CN104844609B - The irreversible bruton's tyrosine kinase inhibitor of dibit point - Google Patents

The irreversible bruton's tyrosine kinase inhibitor of dibit point Download PDF

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CN104844609B
CN104844609B CN201510242552.8A CN201510242552A CN104844609B CN 104844609 B CN104844609 B CN 104844609B CN 201510242552 A CN201510242552 A CN 201510242552A CN 104844609 B CN104844609 B CN 104844609B
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piperidine
compound
2h
1h
tyrosine kinase
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CN201510242552.8A
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CN104844609A (en
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周星露
韩玲
戈震
刘兴国
罗文华
刘冠男
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杭州和正医药有限公司
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Abstract

The invention provides the irreversible bruton's tyrosine kinase inhibitor of a kind of dibit point, containing its pharmaceutical composition and application in antitumor thereof.The tumor cell lines such as A549, SGC7901, MCF 7, PC 9, HL 60 are had antiproliferative inhibitory action by the compounds of this invention, can be applicable in the medicine of the relevant solid tumor for the treatment of human or animal's cell proliferative or leukemia;The compounds of this invention has good pharmacokinetic property, can be applicable to the relevant solid tumor of oral medication human or animal's cell proliferative or leukemia or suffering from autoimmune disease;The compounds of this invention has the response characteristic of dibit point.

Description

The irreversible bruton's tyrosine kinase inhibitor of dibit point

Technical field

The invention belongs to field of medicaments, specifically a kind of irreversible bruton's tyrosine kinase inhibitor of dibit point, containing its pharmaceutical composition and application in antitumor thereof.

Background technology

Little molecule covalent inhibitor (covalentinhibitors), also referred to as irreversible inhibitor (irreversibleinhibitors), it is by covalent bond and target protein residue generation irreversible fixation, thus playing a class inhibitor of its biological function.Covalency inhibitor medicaments in the past few decades in human health is made that significant contribution.Relative to non-covalent inhibitor, covalency inhibitor by being combined, with target protein, the affinity enhanced with target with covalent bond, and this is the basic reason that covalency inhibitor shows its high bioactivity.In recent years, owing to non-covalent anti-tumor drugs targeting is particularly a large amount of for the kinase whose generation for Buddhist nun's class Drug-resistant, people are made to increasingly focus on again covalency inhibitor medicaments.In recent years, many big drug firms have all carried out the research and development of the covalency inhibitor for certain enzyme target spot, and existing some covalent inhibitor enters clinical trial at present, including Afatinib, Cl 1033, HKI-272 etc..Wherein, Afatinib is used for treating the Metastatic Nsclc of EGF-R ELISA (EGFR) gene mutation on July 12nd, 2013 by U.S. FDA official approval, becomes the irreversible inhibitor new drug of the first treatment pulmonary carcinoma ratified by FDA.Additionally, the covalent drug of antiviral is also study hotspot in recent years, and achieved with very big progress, for instance, two anti-hepatitis c virus covalency inhibitor medicaments of FDA approved in 2011, i.e. telaprevir and boceprevir.These researchs demonstrate irreversible inhibitor and can be effectively used for the treatment of disease.

Bruton's tyrosine kinase (Bruton ' styrosinekinase, Btk), the member of a kind of nonreceptor tyrosine kinase Tec family, is the key signal enzyme expressed in all hematopoetic cell types except T lymphocyte and natural killer cell.Btk plays the part of vital role in connection cell surface B-cell receptor (B-cellreceptor, BCR) stimulation to the B cell signal transduction path of response in downstream cellular.Btk is the key regulators of B cell growth, activation, signal conduction and survival.Additionally, Bkt works in other hematopoietic cell signal transduction paths numerous, the TNF-α generation of the such as Toll-like receptor in macrophage (Tolllikereceptor, TLR) and cytokine receptor mediation, IgE receptor (Fc ε R1) the signal conduction in mastocyte, the platelet aggregation of the apoptotic signal conduction of suppression Fas/APO-1 and collagen stimulation in B-pedigree lymphoid cell.Referring to such as C.A.Jeffries etc., J.Bio.Chem. (2003) 278:26258-26264, N.J.Horwood etc., J.Exp.Med. (2003) 197:1603-1611.Recent study shows, Btk signal path is current non-Hodgkin lymphoma (NHL), particularly the new focus in chronic lymphocytic leukemia (CLL), B cell lymphoma and the research of autoimmune disease clinical treatment.Little molecule Btk inhibitor, by acting on BCR signal path, is combined with Btk and suppresses Btk autophosphorylation, stops the activation of Btk, thus blocking cell conductance inducing cell apoptosis.Btk inhibitor selectivity is strong, and toxic and side effects is low, and particularly her cloth replaces the listing of Buddhist nun, is decided to be " breakthrough " new drug by FDA, and its research and development have a extensive future.Yi Bu reacts for the sulfydryl of Buddhist nun with Btk enzyme cysteine (Cys481) residue, and forms covalent bond, makes Btk enzyme deactivation play curative effect.But, Yi Bu replaces Buddhist nun in administration process for Buddhist nun, easily it is metabolized (being metabolized oxydasis be metabolized to dihydroxylated product or inactivated by attacks such as other enzymes containing sulfydryl, cysteine, glutathion) and affects drug effect (see following formula), its clinical administration dosage has reached 560mg/ days, and make patient's burden, therefore still need to the treatment for relevant disease of the development one class highly efficient BTK inhibitor.

Summary of the invention

It is an object of the invention to provide dibit point BTK irreversible inhibitor novel, that have no bibliographical information and optical isomer thereof or its pharmaceutically acceptable salt or solvate.

Based on the passway of metabolism analysis for Buddhist nun of her cloth, if introducing halogen atom at her cloth for the near-end of α, the β-unsaturated double-bond of Buddhist nun so that its metabolism site is occupied and blocks double bond and be metabolized enzyme institute dihydroxylated, and then improves its internal metabolic stability.What is more important, introducing due to halogen atom, recruit is made to be provided with double; two reaction site, (or cell in) is by after other enzymes containing sulfydryl, cysteine, glutathion etc. in vivo, still there is site a---amide group (see following formula) for α halo that can react with the sulfydryl of Btk enzyme cysteine (Cys481) residue, and then play the effect of increased activity.

The present invention adopts the following technical scheme that:

BTK inhibitor provided by the present invention has formula (I) structure:

And optical isomer or its pharmaceutically acceptable salt or solvate, wherein:

Ra, Rb, Rc are independently selected from H, halogen ,-CF3、-CN、-NO2、OH、NH2、-L-C1-C6Alkyl ,-L-C1-C6Thiazolinyl, heteroaryl substituted or non-substituted for-L-or the substituted or non-substituted aryl of-L-,

Wherein L is key, O, S ,-S (=O) ,-S (=O)2、NH、C(O)、CH2,-NHC (O) O ,-NHC (O) or-C (O) NH.

