CN111303155A - 靶向pak1抑制剂及其在抗肿瘤治疗药物中的应用 - Google Patents

靶向pak1抑制剂及其在抗肿瘤治疗药物中的应用 Download PDF

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CN111303155A
CN111303155A CN202010174638.2A CN202010174638A CN111303155A CN 111303155 A CN111303155 A CN 111303155A CN 202010174638 A CN202010174638 A CN 202010174638A CN 111303155 A CN111303155 A CN 111303155A
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姚大红
王金辉
黄健
贺震旦
张瑾
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Abstract

本发明涉及靶向PAK1抑制剂及其在制备抗肿瘤药物中的应用，属于肿瘤治疗药物技术领域。本发明提供结构式如式Ⅰ‑IV所示的化合物或其药学上可接受的盐，其中R1和X如权利要求书和说明书所述。本发明还提供一种药物组合物，包含有效剂量的上述化合物或其药学上可接受的盐。本发明所述的化合物或其药学上可接受的盐或其药物组合物可以作为PAK1抑制剂用于制备抗肿瘤药物。

Description

靶向PAK1抑制剂及其在抗肿瘤治疗药物中的应用
技术领域
本发明涉及靶向PAK1抑制剂及其在制备抗肿瘤药物中的应用，属于肿瘤治疗药物技术领域。
背景技术
乳腺癌是临床上女性常见的高发恶性肿瘤之一，据国际癌症研究中心最新统计表明，全球每年就有170万女性被确诊为乳腺癌，占女性肿瘤发病率的25％，导致50万患者死亡[1]。临床上根据基因表达的差异，将所有乳腺癌分为四大亚型：HER2过表达型，LuminalA型，Luminal B型以及三阴性乳腺癌(TNBC)。TNBC是指雌激素受体(ER)，孕激素受体(PR)和人表皮生长因子受体2(Her-2)均为阴性的乳腺癌，占所有乳腺癌病理类型的15-20％左右，具有特殊的生物学行为和临床病理特征，与其他亚型乳腺癌相比具有细胞分化差、侵袭性病程、远端转移高等特点，因此预后较差，早期复发风险高、五年生存率极低。由于缺乏内分泌治疗和靶向治疗的有效靶标，TNBC的治疗已经成为目前乳腺癌研究领域的难点之一。
根据NCCN(National Comprehensive Cancer Network)指南，目前临床上常用的TNBC治疗手段仍然是手术和常规的全身细胞毒化疗，含蒽环和紫杉烷类药物的方案在已广泛应用于术后辅助治疗，但是密集型，高剂量的化疗会产生巨大的毒性，且肿瘤复发或转移后往往缺乏有效的药物，治疗效果并不理想，预后依然很差。近年来随着医学生物技术的高速发展，通过对TNBC发生发展的信号通路网络的基础研究，小分子靶向治疗成为TNBC治疗研究的热点，并取得了长足的进步。目前针对TNBC处于热点研究领域的相关信号通路，靶标主要有EGFR，MAPK-ERK，PI3K-AKT-mTOR,PARP，热休克蛋白(HSPs)及组蛋白去乙酰化酶(HDAC)等。尤其是PARP抑制剂如Veliparib在体外实验、动物实验以及一、二期临床试验显示出令人鼓舞的抗瘤效果，Veliparib联合化疗较单纯吉西他滨和卡铂化疗改善TNBC患者OS和PFS的相关临床实验也正在开展。因此，在TNBC药物治疗领域，如何发现新的治疗靶标，有针对性的设计出新型靶向治疗小分子药物，并深入探讨其作用机制是解决临床问题的关键，具有重要的研究意义和价值。PAK1作为一个重要的癌基因，在乳腺癌中异常高表达，针对其设计靶向性的小分子抑制剂，是治疗乳腺癌，也别是三阴性乳腺癌重要的策略。
发明内容
本发明解决的技术问题是提供一种作为PAK1抑制剂的化合物。
本发明提供结构式如式Ⅰ-IV所示的化合物或其药学上可接受的盐：
Figure BDA0002410372550000021
其中，R1为氢、C1-C6烷基、C1-C6烷氧基、卤素、硝基、氨基、卤代C1-C6烷基、6-10元芳基或杂芳基，所述的芳基或杂芳基可以被一个或多个R2取代，R2为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基；R1、R2为单取代或多取代；
