CN107759602B - Compound containing conjugated allene structure, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention relates to a compound containing a conjugated allene structure, a pharmaceutical composition and application thereof, in particular to a compound represented by a general formula (I) or a salt thereof, a pharmaceutical composition thereof and application thereof as a BTK inhibitor and/or a B cell activation inhibitor for preventing or treating diseases related to B cell activity abnormity and/or BTK. The compound has better lethal effect on cancer cells such as lymph cancer, breast cancer, liver cancer, intestinal cancer, gastric cancer, lung cancer, cervical cancer and the like, and has potential for treating related cancers and autoimmune diseases.

Description

Compound containing conjugated allene structure, pharmaceutical composition and application thereof

Technical Field

The invention relates to a compound containing conjugated allene structure fragments and application thereof, in particular to a small molecular compound with a conjugated allene structure or salt thereof, a pharmaceutical composition thereof and application thereof as a B cell activation inhibitor for preventing or treating diseases related to abnormal B cell activity.

Background

The chemical structure of carbonyl conjugated alkene, carbonyl conjugated alkyne can form covalent bond with amino acid residue of cysteine, lysine and the like in protein, and is widely applied in the design and development of non-reversible small molecule drugs, small molecule drugs containing carbonyl conjugated alkene have wide application in clinic, for example, the following 7 approved commercially available drugs all contain carbonyl conjugated alkene fragment, EGFR kinase inhibitor Afatinib (Afatinib) and Osimetinib are both approved by the food and drug administration of the United states for treating non-small cell lung cancer, BTK kinase inhibitor Ibrutinib (Ibrutinib) also contains carbonyl conjugated alkene fragment, which is approved for treating lymphoma, 5- α -reductase inhibitor Dutasteride (Dutasteride) is mainly used for treating prostatic hypertrophy, male pattern alopecia, seborrheic alopecia, hereditary alopecia, while 5- α -reductase inhibitor Finasteride mainly contains carbonyl conjugated alkene fragment, Keaparic-kinase (Finarcoside) is mainly used for treating benign prostatic hyperplasia, the two inhibitors also contain carbonyl conjugated alkene-kinase, and the potential of reversible cysteine-conjugated alkyne-kinase for treating benign prostatic hyperplasia, kidney disease has been studied in the clinical stage of benign prostatic hyperplasia, kidney disease, and also has potential for treating benign prostatic hyperplasia.

WO2008039218a2, WO2008121742a2 and WO2010009342a2 disclose carbonyl-conjugated olefin-and carbonyl-conjugated alkyne-containing compounds represented by the general formula (a):

in the general formula (A), La^ARepresents CH₂O, NH or S, Ar^ARepresents a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl group, Y^ARepresents an optionally substituted radical selected from the group consisting of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, Z^ARepresents CO, OCO, NHCO, CS, etc., R^7-AAnd R^8-AIndependently represent H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, or R^7-AAnd R^8-ATaken together to form a bond, R^6-ARepresents H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted aryl, or the like.

WO2008060301a2 discloses carbonyl conjugated alkenes, carbonyl conjugated alkynes containing compounds comprising a 6-hydroxypurine backbone represented by general formula (B):

in the general formula (B), Q^1BAnd Q^2BIndependently selected from CX^1B、CX^2BAnd nitrogen, Q^3BRepresents nitrogen or CH, X^1BAnd X^2BIndependently selected from hydrogen, (C1-C6) alkyl, cyano, halogen and the like, R^1BSelected from hydrogen and (C1-C6) alkyl, yB represents 0 or an integer of 1-3, R^2BAnd R^3BIndependently selected from hydrogen and (C1-C6) alkyl, R^4BSelected from alkyl, heterocycle, aryl and heteroAryl, etc., R^5BSelected from alkyl, heterocyclic, substituted heterocyclic, and the like.

WO2007142755a2 discloses compounds represented by the general formula (C):

in the general formula (C), X^CSelected from nitrogen and CR^8C,R^8CSelected from hydrogen, halogen, and substituted, unsubstituted alkyl, and the like, Q^1CSelected from O, S, etc., Z^CSelected from oxygen, sulfur and NY^5C,Y^5CSelected from hydrogen, substituted or unsubstituted alkyl, and the like, Q^2C、Q^3CAnd Q^4CIndependently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and the like, R^2CSelected from hydrogen, substituted or unsubstituted alkyl, and the like, n^CRepresents 0, 1,2, 3 or 4.

WO2003037890A2 discloses compounds represented by the general formula (D):

in the general formula (D), R^1DSelected from hydrogen, substituted or unsubstituted alkyl,

Etc. R^2DRepresents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group or the like, Y^DSelected from O, C-NO₂And the group of S, Z^DIs selected from

Etc. in which A^D、D^D、E^DAnd M^DEach independently represents CR^12DN and N-oxide, R^12DRepresents a group selected from hydrogen, halogen, amino, hydroxyl, cyano, etc., X^DRepresents a group selected from O, C-NO₂Radicals of S, etc., R^6DRepresents a group selected from a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, etc.; symbol a indicated by a dotted line^DRepresents any one of a single bond or a double bond, n^DRepresents an integer selected from 0, 1 and 2.

CN102918040B discloses a compound represented by the general formula (E):

in the general formula (E), L^Erepresents-O-, -S-, -SO-, -SO₂-、-NH-、-C(O)-、-CH₂-O-、-O-CH₂-、-CH₂-or-CH (OH) -, ring 1 represents a4 to 7-membered cyclic group which may be substituted with 1 to 5 substituents each independently selected from a halogen atom, C1-4 alkyl, C1-4 alkoxy, cyano, C1-4 haloalkyl, C1-4 haloalkoxy, wherein when the number of substituents on ring 1 is 2, the substituents may form a4 to 7-membered cyclic group together with the atom constituting ring 1 to which they are bonded, and ring 2 represents a group which may be substituted with 1 to 3-K-R²Substituted 4-to 7-membered saturated heterocyclic ring, m^ERepresents 0, 1 or 2, n^ERepresents 0, 1,2, 3 or 4, R^1ERepresents a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, a C1-4 haloalkyl group or a C1-4 haloalkoxy group.

WO2013010868A1 discloses compounds represented by the general formula (F):

x in the general formula (F)^FRepresents CH, O, N or S, Y^FRepresents C (R)^6F) N, O or S, Z is CH, N or a bond, A^FRepresents CH or N, B^1FRepresents nitrogen or (CR)^7F),B^2FRepresents nitrogen or (CR)^8F),B^3FRepresents nitrogen or (CR)^9F),B^4FRepresents nitrogen or (CR)^10F),R^1FRepresents R^11FC(O)、R^12FS(O)、R^13FSO₂Or optionally substituted with R^14FSubstituted C1-6 alkyl, R^2FRepresents H, C1-3 alkyl or C3-7 cycloalkyl, R^3FRepresents H, C1-6 alkyl or C3-7 cycloalkyl, R^2FAnd R^3FMay form, together with the N and C atoms to which they are attached, a C3-7 heterocycloalkyl group optionally substituted by one or more fluorine atoms, hydroxy, C1-3 alkyl, C1-3 alkoxy or oxo groups, R^4FRepresents H or C1-3 alkyl, R^5FRepresents H, halogen, cyano, C1-4 alkyl, C1-3 alkoxy, C3-C6 cycloalkyl, any alkyl group optionally substituted by 1 or more halogens, or R^5FRepresents a C6-10 aryl group or a C2-C6 heterocycloalkyl group, R^6FRepresents H or C1-3 alkyl, or R^5FAnd R^6FForm C3-7 cycloalkenyl or C2-C6 heterocycloalkenyl, each of which is optionally substituted with C1-3 alkyl or 1 or more halogens, R^7FRepresents H, halogen and C1-3 alkoxy, R^8FRepresents H and C1-3 alkyl, or R^7FAnd R^8FTo form C6-10 aryl or C1-9 heteroaryl, R^9FAnd R^10FRespectively represent H, halogen and C1-3 alkoxy, R^11FIndependently selected from C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl, each alkyl, alkenyl or alkynyl being optionally substituted with one or more groups selected from hydroxy, C1-4 alkyl, C3-7 cycloalkyl, C1-4 alkylamino, di-C1-4 alkylamino, C1-3 alkoxy, C3-7 cycloalkoxy, C6-10 aryl, C3-7 heterocycloalkyl, or R^11FRepresents C1-3 alkyl-C (O) -S-C1-3 alkyl or C1-5 heteroaryl optionally substituted by one or more groups selected from halogen, cyano, R^12FAnd R^13FIndependently represent C2-6 alkenyl or C2-6 alkynyl, both optionally substituted by one or more groups selected from hydroxy, C1-4 alkyl, C3-7 cycloalkyl, C1-4 alkylamino, di-C1-4 alkylamino, C1-3 alkoxy, C3-7 cycloalkoxy, C6-10 aryl, C3-7 heterocyclic aryl, or R^12FAnd R^13FIndependently represent C1-5 heteroaryl optionally substituted by one or more groups selected from halogen and cyano, R^14FIndependently selected from halogen, cyano, or C3-7 cycloalkyl, C1-4 alkylamino, di-C1-4 alkylamino, C1-3 alkoxy, C3-7 cycloalkoxy, C6-10 aryl, C3-7 heteroaryl, and C3-7 heterocycloalkyl, optionally substituted with one or more groups selected from hydroxy, C1-4 alkyl, C3-7 cycloalkyl, C1-4 alkylamino, di-C1-4 alkylamino, C1-3 alkoxy, C3-7 cycloalkoxy, C6-10 aryl, C3-7 heteroaryl, and C2-6 alkenyl or C2-6 alkynyl.

WO2014025976A1 discloses compounds represented by the general formula (G):

in the general formula (G), wherein A^GOne of the following structures is shown,

R^1Grepresents N (R)^3G)₂Or H, Cy^GRepresents aryl or heteroaryl, each of which is represented by R^2GSubstituted and optionally substituted by halogen, halogeno-C1-4 alkyl, C1-4 alkyl and C1-4 alkoxy, R^2GSelected from the group consisting of optionally substituted halogen, halogenated C1-4 alkyl, C1-4 alkyl, R^3G-S(O)m^G-, cyano, -C (O) -N (R)^3G)₂Or L substituted by C1-4 alkoxy^G-Ar^GC1-6 alkyl and C1-6 alkoxy, L^GRepresents a chemical bond selected from O,>C(O)、-(CH₂)_nG-、-O-(CH₂)_nG-、-N(R^3G)-、-N(R^3G)-(CH₂)_nG-、-(CH₂)_nG-N(R^3G)-、-C(O)-N(R^3G)-、-C(O)-N(R^3G)-(CH₂)_nG-、-N(R^3G)-C(O)-N(R^3G)-、-N(R^3G)-C(O)-、-S(O)m^G-N(R^3G)-、-N(R^3G)-S(O)m^GA linking group of-etc., Ar^GRepresents a carbocyclic, heterocyclic or heteroaryl group, X^1GRepresents a chemical bond or- (CH)₂)_nGA linking group of (A), Y^GRepresents C7-C10 spiro, monocyclic or bicyclic nitrogen-containing heterocycles, carbocycles, aryls optionally containing 0 to 1 ring nitrogen atom, each of which is substituted by one R^4GSubstituted, R^4GRepresents one of the following groups:

n^Grepresents 1,2, 3 or 4, m^GRepresents 0, 1 or 2, R^3GRepresents H or C1-4 alkyl, R^5GRepresents H, C1-4 alkoxy, C1-4 alkyl, C1-4 alkyl, C1-4 alkoxy, - (CH)₂)_nG-heterocycles and heterocycles, each heterocycle being optionally substituted by halogen, hydroxy or R^3G-S(O)_mG-substitution.

However, little research has been done on the biological activity and pharmacological properties of compounds containing conjugated dienyl structural fragments. Conjugated allenes differ from conjugated alkenes and alkynes in chemical structure and properties. Therefore, unlike compounds containing conjugated alkene, or conjugated alkyne moieties, the compounds containing conjugated allene moieties are not known for their biological activity and pharmacological properties, and are subject to uncertainty in all aspects of their properties.

Disclosure of Invention

The invention aims to develop a small molecular compound with a conjugated allene structure.

In order to achieve the aim, the invention firstly synthesizes a compound containing conjugated allene segments, and researches the activity of the compound in tumor cell strains. The results show that the compound containing the conjugated allene structure has a good lethal effect on cancer cells such as B cell lymphoma, breast cancer, liver cancer, intestinal cancer, gastric cancer, lung cancer, cervical cancer and the like for the first time, and the compound containing the conjugated allene structure has potential for treating related cancers and autoimmune diseases, so that the invention is completed.

One aspect of the present invention relates to a compound represented by the general formula (I):

in the general formula (I):

w is selected from the following groups:

preferably selected from

n is 0, 1 or 2;

m is 0, 1,2, 3 or 4;

preferably n + m is different from 0, more preferably n + m is 2,3 or 4;

l represents-O-, -CO-NH-, -NH-CO-, -O-CH₂-、-CO-NH-CH₂-、-NR₆-、-NR₆-CH₂-、-CH₂-NR₆-, -S-, -CH (OH) -or-CH₂-; preferably, L represents-O-or-NH-CO-;

R₁represents a C6-C12 aryl or a 5-10 membered monocyclic or bicyclic heteroaryl group containing 1-4 heteroatoms selected from N, O, S; the aryl and heteroaryl groups are optionally substituted with 1 to 6 substituents selected from halogen, cyano, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, amino, -NH (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl), -NHCO- (C1-C4 alkyl), -NHCO- (C1-C4 alkoxy), preferably optionally substituted with 1 to 2 substituents selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy and amino; the aryl group is preferably phenyl, and the heteroaryl group is preferably pyridyl, benzopyridyl or indolyl;

R₂、R₃and R₄Each independently selected from hydrogen and C1-C4 alkyl, said C1-C4 alkyl being optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, C1-C4 alkoxy, -NH- (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl), -NHCO- (C1-C4 alkyl), -NHCO- (C1-C4 alkoxy), preferably, optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, C1-C4 alkoxy; preferably, R₂、R₃And R₄Are all hydrogen;

or R₂And R₃Together with the carbon to which they are attached form a 4-8 membered ring, optionally containing 1-3 heteroatoms selected from N, O and S, and optionally substituted with 1-3 heteroatoms selected from halogen, hydroxy, amino, C1-C4 alkoxy, (C1-C4 alkyl) -NH₂Substituent of-NH- (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl);

B₁、B₂、B₃and B₄Each independently represents N or-C (R)₅) Preferably, B₁、B₂、B₃And B₄Up to two are N, more preferably up to one is N;

R₅each independently selected from hydrogen, halogen, cyano, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, amino, -NH- (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl), -NHCO- (C1-C4 alkyl) and-NHCO- (C1-C4 alkoxy), preferably each independently selected from hydrogen, halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C4-C4 alkyl, halogen substituted C4-C4 alkoxy and amino, or two adjacent R, C, H, C, N-C-S-C₅Together with the carbon atoms to which they are attached form a phenyl ring or a 5-6 membered heteroaromatic ring containing 1-2 heteroatoms selected from N, O and S;

R₆each independently selected from hydrogen or C1-C4 alkyl, preferably from hydrogen, methyl and ethyl.

In one embodiment, the compound of formula (I) is a compound of formula (I-1):

wherein R is₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m, X and W are as defined for formula (I).

In one embodiment, the compound of formula (I) is a compound of formula (I-2):

wherein R is₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m, X and W are as defined for formula (I).

In one embodiment, the compound of formula (I) is a compound selected from the following formulae (I-3) to (I-7):

wherein, L and R₁Is as defined in formula (I);

B₂represents N or-C (R)₅)；

R₅Each independently selected from hydrogen, halogen, cyano, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, amino, -NH- (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl), -NHCO- (C1-C4 alkyl), and-NHCO- (C1-C4 alkoxy), preferably each independently selected from hydrogen, halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, and amino.

