CN112778282B - 一种嘧啶类小分子化合物及其应用 - Google Patents

一种嘧啶类小分子化合物及其应用 Download PDF

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CN112778282B
CN112778282B CN202110012605.2A CN202110012605A CN112778282B CN 112778282 B CN112778282 B CN 112778282B CN 202110012605 A CN202110012605 A CN 202110012605A CN 112778282 B CN112778282 B CN 112778282B
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CN112778282A (zh
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李校堃
刘志国
林丽
郑小辉
钱建畅
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Wenzhou Medical University
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Abstract

本发明公开了一种嘧啶类小分子化合物及其引用，该嘧啶类小分子化合物对PDGFRα和PDGFRβ激酶具有高效的抑制活性，而对其他激酶抑制活性较弱，因而具有很高的选择性，可以作为一种潜在的抗肿瘤或缓解眼科疾病湿性黄斑变性或葡萄膜炎的药物。

Description

一种嘧啶类小分子化合物及其应用
技术领域
本发明属于生物医药领域，具体涉及一种嘧啶类小分子化合物及其制备方法和应用。
背景技术
血小板衍生生长因子受体(PDGFRs)共有PDGFRα和PDGFRβ两个亚型，它们在生物体的生长、发育、血管形成和组织创伤修复的过程中发挥着重要作用。正常生理条件下，除了在发育过程和组织创伤修复过程中，PDGFRs信号通路在成体中一般处于静息状态。PDGFRs过表达、基因突变和基因重排会导致PDGFRs介导的信号通路的异常激活，并导致一系列疾病如纤维化疾病、肿瘤和各种眼科疾病，如湿性黄斑变性(AMD)以及葡萄膜炎等，因此PDGFRs已经成为治疗这些疾病的重要药物靶点。
目前报道的针对PDGFRs选择性的抑制剂包括伊马替尼、CP-673451等，这些抑制剂通常存在激酶抑制活性差、多靶点抑制导致的毒副作用大以及易导致耐药等多种缺陷。因此，有必要提供一类选择性抑制PDGFRα或PDGFRβ、以及双靶向于PDGFRα和PDGFRβ的抑制剂，以便为精准靶向医疗提供研究基础。
发明内容
本发明的发明人经过实验，发现了一种选择性PDGFRs的抑制剂，该抑制剂可以有效缓解PDGFRs基因高表达的肿瘤及各类眼科疾病。
本发明的技术方案如下：
一种嘧啶类小分子化合物，为式(I)、(II)或(III)所示的化合物、其盐、其溶剂化物、其水合物或其前药中的一种；
Figure BDA0002885726690000021
式(I)～(III)中，R1选自H、-CF3或C1～C5烷基；R2选自H、-CF3、C1～C5烷基、C1～C5烷氧基、卤素、C1～C5烷氧羰基；或者R1、R2与连接R1和R2的两个C形成五元环；
R3为取代或者未取代的芳基，该芳基上的取代基选自C1～C5烷基；
R4为取代或者未取代的芳基，所述芳基上的取代基为C1～C5烷基、C1～C5烷氧基、哌嗪基、取代的哌嗪基、1,1-二氧化硫代吗啉基、哌啶基、取代的哌啶基，所述哌嗪基和哌啶基上的取代基选自C1～C5烷基或者卤素。
作为优选，所述的R1选自H、-CF3或甲基；R2选自H、-CF3、甲基、甲氧基、F、Cl、乙氧羰基；或者R1、R2与连接R1和R2的两个C形成含S五元环。
作为优选，所述的R3为取代或者未取代的吲哚基、取代或者未取代的吲唑基、取代或者未取代的喹喔啉基、取代或者未取代的喹啉基、茚满基；所述吲哚基、吲唑基、喹喔啉基、喹啉基上的取代基为甲基或者乙基。
作为优选，所述R4为取代或者未取代的苯基，所述苯基上的取代基为甲基、甲氧基、哌嗪基、取代的哌嗪基、1,1-二氧化硫代吗啉基、哌啶基、取代的哌啶基，所述哌嗪基和哌啶基上的取代基选自甲基、F或者Cl。
作为优选，为化合物3a、化合物3b、化合物3c、化合物3d、化合物3e、化合物3f、化合物3g、化合物3h、化合物3i、化合物5a、化合物5b、化合物5c、化合物5d、化合物5e、化合物5f、化合物5g、化合物5h、化合物5i、化合物5j、化合物5k、化合物6a、化合物6b、化合物6c、化合物6d、化合物6e、化合物6f、化合物6g、化合物6h、化合物6i、化合物6j、化合物6k、化合物6m、化合物6n、其盐、其溶剂化合物、其水合物或者其前药中的一种。
本发明还提供了一种所述的嘧啶类小分子化合物的应用，所述的嘧啶类小分子化合物用于制备PDGFRs抑制剂。
作为优选，所述的PDGFRs抑制剂能高效抑制PDGFRα和PDGFRβ，而对其他激酶抑制活性中等或较弱。
作为优选，所述的嘧啶类小分子化合物用于制备抗肿瘤药物。
作为优选，所述的抗肿瘤药物用于抑制人青少年骨肉瘤。
作为优选，所述的嘧啶类小分子化合物用于制备治疗眼科疾病的药物；
所述的药物用于抑制眼底血管增生，进而缓解眼科疾病湿性黄斑变性或葡萄膜炎。
同现有技术相比，本发明的有益效果体现在：
(1)本发明的嘧啶类小分子化合物对PDGFRα和PDGFRβ激酶具有高效的抑制活性，而对其他激酶抑制活性中等或较弱，因而具有很高的选择性；
(2)本发明的嘧啶类小分子化合物具有更好的抗骨肉瘤效果，是一种潜在的抗肿瘤药物；
(3)本发明的嘧啶类小分子化合物能有效抑制眼底血管增生，进而缓解眼科疾病湿性黄斑变性或葡萄膜炎。
附图说明
图1为实施例7中活性化合物6i抑制骨肉瘤细胞的黏附和转移实验结果；
图2为实施例8中活性化合物6i对两种骨肉瘤细胞PDGFR信号通路相关蛋白的抑制活性结果。
具体实施方式
实施例1：化合物3a-3i的化学合成
合成路线如下：
Figure BDA0002885726690000031
一般过程如下：
以1a-1i为原料与6-氨基吲唑发生亲核取代反应，生成中间体2a-2i。然后4-(4-甲基哌嗪)苯胺与得到的中间体2a-i发生亲核取代反应生成终产物3a-3i。
以化合物3a合成为例，具体过程如下：
将2,4-二氯-5-氟嘧啶(333.94mg，2mmol)和N,N-二异丙基乙胺(DIPEA)(516.96mg，4mmol)溶解在DMF(4mL)中并冷却至0℃。再向混合液中逐滴加入溶于DMF(2mL)中的6-氨基吲唑(266.3mg，2mmol)。将反应混合物在0℃下搅拌约1小时。接下来，移去冰浴，将反应混合物在室温下搅拌，并在通过TLC监测反应，将所得混合物用乙酸乙酯(3×25mL)萃取，用饱和食盐水洗涤，经无水Na2SO4干燥并浓缩,通过硅胶柱层析得到产物2a.
