WO2023130541A1 - 2-吡唑-3-苯并咪唑衍生物及其制备方法和应用 - Google Patents

2-吡唑-3-苯并咪唑衍生物及其制备方法和应用 Download PDF

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WO2023130541A1
WO2023130541A1 PCT/CN2022/077624 CN2022077624W WO2023130541A1 WO 2023130541 A1 WO2023130541 A1 WO 2023130541A1 CN 2022077624 W CN2022077624 W CN 2022077624W WO 2023130541 A1 WO2023130541 A1 WO 2023130541A1
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pyrazole
benzimidazole derivative
preparation
benzimidazole
receptor
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French (fr)
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李环球
胡庆华
朱一凡
王辉
魏若男
周梦泽
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the invention belongs to the technical field of medicinal chemistry, and in particular relates to 2-pyrazole-3-benzimidazole derivatives and a preparation method and application thereof.
  • P2Y 6 R is a member of eight subtypes of the P2Y receptor family (P2Y 1 R, P2Y 2 R, P2Y 4 R, P2Y 6 R, P2Y 11 R, P2Y 12 R, P2Y 13 R and P2Y 14 R), Expressed in various organs and tissues such as immune organs, cardiovascular system, and nervous tissue, the endogenous ligand of P2Y 6 receptor is an extracellular nucleotide molecule, which specifically activates phospholipase C through its selective agonist UDP (PLC), up-regulate the concentration of intracellular Ca 2+ to achieve the purpose of transmitting intercellular signals and regulating various physiological functions of cells.
  • PLC selective agonist UDP
  • P2Y 6 receptor when the P2Y 6 receptor is stimulated, it can promote the recruitment and chemotaxis of neutrophils and macrophages, and release a variety of inflammatory cytokines, chemokines and mast cell mediators. Studies on mouse models of P2Y 6 receptor gene knockout have shown that P2Y 6 receptors are involved in the occurrence and development of diseases such as cardiovascular disease, respiratory inflammation, and gastrointestinal inflammation.
  • P2Y 6 receptor antagonists have good innovation and application prospects in the field of drug development for inflammatory bowel disease, atherosclerosis and other related inflammatory diseases. It has been pointed out that in the dextran sodium sulfate DSS-induced colitis model, blocking the P2Y 6 receptor alone can protect the intestinal tract from inflammation. Extracellular nucleotide signaling on the surface of intestinal epithelial cells plays an important role in the development of intestinal inflammation and possibly in inflammatory bowel disease IBD. Regulation of P2Y 6 signaling may be a new potential target for the treatment of IBD.
  • a paper published on drug discovery today clarifies that P2Y 6 receptors are widely distributed in various tissues and immune cells, and participate in inflammatory responses by regulating the expression and secretion of cytokines and pro-inflammatory molecules. It causes a range of human diseases and, as such, has become a therapeutic target for the treatment of many diseases, including atherosclerosis and other cardiovascular diseases.
  • the object of the present invention is to provide 2-pyrazole-3-benzimidazole derivatives and their preparation methods and applications, the 2-pyrazole-3-benzimidazole derivatives have better ability to antagonize the P2Y 6 receptor body activity, has obvious inhibitory effect on related inflammatory reactions, and can be used as a drug for preparing P2Y 6 receptor-related inflammatory diseases.
  • the 2-pyrazole-3-benzimidazole derivatives provided by the present invention have the structure of formula I:
  • the R 1 is selected from substituted or unsubstituted phenyl groups, or 5-6 membered heterocyclic groups;
  • the R 2 is selected from substituted or unsubstituted C1-C6 alkyl, alkoxy, hydrogen, heterocyclyl, alkenyl, cyano, hydroxyl, or halogen.
  • the R is selected from furyl, pyrrolyl, phenyl, or substituted phenyl;
  • the R2 is selected from methyl, ethyl, isopropyl, isobutyl, methoxy, hydrogen, phenolic hydroxyl, cyano, trifluoromethyl, nitro or halogen.
