CN110156729B - 一种苯基哌嗪类ube2f小分子抑制剂及其合成方法 - Google Patents

一种苯基哌嗪类ube2f小分子抑制剂及其合成方法 Download PDF

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CN110156729B
CN110156729B CN201910404046.2A CN201910404046A CN110156729B CN 110156729 B CN110156729 B CN 110156729B CN 201910404046 A CN201910404046 A CN 201910404046A CN 110156729 B CN110156729 B CN 110156729B
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孙毅
刘宏民
徐甜甜
栗亚男
余斌
马启胜
侯廷军
潘培辰
熊秀芳
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Zhejiang University ZJU
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Abstract

本发明为一种苯基哌嗪类UBE2F小分子抑制剂及其合成方法，公开了一种通式I所示的苯基哌嗪类化合物或其药学上可接受的盐，并进一步公开了所述通式Ⅰ的合成路线及各步骤的合成方法。本发明苯基哌嗪类化合物为靶向UBE2F的小分子抑制剂，对人源肿瘤细胞通过阻止细胞周期G2/M进程和诱导细胞凋亡，从而有效抑制肿瘤细胞生长。因此该化合物是一类新的、通过靶向UBE2F的特异性抗肿瘤药物。

Description

一种苯基哌嗪类UBE2F小分子抑制剂及其合成方法
技术领域
本发明属于医药技术领域，特别是化学药物合成及药理活性领域，具体涉及一种苯基哌嗪类靶向UBE2F的抗肿瘤小分子抑制剂及其合成方法。
背景技术
Neddylation通路是通过激活酶E1(NAE)，耦联酶E2(UBE2M和UBE2F)和连接酶E3的级联酶促反应，将类泛素分子NEDD8共价结合到底物蛋白分子上的一种蛋白翻译后修饰方式。Cullin-RING ligases(CRLs)是泛素连接酶E3最大的超家族，其活性依赖于Cullin分子的NEDD8修饰。研究表明，CRLs E3泛素连接酶的异常调节可导致细胞的异常增殖、基因组的不稳定性和肿瘤进展，且其过表达与多种肿瘤的不良预后相关。
UBE2M E2耦联酶催化调节Cullin1/2/3/4A/4B的NEDD8修饰，而UBE2F E2耦联酶特异性调控Cullin5的NEDD8修饰。CRL5 E3泛素连接酶可通过K11泛素链修饰促凋亡蛋白NOXA，从而促进NOXA降解；失活CRL5 E3泛素连接酶能促使NOXA积累，从而诱导肿瘤细胞发生NOXA依赖的凋亡。
肿瘤细胞中，常有Neddylation通路关键分子的异常活化。靶向Neddylation通路中的关键分子，是研发新型抗肿瘤药物的新方向。目前，MLN4924(又称Pevonedistat)作为NAE抑制剂，已经进入临床Ib-II阶段，表现出显著的抗肿瘤疗效。而由于其抑制整条Neddylation通路，不可避免地表现出较大的副作用。研究表明，CRL5 E3泛素连接酶活性与肺癌的发生发展密切相关。靶向UBE2F可以选择性抑制CRL5 E3泛素连接酶活性，诱导肺癌细胞发生凋亡，同时尽可能地减少化合物的副作用。
发明内容
本发明针对现有技术的不足，提供一种苯基哌嗪类UBE2F小分子抑制剂，该小分子抑制剂靶向UBE2F，可以选择性抑制CRL5 E3泛素连接酶活性，诱导肺癌细胞发生凋亡，同时尽可能地减少化合物的副作用。
本发明采用如下技术方案：一种苯基哌嗪类UBE2F小分子抑制剂，具有通式I的结构，或具有通式I的结构的盐：
Figure GDA0003900175030000011
其中X，Y选自羰基、磺酰基；
R1选自甲基、苯基、乙烯基、2-萘基、吗啉基、吲哚基，N,N-二甲基、呋喃基、4-硝基苯基、4-甲基-苯基；
R2选自呋喃基、苯基、5-N,N-二甲氨基萘基、4-甲基-苯基、乙烯基、2-萘基、甲基、2-吡啶基、2-吲哚基、N,N-二甲基、吗啉基、3-溴-苯基、4-硝基-苯基、苯乙烯基、2-吡啶-乙烯基、3-吡啶-乙烯基、4-吡啶-乙烯基、4-甲基-苯乙烯基、5-嘧啶乙烯基、5-甲基-呋喃乙烯基、4-硝基-苯乙烯基、2-萘基、3-吲哚基、2-氟-苯乙烯基、4-吡唑基-苯乙烯基、2-氯-苯乙烯基、3-氯-苯乙烯基、4-氟-3-氯-苯乙烯基、4-氟-苯乙烯基、3-三氟甲基-苯乙烯基、1,2-二甲基-3-吲哚乙烯基、5-甲基-3-吲哚乙烯基、6-溴-3-吲哚乙烯基、4-氰基-3-吲哚乙烯基、5-硝基-3-吲哚乙烯基、3-乙基吲哚、4-氮杂-3-吲哚乙烯基、5-氮杂吲哚乙烯基、6-氮杂吲哚乙烯基、7-氮杂吲哚乙烯基、6-甲氧基-3-吲哚乙烯基、7-甲氧基-3-吲哚乙烯基、1-苯基-3-吲哚乙烯基、7-氰基-3-吲哚乙烯基。
上述结构通过以下方案合成：
Figure GDA0003900175030000021
1)e的制备方法
4-氟硝基苯在碱性条件下与Boc保护的哌嗪发生取代反应，反应完全以后萃取、干燥、蒸干得到化合物b，化合物b于醇性溶剂中使用Pd/C还原，抽滤、蒸干后即得到c，c在碱性的有机溶剂中与3-氯苯甲酰氯反应，萃取、干燥、蒸干后得d，将d置于强酸溶剂中，调节pH后即得到化合物e；所述中使用的碱可能为碳酸钾、碳酸氢钠、三乙胺、DIPEA(N,N-二异丙基乙二胺)、氢氧化钠、氢氧化钾，所使用的强酸可能为盐酸、硫酸、三氟乙酸，所使用的溶剂可能为水、二氯甲烷、四氢呋喃、二氧六环、甲醇、乙醇；
2)h的制备方法
b在强酸条件下脱去Boc保护基，碱调节pH即得f，化合物f碱性条件下，有机溶剂中与苯甲酰氯进行作用，萃取、干燥、蒸干后即得到化合物g，醇中使用Pd/C还原g，抽滤、蒸干即得到了化合物h；所述中使用的碱可能为碳酸钾、碳酸氢钠、三乙胺、DIPEA、氢氧化钠、氢氧化钾，所使用的强酸可能为盐酸、硫酸、三氟乙酸，所使用的溶剂可能为水、二氯甲烷、四氢呋喃、二氧六环、甲醇、乙醇；
3)i的制备方法
制备出化合物e或h后，碱性条件下，以T3P(1-丙基环磷酸酐)为缩合剂与相对应羧酸发生缩合反应或与磺酰氯反应，后经萃取、干燥、蒸干、过层析柱得到纯净的i；所述过程中使用的碱可能为碳酸钾、碳酸氢钠、三乙胺、DIPEA，所使用的溶剂为二氯甲烷、乙酸乙酯、DMF(N,N-二甲基甲酰胺)、四氢呋喃、二氧六环。
上述通式I中，X，Y，R1，R2选自以下组合：
Figure GDA0003900175030000031
Figure GDA0003900175030000041
Figure GDA0003900175030000051
合成方案如下：
Figure GDA0003900175030000052
方案二的操作为将化合物f在碱性条件下与对甲基苯甲酰氯反应，经萃取、干燥、蒸干后得j，后在Pd/C条件下还原，抽滤、蒸干后即得k，纯化后得k与Meldrum’s acid进行反应，冷却、抽滤后得到l，l在碱性条件下与各种芳香醛发生Knoevenagel反应，产物经柱层析纯化后即得m；所述过程中使用的碱可能为碳酸钾、碳酸氢钠、三乙胺、DIPEA、哌啶，所使用的溶剂为二氯甲烷、乙酸乙酯、DMF(N,N-二甲基甲酰胺)、四氢呋喃、二氧六环、甲醇、乙醇、甲苯。
本发明的有益效果在于：本发明提供的苯基哌嗪类UBE2F小分子抑制剂靶向UBE2F，可以选择性抑制CRL5 E3泛素连接酶活性，诱导肺癌细胞发生凋亡，同时尽可能地减少化合物的副作用。
附图说明
表1为：随机挑选的本发明包括的不同小分子对人肺鳞癌细胞H2170生长半抑制浓度；
图1为：化合物47小分子抑制剂对人肺鳞癌细胞H2170，人肺腺癌细胞H358和H1650生长半抑制浓度(IC50)；
图2为：人肺鳞癌细胞H2170和人肺腺癌细胞H1650在不同浓度化合物47小分子抑制剂处理24小时后，应用流式细胞仪检测细胞周期分布，绘制细胞周期分布图；
人肺鳞癌细胞H2170和人肺腺癌细胞H1650在不同浓度化合物47小分子抑制剂处理24小时后，Western blot检测凋亡相关蛋白PARP,Caspase3和NOXA蛋白的变化；
图3为：人肺鳞癌细胞H2170和人肺腺癌细胞H1650在20μM化合物47小分子抑制剂处理不同时间，或不同剂量的化合物47小分子抑制剂处理24小时后，Western blot检测Cul5蛋白neddylation修饰水平及底物蛋白NOXA蛋白水平，以及其他cullin蛋白neddylation修饰水平；
图4为：人肺鳞癌细胞H2170在不同浓度化合物47小分子抑制剂处理24小时后，Westernblot检测细胞内UBE2F蛋白质的变化。
具体实施方式
为了对本发明进行更好地说明，特举实施例如下：
实施例1
Figure GDA0003900175030000061
1)b的合成
在250mL圆底烧瓶中依次加入4-氟硝基苯(10g,70.87mmol)，Boc哌嗪(11g,59.06mmol),碳酸钾(12.24g,88.59mmol)，100mL乙腈作为溶剂，加热回流5h；冷却后进行蒸干，分3次加入150mL CH2Cl2转移，水洗三遍，合并有机相后加入无水硫酸镁干燥、蒸干即得到b(14.9g).产率为82.64％，1H NMR(400MHz,CDCl3)δ8.05(d,J＝7.6Hz,2H),7.13–6.69(m,2H),3.64(t,J＝7.1Hz,4H),3.25(t,J＝7.1Hz,4H),1.47(s,9H).