将化合物2a(263.66mg，1mmol)和4-(4-甲基哌嗪)苯胺(191.27mg，1mmol)溶解在甲醇(4ml)中，再加入三氟乙酸(TFA)(148.56μL，2mmol)，升温至80℃，并在通过TLC监测反应。反应完成后冷却至室温，将所得混合物用饱和碳酸氢钠调PH至中性，用乙酸乙酯(3×25mL)萃取，饱和食盐水洗涤，经无水Na2SO4干燥并浓缩，通过硅胶柱层析得到终产物3a。
化合物3a-3i的表征数据如下：
Figure BDA0002885726690000041
5-fluoro-N4-(1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
5-氟-N4-(1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Yellow solid,26.9％yield.1H NMR(500MHz,DMSO-d6)δ12.93(s,1H),9.41(s,1H),8.99(s,1H),8.08(d,J＝3.5Hz,1H),8.01(s,1H),7.91(s,1H),7.69(d,J＝8.5Hz,1H),7.52(d,J＝8.5Hz,3H),6.81(d,J＝8.5Hz,2H),3.06(s,4H),2.57(s,4H),2.30(s,3H).13CNMR(126MHz,DMSO-d6)δ170.28,155.87,149.81,149.73,145.77,140.26,139.40,137.05,133.30,133.15,120.02,119.33,116.55,115.88,101.58,54.61,48.85,14.05；ESI-MS m/z:419.2(M﹢H)+.
Figure BDA0002885726690000051
5-Chloro-N4-(1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
5-氯-N4-(1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺1HNMR(400MHz,DMSO-d6)δ9.06(s,1H),8.89(s,1H),8.10(s,1H),8.04(s,1H),7.76(s,1H),7.71(d,J＝8.6Hz,1H),7.42(d,J＝8.8Hz,2H),7.36(d,J＝8.6Hz,1H),6.69(d,J＝8.8Hz,2H),5.76(s,1H),2.99(s,4H),2.44(s,4H),2.21(s,3H).13C NMR(100MHz,DMSO-d6)δ158.40,156.64,155.15,146.47,137.26,133.78,133.03,120.79,120.44,120.34,118.62,116.20,104.46,104.00,103.67,55.17,49.34,46.27.
Figure BDA0002885726690000052
N4-(1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-5-(trifluoromethyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)-5-(三氟甲基)嘧啶-2,4-二胺
White solid,16.5％yield.1H NMR(400MHz,DMSO-d6)δ9.70(s,1H),8.63(s,1H),8.33(s,1H),7.92(s,1H),7.72(dd,J＝5.6,3.2Hz,1H),7.68–7.65(m,1H),7.48(d,J＝8.8Hz,1H),7.29(d,J＝8.8Hz,3H),7.01(d,J＝8.8Hz,2H),3.18–3.14(m,4H),2.51(d,J＝1.6Hz,4H),2.24(s,3H).13C NMR(126MHz,DMSO-d6)δ166.94,160.70,155.18,148.42,140.43,138.08,133.18,131.68,131.47,129.73,128.62,127.55,126.84,119.81,118.54,115.35,99.13,54.50,48.25,45.65；ESI-MS m/z:469.2(M﹢H)﹢.
Figure BDA0002885726690000061
N4-(1H-indazol-6-yl)-5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
White solid.31.5％yield.1H NMR(500MHz,DMSO-d6)δ12.89(s,1H),8.74(s,1H),8.36(s,1H),8.00(s,1H),7.86(s,1H),7.82(s,1H),7.68(d,J＝9.0Hz,1H),7.51(d,J＝9.0Hz,2H),7.42(d,J＝9.0Hz,1H),6.72(d,J＝9.0Hz,2H),2.99(d,J＝4.0Hz,4H),2.46(d,J＝4.5Hz,4H),2.23(s,3H),2.12(s,3H).13C NMR(126MHz,DMSO-d6)δ159.36,158.45,155.73,145.35,140.38,138.06,133.62,133.23,119.71,119.18,117.52,115.93,105.03,102.29,54.64,48.98,45.61,13.49；ESI-MS m/z:415.2(M﹢H)﹢.
Figure BDA0002885726690000062
N4-(1H-indazol-6-yl)-5-methoxy-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲氧基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
1H NMR(400MHz,DMSO-d6)δ8.77(s,1H),8.65(s,1H),7.96(s,2H),7.83(s,1H),7.65(d,J＝8.7Hz,1H),7.55-7.53(m,3H),6.79(d,J＝9.0Hz,2H),3.86(s,3H),3.01(s,4H),2.45(s,4H),2.22(s,3H).13C NMR(100MHz,DMSO-d6)δ154.60,152.26,145.77,138.16,137.40,134.87,134.48,133.77,120.40,119.79,119.69,119.52,117.10,116.54,101.56,57.42,55.25,49.61,46.27.
Figure BDA0002885726690000071
4-((1H-indazol-6-yl)amino)-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)p yrimidine-5-carboxylate
乙基4--((1氢-吲唑-6-基)氨基)-2-((4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-5-羧酸酯
Yellow solid,95.1％yield.1H NMR(400MHz,DMSO-d6)δ13.05(s,1H),10.34(s,1H),9.86(s,1H),8.73(s,1H),8.08(s,1H),7.76(d,J＝8.4Hz,1H),7.53(d,J＝5.6Hz,2H),7.29(d,J＝7.3Hz,1H),6.81(s,2H),4.34(q,J＝6.8Hz,2H),3.17(s,4H),2.75(s,4H),2.43(s,3H),1.37(t,J＝7.0Hz,3H).13C NMR(100MHz,DMSO-d6)δ166.39,160.64,160.44,153.10,146.23,140.24,136.11,136.03,133.47,133.43,131.57,121.48,120.57,115.83,60.36,53.65,47.55,44.23,14.16；ESI-MS m/z:473.2(M﹢H)﹢.
