CN107383013A

CN107383013A - Pyrazolopyrimidine derivative as BTK inhibitor and preparation method thereof and pharmaceutical composition

Info

Publication number: CN107383013A
Application number: CN201710277829.XA
Authority: CN
Inventors: 吴予川
Original assignee: Zhejiang Chuan Pharmaceutical Technology Co Ltd
Current assignee: Suzhou Sinoway Pharmaceutical Technology Co.,Ltd.
Priority date: 2016-05-16
Filing date: 2017-04-25
Publication date: 2017-11-24
Anticipated expiration: 2037-04-25
Also published as: CN107383013B; CN111153906B; WO2017198049A1; CN111153906A

Abstract

Pyrazolopyrimidine derivative as BTK inhibitor and preparation method thereof and pharmaceutical composition.The invention discloses novel pyrazolopyrimidines compound of the one kind as shown in formula (I), and its stereoisomer, or pharmaceutically acceptable salt, or solvate, or prodrug, wherein the definition of each substituent refers to specification.In addition, the invention also discloses the preparation method of above-claimed cpd, medical composition and its use.

Description

Pyrazolopyrimidine derivative as BTK inhibitor and preparation method thereof and medicine group Compound

Technical field

The invention belongs to field of pharmaceutical chemistry technology, specifically, be related to it is a kind of it is new and effective, selectivity is good, it is good to have Good pyrazolopyrimidine analog derivative pharmacokinetic property, as BTK inhibitor and preparation method thereof and drug regimen Thing.

Background technology

Protein kinase is the maximum family in biology enzyme in human body, including surpasses 500 kinds of protein.Especially, for junket ammonia Acid kinase, the phenol functional group on its tyrosine residue can be phosphorylated, so as to play the effect of important biological signal conduction.Junket Histidine kinase family possesses control cell growth, migration and the member of differentiation.Abnormal kinase activity oneself through being elucidated with being permitted More human body diseases are closely related, and these diseases include cancer, autoimmune disease and inflammatory disease.

Bruton's tyrosine kinase (BTK) is a kind of cytoplasmic, non-receptor EGFR-TK, belongs to TEC kinase families (altogether Have 5 members BTK, TEC, ITK, TXK, BMX) a member.BTK genes are located at the Xq21.33-Xq22 of x-chromosome, share outside 19 Aobvious son, across 37.5kb genomic DNAs.

In addition to T cell and thick liquid cell, BTK is expressed on nearly all hematopoietic cell, is especially occurred in bone-marrow-derived lymphocyte, Essential effect is played in differentiation, signal and existence.B cell is activated by B-cell receptor (BCR), and BTK exists Conclusive effect is served in BCR signal paths.After BCR in B cell is activated, BTK activation can be caused, then caused Phospholipase C (PLC) concentration increase in downstream, and activate IP3 and DAG signal paths.This signal path can promote cell Propagation, adhesion and survival.BTK gene mutations can cause a kind of rare heredity B cell specific immunity deficiency disorders, be claimed For X- linked agammaglobulinemias (X-Iinked agammaglobulinemia, XLA).In this disease, BTK's Function is suppressed, so as to result in the generation of B cell or anacmesis.Male with XLA diseases, it is substantially thin without B in vivo Born of the same parents, circulating antibody is also seldom, serious or even fatal infection easily occurs.This effectively demonstrates BTK in the growth of B cell and divided Played an important role in change.

Small molecule BTK inhibitor can be combined with BTK, suppressed BTK autophosphorylations, prevented BTK activation.This can be blocked The signal transduction of BCR paths, suppress the propagation of B lymphoma cells, the adhesion of oncocyte is destroyed, so as to promote withering for oncocyte Die.And inducing cell apoptosis.This makes BTK turn into noticeable drug target in the relevant cancer of B cell, especially for B cell lymphoma and leukaemia, such as NHL (NHL), chronic lymphocytic leukemia (CLL) and anti-recurrence Property or intractable lymphoma mantle cell (MCL) etc..

BTK inhibitor can also suppress B cell autoantibody and thin except that can resist B cell lymphoma and leukaemia The generation of intracellular cytokine.In autoimmune disease, B cell present autoantigen, promote T cell activation secretion cause inflammation because, Both tissue damage is caused, while activates B cell again and produces lot of antibodies, triggers autoimmune response.T and B cell interaction Feedback regulation chain is formed, causes autoimmune response out of control, aggravates Histopathological lesions.So BTK can be used as autoimmunity Property disease, such as rheumatoid arthritis, systemic loupus erythematosus (SLE), anaphylactia (such as the disease such as esophagitis) Drug target.

In addition also have been reported that BTK inhibitor can be combined with chemotherapeutic or immunologic test point inhibitor, it is right in clinical test A variety of solid tumors show preferable therapeutic effect.

In the medicine listed at present, it is one by Pharmacyclics and Johson ＆ Johnson's joint development to replace Buddhist nun according to Shandong The irreversible BTK inhibitor of kind, oneself obtained FDA approvals respectively in November, 2013 and 2 months 2014, for treating jacket cell lymph Knurl (MCL) and chronic lymphatic leukemia (CLL).Irreversible inhibitor, it is to compare firm covalent bond (covalent Bond) a kind of chemicals combined with the group in zymoprotein, irreversible inhibitor can generally make enzyme lose activity, so as to Play its unique high biological activity." breakthrough " new drug is set to by FDA for Buddhist nun according to Shandong, it by with half Guang ammonia in BTK The sulfydryl of acid reacts, and forms covalent bond, BTK enzymes is inactivated and is played curative effect.However, according to Shandong for Buddhist nun in administration process In, easily it is metabolized and (is metabolized oxydasis and is metabolized to dihydroxylated product or by other enzyme containing sulfydryl, cysteine, gluathiones The attacks such as peptide and inactivate) and influence drug effect.It is daily that its clinical administration dosage has reached 560mg, and makes patient's burden.This Outside, certain inhibitory action, the especially suppression to EGFR also cause tighter some kinases in addition to BTK for Buddhist nun according to Shandong The adverse reactions such as fash, diarrhoea again.Therefore, this area still needs to develop the medicine generation that new class is highly efficient, selectivity is good, good The BTK inhibitor of kinetic property is used for the treatment of relevant disease.

The content of the invention

The present inventor have developed a kind of novel pyrazolopyrimidines analog derivative, and these noval chemical compounds are protein kinase B TK Effectively, the high inhibitor of safety, selectivity.

It is an object of the invention to provide a kind of novel pyrazolopyrimidines analog derivative.It is that a kind of new covalent bond suppresses Agent, by changing itself and cysteine reactivity, to improve the compatibility with target, to improve curative effect, selectivity and security.

Second object of the present invention is to provide the preparation method of said derivative.

Third object of the present invention is to provide the pharmaceutical composition containing said derivative.

Fourth object of the present invention is to provide the purposes of said derivative.

Specifically, in a first aspect, in embodiments of the invention, the invention provides a kind of novel pyrazolopyrimidines Compound, as shown in formula (I), its stereoisomer, dynamic isomer, or pharmaceutically acceptable salt, or solvate, or it is preceding Medicine：

Here,

N, m are independently taken from 0,1 or 2；

L is O ,-C (O)-,-C (O) NH- ,-CH₂-, S, S (O), NH or S (O)₂；

A is taken from substituted or unsubstituted phenyl ring or substituted or unsubstituted hetero-aromatic ring, and with the company of parent nucleus and L Connecing site can be optional；

B is independently taken from substituted or unsubstituted cycloaliphatic ring, substituted or unsubstituted heterocycle, substituted or unsubstituted benzene Ring or substituted or unsubstituted hetero-aromatic ring, and can be optional with L connection site；

R₁And R₂It is each independently selected from hydrogen, unsubstituted C1-C4 alkyl, halogen, cyano group, or R₁And R₂With their phases With carbon atom together with form ternary carbocyclic ring or quaternary carbocyclic ring, or R₁And R₂Merge into oxo base；

Y be selected from cyano group or

R₃、R₄、R₅And R₆It is each independently selected from hydrogen, unsubstituted C1-C4 alkyl, C1-C4 alkyl, the C1- of hydroxyl substitution C4 alkoxy C 1-4 alkyl, halogen, cyano group or-(CH₂)_qN(R^aR^b), here, q 1,2,3 or 4, R^aAnd R^bIndependently of one another Selected from hydrogen, unsubstituted C1-C4 alkyl；

And provide, work as R₁And R₂When being all hydrogen, A is phenyl, and L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- bis- Fluorophenyl；Y is 4- hydroxyl but-2-enes acyl group, butyl- 2- alkynes acyl group or cyano group；

Or, work as R₁And R₂When being all hydrogen, A is phenyl, and L is C (O)-or-CH₂-, B is morpholine -4- bases；Y is cyano group；

Or, work as R₁And R₂When being all hydrogen, A is pyrazolyl, and L is S (O)₂Or-CH₂-, B is phenyl or cyclopropyl；Y is cyanogen Base；

Or, work as R₁And R₂When being all hydrogen, A is pyridine radicals, and L is NH or O, B are pyrans -4- bases or cyclohexyl；Y is cyano group.

In one embodiment of the present invention, one kind Pyrazolopyrimidine compound as shown in formula (I) provided by the invention, Wherein, n, m are independently taken from 0,1 or 2；L is O ,-C (O)-,-C (O) NH- ,-CH₂-, NH or S, more preferably O ,-C (O) NH-, NH.

In one embodiment of the present invention, one kind Pyrazolopyrimidine compound as shown in formula (I) provided by the invention, Wherein, A is taken from substituted or unsubstituted phenyl ring or hetero-aromatic ring, and can be optional with parent nucleus and L connection site；B is independent Ground be taken from substituted or unsubstituted cycloaliphatic ring, substituted or unsubstituted heterocycle, substituted or unsubstituted phenyl ring or substitution or Unsubstituted hetero-aromatic ring, and can be optional with L connection site；Here：

The substituted phenyl ring refers to that optional position is substituted by optional substituents on phenyl ring, the substituent choosing From hydrogen, methyl, methoxyl group, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano group；Preferably, the substituted phenyl takes for fluorine The phenyl of phenyl or the chlorine substitution in generation, more preferably 2,4- difluorophenyls or 4- chlorphenyls；

The unsubstituted hetero-aromatic ring refers to furans, pyrroles, thiophene, oxazole, isoxazole, pyrazoles, imidazoles, thiazole, different thiophene Azoles, oxadiazoles, triazole, thiadiazoles, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine；The substituted hetero-aromatic ring refers to Optional position is substituted by optional substituents on above group, the substituent be selected from hydrogen, methyl, methoxyl group, fluorine, Chlorine, trifluoromethyl, trifluoromethoxy or cyano group；It is highly preferred that the substituted pyridine is chloro-pyridine, particularly preferably is 4- Chloro- pyridine -2- bases；

The unsubstituted cycloaliphatic ring refers to cyclopropane, cyclobutane, pentamethylene, hexamethylene, cycloheptane, cyclooctane；It is described Substituted cycloaliphatic ring refers to that optional position is substituted by optional substituents on above group, the substituent be selected from hydrogen, Methyl, methoxyl group, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano group；

The unsubstituted heterocycle refer to tetrahydrofuran, oxinane, nafoxidine, piperidines, Wherein w It is derived from 0,1 or 2；The substituted heterocycle refers to that optional position is substituted by optional substituents on above group, described Substituent is selected from hydrogen, methyl, methoxyl group, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano group.

In one embodiment of the present invention, one kind Pyrazolopyrimidine compound as shown in formula (I) provided by the invention, Wherein it is preferred to R₁And R₂One of them is hydrogen and another is that unsubstituted C1-C4 alkyl is (methyl, ethyl, n-propyl, different Propyl group, normal-butyl, isobutyl group or the tert-butyl group), or R₁And R₂Cyclopropyl is formed together with the carbon atom being connected with them；More preferably Ground, R₁And R₂All it is hydrogen, either one of them is hydrogen and another is methyl or R₁And R₂The carbon atom one being connected with them Rise and form cyclopropyl.

The present invention a kind of preferred embodiment in, the invention provides one kind as shown in formula (II) pyrazolopyrimidine Compound, its stereoisomer, dynamic isomer, or pharmaceutically acceptable salt, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I)；

And provide, A is phenyl, and L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- difluorophenyls；Y is 4- hydroxyls But-2-ene acyl group, butyl- 2- alkynes acyl group or cyano group；

Or, A is phenyl, L is C (O)-or-CH₂-, B is morpholine -4- bases；Y is cyano group；

Or, A is pyrazolyl, L is S (O)₂Or-CH₂-, B is phenyl or cyclopropyl；Y is cyano group；

Or, A is pyridine radicals, L is NH or O, B are pyrans -4- bases or cyclohexyl；Y is cyano group.