2)g的合成
50mL CH2Cl2中加入上步得到b(13g,42.3mmol)，冰浴条件下20min内缓慢滴加15mLCF3COOH至反应体系，后转至常温下搅拌4h；反应完全后蒸干，加入25mL水溶解固体，使用3mol/L NaOH滴加中和至pH为10出现大量黄色沉淀，抽滤浑浊液即得黄色固体g(7.9g)；产率为88.9％，1H NMR(400MHz,CDCl3)δ8.20–7.88(m,2H),7.14–6.86(m,2H),3.42–3.29(m,4H),3.21–3.06(m,4H),1.92(m,1H).
3)h的合成
冰浴条件下于100mL CH2Cl2中加入g(7g,33.78mmol),三乙胺(4.44g,43.91mmol)，然后逐渐滴加苯甲酰氯(5.46g,38.85mmol)，滴加完成后转至室温条件搅拌2h；反应完全后加水50mL水淬灭，再加入CH2Cl2溶剂50mL，水洗(50mL×3)后有机相加入无水硫酸镁干燥后，蒸干即得h(9.50g)；产率为90.33％
4)i的合成
在50mL乙醇中依次加入h(8g,25.70mmol)，钯碳(1.37g,含量10％)，氢气气氛下常温下搅拌5h，反应完全后过滤除去钯碳，将滤液进行蒸干后得i(6.5g)；产率为89.91％，1HNMR(400MHz,CDCl3)δ7.42(s,5H),6.85–6.77(m,2H),6.67–6.61(m,2H),3.74(d,4H),3.02(d,4H).
5)化合物1的合成
在冰浴条件下5mL CH2Cl2中加入2-呋喃甲酸(47.80mg,0.426mmol)和T3P(169.63mg,0.533mmol),搅拌5min后滴加三乙胺(53.95mg,0.533mmol),加入i(100mg,0.355mmol)，转至常温下继续反应4h；反应完全后，加入15mL水淬灭，后加入30mL的CH2Cl2，水洗(50mL×3)后有机相加入无水硫酸镁干燥，过层析柱纯化(CH2Cl2:MeOH＝20:1,v/v)即得1(78.32mg)；产率为58.46％，1H NMR(400MHz,DMSO-d6)δ10.09(s,1H),7.95(m,2H),7.70–7.61(m,2H),7.60–7.40(m,8H),7.04–6.87(m,2H),3.77(s,4H),3.13(d,J＝9.7Hz,4H).13CNMR(100MHz,DMSO-d6)δ168.98,164.97,147.14,135.79,135.06,131.58,131.27,129.54,128.41,128.28,127.47,126.94,121.45,116.15,48.98.
实施例2化合物2的合成
在冰浴条件下5mL CH2Cl2中加入苯甲酸(52.09mg,0.426mmol)和T3P(169.63mg,0.533mmol),搅拌5min后滴加三乙胺(53.95mg,0.533mmol),加入i(100mg,0.355mmol)，转至常温下继续反应4h；反应完全后，加入15mL水淬灭，后加入30mL的CH2Cl2，水洗(50mL×3)后有机相加入无水硫酸镁干燥，过层析柱纯化(CH2Cl2:MeOH＝20:1,v/v)即得2(72.05mg)；产率为52.55％，1H NMR(400MHz,DMSO-d6)δ10.09(s,1H),7.95(m,2H),7.70–7.61(m,2H),7.60–7.40(m,8H),7.04–6.87(m,2H),3.77(s,4H),3.13(d,J＝9.7Hz,4H).13C NMR(100MHz,DMSO-d6)δ168.98,164.97,147.14,135.79,135.06,131.58,131.27,129.54,128.41,128.28,127.47,126.94,121.45,116.15,48.98.
实施例3化合物3的合成
化合物3的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为52.3％，1H NMR(400MHz,CDCl3)δ8.48(d,J＝8.5Hz,1H),8.35(d,J＝8.6Hz,1H),8.07(dd,J＝7.4,1.3Hz,1H),7.54(t,J＝8.1Hz,1H),7.46–7.33(m,6H),7.17(d,J＝7.5Hz,1H),6.87–6.76(m,2H),6.70–6.57(m,2H),3.69(d,4H),3.04(s,4H),2.87(s,6H).13C NMR(100MHz,CDCl3)δ170.42,152.00,149.00,135.45,134.26,130.56,130.32,129.89,129.73,129.69,128.55,128.47,127.10,124.93,123.12,118.69,116.96,115.14,49.63,45.42.
实施例4化合物4的合成
化合物4的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为62.5％，1H NMR(400MHz,DMSO-d6)δ9.99(s,1H),7.86(d,J＝8.1Hz,2H),7.69–7.59(m,2H),7.52–7.41(m,5H),7.32(d,J＝8.0Hz,2H),7.02–6.90(m,2H),3.77(s,4H),3.14(s,4H),2.38(s,3H).13C NMR(100MHz,DMSO-d6)δ169.01,164.81,147.07,141.24,135.76,132.14,131.63,129.54,128.81,128.42,127.49,126.93,121.46,116.14,48.99,20.95.
实施例5化合物5的合成
化合物5的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为71.6％，1H NMR(400MHz,DMSO-d6)δ9.97(s,1H),7.55(d,J＝8.5Hz,2H),7.46(m,5H),6.94(d,J＝8.4Hz,2H),6.41(m,1H),6.22(m,1H),5.70(m,1H),3.76(s,4H),3.13(s,4H).13C NMR(100MHz,DMSO-d6)δ168.97,162.58,146.98,135.78,131.98,131.49,129.53,128.40,126.92,126.08,120.32,116.33.
