CN116283990B - 取代吡咯烷酮类化合物在作为肿瘤坏死因子抑制剂方面的应用 - Google Patents
取代吡咯烷酮类化合物在作为肿瘤坏死因子抑制剂方面的应用 Download PDFInfo
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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Abstract
取代吡咯烷酮类化合物在作为肿瘤坏死因子抑制剂方面的应用，属于药物化学领域，本发明公开了如式I所示的一系列基于取代吡咯烷酮类化合物的肿瘤坏死因子小分子抑制剂，或其药学上可接受的盐及其在制备治疗或预防自身免疫性、炎症性疾病药物中的应用。
Description
技术领域
本发明属于药物化学领域，具体涉及一种取代吡咯烷酮类化合物在作为肿瘤坏死因子抑制剂方面的应用。
背景技术
肿瘤坏死因子α(TNF-α)是一种强大的免疫调节细胞因子，参与多种疾病的发生发展，如自身免疫性疾病、HIV和癌症等。目前生物大分子抗体药物已成为治疗自身免疫性疾病的有效方法。然而，抗体疗法具有以下不可避免的缺点，包括临床应答率低、感染风险、价格高和静脉给药等。因此，开发TNF-α小分子抑制剂以克服上述缺点是迫切需要的。
2005年，第一个TNF-α拮抗剂(SPD-304)与TNF-α的共结晶结构得以解析，对TNF-α抑制剂的合理设计具有指导意义。尽管已经取得了持续的研究进展，但迄今为止还没有临床批准的TNF-α小分子抑制剂。本发明通过计算机虚拟筛选确定了TNF-α抑制剂先导化合物(如图1所示)，以该化合物的吡咯烷酮环状结构为基本骨架，进行了构效关系研究，合成了一系列TNF-α抑制剂候选化合物。
为TNF-α相关疾病的治疗提供了具有潜力的小分子药物，相比于现有抗体药物，可大大降低疾病治疗和药物生产的成本。本发明化合物可用于制备治疗TNF-α相关的自身免疫性疾病和/或其他疾病的药物。
发明内容
本发明的首要目的是提供如式I所示的一种基于取代吡咯烷酮类化合物的TNF-α小分子抑制剂，或其药学上可接受的盐。
其中：
R1选自H、C1-C4烷基、OH、C1-C4烷基取代的芳基、C1-C4烷基取代的5-10元杂环基，所述杂环基含有1-3个选自N、O、S的杂原子；
R2选自H、嘌呤基、取代苯甲酰基、取代苯乙酰基、取代芳杂酰基，所述芳杂酰基含有1-3个选自N、O的杂原子；
本发明优选如通式I所示的一种基于取代吡咯烷酮类化合物的TNF-α小分子抑制剂，或其药学上可接受的盐：
其中：
R1选自以下取代基团：
R2选自以下取代基团：
本发明最终优选以下化合物，或其药学上可接受的盐：
本发明中，所述的“药学上可接受的盐”，是指所述衍生物与下列酸形成的加成盐：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、萘二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸、苯甲酸等。盐酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、三氟乙酸、马来酸、苯磺酸、琥珀酸以及类似的已知可以接受的酸成盐。
本发明还提供所述取代吡咯烷酮类化合物的制备方法：
目标化合物是由商业来源的N-Boc-天冬氨酸通过四步反应合成的。步骤a：N-Boc天冬氨酸在醋酐存在下发生环化反应得到白色固体酸酐中间体；步骤b：酸酐中间体与相应的胺类原料在甲苯回流条件下发生酰胺化反应，经后续处理得到N-Boc-取代吡咯烷酮母核中间体；步骤c：在室温条件下，使用氯化氢乙酸乙酯溶液进行脱保护，得到母核盐酸盐；步骤d：在碱性条件下，6-氯嘌呤与母核盐酸盐发生亲核取代反应，得到系列一化合物；步骤e：母核盐酸盐与不同的取代羧酸发生缩合反应，得到系列二化合物。
本发明中包含药物组合物的情形，含有治疗有效量的上述任意一项取代吡咯烷酮类化合物或其药学上可接受的盐，以及药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合。
其中，所述的药物组合物的剂型为注射剂、片剂和胶囊剂中的任意一种。
本发明还提供上述的化合物及其药学上可接受的盐或上述的组合物在制备治疗或预防自身免疫性、炎症性疾病药物中的应用。
本发明的有益效果：
TNF-α抑制剂是三大自身免疫疾病：类风湿性关节炎(RA)、强直性脊柱炎(AS)和银屑病性关节炎(PsA)的首选治疗药物。然而，该领域的治疗药物主要以抗体药物为主。抗体药物具有价格昂贵、生产运输储存条件要求高等局限性。因此，TNF-α小分子抑制剂的研发受到国内外科研院所和制药企业的关注。本发明在图1所示化合物结构的基础上进行优化，通过引入羰基构建新的核心结构，使化合物的合成难度降低，通过酰胺键连接R2基团，除了与新引入的羰基一起增加化合物的水溶性之外，还使化合物的灵活性更大，为活性优化提供了便利条件。本发明化合物10展示出优于图1所示化合物的TNF-α抑制活性。此外，进一步的机制研究显示，化合物10阻止TNF-α诱导的caspase信号通路的激活，并且抑制TNF-α诱导的NF-κB信号通路的激活。结合亲和力实验显示化合物10与TNF-α具有直接的相互作用。本发明为TNF-α小分子抑制剂的开发提供了研究基础。
附图说明
图1化合物10直接结合TNF-α；
A：化合物10的结构；
B：MST实验评价化合物10与TNF-α的结合亲和力；
C：CETSA实验评价化合物10对于TNF-α的热稳定性；
D：DARTS实验评价化合物10对于酶解的稳定作用；
图2化合物10阻止TNF-α诱导的caspase信号通路的激活；
A：化合物10抑制TNF-α诱导caspase 8的激活；
B：化合物10抑制TNF-α诱导caspase 3的激活；
图3化合物10阻止TNF-α诱导的NF-κB信号通路的激活；
A：化合物10抑制TNF-α或LPS诱导的IκBα的磷酸化；
B：化合物10抑制TNF-α或LPS诱导的NF-κB p65的入核；
C：化合物10抑制TNF-α或LPS诱导的IL-6和IL-1β的释放；
D：化合物10抑制TNF-α或LPS诱导的IL-6和IL-1β的转录。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容做进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1：叔丁基-(2,5-二氧四氢呋喃-3-基)氨基甲酸酯的合成
将N-Boc天冬氨酸(2.3g，10mmol)添加到Ac2O(10mL)和Et3N(4.2mL，30mmol)的混合溶液中，在80℃下搅拌0.5h，直到反应完成。去除溶剂，得到叔丁基-(2,5-二氧四氢呋喃-3-基)氨基甲酸酯，无需进一步纯化，产率90％。1H NMR(400MHz,DMSO-d6)δ5.99(d,J＝7.3Hz,1H),5.23(dt,J＝7.5,5.7Hz,1H),3.09–2.99(m,2H),1.42(s,9H).
