CN117624126A - 选择性酪氨酸激酶2（tyk2）抑制剂化合物、合成方法及用途 - Google Patents
选择性酪氨酸激酶2（tyk2）抑制剂化合物、合成方法及用途 Download PDFInfo
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- CN117624126A CN117624126A CN202311652197.2A CN202311652197A CN117624126A CN 117624126 A CN117624126 A CN 117624126A CN 202311652197 A CN202311652197 A CN 202311652197A CN 117624126 A CN117624126 A CN 117624126A
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Abstract
本发明公开了选择性酪氨酸激酶2(TYK2)抑制剂化合物、合成方法及用途，所述化合物如通式(I)所示，本发明化合物在H9细胞中具有一定的TYK2抑制活性。本发明化合物能够有效抑制TYK2的活性，随后将给药浓度设置后进行时间依赖性实验，随着给药时间延长，在24h是效果最佳，紧接着进行JAK激酶家族选择性实验，分别对JAK1/JAK2/TYK2通路、JAK1/JAK3通路及JAK2通路相应下游蛋白的磷酸化程度进行了检测，结果显示在不含TYK2的通路中，下游蛋白磷酸化的程度随着浓度增加不变或者变化不明显，这说明化合物B3拥有高的靶标选择性，有较低的脱靶风险，可能具有较高的安全性。进一步说明化合物对银屑病的治疗具有一定的潜力。
Description
技术领域
本发明涉及化合物及其制法和用途，具体涉及选择性酪氨酸激酶2(TYK2)抑制剂化合物、合成方法及用途。
背景技术
银屑病(Psoriasis,PsO)是由免疫系统介导的慢性、复发性、炎症性皮肤病，疾病的折磨给患者带来较大的心理压力和经济压力。而免疫系统异常是诱发银屑病的主要因素之一。现有的银屑病治疗药物主要包括生物制剂和小分子口服药物，其中小分子抑制剂使用最为广泛。药物治疗能抑制银屑病，提高患者生活质量，但是现在一线治疗中重度银屑病的治疗药物存在患者耐受性差、选择性差及耐药性等问题，并且小分子抑制剂存在的不同副作用也不容忽视。因此，开发疗效显著、安全可靠的银屑病药物是药学工作者面临的重大挑战。
传统的小分子抑制剂如甲氨蝶呤、环孢素、阿维A存在严重药物相互作用和长期毒性，磷酸二酯酶(Phosphodiesterases,PDE)抑制剂和Janus激酶(Janus Kinase,JAK)抑制剂在银屑病治疗方面起到关键作用。阿普米司特(Apremilast)是唯一临床批准口服治疗银屑病的PDE-4抑制剂，通过减轻炎症治疗中到重度银屑病。托法替尼(Tofacitinib)、巴瑞克替尼(Baritinib)和鲁索利替尼(Ruxolitinib)属于第一代JAK抑制剂，虽然疗效得到一定程度证实，但是安全性问题导致药物治疗窗口狭窄，不良反应严重，严重的还会导致心脏毒性。乌帕替尼(Upadacitinib)属于第二代JAK抑制剂，由于与一代JAK抑制剂存在一样剂量依赖性的严重不良反应，与银屑病相关的临床试验被终止。研究表明，由于托法替尼、乌帕替尼和巴瑞克替尼在治疗浓度下不抑制TYK2，这可能表明JAK抑制剂典型的不良反应不是由TYK2抑制所致，因此开发高选择性TYK2抑制剂引起了很多学者的关注。选择性TYK2抑制剂Deucravacitinib达到安全性和有效性之间的最佳平衡，目前，TYK2也成为银屑病及其他免疫性疾病治疗药物研发的热门靶点。
在正常和病理状态下，JAK-STAT通路在多种细胞过程中的细胞内信号传递中发挥重要作用，TYK2通过调节IL-12、IL-23、Ⅰ型IFNs受体下游的信号通路影响STAT1和STAT2磷酸化程度。IL-23和IL-17被认为是启动和维持慢性炎症的关键细胞因子，而TYK2是它们之间关键的细胞内信号转导纽带，抑制TYK2的活性可以切断IL23/IL-17之间的联系，从而抑制银屑病发病的关键步骤。阻断TYK2介导的JAK-STAT通路成为研发治疗银屑病的新策略。
目前，银屑病的治疗药物主要为生物制剂和小分子药物，但是生物制剂价格昂贵，小分子药物如泛JAK抑制剂会产生严重的不良反应。氘可来昔替尼(Deucravatinib)作为首款获批上市的TYK2 JH2抑制剂在选择性和安全性上达到了最佳平衡，给银屑病患者带来了新的希望，因此，开发选择性TYK2抑制剂具有重大意义。
发明内容
发明目的：本发明提供一种选择性酪氨酸激酶2(TYK2)抑制剂化合物。本发明的另一目的是提供上述化合物或其可药用盐的制备方法。本发明的另一目的是提供所述的化合物或其可药用的盐在制备TYK2抑制剂中的用途。
技术方案：如通式(I)所示的化合物或其药学上可接受的盐：
其中，R1选自以下结构：
R2为C1-C4烷基或-H；
R3为C1-C4烷基或
所述的化合物或其药学上可接受的盐，
R1选自以下结构：
R2为-CH3或-H；
R3为-CH3、-C2H5或
所述的化合物或其药学上可接受的盐，化合物选自如下结构：
所述的化合物或其药学上可接受的盐的制备方法，包括如下步骤：
路线1：
其中，R1、R2、R3同上所述；
X、Y选自C或N，且两者不相同；
R4：H、-CH3、-OCH3、F；
R5：
或：
路线2：
所述的制备方法，所述路线1中的反应条件为：(a)4,6-二氯烟酸，EDCI，HOBt，DIPEA，DCM，-5～5℃；(b)HATU，DIPEA，DMF，室温；(c)NH4Cl，Fe粉，EtOH，80～90℃；(d)NaHMDS，THF；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，130～150℃。
所述的制备方法，所述路线2中的反应条件为：(a)4,6-二氯烟酸，羰基二咪唑，甲胺盐酸盐，DIPEA，室温；(b)3-硝基-2-(1H)-吡啶酮，醋酸铜，吡啶，1,4-二氧六环，70～90℃；(c)NH4Cl，Fe粉，EtOH，75～95℃；(d)NaHMDS，THF；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，130～150℃。
