CN104086551B

CN104086551B - Compound and its production and use

Info

Publication number: CN104086551B
Application number: CN201410251204.2A
Authority: CN
Inventors: 许勇; 王学海; 李莉娥; 黄璐; 田华; 杨仲文; 夏庆丰; 肖强; 郭涤亮; 涂荣华; 余艳平; 于静; 黄翔; 范昭泽; 何震宇; 张毅; 杨菁
Original assignee: Ren Fu Pharmaceutical Group Stock Co
Current assignee: Hubei Co Ltd Of Bio-Pharmaceutical Industry Institute For Research And Technology
Priority date: 2014-06-06
Filing date: 2014-06-06
Publication date: 2016-09-21
Anticipated expiration: 2034-06-06
Also published as: CN104086551A; WO2015184661A1

Abstract

The invention provides compound and its production and use, this compound is the stereoisomer of compound, tautomer, pharmaceutical active metabolite, officinal salt, hydrate, solvate or prodrug shown in compound shown in Formulas I or Formulas I, wherein, X, R₁、R₂It is defined as in the description.This compound can be as inhibitors of kinases.

Description

Compound and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a compound, a preparation method and application thereof, and more particularly relates to a compound, a preparation method thereof, a pharmaceutical composition, a compound and application of the pharmaceutical composition in preparation of medicines.

Background

Bruton's Tyrosine Kinase (BTK), a non-receptor tyrosine kinase of the Tec kinase family, is a key regulator of B cell development, activation, signaling and survival, playing an important role in B Cell Receptor (BCR) signal transduction. Upon activation of BCR, BTK is first activated by other tyrosine kinases (e.g., Lyn and SYK), resulting in activation of transcription factors essential for B cell proliferation and differentiation. In addition, BTK is involved in receptor signaling associated with B cell migration and adhesion, including chemokine receptors CXCR4, CXCR5, and adhesion molecules (integrins). Uncontrolled activity of BTK kinase leads to irregular cell proliferation that can lead to or enhance carcinogenesis.

The BTK inhibitor ibrutinib was approved by FDA in 2013 at 11 months for the treatment of mantle cell lymphoma, and approved for the treatment of chronic lymphocytic leukemia in 2014 at 2 months. Ibrutinib can form a strong covalent bond with cysteine residue on BTK selectively and irreversibly, and inhibit the transmission of an over-active cell survival signal in malignant B cells so as to achieve the anticancer effect, and Ibrutinib is also the only BTK inhibitor on the market at present. However, ibrutinib has low solubility, high binding rate (PPB) with plasma protein and low oral bioavailability, which directly results in poor therapeutic effect of patients after oral administration.

Thus, current BTK inhibitors remain to be improved.

Disclosure of Invention

The present invention aims to solve at least one of the above technical problems to at least some extent or to at least provide a useful commercial choice. To this end, it is an object of the present invention to propose a compound useful as a kinase inhibitor.

It is another object of the present invention to provide processes and intermediates for preparing compounds of formula I, or stereoisomers, tautomers, pharmaceutically active metabolites, pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof.

It is another object of the present invention to provide a pharmaceutical composition comprising a compound of the present invention, said pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients and a therapeutically effective amount of at least one compound of the present invention, or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof.

It is another object of the present invention to provide the use of a compound of the present invention, or a stereoisomer, tautomer, pharmaceutically active metabolite, pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, for the manufacture of a medicament. According to the embodiment of the invention, the drug can be used as a BTK inhibitor for treating diseases mediated by B cells and is used for preparing the drug for treating tumor diseases, proliferative diseases, allergic diseases, autoimmune diseases and inflammatory diseases.

These and other objects, features and advantages of the compounds of the present invention are disclosed in the following detailed description of the present patent disclosure.