  • benzimidazole compounds have a wide range of biological activities. Studies have shown that benzimidazole heterocyclic compounds have good biological activities such as anti-inflammatory, antibacterial, atherosclerosis, and anti-virus. Due to their unique structure, low toxicity and excellent biological activity, they are widely used in chemistry, medicine, It has been widely used in many fields such as biology and materials science. Using the principle of activity superposition, the activity of many small molecule drugs is greatly improved after introducing benzimidazole groups. It is precisely because benzimidazole derivatives have many excellent properties that they have become a hot spot in drug research and development.
  • the benzimidazole derivative has any of the following structures:
  • the present invention provides a kind of preparation method of 2-pyrazole-3-benzimidazole derivative described in above-mentioned technical scheme, comprises the following steps:
  • the compound of the formula I structure is specifically prepared according to the following route:
  • the present invention provides a 2-pyrazole-3-benzimidazole derivative described in the above technical scheme or a 2-pyrazole-3-benzimidazole derivative prepared by the preparation method described in the above technical scheme in the preparation of P2Y 6 Application of drugs in the treatment of receptor-associated inflammatory diseases.
  • the P2Y 6 receptor-related inflammatory diseases include atherosclerosis, inflammatory bowel disease and the like.
  • the present invention provides a therapeutic drug for P2Y 6 receptor-related inflammatory diseases, which is characterized in that it comprises 2-pyrazole-3-benzimidazole derivatives described in the above technical scheme or 2-pyrazole-3-benzimidazole derivatives prepared by the preparation method described in the above technical scheme. - pyrazole-3-benzimidazole derivatives and pharmaceutically acceptable auxiliary materials.
  • the above-mentioned drugs for treating inflammatory diseases such as atherosclerosis and inflammatory bowel disease provided by the present invention can also be used in combination with other drugs for treating inflammatory diseases.
  • the 2-pyrazole-3-benzimidazole derivative provided by the present invention has a structure represented by formula (I). Experimental results show that the 2-pyrazole-3-benzimidazole derivatives provided by the present invention have better P2Y 6 receptor antagonistic activity and in vitro anti-inflammatory activity, and can be used as the preparation of P2Y 6 receptor-related inflammatory disease therapeutic drugs application.
  • the crude product was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, spin-dried under reduced pressure, and purified by silica gel column chromatography, eluting with petroleum ether: ethyl acetate (4:1) to obtain 4-(furan-2 -yl)-2,4-dioxobutanoic acid ethyl ester.
  • the product was purified by silica gel column chromatography, eluting with petroleum ether: ethyl acetate (50:1 ⁇ 20:1) to give 1-(tert-butyl)-5-(furan-2-yl)-1H-pyrazole -3-Carboxylic acid ethyl ester.
  • the NMR data are as follows:
  • the NMR data are as follows:
  • the NMR data are as follows:
  • the NMR data are as follows:
  • the NMR data are as follows:
  • the NMR data are as follows:
  • Example 1 Using acetophenone as a raw material, see Example 1 for the synthesis method.
  • the NMR data are as follows:
  • Example 1 Using acetophenone and 3,4-diaminotoluene as raw materials, see Example 1 for the synthesis method.
  • the NMR data are as follows:
  • Example 1 Using acetophenone and 4-chloro-1,2-phenylenediamine as raw materials, see Example 1 for the synthesis method.
  • the NMR data are as follows:
  • Example 1 Using p-methylacetophenone and 4-chloro-1,2-phenylenediamine as raw materials, see Example 1 for the synthesis method.
  • the NMR data are as follows:
  • the previously constructed human P2Y 6 R stably transfected HEK293 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin), and inoculated into 6-well culture plates before the experiment.
  • the cells were cultured at 37°C, 95% O 2 , and 5% CO 2 humidity at 5 ⁇ 10 5 cells/ml.
  • the serum-free medium was starved for 12 hours, and 1 ⁇ M compound was added to each well. After 30 minutes of reaction, 10 ⁇ M UDP was added to incubate for 12 hours, and samples were collected to detect the content of intracellular inositol 3-phosphate (IP3).
  • IP3 intracellular inositol 3-phosphate
  • IP3 enzyme-linked immunosorbent assay kit adopts competition ELISA method.
  • the IP3 antigen is coated on the microtiter plate, and the IP3 in the sample or standard competes with the coated IP3 for the binding site on the biotin-labeled anti-IP3 monoclonal antibody during the experiment, and the free components are washed away.