实施例2：叔丁基-(1-(2-(苯并[d][1,3]二恶英-5-基)乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
向20mL甲苯中依次加入叔丁基-(2,5-二氧四氢呋喃-3-基)氨基甲酸酯(1.08g，5mmol)、3,4-亚甲二氧基苯乙胺(1.23g，7.5mmol)和Et3N(2.1mL，15mmol)，回流直到反应完成。减压蒸馏除去溶剂，用1N HCl水溶液酸化至pH 5～6，并用乙酸乙酯萃取，用水和盐水洗涤有机层，无水Na2SO4干燥。最后，通过硅胶柱色谱层析分离，石油醚：乙酸乙酯＝3：1(v/v)，得到白色固体产品，产率76％。1H NMR(400MHz,DMSO-d6)δ6.69(d,J＝8.8Hz,1H),6.65–6.61(m,1H),6.54–6.51(m,1H),5.95(d,J＝7.5Hz,1H),5.92(s,2H),4.76(dt,J＝7.3,4.6Hz,1H),3.76(dq,J＝37.5,6.5Hz,2H),2.98–2.84(m,2H),2.84–2.72(m,2H),1.42(s,9H).
实施例3：叔丁基-(1-苄基-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率78％。1H NMR(400MHz,DMSO-d6)δ7.33–7.27(m,3H),7.26–7.23(m,2H),5.95(d,J＝8.2Hz,1H),4.89(ddd,J＝8.5,5.3,3.5Hz,1H),4.73–4.66(m,2H),3.00(dd,J＝12.5,5.3Hz,1H),2.78(dd,J＝12.3,3.5Hz,1H),1.42(s,9H).
实施例4：叔丁基-(2,5-二氧基-1-苯乙基吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率79％。1H NMR(400MHz,DMSO-d6)δ7.32–7.26(m,2H),7.25–7.18(m,3H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.82–3.67(m,2H),2.99(dd,J＝12.4,5.4Hz,1H),2.82–2.75(m,3H),1.42(s,9H).
实施例5：叔丁基-(2,5-二氧基-1-苯基吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率82％。1H NMR(400MHz,DMSO-d6)δ7.52–7.45(m,1H),7.45–7.38(m,2H),7.35–7.29(m,2H),6.02(d,J＝8.1Hz,1H),4.88(ddd,J＝8.1,5.1,3.2Hz,1H),3.08(dd,J＝12.4,5.0Hz,1H),2.76(dd,J＝12.4,3.2Hz,1H),1.42(s,9H).
实施例6：叔丁基-(1-(4-氟苯乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率68％。1H NMR(400MHz,DMSO-d6)δ7.17(ddt,J＝8.1,3.5,1.0Hz,2H),7.11–7.04(m,2H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.78(dt,J＝12.8,6.4Hz,1H),3.71(dt,J＝13.0,6.5Hz,1H),2.99(dd,J＝12.4,5.4Hz,1H),2.82–2.70(m,3H),1.42(s,9H).
实施例7：叔丁基-(1-(3-氟苯乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率87％。1H NMR(400MHz,DMSO-d6)δ7.29(td,J＝7.7,5.0Hz,1H),7.03(dddd,J＝9.4,7.2,2.3,1.1Hz,1H),6.91(dtt,J＝12.1,2.2,1.0Hz,1H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.76(ddt,J＝44.0,12.8,6.4Hz,2H),3.03–2.85(m,3H),2.79(dd,J＝12.3,3.5Hz,1H),1.42(s,9H).
实施例8：叔丁基-(1-(4-羟基苯乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率75％。1H NMR(400MHz,DMSO-d6)δ7.00(dt,J＝8.5,1.0Hz,2H),6.77–6.71(m,2H),6.52(s,1H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.78(dt,J＝12.8,6.4Hz,1H),3.71(dt,J＝13.0,6.5Hz,1H),2.99(dd,J＝12.4,5.4Hz,1H),2.82–2.76(m,1H),2.80–2.72(m,2H),1.42(s,9H).
实施例9：叔丁基-(1-(2-(2-甲氧基苯氧基)乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率60％。1H NMR(400MHz,DMSO-d6)δ6.93(ddd,J＝7.9,6.3,2.6Hz,1H),6.90–6.82(m,3H),5.95(d,J＝8.2Hz,1H),4.88(ddd,J＝8.2,5.4,3.5Hz,1H),4.36(t,J＝6.4Hz,2H),4.09–4.00(m,2H),3.85(s,2H),2.99(dd,J＝12.5,5.3Hz,1H),2.78(dd,J＝12.5,3.5Hz,1H),1.42(s,9H).
实施例10：叔丁基-(1-(3,4-二甲氧基苯乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率68％。1H NMR(400MHz,DMSO-d6)δ6.77(d,J＝8.2Hz,1H),6.66–6.59(m,2H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.85(d,J＝4.4Hz,6H),3.80(dt,J＝12.7,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),3.02–2.83(m,3H),2.79(dd,J＝12.3,3.5Hz,1H),1.42(s,9H).
实施例11：叔丁基-(2,5-二氧基-1-(4-(三氟甲基)苯乙基)吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率82％。1H NMR(400MHz,DMSO-d6)δ7.56(dq,J＝7.3,1.5Hz,2H),7.11(dt,J＝7.1,1.0Hz,2H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.78(dt,J＝12.8,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.99(dd,J＝12.4,5.4Hz,1H),2.83–2.70(m,3H),1.42(s,9H).
实施例12：叔丁基-(2,5-二氧基-1-(3-(三氟甲基)苯乙基)吡咯烷-3-基)氨基甲酸酯的合成
具体操作及投料配比参考实施例2化合物的制备，产率64％。1H NMR(400MHz,DMSO-d6)δ7.47(dd,J＝11.1,7.2Hz,1H),7.44–7.38(m,2H),7.17(ddq,J＝7.1,2.4,1.1Hz,1H),5.95(d,J＝8.2Hz,1H),4.85(ddd,J＝8.5,5.3,3.5Hz,1H),3.80(dt,J＝12.7,6.4Hz,1H),3.71(dt,J＝12.8,6.3Hz,1H),2.99(dd,J＝12.4,5.4Hz,1H),2.91(tdt,J＝6.4,1.9,0.9Hz,2H),2.79(dd,J＝12.3,3.5Hz,1H),1.42(s,9H).
实施例13：(S)-3-氨基-1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)吡咯烷-2,5-二酮盐酸盐的合成
室温条件下，向叔丁基-(1-(2-(苯并[d][1,3]二恶英-5-基)乙基)-2,5-二氧吡咯烷-3-基)氨基甲酸酯中加入大过量的氯化氢乙酸乙酯溶液，反应1h后，减压蒸馏除去溶剂，即得白色固体产品，未通过1H-NMR进行结构表征。实施例3-12所对应的盐酸盐产品的制备，参考上述操作。
实施例14：(S)-3-((7H-嘌呤-6-基)氨基)-1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)吡咯烷-2,5-二酮(化合物1)的合成
将(S)-3-氨基-1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)吡咯烷-2,5-二酮盐酸盐(597.44mg，2.0mmol)用3mL正丁醇溶解，依次加入6-氯嘌呤(324.58mg，2.1mmol)和DIPEA(523μL，3.0mmol)。回流温度下搅拌6.0h，直到反应完成。减压蒸馏除去正丁醇，用乙酸乙酯和水萃取，饱和食盐水洗涤有机层，无水硫酸钠干燥，通过硅胶柱色谱层析柱层析纯化，二氯甲烷：甲醇＝15：1(v/v)，得到土黄色固体化合物，产率30％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),6.77(d,J＝8.1Hz,1H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.6,0.9Hz,1H),5.92(s,2H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.83(dt,J＝12.7,6.4Hz,1H),3.75(dt,J＝12.8,6.4Hz,1H),2.98–2.84(m,2H),2.81(dd,J＝12.5,3.7Hz,1H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,154.60,151.40,150.59,147.41,145.81,144.11,132.37,122.03,116.96,109.39,109.07,100.90,51.28,40.64,36.31,35.13.HRMS(ESI+):m/zcalculated for C18H17N6O4[M+H]+,381.1311；found,381.1319.
