CN108602834B - Bruton's tyrosine kinase inhibitor - Google Patents

Bruton's tyrosine kinase inhibitor Download PDF

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CN108602834B
CN108602834B CN201780009992.6A CN201780009992A CN108602834B CN 108602834 B CN108602834 B CN 108602834B CN 201780009992 A CN201780009992 A CN 201780009992A CN 108602834 B CN108602834 B CN 108602834B
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thiophen
pyrimidin
pyrazolo
amino
prop
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CN108602834A (en
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孟坤
张建存
王永钢
唐勇
林庆聪
王骏
王宗惠
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Beijing Shengnuoji Pharmaceutical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Abstract

The invention relates to a Bruton's tyrosine kinase inhibitor, which has the structure shown in the following formula (I) and has good selective inhibition effect,

Description

Bruton's tyrosine kinase inhibitor
Technical Field
The invention relates to a Bruton's tyrosine kinase inhibitor, its cis-trans isomer, mixture of cis-trans isomers, optical enantiomer, mixture of enantiomer, raceme or N-oxide, belonging to the field of medicine.
Background
Bruton's tyrosine kinase (hereinafter abbreviated as Btk) is a member belonging to the Tec family of non-receptor tyrosine kinases. It is expressed in all hematopoietic cell types, such as B cells, mast cells and macrophages, except T lymphocytes, natural killer cells and plasma cells, and is an important mediator of at least three key B cell survival mechanisms. Btk is also responsible for a part of the B cell signaling pathway via a B-cell antigen receptor (BCR). This multiplicity of Btk effects can allow it to direct B cell malignancies into lymphoid tissues, allowing tumor cells to contact the necessary microenvironment for survival. Experimental data indicate that Btk also has a role in signaling pathways in monocytes, macrophages, neutrophils, and mast cells. Btk inhibitors can also inhibit Fc-mediated cytokine release from monocytes and macrophages, and can also inhibit FcR-mediated cell degranulation. The lack of Btk has been shown to block B cell antigen receptor signaling, and therefore compounds with Btk inhibitory activity may be useful as agents to block B cell and/or mast cell mediated related diseases, such as: effective treatment methods for cancer, autoimmune diseases, thromboembolic diseases, inflammatory diseases, etc. 'International Reviews of Immunology'. 2012, 31, 119-; study and treatment of Arthritis (Arthritis Research & Therapy), 2011, 13, R115; in clinical immunization (clin. exp. immunol.) 1993,94, 459; chem, medicinal chemistry (MedChem) 2007,2, 58-61. International publication No. WO2008121742 discloses Btk inhibitors having the following formula (i):
Figure GDA0001754238180000021
when L in the structure of the formula (i) is selected from O, Ar is selected from benzene, Y is selected from piperidine, Z is selected from carbonyl, R is selected fromi、RiiAnd RⅲWhen all are selected from H, the existing compound Ibrutinib (Ibrutinib; trade name: Imbruvica) is obtained, and the compound has the structure shown in the following formula (ii):
Figure GDA0001754238180000022
ibrutinib was 11/13/2013, an FDA approved treatment for the treatment of mantle cell lymphoma in the united states. In addition, ibrutinib has shown great potential in the treatment of chronic lymphocytic leukemia and multiple myeloma.
Another Btk inhibitor is disclosed in publication No. CN102918040, having a structure represented by the following formula (iii):
Figure GDA0001754238180000031
the compound having such a structure is excellent in metabolic stability and can avoid hepatotoxicity or the like, in addition to having a Btk selective inhibitory activity, and therefore, is useful as a therapeutic agent for a disease associated with B cells and/or mast cells such as non-hodgkin's lymphoma, which is excellent in safety.
Btk inhibitors having the following formula (iv) are disclosed in patent application publication No. WO 2015048689:
Figure GDA0001754238180000032
the use of the compounds for the treatment of autoimmune, autoimmune and cancer diseases is also disclosed.
With the continuous and intensive research, the structure of the Btk inhibitor and the indication range thereof are further changed.
Disclosure of Invention
The present invention aims to provide a bruton's tyrosine kinase inhibitor having a selective inhibitory effect on non-hodgkin's lymphoma, preferably B-cell non-hodgkin's lymphoma, such as: diffuse large B cell lymphoma, human B cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, Waldenstrom's Macroglobulinemia (WM) and B cell chronic lymphocytic leukemia have good inhibitory effect.
In one aspect, the present invention provides a compound having the general formula (I), its cis-trans isomers, a mixture of cis-trans isomers, optical enantiomers, a mixture of enantiomers, racemate or N-oxide:
Figure GDA0001754238180000041
wherein: w is a 4-6 membered nitrogen-containing saturated heterocyclic group, phenylmethylene (C)3-C6) Cycloalkyl or [3.3-5 ]]A nitrogen-containing saturated heterocyclic group;
R1or R2Each independently selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000042
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl; the substituents on the substituted phenyl or substituted azaphenyl-containing groups can each be independently selected from halogen, (C)1-C4) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One or more of haloalkoxy;
n is an integer of 0 to 4;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomers, mixture of cis-trans isomers, optical enantiomers, mixture of enantiomers, racemate or N-oxide is represented by the formula (II):
Figure GDA0001754238180000051
wherein W is a 4-6 membered nitrogen-containing saturated heterocyclic group, a benzylidene (C)3-C6) Cycloalkyl or [3.3-5 ]]A nitrogen-containing saturated heterocyclic group;
R1or R2Each independently selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkane (I) and its preparation methodOxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000052
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl;
n is an integer of 0 to 4;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomers, a mixture of cis-trans isomers, optical enantiomers, a mixture of enantiomers, racemates or N-oxide thereof is represented by formula (III):
Figure GDA0001754238180000061
wherein R is1Or R2Each independently selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl radical, containingAzophenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000062
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl;
n is an integer of 0 to 4;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomers, mixture of cis-trans isomers, optical enantiomers, mixture of enantiomers, racemate or N-oxide is represented by the formula (IV):
Figure GDA0001754238180000063
wherein, R is2Selected from phenyl, substituted phenyl, benzene (C)1-C4) Alkyl, substituted benzene (C)1-C4) Alkyl, substituted benzene (C)1-C4) Alkoxy, benzylidene (C)3-C6) Cycloalkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000071
one or more of the above; wherein R' is selected from one of phenyl, substituted phenyl, nitrogen-containing hetero phenyl and substituted nitrogen-containing hetero phenylOne or more of the above-mentioned raw materials;
n is an integer of 0 to 4;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomer, mixture of cis-trans isomers, optical enantiomer, mixture of enantiomers, racemate or N-oxide is represented by the formula (V):
Figure GDA0001754238180000072
wherein X, Y is each independently selected from CH or N, R4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, the compound, its cis-trans isomer, mixture of cis-trans isomers, optical enantiomer, mixture of enantiomers, racemate or N-oxide is shown as the formula (VI):
Figure GDA0001754238180000081
wherein R is4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, the compound, its cis-trans isomers, mixture of cis-trans isomers, optical enantiomers, mixture of enantiomers, racemates or N-oxide thereof is represented by formula (VII):
Figure GDA0001754238180000082
wherein R is4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxide thereof is represented by formula (viii):
Figure GDA0001754238180000091
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (IX):
Figure GDA0001754238180000092
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (X):
Figure GDA0001754238180000101
wherein X, Y and Z are each independently selected from CH or N; r5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of halogenated alkoxy.
Preferably, the compound, its cis-trans isomers, a mixture of cis-trans isomers, optical enantiomers, a mixture of enantiomers, racemates or N-oxides is represented by formula (XI):
Figure GDA0001754238180000102
wherein R is1Selected from phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, substituted benzene (C)1-C4) Alkyl, substituted benzene (C)1-C4) Alkoxy, benzylidene (C)3-C6) Cycloalkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000103
one or more of the above; wherein R' is selected from the group consisting of phenyl, substituted phenyl, azaphenyl and substituted azaphenylOne or more of phenyl;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides is represented by formula (XII):
Figure GDA0001754238180000111
wherein X, Y is each independently selected from CH or N, R4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xiii):
Figure GDA0001754238180000121
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xiv):
Figure GDA0001754238180000122
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xv):
Figure GDA0001754238180000131
wherein X, Y and Z are each independently selected from CH or N; r5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xvi):
Figure GDA0001754238180000132
wherein R is4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xvii):
Figure GDA0001754238180000141
wherein R is4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xviii):
Figure GDA0001754238180000142
wherein R is1Or R2Each independently selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, substituted benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000151
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl;
n is an integer of 0 to 4;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xix):
Figure GDA0001754238180000152
wherein R is2Selected from phenyl, substituted phenyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000153
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl;
R1selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl or (C)1-C4) A haloalkoxy group;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocyclic substituted propenyl, wherein the substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xx):
Figure GDA0001754238180000161
wherein R is2Selected from phenyl, substituted phenyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000162
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl;
R3is selected from (C)2-C4) Alkenyl, (C)2-C4) Alkynyl, aminopropyl, N-disubstituted aminopropyl and (C)4-C7) One or more of nitrogen-containing saturated heterocycle substituted propenyl, whereinThe substituent on the substituted amine comprises (C)1-C4) One or more of alkyl and hydroxyl.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xxi):
Figure GDA0001754238180000171
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, the compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, are as shown in formula (XII)
Figure GDA0001754238180000172
Wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xxiii):
Figure GDA0001754238180000181
wherein R is5、R6Each of which isIndependently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of haloalkoxy.
Preferably, said compound, its cis-trans isomers, mixtures of cis-trans isomers, optical enantiomers, mixtures of enantiomers, racemates or N-oxides, is represented by formula (xiv):
Figure GDA0001754238180000182
wherein X, Y and Z are each independently selected from CH or N; r5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of halogenated alkoxy.
Preferably, the compound, its cis-trans isomer, mixture of cis-trans isomers, optical enantiomer, mixture of enantiomers, racemate or N-oxide, wherein the compound is selected from one or more of the following compounds:
1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (5-phenylthiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (5-methyl-4- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((2-methyl) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((m-methyl) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((p-methyl) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((2-methoxy) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((3-methoxy) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (4- ((4-methoxy) phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- ((2-cyano) phenoxymethyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- ((4-cyano) phenoxymethyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- ((3-cyano) phenoxymethyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- (2-methoxybenzene) oxymethylthiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- (3-methoxybenzene) oxymethylthiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5-thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((pyrimidin-4-yloxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] -pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (5- ((2-methoxy-4-methylphenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (5- ((2-methoxy-4-chlorophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((2-methoxy-4-cyanophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((2-chloro-5-methoxyphenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((3-chloro-5-methoxyphenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((3-methoxy-4-cyanophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (2-methylphenoxymethyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-tolyloxymethyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-methylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3, 4-dimethylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-methoxyphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-methoxyphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4-phenylthiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5-phenylthiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-trifluoromethylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-trifluorotoluene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3, 5-xylene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(S) -1- (3- (4-amino-3- (5-phenylthiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (2-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (3-methylphenyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (4-methoxyphenyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-methoxyphenyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((2-fluorophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((3-fluorophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- ((4-fluorophenoxy) methyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(S) -1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (phenethyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -piperidin-1-yl) prop-2-en-1-one;
(R) trans-1- (3- (4-amino-3- (2-styryl-thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
1- (3- (4-amino-3- (2- (3-methylphenoxymethyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (anilinofoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (2-pyridylaminoformyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (3-pyridylaminoformyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (4-pyridylaminoformyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (2- (anilinofoyl) thiophen-4-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (2-pyridylaminoformyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (3-pyridylaminoformyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (4-pyridylaminoformyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (anilinofoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (pyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (pyridin-3-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (pyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (4- (pyridin-3-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(S) -1- (3- (4-amino-3- (5- (pyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (6-cyanopyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) -picolinamide;
(R) -6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) -3-pyridinecarboxamide;
(R) -N- (6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) pyridin-2-yl) acetamide;
(R) -N- (6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) pyridin-3-yl) acetamide;
(R) -1- (3- (4-amino-3- (5- (6-methylpyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (5-methylpyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one.
