CN105418615A - Benzamide derivative, preparation and application - Google Patents

Benzamide derivative, preparation and application Download PDF

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CN105418615A
CN105418615A CN201510906290.0A CN201510906290A CN105418615A CN 105418615 A CN105418615 A CN 105418615A CN 201510906290 A CN201510906290 A CN 201510906290A CN 105418615 A CN105418615 A CN 105418615A
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imidazo
pyrazolyl
reaction
compound
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CN105418615B (en
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胡利明
吕永娟
吴加周
郭爽
卜明
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Beijing University of Technology
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Beijing University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention relates to a benzamide derivative expressed in the formula N-(3-(4-(3-imidazo[1,2-b]pyridazinyl)-1-pyraxolyl)-4-methyl phenyl), preparation and application and belongs to the field of medicinal chemistry. R1 represents -OCH3 or -F or -CF3 or -CH2CH3 or -H or one of other three substances which are shown in the specification, and R2 represents -CF3 or -F or -Cl or -H. Preparation comprises the steps that 2-methyl-5-nitrophenyl hydrazine is prepared, then 4-bromine-1-(2-methyl-5-nitrophenyl)pyrazol is obtained through a reaction, reduction is carried out, Boc anhydride is used for protection, a boric acid pinacol ester reaction is carried out, then coupling is carried out to generate N-Boc-3-4-(3-imidazo[1,2-b]pyridazinyl)-1-pyraxolyl)-4-methylaniline, finally, Boc is removed, and ammonolysis is carried out. The compound has the inhibiting effect on Bcr-Abl kinase.

Description

Benzamide derivatives and Synthesis and applications
Technical field
The invention belongs to medicinal chemistry art, relate to benzamide derivatives, preparations and applicatio, particularly relate to N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives and preparation method thereof and the application as Bcr-Abl tyrosine kinase inhibitor.
Background technology
Chronic myelocytic leukemia (ChronicMyelogenousLeukemia, CML) is pernicious marrow hemopoietic stem cells clonal expansion disease, and the patient of 95% has characteristic Ph karyomit(e) and molecular marker Bcr-Abl fusion gene thereof.Research proves, fusion gene Bcr-Abl is the molecular biological characteristics of chronic myelocytic leukemia, and its product B cr-Abl kinases is the key factor of morbidity.
The micromolecular inhibitor for Bcr-Abl Tyrosylprotein kinase (TKI) the most frequently used clinically comprising at present: first-generation medicine imatinib; S-generation medicine Dasatinib, AMN107 and Bosutinib; Third generation medicine Pu Na is for Buddhist nun.Imatinib (Imatinib) is the oral anti-chronic myelocytic leukemia small molecule tyrosine kinase inhibitors of Novartis Co., Ltd of Switzerland research and development.It has started the New Times taking kinases as target treatment disease, and Imatinib can be combined with the ATP site of Bcr-Abl kinase region, stops amino-acid residue phosphorylation, thus disabling signal pathway, antiproliferative effect, effectively can alleviate CML.After treatment, patient 5 annual survival rate can reach 90%, and it specificly can act on CML cancer cells, and does not almost injure normal cell, and toxic side effect reduces greatly.Along with the use of Imatinib, the sudden change of Bcr-Abl gene result in the appearance of resistance, reduces the curative effect of imatinib to a great extent.Dasatinib (Dasatinib) is a kind of for Bcr-Abl Tyrosylprotein kinase, Src kinase families (Src, Lck, Fyn), the oral kinase inhibitor of Mutiple Targets that all acts on of the multiple kinases such as c-Kit and PDGFR-B, to be researched and developed by Bristol Myers Squibb on June 28th, 2006 and obtain FDA and ratify listing.The same with Imatinib, combine with the ATP site of Bcr-Abl kinase region competitively, but the kinase whose activity of suppression Bcr-Abl is 300 times of Imatinib.Although the resistance that multiple Imatinib occurs can be overcome, invalid to Bcr-Abl.The derivative of a kind of novel aniline pyrimidine class Imatinib of AMN107 (Nilotinib), was approved listing by U.S. FDA on October 29th, 2007.It is stronger than Imatinib 20 times to the kinase whose avidity of Bcr-Abl, and to occurring that patient's (except T315I sudden change) of Imatinib resistance has active widely.The common untoward reaction such as stomach reaction, bone marrow depression, hyperbilirubinemia can be there is in the Most patients using Nilotinib to treat CML.For the resistance that Imatinib, Dasatinib and Nilotinib produce, Wyeth Pharmaceuticals of the U.S. have developed 4-substituted aniline-3-quinoline and adds formonitrile HCN class medicine Bosutinib (Bosutinib), be used for the treatment of CML, on September 4th, 2012 by FDA approval listing.Bosutinib can suppress the autonomous phosphorylation of Scr albumen in various human tumour cell; also the autonomous phosphorylation of Bcr-Abl albumen can be suppressed; and in the antiproliferative for KU812 and K562 cell (the CML cell containing Bcr-Abl) is tested; Bosutinib activity will apparently higher than Imatinib; Bosutinib oral administration biaavailability is higher in addition, but the patient accepting Bosutinib treatment often there will be bad of stomachache, diarrhoea, thrombopenia, heating and fatigue etc. to be answered.Pu Na is that one can act on Abl for Buddhist nun (Ponatinib), PDGFR α, the kinase whose oral Mutiple Targets inhibitor of VEGFR2, FGFR1 and Src.Due to the mechanism of action of its uniqueness, take and can suppress T315I sudden change at interior Bcr-Abl kinase activity.But take the patient of Ponatinib according to FDA latest report, there will be serious vascular problem, so expect to carry out safety evaluation to this medicine.