实施例15：(S)-3-((7H-嘌呤-6-基)氨基)-1-苄基吡咯烷-2,5-二酮(化合物2)的合成
具体操作及投料配比参考实施例14化合物的制备，产率23％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.34–7.21(m,6H),6.78(d,J＝8.0Hz,1H),5.15(ddd,J＝8.1,5.6,3.7Hz,1H),4.78(dt,J＝12.8,1.0Hz,1H),4.70(dt,J＝12.8,1.0Hz,1H),2.86(dd,J＝12.5,3.7Hz,1H),2.74(dd,J＝12.5,5.5Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.34,174.88,154.60,151.40,150.51,144.11,137.28,128.98,127.96,127.92,116.96,51.29,41.33,35.12.HRMS(ESI+):m/z calculated for C16H15N6O2[M+H]+,323.1256；found,323.1222.
实施例16：(S)-3-((7H-嘌呤-6-基)氨基)-1-苯乙基吡咯烷-2,5-二酮(化合物3)的合成
具体操作及投料配比参考实施例14化合物的制备，产率33％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.32–7.26(m,2H),7.26–7.19(m,1H),7.23–7.16(m,2H),6.77(d,J＝8.0Hz,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.84(dt,J＝13.0,6.5Hz,1H),3.72(dt,J＝13.0,6.4Hz,1H),2.81(dd,J＝12.5,3.7Hz,1H),2.81–2.75(m,2H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,154.60,151.40,150.59,144.11,138.31,128.93,128.25,126.48,116.96,51.28,40.79,35.13,34.75.HRMS(ESI+):m/z calculated for C17H17N6O2[M+H]+,337.1413；found,337.1412.
实施例17：(S)-3-((7H-嘌呤-6-基)氨基)-1-苯基吡咯烷-2,5-二酮(化合物4)的合成
具体操作及投料配比参考实施例14化合物的制备，产率27％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.49(ddt,J＝8.8,7.0,1.5Hz,1H),7.45–7.38(m,2H),7.34–7.28(m,2H),6.86(d,J＝7.8Hz,1H),5.29(ddd,J＝7.9,5.1,3.3Hz,1H),2.86(dd,J＝12.4,3.4Hz,1H),2.77(dd,J＝12.5,5.3Hz,1H).13C NMR(400MHz,DMSO-d6)δ174.22,173.31,154.60,151.40,150.06,144.11,133.98,129.48,128.35,127.91,116.98,51.71,35.55.HRMS(ESI+):m/z calculated for C15H13N6O2[M+H]+,309.1100；found,309.1104.
实施例18：(S)-3-((7H-嘌呤-6-基)氨基)-1-(4-氟苯乙基)吡咯烷-2,5-二酮(化合物5)的合成
具体操作及投料配比参考实施例14化合物的制备，产率26％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.17(ddt,J＝8.1,3.5,1.0Hz,2H),7.11–7.04(m,2H),6.77(d,J＝8.1Hz,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.84(dt,J＝12.9,6.5Hz,1H),3.72(dt,J＝13.0,6.5Hz,1H),2.81(dd,J＝12.5,3.7Hz,1H),2.76(tdt,J＝6.4,1.8,1.0Hz,2H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,162.38,160.41,154.60,151.40,150.59,144.11,133.83,130.62,130.55,116.96,115.31,51.28,40.80,35.13,34.33.HRMS(ESI+):m/z calculated for C17H16FN6O2[M+H]+,355.1319；found,355.1320.
实施例19：(S)-3-((7H-嘌呤-6-基)氨基)-1-(3-氟苯乙基)吡咯烷-2,5-二酮(化合物6)的合成
具体操作及投料配比参考实施例14化合物的制备，产率27％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.29(td,J＝7.7,5.0Hz,1H),7.04(ddq,J＝7.5,2.2,1.2Hz,1H),7.00(dddd,J＝10.1,7.9,2.2,1.1Hz,1H),6.91(dtt,J＝12.1,2.2,1.0Hz,1H),6.77(d,J＝8.1Hz,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.87–3.81(m,1H),3.76–3.70(m,1H),2.99–2.86(m,2H),2.81(dd,J＝12.5,3.7Hz,1H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,163.64,154.60,151.40,150.59,144.11,140.54,130.09,124.12,116.96,115.31,114.10,51.28,40.66,36.26,35.13.HRMS(ESI+):m/z calculated for C17H16FN6O2[M+H]+,355.1319；found,355.1322.
实施例20：(S)-3-((7H-嘌呤-6-基)氨基)-1-(4-羟基苯乙基)吡咯烷-2,5-二酮(化合物7)的合成
具体操作及投料配比参考实施例14化合物的制备，产率33％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.00(dt,J＝8.5,1.0Hz,2H),6.77(d,J＝8.1Hz,1H),6.77–6.71(m,2H),6.52(s,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.84(dt,J＝13.0,6.5Hz,1H),3.72(dt,J＝13.0,6.5Hz,1H),2.81(dd,J＝12.5,3.7Hz,1H),2.76(tq,J＝6.4,0.8Hz,2H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,156.09,154.60,151.40,150.59,144.11,130.23,130.01,116.96,116.00,51.28,40.80,35.13,34.62.HRMS(ESI+):m/zcalculated for C17H17N6O3[M+H]+,353.1362；found,353.1360.
实施例21：(S)-3-((7H-嘌呤-6-基)氨基)-1-(4-(三氟甲基)苯乙基)吡咯烷-2,5-二酮(化合物8)的合成
具体操作及投料配比参考实施例14化合物的制备，产率33％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.56(dq,J＝7.3,1.4Hz,2H),7.11(dt,J＝7.1,1.1Hz,2H),6.77(d,J＝8.0Hz,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.84(dt,J＝12.9,6.5Hz,1H),3.72(dt,J＝13.0,6.4Hz,1H),2.85–2.75(m,2H),2.78–2.71(m,1H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,154.60,151.40,150.59,144.11,137.08,130.24,129.54,129.51,125.91,125.88,123.14,116.96,51.28,40.79,35.13,35.08.HRMS(ESI+):m/z calculated for C18H16F3N6O2[M+H]+,405.1287；found,405.1279.