药物组合物，包括所述的化合物或其药学上可接受的盐及药学上可接受的辅料。
所述的化合物或其药学上可接受的盐在制备TYK2抑制剂中的用途。
所述的化合物或其药学上可接受的盐在制备治疗银屑病药物中的用途。
所述的用途，所述药物的剂型包括胶囊剂、片剂、颗粒剂、丸剂、口服液、注射剂。
本发明对先导化合物Deucravacitinib进行结构修饰，通过骨架迁移原理，采用吡啶替换哒嗪，并在酰胺侧链上引入基团以期增加与靶标的作用位点，获得一系列新的化合物。所述新的化合物具有较强的抑制活性及靶标选择性，表现出良好的活性和安全性，有望成为候选化合物进行更深入的机制探索以及体内实验，为后续研究TYK2 JH2抑制剂提供了基础。
有益效果：本发明中的化合物与现有技术相比，本发明化合物B3在H9细胞中具有一定的TYK2抑制活性。本发明化合物B3在1μM的浓度下能够有效抑制TYK2的活性，随后将给药浓度设置为1μM进行时间依赖性实验，随着给药时间延长，在24h是效果最佳，紧接着进行JAK激酶家族选择性实验，分别对JAK1/JAK2/TYK2通路、JAK1/JAK3通路及JAK2通路相应下游蛋白的磷酸化程度进行了检测，结果显示在不含TYK2的通路中，下游蛋白磷酸化的程度随着浓度增加不变或者变化不明显，这说明化合物B3拥有高的靶标选择性，有较低的脱靶风险，可能具有较高的安全性。进一步说明化合物对银屑病的治疗具有一定的潜力。
附图说明
图1为21个化合物对STAT1蛋白的磷酸化程度的实验结果；其中以Deucravacitinib为阳性对照。
具体实施方式
本发明提供选择性TYK2抑制剂化合物合成方法。所述制备方法包括路线1和路线2。
路线1：
其中，R1、R2、R3同上所述；
X、Y选自C或N，且两者不相同；
R4：H、-CH3、-OCH3、F；
R5：
反应条件：(a)4,6-二氯烟酸，EDCI，HOBt，DIPEA，DCM，0℃，15min，30％；(b)HATU，DIPEA，DMF，RT,overnight，80％～90％；(c)NH4Cl，Fe粉，EtOH，85℃，1H，95％；(d)NaHMDS，THF，1H，0℃，25％；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，145℃，2h。
路线2：
反应条件：(a)4,6-二氯烟酸，羰基二咪唑，甲胺盐酸盐，DIPEA，RT，4h，90％～95％，(b)3-硝基-2-(1H)-吡啶酮，醋酸铜，吡啶，1,4-二氧六环，80℃，overnight，60％～70％，(c)NH4Cl，Fe粉，EtOH，85℃，1H，95％；(d)NaHMDS，THF，1H，0℃，25％；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，145℃，2h。
实施例1：4-[4-[[2-[环丙烷甲酰胺基]-5-[甲基胺甲酰基]吡啶-4-基]氨基]苯甲酰胺基]哌啶-1-羧酸叔丁酯(A1)的合成
合成路线：
步骤1：
将4,6-二氯烟酸(5g)以及羰基二咪唑(8.48g)溶于无水THF(100ml)中，N2置换，室温下搅拌30min后，依次加入甲胺盐酸盐(2.1g)、N,N-二异丙基乙胺(DIPEA,7.43g,)，N2置换，室温下反应4h后，TLC监测原料消失，移除N2，加入60mL饱和NaHCO3水溶液淬灭反应，再加入300mL乙酸乙酯萃取，萃取3次合并上层有机相，用饱和NaCl水溶液洗涤3次，最后用无水Na2SO4干燥，浓缩，得到白色固体化合物1a(4.7g)，无需纯化，直接用于下一步。1H NMR(300MHz,DMSO-d6)δ8.62(s,1H),8.48(s,1H),7.91(s,1H),2.78(d,J＝4.7Hz,3H).
步骤2：
对硝基苯甲酸(500mg)、4-氨基-1-叔丁氧羰基哌啶(863mg)溶于无水DMF中，再加入HATU(1.71g)、DIPEA(1.16g)，加上干燥管，室温下过夜反应，TLC监测原料消失，加水淬灭反应，乙酸乙酯萃取(50mL×3)，合并有机相，有机相依次用饱和NaHCO3(100mL×3)、NaCl(100mL×3)洗涤，最后用无水Na2SO4干燥,减压浓缩后柱层析，得到黄色油状化合物1d(900mg,产率86％)。1H NMR(300MHz,Chloroform-d)δ8.31–8.25(m,2H),8.14–8.08(m,2H),7.66(d,J＝7.9Hz,1H),3.86(dp,J＝7.8,4.6Hz,1H),3.59–3.49(m,4H),1.98–1.88(m,2H),1.77–1.67(m,2H),1.45(s,9H).
步骤3：
化合物1d(890mg)溶于10mL EtOH中，再依次加入NH4Cl(1.4g,)水溶液、Fe粉(1.35g)，85℃下回流1h，冷却至室温，用硅藻土过滤，滤饼用EtOH洗涤，减压浓缩滤液，柱层析纯化得到黄色油状化合物1e(770mg,产率94.8％)。1H NMR(300MHz,Chloroform-d)δ7.64–7.52(m,2H),6.70–6.58(m,2H),5.29(t,J＝3.1Hz,1H),4.08(s,4H),2.88(t,J＝13.0Hz,2H),1.98(d,J＝12.4Hz,2H),1.45(q,J＝3.2Hz,9H).
步骤4：
化合物1b(100mg)、化合物1e(172mg)置于两颈瓶中，加入5mL无水THF，N2置换，冰浴下缓缓滴加1M NaHMDS/THF溶液(1.47mL)，反应1h，TLC监测原料消失，移除N2，加水淬灭反应，减压蒸馏除去THF，再用乙酸乙酯萃取(50ml×3)，合并有机相，饱和NaCl水溶液洗涤，最后用无水Na2SO4干燥，减压蒸馏，柱层析纯化，得到白色固体1f(60mg,产率25.1％)。mp:224℃-227℃.1H NMR(300MHz,Chloroform-d)δ10.32(d,J＝3.1Hz,1H),8.36(d,J＝3.3Hz,1H),7.78(dd,J＝8.9,3.2Hz,2H),7.28–7.23(m,2H),7.06(d,J＝3.3Hz,1H),6.88(s,1H),6.20–6.07(m,1H),4.12(s,3H),3.02(d,J＝4.1Hz,3H),2.98–2.83(m,2H),2.03(d,J＝12.5Hz,2H),1.81(s,2H),1.46(d,J＝3.3Hz,9H).