In a kind of more preferred of the present invention, one kind pyrazolopyrimidine as shown in formula (II) provided by the invention Compound is one kind in following compounds：

Here, L, B and Y are as defined above the formula of stating (I) in formula (II-1) or (II-2)；

And provide, L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- difluorophenyls；Y is 4- hydroxyl but-2-ene acyls Base, butyl- 2- alkynes acyl group or cyano group；

Or, L is C (O)-or-CH₂-, B is morpholine -4- bases；Y is cyano group.

In a kind of preferred embodiment of the present invention, the invention provides pyrazolo of the one kind as shown in formula (III) is phonetic Acridine compound, its stereoisomer, dynamic isomer, or pharmaceutically acceptable salt, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I).

In a kind of more preferred of the present invention, one kind pyrazolo as shown in formula (III) provided by the invention is phonetic Acridine compound is one kind in following compounds：

Here, L, B and Y are as defined above the formula of stating (I) in formula (III-1), (III-2), (III-3) and (III-4).

In a kind of preferred embodiment of the present invention, the invention provides pyrazolopyrimidine of the one kind as shown in formula (IV) Compound, its stereoisomer, dynamic isomer, or pharmaceutically acceptable salt, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I).

In a kind of more preferred of the present invention, a kind of pyrazolo as shown in formula (IV) provided by the invention is phonetic Acridine compound is one kind in following compounds：

Here, L, B and Y are as defined above the formula of stating (I) in formula (IV-1) or (IV-2).

In a kind of more preferred of the present invention, formula (I) provided by the invention, (II), (II-1), (II-2), (III), (III-1), (III-2), (III-3), (III-4), (IV), (IV-1) or (IV-2) compound, wherein, L O；

B isWith

Y be selected from cyano group or

R₃、R₄、R₅And R₆It is each independently selected from unsubstituted C1-C4 alkyl, C1-C4 alkyl, the C1-C4 alkane of hydroxyl substitution Epoxide C1-4 alkyl, halogen, cyano group or-(CH₂)_qN(R^aR^b), here, q 1,2,3 or 4, R^aAnd R^bIt is each independently selected from Hydrogen, unsubstituted C1-C4 alkyl.

In a kind of particularly preferred embodiment of the present invention, the present invention provides a kind of one kind in following compounds Pyrazolopyrimidines, or its pharmaceutically acceptable salt, or solvate, or prodrug：

In embodiments of the invention, described " pharmaceutically acceptable salt " refers to pharmaceutically acceptable acid-addition salts With pharmaceutically acceptable base addition salts：

" the pharmaceutically acceptable acid-addition salts " are the biological effectivenesses that refer to retain free alkali and secondary without other Effect, the salt formed with inorganic acid or organic acid.Inorganic acid salt include but is not limited to hydrochloride, hydrobromate, sulfate, Phosphate etc.；Acylate includes but is not limited to formates, acetate, propionate, glycollate, gluconate, lactate, grass Hydrochlorate, maleate, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoic acid Salt, mesylate, tosilate and salicylate etc..These salt can be prepared by methods known in the art.

" the pharmaceutically acceptable base addition salts " be refer to keep free acid biological effectiveness and without other pairs The salt of effect.These salt are by the way that inorganic base or organic base are prepared added to free acid.Salt bag derived from inorganic base Include but be not limited to sodium salt, sylvite, calcium salt and magnesium salts etc..Salt derived from organic base includes but is not limited to ammonium salt, triethylamine salt, relies Propylhomoserin salt, arginine salt etc..These salt can be prepared by methods known in the art.

In embodiments of the invention, described " solvate " refers to the cooperation that the compound of the present invention is formed with solvent Thing.They either reaction or Precipitation or are crystallized out from solvent in a solvent.For example, one is matched somebody with somebody with what water was formed Compound is referred to as " hydrate ".

In embodiments of the invention, compound of the invention can contain one or more chiral centres, and with not Same optical active forms are present.When compound contains a chiral centre, compound includes enantiomter.Present invention bag Include the mixture of both isomers and isomers, such as racemic mixture.Enantiomter can be by known in the art Method is split, such as the methods of crystallization and chiral chromatogram., can when formula (I) compound contains more than one chiral centre Diastereoisomer be present.The present invention includes the mixing of the optically pure specific isomers and diastereoisomer split Thing.Diastereoisomer can be split by means known in the art, such as crystallization and preparation chromatogram.

In embodiments of the invention, described prodrug refers to known amino protecting group and carboxyl-protecting group, in life It is hydrolyzed under the conditions of reason or discharges to obtain parent compound via enzyme reaction.Medicament preparation can refer to before specific (Saulnier,M.G.；Frennesson,D.B.；Deshpande,M.S.；Hansel,S.B and Vysa, D.M.Bioorg.Med.Chem Lett.1994,4,1985-1990.Greenwald,R.B.；Choe,Y.H.；Conover, C.D.；Shum,K.；Wu,D.；Royzen,M.J.Med.Chem.2000,43,475.).

Second aspect, the invention provides the preparation method of the above-mentioned Pyrazolopyrimidine compound as shown in formula (I), including Following steps：

(1) formula (V) compound and formula (VI) compound are reacted, obtains formula (VII) compound；

(2) formula (VII) compound and formula (VIII) compound are reacted, obtains formula (IX) compound；

(3) formula (IX) compound is obtained into formula (X) compound through Deprotection PG；

(4) formula (X) compound and formula (XI) compound are reacted, obtains formula (I) compound；

What is be related in above-mentioned formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI) takes For base R₁、R₂, L, A, B, Y and n, m be as defined above the formula (I) in face, PG is that (suitable amino protecting group includes amino protecting group Acyl group (such as acetyl group), carbon benzamide type (carbamate) (such as 2', 2', 2'- tri-chloroethoxy base carbonyl, Cbz benzyloxies Carbonyl or BOC tert-butoxycarbonyls) and aryl alkyl (such as Bn benzyls), its can in due course by hydrolysis (such as using acid, Such as the trifluoroacetic acid in the hydrochloric acid or dichloromethane in dioxane) or pass through the mode of reducing (such as benzyl or benzyloxycarbonyl Hydrogenolysis or remove 2', 2', 2'- tri-chloroethoxy bases carbonyl using the zinc reproducibility in acetic acid) and remove.Other suitable ammonia Base protection group includes trifluoroacetyl group (- COCF₃), it can remove benzyloxy, carbonyl or tert-butoxy by the hydrolysis of base catalysis Carbonyl, those skilled in the art refer to T.W.Greene ' Protective Groups in Organic Synthesis ' (the 4th edition, J.Wiley and Sons, 2006)), X is chlorine, bromine or hydroxyl.

In embodiments of the invention, the above-mentioned Pyrazolopyrimidine compound as shown in formula (I) provided by the invention Preparation method, wherein, formula (V) compound may be referred to following method to prepare：

In embodiments of the invention, present invention also offers the centre synthesized for above-mentioned Pyrazolopyrimidine compound Body compound, include but is not limited to：

Wherein, Bn is benzyl, and Boc is tertiary butyloxycarbonyl acyl group.

The third aspect, the invention provides a kind of above-mentioned pyrazolopyrimidine of the one or more present invention comprising effective dose Compound or its stereoisomer, dynamic isomer, the pharmaceutical composition of solvate or its pharmaceutically acceptable salt, it is described Pharmaceutical composition also includes pharmaceutically acceptable auxiliary material.

The present invention pharmaceutical composition can be configured to solid-state, semisolid, liquid or gaseous state preparation, as tablet, capsule, Pulvis, granule, paste, solution, suppository, injection, inhalant, gel, microballoon and aerosol.

The pharmaceutical composition of the present invention can be prepared by known method in pharmaceutical field.For example, prepare drug regimen The practical methods of thing are known to the skilled artisan see, for example, The Science and Practice of Pharmacy (Pharmaceutical Sciences are with putting into practice), 20th Edition (Philadelphia College of Pharmacy and Science,2000)。

The method of administration of the pharmaceutical composition of the present invention includes but is not limited to oral, local, percutaneous, muscle, vein, suction Enter, parenteral, sublingual, rectum, vagina and intranasal.For example, be adapted to be administered orally formulation include capsule, tablet, granule with And syrup etc..Formula (I) compound of the invention included in these preparations can be solid powder or particle；It is water-based or non-aqueous Solution or suspension in liquid；Water-In-Oil or oil-in-water emulsion etc..Above-mentioned formulation can by reactive compound with it is a kind of or Variety carrier or auxiliary material are made via general practice of pharmacy.Above-mentioned carrier needs simultaneous with reactive compound or other auxiliary materials Hold.For solid pharmaceutical preparation, conventional non-toxic carrier includes but is not limited to mannitol, lactose, starch, magnesium stearate, cellulose, Portugal Grape sugar, sucrose etc..Carrier for liquid preparation includes but is not limited to water, physiological saline, D/W, ethylene glycol and gathered Ethylene glycol etc..Reactive compound can form solution or suspension with above-mentioned carrier.Specific administering mode and formulation depend on Order of severity of compound physicochemical property in itself and applied disease etc..Those skilled in the art can be according to above-mentioned factor And the knowledge that has with reference to its own determines specific method of administration.It see, for example,：Li Jun,《Clinical pharmacology》, the people Hygienic publishing house, 2008.06；Ding Yufeng, by Clinical Dosage Form Factors and the rational use of medicines, medical Leader, 26 (5), 2007；Howard C.Ansel, Loyd V.Allen, Jr., Nicholas G.Popovich write, and Jiang Zhiqiang master translates,《Pharmaceutical dosage form is with giving medicine body System》, China Medical Science Press, 2003.05.

The unit dosage forms that the pharmaceutical composition of the present invention can contain the active component of scheduled volume with per unit dose are present. Preferable units dosage composition is those of the active component containing daily dose or sub-doses or its appropriate fraction.Therefore, this Kind unit dose can be with administration in one day more than once.Preferable units dosage composition is to contain daily dose described herein above Or those of the active component of sub-doses (administration in one day is more than once) or its appropriate fraction.

The pharmaceutical composition of the present invention is prepared in a manner of meeting medical practice specification, quantitative and administration.Chemical combination of the present invention " therapeutically effective amount " of thing is by the specific illness to be treated, individual, the cause of illness, the target spot of medicine and to prescription for the treatment of The factors such as formula determine.Generally, the dosage through parenteral administration can be 1-200mg/kg, and the dosage of oral administration can be 1- 1000mg/kg。

The scope of effective dose presented herein is not meant to limit the scope of the present disclosure, but represents preferable agent Measure scope.But most preferred dosage can be directed to individual one and be adjusted, this is that those skilled in the art understand and can (such as being write refering to Berkow et al., Merck handbooks, the 16th edition, Merck companies, Rahway, N.J., 1992) determined.

Fourth aspect, the invention provides above-mentioned Pyrazolopyrimidine compound or its stereoisomer, dynamic isomer, molten Agent compound or its pharmaceutically acceptable salt are being prepared for preventing or treating by the purposes in medicine disease mediated BTK.

The invention provides a kind of method of suppression BTK activity, and comprising giving, the above-mentioned pyrazolo of the biosystem present invention is phonetic Acridine compound or its stereoisomer, dynamic isomer, solvate or its pharmaceutically acceptable salt or comprising in the present invention State the medicine of Pyrazolopyrimidine compound or its stereoisomer, dynamic isomer, solvate or its pharmaceutically acceptable salt Compositions.

In some embodiments, the biosystem is enzyme, cell or mammal.

Present invention also offers a kind of method prevented or treat the disease by BTK mediations, it is included to trouble in need Person be given in combination treatment effective dose one or more the present invention above-mentioned Pyrazolopyrimidine compounds or its stereoisomer, Dynamic isomer, solvate or its pharmaceutically acceptable salt and one or more are selected from following medicine：Immunomodulator, Immunologic test point inhibitor, glucocorticoid, NSAIDs, Cox-2 specific inhibitors, TNF-α associated proteins, interference Element, interleukins and chemotherapeutics.

