CN111153906A

CN111153906A - Pyrazolopyrimidine derivatives as BTK inhibitors, process for their preparation and pharmaceutical compositions containing them

Info

Publication number: CN111153906A
Application number: CN202010134019.0A
Authority: CN
Inventors: 吴予川
Original assignee: Suzhou Sinovent Pharmaceuticals Co Ltd
Current assignee: Suzhou Sinoway Pharmaceutical Technology Co ltd
Priority date: 2016-05-16
Filing date: 2017-04-25
Publication date: 2020-05-15
Anticipated expiration: 2037-04-25
Also published as: CN107383013B; CN107383013A; CN111153906B; WO2017198049A1

Abstract

The invention discloses a novel pyrazolopyrimidine compound shown as a formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the definition of each substituent is shown in the specification. In addition, the invention also discloses a preparation method of the compound, a pharmaceutical composition and application thereof.

Description

Pyrazolopyrimidine derivatives as BTK inhibitors, process for their preparation and pharmaceutical compositions containing them

The application is a divisional application of an invention application with the application date of 2017, 4 and 25 months and the application number of 201710277829.X, and the invention name of the invention is 'pyrazolopyrimidine derivative as BTK inhibitor, preparation method thereof and pharmaceutical composition'.

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a novel pyrazolopyrimidine derivative serving as a BTK inhibitor, which is efficient, good in selectivity and good in pharmacokinetic property, and a preparation method and a medicinal composition thereof.

Background

Protein kinases are the largest family of biological enzymes in the human body, including over 500 proteins. In particular, with respect to tyrosine kinases, the phenol function of the tyrosine residue can be phosphorylated, thereby playing an important role in biological signal transduction. The tyrosine kinase family possesses members that control cell growth, migration, and differentiation. Abnormal kinase activity has been elucidated to be closely related to many human diseases including cancer, autoimmune diseases and inflammatory diseases.

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic non-receptor tyrosine kinase belonging to the TEC kinase family (total of 5 members BTK, TEC, ITK, TXK, BMX). The BTK gene is located on the X-chromosome at Xq21.33-Xq22, sharing 19 exons, spanning 37.5kb of genomic DNA.

In addition to T cells and plasma cells, BTK expression plays an essential role on almost all hematopoietic cells, particularly in B-lymphocyte genesis, differentiation, signaling and survival. B cells are activated through the B Cell Receptor (BCR), and BTK plays a crucial role in the BCR signaling pathway. Activation of BCR on B cells leads to activation of BTK, which in turn leads to an increase in downstream phospholipase c (plc) concentration and activation of IP3 and DAG signaling pathways. This signaling pathway can promote proliferation, adhesion and survival of cells. Mutations in the BTK gene result in a rare inherited B-cell specific immunodeficiency disease known as X-linked agammaglobulinemia (XLA). In this disease, BTK function is inhibited, resulting in hindered B cell production or maturation. Men with XLA disease have few B cells in their bodies, have few circulating antibodies, and are susceptible to serious and even fatal infections. This has strongly demonstrated that BTK plays an extremely important role in the growth and differentiation of B cells.

The small-molecule BTK inhibitor can be combined with BTK, inhibit the BTK autophosphorylation and prevent the BTK activation. This can block BCR pathway signaling, inhibit B lymphoma cell proliferation, disrupt tumor cell adhesion, and thereby promote tumor cell apoptosis. And induce apoptosis. This makes BTK an attractive drug target in B-cell related cancers, especially for B-cell lymphomas and leukemias, such as non-hodgkin's lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL), and anti-relapsed or refractory Mantle Cell Lymphoma (MCL), among others.

In addition to being effective against B cell lymphomas and leukemias, BTK inhibitors can also inhibit the production of B cell autoantibodies and cytokines. In autoimmune diseases, B cells present autoantigens that promote T cell activation and secretion to cause inflammatory factors, which not only cause tissue damage, but also activate B cells to produce large amounts of antibodies that trigger autoimmune reactions. T and B cells interact to form a feedback regulatory chain, which leads to uncontrolled autoimmune reaction and aggravates histopathological injury. Therefore, BTK can be used as a drug target of autoimmune diseases, such as rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), and allergic diseases (such as esophagitis).

In addition, it has been reported that BTK inhibitors can be used in combination with chemotherapeutic agents or immune checkpoint inhibitors and show superior therapeutic effects against a variety of solid tumors in clinical trials.

Among the currently marketed drugs, ibrutinib is an irreversible BTK inhibitor developed by Pharmacyclics and hadamard co-company, and has been approved by the FDA for the treatment of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) in 11 months and 2 months in 2014 in 2013, respectively. Irreversible inhibitors, a chemical agent that binds to groups in enzyme proteins with relatively strong covalent bonds (covalentbond), are generally capable of inactivating enzymes and thus exerting their uniquely high biological activities. Ibrutinib is identified by the FDA as a "breakthrough" new drug that exerts its therapeutic effects by reacting with the thiol group of cysteine in BTK and forming a covalent bond, inactivating the BTK enzyme. However, ibrutinib is easily metabolized (oxidized and metabolized into dihydroxylated products by metabolic enzymes or attacked and inactivated by other thiol-containing enzymes, cysteine, glutathione, etc.) during administration, and thus drug efficacy is affected. The clinical administration dose reaches 560mg per day, and the burden of patients is increased. In addition, ibrutinib also has a certain inhibition effect on some kinases except BTK, and especially the inhibition on EGFR can cause more serious adverse reactions such as rash, diarrhea and the like. Therefore, there is still a need in the art to develop a new class of BTK inhibitors with higher efficiency, better selectivity and better pharmacokinetic properties for the treatment of related diseases.

Disclosure of Invention

The inventor develops a novel pyrazolopyrimidine derivative which is an effective, safe and highly selective inhibitor of protein kinase BTK.

The invention aims to provide a novel pyrazolopyrimidine derivative. It is a new covalent bond inhibitor, and can improve the affinity with target by changing its reaction rate with cysteine so as to raise therapeutic effect, selectivity and safety.

The second object of the present invention is to provide a process for the preparation of the above derivatives.

The third purpose of the invention is to provide a pharmaceutical composition containing the derivative.

The fourth purpose of the invention is to provide the application of the derivative.

In particular, in a first aspect, in an embodiment of the present invention, the present invention provides a novel pyrazolopyrimidine compound, represented by formula (I), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug thereof:

here, the first and second liquid crystal display panels are,

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

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

A is taken from a substituted or unsubstituted benzene ring or a substituted or unsubstituted heteroaromatic ring, and the linking site with the parent nucleus and L can be optional;

b is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the site of attachment to L may be optional;

R₁and R₂Each independently selected from hydrogen, unsubstituted C1-C4 alkyl, halogen, cyano, or R₁And R₂Together with the carbon atom to which they are attached form a three-or four-membered carbocyclic ring, or R₁And R₂Combined to oxo;

y is selected from cyano, or

R₃、R₄、R₅And R₆Each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano, or- (CH)₂)_qN(R^aR^b) Where q is 1, 2, 3, or 4, R^aAnd R^bEach independently selected from hydrogen, unsubstituted C1-C4 alkyl;

and specifies when R₁And R₂When both are hydrogen, A is phenyl, L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2, 4-difluorophenyl; y is 4-hydroxybut-2-enoyl, but-2-ynoyl, or cyano;

or, when R is₁And R₂When both are hydrogen, A is phenyl, L is C (O) -, or-CH₂-, B is morpholin-4-yl; y is cyano;

or, when R is₁And R₂When both are hydrogen, A is pyrazolyl and L is S (O)₂or-CH₂-, B is phenyl, or cyclopropyl; y is cyano;

or, when R is₁And R₂When both are hydrogen, A is pyridyl, L is NH, or O, B is pyran-4-yl, or cyclohexyl; y is cyano.