X is selected from halogen, it is preferred to fluorine, chlorine and bromine.

Further, currently preferred compound has formula (II) structure:

And optical isomer or its pharmaceutically acceptable salt or solvate, wherein:

Each Rd is H, halogen ,-CF independently3、-CN、-NO2、-OH、C1-C3Alkoxyl ,-NH2

X is selected from halogen, it is preferred to fluorine, chlorine and bromine.

Further, currently preferred compound has formula (III) structure:

And optical isomer or its pharmaceutically acceptable salt or solvate, wherein:

X is selected from halogen, it is preferred to fluorine, chlorine and bromine.

Term illustrates: term used herein " aryl " refers to full carbon monocycle or the fused polycycle group of 5 to 12 carbon atoms, has the pi-electron system of total conjugated.The limiting examples of aromatic ring has: phenyl ring, naphthalene nucleus and anthracene nucleus.Aromatic ring can be without replacing or replacing.The substituent group of aromatic ring is selected from halogen, nitro, amino, C1-C6Alkyl, C1-C6Alkoxyl, halo C1-C6Alkyl, halo C1-C6Alkoxyl, C3-C6Cycloalkyl, halo C3-C6Cycloalkyl;

Term used herein " heteroaryl " refers to the undersaturated carbocyclic ring of 5 to 12 annular atomses, and wherein one or more carbon are replaced by hetero atom such as oxygen, nitrogen, sulfur etc..Hetero-aromatic ring can be monocycle, it is also possible to be dicyclo, is namely condensed by two rings and forms.Concrete heterocyclic aryl may is that pyridine radicals, pyrimidine radicals, pyrazinyl, isoxazolyl, isothiazolyl, pyrazolyl, thiazolyl, oxazolyl and imidazole radicals etc..Heterocyclic aryl can be without replacing or replacing.The substituent group of heterocyclic aryl is selected from halogen, nitro, amino, C1-C6Alkyl, C1-C6Alkoxyl, halo C1-C6Alkyl, halo C1-C6Alkoxyl, C3-C6Cycloalkyl, halo C3-C6Cycloalkyl;

Term used herein " heterocycle " refers to monocycle or fused ring group, has 5 to 9 annular atomses in ring, and wherein one or two annular atoms is chosen from N, O or S (O)mThe hetero atom of (wherein m is the integer of 0 to 2), all the other annular atomses are C.These rings can have one or more double bond, but these rings do not have the pi-electron system of total conjugated.Without the Heterocyclylalkyl replaced can be that pyrrolidinyl, piperidyl, piperazinyl, morpholino base, thiomorpholine are for base, homopiperazine base etc..Heterocycle can be without replacing or replacing.The substituent group of heterocycle is selected from halogen, nitro, amino, C1-C6Alkyl, C1-C6Alkoxyl, halo C1-C6Alkyl, halo C1-C6Alkoxyl, C3-C6Cycloalkyl, halo C3-C6Cycloalkyl.

Term used herein " alkoxyl " refers to-O-alkyl group, and wherein alkyl is as defined above.The example of " alkoxyl " used herein includes but not limited to methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy and tert-butoxy." alkoxyl " also includes substituted alkoxy.Alkoxyl can optionally be optionally substituted by halogen one or many.

Term " thiazolinyl " refers to a class alkyl, and wherein two initial atoms of alkyl form double bond, and this double bond is not the ingredient of aromatic radical.It is to say, thiazolinyl starts from atom-C (R)=C (R)-R, wherein R refers to the remainder of thiazolinyl, and each R can be identical or different.Alkenyl part can be side chain, straight chain or ring-type (in the case, it also can be referred to as " cycloalkenyl group ").According to structure, thiazolinyl can be monoradical or double; two add group (i.e. alkenylene).Thiazolinyl can be optionally substituted.The limiting examples of thiazolinyl includes-CH=CH2、-C(CH3)=CH2,-CH=CHCH3、-C(CH3)=CHCH3.Alkenylene includes but not limited to-CH=CH-,-C (CH3)=CH-,-CH=CHCH2-,-CH=CHCH2CH2-and-C (CH3)=CHCH2-.Thiazolinyl can have 2-10 carbon atom.Thiazolinyl alternatively has " low-grade alkenyl " of 2-6 carbon atom.

Term used herein " halogen " represents fluorine, chlorine, bromine or iodine, it is preferred to fluorine, chlorine and bromine.

Term " pharmacy can accept derivant " refers to salt and the solvate of selected compounds.

Term used herein " solvate " refers to the complex of the varying chemical metering formed by solute (such as: the formula (I) of the present invention~formula (III) compound) and solvent.For the purposes of the present invention, described solvent can not disturb the biologic activity of solute.The example of suitable solvent includes but not limited to water, methanol, ethanol and acetic acid.The solvent being preferably used is pharmaceutical acceptable solvents.Suitable pharmaceutical acceptable solvents includes but not limited to water, ethanol and acetic acid.It is highly preferred that solvent for use is water.

The present invention adopts method well-known to those skilled in the art can prepare the salt of compound of the present invention.Described salt can be acylate, inorganic acid salt etc., and described acylate includes citrate, fumarate, oxalates, malate, lactate, camsilate, tosilate, mesylate etc.;Described inorganic acid salt includes halogen acid salt, sulfate, phosphate, nitrate etc..Such as, with lower alkanesulfonic acid, such as methanesulfonic acid, trifluoromethanesulfonic acid etc. can form mesylate, fluoroform sulphonate;With aryl sulfonic acid, as benzenesulfonic acid or p-methyl benzenesulfonic acid etc. can form tosilate, benzene sulfonate;With organic carboxyl acid, such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic acid, succinic acid or citric acid etc. can form corresponding salt;With aminoacid, as glutamic acid or aspartic acid can form glutamate, Glu or aspartate.With mineral acid, such as halogen acids (such as Fluohydric acid., hydrobromic acid, hydroiodic acid, hydrochloric acid), nitric acid, carbonic acid, sulphuric acid or phosphoric acid etc. also can form corresponding salt.

Second purpose of the present invention is to provide a kind of pharmaceutical composition, described pharmaceutical composition comprises at least one active component and one or more pharmaceutically acceptable carrier or excipient, described active component can be in the solvate of the BTK inhibitor compound of the present invention, the optical isomer of described compound, described compound or its optical isomer pharmaceutically acceptable salt, described compound or its optical isomer any one or arbitrarily multiple.

Described carrier includes the conventional thinner of pharmaceutical field, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant etc., it may also be necessary to add flavouring agent, sweeting agent etc..Medicine of the present invention can make tablet, powder, granule, capsule, the various ways such as oral liquid and injecting drug use, and the medicine of above-mentioned each dosage form all can be prepared according to the conventional method of pharmaceutical field.