实施例22：(S)-3-((7H-嘌呤-6-基)氨基)-1-(3-(三氟甲基)苯乙基)吡咯烷-2,5-二酮(化合物9)的合成
具体操作及投料配比参考实施例14化合物的制备，产率35％。1H NMR(400MHz,DMSO-d6)δ8.35(s,1H),7.92(d,J＝8.6Hz,1H),7.47(dd,J＝11.1,7.2Hz,1H),7.44–7.38(m,2H),7.17(ddt,J＝7.2,2.4,1.1Hz,1H),6.77(d,J＝8.1Hz,1H),5.17(ddd,J＝8.2,5.5,3.7Hz,1H),3.83(dt,J＝12.7,6.4Hz,1H),3.75(dt,J＝12.8,6.4Hz,1H),2.92(tdt,J＝6.4,3.7,1.0Hz,2H),2.81(dd,J＝12.5,3.7Hz,1H),2.71(dd,J＝12.4,5.6Hz,1H).13C NMR(400MHz,DMSO-d6)δ175.76,174.95,154.60,151.40,150.59,144.11,138.74,131.12,130.01,128.97,126.19,125.24,123.36,116.96,51.28,40.72,36.85,35.13.HRMS(ESI+):m/z calculated for C18H16F3N6O2[M+H]+,405.1287；found,405.1280.
实施例23：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-(叔丁基)苯甲酰胺(化合物10)的合成
将对叔丁基苯甲酸(213.9mg，1.2mmol)用5mL DCM溶解，冰浴条件下，依次向反应体系中加入EDCI(380mg，2.0mmol)、HOBt(135mg，1.0mmol)、(S)-3-氨基-1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)吡咯烷-2,5-二酮盐酸盐(298.7mg，1mmol)和DIPEA(697μL，4mmol)，搅拌0.5h后，继续在室温条件下反应直至反应完全。将混合物倒入冰水中，用乙酸乙酯萃取。用饱和食盐水洗涤有机层，并用无水Na2SO4干燥。最后通过硅胶柱色谱层析纯化，二氯甲烷：甲醇＝40：1(v/v)，得到白色固体产品，产率76％。1H NMR(400MHz,DMSO-d6)δ7.75–7.70(m,2H),7.68(d,J＝8.8Hz,1H),7.47–7.41(m,2H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.5,0.9Hz,1H),5.92(s,2H),4.81(ddd,J＝8.7,5.1,3.3Hz,1H),3.80(dt,J＝12.9,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.98–2.80(m,4H),1.33(s,9H).13C NMR(400MHz,DMSO-d6)δ175.60,173.81,166.55,153.10,147.41,145.81,132.98,132.37,127.93,126.11,122.03,109.39,109.07,100.90,50.33,40.67,36.31,35.43,34.92,31.17.HRMS(ESI+):m/z calculated for C24H26N2O5Na[M+Na]+,445.1739；found,445.1742.
实施例24：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)苯甲酰胺(化合物11)的合成
具体操作及投料配比参考实施例23化合物的制备,产率84.1％。1H NMR(400MHz,DMSO-d6)δ9.18(d,J＝4.0Hz,1H),7.86-7.84(m,2H),7.58-7.56(m,1H),7.52-7.48(m,2H),6.86-6.82(m,2H),6.71-6.69(m,1H),5.98(t,J＝4.0Hz,2H),3.61-3.58(m,2H),3.02-2.96(m,1H),2.75-2.71(m,3H).13C NMR(400MHz,DMSO-d6)δ176.50,175.37,166.70,147.77,146.23,133.54,132.55,132.27,128.94,127.73,122.06,109.52,108.69,101.19,49.31,40.11,35.34,33.23.HRMS(ESI+):m/z calculated for C20H18N2O5Na[M+Na]+,389.1113；found,389.1147.
实施例25：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-羟基苯甲酰胺(化合物12)的合成
具体操作及投料配比参考实施例23化合物的制备，产率80.2％。1H NMR(400MHz,DMSO-d6)δ10.08(s,1H),8.92(d,J＝4.0Hz,1H),7.72-7.70(m,2H),6.85-6.80(m,5H),5.98-5.97(m,2H),4.55-4.54(m,1H),3.60-3.56(m,2H),3.02-2.96(m,1H),2.75-2.71(m,3H).13C NMR(400MHz,DMSO-d6)δ177.97,175.56,167.21,160.96,148.68,146.65,131.67,130.92,125.34,121.20,115.43,109.99,108.70,101.19,50.43,35.07,33.23.HRMS(ESI+):m/z calculated for C20H18N2O6Na[M+Na]+,405.1063；found,405.1082.
实施例26：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-3-羟基苯甲酰胺(化合物13)的合成
具体操作及投料配比参考实施例23化合物的制备，产率71.6％。1H NMR(400MHz,DMSO-d6)δ9.70(s,1H),9.04(d,J＝4.0Hz,1H),7.78-7.22(m,3H),6.95-6.93(m,2H),6.85-6.82(m,2H),6.71-6.68(m,1H),5.97(s,2H),4.55-4.54(m,1H),3.60-3.56(m,2H),3.02-2.96(m,1H),2.72-2.66(m,3H).13C NMR(400MHz,DMSO-d6)δ177.97,175.37,166.75,157.12,148.68,145.60,136.19,133.02,129.95,122.56,118.72,118.20,115.26,110.36,108.69,101.63,49.68,35.35,32.29.HRMS(ESI+):m/z calculated for C20H18N2O6Na[M+Na]+,405.1063；found,405.1066.
实施例27：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-甲氧基苯甲酰胺(化合物14)的合成
具体操作及投料配比参考实施例23化合物的制备，产率64.8％。1H NMR(400MHz,DMSO-d6)δ9.02(d,J＝4.0Hz,1H),7.82(d,J＝4.0Hz,2H),7.03-7.01(m,2H),6.85-6.82(m,2H),6.71-6.68(m,1H),5.97(s,2H),4.55-4.54(m,1H),3.82(s,3H),3.60-3.57(m,2H),3.02-2.96(m,1H),2.74-2.66(m,3H).13C NMR(400MHz,DMSO-d6)δ177.67,175.41,168.26,162.45,148.38,146.65,133.40,129.64,122.94,114.88,109.51,108.69,101.63,56.68,49.29,35.75,33.23.HRMS(ESI+):m/z calculated for C21H20N2O6Na[M+Na]+,419.1219；found,419.1193.
实施例28：(S)-4-乙酰基-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)苯甲酰胺(化合物15)的合成
具体操作及投料配比参考实施例23化合物的制备，产率60.1％。1H NMR(400MHz,DMSO-d6)δ9.37(d,J＝4.0Hz,1H),8.08-8.06(m,2H),7.98-7.96(m,1H),6.86-6.82(m,2H),6.72-6.69(m,1H),5.98-5.98(m,2H),4.55-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.67(m,3H),2.63(s,3H).13C NMR(400MHz,DMSO-d6)δ198.60,177.29,175.32,165.47,150.11,146.23,139.50,137.24,130.62,129.09,121.51,111.79,107.20,102.68,47.95,35.26,34.02,27.46.HRMS(ESI+):m/z calculated for C22H20N2O6Na[M+Na]+,431.1321；found,431.1222.