In one embodiment of the present invention, there is provided a pyrazolopyrimidine compound of formula (I) wherein n, m are independently selected 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, there is provided a pyrazolopyrimidine compound represented by formula (I), wherein a is selected from a substituted or unsubstituted benzene or heteroaromatic ring, and the linking site to the parent nucleus and L is optional; b is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the site of attachment to L may be optional; here:

the substituted benzene ring is substituted by optional substituent groups on the benzene ring at any position, wherein the substituent groups are selected from hydrogen, methyl, methoxy, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano; preferably, the substituted phenyl is fluoro-substituted phenyl, or chloro-substituted phenyl, more preferably 2, 4-difluorophenyl, or 4-chlorophenyl;

the unsubstituted heteroaromatic ring is furan, pyrrole, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole, oxadiazole, triazole, thiadiazole, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine and triazine; the substituted heteroaromatic ring is substituted by optional substituent groups selected from hydrogen, methyl, methoxy, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano at any position on the group; more preferably, the substituted pyridine is chloropyridine, particularly preferably 4-chloro-pyridin-2-yl;

the unsubstituted aliphatic ring refers to cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and cyclooctane; the substituted aliphatic ring is substituted by optional substituent groups selected from hydrogen, methyl, methoxy, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano at any position on the above group;

the unsubstituted heterocycle refers to tetrahydrofuran, tetrahydropyran, tetrahydropyrrole, piperidine, or the like,

Wherein w is taken from 0, 1 or 2; the substituted heterocycle refers to any position on the above groups substituted by optional substituent selected from hydrogen, methyl, methoxy, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano.

In one embodiment of the present invention, there is provided a pyrazolopyrimidine compound of formula (I)Wherein, preferably, R₁And R₂One of which is hydrogen and the other is unsubstituted C1-C4 alkyl (methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl), or R₁And R₂Together with the carbon atom to which they are attached form a cyclopropyl group; more preferably, R₁And R₂Are both hydrogen, or one is hydrogen and the other is methyl, or R₁And R₂Together with the carbon atom to which they are attached form a cyclopropyl group.

In a preferred embodiment of the present invention, the present invention provides a pyrazolopyrimidine compound represented by the formula (II), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug thereof:

wherein L, A, B and Y are as defined above for formula (I);

and provided that A is phenyl, L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2, 4-difluorophenyl; y is 4-hydroxybut-2-enoyl, but-2-ynoyl, or cyano;

or, A is phenyl, L is C (O) -, or-CH₂-, B is morpholin-4-yl; y is cyano;

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

or, A is pyridyl, L is NH, or O, B is pyran-4-yl, or cyclohexyl; y is cyano.

In a more preferred embodiment of the present invention, there is provided a pyrazolopyrimidine compound represented by the formula (II) which is one of the following compounds:

here, L, B and Y in formula (II-1) or (II-2) are as defined above for formula (I);

and provided that L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2, 4-difluorophenyl; y is 4-hydroxybut-2-enoyl, but-2-ynoyl, or cyano;

or, L is C (O) -, or-CH₂-, B is morpholin-4-yl; y is cyano.

In a preferred embodiment of the present invention, the present invention provides a pyrazolopyrimidine compound represented by the formula (III), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug thereof:

here, L, A, B and Y are as defined above for formula (I).

In a more preferred embodiment of the present invention, there is provided a pyrazolopyrimidine compound represented by the formula (III) which is one of the following compounds:

here, L, B and Y in formulae (III-1), (III-2), (III-3) and (III-4) are as defined above for formula (I).

In a preferred embodiment of the present invention, the present invention provides a pyrazolopyrimidine compound represented by the formula (IV), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug thereof:

here, L, A, B and Y are as defined above for formula (I).

In a more preferred embodiment of the present invention, there is provided a pyrazolopyrimidine compound represented by the formula (IV) which is one of the following compounds:

here, L, B and Y in formula (IV-1) or (IV-2) are as defined above for formula (I).

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

b is

Y is selected from cyano, or

R₃、R₄、R₅And R₆Each independently selected from unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano, or- (CH)₂)_qN(R^aR^b) Where q is 1, 2, 3, or 4, R^aAnd R^bEach independently selected from hydrogen, unsubstituted C1-C4 alkyl.

In a particularly preferred embodiment of the present invention, the present invention provides a pyrazolopyrimidine compound selected from one of the following compounds, or a pharmaceutically acceptable salt, or solvate, or prodrug thereof:

in an embodiment of the present invention, said "pharmaceutically acceptable salts" refer to pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts:

the term "pharmaceutically acceptable acid addition salt" refers to a salt with an inorganic or organic acid which retains the biological effectiveness of the free base without any other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, phosphate, and the like; organic acid salts include, but are not limited to, formates, acetates, propionates, glycolates, gluconates, lactates, oxalates, maleates, succinates, fumarates, tartrates, citrates, glutamates, aspartates, benzoates, methanesulfonates, p-toluenesulfonate, salicylates, and the like. These salts can be prepared by methods known in the art.

The term "pharmaceutically acceptable base addition salt" refers to a salt which retains the biological effectiveness of the free acid without other side effects. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, calcium, and magnesium salts, and the like. Salts derived from organic bases include, but are not limited to, ammonium salts, triethylamine salts, lysine salts, arginine salts, and the like. These salts can be prepared by methods known in the art.

In an embodiment of the present invention, the "solvate" refers to a complex formed by the compound of the present invention and a solvent. They either react in a solvent or precipitate out of a solvent or crystallize out. For example, a complex with water is referred to as a "hydrate".

In embodiments of the invention, the compounds of the invention may contain one or more chiral centers and exist in different optically active forms. When the compound contains one chiral center, the compound comprises enantiomers. The present invention includes both isomers and mixtures of isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When the compounds of formula (I) contain more than one chiral center, diastereoisomers may be present. The present invention includes resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers may be resolved by methods known in the art, such as crystallization and preparative chromatography.

In embodiments of the invention, the prodrug refers to known amino protecting groups and carboxy protecting groups, which are hydrolyzed under physiological conditions or released via enzymatic reactions to give the parent compound. Specific prodrug preparation methods are referenced (Saulnier, M.G.; Frannesson, 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.).

In a second aspect, the present invention provides a process for the preparation of a pyrazolopyrimidine compound of formula (I) above, comprising the steps of:

(1) reacting a compound of formula (V) with a compound of formula (VI) to obtain a compound of formula (VII);

(2) reacting a compound of formula (VII) with a compound of formula (VIII) to give a compound of formula (IX);

(3) removing protecting group PG from the compound of formula (IX) to obtain a compound of formula (X);

(4) reacting a compound of formula (X) with a compound of formula (XI) to obtain a compound of formula (I);

the substituent R referred to in the above-mentioned formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI)₁、R₂L, A, B, Y and n, m are as defined for formula (I) above, PG is an amino protecting group (suitable amino protecting groups include acyl groups (e.g. acetyl), carboxamides (e.g. 2',2',2 '-trichloroethoxycarbonyl, Cbz benzyloxycarbonyl or BOC t-butoxycarbonyl) and arylalkyl groups (e.g. Bn benzyl) which may be removed as appropriate by hydrolysis (e.g. using an acid such as hydrochloric acid in dioxane or trifluoroacetic acid in dichloromethane) or by reductive means (e.g. hydrogenolysis of benzyl or benzyloxycarbonyl or reductive removal of 2',2',2' -trichloroethoxycarbonyl using zinc in acetic acid.) other suitable amino protecting groups include trifluoroacetyl (-COCF)₃) Which can remove benzyloxy, carbonyl or tert-butoxycarbonyl Groups by base-catalysed hydrolysis, those skilled in the art can refer to t.w. greene 'Protective Groups in Organic Synthesis' (4 th edition, j.wiley and Sons, 2006)), X being chlorine, bromine or hydroxyl.

In an embodiment of the present invention, there is provided a process for preparing the pyrazolopyrimidine compound represented by the formula (I) above, wherein the compound of the formula (V) may be prepared by referring to the following process:

in embodiments of the present invention, the present invention also provides intermediate compounds useful in the synthesis of the above pyrazolopyrimidine compounds, including, but not limited to:

wherein Bn is benzyl, and Boc is tert-butyloxycarbonyloxy.

In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of one or more of the above pyrazolopyrimidine compounds of the invention or a stereoisomer, a tautomer, a solvate, or a pharmaceutically acceptable salt thereof, which further comprises a pharmaceutically acceptable adjuvant.