实施例6化合物6的合成
化合物6的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为35.2％，1H NMR(400MHz,DMSO-d6)δ9.99(s,1H),8.35(d,J＝2.0Hz,1H),8.08(m,2H),8.00(d,J＝8.1Hz,1H),7.77(m,1H),7.71–7.58(m,2H),7.42(m,5H),7.02–6.92(m,2H),6.84–6.73(m,2H),3.03(s,4H).13C NMR(100MHz,DMSO-d6)δ168.95,147.95,136.66,135.69,134.10,131.48,129.52,129.19,129.09,128.96,128.79,128.38,127.77,127.74,127.58,126.87,122.99,122.17,116.28,53.46,48.42.
实施例7化合物7的合成
化合物7的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为62.5％，1H NMR(400MHz,DMSO-d6)δ9.73(s,1H),7.52–7.39(m,7H),6.96–6.82(m,2H),3.75(s,4H),3.10(s,4H),2.00(s,3H).13C NMR(100MHz,DMSO-d6)δ168.97,167.63,146.65,135.77,131.96,129.53,128.41,126.92,120.07,116.38,49.07,23.76.
实施例8化合物8的合成
化合物8的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为56.3％，1H NMR(400MHz,DMSO-d6)δ10.47(s,1H),8.99–8.56(m,1H),8.19–7.98(m,2H),7.85–7.71(m,2H),7.65(d,1H),7.50–7.43(m,5H),7.02–6.91(m,2H),3.77(s,4H),3.16(s,4H).13CNMR(100MHz,DMSO-d6)δ168.98,161.86,150.05,148.31,147.29,138.02,135.78,130.73,129.53,128.41,126.93,126.64,122.12,121.19,116.15,48.90.
实施例9化合物9的合成
化合物9的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为39.5％，1H NMR(400MHz,CDCl3)δ9.72(s,1H),9.52(s,1H),7.71–7.65(m,4H),7.51–7.43(m,4H),7.47–7.40(m,4H),7.26–7.15(m,4H),6.99–6.94(m,2H),3.63(t,J＝7.1Hz,5H),3.20(t,J＝7.1Hz,5H).13C NMR(100MHz,CDCl3)δ170.10,161.26,145.33,136.01,135.53,134.56,131.72,130.94,128.92,127.59,127.41,124.40,122.81,122.09,114.70,112.28,110.27,49.01,46.37.
实施例10化合物10的合成
化合物10的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为58.3％，1H NMR(400MHz,DMSO-d6)δ9.49(s,1H),7.48–7.40(m,5H),7.12–7.06(m,2H),6.94–6.88(m,2H),3.60(d,4H),2.65(s,6H).13C NMR(100MHz,DMSO-d6)δ168.94,147.51,135.78,130.41,129.53,128.40,126.92,122.00,116.59,48.88,37.76.
实施例11化合物11的合成
化合物11的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为56.8％，1H NMR(400MHz,DMSO-d6)δ8.32(s,1H),7.49–7.40(m,5H),7.34–7.28(m,2H),6.90–6.84(m,2H),3.88–3.44(m,8H),3.41–3.36(m,4H),3.15(s,4H).13C NMR(100MHz,DMSO-d6)δ168.93,155.39,146.09,135.83,132.90,129.51,128.40,126.92,121.06,116.36,65.96,44.09.
实施例12化合物12的合成
化合物12的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为62.5％，1H NMR(400MHz,DMSO-d6)δ10.40(s,1H),8.40–8.30(m,2H),8.22–8.11(m,2H),7.70–7.59(m,2H),7.51–7.39(m,5H),7.03–6.91(m,2H),3.63(d,4H).13C NMR(100MHz,DMSO-d6)δ168.97,163.19,148.97,147.47,140.73,135.79,130.99,129.54,129.01,128.41,126.94,123.47,121.53,116.07,48.83.
实施例13化合物13的合成
化合物13的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为45.6％，1H NMR(400MHz,DMSO-d6)δ9.78(s,1H),7.59–7.52(m,2H),7.43(m,5H),7.31(d,J＝8.0Hz,2H),6.97–6.88(m,2H),6.84–6.77(m,2H),3.77–3.37(m,4H),3.06(s,4H),2.32(s,3H).13C NMR(100MHz,DMSO-d6)δ168.93,147.85,142.83,136.79,135.75,129.52,129.48,129.25,128.39,126.90,126.66,122.71,116.32,48.56,20.91.
实施例14化合物14的合成
化合物14的合成方法参见实施例2中化合物2的合成过程，产品为白色固体。产率为45.8％，1H NMR(400MHz,DMSO-d6)δ10.19(s,1H),8.00(s,1H),7.91(d,J＝7.8Hz,1H),7.64(d,J＝8.2Hz,3H),7.56(t,J＝7.9Hz,1H),7.47(p,J＝6.0,4.6Hz,5H),6.97(d,J＝8.6Hz,2H),3.63(d,J＝111.5Hz,4H),3.25–3.03(m,4H).13C NMR(100MHz,DMSO-d6)δ168.98,163.43,147.30,136.99,135.78,133.14,131.20,131.11,130.30,129.54,128.41,127.23,126.93,126.29,121.51,116.10,48.89.
实施例15
Figure GDA0003900175030000101
1)c的合成
在50mL乙醇中依次加入b(8g,26.03mmol)，钯碳(1.38g,含量10％)，氢气气氛下常温下搅拌5h，反应完全后过滤除去钯碳，将滤液进行蒸干后得c(6.2g)；产率为85.88％。
2)d的合成
冰浴条件下于100mL CH2Cl2中加入c(6g,21.63mmol),三乙胺(3.28g,32.45mmol)，然后逐渐滴加3-氯-苯甲酰氯(4.54g,25.96mmol)，滴加完成后转至室温条件搅拌2h；反应完全后加水50mL水淬灭，再加入CH2Cl2溶剂50mL，水洗(50mL×3)后有机相加入无水硫酸镁干燥后，蒸干即d(7.60g)；产率为84.47％，1H NMR(400MHz,DMSO-d6)δ10.17(s,1H),7.99(t,J＝1.9Hz,1H),7.90(m,1H),7.73–7.49(m,4H),7.05–6.89(m,2H),3.46(d,J＝10.3Hz,4H),3.06(t,J＝5.2Hz,4H),1.43(s,9H).13C NMR(100MHz,DMSO-d6)δ163.39,153.81,147.48,137.01,133.13,131.10,130.30,127.23,126.30,121.47,116.11,78.93,48.74,28.04.
3)e的合成
50mL CH2Cl2中加入上步得到d(7g,16.83mmol)，冰浴条件下20min内缓慢滴加6.5mL CF3COOH至反应体系，后转至常温下搅拌4h；反应完全后蒸干，加入25mL水溶解固体，使用3mol/L NaOH滴加中和至pH为10出现大量黄色沉淀，抽滤浑浊液即得黄色固体g(4.9g)；产率为92.19％。
4)化合物15的合成
冰浴条件下于5mL CH2Cl2中加入e(100mg,0.316mmol),三乙胺(48.06mg,0.475mmol)，然后逐渐滴加乙酰氯(29.83mg,0.380mmol)，滴加完成后转至室温条件搅拌2h；反应完全后加水10mL水淬灭，再加入CH2Cl2溶剂30mL，水洗(15mL×3)后有机相加入无水硫酸镁干燥后，蒸干即15(89.2mg)；产率为78.54％，1H NMR(400MHz,DMSO-d6)δ10.18(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.70–7.49(m,4H),7.04–6.87(m,2H),3.58(m,4H),3.09(m,4H),2.04(s,3H).13C NMR(100MHz,DMSO-d6)δ168.21,163.41,147.39,137.00,133.14,131.11,130.30,127.23,126.29,121.52,116.01,49.07,48.64,45.45,21.15.
实施例16化合物16的合成
化合物16的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为68.3％，1H NMR(400MHz,DMSO-d6)δ10.18(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.67–7.61(m,3H),7.56(t,J＝7.9Hz,1H),7.04–6.94(m,2H),6.86(m,1H),6.15(m,1H),5.78–5.67(m,1H),3.70(m,4H),3.12(s,4H).13C NMR(100MHz,DMSO-d6)δ164.21,163.42,147.30,137.00,133.14,131.16,131.11,130.31,128.10,127.44,127.23,126.29,121.51,116.03,49.32,48.64.