Imatinib and Ponatinib shows extraordinary curative effect in treatment CML process, but they are very expensive, and the patient taking Imatinib and Ponatinib according to statistics needs the expense of 92000 and 138000 dollars every year respectively.Therefore, the low cost alternative medicine of seeking Imatinib and Ponatinib becomes the task of top priority.
Summary of the invention
The object of this invention is to provide N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives and preparation method thereof and the application as Bcr-Abl tyrosine kinase inhibitor.
The invention provides N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives that formula (I) represents,
R 1expression-OCH 3,-F ,-CF 3,-CH 2cH 3,-H,
R 2expression-CF 3,-F ,-Cl or-H.
The compounds of this invention all has the restraining effect to Bcr-Abl Tyrosylprotein kinase, and wherein the inhibition of part of compounds is remarkable.
Present invention also offers the preparation method of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives, comprise the following steps:
A 2-methyl-5-nitro aniline dissolves in ethanol by (), add the concentrated hydrochloric acid of 37%, under above-mentioned system is placed in condition of ice bath, then the aqueous solution of Sodium Nitrite is added drop-wise in above-mentioned system, preferable reaction temperature 0 ~ 5 DEG C, in 0.5 ~ 1 hour reaction times, obtain the 2-methyl-5-nitro hydrazinobenzene hydrochloride salt that formula (II) represents;
B two hydrated stannous chlorides are dissolved in concentrated hydrochloric acid (mass percentage concentration 37%) by (), under being placed in condition of ice bath, then 2-methyl-5-nitro hydrazinobenzene hydrochloride salt is added drop-wise in above-mentioned system, preferred formula (II) compound is 1 ﹕ (1.5 ~ 2) with the amount of substance ratio of two hydrated stannous chlorides, temperature of reaction 0 ~ 10 DEG C, 1 ~ 2 hour reaction times, add NaOH solution (as concentration 1mol/L) neutralization, obtain the 2-methyl-5-nitro phenylhydrazine that formula (III) represents;
C formula (III) compound and 2-bromine mda react by () in ethanol, preferred formula (III) compound is 1 ﹕ (1.1 ~ 1.5) with the amount of substance ratio of 2-bromine mda, temperature of reaction 55 ~ 65 DEG C, in 2 ~ 4 hours reaction times, obtain the bromo-1-of 4-(2-methyl-5-nitrophenyl) pyrazoles that formula (IV) represents;
(d) by formula (IV) compound dissolution in ethanol, add the aqueous solution and the iron powder of ammonium chloride, preferable reaction temperature 80 ~ 90 DEG C, in 2 ~ 3 hours reaction times, obtains 3-(the bromo-1-pyrazolyl of the 4-)-4-monomethylaniline that formula (V) represents;
E formula (V) compound and Boc acid anhydrides react by () in tetrahydrofuran (THF), preferred formula V compound is 1 ﹕ (1.1 ~ 1.4) with the amount of substance ratio of Boc acid anhydrides, temperature of reaction 66 ~ 70 DEG C, in 2 ~ 3 hours reaction times, obtain N-Boc-3-(the bromo-1-pyrazolyl of the 4-)-4-monomethylaniline that formula (VI) represents;
F formula (VI) compound is dissolved in dioxane by (), add connection pinacol borate, Potassium ethanoate and two (triphenylphosphine) palladium chloride, preferred formula (VI) compound, connection pinacol borate, the amount of substance ratio of Potassium ethanoate and two (triphenylphosphine) palladium chloride is 1 ﹕ (1.1 ~ 1.5) ﹕ 3 ﹕ (0.1 ~ 0.2), temperature of reaction 100 ~ 110 DEG C, 15 ~ 18 hours reaction times, obtain the N-Boc-3-(4-(2-(4 that formula (VII) represents, 4, 5, 5-tetramethyl--1, 3, 2-dioxy boron penta cyclic group))-1-pyrazolyl)-4-monomethylaniline,
G () is by imidazo [1,2-b] AIBN of pyridazine, NBS and catalytic amount reacts in chloroform, preferred imidazo [1,2-b] pyridazine, NBS and AIBN amount of substance ratio be 1 ﹕ (1.5 ~ 2) ﹕ (0.1 ~ 0.2), temperature of reaction 65 DEG C, 2 ~ 3 hours reaction times obtained 3-bromine imidazo [1, the 2-b] pyridazine that formula (VIII) represents;
(h) by formula (VII) compound and formula (VIII) compound dissolution in the mixing solutions of dioxane and water, add four (triphenyl phosphorus) palladium and sodium carbonate, the amount of substance ratio of preferred formula (VII) compound, formula (VIII) compound, sodium carbonate and four (triphenyl phosphorus) palladium is 1 ﹕ 2 ﹕ 3 ﹕ (0.1 ~ 0.2), temperature of reaction 110 ~ 120 DEG C, 7 ~ 10 hours reaction times, obtain N-Boc-3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-monomethylaniline that formula (IX) is shown;
I formula (IX) and trifluoroacetic acid are reacted by () in methylene dichloride, 5 ~ 6 hours preferred reaction time, obtain 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-monomethylaniline that formula (X) represents;
J formula (X) compound, 4-chloromethyl benzoic acid chlorides and triethylamine react by () in methylene dichloride, the amount of substance ratio of preferred formula (X) compound, 4-chloromethyl benzoic acid chlorides and triethylamine is 1 ﹕ (2 ~ 3) ﹕ (0.2 ~ 0.5), 2 ~ 3 hours reaction times, obtain 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide that formula (XI) represents;
(K) formula (XI) compound, nucleophilic reagent and potassiumiodide are reacted in DMF, the amount of substance ratio of preferred formula (XI) compound, nucleophilic reagent and potassiumiodide is 1 ﹕ (2 ~ 3) ﹕ (0.1 ~ 0.2), temperature of reaction, 80 ~ 90 DEG C, 2 ~ 3 hours reaction times, obtain N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives that formula (I) represents;
R 1represent
Nucleophilic reagent is:
Or formula (X) compound, substituted benzoyl chloride and triethylamine react by (l) in methylene dichloride, the amount of substance ratio of preferred formula (X) compound, substituted benzoyl chloride and triethylamine is 1 ﹕ (2 ~ 3) ﹕ (0.2 ~ 0.5), 2 ~ 3 hours reaction times, obtain N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives that formula (I) represents;
R 1expression-OCH 3,-F ,-CF 3,-CH 2cH 3or-H, R 2expression-CF 3,-F ,-Cl or-H.