  • Add horseradish peroxidase-labeled avidin, biotin and avidin specifically combine to form an immune complex, and free components are washed away.
  • Chromogenic substrate (TMB) is added, and TMB turns blue under the catalysis of horseradish peroxidase, and turns yellow after adding stop solution.
  • TMB Chromogenic substrate
  • Example 12 Preferred compound in vitro anti-inflammatory activity test method
  • a THP-1 cell model with P2Y 6 R overexpression was constructed using plasmids, normal cells were used as a parallel control group, and cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin ), inoculated to cell culture plates before the experiment, at a seeding density of 1 ⁇ 10 6 cells/ml, and the cells were cultured at 37° C., 95% O 2 , and 5% CO 2 humidity conditions.
  • the 2-pyrazole-3-benzimidazole derivatives prepared by the present invention have better antagonism of P2Y 6 receptor inhibitory activity and P2Y 6- induced in vitro inflammatory response, and can be used to prepare P2Y 6 receptor Drugs for the treatment of related inflammatory diseases.
  • the 2-pyrazole-3-benzimidazole derivatives provided by the present invention have the structure represented by formula (I).
  • Experimental results show that the 2-pyrazole-3-benzimidazole derivatives provided by the present invention have better P2Y 6 receptor antagonistic activity and in vitro anti-inflammatory activity, and can be used as the preparation of P2Y 6 receptor-related inflammatory disease therapeutic drugs application.

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Abstract

本发明提供的2-吡唑-3-苯并咪唑衍生物及其制备方法和应用，2-吡唑-3-苯并咪唑衍生物具有式（Ⅰ）所示结构。实验结果表明，本发明提供的2-吡唑-3-苯并咪唑衍生物具有较好的P2Y6受体拮抗活性和体外抗炎活性，可以作为制备P2Y6受体相关炎症性疾病治疗药物的应用。

Description

2-吡唑-3-苯并咪唑衍生物及其制备方法和应用
本申请要求于2022年01月06日提交中国专利局、申请号为202210012999.6、发明名称为“2-吡唑-3-苯并咪唑衍生物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于药物化学技术领域，尤其涉及2-吡唑-3-苯并咪唑衍生物及其制备方法和应用。
背景技术
P2Y 6R是P2Y受体家族八个亚型(P2Y 1R，P2Y 2R，P2Y 4R，P2Y 6R，P2Y 11R，P2Y 12R，P2Y 13R和P2Y 14R)中的一员，在免疫器官、心血管系统、神经组织等多种器官和组织中表达，P2Y 6受体的内源性配体是胞外核苷酸分子，通过其选择性激动剂UDP特异性激活磷脂酶C(PLC)，上调细胞内Ca 2+浓度，达到传递细胞间信号、调节细胞各种生理功能的目的。目前研究表明，当P2Y 6受体受到激动时，可促进中性粒细胞和巨噬细胞的募集和趋化，并释放多种炎症细胞因子、趋化因子和肥大细胞介质。对P2Y 6受体的基因敲除的小鼠模型的研究表明，P2Y 6受体参与心血管疾病、呼吸道炎症及胃肠道炎症等疾病的发生发展。
