WO2018233655A1 - 一种具有药物活性的杂芳基化合物 - Google Patents

一种具有药物活性的杂芳基化合物 Download PDF

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WO2018233655A1
WO2018233655A1 PCT/CN2018/092122 CN2018092122W WO2018233655A1 WO 2018233655 A1 WO2018233655 A1 WO 2018233655A1 CN 2018092122 W CN2018092122 W CN 2018092122W WO 2018233655 A1 WO2018233655 A1 WO 2018233655A1
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compound
substituted
alkyl
group
unsubstituted
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French (fr)
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刘新
袁哲东
孔锐
陈珊
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上海度德医药科技有限公司
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Priority to EP18819881.6A priority Critical patent/EP3643716B1/en
Priority to JP2020520704A priority patent/JP6859549B2/ja
Publication of WO2018233655A1 publication Critical patent/WO2018233655A1/zh
Priority to US16/724,363 priority patent/US11512085B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • 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
    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of pharmaceutically active heteroaryl compounds.
  • a Bruton-type tyrosine kinase belongs to a Tec family kinase which is a non-receptor type tyrosine kinase, and selectively appears in cells of a B cell line and a bone marrow cell line.
  • Btk plays an important role in the signal transduction of B cells and is a factor that contributes to the survival, differentiation, proliferation and activation of B cells.
  • BCR B cell antigen receptor
  • Btk is thought to be responsible for a part of the signaling pathway to B cells that pass through the BCR. Therefore, it is known that due to the deletion of the human Btk gene, abnormal differentiation of B cells is induced, and the production of immunoglobulin is significantly reduced, thereby producing the onset of X-linked ⁇ -globulinemia (XLA) (refer to Patent Document 1). Examples of the symptoms of the disease include a significant decrease in B cells in peripheral blood and/or an increase in sensitivity to bacterial infection. In addition, it is also known that Btk is involved in the activation of mast cells and/or the physiological function of platelets.
  • a compound having Btk inhibitory activity is useful for the treatment of diseases associated with B cells and/or mast cells, for example, allergic diseases, autoimmune diseases, inflammatory diseases, thromboembolic diseases, cancers, and the like (refer to Non-patent document 2).
  • LaA represents CH2, 0, NH or S
  • ArA represents a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group
  • YA represents an alkyl group, a heteroalkyl group, a cycloalkyl group, a hetero group
  • ZA represents CO, OCO, NHCO, CS
  • R7-A and R8-A independently represent H, unsubstituted Cl-C4 alkyl, substituted Cl- C4 alkyl, unsubstituted Cl-C4 heteroalkyl, substituted Cl-C4 heteroalkyl, unsubstituted C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, unsubstituted C2-C6 heterocycle
  • R1 represents a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, a C1-4 haloalkyl group, or a C1-4 haloalkoxy group; and ring 1 represents each independently selected from a halogen atom, a C1-4 alkane; a 4-7 membered cyclic group substituted with 1 to 5 substituents of a C1-4 alkoxy group, a cyano group, a C1-4 haloalkyl group, or a C1-4 haloalkoxy group, wherein When the substituent is two or more, the substituent may form a 4-7 membered cyclic group together with the atom constituting the ring 1 to which they are bonded; and ring 2 represents a
  • K represents -C(O)-, wherein the bond on the left side is bonded to ring 2; and R2 represents 1-5 which may be independently selected from NR3R4, a halogen atom, CONR5R6, COR7 and OR8, respectively.
  • Patent Document 1 Special Table 2010-504324
  • Patent Document 2 International Publication No. 2008/121742
  • Patent Document 3 International Publication No. 2010/009342
  • Non-Patent Document 1 Nature, Vol. 361, pp. 226-233, 1993
  • Non-Patent Document 2 Anticancer Agents in Medicinal Chemistry, Vol. 7, No. 6, pp. 624-632, 2007
  • R 1 represents H, a substituted or unsubstituted C 1 -C 4 alkyl group, a substituted or unsubstituted C 1 -C 4 heteroalkyl group;
  • R 2 represents R 6 CO, R 7 SO, R 8 SO 2 or a C 1 -C 6 alkyl group substituted by R 9 ;
  • R 3 represents H, one or more substituents such as halogen (fluoro, chloro, bromo, iodo), C 1 -C 4 alkoxy, nitro, cyano, substituted or unsubstituted amino, substituted or unsubstituted Alkyl
  • R 4 represents H, one or more substituents such as halogen (fluoro, chloro, bromo, iodo), C 1 -C 4 alkoxy, nitro, cyano, substituted or unsubstituted amino, substituted or unsubstituted Alkyl
  • R 5 represents a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted C 1 -C 6 heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, substituted or not Substituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 heterocycloalkyl;
  • R 6 is alkenyl, alkynyl, and alkyl, alkenyl, amine or halogen substituted alkenyl, alkynyl;
  • R 7 , R 8 are independently selected from C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, optionally selected from hydroxy, C 1 -C 4 alkyl, C 3 -C 7 cycloalkyl (C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, C 1 -C 3 alkoxy, C 3 -C 7 cycloalkoxy, C 6 -C 10 aryl Or one or more groups of a C 3 -C 7 heterocycloalkyl group; or a C 1 -C 5 heteroaryl group optionally substituted with one or more groups selected from halogen or cyano;
  • R 9 is independently selected from halo, cyano or C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, optionally selected from hydroxy, C 1 -C 4 alkyl, C 3 -C 7 ring Alkyl, (C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, C 1 -C 3 alkoxy, C 3 -C 7 cycloalkoxy, C 6 -C 10 Substituting one or more groups of an aryl group, a C 1 -C 5 heteroaryl group or a C 3 -C 7 heterocycloalkyl group;
  • Z is CH 2 , O, NH, or S
  • n 1 represents 0 or 1
  • n 2 represents an integer of 1-4
  • n 3 represents an integer of 0-1
  • X represents nitrogen or carbon, which may be at any position of the benzene ring; Y represents nitrogen.
  • R 1 represents H, C 1 -C 4 alkyl, C 1 -C 4 heteroalkyl
  • R 2 represents R 6 CO, R 7 SO, R 8 SO 2 or C 1 -C 6 alkyl substituted by R 9 ;
  • R 6 is alkenyl, alkynyl, and alkyl, alkenyl, amine or halogen substituted Alkenyl, alkynyl;
  • R 7 , R 8 are independently selected from C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, and R 9 is independently selected from halo, cyano or C 2 -C 6 alkenyl Or a C 2 -C 6 alkynyl group;
  • R 3 represents H
  • R 4 represents H, halogen (fluoro, chloro, bromo, iodo), substituted or unsubstituted alkyl;
  • R 5 represents a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted C 1 -C 6 heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group;
  • Z is O
  • n 1 represents 0 or 1
  • n 2 represents an integer of 1-4
  • n 3 represents an integer of 0-1
  • X represents nitrogen or carbon and can be in any position of the benzene ring.
  • the compound Ia, Ib, Ic, Id, Ie, If or Ig of the formula is also expressed as a compound represented by the formula (Ia-Ig) or a compound of the formula Ia-Ig.
  • a pharmaceutical composition comprising the compound of the formula Ia-Ig, an optical isomer thereof or a mixture thereof, a salt thereof, a solvate thereof, an N-oxide thereof, or a prodrug thereof.
  • the compound of the formula Ia-Ig, the optical isomer thereof or a mixture thereof, a salt thereof, a solvate thereof, an N-oxide thereof, or a prodrug thereof are preventive and/or as a Btk-related disease
  • Pharmacological use of a therapeutic agent further, as a medicament for preventing and/or treating a Btk-related disease such as an allergic disease, an autoimmune disease, an inflammatory disease, a thromboembolic disease, or cancer; further , as a drug for the prevention and / or treatment of non-Hodgkin's lymphoma drugs.
  • a pharmaceutical composition comprising the above-described compound of the formula Ia-Ig, an optical isomer thereof or a mixture thereof, a salt thereof, a solvate thereof, an N-oxide thereof, or a prodrug thereof, as a B cell activation inhibitor .
  • a method for preventing and/or treating a disease associated with Btk which comprises administering to a mammal a compound represented by the above formula (Ia-Ig), an optical isomer thereof or a mixture thereof, a salt thereof, and a salt thereof.
  • the novel compound provided by the present invention has excellent Btk selective inhibitory activity and is a compound which is excellent in metabolic stability and can avoid hepatotoxicity, and thus can be used as a safety-preserving B cell and other non-Hodgkin's lymphoma and/or A therapeutic agent for mast cell-related diseases.
  • "having a Btk selective inhibitory activity” means a tyrosine kinase other than Btk, particularly Lck (lymphocyte-specific protein tyrosine kinase), and LynA (v-yes-1 Yamaguchi).
  • Lck lymphocyte-specific protein tyrosine kinase
  • LynA v-yes-1 Yamaguchi
  • the sarcoma virus-associated oncogene homologous subtype A) has Btk selective inhibitory activity. From this property, undesired side effects caused by inhibition of other tyrosine kinases can be avoided. For example, it has been found that abnormalities in the retina (oncogene, vol. 16, pp. 2351-2356, 1998) have been found in Lck-deficient mice. Therefore, when Lck is inhibited, side effects on the eye may occur.
  • the halogen atom means fluorine, chlorine, bromine or iodine.
  • the C1-C4 alkyl group means a linear or branched C1-C4 alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a sec-butyl group or a t-butyl group.
  • the C1-C4 alkylene group means a methylene group, an ethylene group, a propylene group, a butylene group, an isomer thereof, and the like.
  • the C1-C4 alkoxy group is a linear or branched Cl- such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group or a t-butoxy group. C4 alkoxy.
  • the C2-C4 alkenyl group means a vinyl group, a 1-propenyl group, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, and a 1,3-butadienyl group.
  • C2-C4 alkynyl means ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1,3-butadiynyl, etc.
  • a chain or branched C2-C4 alkynyl group means a vinyl group, a 1-propenyl group, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, and a 1,3-butadienyl group.
  • an alkyl group includes a linear alkyl group and a branched alkyl group.
  • a geometric isomer (E type, Z type, cis form, trans form) in a double bond, a ring or a fused ring, an optical isomer produced by the presence of an asymmetric carbon atom or the like (R, S Type, ⁇ , ⁇ configuration, enantiomer, diastereomer), optically active (D, L, d, 1 type) having optical activity, polar substance produced by chromatographic separation (high Polar, low polar), equilibrium compounds, rotamers, mixtures of any ratios thereof, racemic mixtures are all included in the present invention. Further, in the present invention, all isomers derived from tautomers are also included.
  • optical isomers in the present invention include not only 100% pure but also less than 50% of other optical isomers.
  • the compound represented by the formula (Ia-Ig) can be converted into the corresponding salt by a known method.
  • the salt is preferably water soluble.
  • suitable salts include salts of alkali metals (potassium, sodium, etc.), salts of alkaline earth metals (calcium, magnesium, etc.), ammonium salts, and pharmaceutically acceptable organic bases (tetramethylammonium, triethylamine).
  • Acid salt trifluoroacetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzene Sulfonic acid salt, toluenesulfonate, isethionate, glucuronate, gluconate, etc.
  • the compound represented by the formula (Ia-Ig) and a salt thereof can also be converted into a solvate.
  • the solvate is preferably low in toxicity and water soluble.
  • Examples of a suitable solvate include a solvate of a solvent such as water or an alcohol (for example, ethanol).
  • the prodrug of the compound represented by the formula (Ia-Ig) means a compound which can be converted into a compound represented by the formula (Ia-Ig) by reacting with an enzyme and/or gastric acid or the like in a living body.
  • the compound represented by the formula (Ia-Ig) has a hydroxyl group as a prodrug of the compound represented by the formula (Ia-Ig)
  • the hydroxyl group is acylated, alkylated, phosphorylated, or boroylated. compound of.
  • the compound of the present invention is effective as a Btk-related disease due to selective Btk inhibitory action, that is, a disease associated with B cells and/or mast cells, for example, an allergic disease, an autoimmune disease, an inflammatory disease, A preventive and/or therapeutic agent for thromboembolic diseases, cancer, graft versus host disease, and the like. Further, the compound of the present invention has an action of selectively inhibiting activation of B cells, and thus is effective as an inhibitor of B cell activation.
  • examples of the allergic diseases include allergy, allergic reaction, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.
  • examples of the autoimmune diseases include inflammatory bowel disease, arthritis, lupus, rheumatism, dry arthritis, osteoarthritis, Still's disease, juvenile arthritis, type I diabetes, Myasthenia gravis, Hashimoto's thyroiditis, ord's thyroiditis, Barthol's disease, Sjogren's syndrome, multiple sclerosis, Guillain-Barre syndrome, acute epidemic encephalomyelitis, Edison's disease, Strabismic cerebral palsy syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, pulmonary hemorrhagic nephropathy, idiopathic thrombocytopenic purpura, optic neuritis , scleroderma, primary biliary cirrhosis, Ritter disease, high arteritis, temporal arteritis, warm-antibody autoimmune hemolytic anemia, Wechsler's granulomato
  • examples of the inflammatory disease include asthma, appendicitis, orbital inflammation, bronchiolitis, bronchitis, synovial inflammation, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, Lacrimal gland inflammation, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, epicondylitis, epididymitis, sarcolemma, connective tissue inflammation, gastritis, gastroenteritis, hepatitis, sweat gland abscess, Laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis, pancreatitis, mumps, meningitis, peritonitis, pharyngitis, pleuris
  • examples of thromboembolic diseases include myocardial infarction, angina pectoris, reocclusion after angioplasty, restenosis after angioplasty, reocclusion after aortic coronary artery bypass, and aortic coronary artery bypass Restenosis after surgery, cerebral infarction, transient ischemia, peripheral vascular occlusion, pulmonary embolism, and deep vein thrombosis.
  • non-Hodgkin's lymphoma is included, and among them, B-cell non-Hodgkin's lymphoma is preferable, and, for example, Burkitt's lymphoma, AIDS-associated lymphoma, marginal zone B-cell lymphoma may be mentioned ( B border cell B-cell lymphoma, extranodal marginal zone B-cell lymphoma, spleen marginal B-cell lymphoma, diffuse large cell B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis , follicular lymphoma, B-cell chronic lymphocytic leukemia, B-cell pro-lymphocytic leukemia, lymphoplasmacytic leukemia/Waldenstrom macroglobulinemia, plasmacytoma, mantle cell lymphoma, mediastinum Cell type B cell lymphoma, intravascular large cell type B cell lymphoma, hairy cell leukemia
  • cancer other than non-Hodgkin's lymphoma including mesangial endocrine tumor
  • VIP-producing tumor VIP-producing tumor
  • PPoma PP-producing tumor
  • the compounds of the invention may be administered alone, however,
  • the combination of the compound of the present invention and other drugs may be administered in the form of a compatibilizer in which the two components are combined in one preparation, or may be formulated in a form to be administered in the respective preparations.
  • a compatibilizer in which the two components are combined in one preparation
  • the combination of simultaneous administration and the difference in interval time is given.
  • the administration of the interval difference may be carried out by first administering the compound of the present invention, then administering another drug, or administering the other drug first, followed by administering the compound of the present invention.
  • Their respective methods of administration may be the same or different.
  • the disease which exerts the preventive and/or therapeutic effect by the above-mentioned combination drug is not particularly limited as long as it is a disease capable of supplementing and/or enhancing the preventive and/or therapeutic effect of the compound of the present invention.
  • drugs for supplementing and/or enhancing the preventive and/or therapeutic effect of the compound of the present invention on allergic diseases for example, antihistamines, leukotriene antagonists, antiallergic agents, thromboxane A2 receptors may be mentioned.
  • drugs for supplementing and/or enhancing the preventive and/or therapeutic effects of the compound of the present invention on autoimmune diseases for example, immunosuppressive agents, steroids, disease-modifying antirheumatic drugs, and elastase inhibitors may be mentioned.
  • cannabinoid-2 receptor stimulating agent prostaglandin, prostaglandin synthetase inhibitor, phosphodiesterase inhibitor, metalloproteinase inhibitor, adhesion molecule inhibitor, anti-TNF- ⁇ preparation, anti-IL-1 preparation, Anti-IL-6 preparations and other anti-cytokine protein preparations, cytokine inhibitors, non-steroidal anti-inflammatory drugs, anti-CD20 antibodies.
  • drugs for supplementing and/or enhancing the preventive and/or therapeutic effect of the compound of the present invention on inflammatory diseases for example, steroids, elastase inhibitors, cannabinoid-2 receptor stimulants, prostates may be mentioned.
  • prostaglandin synthetase inhibitor phosphodiesterase inhibitor, metalloproteinase inhibitor, adhesion molecule inhibitor, anti-leukotriene, anticholinergic, thromboxane A2 receptor antagonist, thromboxane synthase inhibitor, Astragalus derivatives, expectorants, antibacterials, antihistamines, anti-cytokine protein preparations, cytokine inhibitors, forskolin preparations, medium free inhibitors, non-steroidal anti-inflammatory drugs.
  • thrombolytic drugs for supplementing and/or enhancing the preventive and/or therapeutic effect of the compound of the present invention on thromboembolic diseases
  • thrombolytic drugs for example, thrombolytic drugs, heparin, heparin analogs, low molecular weight heparin, warfarin, blood coagulation may be mentioned.
  • an alkylating agent for example, an alkylating agent, a metabolic antagonist, an anticancer antibiotic, a plant organism can be mentioned.
  • an alkylating agent for example, an alkylating agent, a metabolic antagonist, an anticancer antibiotic, a plant organism can be mentioned.
  • antihistamine examples include, for example, azelastine hydrochloride, ebastine, epilisine hydrochloride, ezamestatin fumarate, auranofine, oxamethome, and olopatol hydrochloride.
  • Ding dl-chlorpheniramine maleate, clemastine fumarate, ketotifen fumarate, cetidine, diphenhydramine, diphenhydramine hydrochloride, cyproheptadine hydrochloride, cetirizine hydrochloride Oxazine, desloratadine, terfenadine, famotidine, fexofenadine hydrochloride, betastatin, betaxetine besylate, mizolastine, mequitin, citrate Rice pine, ranitidine, ranitidine hydrochloride, loratadine, promethazine hydrochloride, and perchlorazine hydrochloride.
  • leukotriene antagonist examples include, for example, pulenastate hydrate, montelukast sodium, zafirlukast, aruzide, berbitast, thiorutast, and ilalast.