步骤5：
化合物1f(60mg)溶于1,4-二氧六环中，再依次加入环丙酰胺(31mg)、Cs2CO3(117mg)，N2置换，室温下搅拌15min，再加入Xantphos(10mg)、Pd(OAc)2(4mg)，N2置换，室温下搅拌5min后升温至145℃回流2h，TLC监测原料消失，移除N2，加水淬灭反应，反应液降至室温后硅藻土抽滤，甲醇洗涤滤饼，再将滤液减压旋蒸至干，柱层析纯化，得到白色固体A1(43mg,产率67.2％)。1HNMR(300MHz,DMSO-d6)δ10.80(s,1H),10.68(s,1H),8.65(d,J＝4.8Hz,1H),8.53(s,1H),8.24(d,J＝7.8Hz,1H),8.12(s,1H),7.92–7.83(m,2H),7.30(d,J＝8.6Hz,2H),3.94(d,J＝13.2Hz,3H),2.79(d,J＝4.4Hz,5H),1.97(q,J＝6.1Hz,1H),1.79(d,J＝12.0Hz,2H),1.41(s,11H),0.78(d,J＝6.1Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.42,168.33,165.23,154.50,154.41,151.73,149.21,142.29,129.66,129.27,120.65,109.75,95.97,79.09,46.94,40.53,28.57,26.42,14.78,8.26.HRMS(ESI)m/z calcd forC28H36N6O5[M+H]+536.2720；found 537.2816.HPLC(70％methanol in water):tR＝10.593min,96.2％.
实施例2：4-(3-((2-(环丙烷甲酰胺基)-5-(甲基胺甲酰基)吡啶-4-基)氨基)苯甲酰基)哌啶-1-羧酸叔丁酯(A2)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成临硝基苯甲酸，其他条件不变。1H NMR(400MHz,DMSO-d6)δ10.77(s,1H),10.58(s,1H),8.63(q,J＝4.5Hz,1H),8.52(s,1H),8.18(d,J＝7.9Hz,1H),7.95(s,1H),7.69(t,J＝2.0Hz,1H),7.59(dt,J＝7.5,1.6Hz,1H),7.50–7.40(m,2H),4.12(q,J＝5.7,4.8Hz,1H),4.07–3.87(m,4H),3.17(d,J＝3.0Hz,2H),2.78(d,J＝4.4Hz,3H),1.97(p,J＝6.3Hz,1H),1.77(dd,J＝13.2,3.9Hz,2H),1.40(s,9H),0.86–0.74(m,4H).
实施例3：4-(3-((2-(环丙烷甲酰胺基)-5-(甲基胺甲酰基)吡啶-4-基)氨基)-2-甲氧基苯甲酰胺基)哌啶-1-羧酸叔丁酯(A3)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成2-甲氧基-3-硝基苯甲酸，其他条件不变。1H NMR(300MHz,Chloroform-d)δ10.51(s,1H),9.09(s,1H),8.34(s,1H),8.07(s,1H),7.86–7.74(m,2H),7.63(dd,J＝8.0,1.7Hz,1H),7.33–7.28(m,1H),6.79(d,J＝5.1Hz,1H),4.28–4.05(m,3H),3.83(s,3H),3.06(d,J＝4.7Hz,3H),2.98(d,J＝12.5Hz,2H),2.10–1.99(m,2H),1.63(dt,J＝8.0,3.5Hz,1H),1.10(p,J＝4.3Hz,2H),0.96–0.81(m,4H).13C NMR(151MHz,DMSO-d6)δ173.34,168.36,165.42,154.51,154.34,152.23,149.59,149.07,133.29,131.68,124.53,124.35,123.45,109.88,95.34,79.10,61.88,55.37,46.57,31.60,28.55,26.41,14.76,13.69,8.21,6.66.HRMS(ESI)m/z calcd for C29H39N6O6[M+H]+567.3926；found 567.2917.HPLC(70％methanol in water):tR＝12.810min,95.1％.
实施例4：4-(3-((2-(环丙烷甲酰胺基)-5-(甲基胺甲酰基)吡啶-4-基)氨基)-3-甲氧基苯甲酰胺基)哌啶-1-羧酸叔丁酯(A4)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成3-甲氧基-4-硝基苯甲酸，其他条件不变。mp:232℃-234℃.1H NMR(300MHz,DMSO-d6)δ10.81(s,1H),10.63(s,1H),8.62(d,J＝4.8Hz,1H),8.53(s,1H),8.25(d,J＝7.8Hz,1H),8.14(s,1H),7.57–7.43(m,3H),3.98(d,J＝11.6Hz,3H),3.92(s,3H),2.87(s,2H),2.80(d,J＝4.3Hz,3H),2.00(q,J＝6.0Hz,1H),1.81(d,J＝11.6Hz,2H),1.45(m,2H),1.43(s,9H),0.80(d,J＝6.1Hz,4H).13CNMR(151MHz,DMSO-d6)δ173.40,168.18,165.24,154.45,154.38,151.29,150.37,149.10,131.49,129.78,120.35,118.62,111.02,110.35,96.19,79.10,56.41,47.02,40.54,28.57,26.45,14.78,8.26.HRMS(ESI)m/z calcd for C29H39N6O6[M+H]+567.3926；found567.2914.HPLC(70％methanol in water):tR＝11.647min,98.4％.
实施例5：4-(6-((2-(环丙烷甲酰胺基)-5-甲基胺甲酰基)吡啶-4-基)氨基)烟酰胺基)哌啶-1-羧酸叔丁酯(A5)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成6-氨基烟酸，其他条件不变。mp:240℃-244℃.1H NMR(300MHz,DMSO-d6)δ11.64(s,1H),10.81(s,1H),9.30(s,1H),8.76(d,J＝4.7Hz,1H),8.60(s,1H),8.54(d,J＝7.7Hz,1H),8.41(d,J＝5.2Hz,1H),7.41(s,1H),7.34(dd,J＝5.3,1.3Hz,1H),3.96(d,J＝13.7Hz,3H),2.83(d,J＝4.4Hz,3H),2.04(s,1H),1.82(d,J＝12.5Hz,2H),1.43(s,9H),1.25(s,2H),0.84(t,J＝5.7Hz,4H).13CNMR(151MHz,DMSO-d6)δ173.04,168.51,164.08,154.66,154.45,154.37,150.01,148.85,148.63,144.00,115.27,111.16,109.84,99.72,79.13,47.16,40.54,28.56,26.53,14.82,8.21.HRMS(ESI)m/z calcd for C27H35N7O5[M+H]+538.2772；found 538.2764.HPLC(70％methanol in water):tR＝9.995min,95.7％.