The pharmaceutical composition of the present invention may be formulated into solid, semi-solid, liquid or gaseous preparations such as tablets, capsules, powders, granules, pastes, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.

The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical field. For example, The actual method of preparing a pharmaceutical composition is known to those skilled in The art, see, for example, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

Routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, topical, transdermal, intramuscular, intravenous, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. For example, dosage forms suitable for oral administration include capsules, tablets, granules, and syrups. The compounds of formula (I) of the present invention contained in these formulations may be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, and the like. The above-mentioned dosage forms can be prepared from the active compounds and one or more carriers or adjuvants by customary pharmaceutical methods. The above-mentioned carriers need to be compatible with the active compound or other adjuvants. For solid formulations, non-toxic carriers that are commonly used include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like. Carriers for liquid formulations include, but are not limited to, water, physiological saline, aqueous dextrose, glycols, polyethylene glycols and the like. The active compound may be in solution or suspension with the carrier(s) mentioned above. The particular mode and dosage form of administration will depend, inter alia, on the physicochemical properties of the compound itself and the severity of the condition being treated. The skilled person will be able to determine the particular route of administration based on the factors mentioned above in combination with his own knowledge. See, for example: lijun, clinical pharmacology, national institutes of health, 2008.06; buyufeng, clinical dosage form factor and rational medication, medical guidance, 26(5), 2007; howard c.ansel, Loyd v.allen, jr., Nicholas g.popovich, jiangzhi master translation, pharmaceutical dosage forms and administration systems, chinese medicine science and technology press, 2003.05.

The pharmaceutical compositions of the present invention may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of the active ingredient. Thus, such unit doses may be administered more than once a day. Preferred unit dosage compositions are those containing a daily dose or sub-dose (administered more than once a day), or an appropriate fraction thereof, of the active ingredient as described herein above.

The pharmaceutical compositions of the present invention are formulated, dosed and administered in a manner consistent with medical practice specifications. The "therapeutically effective amount" of a compound of the invention will depend on the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration. Generally, the dose for parenteral administration may be 1-200mg/kg, and the dose for oral administration may be 1-1000 mg/kg.

The effective dosage ranges provided herein are not intended to limit the scope of the invention, but rather represent preferred dosage ranges. However, the most preferred dosage can be adjusted for individual subjects as is known and determinable by those skilled in the art (see, e.g., Berkow et al, Merck Manual, 16 th edition, Merck, Inc., Rahway, N.J., 1992).

In a fourth aspect, the present invention provides the use of the above-mentioned pyrazolopyrimidine compound or a stereoisomer, a tautomer, a solvate, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of a BTK-mediated disease.

The present invention provides a method for inhibiting BTK activity comprising administering to a biological system a pyrazolopyrimidine compound of the invention or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pyrazolopyrimidine compound of the invention or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

In some embodiments, the biological system is an enzyme, a cell, or a mammal.

The present invention also provides a method for preventing or treating BTK-mediated diseases comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the above-described pyrazolopyrimidine compounds of the invention, or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof, in combination with one or more agents selected from the group consisting of an immunomodulator, an immune checkpoint inhibitor, a glucocorticoid, a nonsteroidal anti-inflammatory drug, a Cox-2 specific inhibitor, a TNF- α binding protein, an interferon, an interleukin and a chemotherapeutic agent.

In embodiments of the invention, the BTK-mediated disease includes autoimmune diseases, inflammatory diseases, heteroimmune conditions or diseases, thromboembolic diseases, and cancer. In some embodiments, the cancer comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma, multiple myeloma, mantle cell lymphoma, small lymphocytic lymphoma, fahrenheit macroglobulinemia, solid tumors. In some embodiments, the autoimmune and inflammatory diseases are selected from rheumatoid arthritis, osteoarthritis, juvenile arthritis, chronic obstructive pulmonary disease, multiple sclerosis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, crohn's disease, ulcerative colitis, and irritable bowel syndrome. In some embodiments, the heteroimmune condition or disease comprises graft-versus-host disease, transplantation, transfusion, anaphylaxis, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis, or atopic dermatitis.

Experimental data prove that the pyrazolopyrimidine compound provided by the invention is an effective and safe inhibitor of protein kinase BTK.

Detailed Description

The experiments, synthetic methods and intermediates involved described below are illustrative of the present invention and do not limit the scope of the invention.

The starting materials used in the experiments of the present invention were either purchased from reagent suppliers or prepared from known materials via methods well known in the art. Unless otherwise indicated, the examples herein apply the following conditions:

the units of temperature are degrees Celsius (. degree. C.); room temperature is defined as 18-25 ℃;

drying the organic solvent by using anhydrous magnesium sulfate or anhydrous sodium sulfate; spin-drying under reduced pressure and elevated temperature conditions (e.g., 15mmHg, 30 deg.C) using a rotary evaporator;

the method comprises the following steps of (1) using 200-300-mesh silica gel as a carrier during the rapid column chromatography separation, wherein the thin-layer chromatography represents the thin-layer chromatography;

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

the identification of the final product was done by nuclear magnetic resonance (Bruker AVANCE 300, 300MHz) and LC-MS (Brukerequine 6000, Agilent 1200 series).

Example 1

Preparation of (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile (WS-300)

First step preparation of tert-butyl (R) -3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate:

(S) -3-hydroxypiperidine-1-carboxylic acid tert-butyl ester (1.7g,8.5mmol) and 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (2g,7.7mmol) are dissolved in tetrahydrofuran (100 mL). Triphenylphosphine (2.5g,9.5mmol) was added in one portion in the ice bath and diisopropyl azodicarboxylate (2.1g,10.40mmol) was slowly added dropwise. The ice bath was removed and the reaction was stirred at room temperature for 18 hours and evaporated to dryness under reduced pressure. The crude product was purified by column on silica gel (ethyl acetate: petroleum ether ═ 1:1) to give the product tert-butyl (R) -3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate (3.5g, 100%).

Second step preparation of tert-butyl (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate:

(R) -3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylic acid tert-butyl ester (1.3g,2.9mmol) and 5-chloro-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) pyridine (1g,3.0mmol) were dissolved in 95% ethanol (50 mL). Sodium carbonate (600mg,5.7mmol) and bis (triphenylphosphine) palladium dichloride (200mg,0.28mmol) were added, the reaction was refluxed for 18 hours under argon protection, and evaporated to dryness under reduced pressure. The crude product was extracted with ethyl acetate (50mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by plate preparation (dichloromethane: methanol ═ 50: 1) to give the product (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylic acid tert-butyl ester (960mg, 63%).

Third step preparation of (R) -3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine:

tert-butyl (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate (960mg,1.8mmol) was dissolved in tetrahydrofuran (10mL), concentrated hydrochloric acid (0.5mL) was added, the reaction mixture was stirred at 60 ℃ for 3 hours, and evaporated under reduced pressure to dryness to give the crude product (R) -3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (1.1g, 100%). Directly used for the next reaction.

Fourth step preparation of (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile:

(R) -3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (500mg,1.1mmol) was dissolved in N, N-dimethylformamide (5mL), cesium carbonate (600mg,1.9mmol) and cyanogen bromide (400mg,3.8mmol) were added, the reaction solution was stirred for 5 hours at room temperature, the reaction solution was poured into ethyl acetate (50mL), washed with water (30 mL. multidot.3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product prep. plate was purified (dichloromethane: methanol ═ 50: 1) to give the product (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile (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]

Example 2

Preparation of (R, E) -1- (3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-hydroxybut-2-en-1-one (WS-301)

Example 2 was prepared by the following procedure starting from the product of the third reaction step of example 1:

first step preparation of (R, E) -1- (3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-hydroxybut-2-en-1-one:

(R) -3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (400mg,0.87mmol) and 4-hydroxy-2-butenoic acid (130mg,1.27mmol) were dissolved in tetrahydrofuran (20mL), HATU (550mg,1.45mmol) and triethylamine (200mg,1.98mmol) were added, the reaction solution was stirred at room temperature for 18 hours, and evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product preparation plate was purified (dichloromethane: methanol ═ 10:1) to give the product compound (R, E) -1- (3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-hydroxybut-2-en-1-one (110mg, 22%).