  • pulenastate hydrate montelukast sodium, zafirlukast, aruzide, berbitast, thiorutast, and ilalast.
  • antiallergic agent examples include, for example, ampicillin, azelastine hydrochloride, irapapan, ibudistat, sodium citrate, ebastine, eplesine hydrochloride, and rich. Esomestatin, oxapramide, ozagrel hydrochloride, olopatadine hydrochloride, cromolyn, sodium cromoglycate, ketotifen fumarate, cetuxide, cetirizine hydrochloride, methanesulfonate Stertz, tazaridine, terfenadine, domiquban calcium hydrate, tranilast, nedocromil, fexofenadine, fexofenadine hydrochloride, pyrazolam potassium, mequalin Oxazine, Rematroban, Repeptide, Loratadine.
  • thromboxane A2 receptor inhibitor examples include sertralast, domitriptan calcium hydrate, and rematriptan.
  • thromboxane synthetase inhibitor examples include, for example, sodium mistletide and ozagrel hydrochloride.
  • Examples of the steroid include ancinoxane, hydrocortisone sodium succinate, prednisolone sodium succinate, methylprednisolone sodium succinate, ciclesonide, difluprednione, and propionic acid.
  • immunosuppressive agent examples include azathioprine, ascomycin, everolimus, sulfasalazine, cyclosporin, cyclophosphamide, sirolimus, tirolimem, and bucillamine. , methotrexate, leflunomide.
  • Examples of the disease-modifying antirheumatic drug include D-penicillamine, acetazone, auranofin, sulfasalazine, hydroxychloroquine, bucillamine, methotrexate, and fluoride. Mitt, chlorpheniric acid disodium, gold thioglucose, sodium thiomalate.
  • Examples of the prostaglandins include PGEl preparations (for example, alprostadil ⁇ -cyclodextrin inclusion compound, alprostadil, etc.) and PGI 2 preparations (for example, beraprost sodium, etc.). , PG receptor agonists, PG receptor antagonists, and the like.
  • Examples of the PG receptor include PGE receptors (EP1, EP2, EP3, and EP4), PGD receptors (DP, CRTH2), PGF receptor (FP), PGI2 receptor (IP), and TX receptor (TP). Wait.
  • prostaglandin synthetase inhibitors include sulfasalazine, mesalazine, olsalazine, 4-aminosalicylic acid, auranofin, carprofen, dipyridamole, and flunorolol.
  • Examples of the phosphodiesterase inhibitor include rolipram, cilostazol, and roflumilast (BY-217).
  • adhesion molecule inhibitor for example, an ⁇ 4-integrin antagonist can be mentioned.
  • anti-TNF- ⁇ preparation examples include anti-TNF- ⁇ antibody, soluble TNF- ⁇ receptor, anti-TNF- ⁇ receptor antibody, and soluble TNF- ⁇ -binding protein, and in particular, infliximab , etanercept.
  • anti-IL-1 preparation examples include an anti-IL-1 antibody, a soluble IL-1 receptor, an anti-IL-1Ra and/or an IL-1 receptor antibody, and particularly, anakinra.
  • anti-IL-6 preparation examples include an anti-IL-6 antibody, a soluble IL-6 receptor, and an anti-IL-6 receptor antibody, and in particular, tocilizumab can be mentioned.
  • cytokine inhibitor examples include metformin and T-614.
  • anticholinergic agent examples include trihexyphenidate, trihexyphenidate hydrochloride, biperiden, and biperiden hydrochloride.
  • xanthine derivative examples include aminophylline, theophylline, doxofylline, cetylphylline, and dihydroxypropyl theophylline.
  • peony examples include cumin ammonia alcohol, sodium hydrogencarbonate, bromhexine hydrochloride, carboxymethyl sultanate, ambroxol hydrochloride, methylcysteine hydrochloride, acetylcysteine, and L-hydrochloric acid. Ethylcysteine, tyloxapol.
  • antibacterial agent examples include cefuroxime sodium, meropenem trihydrate, and netilmicin sulfate.
  • mediator free inhibitor examples include tranilast, sodium cromolyn, amlexanox, rispiride, ibudilast, tazarastide, and pyrimilast potassium. .
  • thrombolytic drug examples include alteplase, urokinase, tyrosine kinase, nasapase, nataplase, t-PA, pamitipase, monteplase, and protein kinase. Streptokinase.
  • fondaparin is mentioned, for example.
  • Examples of the low molecular weight heparin include danaparoid sodium, enoxaparin (sodium), nadroparin calcium, bemiparin (sodium), resaparin (sodium), and tinzaparin (sodium).
  • thrombin inhibitors examples include argatroban, ximelagatran, melagatran, dabigatran, bivalirudin, lepirudine, hirudin, and lycopene.
  • ADP receptor antagonist examples include ticlopidine hydrochloride and clopidogrel sulfate.
  • cyclooxygenase inhibitor for example, aspirin is mentioned.
  • alkylating agent examples include, for example, hydrochloric acid mustard-N-oxide, cyclophosphamide, ifosfamide, melphalan, morphine, carbazone, busulfan, and nimustine hydrochloride. , dacarbazine, ramustine and the like.
  • Examples of the metabolic antagonist include methotrexate, guanidine, 6-glycoside, fluorouracil, tegafur, tegafur uracil, carmofur, deoxyfluorouridine, cytarabine, Enentaphine, tegafur, gemester, potassium oxonate, gemcitabine hydrochloride, procarbazine hydrochloride, hydroxyurea, and the like.
  • anticancer antibiotics examples include actinomycin D, lysin C, daunorubicin hydrochloride, doxorubicin hydrochloride, arubicin hydrochloride, neocarcin, and pyridine Bixing, (hydrochloric acid) epirubicin, idarubicin hydrochloride, chromomycin A3, (hydrochloric acid) bleomycin, pilomycin sulfate, pirarubicin, net statin ester and the like.
  • Examples of the vegetable preparations include vinblastine sulfate, vincristine sulfate, vindesine sulfate, irinotecan hydrochloride, etoposide, flutamide, vinorelbine tartrate, docetaxel hydrate, Paclitaxel and the like.
  • hormonal agent examples include estramustine sodium phosphate, metoprolol, cyclostretidine, goserelin acetate, phosphoestrol (diethylstilbestrol diphosphate), tamoxifen citrate, and guanidine.
  • estramustine sodium phosphate metoprolol
  • cyclostretidine goserelin acetate
  • phosphoestrol diethylstilbestrol diphosphate
  • tamoxifen citrate examples include estramustine sodium phosphate, metoprolol, cyclostretidine
  • goserelin acetate examples include phosphoestrol (diethylstilbestrol diphosphate), tamoxifen citrate, and guanidine.
  • Toremifene acid carbazole hydrochloride hydrate, medroxyprogesterone acetate, bicalutamide, leuprolide acetate, anastrozole, exemestane and the like.
  • platinum compound examples include carboplatin, cisplatin, nedaplatin, and the like.
  • anti-CD20 antibody examples include rituximab, temimumab, and orizulimumab.
  • anticancer agents examples include L-asparaginase, octreotide acetate, sodium phenate, and mitoxantrone acetate.
  • combination drug as a combination with the compound of the present invention includes not only drugs which have appeared so far, but also drugs which will appear in the future.
  • the compound of the present invention is usually administered systemically or locally as an active ingredient of the drug in an oral or parenteral form.
  • oral preparation examples include a liquid preparation (for example, an expectorant, a syrup, a pharmaceutically acceptable aqueous preparation, a suspension, an emulsion), and a solid preparation (for example, a tablet (including a sublingual tablet). , orally disintegrating tablets), pills, capsules (including hard capsules, soft capsules, gelatin capsules, microcapsules), powders, granules, troches, etc.
  • parenteral agent examples include liquid preparations (for example, injections (subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drip injections, etc.), eye drops (for example, aqueous eye drops (aqueous drops) Eye drops, water-based teardrop eye drops, viscous eye drops, soluble eye drops, etc.), non-aqueous eye drops (non-aqueous eye drops, non-aqueous teardrop eye drops, etc.), etc.), external preparations ( For example, ointment (eye ointment), ear drops, and the like. These preparations may also be release controlling agents such as immediate release preparations and sustained release preparations. These preparations can be produced by a known method.
  • injections subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drip injections, etc.
  • eye drops for example, aqueous eye drops (aqueous drops) Eye drops, water-based teardrop eye drops, viscous eye drops,
  • the liquid preparation for internal administration as an oral preparation can be prepared, for example, by dissolving, suspending or emulsifying the active ingredient in a commonly used diluent (for example, purified water, ethanol or a mixture thereof). Further, the liquid preparation may further contain a wetting agent, a suspending agent, an emulsifier, a sweetener, a flavor, a fragrance, a preservative, a buffer, and the like.
  • a commonly used diluent for example, purified water, ethanol or a mixture thereof.
  • the liquid preparation may further contain a wetting agent, a suspending agent, an emulsifier, a sweetener, a flavor, a fragrance, a preservative, a buffer, and the like.
  • the solid preparation for internal administration as an oral agent can be, for example, by combining the active ingredient with an excipient (for example, lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), a binder (for example, hydroxypropylcellulose, poly Vinyl pyrrolidone, magnesium metasilicate aluminate, etc., disintegrant (for example, cellulose gluconate, etc.), lubricant (for example, magnesium stearate, etc.), stabilizer, cosolvent (glutamic acid, ascorbic acid) Etc., etc., mixed, and formulated according to a conventional method. Further, if necessary, it may be coated with a coating agent (for example, white sugar, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate or the like), and may be coated with two or more layers.
  • an excipient for example, lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.
  • the external preparation as a parenteral agent can be prepared by a known method or a formulation which is usually used.
  • an ointment can be prepared by grinding or melting an active ingredient in a base.
  • the ointment base can be either known or selected from the bases commonly used. For example, it may be selected from higher fatty acids or higher fatty acid esters (for example, adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipate, myristate, palmitic acid, stearic acid).
  • the oil, the absorbing agent, and the anti-tumor agent may be used alone or in combination of two or more. Further, a
  • the injection as a parenteral agent includes a solid injection which is dissolved or suspended in a solution, a suspension, an emulsion, and a solvent for use.
  • a solvent for use for example, an injection preparation obtained by dissolving, suspending or emulsifying the active ingredient in a solvent can be used.
  • the solvent for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohol such as ethanol, or the like, or a combination thereof can be used.
  • the injection may further contain a stabilizer, a solubilizing agent (for example, glutamic acid, ascorbic acid, polysorbate 80, etc.), a suspending agent, an emulsifier, a pain-relieving agent, a buffering agent, a preservative, and the like.
  • a solubilizing agent for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • a suspending agent for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • an emulsifier for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • a suspending agent for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • an emulsifier for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • a suspending agent for example, glutamic acid, ascorbic acid, polysorbate 80, etc.
  • an emulsifier for example,
  • the amount of the compound of the present invention to be administered as a pharmaceutically active ingredient can be appropriately selected depending on the symptoms, age, dosage form, etc., and if it is an oral preparation, it is preferably administered once to several times (for example, 1 to 3 times) per day to 1 to 500 mg. In the case of an eye drop, it is preferably 1 to several times (for example, 1 to 8 times) or 1 to several drops per one time, and a concentration of 0.000001 to 5% (w/v) is dripped into the eye, and more preferably 0.00001%. An eye drop of 0.05% (w/v) concentration.
  • an ointment of a concentration of 0.000001 to 5% (w/w), more preferably 0.00001 to 0.05% (w/w), once or several times per day (for example, 1 to 4 times).
  • the amount of administration may vary depending on various conditions, and therefore, it may be sufficient to give it in an amount smaller than the above-described amount of administration, and it may be necessary to exceed the above range.
  • Figure 1 shows the inhibitory effect of the test substance on the activation of donor 517T cells.
  • Figure 2 shows the inhibitory effect of the test substance on the activation of donor 517B cells and peripheral blood mononuclear cells.
  • Figure 3 is a graph showing the inhibitory effect of the test substance on the activation of donor 581T cells.
  • Figure 4 shows the inhibitory effect of the test substance on the activation of donor 581B cells and peripheral blood mononuclear cells.
  • Figure 5 is a graph showing the inhibitory effect of the test substance on the activation of donor 956T cells.
  • Figure 6 shows the inhibitory effect of the test substance on the activation of donor 956B cells and peripheral blood mononuclear cells.
  • Figure 7 shows the relative expression levels of p-Btk/t-Btk stimulated by anti-lgG after DOHH2 cells were subjected to sample treatment.
  • Figure 8 shows the relative expression levels of p-PLC ⁇ /t-PLC ⁇ after stimulation with anti-lgG in DOHH2 cells.
  • Figure 9 shows the relative expression levels of p-Erk/t-Erk stimulated by anti-lgG after DOHH2 cells were subjected to sample treatment.
  • Figure 10 shows changes in body weight of male SD rats after taking the test substance.
  • Figure 11 shows changes in body weight of female SD rats after taking the test substance.
  • Figure 12 shows changes in food intake in male SD rats after taking the test substance.
  • Figure 13 is a graph showing changes in food intake in female SD rats after taking the test substance.
  • the position of separation obtained by chromatography and the solvent in parentheses indicated by TLC indicate the elution solvent or developing solvent used, and the ratio indicates the volume ratio.
  • the names of the compounds used in the present specification are generally designated by ACD/Name (registered trademark), a computer program named by the IUPAC Rule, or by the IUPAC nomenclature.
  • the compound 4-aminopyrazolo[3,4-d]pyrimidine (50 g, 0.37 mol) and N,N-dimethylformamide (200 mL) were added to a three-necked flask, and N-brominated amber was added dropwise at room temperature.
  • a solution consisting of an imide (78.1 g, 0.44 mol) and N,N-dimethylformamide (150 mL) was added to a solution of the mixture, and the mixture was stirred at 65 ° C for 4 h. The reaction solution was evaporated to dryness.
  • Example 2 The compound prepared in Example 1 (20 g, 0.093 mol), the compound N-BOC-3-hydroxypiperidine (28.21 g, 0.14 mol) and triphenylphosphine (85.79 g, 0.33 mol) were added to anhydrous. In tetrahydrofuran (200 mL), it was light brown suspension, dropped to 0 ° C, diisopropyl azodicarboxylate (66.14 g, 0.33 mol) was added dropwise, and the temperature was kept below 5 ° C during the dropwise addition, and the solution gradually changed to light.
  • Lithium aluminum hydride (4 eq) was added to anhydrous tetrahydrofuran (20 vol) at N 2 , -10 ° C, maintaining the temperature below -10 °C.
  • Example 5 the compound obtained in Example 5 (0.1 g of) was dissolved in 5ml The 2- methyltetrahydrofuran, 7% NaHCO 3 solution was added. Acryloyl chloride (1.1 eq) was dissolved in 2-methyltetrahydrofuran and slowly added dropwise to the reaction flask. The mixture was stirred at 0 ° C for 2 h, and then EtOAc (EtOAc:EtOAc:EtOAc:EtOAc:EtOAc: The organic layer was concentrated under reduced pressure.
  • EtOAc EtOAc (EtOAc:EtOAc:EtOAc ⁇ 8.51 - 8.54 (d, 1H), 8.41 - 8.42 (m, 2H), 8.11 - 8.32 (m, 2H), 7.85 - 7.89 (m, 2H), 7.78 - 7.82 (m, 2H), 7.18 - 7.21. (m, 2H), 6.52-6.63 (m, 1H), 6.13-6.21 (m, 1H), 5.69-5.77 (m, 1H), 3.81-3.87 (m, 1H), 3.60-3.3.63 (m, 1H), 3.19-3.33 (m, 2H), 1.78-2.08 (m, 2H), 1.47-1.58 (m, 2H)
  • Example 7 The compound obtained in Example 7 was subjected to the procedure of Example 6 to give Compound DD 1-2. ⁇ 8.47-8.55 (d, 1H), 8.39-8.40 (m, 2H), 8.09-8.27 (m, 2H), 7.81-7.85 (m, 2H), 7.75-7.81 (m, 2H) 7.16-7.19 ( m,1H)6.49-6.61(m,1H),6.09-6.15(m,1H),5.66-5.73(m,1H),3.79-3.85(m,1H),3.58-3.61(m,1H),3.11 -3,26(m,2H), 2.57-2.65(s,3H),1.76-2.06(m,2H),1.43-1.55(m,2H)
  • Example 8 The procedure of Example 7 ⁇ Example 8 was carried out by using acetaldehyde instead of paraformaldehyde to obtain Compound DD001-3.
  • Example 8 The procedure of Example 7 ⁇ Example 8 was carried out by using acetone instead of paraformaldehyde to obtain the compound DD001-4.
  • Example 2 The compound obtained in Example 2 (0.2 g) was dissolved in N,N-dimethylformamide (5 ml), styrene (1.2 eq), N,N-diisopropylethylamine (2.5 eq). Nitrogen was added to tris(o-tolyl)phosphine (0.1 eq), palladium acetate (0.1 eq). Incubate at 100 ° C for 10 h. After completion of the reaction, the mixture was concentrated under reduced pressure.
  • Example 12 1-(3-(4-Amino-3-styryl)-1H-pyrazolo[3,4-D]pyrimidin-1-yl)-1-piperidinyl]-2-propene -1-ketone (DD001-5)
  • Example 11 The compound obtained in Example 11 was subjected to the operation of Example 6 to give Compound DD 001.
  • Example 13 1-(3-(4-Amino-3-(2-(2-pyridyl)vinyl)-1H-pyrazolo[3,4-D]pyrimidin-1-yl)-1- Piperidinyl]-2-propen-1-one (DD001-6)
  • Example 11 The styrene was replaced by 2-vinylpyridine, and the procedure of Example 11 ⁇ Example 12 gave Compound DD 1-6.
  • Example 14 1-(3-(4-Amino-3-(4-chlorostyryl)-1H-pyrazolo[3,4-D]pyrimidin-1-yl)-1-piperidinyl] -2-propen-1-one (DD001-7)
  • Example 11 The styrene was replaced by 4-chlorostyrene, and the procedure of Example 11 ⁇ Example 12 gave Compound DD001-7.
  • Example 15 1-(3-(4-Amino-3-(3-chlorostyryl)-1H-pyrazolo[3,4-D]pyrimidin-1-yl)-1-piperidinyl) -2-propen-1-one (DD001-8)
  • the compound DD001-8 was obtained by the operation of Example 11 ⁇ Example 12 using 3-chlorostyrene instead of styrene.