The invention also provides a compound of formula (XXV) or a salt thereof,
Figure GDA0001754238180000241
wherein R is1Or R2Selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000242
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl; r7Is selected from H or
Figure GDA0001754238180000243
Wherein R is3One selected from trifluoromethyl, tert-butoxy and benzyloxy; when R is7In the case of H, the salt of the compound of formula (XXV) may be one of the hydrochloride, sulfate, acetate and trifluoroacetate salts. The compounds of formula (XXV) or salts thereof are intermediates for the compounds of formula (I) -formula (XXIV) according to the invention.
Preferably, wherein the compound is as follows:
Figure GDA0001754238180000251
wherein R is1Or R2Each independently selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000252
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl; r7Is selected from H or
Figure GDA0001754238180000253
Wherein R is3One selected from trifluoromethyl, tert-butoxy and benzyloxy; when R is7When H, the salt of the compound of formula (XXVI) may be one of hydrochloride, sulfate, acetate and trifluoroacetateAnd (4) seed preparation.
Preferably, wherein the compound is as follows:
Figure GDA0001754238180000254
wherein R is1Selected from H, (C)1-C4) Alkyl, (C)1-C4) Alkoxy group, (C)1-C4) Haloalkyl, (C)1-C4) Haloalkoxy, phenyl, substituted phenyl, benzene (C)2-C4) Alkynyl, benzene (C)1-C4) Alkyl, benzylidene (C)3-C6) Cycloalkyl, substituted benzene (C)1-C4) Alkyl, phenoxyalkyl, substituted phenoxyalkyl, benzene (C)1-C4) Alkoxy, substituted benzene (C)1-C4) Alkoxy, benzene (C)2-C4) Alkenyl, substituted benzene (C)2-C4) Alkenyl, azaphenyl, substituted azaphenyl, azaphenyl substituted (C)1-C4) Alkyl, nitrogen-containing hetero phenyl substituted (C)1-C4) Alkoxy and
Figure GDA0001754238180000261
one or more of the above; wherein R' is selected from one or more of phenyl, substituted phenyl, nitrogen-containing hetero-phenyl and substituted nitrogen-containing hetero-phenyl; r7Is selected from H or
Figure GDA0001754238180000262
Wherein R is3One selected from trifluoromethyl, tert-butoxy and benzyloxy; when R is7In the case of H, the salt of the compound of formula (XXVII) may be one of the hydrochloride, sulfate, acetate and trifluoroacetate salts.
The present invention also provides a compound of the formula (xxviii) wherein the compound is as follows:
Figure GDA0001754238180000263
wherein R is8Selected from H, Br or a boronic acid group, R9Selected from H, methyl, cyano, carbamoyl or acetamido. The compounds of formula (XXVIII) are intermediates for the compounds of formula (I) -formula (XXIV).
Preferably, the compounds described therein are of the formula (XXIX):
Figure GDA0001754238180000264
wherein R is8Selected from H, Br or a boronic acid group, R9Selected from H, methyl, cyano, carbamoyl or acetamido.
Preferably, wherein said compound is of formula (XXX):
Figure GDA0001754238180000265
wherein R is8Selected from H, Br or a boronic acid group, R9Selected from H, methyl, cyano, carbamoyl or acetamido.
Preferably, the compounds described therein are of formula (XXXI):
Figure GDA0001754238180000271
wherein R is8Selected from H, Br or a boronic acid group, R9Selected from H, methyl, cyano, carbamoyl or acetamido.
Preferably, the compounds described therein are of the formula (XXXII):
Figure GDA0001754238180000272
wherein R is8Selected from H, Br or a boronic acid group, R9Selected from H, methyl, cyano, and,A carbamoyl group or an acetamide group.
The invention also provides application of the compound, the cis-trans isomer, the mixture of the cis-trans isomers, the optical enantiomer, the mixture of the enantiomers, the racemate or the N-oxide thereof in preparing medicaments for treating malignant tumors, autoimmune diseases and allergic diseases.
Preferably, the malignant tumor comprises one or more of lymphoma, plasmacytoma and leukemia.
Preferably, the lymphoma comprises one or more of non-hodgkin's lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, large cell type B cell lymphoma in blood vessels, burkitt's lymphoma, aids-related lymphoma and marginal zone B cell lymphoma.
Preferably, the non-hodgkin's lymphoma comprises B-cell non-hodgkin's lymphoma.
More preferably, the B-cell non-hodgkin lymphoma comprises one or more of diffuse large B-cell lymphoma and human B-cell lymphoma.
Preferably, the autoimmune disease comprises one or more of arthritis, rheumatism, inflammatory enteritis and lupus erythematosus.
In a further aspect, the present invention provides a bruton's tyrosine kinase inhibitor composition comprising a compound of the present invention, its cis-trans isomers, a mixture of cis-trans isomers, optical enantiomers, a mixture of enantiomers, a racemate or an N-oxide thereof.
Drawings
FIG. 1 shows the improvement of the disease in mice by increasing the duration of action of the compounds of the present invention on rheumatoid arthritis mice.
Detailed Description
The following examples are intended to illustrate the present invention, but are not intended to limit the present invention, and modifications, changes, variations, etc. made within the scope of the present invention are within the scope of the present invention.
Unless otherwise indicated, the term "Bruton's tyrosine kinase inhibitor" as provided herein includes compounds having the structural formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), formula (XIII), formula (XIV), formula (XV), formula (XVI), formula (XVII), formula (XVIII), formula (XIX IX), formula (XIX), formula (XX III), formula (XXXIV), each compound comprising a different stereoisomer having the same structural formula, wherein the stereoisomers also include optical isomers and geometric isomers, the optical isomers are also known as enantiomers, and the geometric isomers are also known as cis-trans isomers.
Optical enantiomers are optical enantiomers with chiral centers that are mirror images of each other.
The mixture of optical enantiomers refers to a mixture obtained by mixing two optical enantiomers which are chiral to each other in different molar ratios.
Racemic modification means that two optical enantiomers chiral to each other are mixed in the same molar ratio, and the resulting mixture is called racemic modification because the optical rotation is offset by intermolecular action.
Cis-trans isomers refer to: the same atoms are respectively positioned in two isomers of the same side and opposite side of the carbon-carbon double bond, the same atoms positioned in the same side of the carbon-carbon double bond are called cis form, and the same atoms positioned in the opposite side of the carbon-carbon double bond are called trans form.
The term "heterocyclic group" herein means, unless otherwise specified, that one or more of the ring-constituting carbon atoms of the cycloalkyl group is replaced with a hetero atom other than carbon to form a heterocyclic group, which includes, without limitation, a nitrogen atom, an oxygen atom, a sulfur atom, and the like. Said "cycloalkyl" includes, but is not limited to, phenyl, cyclohexane, and the like. The "nitrogen-containing heterocyclic group" refers to a heterocyclic group formed by replacing carbon atoms constituting a ring by one or more nitrogen atoms, and when the formed heterocyclic group is a saturated heterocyclic group, the heterocyclic group is referred to as a "nitrogen-containing saturated heterocyclic group"; when the heterocyclic group formed is an unsaturated heterocyclic group, it is referred to as "nitrogen-containing unsaturated heterocyclic group".
The term "benzylidene (C) is used herein unless otherwise indicated3-C6) Cycloalkyl means that one hydrogen atom of the methyl group on benzyl is replaced by (C)3-C6) Cycloalkyl groups are substituted to form the structure of benzylidene cycloalkyl groups wherein the two groups attached to the cycloalkyl group include, but are not limited to, adjacent, alternating and opposing positions.
Unless otherwise indicated, the term "nitrogen-containing saturated heterospirocyclic group" herein refers to two saturated cycloalkyl groups sharing a common carbon atom to form a saturated spirocyclic ring. One carbon atom (an unshared carbon atom) of the saturated spirocyclic ring is replaced by a nitrogen atom, and the saturated spirocyclic ring is bonded to its neighboring groups to form a nitrogen-containing saturated heterospirocyclic group, wherein the number of atoms involved in the designation of the saturated heterospirocyclic ring is determined by the number of carbon atoms or nitrogen atoms forming the backbone of each ring, and does not contain a carbon atom common to both rings.
Unless otherwise indicated, the term "benzene (C) herein1-C4) Alkyl means a hydrogen atom on the phenyl ring is replaced by (C)1-C4) Alkyl substitution, formation of benzene (C)1-C4) Alkyl structures including, without limitation, benzyl, phenethyl, phenylpropyl, phenylisopropyl, phenylbutyl and the like.
Unless otherwise indicated, the term "benzene (C) herein2-C4) Alkynyl means that one hydrogen atom on the phenyl ring is replaced by (C)2-C4) Alkynyl substitution of the resulting benzene (C)2-C4) Alkynyl structures include, without limitation, phenylacetylene, phenylpropyne, phenylbutyyne, and the like. The term "substituted phenyl" as used herein, unless otherwise specified, refers to a phenyl group having a substituent resulting from the substitution of a hydrogen atom on the phenyl group with another atom or group other than a hydrogen atom.
The term "phenoxyalkyl" as used herein, unless otherwise indicated, refers to a group wherein a phenyl group and an alkyl group are bonded through an oxygen atom, and which is bonded through an alkyl group to other external groups, including, but not limited to: phenoxymethyl, phenoxyethyl, phenoxypropyl, and the like.
Unless otherwise indicated, the term "substituted phenoxyalkyl" as used herein refers to a phenoxyalkyl group in which the phenyl group of the phenoxyalkyl group is substituted with an atom or group other than a hydrogen atom to form a phenoxyalkyl group bearing a substituent.
Unless otherwise indicated, "amino-substituted (C) as used herein2-C4) Alkenyl "means (C)2-C4) (C) having an amine group, wherein one or more hydrogen atoms of the alkenyl group are replaced by one or more amine groups2-C4) Alkenyl groups, wherein "hydrogen atom" includes both hydrogen atoms on alkenyl groups and non-alkenyl groups such as: the "amine group" is an organic amine group formed by replacing the hydrogen atom of ammonia by a hydrocarbon group.
Unless otherwise indicated, "benzene (C)2-C4) Alkenyl "means a hydrogen atom on the phenyl ring is replaced by (C)2-C4) To give (C) having a benzene ring2-C4) Alkenyl groups, including without limitation: styryl, phenylpropenyl, phenylisopropenyl, phenylbutenyl, and the like.
Unless otherwise indicated, the term "azaphenyl" herein refers to a phenyl group in which one or more carbon atoms are replaced with nitrogen atoms to form a phenyl group with nitrogen heteroatoms, including, without limitation: pyridyl, m-diazepine, p-diazepine, and the like.
Unless otherwise indicated, the term "plasmacytoma" herein is a group of neoplastic diseases caused by the proliferation of monoclonal plasma cells, including multiple myeloma, primary macroglobulinemia.
In certain embodiments, the subject of the invention may be a mammal, such as a dog, cat, cow, sheep, horse, or a human, preferably a human. The necessary therapeutic amount of the drug of the present invention varies depending on the particular disease and can be readily determined by one of ordinary skill in the art.