In one embodiment, the compound of formula (I) is selected from the following compounds:

another aspect of the present invention relates to a pharmaceutical composition comprising the above-described compound of the present invention or a salt thereof. The pharmaceutical composition may optionally further comprise pharmaceutically acceptable excipients which may be, for example, binders, fillers (diluents), disintegrants, suspensions, suspending agents, slow (controlled) release agents, lyoprotectants, coatings, enteric materials, lubricants (glidants, antiadherents), sweeteners, flavors, plasticizers, opacifiers, solubilizers, humectants, solvents, osmo-regulators, colorants, pigments, surfactants (emulsifiers), water-soluble matrices, fat-soluble matrices, oleaginous matrices, pore-forming agents, gels, preservatives, buffers, chelating agents, antioxidants and the like.

In one embodiment, the pharmaceutical composition is a BTK inhibitor.

In one embodiment, the pharmaceutical composition is a B cell activation inhibitor, in particular a B cell growth inhibitor.

In one embodiment, the pharmaceutical composition is a medicament for preventing or treating diseases associated with B cell activation, particularly B cell growth, and BTK. Another aspect of the present invention relates to a method for preventing and/or treating a disease associated with activated B cells, particularly B cell growth, and/or BTK, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above-described compound of the present invention or a salt thereof. The subject is preferably a mammal, such as a human.

Another aspect of the present invention relates to the use of a compound of the present invention or a salt thereof as described above for the preparation of a medicament for the prevention and/or treatment of diseases associated with activated B cells, in particular B cell growth, and/or BTK.

In one embodiment, the disease associated with activated B cells and/or BTK is an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, or a cancer.

In one embodiment, the cancer is non-hodgkin's lymphoma, breast, liver, bowel, stomach, pancreas, lung, and cervix.

Another aspect of the present invention relates to a compound of the present invention or a salt thereof as described above or a pharmaceutical composition as described above for use in the prevention and/or treatment of diseases associated with activated B cells, in particular B cell growth, and/or BTK.

The present invention will be described in detail below.

In the present invention, halogen means fluorine, chlorine, bromine and iodine.

In the present invention, the C1-C4 alkyl group means a straight-chain or branched C1-C4 alkyl group including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and the like.

In the present invention, the C1-C4 alkoxy group means a straight or branched C1-4 alkoxy group including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.

In the present invention, the halogen-substituted C1-C4 alkyl group means a group in which a C1-C4 alkyl group is substituted with 1 or 2 or more halogen atoms, and examples thereof include a fluoromethyl group, a chloromethyl group, a bromomethyl group, an iodomethyl group, a difluoromethyl group, a trifluoromethyl group, a 1-fluoroethyl group, a 2-fluoroethyl group, a pentafluoroethyl group, a 1-fluoropropyl group, a 2-fluoropropyl group, a 3-fluoropropyl group, a 1-chloropropyl group, a 2-chloropropyl group, a 3-chloropropyl group, a 1-fluorobutyl group, a 2-fluorobutyl group, a 3-fluorobutyl group, a4, 4, 4-trifluorobutyl group, and a 4-bromobutyl group.

In the present invention, the halogen-substituted C1-C4 alkoxy group means a group in which a C1-C4 alkoxy group is substituted with 1 or 2 or more halogen atoms, and examples thereof include trifluoromethoxy group, trichloromethoxy group, chloromethoxy group, bromomethoxy group, fluoromethoxy group, difluoromethoxy group, dimethoxy group, fluoromethoxy group, difluoromethoxy group, dibromomethoxy group, 2-fluoroethoxy group, 2,2, 2-trifluoroethylmethoxy group, 2,2, 2-trichloroethylmethoxy group, 3-chloropropoxy group, 2-fluoro-3-chloropropoxy group, 1-fluorobutoxy group, 2-fluorobutoxy group, 3-fluorobutoxy group and 3-chlorobutoxy group.

In the present invention, the C6-C12 aryl group means a 6-to 12-membered aromatic carbocyclic group, and examples thereof include phenyl, quinonyl, naphthalene-1, 4-diketonyl, naphthyl, anthryl, phenanthryl, azulenyl, acenaphthenyl, fluorenyl and the like.

In the present invention, the 5-10 membered monocyclic or bicyclic heteroaryl group containing 1-4 heteroatoms selected from N, O, S means an aromatic 5-7 membered monocyclic heterocyclic group and 8-10 membered bicyclic heterocyclic group containing 1-4 heteroatoms, for example, examples thereof include pyrrolyl, furyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furazanyl, oxadiazolyl, thiadiazolyl, thiazinyl, benzofuranyl, benzoisothiofuranyl, isoindolyl, benzo [ b ] thienyl, benzo [ c ] thienyl, benzimidazolyl, purinyl, indazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, cinnolinyl and the like.

In the present invention, the 4-to 8-membered ring optionally containing 1 to 3 heteroatoms selected from N, O, S means a 4-to 8-membered saturated carbocyclic ring or a 4-to 8-membered saturated heterocyclic ring containing 1 to 3 heteroatoms selected from N, O, S, for example, examples thereof include a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, an oxepane ring, an acridine ring, a pyrrolidine ring, a piperidine ring, an azepane ring, an azocane ring, a tetrahydrothiophene ring, a1, 4-diazepane ring, a1, 3-dioxepane ring, a1, 4-dioxepane ring, a1, 5-diazacyclooctane ring, a homomorpholine ring, a1, 5-azacyclooctane ring and the like.

In the present invention, the heteroaromatic ring containing 1 to 2 heteroatoms selected from N, O and S means a 5-to 6-membered aromatic heterocycle containing 1 to 2 heteroatoms selected from N, O and S, and examples thereof include a pyrrole ring, a furan ring, an imidazole ring, a pyrazole ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, a pyridazine ring, a thiophene ring, an oxazole ring, an isoxazole ring, a thiazole ring, and an isothiazole ring.

[ isomers ]

In the present invention, unless otherwise specified, the structure of the present invention includes all isomers, for example, alkyl groups include straight-chain alkyl groups and branched-chain alkyl groups, further, geometrical isomers (E-form, Z-form, cis-form, trans-form) in double bonds, rings, fused rings, optical isomers (R and S-forms, α and β forms, enantiomers, diastereomers) resulting from the presence of asymmetric carbon atoms and the like, optically active forms (D, L, D, L-forms), optical isomers, mixtures thereof in any ratio, and racemic mixtures thereof are included in the present invention.

In the present invention, unless otherwise specified, the meanings of the symbols are those familiar to those skilled in the art, for example

Indicating that the chemical bond is directed to the front side of the paper (β configuration);

indicating that the chemical bond is directed toward the back of the paper (α configuration) "/" indicating that the chemical bond may be directed toward the front or back, or a mixture of α and β configurations in any proportion thereof.

The compound represented by the general formula (I) can be converted into a corresponding salt by a known method. The salt is preferably water soluble. Suitable salts include alkali metal (potassium, sodium, etc.) salts, alkaline earth metal (calcium, magnesium, etc.) salts, and ammonium salts, pharmaceutically acceptable salts of organic amines (tetramethylammonium, triethylamine, methylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) aminomethane, lysine, arginine, N-methyl-D-glucamine, etc.), acid adduct salts (inorganic acid salts (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, etc.), organic acid salts (acetate, trifluoroacetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, gluconate, etc.)), and the like.

The compounds represented by the general formula (I) and salts thereof may also exist in the form of solvates thereof. The solvate is preferably low in toxicity and water-soluble. As a suitable solvate, for example, a solvate of a solvent such as water or an alcohol (e.g., ethanol) may be cited, and a plurality of crystal forms may exist.

In addition, the compounds represented by the general formula (I) and salts thereof may also exist in the form of prodrugs thereof. The prodrug is a compound which can be converted into a compound represented by the general formula (I) in vivo by reaction with an enzyme and/or gastric acid or the like. Examples of the prodrug of the compound represented by the general formula (I) include compounds in which a hydroxyl group of the compound represented by the general formula (I) is acylated, alkylated, phosphorylated, borated (for example, compounds in which a hydroxyl group of the compound of the present invention is acetylated, palmitoylated, propionylated, pivaloylated, succinylated, fumarylated, malonylated, dimethylaminocarbonylated, and the like); compounds in which the carboxyl group of the compound represented by the general formula (I) is esterified or amidated (for example, compounds in which the carboxyl group of the compound represented by the general formula (I) is esterified with ethyl, isopropyl, phenyl, hydroxymethyl, dimethylaminomethyl, pivaloyloxymethyl, ethoxycarbonyloxyethyl, peptide, (5-methyl-2-oxo-1, 3-dioxopentyl) methyl, cyclohexyloxycarbonylethyl, formamido, etc.); and the like. These compounds can be prepared by known methods. The prodrug of the compound represented by the general formula (I) may be either a hydrate or a non-hydrate. The prodrug of the compound represented by the general formula (I) may be a compound represented by the general formula (I) under physiological conditions as described in "Guangchua bookstore, 1990, development of pharmaceuticals", volume 7, molecular design, pp 163-198 ".

Further, the compound represented by the general formula (I) may be an isotope (e.g., deuterium, tritium, etc.),¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、^18O、³⁵S、¹⁸F、³⁶Cl、¹²³I、¹²⁵I) And the like.

The isomers (including optical isomers, tautomers and mixtures thereof in any ratio), solvates and prodrugs of the compounds represented by the general formula (I) and salts thereof are included in the present invention as equivalent alternatives. The optical isomers in the present invention include not only compounds with 100% purity but also other optical isomers less than 50%.

[ Process for producing the Compound of the present invention ]

The compound represented by the general formula (I) of the present invention can be prepared by one of the following methods. The method comprises the following steps: compound a1 can be prepared by the condensation reaction of intermediate a2 and intermediate A3, as shown in scheme 1. The condensation reaction conditions may be the synthesis conditions reported in Angew. chem. int. Ed.2014,53, 7491-7494. The reaction conditions after modification and optimization based on the above reaction conditions can also be used as reaction conditions for preparing A1 by condensation of A2 and A3.

Reaction scheme 1

In reaction scheme 1, L, R₁、R₂、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I), R₃And R₄Are both hydrogen.

The second method comprises the following steps: compound a1 can be prepared by condensation reaction of intermediate a2 and intermediate B1 as shown in scheme 2. The condensation reaction conditions may be those reported in Journal of Organic Chemistry 2002,67, 6575-6578. The modified and optimized reaction conditions based on the reaction conditions can also be used as the reaction conditions for preparing A1 by condensation of A2 and B1.

Reaction formula 2

In reaction scheme 2, L, R₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I).

The third method comprises the following steps: compound a1 can be prepared by coupling reaction of intermediate a2, intermediate B2 and carbon monoxide over a catalyst [ e.g. tetrakis (triphenylphosphine) palladium) ]. The coupling reaction conditions may be Organic Letters,2009, Vol.11, No.13, 2900-; reaction conditions reported in Journal of Organic Chemistry,2002,67, 6575-6578. The reaction conditions after modification and optimization based on the above reaction conditions may also be used as the reaction conditions for preparing a 1.

Reaction formula 3

In reaction scheme 3, L, R₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I).

The method four comprises the following steps: compound a1 can be synthesized as shown in equation 4:

(1) under the action of alkali, A1 reacts with chloroacetyl chloride to obtain B3;

(2) b3 reacts with triphenyl phosphine to obtain B4;

(3) b4 can generate a Wittig reagent B5 under the action of alkali;

(6) reaction of B5 with acid chloride B6 affords a 1.

The reaction conditions in the above steps may be those reported in Tetrahedron Letters 56(2015) 3281-3284. Wherein the base is selected from organic and inorganic bases, such as triethylamine, diisopropylethylamine, N-methylmorpholine, sodium ethoxide, and mixtures thereof,

Sodium methoxide, sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide, lithium diisopropylamide, butyl lithium and the like. The reaction conditions after modification and optimization based on the above reaction conditions may also be used as the reaction conditions for preparing a 1.

Reaction formula 4

In reaction formula 4, L, R₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I).

The key intermediate A2 of the compound of the present invention can be a commercially available product or can be synthesized by or by referring to methods in the prior art, for example, the synthesis can be one of the following methods.

The method comprises the following steps: intermediate a2 was synthesized as shown in equation 5:

(1) boric acid A5 or boric acid ester A6 and bromide A7 are subjected to Suzuki coupling reaction to obtain an intermediate A4; the Suzuki coupling reaction conditions can be reaction conditions disclosed in Chemical Reviews 1995,95(7), 2457-2483 and org.processres.Dev.2011,15, 1018-1026, and the modified and optimized reaction conditions based on the reaction conditions can also be used as the reaction conditions for preparing A2;

(2) deprotection reactions were carried out to remove the Cbz protecting group contained in a4 to afford intermediate a 2. For example, the deprotection method described in Protective Grups in Organic Synthesis, Wiley, New York,1999 can be used, or appropriate modifications and optimizations can be made based on literature procedures.

Reaction formula 5

In reaction formula 5, R₁、L、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I).

The second method comprises the following steps: intermediate a2 can be prepared by the following route, as shown in scheme 6:

(1) intermediate A10 and compound A9 are connected through a Mitsunobu reaction to obtain intermediate A8; the Mitsunobu reaction can adopt the method reported in Strategic Applications of Named Reactions in organic Synthesis,2005, Elsevier Inc press and ACS Med. chem. Lett.2016,7,198-203, and can be modified and optimized appropriately based on the literature methods;

(2) deprotection to remove the Boc group of A8 gave A2. The deprotection reaction can be carried out by the method of deprotecting groups described in Protective Grups in Organic Synthesis, Wiley, New York,1999, or can be modified and optimized appropriately based on literature methods.

Reaction formula 6

In reaction formula 6, R₁、L、B₁、B₂、B₃、B₄N, m and W are as defined for formula (I).

Intermediates a5 and a6 can be commercially available products or synthesized using or referencing methods known in the art, for example, they can be synthesized by the following route, as shown in scheme 7: the halogenated aromatic hydrocarbon A11 is transformed and metallized by using n-butyl lithium, and then reacts with triisopropyl borate to obtain boric acid A5 by acidification; or coupling the trifluoromethyl sulfonate A12 of halogenated aromatic ring and aryl alcohol with bis-pinacol borate under the catalysis of a palladium catalyst to obtain the pinacol borate A6. The synthesis methods of the boric acid A5 and the borate A6 can be the methods disclosed in J.org.chem.2002,67,5394-5397 and J.org.chem.1995,60,7508-7510, and can be modified and optimized appropriately based on the literature methods.

Reaction formula 7

In reaction formula 7, R₁、L、B₁、B₂、B₃And B₄Is the same as in formula (I), and X is Br, I or OTf.

Intermediate a7 may be a commercially available product or synthesized using or referencing methods known in the art, for example, it may be synthesized by the following route, as shown in scheme 8:

(1) condensing 2- (3-chloropyrazinyl) methylamine with compound C1(N-Cbz protected α -amino acid, β -amino acid or γ -amino acid) to give amide C2;

(2) amide C2 in phosphorus oxychloride (POCl)₃) Can be dehydrated and cyclized under the action of other reagents to obtain a cyclized product C3;

(3) brominating the cyclization product C3 by N-bromosuccinimide (NBS) to obtain a bromination product C4;

(4) ammonolysis of the brominated product C4 with aqueous ammonia affords compound C5, which is part of a representative a7 structure.

The above-mentioned amide condensation, cyclization, bromination and aminolysis reactions can be prepared by the method described in Comprehensive organic transformations, A Guide to Functional Group Preparations, 2 nd edition (RichardC. Larock, John Wiley & Sons Inc,1999), or by appropriate modification and combination of the methods shown in the examples. The synthetic route can also adopt the reaction conditions in the patent WO2013/010868, and can also be appropriately modified and optimized on the basis of the literature method.