  • Example 2 The compound obtained in Example 2 (10 g) was dissolved in dichloromethane (100 ml), triethylamine (2.1 eq) was added, and di-tert-butyl dicarbonate (1.3 eq) was added at about 10 ° C, and the mixture was stirred for 4-5 h. The reaction is completed. Wash with 10% citric acid overnight. The organic layer was washed with a saturated aqueous
  • Example 17 The compound obtained in Example 17 (12 g), 4-cyanobenzeneboronic acid (1.2 eq), potassium carbonate (2.5 eq) was dissolved in dioxane/water (120 ml / 40 ml) Tetrakistriphenylphosphine palladium (0.05 eq) was added and nitrogen gas was bubbled off for 10 min. Heat to reflux, heat for 4-5h, the reaction is completed. The mixture was cooled to a temperature of about 40 ° C, and the organic layer was concentrated to dryness to give a white compound (10 g).
  • Example 19 0.5 g (1.0 eq) of the compound obtained in Example 19 was suspended in toluene, triethylamine (2.0 eq) was added, acetyl chloride was added dropwise, and a solid precipitated at 100 ° C overnight. After completion of the reaction, 6 vol of H 2 O and 6 vol of ethyl acetate were added. The organic layer was washed with aq. EtOAc (EtOAc)EtOAc. The silica gel column gave a pale white solid (0.2 g).
  • Example 20 The compound obtained in Example 20 (0.2 g) was dissolved in tetrahydrofuran (10 ml), and concentrated hydrochloric acid (1 ml). After completion of the reaction, sodium carbonate was adjusted to pH 7-8, and ethyl acetate (10 ml x 2) was extracted. Dry over anhydrous sodium sulfate and EtOAc (EtOAc)EtOAc.
  • Example 22 1-(3-(4-Amino-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)-1H-pyrazolo[3, 4-D]pyrimidin-1-yl)-1-piperidinyl)-2-propen-1-one (DD001-10)
  • Example 21 The compound obtained in Example 21 was subjected to the operation of Example 6 to give Compound DD001-10.
  • Example 19 The product obtained in Example 19 was subjected to the operation of Example 20 ⁇ Example 21 ⁇ Example 22 using acryloyl chloride instead of acetyl chloride to give Compound DD001-11.
  • Example 24 1-(3-(4-Amino-3-(4-(5-(3-chloropropyl)-1,2,4oxadiazol-3-yl)phenyl)-1H-pyridyl Zoxa[3,4-D]pyrimidin-1-yl)-1-piperidinyl)-2-propen-1-one DD001-12
  • Example 19 The product obtained in Example 19 was subjected to the operation of Example 20 ⁇ Example 21 ⁇ Example 22 using 4-chlorobutyryl chloride instead of acetyl chloride to give Compound DD001-12.
  • Example 19 The product obtained in Example 19 was subjected to the operation of Example 20 ⁇ Example 21 ⁇ Example 22 using benzoyl chloride instead of acetyl chloride to give Compound DD001-13.
  • Example 28 The compound obtained in Example 28 (1.0 g) was subjected to the operation of Example 4 ? ? ? ? ? ? ? ? ? ?
  • Example 28 The compound obtained in Example 28 (1.0 g) was subjected to the operation of Example 4 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
  • Example 28 The compound obtained in Example 28 (1.0 g) was subjected to the procedure of Example 4 ? ? ? ? ? ? ? ?
  • Example 28 The compound obtained in Example 28 (1.0 g) was subjected to the operation of Example 4 ? ? ? ? ? ? ? ? ?
  • Example 26 The compound of Example 26 (10 g) was dissolved in dioxane (150 ml), 4-bromobenzonitrile (1.3 eq) was added, BINAP (1,1 '-binaphthalene-2,2'-bisdiphenylphosphine) (0.2 Eq), potassium tert-butoxide (3.0 eq), palladium acetate (0.2 eq). Nitrogen is deaerated, heated to reflux, and incubated for 4-6 h. The reaction was completed and concentrated to dryness under reduced pressure. Ethyl acetate (100 ml) and water (30 ml) were added to the concentrate. The layers were separated and the aqueous layer was evaporated elut The organic layer was combined, washed with brine and dried over anhydrous sodium sulfate. Concentrate to dryness under reduced pressure to give a pale yellow solid.
  • BINAP 1,1 '-binaphthalene-2,2'-bisdiphenylphosphine
  • Example 33 The compound obtained in Example 33 was subjected to the operation of Example 19 ⁇ Example 20 ⁇ Example 21 ⁇ Example 22 to give Compound DD001-18.
  • Example 35 9-(1-Allylacylpiperidin-3-yl)-6-amino-7-(4-(5-vinyl-1,2,4-oxadiazol-3-yl)benzene Base)-7,9-dihydro-8H-indol-8-one DD001-19
  • Example 33 The compound obtained in Example 33 was subjected to the operation of Example 19 ⁇ Example 23 to give Compound DD001-19.
  • Example 36 9-(1-Allylacylpiperidin-3-yl)-6-amino-7-(4-(5-(3-chloropropyl)-1,2,4-oxadiazole- 3-yl)phenyl)-7,9-dihydro-8H-indol-8-one DD001-20
  • Example 33 The compound obtained in Example 33 was subjected to the operation of Example 19 ⁇ Example 24 to give Compound DD001-20.
  • Example 33 The compound obtained in Example 33 was subjected to the operation of Example 19 ⁇ Example 25 to give Compound DD001-21.
  • Example 40 4-(9-(1-Acrylamidepiperidin-3-yl)-6-amino-8-oxo-8,9-dihydro-7H-indol-7-yl)-N-( Pyridin-2-yl)benzamide DD001-22
  • Example 38 The compound obtained in Example 38 was replaced by 4-bromobenzonitrile, and the procedure of Example 33 was carried out, and the obtained product was subjected to the procedure of Example 28 ⁇ Example 29 to give Compound DD001-22.
  • Example 39 The compound obtained in Example 39 was substituted for 4-phenoxybenzeneboronic acid, and the procedure of Example 3 ⁇ Example 4 ⁇ Example 5 ⁇ Example 6 was carried out to give Compound DD001-23.
  • Example 3 The procedure of Example 3 ⁇ Example 4 ⁇ Example 5 ⁇ Example 6 was carried out by substituting 4-(2-fluorophenoxy)benzeneboronic acid for 4-phenoxybenzeneboronic acid to give Compound DD001-24.
  • Example 11 The styrene was replaced by 5-fluoro-2-vinylpyridine, and the procedure of Example 11 ⁇ Example 12 gave Compound DD001-25.
  • Example 44 The operation of Example 44 was carried out by replacing 4-(dimethylamino)but-2-enoic acid with buty-2-ynoic acid to give the title compound DD001-27. ⁇ 8.6 (d, 1H), 8.21 (d, 1H), 7.98 (d, 1H), 7.83 (d, 1H), 7.6 (brs, 2H), 7.53 (m, 1H), 7.49 (m, 1H) , 7.31 (m, 1H), 6.71-6.91 (m, 1H), 6.07-6.15 (m, 1H), 5.60-5.72 (m, 1H), 4.07-4.24 (m, 2H), 3.70-3.76 (m, 1H), 2.30-2.33 (m, 1H), 2.10-2.18 (m, 1H), 1.61-1.62 (m, 1H), 1.23-1.38 (m, 1H)
  • Example 46 1-(3-(4-Amino-3-(2-(pyridin-2-yl)vinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine -1-yl)-4-(dimethylamino)butan-2-propen-1-one DD001-28
  • Example 13 and Example 44 The procedure of Example 13 and Example 44 was carried out by substituting 4-(dimethylamino)but-2-enoic acid for acryloyl chloride to give the compound 1-(3-(4-amino-3-(2-(pyridine-2). -yl)vinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)butan-2-propen-1-one DD001 -28.
  • Example 46 The operation of Example 46 was carried out by replacing 4-(dimethylamino)but-2-enoic acid with buty-2-ynoic acid to give the title compound (1-(3-(4-amino-3-(2-(pyridine)). -2-yl)vinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-yn-1-one DD001-29.
  • Example 6 The compound 1-(4-phenoxyphenyl) 3-piperidin-3-ylimidazo[1,5-a]pyrazine-8-amine was subjected to the operation of Example 4 ⁇ Example 5 ⁇ Example 6, The compound N-(3-(8-amino-1-(4-phenoxyphenyl)imidazo[1,5-a]pyrazin-3-yl)piperidin-1-yl)acrylamide was obtained.
  • Buffer 50 mM Hepes pH 7.0, 0.1 mM Orthovanadate, 5 mM MgCl 2 , 0.01% BSA;
  • TK-peptide 1 mM TK-peptide, 20 mM ATP, 50 nM SEB;
  • Chromogenic buffer 50 mM Hepes pH 8.0, 0.8 M KF, 20 mM EDTA, 0.01% BSA;
  • the value (IC 50 value) of the 50% inhibition rate of the test compound was calculated from the inhibition curve based on the inhibition rate at each concentration of the test compound.
  • the determination of the inhibitory activity of other kinases uses various kinases in place of Btk, and operates in the same manner as described above.
  • the target compound of the present invention has not only Btk inhibitory activity but also Btk selective inhibitory activity against other kinases.
  • test compound solution (10 mM, 5 ⁇ L) with a methanol solution (495 ⁇ L) to prepare a 100 ⁇ M solution.
  • NADPH regeneration system isocitrate dehydrogenase final concentration 1 unit/mL
  • liver microsome solution final concentration 0.5mg protein/mL
  • the co-culture system is as follows:
  • the co-culture system is as follows:
  • the co-culture system is as follows:
  • T Cell Activation is shown in Figure 1.
  • T Cell Activation is shown in Figure 3.
  • Example Compound 6 and Comparative (Ibrutinib) showed significant inhibition of B cell activity at a concentration of 0.1 uM and showed inhibition of T cell activity at a concentration of 10 uM.
  • test substances were set to three different concentrations, 5 nM, 100 nM and 2000 nM, respectively, and each concentration was repeated once.
  • test substance was added to the wells and DMSO was used as a control, and the final concentration of DMSO was 0.1%.
  • DOHH2 cells were incubated with the test substance for 1 h, and washed three times with 10 volumes of PBS solution, followed by stimulation with anti-IgG (30 ⁇ g/mL; ab98531) for 2 min.
  • Tanon 5200 multi detects chemiluminescence.
  • Positive control drug Amitriptyline hydrochloride (Sigma-Aldrich, internationally accepted standard hERG channel blocker)
  • Extracellular fluid HEPES 10, NaCl 145, KCl 4, CaCl 2 2, MgCl 2 1, Glucose 10, adjusted to pH 7.4 with 1 N sodium hydroxide; osmotic pressure to 290-300 mOsm, filtered, and stored at 4 °C.
  • Intraelectrode solution (in mM): HEPES 10, KOH 31.25, KCl 120, CaCl2 5.374, MgCl2 1.75, EGTA 10, Na2-ATP 4, adjusted to pH 7.2 with 1N hydrogen peroxide; osmotic pressure to 280-290 mOsm, filtered, - Store at 20 ° C.
  • Compound preparation positive control drug amitriptyline hydrochloride, compound 6 and Ibrutinib were first dissolved in 100% DMSO (Sigma-Aldrich, D2650), and each was configured as a 30 mM stock solution. The above stock solution was diluted with DMSO to a solution of 1000 times each test concentration before the experiment, and then diluted 1000 times to the desired concentration with the extracellular liquid. The final concentration of DMSO in the extracellular fluid was 0.1%.
  • the stable cell line CHO-hERG was purchased from AVIVA.
  • the minimum sealing resistance is not less than 500 M ⁇
  • the hERG current is not less than 0.4 nA.
  • the hERG current was recorded using whole cell patch clamp technique.
  • the cell suspension was applied to a 35 mm Petri dish and placed on an inverted microscope stage. After the cells were attached, the cells were perfused with extracellular fluid at a flow rate of 1-2 mL/min.
  • the glass microelectrode is drawn in two steps by a microelectrode drawing device, and its water inlet resistance is 2-5 M ⁇ .
  • the clamping potential was maintained at -80 mV.
  • the voltage was stimulated to depolarize to +60 mV and then repolarized to -50 mV to induce the hERG tail current. All records were performed after the current was stabilized. Extracellular perfusion administration starts from a low concentration, each concentration is 5-10 min until the current is stable, and the next concentration is given.
  • mice Twenty male SD rats, 20 females, SPF grade were selected and divided into 4 groups according to their body weight by simple randomization, 10 in each group and half female.
  • the first group (0 mg/kg) animals were orally administered with blank vehicle, group 2 (400 mg/kg), group 3 (1000 mg/kg), group 4 (2000 mg/kg). Animals were given DD001y by oral gavage. Continuous observation for 14 days.
  • Vehicle control group 30 ml of a solvent (0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate) was pipetted into a suitable container to measure a pH of 7, which was a colorless clear solution.
  • Compound 6 400 mg/kg: Weigh 1507.1 mg of compound 6 into a suitable container, and add a solvent (0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate) to the container to a volume of 30 ml. After 20 minutes, stirring for 15 minutes, using a mixer for 5 minutes, measuring a pH of 7, as a white suspension.
  • a solvent 0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate
  • Compound 6 1000 mg/kg: Weigh 3765.5 mg of compound 6 into a suitable container, and add a solvent (0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate) to the container to a volume of 30 ml. After 25 minutes, the mixture was stirred for 21 minutes, and the mixture was used for 5 minutes, and the pH was measured to be 7 as a white suspension.
  • a solvent 0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate
  • Compound 6 (2000 mg/kg): Weigh 7530.5 mg of compound 6 into a suitable container, and add a solvent (0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate) to a volume of 30 ml, ultrasonication. After 30 minutes, the mixture was stirred for 31 minutes and the pH was measured to be 7 as a white suspension.
  • a solvent 0.5% MC (methylcellulose), 0.4% cremophor EL, 0.1% sodium lauryl sulfate
  • the maximum tolerated dose (MTD) of SD rats after a single oral administration of Compound 6 was 2000 mg/kg.
  • the first group of 3 animals was administered by intravenous injection, and the administration was performed once a day at a dose of 2 mg/kg, and the administration volume was 5 mL/kg.
  • the 3 animals of the second group were administered orally by intragastric administration, and administered once a day on the day of administration, at a dose of 10 mg/kg, and a dose of 10 mL/kg.
  • the first group of 3 animals was administered by intravenous injection, and the administration was performed once a day at a dose of 2 mg/kg, and the administration volume was 2.5 mL/kg.
  • the 3 animals of the second group were administered orally by intragastric administration, and administered once a day on the day of administration, the dose was 30 mg/kg, and the administration volume was 5 mL/kg.
  • the partial pharmacokinetic parameters of compound 6 administered to SD rats are shown in the following table. After intravenous administration of 2 mg/kg of compound 6 to SD rats, Cmax was 658.49 ng/mL, and AUC (0-t) was 270.52 h*ng/ mL; Cmax was 447.09 ng/mL after oral administration of 10 mg/kg of Compound 6, and AUC (0-t) was 449.18 h*ng/mL.
  • Ibrutinib data is from the FDA Database.http://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/205552Orig1s000ClinPharmR.pdf (accessed Jun 2015).
  • the tumor cell lines were cultured in an incubator at 37 ° C, 5% CO 2 according to the respective culture conditions. Passage periodically, taking cells in the logarithmic growth phase for plating.
  • a 400X compound storage plate was prepared: the compound was diluted from the highest concentration gradient to the lowest concentration with DMSO.
  • CellTiter-Glo buffer was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to prepare a CellTiter-Glo working solution.