The present invention is described in detail below:

in a first aspect of the invention, the invention features a compound. According to an embodiment of the invention, the compound is a compound of formula I or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug of a compound of formula I,

wherein X is selected from one of the following: substituted or unsubstituted phenyl, substituted or unsubstituted 3-to 7-membered saturated or unsaturated carbocyclic ring, substituted or unsubstituted 8-to 10-membered saturated or unsaturated bicyclic or aryl ring, substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, substituted or unsubstituted 4-to 7-membered saturated or unsaturated heteroatom having 1 to 3 heteroatomsA ring, a substituted or unsubstituted 7-to 10-membered saturated or unsaturated bicyclic heterocycle having 1 to 5 heteroatoms, a substituted or unsubstituted 8-to 10-membered bicyclic heteroaryl ring having 1 to 5 heteroatoms; r₁Is selected from-R, halogen, -OR, -O (CH)₂)aOR、-CN、-NO₂、-SO₂R、-SO₂N(R)₂、-SOR、-C(O)R、-CO₂R、-C(O)N(R)₂、-NRC(O)R、-NRC(O)NR₂、-NRSO₂R and-N (R)₂Any one of the above; r₂Is selected from-R, halogen, -OR, -O (CH)₂)aOR、-CN、-NO₂、-SO₂R、-SO₂N(R)₂、-SOR、-C(O)R、-CO₂R、-C(O)N(R)₂、-NRC(O)R、-NRC(O)NR₂、-NRSO₂R and-N (R)₂Any one of the above; wherein a is an integer from 1 to 5, and each R group is independently selected from one of the following: hydrogen, substituted or unsubstituted C_1-6An aliphatic group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted 4-to 7-membered heterocyclic ring having 1 to 2 heteroatoms, a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms, each independently selected from any one of nitrogen, oxygen, and sulfur.

Further, according to embodiments of the present invention,is selected from one of the following:

it will be understood by those skilled in the art that, according to the convention used in the art, in the structural formulae of the present application,for delineating chemical bonds, which are the points at which moieties or substituents are attached to a core structure or a backbone structure.

Thus, throughout this specification, those skilled in the art will appreciateThe groups and substituents thereof are selected to provide stable compounds of formula I as described in the examples of the invention or stereoisomers, tautomers, pharmaceutically active metabolites, pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof.

All stereoisomers of the compounds of the invention, whether in admixture or pure form or substantially pure form, are contemplated by the term as used in the present invention. The term "stereoisomer" as used in the present invention may include compounds which are optical isomers by possessing one or more chiral atoms, as well as compounds which are optical isomers by restricted rotation about one or more bonds. The definition of the compounds of the present invention covers all possible stereoisomers and mixtures thereof. Very specifically contemplated are racemic forms and isolated optical isomers having a particular activity. The racemic forms can be resolved by physical methods including, but not limited to, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by conventional methods including, but not limited to, salt formation with an optically active acid followed by crystallization.

As the term is used in the present invention, the compounds of formula I and their salts may exist in their tautomeric form in which hydrogen atoms are transferred to other parts of the molecule and chemical bonds between atoms in the molecule are thus rearranged. It is to be understood that all tautomeric forms, insofar as they may exist, are included herein. In addition, the compounds of formula I of the present invention may have trans isomers and cis isomers.

As used herein, the term "pharmaceutically acceptable salts" refers to the conventional non-toxic salts formed by the reaction of a compound of formula I with an inorganic or organic acid. For example, the conventional non-toxic salts can be prepared by reacting the compounds of formula I with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, and the like; or sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound shown in the general formula I, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid after forming ester and then forming inorganic base; or methylamine salt, ethylamine salt or ethanolamine salt formed by the compound shown in the general formula I and organic alkali; or the compound shown in the general formula I forms ester with lysine, arginine and ornithine and then forms corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid or forms corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

As used herein, the term "prodrug" means that upon administration of the compound to a subject, the compound undergoes chemical transformation by metabolic or chemical processes to yield the compound of formula I and/or a salt and/or solvate thereof. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the present invention. For example, compounds containing a carboxyl group may form physiologically hydrolyzable esters that act as prodrugs by hydrolyzing in vivo to give the compounds of formula I themselves. The prodrugs are preferably administered orally, since hydrolysis in many cases takes place mainly under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.