实施例6：4-(4-((2-((环丙烷甲酰胺基)-5-甲基胺甲酰基)吡啶-4-基)氨基)-3-甲基苯甲酰胺基)哌啶-1-羧酸叔丁酯(A6)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成3-甲基-4-硝基苯甲酸，其他条件不变。mp:187℃-190℃.1H NMR(300MHz,Chloroform-d)δ10.25(s,1H),8.56(s,1H),8.27(s,1H),7.89(s,1H),7.67(d,J＝2.1Hz,1H),7.53(dd,J＝8.4,2.1Hz,1H),7.41(d,J＝8.3Hz,1H),6.53(s,1H),6.20(d,J＝7.8Hz,1H),2.99(d,J＝4.7Hz,3H),2.89(t,J＝12.6Hz,2H),2.33(s,3H),1.97(s,3H),1.47(s,9H),1.05–1.00(m,2H),0.85(dq,J＝7.7,4.0Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.31,168.55,165.41,154.45,154.40,152.48,149.14,140.64,130.68,130.58,130.34,126.35,121.25,109.26,95.70,79.08,55.37,46.93,28.56,26.44,18.15,14.74,8.21.HRMS(ESI)m/z calcd for C29H39N6O5[M+H]+551.2976；found 551.2962.HPLC(70％methanol in water):tR＝12.445min,96.5％.
实施例7：4-(4-((2-((环丙烷甲酰胺基)-5-(甲基胺甲酰基)吡啶-4-基)氨基)-2-甲基苯甲酰胺基)哌啶-1-羧酸叔丁酯(A7)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成2-甲基-3-硝基苯甲酸，其他条件不变。mp:120℃-122℃.1H NMR(300MHz,Chloroform-d)δ10.08(s,1H),8.47(s,1H),8.21(s,1H),7.35(s,1H),6.34(d,J＝4.9Hz,1H),6.21(d,J＝8.3Hz,1H),4.08(t,J＝6.6Hz,2H),2.99(d,J＝4.7Hz,3H),2.92–2.77(m,2H),2.27(s,3H),2.00(d,J＝12.0Hz,2H),1.62(dt,J＝19.9,7.1Hz,2H),1.46(s,9H),1.37–1.31(m,1H),0.95–0.84(m,4H).13C NMR(151MHz,DMSO-d6)δ173.15,169.70,168.66,168.63,154.39,154.35,153.59,149.07,140.06,138.36,129.30,126.70,124.74,123.99,108.75,94.99,79.09,64.97,64.23,55.37,46.56,43.52,30.54,30.45,28.54,26.43,19.03,14.78,14.69,13.99,13.97,8.14.HRMS(ESI)m/z calcd for C29H39N6O5[M+H]+551.2976；found 551.5959.HPLC(70％methanol in water):tR＝9.595min,97.5％.
实施例8：4-(5-((2-(环丙烷甲酰胺基)-5-(甲基胺甲酰基)吡啶-4-基)氨基)吡啶甲酰胺)哌啶-1-羧酸叔丁酯(A8)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成5-氨基烟酸，其他条件不变。mp:156℃-160℃.1H NMR(300MHz,Chloroform-d)δ10.58(s,1H),8.46(d,J＝2.5Hz,2H),8.33(s,1H),8.23–8.11(m,2H),7.85(d,J＝8.3Hz,1H),7.74(dd,J＝8.5,2.6Hz,1H),6.50(d,J＝5.2Hz,1H),4.26–3.85(m,3H),3.01(d,J＝4.7Hz,3H),2.92(d,J＝12.7Hz,2H),1.52(s,2H),1.47(s,9H),1.26(d,J＝8.1Hz,1H),1.07(p,J＝4.3Hz,2H),0.88(dq,J＝7.6,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.53,168.09,163.17,154.60,154.38,151.42,149.30,145.17,141.46,138.94,129.26,123.26,110.26,96.04,79.10,55.38,46.83,31.64,28.57,26.44,14.78,8.32.HRMS(ESI)m/z calcd for C27H35N7O5[M+H]+538.2772；found538.2768.HPLC(70％methanol in water):tR＝12.408min,98.6％.
实施例9：4-(4-((2-((环丙烷甲酰胺基)-5-甲基胺甲酰基)吡啶-4-基)氨基)-3-氟苯甲酰胺基)哌啶-1-羧酸叔丁酯(A9)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成3-氟-4-硝基苯甲酸，其他条件不变。mp:234℃-238℃.1H NMR(300MHz,DMSO-d6)δ10.86(d,J＝14.8Hz,2H),8.72(d,J＝4.8Hz,1H),8.59(s,1H),8.35(d,J＝7.7Hz,1H),8.04(s,1H),7.86–7.72(m,2H),7.61(t,J＝8.2Hz,1H),3.96(d,J＝12.6Hz,3H),2.85(s,2H),2.81(d,J＝4.4Hz,3H),2.00(q,J＝6.1Hz,1H),1.81(d,J＝12.4Hz,2H),1.43(s,9H),1.42–1.22(m,2H),0.81(d,J＝6.1Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.51,168.26,164.12,154.95,154.59,154.40,153.34,151.42,149.13,130.81,130.77,130.43,130.35,124.49,124.47,121.67,115.58,115.44,109.91,96.21,79.09,47.10,28.55,26.43,14.79,8.31.HRMS(ESI)m/z calcd forC28H36FN6O5[M+H]+555.2726；found 555.2718.HPLC(70％methanol in water):tR＝12.635min,98.2％.
实施例10：4-(4-((2-((环丙烷甲酰胺基)-5-甲基胺甲酰基)吡啶-4-基)氨基)-4-氟苯甲酰胺基)哌啶-1-羧酸叔丁酯(A10)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成4-氟-3-硝基苯甲酸，其他条件不变。mp:248℃-252℃.1H NMR(300MHz,DMSO-d6)δ10.81(s,1H),10.55(s,1H),8.68(d,J＝4.6Hz,1H),8.56(s,1H),8.13(d,J＝7.8Hz,1H),8.04–7.95(m,1H),7.73(d,J＝14.7Hz,2H),7.45(t,J＝9.5Hz,1H),3.97(s,2H),3.92(s,1H),2.88(d,J＝13.4Hz,2H),2.81(d,J＝4.3Hz,3H),1.98(m,1H),1.79(d,J＝12.5Hz,2H),1.42(s,9H),0.89(t,J＝7.2Hz,2H),0.81–0.74(m,4H).13C NMR(151MHz,DMSO-d6)δ173.37,169.70,168.32,164.56,158.24,156.58,154.47,154.29,152.54,148.98,131.92,127.04,126.96,125.86,125.80,123.93,116.80,109.37,95.85,79.06,47.02,30.54,28.54,26.41,19.02,14.75,13.99,8.18.HRMS(ESI)m/z calcd for C28H36FN6O5[M+H]+555.2726；found 555.2717.HPLC(70％methanolin water):tR＝15.340min,97.7％.