  • B-Lymphoma Cell Line Growth IC 50 (nM) B-Lymphoma Cell Line Growth IC 50 (nM) TMD8 0.85 OCI-LY19 6788 DOHH-2 ⁇ 3 Granta-519 7065 OCI-LY10 6 Jeko-1 >10000 SU-DHL-4 75 SU-DHL-6 >10000 WSU-DLCL-2 921 SU-DHL-10 >10000 WSU-NHL 1019 DB >10000 Ramos 1398 OCI-LY3 >10000 Mino 2174 Raji >10000

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Abstract

本发明提供了一类具有Btk选择性抑制活性的新化合物，具有更好的代谢稳定性等。

Description

一种具有药物活性的杂芳基化合物 技术领域
本发明涉及具有药物活性的杂芳基化合物技术领域。
背景技术
Bruton型酪氨酸激酶(以下，简称为Btk)属于作为非受体型酪氨酸激酶的Tec家族激酶,选择性出现在B细胞系和骨髓细胞系的细胞中。Btk担负着B细胞的信号传递的重要作用,是有助于B细胞的生存、分化、增殖和活化等的因子。经由B细胞抗原受体(B cell antigen receptor；BCR)的B细胞的信号在广泛范围内诱发生物学反应，在其信号传递异常的情况下，引发B细胞的异常活化和/或病原性的自身抗体的形成等。认为Btk担负着向经由该BCR的B细胞的信号传递通路的一部分的作用。因此，己知，由于人Btk基因的缺失，会诱发B细胞的异常分化，使得免疫球蛋白的产生显著降低，由此产生X连锁性无γ-球蛋白血症(XLA)的发病(参照非专利文献1)。作为该疾病的症状，可举出末梢血中的B细胞显著减少、和/或对细菌感染的感受性增加等。另外，还己知Btk涉及肥大细胞的活化和/或血小板的生理功能。因此，具有Btk抑制活性的化合物对与B细胞和/或肥大细胞相关的疾病，例如，变应性疾病、自身免疫疾病、炎性疾病、血栓栓塞性疾病、癌症等的治疗是有用的(参照非专利文献2)。
但是，作为本发明化合物的现有技术，己知有以下的化合物。
作为具有Btk抑制剂性的化合物，己知有通式(A)
Figure PCTCN2018092122-appb-000001
(式中，LaA表示CH2、0、NH或S,ArA表示取代或未取代的芳基、或者取代或未取代的杂芳基，YA表示选自烷基、杂烷基、环烷基、杂环烷基、芳基和杂芳基的任意取代基，ZA表示CO、OCO、NHCO、CS,R7-A和R8-A独立地表示H、未取代的Cl-C4烷基、取代的Cl-C4烷基、未取代的Cl-C4杂烷基、取代的Cl-C4杂烷基、未取代的C3-C6环烷基、取代的C3-C6环烷基、未取代的C2-C6杂环烷基、和取代的C2-C6杂环烷基，或者R7-A和R8-A一起形成键，R6-A表示H、取代或未取代的Cl-C4烷基、取代或未取代的Cl-C4杂烷基、C1-C6烷氧 基烷基、C1-C8烷基氨基烷基、取代或未取代的C3-C6环烷基、取代或未取代的芳基(其中，各基团的定义是摘选的)。)表示的化合物(参照专利文献1、2和3)。
另外，己知有通式(B)
Figure PCTCN2018092122-appb-000002
(其中L表示-O-、-S-、-SO-、-SO2-、-NH-、-C(O)、-CH2O、-O-CH2-、-CH2-、或-CH(OH)-；R1表示卤素原子、C1-4烷基、C1-4烷氧基、C1-4卤代烷基、或C1-4卤代烷氧基；环1表示可被各自独立地选自卤素原子、C1-4烷基、C1-4烷氧基、氰基、C1-4卤代烷基、或C1-4卤代烷氧基的1-5个取代基取代的4-7元的环状基团，其中，环1上的取代基为2个以上时，该取代基可与它们所结合的构成环1的原子一起形成4-7元的环状基团；环2表示被1-3个-K-R2取代的4-7元的饱和杂环；K表示-C(O)-，其中，左侧的键与环2结合；R2表示可被各自独立地选自NR3R4、卤素原子、CONR5R6、COR7和OR8的1-5个取代基取代的C2-4烯基、或C2-4炔基；R3和R4各自独立地表示氢原子、或可被OR9或CONR10R11取代的C1-4烷基；R3和R4可与所结合的氮原子一起形成可被氧代基或羟基取代的4-7元的含氮饱和杂环；R5和R6各自独立地表示氢原子、C1-4烷基、或苯基；R7表示氢原子、或C1-4烷基；R8表示氢原子、C1-4烷基、苯基、或苯并三唑基；R9表示氢原子、C1-4烷基；R10和R11各自独立地表示氢原子、C1-4烷基；n表示0-4的整数，m表示0-2的整数，n为2个以上时，R1可以相同或不同)
现有技术文献
专利文献
专利文献1：特表2010-504324号公报
专利文献2：国际公开第2008/121742号小册子
专利文献3：国际公开第2010/009342号小册子
非专利文献
非专利文献1：Nature,第361卷，第226-233页，1993年
非专利文献2：Anticancer Agents in Medicinal Chemistry,第7卷，第6号，第624-632页，2007年
发明内容
本发明的目的在于提供一类具有Btk选择性抑制活性的新化合物，具有更好的代谢稳定性等。
为达上述目的，本发明采取的技术方案如下：
如下的通式的化合物Ia、Ib、Ic、Id、Ie、If或Ig，其光学异构体或它们的混合物、其盐、其溶剂合物、其N氧化物、或它们的前药：
Figure PCTCN2018092122-appb-000003
R 1表示H,取代或未取代的C 1-C 4烷基，取代或未取代的C 1-C 4杂烷基；
R 2表示R 6CO、R 7SO、R 8SO 2或被R 9取代的C 1-C 6烷基；
R 3表示H,一个或多个取代基，如卤素(氟、氯、溴、碘)，C 1-C 4烷氧基，硝基，氰基，取代或未取代的氨基，取代或未取代的烷基；
R 4表示H,一个或多个取代基，如卤素(氟、氯、溴、碘)，C 1-C 4烷氧基，硝基，氰基，取代或未取代的氨基，取代或未取代的烷基；
R 5表示取代或未取代的C 1-C 6烷基，取代或未取代的C 1-C 6杂烷基,取代或未取代的芳基，取代或未取代的杂芳基，取代或未取代的C 3-C 6环烷基，取代或未取代的C 2-C 6杂环烷基；
R 6是烯基、炔基，以及烷基、烯基、胺基或卤素取代的烯基、炔基；
R 7，R 8独立的选自C 2-C 6烯基或C 2-C 6炔基，二者任选被选自羟基、C 1-C 4烷基、C 3-C 7环烷基、(C 1-C 4烷基)氨基、二(C 1-C 4烷基)氨基、C 1-C 3烷氧基、C 3-C 7环烷氧基、C 6-C 10芳基或C 3-C 7杂环烷基的一个或多个基团取代；或任选被选自卤素或氰基的一个或多个基团取代的C 1-C 5杂芳基；
R 9独立的选自卤素、氰基或C 2-C 6烯基或C 2-C 6炔基，二者任选被选自羟基、C 1-C 4烷基、C 3-C 7环烷基、(C 1-C 4烷基)氨基、二(C 1-C 4烷基)氨基、C 1-C 3烷氧基、C 3-C 7环烷氧基、C 6-C 10芳基、C 1-C 5杂芳基或C 3-C 7杂环烷基的一个或多个基团取代；
Z是CH 2、O、NH、或S；
n 1表示0或1，n 2表示1-4的整数，n 3表示0-1的整数；
X表示氮或碳，可以在苯环的任一位置；Y表示氮。
优选的，
R 1表示H,C 1-C 4烷基，C 1-C 4杂烷基；
R 2表示R 6CO、R 7SO、R 8SO 2或被R 9取代的C 1-C 6烷基；R 6是烯基、炔基，以及烷基、烯基、胺基或卤素取代的烯基、炔基；R 7，R 8独立的选自C 2-C 6烯基或C 2-C 6炔基，R 9独立的选自卤素、氰基或C 2-C 6烯基或C 2-C 6炔基；
R 3表示H；
R 4表示H、卤素(氟、氯、溴、碘)、取代或未取代的烷基；
R 5表示取代或未取代的C 1-C 6烷基，取代或未取代的C 1-C 6杂烷基,取代或未取代的芳基，取代或未取代的杂芳基；
Z是O；
n 1表示0或1，n 2表示1-4的整数，n 3表示0-1的整数；
X表示氮或碳，可以在苯环的任一位置。
本专利中，通式的化合物Ia、Ib、Ic、Id、Ie、If或Ig亦表述为通式(Ia-Ig)表示 的化合物，或通式化合物Ia-Ig。
一种药物组合物，含有所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药。
所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为Btk抑制剂药物上的应用。
所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为Btk相关的疾病的预防和/或治疗剂的药物上的应用；进一步，在作为预防和/或治疗Btk相关的疾病如变应性疾病、自身免疫疾病、炎性疾病、血栓栓塞性疾病、或癌症的药物上的应用；更进一步，在作为预防和/或治疗非霍奇金淋巴瘤药物上的应用。
含有所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药的药物组合物作为B细胞活化抑制剂。
与Btk相关的疾病的预防和/或治疗方法，是向哺乳动物给予上述[l]所述的通式(Ia-Ig)表示的化合物、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药的有效量。
本发明的有益效果：
本发明提供的新化合物除了具有Btk选择性抑制活性以外，还是代谢稳定性优异、可避免肝脏毒性等的化合物，因此可用作安全性优异、与非霍奇金淋巴瘤等B细胞和/或肥大细胞相关的疾病的治疗剂。
本发明中，“具有Btk选择性抑制活性”是指对Btk以外的酪氨酸激酶，特别是对Lck(淋巴细胞特异性蛋白质酪氨酸激酶)、LynA(v-yes-1山口(Yamaguchi)肉瘤病毒相关癌基因同源亚型A)具有Btk选择性抑制活性。由该特性，可避免由抑制其他酪氨酸激酶而产生的不期望的副作用。例如己知在Lck缺失小鼠中发现了视网膜的异常(癌基因(oncogene)、第16卷、2351-2356页、1998年)，因此，抑制Lck时，有可能产生对眼的副作用。
本发明中，卤素原子是指氟、氯、溴、碘。
本发明中，C1-C4烷基是指甲基、乙基、丙基、异丙基、丁基、仲丁基、叔丁基等直链或支链的C1-C4烷基。
本发明中，C1-C4亚烷基是指亚甲基、亚乙基、亚丙基、亚丁基及它们的异构体等。本发明中，C1-C4烷氧基是指甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基等直链或支链的Cl-C4烷氧基。
本发明中，C2-C4烯基是指乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1，3-丁二烯基等直链或支链的C2-C4烯基。C2-C4炔基是指乙炔基，1-丙炔基，2-丙炔基，1-丁炔基，2-丁炔基，3-丁炔基，1，3-丁二炔基等直链或支链的C2-C4炔基。
异构体
本发明中，只要没有特殊说明，还包括所有的异构体。例如，烷基包括直链烷基和支链烷基。进而，双键、环、稠合环中的几何异构体(E型、Z型、顺式体、反式体)、由存在不对称碳原子等而产生的光学异构体(R,S型、α、β构型、对映异构体、非对映异构体)、具有旋光性的光学活性体(D、L、d、1型)、通过色谱分离产生的极性物(高极性物、低极性物)、平衡化合物、旋转异构体、它们的任意比例的混合物、外消旋混合物均包括在本发明中。另外，本发明中，也包括所有的由互变异构体产生的异构体。
另外，本发明中的光学异构体不仅包括100％纯的，而且包括小于50％的其它光学异构体。
本发明中，只要没有特殊说明，如本领域技术人员所熟知的符号
Figure PCTCN2018092122-appb-000004
表示键合至朝向纸面的一侧(即α构型)，
Figure PCTCN2018092122-appb-000005
表示键合至纸面的观察者眼前侧(即β构型)，
Figure PCTCN2018092122-appb-000006
表示α构型、β构型或它们的任意比例的混合物。
通式(Ia-Ig)表示的化合物可通过公知的方法转化为相应的盐。盐优选为水溶性的。作为适当的盐，可举出碱金属(钾、钠等)的盐、碱土类金属(钙、镁等)的盐、铵盐、药学上可接受的有机碱(四甲基铵、三乙胺、甲胺、二甲胺、环戊胺、苄胺、苯乙胺、哌啶、单乙醇胺、二乙醇胺、三(羟甲基)氨基甲烷、赖氨酸、精氨酸、N-甲基-D-葡糖胺等)的盐、酸加成物盐(无机酸盐(盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、磷酸盐、硝酸盐等)、有机酸盐(乙酸盐、三氟乙酸盐、乳酸盐、酒石酸盐、草酸盐、富马酸盐、马来酸盐、苯甲酸盐、柠檬酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、甲苯磺酸盐、羟乙基磺酸盐、葡糖醛酸盐、葡糖酸盐等)等。
通式(Ia-Ig)表示的化合物及其盐也可以转变为溶剂合物。溶剂合物优选为低毒性且水溶性的。作为适当的溶剂合物，例如可举出水、醇类的溶剂(例如，乙醇等)的溶剂合物。
另外，通式(Ia-Ig)表示的化合物的前药是指能够在生物体内通过酶和/或胃酸等发生反应，由此转变成通式(Ia-Ig)表示的化合物。作为通式(Ia-Ig)表示的化合物的前药，在通式 (Ia-Ig)表示的化合物具有羟基时，可举出该羟基被酰化、烷基化、磷酰化、硼酰化的化合物。
在药品中的用途
本发明化合物由于具有选择性的Btk抑制作用，因此有效用作与Btk相关的疾病，即与B细胞和/或肥大细胞相关的疾病、例如，变应性疾病、自身免疫疾病、炎性疾病、血栓栓塞性疾病、癌症、移植物抗宿主病等的预防和/或治疗剂。另外，本发明化合物还具有选择性抑制B细胞活化的作用，因此有效用作B细胞活化抑制剂。
本发明中，作为变应性疾病，例如可举出过敏、过敏性反应、过敏性结膜炎、过敏性鼻炎、特应性皮炎。
本发明中，作为自身免疫疾病，例如可举出炎性肠病、关节炎、狼疮、风湿、干癣性关节炎、变形性关节炎、斯蒂尔病、青少年性关节炎、I型糖尿病、重症肌无力症、桥本甲状腺炎、ord’s氏甲状腺炎，巴塞多氏病、舍格林氏综合征、多发性硬化症、吉兰-巴雷综合征、急性流行性脑脊髓炎、艾迪生病、斜视性眼阵孪综合征、强直性脊椎炎、抗磷脂抗体综合征、再生不良性贫血、自身免疫性肝炎、腹腔病、肺出血肾综合征、特发性血小板减少性紫癫病、视神经炎、硬皮症、原发性胆汁性肝硬化、赖特尔病、高安动脉炎、颞动脉炎、温抗体型自身免疫性溶血性贫血、韦氏肉芽肿病、干癣、全身性脱毛症、Behcet氏病、慢性疲劳综合征、自律神经失调、子宫内膜疾病、间质性膀胱炎、肌强直、外阴部痛、系统性红斑狼疮。
本发明中，作为炎性疾病，例如可举出哮喘、阑尾炎、眼睑炎、细支气管炎、支气管炎、滑液包炎、宫颈炎、胆管炎、胆囊炎、大肠炎、结膜炎、膀胱炎、泪腺炎、皮炎、皮肌炎、脑炎、心内膜炎、子宫内膜炎、肠炎、上髁炎、附睾炎、肌膜炎、结缔组织炎、胃炎、胃肠炎、肝炎、汗腺脓肿、喉炎、乳腺炎、髓膜炎、脊髓炎、心肌炎、肌炎、肾炎、卵巢炎、睾丸炎、骨炎、胰腺炎、腮腺炎、心膜炎、腹膜炎、咽炎、胸膜炎、静脉炎、肺炎、直肠炎、前列腺炎、肾盂肾炎、鼻炎、耳咽管炎、鼻窦炎、口腔炎、滑膜炎、腱炎、扁桃体炎、葡萄膜炎、阴道炎、血管炎、外阴炎。
本发明中，作为血栓栓塞性疾病，例如可举出心肌梗塞、心绞痛、血管形成术后的再闭塞、血管形成术后的再狭窄、大动脉冠状动脉旁路术后的再闭塞、大动脉冠状动脉旁路术后的再狭窄、脑梗塞、一时性缺血、末梢血管阻塞、肺栓塞、深静脉血栓症。
本发明中，作为癌症，包括非霍奇金淋巴瘤，其中优选B细胞性非霍奇金淋巴瘤，例如可举出伯基特淋巴瘤、AIDS相关性淋巴瘤、边缘区B细胞淋巴瘤(结边缘区B细胞淋巴瘤、结外边缘区B细胞淋巴瘤、脾边缘区B细胞淋巴瘤、弥漫性大细胞型B细胞淋巴瘤、原发性渗出性淋巴瘤、淋巴瘤样肉芽肿病、滤泡性淋巴瘤、B细胞慢性淋巴性白血病、B细胞前淋巴性白血病、淋巴浆细胞性白血病/瓦尔登斯特伦巨球蛋白血症、浆细胞瘤、外套细胞淋巴瘤、 纵膈大细胞型B细胞淋巴瘤、血管内大细胞型B细胞淋巴瘤、毛细胞白血病。另外，作为本发明中的癌症，作为非霍奇金淋巴瘤以外的癌症，包括膜腺内分泌肿瘤，例如可举出胰岛素瘤、胃泌素瘤、胰高血糖素瘤、生长激素瘤、VIP产生肿瘤(VIPoma)、PP产生肿瘤(PPoma)、GRF产生肿瘤。
本发明化合物可以单独给予，但是，为了
1)该化合物的预防和/或治疗效果的补充和/或增强；
2)该化合物的动态·吸收改善、给予量的降低、和/或；
3)该化合物的副作用的减轻；
也可以与其他药物组合，作为联合药物给予。
本发明化合物与其他药物的联合药物可以以在1个制剂中配合两种成分的配伍剂的形式给予，也可以制成在各自的制剂中给予的形式。在以该各自的制剂给予的情况下，包括同时给予和间隔时间差来给予的情况。另外，间隔时间差的给予既可以先给予本发明化合物、后给予其他药物，也可以先给予其他药物、后给予本发明化合物。它们各自的给予方法可以相同或不同。
对由上述联合药物发挥预防和/或治疗效果的疾病没有特殊限定，只要是能够补充和/或增强本发明化合物的预防和/或治疗效果的疾病即可。
作为用于补充和/或增强本发明化合物对变应性疾病的预防和/或治疗效果的其他药物，例如可举出抗组胺药、白三烯拮抗药、抗过敏药、血栓烷A2受体拮抗剂、血栓烷合成酶抑制剂、甾体。
作为用于补充和/或增强本发明化合物对自身免疫疾病的预防和/或治疗效果的其他药物，例如可举出免疫抑制剂、甾体、疾病修饰型抗风湿病药物、弹性硬蛋白酶抑制剂、大麻素-2受体刺激药、前列腺素、前列腺素合成酶抑制剂、磷酸二酯酶抑制剂、金属蛋白酶抑制剂、粘附分子抑制剂、抗TNF-α制剂、抗IL-1制剂、抗IL-6制剂等抗细胞因子蛋白制剂、细胞因子抑制剂、非甾体类抗炎药、抗CD20抗体。
作为用于补充和/或增强本发明化合物对炎性疾病的预防和/或治疗效果的其他药物，例如可举出甾体、弹性硬蛋白酶抑制剂、大麻素-2受体剌激药、前列腺素、前列腺素合成酶抑制剂、磷酸二酯酶抑制剂、金属蛋白酶抑制剂、粘附分子抑制剂、抗白三烯药、抗胆碱药、血栓烷A2受体拮抗剂、血栓烷合成酶抑制剂、黄嘌呤衍生物、祛痰药、抗菌药、抗组胺药、抗细胞因子蛋白制剂、细胞因子抑制剂、弗司扣林制剂、介质游离抑制剂、非甾体类抗炎药。