In embodiments of the present invention, it is described by BTK mediation disease include autoimmune disease, inflammatory disease, Heteroimmune implementations or disease, thromboembolic disorders and cancer.In some embodiments, it is thin to include B for the cancer It is born of the same parents' property chronic lymphocytic leukemia, acute lymphatic leukemia, NHL, Hodgkin lymphoma, acute Myelogenous leukemia, diffusivity large B cell lymphoid tumor, Huppert's disease, lymphoma mantle cell, SLL, Waldenstrom's macroglobulinemia, solid tumor.In some embodiments, the autoimmune disease and inflammatory disease are selected from Rheumatoid arthritis, osteoarthritis, adolescent arthritis, COPD, multiple sclerosis, systemic red yabbi Sore, psoriasis, psoriatic arthritis, Crohn disease, ulcerative colitis and irritable bowel syndrome.In some specific embodiment parties In formula, the heteroimmune implementations or disease include graft versus host disease(GVH disease), transplanting, blood transfusion, allergic reaction, allergy, I Type hypersensitivity, allergic conjunctivitis, allergic rhinitis or atopic dermatitis.

Experimental data proves that above-mentioned Pyrazolopyrimidine compound provided by the invention is protein kinase B TK effective, safety Inhibitor.

Embodiment

Experiment, synthetic method and involved intermediate described below is that the present invention is illustrated, and is not intended to limit The scope of the present invention.

Initiation material used in experiment or purchase are from reagent suppliers or via method well known in the art in the present invention Prepared by known raw material.Unless otherwise indicated, the embodiments herein applies following conditions：

The unit of temperature is degree Celsius (DEG C)；The definition of room temperature is 18-25 DEG C；

Organic solvent uses anhydrous magnesium sulfate or anhydrous sodium sulfate drying；Using Rotary Evaporators in the case where depressurizing Elevated Temperature Conditions Be spin-dried for (such as：15mmHg, 30 DEG C)；

Flash column chromatography uses 200-300 mesh silica gel to represent thin-layered chromatography as carrier, thin-layer chromatography when separating；

Under normal circumstances, the progress of reaction is monitored by thin-layer chromatography or LC-MS；

The identification of final products is by nuclear magnetic resonance (Bruker AVANCE 300,300MHz) and LC-MS (Bruker Esquine 6000, Agilent 1200series) complete.

Embodiment 1

(R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) piperidines -1- nitriles (WS-300) preparation

The first step (R) -3- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-) piperidines -1- t-butyl formates Preparation：

(S) -3- hydroxy piperidines -1- t-butyl formates (1.7g, 8.5mmol) and iodo- 1H- pyrazolos [3,4-d] pyrimidines of 3- - 4- amine (2g, 7.7mmol) is dissolved in tetrahydrofuran (100mL).Ice bath adds triphenylphosphine (2.5g, 9.5mmol) next time, slowly Diisopropyl azodiformate (2.1g, 10.40mmol) is added dropwise.Ice bath is removed, reaction solution is stirred at room temperature 18 hours, and decompression is steamed It is dry.Crude product crosses silicagel column purifying (ethyl acetate:Petroleum ether=1：1) product (R) -3- (iodo- 1H- pyrazoles of 4- amino -3-, is obtained And [3,4-d] pyrimidine -1- bases) piperidines -1- t-butyl formates (3.5g, 100%).

Second step (R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] Pyrimidine -1- bases) piperidines -1- t-butyl formates preparation：

(R) -3- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-) piperidines -1- t-butyl formates (1.3g, 2.9mmol) and the chloro- 2- of 5- (4- (4,4,5,5- tetramethyl -1,3,2- dioxaborinate -2- bases) phenoxy group) pyridine (1g, 3.0mmol) it is dissolved in 95% ethanol (50mL).Add sodium carbonate (600mg, 5.7mmol) and two (triphenylphosphine) palladium chlorides (200mg, 0.28mmol), the lower back flow reaction of argon gas protection 18 hours, evaporated under reduced pressure.Crude product ethyl acetate (50mL) and water (50mL) is extracted.Organic phase anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Residue prepares plate purifying (dichloromethane:First Alcohol=50：1) product (R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-, are obtained D] pyrimidine -1- bases) piperidines -1- t-butyl formates (960mg, 63%).

3rd step (R) -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (piperidines -3- bases) -1H- pyrazolos [3, 4-d] pyrimidine -4- amine preparation：

(R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) piperidines -1- t-butyl formates (960mg, 1.8mmol) are dissolved in tetrahydrofuran (10mL), add concentrated hydrochloric acid (0.5mL), reaction 60 DEG C of liquid stirs 3 hours, evaporated under reduced pressure obtain crude product (R) -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (piperidines - 3- yls) -1H- pyrazolos [3,4-d] pyrimidine -4- amine hydrochlorates (1.1g, 100%).It is directly used in and reacts in next step.

4th step (R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] Pyrimidine -1- bases) piperidines -1- nitriles preparation：

(R) -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (piperidines -3- bases) -1H- pyrazolos [3,4-d] are phonetic Pyridine -4- amine hydrochlorates (500mg, 1.1mmol) are dissolved in DMF (5mL), add cesium carbonate (600mg, 1.9mmol) it is stirred at room temperature 5 hours with cyanogen bromide (400mg, 3.8mmol), reaction solution, reaction solution pours into ethyl acetate (50mL), Wash (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Crude product prepares plate purifying (dichloromethane:Methanol=50： 1), obtain product (R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls) piperidines -1- nitriles (130mg, 27%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.29-8.25(m,2H),8.02-7.99(m,1H),7.72(d,J =8.4,2H), 7.32 (d, J=8.4,2H), 7.18 (d, J=8.8,1H), 4.90-4.85 (m, 1H), 3.64-3.62 (m, 1H),3.56-3.53(m,1H),3.42-3.39(m,1H),3.20-3.15(m,1H),2.17-2.08(m,2H),1.95-1.81 (m,2H).

MS:m/z 446.8[M+1]

Embodiment 2

((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 3- by (R, E) -1- Pyridine -1- bases) piperidin-1-yl) -4- hydroxyl but-2-ene -1- ketone (WS-301) preparation

Using the product of the three-step reaction of embodiment 1 as starting material, embodiment 2 is prepared by following step：

The first step (R, E) -1- (3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3, 4-d] pyrimidine -1- bases) piperidin-1-yl) and -4- hydroxyl but-2-ene -1- ketone preparation：

(R) -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (piperidines -3- bases) -1H- pyrazolos [3,4-d] are phonetic Pyridine -4- amine hydrochlorates (400mg, 0.87mmol) and 4- hydroxyl -2- butenoic acids (130mg, 1.27mmol) are dissolved in tetrahydrofuran (20mL), adds HATU (550mg, 1.45mmol) and triethylamine (200mg, 1.98mmol), reaction solution are stirred at room temperature 18 hours, Evaporated under reduced pressure.Residue is dissolved in ethyl acetate (50mL), washes (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure. Crude product prepares plate purifying (dichloromethane:Methanol=10：1) product compound (R, E) -1- (3- (4- amino -3- (4-, are obtained ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) piperidin-1-yl) -4- hydroxyl butyl- 2- Alkene -1- ketone (110mg, 22%).

H-NMR(400MHz,DMSO-d6):δ ppm 8.43 (s, 1H), 8.26 (d, J=2.8,1H), 8.03-7.99 (m, 1H), 7.72 (d, J=8.0,2H), 7.34 (d, J=8.4,2H), 7.19 (d, J=8.8,2H), 6.80-6.42 (m, 2H), 4.76(brs,1H),4.57-4.54(m,0.5H),4.22-3.98(m,3.3H),3.75(brs,0.3H),3.23-3.21(m, 1H),3.05-3.03(m,0.4H),2.32-2.15(m,2H),1.96-1.93(m,1H),1.62-1.58(m,1H).

MS:m/z 506.2[M+1]

Embodiment 3

(R) -3- (4- amino -3- (4- (4- chlorophenoxies) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) piperidines - The preparation of 1- nitriles (WS-302)

Embodiment 3 is prepared since corresponding starting material using method similar to Example 1.

1H-NMR(400MHz,DMSO-d6):δ ppm 8.27 (s, 1H), 7.67-7.69 (d, J=8Hz, 2H), 7.46- 7.48 (d, J=8Hz, 2H), 7.14-7.20 (m, 4H), 4.84-4.89 (m, 1H), 3.64-3.65 (d, J=4Hz, 1H), 3.50-3.54(m,1H),3.38-3.41(m,1H),3.14-3.20(m,1H),2.08-2.18(m,2H),1.79-1.93(m, 2H).

MS:m/z 446.2[M+1]

Embodiment 4

(R, E) -1- (3- (4- amino -3- (4- (4- chlorophenoxies) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) Piperidin-1-yl) -4- hydroxyl but-2-ene -1- ketone (WS-303) preparation

Embodiment 4 is prepared since corresponding starting material using method similar to Example 2.

1H-NMR(400MHz,DMSO-d6):δ ppm 8.43 (s, 1H), 7.66-7.68 (d, J=8Hz, 2H), 7.47- 7.49 (d, J=8Hz, 2H), 7.14-7.20 (m, 4H), 6.50-6.75 (m, 2H), 4.54-4.72 (m, 2H), 4.21-4.40 (m,6H),4.14(brs,1H),3.18-3.23(brs,1H),2.50(s,1H),2.39-2.42(m,1H),2.21-2.98(m, 1H),1.92(brs,1H),1.58-1.60(m,1H).

MS:m/z 505.2[M+1]

Embodiment 5

(R) -3- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) piperazine The preparation of pyridine -1- nitriles (WS-304)

Embodiment 5 is prepared since corresponding starting material using method similar to Example 1.

¹H-NMR(400MHz,DMSO-d6):δ8.27(s,1H),7.66-7.50(m,5H),7.40-7.36(m,1H), 7.35-7.11(m,3H),4.87-4.85(m,1H),3.65-3.60(m,1H),3.54-3.48(m,1H),3.41-3.38(m, 1H),3.19-3.16(m,1H),2.15-2.17(m,2H),1.90-1.82(m,2H).

MS:m/z 448.2[M+1]

Embodiment 6

(R, E) -1- (3- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) piperidin-1-yl) -4- hydroxyl but-2-ene -1- ketone (WS-305) preparation

Embodiment 6 is prepared since corresponding starting material using method similar to Example 2.

¹H-NMR(400MHz,CDCl₃-d6):δppm 8.46(s,1H),7.62-7.64(m,2H),7.16-7.18(m, 1H),7.06-7.18(m,2H),6.93-7.06(m,3H),6.51-6.63(m,1H),5.41-5.46(brs.,1H),4.82- 4.89(m,2H),4.28-4.38(m,2H),4.19-4.23(m,1H),3.36-3.77(m,1H),2.32-2.39(m,2H), 1.98-2.02(m,2H),2.32-2.39(m,2H).

MS:m/z 506.2[M+1]

Embodiment 7

5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - The preparation of pipecoline -1- nitriles (WS-306)

The preparation of first step 6- methyl piperidine -3- alcohol

5- hydroxy-2-methyls-pyridine (1.5g, 13.7mmol) is dissolved in addition 0.4g platinum dioxides in 20ml acetic acid, mixed Compound stirs 16 hours under the pressure of 50psi hydrogen.Drain solvent and obtain compound.

The preparation of second step 5- hydroxy-2-methyl piperidines -1- t-butyl formates

6- methyl piperidine -3- alcohol (1.10g, 7.8mmol) and sodium carbonate (1.6g, 15.7mmol) are added to dichloromethane In (100mL) solution, then di-tert-butyl dicarbonate (1.71g, 7.8mmol) is added drop-wise in above-mentioned reaction system, reaction solution React 18 hours at ambient temperature, thin-layer chromatography (petroleum ether:Ethyl acetate=3:1) detection reaction is finished, and reaction solution is extracted Take, filtrate is spin-dried for, and crosses post purifying, obtains product 5- hydroxy-2-methyl piperidines -1- t-butyl formates (1.7g).

3rd step 5- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-)-tertiary fourth of pipecoline -1- formic acid The preparation of ester：

5- hydroxy-2-methyl piperidines -1- t-butyl formates (7g, 32.6mmol) and the iodo- 1H- pyrazolos [3,4-d] of 3- are phonetic Pyridine -4- amine (8.5g, 32.6mmol) is dissolved in tetrahydrofuran (100mL).Ice bath add next time triphenylphosphine (11g, 41.5mmol), diisopropyl azodiformate (9.6g, 47.5mmol) is slowly added dropwise.Ice bath is removed, reaction solution is stirred at room temperature 18 Hour, evaporated under reduced pressure.Crude product crosses silicagel column purifying (ethyl acetate:Petroleum ether=1：1), obtaining product 5-, (4- amino -3- is iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- t-butyl formates (12.2g, 82%).