实施例17化合物17的合成
化合物17的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为56.8％，1H NMR(400MHz,DMSO-d6)δ10.16(s,1H),8.49(s,1H),8.22(m,2H),8.09(d,J＝7.9Hz,1H),7.97(s,1H),7.88(d,J＝7.8Hz,1H),7.82–7.69(m,3H),7.59(m,4H),6.89(d,J＝8.7Hz,2H),3.23–3.03(m,8H).13C NMR(100MHz,DMSO-d6)δ163.43,146.85,136.93,134.46,133.12,131.99,131.77,131.39,131.12,130.29,129.39,129.33,129.09,128.86,127.86,127.71,127.21,126.27,122.85,121.44,116.28,48.30,45.85.
实施例18化合物18的合成
化合物18的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为46.9％，1H NMR(400MHz,DMSO-d6)δ10.27(s,1H),7.98(d,J＝1.9Hz,1H),7.90(d,1H),7.65(d,5H),7.54(t,J＝7.8Hz,1H),7.48(d,J＝8.0Hz,2H),7.08(d,2H),3.26(t,J＝4.8Hz,4H),3.06(t,J＝4.8Hz,4H),2.41(s,3H).13C NMR(100MHz,DMSO-d6)δ163.54,143.87,136.84,133.13,131.61,131.21,130.32,129.90,128.04,127.64,127.27,126.35,125.46,121.42,117.16,49.06,45.37,21.02.
实施例19化合物19的合成
化合物19的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为68.5％，1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.00(t,J＝1.9Hz,1H),7.92(m,1H),7.64(m,3H),7.55(t,J＝7.9Hz,1H),7.01–6.91(m,2H),3.58(m,4H),3.31(m,4H),3.18(t,J＝4.6Hz,4H),3.10(m,4H).13C NMR(100MHz,DMSO-d6)δ163.39,162.92,147.53,137.01,133.13,131.09,130.30,127.24,126.31,121.50,115.87,65.89,53.32,48.59,46.87,46.17,17.96,16.67,12.20.
实施例20化合物20的合成
在冰浴条件下5mL CH2Cl2中加入2-吲哚甲酸(61.24mg,0.380mmol)和T3P(151.13mg,0.475mmol),搅拌5min后滴加三乙胺(48.06mg,0.474mmol),加入e(100mg,0.316mmol)，转至常温下继续反应4h；反应完全后，加入15mL水淬灭，后加入30mL的CH2Cl2，水洗(50mL×3)后有机相加入无水硫酸镁干燥，过层析柱纯化(CH2Cl2:MeOH＝20:1,v/v)即得1(56.23mg)；产率为41.66％，1H NMR(400MHz,DMSO-d6)δ11.68–11.57(m,1H),10.19(s,1H),8.00(t,J＝1.9Hz,1H),7.91(d,J＝7.7Hz,1H),7.73–7.60(m,4H),7.56(t,J＝7.9Hz,1H),7.44(d,J＝8.2Hz,1H),7.20(t,J＝7.6Hz,1H),7.09–6.96(m,3H),6.85(d,J＝2.1Hz,1H),3.22(t,J＝5.2Hz,4H).13C NMR(100MHz,DMSO-d6)δ163.41,162.01,147.31,137.02,135.91,133.14,131.17,131.12,130.31,129.77,127.24,126.77,126.31,123.21,121.53,121.31,119.73,115.99,112.03,104.07,48.98.
实施例21化合物21的合成
化合物21的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为45.6％，1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.00(d,J＝2.0Hz,1H),7.91(d,J＝7.7Hz,1H),7.67–7.62(m,3H),7.55(t,J＝7.9Hz,1H),7.00–6.94(m,2H),3.30(dd,J＝6.5,3.6Hz,4H),3.16(m,4H),2.81(s,6H).13C NMR(100MHz,DMSO-d6)δ163.42,147.13,136.98,133.13,131.36,131.12,130.30,127.24,126.31,121.49,116.20,53.38,48.67,45.95,17.98,16.68.
实施例22化合物22的合成
化合物22的合成方法参见实施例20中化合物20的合成过程，产品为白色固体。产率为72.3％，1H NMR(400MHz,DMSO-d6)δ10.19(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.87(d,J＝2.1Hz,1H),7.69–7.62(m,3H),7.56(t,J＝7.8Hz,1H),7.05(d,J＝3.3Hz,1H),7.02–6.95(m,2H),6.65(m,1H),3.19(m,4H).13C NMR(100MHz,DMSO-d6)δ163.41,158.25,147.26,146.96,144.73,137.01,133.14,131.20,131.11,130.30,127.23,126.30,121.51,116.00,115.69,111.30,48.96.
实施例23化合物23的合成
化合物23的合成方法参见实施例20中化合物20的合成过程，产品为白色固体。产率为53.2％，1H NMR(400MHz,DMSO-d6)δ10.18(s,1H),8.35–8.25(m,2H),7.99(t,J＝1.9Hz,1H),7.90(m,1H),7.76–7.69(m,2H),7.66–7.58(m,3H),7.56(t,J＝7.9Hz,1H),7.01–6.92(m,2H),3.62(d,4H),3.11(s,4H).13C NMR(100MHz,DMSO-d6)δ167.05,163.41,147.80,147.20,142.10,137.00,133.13,131.27,131.12,130.31,128.32,127.23,126.30,123.76,121.49,116.14,48.59,46.72.
实施例24化合物24的合成
化合物24的合成方法参见实施例15中化合物15的合成过程，产品为白色固体。产率为67.3％，1H NMR(400MHz,DMSO-d6)δ10.04(s,1H),7.60(t,J＝9.4,2.1Hz,4H),7.53(s,1H),7.49–7.40(m,8H),7.00–6.91(m,2H),6.81(d,1H),3.76(s,4H),3.13(s,4H).13C NMR(100MHz,DMSO-d6)δ168.93,147.96,136.68,135.71,134.11,131.49,129.51,129.19,
实施例25化合物25的合成
Figure GDA0003900175030000131
化合物j，k的合成与实施例1中h，i的方法相同，得到化合物k白色固体；在冰浴条件下5mL CH2Cl2中加入k(100mg,0.338mmol)，肉桂酰氯(67.68mg,0.406mmol)，三乙胺(51.39mg，0.507mmol)，冰浴下反应15min后转至常温下继续反应2h；反应完全后，加入20mL水淬灭，加入30mL CH2Cl2后进行水洗(20mL×3)，有机相用无水硫酸镁干燥后，过层析柱(CH2Cl2:MeOH＝20:1,v/v)即的黄色固体25(73.31mg)；产率为50.67％，1H NMR(400MHz,DMSO-d6)δ10.07(s,1H),7.55(m,5H),7.48–7.38(m,3H),7.34(d,J＝7.9Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(d,J＝8.8Hz,2H),6.81(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,163.42,147.43,139.94,139.73,135.32,133.35,132.32,130.08,129.46,129.37,128.08,127.59,122.97,120.71,116.89,55.38,49.52,21.39.
实施例26
Figure GDA0003900175030000141
1)l的合成
100mL含有k(5g,16.93mmol)的甲苯溶剂中加入Meldrum’s acid(2.93g,20.31mmol)，加热90热条件下搅拌5h，冷却后析出固体，抽滤反应体系，使用***(20mL×3)洗涤三次，烘干后即得到l(4.93g)；产率为75.89％。
2)化合物26的合成
将l(100mg,0.262mmol)，3-吡啶苯甲醛(33.70mg,0.314mmol)，哌嗪(2.23mg,0.0262mmol)加入到5mL无水甲苯溶剂中，后加入100mg无水硫酸镁，加热90℃搅拌10h，减压蒸干后过层析柱(CH2Cl2:MeOH＝40:1,v/v)得到黄色固体26(45.2mg)；产率为40.24％，1HNMR(400MHz,DMSO-d6)δ10.13(s,1H),8.81(d,J＝2.2Hz,1H),8.57(m,1H),8.02(m,1H),7.66–7.52(m,3H),7.48(m,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,162.98,150.71,149.64,147.54,139.73,136.59,134.49,133.35,132.12,131.12,129.37,127.59,124.93,124.50,120.77,116.87,49.47,21.39.
实施例27化合物27的合成
化合物27的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为38.6％，1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.64(d,J＝4.8Hz,1H),7.86(t,J＝7.7Hz,1H),7.59(m,4H),7.32(m,6H),6.96(d,J＝8.5Hz,2H),3.73(s,4H),3.12(s,4H).13CNMR(100MHz,DMSO-d6)δ169.59,163.17,153.41,150.34,147.53,139.73,139.17,137.70,133.35,132.23,129.37,127.59,126.64,124.93,124.59,120.71,116.84,55.38,49.48,21.39.