P2Y 6受体拮抗剂在炎症性肠病，动脉粥样硬化等相关炎症疾病药物开发领域有很好的创新性和应用前景。有文献指出，在葡聚糖硫酸钠DSS诱导的结肠炎模型中，单独阻断P2Y 6受体，可以保护肠道免受炎症的影响。肠上皮细胞表面的细胞外核苷酸信号在肠道炎症的发展以及可能在炎症性肠病IBD中起着重要作用。调控P2Y 6信号可能是治疗IBD的一个新的潜在目标。在drug discovery today上发表的一篇论文，阐明了P2Y 6受体广泛分布于各种组织和免疫细胞中，通过调节细胞因子和促炎分子的表达和分泌参与炎症反应。它会引起一系列人类疾病，因此，已成为治疗许多疾病(包括动脉粥样硬化和其他心血管疾病)的治疗靶点。
发明内容
有鉴于此，本发明的目的在于提供2-吡唑-3-苯并咪唑衍生物及其制备方法和应用，该2-吡唑-3-苯并咪唑衍生物具有较好的拮抗P2Y 6受体活性，对相关炎症反应具有明显的抑制作用，可以作为制备P2Y 6受体相关炎症性疾病治疗药物的应用。
本发明提供的2-吡唑-3-苯并咪唑衍生物具有式Ⅰ结构：
Figure PCTCN2022077624-appb-000001
所述R 1选自取代或非取代苯基、或5～6元杂环基团；
所述R 2选自取代或非取代的C1～C6的烷基、烷氧基、氢、杂环基、烯基、氰基、羟基、或卤素。
在本发明中，所述R 1选自呋喃基、吡咯基、苯基、或取代苯基；
所述R 2选自甲基、乙基、异丙基、异丁基、甲氧基、氢、酚羟基、氰基、三氟甲基、硝基或卤素。
在本发明中，苯并咪唑类化合物具有广泛的生物活性。研究表明，苯并咪唑类杂环化合物具有很好的抗炎抗菌、治疗动脉粥样硬化、抗病毒等生物活性，由于具有独特的结构、低毒性和优良的生物活性，因此在化学、医学、生物学和材料科学等众多领域得到了广泛应用。利用活性叠加原理，在许多小分子药物中引入苯并咪唑基团后，其活性得到很大的提高。正是由于苯并咪唑类衍生物具有众多优良特性，使得它成为药物研发中的一大热点。
在本发明中，所述苯并咪唑衍生物具有以下任一结构：
Figure PCTCN2022077624-appb-000002
Figure PCTCN2022077624-appb-000003
本发明提供了一种上述技术方案所述2-吡唑-3-苯并咪唑衍生物的制备方法，包括以下步骤：
将具有式Ⅱ结构的化合物和具有式Ⅲ结构的化合物反应，得到具有式Ⅰ结构的2-吡唑-3-苯并咪唑衍生物：
Figure PCTCN2022077624-appb-000004
在本发明具体实施例中，所述式Ⅰ结构的化合物具体按照以下路线制得：
Figure PCTCN2022077624-appb-000005
本发明提供了一种上述技术方案所述2-吡唑-3-苯并咪唑衍生物或上述技术方案所述的制备方法制备的2-吡唑-3-苯并咪唑衍生物在制备P2Y 6受体相关炎症性疾病治疗药物中的应用。
在本发明中，所述P2Y 6受体相关炎症性疾病包括动脉粥样硬化，炎症性肠病等。
本发明提供了一种P2Y 6受体相关炎症性疾病治疗药物，其特征在于，包括上述技术方案所述2-吡唑-3-苯并咪唑衍生物或上述技术方案所述制备方法制备的2-吡唑-3-苯并咪唑衍生物以及药学上可接受的辅料。
本发明提供的上述治疗动脉粥样硬化，炎症性肠病等炎症性疾病药物还可以与其他治疗炎症性疾病药物联合使用。
本发明提供的2-吡唑-3-苯并咪唑衍生物，具有式(Ⅰ)所示结构。实验结果表明，本发明提供的2-吡唑-3-苯并咪唑衍生物具有较好的P2Y 6受体拮抗活性和体外抗炎活性，可以作为制备P2Y 6受体相关炎症性疾病治疗药物的应用。
具体实施方式
为了进一步说明本发明，下面结合实施例对本发明提供的2-吡唑-3-苯并咪唑衍生物及其制备方法和应用进行详细地描述，但不能将它们理解为对本发 明保护范围的限定。
实施例1
Figure PCTCN2022077624-appb-000006
2-(1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-基)-苯并[d]咪唑的合成：
将2.8g叔丁醇钾置于在11mL THF中形成悬浮液，在0℃的冰浴中冷却；将1.1g乙酰呋喃和2.7ml草酸二乙酯溶解在11ml乙二醇二甲醚中，将此溶液逐滴加入0℃叔丁醇钾的悬浮液中；将混合物在室温下搅拌1小时。通过TLC监测所有乙酰基呋喃已消耗完后，添加7.5mL的1M HCl溶液。粗产物用乙酸乙酯萃取，有机相经无水硫酸钠干燥，减压旋干，通过硅胶柱色谱法纯化，石油醚：乙酸乙酯(4：1)洗脱，得到4-(呋喃-2-基)-2,4-二氧代丁酸乙酯。
将1.4g 4-(呋喃-2-基)-2,4-二氧代丁酸乙酯溶于乙醇中，加入0.9g叔丁基肼盐酸盐，并将混合物在室温搅拌过夜。当反应完成时，用水和乙酸乙酯萃取粗产物。有机相用无水硫酸钠干燥并在减压下旋干。产物通过硅胶柱色谱法纯化，用石油醚：乙酸乙酯(50：1～20：1)洗脱，得到1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-羧酸乙酯。