In certain embodiments, one or more compounds of the present invention may be used in combination with each other, or optionally in combination with any other active agent, for the preparation of a bruton's tyrosine kinase inhibitor, and if a group of compounds is used, these compounds may be administered to a subject simultaneously, separately or sequentially.
In certain embodiments, the compounds of the present invention may be used in combination with one or more other anti-cancer agents. Anticancer agents that may be used in combination include, but are not limited to, ibrutinib, lestatinib, erlotinib, lapatinib, lenatinib, lapatinib, cediranib, axizumab, alemtuzumab, bevacizumab, panitumumab, trastuzumab, alkylating agents, azadirubes, folic acid antagonists, purine antagonists, pyrimidine antagonists, spindle poisons, topoisomerase inhibitors, apoptosis inducers, angiogenesis inhibitors, podophyllotoxins, nitrosoureas, antimetabolites, protein synthesis inhibitors, kinase inhibitors, antiestrogens, cisplatin, carboplatin, interferons, asparaginase, leuprolide, flutamide, megestrol, mitomycin, bleomycin, doxorubicin, irinotecan, and paclitaxel. In certain embodiments, the anti-cancer agent is an anti-estrogen drug, such as tamoxifen and fulvestrant (ICI182,780).
In certain embodiments, the pharmaceutical compositions of the invention are also useful for treating a disease in an animal. A compound of the invention, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvent or prodrug thereof, may be administered by the ordinary veterinarian in a suitable acceptable formulation. The veterinarian can determine the most appropriate route of administration for a particular animal.
The compounds of the present invention are prepared by the following scheme 1:
Figure GDA0001754238180000311
in the above synthetic route, 5-amino-1H-pyrazole-4-cyano (compound 1 ') as a starting material is reacted with formamidine hydrochloride at 70 to 90 ℃, preferably 80 ℃ to give 1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 2 '), which is reacted with N-iodosuccinimide or iodine chloride in an organic solvent (e.g., dimethylformamide, acetic acid) at 80 ℃ whereby iodination occurs to give 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 3 ').
The reaction in step 3 of scheme 1 is well known, 3-iodo-1H-pyrazolo [3,4-d]Pyrimidin-4-amine (compound 3 ') is reacted with hydroxy compound 4 ' in an organic solvent, such as Tetrahydrofuran (THF), in the presence of triphenylphosphine and diisopropyl azodicarboxylate at 50-70 ℃, whereby a bimolecular nucleophilic substitution (Mitsunobu substitution) reaction occurs to afford compound 5 '. Since the compound 4 ' is a starting material having chiral properties, the compound 4 ' having an appropriate optical activity can be selected as a starting material in the course of synthesis according to the optical activity of the target compound, and the obtained reaction intermediate compound also has the same optical activity as that of the compound 4 '. Compounds 5 'and 6' are reacted in an organic solvent (dimethylformamide, ethylene glycol dimethyl ether, tetrahydrofuran and 1,4-dioxane (1,4-dioxane)) with a metal palladium such as: comprising palladium tetrakis (triphenylphosphine) Pd (PPh)3)4Bis (phenylphosphoryl) dichloropalladium (Pd) (PPh)3)2Cl2And [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride Pd (dppf) Cl2Any one or more of the above-mentioned materials are catalyzed and reacted at 60-80 deg.C, so that the coupling reaction can be implemented to obtain the compound 7'.
The reaction of step 5 in scheme 1 is well known, and compound 7 'is reacted in an organic solvent (dichloromethane, tetrahydrofuran) in the presence of trifluoroacetic acid or hydrochloric acid at 0 ℃ to room temperature, thereby carrying out deprotection reaction to give compound 8'.
The reaction of step 6 in scheme 1 is well known, and compound 8 'is reacted with the corresponding carboxylic acid, or acid chloride, in an organic solvent (dichloromethane or dimethylformamide) at 0 ℃ to room temperature, whereby a condensation reaction occurs to give compound 9'.
Starting material 5-amino-1H-pyrazole-4-cyano CAS number 16617-46-2 was purchased from Shanghai koji chemical Co., Ltd.
Compound 4' is 1-tert-butyloxycarbonyl-3-hydroxypiperidine CAS number 85275-45-2, (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine CAS number 143900-44-1 or (R) -1-tert-butyloxycarbonyl-3-hydroxypiperidine CAS number 143900-43-0, which are commercially available from Shanghai Shaoshan reagent, Inc.
Compound 6' was prepared according to the following procedure: a tetrahydrofuran solution of the corresponding bromo-substituted thiophene compound was added dropwise to a-70 ℃ solution of n-butyllithium (nBuLi) in Tetrahydrofuran (THF) under nitrogen. Reaction at-70 ℃ for 2 hours, followed by addition of methyl borate (B (OMe)3) The reaction was continued at-70 ℃ for 30 minutes, and then the reaction mixture was warmed to room temperature, introduced into 1mol/L hydrochloric acid and stirred for 10 minutes. The organic phase obtained by extraction with ethyl acetate is dried and concentrated under reduced pressure to obtain a crude product of the target boric acid compound, which is not stable and can be directly used for the next reaction without refining.
In the reaction in the present specification, the reaction accompanied by heating may be carried out using a water bath or an oil bath as is common in the art. In the reaction in the present specification, the reaction product can be purified by a usual purification means, for example, a method of distilling a fraction obtained under normal pressure or reduced pressure, high performance liquid chromatography using silica gel, thin layer chromatography, washing, or the like. Purification may be carried out in each reaction or after a plurality of reactions.
Example 1
Preparation of 1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 2')
To a solution of 5-amino-1H-pyrazole-4-cyano (compound 1 ') (20g, 185.2mmol) in ethanol (500mL) was added formamidine hydrochloride (16.4g, 203.7mmol) at room temperature, followed by reaction at 80 ℃ for 10 hours, cooling to room temperature, concentration of the solvent under reduced pressure, and the resulting solid product was washed with water and dried under vacuum to give the desired product (21.3g, 85% yield), i.e., 1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 2').
And (3) detecting by Thin Layer Chromatography (TLC): rf0.5 (dichloromethane: methanol ═ 5:1, open phase)
Mass spectrum detection: MS (ESI) M/z 136(M + 1);
nuclear magnetic resonance: 1HNMR (400MHz, DMSO-d6), 6.96(br s,2H), 7.68(s, 1H), 8.35(s,1H), 13.24(s, 1H).
Example 2: preparation of 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 3')
To 1H-pyrazolo [3,4-d at room temperature]Solution of pyrimidin-4-amine (20g, 148mmol) in acetic acid (500mL) was added iodine chloride ICl (24g, 148mmol), reacted at 80 ℃ for 10 hours, cooled to room temperature, and the solvent was concentrated under reduced pressure to give a solid product, which was in turn Na-substituted2CO3Saturated solution, Na2SO3Washing the saturated solution with water, and vacuum drying to obtain 3-iodine-1H-pyrazole [3,4-d]Pyrimidin-4-amine (compound 3', 30.9g, 80% yield). And (3) thin-layer chromatography detection: TLC: rf0.5 (dichloromethane: methanol ═ 5:1)
Mass spectrum detection: MS (ESI) M/z 262(M + 1);
and (3) nuclear magnetic resonance detection: 1HNMR (400MHz, DMSO-d6), 6.98(br s,2H), 8.35(s,1H),13.20(s, 1H).
Example 3 preparation of (Compound 4')
When ring 2 is piperidine and m ═ 0, compound 4' is: 1-Boc-3-hydroxypiperidine
Compound 5' is: tert-butyl-3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate
To a solution of 3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (compound 3 ') (10g, 38.3 mmol) in tetrahydrofuran (150mL) was added 1-tert-butoxycarbonyl-3-hydroxypiperidine (compound 4'), (9.25g, 46mmol) (optionally, optically active 1-tert-butoxycarbonyl-3-hydroxypiperidine), diisopropyl azodicarboxylate (9.30g, 46mmol), and triphenylphosphine (12.06mmol, 46mmol) in this order at room temperature. Then reacted at 60 ℃ for 5 hours, cooled to room temperature, and the crude product obtained by concentrating the solvent under reduced pressure was separated by liquid chromatography on a silica gel column, the eluting solvent being dichloromethane: methanol (40: 1- >10:1) gave the desired product tert-butyl-3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate (compound 5', 2.8g, yield: 75%).
And (3) thin-layer chromatography detection: TLC: rf0.5 (dichloromethane: methanol 10:1) mass spectrometric detection: MS (ESI) M/z 445(M + 1); and (3) nuclear magnetic resonance detection: 1HNMR (400MHz, CDCl)3)1.67 (s,9H)；1.70-2.40(m，4H)，2.95-4.95(m，5H)，6.95(br s，2H)，8.31 (s，1H) In that respect Example 4
In the product 9' of scheme 1, when R is5、R6Are all H, ring 1 is benzene, L1Is methoxy, wherein the methyl moiety is linked to a thiophene and the oxy group is linked to a benzene, L1Linked to the 5-position of thiophene, the 2-position of thiophene linked to the 3-position of pyrazole ring, m ═ 0, ring 2 is piperidine ring, the 3-position of piperidine ring linked to the 1-position nitrogen of pyrazole ring, the 1-position nitrogen of piperidine ring linked to carbonyl, R is3Is vinyl, which is prepared as follows
Scheme 2:
Figure GDA0001754238180000341
wherein Et3N represents triethylamine and EDCl represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
When R is5、R6Are all H, ring 1 is benzene, L1Is methoxy, wherein the methyl moiety is linked to a thiophene and the oxy group is linked to a benzene, L1Attached to the 5-position of thiophene, boron attached to the 2-position of thiophene, compound 6' is: (5- (phenoxymethyl) thiophen-2-yl) boronic acid.
Compound 6' (5- (phenoxymethyl) thiophen-2-yl) boronic acid to compound 7: 1- (3- (4-amino-3- (5- (phenoxy) benzene Methyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Preparation of pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one
Preparation method of (5- (phenoxymethyl) thiophene-2-yl) boric acid
To a solution of 2-bromothiophene-5-methanol (2g, 10mmol) (CAS 79387-71-6 available from Shanghai Tantake technology, Co., Ltd.) in tetrahydrofuran (50mL) was added phenol (1.46g, 15mmol), diisopropyl azodicarboxylate (3.1g, 15mmol), and triphenylphosphine (4g, 15mmol) in that order at room temperature. Then reacted at room temperature for 10 hours, and the crude product obtained by concentrating the solvent under reduced pressure was separated by liquid chromatography on a silica gel column, the eluting solvent was ethyl acetate: petroleum ether (1: 20- >1:10) gave 1.95g of 2-bromo-5- (phenoxymethyl) thiophene, yield: 70%).
And (3) thin-layer chromatography detection: TLC: rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:15)
Mass spectrum detection: MS (ESI) M/z 269(M + 1);
and (3) nuclear magnetic resonance detection: 1HNMR (400MHz, CDCl)3)5.20(s,2H),6.50-6.75 (m,2H),7.00-7.45(m,5H)。
2-bromo-5- (phenoxymethyl) thiophene (1.90g, 7mmol) was dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-n-butyllithium (5.6mL, 2.5mol/L in n-hexane) at-70 ℃ in tetrahydrofuran (15mL) under a nitrogen atmosphere. After a reaction at-70 ℃ for 2 hours, methyl borate (3.67g, 35mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, 1.25g of crude (5- (phenoxymethyl) thiophen-2-yl) boronic acid was obtained by concentration under reduced pressure, and the product was used in the next reaction without purification because it was easily decomposed.