On the other hand, the present invention is to provide disease, obstacle or disease that the compound described in use formula disclosed herein (I)~formula (III) and optical isomer thereof or its pharmaceutically acceptable salt or solvate benefit from the suppression of bruton's tyrosine kinase (Btk) activity to suppress bruton's tyrosine kinase (Btk) activity or treatment.

In further aspect, provided herein be by giving the compositions of a kind of at least one compound containing therapeutically effective amount of therapist in need thus suppress described curee bruton's tyrosine kinase activity method, the structural formula of wherein said compound is formula (I)~formula (III).In some embodiments, curee's suffering from autoimmune disease in need, for instance inflammatory bowel, arthritis, lupus, rheumatoid arthritis, psoriasis arthropathica, osteoarthritis, Still disease (Still ' sdisease), adolescent arthritis, diabetes, myasthenia gravis, chronic lymphocytic thyroiditis (Hashimoto ' sthyroiditis), Order thyroiditis (Ord ' sthyroiditis), Graves' disease (Graves ' disease), rheumatoid arthritis syndrome (nullsyndrome)、Multiple sclerosis、Guillain-Barre syndrome (Guillain-Barr é syndrome)、Acute disseminated encephalomyelitis、Bronzed disease (Addison ' sdisease)、Opsoclonus-myoclonic syndrome、Mandatory spondylitis、Antiphospholipid antibody syndrome、Aplastic anemia、Autoimmune hepatitis、Celiac disease (coeliacdisease)、Goodpasture's syndrome (Goodpasture ' ssyndrome)、Idiopathic thrombocytopenic purpura、Optic neuritis、Scleroderma、Primary biliary cirrhosis、Reiter syndrome (Reiter ' ssyndrome)、Aortic arch syndrome (Takayasu ' sarteritis)、Temporal arteritis、Warm type autoimmune hemolytic anemia、Wegner granulomatosis (Wegener ' sgranulomatosis)、Psoriasis、Alopecia universalis、Behcet disease (Behcet ' sdisease)、Confirmed fatigue、Familial dysautonomia、Endometriosis、Interstitial cystitis、Neuromyotonia、Scleroderma or vulvodynia.

In further embodiment, curee in need suffers from cancer.In one embodiment, described cancer is B cell proliferative disease, for instance diffusivity large B cell lymphoid tumor, follicular lymphoma, chronic lymphocytic leukemia, chronic lymphocytic leukemia, B cell PL, lymphoplasmacytic lymphoma/macroglobulinemia Waldenstron (Macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmocytoma, extranodal marginal zone B cell lymphoma, lymphoma nodal marginal zone B cell, mantle cell lymphoma, vertical diaphragm (thymus) large B cell lymphoid tumor, intravascular large B cell lymphoma, lymphoma primary effusion, Burkitt lymphoma (Burkittlymphoma)/leukemia or lymphomatoid granulomatosis.

The present invention also provides for the compound or pharmaceutically acceptable salt thereof of the present invention application in preparing BTK inhibitor, particularly the application in preparation treatment cell proliferative diseases.Described cell proliferative diseases includes cancer.In other words, the present invention also provide for the compound described in formula (I)~formula (III) and optical isomer thereof or its pharmaceutically acceptable salt or solvate individually or and other drug application in treatment proliferative diseases (such as cancer) is used in combination.Can and the antineoplastic agent that be used in combination of compound or pharmaceutically acceptable salt thereof provided by the present invention include but and the following kind of non-limiting at least one: mitotic inhibitor (such as vinblastine, vindesine and vinorelbine);Tubulin decomposing inhibitor (such as taxol);Alkylating reagent (such as cisplatin, carboplatin and cyclophosphamide);Antimetabolite (such as 5-fluorouracil, ftorafur, methotrexate, cytosine arabinoside and hydroxyurea);Can be inserted into antibiotic (such as A Leisu, mitomycin and bleomycin A5);Enzyme (such as asparagine enzyme);Topoisomerase inhibitors (as relied on uncle's glycosides and camptothecine);Biological response modifier (such as interferon).

Present invention also offers the method preparing formula (I) and the acceptable derivant of pharmacy thereof, synthesize with the synthetic route shown in following scheme:

As shown in reaction equation, compound 1 (prepared by the method with reference to WO2012158795) and RiB(OH)2Under potassium phosphate, palladium catalyst and suitable solvent or mixed solvent such as dioxane/water exist; back flow reaction 24 hours; the compound 2 obtained is under triphenyl phosphorus, DIAD and suitable solvent such as THF exists; being obtained by reacting compound 3 with the 3-hydroxy piperidine of Boc protection, key intermediate 4 is prepared in hydrolysis in acid condition subsequently.Under this key intermediate DCC and suitable solvent such as DCM exists, and substitutional crylic acid fragment condensation reaction, obtain formula (I) compound.

Inventor experiments prove that, the tumor cell lines such as A549, SGC7901, MCF-7, PC-9, HL-60 are had antiproliferative inhibitory action by the compounds of this invention, can be applicable in the medicine of the relevant solid tumor for the treatment of human or animal's cell proliferative or leukemia.

Inventor experiments prove that, the compounds of this invention has good pharmacokinetic property, can be applicable to the relevant solid tumor of oral medication human or animal's cell proliferative or leukemia or suffering from autoimmune disease.

Inventor experiments prove that, the compounds of this invention has the response characteristic of dibit point.

Detailed description of the invention

The exploitativeness of the present invention is described by the examples below, it will be understood by those of skill in the art that the instruction according to prior art, corresponding technical characteristic modified or replaces, still falling within the scope of protection of present invention.

The preparation of embodiment 1. key intermediate 4a

The synthesis of step 1.3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2a)

By iodo-for 3-1H-pyrazolo [3; 4-d] pyrimidine-4-amine (compound 1) (2.61g; 10mmol), 4-phenoxy group phenylboric acid (3.85g, 18mmol) and potassium phosphate (5.375g, 25mmol) are sequentially added in single neck bottle; add 1; 4-dioxane (40mL) and water (10mL), under nitrogen protection, add triphenyl phosphorus palladium (1.76g; 1.5mmol), back flow reaction 24h.React complete and be cooled to room temperature, stirring is overnight, precipitate out yellow mercury oxide, sucking filtration, washing (50ml*3), obtains yellow solid 2.18g, for 3-(4-Phenoxyphenyl)-1H-pyrazolo [3 after dry 24 hours, 4-d] pyrimidine-4-amine (compound 2a), productivity 72%.