Substituted benzoyl chloride structural formula is:
The inventive method uses industrial common reagent and conventional working condition, and reaction conditions is gentle, and step is simple.
Chemical equation in above-mentioned building-up process is as follows:
Wherein, R 1expression-OCH 3,-F ,-CF 3,-CH 2cH 3or-H, represent further
r 2expression-CF 3,-F ,-Cl or-H.
The inventive method be first with 2-methyl-5-nitro aniline for raw material, 2-methyl-5-nitro phenylhydrazine is generated through diazotization and reduction reaction, then carry out condensation with 2-bromine mda and close ring, obtain the bromo-1-of 4-(2-methyl-5-nitrophenyl) Pyrazol intermediate, then through reduction, Boc acid anhydrides is protected, with the reaction of connection pinacol borate under palladium chtalyst, obtain N-Boc-3-(4-(2-(4, 4, 5, 5-tetramethyl--1, 3, 2-dioxy boron penta cyclic group))-1-pyrazolyl)-4-monomethylaniline, afterwards with 3-bromine imidazo [1, 2-b] pyridazine carries out palladium chtalyst coupling and generates N-Boc-3-(4-(3-imidazo [1, 2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline intermediate, this intermediate last is through de-Boc, ammonia solution in the basic conditions, production (I) compound.The compounds of this invention is inhibited to Bcr-Abl kinases.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, but the present invention is not limited to following examples.
Embodiment 1
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-3-trifluoromethyl benzamide
The preparation of (a) 2-methyl-5-nitro hydrazinobenzene hydrochloride salt
By raw material 2-methyl-5-nitro aniline (6.0g, 39.4mmol) be dissolved in three mouthfuls of round-bottomed flasks containing 250mL ethanol, add the concentrated hydrochloric acid of 10mL37%, under above-mentioned system is placed in condition of ice bath, again by Sodium Nitrite (2.8g, 40.6mmol) be dissolved in 15mL distilled water, with constant pressure funnel, it is slowly joined in above-mentioned reaction system, in dropping process, temperature controls at 0 ~ 5 DEG C, drip and finish, reaction system is continued react 0.5 ~ 1 hour under condition of ice bath, TLC detection reaction process, stopped reaction after raw material disappears, directly enters next step.
The preparation of (b) 2-methyl-5-nitro phenylhydrazine
The concentrated hydrochloric acid of 14mL37% is joined in there-necked flask, adding 20mL distilled water wherein, take two hydrated stannous chloride (18.0g, 79.6mmol) add in flask, under above-mentioned system is placed in condition of ice bath, again the 2-methyl-5-nitro hydrazinobenzene hydrochloride salt obtained in step (a) is slowly joined in above-mentioned reaction system by constant pressure funnel, in dropping process, temperature controls at 0 ~ 10 DEG C, drip and finish, reaction system is continued react 1 ~ 2 hour under condition of ice bath, TLC detection reaction process, stopped reaction after raw material disappears.Add the neutralization of 1mol/LNaOH solution, decompression distilled removes partial solvent, adds 50mL water, with 50mL × 3 extraction into ethyl acetate, merges organic phase, anhydrous magnesium sulfate drying, filters, and decompression precipitation, re-crystallizing in ethyl acetate, obtains yellow solid product.
Sterling is yellow solid, two step associating yields: 65%
1HNMR(DMSO-d 6,400MHz,δppm):7.84(d,J＝2.4Hz,1H),7.37(dd,J＝8.1,2.4Hz,1H),7.16(d,J＝8.1Hz,1H),6.88(s,1H),4.22(s,2H),2.13(s,3H).
The preparation of the bromo-1-of (c) 4-(2-methyl-5-nitrophenyl) pyrazoles
2-methyl-5-nitro phenylhydrazine (12.0g, 71.9mmol) and 2-bromine mda (13.0g, 86.3mmol) are dissolved in 50mL ethanol, system is warming up to 55 ~ 65 DEG C, react after 2 ~ 4 hours, TLC detection reaction process, stopped reaction after raw material disappears.Reduce pressure system precipitation, and residue obtains light yellow solid through column chromatography (elutriant: petrol ether/ethyl acetate) purifying.
Sterling is light yellow solid, yield: 40%
1HNMR(CDCl 3,400MHz,δppm):8.10-8.36(m,2H),7.74(d,J＝8.2Hz,2H),7.53(d,J＝8.7Hz,1H),2.43(s,3H).
ESI-MSm/z:281.4[M+H] +.