It is also understood that hydrates, solvates (e.g., methanolate, DMSO) of the compounds of formula I of the present invention are also within the scope of the present invention. Methods of solvation are well known in the art.

In a second aspect of the present invention, the present invention provides a method and an intermediate for preparing a compound shown in formula i, or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof.

According to the embodiment of the invention, the method for preparing the compound shown in formula i or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof comprises the following steps: (1) contacting the compound represented by the formula 1 with a compound represented by the formula 2 to obtain a compound represented by the formula 3; (2) contacting the compound represented by the formula 3 with a compound represented by the formula 4 (acryloyl chloride) to obtain a compound represented by the formula 5; (3) contacting the compound represented by the formula 5 with a compound represented by the formula 6a so as to obtain the compound represented by the formula I.

Wherein, X, R₁And R₂As defined above.

The inventor finds that the compound shown in the formula I or the stereoisomer, the tautomer, the pharmaceutically active metabolite, the pharmaceutically acceptable salt, the hydrate, the solvate or the prodrug thereof can be quickly and effectively prepared by the method, the synthetic route is short, the environment is friendly, the yield and the purity of the target product are high, the raw materials are easy to obtain, the operation and the post-treatment are simple, and the method is suitable for industrial production.

The general process used in the examples of the invention for preparing compounds of formula I is described below:

1) preparation of Compound (intermediate) represented by formula 3

Contacting a compound represented by formula 1 with a compound represented by formula 2 to obtain a compound represented by formula 3;

specifically, the compound shown in the formula 1, the compound shown in the formula 2 and Tetrahydrofuran (THF) are added into a three-necked bottle, cesium carbonate is added, reflux is carried out for 12-24 hours, after the reaction is finished, an oily liquid is obtained through concentration, methanol is firstly used for dissolution, acetone is then used for recrystallization, a solid is separated out, and the compound shown in the formula 3 is obtained through filtration and drying without further purification.

2) Preparation of Compound (intermediate) represented by formula 5

Contacting the compound represented by the formula 3 with a compound represented by the formula 4 to obtain a compound represented by the formula 5;

specifically, the compound shown in the formula 3 and N-methylpyrrolidone (NMP) are mixed at the temperature of between 5 ℃ below zero and 5 ℃, the compound shown in the formula 4 is slowly added, the mixture is subjected to heat preservation and stirring reaction at the temperature of 0 ℃ for 60 minutes, then water is added, the stirring is continued for half an hour, and then saturated NaHCO is added₃The solution was basified in aqueous solution and then extracted 3 times with ethyl acetate. The combined ethyl acetate extracts were washed with water, then brine, Na₂SO₄Drying and concentration under reduced pressure gave the compound represented by formula 5.

3) Preparation of Compounds of formula I

Contacting the compound represented by the formula 5 with a compound represented by the formula 6a so as to obtain a compound represented by the formula I;

specifically, the compound represented by formula 5 and the compound represented by formula 6a (the molar ratio of the compound represented by formula 5 to the compound represented by formula 6a is 1: 1.5) are added to a solution of N-methylpyrrolidone, and microwave irradiation is performed (100 ℃ to 200 ℃ for 5 to 30 minutes). The reaction mixture was cooled, diluted with water, and extracted 3 times with ethyl acetate. The combined ethyl acetate extracts were washed with water, then brine, Na₂SO₄Drying and concentrating under reduced pressure, dissolving with isopropanol, recrystallizing at-5 deg.C, filtering to obtain white solid, and drying under reduced pressure to obtain the compound shown in formula I.

In embodiments of the invention, the compound of formula I may be: a compound of formula I or a stereoisomer, tautomer, pharmaceutically active metabolite, pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

The inventors of the present invention found that the compounds of the present invention also show improved solubility and less efflux from cells, thereby improving bioavailability.