实施例29：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-(1,1'-联苯基)-4-甲酰胺(化合物16)的合成
具体操作及投料配比参考实施例23化合物的制备，产率56.1％。1H NMR(400MHz,DMSO-d6)δ7.96-7.94(m,1H),7.83-7.80(m,2H),7.76-7.74(m,2H),7.52-7.48(m,2H),7.42(s,1H),6.87-6.83(m,2H),6.72-6.71(m,1H),5.98(s,2H),4.55-4.54(m,1H),3.61-3.60(m,2H),3.10-3.00(m,1H),2.76-2.72(m,3H).13C NMR(400MHz,DMSO-d6)δ176.51,175.39,166.37,147.78,146.24,143.72,139.46,132.55,132.34,129.52,128.65,128.43,127.37,127.13,122.08,109.53,108.70,101.20,49.33,35.38,33.24.HRMS(ESI+):m/zcalculated for C26H22N2O5Na[M+Na]+,431.1321；found,431.1331.
实施例30：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-甲基苯甲酰胺(化合物17)的合成
具体操作及投料配比参考实施例23化合物的制备，产率53.3％。1H NMR(400MHz,DMSO-d6)δ9.09(d,J＝4.0Hz,1H),7.76-7.29(m,2H),6.86-6.82(m,2H),6.72-6.69(m,1H),5.98-5.97(m,2H),4.55-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.67(m,3H),2.36(s,3H).13C NMR(400MHz,DMSO-d6)δ176.57,175.39,166.59,147.77,146.23,142.24,132.55,130.79,129.45,127.76,122.06,109.51,108.69,49.27,33.38,33.23,21.45.HRMS(ESI+):m/zcalculated for C21H20N2O5Na[M+Na]+,403.1270；found,403.1290.
实施例31：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-氯苯甲酰胺(化合物18)的合成
具体操作及投料配比参考实施例23化合物的制备，产率44.5％。1H NMR(400MHz,DMSO-d6)δ9.09(d,J＝3.0Hz,1H),7.52-7.42(m,4H),6.84-6.81(m,2H),6.70-6.68(m,1H),5.98-5.97(m,2H),4.55-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.67(m,3H).13C NMR(400MHz,DMSO-d6)δ175.95,175.17,166.94,147.76,146.22,135.89,132.51,130.29,129.44,127.69,122.07,109.50,108.69,101.19,49.09,40.14,35.25,33.17.HRMS(ESI+):m/z calculated for C20H17ClN2O5Na[M+Na]+,423.0724；found,423.0716.
实施例32：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-3-氯苯甲酰胺(化合物19)的合成
具体操作及投料配比参考实施例23化合物的制备，产率46.9％。1H NMR(400MHz,DMSO-d6)δ9.30(d,J＝4.0Hz,1H),7.89-7.88(m,2H),7.82-7.80(m,1H),7.65-7.65(m,1H),7.57-7.55(m,1H),5.98-5.97(m,2H),4.55-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.69(m,3H).13C NMR(400MHz,DMSO-d6)δ176.28,175.31,165.32,147.77,146.23,135.54,133.82,132.51,132.13,131.03,127.54,126.51,122.06,109.52,108.70,101.19,49.33,35.24,33.22.HRMS(ESI+):m/z calculated for C20H17ClN2O5Na[M+Na]+,423.0724；found,423.0738.
实施例33：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-(叔丁氧基)苯甲酰胺(化合物20)的合成
具体操作及投料配比参考实施例23化合物的制备，产率73.8％。1H NMR(400MHz,DMSO-d6)δ9.06(d,J＝2.0Hz,1H),7.79-7.77(m,2H),7.07-7.05(m,2H),6.85-6.82(m,2H),6.71-6.70(m,1H),5.98-5.97(m,2H),4.55-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.69(m,3H),1.35(s,9H).13C NMR(400MHz,DMSO-d6)δ176.58,175.40,166.30,158.94,147.77,132.56,129.05,122.81,122.06,109.51,108.69,101.19,79.37,49.29,35.38,33.22,29.00.HRMS(ESI+):m/z calculated for C24H26N2O6Na[M+Na]+,461.1689；found,461.1673.
实施例34：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-(三氟甲氧基)苯甲酰胺(化合物21)的合成
具体操作及投料配比参考实施例23化合物的制备，产率65.5％。1H NMR(400MHz,DMSO-d6)δ9.29(d,J＝4.0Hz,1H),7.99-7.96(m,2H),7.52-7.49(m,2H),6.85-6.82(m,2H),6.71-6.69(m,1H),5.98-5.97(m,2H),4.56-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.69(m,3H).13C NMR(400MHz,DMSO-d6)δ176.33,175.30,165.52,147.77,146.24,132.63,132.52,130.13,122.06,121.23,109.51,108.69,101.19,49.36,35.26,33.22.HRMS(ESI+):m/z calculated for C21H17F3N2O6Na[M+Na]+,473.0925；found,473.0936.
实施例35：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-3-(三氟甲基)苯甲酰胺(化合物22)的合成
具体操作及投料配比参考实施例23化合物的制备，产率48.8％。1H NMR(400MHz,DMSO-d6)δ9.46(d,J＝4.0Hz,1H),8.19-8.15(m,2H),7.98-7.96(m,1H),7.77(s,1H),6.86-6.82(m,2H),6.72-6.69(m,1H),5.98-5.97(m,2H),4.56-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,1H),2.75-2.69(m,3H).13C NMR(400MHz,DMSO-d6)δ176.27,175.31,165.27,147.77,146.24,134.42,132.50,131.89,130.39,128.87,124.86,122.06,109.52,108.69,101.19,49.86,35.97,33.23.HRMS(ESI+):m/z calculated for C21H17F3N2O5Na[M+Na]+,457.0987；found,457.0972.
实施例36：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-(二甲氨基)苯甲酰胺(化合物23)的合成
具体操作及投料配比参考实施例23化合物的制备，产率73.7％。1H NMR(400MHz,DMSO-d6)δ8.80(d,J＝4.0Hz,1H),7.71-7.68(m,2H),6.86-6.82(m,2H),6.73-6.68(m,3H),5.98-5.97(m,2H),4.56-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,7H),2.75-2.68(m,3H).13C NMR(400MHz,DMSO-d6)δ176.88,175.49,166.62,152.85,147.75,146.20,132.58,129.15,122.04,120.01,111.22,109.50,108.68,101.18,49.24,35.56,33.24.HRMS(ESI+):m/z calculated for C22H23N3O5Na[M+Na]+,432.1535；found,432.1507.
实施例37：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-丁基苯甲酰胺(化合物24)的合成
具体操作及投料配比参考实施例23化合物的制备，产率53.2％。1H NMR(400MHz,DMSO-d6)δ7.77-7.75(m,2H),7.32-7.30(m,2H),6.85-6.68(m,3H),5.98-5.97(m,2H),4.56-4.54(m,1H),3.62-3.57(m,2H),3.02-2.96(m,7H),2.75-2.61(m,5H),1.58-1.55(m,2H),1.32-1.27(m,2H),0.89(t,J＝8.0Hz,3H).13C NMR(400MHz,DMSO-d6)δ176.54,175.37,166.61,147.76,146.99,146.21,132.54,131.02,128.79,127.77,122.05,109.50,108.68,101.18,49.27,35.37,35.09,33.27,33.23,22.15,14.21.HRMS(ESI+):m/z calculatedfor C24H26N2O5Na[M+Na]+,445.1739；found,445.1728.