将200mg 1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-羧酸乙酯溶解在3mL四氢呋喃中，然后在0℃下将其滴加到氢化锂铝(50mg)在10mL四氢呋喃中的浆液中。在此温度下保持30分钟后，将反应加热至回流2小时。将反应冷却至室温，并向反应中加入10mL乙酸乙酯。向反应液中加入5N氢氧化钠，直到出现白色沉淀为止。过滤，滤液用乙酸乙酯和水萃取。用硫酸镁干燥有机层，过滤，减压旋干，得到粗制的(1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-基)甲醇。
将188mg(1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-基)甲醇溶于DCM中，冰浴下加入434mg Dess-Martin试剂，室温下搅拌1h，用饱和碳酸钠淬灭，硅藻土过滤，乙酸乙酯萃取滤液，无水硫酸钠干燥，减压旋干得到粗制的1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-甲醛。
将80mg1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-甲醛，60mg邻苯二胺， 80mg亚硫酸氢钠溶于乙醇：水＝2：1的溶液中，回流1h。乙酸乙酯萃取，无水硫酸钠干燥，减压旋干，通过薄层色谱法纯化，石油醚：乙酸乙酯(3：1)，得到纯净目标产物。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.61(s,1H),7.92(s,1H),7.57(d,J＝35.0Hz,2H),7.18(d,J＝6.4Hz,2H),7.01(s,1H),6.77(d,J＝3.3Hz,1H),6.68(t,J＝2.5Hz,1H),1.56(s,9H).
13C NMR(101MHz,DMSO-d 6)δ152.42,152.17,142.66,142.19,137.03,135.49,129.92,123.10,123.04,119.30,112.40,112.13,111.52,104.50,60.49,28.94.
实施例2
Figure PCTCN2022077624-appb-000007
2-(1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-基)-5-甲基-1H-苯并[d]咪唑的合成：
以3,4-二氨基甲苯为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.49(s,1H),7.92(d,J＝1.7Hz,1H),7.45(d,J＝8.1Hz,1H),7.36(s,1H),7.00(s,2H),6.77(d,J＝3.2Hz,1H),6.68(dd,J＝3.3,1.9Hz,1H),2.43(s,3H),1.56(s,9H).
13C NMR(101MHz,DMSO-d 6)δ153.16,152.16,142.66,137.16,137.03,135.12,129.92,129.80,127.10,113.83,113.30,112.40,111.52,104.50,60.49,28.94,20.92.
实施例3
Figure PCTCN2022077624-appb-000008
2-(1-(叔丁基)-5-(呋喃-2-基)-1H-吡唑-3-基)-5-氯-1H-苯并[d]咪唑的合成：
以4-氯-1,2-苯二胺为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.80(d,J＝25.1Hz,1H),7.91(d,J＝1.8Hz,1H),7.70–7.61(m,1H),7.52(s,1H),7.21(s,1H),7.03(s,1H),6.77(d,J＝3.2Hz,1H),6.67(dd,J＝3.3,1.9Hz,1H),1.55(s,9H).
13C NMR(101MHz,DMSO-d 6)δ153.16,152.16,142.66,137.03,135.15,135.06,129.92,128.37,125.20,115.61,113.93,112.40,111.52,104.50,60.49,28.94.
实施例4
Figure PCTCN2022077624-appb-000009
2-(1-(叔丁基)-5-(1H-吡咯-2-基)-1H-吡唑-3-基)-1H-苯并[d]咪唑的合成：
以2-乙酰基吡咯为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ11.27–11.24(m,1H),7.58(dd,J＝5.9,3.3Hz,2H),7.18(dd,J＝6.0,3.2Hz,2H),6.91(q,J＝2.4Hz,1H),6.86(s,1H),6.25(dt,J＝3.7,2.2Hz,1H),6.16(q,J＝2.8Hz,1H),1.50(s,9H).