And (3) thin-layer chromatography detection: TLC: rf0.5 (ethyl acetate: petroleum ether ═ 1:5) mass spectrometric detection: MS (ESI) M/z 235(M + 1);
II, tert-butyl-3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidine-1- Process for preparing 1-formylpiperidine
To tert-butyl-3- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] at room temperature]To a solution of pyrimidin-1-yl) piperidine-1-carboxylate (200mg, 0.45mmol) in ethylene glycol dimethyl ether (5mL) and water (2mL) were added in this order (5- (phenoxymethyl) thiophen-2-yl) boronic acid (211mg, 0.90mmol), tetrakis (triphenylphosphine) palladium (26mg, 0.02mmol), Na2CO3(143mg, 1.35 mmol). Then reacting at 80 deg.C for 16 hr, cooling to room temperature, pouring into 50mL water, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (8: 1 → 2:1) to obtain the target product tert-butyl 3- (4-amino-3- (5- (phenoxymethyl) thiophene-2-yl) -1H-pyrazole [3,4-d]Pyrimidin-1-yl) piperidine-1-Formate (160mg, yield 75%).
Thin-layer chromatography: (TLC) detection: rf0.5 (Petroleum ether: ethyl acetate ═ 2:1 development phase)
Mass spectrum detection: MS (ESI) M/z 507(M + 1);
nuclear magnetic resonance: 1HNMR (400MHz, CDCl)3)、1.68(s,9H)、0(m,1H)、 2.28-2.45(m,2H)、2.90-3.25(m,1H)、3.40-3.80(m,1H)、4.05-4.25(m, 2H)、4.65-4.90(m,2H)、5.31(s,2H)、6.30-6.40(m,1H)、6.60-6.70(m,1H)、 7.05-7.15(m,2H)、7.20-7.25(m,1H)、7.30-7.40(m,4H)、8.41(s,1H)。
Ⅲ.3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Process for preparing pyrimidin-1-yl) piperidines Preparation method
To tert-butyl 3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] at 0 DEG C]After trifluoroacetic acid (2mL) was added to a solution of pyrimidin-1-yl) piperidine-1-carboxylate (160mg, 0.32mmol) in dichloromethane (4mL), the mixture was warmed to room temperature and reacted for 4 hours to give 3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3, 4-d%]The trifluoroacetate of the pyrimidine-1-yl) piperidine can also be added with hydrochloric acid, sulfuric acid or acetic acid to obtain the corresponding hydrochloride, sulfate or acetate. The crude product obtained by concentrating the solvent under reduced pressure was dissolved in ethyl acetate (20mL) over Na2CO3Washing with saturated solution and then with anhydrous Na2SO4Drying, and concentrating the solvent under reduced pressure to obtain 3- (4-amino-3- (5- (phenoxymethyl) thiophene-2-yl) -1H-pyrazolo [3,4-d]Crude pyrimidin-1-yl) piperidine (80 mg) was used directly in the next reaction to give the corresponding hydrochloride, sulfate or acetate salt.
And (3) thin-layer chromatography detection: TLC: rf0.5 (dichloromethane: methanol ═ 5:1)
Mass spectrum detection: MS (ESI) M/z 407(M + 1);
IV, 3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidines to Compound 1: 1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine- Preparation of 1-yl) prop-2-en-1-oneMethod of producing a composite material
To 3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] at 0 DEG C]To a solution of pyrimidin-1-yl) -1-piperidine (80mg, 0.20mmol) in dichloromethane (5mL) was added acrylic acid (23mg, 0.30mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (64mg, 0.30mmol) in that order (CAS number 25952-53-8, purchase: shanghai Bizhao Biotech Co., Ltd.), 4-Dimethylaminopyridine (DMAP) (13mg, 0.10mmol), followed by reaction at room temperature for 2 hours, washing with water and then with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure was purified by thin layer chromatography (ethyl acetate: methanol ═ 40: 1) to give compound 1: 1- (3- (4-amino-3- (5- (phenoxymethyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one (54mg, yield 60%).
TLC：Rf0.5 (ethyl acetate: methanol ═ 30:1) ms (esi) M/z 461(M + 1);1HNMR(400MHz,CDCl3)(m,1H),2.27-2.41(m,2H),2.89-3.24(m, 1H),3.40-3.78(m,1H),4.07-4.28(m,2H),4.65-4.91(m,2H),5.31(s,2H), 5.70-5.77(m,1H),5.83(s,2H),6.31-6.40(m,1H),6.59-6.67(m,1H), 7.04-7.10(m,2H),7.19-7.25(m,1H),7.30-7.32(m,2H),7.33-7.40(m,2H), 8.41(s,1H)。
v. intermediate compound and preparation method thereof
Intermediate compound 6 'was modified in a similar manner to example 4 to give the following table compounds, wherein intermediate compound 6' may be selected from the following compounds:
(4- (Phenoxymethyl) thiophen-2-yl) boronic acid
(4- ((5-methylphenoxy) methyl) thiophen-2-yl) boronic acid
(5- (phenoxymethyl) thiophen-3-yl) boronic acid
(4- ((o-tolyloxy) methyl) thiophen-2-yl) boronic acid
(4- ((m-tolyloxy) methyl) thiophen-2-yl) boronic acid
(4- ((p-tolyloxy) methyl) thiophen-2-yl) boronic acid
(4- ((o-methoxyphenoxy) methyl) thiophen-2-yl) boronic acid
(4- ((m-methoxyphenoxy) methyl) thiophen-2-yl) boronic acid
(4- ((p-methoxyphenoxy) methyl) thiophen-2-yl) boronic acid
(5- ((o-cyanophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((p-cyanophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((m-cyanophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((o-methoxyphenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((m-methoxyphenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((pyrimidin-4-yloxy) methyl) thiophen-3-yl) boronic acid
(5- ((2-methoxy-4-methylphenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((2-methoxy-4-chlorophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((2-methoxy-4-cyanophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((2-chloro-5-methoxyphenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((3-chloro-5-methoxyphenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((3-methoxy-4-cyanophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((o-tolyloxy) methyl) thiophen-3-yl) boronic acid
(5- ((p-tolyloxy) methyl) thiophen-3-yl) boronic acid
(5- ((o-fluorophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((m-fluorophenoxy) methyl) thiophen-3-yl) boronic acid
(5- ((p-fluorophenoxy) methyl) thiophen-3-yl) boronic acid
((5-phenoxymethyl) thiophen-3-yl) boronic acid
The synthesis of the boronic acid compounds described above is similar to the preparation of the compound 6' of example 4, which is (5- (phenoxymethyl) thiophen-2-yl) boronic acid.
Example 5
When R is5、R6Are all made ofH, ring 1 is benzene, L1Is a single bond, the benzene ring is connected with the 5-position of thiophene, 1H-pyrazole [3,4-d]Pyrimidin-1-yl attached to the 3-position of the thiophene, compound 6' is: ((5-phenyl) thiophen-3-yl) boronic acid
5.1Preparation method of intermediate ((5-phenyl) thiophene-3-yl) boric acid：
3-bromo-5- (phenyl) thiophene (CAS number 38071-58-8, available from Shanghai Biao pharmaceutical science, Ltd.) (2g, 8.3mmol) was dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-n-butyllithium (6.7mL, 2.5mol/L in n-hexane) in tetrahydrofuran (15mL) at-70 ℃ under an argon atmosphere. After reaction at-70 ℃ for 2 hours, methyl borate (4.35g, 42mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, 1.30g of crude (5- (phenyl) thiophen-3-yl) boronic acid was obtained by concentration under reduced pressure and used in the next reaction without purification since the product was easily decomposed.
And (3) thin-layer chromatography detection: TLC: rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
Mass spectrum detection: MS (ESI) M/z 205(M + 1);
5.2 preparation of intermediates ((5-phenyl) thiophen-2-yl) boronic acid and ((4-phenyl) thiophen-2-yl) boronic acid
The synthesis of ((5-phenyl) thiophen-2-yl) boronic acid and ((4-phenyl) thiophen-2-yl) boronic acid is the same as the preparation of (5- (phenyl) thiophen-3-yl) boronic acid.
Process for preparing 5, 32-bromo-4- (m-tolyl) thiophene
When R is5、R6Are all H, ring 1 is toluene, L1Is a single bond, the benzene ring is connected with the 5-position of thiophene, 1H-pyrazole [3,4-d]Pyrimidin-1-yl attached to the 3-position of the thiophene, compound 6' is: ((5- (3-methylphenyl)) thiophen-3-yl) boronic acid prepared by the method comprising:
to a solution of 2, 4-dibromothiophene (4g, 16mmol) (purchased from Sea reagent under the trade name 2, 4-dibromothiophene, CAS No. 3140-92-9, trade name: 1993) in toluene (5)0mL) was added to m-tolueneboronic acid (2.25g, 16mmol), (CAS No.: 17933-03-8, trade name 3-toluic acid, available from carbofuran technologies, product number: 256729) Na2CO3(3.5g，33mmol)，Pd(PPh3)4(380mg, 0.3mmol) and H2O (50mL), then replaced with argon for protection, heated to 100 ℃ for 12 hours, and followed by TLC for the end of the reaction. After the reaction is finished, cooling to room temperature, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product is obtained by decompression concentration and is chromatographed by a silica gel column, and the elution solvent is ethyl acetate: petroleum ether (1: 50-)>1:20) 2g (yield 50%) of 2-bromo-4- (m-tolyl) thiophene were obtained as a white solid.
TLC：Rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:30)
MS(ESI)m/z 253(M+1)；1HNMR(400MHz,CDCl3)2.38(s,3H), 7.15-7.60(m,6H)。
5.4 preparation of intermediate (4- (3-tolyl) thiophen-2-yl) boronic acid
2-bromo-4- (3-tolyl) thiophene (2g, 8mmol) (prepared as described in 5.3) was dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-n-butyllithium (6.4mL, 2.5mol/L in n-hexane) at-70 ℃ in tetrahydrofuran (15mL) under a nitrogen atmosphere. After a reaction at-70 ℃ for 2 hours, trimethyl borate (4g, 40mmol), (CAS No.: 121-43-7, available from Bester reagent under the trade name trimethyl borate, trade name: B00269101) was added thereto, and the reaction was continued at-70 ℃ for 30 minutes, and then heated to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product (1.30 g) of (4- (3-tolyl) thiophen-2-yl) boronic acid was obtained by concentration under reduced pressure and used in the next reaction without purification since the product was easily decomposed.
TLC：Rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
MS(ESI)m/z 219(M+1)。
5.5 intermediates ((5- (3-methoxyphenyl)) thiophen-3-yl) boronic acid and ((5- (4-methoxyphenyl)) thiophen- Process for preparing 3-yl) boronic acids
The synthesis methods of the above-mentioned boronic acid compounds are the same as those for the preparation of (4- (3-tolyl) thiophen-2-yl) boronic acid.
5.6 preparation of the intermediate (5- (m-tolyl) thiophen-2-yl) boronic acid
When R is5、R6Are each H, ring 1 is 3-tolyl, L1Is a single bond, the 3-tolyl group is connected with the 5-position of the thiophene, and the 1H-pyrazolo [3,4-d]The pyrimidine-1-group is connected with the 2-position of thiophene, the compound 6' is (5- (m-tolyl) thiophene-2-yl) boric acid, and the preparation method comprises the following steps: to a toluene solution (50mL) of 2, 5-dibromothiophene (4g, 16mmol) (available from Qingdao Tongyuan medicine Co., Ltd., trade name 2, 5-dibromothiophene, CAS No.: 3141-27-3) was added in sequence m-tolylboronic acid (2.25g, 16mmol) (CAS No.: 17933-03-8, available from Shanghai Qu chemical Co., Ltd., trade name m-tolylboronic acid), Na2CO3(3.5g，33mmol)，Pd(PPh3)4(380mg, 0.3mmol) and H2O (50mL), then replaced with argon for protection, heated to 100 ℃ for 12 hours, and followed by TLC for the end of the reaction. After the reaction is finished, cooling to room temperature, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product is obtained by decompression concentration and is chromatographed by a silica gel column, and the elution solvent is ethyl acetate: petroleum ether (1: 50-)>1:20) 3.1g (yield 75%) of 2-bromo-5- (m-tolyl) thiophene were obtained as a white solid.