实施例11：6-(环丙烷甲酰胺基)-4-((2-甲氧基-3-(吗啉基-4-羰基)苯基)胺基)-N-甲基烟酰胺(B1)的合成
参照实施例3的合成方法，将4-氨基-1-叔丁氧羰基哌啶替换成吗啉，其他条件不变。mp:158℃-161℃.1H NMR(300MHz,Chloroform-d)δ10.27(s,1H),9.04(s,1H),8.22(s,1H),7.84(s,1H),7.43(dd,J＝8.0,1.6Hz,1H),7.15(t,J＝7.8Hz,1H),7.02(dd,J＝7.6,1.6Hz,1H),6.91(d,J＝5.3Hz,1H),3.75(d,J＝8.8Hz,7H),3.69–3.48(m,2H),3.42–3.19(m,2H),2.93(d,J＝4.6Hz,3H),1.56(dq,J＝7.9,4.5,4.0Hz,1H),0.99(p,J＝4.2Hz,2H),0.82(dq,J＝7.5,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.28,168.37,166.63,154.44,152.38,148.98,148.80,132.92,132.11,132.04,131.28,125.04,123.58,123.40,109.68,95.59,66.72,66.48,61.66,55.37,47.58,42.13,26.42,14.74,8.19,8.16.HRMS(ESI)m/zcalcd for C23H27N5O5[M+H]+454.2085；found 454.2077.HPLC(75％methanol inwater):tR＝3.902min,97.7％.
实施例12：6-(环丙烷甲酰胺基)-4-((3-(二丁基氨甲酰基)-2-甲氧基苯基)胺基)-N-甲基烟酰胺(B2)的合成
参照实施例3的合成方法，将4-氨基-1-叔丁氧羰基哌啶替换成二正丁胺，其他条件不变。mp:174℃-179℃.1H NMR(300MHz,Chloroform-d)δ10.36(s,1H),8.39(s,1H),8.27(s,1H),8.07(s,1H),7.53(dd,J＝8.1,1.6Hz,1H),7.21(t,J＝7.8Hz,1H),7.01(dd,J＝7.6,1.6Hz,1H),6.42(d,J＝5.2Hz,1H),3.84(s,3H),3.16(dd,J＝30.9,11.7Hz,4H),3.04(d,J＝4.7Hz,3H),1.67(d,J＝7.6Hz,1H),1.54–1.41(m,4H),1.19–1.07(m,4H),1.01(t,J＝7.3Hz,3H),0.90(td,J＝6.9,6.0,3.6Hz,4H),0.80(t,J＝7.3Hz,3H).13CNMR(151MHz,DMSO-d6)δ173.25,168.39,167.87,154.51,152.38,149.01,148.17,133.03,124.95,122.83,122.77,109.70,95.51,61.64,47.97,43.70,30.45,29.48,26.41,20.02,19.58,14.73,14.28,13.87,8.17.HRMS(ESI)m/z calcd for C27H38N5O4[M+H]+496.2918；found498.2910.HPLC(75％methanol in water):tR＝13.332min,99.1％.
实施例13：4-((3-(丁基氨甲酰基)-2-甲氧基苯基)胺基)-6-(环丙烷甲酰胺基)-N-甲基烟酰胺(B3)的合成
参照实施例3的合成方法，将4-氨基-1-叔丁氧羰基哌啶替换成一正丁胺，其他条件不变。mp:164℃-168℃.1H NMR(300MHz,DMSO-d6)δ10.80(s,1H),10.67(s,1H),8.64(q,J＝4.4Hz,1H),8.52(s,1H),8.28(t,J＝5.7Hz,1H),8.04(s,1H),7.54–7.51(m,1H),7.22–7.19(m,1H),6.78(s,1H),3.70(s,3H),3.26(q,J＝6.6Hz,2H),2.79(d,J＝4.4Hz,3H),2.05–1.91(m,1H),1.51(td,J＝5.0,2.2Hz,2H),0.91(t,J＝7.2Hz,3H),0.77(s,2H),0.63–0.60(m,4H).13C NMR(151MHz,DMSO-d6)δ175.12,173.34,168.37,165.94,154.52,152.24,149.66,149.07,133.29,131.66,124.55,123.46,109.88,95.35,61.88,39.12,31.59,26.41,20.08,14.16,13.69,6.66.HRMS(ESI)m/z calcd for C23H29N5O4[M+H]+440.2292；found 440.2283.HPLC(75％methanol in water):tR＝5.852min,97.6％.
实施例14：6-(环丙烷甲酰胺基)-4-((2-甲氧基-3-((四氢-2H-吡喃-4-基)胺甲酰基)苯基)胺基)-N-甲基烟酰胺(B4)的合成
参照实施例3的合成方法，将4-氨基-1-叔丁氧羰基哌啶替换成4-氨基四氢吡喃盐酸盐，其他条件不变。mp:138℃-140℃.1H NMR(300MHz,Chloroform-d)δ10.43(s,1H),9.80(s,1H),8.28(s,1H),7.99(s,1H),7.77(d,J＝7.8Hz,1H),7.65(dd,J＝7.9,1.6Hz,1H),7.52(dd,J＝8.0,1.6Hz,1H),7.22–7.08(m,2H),4.17–4.09(m,1H),3.97(dt,J＝11.9,3.6Hz,2H),3.78(s,3H),3.51(td,J＝11.5,2.2Hz,2H),2.98(d,J＝4.5Hz,3H),1.93(d,J＝4.0Hz,1H),1.61–1.50(m,2H),1.24(t,J＝7.1Hz,2H),1.06(p,J＝4.1Hz,2H),0.86(dq,J＝7.6,4.5,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.35,168.38,165.39,154.52,152.25,149.61,149.08,133.30,131.66,124.52,124.37,123.46,109.90,95.35,66.40,61.87,60.22,45.81,32.70,26.41,14.77,8.21.HRMS(ESI)m/z calcd for C24H30N5O5[M+H]+468.2242；found 468.2234.HPLC(75％methanol in water):tR＝4.145min,99.1％.
实施例15：6-[环丙烷甲酰胺基]-4-[[2-甲氧基-3-[哌啶-4-氨甲酰基]苯基]胺基]-N-甲基烟酰胺(B5)的合成
步骤1：
A3(100mg)溶于3mL乙酸乙酯中，加入0.1mL36％HCl，室温下反应1h，TLC监测原料消失，加饱和NaHCO3溶液调节PH至7-8，减压浓缩，柱层析纯化，得到白色固体B5(78mg,95％)。mp:236℃-238℃.1H NMR(300MHz,DMSO-d6)δ11.05(s,1H),9.18(d,J＝4.8Hz,1H),9.06(s,1H),8.84(d,J＝9.9Hz,1H),8.58(d,J＝7.5Hz,1H),8.51(s,1H),7.53(dd,J＝7.8,1.8Hz,1H),7.35(dd,J＝7.7,1.8Hz,1H),7.30–7.23(m,2H),4.06(s,1H),3.70(s,3H),3.32(s,2H),3.05–2.95(m,2H),2.81(d,J＝4.4Hz,3H),2.00(ddt,J＝10.2,7.3,4.4Hz,3H),1.86–1.73(m,2H),1.00–0.90(m,4H).13C NMR(151MHz,DMSO-d6)δ165.78,150.87,132.25,130.94,124.82,110.52,94.84,62.12,55.40,44.54,42.29,28.31,26.52,15.23,9.50.HRMS(ESI)m/z calcd for C24H30N6O4[M+H]+467.2401；found467.2395.