In a third aspect of the present invention, the present invention provides a pharmaceutical composition comprising a compound of formula I or at least one of a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof. According to an embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise conventional additives such as flavoring agents, etc.

The pharmaceutical composition provided by the invention preferably contains 0.1-99% of the compound shown in the formula I as an active ingredient, more preferably, the compound shown in the formula I as an active ingredient accounts for 10-40% of the total weight of the pharmaceutical composition, and the balance is a pharmaceutically acceptable carrier and/or a conventional additive.

The compounds and pharmaceutical compositions provided herein may be in a variety of forms such as tablets, capsules, injections, injectable powders, syrups, solutions, suspensions, aerosols, and the like, and may be presented in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or instillation.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The compounds and pharmaceutical compositions of the present invention may be administered to mammals in clinical use, including humans and animals, by oral, nasal, dermal, pulmonary or gastrointestinal routes, among others. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment regimen. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found. The most preferred route of administration is oral.

In a fourth aspect of the invention, the invention provides a use of the compound shown in formula i or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof in preparing a medicament. According to an embodiment of the invention, the medicament is for use as a kinase inhibitor. According to embodiments of the invention, the compounds of the invention may modulate kinase activity, including modulation of BTK, and may be useful as kinase inhibitors, and other types of kinase activity that may be modulated by the compounds of the invention include, but are not limited to, members of the Tec family, such as BMX, BTK, ITK, Txk, and Tec, and mutants thereof. Therefore, the medicament can be effectively used as a BTK inhibitor, and further can treat diseases mediated by B cells, and can be used for treating tumor diseases, proliferative diseases, allergic diseases, autoimmune diseases and inflammatory diseases.

Furthermore, compared with the existing BTK inhibitor ibrutinib, the compound shown in the formula I has higher BTK kinase inhibition activity. Furthermore, after pretreatment of recombinant BTK with the compound of formula I of the present invention, repeated washing with inhibitor-free medium did not restore activity, indicating irreversible inhibition of BTK by the compound of the present invention.

In particular, the agents of the invention are useful for the treatment of one or more diseases associated with BTK activity including, but not limited to, solid tumors, lymphomas, soft tissue sarcomas, lymphocytic lymphomas, mantle cell lymphomas, melanomas, multiple myelomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, rheumatoid spondylitis, gouty arthritis, and the like.

Further, in a bovine collagen-induced murine arthritis model (CIA) experiment, the compounds of the examples of the present invention showed better therapeutic effects than ibrutinib.

The compound shown as the formula I used as the kinase inhibitor is used as a BTK inhibitor, successfully overcomes the defects of low solubility and low oral bioavailability of the existing BTK inhibitor ibrutinib medicament, and has good clinical application and medical application.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The embodiment of the invention provides a compound shown in a formula I or a stereoisomer, a tautomer, a pharmaceutically active metabolite, a pharmaceutically acceptable salt, a hydrate, a solvate or a prodrug thereof, a method for preparing the compound shown in the formula I or the stereoisomer, the tautomer, the pharmaceutically active metabolite, the pharmaceutically acceptable salt, the hydrate, the solvate or the prodrug thereof, an intermediate and a pharmaceutical composition thereof, and application of the compound and the pharmaceutical composition in preparing medicaments.

Example 1: preparation of the Compound (intermediate) of formula 3

A compound represented by formula 1 (17.0 g, 0.1 mol), a compound represented by formula 2 (11.1 g, 0.11 mol), and tetrahydrofuran (800 ml) were added to a three-necked flask, cesium carbonate (39.1 g, 0.12 mol) was added, the mixture was refluxed for 15 hours, and after the reaction was completed, the mixture was concentrated to give an oily liquid, which was dissolved with methanol (60 ml), and then stirred with acetone (180 ml) to recrystallize, precipitate a solid, which was filtered, and dried to give a compound represented by formula 3 (18.7 g, yield 74.0%) without further purification.