MS:m/z 459.2[M+1]

4th step 5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-pipecoline -1- t-butyl formates preparation：

5- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-)-pipecoline -1- t-butyl formates (1g, 2.2mmol) and the chloro- 2- of 5- (4- (4,4,5,5- tetramethyl -1,3,2- dioxaborinate -2- bases) phenoxy group) pyridine (800mg, 2.4mmol) it is dissolved in 95% ethanol (50mL).Add sodium carbonate (500mg, 4.7mmol) and two (triphenylphosphine) palladium chlorides (150mg, 0.21mmol), the lower back flow reaction of argon gas protection 18 hours, evaporated under reduced pressure.Crude product is in dichloromethane (50mL) and water Extracted in (50mL).Organic phase anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Residue prepares plate purifying (dichloromethane Alkane:Methanol=50：1) product 5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-, are obtained D] pyrimidine -1- bases)-pipecoline -1- t-butyl formates (700mg, 60%).

MS:m/z 536.3[M+1]

5th step 3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperidine -3- bases) -1H- pyrazolos The preparation of [3,4-d] pyrimidine -4- amine hydrochlorates：

5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - Pipecoline -1- t-butyl formates (700mg, 1.3mmol) are dissolved in tetrahydrofuran (10mL), add concentrated hydrochloric acid (1mL), reaction 60 DEG C of liquid stirs 3 hours, and evaporated under reduced pressure obtains crude product 3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperazines Pyridine -3- bases) -1H- pyrazolos [3,4-d] pyrimidine -4- amine hydrochlorates (550mg, 89%).It is directly used in and reacts in next step.

MS:m/z 436.4[M+1]

6th step 5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-pipecoline -1- nitriles preparation：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperidine -3- bases) -1H- pyrazolos [3,4-d] Pyrimidine -4- amine hydrochlorates (250mg, 0.53mmol) are dissolved in DMF (3mL), add cesium carbonate (250mg, 0.77mmol) it is stirred at room temperature 6 hours with cyanogen bromide (250mg, 2.36mmol), reaction solution, reaction solution pours into ethyl acetate (50mL), wash (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Crude product prepares plate purifying (dichloromethane:First Alcohol=50：1), obtaining product 5-, (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic Pyridine -1- bases)-pipecoline -1- nitriles (120mg, 49%).

¹H-NMR(400MHz,DMSO-d6):δ ppm 11.67 (brs, 1H), 8.20 (s, 1H), 8.06 (d, J=2.4, 1H), 7.68-7.64 (m, 3H), 7.28 (d, J=8.8,2H), 6.95 (d, J=8.4,1H), 6.43 (brs, 1H), 4.94- 4.91(m,1H),3.92-3.87(m,1H),3.48-3.44(m,3H),2.49-2.46(m,1H),2.02-1.88(m,3H), 1.37 (d, J=6.8,3H)

MS:m/z 461.4[M+1]

Embodiment 8

(E) ((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 5- by -1- Pyridine -1- bases)-pipecoline -1- bases) -4- hydroxyl but-2-ene -1- ketone (WS-307) preparation

Using the product of the step of embodiment 7 the 5th reaction as starting material, embodiment 8 is prepared by following step：

The first step (E) -1- (5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4- D] pyrimidine -1- bases)-pipecoline -1- bases) and -4- hydroxyl but-2-ene -1- ketone preparation：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperidine -3- bases) -1H- pyrazolos [3,4-d] Pyrimidine -4- amine hydrochlorates (250mg, 0.53mmol) and 4- hydroxyl -2- butenoic acids (100mg, 0.98mmol) are dissolved in tetrahydrofuran (10mL), adds HATU (300mg, 0.79mmol) and triethylamine (100mg, 0.99mmol), reaction solution are stirred at room temperature 18 hours, Evaporated under reduced pressure.Residue is dissolved in ethyl acetate (50mL), washes (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure. Crude product prepares plate purifying (dichloromethane:Methanol=10：1), obtain product (E) -1- (5- (4- amino -3- (4- ((5- chloropyridines - 2- yls) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -4- hydroxyl but-2-enes -1- Ketone (155mg, 50%).

¹H-NMR(400MHz,DMSO-d6):δ ppm 8.35 (d, J=7.6,1H), 8.14 (d, J=2.8,1H), 7.75- 7.69 (m, 3H), 7.31 (d, J=7.6,2H), 6.99-6.95 (m, 2H), 6.60-6.50 (m, 1H), 5.75 (brs, 1.7H), 5.05(brs,0.4H),4.82-4.70(m,1.6H),4.50-4.35(m,2.5H),4.10-4.05(m,0.5H),3.90- 3.80(m,0.5H),3.50-3.45(m,0.7H),3.10-3.00(m,0.2H),2.60-2.50(m,1.3H),2.07-1.75 (m,5.4H),1.43-1.25(m,3.2H).

MS:m/z 520.4[M+1]

Embodiment 9

5- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -2- first The preparation of phenylpiperidines -1- nitriles (WS-308)

Embodiment 9 is prepared since corresponding starting material using method similar to Example 7.

¹H-NMR(400MHz,DMSO-d6):8.39 (s, 1H), 7.68 (d, J=8.8,1H), 7.19-7.08 (m, 3H), 7.03-6.90(m,2H),5.57(brs,2H),4.98-4.94(m,1H),3.97-3.92(m,1H),3.57-3.46(m,2H), 2.56-2.51 (m, 1H), 2.06-1.95 (m, 3H), 1.43 (d, J=7.8,3H)

MS:m/z 462.3[M+1]

Embodiment 10

(4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by (2S, 5R) -5- Pyridine -1- bases)-pipecoline -1- nitriles (WS-309) and (2R, 5S) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) oxygen Base) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) and-pipecoline -1- nitriles (WS-310) preparation

Embodiment 10 be embodiment 7 the chiral fractionation of product after be made.Splitting condition is：Supercritical Fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-309 spectral datas：

MS:m/z 461.4[M+1]

WS-310 spectral datas：

MS:m/z 461.4[M+1]

Embodiment 11

(E) -1- ((2S, 5R) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3, 4-d] pyrimidine -1- bases)-pipecoline -1- bases) -4- hydroxyl but-2-ene -1- ketone (WS-311) and (E) -1- ((2R, 5S) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -2- methyl piperazines Pyridine -1- bases) -4- hydroxyl but-2-ene -1- ketone (WS-312) preparation

Embodiment 11 be embodiment 8 the chiral fractionation of product after be made.Splitting condition is：Supercritical Fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-311 spectral datas：

MS:m/z 520.4[M+1]

WS-312 spectral datas：

MS:m/z 520.4[M+1]

Embodiment 12

(2S, 5R) -5- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base)-pipecoline -1- nitriles (WS-313) and (2R, 5S) -5- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- Pyrazolo [3,4-d] pyrimidine -1- bases)-pipecoline -1- nitriles (WS-314) preparation

Embodiment 12 be embodiment 9 the chiral fractionation of product after be made.Splitting condition is：Supercritical Fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-313 spectral datas：

MS:m/z 462.3[M+1]

WS-314 spectral datas：

MS:m/z 462.3[M+1]

Embodiment 13

6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - The preparation of 4- azaspiros [2.5] octane -4- nitriles (WS-315)

The preparation of first step 4 hydroxybutyric acid methyl esters：

Dihydrofuran -2 (3H) -one (100g, 1.163mol) and triethylamine (460g, 4.65mol) are added to methanol In (1L) solution, reaction solution reacts 24 hours under conditions of 60 DEG C, thin-layer chromatography (petroleum ether:Ethyl acetate=2:1) detect Reaction is finished, and reaction solution is spin-dried for, and obtains 4 hydroxybutyric acid methyl esters (120g, 87.6%, yellow liquid), is directly used in next step Reaction.

The preparation of second step 4- oxobutyrates：

4 hydroxybutyric acid methyl esters (120g, 1.02mol) is added in dichloromethane (1.2L) solution, then by chloro-chromic acid Pyridine (330g, 1.53mol) is added in above-mentioned reaction solution, is reacted 12 hours under room temperature condition, thin-layer chromatography (petroleum ether:Second Acetoacetic ester=3:1) detection reaction finish, reaction solution is filtered by diatomite, is spin-dried for, obtain 4- oxobutyrates (60g, 50%, yellow liquid), it is directly used in and reacts in next step.

The preparation of 3rd step 4- hydroxyl -5- nitro methyl valerates：

In ice-water bath, by 4- oxobutyrates (60g, 0.46mol), nitromethane (42g, 0.69mol), tetrahydrofuran (300mL) and the tert-butyl alcohol (300mL) are added in reaction bulb, and potassium tert-butoxide (5g) then is slowly added into above-mentioned reaction system In, it is warmed to room temperature, reacts 2 hours, thin-layer chromatography (petroleum ether:Ethyl acetate=3:1) detection reaction finishes, and adds water (30mL) to quench Go out reaction, be spin-dried for solvent, add water (300mL) and ethyl acetate (300mL) liquid separation, organic phase saturated common salt water washing, nothing Aqueous sodium persulfate is dried, and is spin-dried for, is obtained crude product 4- hydroxyl -5- nitros methyl valerates (45g, pale yellow oily liquid) and be directly used in down One step.

The preparation of 4th step 5- hydroxy piperidine -2- ketone：

4- hydroxyl -5- nitros methyl valerates (45g, 0.23mol) and palladium carbon (2.1g) are added to methanol (500mL) solution In, reaction solution reacts 24 hours, thin-layer chromatography (petroleum ether at 60 DEG C under the conditions of hydrogen is existing:Ethyl acetate=1:1) detect Reaction is finished, and reaction solution is filtered by diatomite, and filtrate is spin-dried for, obtain 5- hydroxy piperidine -2- ketone (10g, yellow solid, 38%), it is directly used in and reacts in next step.

The preparation of the 5th step 1- benzyls -5- (benzyloxy) piperidines -2- ketone：

At room temperature, 5- hydroxy piperidine -2- ketone (10g, 0.1mol) is added in dimethyl sulfoxide (DMSO) (100mL), then will Sodium hydride (10g, 0.25mol) is slowly added into above-mentioned reaction system, after addition, by benzyl bromine (43.5g, 0.25mol) It is added in reaction solution, is stirred overnight, thin-layer chromatography (petroleum ether:Ethyl acetate=1:1) detection reaction finishes, to reaction system Reaction is quenched in middle addition saturated ammonium chloride (100ml), and ethyl acetate (100mL*3) extracts three times, and saturated common salt water washing is anhydrous Sodium sulphate is dried, and is spin-dried for, and is crossed post purifying, is obtained 1- benzyls -5- (benzyloxy) piperidines -2- ketone (16g, yellow solid, 54%).

The preparation of the 6th step 4- benzyls -6- (benzyloxy) -4- azaspiros [2.5] octane：

Under -78 DEG C of nitrogen protections, 1- benzyls -5- (benzyloxy) piperidines -2- ketone (15g, 50mmol) is dissolved in anhydrous tetrahydrochysene In furans (150mL), ethylmagnesium bromide (150mL) is then slowly added dropwise into reaction bulb, after being added dropwise, then by metatitanic acid 4 third Ester (45g, 150mmol) is added in above-mentioned reaction system, is added dropwise, reaction solution is warmed to room temperature, and is stirred 2 hours, thin layer Chromatogram (petroleum ether:Ethyl acetate=10:1) detection reaction is finished, and saturated ammonium chloride (100ml) is added into reaction system and is quenched Reaction, ethyl acetate (100mL*3) are extracted three times, saturated common salt water washing, anhydrous sodium sulfate drying, are spin-dried for, and are crossed post purifying, are obtained To 4- benzyls -6- (benzyloxy) -4- azaspiros [2.5] octane (5.1g, yellow solid, 31%).

The preparation of the 7th step 4- azaspiros [2.5] octyl- 6- alcohol：

By 4- benzyls -6- (benzyloxy) -4- azaspiros [2.5] octane (5.5g, 18mmol) and palladium carbon (2g, 1.8mmol) It is added in methanol (200mL) and hydrogen chloride (2mL) solution, reaction solution reacts 48 hours, thin layer color at 60 DEG C under hydrogen atmosphere Compose (petroleum ether:Ethyl acetate=10:1) detection reaction finishes, and reaction solution is filtered by diatomite, filtrate is spin-dried for, and obtains 4- Azaspiro [2.5] octyl- 6- alcohol (2.5g, yellow solid), it is directly used in and reacts in next step.