实施例28化合物28的合成
化合物28的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为45.3％，1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.64(d,J＝5.2Hz,2H),7.69–7.43(m,5H),7.34(d,J＝7.8Hz,2H),7.27(d,J＝7.7Hz,2H),7.06–6.91(m,3H),3.66(m,4H),3.13(s,4H),2.36(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,162.65,150.87,147.65,142.52,139.73,137.36,133.35,131.96,129.37,127.59,127.44,122.15,120.82,116.84,49.42,21.39.
实施例29化合物29的合成
化合物29的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为42.4％，1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),7.55(dd,J＝32.2,8.2Hz,5H),7.40–7.16(m,6H),6.95(d,J＝8.6Hz,2H),6.75(d,J＝15.6Hz,1H),3.62(m,4H),3.12(s,4H),2.34(s,6H).13C NMR(100MHz,DMSO-d6)δ169.59,163.59,147.39,139.90,139.86,139.72,133.35,132.58,132.38,130.06,129.37,128.06,127.59,121.92,120.68,116.89,49.52,21.43,21.39.
实施例30化合物30的合成
化合物30的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为65.3％，1H NMR(400MHz,DMSO-d6)δ10.20(s,1H),9.17(s,1H),9.06(s,2H),7.68–7.48(m,3H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.7Hz,2H),7.05–6.85(m,3H),3.73(s,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,162.58,158.81,155.98,147.65,139.73,133.35,133.28,131.96,129.37,127.59,126.65,120.85,116.85,49.44,21.39.
实施例31化合物31的合成
化合物31的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为52.1％，1H NMR(400MHz,DMSO-d6)δ9.98(s,1H),7.56(d,J＝8.8Hz,2H),7.33(d,J＝7.8Hz,2H),7.31–7.20(m,3H),6.93(d,J＝8.6Hz,2H),6.70(d,J＝3.2Hz,1H),6.52(d,J＝15.4Hz,1H),6.24(d,J＝3.2Hz,1H),3.73(s,4H),3.11(s,4H),2.34(d,J＝4.9Hz,6H).13CNMR(100MHz,DMSO-d6)δ169.59,163.50,154.70,150.17,147.32,139.72,133.35,132.48,129.37,127.59,127.07,120.49,118.68,116.88,116.12,109.41,49.54,21.38,13.99.
实施例32化合物32的合成
化合物32的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为35.6％，1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.32–8.25(m,2H),7.93–7.84(m,2H),7.70–7.55(m,3H),7.52–7.21(m,6H),7.06–6.92(m,3H),3.77(s,4H),3.22–3.04(m,4H).13CNMR(100MHz,DMSO-d6)δ169.47,162.69,148.02,141.95,137.48,136.30,132.07,131.10,130.04,129.11,128.91,127.44,124.74,124.63,120.79,116.87,49.44.
实施例33化合物33的合成
化合物33的合成方法参见实施例26中化合物25的合成过程，产品为白色固体。产率为65.33％，1H NMR(400MHz,DMSO-d6)δ9.73(s,1H),7.47–7.39(m,2H),7.33(d,J＝7.9Hz,2H),7.26(d,J＝7.9Hz,2H),6.94–6.85(m,2H),3.71(s,4H),3.08(s,4H),2.35(s,3H),1.99(s,3H).13C NMR(100MHz,DMSO-d6)δ169.58,168.07,147.17,139.71,133.36,132.49,129.36,127.58,120.55,116.88,49.64,24.28,21.39.
实施例34化合物34的合成
化合物34的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为56.2％，1H NMR(400MHz,DMSO-d6)δ10.10(s,1H),8.13(s,1H),8.04–7.89(m,3H),7.82–7.67(m,2H),7.66–7.52(m,4H),7.39–7.22(m,4H),7.03–6.86(m,3H),3.74(s,4H),3.13(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.60,163.46,147.46,139.90,139.73,133.88,133.51,133.36,132.91,132.34,129.37,129.33,129.09,128.79,128.15,127.59,127.45,127.23,124.01,123.40,120.71,116.90,55.37,49.51,21.39.
实施例35化合物35的合成
化合物35的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为41.5％，1H NMR(400MHz,DMSO-d6)δ11.63(s,1H),9.83(s,1H),7.96(d,J＝7.1Hz,1H),7.81(d,J＝2.8Hz,1H),7.72(d,J＝15.6Hz,1H),7.59(d,J＝8.5Hz,2H),7.51–7.44(m,1H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.8Hz,2H),7.24–7.18(m,2H),6.95(d,J＝8.7Hz,2H),6.78(d,J＝15.7Hz,1H),3.65(m,4H),3.13(m,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,164.94,147.05,139.72,137.91,134.49,133.37,133.00,131.26,129.37,127.59,125.32,122.76,120.87,120.31,117.04,116.77,112.70,49.07,21.39.
实施例36化合物36的合成
化合物36的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为35.6％，1H NMR(400MHz,DMSO-d6)δ10.09(s,1H),7.88–7.67(m,1H),7.65–7.51(m,3H),7.46(m,1H),7.30(m,5H),7.09(d,J＝8.6Hz,1H),6.74(d,J＝15.8Hz,3H),3.62(d,4H),3.12(s,4H),2.35(s,3H),1.74–1.22(m,2H).13C NMR(100MHz,DMSO-d6)δ170.05,165.28,162.67,160.15,145.48,141.33,135.18,133.91,133.19,133.11,130.65,130.57,129.72,129.64,129.39,129.11,125.53,125.50,122.80,122.51,122.31,121.86,121.83,116.17,115.97,114.60,48.93,46.30,21.35.
实施例37化合物37的合成
化合物37的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为36.3％，1H NMR(400MHz,DMSO-d6)δ10.06(s,1H),8.59(d,J＝2.6Hz,1H),7.94(d,J＝8.2Hz,2H),7.79(s,1H),7.74(d,J＝8.3Hz,2H),7.63–7.54(m,3H),7.34(d,J＝7.8Hz,2H),7.28(s,2H),6.96(d,J＝8.5Hz,2H),6.82(d,J＝15.7Hz,1H),6.59(t,J＝2.2Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,163.41,147.44,141.86,140.71,139.73,138.95,133.35,133.09,132.32,129.37,128.31,127.59,122.80,120.69,119.04,116.89,108.71,55.38,49.53,21.39.
实施例38化合物38的合成
化合物38的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为41.3％，1H NMR(400MHz,DMSO-d6)δ10.17(s,1H),7.91–7.73(m,2H),7.68–7.52(m,3H),7.49–7.23(m,6H),6.91(m,3H),3.62(m,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.58,162.87,147.57,139.73,135.13,133.82,133.35,133.13,132.06,131.51,130.52,129.37,128.31,128.08,127.59,126.07,120.78,116.84,55.38,49.43,21.39.
实施例39化合物39的合成
化合物39的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为45.6％，1H NMR(400MHz,DMSO-d6)δ10.08(s,1H),7.69(s,1H),7.63–7.40(m,6H),7.39–7.22(m,4H),6.96(d,J＝8.5Hz,2H),6.86(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.59,163.05,147.52,139.73,138.28,137.65,134.17,133.34,132.15,131.29,129.66,129.37,127.76,127.59,126.52,124.72,120.72,116.87,99.99,49.47,21.39.
实施例40化合物40的合成
化合物40的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为39.6％，1H NMR(400MHz,DMSO-d6)δ10.06(s,1H),7.93–7.82(m,1H),7.76–7.62(m,3H),7.61–7.46(m,4H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.7Hz,2H),6.95(d,J＝8.5Hz,2H),6.80(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H).13C NMR(100MHz,DMSO-d6)δ169.99,164.16,159.08,156.56,145.48,141.33,141.19,135.18,133.89,131.79,131.76,129.48,129.40,129.37,129.11,129.04,123.34,123.14,122.80,119.94,117.27,117.07,114.65,48.73,45.23,21.35.