Example 2: preparation of the Compound (intermediate) of formula 3

A compound represented by formula 1 (17.0 g, 0.1 mol), a compound represented by formula 2 (15.2 g, 0.15 mol), and tetrahydrofuran (800 ml) were added to a three-necked flask, cesium carbonate (48.9 g, 0.15 mol) was added, the mixture was refluxed for 12 hours, and after the reaction was completed, the mixture was concentrated to give an oily liquid, which was dissolved with methanol (60 ml), and then stirred with acetone (180 ml) to recrystallize, precipitate a solid, which was filtered, and dried to give a compound represented by formula 3 (18.2 g, yield 72.1%) without further purification.

Example 3: preparation of the Compound (intermediate) of formula 3

A compound represented by formula 1 (17.0 g, 0.1 mol), a compound represented by formula 2 (10.6 g, 0.105 mol), and tetrahydrofuran (800 ml) were added to a three-necked flask, cesium carbonate (39.1 g, 0.12 mol) was added, the mixture was refluxed for 24 hours, and after the reaction was completed, the mixture was concentrated to give an oily liquid, which was dissolved with methanol (60 ml), and then stirred with acetone (180 ml) to recrystallize, precipitate a solid, which was filtered, and dried to give a compound represented by formula 3 (18.4 g, yield 72.8%) without further purification.

Example 4: preparation of the Compound (intermediate) of formula 5

A compound of formula 3 (25.3 g, 0.1 mol) was reacted with N-methylpyrrolidone (300 mL) at-5 deg.CMixing at 5 deg.C, slowly adding the compound of formula 4 (19.9 g, 0.22 mol), stirring at 0 deg.C for 60 min, adding water (50 ml), stirring for half an hour, and adding saturated NaHCO₃The solution was basified in aqueous solution and then extracted 3 times with 300 ml of ethyl acetate. The combined ethyl acetate extracts were washed with water, then brine, Na₂SO₄Drying and concentration under reduced pressure gave the compound represented by formula 5 (23.9 g, yield 66.3%).

Example 5: preparation of the Compound of formula I-1

The compound of formula I-1 was prepared according to the scheme, procedure described below. The method comprises the following specific steps:

a compound represented by the formula 5 (36.1 g, 0.1 mol) and a compound represented by the formula 6a-1 (15.2 g, 0.15 mol) were added to a solution of N-methylpyrrolidone (600 ml), and microwave irradiation was carried out (100 ℃ C., 30 minutes). The reaction mixture was cooled, diluted with 500 ml of water and extracted 3 times with 400 ml of ethyl acetate. The combined ethyl acetate extracts were washed with water, then brine, Na₂SO₄Drying and concentrating under reduced pressure, dissolving with 300 ml of isopropanol, recrystallizing at-5 ℃, filtering to obtain white solid, and drying under reduced pressure to obtain the compound shown in the formula I-1 (the yield is 34.5 g, and the yield is 78.0%).

Example 6: preparation of the Compound of formula I-2

A compound represented by the formula 5 (36.1 g, 0.1 mol) and a compound represented by the formula 6a-2 (25.1 g, 0.15 mol) were added to a solution of N-methylpyrrolidone (600 ml), and microwave irradiation was carried out (160 ℃ C., 10 minutes). The subsequent treatment was conducted in the same manner as in example 5 to obtain a compound represented by the formula I-2 (yield 40.2 g, yield 81.6%).

Example 7: preparation of the Compound of formula I-3

A compound represented by the formula 5 (36.1 g, 0.1 mol) and a compound represented by the formula 6a-2 (30.8 g, 0.15 mol) were added to a solution of N-methylpyrrolidone (500 ml) and subjected to microwave irradiation (200 ℃ C., 5 minutes). The subsequent treatment was conducted in the same manner as in example 5 to obtain a compound represented by the formula I-3 (yield 38.1 g, yield 72.0%).