TLC：Rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:30)
MS(ESI)m/z 253(M+1)；1HNMR(400MHz,CDCl3)2.33(s,3H), 7.10-7.70(m,6H)。
2-bromo-5- (m-tolyl) thiophene (2g, 8mmol) was dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-70 ℃ n-butyllithium (6.4mL, 2.5mol/L in n-hexane) in tetrahydrofuran (15mL) under a nitrogen atmosphere. After reaction at-70 ℃ for 2 hours, methyl borate (4g, 40mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product (1.20 g) of (5- (m-tolyl) thiophen-2-yl) boronic acid was obtained by concentration under reduced pressure and used in the next reaction without purification since the product was easily decomposed.
TLC：Rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
MS(ESI)m/z 219(M+1)。
5.7. Preparation method of intermediate similar to 5.6
The following intermediates were prepared analogously to the intermediate of 5.6:
(5- (3, 4-dimethylphenyl) thiophen-2-yl) boronic acid
(5- (m-methoxyphenyl) thiophen-2-yl) boronic acid
(5- (p-methoxyphenyl) thiophen-2-yl) boronic acid
(5- (4-phenyl) thiophen-2-yl) boronic acid
(5- (p-trifluoromethylphenyl) thiophen-2-yl) boronic acid
(5- (m-trifluoromethylphenyl) thiophen-2-yl) boronic acid
(5- (3, 5-dimethylphenyl) thiophen-2-yl) boronic acid
(5-phenyl) thiophen-2-yl) boronic acid
(5- (o-fluorophenyl) thiophen-2-yl) boronic acid
(5- (m-fluorophenyl) thiophen-2-yl) boronic acid
(5- (p-fluorophenyl) thiophen-2-yl) boronic acid
The synthesis methods of the above boric acid compounds are the same as those for the preparation of (5- (m-tolyl) thiophen-2-yl) boric acid.
5.8 preparation of intermediate (2- (styryl) thiophen-4-yl) boronic acid
When R is5、R6Are all H, ring 1 is styryl, L1Is a single bond, the styryl is connected with the 2 position of the thiophene through the single bond, and the 1H-pyrazole [3,4-d ]]Pyrimidin-1-yl attached to the 4-position of thiophene, m ═ 0, ring 2 is piperidine, compound 6' is (2- (styryl) thiophen-4-yl) boronic acid, prepared by the method of: 4-bromo-2-thiophene-carbaldehyde (4g, 21mmol) (CAS18791-75-8, available from Bester reagent Ltd.) and benzyltriphenylphosphonium chloride (10.9g,25mmol) was dissolved in 50mL of isopropanol and then lithium hydroxide monohydrate (1.32g, 32mmol) was added. Heating to 85 deg.C, reacting for 4 hr, and TLC (thin layer chromatography, R)f0.5 (ethyl acetate: petroleum ether: 1:50)) followed the end of the reaction. After the reaction, 50mL of ethyl acetate was added, the mixture was washed with 50mL of water 2 times, the organic layer was separated, dried, and the crude product obtained was dissolved in 30mL of tetrahydrofuran by spin-drying under reduced pressure, followed by addition of iodine (0.78g, 3mmol) and stirring at room temperature for 10 hours. Adding 50mL of ethyl acetate, washing with saturated sodium bicarbonate (50mL) and sodium sulfite (50mL) in sequence, separating the organic layer, drying, and carrying out liquid chromatography on the obtained crude product by using a silica gel column under reduced pressure, wherein the elution solvent is ethyl acetate: petroleum ether (1: 100-)>1:20) to obtain 4.7g of trans 2- (styryl) thiophene-4-bromine, yield: 85%).
And (3) thin-layer chromatography detection: TLC: rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:15)
Mass spectrum detection: MS (ESI) M/z 265(M + 1);
and (3) nuclear magnetic resonance detection: 1HNMR (400MHz, CDCl)3)6.80-7.00(m,2H),7.08 (s,1H),7.20(s,1H),7.25-7.65(m,5H)。
Trans 2- (styryl) thiophene-4-bromo (2g, 7.5mmol) is dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-n-butyllithium (6mL, 2.5mol/L in n-hexane) at-70 ℃ in tetrahydrofuran (15mL) under a nitrogen atmosphere. After reaction at-70 ℃ for 2 hours, methyl borate (3.85g, 38mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, crude trans (2- (styryl) thiophen-4-yl) boronic acid (1.60 g) is obtained by vacuum concentration, and the product is easy to decompose and is directly used for the next reaction without refining.
TLC：Rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
MS(ESI)m/z 231(M+1)。
Process for the preparation of 5.9(2- (phenylethyl) thiophen-4-yl) boronic acid
When R is5、R6Are all H, ring 1 is phenethyl, L1Is a single bond, phenethylRadical attached to 2-position of thiophene, 1H-pyrazolo [3,4-d]Pyrimidin-1-yl is attached to the 4-position of thiophene, m ═ 0, ring 2 is piperidine, and compound 6' is (2- (phenethyl) thiophen-4-yl) boronic acid.
Trans 2- (styryl) thiophene-4-bromo (2.5g, 9.5mmol) dissolved in ethyl acetate (30mL), with 10% palladium on charcoal (1g) added for 2h at room temperature under hydrogen atmosphere, TLC (thin layer chromatography, R)f0.5 (ethyl acetate: petroleum ether: 1:50)) followed the end of the reaction. The organic phase obtained with palladium carbon filtered off was subjected to liquid chromatography on a silica gel column for the crude product obtained by concentration under reduced pressure, and the elution solvent was ethyl acetate: petroleum ether (1: 100-)>1:20) to obtain 2- (phenethyl) thiophene-4-bromine 2g, yield: 80 percent.
And (3) thin-layer chromatography detection: TLC: rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:15)
Mass spectrum detection: MS (ESI) M/z 267(M + 1);
and (3) nuclear magnetic resonance detection: 1HNMR (400MHz, CDCl)3)2.65-2.90(m,4H),6.54 (s,1H),6.66(s,1H),7.20-7.45(m,5H)。
1.20g of crude product, which is easily decomposed, was used in the next reaction without purification.
Thin Layer Chromatography (TLC): rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
MS(ESI)m/z 267(M+1)。
2- (phenylethyl) thiophene-4-bromo (2g, 7.5mmol) was dissolved in tetrahydrofuran (10mL) and added dropwise to a solution of-70 ℃ n-butyllithium (6mL, 2.5mol/L in n-hexane) in tetrahydrofuran (15mL) under a nitrogen atmosphere. After reaction at-70 ℃ for 2 hours, methyl borate (3.85g, 38mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, 1.40g of crude (2- (phenethyl) thiophen-4-yl) boronic acid was obtained by concentration under reduced pressure and used in the next reaction without purification since the product was easily decomposed.
TLC：Rf0.5 (ethyl acetate: petroleum ether ═ 1:5) ms (esi) M/z 233(M + 1).
The compounds of the present invention are also prepared by the following scheme 3:
Figure GDA0001754238180000441
the reaction of step 1 in scheme 3 is well known, and in the above synthetic scheme, compound 5' as a starting material is prepared by the same method as in scheme 1. Compound 5 'with compound 10' in an organic solvent (dimethylformamide, ethylene glycol dimethyl ether, tetrahydrofuran and 1,4-dioxane) in the presence of a metal palladium, for example: comprising palladium tetrakis (triphenylphosphine) Pd (PPh)3)4Bis (phenylphosphoryl) dichloropalladium (Pd) (PPh)3)2Cl2And [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride Pd (dppf) Cl2Any one or more of the above-mentioned materials are catalyzed and reacted at 60-80 deg.C, so that the coupling reaction can be implemented to obtain the compound 11'.
The reaction of step 2 in scheme 3 is well known, and compound 11 'is reacted in tetrahydrofuran in the presence of tetrabutylammonium fluoride at 0 ℃ to room temperature to remove the tert-butyldimethylsilane protecting group to give compound 12'.
The reaction of step 3 in scheme 3 is well known, compound 12' is added to acetonitrile in sequence with phosphate buffer (pH 6.7), 2,6, 6-tetramethylpiperidine-nitroxide (TEMPO), NaClO2(sodium chlorite) and NaClO (sodium hypochlorite) were reacted at 35 ℃ to conduct a hydroxyl oxidation reaction, thereby obtaining a carboxylic acid compound 13'.
The reaction of step 4 in scheme 3 is well known, compound 13 'is reacted with compound 14' in an organic solvent (dichloromethane or dimethylformamide) in the presence of the condensing agent 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCL) (CAS No. 25952-53-8, available from Jinnan Pano chemical Co., Ltd.), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (CAS No. 148893-10-1, available from Shanghai Zine laboratory) or O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU) (CAS No. 125700-67-6, available from Nanjing peptide industries, Tech., Ltd.) at room temperature, by doing so, a condensation reaction occurs to give compound 15'. Wherein compound 14' may be selected from 2-aminopyridine, 3-aminopyridine, 4-aminopyridine or aniline.
The reaction of step 5 in scheme 3 is well known in the same manner as the operation of step 5 in scheme 1. The reaction of step 6 in scheme 3 is well known in the same manner as in step 6 in scheme 1, wherein the starting aminopyridines are available from Beijing Vida chemical Co., Ltd, with 3-aminopyridine having a commercial number of 462-08-8, 2-aminopyridine having a commercial number of 504-29-0, and 4-aminopyridine having a commercial number of 504-24-5.
Example 6
When compound 10 'is ((5-tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid, compound 11' in which tert-butyldimethylsilyloxymethylene is attached to the 5-position of thiophene, 1H-pyrazolo [3,4-d ] in scheme 3, step 1]Pyrimidin-1-yl attached to the 2-position of thiophene, m ═ 0, ring 2 is piperidine, and compound 11' is tert-butyl-3- (4-amino-3- (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine-1-carboxylate. The preparation method comprises the following steps: to (R) tert-butyl-3- (-4-amino-3-iodo-1H-pyrazolo [3, 4-d) at room temperature]To a solution of pyrimidin-1-yl) piperidine-1-carboxylate (300mg, 0.68mmol) in ethylene glycol dimethyl ether (5mL) and water (2mL) were added in sequence (5- (tert-butyldimethylsilyloxymethylene)) thiophen-2-yl) boronic acid (345mg, 1.35mmol), tetrakistriphenylphosphine (39mg, 0.03mmol), Na2CO3(215mg, 2.1 mmol). Then reacting at 80 deg.C for 16 hr, cooling to room temperature, pouring into 50mL water, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (8: 1 → 2:1) to obtain the target product (R) tert-butyl-3- (4-amino-3- (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine-1-carboxylate (275mg, 78% yield).