实施例41化合物41的合成
化合物41的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为56.2％，1H NMR(400MHz,DMSO-d6)δ10.05(s,1H),7.68(m,2H),7.62–7.51(m,3H),7.38–7.24(m,6H),6.95(d,J＝8.7Hz,2H),6.74(d,J＝15.7Hz,1H),3.73(s,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.58,163.35,147.45,139.73,138.73,133.35,132.28,131.98,130.29,130.20,129.37,127.59,122.87,120.71,116.88,116.55,116.33,55.38,49.51,21.39.
实施例42化合物42的合成
化合物42的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为49.3％，1H NMR(400MHz,DMSO-d6)δ10.11(s,1H),7.97(s,1H),7.93(d,J＝7.8Hz,1H),7.76(d,J＝7.8Hz,1H),7.70(d,J＝7.8Hz,1H),7.64(d,J＝15.8Hz,1H),7.59(d,J＝8.9Hz,2H),7.34(d,J＝8.0Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(s,2H),6.91(s,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.58,162.97,147.55,139.73,138.12,136.54,133.35,132.14,131.89,130.61,130.39,129.37,127.59,125.86,125.18,124.32,120.69,116.88,55.38,21.39.
实施例43化合物43的合成
化合物43的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为45.6％，1H NMR(400MHz,DMSO-d6)δ10.06(s,1H),7.82(s,1H),7.67–7.49(m,5H),7.41(t,J＝7.8Hz,1H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(d,J＝8.5Hz,2H),6.84(d,J＝15.7Hz,1H),3.73(s,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.58,163.03,147.52,139.73,138.19,137.92,133.35,132.55,132.14,131.56,130.60,129.37,127.59,126.92,124.72,122.75,120.70,116.87,55.38,49.47,21.39.
实施例44化合物44的合成
Figure GDA0003900175030000181
1)44a的合成
冰浴条件下取三氯氧磷(633.54mg,4.13mmol)缓慢滴加入DMF(302.04mg,4.13mmol)中，冰浴下搅拌15min，将溶于5mL DMF的1,2-二甲基吲哚(500mg,3.44mmol)缓慢滴入，而后转至常温下搅拌3h；反应完全后加入5mL水后震摇后白色固体出现，抽滤后干燥固体即为化合物44a(378.2mg),产率63.37％。
2)化合物44的合成
化合物44的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为53.2％，1H NMR(400MHz,DMSO-d6)δ9.75(s,1H),7.85(m,1H),7.68(d,J＝15.5Hz,1H),7.52(d,J＝8.9Hz,2H),7.47–7.41(m,1H),7.27(d,J＝8.1Hz,2H),7.20(d,J＝7.9Hz,2H),7.18–7.15(m,1H),7.15–7.08(m,1H),6.93–6.85(m,2H),6.70(d,J＝15.6Hz,1H),3.66(s,3H),3.49(m,4H),2.47(s,3H),2.28(s,3H).13C NMR(100MHz,DMSO-d6)δ169.07,164.61,146.49,141.54,139.22,137.23,132.85,132.56,128.87,127.08,125.03,121.59,120.58,119.73,119.35,116.54,115.45,110.03,108.04,29.75,20.89,10.24.
实施例45化合物45的合成
Figure GDA0003900175030000191
45a的合成方法参见实施例44中44a的合成过程，化合物45的合成方法参见实施例26中化合物26的合成过程，产品为黄色固体。产率为37.6％，1H NMR(400MHz,DMSO-d6)δ11.43(d,J＝2.7Hz,1H),9.75(s,1H),7.68(d,J＝3.5Hz,2H),7.63(s,1H),7.53(d,J＝8.9Hz,2H),7.31–7.27(m,2H),7.26(d,J＝2.0Hz,1H),6.97(m,1H),6.90–6.85(m,2H),6.66(d,J＝15.6Hz,1H),3.55(m,4H),3.04(s,4H),2.41(s,3H),2.28(s,3H).13C NMR(100MHz,DMSO-d6)δ169.08,164.53,146.52,139.21,135.77,134.30,132.85,132.53,130.97,129.12,128.87,127.08,125.08,123.76,119.86,119.73,116.52,115.74,111.94,111.76,59.71,49.18,21.43,20.88.
实施例46化合物46的合成
化合物46的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为53.6％，1H NMR(400MHz,DMSO-d6)δ11.78–11.71(m,1H),9.86(s,1H),7.90(d,J＝8.6Hz,1H),7.85(d,J＝2.2Hz,1H),7.71(d,J＝15.8Hz,1H),7.67(d,J＝1.8Hz,1H),7.62–7.57(m,2H),7.37–7.34(m,2H),7.33(d,J＝2.0Hz,1H),7.27(d,J＝7.9Hz,2H),6.98–6.93(m,2H),6.78(d,J＝15.7Hz,1H),3.73(s,4H),3.11(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.08,164.19,146.61,139.21,138.28,133.29,132.84,132.36,131.48,128.86,127.08,123.83,123.14,121.52,119.84,117.12,116.52,114.94,114.89,112.31,49.14,20.89.
实施例47化合物47的合成
化合物47的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为53.1％，1H NMR(400MHz,DMSO-d6)δ12.15(d,J＝2.7Hz,1H),9.84(s,1H),8.36–8.30(m,2H),7.96(d,J＝2.8Hz,1H),7.70(d,J＝15.7Hz,1H),7.59(d,J＝8.8Hz,2H),7.34(d,J＝7.9Hz,2H),7.29–7.24(m,3H),6.99–6.93(m,2H),6.82(d,J＝15.8Hz,1H),3.62(m,4H),3.14(m,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.08,164.10,149.55,146.65,143.60,139.22,133.44,132.84,132.29,130.84,128.86,128.09,127.08,119.86,117.51,117.10,116.58,116.53,110.99,49.13,20.88.
实施例48化合物48的合成
化合物48的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为45.1％，1H NMR(400MHz,DMSO-d6)δ12.26–12.06(m,1H),9.85(s,1H),8.42(s,1H),8.03(d,J＝2.6Hz,1H),7.73(d,J＝15.8Hz,1H),7.68–7.63(m,1H),7.59(m,3H),7.38–7.32(m,2H),7.27(d,J＝7.9Hz,2H),7.00–6.93(m,2H),6.87(d,J＝15.8Hz,1H),3.63(m,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.08,163.96,146.70,139.22,132.91,132.84,132.61,132.18,128.87,127.08,125.16,125.05,124.51,120.52,119.89,118.13,116.52,113.66,112.82,102.54,54.87,49.12.
实施例49化合物49的合成
化合物49的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为48.6％，1H NMR(400MHz,DMSO-d6)δ12.29(s,1H),10.06(s,1H),8.87(d,J＝2.2Hz,1H),8.13(m,1H),8.10(s,1H),7.78(d,J＝15.8Hz,1H),7.67(d,J＝9.0Hz,1H),7.64–7.54(m,2H),7.34(d,J＝8.1Hz,2H),7.27(d,J＝7.8Hz,2H),7.00–6.93(m,2H),6.87(d,J＝15.8Hz,1H),3.63m,4H),3.12(s,4H),2.36(s,3H).13C NMR(100MHz,DMSO-d6)δ169.09,163.87,146.72,141.68,140.58,139.23,134.07,132.83,132.56,132.18,128.87,127.08,123.98,120.05,118.13,117.71,116.55,116.47,114.22,112.87,49.09,20.88.
实施例50化合物50的合成
化合物50的合成方法参见实施例26中化合物26的合成过程，产品为白色固体。产率为75.3％，1H NMR(400MHz,DMSO-d6)δ10.77(s,1H),9.76(s,1H),7.57(d,J＝7.9Hz,1H),7.52–7.44(m,2H),7.37–7.30(m,3H),7.27(d,J＝7.9Hz,2H),7.12(d,J＝2.3Hz,1H),7.10–7.04(m,1H),7.03–6.87(m,3H),3.11(s,4H),3.01(t,J＝7.6Hz,2H),2.64(m,2H),2.35(s,3H).13CNMR(100MHz,DMSO-d6)δ169.10,164.11,149.53,146.65,143.60,139.23,133.46,132.81,132.27,130.84,128.87,128.11,127.08,119.87,117.48,117.10,116.59,116.52,110.98,49.13,20.88.