The preparation of (d) 3-(the bromo-1-pyrazolyl of 4-)-4-monomethylaniline
By bromo-for 4-1-(2-methyl-5-nitrophenyl) pyrazoles (3.5g, 12.5mmol) be dissolved in ethanol, then by ammonium chloride (2.67g, 49.9mmol) be dissolved in 6mL water, then add in above-mentioned system by the aqueous solution of ammonium chloride, system is warming up to 55 ~ 60 DEG C, add 2.8g iron powder, back flow reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.With diatomite filtration and by filtrate reduced in volume, residue obtains pale yellowish oil liquid through column chromatography (elutriant: petrol ether/ethyl acetate) purifying.
Sterling is pale yellowish oil liquid, yield: 80%
ESI-MSm/z:251.3[M+H] +.
The preparation of (e) N-Boc-3-(the bromo-1-pyrazolyl of 4-)-4-monomethylaniline
By 3-(the bromo-1-pyrazolyl of 4-)-4-monomethylaniline (3.2g, 12.7mmol) be dissolved in tetrahydrofuran solution, under above-mentioned system is placed in condition of ice bath, 3.5mLBoc acid anhydrides is being added drop-wise in above-mentioned system, is dripping and finish, back flow reaction 2 ~ 3 hours, TLC detection reaction process, reaction solution is cooled to room temperature after disappearing by raw material, and decompression precipitation, residue obtains light yellow solid through column chromatography (elutriant: petrol ether/ethyl acetate) purifying.
Sterling is light yellow solid, yield: 90%
1HNMR(DMSO-d 6,400MHz,δppm):9.55(s,1H),8.32(s,1H),7.83(s,1H),7.55(s,1H),7.38(d,J＝8.3Hz,1H),7.26(d,J＝8.4Hz,1H),2.10(s,3H),1.47(s,9H).
ESI-MSm/z:351.3[M+H] +.
The preparation of (f) N-Boc-3-(4-(2-(4,4,5,5-tetramethyl--1,3,2-dioxy boron penta cyclic group))-1-pyrazolyl)-4-monomethylaniline.
By N-Boc-3-(the bromo-1-pyrazolyl of 4-)-4-monomethylaniline (3.0g, 8.5mmol) be dissolved in 20mL dioxane, two (triphenylphosphine) palladium chloride (0.6g is added again in flask, 0.85mmol), Potassium ethanoate (2.5g, 25.5mmol) with connection pinacol borate (2.6g, 10.2mmol).Under argon shield, be heated to back flow reaction 15 ~ 18 hours, TLC detection reaction process, reaction solution is cooled to room temperature after disappearing by raw material, and decompression precipitation, residue obtains light yellow solid through column chromatography (elutriant: petrol ether/ethyl acetate) purifying.
Sterling is light yellow solid, yield: 50%
ESI-MSm/z:400.2[M+H] +.
The preparation of (g) 3-bromine imidazo [1,2-b] pyridazine
By imidazo [1,2-b] pyridazine (6.0g, 50.4mmol), NBS (6.0g, 75.6mmol) join in 50mL chloroform with the AIBN of catalytic amount, be heated to back flow reaction 2 hours, TLC detection reaction process, after raw material disappears, reaction solution be cooled under room temperature is placed in condition of ice bath and stir, there is Precipitation, filter and filtrate reduced in volume can be obtained sterling.
Sterling is light yellow solid, yield: 75%
1HNMR(CDCl 3,400MHz,δppm):8.50(d,J＝3.6Hz,1H),8.01-7.98(m,1H),7.84(s,1H),7.15-7.12(m,1H).
ESI-MSm/z:200.1[M+H] +.
The preparation of (h) N-Boc-3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline
By N-Boc-3-(4-(2-(4; 4; 5; 5-tetramethyl--1; 3,2-dioxy boron penta cyclic group))-1-pyrazolyl)-4-monomethylaniline (2.5g, 6.3mmol) adds in flask; add 30mL dioxane, 6mL water stirring and dissolving; add 3-bromine imidazo [1,2-b] pyridazine (2.5g, 12.6mmol), four (triphenyl phosphorus) palladium (0.73g; 0.63mmol), sodium carbonate (2.67g; after 25.2mmol) stirring, reflux under argon shield, TLC detection reaction process; react 7 ~ 10 hours, be cooled to room temperature.Reaction solution diatomite filtration, decompression precipitation, residue is through column chromatography (eluent: petrol ether/ethyl acetate) purifying.
Sterling is light yellow solid, yield: 65%
1HNMR(DMSO-d 6,400MHz,δppm):9.53(s,1H),8.69(s,1H),8.66(d,J＝4.4Hz,1H),8.45(s,1H),8.21(dd,J＝13.8,6.9Hz,2H),7.66(s,1H),7.42(d,J＝8.4Hz,1H),7.31–7.24(m,2H),2.20(s,3H),1.48(s,8H).
ESI-MSm/z:391.2[M+H] +.
The preparation of (i) 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline
By N-Boc-3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (1.0g, 2.56mmol) be dissolved in 30mL methylene dichloride, under being placed in condition of ice bath, 3mL trifluoroacetic acid is added drop-wise in above-mentioned system, drips and finish, normal-temperature reaction 5 ~ 6 hours, TLC detection reaction process, stopped reaction after raw material disappears.Decompression distilled removes partial solvent, and residue saturated sodium bicarbonate aqueous solution neutralizes, with 30mL × 3 dichloromethane extraction, merge organic phase, anhydrous magnesium sulfate drying, filter, decompression precipitation, residue is through column chromatography (eluent: petrol ether/ethyl acetate) purifying.