作为用于补充和/或增强本发明化合物对血栓栓塞性疾病的预防和/或治疗效果的其他药物，例如可举出溶栓药物、肝素、肝素类似物、低分子量肝素、华法林、凝血酶抑制剂、因 子Xa抑制剂、ADP受体拮抗剂、环氧合酶抑制剂。
作为用于补充和/或增强本发明化合物对非霍奇金淋巴瘤的预防和/或治疗效果的其他药物，例如可举出烷基化剂、代谢拮抗剂、抗癌性抗生素、植物性生物碱药、激素药、铂化合物、抗CD20抗体、其他抗癌剂。
作为抗组胺药的例子，例如可举出盐酸氮卓斯汀、依巴斯汀、盐酸依匹斯汀、富马酸依美斯汀、金诺芬、奥沙米特、盐酸奥洛他定、dl-马来酸氯苯那敏、富马酸氯马斯汀、富马酸酮替芬、西眯替丁、茶苯海明、盐酸苯海拉明、盐酸赛庚皖、盐酸西替利嗪、地氯雷他定、特非那定、法莫替丁、盐酸非索非那定、贝他斯汀、苯磺酸贝他斯汀、咪唑斯汀、美喹他嗦、糠酸莫米松、雷尼替丁、盐酸雷尼替丁、氯雷他定、盐酸异丙嗪、盐酸高氯环嗪。
作为白三烯拮抗药的例子，例如可举出普仑司特水合物、孟鲁司特钠、扎鲁司特、阿鲁司特、泊比司特、硫鲁司特、伊拉司特钠、维鲁司特、利托司特、因那司特、吡咯司特、托鲁司特、多夸司特。
作为抗过敏药的例子，例如可举出氨来呫诺、盐酸氮卓斯汀、伊拉帕泛、异丁司特、咪曲司特钠、依巴斯汀、盐酸依匹斯汀、富马酸依美斯汀、奥沙米特、盐酸奥扎格雷、盐酸奥洛他定、色甘酸、色甘酸钠、富马酸酮替芬、塞曲司特、盐酸西替利嗪、甲磺司特、他扎司特、特非那定、多米曲班钙水合物、曲尼司特、奈多罗米、非索非那定、盐酸非索非那定、吡嘧司特钾、美喹他嗪、雷马曲班、瑞吡司特、氯雷他定。
作为血栓烷A2受体抑制剂的例子，例如可举出塞曲司特、多米曲班钙水合物、雷马曲班。
作为血栓烷合成酶抑制剂的例子，例如可举出咪曲司特钠、盐酸奥扎格雷。
作为甾体的例子，例如可举出安西奈德、氢化可的松琥珀酸钠、泼尼松龙琥珀酸钠、甲泼尼龙琥珀酸钠、环索奈德、二氟泼尼醋、丙酸倍他米松、地塞米松、地夫可特、曲安西龙、曲安奈德、氯氟舒松、棕榈酸地塞米松、氢化可的松、氟米松新戊酸醋、丁乙酸泼尼松龙、布地奈德、硫酸普拉睾酮、糠酸莫米松、醋酸氟轻松、氟轻松、氟氢缩松、氟尼缩松、泼尼松龙、丙酸阿氯米松、丙酸氯倍他索、丙酸地塞米松、丙酸地泼罗酮、丙酸氟替卡松、丙酸倍氯米松、倍他米松、甲泼尼龙、磺庚甲泼尼龙、甲泼尼龙琥珀酸钠、磷酸地塞米松钠、氢化可的松磷酸钠、泼尼松龙磷酸钠、戊酸二氟可龙、戊酸地塞米松、戊酸倍他米松、泼尼松龙醋酸戊酸酯、醋酸可的松、双醋二氟拉松、醋酸地塞米松、醋酸曲安西龙、醋酸帕拉米松、醋酸卤泼尼松、醋酸氟氢可的松、泼尼松龙醋酸酯、甲泼尼龙醋酸酯、丁酸氯倍他松、丁酸氢化可的松、丁酸丙酸氢化可的松、丁酸丙酸倍他米松。
作为免疫抑制剂的例子，例如可举出硫唑嘌呤、子囊霉素、依维莫司、柳氮磺吡啶、环抱素、环磷酰胺、西罗莫司、他罗利姆、布西拉明、甲氨蝶呤、来氟米特。
作为疾病修饰型抗风湿病药物的例子，例如可举出D-青霉胺、阿克他利、金诺芬、柳氮磺吡啶、羟氯喹、布西拉明、甲氨蝶呤、来氟米特、氯苯扎利二钠、金硫葡糖、硫代苹果酸金钠。
作为前列腺素类(以下，简称为PG)，例如可举出PGEl制剂(例如：前列地尔α-环糊精包合物、前列地尔等)、PGI2制剂(例如：贝前列素钠等)、PG受体激动剂、PG受体拮抗剂等。作为PG受体，可举出PGE受体(EPl、EP2、EP3、EP4)、PGD受体(DP、CRTH2)、PGF受体(FP)、PGI2受体(IP)、TX受体(TP)等。
作为前列腺素合成酶抑制剂的例子，例如可举出柳氮磺吡啶、美沙拉嗪、奥沙拉秦、4-氨基水杨酸、金诺芬、卡洛芬、联苯吡胺、氟诺洛芬、氟比洛芬、吲哚美辛、酮洛芬、氯诺昔康、洛索洛芬、美洛昔康、奥沙普秦、帕沙米特、哌普生、吡罗昔康、吡罗昔康肉桂酸、扎托洛芬、普拉洛芬。
作为磷酸二醋酶抑制剂的例子，例如可举出咯利普兰、西洛司特、罗氟司特(BY-217)。
作为粘附分子抑制剂的例子，例如可举出α4-整联蛋白拮抗剂。
作为抗TNF-α制剂的例子，例如可举出抗TNF-α抗体、可溶性TNF-α受体、抗TNF-α受体抗体、可溶性TNF-α结合蛋白质，特别是可举出英夫利西单抗、依那西普。
作为抗IL-1制剂的例子，可举出抗IL-1抗体、可溶性IL-1受体、抗IL-lRa和/或IL-1受体抗体，特别是可举出阿那白滞素。
作为抗IL-6制剂的例子，可举出抗IL-6抗体、可溶性IL-6受体、抗IL-6受体抗体，特别是可举出托珠单抗。
作为细胞因子抑制剂的例子，例如可举出甲磺司特、T-614。
作为抗胆碱药的例子，例如可举出苯海索、盐酸苯海索、比哌立登、盐酸比哌立登。
作为黄嘌呤衍生物，例如可举出氨茶碱、茶碱、多索茶碱、西潘茶碱、二羟丙茶碱。
作为祛痰药，可举出小茴香氨酒精、碳酸氢钠、盐酸溴己新、羧甲司坦、盐酸氨溴索、盐酸甲基半胱氨酸、乙酰基半胱氨酸、L-盐酸乙基半胱氨酸、泰洛沙泊。
作为抗菌药的例子，例如可举出头孢呋辛钠、美罗培南三水合物、硫酸奈替米星、
硫酸西梭霉素、头孢布烯、PA-1806、IB-367、妥布霉素、PA-1420、多柔比星、硫酸阿司米星、盐酸头孢他美酯。
作为介质(mediator)游离抑制剂的例子，例如可举出曲尼司特、色甘酸钠、氨来呫诺、瑞吡司特、异丁司特、他扎司特、吡嘧司特钾等。
作为溶栓药物的例子，例如可举出阿替普酶、尿激酶、替来激酶、那沙普酶、那替普酶、t-PA、帕米普酶、孟替普酶、蛋白激酶、链激酶。
作为肝素类似物的例子，例如可举出磺达肝素。
作为低分子量肝素的例子，例如可举出达那肝素钠、依诺肝素(钠)、纳屈肝素钙、贝米肝素(钠)、瑞肝素(钠)、亭扎肝素(钠)。
作为凝血酶抑制剂的例子，例如可举出阿加曲班、希美加群、美拉加群、达比加群、比伐卢定、来匹卢定、水蛭素、地回卢定。
作为ADP受体拮抗剂的例子，例如可举出盐酸噻氯匹定、硫酸氯吡格雷。
作为环氧合酶抑制剂的例子，例如可举出阿司匹林。
作为烷基化剂的例子，例如可举出盐酸氮芥-N-氧化物、环磷酰胺、异环磷酰胺、美法仑、睡替派、卡巴琨、白消安、盐酸尼莫司汀、达卡巴嗪、雷莫司汀等。
作为代谢拮抗剂的例子，例如可举出甲氨蝶岭、巯嘌呤、6-巯嘌呤苷、氟尿嘧啶、替加氟、替加氟尿嘧啶、卡莫氟、去氧氟尿苷、阿糖胞苷、依诺他滨、替加氟·吉美斯特·氧嗪酸钾、盐酸吉西他滨、盐酸丙卡巴肼、羟基脲等。
作为抗癌性抗生素的例子，例如可举出放线菌素D、丝裂菌素C、盐酸柔红霉素、盐酸多柔比星、盐酸阿柔比星、新制癌菌素、盐酸吡柔比星、(盐酸)表柔比星、盐酸伊达比星、色霉素A3、(盐酸)博来霉素、硫酸培洛霉素、吡柔比星、净司他丁斯酯等。
作为植物性制剂的例子，例如可举出硫酸长春碱、硫酸长春新碱、硫酸长春地辛、盐酸伊立替康、依托泊苷、氟他胺、酒石酸长春瑞滨、多西他赛水合物、紫杉醇等。
作为激素剂的例子，例如可举出雌莫司汀磷酸钠、美雄烷、环硫雄醇、醋酸戈舍瑞林、磷雌酚(二磷酸己烯雌酚)、枸橼酸他莫昔芬、枸橼酸托瑞米芬、盐酸法倔唑水合物、醋酸甲羟孕酮、比卡鲁胺、醋酸亮丙瑞林、阿那曲唑、依西美坦等。
作为铂化合物的例子，例如可举出卡铂、顺铂、奈达铂等。
作为抗CD20抗体的例子，例如可举出利妥昔单抗、替伊莫单抗、奥瑞珠单抗。
作为其他抗癌剂的例子，例如可举出L-天冬酰胺酶、醋酸奥曲肽、卟菲尔钠、醋酸米托蒽醌。
另外，作为与本发明化合物组合的联合药物，不仅包括迄今为止己经出现的药物，还包括今后将会出现的药物。
本发明化合物通常作为药物有效成分以口服或非口服的形式全身或局部给予。作为口服剂，例如可举出内服用液体制剂(例如，酏剂、糖浆剂、药剂学可接受的水剂、混悬剂、乳剂)、内服用固体制剂(例如，片剂(包括舌下片、口腔崩解片)、丸剂、胶囊剂(包括硬胶囊、软胶囊、明胶胶囊、微囊)、散剂、颗粒剂、糖锭剂)等。作为非口服剂，例如可举出液体制剂(例如，注射剂(皮下注射剂、静脉内注射剂、肌内注射剂、腹腔内注射剂、滴 注剂等)、滴眼剂(例如，水性滴眼剂(水性滴眼液、水性泪悬滴眼液、粘性滴眼液、可溶性滴眼液等)、非水性滴眼剂(非水性滴眼液、非水性泪悬滴眼液等))等)、外用剂(例如，软膏(眼膏剂))、滴耳剂等。这些制剂也可以是速释制剂、缓释制剂等释放控制剂。这些制剂可以通过公知方法来制备。
作为口服剂的内服用液体制剂例如可通过将有效成分在通常使用的稀释剂(例如，纯化水、乙醇或它们的混合液等)中溶解、混悬或乳化来制备。进而，该液体制剂还可以含有湿润剂、助悬剂、乳化剂、甜味剂、风味剂、芳香剂、防腐剂、缓冲剂等。
作为口服剂的内服用固体制剂例如可通过将有效成分与赋形剂(例如，乳糖、甘露醇、葡萄糖、微晶纤维素、淀粉等)、粘合剂(例如，羟丙基纤维素、聚乙烯吡咯烷酮、偏硅酸铝酸镁等)、崩解剂(例如，纤维素葡糖酸钙等)、润滑剂(例如，硬脂酸镁等)、稳定剂、助溶剂(谷氨酸、抗坏血酸等)等混合，根据常规方法来制剂化。另外，根据需要还可以用包衣剂(例如，白糖、明胶、羟丙基纤维素、羟丙甲基纤维素酞酸酯等)包覆，并且可以用2个以上的层来包覆。
作为非口服剂的外用剂可以通过公知的方法或通常使用的配方来制备。例如，软膏剂可以通过将有效成分在基剂中研磨或熔融来制备。软膏基剂可以是公知的或者选自通常使用的基剂。例如，可以将选自高级脂肪酸或高级脂肪酸酯(例如，己二酸、肉豆蔻酸、棕榈酸、硬脂酸、油酸、己二酸酯、肉豆寇酸酯、棕榈酸、硬脂酸酯、油酸酯等)、蜡类(例如，蜜蜡、鲸蜡、地蜡等)、表面活性剂(例如，聚氧乙烯烷基醚磷酸酯等)、高级醇(例如，鲸蜡醇、硬脂醇、十六十八醇等)、硅油(例如，二甲基聚硅氧烷等)、烃类(例如，亲水凡士林、白色凡士林、纯化羊毛脂、液体石蜡等)、二醇类(例如，乙二醇、二甘醇、丙二醇、聚乙二醇、聚乙二醇等)、植物油(例如，蓖麻油、橄榄油、芝麻油、松节油等)、动物油(例如，貂油、蛋黄油、角鲨烷、角鲨烯等)、水、吸收促进剂、抗肿剂中的物质单独或2种以上混合使用。进而，还可以含有保湿剂、防腐剂、稳定剂、抗氧化剂、增香剂等。
作为非口服剂的注射剂包括溶解或混悬在溶液、混悬液、乳浊液和用时溶剂中使用的固态的注射剂。注射剂例如可以使用将有效成分在溶剂中溶解、混悬或乳化而成的注射剂。作为溶剂，例如可使用注射用蒸馏水、生理盐水、植物油、丙二醇、聚乙二醇、乙醇之类的醇类等及它们的组合。进而，该注射剂还可以含有稳定剂、助溶剂(例如，谷氨酸、抗坏血酸、聚山梨酯80等)、助悬剂、乳化剂、无痛化剂、缓冲剂、防腐剂等。它们可以在最终工序中通过灭菌或无菌操作法来制备。另外，还可以制备无菌的固体制剂，例如冻干品，可以在使用前将其溶解在无菌化或无菌的注射用蒸馏水或其他溶剂中来使用。
本发明化合物作为药物有效成分使用时的给予量可以根据症状、年龄、剂型等来适宜选 择，如果是口服剂，优选每日1次～数次(例如，1～3次)给予1～5OOmg。如果是滴眼剂，优选每日1～数次(例如，1～8次)、1～数滴/1次量，向眼睛点滴0.000001～5％(w/v)的浓度、更优选0.00001～0.05％(w/v)的浓度的滴眼剂。另外，如果是眼膏，优选每日1～数次(例如，1～4次)涂布0.000001～5％(w/w)、更优选0.00001～0.05％(w/w)的浓度的眼膏。
当然，如上所述，给予量可以根据各种条件而变化，因此，既存在以比上述给予量更少的量给予就足够的情况，也存在必须超出上述范围的情况。
说明书附图
图1为受试物对供体517T细胞活化的抑制作用
图2为受试物对供体517B细胞和外周血单核细胞活化的抑制作用
图3为受试物对供体581T细胞活化的抑制作用
图4为受试物对供体581B细胞和外周血单核细胞活化的抑制作用
图5为受试物对供体956T细胞活化的抑制作用
图6为受试物对供体956B细胞和外周血单核细胞活化的抑制作用
图7为DOHH2细胞经受试物处理后，用anti-lgG刺激，其p-Btk/t-Btk的相对表达水平
图8为DOHH2细胞经受试物处理后，用anti-lgG刺激，其p-PLCγ/t-PLCγ的相对表达水平
图9为DOHH2细胞经受试物处理后，用anti-lgG刺激，其p-Erk/t-Erk的相对表达水平
图10为雄性SD大鼠服用受试物后体重变化
图11为雌性SD大鼠服用受试物后体重变化
图12为雄性SD大鼠服用受试物后进食量的变化
图13为雌性SD大鼠服用受试物后进食量的变化
具体实施方式
以下，通过实施例详述本发明，但本发明并不限定于这些实施例。
由色谱得到的分离的位置和由TLC表示的括号内的溶剂表示所使用的洗脱溶剂或展开溶剂，比例表示体积比。
本说明书中使用的化合物的名称，一般采用基于IUPAC法则进行命名的计算机程序--Advanced Chemistry Development公司的ACD/Name(注册商标)，或者根据IUPAC命名法来命名。
实施例1：3-溴-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000007
向三口烧瓶中加入化合物4-氨基吡唑并[3,4-d]嘧啶(50g，0.37mol)和N,N-二甲基甲酰胺(200mL)，室温下滴加入由N-溴代琥珀酰亚胺(78.1g，0.44mol)和N,N-二甲基甲酰胺(150mL)组成的溶液，滴毕升至65℃搅拌反应4h。反应液蒸干，加入水(500mL)，0℃下搅拌1h，析出土黄色固体，调节pH＝7，过滤，滤饼依次以水(250mL)、冷乙醇(250mL)洗涤，真空干燥得土黄色固体65g，收率82％。
实施例2：3-溴-1-(3-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000008
氮气保护，将实施例1所制备化合物(20g，0.093mol)、化合物N-BOC-3-羟基哌啶(28.21g，0.14mol)和三苯基膦(85.79g，0.33mol)加入至无水四氢呋喃(200mL)中，呈淡棕色悬浊液，降至0℃，滴加入偶氮二甲酸二异丙酯(66.14g，0.33mol)，滴加过程保持温度5℃以下，溶液逐渐转变为淡黄色澄清，滴毕逐渐升至0-10℃搅拌反应3h，加入浓盐酸(78mL)，升至50℃搅拌反应2h，降至室温，过滤，滤饼加水溶解，以6N氢氧化钠溶液调节pH至8，二氯甲烷提取，无水硫酸钠干燥有机相，过滤，真空浓缩至干得类白色固体19.5g，收率70％。m/z(MH +)297,1H NMR(400MHz,DMSO)δ1.94-2.11,2.92-2.98,3.01-3.36，3.45-3.47，5.12-5.19,8.50-8.51,9.61-9.87。
实施例3：3-(4-苯氧基苯基)-1-(3-哌啶基)-1H-吡唑并[3,4-d]嘧啶-4-胺
Figure PCTCN2018092122-appb-000009
将实施例2所得化合物(1.0g，1.0eq)，4-苯氧基苯硼酸(1.7g，1.3eq)，碳酸钾(1.6g，4.0eq)悬浮于dioxane(21ml)/H 2O(9ml)，氮气鼓泡除氧10min。加入四三苯基膦钯(0.1g，0.05eq)，继续鼓泡5min。加热至回流，4h后取样TLC(二氯甲烷:甲醇＝9:1)。反应完毕，降至室温，分液，有机层浓缩得油状物。加入10ml水，15ml乙酸乙酯，有不溶物。4N盐酸调节pH至2-3，溶清。分液，去有机层，水层用乙酸乙酯10ml x 2洗涤。4N氢氧化钠溶液调节pH至8-9，加入乙酸乙酯20ml溶清，萃取。饱和食盐水10ml洗涤，无水硫酸钠3g干燥。减压浓缩，得淡黄色固体0.5g。
实施例4：1-(3-(1-亚硝基哌啶基))-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-4-胺
Figure PCTCN2018092122-appb-000010
将实施例3所得化合物(4.5g)悬浮于20ml水中，加入醋酸,溶清。N 2，10℃下滴加NaNO 2溶液20ml，有固体析出。0-10℃搅拌6h，取样TLC(乙酸乙酯:甲醇＝10:1)，反应完毕，加入碳酸钠固体消耗掉多余的醋酸，加入40ml乙酸乙酯。分液，水层用10ml x 2乙酸乙酯萃取，合并有机层，饱和食盐水洗涤。减压浓缩至干，得淡黄色固体4.5g。
δ8.25-8.3(d,1H),7.38-7.47(m,4H),7.18-7.21(m,2H),7.13-7.22(m,5H),5.79-5.82(s,2H),5.03-5.08(m,1H),4.84-4.91(m,1H),4.47-4.56(m,1H),4.11-4.15(m,1H),2.65-2.74(m,1H),2.60-2.64(m,1H)1.99-2.09(m,1H),1.72-1.75(m,1H)
实施例5：1-(3-(1-亚硝基哌啶基))-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶-4-胺
Figure PCTCN2018092122-appb-000011
N 2，-10℃,将氢化铝锂(4eq)加入无水四氢呋喃(20vol)中，维持温度-10℃以下。将实施例4所得化合物(2g)悬浮于无水四氢呋喃(10vol)中，将悬液缓慢滴加入反应瓶中，维持温度小于零摄氏度。滴毕，保温4h，取样TLC(乙酸乙酯:甲醇＝2:1)。Na 2SO 4·10H 2O，淬灭反应液，硅藻土过滤，滤饼用10ml四氢呋喃淋洗，减压浓缩至干，所得白色泡沫状固体过硅胶柱，得目标产物(1g)。
实施例6：N-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)丙烯酰胺(化合物DD001-1)
Figure PCTCN2018092122-appb-000012
N 2，将实施例5所得化合物(0.1g)溶于5ml 2-甲基四氢呋喃中，加入7％NaHCO 3溶液。将丙烯酰氯(1.1eq)溶解于2-甲基四氢呋喃中，缓慢滴加入反应瓶中。0℃以下搅拌2h，取样TLC(乙酸乙酯:二氯甲烷：甲醇＝10:5:1)静置分液，水层用5ml x 2乙酸乙酯萃取，合并有机层，饱和食盐水洗涤。减压浓缩，过硅胶柱(洗脱液乙酸乙酯:二氯甲烷:甲醇＝10:5:1)，得白色固体DD001-1(0.1g)。δ8.51-8.54(d,1H),8.41-8.42(m,2H),8.11-8.32(m,2H),7.85-7.89(m,2H),7.78-7.82(m,2H),7.18-7.21(m,2H),6.52-6.63(m,1H),6.13-6.21(m,1H),5.69-5.77(m,1H),3.81-3.87(m,1H),3.60-3.3.63(m,1H),3.19-3.33(m,2H),1.78-2.08(m,2H),1.47-1.58(m,2H)
实施例7：1-(3-(1-甲基氨基哌啶基))-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d]嘧啶 -4-胺