The synthesis of step 2.3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3a)

By 3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2a) (2.12g, 7mmol), 3-hydroxy piperidine-1-t-butyl formate (1.55g, 7.7mmol), triphenyl phosphorus (2.75g, 10.5mmol) and azodiisobutyronitrile (2.12g, 10.5mmol) be dissolved in THF (250ml), room temperature reaction 12h (whether the monitoring reaction of TLC thin layer chromatography is complete).After completion of the reaction, decompression and solvent recovery.Adding 100 ethyl acetate in residue reactant mixture, extract organic layer 3 times with 100ml*3 saturated sodium carbonate solution, merge organic facies, after saturated sodium-chloride washes 1 time, anhydrous sodium sulfate dries.Decompression and solvent recovery obtains brown solid 1.12g, productivity 33%.

The synthesis of step 3.3-(4-Phenoxyphenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4a)

By 3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3a) (0.97g, 2mmol) it is dissolved in 15ml dioxane, dropping 10ml2NHCl, stirred overnight at room temperature.The crude product recrystallizing methanol that decompression and solvent recovery obtains, obtains off-white color solid 4a (0.56g, productivity 66%, [M+H]=423.1).

The preparation of embodiment 2. key intermediate 4b

The synthesis of step 1.3-(4-(4-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2b)

Synthesis step reference example 1 step 1. synthetic method being similar to compound 2a, prepares compound 2b (2.56g, productivity 77%) from 4-(4-methoxyphenoxy) phenylboric acid (4.39g, 18mmol).

The synthesis of step 2.3-(4-amino-3-(4-(4-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3b)

Synthesis step reference example 1 step 2. synthetic method being similar to compound 3a prepares compound 3b (1.26g, productivity 35%).

The synthesis of step 3.3-(4-(4-methoxyphenoxy) phenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4b)

Synthesis step reference example 1 step 3. synthetic method being similar to compound 4a prepares compound 4b (0.59, productivity 65%, [M+H]=453.3).

The preparation of embodiment 3. key intermediate 4c

The synthesis of step 1.3-(4-(3-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2c)

Synthesis step reference example 1 step 1. synthetic method being similar to compound 2a, prepares compound 2c (2.49g, productivity 75%) from 4-(3-methoxyphenoxy) phenylboric acid (4.39g, 18mmol).

The synthesis of step 2.3-(4-amino-3-(4-(3-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3c)

Synthesis step reference example 1 step 2. synthetic method being similar to compound 3a prepares compound 3c (1.25g, productivity 35%).

The synthesis of step 3.3-(4-(3-methoxyphenoxy) phenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4c)

Synthesis step reference example 1 step 3. synthetic method being similar to compound 4a prepares compound 4c (0.61g, productivity 68%, [M+H]=453.3).

The preparation of embodiment 4. key intermediate 4d

The synthesis of step 1.3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2d)

Synthesis step reference example 1 step 1. synthetic method being similar to compound 2a, prepares compound 2d (2.62g, productivity 72%) from 4-(3,4-dimethoxy phenoxy group) phenylboric acid (4.93g, 18mmol).

The synthesis of step 2.3-(4-amino-3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3d)

Synthesis step reference example 1 step 2. synthetic method being similar to compound 3a prepares compound 3d (1.04g, productivity 30%).

The synthesis of step 3.3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4d)

Synthesis step reference example 1 step 3. synthetic method being similar to compound 4a prepares compound 4d (0.53g, productivity 64%, [M+H]=483.1).

The preparation of embodiment 5. key intermediate 4e

The synthesis of step 1.3-(4-(4-fluorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2e)

Synthesis step reference example 1 step 1. synthetic method being similar to compound 2a, prepares compound 2e (2.37g, productivity 74%) from 4-(4-fluorophenoxy) phenylboric acid (4.18g, 18mmol).

The synthesis of step 2.3-(4-amino-3-(4-(4-fluorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3e)

Synthesis step reference example 1 step 2. synthetic method being similar to compound 3a prepares compound 3e (1.30g, productivity 37%).

The synthesis of step 3.3-(4-(4-fluorophenoxy) phenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4e)

Synthesis step reference example 1 step 3. synthetic method being similar to compound 4a prepares compound 4e (0.65g, productivity 74%, [M+H]=441.1).

The preparation of embodiment 6. key intermediate 4f

The synthesis of step 1.3-(4-(4-chlorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-4-amine (compound 2f)

Synthesis step reference example 1 step 1. synthetic method being similar to compound 2a, prepares compound 2f (2.29g, productivity 68%) from 4-(4-chlorophenoxy) phenylboric acid (4.46g, 18mmol).

The synthesis of step 2.3-(4-amino-3-(4-(4-chlorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidines-1-t-butyl formate (compound 3f)

Synthesis step reference example 1 step 2. synthetic method being similar to compound 3a prepares compound 3f (1.46g, productivity 40%).

The synthesis of step 3.3-(4-(4-chlorophenoxy) phenyl)-1-(piperidines-3-base)-1H-pyrazolo [3,4-d] pyrimidine-4-amine hydrochlorate (compound 4f)

Synthesis step reference example 1 step 3. synthetic method being similar to compound 4a prepares compound 4f (0.72g, productivity 79%, [M+H]=457.2).

The preparation (compound 5-1) of embodiment 7.1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-fluoropropyl-2-alkene-1-ketone

4a (548mg, 1.3mmol), 2-perfluoroalkyl acrylate (117mg, 1.3mmol) is dissolved in 5ml dichloromethane, dichloromethane (5ml) solution of room temperature dropping DCC (267mg, 1.3mmol), back flow reaction 4 hours.After reaction terminates, being cooled to room temperature, sucking filtration, after filtrate concentration, column chromatography for separation (petroleum ether: ethyl acetate: triethylamine=2: 1: 0.1) obtains white solid 207mg, productivity 35%, [M+H]=459.2.1H-NMR (δ, CDCl3-d6): 8.34 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.39 (m, 2H, ArH), 7.16 (m, 3H, ArH), 7.08 (d, 2H, J=8.0Hz, ArH), 6.12 (s, 2H ,-NH2), 5.30 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.18 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.66(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.12(m,0.5H,piperidine),3.76(m,0.5H,piperidine),3.47(m,0.5H,piperidine),3.17(m,0.5H,piperidine),2.87(m,0.5H,piperidine),2.81(m,0.5H,piperidine),2.30(m,2H,piperidine),1.75(m,2H,piperidine).

The preparation (compound 5-2) of embodiment 8.1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-2 (257mg, productivity 42%, [M+H]=475.2).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.40 (m, 2H, ArH), 7.17 (m, 3H, ArH), 7.09 (d, 2H, J=8.0Hz, ArH), 5.67 (s, 2H ,-NH2), 5.62 (m, 2H ,-C=CH2null),4.93(m,1H,piperidine),4.68(m,0.5H,piperidine),4.52(m,0.5H,piperidine),4.13(m,0.5H,piperidine),3.98(m,0.5H,piperidine),3.80(m,0.5H,piperidine),3.50(m,0.5H,piperidine),3.23(m,0.5H,piperidine),2.91(m,0.5H,piperidine),2.30(m,2H,piperidine),1.77(m,2H,piperidine).