Sterling is light yellow solid, yield: 85%
1HNMR(DMSO-d 6,400MHz,δppm):8.66(dd,J＝4.4,1.4Hz,1H),8.64(s,1H),8.41(s,1H),8.21(s,1H),8.21–8.18(m,1H),7.25(dd,J＝9.2,4.5Hz,1H),7.04(d,J＝8.2Hz,1H),6.66(d,J＝2.3Hz,1H),6.60(dd,J＝8.2,2.3Hz,1H),5.23(s,2H),2.09(s,3H).
ESI-MSm/z:291.1[M+H] +.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-3-trifluoromethyl benzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 3-trifluoromethyl benzoyl chloride (0.43g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):8.94(d,J＝4.0Hz,1H),8.81(s,1H),8.51(d,J＝10.2Hz,2H),8.39(d,J＝9.3Hz,1H),8.27–8.20(m,2H),7.95(d,J＝7.0Hz,2H),7.76(dd,J＝14.6,7.2Hz,2H),7.66(dd,J＝9.3,4.5Hz,1H),7.44(d,J＝8.4Hz,1H),2.23(s,3H).
HRMS(ESI)m/z:C 24H 17F 3N 6O,calculated463.1494[M+H] +,found463.1513[M+H] +.
Embodiment 2
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-trifluoromethyl benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-trifluoromethyl benzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 4-trifluoromethyl benzoyl chloride (0.43g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):8.92(d,J＝3.5Hz,1H),8.80(s,1H),8.50(d,J＝2.6Hz,2H),8.38(d,J＝9.2Hz,1H),8.11(d,J＝8.1Hz,2H),7.98(d,J＝1.9Hz,1H),7.89(d,J＝8.3Hz,2H),7.76–7.71(m,1H),7.63(dd,J＝9.2,4.5Hz,1H),7.44(d,J＝8.5Hz,1H),2.23(s,3H).
HRMS(ESI)m/z:C 24H 17F 3N 6O,calculated463.1494[M+H] +,found463.1512[M+H] +.
Embodiment 3
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-fluorobenzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-fluorobenzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 4-fluorobenzoyl chloride (0.33g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.50(s,1H),8.78(s,1H),8.75(d,J＝4.0Hz,1H),8.50(s,1H),8.34(s,1H),8.27(d,J＝9.5Hz,1H),8.08(dd,J＝8.5,5.6Hz,2H),8.01(s,1H),7.82(d,J＝8.4Hz,1H),7.45–7.34(m,4H),2.27(s,3H).
HRMS(ESI)m/z:C 23H 17FN 6O,calculated413.1526[M+H] +,found413.1541[M+H] +.
Embodiment 4
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-3-chlorobenzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-3-chlorobenzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 3-chloro-benzoyl chloride (0.36g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.55(s,1H),8.76(s,1H),8.67(d,J＝3.4Hz,1H),8.49(s,1H),8.25(s,1H),8.20(d,J＝9.0Hz,1H),8.04(s,1H),7.98(s,1H),7.94(d,J＝7.6Hz,1H),7.82(d,J＝8.3Hz,1H),7.68(d,J＝7.5Hz,1H),7.58(t,J＝7.9Hz,1H),7.43(d,J＝8.4Hz,1H),7.26(dd,J＝9.1,4.4Hz,1H),2.28(s,3H).
HRMS(ESI)m/z:C 23H 17FN 6O,calculated413.1526[M+H] +,found413.1541[M+H] +.
Embodiment 5
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-methoxy benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-methoxy benzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 4-methoxy benzoyl chloride (0.35g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.28(s,1H),8.75(s,1H),8.68(d,J＝3.2Hz,1H),8.49(s,1H),8.25(s,1H),8.21(d,J＝9.1Hz,1H),7.99(d,J＝8.2Hz,3H),7.81(d,J＝8.3Hz,1H),7.40(d,J＝8.4Hz,1H),7.27(dd,J＝9.2,4.4Hz,1H),7.08(d,J＝8.7Hz,2H),3.84(s,3H),2.27(s,3H).
HRMS(ESI)m/z:C 24H 21N 6O 2,calculated425.1726[M+H] +,found425.1742[M+H] +.
Embodiment 6
The preparation of N-(3-(4-(3-(imidazo [1,2-b] pyridazinyl))-1-pyrazolyl)-4-aminomethyl phenyl)-4-ethyl benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-ethyl benzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 4-ethylamino benzonitrile acyl chlorides (0.35g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.34(s,1H),8.75(s,1H),8.67(d,J＝4.1Hz,1H),8.48(s,1H),8.24(s,1H),8.21(d,J＝9.0Hz,1H),8.00(s,1H),7.91(d,J＝8.0Hz,2H),7.82(d,J＝8.4Hz,1H),7.43–7.34(m,3H),7.26(dd,J＝9.1,4.5Hz,1H),2.69(d,J＝7.4Hz,2H),2.27(s,3H),1.23(d,J＝7.5Hz,3H).
HRMS(ESI)m/z:C 25H 23N 6O,calculated423.1933[M+H] +,found423.1949[M+H] +.
Embodiment 7
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-3-fluorobenzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) N-(3-(4-(3-(imidazo [1,2-b] pyridazinyl))-1-pyrazolyl)-4-aminomethyl phenyl)-3-fluorobenzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.2g, 0.69mmol) be dissolved in 5mL methylene dichloride, add 50 μ L triethylamines, under above-mentioned system is placed in condition of ice bath, again by 3-fluorobenzoyl chloride (0.33g, 2.07mmol) be dissolved in 3mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):8.91(d,J＝4.1Hz,1H),8.80(s,1H),8.49(d,J＝2.4Hz,2H),8.37(d,J＝9.4Hz,1H),7.97(s,1H),7.79(d,J＝7.9Hz,1H),7.74(d,J＝8.4Hz,2H),7.64–7.54(m,2H),7.49–7.39(m,2H),2.23(s,3H).