H-NMR(400MHz,DMSO-d6):δppm 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]

Example 3

Preparation of (R) -3- (4-amino-3- (4- (4-chlorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile (WS-302)

Example 3 was prepared in analogy to example 1, starting from the corresponding starting material.

1H-NMR(400MHz,DMSO-d6):δppm 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]

Example 4

Preparation of (R, E) -1- (3- (4-amino-3- (4- (4-chlorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-hydroxybut-2-en-1-one (WS-303)

Example 4 was prepared analogously to example 2, starting from the corresponding starting materials.

1H-NMR(400MHz,DMSO-d6):δppm 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]

Example 5

Preparation of (R) -3- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile (WS-304)

Example 5 was prepared analogously to example 1, starting from the corresponding starting materials.

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]

Example 6

Preparation of (R, E) -1- (3- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -4-hydroxybut-2-en-1-one (WS-305)

Example 6 was prepared in analogy to example 2, starting from the corresponding starting material.

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]

Example 7

Preparation of 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-306)

First step preparation of 6-methylpiperidin-3-ol

5-hydroxy-2-methyl-pyridine (1.5g,13.7mmol) was dissolved in 20ml of acetic acid, 0.4g of platinum dioxide was added, and the mixture was stirred under a pressure of 50psi of hydrogen for 16 hours. And (5) pumping the solvent to obtain the compound.

Second step preparation of 5-hydroxy-2-methylpiperidine-1-carboxylic acid tert-butyl ester

6-methylpiperidine-3-ol (1.10g, 7.8mmol) and sodium carbonate (1.6g,15.7mmol) were added to a dichloromethane (100mL) solution, and di-tert-butyl dicarbonate (1.71g, 7.8mmol) was added dropwise to the reaction system, and the reaction mixture was reacted at room temperature for 18 hours, and then the reaction completion was detected by thin layer chromatography (petroleum ether: ethyl acetate ═ 3:1), followed by extraction of the reaction mixture, spin-drying of the filtrate, and purification by column chromatography, to obtain a product, i.e., tert-butyl 5-hydroxy-2-methylpiperidine-1-carboxylate (1.7 g).

Third step preparation of tert-butyl 5- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate:

tert-butyl 5-hydroxy-2-methylpiperidine-1-carboxylate (7g,32.6mmol) and 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (8.5g,32.6mmol) are dissolved in tetrahydrofuran (100 mL). Triphenylphosphine (11g,41.5mmol) was added in one portion in the ice bath and diisopropyl azodicarboxylate (9.6g,47.5mmol) was slowly added dropwise. The ice bath was removed and the reaction was stirred at room temperature for 18 hours and evaporated to dryness under reduced pressure. The crude product was purified by column on silica gel (ethyl acetate: petroleum ether ═ 1:1) to give the product tert-butyl 5- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate (12.2g, 82%).

MS:m/z 459.2[M+1]

Fourth step preparation of tert-butyl 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate:

tert-butyl 5- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate (1g,2.2mmol) and 5-chloro-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) phenoxy) pyridine (800mg,2.4mmol) are dissolved in 95% ethanol (50 mL). Sodium carbonate (500mg,4.7mmol) and bis (triphenylphosphine) palladium dichloride (150mg,0.21mmol) were added, the reaction was refluxed for 18 hours under argon protection, and evaporated to dryness under reduced pressure. The crude product was extracted in dichloromethane (50mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified on plate (dichloromethane: methanol ═ 50: 1) to give the product tert-butyl 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate (700mg, 60%).

MS:m/z 536.3[M+1]

Fifth step preparation of 3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride:

tert-butyl 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carboxylate (700mg,1.3mmol) was dissolved in tetrahydrofuran (10mL), concentrated hydrochloric acid (1mL) was added, the reaction mixture was stirred at 60 ℃ for 3 hours, and evaporated under reduced pressure to dryness to give a crude product, 3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (550mg, 89%). Directly used for the next reaction.

MS:m/z 436.4[M+1]

Sixth step preparation of 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile:

3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (250mg,0.53mmol) was dissolved in N, N-dimethylformamide (3mL), cesium carbonate (250mg,0.77mmol) and cyanogen bromide (250mg,2.36mmol) were added, the reaction solution was stirred at room temperature for 6 hours, the reaction solution was poured into ethyl acetate (50mL), washed with water (30 mL. multidot.3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on plate (dichloromethane: methanol ═ 50: 1) to give product 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (120mg, 49%).

¹H-NMR(400MHz,DMSO-d6):δppm 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]

Example 8

(E) Preparation of (E) -1- (5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -4-hydroxybut-2-en-1-one (WS-307)

Example 8 was prepared by the following procedure starting with the product of the fifth reaction step of example 7:

first step preparation of (E) -1- (5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -4-hydroxybut-2-en-1-one:

3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (250mg,0.53mmol) and 4-hydroxy-2-butenoic acid (100mg,0.98mmol) were dissolved in tetrahydrofuran (10mL), HATU (300mg,0.79mmol) and triethylamine (100mg,0.99mmol) were added, the reaction solution was stirred at room temperature for 18 hours, and evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. Crude product prep. plate purification (dichloromethane: methanol ═ 10:1) gave product (E) -1- (5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -4-hydroxybut-2-en-1-one (155mg, 50%).

¹H-NMR(400MHz,DMSO-d6):δppm 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]

Example 9

Preparation of 5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-308)

Example 9 was prepared analogously to example 7, starting from the corresponding starting materials.

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]

Example 10

Preparation of (2S,5R) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-309) and (2R,5S) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-310)

Example 10 was prepared by chiral resolution of the product of example 7. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-309 spectrum data:

MS:m/z 461.4[M+1]

WS-310 spectrogram data:

MS:m/z 461.4[M+1]

example 11

(E) -1- ((2S,5R) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -4-hydroxybut-2-en-1-one (WS-311) and (E) -1- ((2R,5S) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -4-hydroxybut-2- Preparation of en-1-one (WS-312)

Example 11 was prepared by chiral resolution of the product of example 8. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-311 spectrum data:

MS:m/z 520.4[M+1]

WS-312 spectrogram data:

MS:m/z 520.4[M+1]

example 12

Preparation of (2S,5R) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-313) and (2R,5S) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-314)

Example 12 was prepared by manual resolution of the product of example 9. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-313 spectrogram data:

MS:m/z 462.3[M+1]

WS-314 spectrogram data:

MS:m/z 462.3[M+1]

example 13

Preparation of 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile (WS-315)

First step preparation of methyl 4-hydroxybutyrate:

dihydrofuran-2 (3H) -one (100g, 1.163mol) and triethylamine (460g,4.65mol) were added to a methanol (1L) solution, the reaction solution was reacted at 60 ℃ for 24 hours, the completion of the reaction was detected by thin layer chromatography (petroleum ether: ethyl acetate ═ 2:1), and the reaction solution was spin-dried to obtain methyl 4-hydroxybutyrate (120g, 87.6%, yellow liquid) which was used directly in the next reaction.

Second step preparation of methyl 4-oxobutyrate:

methyl 4-hydroxybutyrate (120g, 1.02mol) was added to a dichloromethane (1.2L) solution, pyridine chlorochromate (330g, 1.53mol) was added to the reaction solution, the reaction was carried out at room temperature for 12 hours, the completion of the reaction was detected by thin layer chromatography (petroleum ether: ethyl acetate 3:1), and the reaction solution was filtered through celite and dried by spinning to obtain methyl 4-oxobutyrate (60g, 50%, yellow liquid) which was used directly in the next reaction.

The third step is the preparation of methyl 4-hydroxy-5-nitropentanoate:

in an ice-water bath, methyl 4-oxobutyrate (60g, 0.46mol), nitromethane (42g, 0.69mol), tetrahydrofuran (300mL) and tert-butanol (300mL) are added into a reaction bottle, potassium tert-butoxide (5g) is slowly added into the reaction system, the temperature is raised to room temperature, the reaction is carried out for 2 hours, thin layer chromatography (petroleum ether: ethyl acetate: 3:1) detects that the reaction is finished, water (30mL) is added to quench the reaction, water (300mL) and ethyl acetate (300mL) are added to carry out liquid separation, an organic phase is washed by saturated saline, dried by anhydrous sodium sulfate and spun to obtain crude methyl 4-hydroxy-5-nitropentanoate (45g, light yellow oily liquid) which is directly used for the next step.