实施例38：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-(4-(叔丁基)苯基)乙酰胺(化合物25)的合成
具体操作及投料配比参考实施例23化合物的制备，产率76.4％。1H NMR(400MHz,DMSO-d6)δ8.69-8.68(m,1H),7.34-7.30(m,2H),7.17-7.15(m,2H),6.81-6.68(m,2H),6.66-6.64(m,1H),5.97-5.96(m,2H),4.46-4.38(m,1H),3.55-3.51(m,2H),3.42(s,2H),3.00-2.90(m,2H),2.69-2.45(m,3H),1.26(s,9H).13C NMR(400MHz,DMSO-d6)δ176.36,175.19,171.21,149.23,147.74,146.20,133.04,132.48,129.13,125.49,122.01,109.45,108.66,101.17,48.97,41.65,35.39,34.58,33.15,31.63.HRMS(ESI+):m/z calculatedfor C25H28N2O5Na[M+Na]+,459.1896；found,459.1890.
实施例39:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-(3-(三氟甲氧基)苯基)乙酰胺(化合物26)的合成
具体操作及投料配比参考实施例23化合物的制备，产率44.2％。1H NMR(400MHz,DMSO-d6)δ8.80-8.78(m,1H),7.47-7.43(m,1H),7.29-7.22(m,3H),6.81-6.79(m,2H),6.67-6.64(m,1H),5.97(s,2H),4.47-4.40(m,1H),3.56-3.51(m,4H),2.98-2.90(m,1H),2.60-2.52(m,2H),2.52-2.47(m,1H).13C NMR(400MHz,DMSO-d6)δ176.28,175.14,170.46,148.76,147.74,146.20,138.80,132.47,130.56,128.72,122.01,121.96,119.48,108.64,101.17,49.01,41.44,35.52,33.13.HRMS(ESI+):m/z calculated for C22H19F3N2O6Na[M+Na]+,487.1093；found,487.1103.
实施例40:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-氟异烟酰胺(化合物27)的合成
具体操作及投料配比参考实施例23化合物的制备，产率48.7％。1H NMR(400MHz,DMSO-d6)δ9.58-9.56(m,1H),8.45-8.43(m,1H),7.74-7.72(m,1H),7.53-7.52(m,1H),6.85-6.82(m,2H),6.71-6.69(m,1H),5.98(s,2H),4.72-4.66(m,1H),3.62-3.58(m,2H),3.08-3.02(m,1H),2.74-2.68(m,3H).13C NMR(400MHz,DMSO-d6)δ175.91,175.16,163.85,162.79,149.31,149.16,147.76,146.23,132.45,122.06,120.20,120.16,109.50,108.69,108.35,107.96,101.19,49.38,35.05,33.21.HRMS(ESI+):m/z calculated forC19H16FN3O5Na[M+Na]+,408.0972；found,408.0958.
实施例41:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)异烟酰胺(化合物28)的合成
具体操作及投料配比参考实施例23化合物的制备，产率56.6％。1H NMR(400MHz,DMSO-d6)δ9.49-9.47(m,1H),8.77-8.76(m,2H),7.76-7.74(m,2H),6.85-6.82(m,2H),6.71-6.69(m,1H),5.98(s,2H),4.72-4.65(m,1H),3.08-3.02(m,1H),2.74-2.67(m,3H).13CNMR(400MHz,DMSO-d6)δ176.10,175.22,165.22,150.92,147.76,146.23,132.48,122.06,121.58,109.50,108.69,101.19,49.33,35.14,33.21.HRMS(ESI+):m/z calculated forC19H17N3O5Na[M+Na]+,390.1066；found,390.1097.
实施例42:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)异喹啉-1-甲酰胺(化合物29)的合成
具体操作及投料配比参考实施例23化合物的制备，产率66％。1H NMR(400MHz,DMSO-d6)δ8.85(d,J＝4.6Hz,1H),8.28–8.23(m,1H),8.06–7.98(m,2H),7.68(d,J＝9.7Hz,1H),7.62(ddd,J＝8.6,7.1,1.3Hz,1H),7.59–7.52(m,1H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.5,0.9Hz,1H),5.92(s,2H),4.86(ddd,J＝9.7,5.3,3.5Hz,1H),3.80(dt,J＝12.9,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.98–2.80(m,4H).13C NMR(400MHz,DMSO-d6)δ175.50,173.88,166.57,147.41,146.20,145.81,145.24,137.18,132.37,131.93,130.21,128.59,127.57,126.26,122.19,122.03,109.39,109.07,100.90,50.17,40.67,36.31,35.35.HRMS(ESI+):m/z calculated forC23H19N3O5Na[M+Na]+,440.1222；found,440.1230.
实施例43:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)喹啉-4-甲酰胺(化合物30)的合成
具体操作及投料配比参考实施例23化合物的制备，产率70％。1H NMR(400MHz,DMSO-d6)δ8.73(d,J＝4.6Hz,1H),8.26–8.21(m,1H),8.16(dd,J＝8.1,1.5Hz,1H),7.86–7.79(m,2H),7.67(dddd,J＝25.6,8.3,7.0,1.5Hz,2H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.7,0.9Hz,1H),5.92(s,2H),4.84(ddd,J＝8.9,5.3,3.5Hz,1H),3.80(dt,J＝12.9,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.98–2.80(m,4H).13C NMR(400MHz,DMSO-d6)δ175.60,173.79,167.83,149.60,147.41,146.68,145.81,137.97,132.37,129.86,129.82,128.30,127.92,125.16,122.05,122.03,109.39,109.07,100.91,50.34,40.67,36.31,35.41.HRMS(ESI+):m/z calculated for C23H19N3O5Na[M+Na]+,440.1222；found,440.1232.
实施例44:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)喹啉-5-甲酰胺(化合物31)的合成
具体操作及投料配比参考实施例23化合物的制备，产率72％。1H NMR(400MHz,DMSO-d6)δ8.89(dd,J＝4.2,2.2Hz,1H),8.52(dd,J＝7.9,2.2Hz,1H),8.19–8.14(m,1H),7.92(dd,J＝8.6,1.4Hz,1H),7.85(d,J＝9.0Hz,1H),7.66(dd,J＝8.5,7.6Hz,1H),7.59(dd,J＝7.8,4.1Hz,1H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.6,0.9Hz,1H),5.92(s,2H),4.84(ddd,J＝8.7,5.3,3.5Hz,1H),3.80(dt,J＝12.9,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.99–2.80(m,4H).13C NMR(400MHz,DMSO-d6)δ175.60,173.79,167.64,150.12,149.09,147.41,145.81,134.01,133.03,132.37,130.32,128.39,127.90,127.49,124.47,122.03,109.39,109.07,100.90,50.45,40.67,36.31,35.40.HRMS(ESI+):m/z calculated for C23H19N3O5Na[M+Na]+,440.1222；found,440.1230.