X为O或S。
本发明优选如下结构的化合物或其药学上可接受的盐：
其中，R1为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基、苯基、萘基，所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基；R1、R2为单取代或多取代；
X为O或S。
本发明优选如下结构的化合物或其药学上可接受的盐：
其中，R1为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基、苯基、萘基，所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；R1、R2为单取代或多取代；
X为O或S。
本发明优选如下结构的化合物或其药学上可接受的盐：
其中，
式(I)中：
R1为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O；
式(II)中：
R1为苯基、萘基；所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O或S；
式(III)中：
R1为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O；
式(IV)中：
R1为苯基、萘基；所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O；
本发明优选如下结构的化合物或其药学上可接受的盐：
Figure BDA0002410372550000041
Figure BDA0002410372550000051
Figure BDA0002410372550000061
本发明还提供了所述的化合物的药学上可接受的盐。药学上可接受的盐可以为所述化合物的硝酸盐、盐酸盐、硫酸盐、磷酸盐或柠檬酸盐等。
本发明还提供一种药物组合物，它是包含有效剂量的上述化合物或其药学上可接受的盐的制剂。可以通过本领域已知的方法可将本发明化合物制成以下形式：片剂、胶囊剂、水性或油性溶液剂、混悬剂、乳剂、乳膏剂、软膏剂、凝胶剂、喷鼻剂、栓剂、用于吸入的细小分散的粉剂或气雾剂或喷雾剂、用于胃肠道外(包括静脉内、肌内或输注)的无菌水性或油性溶液或混悬剂或无菌乳剂。可采用无菌水或水-丙二醇溶液作为溶剂来制备液体制剂，还可将活性组分配制在聚乙二醇水溶液中。用于口服给予的水性溶液可通过将活性组分溶解在水中并按需要加入合适的着色剂、矫味剂、稳定剂和增稠剂来制备。口服使用的水性混悬剂可通过将细小分散的活性组分与粘性物质一道分散在水中，所述粘性物质如为天然合成胶、树脂、甲基纤维素、羧甲基纤维素和其他药剂领域已知的悬浮剂。
药物组合物可为单位剂量形式。在这些形式中，将所述组合物分成含适量活性组分的单位剂量。该单位剂量形式可为包装制剂，包装中包括分隔量的制剂，例如盒装片剂、胶囊剂和在管形瓶或安瓿中的粉剂。单位剂量形式还可为胶囊剂、扁囊剂或片剂或其可为适当数量的任何这些包装形式。
本发明的药物组合物，其活性成分可仅为本发明的化合物，也可与其他抗肿瘤化合物组合作为活性成分。
本发明还提供上述化合物或其药学上可接受的盐或其药物组合物在制备PAK抑制剂中的应用。
本发明进一步提供了上述化合物或其药学上可接受的盐或其药物组合物在制备PAK1抑制剂中的应用。
进一步地，本发明还提供了上述化合物或其药学上可接受的盐或其药物组合物在制备治疗抗肿瘤药物中的应用。
所述的肿瘤为肝癌、肺癌、乳腺癌、肾癌、
以本发明的化合物治疗肿瘤过程中，可采用本发明的药物组合物与其他抗肿瘤药进行联合治疗。
在治疗肿瘤时，可通过同时、顺序或单独给予各种治疗成分可实现这种联合治疗。此类组合产品应用有效剂量范围内的本发明化合物和准许剂量范围内的其他药学活性剂。
附图说明
图1为MDA-MB-231细胞用化合物49处理24和48小时，MTT检测细胞存活率。
图2为MDA-MB-231细胞用0，10，20，40μM化合物49处理24小时，Hoechst33258荧光染色图。
图3为MDA-MB-231细胞用0，10，20，40μM化合物49处理24小时，Annexin V-PI流式检测细胞凋亡率。
图4为MDA-MB-231细胞用0，10，20，40μM化合物49处理24小时，Transwell检测细胞的迁移。
具体实施方式
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实施例范围之中。