13C NMR(101MHz,DMSO-d 6)δ153.16,152.16,142.66,137.03,135.15,135.06,129.92,128.37,125.20,115.61,113.93,112.40,111.52,104.50,60.49,28.94.
实施例5
Figure PCTCN2022077624-appb-000010
2-(1-(叔丁基)-5-(1H-吡咯-2-基)-1H-吡唑-3-基)-5-甲基-1H-苯并[d]咪唑的 合成：
以2-乙酰基吡咯和3,4-二氨基甲苯为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.42(s,1H),11.25–11.19(m,1H),7.44(d,J＝8.2Hz,1H),7.34(s,1H),6.99(d,J＝8.1Hz,1H),6.90(q,J＝2.4Hz,1H),6.80(s,1H),6.23(q,J＝2.6,1.9Hz,1H),6.15(q,J＝2.8Hz,1H),2.42(s,3H),1.49(s,9H).
13C NMR(101MHz,DMSO-d 6)δ153.16,152.16,142.66,137.03,135.15,135.06,129.92,128.37,125.20,115.61,113.93,112.40,111.52,104.50,60.49,28.94.
实施例6
Figure PCTCN2022077624-appb-000011
2-(1-(叔丁基)-5-(1H-吡咯-2-基)-1H-吡唑-3-基)-5-氯-1H-苯并[d]咪唑的合成：
以2-乙酰基吡咯和4-氯-1,2-苯二胺为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.73(s,1H),11.23(d,J＝2.9Hz,1H),7.63–7.51(m,2H),7.20(dd,J＝8.5,2.1Hz,1H),6.90(q,J＝2.4Hz,1H),6.84(s,1H),6.23(dt,J＝3.9,1.8Hz,1H),6.15(q,J＝2.7Hz,1H),1.49(s,9H).
13C NMR(101MHz,DMSO-d 6)δ153.16,152.16,142.66,137.03,135.15,135.06,129.92,128.37,125.20,115.61,113.93,112.40,111.52,104.50,60.49,28.94.
实施例7
Figure PCTCN2022077624-appb-000012
2-(1-(叔丁基)-5-苯基-1H-吡唑-3-基)-1H-苯并[d]咪唑的合成：
以苯乙酮为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.59(s,1H),7.48(s,7H),7.17(d,J＝6.5Hz,2H),6.74(s,1H),1.49(s,9H).
13C NMR(101MHz,DMSO-d6)δ152.42,144.60,142.19,137.14,135.49,131.16,128.86,128.52,127.10,123.10,123.04,119.30,112.13,105.02,60.32,28.94.
实施例8
Figure PCTCN2022077624-appb-000013
2-(1-(叔丁基)-5-苯基-1H-吡唑-3-基)-5-甲基-1H-苯并[d]咪唑的合成：
以苯乙酮和3,4-二氨基甲苯为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d6)δ12.44(d,J＝10.7Hz,1H),7.48(s,6H),7.42–7.36(m,1H),6.99(dd,J＝15.6,8.1Hz,1H),6.71(s,1H),2.41(d,J＝7.2Hz,3H),1.48(s,9H).
13C NMR(101MHz,DMSO-d6)δ153.16,144.60,137.15,135.12,131.16,129.80,128.86,128.52,127.10,113.83,113.30,105.02,60.32,28.94,20.92.
实施例9
Figure PCTCN2022077624-appb-000014
2-(1-(叔丁基)-5-苯基-1H-吡唑-3-基)-5-氯-1H-苯并[d]咪唑的合成：
以苯乙酮和4-氯-1,2-苯二胺为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.76(d,J＝25.0Hz,1H),7.69–7.60(m,1H),7.48(d,J＝2.2Hz,6H),7.20(dd,J＝14.0,8.3Hz,1H),6.75(s,1H),1.48(s,9H).
13C NMR(101MHz,DMSO-d6)δ153.16,144.60,137.14,135.15,135.06,131.16,128.86,128.52,128.37,127.10,125.20,115.61,113.93,105.02,60.32,28.94.