The preparation of the 8th step 6- hydroxyl -4- azaspiros [2.5] octane -4- t-butyl formates：

4- azaspiros [2.5] octyl- 6- alcohol (1g, 7.8mmol) and sodium carbonate (1.6g, 15.7mmol) are added to dichloromethane In alkane (100mL) solution, then di-tert-butyl dicarbonate (1.71g, 7.8mmol) is added drop-wise in above-mentioned reaction system, reacted Liquid reacts 18 hours at ambient temperature, thin-layer chromatography (petroleum ether:Ethyl acetate=3:1) detection reaction finishes, by reaction solution Extraction, filtrate are spin-dried for, and cross post purifying, obtain 6- hydroxyl -4- azaspiros [2.5] octane -4- t-butyl formates (1.7g).

9th step 6- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-) -4- azaspiros [2.5] octane -4- The preparation of t-butyl formate：

6- hydroxyl -4- azaspiros [2.5] octane -4- t-butyl formates (2.5g, 11.0mmol) and the iodo- 1H- pyrazolos of 3- [3,4-d] pyrimidine -4- amine (3.4g, 13.0mmol) is dissolved in tetrahydrofuran (200mL).Ice bath adds triphenylphosphine next time (4.5g, 17.2mmol), diisopropyl azodiformate (3.5g, 17.3mmol) is slowly added dropwise.Remove ice bath, reaction solution room temperature Stirring 18 hours, evaporated under reduced pressure.Crude product crosses silicagel column purifying (EA:PE=1：1) product 6- (the iodo- 1H- pyrroles of 4- amino -3-, are obtained Azoles simultaneously [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- t-butyl formates (3.6g, 70%).

Tenth step 6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls) -4- azaspiros [2.5] octane -4- t-butyl formates preparation：

6- (iodo- 1H- pyrazolos [3,4-d] pyrimidine -1- bases of 4- amino -3-) -4- azaspiros [2.5] octane -4- formic acid uncles Butyl ester (500mg, 1.1mmol) and the chloro- 2- of 5- (4- (4,4,5,5- tetramethyl -1,3,2- dioxaborinate -2- bases) phenoxy group) Pyridine (500mg, 1.5mmol) is dissolved in 95% ethanol (20mL).Add sodium carbonate (230mg, 2.2mmol) and two (triphenyls Phosphine) palladium chloride (120mg, 0.2mmol), the lower back flow reaction of argon gas protection 18 hours, evaporated under reduced pressure.Crude product dichloromethane (50mL) and water (50mL) extract.Organic phase anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Residue prepares plate purifying (two Chloromethanes:Methanol=50：1) product 6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos, are obtained [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- t-butyl formates (420mg, 76%).

11st step 3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (4- azaspiros [2.5] octyl- 6- yls) -1H- The preparation of pyrazolo [3,4-d] pyrimidine -4- amine hydrochlorates：

6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - 4- azaspiros [2.5] octane -4- t-butyl formates (420mg, 0.8mmol) are dissolved in tetrahydrofuran (10mL), add concentrated hydrochloric acid (1mL), 60 DEG C of reaction solution stir 5 hours, and evaporated under reduced pressure obtains crude product 3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (4- azaspiros [2.5] octyl- 6- yls) -1H- pyrazolos [3,4-d] pyrimidine -4- amine hydrochlorates (450mg).It is directly used in next step Reaction.

(4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 12nd step 6- Pyridine -1- bases) -4- azaspiros [2.5] octane -4- nitriles preparation：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (4- azaspiros [2.5] octyl- 6- yls) -1H- pyrazolos [3, 4-d] pyrimidine -4- amine hydrochlorates (200mg, 0.44mmol) are dissolved in DMF (2mL), add cesium carbonate (200mg, 0.62mmol) and cyanogen bromide (200mg, 1.89mmol), reaction solution are stirred at room temperature 6 hours, and reaction solution pours into acetic acid second Ester (50mL), wash (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Crude product prepares plate purifying (dichloromethane: Methanol=50：1) product compound 6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos, are obtained [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- nitriles (31mg, 15.9%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.30-8.26(m,2H),8.02-7.99(m,1H),7.73(d,J =8.4,2H), 7.32 (d, J=8.4,2H), 7.18 (d, J=8.8,1H), 5.03-5.01 (m, 1H), 3.67-3.57 (m, 2H),2.39-2.36(m,1H),2.19-2.15(m,2H),1.57-1.53(m,1H),0.98-0.77(m,4H).

MS:m/z 473.2[M+1]

Embodiment 14

(R) -6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) -4- azaspiros [2.5] octane -4- nitriles (WS-316) and (S) -6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) Phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- nitriles (WS-317) preparation

The compound of embodiment 14 can be obtained after the chiral fractionation of product of the step of embodiment 13 the 12nd reaction.Splitting condition is： Supercritical fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-316 spectral datas：

MS:m/z 473.2[M+1]

WS-317 spectral datas：

MS:m/z 473.2[M+1]

Embodiment 15

(E) ((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 6- by -1- Pyridine -1- bases) -4- azaspiros [2.5] octyl- 4- yls) -4- hydroxyl but-2-ene -1- ketone (WS-318) preparation

Using the product of the single step reaction of embodiment 13 the tenth as starting material, embodiment 15 is prepared by following step：

The first step (E) -1- (6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4- D] pyrimidine -1- bases) -4- azaspiros [2.5] octyl- 4- yls) and -4- hydroxyl but-2-ene -1- ketone preparation：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (4- azaspiros [2.5] octyl- 6- yls) -1H- pyrazolos [3, 4-d] pyrimidine -4- amine hydrochlorates (220mg, 0.48mmol) and 4- hydroxyl -2- butenoic acids (80mg, 0.78mmol) be dissolved in tetrahydrochysene furan Mutter (5mL), adds HATU (230mg, 0.61mmol) and triethylamine (100mg, 0.99mmol), it is small that reaction solution is stirred at room temperature 18 When, evaporated under reduced pressure.Residue is dissolved in ethyl acetate (50mL), washes (30mL*3), anhydrous sodium sulfate drying, and filtering, decompression is steamed It is dry.Crude product prepares plate purifying (dichloromethane:Methanol=10：1) product (E) -1- (6- (4- amino -3- (4- ((5- chlorine pyrroles, are obtained Pyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octyl- 4- yls) -4- hydroxyl butyl- 2- alkene -1- ketone (30mg, 12%).

¹H-NMR(400MHz,DMSO-d6):δ ppm 8.32 (s, 1H), 8.26 (d, J=2.8,1H), 8.02-7.99 (m, 1H),7.73-7.71(m,2H),7.34-7.31(m,2H),7.21-7.17(m,2H),7.08-6.81(m,3H),4.80-4.50 (m,3H),4.20-4.00(m,2H),3.55-3.53(m,2H),2.20-2.00(m,2H),1.40-1.10(m,2H),0.90- 0.70(m,2H)

MS:m/z 532.2[M+1]

Embodiment 16

((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 6- by (R, E) -1- Pyridine -1- bases) -4- azaspiros [2.5] octyl- 4- yls) -4- hydroxyl but-2-ene -1- ketone (WS-319) and (S, E) -1- (6- (4- ammonia Base -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] Octyl- 4- yls) -4- hydroxyl but-2-ene -1- ketone (WS-320) preparation

The compound of embodiment 16 can be obtained after the chiral fractionation of compound of embodiment 15.Splitting condition is： Supercritical fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-319 spectral datas：

MS:m/z 532.2[M+1]

WS-320 spectral datas：

MS:m/z 532.2[M+1]

Embodiment 17

6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- nitrogen The preparation of miscellaneous spiral shell [2.5] octane -4- nitriles (WS-321)

Embodiment 17 is prepared since corresponding starting material using method similar to Example 13.

¹H-NMR(400MHz,DMSO-d6):δppm 8.28(s,1H),7.69-7.53(m,6H),7.39-7.37(m, 1H),7.18-7.11(m,2H),5.03-4.98(m,1H),3.64-3.56(m,2H),2.19-2.16(m,1H),2.15-2.06 (m,2H),1.34-1.32(m,1H),0.97-0.78(m,4H).

MS:m/z 474.2[M+1]

Embodiment 18

(E) -1- (6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) -4- azaspiros [2.5] octyl- 4- yls) -4- hydroxyl but-2-ene -1- ketone preparation (WS-322) preparation

Embodiment 18 is prepared since corresponding starting material using method similar to Example 15.

¹H-NMR(400MHz,DMSO-d6):δppm 8.40(s,1H),7.70-7.68(m,2H),7.30-7.04(m, 7H),4.75-4.70(m,0.73H),4.55-4.50(m,0.17H),4.13-4.10(m,2H),3.61-3.58(m,2H), 3.01-2.95(m,0.65H),2.72-2.60(m,0.81H),2.43-2.38(m,1H),1.84-1.78(m,3H),1.31- 1.26(m,2H),0.98-0.70(m,2H)

MS:m/z 533.2[M+1]

Embodiment 19

(2S, 5R) -5- (4- amino -3- (4- (4- chlorobenzenes oxygen) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -2- Methyl piperidine -1- nitriles (WS-323) and (2R, 5S) -5- (4- amino -3- (4- (4- chlorobenzenes oxygen) phenyl) -1H- pyrazolos [3,4- D] pyrimidine -1- bases)-pipecoline -1- nitriles (WS-324) preparation

5- (4- ammonia is first prepared since corresponding starting material, using method similar to Example 7 in embodiment 19 Base -3- (4- (4- chlorobenzenes oxygen) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- nitriles, compound warp Compound WS-323 and WS-324 can be obtained after chiral resolution.Splitting condition is：Supercritical fluid Chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-323 spectral datas：

¹H-NMR(400MHz,CDCl3):δ ppm 8.35 (d, J=11.2Hz, 1H), 7.72-7.67 (m, 2H), 7.36- 7.31(m,2H),7.16-7.04(m,2H),7.02-6.99(m,2H),5.54(s,2H),4.96-4.94(m,1H),3.99- 3.91(m,1H),3.54-3.45(m,2H),2.54-2.53(m,1H),2.07-1.97(m,3H),1.44-1.40(m,3H).

MS:m/z 460.4[M+H]

WS-324 spectral datas：

MS:m/z 460.4[M+H]

Embodiment 20

(2S, 5R) -5- (4- amino -3- (4- phenoxy phenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -2- methyl piperazines Pyridine -1- nitriles (WS-325) and (2S, 5R) -5- (4- amino -3- (4- phenoxy phenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) - The preparation of pipecoline -1- nitriles (WS-326)

5- (4- ammonia is first prepared since corresponding starting material, using method similar to Example 9 in embodiment 20 Base -3- (4- phenoxy phenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- nitriles, the compound is chiral to tear open Compound WS-325 and WS-326 can be obtained after point.Splitting condition is：Supercritical fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-325 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.37(s,1H),7.70-7.66(m,2H),7.42-7.38(m,2H), 7.20-7.15(m,3H),7.11-7.08(m,2H),5.51(s,2H),4.99-4.94(m,1H),3.99-3.93(m,1H), 3.58-3.47 (m, 2H), 2.56-2.52 (m, 1H), 2.06-1.96 (m, 3H), 1.43 (d, J=6.8,3H)

MS:m/z 426.5[M+H]

WS-326 spectral datas：

MS:m/z 426.5[M+1]

Embodiment 21

1- ((2S, 5R) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] Pyrimidine -1- bases)-pipecoline -1- bases) -2- acrylic -1- ketone (WS-327) and 1- ((2R, 5S) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -2- propylene The preparation of base -1- ketone (WS-328)

Using the product of the step of embodiment 7 the 5th reaction as starting material, embodiment 21 is prepared by following step：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperidine -3- bases) -1H- pyrazolos [3,4-d] Pyrimidine -4- amine hydrochlorates (300mg, 0.63mmol) and acrylic acid (70mg, 0.98mmol) are dissolved in THF (10mL), add HATU 18h, evaporated under reduced pressure is stirred at room temperature in (362mg, 0.95mmol) and triethylamine (192mg, 1.91mmol), reaction solution.Residue is molten In ethyl acetate (50mL), wash (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.Crude product prepares plate purifying (DCM:MeOH=10：1) 5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d], are obtained Pyrimidine -1- bases)-pipecoline -1- bases) -2- acrylic -1- ketone (58mg, 29%).It can be obtained after the chiral fractionation of the compound Embodiment 21 compound WS-327 and WS-328.Splitting condition is：Supercritical fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-327 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.38(s,1H),8.14(s,1H),7.75-7.73(m,3H),7.31- 7.26 (m, 2H), 6.98 (d, J=8.8Hz, 1H), 6.62-6.59 (m, 1H), 6.30 (d, J=8.4Hz, 1H 1H), 5.72- 5.65(m,1H),5.55(s,2H),4.87-4.80(m,2H),4.14-4.03(m,1H),3.53-3.47(m,1H),2.67- 2.57 (m, 1H), 2.08-1.96 (m, 2H), 1.86-1.83 (m, 1H), 1.34 (d, J=8.8Hz, 3H)