Thin-layer chromatography: (TLC) detection: rf0.5 (Petroleum ether: ethyl acetate ═ 3:1 developing phase))
Mass spectrum detection: MS (ESI) M/z 545(M + 1);
nuclear magnetic resonance:1HNMR(400MHz,CDCl3):0.20(s,3H),0.21(s,3H), 1.59(s,9H)、1.69(s,9H)、1.60-2.45(m,4H)、2.90-4.90(m,5H)、5.31(s, 2H)、6.30-6.40(m,1H)、6.60-6.70(m,1H)、7.00-7.15(m,2H)、8.40(s, 1H)。
in step 2, scheme 3, compound 11 ' is tert-butyldimethylsilyloxymethylene attached to the 5-position of thiophene, 1H-pyrazolo [3,4-d ] pyrimidin-1-yl is attached to the 2-position of thiophene, m ═ 0, ring 2 is piperidine, compound 11 ' is (R) tert-butyl-3- (4-amino-3- (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate, compound 12 ' is (R) tert-butyl-3- (4-amino-3- (5- (hydroxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidine-1-yl) piperidine-1-formic ether, and the preparation method comprises the following steps:
to the compound 11', (R) tert-butyl-3- (4-amino-3- (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d at room temperature]Pyrimidin-1-yl) piperidine-1-carboxylate (250mg, 0.46mmol) in tetrahydrofuran (20mL) was added tetrabutylammonium fluoride (180mg, 0.61 mmol). Then reacted at room temperature for 3 hours, a saturated ammonium chloride solution (50mL) was added thereto, extraction was performed with ethyl acetate, and the organic solution was extracted with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (4: 1 → 1:1) to obtain the target product (R) tert-butyl-3- (4-amino-3- (5- (hydroxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine-1-carboxylate (158mg, yield 80%).
Thin-layer chromatography: (TLC) detection: rf0.5 (Petroleum ether: ethyl acetate ═ 1:1 development phase)
Mass spectrum detection: MS (ESI) M/z 431(M + 1);
nuclear magnetic resonance:1HNMR(400MHz,CDCl3):1.68(s,9H)、1.60-2.45(m, 4H)、2.90-4.90(m,5H)、5.34(s,2H)、6.30-6.45(m,1H)、6.62-6.70(m, 1H)、7.00-7.15(m,2H)、8.41(s,1H)。
the hydroxymethylene group of compound 12' in step 3 of scheme 3 is attached to the 5-position of the thiophene, 1H-pyrazolo [3,4-d]Pyrimidin-1-yl attached to the 2-position of thiophene, m ═ 0, ring 2 is piperidine, and compound 12' is (R) tert-butyl-3- (4-amino-3- (5- (hydroxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine-1-carboxylate compound 13' is (R) tert-butyl-3- (3- (5-carboxythien-2-yl) -4-amino-1H-pyrazolo [3,4-d]The preparation method of the pyrimidine-1-yl) piperidine-1-formic ether comprises the following steps: to compound 12', (R) tert-butyl-3- (4-amino-3- (5- (hydroxymethylene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] at room temperature]To an acetonitrile solution (5mL) of pyrimidin-1-yl) piperidine-1-carboxylate (150mg,0.34mmol) was added phosphate buffer (1.5 mL, pH 6.7), 2,2,6, 6-tetramethylpiperidine nitroxide (5mg, 0.03mmol), NaClO2(80mg, 80% pure in 0.5mL water) and NaClO (0.015mL, 5% aqueous solution). Then, the reaction mixture was reacted at 35 ℃ for 5 hours, a phosphate buffer (10mL, pH 3.6) was added, extraction was performed with ethyl acetate, and the organic solution was extracted with anhydrous Na2SO4After drying, the crude target product (R) tert-butyl-3- (3- (5-formic acid thiophen-2-yl) -4-amino-1H-pyrazole [3, 4-d) is obtained by concentrating the solvent under reduced pressure]Pyrimidin-1-yl) piperidine-1-carboxylate (120mg), (compound 13') was used in the next reaction without further purification.
Thin-layer chromatography: (TLC) detection: rf0.5 (dichloromethane: ethyl acetate ═ 1:2 development phase)
Mass spectrum detection: MS (ESI) M/z 445(M + 1).
Compound 13 ' is (R) tert-butyl-3- (3- (5-carboxylic acid thiophen-2-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylate, compound 14 ' is (R) -2-aminopyridine, compound 15 ' is (R) -3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidine-1-carboxylic acid tert-butyl ester in step 4 of scheme 3, prepared by the following method:
to the compound 13', tert-butyl-3- (3- (5-carboxylic acid thiophen-2-yl) -4-amino-1H-pyrazolo [3,4-d at room temperature]Pyrimidin-1-yl) piperidine-1-carboxylic acid ester (120mg, 0.27mmol) in N, N-dimethylformamide (5mL) was added compound 14', 2-aminopyridine(76mg, 0.81mmol) (CAS No. 504-29-0, available from Afahesar chemical Co., Ltd., China), HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) (154mg, 0.41mmol) (CAS No: 148893-10-1, available from Peng Biotech Co., Ltd., Shanghai) and N, N-diisopropylethylamine (172mg, 1.1 mmol). Then, the reaction mixture was reacted at room temperature for 10 hours, a saturated ammonium chloride solution (20mL) was added thereto, extraction was performed with ethyl acetate, and the organic solution was extracted with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (4: 1 → 1:1) to obtain the target product compound 15' (R) -tert-butyl-3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophene-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidine-1-carboxylate (98mg, yield 70%).
Thin-layer chromatography: (TLC) detection: rf0.5 (Petroleum ether: ethyl acetate ═ 1:1 development phase)
Mass spectrum detection: MS (ESI) M/z 521(M + 1);
nuclear magnetic resonance:1HNMR(400MHz,CDCl3) 1.66(s,9H),1.60-2.45(m,4H), 2.85-4.95(m,5H),6.45-6.65(m,2H),6.70-6.90(m,2H),7.40-8.15(m, 4H), 8.42(s, 1H), 9.18(s, 1H). Compound 16' in step 5, scheme 3 is (R) -3- (4-amino-3- (5- (2-pyridylaminoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]The preparation method of the pyrimidine-1-yl) -1-piperidine comprises the following steps: to the compound 15', (R) tert-butyl-3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d at room temperature]To a solution of pyrimidin-1-yl) piperidine-1-carboxylate (90mg, 0.17mmol) in dichloromethane (5mL) was added 2mL of trifluoroacetic acid. Then reacted at room temperature for 4 hours to give (R) -3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]The trifluoroacetic acid salt of pyrimidine-1-yl) -1-piperidine can also be added with hydrochloric acid, sulfuric acid or acetic acid to obtain corresponding hydrochloride, sulfate or acetate. The crude product obtained by concentrating the solvent under reduced pressure was dissolved in ethyl acetate (20mL) over Na2CO3Washing with saturated solution and then with anhydrous Na2SO4Drying, and concentrating the solvent under reduced pressure to obtain compound (R) -3- (4-amino-3- (5)- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Crude pyrimidin-1-yl) -1-piperidine (compound 16') 70mg was used directly in the next reaction.
And (3) thin-layer chromatography detection: TLC: rf0.5 (dichloromethane: methanol ═ 5:1)
Mass spectrum detection: MS (ESI) M/z 421(M + 1).
Compound 17' in step 6, scheme 3 is (R) -1- (3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one, prepared by a process which comprises:
to (R) -3- (4-amino-3- (5- (2-pyridylcarbamoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] at 0 DEG C]To a solution of pyrimidin-1-yl) -1-piperidine (compound 16') (70mg, 0.17mmol) in dichloromethane (5mL) was added acrylic acid (23mg, 0.30mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (64mg, 0.30mmol), 4-dimethylaminopyridine (13mg, 0.10mmol) in that order, followed by reaction at room temperature for 2 hours, washing with water and then anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure was purified by thin layer chromatography (ethyl acetate: methanol ═ 40: 1) to give compound 17' (R) -1- (3- (4-amino-3- (5- (2-pyridylaminoyl) thiophen-2-yl) -1H-pyrazolo [3,4-d]Pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one (51mg, yield 63%).
TLC：Rf0.5 (Ethyl acetate: methanol ═ 30:1)
MS(ESI)m/z 475(M+1)；1HNMR(400MHz,CDCl3)
1.60-2.45(m,4H),2.89-4.95(m,5H),5.25-5.65(m,3H),6.55-6.95(m, 4H),7.30-7.32(m,2H),7.30-8.15(m,4H),8.42(s,1H),9.14(s,1H)。
By modifying compound 10 'in a manner similar to that of example 6, compounds 51-62 shown in table 1 below can be obtained, wherein compound 10' can be selected from the following compounds:
(5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid;
(4- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid;
(2- (tert-butyldimethylsilyloxymethylene) thiophen-4-yl) boronic acid
When tert-butyldimethylsilyloxymethylene is linked to the 5-position of thiophene and the 2-position of thiophene is linked to the 3-position of 1H-pyrazolo [3,4-d ] pyrimidinyl, compound 10' is (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid.
The compound 10' (5- (tert-butyl dimethyl silica methylene) thiophene-2-yl) boric acid and the synthesis method thereof are as follows:
to a solution of 2-bromothiophene-5-methanol (4g, 20mmol) (CAS No. 79387-71-6, available from Shanghai Tantake technology, Ltd.) in N, N-dimethylformamide (50mL) was added imidazole (2g, 30mmol) and tert-butyldimethylsilyl chloride (3.9g, 25mmol) (CAS No. 18162-48-6, available from Haimenbett Fine chemical Co., Ltd.) in this order at room temperature. Then reacted at room temperature for 2 hours, 200mL of water was added, followed by extraction with ethyl acetate, and the separated organic layer was washed with water (100mL) 2 times, and then the organic phase was dried over anhydrous sodium sulfate, and the crude product obtained by concentrating the solvent under reduced pressure was separated by liquid chromatography on a silica gel column, the eluting solvent being ethyl acetate: petroleum ether (1: 30- >1:15) gave 2-bromo- (5-tert-butyldimethylsilyloxymethylene) thiophene) (5.5g, yield: 90%).
And (3) thin-layer chromatography detection: TLC: rf0.5 (ethyl acetate: petroleum ether ═ 1:10) mass spectrometric detection: MS (ESI) M/z 307(M + 1);
and (3) nuclear magnetic resonance detection:1HNMR(400MHz,CDCl3):0.20(s,3H),0.21(s,3 H),1.60(s,9H),5.21(s,2H),6.55-6.75(m,2H)。
2-bromo- (5-tert-butyldimethylsilyloxymethylene) thiophene (1.8g, 5.9mmol) was dissolved in tetrahydrofuran (15mL) and added dropwise to a solution of-n-butyllithium (4.8mL, 2.5mol/L in n-hexane) at-70 ℃ in tetrahydrofuran (10mL) under a nitrogen atmosphere. After reaction at-70 ℃ for 2 hours, methyl borate (3.15g, 30mmol) was added, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and stirred in 1mol/L hydrochloric acid (30mL) for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the mixture was concentrated under reduced pressure to give compound 10' (5- (tert-butyldimethylsilyl) silaneOxymethylene) thiophen-2-yl) boronic acid crude product 1.20g was used in the next reaction without purification because the product was easily decomposed.
And (3) thin-layer chromatography detection: TLC: rf0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
Mass spectrum detection: MS (ESI) M/z 273(M + 1);
(4- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid
(2- (tert-butyldimethylsilyloxymethylene) thiophen-4-yl) boronic acid
The synthesis methods of the above-mentioned boronic acid compounds are the same as the preparation method of the compound 10' (5- (tert-butyldimethylsilyloxymethylene) thiophen-2-yl) boronic acid.