Reaction formula 8

In the reaction formula 8, n, m and W are as defined in the general formula (I).

Intermediate a10 may be a commercially available product or synthesized using or referencing methods known in the art, for example, it may be synthesized by the following route, as shown in scheme 9: boronic acid a5, or boronic esters a6 and a13, were subjected to Suzuki coupling to afford intermediate a 10. The Suzuki coupling reaction can be carried out by the methods disclosed in Chemical Reviews 1995,95(7): 2457-2483 and documents such as org. Process Res. Dev.2011,15, 1018-1026, and can be appropriately modified and optimized on the basis of the documents.

Reaction formula 9

In reaction formula 9, R₁、L、B₁、B₂、B₃、B₄And W is as defined for formula (I), and X is Br, I or OTf.

A13 is aryl ring compound substituted by halogeno or trifluoromethyl sulfonate, and the aryl rings are 5-6 membered monocyclic heterocycle or 8-10 fused ring heterocycle compound. A13 can be a commercially available product or can be synthesized by methods known in the art, for example, according to Comprehensive Heterocyclic Chemistry III. Editors-in-Chief AlanR.Katritzky, Christopher A.Ramsden, Eric F.V.Scriven, and Richard J.K.Taylor, Elsevier Ltd. Press, 2008 and synthetic methods in the literature cited in this document.

Drawings

Fig. 1 is a graph showing the cellular activities of ibrutinib and compound 4 according to the present invention measured in B-cell lymphoma Raji cell lines.

Detailed Description

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

General purification of compounds preparative chromatographic conditions: the purification instrument is used for preparing high performance liquid phase by Shimadzu semi-preparationChromatography (by HPLC, model LC-20AR) on a SunAire Prep C18OBR column^TM5 uM. The mobile phases used for the HPLC work were water (0.1% trifluoroacetic acid) and acetonitrile (0.1% trifluoroacetic acid), the mobile phase gradient being a 1% increase in the proportion of acetonitrile per minute. During purification, the initial acetonitrile ratio is changed correspondingly according to the property of the compound.

¹H NMR represents hydrogen spectrum of hydrogen atom signal spectrum measured by a nuclear magnetic resonance apparatus.¹H NMR immediately following in brackets, CDCl₃Representing the dissolution sample solvent as deuterated chloroform, MeOD-d4 representing the dissolution sample solvent as tetradeuterated methanol, DMSO-d₆Representative of the dissolution sample solvent is hexadeuterodimethylsulfoxide. 400MHz represents the frequency of the electromagnetic waves used by the NMR spectrometer used for the test.

ESI-MS represents the mass spectrometer instrument using a probe in electrospray ionization (ESI) mode. [ M + H ]]⁺The latter figure is the theoretical value of mass-to-charge ratio after ionization of the species.

The specific rotation was measured by a Rudolph Autopol VI polarimeter, model INS-QC-76.

CH₂Cl₂Represents dichloromethane, MeOH represents methanol, Cbz represents a benzyloxycarbonyl protecting group, and Boc represents a tert-butoxycarbonyl protecting group.

The names of compounds used in this specification are generally named according to IUPAC nomenclature, and also according to common names.

Preparative example 1 synthesis of boronic acid, boronic ester:

the method comprises the following steps: synthesis of N- (2-pyridyl) 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) -benzamide (C007)

Step one, synthesizing p-bromobenzoyl chloride: in a 250mL glass flask, p-bromobenzoic acid (3g,15mmol), N, N-dimethylformamide (DMF, 0.4mL) and dichloromethane (CH) were added₂ Cl ₂50 mL). Cooling the reaction solution to zero degree with ice water bath, adding thionyl chloride (SOCl)₂，1.32mL,18mmol). The reaction was warmed to room temperature and stirred overnight. Evaporation to dryness was carried out by rotary evaporation and the crude product was purified by co-evaporation with toluene (30 mL. times.2) to give a colorless liquid which was used directly in the next reaction.

Step two, synthesizing N- (2-pyridyl) p-bromobenzamide (C004): all colorless liquid obtained in the previous step was put into a 250mL glass flask and dissolved in dry tetrahydrofuran (THF, 20 mL). This solution was transferred to a 250mL flask containing 4-N, N-dimethylaminopyridine (DMAP, 0.183g,1.5mmol), 2-aminopyridine (1.41g,15mmol), diisopropylethylamine (DIEA, 3.2mL,18mmol) and dry tetrahydrofuran (30 mL). The mixed solution was heated to 50 ℃ and stirred overnight. Tetrahydrofuran was evaporated by rotary evaporation and the residue was dissolved in ethyl acetate (300 mL). The organic phase was extracted twice with hydrochloric acid solution (0.2N,100mL) and twice with aqueous sodium hydroxide solution (0.2N,100 mL). Finally, the organic phase was washed with saturated brine, dried and concentrated to give the title compound C004 as a solid (2.67 g). The product was used directly in the next reaction without further purification.¹H NMR(CDCl₃400MHz) 8.37(d, J-8.40 Hz,1H),8.30-8.26(m,1H),7.81(d, J-8.40 Hz,2H),7.80-7.72(m,1H),7.64(d, J-8.40 Hz,2H),7.21-7.06(m,1H), ESI-MS theoretical calculation C₁₂H₁₀ ⁷⁹BrN₂O[M+H]⁺277.0; the experiment found that 277.0.

Step three: synthesis of N- (2-pyridyl) 4- (4,4,5,5-tetramethyl- [1,3,2] dioxolane boryl) benzamide (C007): in a 250mL glass flask, C004(960mg,3mmol), bis-pinacolato borate (1.5g,6.0mmol), potassium acetate (KOAc, 1.2g,12mmol) and dry 1, 4-dioxane (20mL) were added. The reaction solution was deoxygenated, and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (210mg,0.3mmol) was added, and deoxygenated again. The reaction solution was heated to 90 ℃ and stirred overnight. The reaction was quenched with 50mL of water and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with saturated brine, dried, concentrated and purified over silica gel column. Purification afforded the title compound, C007, as a solid (925 mg).

¹H NMR(CDCl₃,400MHz):8.86(s,br,1H),8.42(d,J＝8.39Hz,1H),8.29(d,J＝4.62Hz,1H),793(s,4H),7.81-7.74(m,1H),7.09(dd, J ═ 6.78,5.37Hz,1H),1.37(s, 12H). Theoretical calculation of ESI-MS C₁₈H₂₂BN₂O₃[M+H]⁺325.2; the test result is 325.3.

The second method comprises the following steps: synthesis of N- (2- (4-trifluoromethyl) pyridyl) 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) -benzamide (C012)

Step one, synthesizing p-bromobenzoyl chloride: in a 250mL glass flask, p-bromobenzoic acid (3g,15mmol), N, N-dimethylformamide (0.4mL) and dichloromethane (50mL) were added. The reaction solution was cooled to zero degrees with an ice-water bath, and thionyl chloride (1.32mL,18mmol) was added. The reaction was warmed to room temperature and stirred overnight. Evaporation to dryness was carried out by rotary evaporation and the crude product was purified by co-evaporation with toluene (30 mL. times.2) to give a colorless liquid which was used directly in the next reaction.

Step two, synthesizing N- (2- (4-trifluoromethyl) pyridyl) p-bromobenzamide (C009): all colorless liquid obtained in the above reaction step was placed in a 250mL glass flask and dissolved in dry tetrahydrofuran (20 mL). This solution was transferred to a 250mL flask containing 4-N, N-dimethylaminopyridine (0.183g,1.5mmol), 2-amino-4-trifluoromethylpyridine (1.41g,15mmol), diisopropylethylamine (3.2mL,18mmol) and dry tetrahydrofuran (30 mL). The mixed solution was heated to 50 ℃ and stirred overnight. Tetrahydrofuran was evaporated by rotary evaporation and dissolved in ethyl acetate (300 mL). The organic phase was extracted twice with hydrochloric acid solution (0.2N,100mL) and twice with aqueous sodium hydroxide solution (0.2N,100 mL). Finally, the organic phase was washed with saturated brine, dried and concentrated to obtain the objective compound C009 as a solid (3.93 g). The product was used directly in the next reaction without further purification.

¹H NMR(CDCl₃400MHz) 8.99(s,1H),8.67(s,1H),8.39(d, J ═ 5.00Hz,1H),7.80(d, J ═ 8.35Hz,2H),7.64(d, J ═ 8.35Hz,2H),7.29(d, J ═ 4.75Hz, 1H). Theoretical calculation of ESI-MS C₁₃H₉ ⁷⁹BrF₃N₂O[M+H]⁺345.0; experimental survey345.1 percent.

Step three, synthesizing N- (2- (4-trifluoromethyl) pyridyl) 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) benzamide (C012): in a 250mL glass flask, C009(1.70g,5mmol), bis-pinacolato borate (2.54g,10.0mmol), potassium acetate (2.0g,20mmol) and dry 1, 4-dioxane (30mL) were added. The reaction solution was deoxygenated, and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (350mg,0.5mmol) was added, and deoxygenated again. The reaction solution was heated to 90 ℃ and stirred overnight. The reaction was quenched with 50mL of water and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried, concentrated and purified by means of a silica gel column to give the objective compound C012 as a viscous liquid (1.65g), which was gradually solidified by leaving it in a refrigerator at-20 ℃.

¹H NMR(CDCl₃400MHz) 9.00-8.90(br,1H),8.73(s,1H),8.47(d, J-5.08 Hz,1H),8.00-7.90(m,2H),7.31(d, J-5.08 Hz,1H),1.37(s, 12H). Theoretical calculation of ESI-MS C₁₉H₂₁BF₃N₂O₃[M+H]⁺393.2; the experiment found that 393.3.

The third method comprises the following steps: synthesis of N- (2- (4-trifluoromethyl) pyridyl) 2-fluoro-4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) benzamide (C019)

Step one, synthesizing 2-fluoro-4-bromobenzoyl chloride: in a 250mL glass flask, 2-fluoro-4-bromobenzoic acid (3.28g,15mmol), N, N-dimethylformamide (0.4mL) and dichloromethane (50mL) were added. The reaction solution was cooled to zero degrees with an ice-water bath, and thionyl chloride (1.32mL,18mmol) was added. The reaction was warmed to room temperature and stirred overnight. Evaporation to dryness was carried out by rotary evaporation and the crude product was purified by co-evaporation with toluene (30 mL. times.2) to give a colorless liquid which was used directly in the next reaction.

Step two, synthesizing N- (2- (4-trifluoromethyl) pyridyl) 2-fluoro-4-bromobenzamide (C016): all colorless liquid obtained in the above reaction step was placed in a 250mL glass flask and dissolved in dry tetrahydrofuran (20 mL). This solution was transferred to a 250mL flask containing 4-N, N-dimethylaminopyridine (0.183g,1.5mmol), 2-amino-4-trifluoromethylpyridine (2.43g,15mmol), diisopropylethylamine (3.2mL,18mmol) and dry tetrahydrofuran (30 mL). The mixed solution was heated to 50 ℃ and stirred overnight. Tetrahydrofuran was evaporated by rotary evaporation and dissolved in ethyl acetate (300 mL). The organic phase was extracted twice with hydrochloric acid solution (0.2N,100mL) and twice with aqueous sodium hydroxide solution (0.2N,100 mL). Finally, the organic phase was washed with saturated brine, dried and concentrated to give the objective compound C016 as a solid (3.51 g). The product was used directly in the next reaction without further purification.

¹H NMR(CDCl₃400MHz) 9.20-9.10(br,1H),8.67(s,1H),8.51(d, J-5.10 Hz,1H),8.06(t, J-8.39 Hz,1H),7.51(d, J-8.45 Hz,1H),7.44(d, J-11.56 Hz,1H),7.33(d, J-5.01 Hz, 1H). Theoretical calculation of ESI-MS C₁₃H₈ ⁷⁹BrF₄N₂O[M+H]⁺363.0; the test result is 363.1.

Step three, N- (2- (4-trifluoromethyl) pyridyl) 2-fluoro-4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) benzamide (C019): in a 250mL glass flask, C016(1.81g,5mmol), bis-pinacolato borate (2.54g,10.0mmol), potassium acetate (2.0g,20mmol) and dry 1, 4-dioxane (30mL) were added. The reaction solution was deoxygenated, and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (350mg,0.5mmol) was added, and deoxygenated again. The reaction solution was heated to 90 ℃ and stirred overnight. The reaction was quenched with 50mL of water and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried, concentrated and purified by silica gel column to give the objective compound C019 as a solid (2.46 g).

¹H NMR(CDCl₃400MHz) 9.40-9.22(br,1H),8.70(s,1H),8.56-8.40(m,1H),8.14(t, J ═ 6.76Hz,1H),7.74(d, J ═ 7.30Hz 1H),7.63(d, J ═ 12.34Hz,1H),7.36-7.22(m,1H),1.36(s, 12H). Theoretical calculation of ESI-MS C₁₉H₂₀BF₄N₂O₃[M+H]⁺411.2; the test result is 444.3.

The method four comprises the following steps: synthesis of 2- (4-phenoxyphenyl) -4,4,5, 5-Tetramethyl- [1,3,2] dioxaborolan (4,4,5, 5-tetramethylyl-2- (4-phenoxy-phenyl) - [1,3,2] dioxaborolane, A)

4-Bromobiphenyl ether (6.2g,25.0mmol) and isopropanol pinacol borate (10.3mL,50mmol) were dissolved in dry tetrahydrofuran, cooled well to-78 ℃ under nitrogen, n-butyllithium (2.5M,18mL,45mmol) was added dropwise, stirred for 6 hours after the addition was completed, and slowly warmed to-40 ℃ for 12 hours. The reaction solution was added to a saturated ammonium chloride solution, and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness by rotary evaporation and purified on a column to give the desired compound 5.4g with a yield of 72%.¹H NMR(CDCl₃,400MHz):7.78(d,J＝8.5Hz,2H),7.35(t,J＝8.0Hz,2H),7.13(t,J＝7.3Hz,1H),7.0 3(d,J＝8.3Hz,2H),6.98(d,J＝8.6Hz,2H),1.34(s,12H)。

Preparation example 2 Synthesis of Key intermediate N-Cbz- (S) -3- (1-bromo, 8-aminoimidazo [1,5-a ] pyrazinyl) substituted-2-pyrrolidines (S8)

2- (3-Chloropyrazin-2-yl) methanamine (S4)

Step one, synthesizing 2- (3-chloropyrazinyl) methyl bromide (S2): 2-chloro-3-methylpyrazine (S1,7.72g,60mmol) and N-bromosuccinimide (NBS, 14.87g,84mmol) were dissolved in carbon tetrachloride (CCl)₄100 mL). Adding benzoyl peroxide (Bz) in one time₂O₂1.45g,6mmol) and the reaction was refluxed overnight. Then, the reaction solution was cooled to room temperature, filtered to remove the solid, and the solid was washed with carbon tetrachloride to obtain CCl₄And (3) solution. The carbon tetrachloride solutions were combined and concentrated to give 13.27g of a mixture containing S2, which was used in the next reaction without purification.

Step two, synthesizing 2- (3-chloropyrazinyl) methyl-substituted-2-isoindole-1, 3-dione (S3): 13.27g of the mixture containing S2 in the previous step was dissolved in anhydrous N, N-dimethylformamide (150 mL). To the solution was added potassium phthalimide (22.2g), and the solution was heated to 60 ℃ for reaction overnight. After the reaction was completed, N-dimethylformamide was distilled off by a rotary evaporator, and water and methylene chloride were added. The aqueous phase was extracted with dichloromethane. The organic phases were combined, dried and concentrated to give a mixture containing S3. The mixture was used in the next reaction without further purification.