实施例16：6-(环丙烷甲酰胺基)-4-((2-甲氧基-3-(4-甲基哌嗪-1-羰基)苯基)胺基)-N-甲基烟酰胺(B6)的合成
参照实施例1的合成方法，将对硝基苯甲酸替换成(3-氨基-2-甲氧基苯基)(4-甲基-1-哌嗪基)甲酮，其他条件不变。mp:226℃-228℃.1H NMR(300MHz,DMSO-d6)δ10.78(s,1H),10.60(s,1H),8.65(d,J＝4.7Hz,1H),8.53(s,1H),7.96(d,J＝7.6Hz,1H),7.46(dd,J＝8.1,1.6Hz,1H),7.21(t,J＝7.8Hz,1H),6.99(dd,J＝7.6,1.5Hz,1H),3.67(s,3H),3.20(d,J＝5.0Hz,2H),3.07(q,J＝7.3Hz,2H),2.80(d,J＝4.3Hz,3H),2.48–2.27(m,4H),2.24(s,3H),2.04–1.94(m,1H),1.20(t,J＝7.3Hz,2H),0.78(d,J＝4.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.27,168.37,166.46,154.46,152.31,149.00,148.65,132.95,131.60,124.96,123.19,123.14,109.72,95.59,61.61,55.13,46.04,41.41,26.43,14.73,8.16.HRMS(ESI)m/z calcd for C24H30N6O4[M+H]+467.2401；found 467.2395.HPLC(75％methanol in water):tR＝4.067min,97.1％.
实施例17：4-((3-(丁基氨甲酰基)-2-甲氧基苯基)胺基)-6-(环丙烷甲酰胺基)-N-乙基烟酰胺(C1)的合成
参照实施例1的合成方法，将甲胺盐酸盐替换成乙胺盐酸盐，其他条件不变。mp:142℃-148℃.1H NMR(300MHz,Chloroform-d)δ10.49(s,1H),9.61(s,1H),8.31(s,1H),8.10(s,1H),7.82(dd,J＝16.9,6.8Hz,2H),7.62(d,J＝7.9Hz,1H),7.35–7.25(m,1H),6.82(t,J＝5.5Hz,1H),3.82(s,3H),3.50(dq,J＝13.9,6.6Hz,4H),1.72(dt,J＝7.9,3.6Hz,1H),1.63(t,J＝7.5Hz,2H),1.48(tt,J＝8.2,4.9Hz,4H),1.31(t,J＝7.2Hz,3H),1.08(p,J＝4.2Hz,2H),0.90(dq,J＝6.4,3.5Hz,2H),0.81(dt,J＝7.3,3.7Hz,4H).13CNMR(151MHz,DMSO-d6)δ175.11,173.35,167.71,165.93,154.52,152.27,149.63,133.31,131.64,124.54,123.40,110.02,95.34,61.87,39.12,34.27,31.60,20.08,15.08,14.16,13.69,8.21,6.66.HRMS(ESI)m/z calcd for C24H31N5O4[M+H]+454.2449；found454.2449.HPLC(75％methanol in water):tR＝6.767min,99.2％.
实施例18：4-((3-(丁基氨甲酰基)-2-甲氧基苯基)胺基)-6-(环丙烷甲酰胺基)-N-环丙基烟酰胺(C2)的合成
参照实施例1的合成方法，将甲胺盐酸盐替换成环丙胺，其他条件不变。1H NMR(300MHz,Chloroform-d)δ10.44(s,1H),8.77(s,1H),8.23(s,1H),8.04(s,1H),7.84–7.71(m,2H),7.59(dd,J＝8.0,1.7Hz,1H),7.27(s,1H),6.64(s,1H),3.79(s,3H),3.52–3.40(m,2H),2.90(dq,J＝7.2,3.6Hz,1H),1.60(s,1H),1.42(dt,J＝8.0,4.6Hz,4H),1.07–1.01(m,2H),0.98(d,J＝2.7Hz,3H),0.91–0.88(m,2H),0.79(dd,J＝5.8,2.2Hz,4H).13C NMR(151MHz,DMSO-d6)δ175.12,173.36,169.28,165.94,154.56,152.16,149.63,149.31,133.28,131.65,124.54,123.38,109.77,95.33,61.90,39.13,31.59,23.26,20.08,14.16,13.69,8.22,6.66,6.16.HRMS(ESI)m/z calcd for C25H32N5O4[M+H]+466.2449；found466.2453.HPLC(75％methanol in water):tR＝6.990min,98.2％.
实施例19：4-((3-(丁基氨甲酰基)-2-甲氧基苯基)胺基)-6-(环丙烷甲酰胺基)-N，N-二甲基烟酰胺(C3)的合成
参照实施例1的合成方法，将甲胺盐酸盐替换成二甲胺，其他条件不变。mp:158℃-162℃.1H NMR(300MHz,Chloroform-d)δ9.50(s,1H),8.85(s,1H),8.18(s,1H),8.06(s,1H),7.84–7.72(m,2H),7.62(dd,J＝8.0,1.7Hz,1H),7.28(t,J＝7.5Hz,1H),3.81(s,3H),3.48(td,J＝7.0,5.6Hz,2H),3.18(s,6H),1.62–1.57(m,1H),1.47–1.41(m,2H),1.07(dt,J＝6.5,3.4Hz,2H),0.98(d,J＝3.8Hz,4H),0.87(dq,J＝7.4,3.9Hz,2H),0.79(dt,J＝8.0,3.4Hz,3H).13C NMR(151MHz,DMSO-d6)δ175.10,173.25,168.25,165.88,153.72,150.70,149.68,148.33,133.41,131.36,124.53,124.47,123.65,113.42,96.85,61.83,39.13,31.60,20.08,14.73,14.17,13.69,8.14,6.66.HRMS(ESI)m/z calcd for C24H31N5O4[M+H]+454.2449；found 454.2446.HPLC(75％methanol in water):tR＝5.140min,94.7％.