According to the examples of the present invention, the compounds represented by the formula I-4 to the compounds represented by the formula I-26 were prepared in the same manner as in the examples 5 to 7 from the compounds represented by the formula I-1 to the compounds represented by the formula I-3. Wherein the feeding molar ratio of the compound shown in the formula 5 to the compound shown in the formula 6a is 1: 1.5. for example, starting from a compound of formula 6a-15, a compound of formula 5 is contacted with a compound of formula 6a-15 to obtain the corresponding compound of formula I-15. Specific compounds and data are shown in table 1 below.

Table 1:

example 8: human B cell stimulation protocol

Human B cells were purified from 150ml blood. Specifically, the blood was diluted 1/2 with PBS and centrifuged through a Ficoll density gradient. B cells were isolated from monocytes by negative selection using B cell isolation kit II from miltenyi (Auburn, CA). Then, 50000B cells were stimulated with 10. mu.g/ml goat F (ab') 2 anti-human IgM antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.) per well in a 96-well plate. The compounds represented by formulae I-1 to I-26 prepared in examples 1 to 7 were diluted with DMSO (dimethyl sulfoxide) and added to the cells. The final concentration of DMSO was 0.5%. Proliferation was measured after 3 days using Promega CellTiter-Glo (Madison, Wis.). As a result, the compounds shown in the formula I are tested to be active.

Example 9: human recombinant BTK enzyme assay

To a V-bottom 384-well plate, test compound, human recombinant BTK (1nM, Invitrogen corporation), fluorescein peptide (1.5. mu.M), ATP (20. mu.M), and assay buffer (20mM HEPES (pH 7.4), 10mM MgCl₂0.015% Brij35 and 4mM DTT in 1.6% DMSO), with a final volume of 30 μ L. After 60 minutes incubation at room temperature, the reaction was stopped by adding 45. mu.l of 35mM EDTA to each sample. The reaction mixture was subjected to electrophoretic separation of the fluorescent substrate and phosphorylated product on a Caliper LabChip3000(Caliper, Hopkinton, Mass.)And (6) analyzing. Inhibition data was calculated by comparison with the no enzyme control reaction as 100% inhibition and the no inhibitor control as 0% inhibition. Dose response curves were generated to determine the concentration required to inhibit kinase activity by 50% (IC 50). Compounds were dissolved in dimethyl sulfoxide (DMSO) at 10mM and then evaluated at 11 concentrations. Using this assay, IC50 values were determined for the compounds of the invention as well as for the existing BTK inhibitor compound ibrutinib. The results are shown in Table 2.

Table 2 shows the activity of selected compounds of the invention in an in vitro BTK kinase inhibition assay. The compound numbers correspond to the compound numbers in table 1. The compound with activity designated "A" provides an IC50 ≦ 10 nM; the compound activity designated "B" provided an IC50 of 10-100 nM; compounds with activity designated "C" provided an IC50 of 100-1000 nM; compounds with activity designated "D" provided an IC50 of 1000-10000 nM; whereas the compound with activity designated "E" provided an IC50 of > 10000 nM. Compared with ibrutinib, the compound shown in the formula I has higher BTK kinase inhibition activity. Furthermore, after pretreatment of recombinant BTK with the compounds of the invention, repeated washing with inhibitor-free medium did not restore activity, indicating irreversible inhibition of BTK by these compounds.