Step three, synthesizing 2- (3-chloropyrazinyl) methylamine: the mixture containing S3 in the previous step was placed in a 250mL glass bottle, and 100mL of methanol was added and mixed. Add 85% hydrazine hydrate (H) to the flask₂N-NH₂)6.6g, and the reaction solution was heated to 60 ℃ to react for 2 hours. Cooling, the solid was filtered on a Buchner funnel, the filter cake was washed with methanol (500mL), the resulting methanol solution was concentrated and the resulting mixture was dissolved in dichloromethane (300 mL). The mixture was filtered again through a Buchner funnel, the filter cake was washed with dichloromethane, and the resulting dichloromethane solution was concentrated and purified with a silica gel column to give 3-chloro-2-pyrazinylmethylamine (S4) (3.53g, 21% overall yield over 3 steps).¹HNMR(CDCl₃,400MHz):8.48(d,J＝2.31Hz,2H),8.26(d,J＝2.31Hz,2H),4.14(s,3H)。

N- (3-Chloropyrazin-2-ylmethyl) N-Cbz-prolinamide (S5)

Step four, S4(0.90g,7.3mmol), N-Cbz proline (2.74g,11mmol) and dichloromethane (20mL) were added to a 100mL glass vial. The reaction flask was cooled with ice water and then triethylamine (Et) was added₃N, 4mL,38mmol) and stirred for 10 min. 2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,3.33g,8.8mmol) was added in one portion and stirred at room temperature overnight. The reaction was quenched with water, the aqueous phase was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried, concentrated and purified by silica gel column to give the objective compound S5 as an oily liquid (4.35 g).¹HNMR(CDCl₃400MHz) 8.40-8.24(m,1H),8.28(s,1H),7.46-7.00(m,5H),5.30-4.95(m,2H),4.80-4.30(m,3H),3.75-3.40(m,2H),2.40-2.10(m,2H),2.10-1.75(m, 2H). Theoretical calculation of ESI-MS C₁₈H₁₉ ³⁵ClN₄NaO₃[M+Na]⁺397.1; the test result is 397.1.

N-Cbz- (S) -3- (8-chloroimidazo [1,5-a ] pyrazinyl) substituted-2-pyrrolidines (S6)

Step five, in a 250mL glass vial, S5(8.64g,23.1mmol),1, 3-dimethyl-2-imidazolidinone (7.5mL,69.3mmol) and dry acetonitrile (50mL) were added. Cooling the reaction flask and the reaction solution in the flask to 0 deg.C, and adding phosphorus oxychloride (POCl)₃13.5mL,92.4mmol) and warmed to 60-65 deg.c for reaction overnight. Finally, the reaction solution was poured into 25% aqueous ammonia (50mL), and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, concentrated and purified by silica gel column to give the title compound S6 as a white solid (5.89 g).

¹H NMR(CDCl₃400MHz) 8.15(d, J ═ 4.40Hz,0.5H),7.79(s,0.5H),7.68(s,0.5H),7.62-7.54(m,0.5H),7.36-7.16(m,3H),7.15-7.05(m,1H),6.96(d, J ═ 5.65Hz,0.5H),6.73(d, J ═ 7.54Hz,1H),5.32-5.23(m,0.5H),5.15(t, J ═ 6.29Hz,0.5H),5.06(d, J ═ 12.26Hz,0.5H),4.96(d, J ═ 11.89Hz,1H),4.64(d, J ═ 11.89, 0.5H), 3.80H, 3.5H, 3.58H, 2.2H), 2.15-1H (m,2H), 2.5H, 1H, 2H, 1H, 15(d, 2H). Theoretical calculation of ESI-MS C₁₈H₁₈ ³⁵ClN₄O₂[M+H]⁺357.1; the test result is 357.3.

N-Cbz- (S) -3- (1-bromo, 8-chloroimidazo [1,5-a ] pyrazinyl) substituted-2-pyrrolidines (S7)

Step six, in a 250mL glass bottle, S6(5.89g,16.5mmol) and dry N, N-dimethylformamide (40mL) were added. The flask and the reaction solution in the flask were cooled to 0 ℃ and N-bromosuccinimide (3.22g,18.2mmol) was added. The reaction was slowly warmed to room temperature and reacted overnight. The reaction was quenched with saturated sodium thiosulfate, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine, dried, concentrated, and purified by silica gel column to give the title compound S7 as a yellow solid (3.19 g).

¹H NMR(CDCl₃400MHz) 8.21(d, J-4.84 Hz,0.5H),7.41(d, J-4.63 Hz,0.5H),7.39-7.14(m,5H),6.95-6.80(m,1H),5.25-4.90(m,2.6H),4.68(d, J-11.44 Hz,0.4H),3.80-3.56(m,2H),2.60-2.20(m,3H),2.10-1.90(m, 1H). Theoretical calculation of ESI-MS C₁₈H₁₇ ⁷⁹Br³⁵ClN₄O₂[M+H]⁺435.0; 435.1 is measured.

N-Cbz- (S) -3- (1-bromo, 8-aminoimidazo [1,5-a ] pyrazinyl) substituted-2-pyrrolidines (S8)

Step seven, into a 25mL thick-walled glass vial, S7(0.5g,1.1mmol) and isopropanol (16mL) were added. To the solution was added 4mL of concentrated ammonia, then the tube was closed and heated in an oil bath at 80 ℃ overnight. The reaction solution was cooled and then concentrated, and the resulting mixture was purified by a silica gel column to obtain the objective compound S8 as a white solid (137 mg).

¹H NMR(CDCl₃400MHz) 7.60(d, J-4.82 Hz,0.5H),7.40-7.10(m,4.5H),7.04-6.92(m,1H),6.88(d, J-6.37 Hz,0.6H),6.69(d, J-4.71 Hz,0.4H),5.20-4.90(m,2.6H),4.75(d, J-11.90 Hz,0.4H),3.80-3.50(m,2H),2.40-2.10(m,3H),2.06-1.86(m, 1H). Theoretical calculation of ESI-MS C₁₈H₁₉ ⁷⁹BrN₅O₂[M+H]⁺416.1; the experiment found that 416.3.

EXAMPLE 1 Synthesis of the Compound (S) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butynoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (1)

The method comprises the following steps: synthesis of benzyl (S) -2- ((1- (4- (pyridin-2-ylcarbamoyl) phenyl) -8-amino) imidazo [1,5-a ] pyrazin-2-yl) pyrrolidin-1-ylcarboxylate (C018)

In a 100mL glass bottle, 4- (4,4,5,5-tetramethyl- [1,3,2] is added]Dioxoboronyl) -benzoic acid 2-pyridylamine amide (213mg,0.66mmol), S8(137mg,0.33mmol) and 1, 2-dimethoxyethane (4 mL). To the above solution was added aqueous sodium carbonate (2M,2mL) and the solution was deoxygenated. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (24mg) and oxygen removed again. The reaction was refluxed overnight, cooled to room temperature and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, concentrated and purified by silica gel column to give the title compound C018(110 mg). ESITheoretical calculation of MS C₃₀H₂₈N₇O₃[M+H]⁺534.2; the experiment result shows that 534.2.

Step two: synthesis of (S) -N- (pyridin-2-yl) -4- (3- (pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (C020)

In a 50mL glass vial, C018(110mg) was added. A solution of hydrobromic acid in acetic acid (33%, 3mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 10mL of water. This solution was purified by HPLC to give the trifluoroacetate salt of the target compound C020, which was lyophilized to give 60mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 10%, retention time t_1/27.6 min.

¹H NMR (MeOD-d4,400mhz):8.47(d, J ═ 5.53Hz,1H),8.40-8.32(m,1H),8.28(d, J ═ 8.24Hz,2H),8.01(d, J ═ 8.60Hz,1H),7.98-7.92(m,3H),7.58(t, J ═ 6.62Hz,1H),7.17(d, J ═ 5.86Hz,1H),5.43(t, J ═ 7.41Hz,1H),3.70-3.60(m,1H),3.60-3.50(m,1H),2.70-2.60(m,1H),2.55-2.42(m,1H),2.42-2.32(m,1H),2.32-2.20(m, 1H). ESI-MS on C₂₂H₂₂N₇Theoretical calculation of O [ M + H]⁺400.5; the test result is 400.3.

Step three: synthesis of (S) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butynoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (1)

In a 50mL glass bottle, 3-butynoic acid (9mg,0.1mmol), 2-chloro-1-methylpyridine iodide (24mg,0.1mmol) and dry dichloromethane (3mL) were added and the solution was stirred at room temperature for 1 hour. A solution of C020 in trifluoroacetate (30mg) and triethylamine (0.3mL,2.1mmol) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 1 hour. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 1, and lyophilized to give 2.4mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 10%, retention time t_1/214.6 min.

¹H NMR(MeOD-d4,400MHz):8.47(d,J＝6.10Hz,1H),8.41-8.35(m,1H),8.30-8.22(m,2H),8.04-7.95(m,2H),7.92-7.86(m,2H),7.62-7.55(m,1H),7.09-7.05(m,1H),6.09(t,J＝6.48Hz,1H),5.57-5.48(m,1H),5.305.15(m,2H),4.00-3.90(m,1H),3.90-3.80(m,1H),2.54-2.40(m,2H),2.35-2.25(m,1H),2.25-2.10(m, 1H). Theoretical calculation of ESI-MS C₂₆H₂₄N₇O₂[M+H]⁺466.2; 466.3 is measured by experiments. Specific rotation degree

(MeOH)。

EXAMPLE 2 Synthesis of the Compound (S) -N- (4-trifluoromethylpyridin-2-yl) para- (3- (1- (2, 3-Butadienoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (2)

The method comprises the following steps: synthesis of benzyl (S) -2- ((1- (4- (4-trifluoromethylpyridin-2-ylcarbamoyl) phenyl) -8-amino) imidazo [1,5-a ] pyrazin-2-yl) pyrrolidin-1-ylcarboxylate (C014)

In a 100mL glass bottle, 4- (4,4,5,5-tetramethyl- [1,3,2] is added]Dioxoboronyl) -benzoic acid 2- (4-trifluoromethyl) pyridylamine amide (470mg,1.2mmol), S8(246mg,0.6mmol) and 1, 2-dimethoxyethane (6 mL). Adding Na into the solution₂CO₃Aqueous (2M,3mL) and deoxygenated the solution. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (42mg) and oxygen removed again. The reaction was refluxed overnight, cooled to room temperature and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, concentrated and purified by silica gel column to give the objective compound C014(350 mg). Theoretical calculation of ESI-MS C₃₁H₂₇F₃N₇O₃[M+H]⁺602.2; the test result is 602.3.

Step two: synthesis of (S) -N- (4-trifluoromethylpyridin-2-yl) 4- (3- (pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (C017)

To a 50mL glass bottle, C014(210mg) was added. A solution of hydrobromic acid in acetic acid (33%, 4mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 10mL of water. This is achieved byThe solution was purified by HPLC to give the trifluoroacetate salt of the target compound C017, and lyophilized to give 150mg of solid. HPLC purification conditions: initial acetonitrile proportion 15%, retention time t_1/215.0 min.

¹H NMR (MeOD-d4,400mhz) 8.65 to 8.86(m,2H),8.18(d, J ═ 8.19Hz,2H),7.95(d, J ═ 5.89Hz,1H),7.90(d, J ═ 8.19Hz,2H),7.43(d, J ═ 4.96Hz,1H),7.15(d, J ═ 5.89Hz,1H),5.44(t, J ═ 7.43Hz,1H),3.70 to 3.60(m,1H),3.60 to 3.50(m,1H),2.74 to 2.60(m,1H),2.54 to 2.42(m,1H),2.42 to 2.32(m,1H),2.32 to 2.20(m, 1H). Theoretical calculation of ESI-MS C₂₃H₂₁F₃N₇O[M+H]⁺468.2; the test result is 468.2.

Step three: synthesis of (S) -N- (4-trifluoromethylpyridin-2-yl) para- (3- (1- (2, 3-butynoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (2)

In a 50mL glass bottle, 3-butynoic acid (10mg,0.12mmol), 2-chloro-1-methylpyridine iodide (38mg,0.15mmol) and dry dichloromethane (3mL) were added and the solution was stirred at room temperature for 1 hour. A solution of C017 trifluoroacetate (40mg) and triethylamine (0.3mL,2.1mmol) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 1 hour. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 2, which was lyophilized to give 8.0mg of a solid. HPLC purification conditions: initial acetonitrile proportion 15%, retention time t_1/226.2 minutes.

¹H NMR (MeOD-d4,400mhz) 8.65 to 8.55(m,2H),8.18(d, J ═ 8.24Hz,2H),8.02(d, J ═ 5.92Hz,1H),7.85(d, J ═ 8.24Hz,2H),7.44(d, J ═ 5.08,1H),7.06(d, J ═ 5.92Hz,1H),6.09(t, J ═ 6.52Hz,1H),5.60 to 5.50(m,1H),5.30 to 5.20(m,2H),4.00 to 3.90(m,1H),3.90 to 3.80(m,1H),2.54 to 2.40(m,2H),2.36 to 2.22(m,1H),2.22 to 2.10(m, 1H). Theoretical calculation of ESI-MS C₂₇H₂₃F₃N₇O₂[M+H]⁺534.2; the experiment result shows 534.1. Specific rotation degree

EXAMPLE 3 Synthesis of the Compound (S) -N- (4-trifluoromethylpyridin-2-yl) 2-fluoro para- (3- (1- (2, 3-butenoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (3)

The method comprises the following steps: synthesis of benzyl (S) -2- ((1- (3-fluoro-4- (4-trifluoromethylpyridin-2-ylcarbamoyl) phenyl) -8-amino) imidazo [1,5-a ] pyrazin-2-yl) -pyrrolidin-1-ylcarboxylate (C022)

In a 100mL glass bottle, 2-fluoro-4- (4,4,5,5-tetramethyl- [1,3, 2)]Dioxoboronyl) -benzoic acid 2- (4-trifluoromethyl) pyridylamine amide (220mg,0.5mmol), S8(100mg,0.24mmol) and 1, 2-dimethoxyethane (6 mL). To the above solution was added aqueous sodium carbonate (2M,4mL) and the solution was deoxygenated. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (24mg) and oxygen removed again. The reaction was refluxed overnight, cooled to room temperature and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, concentrated and purified by silica gel column to give the title compound C022(156 mg). Theoretical calculation of ESI-MS C₃₁H₂₆F₄N₇O₃[M+H]⁺620.2; the experiment shows that 620.0

Step two: synthesis of (S) -N- (4-trifluoromethylpyridin-2-yl) 2-fluoro-4- (3- (pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (C024)

In a 50mL glass bottle, C022(156mg) was added. A solution of hydrobromic acid in acetic acid (33%, 3mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 10mL of water. This solution was purified by HPLC to give the trifluoroacetate salt of target compound C024, which was lyophilized to give 62.9mg of solid. HPLC purification conditions: initial acetonitrile proportion of 20%, retention time t_1/213.5 min.

¹H NMR(MeOD-d4,400MHz):8.64-8.58(m,2H),8.05(t,J＝7.77Hz,1H),7.95(d,J＝5.88Hz,1H),7.75-7.65(m,2H),7.46(d,J＝5.04Hz,1H),7.17(d,J＝5.88Hz,1H),5.43(t,J＝7.41Hz,1H),3.70-3.60(m,1H),3.60-3.50(m,1H),2.72-2.60(m,1H),2.52-2.40(m,1H),2.40-2.32(m,1H),2.32-2.20(m, 1H). Theoretical calculation of ESI-MS C₂₃H₂₀F₄N₇O[M+H]⁺486.2; the test result is 486.2.