HRMS(ESI)m/z:C 23H 18FN 6O,calculated413.1526[M+H] +,found413.1552[M+H] +.
Embodiment 8
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4 – (1-(4-methylpiperazine base) methyl) benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1.
The preparation of (j) 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide
By 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-monomethylaniline (0.6g, 2.07mmol) be dissolved in 10mL methylene dichloride, add 0.1mL triethylamine, under above-mentioned system is placed in condition of ice bath, again by 4-chloromethyl benzoic acid chlorides (0.99g, 6.21mmol) be dissolved in 5mL methylene dichloride, then it is slowly added drop-wise in above-mentioned ice bath system, drip and finish, continue ice bath reaction 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.By reacting liquid filtering, insolubles uses water, washed with dichloromethane respectively, obtains sterling finally by column chromatography (elutriant: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.52(s,1H),8.89(d,J＝3.8Hz,1H),8.82(s,1H),8.52(d,J＝4.0Hz,2H),8.40(d,J＝9.0Hz,1H),8.04(s,1H),8.00(d,J＝8.1Hz,2H),7.82(d,J＝7.2Hz,1H),7.60(d,J＝8.1Hz,2H),7.56(dd,J＝9.3,4.4Hz,1H),7.43(d,J＝8.3Hz,1H),4.85(s,2H),2.27(s,3H).
ESI-MSm/z:443.1[M+H] +.
The preparation of (k) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4 – (1-(4-methylpiperazine base) methyl) benzamide
By 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide (0.2g, 0.45mmol) be dissolved in 5mLN, in dinethylformamide, add the potassiumiodide of catalytic amount, stirring at normal temperature 15 minutes, then in reaction system, 1-methylpiperazine (0.13g is added, 1.35mmol), system is warming up to 90 DEG C, react 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.Reaction solution is cooled to room temperature, adds 20mL water wherein, with 20 × 3mL dichloromethane extraction, merge organic phase.Then the saturated ammonium chloride solution of organic phase washs 3 times, anhydrous magnesium sulfate drying, and decompression precipitation, residue obtains sterling through column chromatography (eluent: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.42(s,1H),8.76(s,1H),8.68(d,J＝3.3Hz,1H),8.49(s,1H),8.28–8.17(m,2H),8.01(s,1H),7.95(d,J＝8.0Hz,2H),7.82(d,J＝8.2Hz,1H),7.44(dd,J＝17.4,8.2Hz,3H),7.27(dd,J＝9.1,4.3Hz,1H),3.56(s,2H),2.45(s,6H),2.27(s,8H).
HRMS(ESI)m/z:C 29H 31N 8O,calculated507.2621[M+H] +,found507.2619[M+H] +.
Embodiment 9
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-(1-morpholinyl) methyl benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1, and step (j) is with embodiment 8
The preparation of (k) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-(1-morpholinyl) methyl benzamide
By 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide (0.2g, 0.45mmol) be dissolved in 5mLN, in dinethylformamide, add the potassiumiodide of catalytic amount, stirring at normal temperature 15 minutes, then in reaction system, morpholine (0.12g is added, 1.35mmol), system is warming up to 90 DEG C, react 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.Reaction solution is cooled to room temperature, adds 20mL water wherein, with 20 × 3mL dichloromethane extraction, merge organic phase.Then the saturated ammonium chloride solution of organic phase washs 3 times, anhydrous magnesium sulfate drying, and decompression precipitation, residue obtains sterling through column chromatography (eluent: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):10.40(s,1H),8.76(s,1H),8.68(d,J＝3.8Hz,1H),8.49(s,1H),8.25(s,1H),8.21(d,J＝9.3Hz,1H),8.00(s,1H),7.94(d,J＝8.0Hz,2H),7.81(d,J＝7.9Hz,1H),7.48(d,J＝7.9Hz,2H),7.42(d,J＝8.7Hz,1H),7.27(dd,J＝8.9,4.1Hz,1H),3.59(s,4H),3.55(s,2H),2.37(s,4H),2.27(s,3H).
HRMS(ESI)m/z:C 28H 28N 7O 2,calculated494.2304[M+H] +,found494.2303[M+H] +.
Embodiment 10
The preparation of N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-(piperidino) methyl benzamide
Step (a), (b), (c), (d), (e), (f), (g), (h), (i) are with embodiment 1, and step (j) is with embodiment 8
The preparation of (k) N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl)-4-(piperidino) methyl benzamide
By 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide (0.2g, 0.45mmol) be dissolved in 5mLN, in dinethylformamide, add the potassiumiodide of catalytic amount, stirring at normal temperature 15 minutes, then in reaction system, piperidines (0.11g is added, 1.35mmol), system is warming up to 90 DEG C, react 2 ~ 3 hours, TLC detection reaction process, stopped reaction after raw material disappears.Reaction solution is cooled to room temperature, adds 20mL water wherein, with 20 × 3mL dichloromethane extraction, merge organic phase.Then the saturated ammonium chloride solution of organic phase washs 3 times, anhydrous magnesium sulfate drying, and decompression precipitation, residue obtains sterling through column chromatography (eluent: methylene chloride/methanol) purifying.