Figure BDA0002885726690000072
N4-(1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-6-(trifluoromethyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)-6-(三氟甲基)嘧啶-2,4-二胺
Yellow solid,50.7％yield.1H NMR(500MHz,DMSO-d6)δ13.07(s,1H),9.47(s,1H),8.81(s,1H),8.31(s,1H),8.08(s,1H),7.74(d,J＝8.5Hz,1H),7.57(s,1H),7.26(s,2H),7.17(s,1H),6.43(s,2H),2.93(s,4H),2.41(s,4H),2.20(s,3H).13C NMR(126MHz,DMSO-d6)δ160.52,157.77,155.55,155.51,146.34,140.18,136.35,133.20,131.54,126.00,123.86,120.76,120.73,120.52,119.94,115.31,54.58,48.62,45.70；ESI-MS m/z:469.2(M﹢H)﹢.
Figure BDA0002885726690000081
N4-(1H-indazol-6-yl)-6-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-6-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Yellow liquid,29.3％yield.1H NMR(500MHz,DMSO-d6)δ12.76(s,1H),9.24(s,1H),8.85(s,1H),7.96(s,1H),7.82(s,1H),7.64(d,J＝8.5Hz,1H),7.60(d,J＝8.5Hz,2H),7.32(d,J＝8.5Hz,1H),6.84(d,J＝8.0Hz,2H),6.07(s,1H),3.05(s,4H),2.50(s,4H),2.24(s,3H),2.20(s,3H).13C NMR(126MHz,DMSO-d6)δ164.46,161.29,159.59,146.55,140.88,139.50,133.04,132.11,121.71,120.21,119.76,117.86,116.03,115.10,97.80,54.45,48.48,45.62,23.61；ESI-MS m/z:415.2(M﹢H)﹢.
Figure BDA0002885726690000082
N4-(1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)thieno[2,3-d]pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-6-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)噻吩[2,3-d]嘧啶-2,4-二胺
1H NMR(400MHz,DMSO-d6)δ9.51(s,1H),8.86(s,1H),8.05(d,J＝5.4Hz,1H),8.03(s,1H),7.93(s,1H),7.71(d,J＝8.7Hz,1H),7.62(d,J＝9.0Hz,2H),7.54-7.49(m,1H),7.18(d,J＝5.4Hz,1H),6.80(d,J＝9.0Hz,2H),3.03(s,4H),2.44(s,4H),2.20(S,3H).13CNMR(100MHz,DMSO-d6)δ162.66,158.62,155.98,146.08,140.80,137.97,134.18,134.11,133.81,123.85,120.58,120.51,120.40,119.98,117.83,116.43,107.67,103.16,55.24,49.54,46.29.
实施例2：化合物5a-5j的化学合成
合成路线如下：
Figure BDA0002885726690000091
一般过程如下：
2,4-二氯-5-甲基嘧啶(1d)与各种取代胺发生亲核取代反应，生成中间体4a-4j。然后4-(4-甲基哌嗪)苯胺与得到的中间体4a-4j发生亲核取代反应生成终产物5a-5j。
以化合物5a合成为例，具体过程如下：
N,N-二异丙基乙胺(DIPEA)(516.96mg，4mmol)溶解在DMF(4mL)中并冷却至0℃。再向混合液中逐滴加入溶于DMF(2mL)中的5-氨基吲哚(264.3mg，2mmol)。将反应混合物在0℃下搅拌约1小时。接下来，移去冰浴，将反应混合物在室温下搅拌，并在通过TLC监测反应。反应完成后将所得混合物用乙酸乙酯(3×25mL)萃取，用饱和食盐水洗涤，经无水Na2SO4干燥并浓缩，通过硅胶柱层析得到中间体4a。将4a(258.7mg，1mmol)和4-(4-甲基哌嗪)苯胺(191.27mg，1mmol)溶解在甲醇(4ml)中，再加入TFA(148.56μL，2mmol)，升温至80℃，并在通过TLC监测反应。反应完成后冷却至室温,将所得混合物用饱和碳酸氢钠调PH至中性，用乙酸乙酯(3×25mL)萃取，饱和食盐水洗涤，经无水Na2SO4干燥并浓缩，通过硅胶柱层析得到终产物5a。
得到的化合物5a-5k的结构和表征数据如下：
Figure BDA0002885726690000101
N4-(1H-indol-5-yl)-5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲哚-5-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Pink solid,41.91％yield.1H NMR(500MHz,DMSO-d6)δ11.00(s,1H),8.54(s,1H),8.09(s,1H),7.83(s,1H),7.76(s,1H),7.47(d,J＝9.0Hz,2H),7.39–7.30(m,2H),7.25(d,J＝8.5Hz,1H),6.65(d,J＝9.0Hz,2H),6.39(s,1H),2.96(d,J＝4.5Hz,4H),2.47–2.38(m,4H),2.21(s,3H),2.09(s,3H).13C NMR(126MHz,DMSO-d6)δ159.95,158.57,154.90,145.17,133.84,133.01,131.42,127.55,125.44,119.65,118.64,115.98,114.77,110.70,104.30,101.05,54.74,49.16,45.75,13.52；ESI-MS m/z:414.2(M﹢H)﹢.
Figure BDA0002885726690000102
N4-(1H-indol-4-yl)-5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲哚-4-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Gray solid,74.49％yield.1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.60(s,1H),8.08(s,1H),7.85(s,1H),7.43–7.35(m,3H),7.31(t,J＝2.8Hz,1H),7.26(d,J＝8.1Hz,1H),7.12(t,J＝8.0Hz,1H),6.65(d,J＝8.8Hz,2H),6.49–6.33(m,1H),3.02–2.96(m,4H),2.50–2.44(m,4H),2.25(s,3H),2.19(s,3H).13C NMR(126MHz,DMSO-d6)δ159.94,158.51,155.20,145.12,136.81,133.78,131.53,124.20,123.06,120.90,119.42,115.74,114.12,107.71,104.64,99.56,54.67,49.10,45.67,13.38；ESI-MS m/z:414.2(M﹢H)﹢.
Figure BDA0002885726690000111
5-methyl-N4-(1-methyl-1H-indol-5-yl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
5甲基-N4-(1-甲基-1氢-吲哚-5-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Gray solid,52.1％yield.1H NMR(400MHz,DMSO-d6)δ8.63(s,1H),8.18(s,1H),7.90(d,J＝2.0Hz,1H),7.80(s,1H),7.50(d,J＝9.2Hz,2H),7.43(d,J＝8.8Hz,1H),7.33(dd,J＝7.2,2.8Hz,2H),6.69(d,J＝9.2Hz,2H),6.40(d,J＝2.8Hz,1H),3.83(s,3H),3.04–2.97(m,4H),2.49–2.43(m,4H),2.24(s,3H),2.12(s,3H).13C NMR(126MHz,DMSO-d6)δ159.88,158.55,154.96,145.17,133.76,133.55,131.65,129.71,127.89,119.73,118.59,115.82,114.93,109.00,104.29,100.22,54.73,49.02,45.75,32.51,13.52；ESI-MS m/z:428.6(M﹢H)﹢.