Step three: synthesis of (S) -N- (4-trifluoromethylpyridin-2-yl) 2-fluoro-para- (3- (1- (2, 3-butynoyl) pyrrolidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (3)

In a 50mL glass bottle, 3-butynoic acid (8.4mg,0.1mmol), 2-chloro-1-methylpyridine iodide (24mg,0.1mmol) and dry dichloromethane (3mL) were added and the solution was stirred at room temperature for 1 hour. A solution of C024 in trifluoroacetate (40mg) and triethylamine (0.2mL,1.4mmol) in dichloromethane (3mL) was added to the above reaction flask and stirred at room temperature for 1 hour. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 3, and lyophilized to give 13.0mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 20%, retention time t_1/222.9 min.

¹H NMR (MeOD-d4,400mhz) 8.65-8.55(m,2H),8.10-7.96(m,2H),7.70-7.60(m,2H),7.50-7.42(m,1H),7.07(d, J ═ 5.93Hz,1H),6.09(t, J ═ 6.53Hz,1H),5.60-5.50(m,1H),5.30-5.18(m,2H),4.00-3.90(m,1H),3.90-3.80(m,1H),2.54-2.40(m,2H),2.34-2.22(m,1H),2.20-2.10(m, 1H). Theoretical calculation of ESI-MS C₂₇H₂₂F₄N₇O₂[M+H]⁺552.2; the experiment found that the product is 552.1.

EXAMPLE 4 Synthesis of the Compound (R) -2, 3-butenoyl 3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (4)

The method comprises the following steps: 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (B)

1H- [3,4-d]Pyrazolopyrimidine-4-amine (5.3g,39mmol) and N-iodosuccinimide (13.3g,59mmol) were suspended in N, N-dimethylformamide and heated to 80 ℃ under nitrogen for 12 hours. After the reaction is finished, cooling to room temperature, adding water, precipitating a large amount of solid,the mixture was filtered through a buchner funnel, and the solid was washed twice with a saturated sodium thiosulfate solution, water, and hot ethanol, respectively, and then dried by an oil pump to obtain 7.8g of the target compound with a yield of 77%.¹H NMR(DMSO-d₆,400MHz):8.15(s,1H)。

Step two: 3- (4-Phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine (C)

Mixing 3-iodo-1H- [3,4-d]Pyrazolopyrimidine-4-amine (435mg,1.7mmol) in N, N-dimethylformamide/H₂Adding 2- (4-phenoxyphenyl) -4,4,5, 5-tetramethyl- [1,3,2] into the mixed solvent of O]Cyclopentaborane dioxide (1.5g,5.0mmol), potassium phosphate (531mg,2.5mmol) and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane complex (69mg,0.08mmol) was heated to 120 ℃ under nitrogen for 12 hours. After the reaction, water was added and a large amount of solid was precipitated. Extraction with ethyl acetate was carried out three times, the organic phases were combined, washed with water three times, dried by adding anhydrous sodium sulfate, the solvent was dried by spinning, and column purification was carried out to obtain the objective compound 187mg in 37% yield.¹H NMR(DMSO-d₆400MHz) 13.56(s,1H),8.22(s,1H),7.66(d, J ═ 8.7Hz,2H),7.43(t, J ═ 8.1Hz,2H),7.21-7.11(m, 5H). Theoretical calculation of ESI-MS C₁₇H₁₃N₅O[M+H]⁺304.1, the experiment found: 304.3.

step three: (R) -1- (piperidin-3-yl) -3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-D ] pyrimidine-4-amine (D)

Reacting 3- (4-phenoxyphenyl) -1H- [3,4-d]Pyrazolopyrimidine-4-amine (185mg,0.61mmol), (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine (246mg,1.22mmol) and triphenylphosphine (640mg,2.44mmol) were dissolved in dry tetrahydrofuran, diisopropyl azodicarboxylate (0.6mL,3.05mmol) was added dropwise, and the mixture was heated to 40 ℃ for 12 hours. After the reaction is finished, the tetrahydrofuran is removed by spinning, and the 3- [ 4-amino-3- (4-phenoxyphenyl) -1- [3,4-d is obtained by column purification]Pyrazolopyrimidinyl]Tert-butyl piperidinecarboxylate. This was dissolved in methylene chloride (6mL), and trifluoroacetic acid (6mL) was added to the solution to conduct a reaction at room temperature for 12 hours. The reaction progress was monitored by HPLC. After the reaction is finished, removing trifluoroacetic acid by spinning, adding water and methanol, separating by an HPLC preparative column, and freeze-drying to obtain 111mg of the trifluoroacetate salt of the target compound with the yield of 38%. HPLC purification conditions: initial acetonitrile ratio25% retention time t _1/210 minutes.

¹H NMR(MeOD-d₄400MHz) 8.43(s,1H),7.72(d, J-8.5 Hz,2H),7.42(t, J-8.0 Hz,2H),7.23-7.09(m,5H),5.31-5.22(m,1H),3.77-3.65(m,2H),3.45-3.38(m,1H),3.29-3.20(m,1H),2.40-2.23(m,2H),2.19-2.09(m,1H),2.02-1.90(m, 1H). Theoretical calculation of ESI-MS C₂₂H₂₂N₆O[M+H]⁺387.2, the experiment found: 387.1.

step four: (R) -2, 3-butenoyl 3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (4)

In a 50mL glass bottle, 3-butynoic acid (10mg,0.12mmol), 2-chloro-1-methylpyridine iodide (38mg,0.15mmol) and dry dichloromethane (3mL) were added, and the reaction solution was stirred at room temperature for 1 hour. 1-3-piperidine- (4-phenoxyphenyl) -1H- [3,4-d]A solution of pyrazolopyrimidine-4-amine trifluoroacetate (40mg,0.1mmol) and triethylamine (0.1mL,0.72mmol) in dichloromethane (3mL) was added to the above reaction flask and stirred at room temperature for 1 hour. The dichloromethane was evaporated using a rotary evaporator. The resulting dope was purified by HPLC to give the trifluoroacetate salt of target compound 4, and lyophilized to give 7mg (15%) of a solid. HPLC purification conditions: initial acetonitrile proportion of 20%, retention time t_1/230.0 min.

¹H NMR(MeOD-d₄400 MHz: 8.39-8.45(m,1H),7.65-7.72(m,2H),7.39-7.45(m,2H),7.14-7.23(m,3H),7.08-7.14(m,2H),6.26(t, J ═ 5.97Hz,0.5H),6.09(t, J ═ 6.64Hz,0.5H),5.198-5.26(m,2H),4.60-4.45(m,0.7H),4.40-4.15(m,1H),4.10-3.95(m,0.6H), 3.95-385 (m,0.3H),3.85-3.70(m,0.3H),3.65-3.56(m,0.5H),3.56-3.40(m,0.5H), 2.30H, 1.20-20H), theoretical calculation values (m,1H, 2H), 1.5H, 1H, 20H, 1H, 20-20H, 1H, 20H, 1₂₆H₂₅N₆O₂[M+H]⁺453.2, the experiment found: 453.1. specific rotation degree

(MeOH)。

Synthetic route to key intermediate C044 of preparation example 3:

step one, synthesizing (R) -3- [ (3-chloro-pyrazine-2-methylene) -carbamoyl ] -piperidine-1-formic acid tert-butyl ester (C032)

In a 100mL glass bottle, S4(0.60g,4.8mmol), N-Boc- (R) -3-carboxylic acid piperidine (1.37g,5.9mmol) and dichloromethane (30mL) were added. The reaction flask was cooled with ice water, and then triethylamine (3mL,19.2mmol) was added and stirred for 10 min. 2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,2.24g,5.9mmol) was added in one portion and stirred at room temperature overnight. The reaction was quenched with water, the aqueous phase was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried, concentrated and purified by silica gel column. Purification gave the title compound C032 as an oily liquid (1.52 g).¹H NMR (CDCl3,400MHz) 8.45(s,1H),8.32(s,1H),7.10-6.90(br,1H),4.80-4.60(m,2H),4.00-3.80(m,1H),3.20-3.00(m,1H),3.00-2.80(m,1H),2.50-2.30(m,1H),2.00-1.90(m,1H),1.90-1.60(m,4H),1.45(s, 9H). Theoretical calculation of ESI-MS C₁₆H₂₃ ³⁵ClN₄NaO₃[M+Na]⁺377.1, found experimentally: 377.1.

step two, synthesizing (R) -3- [ (3-chloro-pyrazine-2-methylene) -carbamoyl ] -piperidine-1-carboxylic acid benzyl ester (C036)

In a 100mL glass vial, C032(1.52g,4.3mmol) and dichloromethane (5mL) were added. Trifluoroacetic acid (5mL) was added by syringe, followed by stirring at room temperature for 1 hour. The solvent and trifluoroacetic acid were evaporated using a rotary evaporator. The resulting crude was placed in a 100mL glass vial and anhydrous tetrahydrofuran (30mL) and triethylamine (2.5mL,18mmol) were added. The reaction flask was cooled with ice water, benzyl chloroformate (1.1g,6.5mmol) was added via syringe and stirred at room temperature overnight. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate and the organic phases were combined. The organic phase was washed with brine, then dried over anhydrous sodium sulfate and concentrated using a rotary evaporator. The remaining crude product was purified by silica gel column to give C036(1.1 g).

¹H NMR(CDCl₃,400MHz):8.40(s,1H),8.31(s,1H),7.45-7.25(m,5H),5.20-5.04(m,2H),4.804.57(m,2H),4.23-4.10(m,1H),4.10-3.80(m,1H),3.28-3.15(m,1H),3.12-2.84(m,1H),2.54-2.35(m,1H),2.22-2.06(m,1H),2.06-1.65(m,3H),1.62-1.40(m, 1H). Theoretical calculation of ESI-MS C₁₉H₂₁ ³⁵ClN₄NaO₃[M+Na]⁺As 411.1, experimentally determined: 411.3.

step three, synthesizing (R) -3- (8-chloro- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-benzyl formate (CDA-114)

Reacting 3- [ (3-chloro-pyrazine-2-methylene) -carbamoyl]-piperidine-1-carboxylic acid benzyl ester (C036, 1.10g,2.84mmol) was dissolved in acetonitrile, 1, 3-dimethyl-2-imidazolidinone (0.97g,8.51mmol) was added, cooled to 0 deg.C, phosphorus oxychloride (1.74g,11.40mmol) was added, and after the addition, the temperature was raised to 60 deg.C for 12 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and then slowly added to 50mL of 25% aqueous ammonia ice. Extraction was performed with ethyl acetate (100 mL. times.3), and the organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, the solvent was removed by rotation, and the mixture was passed through a silica gel column to obtain the objective compound CDA-114(524 mg).¹H NMR(CDCl₃400MHz) of 7.85 to 7.65(m,2H),7.50 to 7.30(m,5H),7.25 to 7.10(m,1H),5.24 to 5.04(m,2H),4.50 to 4.18(m,2H),3.28 to 2.84(m,3H),2.24 to 2.07(m,2H),1.97 to 1.83(m,1H),1.80 to 1.55(m, 1H). Theoretical calculation of ESI-MS C₁₉H₁₉ ³⁵ClN₄O₂[M+H]⁺371.1, the experiment found: 371.4.

step four, synthesizing (R) -3- (1-bromo-8-chloro- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-carboxylic acid benzyl ester (CDA-116)

Reacting 3- (8-chloro- [1, 5-a)]Imidazopyrazin-3-yl) -piperidine-1-carboxylic acid benzyl ester (CDA-114, 514mg,1.39mmol) was dissolved in 5mL of DMF, cooled to 0 deg.C and NBS (272mg,1.53mmol) added overnight at room temperature. After completion of the reaction, 30mL of a saturated sodium thiosulfate solution was added to the reaction mixture, extraction was performed with ethyl acetate (100 mL. times.3), the organic phases were combined, washed with water (50 mL. times.3), dried over anhydrous sodium sulfate, the solvent was removed by rotation, and the mixture was purified by a silica gel column to obtain the objective compound CDA-116(628 mg).¹H NMR(CDCl₃,400MHz):7.80-7.65(m,1H),7.50-7.25(m,5H),7.20-7.05(m,1H),5.27-5.04(m,2H),4.49-4.19(m,2H),3.24-2.83(m,3H),2.18-2.10(m,2H),1.95-1.82(m,1H),1.70-1.50(m,1H) In that respect Theoretical calculation of ESI-MS C₁₉H₁₈ ³⁵Cl⁷⁹BrN₄O₂[M+H]⁺449.0, the experiment found: 449.2.

step five, synthesizing (R) -3- (1-bromo-8-amino- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-carboxylic acid benzyl ester (C044)

To a 25mL thick-walled glass vial, CDA-116(0.53g,1.1mmol), DMSO (2mL) and isopropanol (12mL) were added. Concentrated ammonia (6mL) was added to the solution, then the tube was closed and heated in an oil bath at 85 ℃ overnight. The reaction solution was cooled and then concentrated, and the resulting mixture was purified by a silica gel column to obtain the objective compound C044(389 mg).

¹H NMR(CDCl₃400MHz) of 7.50 to 7.20(m,5H),7.10 to 6.80(m,2H),6.10 to 5.80(m,2H),5.20 to 5.00(m,2H),4.50 to 4.10(m,2H),3.20 to 2.70(m,3H),2.20 to 1.70(m,3H),1.70 to 1.50(m, 1H). Theoretical calculation of ESI-MS C₁₉H₂₁ ⁷⁹BrN₅O₂[M+H]430.1, experimentally determined: 430.2.

synthetic route to key intermediate C049 of preparation example 4:

step one, synthesizing (S) -2- [ (3-chloro-pyrazine-2-methylene) -carbamoyl ] -piperidine-1-carboxylic acid tert-butyl ester (C038)

In a 100mL glass bottle, S4(0.60g,4.8mmol), N-Boc- (S) -2-carboxylic acid piperidine (1.37g,6.0mmol) and dichloromethane (30mL) were added. The reaction flask was cooled with ice water, and then triethylamine (3mL,19.2mmol) was added and stirred for 10 min. 2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,2.28g,6.0mmol) was added in one portion and stirred at room temperature overnight. The reaction was quenched with water, the aqueous phase was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried, concentrated and purified by silica gel column to give the objective compound C038 as an oily liquid (1.59 g).¹H NMR(CDCl₃,400MHz):8.42(s,1H),8.31(s,1H),7.40-7.25(br,1H),5.00-4.40(m,3H),4.20-4.00(m,1H),3.00-2.73(m,1H),2.40-2.20(m,1H),1.80-1.20(m5H),1.50(s, 9H). Theoretical calculation of ESI-MS C₁₆H₂₃ ³⁵ClN₄NaO₃[M+Na]⁺377.1, found experimentally: 377.4.

step two, synthesizing (S) -2- [ (3-chloro-pyrazine-2-methylene) -carbamoyl ] -piperidine-1-carboxylic acid benzyl ester (C040)

In a 100mL glass bottle, C038(1.59g,4.5mmol) and dichloromethane (5mL) were added. Trifluoroacetic acid (5mL) was added by syringe, followed by stirring at room temperature for 1 hour. The solvent and trifluoroacetic acid were evaporated using a rotary evaporator. The resulting crude was placed in a 100mL glass vial and anhydrous tetrahydrofuran (20mL) and triethylamine (2.5mL,18mmol) were added. The reaction flask was cooled with ice-water, benzyl chloroformate (1.2g,6.75mmol) was added via syringe and stirred at room temperature overnight. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate and the organic phases were combined. The organic phase was washed with brine, then dried over anhydrous sodium sulfate and concentrated using a rotary evaporator. The remaining crude product was purified by silica gel column to give C040(1.29 g).