实施例45:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-甲基喹啉-6-甲酰胺(化合物32)的合成
具体操作及投料配比参考实施例23化合物的制备，产率56％。1H NMR(400MHz,DMSO-d6)δ8.28(t,J＝1.8Hz,1H),8.13–8.04(m,2H),7.99(dd,J＝8.3,1.5Hz,1H),7.93(d,J＝8.8Hz,1H),7.33(dt,J＝7.9,0.8Hz,1H),6.69(d,J＝8.8Hz,1H),6.63(ddt,J＝8.6,1.7,1.0Hz,1H),6.52(dd,J＝1.7,0.9Hz,1H),5.92(s,2H),4.81(ddd,J＝8.7,5.1,3.4Hz,1H),3.80(dt,J＝12.9,6.4Hz,1H),3.71(dt,J＝12.8,6.4Hz,1H),2.98–2.89(m,2H),2.92–2.84(m,1H),2.83(dd,J＝12.5,3.5Hz,1H),2.73(d,J＝0.7Hz,3H).13C NMR(400MHz,DMSO-d6)δ175.60,173.79,167.10,157.57,149.90,147.41,145.81,135.42,132.37,130.99,128.82,128.44,128.26,126.87,122.57,122.03,109.39,109.07,100.90,50.33,40.67,36.31,35.43,24.78.HRMS(ESI+):m/z calculated for C24H21N3O5Na[M+Na]+,454.1379；found,454.1362.
实施例46:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)喹啉-2-甲酰胺(化合物33)的合成
具体操作及投料配比参考实施例23化合物的制备，产率78.9％。1H NMR(400MHz,DMSO-d6)δ8.65-8.59(m,1H),8.17-8.10(m,3H),7.93-7.88(m,1H),7.77-7.73(m,1H),6.87-6.83(m,1H),6.73-6.70(m,1H),5.98(s,2H),4.98-4.93(m,1H),3.63-3.59(m,1H),3.10-3.03(m,1H),2.78-2.67(m,3H).13C NMR(400MHz,DMSO-d6)δ176.52,175.40,164.79,149.79,147.77,146.49,146.23,138.51,132.57,131.19,129.56,129.42,128.77,128.67,122.05,119.17,109.50,108.71,101.20,48.78,35.52,33.24.HRMS(ESI+):m/zcalculated for C23H19N3O5Na[M+Na]+,440.1222；found,440.1265.
实施例47:(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)喹啉-2-甲酰胺(化合物34)的合成
具体操作及投料配比参考实施例23化合物的制备，产率66.7％。1H NMR(400MHz,DMSO-d6)δ9.75-9.73(m,1H),9.47(s,1H),8.21-8.19(m,2H),8.02-8.00(m,2H),6.86-6.82(m,1H),5.98-5.97(m,2H),5.02-4.93(m,1H),3.63-3.59(m,2H),3.10-3.03(m,1H),2.78-2.72(m,3H).13C NMR(400MHz,DMSO-d6)δ176.31,175.36,163.84,147.78,146.24,144.15,143.95,143.61,140.24,132.67,132.54,132.00,129.85,129.68,122.07,109.51,108.73,101.21,48.71,35.43,33.24.HRMS(ESI+):m/z calculated for C22H18N4O5Na[M+Na]+,441.1175；found,441.1175.
实施例48：N-((S)-1-(2-(苯并[d][1,3]二恶英-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2,3,4a，8a-四氢苯并[b][1,4]二恶英-2-甲酰胺(化合物35)的合成
具体操作及投料配比参考实施例23化合物的制备，产率76.2％。1H NMR(400MHz,DMSO-d6)δ6.99-6.97(m,1H),6.91-6.80(m,5H),6.68-6.66(m,1H),5.97(s,1H),4.87-4.86(m,1H),4.78-4.74(m,1H),4.44-4.30(m,1H),4.20-4.14(m,1H),3.56-3.50(m,2H),3.00-2.90(m,1H),2.70-2.68(m,2H).13C NMR(400MHz,DMSO-d6)δ176.04,176.00,175.17,167.88,167.75,147.76,146.23,143.47,142.41,132.48,122.21,122.03,117.76,117.63,109.47,108.69,101.20,72.87,65.16,48.51,48.31,35.35,33.16.HRMS(ESI+):m/zcalculated for C22H22N2O7Na[M+Na]+,449.1325；found,449.1332.
实施例49：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-2-(1H-吲哚-3-基)乙酰胺(化合物36)的合成
具体操作及投料配比参考实施例23化合物的制备，产率42.6％。1H NMR(400MHz,DMSO-d6)δ10.90(s,1H),8.53-8.51(m,1H),7.52-7.50(m,1H),7.36-7.33(m,1H),7.21-7.20(m,1H),7.07-6.98(m,1H),6.81-6.79(m,1H),6.66-6.64(m,1H),5.96(s,2H),4.87-4.86(m,1H),3.56-3.50(m,4H),3.00-2.90(m,1H),2.80-2.75(m,2H),2.70-2.64(m,1H).13CNMR(400MHz,DMSO-d6)δ176.49,175.26,171.73,147.74,146.20,136.60,132.52,127.64,124.39,122.02,121.47,118.98,111.81,109.46,108.66,108.54,101.18,48.95,35.38,33.14,32.69.HRMS(ESI+):m/z calculated for C23H21N3O5Na[M+Na]+,442.1379；found,442.1362.
实施例50：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-1H-吲哚-5-甲酰胺(化合物37)的合成
具体操作及投料配比参考实施例23化合物的制备，产率72.4％。1H NMR(400MHz,DMSO-d6)δ11.45(s,1H),9.07-9.05(m,1H),7.96(s,1H),7.62-7.60(m,1H),7.54-7.50(m,2H),6.87-6.83(m,2H),6.73-6.70(m,1H),5.98(s,2H),4.87-4.86(m,1H),3.62-3.59(m,2H),3.00-2.90(m,1H),2.80-2.72(m,3H).13C NMR(400MHz,DMSO-d6)δ176.82,175.50,168.26,148.38,146.95,136.19,132.60,131.26,128.85,126.78,120.04,117.36,111.85,109.99,108.70,100.58,50.06,35.52,33.64,21.45.HRMS(ESI+):m/z calculated forC22H19N3O5Na[M+Na]+,428.1222；found,428.1234.
实施例51：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-1H-吲唑-3-甲酰胺(化合物38)的合成
具体操作及投料配比参考实施例23化合物的制备，产率52.8％。1H NMR(400MHz,DMSO-d6)δ9.04-9.02(m,1H),8.14-8.12(m,1H),7.76-7.74(m,1H),7.30-7.28(m,1H),6.85-6.81(m,1H),6.71-6.68(m,1H),5.97(s,2H),4.87-4.86(m,1H),4.14(s,3H),3.60-3.56(m,2H),3.01(s,1H),2.74-2.68(m,3H).13C NMR(400MHz,DMSO-d6)δ176.70,175.42,147.77,141.39,132.60,127.15,123.07,122.62,122.08,110.99,109.53,108.71,101.20,48.29,36.47,35.61,33.22.HRMS(ESI+):m/z calculated for C22H20N4O5Na[M+Na]+,443.1331；found,443.1335.