Sterling is yellow-green fluorescence solid, yield: 80%
1HNMR(DMSO-d 6,400MHz,δppm):8.76(s,1H),8.68(s,1H),8.49(s,1H),8.25(s,1H),8.21(d,J＝9.1Hz,1H),8.00(s,1H),7.92(d,J＝8.0Hz,2H),7.81(d,J＝8.1Hz,1H),7.47–7.41(m,3H),7.27(dd,J＝9.1,4.4Hz,1H),3.50(s,2H),2.33(s,4H),2.27(s,3H),1.50(s,6H).
HRMS(ESI)m/z:C 29H 30N 7O,calculated492.2512[M+H] +,found492.2510[M+H] +.
Embodiment 11
External test protein kinase activity uses Kinase-Glo kinases luminescence method, and concrete grammar is as follows.
Prepared by sample determination:
(1) sample compound is dissolved in DMSO, is then quantitatively 500 μMs of concentration and is transferred to solution in quantifying board to transfer to dosage plate with DMSO dilution.Compound is with 5 times of concentration DMSO serial dilutions.Then each concentration reaction buffered soln obtains 10 × ultimate density by 10 times of concentration dilutions.Be transferred to by the compound of concentration range 0.03 ~ 50 μM in Bcr-Abl determination of activity plate, dosage is 1 μ L/ hole.
(2) with DMSO, positive control STSP is configured to the mother liquor of 10mM concentration, is diluted to 100 μMs with DMSO.First with 5 times of concentration DMSO serial dilutions.Then each concentration reaction buffered soln obtains 10 × ultimate density by 10 times of concentration dilutions.Be transferred to by the compound of concentration range 0.00064 ~ 10 μM in Bcr-Abl determination of activity plate, dosage is 1 μ L/ hole.
(3) to HPE and ZPE hole, with reaction buffered soln, 2 μ LDMSO are diluted 10 times and obtain 10%DMSO dilution, then transfer them in determination of activity plate, dosage is 1 μ L/ hole.
Compound suppresses the IC of Bcr-Abl 50measure:
(1) measure the preparation of required sample, there is no the kinase whose compound of HPE, but containing ATP, substrate and 1%DMSO; ZPE (0% effect): do not have compound but contain kinases, ATP, substrate and 1%DMSO solution; Positive compound hole: the positive compound containing kinases, ATP, substrate and different concns.Test compounds: the testing compound containing kinases, ATP, substrate and different concns.
(2) preparation of required reagent is measured, 4 × ATP: ATP dilution 4 times in test buffered soln is obtained working solution; 4 × substrate: Poly (glucose: tyrosine) dilution 4 times in test buffered soln is obtained working solution; 2.5 × Bcr-Abl kinases: kinases mensuration dilution buffer is diluted 2.5 times and obtained working solution.
(3) kinase reaction adds 10 × compound to 384 hole assay plate, 1 μ L/ hole.For HPE and ZPE hole, add the 10%DMSO solution in same volume (1 μ L/ hole); Add 2.5 × Bcr-Abl kinases to assay plate, 4 μ L/ holes.For HPE and ZPE hole, add the mensuration buffered soln in same volume (4 μ L/ hole); Assay plate is put into whizzer with centrifugal 1 minute of 1000rpm so that mixed system; Assay plate preincubation at 30 DEG C is 30 minutes; 4 × ATP and the 4 × substrate of mixing equal volume obtain 2 × ATP-substrate mixture.This mixture is the reaction mixture that Bcr-Abl kinase activity measures acceptor; Add 2 × ATP-substrate mixture to assay plate, 5 μ L/ holes; Assay plate is put into whizzer with centrifugal 1 minute of 1000rpm so that mixed system; Assay plate preincubation at 30 DEG C is 1h; Kinasegloplus is joined in respective hole, 10 μ L/ holes, then at 27 DEG C, assay plate is hatched 20 minutes; Fluorescence intensity level is read with Envision.Kinasegloplus reagent places 30 minutes before using at room temperature.
(4) the analyzing and processing Prism5.0 of raw data analyzes raw data; Calculating CompoundinHibitioryrate=(" Compound " reading-ZEP)/(HPE-ZPE) × 100% of the inhibiting rate of compound, measurement result is in table 1.
Table 1: take STSP as positive control, has carried out determination of activity to 10 target compounds of synthesis

Claims (6)

1.N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives, it is characterized in that, structural formula is as follows
R 1expression-OCH 3,-F ,-CF 3,-CH 2cH 3,-H,
R 2expression-CF 3,-F ,-Cl or-H.