MS:m/z 490.2[M+H]

WS-328 spectral datas：

MS:m/z 490.2[M+H]

Embodiment 22

1- ((2S, 5R) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] Pyrimidine -1- bases)-pipecoline -1- bases) -2- butynyl -1- ketone (WS-329) and 1- ((2R, 5S) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -2- butine The preparation of base -1- ketone (WS-330)

Using the product of the step of embodiment 7 the 5th reaction as starting material, embodiment 22 is prepared by following step：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (6- methyl piperidine -3- bases) -1H- pyrazolos [3,4-d] Pyrimidine -4- amine hydrochlorates (230mg, 0.53mmol) and butyl- 2- acetylenic acids (80mg, 0.98mmol) are dissolved in THF (10mL), add 18h, evaporated under reduced pressure is stirred at room temperature in HATU (300mg, 0.79mmol) and triethylamine (100mg, 0.99mmol), reaction solution.It is remaining Thing is dissolved in ethyl acetate (50mL), washes (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.It is pure that crude product prepares plate Change (DCM:MeOH=10：1) 1- ((2S, 5R) -5- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H-, are obtained Pyrazolo [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -2- butynyl -1- ketone (130mg, 50%).The compound passes through Embodiment 22 compound WS-329 and WS-330 can be obtained after chiral resolution.Splitting condition is：Supercritical fluid Chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-329 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.38(s,1H),8.14(s,1H),7.77-7.69(m,3H),7.33- 7.29(m,2H),7.00-6.96(m,1H),5.50(s,2H),4.98-4.81(m,3H),3.89-3.87(m,1H),2.57- 2.53 (m, 1H), 2.05 (s, 3H), 1.85-1.76 (m, 3H), 1.41 (d, J=6.8Hz, 3H)

MS:m/z 502.3[M+H]

WS-330 spectral datas：

MS:m/z 502.3[M+H]

Embodiment 23

1- ((2S, 5R) -5- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-pipecoline -1- bases) -2- acrylic -1- ketone (WS-331) and 1- ((2R, 5S) -5- (4- amino -3- (4- (2,4- Difluoro phenoxy group) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -2- acrylic -1- ketone (WS-332) preparation

Embodiment 23 is prepared since corresponding starting material using the method similar with embodiment 21.

WS-331 spectral datas：

¹H-NMR(400MHz,CDCl3):δ ppm 8.37 (s, 1H), 7.64 (d, J=8.8Hz, 2H), 7.19-7.13 (m, 1H), 7.09 (d, J=8.4Hz, 2H), 7.04-6.97 (m, 1H), 6.94-6.90 (m, 1H), 6.66-6.54 (m, 1H), 6.31- 6.25(m,1H),5.72-5.65(m,1H),5.45(s,2H),4.85-4.78(m,1H),3.85-3.44(m,2H),2.59- 2.53 (m, 1H), 2.12-1.89 (m, 2H), 1.85-1.77 (m, 1H), 1.33 (d, J=8.8Hz, 3H)

MS:m/z 491.4[M+H]

WS-332 spectral datas：

MS:m/z 491.4[M+H]

Embodiment 24

1- ((2S, 5R) -5- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine - 1- yls)-pipecoline -1- bases) -2- butynyl -1- ketone (WS-333) and 1- ((2R, 5S) -5- (4- amino -3- (4- (2,4- Difluoro phenoxy group) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- bases) -2- butynyl -1- ketone (WS-334) preparation

Embodiment 24 is prepared since corresponding starting material using the method similar with embodiment 22.

WS-333 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.37(s,1H),7.67-7.62(m,2H),7.19-7.13(m,1H), 7.11-7.08(m,2H),7.02-6.98(m,1H),6.97-6.90(m,1H),5.49(s,1H),4.81-4.77(m,2H), 3.87-3.46 (m, 1H), 2.56-2.53 (m, 1H), 1.98 (s, 3H), 1.32 (d, J=7.2Hz, 3H)

MS:m/z 503.4[M+H]

WS-334 spectral datas：

MS:m/z 503.4[M+H]

Embodiment 25

(R) ((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 6- by -1- Pyridine -1- bases) -4- azaspiros [2.5] octane -4- bases) -2- acrylic -1- ketone (WS-335) and (S) -1- (6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- Base) -2- acrylic -1- ketone (WS-336) preparation

Using the product of the single step reaction of embodiment 13 the tenth as starting material, embodiment 25 is prepared by following step：

3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1- (4- azaspiros [2.5] octyl- 6- yls) -1H- pyrazolos [3, 4-d] pyrimidine -4- amine hydrochlorates (50mg, 0.11mmol) and acrylic acid (8.03mg, 0.11mmol) is dissolved in THF (5mL), adds 18h, evaporated under reduced pressure is stirred at room temperature in HATU (63.67mg, 0.17mmol) and triethylamine (33.89mg, 0.33mmol), reaction solution. Residue is dissolved in ethyl acetate (50mL), washes (30mL*3), anhydrous sodium sulfate drying, filtering, evaporated under reduced pressure.It is prepared by crude product Plate purifies (DCM:MeOH=10：1) 1- (6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazoles, is obtained And [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- bases) -2- acrylic -1- ketone (38mg, 67%).The compound Embodiment 25 compound WS-335 and WS-336 can be obtained after chiral fractionation.Splitting condition is：Supercritical fluid Chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% methanol, 70mL/ minutes).

WS-335 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.40(s,1H),8.17(s,2H),7.78-7.71(m,2H),7.21 (d, J=8.4Hz, 2H), 6.99 (d, J=8.8Hz, 1H), 6.91-6.90 (m, 1H), 6.42-6.38 (m, 1H), 5.75-5.73 (m,1H),5.61(s,2H),4.98-4.94(m,1H),3.78-3.75(m,1H),2.54-2.49(m,1H),2.30-2.22 (m,2H),1.28-1.20(m,3H),1.14-1.10(m,1H),0.90-0.82(m,2H).

MS:m/z 502.3[M+H]

WS-336 spectral datas：

MS:m/z 502.4[M+H]

Embodiment 26

(R) ((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 6- by -1- Pyridine -1- bases) -4- azaspiros [2.5] octane -4- bases) -2- butynyl -1- ketone (WS-337) and (S) -1- (6- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- Base) -2- butynyl -1- ketone (WS-338) preparation

Embodiment 26 is prepared since corresponding starting material using the method similar with embodiment 22.

WS-337 spectral datas：

¹H-NMR(400MHz,CDCl₃-d6):δppm 8.40(s,1H),8.15(s,1H),7.79-7.69(m,3H), 7.32-7.29(m,2H),6.99-6.96(m,1H),5.54(s,1H),5.04-5.02(m,1H),4.58-4.56(m,1H), 4.07-4.05(m,1H),2.53-2.44(m,1H),2.26-2.20(m,2H),2.06(s,3H),2.01-1.98(s,1H), 1.43-1.41(m,1H),1.06-1.05(m,1H),1.05-1.04(m,2H).

MS:m/z 514.3[M+H]

WS-338 spectral datas：

MS:m/z 514.3[M+H]

Embodiment 27

(R) ((4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] are phonetic by 6- by -1- Pyridine -1- bases) -4- azaspiros [2.5] octane -4- bases) -4- hydroxyls -2- butynyl -1- ketone (WS-339) and (S) -1- (6- (4- ammonia Base -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] are pungent Alkane -4- bases) -4- hydroxyl -2- butynyl -1- ketone (WS-340) preparation

Embodiment 27 is prepared since corresponding starting material using the method similar with embodiment 21.

WS-339 spectral datas：

¹H-NMR(400MHz,CDCl₃):δ ppm 8.38 (s, 1H), 8.13 (d, J=2.4Hz, 1H), 7.76-7.69 (m 3H), 7.31-7.29 (m, 2H), 6.97 (d, J=8.8Hz, 1H), 5.63-5.53 (m, 1H), 5.01-4.96 (m, 1H), 4.46- 4.40(m,1H),4.26-4.23(m,1H),3.84-3.83(m,1H),2.27-2.21(m,1H),2.12-2.08(m,3H), 1.28-1.25(m,1H),1.04-1.02(m,1H),0.93-0.75(m,2H).

MS:m/z 530.3[M+H]

WS-340 spectral datas：

MS:m/z 530.3[M+H]

Embodiment 28

4- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) The preparation of piperidines -1- nitriles (WS-341)

Embodiment 28 is prepared since corresponding starting material using method similar to Example 1.

¹H-NMR(400MHz,CDCl₃) δ ppm 8.37 (s, 1H), 7.64 (d, J=8.4Hz, 2H), 7.19-7.13 (m, 1H), 7.09 (d, J=8.4Hz, 2H), 7.02-6.97 (m, 1H), 6.94-6.90 (m, 1H), 5.63 (s, 2H), 4.92-4.86 (m,1H),3.66-3.63(m,2H),3.31-3.25(m,2H),2.54-2.44(m,2H),2.10-2.01(m,2H).

MS:m/z 448.3[M+H]

Embodiment 29

5- (4- amino -3- (1- benzyl -1H- pyrazoles -4- bases) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -2- methyl piperazines The preparation of pyridine -1- nitriles (WS-342)

Embodiment 29 is prepared since corresponding starting material using method similar to Example 7.

¹H-NMR(400MHz,CDCl3):δ ppm 8.34 (s, 1H), 7.85 (d, J=16.8Hz, 2H), 7.41-7.26 (m,5H),5.54(s,2H),5.39(s,2H),4.92-4.90(m,1H),3.94-3.88(m,1H),3.74-3.72(m,1H), 3.57-3.49 (m, 1H), 3.48-3.42 (m, 1H), 2.49 (m, 1H), 2.00-1.90 (m, 1H), 1.63-1.54 (m, 3H)

MS:m/z 414.3[M+H]

Embodiment 30

5- (4- amino -3- (4- (morpholine methyl) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline - The preparation of 1- nitriles (WS-343)

Embodiment 30 is prepared since corresponding starting material using method similar to Example 7.

¹H-NMR(400MHz,CDCl3):δ ppm 8.34 (s, 1H), 7.69 (d, J=7.6Hz, 2H), 7.53 (d, J= 7.6Hz, 2H), 5.54 (s, 2H), 4.97 (m, 1H), 3.97-3.93 (m, 1H), 3.75-3.74 (m, 4H), 3.60-3.47 (m, 5H), 2.56-2.52 (m, 4H), 2.49 (m, 1H), 2.01-1.96 (m, 3H), 1.44-1.52 (d, J=6.8Hz, 3H)

MS:m/z 433.3[M+H]

Embodiment 31

5- (4- amino -3- (4- benzyl phenyls) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- nitriles (WS-344) preparation

Embodiment 31 is prepared since corresponding starting material using method similar to Example 7.

¹H-NMR(400MHz,CDCl3):δ ppm 8.36 (s, 1H), 7.65-7.63 (m, 2H), 7.38-7.36 (m, 2H), 7.25-7.23(m,2H),5.55(s,2H),4.97-4.94(m,1H),3.97-3.92(m,1H),3.74-3.72(m,1H), 3.58-3.52 (m, 1H), 2.54-2.52 (m, 1H), 2.05-1.96 (m, 2H), 1.61-1.56 (m, 3H)

MS:m/z 424.3[M+H]

Embodiment 32

5- (4- amino -3- (4- (phenylamino) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases)-pipecoline -1- The preparation of nitrile (WS-345)

Embodiment 32 is prepared since corresponding starting material using method similar to Example 7.

¹H-NMR(400MHz,CDCl3):δ ppm 8.36 (s, 1H), 7.60 (d, J=8.4Hz, 2H), 7.35-7.29 (m, 2H),7.20-7.15(m,4H),7.04-7.00(m,1H),5.91(s,2H),5.57(s,1H),4.97-4.93(m,1H), 3.98-3.93(m,1H),3.59-3.55(m,1H),3.50-3.46(m,1H),2.58-2.52(m,1H),2.05-2.03(m, 2H), 2.00-1.97 (m, 2H), 1.44 (d, J=6.8Hz, 3H)

MS:m/z 425.4[M+H]

Embodiment 33

(R) -1- (6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) -4- azaspiros [2.5] octane -4- bases) -2- acrylic -1- ketone (WS-346) and (S) -1- (6- (4- amino -3- (4- (2, 4- difluoros phenoxy group) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- bases) -2- propylene The preparation of base -1- ketone (WS-347)

Embodiment 33 is prepared since corresponding starting material using the method similar with embodiment 21.