实施例1化合物1～29的合成
化合物1～29采用如下反应式合成：
Figure BDA0002410372550000081
流程1合成路线和条件(i)N,N-二异丙基乙胺，异丙醇，回流，4h；(ii)甲醇，HCl，室温，3h；(iii)酰氯衍生物，三乙胺，二氯甲烷，室温或者异氰酸酯衍生物(异硫氰酸酯衍生物)，三乙胺，氯仿，60℃。
中间体3a制备
将4-chloro-1H-pyrazolo[3,4-d]pyrimidine(30mmol)溶于异丙醇(60ml)中，加入DIEA(45mmol)和叔丁基4-氨基哌啶-1-羧酸盐(39mmol)，使混合物连续升温回流4小时。反应完成后，将残渣蒸发并加入50ml水，用三氯甲烷萃取。有机层用饱和的碳酸氢钠和盐水清洗，然后在无水硫酸钠上干燥。将溶剂在减压下除去后得粗品。采用硅胶色谱(二氯甲烷/甲醇30:1)对进行纯化，得到白色固体，收率89％。1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.23(1H,s),8.19(1H,d,J＝7.7Hz),8.18(1H,s),4.40(2H,m),3.21(1H,m),3.03(2H,m),1.98(2H,m),1.50(2H,m)；1.48(9H,s)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.9,156.0,154.8,137.1,132.8,100.3,84.1,45.4,45.4,31.8,31.8,33.3,33.3,33.3.
中间体4a合成
取250ml圆底烧瓶，加入90ml HCl气饱和甲醇，0℃，充分搅拌4小时，加入适量中间3a(20mmol)。实验完成后，过滤出中间产物4a为白色固体，直接用于下一步反应，不进行纯化，收率为95％。
化合物1-29合成通法
在二氯甲烷(10ml)中加入中间体4a(1mmol)和DIEA(1.1mmol)，室温滴加二氯甲烷(2ml)中酰氯衍生物或异氰酸酯衍生物(1.1eq)溶液3小时。用水、饱和的碳酸氢钠和盐水清洗混合物，然后在无水硫酸钠上干燥。减压脱除溶剂后，用硅胶闪蒸层析法纯化粗品，用二氯甲烷/甲醇(10-20％)洗脱，得率70-90％。
以下为化合物1-29的核磁检测结果。
化合物1,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.23(1H,s),8.19(1H,d,J＝7.7Hz),8.18(1H,s),7.30(2H,d,J＝8.3Hz),7.26(2H,d,J＝8.3Hz),4.40(2H,m),3.21(1H,m),3.03(2H,m),2.34(3H,s),1.98(2H,m),1.50(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):169.6,156.0,154.8,139.5,133.7,132.9,129.8,129.4,129.4,127.2,127.2,100.3,47.4,46.5,46.5,32.2,32.2,21.4；HRMS(ESI)+calculated for C18H21N6O,[M+H]+:m/z 337.1771,found 337.1750；
化合物2,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.22(1H,s),8.12(1H,s),8.10(1H,d,J＝7.7Hz),7.34-7.25(3H,m),7.16(1H,m),4.56(1H,d,J＝12.8Hz),4.39(1H,m),3.35(1H,d,J＝12.8Hz),3.16(1H,t,J＝12.4Hz),2.99(1H,t,J＝12.4Hz),2.25(3H,s),2.07(1H,d,J＝10.9Hz),1.88(1H,d,J＝10.9Hz),1.53(1H,m),1.37(1H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.9,156.0,154.8,137.1,134.0,132.8,130.6,129.0,126.3,126.0,125.7,100.3,47.4,45.4,45.4,32.7,31.8,19.1；HRMS(ESI)+calculatedfor C20H25N6O,[M+H]+:m/z 337.1771,found 337.1750.