表1本发明的以下其他化合物
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实施例20：6-(环丙烷甲酰胺基)-4-((1-(4-氟苯基)-2-氧-1,2-二氢吡啶-3-基)氨基)-N-甲基烟酰胺(A11)的合成
合成路线：
步骤1：
合成方法同实施例1步骤1。
步骤2：
3-硝基-2-(1H)-吡啶酮(1g)、4-氟苯硼酸(1.5g)、醋酸铜(2.13g)以及吡啶(8.6mL)依次加入24mL1,4-二氧六环中，80℃下反应过夜，加水淬灭反应，减压蒸干溶剂，用乙酸乙酯萃取(80mL×3)，合并有机相，依次用10％HCl水溶液、饱和NaCl水溶液洗涤，无水Na2SO4干燥，减压蒸去溶剂，得到白色固体11d，直接下投。
步骤3：
合成方法同实施例1步骤3得白色固体11e(299mg,产率67.3％)。1H NMR(300MHz,Chloroform-d)δ7.45–7.32(m,2H),7.16(t,J＝8.6Hz,2H),6.75(dd,J＝6.9,1.7Hz,1H),6.58(dd,J＝7.2,1.7Hz,1H),6.14(t,J＝7.0Hz,1H),4.30(s,2H)。
步骤4：
合成方法同实施例1步骤4得浅黄色固体化合物11f(274mg,50.2％)。1H NMR(300MHz,Chloroform-d)δ9.80(s,1H),8.76(s,1H),7.76(dd,J＝7.3,1.3Hz,1H),7.51(q,J＝5.1Hz,1H),7.48–7.42(m,2H),7.16–7.08(m,3H),7.00(s,1H),6.82(s,1H),2.91(d,J＝4.9Hz,3H)。
步骤5
合成方法同实施例1步骤5得白色固体化合物A11(168mg,54％)。mp:260℃-268℃.1H NMR(300MHz,DMSO-d6)δ10.88(s,1H),10.73(s,1H),8.56(q,J＝4.5Hz,1H),8.51(s,1H),8.25(s,1H),7.60–7.48(m,2H),7.45(dd,J＝7.3,1.6Hz,1H),7.41–7.33(m,2H),7.31(dd,J＝7.0,1.6Hz,1H),6.41(t,J＝7.1Hz,1H),2.76(d,J＝4.3Hz,3H),2.03(tt,J＝7.6,3.7Hz,1H),0.83(tq,J＝8.4,5.6,4.2Hz,4H).13C NMR(75MHz,DMSO-d6)δ173.61,167.70,163.53,160.28,158.04,154.56,150.26,149.03,137.48,137.44,131.19,130.93,129.50,129.38,119.23,116.50,116.20,111.37,105.87,96.98,26.37,14.86,8.36.HRMS(ESI)m/zcalcd for C22H21FN5O3[M+H]+422.1623；found 422.1616.HPLC(75％methanol in water):tR＝4.698min,97.7％。
实施例21：A12的合成
参照实施例20的合成方法，将4-氟苯硼酸替换成苯硼酸，其他条件不变。1HNMR(300MHz,DMSO-d6)δ10.88(s,1H),10.73(s,1H),8.54(d,J＝18.7Hz,2H),8.25(s,1H),7.61–7.50(m,2H),7.50–7.41(m,4H),7.32(dd,J＝6.9,1.6Hz,1H),6.41(t,J＝7.1Hz,1H),2.76(d,J＝4.4Hz,3H),2.03(h,J＝5.5,5.0Hz,1H),0.90–0.76(m,4H).13C NMR(75MHz,DMSO-d6)δ173.61,167.70,157.95,154.56,150.28,149.03,141.26,131.20,130.94,129.57,128.79,127.18,119.20,111.37,105.84,96.97,26.37,14.86,8.35.HRMS(ESI)m/zcalcd for C22H22N5O3[M+H]+404.1717；found 404.1713.HPLC(75％methanol in water):tR＝4.690min,98.4％.
表2本发明的以下其他化合物
实施例22：活性测试
表3是以下活性测试所用到的仪器来源。
表3仪器来源
(一)Cell Counting Kit-8(CCK-8)试剂盒检测化合物的细胞安全性
1、实验方法：H9细胞采用RPMI-1640完全培养基(含10％FBS)培养，置于含5％CO237℃培养箱中培养，当细胞密度达到80％-90％时，采取半量换液法进行传代，即向T25培养瓶中加入5mL新的培养基，吹打均匀后吸取一半细胞悬浮液移入新的培养瓶中继续培养，半量换液法2-3次之后需要将细胞悬浮液移入离心管中，1000rpm下离心5min，倒去上清液除去死细胞及多次使用的培养基，最后加入新的培养基，用移液枪吹打均匀后，取同体积细胞悬浮液分别置于2个培养瓶中，于5％CO237℃的培养箱中培养。当细胞密度达到80％时，取对数生长期的待测试细胞，配制成浓度为6×104个/mL的细胞悬浮液接种于96孔板中，每孔100μL，置于培养箱中培养4h。将提前配制好的10mM化合物DMSO储备液用培养基分别稀释为100μM、40μM、20μM，再将化合物稀释液加入96孔板中，每孔100μL，使给药终浓度分别为50μM、20μM、10μM，每个浓度3个复孔，将加入相同体积培养基替代含药培养基的复孔作为对照组，将无细胞也不含药培养基液的培养基复孔作为空白组。置于培养箱中培养24h后，每孔加入20μL的CCK-8试剂，避光孵育4h，用酶标仪检测450nm下各孔的吸光度(OD值)，用以下公式计算细胞存活率：细胞存活率(％)＝(实验组OD值-空白组OD值)/(对照组OD值-空白组OD值)×100％
2、实验结果：
以Deucravacitinib为阳性对照，对合成的烟酰胺类化合物进行了CCK8细胞安全性测试，研究结果表明，大部分化合物对H9细胞的安全性良好，并且发现在10μM浓度下，化合物A1、A4、A8及A9明显抑制H9细胞生长，其余化合物的细胞存活率均大于80％，且优于阳性药Deucravacitinib，在50μM下大部分化合物导致H9细胞的存活率低于50％，因此选择10μM作为后续磷酸化STAT1蛋白含量测试的给药浓度。
(二)Western Blot实验检测STAT1蛋白的磷酸化程度
1、实验方法：
(1)蛋白提取