Figure BDA0002885726690000112
N4-(1H-indazol-5-yl)-5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-5-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Yellow oil,63.5％yield.1H NMR(500MHz,DMSO-d6)δ8.80(s,1H),8.76(d,J＝3.5Hz,1H),8.56(s,1H),8.50(s,1H),8.12(d,J＝8.0Hz,1H),8.04(dd,J＝9.0,2.0Hz,1H),7.96–7.89(m,3H),7.48(d,J＝8.5Hz,2H),6.78(d,J＝9.0Hz,1H),3.06–3.00(m,4H),2.48–2.43(m,4H),2.23(s,2H),2.16(s,3H).13C NMR(126MHz,DMSO-d6)δ158.89,158.64,156.12,148.42,145.85,144.42,138.08,135.19,133.13,128.64,128.28,125.73,121.23,120.85,116.54,115.85,105.27,54.73,48.98,45.76,13.48；ESI-MS m/z:414.3(M﹢H)﹢.
Figure BDA0002885726690000121
5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N4-(quinoxalin-6-yl)pyrimidine-2,4-diamine
5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)-N4-(苯并吡嗪-6-基)-嘧啶-2,4-二胺
Yellow solid,68.8％yield.1H NMR(500MHz,DMSO-d6δ8.90(s,1H),8.86(s,1H),8.79(d,J＝1.5Hz,1H),8.76(s,1H),8.57(s,1H),8.32(dd,J＝9.0,2.0Hz,1H),8.00(d,J＝9.0Hz,1H),7.96(s,1H),7.53(d,J＝8.5Hz,2H),6.80(d,J＝8.5Hz,2H),3.02(s,4H),2.45(s,4H),2.22(s,3H),2.20(s,3H).13C NMR(126MHz,DMSO-d6)δ158.69,158.50,156.56,145.72,145.46,143.27,143.18,141.62,138.81,133.13,128.52,126.27,120.34,116.74,115.96,105.74,54.72,49.02,45.76,13.56；ESI-MS m/z:427.2(M﹢H)﹢.
Figure BDA0002885726690000131
5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N4-(quinolin-7-yl)pyrimidine-2,4-diamine
5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)-N4-(喹喔啉-7-基)-嘧啶-2,4-二胺
Gray solid,44.6％yield.1H NMR(500MHz,DMSO)δ8.75(dd,J＝6.0,2.5Hz,2H),8.53(s,1H),8.50(s,1H),8.11(d,J＝7.5Hz,1H),8.02(d,J＝9.0Hz,1H),7.95–7.89(m,2H),7.45(t,J＝7.0Hz,3H),6.78(d,J＝8.5Hz,2H),3.02(d,J＝4.0Hz,4H),2.45(d,J＝4.5Hz,4H),2.22(s,3H),2.15(s,3H).13C NMR(126MHz,DMSO-d6)δ158.90,158.62,156.10,148.45,145.88,144.29,138.04,135.31,133.03,128.55,128.27,125.76,121.29,120.98,116.56,115.86,105.31,54.64,48.89,45.67,13.42；ESI-MS m/z:426.2(M﹢H)﹢.
Figure BDA0002885726690000132
5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N4-(5,6,7,8-tetrahydronaphthalen-2-yl)pyrimidine-2,4-diamine
5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)-N4-(5,6,7,8-四氢萘-2-基)-嘧啶-2,4-二胺
Gray solid,71.2％yield.1H NMR(400MHz,DMSO-d6)δ8.62(s,1H),7.96(s,1H),7.79(s,1H),7.37(d,J＝9.2Hz,2H),7.23–7.14(m,2H),7.08–7.01(m,1H),6.64(d,J＝8.8Hz,2H),3.03–2.95(m,4H),2.83(s,2H),2.61(s,2H),2.51–2.47(m,4H),2.26(s,3H),2.10(s,3H),1.69(s,4H).13C NMR(126MHz,DMSO-d6)δ160.32,158.46,154.96,144.92,137.86,137.35,134.04,133.50,126.21,125.11,124.56,118.99,115.78,104.01,54.60,49.08,45.58,29.32,24.52,22.43,22.36,13.28；ESI-MS m/z:429.3(M﹢H)﹢.
Figure BDA0002885726690000141
N4-(2,3-dihydro-1H-inden-5-yl)-5-methyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(2,3-二氢-1H-茚-5-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)-嘧啶-2,4-二胺
Yellow solid,43.4％yield.1H NMR(400MHz,DMSO)δ8.70(s,1H),8.12(s,1H),7.83(s,1H),7.65(d,J＝1.2Hz,1H),7.51(d,J＝9.2Hz,2H),7.38(dd,J＝8.0,1.6Hz,1H),7.18(d,J＝8.0Hz,1H),6.79(d,J＝9.2Hz,2H),3.07–3.01(m,4H),2.87(t,J＝7.6Hz,4H),2.50–2.45(m,4H),2.24(s,3H),2.10(s,3H),2.05(dd,J＝14.8,7.6Hz,2H).13C NMR(126MHz,DMSO)δ159.24,158.51,155.42,145.43,143.59,138.01,137.82,133.56,123.63,120.23,120.00,118.48,115.84,104.63,54.71,49.08,45.73,32.58,31.81,25.29,13.48；ESI-MS m/z:415.3(M﹢H)﹢.
Figure BDA0002885726690000142
5-methyl-N4-(3-methyl-1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-y l)phenyl)pyrimidine-2,4-diamine
5-甲基-N4-(3-甲基-1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)-嘧啶-2,4-二胺
White solid,35.4％yield.1H NMR(500MHz,DMSO-d6)δ12.48(s,1H),8.73(s,1H),8.37(s,1H),7.85(s,1H),7.80(s,1H),7.59(d,J＝8.0Hz,1H),7.51(d,J＝7.5Hz,2H),7.36(d,J＝8.0Hz,1H),6.72(d,J＝8.0Hz,2H),3.01(s,4H),2.47(s,4H),2.25(s,4H),2.12(s,3H).13C NMR(126MHz,DMSO-d6)δ159.41,158.41,155.63,145.20,141.34,138.09,133.68,119.71,119.10,118.55,116.73,115.95,105.08,102.37,54.45,48.77,45.36,13.52,11.69；ESI-MS m/z:429.3(M﹢H)﹢.