The preparation (compound 5-3) of embodiment 9.1-(3-(4-amino-3-(4-Phenoxyphenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-3 (225mg, productivity 33%, [M+H]=519.2).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.64 (d, 2H, J=8.0Hz, ArH), 7.40 (m, 2H, ArH), 7.16 (m, 3H, ArH), 7.09 (d, 2H, J=8.0Hz, ArH), 6.05 (s, 1H ,-NH2), 5.79 (m, 3H ,-C=CH2,-NH2null),4.93(m,1H,piperidine),4.66(m,0.5H,piperidine),4.51(m,0.5H,piperidine),4.11(m,0.5H,piperidine),3.97(m,0.5H,piperidine),3.77(m,0.5H,piperidine),3.22(m,0.5H,piperidine),3.11(m,0.5H,piperidine),2.91(m,0.5H,piperidine),2.28(m,2H,piperidine),1.71(m,2H,piperidine).

The preparation (compound 5-4) of embodiment 10.1-(3-(4-amino-3-(4-(4-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-fluoropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-4 (212mg, productivity 33%, [M+H]=489.2).1H-NMR (δ, CDCl3-d6): 8.35 (s, 1H, ArH), 7.66 (d, 2H, J=8.0Hz, ArH), 7.38 (d, 2H, J=8.0Hz, ArH), 7.17 (d, 2H, J=8.0Hz, ArH), 7.08 (d, 2H, J=8.0Hz, ArH), 6.15 (s, 2H ,-NH2), 5.31 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.18 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2), 4.92 (m, 1H, piperidine), 4.66 (m, 0.5H, piperidine), 4.44 (m, 0.5H, piperidine), 4.12 (m, 0.5H, piperidine), 3.76 (m, 0.5H, piperidine), 3.62 (s, 3H ,-OCH3), 3.47 (m, 0.5H, piperidine), 3.17 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.81 (m, 0.5H, piperidine), 2.30 (m, 2H, piperidine), 1.75 (m, 2H, piperidine).

The preparation (compound 5-5) of embodiment 11.1-(3-(4-amino-3-(4-(4-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-5 (212mg, productivity 38%, [M+H]=505.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.38 (d, 2H, J=8.0Hz, ArH), 7.17 (d, 2H, J=8.0Hz, ArH), 7.09 (d, 2H, J=8.0Hz, ArH), 6.03 (s, 2H ,-NH2), 5.24 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.07 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.01 (d, 1H ,-C=CH2), 4.87 (m, 1H, piperidine), 4.62 (m, 0.5H, piperidine), 4.39 (m, 0.5H, piperidine), 4.22 (m, 0.5H, piperidine), 3.77 (m, 0.5H, piperidine), 3.68 (s, 3H ,-OCH3), 3.45 (m, 0.5H, piperidine), 3.14 (m, 0.5H, piperidine), 2.86 (m, 0.5H, piperidine), 2.78 (m, 0.5H, piperidine), 2.28 (m, 2H, piperidine), 1.74 (m, 2H, piperidine).

The preparation (compound 5-6) of embodiment 12.1-(3-(4-amino-3-(4-(4-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-6 (234mg, productivity 33%, [M+H]=551.1).1H-NMR (δ, CDCl3-d6): 8.36 (s, 1H, ArH), 7.66 (d, 2H, J=8.0Hz, ArH), 7.39 (d, 2H, J=8.0Hz, ArH), 7.17 (d, 2H, J=8.0Hz, ArH), 7.09 (d, 2H, J=8.0Hz, ArH), 6.18 (s, 2H ,-NH2), 5.33 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.19 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.12 (d, 1H ,-C=CH2), 4.96 (m, 1H, piperidine), 4.68 (m, 0.5H, piperidine), 4.45 (m, 0.5H, piperidine), 4.16 (m, 0.5H, piperidine), 3.77 (m, 0.5H, piperidine), 3.65 (s, 3H ,-OCH3), 3.48 (m, 0.5H, piperidine), 3.18 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.81 (m, 0.5H, piperidine), 2.31 (m, 2H, piperidine), 1.75 (m, 2H, piperidine).

The preparation (compound 5-7) of embodiment 13.1-(3-(4-amino-3-(4-(3-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-fluoropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-7 (205mg, productivity 32%, [M+H]=489.2).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.63 (d, 2H, J=8.0Hz, ArH), 7.12 (m, 1H, ArH), 7.05 (d, 2H, J=8.0Hz, ArH), 6.52 (m, 2H, ArH), 6.39 (s, 1H, ArH), 6.14 (s, 2H ,-NH2), 5.27 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.06 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.03 (d, 1H ,-C=CH2), 4.91 (m, 1H, piperidine), 4.62 (m, 0.5H, piperidine), 4.40 (m, 0.5H, piperidine), 4.11 (m, 0.5H, piperidine), 3.73 (m, 0.5H, piperidine), 3.64 (s, 3H ,-OCH3), 3.43 (m, 0.5H, piperidine), 3.13 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.80 (m, 0.5H, piperidine), 2.17 (m, 2H, piperidine), 1.77 (m, 2H, piperidine).

The preparation (compound 5-8) of embodiment 14.1-(3-(4-amino-3-(4-(3-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-8 (272mg, productivity 41%, [M+H]=505.2).1H-NMR (δ, CDCl3-d6): 8.36 (s, 1H, ArH), 7.64 (d, 2H, J=8.0Hz, ArH), 7.16 (m, 1H, ArH), 7.05 (d, 2H, J=8.0Hz, ArH), 6.53 (m, 2H, ArH), 6.38 (s, 1H, ArH), 6.14 (s, 2H ,-NH2), 5.32 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.16 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2), 4.92 (m, 1H, piperidine), 4.63 (m, 0.5H, piperidine), 4.40 (m, 0.5H, piperidine), 4.12 (m, 0.5H, piperidine), 3.73 (m, 0.5H, piperidine), 3.66 (s, 3H ,-OCH3), 3.43 (m, 0.5H, piperidine), 3.13 (m, 0.5H, piperidine), 2.85 (m, 0.5H, piperidine), 2.83 (m, 0.5H, piperidine), 2.27 (m, 2H, piperidine), 1.76 (m, 2H, piperidine).