¹H NMR(CDCl₃400MHz) 8.36(s,1H),8.30(s,1H),7.48-7.05(m,5H),5.30-5.10(m,2H),5.05-4.85(m,1H),4.85-4.70(m,1H),4.70-4.50(m,1H),4.30-4.00(m,1H),3.10-2.90(m,1H),2.50-2.30(m,1H),1.80-1.37(m, 5H). Theoretical calculation of ESI-MS C₁₉H₂₁ ³⁵ClN₄NaO₃[M+Na]⁺As 411.1, experimentally determined: 411.1.

step three, synthesizing (S) -2- (8-chloro- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-benzyl formate (C042)

2- [ (3-chloro-pyrazine-2-methylene) -carbamoyl ] -piperidine-1-carboxylic acid benzyl ester (C036, 1.29g,3.5mmol) was dissolved in acetonitrile, 1, 3-dimethyl-2-imidazolidinone (1.14mL,10.5mmol) was added, cooling to 0 deg.C, phosphorus oxychloride (2.05mL,14mmol) was added, and after the addition, the temperature was raised to 60 deg.C for 12 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the reaction mixture was slowly added to a mixture of 30mL of concentrated aqueous ammonia and ice. Extraction with ethyl acetate (100 mL. times.3) was performed, and the organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, the solvent was removed by rotation, and the mixture was subjected to silica gel column to obtain the objective compound C042(756 mg).

¹H NMR(CDCl₃400MHz, 8.10-7.80(m,1H),7.80(s,1H),7.45-7.30(m,5H),7.26-7.05(m,1H),5.90-5.75(br,1H),5.30-5.05(m,2H),4.10-3.90(m,1H),2.70(td, J ═ 13.25,2.29Hz,1H),2.55-2.30(m,2H),2.10-1.90(m,1H),1.90-1.75(m,1H),1.76-1.65(m,1H),1.65-1.50(m, 1H). Theoretical calculation of ESI-MS C₁₉H₂₀ ³⁵ClN₄O₂[M+H]⁺371.1, the experiment found: 371.3.

step four, synthesizing (S) -2- (1-bromo-8-chloro- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-carboxylic acid benzyl ester (C048)

Benzyl 2- (8-chloro- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-carboxylate (C042, 969mg,2.6mmol) was dissolved in 25mL of DMF, cooled to 0 deg.C and NBS (513mg,2.9mmol) added overnight at room temperature. After completion of the reaction, 30mL of a saturated sodium thiosulfate solution was added to the reaction mixture, extraction was performed with ethyl acetate (100 mL. times.3), the organic phases were combined, washed with water (50 mL. times.3), dried over anhydrous sodium sulfate, the solvent was removed by rotation, and the mixture was purified by a silica gel column to obtain the objective compound C048(650 mg).

¹H NMR(CDCl₃400MHz) 8.10-7.80(m,1H),7.50-7.30(m,5H),7.23-7.07(m,1H),5.85-5.70(m,1H),5.28-5.10(m,2H),4.06-3.94(m,1H),2.73(td, J ═ 13.43,1.84Hz,1H),2.53-2.38(m,1H),2.38-2.25(m,1H),2.03-1.89(m,1H),1.86-1.67(m,2H),1.63-1.46(m, 1H). Theoretical calculation of ESI-MS C₁₉H₁₉ ⁷⁹Br³⁵ClN₄O₂[M+H]⁺449.1, the experiment found: 449.1.

step five, synthesizing (S) -2- (1-bromo-8-amino- [1,5-a ] imidazopyrazin-3-yl) -piperidine-1-carboxylic acid benzyl ester (C049)

To a 25mL thick-walled glass vial, C048(0.50g,1.0mmol), DMSO (2mL) and isopropanol (12mL) were added. Concentrated ammonia (6mL) was added to the solution, then the tube was closed and heated in an oil bath at 85 ℃ overnight. The reaction solution was cooled and then concentrated, and the resulting mixture was purified by a silica gel column to obtain the objective compound C049.

¹H NMR(CDCl₃,400MHz):7.60-7.25(m,5H),7.05-6.80(m,1H),6.00-5.60(m,3H),5.28-5.09(m,2H),4.00(d,J＝13.2Hz,1H),2.79(dt,J＝13.3Hz,J＝2.1Hz,1H),2.50-2.22(m,2H),2.00-1.84(m,1H),1.82-1.64(m,2H),1.62-1.45(m, 1H). Theoretical calculation of ESI-MS C₁₉H₂₁ ⁷⁹BrN₅O₂[M+H]⁺430.1, the experiment found: 430.2.

example 5: synthesis of (R) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butenoyl) piperidin-3-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (5)

The method comprises the following steps: (R) -benzyl 3- ((1- (4- (pyridin-2-ylcarbamoyl) phenyl) -8-amino) imidazo [1,5-a ] pyrazin-3-yl) -piperidin-1-ylcarboxylate (C047)

In a 100mL glass bottle, 4- (4,4,5,5-tetramethyl- [1,3,2] is added]Dioxocyclopentanobutyl) -benzoic acid 2-pyridylamine amide (583mg,1.8mmol), C044(389mg,0.90mmol) and 1, 2-dimethoxyethane (6 mL). To the above solution was added aqueous sodium carbonate (2M,6mL) and the solution was deoxygenated. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (73mg.0.09mmol) and oxygen again. The reaction was refluxed overnight, cooled to room temperature and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried, concentrated and purified over silica gel column. Purification gave the title compound C047(498 mg). Theoretical calculation of ESI-MS C₃₁H₃₀N₇O₃[M+H]⁺548.2; the test result is 548.2.

Step two: synthesis of (R) -N- (pyridin-2-yl) para (3- (piperidin-3-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (CDA-118)

In a 50mL glass vial, C047(498mg) was added. A solution of hydrobromic acid in acetic acid (33%, 6mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 20mL of water. This solution was purified by HPLC to give the trifluoroacetate salt of the desired compound CDA-118, which was lyophilized to give 170mg of solid.

HPLC purification conditions: initial acetonitrile proportion of 5%, retention time t_1/212.5 min.¹H NMR(MeOD-d4,400MHz):8.55-8.43(m,2H),8.31(d, J ═ 8.2Hz,2H),8.02-7.92(m,4H),7.66(t, J ═ 6.7Hz,1H),7.11(d, J ═ 5.9Hz,1H),3.92-3.81(m,1H),3.69(dd, J ═ 12.6Hz, J ═ 3.5Hz,1H)3.60(dd, J ═ 12.6Hz, J ═ 9.6Hz,1H),3.50-3.40(m,1H),3.30-3.18(m,1H),2.36-2.20(m,1H),2.10-1.90(m, 3H). Theoretical calculation of ESI-MS C₂₃H₂₄N₇O[M+H]⁺414.2; the test result is 414.3.

Step three: synthesis of (R) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butenoyl) piperidin-3-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (5)

In a 50mL glass bottle, 3-butynoic acid (8mg,0.09mmol), 2-chloro-1-methylpyridine iodide (38mg,0.17mmol) and dry dichloromethane (3mL) were added and the solution was stirred at room temperature for 1 hour. A solution of CDA-118 in trifluoroacetate (40mg,0.076mmol) and triethylamine (0.1mL) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 1 hour. The viscous product obtained by evaporating dichloromethane with a rotary evaporator was purified by HPLC to obtain the trifluoroacetate salt of the objective compound 5, which was then lyophilized to obtain 11.2mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 10%, retention time t_1/216.5 min.¹HNMR (MeOD-d4,400mhz):8.47(d,1H, J ═ 5.4Hz),8.33(t,1H, J ═ 7.8Hz),8.26(d,2H, J ═ 8.2Hz),7.99(d,1H, J ═ 8.6Hz),7.96-7.85(m,3H),7.55(t,1H, J ═ 6.5Hz),7.06(d,1H, J ═ 6.0Hz),6.30-6.15(m,1H),5.30-5.10(m,2H),4.65-4.55(m,1H),4.35-4.22(m,1H),4.17-4.09(m,1H),3.87-3.76(m,1H),3.45-3.35(m,1H), 1.84-1H, 1.84(m,1H), 1.17-4.84 (m,1H). Theoretical calculation of ESI-MS C₂₇H₂₆N₇O₂[M+H]⁺480.2; the test result is 480.2.

Example 6: synthesis of (S) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butenoyl) piperidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (6)

The method comprises the following steps: (S) -benzyl 2- ((1- (4- (pyridin-2-ylcarbamoyl) phenyl) -8-amino) imidazo [1,5-a ] pyrazin-3-yl) piperidin-1-ylcarboxylate (C050)

In a 100mL glass vial, 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaboronyl) -benzoic acid 2-pyridylaminamide (608mg,2.0mmol), C049(400mg,1.0mmol) and 1, 2-dimethoxyethane (6mL) were added. To the above solution was added aqueous sodium carbonate (2M,6mL) and the solution was deoxygenated. Then, the [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (81mg.0.1mmol) was added to the reaction flask and oxygen was again removed. The reaction was refluxed overnight, cooled to room temperature and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried, concentrated and purified over silica gel column. Purification yielded the target compound C050(515 mg).

¹H NMR(CDCl₃400MHz) 8.90-8.80(br,1H),8.42(d, J ═ 8.31Hz,1H),8.31(d, J ═ 4.35Hz,1H),8.06(d, J ═ 7.98Hz,2H),7.85(d, J ═ 7.90Hz,2H),7.79(t, J ═ 7.62Hz,1H),7.44-7.30(m,5H),7.15-7.06(m,1H),7.05-6.90(m 1H),5.90-5.72(br,1H),5.30-5.10(m,3H),4.04(d, J ═ 12.56Hz,1H),2.91(t, J ═ 12.88Hz,1H),2.60-2.44(m, 2.44H), 2.04 (d, J ═ 12.56Hz,1H),2.91(t, J ═ 12.88, 1H),2.60-2.44(m, 6H), 6.66H, 1H), 1H, 6H, 1H, 6, 1. Theoretical calculation of ESI-MS C₃₁H₃₀N₇O₃[M+H]⁺548.2; the test result is 548.1.

Step two: synthesis of (S) -N- (pyridin-2-yl) para- (3- (piperidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (C053)

In a 50mL glass bottle, C050(500mg) was added. A solution of hydrobromic acid in acetic acid (33%, 10mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 20mL of water. This solution was purified by HPLC to give the trifluoroacetate salt of the objective compound C053 (515 mg).

HPLC purification conditions: initial acetonitrile proportion of 5%, retention time t_1/213 minutes.¹H NMR (MeOD-d4,400mhz) 8.48(d,1H, J ═ 5.5Hz),8.39(t,1H, J ═ 8.0Hz),8.28(d,2H, J ═ 8.2Hz),8.05-7.92(m,4H),7.59(t,1H, J ═ 6.6Hz),7.15(d,1H, J ═ 5.8Hz),3.64-3.56(m,1H),3.34-3.26(m,2H),2.40-2.25(m,1H),2.15-1.80(m, 5H). Theoretical calculation of ESI-MS C₂₃H₂₄N₇O[M+H]⁺414.2; the experiment found that 414.2.

Step three: synthesis of (S) -N- (pyridin-2-yl) para- (3- (1- (2, 3-butenoyl) piperidin-2-yl) -8-aminoimidazo [1,5-a ] pyrazin-1-yl) benzamide (6)

In a 50mL glass vial, 3-butynoic acid (13mg) and dry dichloromethane (3mL) were added. To the above solution was added 2-chloro-1-methylpyridine iodide (23 mg). The solution was stirred at room temperature for 1 hour. A solution of C053 in trifluoroacetate (44mg) and triethylamine (0.1mL) in dichloromethane (3mL) was added to the above reaction flask and stirred at room temperature for 40 minutes. The viscous product obtained by evaporating dichloromethane with a rotary evaporator was purified by HPLC to obtain the trifluoroacetate salt of the objective compound 6, which was then lyophilized to obtain 6.8mg of a solid.

HPLC purification conditions: initial acetonitrile proportion of 10%, retention time t_1/220.5 min.¹H NMR (MeOD-d4,400mhz):8.48(d,1H, J ═ 5.7Hz),8.36(t,1H, J ═ 8.0Hz),8.27(d,2H, J ═ 8.2Hz),8.00-7.90(m,3H),7.91-7.81(m,1H),7.57(t,1H, J ═ 6.6Hz),7.04(d,1H, J ═ 5.8Hz),6.27(t,2H, J ═ 6.55Hz),5.26(d,2H, J ═ 6.55Hz),4.10-3.90(m,1H),3.50-3.20(m,1H),2.50-2.30(m,2H),2.10-1.90(m,1H), 1.91-76 (m,1H), 1.58(m, 1H). Theoretical calculation of ESI-MS C₂₇H₂₆N₇O₂[M+H]⁺480.2; the experiment found that 480.3. Specific rotation degree

(MeOH)。

EXAMPLE 7 Synthesis of the Compound (R) -2, 3-butenoyl 3- (3- (4-phenoxy-2-fluoro-phenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (7)

The method comprises the following steps: synthesis of 2-fluoro-4-bromophenyl-phenyl ether (C041)

In a 50mL dry glass bottle, p-bromom-fluorophenol (400mg,2mmol), phenylboronic acid (490mg,4mmol), anhydrous cupric acetate (437mg,2.4mmol) and dry molecular sieve (C: (R) (R))

2g) In that respect Dry dichloromethane (10mL) was added to the bottle and finally triethylamine (1.5mL) was added. After the reaction was stirred at room temperature for 24 hours, the reaction solution was filtered through celite to remove solids. The celite was rinsed with dichloromethane (200mL), the organic phases were combined and concentrated, and the resulting reaction crude was purified with normal phase silica gel column to give the title compound C041(140 mg).¹H NMR(CDCl₃,400MHz):7.46(t,1H,J＝8.34Hz),7.42-7.36(m,2H),7.18(t,1H,J＝7.41Hz),7.04(d,2H,J＝7.86Hz),6.77(dd,1H,J＝2.57Hz,J＝9.76Hz),6.70(d,1H,J＝8.81Hz)。

Step two: synthesis of 2- (4-phenoxy-2-fluoro-phenyl) -4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan (C051)

In a 100mL dry glass bottle, 2-fluoro-4-bromophenyl-phenyl ether (700mg,2.62mmol), bis-pinacol boronate (1.32g,5.2mmol), potassium acetate (KOAc, 1.03g,10.5mmol) and dry 1, 4-dioxane (15mL) were added. Removing oxygen from the reaction solution, and adding [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane complex (182mg,0.26mmol) and oxygen removed again. The reaction solution was heated to 90 ℃ and stirred overnight. The reaction was quenched with 50mL of water and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with saturated brine, dried, concentrated and purified over silica gel column. Purification afforded the title compound C051 as a solid (1.30 g).¹H NMR(CDCl₃400MHz) 7.68(t,1H, J ═ 7.61Hz),7.38(d,2H, J ═ 7.71Hz),7.17(t,1H, J ═ 7.46Hz),7.04(d,2H, J ═ 8.05Hz),6.76(d,1H, J ═ 8.21Hz),6.61(d,1H, J ═ 10.71Hz),1.35(s, 12H). Theoretical calculation of ESI-MS C₁₈H₂₁BFO₃[M+H]315.2, found experimentally.

Step three: synthesis of 3- (4-phenoxy-2-fluoro-phenyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine (C054)

Mixing 3-iodo-1H- [3,4-d]Pyrazolopyrimidine-4-amine (520mg,2.0mmol) in N, N-dimethylformamide/H₂O (12mL-8mL), C051(1.30g,4.1mmol), potassium phosphate (1.27g,6mmol) and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane complex (163mg,0.2mmol) was heated to 120 ℃ under nitrogen for 12 hours. After the reaction is finished, adding water and a large amount of solidAnd (4) precipitating. Extracting with ethyl acetate (50mL) for three times, combining organic phases, washing with water for three times, adding anhydrous sodium sulfate for drying, spin-drying the solvent, and purifying by column chromatography to obtain the target compound C054184 mg.