Figure BDA0002885726690000151
(2-(6-((5-methyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-2H-indazol-2-yl)ethyl)carbamate
叔丁基(2-(6-((5-甲基-2-((4-(4-甲基哌嗪-1-基)苯基)氨基)-嘧啶-4基)氨基)-2氢-吲唑-2-基)乙基)氨基甲酸酯
Gray solid,12.7％yield.1H NMR(500MHz,DMSO-d6)δ8.75(s,1H),8.21(s,1H),8.14(s,2H),7.84(s,1H),7.61(d,J＝8.5Hz,1H),7.55(d,J＝8.5Hz,1H),7.28(d,J＝8.0Hz,1H),7.05(s,1H),6.79(d,J＝8.5Hz,2H),4.41(d,J＝6.0Hz,2H),3.49–3.45(m,2H),3.02(s,4H),2.45(s,4H),2.21(s,3H),2.12(s,3H),1.37(s,9H).13C NMR(126MHz,DMSO-d6)δ159.25,158.53,155.58,155.51,148.76,145.43,137.09,133.50,123.78,119.93,119.78,119.28,118.05,116.03,107.06,105.02,77.95,54.71,52.05,49.09,45.73,40.51,28.17,13.52；ESI-MS m/z:558.3(M﹢H)﹢.
Figure BDA0002885726690000152
5-methyl-N4-(1-methyl-1H-indazol-6-yl)-N2-(4-(4-methylpiperazin-1-y l)phenyl)pyrimidine-2,4-diamine
5-甲基-N4-(1-甲基-1氢-吲唑-6-基)-N2-(4-(4-甲基哌嗪-1-基)苯基)-嘧啶-2,4-二胺
Gray solid,54.1％yield.1H NMR(400MHz,DMSO-d6)δ8.83(s,1H),8.44(s,1H),8.14(s,1H),7.97(s,1H),7.91(s,1H),7.68(s,1H),7.52(s,2H),7.44(s,1H),6.75(s,2H),3.91(s,3H),3.02(s,4H),2.46(s,4H),2.24(s,3H),2.17(s,3H).13C NMR(100MHz,DMSO-d6)δ159.17,158.41,155.84,145.55,140.11,138.42,133.40,132.08,120.14,119.46,117.03,115.78,105.23,100.67,54.71,48.97,45.73,35.20,13.54；ESI-MS m/z:429.2(M﹢H)﹢.
实施例3：化合物6a-6m的化学合成
化合物6a-6m的合成路线如下：
Figure BDA0002885726690000161
一般合成路线如下：
2,4-二氯-5-甲基嘧啶(1d)与6氨基吲唑反应生产中间体2d，中间体2d再与各种取代胺发生亲核取代反应，生成终产物6a-6m。
以化合物6a合成为例，具体合成如下：
化合物2d按照实施例1制备，将化合物2d(259.7mg，1mmol)和4-(4-甲基哌嗪)苯胺(191.27mg，1mmol)溶解在4ml甲醇中,再加入TFA(148.56μL，2mmol)，升温至80℃，并在通过TLC监测反应。反应完成后冷却至室温,将所得混合物用饱和碳酸氢钠调PH至中性，用乙酸乙酯(3×25mL)萃取，饱和食盐水洗涤，经无水Na2SO4干燥并浓缩,通过硅胶柱层析得到产物6a。
化合物6a-6m的结构和表征数据如下：
Figure BDA0002885726690000162
N4-(1H-indazol-6-yl)-5-methyl-N2-(4-morpholinophenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲基-N2-(4-***啉苯基)-嘧啶-2,4-二胺
Yellow solid,33.9％yield.1H NMR(500MHz,DMSO-d6)δ12.89(s,1H),8.72(s,1H),8.35(s,1H),8.00(s,1H),7.86(s,1H),7.82(s,1H),7.67(d,J＝8.5Hz,1H),7.50(d,J＝8.5Hz,2H),7.41(d,J＝8.5Hz,1H),6.70(d,J＝9.0Hz,2H),2.89(d,J＝4.5Hz,4H),2.82(d,J＝4.5Hz,4H),2.12(s,3H).13C NMR(126MHz,DMSO-d6)δ159.35,158.46,155.74,146.12,140.43,138.06,133.52,133.18,119.72,119.17,117.53,115.92,104.99,102.32,50.38,45.62,13.48；ESI-MS m/z:402.2(M﹢H)﹢.
Figure BDA0002885726690000171
N4-(1H-indazol-6-yl)-5-methyl-N2-(4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲基-N2-(4-(哌嗪-1-基)苯基)-嘧啶-2,4-二胺
Yellow solid,29.4％yield.1H NMR(500MHz,DMSO-d6)δ12.93(s,1H),8.93(s,1H),8.55(s,1H),8.01(s,1H),7.84(d,J＝18.5Hz,2H),7.69(d,J＝8.5Hz,1H),7.50(d,J＝9.0Hz,2H),7.41(d,J＝8.0Hz,1H),6.73(d,J＝8.5Hz,2H),3.74–3.67(m,4H),3.01–2.92(m,4H),2.13(s,3H).13C NMR(126MHz,DMSO-d6)δ159.84,156.85,152.41,146.01,140.31,137.43,133.25,132.61,120.52,119.89,119.51,117.78,115.57,105.57,103.16,66.11,49.26,13.41；ESI-MS m/z:401.2(M﹢H)﹢.
Figure BDA0002885726690000172
N2-(4-(4,4-difluoropiperidin-1-yl)phenyl)-N4-(1H-indazol-6-yl)-5-methylpyrimidine-2,4-diamine
N2-(4-(4,4-二氟哌嗪-1-基)苯基)-N4-(1氢-吲唑-6-基)-5-甲基嘧啶-2,4-二胺
Gray solid,61.1％yield.1H NMR(500MHz,DMSO-d6)δ12.98(s,1H),8.79(s,1H),8.44(s,1H),7.99(s,1H),7.92(s,1H),7.86(s,1H),7.66(d,J＝8.5Hz,1H),7.55(d,J＝9.0Hz,2H),7.44(d,J＝8.5Hz,1H),6.79(d,J＝9.0Hz,2H),3.19–3.10(m,4H),2.14(s,3H),2.07–1.99(m,4H).13C NMR(126MHz,DMSO-d6)δ159.38,158.36,155.70,144.03,140.58,140.41,138.04,134.15,133.18,119.67,119.14,117.52,116.94,105.19,102.39,54.87,46.85,33.07,13.54；ESI-MS m/z:436.2(M﹢H)﹢.