Figure PCTCN2018092122-appb-000013
将实施例5所得化合物(0.2g)悬浮于甲醇(5ml)中。N 2，加入多聚甲醛(1.0eq)，64℃回流4h，取样TLC(乙酸乙酯:甲醇＝2:1)。降温至室温，加入氰基硼氢化钠/乙醇溶液。加毕，64摄氏度回流2h，取样TLC(乙酸乙酯:甲醇＝2:1)。减压浓缩至干，过硅胶柱(洗脱液乙酸乙酯:二氯甲烷:甲醇＝10:5:1)，得白色泡沫状固体(0.1g)。
实施例8：N-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-N-甲基丙烯酰胺(DD001-2)
Figure PCTCN2018092122-appb-000014
将实施例7所得化合物按实施例6的操作得到化合物DD001-2。δ8.47-8.55(d,1H),8.39-8.40(m,2H),8.09-8.27(m,2H),7.81-7.85(m,2H),7.75-7.81(m,2H)7.16-7.19(m,1H)6.49-6.61(m,1H),6.09-6.15(m,1H),5.66-5.73(m,1H),3.79-3.85(m,1H),3.58-3.61(m,1H),3.11-3,26(m,2H),2.57-2.65(s,3H),1.76-2.06(m,2H),1.43-1.55(m,2H)
实施例9：N-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-N-乙基丙烯酰胺(DD001-3)
Figure PCTCN2018092122-appb-000015
用乙醛代替多聚甲醛，进行实施例7→实施例8的操作，得到化合物DD001-3。
实施例10：N-(3-(4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-N-异丙基丙烯酰胺(DD001-4)
Figure PCTCN2018092122-appb-000016
用丙酮代替多聚甲醛，进行实施例7→实施例8的操作，得到化合物DD001-4。
实施例11：3-苯乙烯基-1-(3-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
将实施例2所得化合物(0.2g)溶于N,N-二甲基甲酰胺(5ml)中，加入苯乙烯(1.2eq)，N,N-二异丙基乙胺(2.5eq)。氮气，加入三(邻甲苯基)膦(0.1eq),醋酸钯(0.1eq)。100℃保温10h。反应完毕，减压浓缩，过硅胶柱，得到淡黄色固体0.1g。
实施例12：1-(3-(4-氨基-3-苯乙烯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基]-2-丙烯-1-酮(DD001-5)
Figure PCTCN2018092122-appb-000018
将实施例11所得化合物通过实施例6的操作得到化合物DD001-5。
实施例13：1-(3-(4-氨基-3-(2-(2-吡啶基)乙烯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基]-2-丙烯-1-酮(DD001-6)
Figure PCTCN2018092122-appb-000019
用2-乙烯基吡啶代替苯乙烯，通过实施例11→实施例12的操作，得到化合物DD001-6。
δ8.57-8.61(d,1H),8.19-8.21(s,1H),7.98-8.01(d,1H),7.83-7.79(d,1H),7.55-7.60(brs,2H),7.53-7.57(m,1H),7.49-7.51(m,1H),7.31-7.34(m,1H),6.71-6.91(m,1H),6.07-6.15(m,1H),5.60-5.72(m,1H),4.07-4.24(m,2H),3.70-3.76(m,1H),2.30-2.33(m,1H),2.10-2.18(m,1H),1.61-1.62(m,1H),1.23-1.38(m,1H)
实施例14：1-(3-(4-氨基-3-(4-氯苯乙烯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基]-2-丙烯-1-酮(DD001-7)
Figure PCTCN2018092122-appb-000020
用4-氯苯乙烯代替苯乙烯，通过实施例11→实施例12的操作，得到化合物DD001-7。
实施例15：1-(3-(4-氨基-3-(3-氯苯乙烯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-2-丙烯-1-酮(DD001-8)
Figure PCTCN2018092122-appb-000021
用3-氯苯乙烯代替苯乙烯，通过实施例11→实施例12的操作，得到化合物DD001-8。
实施例16：1-(3-(4-氨基-3-(2-氯苯乙烯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基]-2-丙烯-1-酮(DD001-9)
Figure PCTCN2018092122-appb-000022
用2-氯苯乙烯代替苯乙烯，通过实施例11→实施例12的操作，得到化合物DD001-9。
实施例17：3-溴-1-(3-(1-(叔丁氧基羰基)-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000023
氮气，将实施例2所得化合物(10g)溶于二氯甲烷(100ml)中，加入三乙胺(2.1eq),10℃左右加入二碳酸二叔丁酯(1.3eq),保温搅拌4-5h，反应完毕。10％柠檬酸洗涤，分夜。有机层用饱和碳酸钠溶液洗涤，饱和食盐水洗，干燥，减压浓缩得白色固体12g。
实施例18：3-(4-氰基苯基)-1-(3-1-(叔丁氧基羰基)-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000024
将实施例17所得化合物(12g),4-氰基苯硼酸(1.2eq)碳酸钾(2.5eq)溶于二噁烷/水(120ml/40ml)中，氮气鼓泡除氧20min。加入四三苯基膦钯(0.05eq)，氮气继续鼓泡除氧10min。加热至回流，保温4-5h，反应完毕。降温至40℃左右趁热分液，有机层减压浓缩至干，得到白色化合物(10g)。
实施例19：3-(4-(氨基(羟基亚氨基)甲基)苯基)-1-(3-1-(叔丁氧基羰基)-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000025
将实施18所得化合物10g(1.0eq)悬浮于乙醇中，依次加入三乙胺(2.1eq)，盐酸羟胺(2.0eq)，加热至回流，3h，取样TLC(乙酸乙酯:甲醇＝10:1)。反应完毕，减压浓缩至干，得白色固体，加入水和乙酸乙酯，溶清，分液，去水层，有机层用饱和食盐水洗涤，分液。减压浓缩得白色固体10g。
实施例20：3-(4-(5-甲基-1,2,4-恶二唑基)苯基)-1-(3-1-(叔丁氧基羰基)-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000026
将实施例19所得化合物0.5g(1.0eq)悬浮于甲苯中，加入三乙胺(2.0eq)，滴加乙酰氯，有固体析出,100℃过夜。反应完毕，加入6vol H 2O,6vol乙酸乙酯。分液，取有机层，10％柠檬酸洗涤有机层，饱和碳酸氢钠洗涤，饱和食盐水洗涤，减压浓缩至干。过硅胶柱，得淡白色固体(0.2g)。
实施例21：3-(4-(5-甲基-1,2,4-恶二唑基)苯基)-1-(3-哌啶基)-1H-吡唑并[3,4-D]嘧啶-4-胺
Figure PCTCN2018092122-appb-000027
将实施例20所得化合物(0.2g)溶于四氢呋喃(10ml)中，加入浓盐酸(1ml),50℃保温2h。反应完毕，碳酸钠调节pH至7-8，乙酸乙酯(10mlx2)萃取。无水硫酸钠干燥，减压浓缩至干，得白色固体(0.1g)。
实施例22：1-(3-(4-氨基-3-(4-(5-甲基-1,2,4恶二唑-3-基)苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-2-丙烯-1-酮(DD001-10)
Figure PCTCN2018092122-appb-000028
将实施例21所得化合物通过实施例6的操作，得到化合物DD001-10。
实施例23：1-(3-(4-氨基-3-(4-(5-乙烯基-1,2,4恶二唑-3-基)苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-2-丙烯-1-酮(DD001-11)
Figure PCTCN2018092122-appb-000029
用丙烯酰氯代替乙酰氯，将实施例19所得产物进行实施例20→实施例21→实施例22的操作，得到化合物DD001-11。
实施例24：1-(3-(4-氨基-3-(4-(5-(3-氯丙基)-1,2,4恶二唑-3-基)苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-2-丙烯-1-酮DD001-12
Figure PCTCN2018092122-appb-000030
用4-氯丁酰氯代替乙酰氯，将实施例19所得产物进行实施例20→实施例21→实施例22的操作，得到化合物DD001-12。
实施例25：1-(3-(4-氨基-3-(4-(5-苯基-1,2,4恶二唑-3-基)苯基)-1H-吡唑并[3,4-D]嘧啶-1-基)-1-哌啶基)-2-丙烯-1-酮DD001-13
Figure PCTCN2018092122-appb-000031
用苯甲酰氯代替乙酰氯，将实施例19所得产物进行实施例20→实施例21→实施例22的操作，得到化合物DD001-13。
实施例26：3-(6-氨基-8-氧代-7，8-二氢-9H-嘌呤-9-基)哌啶-1-甲酸叔丁酯
Figure PCTCN2018092122-appb-000032
以4,6-二氯-5-硝基嘧啶为起始原料，参照专利CN201180026837的合成方法，得到目标化合物30.6g。
实施例27：3-[6-氨基-8-氧代-7-(4-苯氧基苯基)-7，8-二氢-9H-嘌呤-9-基]哌啶-1-甲酸叔丁酯
Figure PCTCN2018092122-appb-000033
将实施例26所得化合物(9.0g)悬浮于二氯甲烷(200ml)中，加入4-苯氧基苯硼酸(13.3g,2.3eq)，4A分子筛(9.0g)，加入吡啶(2.0eq)，溶清，加入醋酸铜(2.0eq)，敞口,8-10℃反应，72h，取样TLC(乙酸乙酯:PE＝1：2)。反应完毕，10％柠檬酸溶液洗涤。分液，去水层，有机层用饱和碳酸钠溶液洗涤，饱和食盐水洗，所得溶液直接用于下一步。
实施例28：6-氨基-7-(4-苯氧基苯基)-9-(哌啶-3-基)-7，9-二氢-8H-嘌呤-8-酮
Figure PCTCN2018092122-appb-000034
向实施例27所得溶液中滴加10ml浓盐酸，25-30℃)搅拌4h，取样TLC(乙酸乙酯:甲醇＝10:1)。反应完毕，加入20ml水，分液，得水层。4N氢氧化钠溶液调节pH至9-10，50ml乙酸乙酯萃取。分液，水层用乙酸乙酯25ml x 2萃取。合并有机层，饱和食盐水洗涤，无水硫酸钠干燥，得固体6.0g。
实施例29：N-(3-(6-氨基-8-氧代-7-(4-苯氧基苯基)-7，8-二氢-9H-嘌呤-9-基)哌啶-1-基)丙烯酰胺DD001-14
Figure PCTCN2018092122-appb-000035
将实施例28所得化合物(1.0g)进行实施例4→实施例5→实施例6的操作，得到化合物DD001-14(0.1g)。
实施例30：N-(3-(6-氨基-8-氧代-7-(4-苯氧基苯基)-7，8-二氢-9H-嘌呤-9-基)哌啶-1-基)-N-甲基丙烯酰胺DD001-15
Figure PCTCN2018092122-appb-000036
将实施例28所得化合物(1.0g)进行实施例4→实施例5→实施例7→实施例8的操作，得到化合物DD001-15(50mg)。
实施例31：N-(3-(6-氨基-8-氧代-7-(4-苯氧基苯基)-7，8-二氢-9H-嘌呤-9-基)哌啶-1-基)-N-乙基丙烯酰胺DD001-16
Figure PCTCN2018092122-appb-000037
将实施例28所得化合物(1.0g)进行实施例4→实施例5→实施例9的操作，得到化合物DD001-16(40mg)。
实施例32：N-(3-(6-氨基-8-氧代-7-(4-苯氧基苯基)-7，8-二氢-9H-嘌呤-9-基)哌啶-1-基)-N-异丙基丙烯酰胺DD001-17
Figure PCTCN2018092122-appb-000038
将实施例28所得化合物(1.0g)进行实施例4→实施例5→实施例10的操作，得到化合物DD001-17(60mg)。
实施例33：3-[6-氨基-7-(4-氰基苯基)-8-氧代-7，8-二氢-9H-嘌呤-9-基]哌啶-1-甲酸叔丁酯
Figure PCTCN2018092122-appb-000039
将实施例26所的化合物(10g)溶于dioxane(150ml)，加入4-溴苯腈(1.3eq)，BINAP(1,1'-联萘-2,2'-双二苯膦)(0.2eq),叔丁醇钾(3.0eq)，醋酸钯(0.2eq)。氮气除氧，加热至回流，保温4-6h。反应完毕，减压浓缩至干。向浓缩物中加入乙酸乙酯(100ml),水(30ml)。分液，水层用乙酸乙酯(20ml)萃取。合并有机层，饱和食盐水洗，无水硫酸钠干燥。减压浓缩至干，得淡黄色固体。
实施例34：9-(1-烯丙酰基哌啶-3-基)-6-氨基-7-(4-(5-甲基-1，2，4-恶二唑-3- 基)苯基)-7，9-二氢-8H-嘌呤-8-酮DD001-18
Figure PCTCN2018092122-appb-000040
将实施例33所得化合物进行实施例19→实施例20→实施例21→实施例22的操作，得到化合物DD001-18。
实施例35：9-(1-烯丙酰基哌啶-3-基)-6-氨基-7-(4-(5-乙烯基-1，2，4-恶二唑-3-基)苯基)-7，9-二氢-8H-嘌呤-8-酮DD001-19
Figure PCTCN2018092122-appb-000041
将实施例33所得化合物进行实施例19→实施例23的操作，得到化合物DD001-19。
实施例36：9-(1-烯丙酰基哌啶-3-基)-6-氨基-7-(4-(5-(3-氯丙基)-1，2，4-恶二唑-3-基)苯基)-7，9-二氢-8H-嘌呤-8-酮DD001-20
Figure PCTCN2018092122-appb-000042
将实施例33所得化合物进行实施例19→实施例24的操作，得到化合物DD001-20。
实施例37：9-(1-烯丙酰基哌啶-3-基)-6-氨基-7-(4-(5-苯基-1，2，4-恶二唑-3-基)苯基)-7，9-二氢-8H-嘌呤-8-酮DD001-21
Figure PCTCN2018092122-appb-000043
将实施例33所得化合物进行实施例19→实施例25的操作，得到化合物DD001-21。
实施例38：4-溴-N-(吡啶-2-基)苯甲酰胺
Figure PCTCN2018092122-appb-000044
将2-氨基吡啶(0.65g,1.0eq),吡啶(0.8g，1.5eq)溶于二氯甲烷(15ml),缓慢滴加4-溴苯甲酰氯(1.5g)，滴毕，室温搅拌4h，反应完毕。水洗涤，10％柠檬酸洗涤。减压干燥，得固体4-溴-N-(2-吡啶基)-苯甲酰胺1.5g(79％)。
实施例39：N-(吡啶-2-基)-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)苯甲酰胺
Figure PCTCN2018092122-appb-000045
将实施例38所得化合物(1.0eq)溶于二噁烷中，加入联硼酸频那醇酯(1.3eq),醋酸钾(1.6eq)。反应混合物用氮气脱气，随后添加1,1'-双(二苯基膦)二茂铁二氯化钯二氯甲烷(0.05eq)。回流反应8h，反应完毕，减压浓缩大部分溶剂，乙酸乙酯溶解，水洗，盐水洗，无水硫酸钠干燥，减压浓缩。柱层析(洗脱剂二氯甲烷/甲醇＝20：1)得白色固体N-(吡啶-2- 基)-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)苯甲酰胺(75.3％)。
实施例40：4-(9-(1-丙烯酰胺哌啶-3-基)-6-氨基-8-氧代-8,9-二氢-7H-嘌呤-7-基)-N-(吡啶-2-基)苯甲酰胺DD001-22
Figure PCTCN2018092122-appb-000046
实施例38所得化合物代替4-溴苯腈，进行实施例33的操作，所得产物进行实施例28→实施例29的操作，得到化合物DD001-22。
实施例41：4-(1-(1-丙烯酰胺哌啶-3-基)-4-氨基-1H-吡唑并[3,4-d]嘧啶-3-基)-N-(吡啶-2-基)苯甲酰胺DD001-23
Figure PCTCN2018092122-appb-000047
实施例39所得化合物代替4-苯氧基苯硼酸，进行实施例3→实施例4→实施例5→实施例6的操作，得到化合物DD001-23。
实施例42：N-(3-(4-氨基-3-(4-(2-氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丙烯酰胺DD001-24
Figure PCTCN2018092122-appb-000048
由4-(2-氟苯氧基)苯硼酸代替4-苯氧基苯硼酸，进行实施例3→实施例4→实施例5→实施例6的操作，得到化合物DD001-24。
实施例43：1-(3-(4-氨基-3-(4-氟苯乙烯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-2-丙烯-1-酮DD001-25
Figure PCTCN2018092122-appb-000049
用5-氟-2-乙烯基吡啶代替苯乙烯，通过实施例11→实施例12的操作，得到化合物DD001-25。
实施例44：N-(3-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-(二甲氨基)丁-2-烯酰胺DD001-26