Fourth step 5-hydroxypiperidine-2-one preparation:

methyl 4-hydroxy-5-nitropentanoate (45g, 0.23mol) and palladium on carbon (2.1g) were added to a methanol (500mL) solution, the reaction solution was reacted at 60 ℃ for 24 hours in the presence of hydrogen, the completion of the reaction was detected by thin layer chromatography (petroleum ether: ethyl acetate 1:1), the reaction solution was filtered through celite, and the filtrate was spin-dried to obtain 5-hydroxypiperidin-2-one (10g, yellow solid, 38%) which was used directly in the next reaction.

Fifth step preparation of 1-benzyl-5- (benzyloxy) piperidin-2-one:

at room temperature, 5-hydroxypiperidine-2-one (10g, 0.1mol) was added to dimethyl sulfoxide (100mL), sodium hydride (10g, 0.25mol) was slowly added to the above reaction system, after the addition was completed, benzyl bromide (43.5g, 0.25mol) was added to the reaction solution, the mixture was stirred overnight, thin layer chromatography (petroleum ether: ethyl acetate 1:1) was performed to detect completion of the reaction, saturated ammonium chloride (100mL) was added to the reaction system to quench the reaction, ethyl acetate (100mL 3) was extracted three times, saturated common salt was added, anhydrous sodium sulfate was dried, spin-dried, and column-purified to obtain 1-benzyl-5- (benzyloxy) piperidin-2-one (16g, yellow solid, 54%).

Sixth step preparation of 4-benzyl-6- (benzyloxy) -4-azaspiro [2.5] octane:

dissolving 1-benzyl-5- (benzyloxy) piperidin-2-one (15g, 50mmol) in anhydrous tetrahydrofuran (150mL) at 78 ℃ under the protection of nitrogen, slowly dropwise adding ethylmagnesium bromide (150mL) into a reaction bottle, adding tetrapropyl titanate (45g, 150mmol) into the reaction system after dropwise adding, heating the reaction solution to room temperature after dropwise adding, stirring for 2 hours, detecting the completion of the reaction by thin layer chromatography (petroleum ether: ethyl acetate ═ 10:1), adding saturated ammonium chloride (100mL) into the reaction system to quench the reaction, extracting ethyl acetate (100mL × 3) for three times, washing with saturated common salt water, drying over anhydrous sodium sulfate, spin-drying, purifying by a column to obtain 4-benzyl-6- (benzyloxy) -4-azaspiro [2.5] octane (5.1 g), yellow solid, 31%).

Seventh step preparation of 4-azaspiro [2.5] octan-6-ol:

4-benzyl-6- (benzyloxy) -4-azaspiro [2.5] octane (5.5g, 18mmol) and palladium on carbon (2g,1.8mmol) were added to a solution of methanol (200mL) and hydrogen chloride (2mL), the reaction mixture was reacted at 60 ℃ for 48 hours under hydrogen conditions, the completion of the reaction was detected by thin layer chromatography (petroleum ether: ethyl acetate 10:1), the reaction mixture was filtered through celite, and the filtrate was dried by spinning to give 4-azaspiro [2.5] octan-6-ol (2.5g, yellow solid) which was used directly in the next reaction.

Eighth step preparation of tert-butyl 6-hydroxy-4-azaspiro [2.5] octane-4-carboxylate:

4-azaspiro [2.5] octan-6-ol (1g, 7.8mmol) and sodium carbonate (1.6g,15.7mmol) were added to a dichloromethane (100mL) solution, di-tert-butyl dicarbonate (1.71g, 7.8mmol) was added dropwise to the reaction system, the reaction mixture was reacted at room temperature for 18 hours, the reaction was detected by thin layer chromatography (petroleum ether: ethyl acetate ═ 3:1), the reaction mixture was extracted, the filtrate was dried by spinning, and column-purified to obtain 6-hydroxy-4-azaspiro [2.5] octane-4-carboxylic acid tert-butyl ester (1.7 g).

Ninth step preparation of tert-butyl 6- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate:

tert-butyl 6-hydroxy-4-azaspiro [2.5] octane-4-carboxylate (2.5g,11.0mmol) and 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (3.4g,13.0mmol) were dissolved in tetrahydrofuran (200 mL). Triphenylphosphine (4.5g,17.2mmol) was added in one portion in the ice bath and diisopropyl azodicarboxylate (3.5g,17.3mmol) was slowly added dropwise. The ice bath was removed and the reaction was stirred at room temperature for 18 hours and evaporated to dryness under reduced pressure. The crude product was purified by column on silica gel (EA: PE ═ 1:1) to give the product tert-butyl 6- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate (3.6g, 70%).

Tenth step preparation of tert-butyl 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate:

tert-butyl 6- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate (500mg,1.1mmol) and 5-chloro-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) pyridine (500mg,1.5mmol) were dissolved in 95% ethanol (20 mL). Sodium carbonate (230mg,2.2mmol) and bis (triphenylphosphine) palladium dichloride (120mg,0.2mmol) were added, the reaction was refluxed for 18 hours under argon protection, and evaporated to dryness under reduced pressure. The crude product was extracted with dichloromethane (50mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified using plate prep (dichloromethane: methanol ═ 50: 1) to give the product tert-butyl 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate (420mg, 76%).

Eleventh step preparation of 3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (4-azaspiro [2.5] oct-6-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride:

tert-butyl 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carboxylate (420mg,0.8mmol) was dissolved in tetrahydrofuran (10mL), concentrated hydrochloric acid (1mL) was added, the reaction mixture was stirred at 60 ℃ for 5 hours, and the mixture was evaporated to dryness under reduced pressure to give a crude product, 3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (4-azaspiro [2.5] oct-6-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (450 mg). Directly used for the next reaction.

Preparation of twelfth step 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile:

3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1- (4-azaspiro [2.5] oct-6-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (200mg,0.44mmol) was dissolved in N, N-dimethylformamide (2mL), cesium carbonate (200mg,0.62mmol) and cyanogen bromide (200mg,1.89mmol) were added, the reaction solution was stirred for 6 hours at room temperature, the reaction solution was poured into ethyl acetate (50mL), washed with water (30 mL. multidot.3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on plate (dichloromethane: methanol ═ 50: 1) to give the product compound 6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile (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]

Example 14

Preparation of (R) -6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile (WS-316) and (S) -6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile (WS-317)

Example 13 the product of the twelfth reaction was resolved by hand to give the compound of example 14. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-316 spectrogram data:

MS:m/z 473.2[M+1]

WS-317 spectrogram data:

MS:m/z 473.2[M+1]

example 15

(E) Preparation of (E) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one (WS-318)

Example 15 was prepared by the following procedure starting from the product of the eleventh reaction of example 13:

first step preparation of (E) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one:

3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (4-azaspiro [2.5] oct-6-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (220mg,0.48mmol) and 4-hydroxy-2-butenoic acid (80mg,0.78mmol) were dissolved in tetrahydrofuran (5mL), HATU (230mg,0.61mmol) and triethylamine (100mg,0.99mmol) were added, the reaction solution was stirred warm for 18 hours, and evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product prep. plate was purified (dichloromethane: methanol ═ 10:1) to give the product (E) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one (30mg, 12%).

¹H-NMR(400MHz,DMSO-d6):δppm 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]

Example 16

(R, E) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one (WS-319) and (S, E) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one (S, E) ((S, E) -1- (6-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4 WS-320) preparation

The compound of example 15 can be resolved by chiral resolution to give the compound of example 16. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-319 spectrum data:

MS:m/z 532.2[M+1]

WS-320 spectrogram data:

MS:m/z 532.2[M+1]

example 17

Preparation of 6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octane-4-carbonitrile (WS-321)

Example 17 was prepared in analogy to example 13, starting from the corresponding starting material.

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]

Example 18

(E) Preparation of (WS-322) 1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] oct-4-yl) -4-hydroxybut-2-en-1-one

Example 18 was prepared in analogy to example 15, starting from the corresponding starting material.