Figure BDA0002885726690000181
4-(4-((4-((1H-indazol-6-yl)amino)-5-methylpyrimidin-2-yl)amino)phenyl)thiomor-pholine 1,1-dioxide
4-(4-((4-((1氢-吲唑-6-基)氨基)-5-甲基嘧啶-2-基)氨基)苯基)硫代吗啉1,1-二氧化物
Gray solid,81.4％yield.1H NMR(500MHz,DMSO-d6)δ13.00(s,1H),8.83(s,1H),8.45(s,1H),8.01–7.84(m,3H),7.67(d,J＝8.5Hz,1H),7.57(d,J＝8.0Hz,2H),7.43(d,J＝8.0Hz,1H),6.82(d,J＝8.0Hz,2H),3.62(s,4H),3.11(s,4H),2.14(s,3H).13C NMR(126MHz,DMSO-d6)δ159.39,158.85,158.29,155.68,142.01,138.02,134.28,133.10,119.84,119.67,119.11,117.52,116.69,105.32,102.49,49.91,47.86,13.56；ESI-MS m/z:450.2(M﹢H)﹢.
Figure BDA0002885726690000182
N4-(1H-indazol-6-yl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5-methylpyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-N2-(2-甲氧基-4-(4-甲基哌嗪-1-基)苯基)-5-甲基嘧啶-2,4-二胺
Gray solid,26.6％yield.1H NMR(500MHz,DMSO-d6)δ12.92(s,1H),8.45(s,1H),7.98(s,1H),7.91–7.81(m,3H),7.64(d,J＝9.0Hz,1H),7.41(d,J＝8.5Hz,1H),7.26(s,1H),6.61(s,1H),6.31(d,J＝7.5Hz,1H),3.81(s,3H),3.07(s,4H),2.48(s,4H),2.24(s,3H),2.13(s,3H).13C NMR(126MHz,DMSO-d6)δ159.34,158.44,155.70,149.45,146.79,140.43,138.05,133.17,121.81,120.86,119.72,119.07,117.28,106.97,105.48,101.85,100.14,55.60,54.71,48.97,45.74,13.47；ESI-MS m/z:445.3(M﹢H)﹢.
Figure BDA0002885726690000191
N4-(1H-indazol-6-yl)-N2-(3-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5-methylpyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-N2-(3-甲氧基-4-(4-甲基哌嗪-1-基)苯基)-5-甲基嘧啶-2,4-二胺
Yellow solid,55.9％yield.1H NMR(500MHz,DMSO-d6)δ12.85(s,1H),8.79(s,1H),8.37(s,1H),7.99(s,1H),7.89(s,1H),7.81(s,1H),7.66(d,J＝8.5Hz,1H),7.40(d,J＝8.5Hz,1H),7.29(s,1H),7.24(d,J＝9.5Hz,1H),6.63(s,1H),3.43(s,3H),2.86(s,4H),2.47(s,4H),2.24(s,3H),2.13(s,3H).13C NMR(126MHz,DMSO-d6)δ159.28,158.30,155.70,151.91,140.40,138.05,136.65,134.85,133.21,119.78,119.18,117.76,117.43,110.62,105.47,103.75,102.14,55.01,54.84,50.31,45.84,13.50；ESI-MS m/z:445.2(M﹢H)﹢.
Figure BDA0002885726690000192
N4-(1H-indazol-6-yl)-5-methyl-N2-(4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲基-N2-(4-(4-甲基哌嗪-1-基)苯基)嘧啶-2,4-二胺
Yellow solid,29.4％yield.1H NMR(500MHz,DMSO-d6)δ12.93(s,1H),8.93(s,1H),8.55(s,1H),8.01(s,1H),7.84(d,J＝18.5Hz,2H),7.69(d,J＝8.5Hz,1H),7.50(d,J＝9.0Hz,2H),7.41(d,J＝8.0Hz,1H),6.73(d,J＝8.5Hz,2H),3.74–3.67(m,4H),3.01–2.92(m,4H),2.13(s,3H).13C NMR(126MHz,DMSO-d6)δ159.84,156.85,152.41,146.01,140.31,137.43,133.25,132.61,120.52,119.89,119.51,117.78,115.57,105.57,103.16,66.11,49.26,13.41；ESI-MS m/z:401.2(M﹢H)﹢.
Figure BDA0002885726690000201
N2-(4-(4-ethylpiperazin-1-yl)-2-methoxyphenyl)-N4-(1H-indazol-6-yl)-5-methylpyrimidine-2,4-diamine
N2-(4-(4-乙基哌嗪-1-基)-2-甲氧基苯基)-N4-(1氢-吲唑-6-基)-5-甲基嘧啶-2,4-二胺
White solid,19.2％yield.1H NMR(500MHz,DMSO-d6)δ12.82(s,1H),8.38(s,1H),7.99(s,1H),7.91–7.82(m,3H),7.65(d,J＝8.5Hz,1H),7.39(d,J＝9.5Hz,1H),7.28(s,1H),6.61(d,J＝2.0Hz,1H),6.31(d,J＝10.0Hz,1H),3.81(s,3H),3.06(s,4H),2.51(s Hz,4H),2.38(q,J＝7.0Hz,2H),2.12(s,3H).13C NMR(126MHz,DMSO-d6)δ159.34,158.42,155.69,149.45,146.81,140.40,138.03,133.19,121.81,120.86,119.71,119.06,117.28,106.95,105.47,101.86,100.16,55.61,52.37,51.58,49.06,13.47,11.92；ESI-MS m/z:459.3(M﹢H)﹢.
Figure BDA0002885726690000202
N2-(4-(4-ethylpiperazin-1-yl)-3-methoxyphenyl)-N4-(1H-indazol-6-yl)-5-methylpyrimidine-2,4-diamine
N2-(4-(4-乙基哌嗪-1-基)-3-甲氧基苯基)-N4-(1氢-吲唑-6-基)-5-甲基嘧啶-2,4-二胺
Colorless oil,65.1％yield.1H NMR(400MHz,DMSO-d6)δ12.90(s,1H),8.82(s,1H),8.42(s,1H),7.99(s,1H),7.89(s,1H),7.85(s,1H),7.64(d,J＝8.4Hz,1H),7.41(d,J＝8.8Hz,1H),7.30(s,1H),7.25(d,J＝8.8Hz,1H),6.63(d,J＝8.8Hz,1H),5.33(s,1H),3.43(s,3H),2.86(s,4H),2.39-2.37(m,2H),2.13(s,4H),1.77(s,3H),1.22(t,J＝6.8Hz,1H).13C NMR(126MHz,DMSO-d6)δ159.29,158.27,155.68,151.90,140.42,138.07,136.66,134.84,133.18,119.74,119.12,117.71,117.39,110.60,105.52,103.74,102.17,52.63,52.07,51.67,50.36,13.53,11.87；ESI-MS m/z:459.3(M﹢H)﹢.