Figure PCTCN2018092122-appb-000050
向实施例5所得化合物1-(3-(1-亚硝基哌啶基))-3-(4-苯氧基苯基)-1H-吡唑并[3,4-d] 嘧啶-4-胺(0.5g,1.0eq)、三乙胺(0.38g,3.0eq)和4-(二甲基氨基)丁-2-烯酸盐酸盐(0.21g,1.0eq)的二氯甲烷溶液中加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(0.47g,1.0eq)。室温搅拌1.5h。反应完毕，混合物用水洗，硫酸镁干燥得到，减压浓缩。残余物硅胶柱纯化(洗脱液二氯甲烷/甲醇＝10：1至5：1)，得到目标化合物N-(3-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-(二甲氨基)丁-2-烯酰胺0.25g(40％)DD001-26。
实施例45：N-(3-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁-2-炔酰胺DD001-27
Figure PCTCN2018092122-appb-000051
由丁-2-炔酸代替4-(二甲基氨基)丁-2-烯酸进行实施例44的操作，得到目标化合物DD001-27。δ8.6(d,1H),8.21(d,1H),7.98(d,1H),7.83(d,1H),7.6(brs,2H),7.53(m,1H),7.49(m,1H),7.31(m,1H),6.71-6.91(m,1H),6.07-6.15(m,1H),5.60-5.72(m,1H),4.07-4.24(m,2H),3.70-3.76(m,1H),2.30-2.33(m,1H),2.10-2.18(m,1H),1.61-1.62(m,1H),1.23-1.38(m,1H)
实施例46：1-(3-(4-氨基-3-(2-(吡啶-2-基)乙烯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-(二甲氨基)丁-2-丙烯-1-酮DD001-28
Figure PCTCN2018092122-appb-000052
用4-(二甲基氨基)丁-2-烯酸替代丙烯酰氯进行类似实施例13与实施例44的操作得到化合物1-(3-(4-氨基-3-(2-(吡啶-2-基)乙烯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-(二甲氨基)丁-2-丙烯-1-酮DD001-28。
实施例47：(1-(3-(4-氨基-3-(2-(吡啶-2-基)乙烯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁-2-炔-1-酮DD001-29
Figure PCTCN2018092122-appb-000053
由丁-2-炔酸代替4-(二甲基氨基)丁-2-烯酸进行实施例46的操作，得到目标化合物(1-(3-(4-氨基-3-(2-(吡啶-2-基)乙烯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)丁-2-炔-1-酮DD001-29。δ8.51-8.55(d,1H),8.21-8.27(s,1H),7.83-7.79(d,1H),7.68-7.72(d,1H),7.55-7.60(m,2H),7.53-7.57(m,1H),7.49-7.51(m,1H),7.31-7.34(m,1H),6.71-6.91(m,1H),6.07-6.15(m,1H),5.60-5.72(m,1H),4.07-4.24(m,2H),3.70-3.76(m,1H),1.92-1.95(m,3H),1.61-1.62(m,1H)
实施例48：3-(8-氨基-1-碘咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯
Figure PCTCN2018092122-appb-000054
由1-(苄氧羰基)哌啶-3-甲酸代替Z-Pro-OH，N-碘代琥珀酰亚胺代替N-溴代琥珀酰亚胺，参照专利CN201280045383中间体1的合成方法，生成化合物2-(8-氨基-1-碘咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯。
实施例49：3-(8-氨基-1-(4-苯氧苯基)咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯
Figure PCTCN2018092122-appb-000055
将3-(8-氨基-1-碘咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯(5g,10.48mmol),4-苯氧基苯硼酸(2.69，1.2eq)，碳酸钾(5.07g，3.5eq)悬浮于dioxane(21ml)/H2O(9ml)，氮气鼓泡除氧10min。加入四三苯基膦钯(0.24g，0.02eq)，继续鼓泡5min。加热至回流，4h后取样TLC(二氯甲烷:甲醇＝9:1)。反应完毕，降至室温，分液，有机层浓缩得油状物。加入10ml水，15ml乙酸乙酯，有不溶物。4N盐酸调节pH至2-3，溶清。分液，去有机层，水层用乙酸乙酯10ml x 2洗涤。4N氢氧化钠溶液调节pH至8-9，加入乙酸乙酯20ml溶清，萃取。饱和食盐水10ml洗涤，无水硫酸钠3g干燥。减压浓缩，得3-(8-氨基-1-(4-苯氧苯基)咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯4.2g。
实施例50：1-(4-苯氧苯基)3-哌啶-3-基咪唑并[1，5-a]吡嗪-8-胺
Figure PCTCN2018092122-appb-000056
将3-(8-氨基-1-(4-苯氧苯基)咪唑并[1，5-a]吡嗪-3基)哌啶-1-甲酸苄酯(2g，3.85mmol)中添加33％氢溴酸/乙酸溶液(38.5mmol,7ml)，室温搅拌2h。混合物中加入水/二氯甲烷(1:1,30ml)。2N氢氧化钠溶液调节pH至8-9，分液，水相用二氯甲烷(10ml)萃取。合并有机层，无水硫酸镁干燥，过滤浓缩，得1-(4-苯氧苯基)3-哌啶-3-基咪唑并[1，5-a]吡嗪-8-胺。
实施例51：N-(3-(8-氨基-1-(4-苯氧苯基)咪唑并[1，5-a]吡嗪-3基)哌啶-1-基)丙烯酰胺
Figure PCTCN2018092122-appb-000057
将化合物1-(4-苯氧苯基)3-哌啶-3-基咪唑并[1，5-a]吡嗪-8-胺进行实施例4→实施例5→实施例6的操作，得到化合物N-(3-(8-氨基-1-(4-苯氧苯基)咪唑并[1，5-a]吡嗪-3基)哌啶-1-基)丙烯酰胺。
目标化合物列表
Figure PCTCN2018092122-appb-000058
Figure PCTCN2018092122-appb-000059
Figure PCTCN2018092122-appb-000060
Figure PCTCN2018092122-appb-000061
Figure PCTCN2018092122-appb-000062
Figure PCTCN2018092122-appb-000063
Figure PCTCN2018092122-appb-000064
Figure PCTCN2018092122-appb-000065
生物学实施例1：Btk抑制活性和对Btk选择性的测定(体外试验)
Btk酶抑制活性的测定
激酶反应：
缓冲液:50mM Hepes pH7.0,0.1mM Orthovanadate,5mM MgCl 2,0.01％BSA；
2nM BTK；
1mM TK-peptide,20mM ATP,50nM SEB；
预保温15minutes；
23℃反应90minutes；
显色反应：
显色缓冲液:50mM Hepes pH8.0,0.8M KF,20mM EDTA,0.01％BSA；
6.7nM TK-Antibody,62.5nM XL665；
23℃反应60minutes；
检测设备：
Envision(PerkinElmer#2104)
由基于受试化合物的各浓度下的抑制率的抑制曲线，计算受试化合物的50％抑制率的值(IC 50值)。
其他激酶(例如，Lck、LynA的抑制活性的测定使用各种激酶代替Btk，与上述方法同样操作。
结果，对于本发明目标化合物的IC 50值，如下表1所示：
表1
实施例编号 IC 50(nM)
6 0.27
8 0.64
13 0.46
14 5.82
15 6.90
16 16.7
22 145
24 78
29 >1000
30 76.8
34 209
35 176
36 >1000
40 23
42 0.9
51 5.1
Ibrutinib 0.19
另外，本发明化合物对其他激酶，特别是对Lck、LynA的Btk选择性抑制活性基于各种激酶的IC 50值之比而计算，如下表2所示：
表2
实施例编号 Lck[IC 50]/Btk[IC 50] LynA[IC 50]/Btk[IC 50]
6 285 137
8 139 62.5
13 420 507
Ibrutinib 158 320
由该结果可知，本发明目标化合物不仅具有Btk抑制活性，而且对其他激酶具有Btk选 择性抑制活性。
生物学实验例2：人和大鼠肝微粒体稳定性试验
配制缓冲液:
1. 100mM磷酸钾盐缓冲液,pH 7.4
2. 10mM MgCl 2
受试化合物溶液的制备:
1.用甲醇溶液(495μL)稀释受试化合物溶液(10mM，5μL)，制备100μM溶液
2.工作液配制：将上述溶液(50μL)用磷酸钾缓冲液(450μL，100mM)稀释，得稀释液(10μM)
NADPH再生体系(异柠檬酸脱氢酶终浓度1unit/mL):
1.β-NADP,供应商:sigma Cat.No.N0505
2.异柠檬酸供应商:Sigma Cat.No.I1252
3.异柠檬酸脱氢酶,供应商:sigma Cat.No.I2002
肝微粒体溶液制备(终浓度0.5mg protein/mL)
Figure PCTCN2018092122-appb-000066
终止液:
含100ng/mL甲苯磺丁脲和100ng/mL拉贝洛尔的冷乙腈作内标
1..向空白以外的平板(T0，T5，T10，T20，T30，T60，NCF60)加10μL/孔工作液
2..加80μL/孔微粒体溶液，37℃孵育10min。
3.向NCF60平板中每孔加入10μL 100mM磷酸盐缓冲液，37℃温浴，开始计时
Figure PCTCN2018092122-appb-000067
4.预加热后，加10μL/孔NADPH再生体系，开始反应
5.. 37℃孵育，开始计时
Figure PCTCN2018092122-appb-000068
6..加300μL/孔终止液(4℃，含100ng/mL甲苯磺丁脲和100ng/mL拉贝洛尔)终止反应
7..将平板震荡10min
8.. 4℃离心，转速4000rpm
9.样品LC/MS/MS检测
数据分析Use equation of first order kinetics to calculate t1/2and Clint(mic):运用一级动力学方程计算t1/2和Clint(mic)
一级动力学方程:
Figure PCTCN2018092122-appb-000069
when
Figure PCTCN2018092122-appb-000070
Figure PCTCN2018092122-appb-000071
Figure PCTCN2018092122-appb-000072
Figure PCTCN2018092122-appb-000073
实验结果如下表3所示：
表3
Figure PCTCN2018092122-appb-000074
Figure PCTCN2018092122-appb-000075
由实验结果比较各化合物CL int(liver)(ml/min/kg)值可知，本发明化合物较Ibrutinib有更好的稳定性。
生物学实施例3：体外受试化合物对PBMC/B/T细胞活化的影响
1.试剂、仪器
1)
Figure PCTCN2018092122-appb-000076
2)
  Instrument Vendor Model#
1 BD FACSCanto TM II BD FACSCanto TM II
2.抗体
Figure PCTCN2018092122-appb-000077
3.样品表
Figure PCTCN2018092122-appb-000078
4.Method
4.1准备样品
ID concentration ID concentration
C1 40uM C4 20uM
C2 4uM C5 2uM
C3 0.4uM C6 0.2uM
Note:C1,C2,C3is used for PBMC/B cell activation；C4,C5,C6is used for T cell activation
4.2准备Anti-human IgM
ID Concentration Note
Stock 1.3mg/ml NA
C1 20ug/ml For B cell activation
C2 40ug/ml For PBMC activation
4.3分离B细胞和T细胞
细胞分离程序参照STEMCELL实验盒
4.4共培养
4.4.1激活B细胞
共培养体系如下:
component Final Concentration
Anti-human IgM 5ug/ml
CPD 10uM,1uM,0.1uM
4.4.2激活PBMC
共培养体系如下:
component Final Concentration
Anti-human IgM 10ug/ml
CPD 10uM,1uM,0.1uM
4.4.3激活T细胞
共培养体系如下:
component Final Concentration
CD3/CD28dynabeads Beads:T＝1:1
CPD 10uM,1uM,0.1uM
4.5抗体染色与数据分析
在共培养前24小时按照标准方法进行抗体染色
4.5.1.PBMC和B细胞激活
PE Mouse Anti-Human CD20
APC anti-human CD69
LIVE/DEAD Fixable Violet Dead Cell Stain Kit
4.5.2 T细胞激活
FITC Mouse Anti-Human CD4
PerCP-Cy TM5.5Mouse Anti-Human CD8
APC anti-human CD69
LIVE/DEAD Fixable Violet Dead Cell Stain Kit
5.结果
Donor 517
T Cell Activation见附图1。
Donor 517
B and PBMC Activation见附图2。
Donor 581
T Cell Activation见附图3。
Donor 581
B and PBMC Activation见附图4。
Donor 956
T Cell Activation见附图5。
Donor 956
B and PBMC Activation见附图6。
实施例化合物6和对比物(Ibrutinib)在0.1uM浓度下表现出明显的B细胞活性抑制作用，在10uM浓度下显示出对T细胞活性抑制作用。
生物学实施例4：通过Western blotting分析受试化合物对DOHH2细胞p-Btk/t-Btk,p-PLCγ/t-PLCγ，p-Erk/t-Erk表达水平的影响
1.试剂
Figure PCTCN2018092122-appb-000079
Figure PCTCN2018092122-appb-000080
2.仪器
Figure PCTCN2018092122-appb-000081
3.抗体
  Antibody Vendor Catalog# MW(kDa) Source
1 p-BTK Antibody Cell Signaling Technology 87141S 78 Rabbit
2 t-BTKAntibody Cell Signaling Technology 8547S 77 Rabbit
3 p-PLCγAntibody Cell Signaling Technology 2821S 155 Rabbit
4 t-PLCγAntibody Cell Signaling Technology 2822S 155 Rabbit
5 p-ERK Antibody Cell Signaling Technology 4377S 42,44 Rabbit
6 t-ERK Antibody Cell Signaling Technology 9107S 42,44 Mouse
7 Actin Antibody Cell Signaling Technology 4967S 45 Rabbit
4.准备样品:
Serial Sample name
1 DMSO
2 DMSO
3 化合物6,5nM
4 化合物6,5nM
5 化合物6,100nM
6 化合物6,100nM
7 化合物6,2000nM
8 化合物6,2000nM
9 化合物13,5nM
10 化合物13,5nM
11 化合物13,100nM
12 化合物13,100nM
13 化合物13,2000nM
14 化合物13,2000nM
15 化合物45,5nM
16 化合物45,5nM
17 化合物45,100nM
18 化合物45,100nM
19 化合物45,2000nM
20 化合物45,2000nM
21 化合物47,5nM
22 化合物47,5nM
23 化合物47,100nM
24 化合物47,100nM
25 化合物47,2000nM
26 化合物47,2000nM
27 对比物(Ibrutinib),5nM
28 对比物(Ibrutinib),5nM
29 对比物(Ibrutinib),100nM
30 对比物(Ibrutinib),100nM
31 对比物(Ibrutinib),2000nM
32 对比物(Ibrutinib),2000nM
5.细胞培养
1)收集对数期DOHH2细胞.
2)调整细胞浓度至所需浓度.