实施例10
Figure PCTCN2022077624-appb-000015
2-(1-(叔丁基)-5-(对甲苯基)-1H-吡唑-3-基)-5-氯-1H-苯并[d]咪唑的合成：
以对甲基苯乙酮和4-氯-1,2-苯二胺为原料，合成方法参见实施例1。
核磁数据如下：
1H NMR(400MHz,DMSO-d 6)δ12.76(d,J＝25.0Hz,1H),7.69–7.60(m,1H),7.48(d,J＝2.2Hz,6H),7.20(dd,J＝14.0,8.3Hz,1H),6.75(s,1H),1.48(s,9H).
13C NMR(101MHz,DMSO-d6)δ153.16,144.60,137.74,137.14,135.15,135.06,131.02,129.03,128.37,127.15,125.20,115.61,113.93,105.02,60.32,28.94,21.42.
实施例11 化合物对P2Y 6受体体外拮抗活性测试方法
将前期构建的人P2Y 6R稳转HEK293细胞培养于DMEM培养基中(含10％胎牛血清、100U/ml青霉素和100μg/ml链霉素)，实验前接种至6孔培养板，接种密度为5×105cells/ml，细胞于37℃、95％O 2、5％CO 2湿度条件下培养。实验前换无血清培养基饥饿12h，每孔加入1μM化合物，反应30min后加入10μM UDP孵育12h后收集样品检测胞内3磷酸肌醇(IP3)含量。
3磷酸肌醇(IP3)酶联免疫吸附测定试剂盒采用竞争ELISA法。用IP3抗原包被于酶标板上，实验时样品或标准品中的IP3与包被的IP3竞争生物素标记的抗IP3单抗上的结合位点，游离的成分被洗去。加入辣根过氧化物酶标记的亲和素，生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。加入显色底物(TMB)，TMB在辣根过氧化物酶的催化下呈现蓝色，加终止液后变成黄色。用酶标仪在450nm波长处测OD值，IP3浓度与OD450值之间呈反比，通过绘制标准曲线计算出样品中IP3的浓度。
最后计算每组复孔的平均OD值。以浓度为横坐标，OD值为纵坐标，在双对数坐标纸上绘出四参数逻辑函数的标准曲线；通过标准曲线计算出样品中IP3的浓度。实验重复三次，取平均值并计算化合物对P2Y 6R的IC 50。
实验结果如表1所示：
表1 化合物对P2Y 6R体外拮抗活性测试结果
化合物 ％inhibition at 10μM hP2Y 6R IC 50(nM)
Ⅰ-1 69.12 878.54
Ⅰ-2 67.45 765.32
Ⅰ-3 132.71 5.75
Ⅰ-4 78.76 920.35
Ⅰ-5 69.56 278.56
Ⅰ-6 100.62 132.54
Ⅰ-7 73.92 335.09
Ⅰ-8 86.39 97.27
Ⅰ-9 106.39 18.30
Ⅰ-10 112.58 54.23
实施例12 优选化合物体外抗炎活性测试方法
采用质粒构建P2Y 6R过表达的THP-1细胞模型，用正常细胞作为平行对照组，培养于RPMI-1640培养基中(含10％胎牛血清、100U/ml青霉素和100μg/ml链霉素)，实验前接种至细胞培养板，接 种密度为1×10 6cells/ml，细胞于37℃、95％O 2、5％CO 2湿度条件下培养。实验前预先向培养基中分别加入U73122(PLCβ抑制剂)和BAPTA-AM(Ca 2+螯合剂)，1h后向细胞中加入终浓度为50μg/ml的ox-LDL，24h后显微镜下观察细胞形态，检测细胞培养上清液中NLRP3炎症小体，IL-1β、TNF-α等炎症因子的表达含量，计算IC 50。
实验结果如表2所示：
表2 化合物对体外抗炎活性测试结果
Figure PCTCN2022077624-appb-000016
由上述实施例可知，本发明制备的2-吡唑-3-苯并咪唑衍生物具有较好的拮抗P2Y 6受体抑制活性及P2Y 6诱导的体外炎症反应，可以用于制备P2Y 6受体相关炎症性疾病治疗药物。
由以上实施例可知，本发明提供的2-吡唑-3-苯并咪唑衍生物，具有式(Ⅰ)所示结构。实验结果表明，本发明提供的2-吡唑-3-苯并咪唑衍生物具有较好的P2Y 6受体拮抗活性和体外抗炎活性，可以作为制备P2Y 6受体相关炎症性疾病治疗药物的应用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (6)

  1. 2-吡唑-3-苯并咪唑衍生物，具有式Ⅰ结构：
    Figure PCTCN2022077624-appb-100001
    所述R 1选自取代或非取代苯基、或5～6元杂环基团；
    所述R 2选自取代或非取代的C1～C6的烷基、烷氧基、氢、杂环基、烯基、氰基、羟基、或卤素。
  2. 根据权利要求1所述的2-吡唑-3-苯并咪唑衍生物，其特征在于，所述R 1选自呋喃基、吡咯基、苯基、或取代苯基；