Figure BDA0002885726690000211
N4-(1H-indazol-6-yl)-5-methyl-N2-(4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)pyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-5-甲基-N2-(4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)嘧啶-2,4-二胺
White solid,73.6％yield.1H NMR(500MHz,DMSO-d6)δ12.89(s,1H),8.71(s,1H),8.35(s,1H),7.99(s,1H),7.84(d,J＝12.5Hz,2H),7.67(d,J＝8.7Hz,1H),7.49(d,J＝8.9Hz,2H),7.42(d,J＝8.6Hz,1H),6.71(d,J＝8.9Hz,2H),3.52(d,J＝12.0Hz,2H),2.54(s,1H),2.30(s,4H),2.26–2.18(m,2H),1.80(d,J＝11.6Hz,3H),1.54–1.42(m,3H).13C NMR(126MHz,DMSO-d6)δ159.35,158.46,155.74,145.54,138.07,133.40,133.21,119.72,119.15,117.51,116.40,105.01,102.28,83.88,79.19,60.85,55.15,49.26,48.54,45.74,27.88,13.51；ESI-MS m/z:498.3(M﹢H)﹢.
Figure BDA0002885726690000212
N4-(1H-indazol-6-yl)-N2-(2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-5-methylpyrimidine-2,4-diamine
N4-(1氢-吲唑-6-基)-N2-(2-甲氧基-4-(4-(4-甲基哌嗪-1-基)哌啶-1-基)苯基)-5-甲基嘧啶-2,4-二胺
Yellow solid,68.9％yield.1H NMR(500MHz,DMSO-d6)δ12.83(s,1H),8.38(s,1H),7.98(s,1H),7.85(d,J＝7.0Hz,3H),7.64(d,J＝8.5Hz,1H),7.38(d,J＝8.0Hz,1H),7.27(s,1H),6.60(s,1H),6.31(d,J＝7.0Hz,1H),3.80(s,3H),3.61(d,J＝12.0Hz,2H),2.58(t,J＝11.5Hz,3H),2.36–2.20(m,4H),2.13(d,J＝9.0Hz,6H),1.83(d,J＝11.3Hz,2H),1.56–1.42(m,2H).13C NMR(126MHz,DMSO-d6)δ159.36,158.47,155.66,150.46,149.64,146.99,138.02,133.30,121.54,121.21,119.78,119.04,117.34,107.47,105.40,101.95,100.62,60.84,55.57,54.98,49.11,48.46,45.63,27.76,13.39；ESI-MS m/z:528.3(M﹢H)﹢.
Figure BDA0002885726690000221
(4-((4-((1H-indazol-6-yl)amino)-5-methylpyrimidin-2-yl)amino)pheny l)(4-methylpiperazin-1-yl)methanone
(4-((4-((1氢-吲唑-6-基))氨基)-5-甲基嘧啶-2-基)氨基)苯基)(4-甲基哌嗪-1-基)甲酮
Colorless liquid,13.8％yield.1H NMR(400MHz,DMSO-d6)δ9.30(s,1H),8.53(s,1H),7.96(s,1H),7.88(s,1H),7.79(s,1H),7.71(d,J＝8.8Hz,2H),7.64(d,J＝8.4Hz,1H),7.33(d,J＝8.8Hz,1H),7.11(d,J＝8.8Hz,2H),2.84(s,4H),2.44(s,4H),2.10(s,3H),1.70(s,3H).13C NMR(126MHz,DMSO-d6)δ169.22,159.54,157.86,155.63,142.65,140.46,137.79,133.12,127.81,126.85,119.73,119.34,117.88,117.29,106.37,103.04,54.55,53.86,45.59,13.57；ESI-MS m/z:473.2(M﹢H)﹢.
Figure BDA0002885726690000231
4-((4-((1H-indazol-6-yl)amino)-5-methylpyrimidin-2-yl)amino)-N-(2-morpholinoethyl)benzamide
(4-((4-((1氢-吲唑-6-基))氨基)-5-甲基嘧啶-2-基)氨基)-N-(2-***啉乙基)苯甲酰胺
White solid,60.2％yield.1H NMR(500MHz,DMSO-d6)δ13.02(s,1H),9.33(s,1H),8.59(s,1H),8.24(s,1H),8.15–7.88(m,3H),7.79(d,J＝8.0Hz,2H),7.71(d,J＝8.5Hz,1H),7.63(d,J＝8.0Hz,2H),7.46(d,J＝8.0Hz,1H),3.57(s,4H),2.43(d,J＝13.5Hz,6H),2.17(s,3H).13C NMR(126MHz,DMSO-d6)δ165.92,159.44,157.76,155.56,143.90,140.41,137.84,133.18,127.69,125.85,119.69,119.25,117.63,116.82,106.65,102.50,66.14,57.44,53.26,36.35,13.64；ESI-MS m/z:473.2(M﹢H)﹢.
实施例4：化合物的激酶抑制试验
实验采用方法为Caliper Mobility Shift Assay，该方法是以微流体芯片技术的迁移率检测技术为核心的检测平台。实验步骤：配置1.25x激酶反应缓冲液(62.5mmol/LHEPES，pH 7.5；0.001875％Brij-35；12.5mmol/LMgCl2；2.5mM DTT)和激酶反应终止液(100mmol/L HEPES，pH 7.5；0.015％Brij-35；0.2％Coating Reagent#3)；在5μl的5x浓度的化合物溶液中(用DMSO溶解，用水稀释10倍)加入10μl的2.5x的激酶溶液(在1.25x激酶反应缓冲液中加激酶)，室温孵育10min后再加入10μl的2.5x底物肽溶液(在1.25x激酶反应缓冲液中加FAM标记肽和ATP)，在28℃下反应特定的时间后加入25μl激酶反应终止液。在Caliper上测试收集数据，对激酶活性的抑制率＝(max-conversion)/(max-min)*100。“max”为未加化合物的DMSO对照，“min”为低对照。测定IC50时每种样品设10个稀释度各2个复孔，3次重复，结果见表1。