WS-346 spectral datas：

¹H-NMR(400MHz,CDCl3):δ ppm 8.37 (s, 1H), 7.65 (d, J=8.4Hz, 2H), 7.19-7.13 (m, 1H),7.02-6.97(m,1H),6.94-6.90(m,1H),6.39-6.35(m,1H),5.72-5.69(m,1H),5.49-5.46 (m,2H),4.95-4.94(m,1H),3.78-3.74(m,1H),2.52-2.47(m,1H),2.30-2.22(m,1H),1.82- 1.69(m,2H),1.28-1.20(m,1H),1.14-1.10(m,1H),0.90-0.79(m,2H).

MS:m/z 503.4[M+H]

WS-347 spectral datas：

MS:m/z 503.4[M+H]

Embodiment 34

(R) -1- (6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) -4- azaspiros [2.5] octane -4- bases) -2- butynyl -1- ketone (WS-348) and (S) -1- (6- (4- amino -3- (4- (2, 4- difluoros phenoxy group) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- bases) -2- butine The preparation of base -1- ketone (WS-349)

Embodiment 34 is prepared since corresponding starting material using the method similar with embodiment 26.

WS-348 spectral datas：

¹H-NMR(400MHz,CDCl3):δppm 8.37(s,1H),7.69-7.65(m,2H),7.16-7.13(m,1H), 7.11-7.07(m,2H),7.02-6.97(m,1H),6.94-6.92(m,1H),5.47(s,2H),5.04-4.91(m,1H), 4.59-4.30(m,1H),4.06-3.65(m,1H),2.52-2.45(m,1H),2.25-2.17(m,2H),2.05(s,3H), 1.09-0.95(m,1H),0.89-0.86(m,2H),0.77-0.72(m,1H).

MS:m/z 515.4[M+H]

WS-349 spectral datas：

MS:m/z 515.4[M+H]

Embodiment 35

(R) -1- (6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) -4- azaspiros [2.5] octane -4- bases) -4- hydroxyls -2- butynyl -1- ketone (WS-350) and (S) -1- (6- (4- amino -3- (4- (2,4 difluorobenzene epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) -4- azaspiros [2.5] octane -4- bases) - The preparation of 4- hydroxyl -2- butynyl -1- ketone (WS-351)

Embodiment 35 is prepared since corresponding starting material using the method similar with embodiment 21.

WS-350 spectral datas：

¹H-NMR(400MHz,CDCl3):δ ppm 8.37 (s, 1H), 7.66 (d, J=8,2H), 7.21-7.15 (m, 1H), 7.11-7.08(m,2H),7.02-6.97(m,1H),6.94-6.86(m,1H),5.44(s,2H),5.03-4.92(m,1H), 4.56-4.39(m,2H),4.32-4.13(m,2H),3.85-3.72(m,1H),2.70-2.43(m,1H),2.33-2.24(m, 1H),2.09-2.03(m,2H),1.11-1.00(m,2H),0.88-0.68(m,2H).

MS:m/z 531.4[M+H]

WS-351 spectral datas：

MS:m/z 531.4[M+H]

Embodiment 36

3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- bases) It is prepared by pyrrolidines -1- nitriles (WS-352)

Embodiment 36 is prepared since corresponding starting material using method similar to Example 1.

¹H-NMR(400MHz,CD₃OD) δ ppm 8.37 (s, 1H), 7.72 (d, J=8.8Hz, 2H), 7.32-7.26 (m, 1H), 7.21-7.18 (m, 1H), 7.16 (d, J=8.4Hz, 2H), 7.06-7.01 (m, 1H), 5.62-5.32 (s, 1H), 3.97- 3.92(m,1H),3.87-3.80(m,2H),3.71-3.68(m,1H),2.53-2.48(m,2H),2.20-2.00(m,1H).

MS:434.2[M+H]

Embodiment 37

(R) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazolos [3,4-d] pyrimidine -1- Base) pyrrolidines -1- nitriles (WS-353) and (S) -3- (4- amino -3- (4- ((5- chloropyridine -2- bases) epoxide) phenyl) -1H- pyrazoles And [3,4-d] pyrimidine -1- bases) pyrrolidines -1- nitriles (WS-354) preparation

The compound WS-353 and WS-354 of embodiment 37 can be obtained after the chiral fractionation of compound of embodiment 36.Split bar Part is：Supercritical fluid chromatography (ChiralPak AD 5 μ, 21x 250mm col, 27% first Alcohol, 70mL/ minutes).

WS-353 spectral datas：

MS:434.2[M+H]

WS-354 spectral datas：

MS:434.2[M+H]

Embodiment 38~47

Using the method similar with this patent embodiment 1~37, the compound of structure shown in embodiment 38~47 can be prepared.

Embodiment 48

BTK kinase activity Inhibition tests

In the experiment based on time-resolved fluorescence Resonance energy transfer method, compound disclosed herein is tested to BTK The inhibitory action of kinase activity.The Btk of restructuring and compound disclosed herein at room temperature containing 50mM Tris pH7.4, 10mM MgCl₂、2mM MnCl₂, 0.1mM EDTA, 1mM DTT, 20nM SEB, 0.1%BSA, 0.005%tween-20 examination Test in buffer solution and be pre-incubated 1 hour.By adding ATP (under ATP Km concentration) and peptide substrates (Biotin- AVLESEEELYSSARQ-NH2 initiation reaction) is carried out.After incubating 1 hour at room temperature, add and isometric contain 50mM HEPES PH7.0,800mM KF, 20mM EDTA, 0.1%BSA, the p-Tyr66 antibody and marked by streptavidin for connecting Eu cryptate compounds XL665 terminate liquid is with terminating reaction.Disk incubates 1 hour again at room temperature, is then read on BMG PHERAstar FS instruments Take TR-FRET signals (ex337nm, em 620nm/665nm).Ratio based on the fluorescence at 615nm with the fluorescence at 665nm, Residual enzymic activities in the case of calculating compound concentration increase.Four ginsengs that the IC50 of each compound passes through Graphpad Prism softwares Count logical equation fitting data and obtain.

According to above-mentioned experimental method, the part of compounds in the present invention shows stronger BTK kinase inhibiting activities (IC50<1000nM), the very strong (IC50 of the kinase inhibiting activity of some of preferred compounds<100nM).Concrete outcome is seen below Table.

Kinase inhibiting activity grade is divided into A, B, C, specifically A (IC₅₀<100nM), B (100nM<IC₅₀<1000nM), C (IC₅₀>1000nM)

Embodiment 49

Vitro kinase is selectively tested

EGFR, ITK kinase activity detection platforms are established using based on time-resolved fluorescence Resonance energy transfer method；Adopt LCK, SRC, LYN kinase activity detection platforms are established with Z '-Lyte methods；Using Lance Ultra methods establish TEC and JAK3 kinase activity detection platforms, inhibitory action of the compound disclosed herein to different kinase activities is tested respectively.Eachization Compound determines enzymatic activity data under 11 concentration respectively, and the IC of the compound is calculated with Graphpad Prism softwares₅₀Value.

According to above-mentioned experimental method, some compounds in the present invention show very strong Kinase Selectivity, hence it is evident that excellent In control compound Buddhist nun is replaced according to Shandong.As a result see the table below.

Compound number

LCK

SRC

LYN

EGFR

ITK

TEC

WS-311

C

B

C

A

WS-349

B

C

A

Buddhist nun is replaced according to Shandong

A

Embodiment 50

B cell Inhibition test

The of short duration B cell activation for being enough to suppress exposed to BTK inhibitor in normal human subject B cell in vitro.This scheme Live body inner cell is simulated to the predicted exposure of inhibitor, and shows that the suppression to B cell although being cleaned inhibitor is still able to Keep.

If CD B cells be using Saite match general (RosetteSep) human B cell enrichment cocktail by Solid phase from Healthy donors Blood purification obtains.By plating cells in growth medium (10%RPMI+10% hyclones) and add refer to Determine the inhibitor of concentration.After being cultivated 1 hour at 37 DEG C, by cell washing three times, washing every time is used in growth medium It is middle to carry out 8 times of dilutions.Then cell is irritated 18 hours with 10 μ g/mL IgM F (ab') 2 at 37 DEG C.Then use anti-CD 6 9- PE antibody on cell is dyed and analyzed by flow cytometry using standard conditions.Method according to more than is surveyed Fixed, preferable compound has stronger inhibitory activity to B cell in the present invention, and its IC50 value is less than 1nM.

Embodiment 51

T cell Inhibition test

If CD T cells be using Saite match general (RosetteSep) human T cells enrichment cocktail by Solid phase from Healthy donors Blood purification obtains.By plating cells in growth medium (10%RPMI+10% hyclones) and add refer to Determine the inhibitor of concentration.After being cultivated 1 hour at 37 DEG C, by cell washing three times, washing every time is used in growth medium It is middle to carry out 10 times of dilutions.Cell then is coated with into pearl with anti-CD3/CD28, and (pearl/cell proportion is 1:1) irritated at 37 DEG C 18 hours.Then dyed with anti-CD 6 9-PE antibody on cell and analyzed by flow cytometry using standard conditions.

Method according to more than is measured, and preferable compound has very weak inhibitory activity to T cell in the present invention Or unrestraint, its IC50 value are more than 4000nM.

Embodiment 52

People's Whole blood B Cell Inhibition test

People's whole blood (hWB) is obtained from healthy volunteer, is collected blood into by venipuncture anti-coagulated with liquaemin In Vacutainer pipes.Test compound is diluted to initial drug concentrations needed for 10 times in PBS), then 10% in PBS In DMSO in three times be serially diluted, to obtain 9 points of dose response curve.By the 5.5 μ L formula of every kind of chemical compound diluted liquid one Two parts are added on the plate at the hole V-type bottoms of aiil 96；The 10% of 5.5 μ L is added in PBS into control and non-stimulated hole DMSO.To every hole addition people's whole blood (100 μ L), upon mixing by plate in 37C, 5%CO₂, 100% humidity incubation 30 minutes. Under stirring sheep F (ab') 2 anti-human IgM (Southern Biotech) (10 μ L 500 μ g/ are added to every hole (except non-stimulated hole) ML solution, 50 μ g/mL ultimate densities), and plate is incubated other 20 hours.20 hours incubate at the end of, by sample with it is glimmering 20 μ L APC mouse anti humans CD69 (BD Pharmingen) of light probe mark are in 37C, 5%CO₂, 30 points of 100% humidity incubation Clock.Including induction control, the undyed and simple stain agent for compensation adjustment and initial voltage setting.Then sample is used 1ml IX Pharmingen Lyse Buffer (BD Pharmingen) cracking, and plate is centrifuged 5 minutes in 1500rpm. Supernatant is removed by aspirating, the granule of residual is cracked again with other 1ml IX Pharmingen Lyse Buffer, And plate is centrifuged as preceding.Supernatant is suctioned out, the granule of residual (is washed in FACs buffer solutions in PBS+1%FBQ.After centrifugation And after removing supernatant, granule is resuspended in 150 μ L FACs buffer solutions.Sample is transferred to suitable in BD LSR II 96 orifice plates run in the pore systems of HTS 96 of flow cytometer.Using be adapted to fluorogen used excite and launch wavelength, obtain Access obtains percentage positive cell value according to and using Cell Quest Software.As a result initially facs analysis software is used (Flow Jo) is analyzed.IC50 values use XLfit v3, and formula 201 calculates.

Method according to more than is measured, and preferable compound has stronger to B cell in people's whole blood in the present invention Inhibitory activity, its IC50 value are less than 200nM.

Embodiment 53

Stability study of the compound in hepatomicrosome

1. testing compound is dissolved in acetonitrile, the storing solution that concentration is 0.5mM is made.

2.2 μ L storing solutions add 1.5ml centrifuge tubes in, then add 148 μ L phosphate buffers (100mM, pH 7.4) and 10 μ L hepatomicrosomes (protein concentration 20mg/ml) suspensions (BD Gentest companies), the kind of hepatomicrosome is people respectively, Dog, rat, mouse；Control group adds 158 μ L phosphate buffers (100mM, pH 7.4).

3. the mixed system prepared in step 2, being incubated in advance in 37 DEG C of water-baths 3 minutes, 40 μ L NADPH hairs are then added NADP+ (is contained in raw body system：6.5mM, glucose 6- phosphoric acid：16.5mM,MgCl₂：16.5mM G 6 PD: 2U/ml) start reaction, and be incubated 1 hour in 37 DEG C of water-baths.