The preparation (compound 5-9) of embodiment 15.1-(3-(4-amino-3-(4-(3-methoxyphenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-9 (222mg, productivity 31%, [M+H]=551.1).1H-NMR (δ, CDCl3-d6): 8.36 (s, 1H, ArH), 7.64 (d, 2H, J=8.0Hz, ArH), 7.13 (m, 1H, ArH), 7.05 (d, 2H, J=8.0Hz, ArH), 6.52 (m, 2H, ArH), 6.38 (s, 1H, ArH), 6.14 (s, 2H ,-NH2), 5.27 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.03 (d, 1H ,-C=CH2), 4.91 (m, 1H, piperidine), 4.62 (m, 0.5H, piperidine), 4.40 (m, 0.5H, piperidine), 4.11 (m, 0.5H, piperidine), 3.76 (m, 0.5H, piperidine), 3.67 (s, 3H ,-OCH3), 3.44 (m, 0.5H, piperidine), 3.13 (m, 0.5H, piperidine), 2.85 (m, 0.5H, piperidine), 2.77 (m, 0.5H, piperidine), 2.33 (m, 2H, piperidine), 1.77 (m, 2H, piperidine).

Embodiment 16.1-(3-(4-amino-3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl) preparation (compound 5-10) of-2-fluoropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-10 (211mg, productivity 31%, [M+H]=519.2).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.63 (d, 2H, J=8.0Hz, ArH), 7.05 (d, 2H, J=8.0Hz, ArH), 6.62 (d, 1H, J=8.0Hz, ArH), 6.41 (d, 1H, J=8.0Hz, ArH), 6.27 (s, 1H, ArH), 5.96 (s, 2H ,-NH2), 5.27 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.06 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.03 (d, 1H ,-C=CH2), 4.91 (m, 1H, piperidine), 4.62 (m, 0.5H, piperidine), 4.40 (m, 0.5H, piperidine), 4.11 (m, 0.5H, piperidine), 3.74 (m, 3.5H ,-OCH3, piperidine), 3.66 (s, 3H ,-OCH3), 3.43 (m, 0.5H, piperidine), 3.12 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.85 (m, 0.5H, piperidine), 2.17 (m, 2H, piperidine), 1.75 (m, 2H, piperidine).

Embodiment 17.1-(3-(4-amino-3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl) preparation (compound 5-11) of-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-11 (232mg, productivity 33%, [M+H]=535.2).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.64 (d, 2H, J=8.0Hz, ArH), 7.04 (d, 2H, J=8.0Hz, ArH), 6.62 (d, 1H, J=8.0Hz, ArH), 6.42 (d, 1H, J=8.0Hz, ArH), 6.27 (s, 1H, ArH), 5.96 (s, 2H ,-NH2), 5.27 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.06 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.06 (d, 1H ,-C=CH2), 4.92 (m, 1H, piperidine), 4.61 (m, 0.5H, piperidine), 4.40 (m, 0.5H, piperidine), 4.11 (m, 0.5H, piperidine), 3.75 (m, 3.5H ,-OCH3, piperidine), 3.66 (s, 3H ,-OCH3), 3.43 (m, 0.5H, piperidine), 3.12 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.75 (m, 0.5H, piperidine), 2.31 (m, 2H, piperidine), 1.73 (m, 2H, piperidine).

Embodiment 18.1-(3-(4-amino-3-(4-(3,4-dimethoxy phenoxy group) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl) preparation (compound 5-12) of-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-12 (197mg, productivity 26%, [M+H]=581.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.63 (d, 2H, J=8.0Hz, ArH), 7.05 (d, 2H, J=8.0Hz, ArH), 6.62 (d, 1H, J=8.0Hz, ArH), 6.40 (d, 1H, J=8.0Hz, ArH), 6.27 (s, 1H, ArH), 6.08 (s, 2H ,-NH2), 5.27 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.06 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.03 (d, 1H ,-C=CH2), 4.91 (m, 1H, piperidine), 4.62 (m, 0.5H, piperidine), 4.45 (m, 0.5H, piperidine), 4.11 (m, 0.5H, piperidine), 3.75 (m, 3.5H ,-OCH3, piperidine), 3.66 (s, 3H ,-OCH3), 3.43 (m, 0.5H, piperidine), 3.12 (m, 0.5H, piperidine), 2.87 (m, 0.5H, piperidine), 2.76 (m, 0.5H, piperidine), 2.27 (m, 2H, piperidine), 1.75 (m, 2H, piperidine).

The preparation (compound 5-13) of embodiment 19.1-(3-(4-amino-3-(4-(4-fluorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-fluoropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-13 (223mg, productivity 36%, [M+H]=477.2).1H-NMR (δ, CDCl3-d6): 8.35 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.47 (d, 2H, J=8.0Hz, ArH), 7.25 (d, 2H, J=8.0Hz, ArH), 7.02 (d, 2H, J=8.0Hz, ArH), 6.12 (s, 2H ,-NH2), 5.31 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.18 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.66(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.12(m,0.5H,piperidine),3.75(m,0.5H,piperidine),3.44(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.87(m,0.5H,piperidine),2.75(m,0.5H,piperidine),2.30(m,2H,piperidine),1.77(m,2H,piperidine).

The preparation (compound 5-14) of embodiment 20.1-(3-(4-amino-3-(4-(4-fluorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-14 (254mg, productivity 40%, [M+H]=493.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.47 (d, 2H, J=8.0Hz, ArH), 7.25 (d, 2H, J=8.0Hz, ArH), 7.02 (d, 2H, J=8.0Hz, ArH), 6.12 (s, 2H ,-NH2), 5.32 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.19 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.65(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.12(m,0.5H,piperidine),3.75(m,0.5H,piperidine),3.44(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.89(m,0.5H,piperidine),2.77(m,0.5H,piperidine),2.32(m,2H,piperidine),1.76(m,2H,piperidine).

The preparation (compound 5-15) of embodiment 21.1-(3-(4-amino-3-(4-(4-fluorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-15 (213mg, productivity 30%, [M+H]=538.1).1H-NMR (δ, CDCl3-d6): 8.36 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.47 (d, 2H, J=8.0Hz, ArH), 7.24 (d, 2H, J=8.0Hz, ArH), 7.06 (d, 2H, J=8.0Hz, ArH), 6.09 (s, 2H ,-NH2), 5.31 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.18 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.63(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.12(m,0.5H,piperidine),3.75(m,0.5H,piperidine),3.43(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.87(m,0.5H,piperidine),2.79(m,0.5H,piperidine),2.31(m,2H,piperidine),1.72(m,2H,piperidine).

The preparation (compound 5-16) of embodiment 22.1-(3-(4-amino-3-(4-(4-chlorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-fluoropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-16 (211mg, productivity 33%, [M+H]=493.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.43 (d, 2H, J=8.0Hz, ArH), 7.11 (d, 2H, J=8.0Hz, ArH), 6.89 (d, 2H, J=8.0Hz, ArH), 6.02 (s, 2H ,-NH2), 5.26 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.13 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.01 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.66(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.11(m,0.5H,piperidine),3.73(m,0.5H,piperidine),3.44(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.86(m,0.5H,piperidine),2.73(m,0.5H,piperidine),2.30(m,2H,piperidine),1.74(m,2H,piperidine).