¹H NMR(CDCl₃400MHz) 7.56-7.40(m,4H),7.21(t,1H, J ═ 7.72Hz),7.18(d,2H, J ═ 8.20Hz),7.01(d,1H, J ═ 11.61Hz),6.92(d,1H, J ═ 9.10 Hz). Theoretical calculation of ESI-MS C₁₇H₁₃FN₅O[M+H]⁺322.1, found experimentally: 322.1.

step four: synthesis of (R) -1- (piperidin-3-yl) -3- (4-phenoxy-2-fluoro-phenyl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine (C065)

C054(184mg,0.60mmol), (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine (241mg,1.2mmol) and triphenylphosphine (628mg,2.4mmol) were dissolved in dry tetrahydrofuran, diisopropyl azodicarboxylate (2.42g,12mmol) was added dropwise, and the mixture was heated to 40 ℃ for 12 hours. After the reaction is finished, the tetrahydrofuran is removed by spinning, and the tert-butyl 3- [ 4-amino-3- (4-phenoxy-2-fluoro-phenyl) -1- [3,4-d ] pyrazolopyrimidinyl ] piperidinecarboxylate is obtained by column purification. This was dissolved in methylene chloride (3mL), and trifluoroacetic acid (8mL) was added to the solution to conduct a reaction at room temperature for 12 hours. After the reaction is finished, removing trifluoroacetic acid by spinning, adding water and methanol, separating by an HPLC (high performance liquid chromatography) preparative column, and freeze-drying to obtain 95mg of trifluoroacetate of the target compound C065.

HPLC purification conditions: initial acetonitrile proportion 25%, retention time t_1/214 minutes.¹H NMR(MeOD-d₄400MHz) 8.43(s,1H),7.65(t,1H, J ═ 8.5Hz),7.46(t,2H, J ═ 7.7Hz),7.25(t,1H, J ═ 7.4Hz),7.15(d,2H, J ═ 8.0Hz),6.97(d,1H, J ═ 8.9Hz),6.91(d,1H, J ═ 11.5Hz),5.38-5.24(m,1H),3.78-3.68(m,2H),3.50-3.40(m,1H),3.30-3.16(m,1H),2.40-2.24(m,2H),2.22-2.10(m,1H),2.06-1.90(m, 1H). Theoretical calculation of ESI-MS C₂₂H₂₂FN₆O[M+H]405.2, experimentally determined: 405.1.

step five: (R) -2, 3-butenoyl 3- (3- (4-phenoxy-2-fluoro-phenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (7)

3-Butynoic acid (16.8mg,0.2mmol) was placed in a 50mL glass vial, dried dichloromethane (3mL) was added to dissolve the substrate, then 2-chloro-1-methylpyridine iodide (29mg,0.13mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. A solution of C065(50mg,0.1mmol) and triethylamine (0.1mL,0.72mmol) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 40 minutes. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 7, which was lyophilized to give 18mg of a solid.

HPLC purification conditions: initial acetonitrile proportion of 30%, retention time t_1/218 minutes.¹H NMR(MeOD-d₄400MHz):8.45-8.37(m,1H),7.68-7.58(m,1H),7.66-7.58(m,1H),7.46(t,2H, J ═ 7.85Hz),7.25(t,1H, J ═ 7.43Hz),7.16(d,2H, J ═ 7.98Hz),6.97(d,1H, J ═ 8.68Hz),6.91(d,1H, J ═ 11.54Hz),6.24(t,0.5H, J ═ 6.15Hz),6.08(t,0.5H, J ═ 6.15Hz),5.22-5.05(m,1H),4.60-4.40(m,0.6H),4.40-4.10(m,0.8H),4.10-3.80(m, 3.70H), 3.70H, 3.70 (m-3.70H), 3.3.70H, 3.70 (m-3.3.50H), 3.3.50H, 3.70H, 3.3, 3.5H, 3.1H, 3₂₆H₂₄FN₆O₂[M+H]⁺471.2, experimentally determined: 471.3.

example 8: synthesis of the Compound (S) -2, 3-butenoyl-2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) -1-pyrrolidine (8)

The method comprises the following steps: synthesis of the Compound benzyl (S) -2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) -pyrrolidin-1-ylcarboxylate (C063)

In a 100mL glass bottle, 2- (4-phenoxyphenyl) -4,4,5, 5-tetramethyl- [1,3,2]Dioxolane (580mg,2.0mmol), S8(400mg,1.0mmol) and 1, 2-dimethoxyethane (6 mL). To the above solution was added aqueous sodium carbonate (2M,6mL) and the solution was deoxygenated. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (70mg.0.1mmol) and deoxygenated again. The reaction was refluxed overnight, cooled to room temperature, and the aqueous phase was extracted three times with ethyl acetate (50 mL. times.3). The organic phases were combined, washed with saturated brine and driedDried, concentrated and purified over silica gel column. Purification gave the title compound C063(320 mg). Theoretical calculation of ESI-MS C₃₀H₂₈N₅O₃[M+H]⁺506.2; the test result is 506.1.

Step two: synthesis of 1- (4-phenoxyphenyl) -3- (pyrrolidin-2-yl) -8-amino-imidazo [1,5-a ] pyrazine (C067)

In a 50mL glass bottle, C063(320mg) was added. A solution of hydrobromic acid in acetic acid (33%, 6mL) was added via syringe. The reaction was stirred at room temperature for 1 hour and quenched by the addition of 20mL of water. This solution was purified by HPLC to give the trifluoroacetate salt of the target compound C067, which was lyophilized to give 148mg of solid.

HPLC purification conditions: initial acetonitrile proportion 15%, retention time t_1/216 minutes.¹H NMR (MeOD-d4,400mhz):7.90(d,1H, J ═ 5.90Hz),7.70(d,2H, J ═ 8.49Hz),7.41(t,2H, J ═ 7.85Hz),7.24-7.05(m,6H),5.41(t,1H, J ═ 7.41Hz),3.70-3.59(m,1H),3.59-3.50(m,1H),2.70-2.58(m,1H),2.54-2.40(m,1H),2.40-2.18(m, 2H). Theoretical calculation of ESI-MS C₂₂H₂₂N₅O[M+H]⁺372.2; the test result is 372.3.

Step three: synthesis of the Compound (S) -2, 3-butenoyl-2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) -1-pyrrolidine (8)

In a 50mL glass vial, 3-butynoic acid (17mg,0.2mmol) and dry dichloromethane (3mL) were added followed by 2-chloro-1-methylpyridine iodide (30mg,0.13mmol) and the solution was stirred at room temperature for 1 hour. A solution of C067 trifluoroacetate salt (50mg,0.1mmol) and diisopropylethylamine (0.2mL) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 40 minutes. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of target compound 8, and lyophilized to give 14.8mg of a solid. HPLC purification conditions: initial acetonitrile proportion 25%, retention time t_1/218 minutes.¹H NMR(MeOD-d4,400MHz):7.96(d,1H,J＝5.94Hz),7.63(d,2H,J＝8.50Hz),7.41(t,2H,J＝7.83Hz),7.22-7.08(m,5H),7.00(d,1H,J＝5.91Hz),6.07(t,1H,J＝6.53Hz),5.20-4.80(m,1H),5.30-5.18(m,2H),3.98-3.88(m,1H),3.88-3.78(m,1H),2.60-2.33(m,2H) 2.30-2.20(m,1H),2.20-2.08(m, 1H). Theoretical calculation of ESI-MS C₂₆H₂₄N₅O₂[M+H]⁺438.2; the test result is 438.1.

EXAMPLE 9 Synthesis of the Compound (R) -2, 3-butynenoyl 3- (3- (4- (pyridin-2-ylcarbamoyl) phenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (9)

Step one, synthesizing (R) -3- (3-iodo-4-amino-1H-pyrazolo [3,4-d ] pyrimidine-1-yl) piperidine carboxylic acid tert-butyl ester (CDA-132)

3-iodo-1H- [3,4-d ] pyrazolopyrimidin-4-amine (B,200mg,0.77mmol) was dissolved in dry tetrahydrofuran, and triphenylphosphine (603mg,2.30mmol), (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine (310mg,1.54mmol) was added. Diisopropyl azodicarboxylate (0.92mL,4.60mmol) was added dropwise under nitrogen. After the addition was complete, the mixture was stirred at 40 ℃ for 12 hours. After the reaction, tetrahydrofuran was removed by rotation, and the mixture containing CDA-132 was purified by column chromatography to obtain 580 mg.

Step two, synthesizing (R) -3- (3- (4- (pyridine-2-yl carbamoyl) phenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidine-1-yl) piperidine carboxylic acid tert-butyl ester (CDA-133):

the reaction flask was charged with the mixture containing CDA-132 of the previous step (580mg), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (500mg,1.54mmol), ethylene glycol dimethyl ether (6mL,2mol/L), and sodium carbonate solution (3mL, 2M). After oxygen was removed and nitrogen purged, [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (63mg,0.08mmol) was added, heated to 95 ℃ under nitrogen protection and stirred for 12 hours. After cooling, 50mL of water was added, extraction was carried out three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and after rotary removal of the solvent, column purification was carried out to obtain 360mg of a mixture containing CDA-133.

Step three Synthesis of (R) -4- (4-amino-1- (piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (pyridin-2-yl) benzamide (CDA-136):

the last step comprisesThe mixture of CDA-133 was dissolved in 3mL of dichloromethane, 6mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 12 hours. After the solvent was removed by evaporation, water and methanol were added, and HPLC purification gave the trifluoroacetate salt of CDA-136, which was lyophilized to give 254mg of solid. The total yield of the three steps is 63 percent. HPLC purification conditions: initial acetonitrile proportion of 10%, retention time t _1/210 minutes.¹HNMR(MeOD-d₄400MHz 8.50(s,1H),8.48(s,1H),8.37(t,1H, J ═ 7.94Hz),8.26(d,2H, J ═ 7.94Hz),8.04(d,1H, J ═ 8.53Hz),7.94(d,2H, J ═ 7.94Hz),7.58(t,1H, J ═ 6.44Hz),5.44-5.28(m,1H),3.81-3.74(m,2H),3.54-3.42(m,1H),3.32-3.22(m,1H),2.46-2.28(m,2H),2.24-2.14(m,1H),2.12-1.98(m, 1H). Theoretical calculation of ESI-MS C₂₂H₂₂N₈O[M+H]⁺415.2; the experiment shows that: 415.2.

step four Synthesis of the Compound (R) -2, 3-butenoyl-3- (3- (4- (pyridin-2-ylcarbamoyl) phenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine (9):

3-Butynoic acid (10mg,0.13mmol) was dissolved in dry dichloromethane, 2-chloro-1-methylpyridine iodide (25mg,0.11mmol) was added, and the mixture was stirred at room temperature for 1 hour. A solution of CDA-136 trifluoroacetate (50mg,0.1mmol) and diisopropylethylamine (0.2mL) in dry dichloromethane (3mL) was added and the reaction was allowed to proceed at room temperature for 1 hour. After removal of the solvent by evaporation, water and methanol were added and purified by HPLC to give the trifluoroacetate salt of compound 9, which was lyophilized to give 23mg of solid. The yield of this step was 41%. HPLC purification conditions: the initial acetonitrile proportion was 10%, the retention time t ═ 18 minutes.¹H NMR(MeOD-d₄400MHz 8.44(s,1H),8.42(s,1H),8.22(d,2H, J-8.5 Hz),8.18-8.08(m,2H),7.92(d,2H, J-6.76 Hz),7.45-7.35(m,1H),6.30-6.23(m,0.6H),6.12-6.04(m,0.4H),5.28-4.93(m,3H),4.53-4.45(m,0.5H),4.32-4.22(m,0.5H),4.21-4.10(m,0.5H),4.08-4.01(m,0.5H),3.99-3.87(m,0.5H),3.71-3.62(m,0.5H), 3.52-3.52 (m, 3.83-2H), 2.06-1H, 2H, 1H, 4.3H, 4H, 4.3H, 4H, 4.5H, 4.3. Theoretical calculation of ESI-MS C₂₆H₂₄N₈O₂[M+H]⁺481.2; the experiment shows that: 481.2.

example 10: synthesis of (S) -2, 3-butenoyl-2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) piperidine (10)

The method comprises the following steps: synthesis of the Compound benzyl (S) -2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) -piperidin-1-ylcarboxylate (C092)

In a 100mL glass bottle, 2- (4-phenoxyphenyl) -4,4,5, 5-tetramethyl- [1,3,2]Dioxolane (414mg,1.4mmol), C049(280mg,0.65mmol) and 1, 2-dimethoxyethane (8 mL). To the above solution was added aqueous sodium carbonate (2M,4mL) and the solution was deoxygenated. Then, add [1,1' -bis (diphenylphosphino) ferrocene to the reaction flask]Palladium dichloride dichloromethane complex (50mg.0.07mmol) and deoxygenated again. The reaction was refluxed overnight, cooled to room temperature, and the aqueous phase was extracted three times with ethyl acetate (50 ml. reaction. the organic phases were combined and washed with saturated brine, dried, concentrated and purified by silica gel column to give the objective compound C092(220 mg).¹H NMR(CDCl₃400MHz) 7.63(d,2H, J ═ 8.17Hz),7.43-7.30(m,7H),7.20-7.05(m,5H),7.00-6.85(m,1H),5.27-4.97(m,4H),4.08-3.96(m,1H),2.88(t,1H, J ═ 12.80Hz),2.61-2.45(m,1H),2.45-2.32(m,1H),2.02-1.50(m, 5H). Theoretical calculation of ESI-MS C₃₁H₃₀N₅O₃[M+H]⁺520.2; the experiment result is 520.2.

Step two: synthesis of 1- (4-phenoxyphenyl) -3- (piperidin-2-yl) -8-amino-imidazo [1,5-a ] pyrazine (C096)

In a 50mL glass bottle, C092(220mg) was added. A solution of hydrobromic acid in acetic acid (33%, 8mL) was added via syringe. After stirring at room temperature for 1 hour, 20mL of water was added to quench. This solution was purified by HPLC to give the trifluoroacetate salt of the target compound C096, which was lyophilized to give 148mg of solid. HPLC purification conditions: initial acetonitrile proportion 15%, retention time t_1/216 minutes.¹H NMR (MeOD-d4,400mhz):7.92(d,1H, J ═ 5.94Hz),7.70(d,2H, J ═ 8.56Hz),7.43(t,2H, J ═ 7.89Hz),7.25 to 7.06(m,6H),5.16 to 4.90(m,1H),3.65 to 3.54(m,1H),3.40 to 3.21(m,1H),2.37 to 2.26(m,1H),2.14 to 1.97(m,3H),1.94 to 1.78(m, 2H). Theoretical calculation of ESI-MS C₂₃H₂₄N₅O[M+H]⁺386.2; the test result is 386.2.

Step three: synthesis of the Compound (S) -2, 3-butenoyl-2- (1- (4-phenoxyphenyl) -8-aminoimidazo [1,5-a ] pyrazin-3-yl) -1-piperidine (10)

In a 50mL glass vial, 3-butynoic acid (17mg,0.2mmol) and dry dichloromethane (3mL) were added followed by 2-chloro-1-methylpyridine iodide (30mg,0.13mmol) and the solution was stirred at room temperature for 1 hour. A solution of C096 trifluoroacetate salt (50mg,0.1mmol) and diisopropylethylamine (0.2mL) in dichloromethane (3mL) was added to the above reaction flask and stirred at room temperature for 40 minutes. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 10, which was lyophilized to give 18.3mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 30%, retention time t_1/219.5 min.¹H NMR (MeOD-d4,400mhz) 7.85 to 7.75(m,1H),7.73 to 7.65(m,2H),7.45 to 7.38(m,2H),7.24 to 7.14(m,3H),7.14 to 7.07(m,2H),7.01 to 6.94(m,1H),6.26(t,1H, J ═ 6.62Hz),6.29 to 6.14(m,1H),5.25(d,2H, J ═ 6.50Hz),4.10 to 3.83(m,1H),2.50 to 2.27(m,2H),2.10 to 1.93(m,1H),1.90 to 1.73(m, 3H). Theoretical calculation of ESI-MS C₂₇H₂₆N₅O₂[M+H]⁺452.2; the experiment found that 452.1.