3)向6孔板中加入2ml细胞悬液进行培养。受试物分别设置三个不同浓度,5nM,100nM和2000nM,每个浓度重复一次。
Plate 1:
Figure PCTCN2018092122-appb-000082
Plate 2:
Figure PCTCN2018092122-appb-000083
Plate 3:
Figure PCTCN2018092122-appb-000084
Plate 4:
Figure PCTCN2018092122-appb-000085
Plate 5:
Figure PCTCN2018092122-appb-000086
Plate 6:
Figure PCTCN2018092122-appb-000087
4)将受试物加入板孔中并且DMSO作对照，DMSO终浓度为0.1％。
5)培育1h。
6)DOHH2cells与受试物培育1h后,用10倍体积的PBS溶液洗涤三次,随后用anti-IgG(30μg/mL；ab98531)刺激2min。
6.蛋白提取和定量
1)收集细胞，1200rpm离心5min。
2)冰浴的1X PBS淋洗细胞1次。
3)每管加入0.5ml冰浴的1X RIPA缓冲液(含1％蛋白酶抑制剂Cocktail和1％磷酸酶抑制剂Cocktail 2)，冰浴培育30min。
4)4℃，14000rpm离心10min，收集上清液。
5)用Pierce TM BCA Protein Assay Kit测定蛋白浓度。
6)根据BCA蛋白定量结果,dilute all samples to the same final concentration用RIPA缓冲液加4X LDS样品缓冲液和10X样品还原剂将所有样品稀释至相同的终浓度.100℃加热10min。
6.Western blotting
1)将样品加入
Figure PCTCN2018092122-appb-000088
Novex 4-12％Bis-Tris gel,10μL每槽孔。80V电压30min，120V电压90min。
Gel 1:
Serial Sample name
1 DMSO
2 DMSO
3 化合物6,5nM
4 化合物6,5nM
5 化合物6,100nM
6 化合物6,100nM
7 化合物6,2000nM
8 化合物6,2000nM
9 化合物13,5nM
10 化合物13,5nM
11 化合物13,100nM
12 化合物13,100nM
13 化合物13,2000nM
14 化合物13,2000nM
15 化合物45,5nM
16 化合物45,5nM
17 化合物45,100nM
18 化合物45,100nM
19 化合物45,2000nM
20 化合物45,2000nM
Gel 2:
Serial Sample name
1 DMSO
2 DMSO
3 化合物47,5nM
4 化合物47,5nM
5 化合物47,100nM
6 化合物47,100nM
7 化合物47,2000nM
8 化合物47,2000nM
9 对比物(Ibrutinib),5nM
10 对比物(Ibrutinib,5nM
11 对比物(Ibrutinib,100nM
12 对比物(Ibrutinib,100nM
13 对比物(Ibrutinib,2000nM
14 对比物(Ibrutinib,2000nM
2)用
Figure PCTCN2018092122-appb-000089
Gel转移装置将蛋白转至硝化纤维膜,7min。
3)将蛋白与1xTBST含5％脱脂牛奶室温培育1h。
4)1xTBST洗涤三次，每次5min。
5)将膜与5-10mL稀释的初始抗体4℃培育,缓慢震荡过夜。
6)1xTBST洗涤三次，每次10min。
7)将膜与HRP-conjugated次级抗体室温培育，缓慢震荡1h。
8)1xTBST洗涤三次，每次10min。
9)加入West Femto Maximum Sensitivity试剂盒中的HRP底物。
10)Tanon 5200 multi检测化学发光。
7.定量条带强度
用Image quant densitometry软件定量每条色带强度。
8.结果
DOHH2细胞经受试物处理后，用anti-lgG刺激，Western blotting分析其p-Btk/t-Btk的相对表达水平，结果见图7。
DOHH2细胞经受试物处理后，用anti-lgG刺激，Western blotting分析其p-PLCγ/t-PLCγ的相对表达水平，结果见图8。
DOHH2细胞经受试物处理后，用anti-lgG刺激，Western blotting分析其p-Erk/t-Erk的相对表达水平，结果见图9。
实验结果表明，DOHH2细胞经过化合物6与Ibrutinib处理后其p-Btk/t-Btk，p-PLCγ/t-PLCγ，p-Erk/t-Erk相对水平都有明显下降。
生物学实施例5：hERG电流阻断试验
1.阳性对照药：盐酸阿米替林(Amitriptyline hydrochloride)(Sigma-Aldrich，国际通用的标准hERG通道阻断剂)
2.溶液及化合物的配制
细胞外液(mM):HEPES 10、NaCl 145、KCl 4、CaCl2 2、MgCl2 1、Glucose 10，用1N 氢氧化钠调节pH至7.4；渗透压至290-300mOsm，过滤，4℃保存。
电极内液(in mM)：HEPES 10、KOH 31.25、KCl 120、CaCl2 5.374、MgCl2 1.75、EGTA 10、Na2-ATP 4，用1N氢氧化调节pH至7.2；渗透压至280-290mOsm，过滤，-20℃保存。
化合物配制：阳性对照药盐酸阿米替林，化合物6和Ibrutinib先溶于100％DMSO(Sigma-Aldrich,D2650)，均配置成30mM的储备液。实验前用DMSO将上述储备液稀释为各个试验浓度1000倍的溶液，再用细胞外液稀释1000倍到所需浓度。细胞外液中DMSO终浓度为0.1％。
3.细胞株
稳定细胞株CHO-hERG购自AVIVA公司。为了质量控制，最小的封接电阻不小于500MΩ，并且hERG电流不小于0.4nA。
4.电生理试验
采用全细胞膜片钳技术记录hERG电流。取细胞悬液加于35mm的培养皿中，置于倒置显微镜载物台上。待细胞贴壁后，用细胞外液灌流，流速为1-2mL/min。玻璃微电极由微电极拉制仪两步拉制，其入水电阻值为2-5MΩ。建立全细胞记录后，保持钳制电位为-80mV。给予电压刺激时去极化至+60mV，然后复极化至-50mV引出hERG尾电流。所有记录均在电流稳定后进行。胞外灌流给药从低浓度开始，每个浓度5-10min至电流稳定，再给下一个浓度。
5.数据采集和分析
通过Digidata 1440(Molecular Devices)和pCLAMP软件(10.2版，Molecular Devices)A/D–D/A数模转换，进行刺激发放及信号采集；膜片钳放大器(Multiclamp 700B，Molecular Devices)放大信号。
使用Clampfit(10.2版，Molecular Devices)，EXCEL(2013版，Microsoft)和GraphPad Prism进行进一步数据分析和曲线拟合。数据均以均值±标准差表示。
在数据处理中，判断对hERG的阻断效应时，将尾电流的峰值和其基线进行校正。用尾流的抑制率表示不同浓度下各化合物的作用。IC50数值由Hill方程进行拟合所得：
Figure PCTCN2018092122-appb-000090
y：I/I control；max：为100％；min：为0％；[drug]：测试物浓度；n H：Hill斜率；IC 50：测试物的最大半数抑制浓度。
6.结果
本试验利用全细胞膜片钳技术，在稳定表达hERG通道的CHO-K1细胞株上检测了化合物6和Ibrutinib对hERG电流的阻断作用。测试化合物的半数抑制浓度(IC50)由Logistic方程最佳拟合得出。化合物对hERG的阻断效应见下表。Amitriptyline是使用最为广泛的阻断hERG电流工具药物之一，故在本次研究中作为阳性对照药物，结果如下
在CHO-K1稳定细胞株上所记录到的化合物对hERG电流的IC50数值
样本 IC50(uM)
Amitriptyline 4.48
实施例化合物6 8.06
Ibrutinib 0.97
生物学实施例6：单剂量毒性试验
挑选健康SD大鼠雄性20只，雌性20只，SPF级，按体重通过简单随机化方式分为4组，每组10只，雌性各半。在实验第1天，对第1组(0mg/kg)动物经口灌胃给予空白溶媒、第2组(400mg/kg)，第3组(1000mg/kg)、第4组(2000mg/kg)动物经口灌胃给予DD001y。连续观察14天。
实验期间所有动物，实验第1天进行1次(给药后2小时)，第2～14天每天1次进行详细临床观察。体重在动物分组当日，给药当天(给药前)、给药后第4、7、10、13天及解剖当天进行称量和记录。实验中第2、6、9、13天24小时内称取每笼耗食量并记录。所有存活动物在第15天进行大体解剖检查。
1.供试品和对照品制备
溶媒对照组：吸取30ml的溶媒(0.5％MC(methylcellulose),0.4％cremophor EL,0.1％sodium lauryl sulfate)到合适的容器中，测量pH值为7，为无色澄清溶液。
化合物6(400mg/kg)：称量1507.1mg化合物6到合适容器中，向该容器中加入溶媒(0.5％MC(methylcellulose),0.4％cremophor EL,0.1％sodium lauryl sulfate)定容至30ml，超声20分钟，搅拌15分钟，使用混合仪5分钟，测量pH值为7，为白色混悬液。
化合物6(1000mg/kg)：称量3765.5mg化合物6到合适容器中，向该容器中加入溶媒(0.5％MC(methylcellulose),0.4％cremophor EL,0.1％sodium lauryl sulfate)定容至30ml，超声25分钟，搅拌21分钟，使用混合仪5分钟，测量pH值为7，为白色混悬液。
化合物6(2000mg/kg)：称量7530.5mg化合物6到合适容器中，向该容器中加入溶媒(0.5％MC(methylcellulose),0.4％cremophor EL,0.1％sodium lauryl sulfate)定 容至30ml，超声30分钟，搅拌31分钟，测量pH值为7，为白色混悬液。
实验设计表
Figure PCTCN2018092122-appb-000091
动物灌胃口服给药前禁食10～16h，给药后2h恢复给食。
3.结果
受试对象具体情况和表现见说明书附图10-13所示。
试验过程中未见动物死亡。所有存活至实验结束的动物体重在试验过程中均逐渐增加，与溶剂对照组比较，未见异常。400mg/kg组和1000mg/kg组中个别动物出现红色干性分泌物(鼻周)，2000mg/kg组中个别动物出现红色干性分泌物(鼻周)、红色湿性分泌物(鼻周)、红色干性分泌物(左眼眼周)，以上异常可能与供试品给药相关。给药后第2天1000mg/kg组和2000mg/kg组动物耗食量低于溶剂对照组，第6天均恢复正常。各只动物大体解剖时未见异常。
在本试验条件下，SD大鼠单次经口给予化合物6后，最大耐受量(MTD)为2000mg/kg。
Figure PCTCN2018092122-appb-000092
Ibrutinib数据来自FDA Database.http://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/205552Orig1s000ClinPharmR.pdf(accessed Jun 2015)
生物学实施例7：SD大鼠、比格犬分别单次静脉和口服给予受试物药代动力学研究
挑选6只雄性SD大鼠，按照实验设计表进行分组及给药。第一组的3只动物通过静脉注射给药，给药当天给药1次，给药剂量为2mg/kg，给药体积为5mL/kg。第二组的3只动物 通过口服灌胃给药，给药当天给药1次，给药剂量为10mg/kg，给药体积为10mL/kg。
对所有动物每天进行2次笼边观察，以确定其是否有发病、损害或死亡等状况出现，以及供食和供水是否充足。试验过程中所有给药动物在给药前及采血点进行详细临床观察。
在给药前(0h)，给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h和24h，共10个时间点从股静脉采集血样用于血药浓度检测。
挑选6只雄性比格犬，按照实验设计表进行分组及给药。第一组的3只动物通过静脉注射给药，给药当天给药1次，给药剂量为2mg/kg，给药体积为2.5mL/kg。第二组的3只动物通过口服灌胃给药，给药当天给药1次，给药剂量为30mg/kg，给药体积为5mL/kg。
对所有动物每天进行2次笼边观察，以确定其是否有发病、损害或死亡等状况出现，以及供食和供水是否充足。试验过程中所有给药动物在给药前及采血点进行详细临床观察。
在给药前(0h)，给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h和24h，共10个时间点从股静脉采集血样用于血药浓度检测。
实验设计表
Figure PCTCN2018092122-appb-000093
*在口服给药前，所有动物禁食过夜(10-14小时)，给药后2小时给食。
Figure PCTCN2018092122-appb-000094
*在口服给药前，所有动物禁食过夜(10-18小时)，给药后2小时给食。
结果
SD大鼠给予化合物6的部分药代动力学参数见下表，SD大鼠静脉注射给予2mg/kg的化合物6后C max为658.49ng/mL，AUC (0-t)为270.52h*ng/mL；口服灌胃给予10mg/kg的化合物6后C max为447.09ng/mL，AUC (0-t)为449.18h*ng/mL。
比格犬给予化合物6的部分药代动力学参数见下表。比格犬静脉注射给予2mg/kg的化合物6后Cmax为1225.90ng/mL，AUC(0-t)为888.22h*ng/mL；口服灌胃给予30mg/kg的 化合物6后Cmax为3828.63ng/mL，AUC(0-t)为7419.16h*ng/mL。
Figure PCTCN2018092122-appb-000095
Figure PCTCN2018092122-appb-000096
Ibrutinib数据来自FDA Database.http://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/205552Orig1s000ClinPharmR.pdf(accessed Jun 2015).
生物学实施例8：研究活性化合物抑制体外肿瘤细胞增殖的作用
1.细胞系及培养方法
Figure PCTCN2018092122-appb-000097
2.培养基
表.培养基及试剂
培养基及试剂 生产商 货号
RPMI 1640 GIBCO 22400-089
DMEM GIBCO 11995-065
Dulbecco's PBS Thermo SH30028.02B
FBS Hyclone SH30084.03
Antibiotic-antimycotic GIBCO 15240-062
DMSO SIGMA D2650
L-glutamine Invitrogen 25030164
3.细胞活性实验所用试剂及仪器
Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573).
2104 EnVision读板器，PerkinElmer.
4.实验方法及步骤
4.1细胞培养
将肿瘤细胞系按各自的培养条件在37℃，5％CO 2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。
4.1.1细胞铺板
1)用台盼兰进行细胞染色并计数活细胞。
2)将细胞浓度调整至合适浓度。
3)在培养板中每孔加入90μL细胞悬液，在空白对照空中加入不含细胞的培养液。
4)将培养板在37℃,5％CO 2,及100％相对湿度的培养箱中培养过夜。
4.2化合物存储板制备
制备400X化合物存储板：将化合物用DMSO从最高浓度梯度稀释至最低浓度。
4.3 10X化合物工作液的配制及化合物处理细胞
1)10X化合物工作液的配制：在V形底的96孔板中加入76μL细胞培养液，从400X化合物存储板中吸取4μL化合物加入96孔板的细胞培养液中。在溶媒对照和空白对照中加入4μL DMSO。加入化合物或DMSO后用排枪吹打混匀。
2)加药：取10μL的10X化合物工作液按表1所示加入到细胞培养板中。在溶媒对照和空白对照中加入10μL DMSO-细胞培养液混合液。DMSO终浓度为0.50％。
3)将96孔细胞板放回培养箱中培养72h。
4.4 CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
1)将CellTiter-Glo缓冲液融化并放置至室温。
2)将CellTiter-Glo底物放置至室温。
3)在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
4)缓慢涡旋震荡使充分溶解。
5)取出细胞培养板放置30分钟使其平衡至室温。
6)在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。
7)将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
8)培养板在室温放置10分钟以稳定发光信号。
9)在2104 EnVision读板器上检测发光信号。
5.数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(1–(RLU化合物–RLU空白对照)/(RLU溶媒对照–RLU空白对照))*100％。
化合物6对体外肿瘤细胞增殖的抑制作用
B-Lymphoma Cell Line Growth IC 50(nM) B-Lymphoma Cell Line Growth IC 50(nM)
TMD8 0.85 OCI-LY19 6788
DOHH-2 <3 Granta-519 7065
OCI-LY10 6 Jeko-1 >10000
SU-DHL-4 75 SU-DHL-6 >10000
WSU-DLCL-2 921 SU-DHL-10 >10000
WSU-NHL 1019 DB >10000
Ramos 1398 OCI-LY3 >10000
Mino 2174 Raji >10000

Claims (8)

  1. 如下的通式的化合物Ia、Ib、Ic、Id、Ie、If或Ig，其光学异构体或它们的混合物、其盐、其溶剂合物、其N氧化物、或它们的前药：
    Figure PCTCN2018092122-appb-100001
    R 1表示H,取代或未取代的C 1-C 4烷基，取代或未取代的C 1-C 4杂烷基；
    R 2表示R 6CO、R 7SO、R 8SO 2或被R 9取代的C 1-C 6烷基；
    R 3表示H,一个或多个取代基，如卤素(氟、氯、溴、碘)，C 1-C 4烷氧基，硝基，氰基，取代或未取代的氨基，取代或未取代的烷基；
    R 4表示H,一个或多个取代基，如卤素(氟、氯、溴、碘)，C 1-C 4烷氧基，硝基，氰基，取 代或未取代的氨基，取代或未取代的烷基；
    R 5表示取代或未取代的C 1-C 6烷基，取代或未取代的C 1-C 6杂烷基,取代或未取代的芳基，取代或未取代的杂芳基，取代或未取代的C 3-C 6环烷基，取代或未取代的C 2-C 6杂环烷基；
    R 6是烯基、炔基，以及烷基、烯基、胺基或卤素取代的烯基、炔基；
    R 7，R 8独立的选自C 2-C 6烯基或C 2-C 6炔基，二者任选被选自羟基、C 1-C 4烷基、C 3-C 7环烷基、(C 1-C 4烷基)氨基、二(C 1-C 4烷基)氨基、C 1-C 3烷氧基、C 3-C 7环烷氧基、C 6-C 10芳基或C 3-C 7杂环烷基的一个或多个基团取代；或任选被选自卤素或氰基的一个或多个基团取代的C 1-C 5杂芳基；
    R 9独立的选自卤素、氰基或C 2-C 6烯基或C 2-C 6炔基，二者任选被选自羟基、C 1-C 4烷基、C 3-C 7环烷基、(C 1-C 4烷基)氨基、二(C 1-C 4烷基)氨基、C 1-C 3烷氧基、C 3-C 7环烷氧基、C 6-C 10芳基、C 1-C 5杂芳基或C 3-C 7杂环烷基的一个或多个基团取代；
    Z是CH 2、O、NH、或S；
    n 1表示0或1，n 2表示1-4的整数，n 3表示0-1的整数；
    X表示氮或碳，可以在苯环的任一位置；Y表示氮。
  2. 如权利要求1所述的通式化合物Ia、Ib、Ic、Id、Ie、If或Ig，其光学异构体或它们的混合物、其盐、其溶剂合物、其N氧化物、或它们的前药，其特征在于：
    R 1表示H,C 1-C 4烷基，C 1-C 4杂烷基；
    R 2表示R 6CO、R 7SO、R 8SO 2或R 9取代的C 1-C 6烷基；R 6是烯基、炔基，以及烷基、烯基、胺基或卤素取代的烯基、炔基；R 7，R 8独立的选自C 2-C 6烯基或C 2-C 6炔基，R 9独立的选自卤素、氰基或C 2-C 6烯基或C 2-C 6炔基；
    R 3表示H；
    R 4表示H、卤素(氟、氯、溴、碘)、取代或未取代的烷基；
    R 5表示取代或未取代的C 1-C 6烷基，取代或未取代的C 1-C 6杂烷基,取代或未取代的芳基，取代或未取代的杂芳基；
    Z是O；
    n 1表示0或1，n 2表示1-4的整数，n 3表示0-1的整数；
    X表示氮或碳，可以在苯环的任一位置。
  3. 一种药物组合物，含有权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药。
  4. 权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为Btk抑制剂药物上的应用。
  5. 权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为Btk相关的疾病的预防和/或治疗剂的药物上的应用。
  6. 权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为预防和/或治疗变应性疾病、自身免疫疾病、炎性疾病、血栓栓塞性疾病、或癌症的药物上的应用。
  7. 权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药在作为预防和/或治疗非霍奇金淋巴瘤药物上的应用。
  8. 权利要求1或2所述的通式化合物Ia-Ig、其光学异构体或它们的混合物、其盐、其溶剂合物、其N-氧化物、或它们的前药的药物组合物作为B细胞活化抑制剂。
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WO2021089791A1 (en) 2019-11-08 2021-05-14 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment of cancers that have acquired resistance to kinase inhibitors
WO2021148581A1 (en) 2020-01-22 2021-07-29 Onxeo Novel dbait molecule and its use
WO2022140246A1 (en) 2020-12-21 2022-06-30 Hangzhou Jijing Pharmaceutical Technology Limited Methods and compounds for targeted autophagy

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JP2020525544A (ja) 2020-08-27
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