The preparation (compound 5-17) of embodiment 23.1-(3-(4-amino-3-(4-(4-chlorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-chloropropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-17 (245mg, productivity 37%, [M+H]=509.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.43 (d, 2H, J=8.0Hz, ArH), 7.11 (d, 2H, J=8.0Hz, ArH), 6.89 (d, 2H, J=8.0Hz, ArH), 6.02 (s, 2H ,-NH2), 5.19 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.10 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.00 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.66(m,0.5H,piperidine),4.44(m,0.5H,piperidine),4.11(m,0.5H,piperidine),3.73(m,0.5H,piperidine),3.44(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.86(m,0.5H,piperidine),2.77(m,0.5H,piperidine),2.31(m,2H,piperidine),1.74(m,2H,piperidine).

The preparation (compound 5-18) of embodiment 24.1-(3-(4-amino-3-(4-(4-chlorophenoxy) phenyl)-1H-pyrazolo [3,4-d] pyrimidine-1-base) piperidin-1-yl)-2-bromopropyl-2-alkene-1-ketone

Synthesis step reference example 7. synthetic method being similar to compound 5-1 prepares compound 5-18 (226mg, productivity 31%, [M+H]=554.1).1H-NMR (δ, CDCl3-d6): 8.37 (s, 1H, ArH), 7.65 (d, 2H, J=8.0Hz, ArH), 7.43 (d, 2H, J=8.0Hz, ArH), 7.11 (d, 2H, J=8.0Hz, ArH), 6.89 (d, 2H, J=8.0Hz, ArH), 6.12 (s, 2H ,-NH2), 5.19 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.12 (d, 0.5H, J=4.0Hz ,-C=CH2), 5.00 (d, 1H ,-C=CH2null),4.92(m,1H,piperidine),4.66(m,0.5H,piperidine),4.40(m,0.5H,piperidine),4.11(m,0.5H,piperidine),3.75(m,0.5H,piperidine),3.39(m,0.5H,piperidine),3.19(m,0.5H,piperidine),2.89(m,0.5H,piperidine),2.74(m,0.5H,piperidine),2.27(m,2H,piperidine),1.75(m,2H,piperidine).

The external Btk kinase inhibiting activity of embodiment 25. and anti tumor activity in vitro test

The external Btk kinase inhibiting activity assay method of the compounds of this invention:

Be dissolved in DMSO by medicine making the storing solution of 10mM, and the test concentrations medicinal liquid being diluted to 50x be standby, test concentrations with 3 by gradient dilution, respectively 25nM, 8.33nM, 2.78nM, 0.93nM, 0.31nM, 0.10nM.In 96 orifice plates, add 10 μ L50x medicine reserve liquids, add 90 μ L1x kinase buffer liquid, on the oscillator concussion 10 minutes.Take 5 μ L from the 96 each holes of orifice plate and be transferred to 384 orifice plates, in 384 orifice plates, set 2 multiple holes.Kinase reaction: prepare 2.5x kinase buffer liquid: added by enzyme in 1x kinases basis buffer.Prepare 2.5x small peptide solution: the small peptide of FAM labelling and ATP are added in 1x kinases basis buffer.Added with, in 5 μ L medicinal liquid 384 orifice plates, adding 10 μ L2.5x kinase buffer liquid, incubated at room 10 minutes.384 orifice plates add 10 μ L2.5x small peptide solution, hatches 1 hour for 28 DEG C.Add 25 μ L stop buffer stopped reaction.Reading, and the suppression ratio that computerized compound is to enzyme, the Fitting Calculation goes out the kinase whose IC of BTK50

Select different solid tumors and leukemia cell line that synthesized compound has carried out the mensuration of anti tumor activity in vitro:

Cell strain: human lung carcinoma cell (A549, PC9), human breast cancer cell (MCF7), gastric carcinoma cells (SGC7901), acute promyelocytic leukemia cell (HL60).

Culture medium: A549:RPMI1640+ hyclone

MCF7:DMEM+ hyclone

SGC7901:RPMI1640+ new-born calf serum

HL60:RPMI1640+ hyclone

PC9:DMEM+ hyclone

Method for preparation of drug: be dissolved in DMSO by medicine making the storing solution of 10mM, and dilution obtains 5 variable concentrations (test concentrations 100x) by a certain percentage.

Tumor cell In vitro culture:

By four selected strain tumor cell A549, MCF7, SGC7901, HL60, PC9, in 37 DEG C, 5%CO2Hatching in cell culture incubator, go down to posterity when cell density grows to 70~90% (going down to posterity after attached cell Duck ' sEDTA digestion), needed for experiment later.

Tumor cell A549, MCF7, SGC7901, HL60, PC9, plant into 4000/200 μ L/ holes, in 37 DEG C, 5%CO on 96 orifice plates2Overnight incubation in cell culture incubator.Every hole adds compound 2 μ L, final concentration of 50 μMs, 10 μMs, 2 μMs, 0.4 μM, and 0.08 μM common in 37 DEG C, 5%CO2Cell culture incubator is hatched 72 hours, with DMSO (2%) for matched group.After 72 hours, add 20 μ LCCK-8 solution, be placed in 37 DEG C, 5%CO2In cell culture incubator 4 hours.With having added respective amount cell culture fluid and CCK-8 solution but not added the hole of cell as blank.Measuring absorbance (OD value) by microplate reader at 450nm, the data obtained is used for calculating IC50

The computing formula of cell inhibitory rate is: cell inhibitory rate %=[(matched group OD value-blank group OD value)-(medication group OD value-blank group OD value)]/(compared with control cells OD value-blank group OD value) × 100%, tries to achieve half-inhibition concentration (IC by CalcuSyn computed in software50)。

Table 1 part of compounds is to BTK kinase inhibiting activity and tumor cell in vitro proliferation inhibition activity

As can be seen from the table, BTK is had obvious inhibitory activity by all of compound, and activity is respectively less than 10nM, suitable for Buddhist nun's activity with her cloth of positive control, illustrate that introducing halogen is substantially free of impact in the α position of acrylamide to its activity, still shows potent BTK kinase inhibiting activity.But, on a cellular level, compound 5-1~5-6 and the 5-1~5-18 tested all shows the tumor cell proliferation inhibition activity more more potent than positive control, not only to solid tumor cell activity higher (A549, SGC-7901, MCF-7, PC-9), and blood tumor cell (HL-60) is same.Such as, compound 5-2 activity on SGC-7901 cell is high 5 times.Therefore, the involved in the present invention BTK inhibitor that can be used as has wide antitumor application prospect.