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]

Example 19

Preparation of (2S,5R) -5- (4-amino-3- (4- (4-chlorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-323) and (2R,5S) -5- (4-amino-3- (4- (4-chlorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-324)

Example 19 starting from the corresponding starting material, 5- (4-amino-3- (4- (4-chlorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile, which is prepared by a chiral resolution, is prepared in a similar manner to example 7 and gives the compounds WS-323 and WS-324. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-323 spectrogram data:

¹H-NMR(400MHz,CDCl3):δppm 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 spectrogram data:

MS:m/z 460.4[M+H]

example 20

Preparation of (2S,5R) -5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-325) and (2S,5R) -5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-326)

Example 20 starting from the corresponding starting material, 5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile, which is prepared by a chiral resolution, is prepared in a similar manner to example 9 and gives the compounds WS-325 and WS-326. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-325 spectrogram data:

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 spectrum data:

MS:m/z 426.5[M+1]

example 21

Preparation of 1- ((2S,5R) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-propen-1-one (WS-327) and 1- ((2R,5S) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-propen-1-one (WS-328).

Example 21 was prepared by the following procedure starting with the product of the fifth reaction step of example 7:

3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (300mg,0.63mmol) and acrylic acid (70mg,0.98mmol) were dissolved in THF (10mL), HATU (362mg,0.95mmol) and triethylamine (192mg,1.91mmol) were added, and the reaction solution was stirred warm for 18H and evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on a plate (DCM: MeOH ═ 10:1) to give 5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-propenyl-1-one (58mg, 29%). This compound was resolved by hand to give the compounds WS-327 and WS-328 of example 21. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-327 spectrum data:

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 spectrogram data:

MS:m/z 490.2[M+H]

example 22

Preparation of 1- ((2S,5R) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-butyn-1-one (WS-329) and 1- ((2R,5S) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-butyn-1-one (WS-330).

Example 22 was prepared by the following procedure starting from the product of the fifth reaction step of example 7:

3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (6-methylpiperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (230mg,0.53mmol) and but-2-ynoic acid (80mg,0.98mmol) were dissolved in THF (10mL), HATU (300mg,0.79mmol) and triethylamine (100mg,0.99mmol) were added, the reaction solution was stirred at room temperature for 18H, evaporated under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on a plate (DCM: MeOH ═ 10:1) to give 1- ((2S,5R) -5- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-butyn-1-one (130mg, 50%). This compound was resolved by hand to give the compounds WS-329 and WS-330 of example 22. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-329 spectrogram data:

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 spectrogram data:

MS:m/z 502.3[M+H]

example 23

Preparation of 1- ((2S,5R) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-propenyl-1-one (WS-331) and 1- ((2R,5S) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-propenyl-1-one (WS-332).

Example 23 was prepared in analogy to example 21, starting from the corresponding starting material.

WS-331 spectrum data:

¹H-NMR(400MHz,CDCl3):δppm 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 spectrum data:

MS:m/z 491.4[M+H]

example 24

Preparation of 1- ((2S,5R) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-butyn-1-one (WS-333) and 1- ((2R,5S) -5- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidin-1-yl) -2-butyn-1-one (WS-334).

Example 24 was prepared in analogy to example 22, starting from the corresponding starting material.

WS-333 spectrogram data:

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 spectrogram data:

MS:m/z 503.4[M+H]

example 25

Preparation of (R) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-propenyl-1-one (WS-335) and (S) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-propenyl-1-one (WS-336).

Example 25 was prepared by the following procedure starting from the product of the eleventh reaction of example 13:

3- (4- ((5-Chloropyridin-2-yl) oxy) phenyl) -1- (4-azaspiro [2.5] oct-6-yl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine hydrochloride (50mg,0.11mmol) and acrylic acid (8.03mg,0.11mmol) were dissolved in THF (5mL), HATU (63.67mg,0.17mmol) and triethylamine (33.89mg,0.33mmol) were added, the reaction solution was stirred warm for 18H, evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (50mL), washed with water (30mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on a plate (DCM: MeOH ═ 10:1) to give 1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-propenyl-1-one (38mg, 67%). This compound was resolved by hand to give the compounds WS-335 and WS-336 of example 25. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-335 spectrogram data:

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 spectrum data:

MS:m/z 502.4[M+H]

example 26

Preparation of (R) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-butyn-1-one (WS-337) and (S) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-butyn-1-one (WS-338).

Example 26 was prepared in analogy to example 22, starting from the corresponding starting material.

WS-337 spectrogram data:

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 spectrum data:

MS:m/z 514.3[M+H]

example 27

(R) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -4-hydroxy-2-butyn-1-one (WS-339) and (S) -1- (6- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -4-hydroxy-2-butyn-1-one (S) WS-340) preparation

Example 27 was prepared in analogy to example 21, starting from the corresponding starting material.

WS-339 spectrogram data:

¹H-NMR(400MHz,CDCl₃):δppm 8.38(s,1H),8.13(d,J＝2.4Hz,1H),7.76-7.69(m3H),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 spectrogram data:

MS:m/z 530.3[M+H]

example 28

Preparation of 4- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carbonitrile (WS-341)

Example 28 was prepared in analogy to example 1, starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃)δppm 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]

Example 29

Preparation of 5- (4-amino-3- (1-benzyl-1H-pyrazol-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-342)

Example 29 was prepared analogously to example 7, starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl3):δppm 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]

Example 30

Preparation of 5- (4-amino-3- (4- (morpholinomethyl) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-343)

Example 30 was prepared in analogy to example 7, starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl3):δppm 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]

Example 31

Preparation of 5- (4-amino-3- (4-benzylphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-344)

Example 31 was prepared analogously to example 7, starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl3):δppm 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]

Example 32

Preparation of 5- (4-amino-3- (4- (phenylamino) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -2-methylpiperidine-1-carbonitrile (WS-345)

Example 32 was prepared analogously to example 7, starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl3):δppm 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]

Example 33

Preparation of (R) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-propenyl-1-one (WS-346) and (S) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-propenyl-1-one (WS-347).

Example 33 was prepared in analogy to example 21, starting from the corresponding starting material.

WS-346 spectrogram data:

¹H-NMR(400MHz,CDCl3):δppm 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 Spectrum data:

MS:m/z 503.4[M+H]

example 34

Preparation of (R) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-butyn-1-one (WS-348) and (S) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -2-butyn-1-one (WS-349).

Example 34 was prepared in analogy to example 26, starting from the corresponding starting material.

WS-348 spectrogram data:

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 profile data:

MS:m/z 515.4[M+H]

example 35

Preparation of (R) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -4-hydroxy-2-butyn-1-one (WS-350) and (S) -1- (6- (4-amino-3- (4- (2, 4-difluorophenoxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -4-azaspiro [2.5] octan-4-yl) -4-hydroxy-2-butyn-1-one (WS-351).

Example 35 was prepared in analogy to example 21, starting from the corresponding starting material.

WS-350 spectrum data:

¹H-NMR(400MHz,CDCl3):δppm 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 spectrogram data:

MS:m/z 531.4[M+H]

example 36

Preparation of 3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidine-1-carbonitrile (WS-352)

Example 36 was prepared in analogy to example 1, starting from the corresponding starting material.

¹H-NMR(400MHz,CD₃OD)δppm 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]

Example 37

Preparation of (R) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidine-1-carbonitrile (WS-353) and (S) -3- (4-amino-3- (4- ((5-chloropyridin-2-yl) oxy) phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidine-1-carbonitrile (WS-354)

The compound of example 36 was resolved by hand to give compounds WS-353 and WS-354 of example 37. The splitting conditions are as follows: supercritical fluid chromatography (ChiralPak AD 5. mu. 21X 250mm col, 27% methanol, 70 mL/min).

WS-353 spectrum data:

MS:434.2[M+H]

WS-354 spectrogram data:

MS:434.2[M+H]

examples 38 to 47

By a method similar to that of examples 1 to 37 of the present patent, compounds having the structures shown in examples 38 to 47 can be produced.