Example 11: synthesis of the Compound (R) -2, 3-butynenoyl 3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidine

Step one, synthesizing (R) -3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidine-1-yl) pyrrolidine carboxylic acid tert-butyl ester (CDA-148-1):

intermediate C (100mg,0.33mmol) was dissolved in dry tetrahydrofuran and triphenylphosphine (346mg,1.32mmol) and (S) -1-tert-butoxycarbonyl-3-hydroxypyrrolidine (125mg,0.66mmol) were added. Diisopropyl azodicarboxylate (0.40mL,1.98mmol) was added dropwise under nitrogen. After the addition was complete, the mixture was stirred at 40 ℃ for 12 hours. After the reaction, tetrahydrofuran was removed by rotation, and the mixture containing CDA-148-1 (400 mg) was obtained by column purification.

Step two, synthesizing (R) -3- (4-phenoxyphenyl) -1- (pyrrolidine-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine (CDA-148):

the mixture containing CDA-148-1 obtained in the previous step was dissolved in 4mL of dichloromethane, 4mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 12 hours. After the solvent was removed by evaporation, water and methanol were added, and HPLC purification gave the trifluoroacetate salt of CDA-148, which was lyophilized to give 120mg of solid. The yield of the two steps is 74 percent. HPLC purification conditions: the initial acetonitrile proportion was 15%, and the retention time t was 15 minutes.¹HNMR(MeOD-d₄400MHz) 8.45(s,1H),7.73(d,2H, J-8.50 Hz),7.39(t,2H, J-7.92 Hz),7.20-7.05(m,5H),5.89-5.81(m,1H),3.96-3.85(m,2H),3.83-3.74(m,1H),3.67-3.57(m,1H),2.73-2.61(m,1H),2.60-2.50(m, 1H). Theoretical calculation of ESI-MS C₂₁H₂₀N₆O[M+H]⁺373.2; the experiment shows that: 373.1.

step three: synthesis of the Compound (R) -2, 3-butynoyl3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidine (11)

In a 50mL glass vial, 3-butynoic acid (11mg,0.14mmol) and dry dichloromethane (3mL) were added followed by 2-chloro-1-methylpyridine iodide (31.5mg,0.14mmol) and the solution was stirred at room temperature for 1 hour. A solution of CDA-148 in trifluoroacetate (60mg,0.12mmol) and diisopropylethylamine (0.2mL) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 30 min. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of the target compound 11, which was lyophilized to give 23.8mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 20%, retention time t_1/222.5 minutes.¹H NMR (MeOD-d4,400MHz):8.45-8.40(m,1H),7.71-7.64m,2H),7.44-7.37(m,2H),7.22-7.12(m,3H),7.12-7.05(m,2H),6.10(t,0.5H, J ═ 6.53Hz),6.02(t,0.5H, J ═ 6.53Hz),5.75-5.62(m,1H),5.30-5.22(m,2H),4.22-3.82(m,3.5H),3.81-3.66(m,0.5H),2.69-2.47(m, 2H). Theoretical calculation of ESI-MS C₂₅H₂₃N₆O₂[M+H]⁺439.2; the test result is 439.1.

Example 12: synthesis of the Compound 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) azetidin-1-yl) -2, 3-buty-len-1-one

Step one, synthesis of tert-butyl 3- (3- (4-phenoxyphenyl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) azetidinecarboxylate (CDB 004):

intermediate C (100mg,0.33mmol) and cesium carbonate (215mg,0.66mmol) were dissolved in dry DMF. Tert-butyl 3- (methylsulfonyloxy) azetidinecarboxylate (166mg,0.66mmol) was dissolved in 2mL dry DMF under nitrogen, added dropwise to the system and heated to 75 deg.C with stirring for 24 h. After the reaction, water was added, and extraction was carried out three times with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column to obtain 45mg of the objective compound CDB 004.

Step two, synthesis of 3- (4-phenoxyphenyl) -1- (azetidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidine-4-amine (CDB 007):

CDB004(45mg,0.10mmol) was dissolved in 2mL of dichloromethane, 2mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 12 hours. After removal of the solvent by evaporation, water and methanol were added and purified by HPLC to give the trifluoroacetate salt of CDB007 which was lyophilized to give 45mg of solid. The yield of the two steps is 29 percent. HPLC purification conditions: the initial acetonitrile proportion was 20%, the retention time t ═ 11 minutes.¹H NMR(MeOD-d₄400MHz) 8.43(s,1H),7.76(d,2H, J ═ 8.56Hz),7.44-7.38(m,2H),7.22-7.15(m,3H),7.13-7.08(m,2H),6.08-5.99(m,1H),4.80-4.63(m, 4H). Theoretical calculation of ESI-MS C₂₀H₁₈N₆O[M+H]⁺359.2; the experiment shows that: 359.1.

step three, synthesizing 1- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidine-1-yl) azetidin-1-yl) -2, 3-butralin-1-one (12):

in a 25mL glass vial, 3-butynoic acid (10mg,0.12mmol) and dry dichloromethane (3mL) were added followed by 2-chloro-1-methylpyridine iodide (28mg,0.12mmol) and the solution was stirred at room temperature for 1 hour.A solution of the trifluoroacetate salt of CDB007 (45mg,0.09mmol) and diisopropylethylamine (0.2mL) in dichloromethane (3mL) was added to the reaction flask and stirred at room temperature for 60 minutes. The dichloromethane was evaporated using a rotary evaporator and the resulting dope was purified by HPLC to give the trifluoroacetate salt of target compound 12, and lyophilized to give 15mg of a solid. HPLC purification conditions: initial acetonitrile proportion of 20%, retention time t_1/222.5 minutes.¹H NMR (MeOD-d4,400mhz):8.41(s,1H),7.73(d,2H, J ═ 8.72Hz),7.45-7.38(m,2H),7.23-7.15(m,3H),7.14-7.09(m,2H),5.93-5.85(m,1H),5.81(t,1H, J ═ 6.68Hz),5.32(d,2H, J ═ 6.72Hz),4.83-4.74(m,2H),4.64-4.53(m, 2H). Theoretical calculation of ESI-MS C₂₄H₂₀N₆O₂[M+H]⁺425.3; the test result is 425.3.

The structures of the above compounds are summarized as follows:

by the above-described synthesis method, the following compounds can also be synthesized:

experimental example 1: method for testing cell activity of compound

A20 mM compound stock solution was prepared by dissolving a sample to be tested in 100% dimethyl sulfoxide. Compounds were diluted with 100% dimethylsulfoxide to the highest concentration required for the experiment (2.5mM or 62.5 μ M).

Add 5. mu.L of compound solution to 96-well flat bottom clear cell culture plates and dilute 2-fold sequentially with cell culture medium (containing 10% fetal calf serum) to a gradient of desired compound concentration (44.6. mu.M-0.0218. mu.M or 1.116-0.544 nM). Finally, 70 μ L of the cell solution to be tested was added to each well at a cell density of 3000-. In the experiment, two control groups were set, in addition to the compound tested: 1) cells and medium were added, but no compound control; 2) the medium containing 10% fetal bovine serum was added, but the cell-free and compound-free group. After culturing the 96-well plate in a 37 ℃ cell culture chamber containing 5% carbon dioxide for 4 days, 14. mu.L of CCK-8 reagent was added to each well, followed by incubation at 37 ℃ for 2 to 4 hours. The 96-well plate was read with a TECAN microplate reader, and the absorbance at a wavelength of 450nm was taken.

The effect of different concentrations of compound on cell activity was calculated using the following formula: [ experimental group absorbance-absorbance of medium containing 10% fetal bovine serum (cell-free and compound-free group) ]/[ absorbance of cell-free and compound-free group-absorbance of medium containing 10% fetal bovine serum (cell-free and compound-free group) ] × 100%.

The data were processed using GraphPad Prism 6 software, and the IC was obtained₅₀The values are the compound concentrations corresponding to a 50% inhibition of cell growth.

For example: based on the above test method for cell growth inhibitory activity of compounds, the cell growth inhibitory activity measured in the Raji cell line of B-cell lymphoma is shown in fig. 1. Wherein, IC of Compound 4₅₀IC value of 0.36 + -0.05 μ M and Ibrutinib (Ibrutinib)₅₀The value is 3.42 +/-1.61 mu M, and the activity of the compound 4 is superior to that of ibrutinib.

For example, based on the above test methods for cell growth inhibitory activity of compounds 2-4 and Ibrutinib (Ibrutinib) in B-cell lymphoma Raji cell line, B-cell lymphoma NAMALWA cell line, intestinal cancer cell lines LOVO, HCT-116, liver cancer cell line HepG2, breast cancer cell line MDA-MB-231, cervical cancer cell lineCell growth inhibitory Activity IC in Hela cell line, gastric cancer cell line NCI-N87 and Lung cancer cell lines NCI-H1975 and NCI-H1650₅₀The values are given in the table below. On the whole, the growth inhibition activity of the compounds 2-4 on the tumor cell strains is better than that of ibrutinib.

As can be seen from the data in the table, the growth inhibitory activity of the compounds 2 and 3 on B cells is significantly higher than that of the marketed drug Ibrutinib (Ibrutinib), and the average value of IC50 of Ibrutinib is 85 times of the IC50 value of the compound 2, which indicates that the cell growth inhibitory activity of the compound 2 in the B cell lymphoma Raji cell strain has significant superiority.

Compared with ibrutinib, the cell growth inhibitory activity (IC50) of the compounds 2 and 3 in B-cell lymphoma cell strains NAMALWA, liver cancer cell strains HepG2, breast cancer cell strains MDA-MB-231, intestinal cancer cell strains HCT-116, cervical cancer cell strains Hela, stomach cancer cell strains NCI-N87, lung cancer cell strains NCI-H1975 and NCI-H1650 is improved by more than 20-100, which shows that the cell growth inhibitory activity of the compounds 2 and 3 is obviously improved in other cancer cell strains as well as B-cell lymphoma cell strains.

Based on the cell activity test method of the compounds, IC of the compounds 1-10 and ibrutinib in Raji cell line of B cell lymphoma, NAMALWA cell line of B cell lymphoma and HCT-116 cell line of intestinal cancer is tested₅₀The values are given in the table below.

Experimental example 2: method for testing kinase activity of compound BTK

The test method is based on the principle of solid-phase bead adsorption induced fluorescence deviation and is used for testing the kinase activity.

First, KR buffer solution (10mM Tris-HCl,10mM MgCl) was used₂,0.01％Tween-20,0.05％NaN₃,1mM DTT,2mM MnCl₂pH 7.2) to a concentration of 0.4U/mL. Secondly, benefit fromTest compound concentrations were 2mM to 63.2nM diluted with DMSO gradient. The DMSO solution of the diluted test compound described above was then diluted 50-fold with KR buffer solution on a 384-well plate. The gradient of test compound formulated as described above ensures a final concentration gradient of test compound in the 384 well plates of 10. mu.M to 0.316nM per well.

In a 384 well plate, 5. mu.L of the above test compound concentration gradient solution and 5. mu.L of 0.4U/mL BTK kinase solution were added to each well. The test compound and BTK kinase were mixed well for 60 minutes at room temperature, and then 5 μ L of fluorescently labeled polypeptide substrate (200nM) in KR buffer was added to each well, so that the final concentration of polypeptide substrate per well was 50 nM. Finally, 5. mu.L of 20. mu.M ATP in KR buffer was added to each well to give a final ATP concentration of 5. mu.M per well. After the phosphorylation reaction was carried out at room temperature for 2 hours, the reaction was quenched. After incubation at room temperature for 60 minutes, the fluorescence polarization values were read using a microplate reader. Since the phosphorylated substrate is adsorbed and immobilized by the solid phase bead, the difference (mP) in the fluorescence polarization value of 535nM can be read both horizontally and vertically. Based on the value of mP for the reference well without ATP added, Δ mP per well values can be calculated. The IC of the test compound can be determined by processing the data with GraphPad Prism 6 software and plotting Δ mP against the concentration gradient₅₀The value is obtained.

Based on the above method, the BTK kinase inhibitory IC of the synthesized compound was determined₅₀The values are as follows:

Claims (18)

1. a compound represented by the general formula (I):

in the general formula (I):

w is selected from the following groups:

n is 0, 1 or 2;

m is 0, 1,2, 3 or 4;

n + m is 2,3 or 4;

l represents-O-, -CO-NH-, -NH-CO-, -NR₆-, -S-or-CH₂-；

R₁Represents a C6-C12 aryl or a 5-10 membered monocyclic heteroaryl group containing 1-4 heteroatoms selected from N, O, S; the aryl and heteroaryl groups are optionally substituted with 1-2 substituents selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen-substituted C1-C4 alkyl, halogen-substituted C1-C4 alkoxy, and amino;

R₂、R₃and R₄Each independently selected from hydrogen and C1-C4 alkyl, said C1-C4 alkyl being optionally substituted with 1-3 substituents selected from halogen, hydroxy, amino, C1-C4 alkoxy, -NH- (C1-C4 alkyl), -N (C1-C4 alkyl) (C1-C4 alkyl);

B₁、B₂、B₃and B₄Each independently represents N or-C (R)₅)；

R₅Each independently selected from hydrogen, halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, and amino;

R₆each independently selected from hydrogen or C1-C4 alkyl.

2. The compound or salt according to claim 1, wherein,

w is selected from

3. A compound or a salt thereof as claimed in claim 1 wherein L represents-O-or-NH-CO-.

4. The compound according to claim 1, wherein the aryl group is a phenyl group and the heteroaryl group is a pyridyl group, or a salt thereof.

5. The compound or salt according to claim 1, wherein R is₂、R₃And R₄Are both hydrogen.

6. The compound or salt according to claim 1, wherein B is₁、B₂、B₃And B₄At most two of which are N.

7. The compound or salt according to claim 6, wherein B is₁、B₂、B₃And B₄At most one of which is N.

8. The compound or salt according to claim 1, wherein R is₆Each independently selected from hydrogen, methyl and ethyl.

9. The compound according to any one of claims 1 to 8, wherein the compound of the general formula (I) is a compound of the general formula (I-1):

wherein R is₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m, X and W are as defined in the corresponding claims.

10. The compound according to any one of claims 1 to 8, wherein the compound of the general formula (I) is a compound of the general formula (I-2):

wherein R is₁、R₂、R₃、R₄、B₁、B₂、B₃、B₄N, m, X and W are as defined in the corresponding claims.

11. The compound according to any one of claims 1 to 8, wherein the compound of the general formula (I) is a compound selected from the following general formulae (I-3) to (I-7):

wherein, L and R₁Are as defined in the corresponding claims;

B₂represents N or-C (R)₅)；

R₅Each independently selected from hydrogen, halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, halogen substituted C1-C4 alkyl, halogen substituted C1-C4 alkoxy, and amino.

12. A compound according to claim 1, or a salt thereof, wherein the compound of formula (I) is selected from the following compounds:

13. a pharmaceutical composition comprising a compound of any one of claims 1-12 or a salt thereof.

14. The pharmaceutical composition of claim 13, wherein said pharmaceutical composition is a BTK inhibitor.

15. The pharmaceutical composition of claim 13, wherein said pharmaceutical composition is a B cell activation inhibitor.

16. Use of a compound of any one of claims 1 to 12 or a salt thereof for the manufacture of a medicament for the prevention and/or treatment of a disease associated with abnormal B cell activity and/or BTK.

17. The use of claim 16, wherein the disease associated with abnormal B cell activity and/or BTK is an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, or a cancer.

18. The use of claim 17, wherein the cancer is one or more selected from non-hodgkin's lymphoma, breast cancer, liver cancer, intestinal cancer, stomach cancer, pancreatic cancer, lung cancer and cervical cancer.

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