实施例51化合物51的合成
化合物51的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为56.3％，1H NMR(400MHz,DMSO-d6)δ11.74(s,1H),10.00(s,1H),8.48(m,1H),8.03(s,1H),7.85(m,1H),7.73(d,J＝15.3Hz,1H),7.69–7.62(m,2H),7.47(d,J＝15.3Hz,1H),7.34(d,J＝8.1Hz,2H),7.30–7.21(m,3H),6.95–6.90(m,2H),3.62(m,4H),3.10(s,4H),2.35(s,3H).13CNMR(100MHz,DMSO-d6)δ170.05,165.13,145.48,142.87,141.39,139.81,135.07,133.86,133.01,129.84,129.37,129.15,129.11,122.90,120.84,118.97,117.81,114.71,110.98,48.85,45.46,21.36.
实施例52化合物52的合成
化合物52的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为41.2％，1H NMR(400MHz,DMSO-d6)δ11.79(s,1H),10.05(s,1H),8.52(m,1H),8.07(d,J＝2.9Hz,1H),7.94–7.87(m,2H),7.79(d,J＝15.3Hz,1H),7.69(d,J＝8.7Hz,2H),7.51(d,J＝15.3Hz,1H),7.38(d,J＝7.7Hz,2H),7.34–7.25(m,4H),6.97(d,J＝8.8Hz,2H),3.77(s,4H),3.21–3.03(m,4H),2.39(s,3H).13C NMR(100MHz,DMSO-d6)δ170.05,165.08,145.48,144.46,143.61,141.39,139.92,135.07,133.86,132.63,129.37,129.11,128.50,123.95,122.90,120.42,114.71,110.15,106.19,48.85,45.46,21.36.
实施例53化合物53的合成
化合物53的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为47.1％，1H NMR(400MHz,DMSO-d6)δ12.10(s,1H),9.89(s,1H),8.83(s,1H),8.30(d,J＝5.5Hz,1H),8.04(s,1H),7.90(d,J＝5.5Hz,1H),7.74(d,J＝15.7Hz,1H),7.62–7.56(m,2H),7.34(d,J＝8.1Hz,2H),7.27(d,J＝7.9Hz,2H),6.98–6.93(m,2H),6.84(d,J＝15.7Hz,1H),3.72(s,4H),3.12(s,4H),2.35(s,3H),1.99(s,1H).13C NMR(100MHz,DMSO-d6)δ169.09,164.02,146.66,139.23,139.13,135.13,134.28,133.64,132.90,132.83,132.26,129.16,128.87,127.08,119.87,118.04,116.51,114.52,111.70,59.72,49.11,43.68,22.16,20.89,20.73,14.05.
实施例54化合物54的合成
化合物54的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为51.3％，1H NMR(400MHz,DMSO-d6)δ11.54(s,1H),9.85(s,1H),7.87(d,J＝8.7Hz,1H),7.72(m,2H),7.63(d,J＝8.6Hz,2H),7.38(d,J＝7.7Hz,2H),7.31(d,J＝7.8Hz,2H),7.06–6.96(m,3H),6.89(m,1H),6.78(d,J＝15.7Hz,1H),3.85(s,3H),3.67(m,4H),3.15(s,4H),2.40(s,3H).13CNMR(100MHz,DMSO-d6)δ169.60,164.99,156.57,147.04,139.72,138.88,134.53,133.37,133.01,130.28,129.37,127.59,121.07,120.30,119.46,117.04,116.47,112.83,110.76,95.87,55.72,49.68,21.39.
实施例55化合物55的合成
化合物55的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为62.4％，1H NMR(400MHz,DMSO-d6)δ11.74(d,J＝2.8Hz,1H),9.82(s,1H),7.77–7.68(m,2H),7.62–7.56(m,2H),7.54(d,J＝8.1Hz,1H),7.34(d,J＝8.0Hz,2H),7.27(d,J＝7.9Hz,2H),7.13(t,J＝7.9Hz,1H),6.97–6.91(m,2H),6.80(d,J＝7.8Hz,1H),6.75(d,J＝15.6Hz,1H),3.94(s,3H),3.62(d,J＝80.2Hz,4H),3.11(s,4H),2.35(s,3H).13C NMR(101MHz,CDCl3)δ174.35,169.69,151.80,151.66,144.45,139.36,138.13,137.75,135.33,134.11,132.62,132.33,131.58,126.35,125.09,121.77,121.59,118.03,108.24,60.50,54.43,26.13.
实施例56化合物56的合成
化合物56的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为31.2％，1H NMR(400MHz,DMSO-d6)δ9.95(s,1H),8.13(s,1H),8.11–8.05(m,1H),7.79(d,J＝15.8Hz,1H),7.70–7.53(m,8H),7.50–7.45(m,1H),7.36–7.32(m,4H),7.27(d,J＝7.9Hz,2H),7.01–6.89(m,3H),3.72(s,4H),3.12(s,4H),2.35(s,3H).13C NMR(100MHz,DMSO-d6)δ169.15,164.12,146.68,139.27,138.07,136.64,132.90,132.84,132.77,132.23,129.92,128.88,127.25,127.08,126.05,124.22,123.51,121.68,120.54,119.93,118.15,116.52,113.72,111.16,59.75,49.11,28.96,20.88.
实施例57化合物57的合成
化合物57的合成方法参见实施例45中化合物45的合成过程，产品为黄色固体。产率为49.2％，1H NMR(400MHz,DMSO-d6)δ9.94(s,1H),8.29(d,J＝8.1Hz,1H),7.97(s,1H),7.77(d,J＝15.8Hz,1H),7.72(d,J＝7.4Hz,1H),7.60(d,J＝8.7Hz,2H),7.34(m,4H),7.25(d,J＝7.8Hz,2H),6.97–6.92(m,2H),6.88(d,J＝15.9Hz,1H),3.79–3.62(m,4H),3.10(s,4H),2.33(s,3H).13C NMR(100MHz,DMSO-d6)δ169.16,164.02,146.71,139.26,136.87,132.74,132.57,132.15,131.80,128.86,127.37,127.07,125.88,125.37,120.53,119.99,118.32,116.95,116.50,113.26,94.73,49.09,20.86.
实施例58化合物58的合成
化合物58的合成方法参见实施例45中化合物45的合成过程，产品为白色固体。产率为85.1％，1H NMR(400MHz,CDCl3)δ7.37–7.32(m,2H),7.30–7.26(m,2H),7.22(d,J＝8.1Hz,2H),6.98–6.85(m,3H),3.77(m,4H),3.19(s,4H),2.39(s,3H).13C NMR(100MHz,CDCl3)δ170.59,151.00,140.00,132.68,129.23,129.11,127.25,120.58,116.71,49.79,21.38.