实施例52：(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-1H-苯并[d]咪唑-5-甲酰胺(化合物39)的合成
具体操作及投料配比参考实施例23化合物的制备，产率82.5％。1H NMR(400MHz,DMSO-d6)δ9.17-9.15(m,1H),8.36-8.34(m,1H),8.25-8.00(m,1H),7.76-7.50(m,2H),6.87-6.83(m,2H),6.72-6.70(m,1H),5.98(s,2H),4.65-4.60(m,1H),3.63-3.56(m,2H),3.07-3.01(m,1H),2.74-2.68(m,3H).13C NMR(400MHz,DMSO-d6)δ176.70,175.46,167.22,147.77,146.23,132.58,122.07,109.53,108.69,101.19,49.39,40.11,35.46,33.24.HRMS(ESI+):m/z calculated for C21H18N4O5Na[M+Na]+,429.1175；found,429.1175.
实施例53：CCK8法检测化合物对于TNF-α和放线菌素D诱导的细胞调亡的抑制作用
取对数期生长的L929细胞，用胰酶消化，铺96孔板，密度20000个/孔。不同浓度梯度(0-200μM)的化合物0-54与10ng/mL TNF-α在37℃孵育10h后，96孔板给予预孵育的TNF-α-化合物复合物和1μg/mL放线菌素D。在给药24h后用CCK-8在450nm波长下，检测活细胞数目。结果显示，(S)-N-(1-(2-(苯并[d][1,3]二氧醇-5-基)乙基)-2,5-二氧吡咯烷-3-基)-4-(叔丁基)苯甲酰胺(化合物10)对TNF-α和放线菌素D诱导的细胞调亡具有强烈的抑制作用，IC50为13.11±1.54μM。
实施例54：TNF-α蛋白的克隆、表达和纯化
将人类TNF-α克隆到pet28a中，在大肠杆菌BL21(DE3)中表达。转染了重组质粒的BL21菌株在37℃培养3-4h,后加1mM IPTG，在20℃时促进蛋白表达。20℃过夜生长后，3000g离心10min收集细菌。细菌在Lysis Buffer中超声破碎，21500g离心1h，收集上清为蛋白粗品。先经过Ni-NTA亲和层析柱，Binding buffer洗杂，Elution buffer洗脱。
Lysis Buffer配方：20mM Tris,pH 8.5,200mM NaCl,5mMβ-巯基乙醇,0.1％TritonX-100,and 5％甘油
Binding buffer配方：20mM Tris,pH 8.5,200mM NaCl,10mM咪唑
Elution buffer配方：20mM Tris,pH 8.5,200mM NaCl,150mM咪唑
将目的蛋白溶液转移至30kDa的蛋白浓缩管中，4℃，3000rpm浓缩至0.5mL后，使用的AKTA Pure蛋白纯化系统中的凝胶过滤色谱进行纯化，纯化体系为20mM Tris,pH 8.5,200mM NaCl，2mM DTT。
实施例55：MST测定化合物10与TNF-α的结合亲和力
将纯化得到的TNF-α蛋白用蛋白缓冲液(20mM Hepes,pH 7.5,200mM NaCl)配制成10μM，加入2μL MST荧光染料，室温避光孵育30min。将染色混合液加入到MST试剂盒中的B柱中，分离出已荧光标记的蛋白质溶液。用蛋白缓冲液梯度稀释化合物10，配成12个浓度梯度，在与荧光标记蛋白等体积混匀。通过标准毛细管吸取样品置于微量热泳动仪上检测。参数设置为LED Power 100％，MST Power 20％。最后结果通过Nano Temper Analysis1.2.20软件进行分析，拟合得到解离常数(Kd)值，化合物10与TNF-α结合亲和力的Kd为：12.06±4.32μM(图1A-1B)。
实施例56：CETSA实验评价化合物10与TNF-α的结合
纯化的TNF-α蛋白与DMSO或者10μM化合物10于37℃孵育4h后，等分为6份，分别于82，84，86，88，90和92℃加热15min，加入5×SDS-PAGE上样缓冲液，95℃加热变性5min后，进行免疫印迹检测。结果显示化合物10提高了TNF-α蛋白的热稳定性，耐受温度从90℃提高至92℃(图1C)。
实施例57：DARTS实验评价化合物10与TNF-α的结合
纯化的TNF-α蛋白均分为4等份，分为对照组、酶解组、给药组(5、10μM)，先于室温使化合物与蛋白孵育1h，然后酶解组与给药组中加入1:1000稀释的蛋白酶Pronase，对照组给予等体积的PBS，混合均匀后，于37℃水浴锅中孵育15min。立即取出，加入SDS-PAGE上样缓冲液，95℃加热变性5min后，进行免疫印迹检测。结果显示，约60％的TNF-α蛋白在用Pronase处理后15分钟内被降解，而在化合物10处理后TNF-α的降解明显减少(图1D)。
实施例58：化合物10阻止TNF-α诱导的caspase信号通路的激活
化合物10在TNF-α和放线菌素D诱导的L929细胞凋亡中剂量依赖性地抑制caspase8和caspase 3的切割和活化(图2)。
实施例59：化合物10阻止TNF-α诱导的NF-κB信号通路的激活
化合物10对TNF-α诱导的NF-κB信号通路和LPS诱导的NF-κB信号通路的激活具有明显的抑制作用。具体表现在化合物10能够以剂量依赖性方式抑制IκBα的磷酸化并且抑制NF-κB p65的核转位(图3A-3B)。
实施例60：化合物10能够抑制TNF-α诱导的炎性细胞因子的表达和释放
RT-PCR和ELISA法检测了TNF-α或者LPS刺激的RAW264.7细胞上清中IL-6和IL-1β的转录和表达。结果表明，化合物10显著下调了TNF-α或者LPS诱导的IL-6和IL-1β的mRNA水平和蛋白水平(图3C-3D)。因此，以上结果表明，化合物10抑制了TNF-α或者LPS诱导的炎症细胞因子IL-6和IL-1β的转录和表达。
表1化合物1-39半最大效应浓度(EC50)数值
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Claims (7)
1.一种取代吡咯烷酮类化合物，其特征在于，其为如下所示化合物或其药学上可接受的盐；
2.一种药物组合物，其特征在于，包含权利要求1所述的取代吡咯烷酮类化合物或其药学上可接受的盐，以及药学上可接受的载体、稀释剂、辅剂、媒介物或它们的组合。
3.根据权利要求2所述的药物组合物，其特征在于，所述的药物组合物的剂型为注射剂、片剂和胶囊剂中的任一种。
4.权利要求1所述的取代吡咯烷酮类化合物或其药学上可接受的盐在制备肿瘤坏死因子抑制剂方面的应用。
5.根据权利要求4所述的应用，其特征在于，在制备治疗或预防自身免疫性、炎症性疾病药物中的应用。
6.权利要求2-3任一项所述的药物组合物在制备肿瘤坏死因子抑制剂方面的应用。
7.根据权利要求6所述的应用，其特征在于，在制备治疗或预防自身免疫性、炎症性疾病药物中的应用。
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