Example 48

BTK kinase Activity inhibition assay

The compounds disclosed herein were tested for inhibition of BTK kinase activity in assays based on time-resolved fluorescence resonance energy transfer methods. Recombinant Btk and compounds disclosed herein were prepared at room temperature in the presence of 50mM Tris pH7.4, 10mM MgCl₂、2mM MnCl₂0.1mM EDTA, 1mM DTT, 20nM SEB, 0.1% BSA, 0.005% tween-20 in assay buffer were incubated for 1 hour in advance. The reaction was initiated by the addition of ATP (at ATP Km concentration) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH 2). After 1 hour incubation at room temperature, an equal volume of stop solution containing 50mM HEPES pH7.0, 800mM KF, 20mM EDTA, 0.1% BSA, Eu-cryptand-linked p-Tyr66 antibody, and streptavidin-labeled XL665 was added to stop the reaction. The plates were incubated for an additional 1 hour at room temperature, and then the TR-FRET signal (ex337nm, em 620nm/665nm) was read on a BMG PHERAStar FS instrument. The residual enzyme activity at increasing compound concentration was calculated based on the ratio of the fluorescence at 615nm to the fluorescence at 665 nm. IC50 for each compound was obtained by fitting the data to the four parameter logistic equation of Graphpad Prism software.

According to the above experimental methods, some of the compounds of the present invention showed strong BTK kinase inhibitory activity (IC50<1000nM), and some of the preferred compounds showed strong kinase inhibitory activity (IC50<100 nM). Specific results are shown in the following table.

The kinase inhibitory activity was ranked A, B, C, specifically A (IC)₅₀<100nM)，B(100nM<IC₅₀<1000nM)，C(IC₅₀>1000nM)

Example 49

In vitro kinase selectivity assay

Establishing an EGFR and ITK kinase activity detection platform by adopting a time-resolved fluorescence resonance energy transfer method; an LCK, SRC and LYN kinase activity detection platform is established by adopting a Z' -Lyte method; TEC and JAK3 kinase activity detection platforms are established by adopting a Lance Ultra method, and the inhibition effect of the compound disclosed by the invention on the activity of different kinases is respectively tested. The enzyme activity data was determined at 11 concentrations for each compound and the IC of the compound was calculated using Graphpad Prism software₅₀The value is obtained.

According to the experimental methods, some of the compounds of the present invention showed strong kinase selectivity, which is significantly superior to that of the control compound ibrutinib. The results are shown in the following table.

Compound numbering

LCK

SRC

LYN

EGFR

ITK

TEC

WS-311

C

B

C

A

WS-349

B

C

A

Ibrutinib

A

Example 50

B cell inhibition assay

Brief exposure to BTK inhibitors in vitro is sufficient to inhibit B cell activation in normal human B cells. This protocol mimics the predicted exposure of cells to the inhibitor in vivo and shows that inhibition of B cells is maintained despite washing of the inhibitor.

CD B cells were purified from healthy donor blood by negative selection using a cocktail of human B cell enrichment in RosetteSep (RosetteSep). Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and added with the indicated concentration of inhibitor. After incubation for 1 hour at 37 ℃, cells were washed three times, each wash being diluted 8-fold in growth medium. Cells were then stimulated with 10. mu.g/mL IgM F (ab')2 for 18 hours at 37 ℃. Cells were then stained with anti-CD 69-PE antibody and analyzed by flow cytometry using standard conditions. Preferred compounds of the invention have a strong inhibitory activity against B cells, as measured by the above method, with an IC50 value of less than 1 nM.

Example 51

T cell suppression assay

CD T cells were purified from healthy donor blood by negative selection using a cocktail of human T cell enrichment in RosetteSep (RosetteSep). Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and added with the indicated concentration of inhibitor. After incubation for 1 hour at 37 ℃, cells were washed three times, each wash being diluted 10-fold in growth medium. Cells were then stimulated with anti-CD3/CD28 coated beads (bead/cell ratio 1:1) for 18 hours at 37 ℃. Cells were then stained with anti-CD 69-PE antibody and analyzed by flow cytometry using standard conditions.

Preferred compounds of the invention have weak or no inhibitory activity against T cells, as measured by the above method, with IC50 values greater than 4000 nM.

Example 52

Human Whole blood B cell inhibition assay

Human whole blood (hWB) was obtained from healthy volunteers and collected by venipuncture into Vacutainer tubes anticoagulated with sodium heparin. Test compounds were diluted 10-fold in PBS to the desired initial drug concentration) followed by three-fold serial dilutions in 10% DMSO in PBS to give a 9-point dose response curve. 5.5 μ L of each compound dilution was added in duplicate to a plate with aiil 96 wells in V-bottom; to control and non-stimulated wells 5.5 μ L of 10% DMSO in PBS was added. Human whole blood (100. mu.L) was added to each well and the plates were mixed at 37C, 5% CO₂，100％Humidity incubation for 30 minutes. To each well (except for non-irritating wells) sheep F (ab')2 anti-human igm (southern biotech) (10 μ L of 500 μ g/mL solution, 50 μ g/mL final concentration) was added with stirring and the plates were incubated for an additional 20 hours. At the end of the 20 hour incubation, the samples were mixed with 20. mu.L of APC mouse anti-human CD69(BD Pharmingen) labeled with fluorescent probes at 37C, 5% CO₂Incubate at 100% humidity for 30 minutes. Including induced control, unstained and single stain for compensation of adjustments and initial voltage settings. The sample was then lysed with 1ml of IX Pharmingen Lyse Buffer (BD Pharmingen) and the plate was centrifuged at 1500rpm for 5 minutes. The supernatant was removed by aspiration, the remaining pellet was re-lysed with another 1ml of IX Pharmingen Lyse Buffer, and the plates were centrifuged as before. The supernatant was aspirated, the residual pellet was washed in FACs buffer (PBS + 1% FBQ. after centrifugation and removal of supernatant, the pellet was resuspended in 150. mu.L of FACs buffer.

Preferred compounds of the invention have a strong inhibitory activity against B cells in human whole blood, as measured by the above method, with an IC50 value of less than 200 nM.

Example 53

Stability study of Compounds in liver microsomes

1. The test compound was dissolved in acetonitrile to prepare a stock solution having a concentration of 0.5 mM.

mu.L of the stock solution was added to a 1.5ml centrifuge tube, followed by 148. mu.L of phosphate buffer (100mM, pH 7.4) and 10. mu.L of liver microsome (protein concentration 20mg/ml) suspension (BD Gentest Co., Ltd.) of human, dog, rat, mouse species, respectively; to the control group, 158. mu.L of phosphate buffer (100mM, pH 7.4) was added.

3. The mixed system prepared in the step 2 is pre-incubated for 3 minutes in water bath at 37 ℃, and then 40 mu L of NADPH generating system (containing NADP +: 6.5 m) is addedM, glucose 6-phosphate: 16.5mM, MgCl₂: 16.5mM, glucose 6-phosphate dehydrogenase: 2U/ml) and incubated in a water bath at 37 ℃ for 1 hour.

4. After the reaction was carried out for 1 hour, the centrifuge tube was taken out from the water bath, and 400. mu.L of acetonitrile was added to terminate the reaction, followed by vortex shaking for 3 minutes and finally centrifugation (13000rpm, 4 ℃) for 5 minutes, and the supernatant was taken to measure the remaining drug concentration Cr by HPLC.

5. Preparation of 0 min reaction samples prepared in parallel: and (3) pre-incubating the mixed system prepared in the step 2 in a water bath at 37 ℃ for 3 minutes, taking out, adding 400 mu L of acetonitrile, and then adding 40 mu L of NADPH to generate the system. After vortexing for 3 min, the cells were centrifuged (13000rpm, 4 ℃) for 5min, and the supernatant was analyzed by HPLC for the drug concentration C0.

6. After 60 minutes incubation, the remaining percentage of drug in the incubation system was calculated as follows:

the drug residue (%) ═ Cr/C0X 100%

Preferred compounds of the invention exhibit better microsomal stability according to the experimental procedure described above, with a residual percentage of > 30% in liver microsomes of various species.