After 4. reaction is carried out 1 hour, centrifuge tube is taken out from water-bath, and adds 400 μ L acetonitrile terminating reactions, then whirlpool Rotation concussion 3 minutes, finally centrifuges (13000rpm, 4 DEG C) 5 minutes, takes supernatant HPLC detection residual drug concentration Cs r.

5. the parallel preparation method for preparing 0 minute response sample：The mixed system prepared in step 2, in 37 DEG C of water-baths In incubate 3 minutes in advance after take out, add 400 μ L acetonitriles, then add 40 μ L NADPH occur system.It is vortexed after shaking 3 minutes, from The heart (13000rpm, 4 DEG C) 5 minutes, take supernatant HPLC detection drug concentrations C0.

6. after 60 minutes are incubated, remaining percentage of the medicine in incubation system calculates according to the following formula：

Medicine residue (%)=Cr ÷ C0 × 100%

According to above-mentioned experimental method, the preferred compound in the present invention shows preferable Microsomal Stability, its Remaining percentage in the hepatomicrosome of each kind>30%.

Embodiment 54

Compound is evaluated to CYP enzyme inhibitions

The metabolism of CYP enzymes is the main path of drug biotransformation, and its quantity and active size directly affect medicine in vivo Activation and metabolism.As the main metabolic enzyme of exogenous compounds, cromoci YP is important medicine I phase metabolic enzymes, can To be catalyzed the oxidation of a variety of exogenous compounds and reductive metabolism.CYP enzymes play very important during the elimination of medicine Effect, while be also principal element caused by drug interaction when causing drug combination.

Method：This experiment determines compound to five kinds in the hepatomicrosome of people source simultaneously using cocktail probe medicaments method The inhibitory action of CYP450 enzymes, people source microsome come from BD Gentest companies.

Experimental procedure is as follows：

Reaction is carried out in 100mM phosphate buffers, the μ L of cumulative volume 200.Concentration of microsomes is in reaction system 0.25mg/mL, testing compound concentration are 20 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.25 μM, Specific probe and dense Degree is respectively 40 μM of phenaetin (CYP1A2), 5 μM of dextromethorphan (CYP2D6), 10 μM of Diclofenac (CYP2C9), the U.S. sweet smell of S- Appropriate 40 μM of English (CYP2C19), 80 μM of testosterone (CYP3A4).Incubation system preincubate 5 minutes in 37 degree of constant temperature oscillators, add System (NADP+ containing 1.3mM, 3.3mM glucose 6- phosphoric acid, 0.4U/L G 6 PDs, 3.3mM occur for NADPH MgCL2) start to react.Isometric acetonitrile terminating reaction is added after being incubated 45min, is vortexed, 13000rpm centrifugations, takes supernatant LC-MS-MS sample introductions determine metabolite growing amount.Specific metabolic product is respectively paracetamol (CYP1A2), right coffee Alkane (CYP2D6), 4- hydroxyls Diclofenac (CYP2C9), 4- hydroxyls mephenytoin (CYP2C19), 6 beta-hydroxy testosterones (CYP3A4).Specific inhibitor is respectively furafylline (CYP1A2), quinindium (CYP2D6), sulfaphenazolum (CYP2C9), parnitene (CYP2C19), ketoconazole (CYP3A4).This experiment final result is calculation of half inhibitory concentration IC50 values.IC50=((the low inhibiting rate % of 50%-)/(high inhibiting rate %- low inhibiting rate %)) × (high concentrations-low concentration)+low Concentration.According to above-mentioned experimental method, preferred compounds of the invention only has not strong suppression to various CYP enzymes or without suppression System, illustrates that its metabolic effect to other medicines is smaller.

Embodiment 55

Pharmacokinetics research method of the compound in rat body

1. male SD rat【Magnificent Fukang】After buying in, raised 7 days in this laboratory adaptability.

2.9 SD rats are randomly divided into 3 groups, and every group 3, one group is used for gastric infusion, and another group is used for tail vein injection Administration.The rat of gastric infusion group, overnight fast is needed before administration.

3. after rat administration, blood sample is gathered at following time point using the method for orbital venous plexus blood sampling：I.V.:(administration Before), 0.08 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours.P.O.:0.08 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours.Each blood sampling time point blood sampling volume is about 300 μ l。

4. the blood sample of collection is centrifuged 5 minutes at 4 DEG C with 12000rpm rotating speeds, upper plasma sample is then gathered, and in- Preserved in 20 DEG C of refrigerators to be measured.

5. experimental implementation is summarized and is shown in Table 4：

The pharmacokinetics experimental design of table 4, compound in rat body

6. use LC-MS/MS (UPLC-MS/MS:Liquid phase Waters Acquity UPLC (USA) and mass spectrum 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS MS:Liquid phase Agilent 1200series (USA) and Mass spectrum API 4000 (Applied Biosystem/MDS SCIEX)) detection blood plasma in compound concentration.Typical detector bar Part see the table below.

Use pharmacokinetics professional software WinNonlin【Model：Phoenix^TM 6.1 producer： Pharsight Corporation】Calculate pharmacokinetic parameter.【Phoenix 1.1User’s Guide:p251-p300】

According to above-mentioned experimental method, the compound that has been determined in the present invention show preferable bioavilability (> 40%).

Embodiment 56

HERG Binding experiments (Dofetillide methods)

According to the method described on patent US20050214870A1, the IC50 values that compound suppresses to hERG can be determined.This Preferable compound only has very weak inhibitory action or unrestraint to act on to hERG in invention, and its IC50 value is more than 1000nM.

Claims

1. a kind of novel pyrazolopyrimidines compound, as shown in formula (I), its stereoisomer, dynamic isomer, or pharmaceutically may be used The salt of receiving, or solvate, or prodrug：

Here,

N, m are independently taken from 0,1 or 2；

L is O ,-C (O)-,-C (O) NH- ,-CH₂-, S, S (O), NH or S (O)₂；

A is taken from substituted or unsubstituted phenyl ring or substituted or unsubstituted hetero-aromatic ring, and with the connection position of parent nucleus and L Point is optional；

B be independently taken from substituted or unsubstituted cycloaliphatic ring, substituted or unsubstituted heterocycle, substituted or unsubstituted phenyl ring, Or substituted or unsubstituted hetero-aromatic ring, and be optional with L connection site；

R₁And R₂It is each independently selected from hydrogen, unsubstituted C1-C4 alkyl, halogen, cyano group, or R₁And R₂The carbon being connected with them Atom forms ternary carbocyclic ring or quaternary carbocyclic ring, or R together₁And R₂Merge into oxo base；

Y be selected from cyano group orHere, R₃、R₄、R₅And R₆It is taken from independently of one another Hydrogen, unsubstituted C1-C4 alkyl, hydroxyl substitution C1-C4 alkyl, C1-C4 alkoxy C 1-4 alkyl, halogen, cyano group or- (CH₂)_qN(R^aR^b), here, q 1,2,3 or 4, R^aAnd R^bIt is each independently selected from hydrogen, unsubstituted C1-C4 alkyl；

And provide, work as R₁And R₂When being all hydrogen, A is phenyl, and L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- difluorobenzenes Base；Y is 4- hydroxyl but-2-enes acyl group, butyl- 2- alkynes acyl group or cyano group；

Or, work as R₁And R₂When being all hydrogen, A is pyrazolyl, and L is S (O)₂Or-CH₂-, B is phenyl or cyclopropyl；Y is cyano group；

2. compound as claimed in claim 1, wherein, n, m are independently taken from 0,1 or 2；L is O ,-C (O)-,-C (O) NH- ,-CH₂-, NH or S, more preferably O ,-C (O) NH-, NH.

3. compound as claimed in claim 1, wherein, A is taken from substituted or unsubstituted phenyl ring or substitution or unsubstituted Hetero-aromatic ring, and be optional with the connection site of parent nucleus and L；B is independently taken from substituted or unsubstituted cycloaliphatic ring, taken Generation or unsubstituted heterocycle, substituted or unsubstituted phenyl ring or substituted or unsubstituted hetero-aromatic ring, and with L connection position Point is optional；Here：

The substituted phenyl ring refers to that optional position is substituted by optional substituents on phenyl, and the substituent is selected from Hydrogen, methyl, methoxyl group, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano group；Preferably, the substituted phenyl ring substitutes for fluorine Phenyl or chlorine substitution phenyl, more preferably 2,4- difluorophenyls or 4- chlorphenyls；

The unsubstituted hetero-aromatic ring refers to furans, pyrroles, thiophene, oxazole, isoxazole, pyrazoles, imidazoles, thiazole, isothiazole, evil Diazole, triazole, thiadiazoles, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, triazine；The substituted hetero-aromatic ring refers to above base Optional position is substituted by optional substituents in group, and the substituent is selected from hydrogen, methyl, methoxyl group, fluorine, chlorine, trifluoro Methyl, trifluoromethoxy or cyano group；It is highly preferred that the substituted pyridine is chloro-pyridine, particularly preferably is the chloro- pyrroles of 4- Pyridine -2- bases；

The unsubstituted cycloaliphatic ring refers to cyclopropane, cyclobutane, pentamethylene, hexamethylene, cycloheptane, cyclooctane；The substitution Cycloaliphatic ring refer to that optional position is substituted by optional substituents on above group, the substituent be selected from hydrogen, methyl, Methoxyl group, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano group；

4. compound as claimed in claim 1, wherein, R₁And R₂One of them is hydrogen and another is C1-C4 alkyl, or R₁And R₂Cyclopropyl is formed together with the carbon atom being connected with them；It is highly preferred that R₁And R₂All it is hydrogen, or one of them is hydrogen And another is methyl, or R₁And R₂Cyclopropyl is formed together with the carbon atom being connected with them.

5. compound as claimed in claim 1, wherein it is preferred to, the Pyrazolopyrimidine compound as shown in formula (II), it is vertical Body isomers, dynamic isomer, or pharmaceutically acceptable salt, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I)；

And provide, A is phenyl, and L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- difluorophenyls；Y is 4- hydroxyl butyl- 2- Enoyl-, butyl- 2- alkynes acyl group or cyano group；

Or, A is pyridine radicals, L is NH or O, B are pyrans -4- bases or cyclohexyl；Y is cyano group；

It is highly preferred that the Pyrazolopyrimidine compound shown in formula (II) is one kind in following compounds：

And provide, L is O, and B is 4- chlorphenyls, 3- chloropyridine -6- bases, 2,4- difluorophenyls；Y be 4- hydroxyl but-2-enes acyl group, Butyl- 2- alkynes acyl group or cyano group；

Or, L is C (O)-or-CH₂-, B is morpholine -4- bases；Y is cyano group；

Or：Preferably, the Pyrazolopyrimidine compound as shown in formula (III), its stereoisomer, dynamic isomer, or pharmacy Upper acceptable salt, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I)；

It is highly preferred that the Pyrazolopyrimidine compound shown in formula (III) is one kind in following compounds：

Here, L, B and Y are as defined above the formula of stating (I) in formula (III-1), (III-2), (III-3) and (III-4)；Or：

Preferably, the Pyrazolopyrimidine compound as shown in formula (IV), its stereoisomer, dynamic isomer, or pharmaceutically may be used The salt of receiving, or solvate, or prodrug：

Here, L, A, B and Y are as defined above the formula of stating (I)；

It is highly preferred that the Pyrazolopyrimidine compound shown in formula (IV) is one kind in following compounds：

6. compound as claimed in claim 5, wherein, L O；

B isWith

Y be selected from cyano group or

R₃、R₄、R₅And R₆It is taken from hydrogen, unsubstituted C1-C4 alkyl, C1-C4 alkyl, the C1-C4 alkoxy Cs 1-4 of hydroxyl substitution Alkyl, halogen, cyano group or-(CH₂)_qN(R^aR^b), here, q 1,2,3 or 4, R^aAnd R^bIt is each independently selected from hydrogen, unsubstituted C1-C4 alkyl.

7. one kind in following compounds, or its pharmaceutically acceptable salt, or solvate, or prodrug：

8. the preparation method of compound, comprises the following steps any one of claim 1-7：

The substituent being related in above-mentioned formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI) R₁、R₂, L, B, Y and n, m be as defined above the formula (I) in face, PG is amino protecting group, and X is chlorine, bromine or hydroxyl.

9. include compound any one of claim 1-7 or the pharmaceutical composition of its pharmaceutically acceptable salt.

10. compound any one of claim 1-7 or claim 9 described pharmaceutical composition are preparing BTK inhibitor Application in medicine.

11. application as claimed in claim 10, wherein, BTK inhibitor refers to prevent or treated by the disease of BTK mediations, institute State disease and be selected from autoimmune disease, inflammatory disease, heteroimmune implementations or disease, thromboembolic disorders and cancer.