Table 2: BTK inhibition data

Compound numbering	IC50(μ M) inhibiting BTK	Activity assignment
			I-1	0.018	A
I-2	0.025	A
			I-3	0.016	A
I-4	0.54	A
			I-5	1.2	A
I-6	0.41	A
			I-7	0.94	A
I-8	1.2	A

I-9	1.7	A
			I-10	0.45	A
I-11	0.58	A
			I-12	0.028	A
I-13	0.049	A
			I-14	0.80	A
I-15	1.7	A
			I-16	2.3	A
I-17	0.44	A
			I-18	0.050	A
I-19	0.045	A
			I-20	0.39	A
I-21	0.038	A
			I-22	1.3	A
I-23	0.50	A
			I-24	0.80	A
I-25	0.016	A
			I-26	0.48	A
Ibrutinib	2.5	A

Example 10: mouse arthritis model induced by bovine Collagen (CIA)

1. Preparing an emulsion: weighing bovine collagen, dissolving in acetic acid to prepare a bovine collagen solution with the concentration of 8mg/ml, and standing overnight at 4 ℃. An equal volume of complete Freund's adjuvant (CFA, 1mg/ml) was added to the bovine collagen solution to prepare a mixture of bovine collagen CFA at a final concentration of 4mg/ml, which was then homogenized on wet ice with a high speed homogenizer until a white emulsion was formed.

On day 0, rats were injected with an emulsion of type II bovine collagen in Complete Freund's Adjuvant (CFA), and 50 μ l were injected intradermally (i.d.) at several sites on the back. A booster injection (i.d.) of 50 microliters of collagen emulsion was provided at an alternate site on the base or back of the tail on about day 7. Arthritis is typically observed 12 to 14 days after the initial collagen injection. From day 14 onwards, the progression of arthritis in the animals can be evaluated as described below (evaluation of arthritis).

Evaluation of arthritis:

in both models, the inflammatory development of the paw and limb joints was quantified using a scoring system comprising the evaluation of 4 paws according to the criteria described below, see table 3.

Table 3: scoring criteria

Score value	Corresponding symptoms
		0 point (min)	No erythema and redness/or redness
1 minute (1)	Erythema or mild redness near the tarsal bones or at the ankle or metatarsal bones, swelling and/or redness of the paw or one of the toes
		2 is divided into	Ankle and metatarsal jointSlight erythema and swelling of bone, or swelling of two or more joints
3 points of	Moderate erythema and swelling of the ankle, wrist and metatarsals, or total swelling of the paw, involving more than two joints
		4 is divided into	Severe inflammation of the entire paw and toe, severe arthritis

On day 21, all animals were grouped so that the weight and incidence (i.e., the number of animals in the total) were substantially the same for each group. Oral gavage was started on day 21, once daily (QD), and the doses administered to each group of animals are shown in table 4. 14 days after administration, the inhibition rate of each group of drugs on arthritic rats was calculated.

Table 4:

group of

Number of animals

Administration of drugs

Dosage to be administered

Time of administration

Frequency of administration

Inhibition rate

1

10

Physiological saline

10mg/kg

14 days

QD

/

2

10

Compounds of formula I-2

5mg/kg

14 days

QD

58.22％

3

10

Compounds of formula I-11

3mg/kg

14 days

QD

42.78％

4

10

Compounds of formula I-11

10mg/kg

14 days

QD

72.31％*

5

10

Compounds of formula I-18

10mg/kg

14 days

QD

54.46％

6

10

Ibrutinib

10mg/kg

14 days

QD

56.20％

Note: p < 0.01

As can be seen from Table 4, group 6 (10 mg/kg of ibrutinib) showed comparable inhibition rates to those of groups 2 (5 mg/kg of the compound of formula I-2) and 5 (10 mg/kg of the compound of formula I-18) of the present invention. However, the inhibition rate of group 4 (compound of formula I-11, 10mg/kg) of the present invention was significantly higher than that of group 6 (ibrutinib, 10 mg/kg).