Example 54

Evaluation of Compound on inhibition of CYP enzymes

CYP enzyme metabolism is the main pathway for drug biotransformation, and the number and activity directly affect the activation and metabolism of drugs in vivo. Cytochrome CYP, a major metabolic enzyme of exogenous compounds, is an important drug phase I metabolic enzyme, and can catalyze oxidative and reductive metabolism of a variety of exogenous compounds. CYP enzymes play a very important role in the elimination process of drugs and are also the main factors causing drug interaction in drug combination.

The method comprises the following steps: in the experiment, a cocktail probe pharmaceutical method is adopted to simultaneously determine the inhibition effect of the compound on five CYP450 enzymes in human liver microsomes, wherein the human microsomes come from BD Gentest company.

The experimental procedure was as follows:

the reaction is carried out in 100mM phosphate buffer solution, the total volume is 200 mu L, the microsome concentration in the reaction system is 0.25mg/mL, the concentration of the compound to be tested is 20 mu M, 6.67 mu M, 2.22 mu M, 0.74 mu M and 0.25 mu M, the specific probe substrate and the concentration are respectively phenacetin (CYP1A2)40 mu M, dextromethorphan (CYP2D6)5 mu M, diclofenac (CYP2C9)10 mu M, S-mefenstazin (CYP2C19)40 mu M and testosterone (CYP3A4)80 mu M, the incubation system is pre-incubated for 5 minutes in a 37-degree constant temperature oscillator, acetonitrile with equal volume is added after the incubation is carried out for 5 minutes, an NADPH generation system (containing 1.3mM NADP +, 3.3mM glucose 6-phosphate, 0.4U/L glucose 6-phosphate dehydrogenase, 3.3mM MMCL 85) is added for stopping the reaction, the reaction is carried out after the NADPH generation system is incubated for 45min, the final inhibition rate is measured by taking the concentration of the low fenpropiconazole (CYP 2A) of the CYP 6-CYP 6 inhibiting rate (CYP 6-CYP 8 percent), the concentration of the low fenpropiconazole, the final inhibition rate of the metabolite (CYP 2) and the final inhibition rate of the CYP 3-fenbuconazole (CYP 3-fenpropiconazole), the final inhibition rate of the metabolite inhibition rate is calculated by the method, the CYP 2A-3-fenhydrazole (CYP 2-3-fenbuconazole (CYP 2) and the concentration of the test results are respectively, the low-inhibition rate of the CYP 6-inhibition rate of the test results of the CYP 2-inhibition rate of the CYP 6-inhibition rate of the CYP 2-inhibition rate of the CYP 6-CYP 2-fencloquinconazole (CYP 2-fencloquinconazole), the test is calculated by the test is showed that the test is lower than the test is.

Example 55

Pharmacokinetics research method of compound in rat body

1. Male SD rats [ Huafukang ] were purchased and then adaptively bred in the laboratory for 7 days.

2.9 SD rats were randomly divided into 3 groups of 3 rats, one group for intragastric administration and the other group for tail vein injection. Rats in the gavage group were fasted overnight before dosing.

3. Following administration to rats, blood samples were collected at the following time points using the orbital venous plexus blood collection method: i.v. (before administration), 0.08 hours, 0.25 hours, 0.5 hours, 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. The amount of blood collected at each blood collection time point was about 300. mu.l.

4. The collected blood samples were centrifuged at 12000rpm for 5 minutes at 4 ℃ and then the upper plasma samples were collected and stored in a-20 ℃ freezer for testing.

5. The experimental procedures are summarized in table 4:

TABLE 4 pharmacokinetic test design of Compounds in rats

6. The concentration of the compound in plasma was measured using LC-MS/MS (UPLC-MS/MS: liquid phase Waters Acquity UPLC (USA) and Mass Spectrometry 5500QTrap (Applied Biosystem/MDS SCIEX) or HPLC-MS \ MS: liquid phase Agilent 1200 servers (USA) and Mass Spectrometry API 4000(Applied Biosystem/MDS SCIEX)). Typical assay conditions are shown in the following table.

Pharmacokinetic professional software WinNonlin model no: phoenix^TMWinNonlin

6.1 manufacturer: pharsight Corporation calculates pharmacokinetic parameters. [ Phoenix 1.1 User's Guide: p251-p300 ]

According to the experimental procedure described above, the compounds determined in the present invention show better bioavailability (> 40%).

Example 56

hERG binding assay (Dofetilide method)

IC50 values of compounds for hERG inhibition can be determined according to the method described on patent US20050214870 a 1. Preferred compounds of the invention have little or no inhibition of hERG, with IC50 values greater than 1000 nM.

Claims

1. A pyrazolopyrimidine compound of formula (I), a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

here, the first and second liquid crystal display panels are,

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

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

A is taken from a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to the parent nucleus and L is optional;

b is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the site of attachment to L is optional;

y is selected from cyano, or

Here, R₃、R₄、R₅And R₆Each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano, or- (CH)₂)_qN(R^aR^b) Where q is 1, 2, 3, or 4, R^aAnd R^bEach independently selected from hydrogen, unsubstituted C1-C4 alkyl;

drawing instrumentWhen R is fixed₁And R₂When both are hydrogen, A is phenyl, L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2, 4-difluorophenyl; y is 4-hydroxybut-2-enoyl, but-2-ynoyl, or cyano;

2. The compound of 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. The compound of claim 1, wherein a is taken from a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to the parent nucleus and L is optional; b is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted phenyl ring, or a substituted or unsubstituted heteroaryl ring, and the site of attachment to L is optional; here:

the substituted benzene ring is substituted by optional substituent groups on the phenyl at any position, wherein the substituent groups are selected from hydrogen, methyl, methoxy, fluorine, chlorine, trifluoromethyl, trifluoromethoxy or cyano; preferably, the substituted phenyl ring is fluoro-substituted phenyl, or chloro-substituted phenyl, more preferably 2, 4-difluorophenyl, or 4-chlorophenyl;

4. The compound of claim 1, wherein R₁And R₂One of which is hydrogen and the other is C1-C4 alkyl, or R₁And R₂Together with the carbon atom to which they are attached form a cyclopropyl group; more preferably, R₁And R₂Are both hydrogen, or one is hydrogen and the other is methyl, or R₁And R₂Together with the carbon atom to which they are attached form a cyclopropyl group.

5. The compound of claim 1, wherein, preferably, the pyrazolopyrimidine compound of formula (II), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein L, A, B and Y are as defined above for formula (I);

or, A is phenyl, L is C (O) -, or-CH₂-, B is morpholin-4-yl; y is cyano;

or, A is pyridyl, L is NH, or O, B is pyran-4-yl, or cyclohexyl; y is cyano;

more preferably, the pyrazolopyrimidine compound of formula (II) is one of the following compounds:

or, L is C (O) -, or-CH₂-, B is morpholin-4-yl; y is cyano;

or: preferably, the pyrazolopyrimidine compound represented by formula (III), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein L, A, B and Y are as defined above for formula (I);

more preferably, the pyrazolopyrimidine compound of formula (III) is one of the following compounds:

here, L, B and Y in formulae (III-1), (III-2), (III-3) and (III-4) are as defined above for formula (I); or:

preferably, the pyrazolopyrimidine compound represented by formula (IV), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein L, A, B and Y are as defined above for formula (I);

more preferably, the pyrazolopyrimidine compound of formula (IV) is one of the following compounds:

6. The compound of claim 5, wherein L is O;

b is

And

y is selected from cyano, or

R₃、R₄、R₅And R₆Is selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano, or- (CH)₂)_qN(R^aR^b) Where q is 1, 2, 3, or 4, R^aAnd R^bEach independently selected from hydrogen, unsubstituted C1-C4 alkyl.

7. One selected from the following compounds, or a pharmaceutically acceptable salt thereof:

8. a process for the preparation of a compound according to any one of claims 1 to 7, comprising the steps of:

the substituent R referred to in the above-mentioned formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI)₁、R₂L, B, Y and n, m are as defined above for formula (I), PG is an amino protecting group and X is chlorine, bromine or hydroxy.

9. A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof.

10. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 9 for the manufacture of a BTK inhibitor medicament.

11. The use according to claim 10, wherein the BTK inhibitor is for the prevention or treatment of a BTK mediated disease selected from the group consisting of autoimmune diseases, inflammatory diseases, heteroimmune conditions or diseases, thromboembolic diseases, and cancer.