Example 7
When R is5、R6Are each H, ring 1 is 2-pyridyl, L1Is a single bond, the 2-pyridyl is connected with the 5-position of the thiophene, and the 1H-pyrazole [3,4-d]-pyrimidin-1-yl is attached to the 2-position of thiophene, m ═ 0, ring 2 is piperidine, compound 6' is (5- (2-pyridyl) thiophen-2-yl) boronic acid, prepared by the method of: to a solution of 2-bromopyridine (3g, 19.1mmol) (CAS No: 109-04-6, ex Bester reagent, trade name B012654) in ethylene glycol dimethyl ether (60mL) at room temperature were added, in order, water (30mL), (thien-2-yl) boronic acid (3.4g, 26.7mmol), tetratriphenylphosphadil (454mg, 3.8mmol), and Na2CO3(6.1g, 57.3 mmol). Then replacing with argon for 3 times, reacting at 80 deg.C for 6 hr, cooling to room temperature, pouring into 50mL water, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (100: 1 → 60:1) gave the desired product, 2- (2-pyridyl) thiophene (2.5g, yield 83%).
Thin-layer chromatography: (TLC) detection: rf0.5 (petroleum ether: ethyl acetate ═ 8:1 development phase); mass spectrum detection: MS (ESI) M/z 162(M + 1); nuclear magnetic resonance:1HNMR(400MHz, CDCl3)：7.10-7.45(m,2H)、7.60-7.95(m,4H)、8.50-8.60(m,1H)。
7.1 preparation method of 2-bromo-5- (2-pyridyl) thiophene：
To 2- (2-pyridyl) thiophene (2.5g, 15.5mmol) in dichloromethane (100mL) at 0 deg.C was added dropwise a solution of bromine (2.50g, 15.6mmol) in dichloromethane (50 mL). After the dropwise addition is finished for 1 hour, the temperature is raised to room temperature, the mixture is stirred for 2 hours, and saturated Na is poured into the mixture2CO3(200mL), the organic phase was separated and washed with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is separated by liquid chromatography on a silica gel column, and the elution solvent is petroleum ether: ethyl acetate (100: 1 → 80:1) gave the desired product, 2-bromo-5- (2-pyridyl) thiophene (3g, yield 81%).
Thin-layer chromatography: (TLC) detection: rf0.5 (petroleum ether: ethyl acetate ═ 8:1 development phase); mass spectrum detection: MS (ESI) M/z 240(M + 1); nuclear magnetic resonance: 1HNMR (400MHz, CDCl 3): 7.15-7.50(m,3H), 7.65-7.95(m,2H), 8.55-8.60(m, 1H).
The preparation method of the (5- (2-pyridyl) thiophene-2-yl) boric acid comprises the following steps: 2-bromo-5- (2-pyridyl) thiophene (2g, 8.3mmol) was dissolved in tetrahydrofuran (15mL) and added dropwise to a solution of-n-butyllithium (6.6mL, 2.5mol/L in n-hexane) at-70 ℃ below zero in tetrahydrofuran (15mL) under a nitrogen atmosphere. After the completion of the dropwise addition for 30 minutes, the reaction was continued at-70 ℃ for 2 hours, and then trimethyl borate (5.2g, 50mmol) was added thereto, the reaction was continued at-70 ℃ for 30 minutes, and the mixture was warmed to room temperature and introduced into 1mol/L hydrochloric acid (20mL) and stirred for 10 minutes. Extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, 1.50g of crude (5- (2-pyridyl) thiophen-2-yl) boronic acid was obtained by concentration under reduced pressure and used in the next reaction without purification since the product was easily decomposed. Route 1, steps 4-6, are then repeated.
TLC: rf 0.5 (Ethyl acetate: Petroleum Ether ═ 1:5)
MS(ESI)m/z 206(M+1)。
Scheme 4 for preparing the above intermediates is as follows:
Figure GDA0001754238180000511
in the above scheme, R7Selected from methyl, cyano, carboxamido orAcetylamino, R7May be located at the 5-or 6-position of the pyridyl group. By selecting thiophene boric acid substituted at different positions and selecting bromopyridyl substituted at different positions, the pyridyl and the thienyl at the desired connecting position can be obtained.
7.2. Intermediates of preparation analogous to 7.1
(5- (3-pyridinyl) thiophen-2-yl) boronic acid
5- (2- (6-methylpyridyl)) thiophen-2-yl) boronic acid
5- (2- (5-methylpyridyl)) thiophen-2-yl) boronic acid
(4- (2-pyridinyl) thiophen-2-yl) boronic acid
(4- (3-pyridinyl) thiophen-2-yl) boronic acid
The synthesis of the boronic acid compounds described above is similar to the preparation of the compound 6' (5- (2-pyridyl) thiophen-2-yl) boronic acid.
7.3 when R is5、R6Are both H, ring 1 is 2- (6-cyanopyridyl), L1A single bond, the 2- (6-cyano) pyridyl group is connected with the 5-position of the thiophene, and the 1H-pyrazolo [3,4-d]Pyrimidin-1-yl attached to the 2-position of thiophene, m ═ 0, ring 2 is piperidine, compound 6' is (5- (6-cyanopyridin-2-yl) thiophen-2-yl) boronic acid, prepared by the method of: the preparation of 5-bromo- ((6-cyanopyridin-2-yl) thiophen-2-yl) is similar to the preparation of 2-bromo-5- (2-pyridinyl) thiophene. To a solution of 5-bromo- ((6-cyanopyridin-2-yl) thiophen-2-yl) (3g, 11.3mmol) in ethylene glycol dimethyl ether (60mL) was added water (30mL), bis (pinacolato) diboron (2.88g, 11.3mmol), 1-bis (diphenylphosphino) ferrocene dichloropalladium dichloromethane complex (920mg, 1.1mmol) and NaOAc (3.1g, 22.6mmol) in that order at room temperature. Then replacing with argon for 3 times, reacting at 80 deg.C for 12 hr, cooling to room temperature, pouring into 50mL water, extracting with ethyl acetate, extracting organic liquid with anhydrous Na2SO4After drying, the crude product obtained by concentrating the solvent under reduced pressure is dissolved in 30mL of ethyl acetate, 0.1M hydrochloric acid 10mL is added and stirred at room temperature for 5h, saturated Na is added2CO3Adjusting pH to about 6, extracting with ethyl acetate, and extracting organic liquid with anhydrous Na2SO4After drying, the crude target product (5- (6-cyanopyridin-2-yl) is obtained by concentrating the solvent under reduced pressure) Thiophen-2-yl) boronic acid, which was used in the next reaction without purification due to instability of the product.
Thin-layer chromatography: (TLC) detection: rf 0.5 (petroleum ether: ethyl acetate ═ 6:1 developing phase); mass spectrum detection: MS (ESI) M/z 231(M + 1).
7.4 preparation of intermediates analogous to 7.3
5- (2- (5-cyanopyridyl) thiophen-2-yl) boronic acid
5- (2- (5-carboxamidopyridyl) thiophen-2-yl) boronic acid
5- (2- (6-carboxamidopyridyl) thiophen-2-yl) boronic acid
5- (2- (5- (aminoacetyl) pyridinyl) thiophen-2-yl) boronic acid
5- (2- (6- (aminoacetyl) pyridinyl) thiophen-2-yl) boronic acid
The preparation of the intermediate disclosed in example 5 in combination with the synthetic route of the present invention gives the following compounds 1 to 50 as shown in table 1, and the preparation of the intermediate disclosed in example 6 in combination with the synthetic route of the present invention gives the following compounds 51 to 62 as shown in table 1:
TABLE 1
Figure GDA0001754238180000531
Figure GDA0001754238180000541
Figure GDA0001754238180000551
Figure GDA0001754238180000561
Figure GDA0001754238180000571
Figure GDA0001754238180000581
Figure GDA0001754238180000591
Figure GDA0001754238180000601
Figure GDA0001754238180000611
Figure GDA0001754238180000621
Figure GDA0001754238180000631
Figure GDA0001754238180000641
Figure GDA0001754238180000651
Figure GDA0001754238180000661
Figure GDA0001754238180000671
Figure GDA0001754238180000681
Figure GDA0001754238180000691
Figure GDA0001754238180000701
Figure GDA0001754238180000711
Figure GDA0001754238180000721
Figure GDA0001754238180000731
Example 8
Determination of BTK inhibitory Activity and selectivity for Btk
Experimental Material：
1. Bruton kinase inhibitors (BTK)
Invitrogen, trade number: PR5442A (Invitrogen-PR5442A)
2. Detection kit
Sece Bio Inc. (Cisbio), trade number 62TK0PEJ
3. Detection board
Platinum elmer, trade number: 6007299
(PerkinElmer-6007299)
4. Fluorescence plate reader/container and universal microplate reader
Platinum elmer, trade number: 2104(PerkinElmer-2104)
The experimental steps are as follows:
1. compound dilution: the test compound and the positive compound Ibrutinib (Ibrutinib) are diluted by 3 times with dimethyl sulfoxide (DMSO) for 11 concentrations, and the final system concentration is from 10 μ M to 0.17 nM.
2. In a buffer of 50mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes) (pH7.5), 5mM MgCl2，0.01mM Na3VO41% Bovine Serum Albumin (BSA) in a 10L reaction containing 1nM Bruton's tyrosine kinase (Btk), 1M biotin polypeptide (biotin-TK peptide), 20M ATP, in 50mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes) buffer (pH7.5), and a diluted solution of the test compound in Dimethylsulfoxide (DMSO) was added to a 96 well test plate and incubated for 90 minutes at 23 deg.C. Then, 10. mu.l of a solution containing 20mM ethylenediaminetetraacetic acid (EDTA), 6.7 nM thymidine Btk-deficient antibody (TK) antibody, 62.5nM stop solution (SA-XL665, available from Shanghai Bayer bioscience, Inc. (cisbio)) was added and incubated at 23 ℃ for 60 minutes.
3. The fluorescence intensity at 445nm and 520nm was measured for each well using a fluorescence plate reader/container and a universal microplate reader. The ratio of phosphorylation was determined by the ratio of coloration at 445nm (coumarin coloration) to 520nm (fluorescein coloration) according to the instructions attached to the kit.
4. Calculating the inhibition rate of the compound from the data read by the instrument, and then calculating the IC50The value is obtained. (XLFIT 5 Medium mode 205 using IDBS)
The inhibition (%) of the test compound was calculated using the following formula:
phosphorylation inhibition rate (%) ═ 1- { (a)C-AX)/(AC-AB)}X 100
AC: phosphorylation rate when dimethyl sulfoxide alone (control) was added
AX: phosphorylation rate upon addition of test compound
AB: phosphorylation rate when ATP (blank) was added
The value of 50% Inhibition (IC) of the test compound was calculated from the inhibition curve based on the inhibition at each concentration of the test compound50Value).
Measurement of inhibitory Activity of other tyrosine kinase groups such as Lck the same procedure as described above was carried out using various kinases in place of Btk.
TABLE 2 inhibition of tyrosine kinases by representative compounds of the invention
Figure GDA0001754238180000741
Figure GDA0001754238180000751
Figure GDA0001754238180000761
The results show that the compound has good selective inhibition effect on Btk.