2. the preparation method of N-according to claim 1 (3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives, is characterized in that, comprise the following steps:
A 2-methyl-5-nitro aniline dissolves in ethanol by (), add concentrated hydrochloric acid, under above-mentioned system is placed in condition of ice bath, then the aqueous solution of Sodium Nitrite is added in above-mentioned system, obtain the 2-methyl-5-nitro hydrazinobenzene hydrochloride salt that formula (II) represents;
B two hydrated stannous chlorides are dissolved in the concentrated hydrochloric acid of 37% by (), under being placed in condition of ice bath, then formula (II) compound is added drop-wise in above-mentioned system, react after 0.5 ~ 2 hour, add NaOH solution neutralization, obtain the 2-methyl-5-nitro phenylhydrazine that formula (III) represents;
C formula (III) compound and 2-bromine mda react by () in ethanol, obtain the bromo-1-of 4-(2-methyl-5-nitrophenyl) pyrazoles that formula (IV) represents;
D formula (IV) compound dissolution in ethanol, is added the aqueous solution and the iron powder of ammonium chloride by (), obtain 3-(the bromo-1-pyrazolyl of the 4-)-4-monomethylaniline that formula (V) represents;
E formula (V) compound and Boc acid anhydrides react by () in tetrahydrofuran (THF), obtain N-Boc-3-(the bromo-1-pyrazolyl of the 4-)-4-monomethylaniline that formula (VI) represents;
F formula (VI) compound is dissolved in dioxane by (), add connection pinacol borate, Potassium ethanoate and two (triphenylphosphine) palladium chloride, be obtained by reacting the N-Boc-3-(4-(2-(4 that formula (VII) represents, 4,5,5-tetramethyl--1,3,2-dioxy boron penta cyclic group))-1-pyrazolyl)-4-monomethylaniline;
G the AIBN of imidazo [1,2-b] pyridazine, NBS and catalytic amount reacts by () in chloroform, obtain 3-bromine imidazo [1, the 2-b] pyridazine that formula (VIII) represents;
(h) by formula (VII) compound and formula (VIII) compound dissolution in the mixing solutions of dioxane and water, add four (triphenyl phosphorus) palladium and sodium carbonate, be obtained by reacting N-Boc-3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-monomethylaniline that formula (IX) represents;
I formula (IX) and trifluoroacetic acid are reacted by () in methylene dichloride, obtain 3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-monomethylaniline that formula (X) represents;
J formula (X) compound, 4-chloromethyl benzoic acid chlorides and triethylamine react by () in methylene dichloride, obtain 4-chloromethyl-N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide that formula (XI) represents;
K () is by formula (XI) compound, nucleophilic reagent and potassiumiodide react in DMF, obtain the N-(3-(4-(3-(imidazo [1 that formula (I) represents, 2-b] pyridazinyl))-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives
R 1represent
Or formula (X) compound, substituted benzoyl chloride and triethylamine react by (l) in methylene dichloride, obtain N-(3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-the pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives that formula (I) represents;
R 1expression-OCH 3,-F ,-CF 3,-CH 2cH 3or-H,
R 2expression-CF 3,-F ,-Cl or-H.
3. method according to claim 2, is characterized in that, step (a) temperature of reaction 0 ~ 5 DEG C, 0.5 ~ 1 hour reaction times; Step (b) formula (II) compound is 1 ﹕ (1.5 ~ 2) with the amount of substance ratio of two hydrated stannous chlorides, temperature of reaction 0 ~ 10 DEG C, 1 ~ 2 hour reaction times; Step (c) formula (III) compound is 1 ﹕ (1.1 ~ 1.5) with the amount of substance ratio of 2-bromine mda, temperature of reaction 55 ~ 65 DEG C, 2 ~ 4 hours reaction times; Step (d) temperature of reaction 80 ~ 90 DEG C, 2 ~ 3 hours reaction times; Step (e) formula (V) compound is 1 ﹕ (1.1 ~ 1.4) with the amount of substance ratio of Boc acid anhydrides, temperature of reaction 66 ~ 70 DEG C, 2 ~ 3 hours reaction times; The amount of substance ratio that step (f) formula (VI) Hua He Wu ﹕ joins which Chun Zhi ﹕ Cu Suan Jia ﹕ bis-(triphenylphosphine) palladium chloride of boric acid Pin is 1 ﹕ (1.1 ~ 1.5) ﹕ 3 ﹕ (0.1 ~ 0.2), temperature of reaction 100 ~ 110 DEG C, 15 ~ 18 hours reaction times; [1,2-b] Da Qin ﹕ NBS ﹕ AIBN amount of substance ratio is 1 ﹕ (1.5 ~ 2) ﹕ (0.1 ~ 0.2), temperature of reaction 65 DEG C, 2 ~ 3 hours reaction times to step (g) imidazo; The amount of substance ratio of step (h) formula (VII) Hua He Wu ﹕ formula (VIII) Hua He Wu ﹕ Tan Suan Na ﹕ tetra-(triphenyl phosphorus) palladium is 1 ﹕ 2 ﹕ 3 ﹕ (0.1 ~ 0.2), temperature of reaction 110 ~ 120 DEG C, 7 ~ 10 hours reaction times; Step (i) normal-temperature reaction 5 ~ 6 hours; It is 1 ﹕ (2 ~ 3) ﹕ (0.2 ~ 0.5) that step (j) formula (X) changes the amount of substance ratio closing thing ﹕ 4-chloromethyl Benzoyl chloride ﹕ triethylamine, 2 ~ 3 hours reaction times; The amount of substance ratio of step (k) formula (XI) Hua He Wu ﹕ nucleophilic Shi Ji ﹕ potassiumiodide is 1 ﹕ (2 ~ 3) ﹕ (0.1 ~ 0.2), temperature of reaction 80 ~ 90 DEG C, 2 ~ 3 hours reaction times; It is 1 ﹕ (2 ~ 3) ﹕ (0.2 ~ 0.5) that step (l) formula (X) changes the amount of substance ratio closing thing ﹕ substituted benzoyl acyl chlorides ﹕ triethylamine, 2 ~ 3 hours reaction times.
4. method according to claim 2, is characterized in that, step (k) nucleophilic reagent is:
5. method according to claim 2, is characterized in that, step (I) substituted benzoyl chloride structural formula is:
6. the application of N-according to claim 1 (3-(4-(3-imidazo [1,2-b] pyridazinyl)-1-pyrazolyl)-4-aminomethyl phenyl) benzamide derivatives in preparation Bcr-Abl tyrosine kinase inhibitor.
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