    所述R 2选自甲基、乙基、异丙基、异丁基、甲氧基、氢、酚羟基、氰基、三氟甲基、硝基或卤素。
  3. 根据权利要求1所述的2-吡唑-3-苯并咪唑衍生物，其特征在于，所述苯并咪唑衍生物具有以下任意一种结构：
    Figure PCTCN2022077624-appb-100002
    Figure PCTCN2022077624-appb-100003
  4. 一种权利要求1～3任一项所述2-吡唑-3-苯并咪唑衍生物的制备方法，包括以下步骤：
    将具有式Ⅱ结构的化合物和具有式Ⅲ结构的化合物反应，得到具有式Ⅰ结构的2-吡唑-3-苯并咪唑衍生物：
    Figure PCTCN2022077624-appb-100004
  5. 一种权利要求1～3任一项所述2-吡唑-3-苯并咪唑衍生物或权利要求4所述的制备方法制备的2-吡唑-3-苯并咪唑衍生物在制备P2Y 6受体相关炎症性疾病治疗药物中的应用。
  6. 一种P2Y 6受体相关炎症性疾病治疗药物，其特征在于，包括权利要求1～3任一项所述2-吡唑-3-苯并咪唑衍生物或权利要求4所述制备方法制备的2-吡唑-3-苯并咪唑衍生物以及药学上可接受的辅料。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958950A (en) * 1995-10-05 1999-09-28 Warner-Lambert Company Benzimidazole compounds useful for the treatment of inflammatory disease, atherosclerosis, restenosis or inhibiting lipoxygenase
WO2005085230A1 (de) * 2004-03-02 2005-09-15 Sanofi-Aventis Deutschland Gmbh 4-benzimidazol-2-yl-pyridazin-3-on-derivative, ihre herstellung und verwendung in arzneimitteln
WO2007056155A1 (en) * 2005-11-03 2007-05-18 Chembridge Research Laboratories, Inc. Heterocyclic compounds as tyrosine kinase modulators
CN1993124A (zh) * 2004-07-22 2007-07-04 默克公司 取代吡唑、含有这种化合物的组合物及其应用
WO2020150545A1 (en) * 2019-01-17 2020-07-23 Samumed, Llc Pyrazole derivatives as modulators of the wnt/b-catenin signaling pathway

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958950A (en) * 1995-10-05 1999-09-28 Warner-Lambert Company Benzimidazole compounds useful for the treatment of inflammatory disease, atherosclerosis, restenosis or inhibiting lipoxygenase
WO2005085230A1 (de) * 2004-03-02 2005-09-15 Sanofi-Aventis Deutschland Gmbh 4-benzimidazol-2-yl-pyridazin-3-on-derivative, ihre herstellung und verwendung in arzneimitteln
CN1993124A (zh) * 2004-07-22 2007-07-04 默克公司 取代吡唑、含有这种化合物的组合物及其应用
WO2007056155A1 (en) * 2005-11-03 2007-05-18 Chembridge Research Laboratories, Inc. Heterocyclic compounds as tyrosine kinase modulators
WO2020150545A1 (en) * 2019-01-17 2020-07-23 Samumed, Llc Pyrazole derivatives as modulators of the wnt/b-catenin signaling pathway

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