Example 11: water solubility determination

Taking compound I-2 as an example, the water solubility of compound I-2 of the present invention was compared with that of ibrutinib. The measurement method is as follows:

the equilibrium solubility was determined in an aqueous buffer solution at pH 7.4. The pH of the buffer solution at pH 7.4 was adjusted to pH 7.4 by adjusting the pH of a 0.07M sodium dihydrogen phosphate solution, which had an ionic strength of 0.15, with 10 moles/liter sodium hydroxide. At least 1mg of the powder was mixed with 1 ml of buffer to prepare a mixture with a concentration greater than 1 mg/ml. These samples were shaken for more than 2 hours and left overnight at room temperature. The sample was then filtered through a 0.45 μm nylon syringe filter first saturated with the sample, and 2 consecutive samples were taken from the filtrate. The filtrate was measured by HPLC against a standard solution prepared in 50% methanol. The solubility of compound I-2 was 44 mg/ml and the solubility of ibrutinib was 12 mg/ml.

Example 12: capsules for oral administration

Composition (I)	% weight/weight
		Active ingredient	20.0％
Lactose	79.5％
		Magnesium stearate	0.5％

The ingredients in the above table were mixed and the active ingredient (compound of the invention) was dispensed into capsules, prepared as a total of 1000 grams of powder, and filled into capsules, each capsule containing 200mg of active ingredient.

Example 13: capsules for oral administration

Composition (I)	% weight/weight
		Active ingredient	40.0％
Pregelatinized corn starch	59.5％
		Magnesium stearate	0.5％

The ingredients in the above table were mixed and the active ingredient (compound of the invention) was dispensed into capsules, prepared as a total of 1000 grams of powder, and filled into capsules, each capsule containing 400mg of active ingredient.

Example 14: tablet for oral administration

Composition (I)	% weight/weight
		Active ingredient	20.0％
Lactose	75.5％
		Croscarmellose sodium	3.0％
Polyvinylpyrrolidone	1.0％
		Magnesium stearate	0.5％

The ingredients in the above table are mixed and granulated using a solvent such as ethanol. The formulation is then dried to a total of 1000 grams of powder and formed into tablets using a suitable tablet press, each tablet having 200mg of the active ingredient (compound of the invention).

Example 15: injection for injection administration

Composition (I)	% weight/weight
		Active ingredient	0.20 g
Sodium chloride	Proper amount of the drug to be isotonic
		Croscarmellose sodium	100 ml of

The active ingredient (compound of the invention) is dissolved in a portion of the water for injection. Sufficient sodium chloride is then added with stirring to make the solution isotonic. The solution was made up to weight with the remaining water for injection, filtered through a 0.2 micron membrane filter, and packaged under sterile conditions to give an injection.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example" or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A compound of formula I or a stereoisomer or tautomer of a compound of formula I,

wherein,

is selected from one of the following:

2. a process for preparing the compound of claim 1, comprising:

(1) contacting a compound represented by formula 1 with a compound represented by formula 2 to obtain a compound represented by formula 3;

(2) contacting the compound represented by the formula 3 with a compound represented by the formula 4 to obtain a compound represented by the formula 5;

(3) contacting the compound of formula 5 with a compound of formula 6a to obtain a compound of formula I,

wherein,as defined in claim 1.

3. A pharmaceutical composition, comprising:

the compound of claim 1.

4. The pharmaceutical composition of claim 3, further comprising:

a pharmaceutically acceptable excipient.

5. Use of a compound according to claim 1 or a pharmaceutical composition according to claim 3 or 4 in the manufacture of a medicament for use as a kinase inhibitor.

6. Use according to claim 5, characterized in that the medicament is for use as a BTK inhibitor.

7. The use according to claim 5, wherein the medicament is for the treatment of a disease mediated by B cells.

8. The use according to claim 5, wherein the medicament is for the treatment of at least one of a neoplastic disease, a proliferative disease, an allergic disease, an autoimmune disease and an inflammatory disease.

9. Use according to claim 5, wherein the medicament is for the treatment of at least one of solid tumors, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, rheumatoid spondylitis and gouty arthritis.

10. The use according to claim 9, wherein the solid tumor is at least one selected from the group consisting of lymphoma, soft tissue sarcoma, lymphocytic lymphoma, mantle cell lymphoma, melanoma, and multiple myeloma.