The sign of the external dibit point irreversible inhibitor character of embodiment 26.:

First, after adopting compound 5-3 pretreatment restructuring Btk, with the culture medium repeated washing without inhibitor, its activity will not recover (referring to such as J.B.Smaill etc., J.Med.Chem.1999,42,1803).Secondly, mass spectrum is passed through, it was observed that the ratio of the molecular weight of the covalent complex between main mass spectra peak correspondence compound 5-3 and Btk is 1: 1 (compound 4:518, dalton, Btk kinases binding domain of recombinating: 33487;Complex predictive value is 34005, and measured value is 34005).

Secondly, after adopting compound 5-3 and cysteine to incubate bath altogether 3 hours, detect through HPLC-MS, find that majority of compounds 5-3 is changed into by the product of two cysteine additions that (predicted molecular weight is: 680), actual molecular weight is (680), experiment proves, the compound tested has by the ability of bimolecular compounds containing thiol groups addition, although it is suitable for Buddhist nun that this also illustrates her cloth of this compounds Btk kinase inhibiting activity in vitro and positive drug, but her cloth can be substantially better than in cell aspect and replace Buddhist nun.

Embodiment 27. is administered orally pharmacokinetic studies

Replacing Buddhist nun for reference with her cloth, investigated compound 5-2 and 5-3 pharmacokinetic property in rat body respectively, concrete grammar is as follows: with SD rat for laboratory animal, gastric infusion 20mg/kg, tail vein Bolos intravenous administration 5mg/kg.The tail venous blood sampling time point of gastric infusion is 0.17,0.33,0.5,1,1.5,2,4,6,8,12,24 hour;It is 0.05,0.1,0.17,0.5,1,2,4,6,8,12,24 hour that intravenously administrable takes blood time point.Take whole blood 0.3ml, take blood plasma 0.1ml after centrifugal and adopt LC-MS to be analyzed.It is shown that the oral administration biaavailability of compound 5-2 and 5-3 respectively 25% and 17%, and her cloth is 12% for the bioavailability of Buddhist nun, and therefore compound 5-2 and 5-3 stability in vivo relatively her cloth has had for Buddhist nun and significantly significantly improves.After its reason may is that the α position of acrylamide introduces halogen introducing, under the premise not affecting drug absorption so that the metabolic rate of double bond is subject to a degree of suppression, and then improves blood drug level and improve bioavailability.

Embodiment 28: use compound 5-2 to treat class and divide wet arthritis

In the mouse model of rheumatoid arthritis, have rated the in vivo efficacy of compound 5-2, in Balb/c mice, by giving anti-collagen antibody and lipopolysaccharide-induced arthritis (Nandakumar etc., Am.J.Pathol.2003,163:1827-1837).Concrete grammar was as follows: at the 0th day, injected the ChemicomAb mixture of the anti-II collagen type of 100mg/kg in female Balb/c mouse vein, at the 1st day, and peritoneal injection 1.25mg/kg lipopolysaccharide.At the 2nd day to 12 days, by the compound 5-2 of 10mg/kg, every day oral administration 1 time.Result shows, compound 5-2 has obvious internal anti-class and divides the effect of wet arthritis, specifically, the inflammatory cell infiltration that occurs in model group, synovial hyperplasia, inflammatory granulation tissue are formed, the phenomenon such as slough appearance after compound 5-2 treats be improved significantly.

Claims (10)

1. a bruton's tyrosine kinase inhibitor, it is characterised in that it has the structure of formula I:
And optical isomer or its pharmaceutically acceptable salt or solvate, wherein: Ra, Rb, Rc are independently selected from H, halogen ,-CF3、-CN、-NO2、OH、NH2、-L-C1-C6Alkyl ,-L-C1-C6Thiazolinyl, heteroaryl substituted or non-substituted for-L-or the substituted or non-substituted aryl of-L-, wherein L is key, O, S ,-S (=O) ,-S (=O)2、NH、C(O)、CH2,-NHC (O) O ,-NHC (O) or-C (O) NH,
X is selected from fluorine, chlorine and bromine.
2. a bruton's tyrosine kinase inhibitor, it is characterised in that it has the structure of formula II:
And optical isomer or its pharmaceutically acceptable salt or solvate, wherein: each Rd is H, halogen ,-CF independently3、-CN、-NO2、-OH、C1-C3Alkoxyl or-NH2, X is selected from fluorine, chlorine and bromine.
3. a bruton's tyrosine kinase inhibitor, it is characterised in that it has the structure of general formula III:
And optical isomer or its pharmaceutically acceptable salt or solvate, wherein: X is selected from fluorine, chlorine and bromine.
4. a pharmaceutical composition, described pharmaceutical composition comprises at least one active component and one or more pharmaceutically acceptable carrier or excipient, described active component be in claims 1 to 3 bruton's tyrosine kinase inhibitor compound as described in any one, as described in compound optical isomer, as described in compound or its optical isomer pharmaceutically acceptable salt, as described in compound or its optical isomer solvate in any one or arbitrarily multiple.
5. in claims 1 to 3, bruton's tyrosine kinase inhibitor compound as described in any one and optical isomer thereof or its pharmaceutically acceptable salt or solvate suppress the application in the medicine of disease, obstacle or disease that bruton's tyrosine kinase activity or treatment benefit from the suppression of bruton's tyrosine kinase activity in preparation.
6. in claims 1 to 3 bruton's tyrosine kinase inhibitor compound as described in any one and optical isomer thereof or its pharmaceutically acceptable salt or solvate preparation individually or and other drug application in the medicine for the treatment of cell proliferative diseases is used in combination.
7. in claims 1 to 3 bruton's tyrosine kinase inhibitor compound as described in any one and optical isomer thereof or its pharmaceutically acceptable salt or solvate preparation individually or and other drug application in the medicine for the treatment of cancer is used in combination.
8. in claims 1 to 3 bruton's tyrosine kinase inhibitor compound as described in any one and optical isomer thereof or its pharmaceutically acceptable salt or solvate preparation individually or and other drug application in the medicine for the treatment of autoimmune disease is used in combination.
9. in claims 1 to 3 bruton's tyrosine kinase inhibitor compound as described in any one and optical isomer thereof or its pharmaceutically acceptable salt or solvate preparation individually or and other drug application in the medicine for the treatment of lupus erythematosus is used in combination.
10. the method preparing bruton's tyrosine kinase inhibitor as claimed in claim 1, it is characterised in that comprise the steps:
Compound 1 and R1B(OH)2Under potassium phosphate, palladium catalyst and suitable solvent or mixed solvent such as dioxane/water exist; back flow reaction 24 hours; the compound 2 obtained is under triphenyl phosphorus, DIAD and suitable solvent such as THF exists; it is obtained by reacting compound 3 with the 3-hydroxy piperidine of Boc protection; key intermediate 4 is prepared in hydrolysis in acid condition subsequently; this key intermediate under DCC and suitable solvent such as DCM exist, and substitutional crylic acid fragment condensation reaction, obtain compound of Formula I.
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