Example 9
Determination of Btk-specific signalling pathway Activity in human lymphoma Lamos cells (Ramos cells)
1. Test materials
Ramos cells
Cell culture Medium (RPMI1640) Invitrogen # 11875093
Invitrogen #10099-141 from Fetal Bovine Serum (FBS)
Invitrogen # F10471 of Fluo-4 calcium flux detection kit
Immunoglobulin IgM Immunity technologies Ltd (southern Biotech) #2020-01
384-well plate (384-well plate) Glanner corporation (Greiner) #781946
2. Test procedure
IgM EC80And (3) detection: cells were harvested, resuspended in 1640 medium containing 0.1% FBS (fetal bovine serum) and adjusted to a concentration of 5x106and/mL. 20L/well of cell suspension was added to the cell plate, 40L Fluo-4 loaded dye (loading dye) was added, and incubation was carried out at 37 ℃ for 50 minutes. IgM was diluted 3-fold in a gradient to a final concentration of 10g/mL to 0.0046g/mL, and 10L of IgM was transferred to using a high throughput cell level screening system (FLIPR)In the cell plate, and the fluorescence value was read. Calculation of drug concentration (EC) for IgM80)。
Compound IC50And (3) detection: cells were harvested, resuspended in 1640 medium containing 0.1% FBS and adjusted to a concentration of 5 × 106and/mL. 20L/well of cell suspension was added to the cell plate. The test compound and the positive compound Ibrutinib (Ibrutinib) were diluted in 3-fold gradient to a final concentration of 10M to 0.0046M, 10L of the compound was transferred to a cell plate, and incubated at 37 ℃ for 60 minutes. 40L of fluorescent loading dye (Fluo-4loading dye) was added and incubated at 37 ℃ for 50 min. Transfer of 10L 8 × EC Using high throughput cell transfer screening System (FLIPR)80IgM was plated onto cell plates and fluorescence was read. Inhibition was plotted using plotting Software (Prism) GraphPad Software) to calculate compound IC50。
TABLE 3 inhibition of Btk-specific signaling pathways in Ramos cells by representative compounds of the invention
Figure GDA0001754238180000771
The results show that the compound has better inhibition effect on the Ramos cell Btk specific signal transduction pathway than ibrutinib.
Example 10
Determination of proliferative Activity of Non-Hodgkin lymphoma cell line
1. Test materials
Ramos (human Burkitts lymphoma cells)
HBL-1 (human diffuse large B lymphoma cell)
Daudi (human Burkitts lymphoma cells)
DOHH-2 (human follicular lymphoma cells)
JeKo-1 (human mantle cell lymphoma cell)
OCI-LY-19 (Burkitts lymphoma cells)
Z-138 (human mantle cell lymphoma cell)
SU-DHL-4 (human diffuse large B lymphoma cell)
SU-DHL-10 (human diffuse large B lymphoma cell)
WSU-DLCL2 (human follicular lymphoma cells)
Microplate reader Molecular Devices Spectra MAX I3
Cell culture Medium (RPMI1640) Saimer Feisha (Gibco) # C11875500BT
Invitrogen #10099-141 of fetal bovine serum FBS
Cell proliferation and activity detection kit (CCK-8) Dojindo chemical industry (Dojindo) # CK04B
384-well plate (384-well plate) Corning corporation (Corning) #3701
2. Test procedure
Cells were harvested, resuspended in 1640 medium containing 10% FBS (fetal bovine serum) and adjusted to a concentration of 3 × 104and/mL. Add 50. mu.L/well of cell suspension to the cell plate. The test compound and the positive compound Ibrutinib (Ibrutinib) were diluted in a 3-fold gradient, 5. mu.L of the compound solution was transferred to a cell plate to a final concentration of 50. mu.M or 1. mu.M to 0.128nM or 0.0026nM, and incubated at 37 ℃ for 72 hours. 5. mu.L of a 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonated benzene) -2H-tetrazole monosodium salt solution, i.e., a CCK-8 solution (purchased from Shanghai Probiotics, Ltd., trade name CCK-8 kit, trade name 40203ES60), was added and incubated at 37 ℃ for 3 hours. Fluorescence was read using a microplate reader. Cell proliferation profiles were generated using the mapping Software prism5.0(GraphPad Software) to calculate Compound IC50。
Cell growth inhibition rate (%) ═ 1- { (AC-AX)/(AC-AB) } X100
AC: absorbance value of negative control when dimethyl sulfoxide alone (control) was added
AX: absorbance values of wells to which test compounds were added
AB: absorbance values of blank control (blank)
TABLE 4 inhibitory Effect of representative Compounds of the present invention on non-Hodgkin lymphoma cell proliferation
Figure GDA0001754238180000791
TABLE 5
Figure GDA0001754238180000801
The results show that the compound of the invention has obvious inhibition effect on the proliferation of Non-Hodgkin lymphoma cells, and the activity of part of the compound is obviously superior to that of ibrutinib.
Example 11
Pharmacodynamic study of the compound in type II collagen-induced arthritis model of mice
Collagen-induced arthritis is an experimental animal model induced by species-specific collagen type II immunization. Because the genetic background and the immunopathology change of the model are very similar to the clinical rheumatoid arthritis, the model becomes a more ideal animal model for researching the rheumatoid arthritis at present.
The model making method comprises the following steps: DBA/1J mice, 7 weeks old, 18-22g in weight, male, (purchased from Jinan Ono bioengineering, Inc., trade name DBA/1J mice, model: DBA/1J). Appropriate amount of bovine type II collagen was dissolved in 0.01mol/L acetic acid (4mg collagen/ml), and emulsified with equal amount of Freund's complete adjuvant in ice bath, 0.1ml (containing 200. mu.g collagen) emulsion was injected into the tail root of each mouse, and on day 21, equal amount of collagen was emulsified with Freund's incomplete adjuvant to enhance immunity for 1 time.
Materials and methods: test compound 17, compound 63, compound 64 were dissolved in polyethylene glycol 400, castor oil ethoxylate (KolliphorRH40) at a concentration of 200 mg/ml-8: 2. (ethoxylate of Castor oil CAS number 61788-85-0, from Pasteur, Germany) mice were orally fed type II collagen-induced arthritic DBA/1J mice once daily at a dose of 25 mg/kg. The mice were divided into four groups of test compound 17, compound 63, compound 64 and vehicle, wherein the vehicle group was administered once daily to the mice for 14 consecutive days at a dose of 100mg/kg by the ratio of ethylene glycol 400 to castor oil ethoxylate of 8: 2.
Observation indexes and analysis: arthritis index score. The arthritis index was scored according to Wood's arthritis scoring criteria. Score 0, normal; 1 point, red and swollen relates to 1 finger joint; 2 points, the red swelling refers to mild red swelling of more than 2 finger joints or the whole paw; 3 points, the paw is red and swollen seriously; 4 points, severe swelling of the paw, stiff joints and lack of elasticity. Lesions were scored for each of the 4 paws as 0-4 total points of the extremities, the number of arthritic limbs per group of mice was expressed as a percentage, and the points at different times (arthritic index) were compared, and the incidence of arthritis and the onset time of arthritis were also recorded. As shown in FIG. 1, it can be seen from FIG. 1 that the arthritis status of the mice improved with the increase of the administration time.
EXAMPLE 12 in vitro growth inhibition of Chronic leukemia K562 cells by Compounds
In vitro CellTiter-
Figure GDA0001754238180000812
ATP luminescent cell viability assay
Leukemia K562 cells were obtained from ATCC and maintained at 37 ℃ with 5% CO2Atmosphere and in dulbecco medium (IMDM) and 10% fetal calf serum. Cells with 6 x103The test compounds were dissolved in DMSO at concentrations of 0. mu.M, 0.3. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M and 100. mu.M for 72 hours, and then cells affected with the compounds were detected using CellTiter-Glo luminescence cell viability assay kit, and luminescence values were recorded.
The results of the in vitro effect of some compounds of the invention on the viability of leukemic K562 cells are listed in Table 6 below.
TABLE 6
Figure GDA0001754238180000811

Claims (11)

1. A compound having the general formula (I):
Figure FDA0002744229850000011
wherein X, Y is each independently selected from CH or N, R4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
2. The compound of claim 1, of formula (ii):
Figure FDA0002744229850000012
wherein R is4Selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl, carbamoyl, acetamido and (C)1-C4) One of the haloalkoxy groups.
3. The compound of claim 1, of formula (iii):
Figure FDA0002744229850000021
wherein R is5、R6Each independently selected from H, halogen, (C)1-C3) Alkoxy group, (C)1-C4) Alkyl, cyano, (C)1-C4) Haloalkyl and (C)1-C4) One or more of halogenated alkoxy.
4. A compound according to any one of claims 1 to 3, wherein the compound is selected from one or more of the following compounds:
1- (3- (4-amino-3- (5-phenylthiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5-thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-methylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3, 4-dimethylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-methoxyphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-methoxyphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-trifluoromethylphenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-trifluorotoluene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3, 5-xylene) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(S) -1- (3- (4-amino-3- (5-phenylthiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (2-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (3-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (4-fluorophenyl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (pyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (pyridin-3-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(S) -1- (3- (4-amino-3- (5- (pyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (6-cyanopyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) -picolinamide;
(R) -6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) -3-pyridinecarboxamide;
(R) -N- (6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) pyridin-2-yl) -acetamide;
(R) -N- (6- (5- (1- (1-acryloylpiperidin-3-yl) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) thiophen-2-yl) pyridin-3-yl) acetamide;
(R) -1- (3- (4-amino-3- (5- (6-methylpyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one;
(R) -1- (3- (4-amino-3- (5- (5-methylpyridin-2-yl) thiophen-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) prop-2-en-1-one.
5. Use of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a disease associated with malignancy, autoimmune disease and allergic disease.
6. The use according to claim 5, wherein the malignant tumor is selected from one or more of lymphoma, plasmacytoma and leukemia.
7. The use according to claim 6, wherein the lymphoma is selected from one or more of non-Hodgkin's lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, intravascular large cell B-cell lymphoma, Burkitt's lymphoma, AIDS-related lymphoma and marginal zone B-cell lymphoma.
8. The use of claim 7, wherein said non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma.
9. The use of claim 8, wherein said B-cell non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.
10. The use according to claim 5, wherein the autoimmune disease is selected from one or more of arthritis, rheumatism, inflammatory bowel disease and lupus erythematosus.
11. A bruton's tyrosine kinase inhibitor composition comprising a compound of any one of claims 1-4.
CN201780009992.6A 2016-02-05 2017-01-26 Bruton's tyrosine kinase inhibitor Active CN108602834B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101610676A (en) * 2006-09-22 2009-12-23 药品循环公司 The inhibitor of bruton's tyrosine kinase
CN103319488A (en) * 2007-03-28 2013-09-25 环状药物公司 Inhibitors of bruton's tyrosine kinase
CN103857396A (en) * 2011-07-13 2014-06-11 药品循环公司 Inhibitors of bruton's tyrosine kinase
WO2014188173A1 (en) * 2013-05-20 2014-11-27 Redx Pharma Limited Pyrazolopyrimidine derivatives useful as inhibitors of bruton's tyrosine kinase
WO2015048689A1 (en) * 2013-09-30 2015-04-02 Pharmacyclics, Inc. Inhibitors of bruton's tyrosine kinase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101610676A (en) * 2006-09-22 2009-12-23 药品循环公司 The inhibitor of bruton's tyrosine kinase
CN103319488A (en) * 2007-03-28 2013-09-25 环状药物公司 Inhibitors of bruton's tyrosine kinase
CN103857396A (en) * 2011-07-13 2014-06-11 药品循环公司 Inhibitors of bruton's tyrosine kinase
WO2014188173A1 (en) * 2013-05-20 2014-11-27 Redx Pharma Limited Pyrazolopyrimidine derivatives useful as inhibitors of bruton's tyrosine kinase
WO2015048689A1 (en) * 2013-09-30 2015-04-02 Pharmacyclics, Inc. Inhibitors of bruton's tyrosine kinase

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