functional microplate reader of PerkinElmer measures the luminous value in every hole, calculates inhibitor to the suppression ratio of ABL1 and IC50。
The structural formula of 6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention is:
6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention is to Bcr-Abl tyrosine-kinase enzyme inhibition activity result, as shown in table 1.
The table 1 inhibitor inhibitory activity result (IC to Bcr-Abl tyrosine kinase50)
Bcr-Abl kinases is had stronger inhibitory activity by result display 6-phenylpyridine-2-amine Bcr-Abl inhibitor, Bcr-Abl kinases plays a significant role in cell signalling and conversion, it is by phosphorylation and activates a series of stream substrates, promote the CML unlimited hypertrophy of maturation granulocyte, Bcr-Abl does not express in normal cell, is the ideal targets for the treatment of CML.Bcr-Abl inhibitor can reach the purpose for the treatment of CML by suppressing Bcr-Abl kinase activity.
Measure the 6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention growth inhibitory activity to tumor cell below.
Adopt the mtt assay inspection 6-phenylpyridine-2-amine Bcr-Abl inhibitor growth inhibitory activity to tumor cell:
6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention has antitumor action, tumor cell is had vitro inhibition proliferation activity, human leukemia cell's (K562 cell) has the proliferation activity suppressing tumor cell, it is possible to for leukemic treatment.
Take the logarithm human leukemia cell's (K562 cell) of trophophase, is diluted to 10 by RPMI1640 culture medium4The cell solution of individual/mL, parallel is inoculated in 96 well culture plates (2000/hole), and every hole inoculation volume is 200 μ L, 37 DEG C, 5%CO2Incubator is cultivated 12h；
Every hole adds the testing compound 20 μ L of variable concentrations, makes the final concentration of of compound in hole: 1.5 × 10-7Mol/L, 1.5 × 10-6Mol/L, 1.5 × 10-5Mol/L, 1.5 × 10-4Mol/L, each concentration sets 3 multiple holes, and negative control adds cell and is not added with compound, if 6 multiple holes, nilotinib is positive control, continues to cultivate 48h；
Every hole adds MTT (5mg/mL) 10 μ L, make the final concentration 0.5mg/mL of MTT in hole, 37 DEG C of incubators hatch 4h, careful suction abandons supernatant, every hole adds DMSO150 μ L, and vibrate 15min, and enzyme-linked immunosorbent assay instrument measures the ultraviolet absorption value (OD value) at 490nm place, each hole, then calculate cell inhibitory rate, and obtain IC50 value according to suppression ratio；
The computing formula of cell inhibitory rate is:
Suppression ratio %=(control wells mean OD value-medication group mean OD value)/control wells mean OD value × 100%
Testing result shows: compared with negative control group, and above-mentioned tumor cell is had In-vitro Inhibitory Effect in various degree by 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
6-phenylpyridine-2-amine Bcr-Abl inhibitor provided by the invention is as shown in table 2 to the suppression result of tumor cell.
Table inhibitor 2 inhibitory activity result IC50 (μM) to K562
Result display 6-phenylpyridine-2-amine Bcr-Abl inhibitor prepared by the present invention can suppress the growth of tumor cell, illustrate that 6-phenylpyridine-2-amine Bcr-Abl inhibitor is good to the proliferation inhibiting effect of tumor cell, can be applicable to the preparation of antitumor drug.
Claims (4)
1. the preparation method of a 6-phenylpyridine-2-amine Bcr-Abl inhibitor, it is characterised in that comprise the following steps:
1) 2-amino-6-bromopyridine and acyl chloride reaction obtain compound 6；
2) compound 6 and Carboxybenzeneboronic acid is obtained compound 7 by SUZUKI coupling reaction；
3) preparation of the benzoyl piperazine containing substituent group: compound 8 and pivaloyl chloride reaction generate mixed acid anhydride intermediate, then are obtained by reacting compound 10 with piperazine；
4) compound 7 and compound 10 obtain 6-phenylpyridine-2-amine Bcr-Abl inhibitor by mixed acid anhydride method；
Wherein, the structural formula of compound 6 is
The structural formula of compound 7 is
The structural formula of compound 8 is
The structural formula of compound 10 is
The structural formula of the 6-phenylpyridine-2-amine Bcr-Abl inhibitor prepared is
R1For
R2For 3-CF3、4-Cl-3-CF3Or 4-C (CH3)3。
2. the preparation method of 6-phenylpyridine-2-amine Bcr-Abl inhibitor according to claim 1, it is characterized in that, described step 1) concrete operations be: 2-amino-6-bromopyridine is dissolved in anhydrous methylene chloride, add anhydrous triethylamine, ice bath stirs 30 minutes, then drips the anhydrous methylene chloride solution of acyl chlorides under condition of ice bath, after dripping, remove ice bath, rise to room temperature reaction overnight；After reaction terminates, with dchloromethane, washing, then dry with anhydrous sodium sulfate, then through pillar layer separation, obtain compound 6；
Described step 2) concrete operations be: by compound 6, Carboxybenzeneboronic acid, tetra-triphenylphosphine palladium and cesium carbonate are dissolved in the mixed solution of acetonitrile and water, it is warming up to 90 DEG C to react 48 hours, react rear sucking filtration, filtrate be cooled to room temperature after with salt acid for adjusting pH value to precipitating out solid, filter, obtain compound 7.
3. the preparation method of 6-phenylpyridine-2-amine Bcr-Abl inhibitor according to claim 1, it is characterized in that, described step 3) concrete operations be: compound 8 is dissolved in anhydrous methylene chloride, add triethylamine and pivaloyl chloride, at room temperature stir to clarify, then reaction 4 hours, obtain mixed acid anhydride intermediate；After having reacted, the ethanol solution of disposable addition piperazine, at room temperature reacts overnight, after reaction terminates, add concentrated hydrochloric acid, then with dichloromethane extraction, discarding dichloromethane phase, aqueous phase sodium hydroxide is adjusted to alkalescence, it is extracted with ethyl acetate again, merge organic facies, by the dry organic facies of anhydrous sodium sulfate, obtain compound 10；
Described step 4) concrete operations be: compound 7 and 4-methyl morpholine are added sequentially in anhydrous methylene chloride, under condition of ice bath, dropping isobutyl chlorocarbonate dichloromethane solution, at 0 DEG C react 30～40min；After having reacted, under condition of ice bath, the dichloromethane solution of dropping compound 10 and 4-methyl morpholine, after dripping, remove ice bath, rise to room temperature reaction overnight, after reaction terminates, add dchloromethane, washing, then organic facies anhydrous slufuric acid ammonium dries, and then through column chromatography separating purification, obtains 6-phenylpyridine-2-amine Bcr-Abl inhibitor.
4. the preparation method of 6-phenylpyridine-2-amine Bcr-Abl inhibitor according to claim 1, it is characterized in that, the described preparation process to Carboxybenzeneboronic acid is: parabromotoluene prepares Grignard reagent by grignard reaction, Grignard reagent and methyl borate. are obtained by reacting methylphenylboronic acid ester, methylphenylboronic acid Ester hydrolysis is obtained methylphenylboronic acid, methylphenylboronic acid oxidation is obtained Carboxybenzeneboronic acid.
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