上述化合物1-58的结构式如下表所示：
Figure GDA0003900175030000231
Figure GDA0003900175030000241
Figure GDA0003900175030000251
Figure GDA0003900175030000261
Figure GDA0003900175030000271
Figure GDA0003900175030000281
Figure GDA0003900175030000291
生物学试验例1：肿瘤细胞增殖抑制实验
检测本发明的小分子抑制剂抑制人肿瘤细胞增殖的活性，采用CCK8法(美国MCE)或ATPlite法(美国Perkin-Elmer)。
细胞株采用人肺鳞癌细胞株H2170和人肺腺癌细胞株H358和H1650。培养基为RPMI1640(美国Gibco)+10％FBS(胎牛血清，美国Gibco)+双抗。
试剂配制：母液浓度为20mM，溶剂为DMSO(美国Sigma-Aldrich)，储存于-20℃，使用时室温化开，应用细胞培养基稀释至所需浓度。
CCK8检测方法：96孔板每孔加入浓度为5×104个/mL的细胞悬液100μL，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基100μL，使终浓度分别为0，0.0046，0.0137，0.0412，0.1235，0.3704，1.1111，3.3333，10和30μM。设三个复孔，继续培养72小时后弃去培养基溶液，每孔加入100μL含10％CCK8溶液的培养基，继续培养1小时后，使用酶标仪SpectraMax iD3检测450nm OD值。计算细胞半数抑制浓度IC50值。
ATPlite检测方法：96孔板每孔加入浓度为5×104个/mL的细胞悬液100μL，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基100μL，使终浓度分别为0，0.0046，0.0137，0.0412，0.1235，0.3704，1.1111，3.3333，10和30μM。设三个复孔，继续培养72小时后弃去培养基溶液，每孔加入50μL ATPlite试剂，室温700转/分钟振荡2分钟后，转移至不透明白板。室温暗处理10分钟后，使用酶标仪SpectraMax iD3检测化学发光值。计算细胞半数抑制浓度IC50值。
结果见表1及图1。
表1：本发明小分子抑制剂对人肺鳞癌细胞H2170 IC50值
样品名称 IC<sub>50</sub>值(μM) 样品名称 IC<sub>50</sub>值(μM)
化合物13 69.8 化合物35 9.9
化合物20 91.1 化合物36 109.8
化合物24 8.8 化合物41 9.0
化合物31 12.9 化合物47 1.8
化合物32 9.4 化合物48 11.6
表1为随机挑选本发明包括的不同小分子，检测其对人肺鳞癌细胞H2170生长的抑制情况。将肺癌细胞H2170以5000个/孔种于96孔板，以浓度梯度(每种浓度3个复孔)的小分子抑制剂处理72小时后，应用CCK8试剂盒检测细胞生长，计算肿瘤生长半抑制浓度(IC50)
图1为化合物47小分子抑制剂抑制人肺癌细胞生长。将肺癌细胞H2170、H358和H1650以5000个/孔种于96孔板，以浓度梯度(每种浓度3个复孔)的化合物47小分子抑制剂处理72小时后，应用ATPlite试剂盒检测细胞生长，计算肿瘤生长半抑制浓度(IC50)
以上实验结果显示，随机挑选本发明包含的化合物，有良好的抑制肺癌细胞生长的活性，尤其化合物47，IC50值在不同肺癌细胞系均小于10μM，通过分析上述化合物的结构式，可以确定，具有通式I的结构，或具有通式I的结构的盐可作为潜在的抗肿瘤小分子抑制剂。
下面以化合物47为例，对具有通式I的结构的物质的作用机制进行说明。
生物学试验例2：诱导细胞周期G2/M期阻滞和细胞凋亡实验
检测本发明的小分子抑制剂对细胞周期进程的影响，以及对细胞凋亡的影响，采用流式细胞技术及Western blot法。
细胞株采用人肺鳞癌细胞株H2170和人肺腺癌细胞株H1650。培养基为RPMI 1640(美国Gibco)+10％FBS(胎牛血清，美国Gibco)+双抗。
试剂配制：母液浓度为20mM，溶剂为DMSO，储存于-20℃，使用时室温化开，应用细胞培养基稀释至所需浓度。
流式细胞技术检测方法：肺癌细胞以50-60％密度种于60mm细胞培养皿，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基，终浓度分别为2.5，5，10和20μM，溶剂DMSO作为阴性对照。继续培养24小时后胰酶消化收获细胞，70％酒精固定过夜，碘化丙啶/核糖核酸酶染色溶液(PI/RNase)(英国BD Biosciences)染色15分钟，Cytoflex S流式细胞仪检测细胞周期。
Western blot检测方法：肺癌细胞以50-60％密度种于60mm细胞培养皿，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基，终浓度分别为2.5，5，10和20μM，溶剂DMSO作为阴性对照，继续培养24小时后收获细胞，应用Western blot法检测凋亡水平。应用抗体分别为PARP(美国Cell Signaling Technology)，Caspase-3(美国CellSignaling Technology)，NOXA(德国Merck Millipore)，β-Actin抗体(美国Sigma-Aldrich)。结果见图2。
图2为化合物47小分子抑制剂以剂量依赖的方式诱导细胞周期G2/M期阻滞和细胞凋亡。肺癌细胞H2170或H1650经化合物47小分子抑制剂处理24小时后，固定过夜，PI/RNase染液染色，并用Cytoflex S流式细胞仪检测细胞周期；应用PARP，Caspase-3和NOXA抗体，检测细胞凋亡水平。β-Actin作为上样量的参照。
以上实验结果显示，本发明的具有通式I的结构的化合物能诱导肺癌细胞周期G2/M期阻滞和肺癌细胞凋亡,可作为潜在的抗肿瘤小分子抑制剂。
生物学试验例3：Cullin neddylation修饰抑制实验
检测本发明的小分子抑制剂抑制肺癌细胞内cullin分子发生neddylation修饰的活性，采用Western blot法。
细胞株采用人肺鳞癌细胞株H2170和人肺腺癌细胞株H1650。培养基为RPMI 1640(美国Gibco)+10％FBS(胎牛血清，美国Gibco)+双抗。
试剂配制：母液浓度为20mM，溶剂为DMSO，储存于-20℃，使用时室温化开，应用细胞培养基稀释至所需浓度。
Western blot检测方法：肺癌细胞以50-60％密度种于60mm细胞培养皿，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基，终浓度分别为0.3，1，3，10和20μM，溶剂DMSO和MLN4924(0.3μM)分别作为阴性和阳性对照，继续培养一定时间后收获细胞，应用Western blot法检测多个cullin蛋白的neddylation水平。应用抗体分别为Cul1抗体(美国Santa Cruz)，Cul2抗体(美国Santa Cruz)，Cul3抗体(美国Cell SignalingTechnology),Cul4A抗体(美国Proteintech)，Cul4B抗体(美国Proteintech)，Cul5抗体(美国Santa Cruz)，NOXA(德国Merck Millipore)，β-Actin抗体(美国Sigma-Aldrich)。结果见图3。
图3为化合物47小分子抑制剂以时间和剂量依赖的方式抑制Cul5蛋白的neddylation修饰，诱导其底物蛋白NOXA的积累。肺癌细胞H2170或H1650经化合物47小分子抑制剂处理后，应用特异的cullin抗体及NOXA抗体，检测多个cullin蛋白的neddylation修饰水平及底物NOXA蛋白水平。β-Actin作为上样量的参照。SE：短曝光；LE：长曝光。
以上实验结果显示，本发明的具有通式I的结构的化合物均以时间和剂量依赖的方式特异性抑制Cul5蛋白发生neddylation修饰，诱导其底物蛋白NOXA积累，而对其他cullin的neddylation没有作用。提示作用靶点为NEDD8 E2耦联酶UBE2F。
生物学试验例4：UBE2F蛋白抑制试验
检测本发明的小分子抑制剂对肺癌细胞内UBE2F蛋白水平的影响，采用Westernblot法。细胞株采用人肺鳞癌细胞株H2170。培养基为RPMI 1640(美国Gibco)+10％FBS(胎牛血清，美国Gibco)+双抗。
试剂配制：母液浓度为20mM，溶剂为DMSO，储存于-20℃，使用时室温化开，应用细胞培养基稀释至所需浓度。
Western blot检测方法：肺癌细胞以50-60％密度种于60mm细胞培养皿，于细胞培养箱(37℃，5％CO2)培养。24小时后，加入含有小分子试剂的培养基，终浓度分别为0.3，1，3，10和20μM，溶剂DMSO和MLN4924(0.3μM)分别作为阴性和阳性对照，继续培养24小时后收获细胞，应用Western blot法检测UBE2F蛋白水平。应用抗体分别为UBE2F抗体(英国Abcam)，UBE2M(美国Cell Signaling Technology)和β-Actin抗体(美国Sigma-Aldrich)。结果见图4。
图4为化合物47小分子抑制剂能选择性降低细胞内UBE2F蛋白质水平。肺癌细胞H2170经化合物47小分子化合物处理24小时后，应用UBE2F和UBE2M抗体，检测其蛋白水平。β-Actin作为上样量的参照。SE：短曝光；LE：长曝光。
以上实验结果显示，本发明的具有通式I的结构的化合物以剂量依赖方式选择性的降低UBE2F蛋白质水平，而对UBE2F的家族成员UBE2M没有作用，是靶向UBE2F的特异性抑制剂。

Claims (3)

1.一种苯基哌嗪类UBE2F小分子抑制剂，其特征在于，具有通式I的结构，或具有通式I的结构的盐：
Figure FDA0003900175020000011
其中X，Y为羰基；
R1为4-甲基-苯基；
R2为7-氮杂吲哚乙烯基。
2.一种权利要求1所述的小分子抑制剂的合成方法，其特征在于，该方法包括以下步骤：
Figure FDA0003900175020000012
3.一种药物组合物，该组合物含有权利要求1所述的小分子抑制剂。
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