取对数生长的H9细胞用RPMI-1640培养基配制成8×106个/mL的悬浮液，接种至6孔板中，每孔接种1mL细胞悬浮液，置于培养箱中培养4h后加入1mL 20μM浓度的待测化合物稀释液，每个孔中化合物终浓度为10μM，给药1H后加入5μL 20μg/mL的IFN-α刺激细胞，30min后将6孔板中的细胞悬浮液移至2mL EP管中，4℃1000rpm离心5min，弃上清液，每个EP管加入1mL4℃PBS洗涤细胞，4℃1000rpm离心5min，弃上清液，每管加入50μL提前配制好的RIPA裂解液(RIPA：PMSF：磷酸化酶抑制剂＝100：1：2)裂解细胞，冰上裂解30min，每15min于涡旋机上涡旋一次，裂解结束后于4℃12000rpm下离心15min，取上清用BCA试剂盒进行蛋白定量，562nm下测定吸光度，计算30μg蛋白所需上样体积，加入Loading Buffer后进行蛋白变性，保存于-80℃冰箱中。
(2)制胶
选用10％SDS-PAGE凝胶，首先配制分离胶(下层胶溶液：下层胶缓冲液：促凝剂＝1mL:1mL:20μL)混匀，灌入制胶板内，加入适量的甲醇或者水，压平分离胶，待下层胶凝固后(15min～30min)，弃去上层甲醇或者水，并用滤纸吸取多余的甲醇或水；接着配制上层胶(下层胶溶液：下层胶缓冲液：促凝剂＝1mL:1mL:20μL)，混匀后灌入制胶板内，缓慢插入梳子，凝固后置于Tris-Glycine电泳缓冲液中取出梳子待上样。
(3)上样-电泳
将制备好的蛋白样品按计算得到的上样体积加入至预制胶的上样孔中，60V浓缩40min后，120V恒压电泳观察marker看蛋白是否分离，通常溴酚蓝移至凝胶底部后停止电泳。
(4)转膜
电泳结束后，在转膜液中切割相应目标蛋白凝胶条带，将凝胶置于转印滤纸上，再剪下相应大小的PVDF膜，放入甲醇中活化10min，再放于相应的凝胶条带上并排除气泡，最后再盖上转印滤纸及海绵，最后放入转膜夹中在200mA恒流及冰浴下进行转膜，70min后结束转膜。
(5)封闭
转膜完成后，将PVDF膜转移至抗体孵育盒中，用TBST溶液洗涤3次，再加入5mL 5％脱脂牛奶或者5％BSA溶液在室温下封闭1h，以封闭膜上的非特异性蛋白位点。
(6)抗体孵育及显影
封闭结束后吸出封闭液并用TBST洗涤条带3次，再加入稀释后的一抗，4℃下孵育12h-16h，结束后回收一抗，再用TBST洗涤条带3次，每次5min，最后加入适宜浓度的二抗稀释液，室温下孵育2h，吸出二抗稀释液，用TBST洗涤3次条带，每次5min，最后用化学发光成像系统进行显影。
2、实验结果
如图1所示，以Deucravacitinib为阳性对照，检测了21个化合物对STAT1蛋白的磷酸化程度，选择化合物B3作为候选化合物，进行浓度、时间依赖实验以及JAK家族选择性实验。
(三)化合物B3浓度及时间依赖性实验
1、实验方法
(1)浓度依赖性实验
取对数生长的H9细胞用RPMI-1640培养基配制成8×106个/mL的悬浮液，接种至6孔板中，每孔接种1mL细胞悬浮液，置于培养箱中培养4h后分别加入1mL 20μM、10μM、2μM、1μM浓度的待测化合物稀释液，每个孔中化合物终浓度为10μM、5μM、1μM、0.5μM，给药1h后加入5μL 20μg/mL的IFN-α刺激细胞，30min后提取蛋白。
(2)时间依赖性实验
H9细胞用RPMI-1640培养基配制成8×106个/mL的悬浮液，接种至6孔板中，每孔接种1mL细胞悬浮液，置于培养箱中培养4h后加入1mL 2μM浓度的待测化合物稀释液，每个孔中化合物终浓度为1μM，分别给药24h、10h、6h、2h、1h后加入5μL 20μg/mL的IFN-α刺激细胞，30min后提取蛋白。
2、实验结果
总共设计了4个给药浓度，化合物B3在1μM浓度下明显抑制JAK1/TYK2通路，化合物B3抑制作用随着浓度增大而增强；根据浓度依赖性实验结果，时间依赖性实验给药浓度为1μM，总共采用了5个给药时间，达到相应给药时间后再给予IFN-α刺激细胞，30min后提取蛋白进行Western Blot实验，在1-10h间对信号通路的抑制作用相似，24h时仍然存在抑制作用且效果优于其他给药时间。
B3具有良好的TYK2抑制活性。通过CCK-8试剂盒检测化合物的细胞安全性、Western Blot实验检测STAT1蛋白的磷酸化程度、化合物B3浓度及时间依赖性实验结果表明上述21个化合物可作为TYK2选择性抑制剂，用于治疗自身免疫性疾病，特别是适用于治疗银屑病。
Claims (10)
1.如通式(I)所示的化合物或其药学上可接受的盐：
其中，R1选自以下结构：
R2为C1-C4烷基或-H；
R3为C1-C4烷基或
2.根据权利要求1所述的化合物或其药学上可接受的盐，其特征在于，R1选自以下结构：
R2为-CH3或-H；
R3为-CH3、-C2H5或
3.根据权利要求1所述的化合物或其药学上可接受的盐，其特征在于，化合物选自如下结构：
4.一种权利要求1所述的化合物或其药学上可接受的盐的制备方法，其特征在于，包括如下步骤：
路线1：
其中，R1、R2、R3同权利要求1所述；
X、Y选自C或N，且两者不相同；
R4：H、-CH3、-OCH3、F；
R5：
或：
路线2：
R6：
5.根据权利要求4所述的制备方法，其特征在于，所述路线1中的反应条件为：(a)4,6-二氯烟酸，EDCI，HOBt，DIPEA，DCM，-5～5℃；(b)HATU，DIPEA，DMF，室温；(c)NH4Cl，Fe粉，EtOH，80～90℃；(d)NaHMDS，THF；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，130～150℃。
6.根据权利要求4所述的制备方法，其特征在于，所述路线2中的反应条件为：(a)4,6-二氯烟酸，羰基二咪唑，甲胺盐酸盐，DIPEA，室温；(b)3-硝基-2-(1H)-吡啶酮，醋酸铜，吡啶，1,4-二氧六环，70～90℃；(c)NH4Cl，Fe粉，EtOH，75～95℃；(d)NaHMDS，THF；(e)环丙酰胺，Cs2CO3，Xantphos，Pd(OAc)2，1,4-二氧六环，130～150℃。
7.一种药物组合物，其特征在于，包括权利要求1所述的化合物或其药学上可接受的盐及药学上可接受的辅料。
8.权利要求1所述的化合物或其药学上可接受的盐在制备TYK2抑制剂中的用途。
9.权利要求1所述的化合物或其药学上可接受的盐在制备治疗银屑病药物中的用途。
10.权利要求9所述的用途，其特征在于，所述药物的剂型包括胶囊剂、片剂、颗粒剂、丸剂、口服液、注射剂。
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