表1所合成化合物的化学结构及在100nM浓度下对PDGFRα和PDGFRβ激酶的抑制率(％)。
Figure BDA0002885726690000241
Figure BDA0002885726690000251
表2.部分活性化合物对PDGFRα和PDGFRβ激酶抑制的IC50值
Figure BDA0002885726690000252
表3.部分化合物对相关激酶抑制的IC50值
Figure BDA0002885726690000253
结果表明，10个活性化合物可以高效抑制激酶PDGFRα和PDGFRβ，而对其他激酶抑制活性相对较弱，表明具有良好的激酶选择性。特别是化合物6g、6i和6f对PDGFRα和PDGFRβ抑制活性显著，其中6i抑制活性最强。
实施例5活性化合物的激酶选择性实验
依照上述激酶抑制活性测定方法，测定活性化合物对其他多种激酶的抑制活性，以表征活性抑制剂的激酶选择性。
表4.活性化合物在100nM浓度下6i对16种激酶的选择性抑制活性Compounds％inhibition@100nM
Figure BDA0002885726690000261
结果表明，活性化合物6i在100nM浓度下可以高效抑制激酶PDGFRα和PDGFRβ，抑制率达到100％以上，而对其他激酶抑制活性相对较弱，表明具有良好的激酶选择性。
实施例6活性化合物对骨肉瘤细胞的抑制试验
采用MTT法，将对数期的骨肉瘤细胞以1500cell/孔的细胞浓度接种于96孔板，每孔200μL细胞悬液，培养6h。将样品配成0.5,2.5,5,10,25μmol/L，5个浓度梯度的溶液，每个样品设5个复孔，置于培养箱培养48h(37℃，5％CO2)，加MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)每孔20μL，继续培养4h后，吸弃培养液，每孔加入200μL DMSO，震荡溶解10min后，用多功能酶标仪在490nM处测定各孔的OD值并计算抑制率。IC50值计算方法：利用GraphPad Prism软件通过曲线拟合得到函数计算样品的IC50值。
表5.MTT法测定活性化合物对四种骨肉瘤细胞的抑制活性(IC50)
Figure BDA0002885726690000271
结果表明，三个活性化合物相对于三个阳性药物均具有强烈的抑制四种骨肉瘤瘤细胞株增殖的活性，表明这些化合物具有优异的抗骨肉瘤效果。
实施例7活性化合物6i抑制骨肉瘤细胞的黏附和转移实验
96孔板预孵人血浆纤连蛋白(Human Plasma Fibronectin),接种分别经、0,0.1,0.2,0.4μM 6i和0.4μM Pazopanib,0.4μM Imatinib作用48小时的骨肉瘤细胞，每孔5×104个，每组设6个复孔，37℃，5％的CO2培养40min，PBS洗去未黏附的细胞，4％多聚甲醛100μL每孔,室温固定15min,100uL,每孔200μLPBS洗涤三次，弃掉PBS，每孔50μL结晶紫室温染色5min,染色结束后每孔200μL超纯水洗涤三次，弃超纯水并拍干，每孔加入100μL 33％冰醋酸，震荡10min溶解结晶紫，酶标仪570nM测吸光度，黏附率(％)＝(加药处理组肿瘤细胞OD值-Hu FN OD值)/(未处理肿瘤细胞OD值-Hu FN OD值)×100％。
将密度为5×104个0,0.1,0.2,0.4μM 6i和0.4μM Pazopanib,0.4μM Imatinib处理48h后的骨肉瘤细胞接种到6孔板中，加入含10％血清的RMPL 1640培养液，放入培养箱使其贴壁形成单层细胞，用200uL枪头作“+”十字划痕，用无菌PBS洗涤3次，分别加入含2.5μLTCS的基础培养基，分别于0h,24,48h在倒置显微镜下拍照，用Imag J软件测量其迁移距离。
结果见图1，结果表明，0.1、0.2和0.4uM的活性化合物6i相对于阳性药物均具有强烈的剂量依赖性抑制骨肉瘤细胞MG63和MNNG黏附(4A)和转移(4B)的活性。
实施例8活性化合物6i对两种骨肉瘤细胞PDGFR信号通路相关蛋白的抑制活性
将化合物预处理骨肉瘤细胞株48h后，吸掉培养基，PBS洗涤3次，裂解后提取总蛋白，利用Western Blot检测PDGFR信号通路相关蛋白的磷酸化水平。
结果见图2，结果表明，0.1、0.2和0.4uM的活性化合物6i相对于阳性药物均具有强烈的剂量依赖性抑制骨肉瘤细胞MG63和MNNG相关信号通路蛋白磷酸化活性。
实施例8活性化合物对两种人新生血管细胞的抑制活性试验
采用与实施例6相同的MTT法，测定活性化合物6i对两种视网膜细胞的抑制活性。
表6.MTT法测定活性化合物对三种人视网膜细胞的抑制活性(IC50，uM)
Figure BDA0002885726690000281
结果表明，活性化合物6i相对于多个阳性药物均具有强烈的抑制人永生化人脑微血管内皮细胞(HCMEC/D3)和人脑血管周细胞(HBVP)的活性，而低浓度下对人的正常视网膜细胞ARPE-19几乎无抑制活性，表明活性化合物6i可以有效抑制眼底血管增生，进而缓解眼科疾病湿性黄斑变性或葡萄膜炎，且毒性较低。

Claims (7)

1.一种嘧啶类小分子化合物，其特征在于，为式（III）所示的化合物或其盐中的一种；
Figure DEST_PATH_IMAGE002
（III）
式（III）中，R4为取代或者未取代的苯基，所述苯基上的取代基为C1~C5烷基、C1~C5烷氧基、哌嗪基、取代的哌嗪基、1,1-二氧化硫代吗啉基、哌啶基、取代的哌啶基，所述哌嗪基和哌啶基上的取代基选自卤素；
或者化合物6a、化合物6e、化合物6f、化合物6g、化合物6h、化合物6i、化合物6j、化合物6k、化合物6m、化合物6n或其盐中的一种；
结构式如下：
Figure DEST_PATH_IMAGE004
Figure DEST_PATH_IMAGE006
Figure DEST_PATH_IMAGE008
。
2.根据权利要求1所述的嘧啶类小分子化合物，其特征在于，所述苯基上的取代基为甲基、甲氧基、哌嗪基、取代的哌嗪基、1,1-二氧化硫代吗啉基、哌啶基、取代的哌啶基，所述哌嗪基和哌啶基上的取代基选自F或者Cl。
3.根据权利要求1所述的嘧啶类小分子化合物，其特征在于，为化合物6a、化合物6b、化合物6c、化合物6d、化合物6e、化合物6f、化合物6g、化合物6h、化合物6i、化合物6j、化合物6k、化合物6m、化合物6n或其盐中的一种；
结构式如下：
Figure DEST_PATH_IMAGE010
Figure DEST_PATH_IMAGE012
Figure DEST_PATH_IMAGE014
Figure DEST_PATH_IMAGE016
。
4.一种如权利要求1~3任一项所述的嘧啶类小分子化合物的应用，其特征在于，所述的嘧啶类小分子化合物用于制备PDGFRs抑制剂。
5.根据权利要求4所述的嘧啶类小分子化合物的应用，其特征在于，所述的嘧啶类小分子化合物用于制备抗肿瘤药物。
6.根据权利要求5所述的嘧啶类小分子化合物的应用，其特征在于，所述的抗肿瘤药物用于抑制骨肉瘤细胞。
7.根据权利要求4所述的嘧啶类小分子化合物的应用，其特征在于，所述的嘧啶类小分子化合物用于制备治疗眼科疾病的药物；
所述的药物用于抑制眼底血管增生，进而缓解眼科疾病湿性黄斑变性或葡萄膜炎。
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