化合物3,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.22(1H,s),8.12(1H,s),8.10(1H,d,J＝7.7Hz),6.90(1H,s),6.88(1H,s),4.61(1H,d,J＝13.1Hz),4.38(1H,m),3.28(1H,d,J＝13.1Hz),3.13(1H,t,J＝12.0Hz),2.97(1H,t,J＝12.0Hz),2.24(3H,s),2.21(3H,s),2.10(3H,s),2.07(1H,d,J＝10.8Hz),1.89(1H,d,J＝10.8Hz),1.49(1H,m),1.38(1H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.4,156.0,154.8,137.5,134.2,133.4,133.7,128.5,128.5,128.3,128.3,100.3,47.4,44.9,44.9,32.6,31.9,21.1,19.2,19.0；HRMS(ESI)+calculated for C20H25N6O,[M+H]+:m/z 365.2084,found 365.2144.
化合物4,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.41(1H,s),8.22(1H,s),8.20(1H,d,J＝7.6Hz),8.12(1H,s),7.48(2H,d,J＝7.8Hz),7.30(2H,d,J＝7.8Hz),4.41(2H,m),3.22(1H,m),3.03(2H,m),1.97(2H,m),1.53(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.6,164.2,161.2,155.9,154.8,135.1,133.0,129.8,129.7,116.0,115.8,100.3,47.4,46.5,46.5,31.7,31.7；HRMS(ESI)+calculated for C17H18FN6O,[M+H]+:m/z 341.1521,found 341.1535.
化合物5,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.23(1H,s),8.14(1H,s),8.08(1H,d,J＝7.4Hz),7.54(2H,d,J＝8.5Hz),7.44(2H,d,J＝8.5Hz),4.40(2H,m),3.61(1H,m),3.24(1H,m),3.02(1H,m),2.06(1H,m),1.96(1H,m),1.50(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.5,156.0,154.9,135.4,134.6,132.8,132.8,129.1,129.1,129.0,129.0,100.3,47.4,46.5,41.1,32.4,31.6；HRMS(ESI)+calculated forC17H18ClN6O,[M+H]+:m/z 357.1225,found 357.1278.
化合物6,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.22(1H,s),8.19(1H,s),8.17(1H,d,J＝7.7Hz),7.57-7.50(1H,m),7.48-7.41(2H,m),7.40–7.34(1H,m),4.52(1H,d,J＝13.8Hz),4.39(1H,m),3.32(1H,d,J＝13.8Hz),3.16(1H,t,J＝11.0Hz),2.99(1H,t,J＝11.0Hz),2.07(1H,d,J＝12.8Hz),1.90(1H,d,J＝12.8Hz),1.56(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.9,165.7,156.0,154.9,136.6,136.4,132.9,130.9,130.8,129.9,129.5,100.3,47.3,46.0,45.4,32.3,31.6；HRMS(ESI)+calculated forC17H18ClN6O,[M+H]+:m/z 357.1225,found 357.1278.
化合物7,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.23(1H,s),8.20(1H,d,J＝7.9Hz),8.18(1H,s),7.67(2H,d,J＝8.4Hz),7.36(2H,d,J＝8.4Hz),4.40(2H,m),3.23(1H,m),3.03(2H,m),2.00(2H,m),1.52(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.5,156.0,135.8,132.9,132.1,131.9,131.9,131.8,129.4,129.4,123.3,100.3,47.4,46.5,46.5,32.1,32.1；HRMS(ESI)+calculated for C17H18BrN6O,[M+H]+:m/z 403.0720,found 403.0707.
化合物8,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.22(1H,s),8.20(1H,s),8.18(1H,d,J＝7.7Hz),7.85(2H,d,J＝8.1Hz),7.63(2H,d,J＝8.1Hz),4.48(1H,d,J＝11.8Hz),4.39(1H,m),3.55(1H,d,J＝11.8Hz),3.24(1H,t,J＝11.0Hz),2.99(1H,t,J＝11.0Hz),2.08(1H,d,J＝12.8Hz),1.94(1H,d,J＝12.8Hz),1.58(1H,d,J＝10.0Hz),1.48(1H,d,J＝10.0Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.5,156.0,154.9,135.4,134.6,132.9,132.8,127.9,127.9,126.4,126.0,126.0,100.3,47.4,46.5,41.1,32.4,31.6；HRMS(ESI)+calculated for C18H18F3N6O,[M+H]+:m/z 391.1489,found 391.1474.
化合物9,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.41(1H,s),8.24(1H,s),8.22(1H,d,J＝7.7Hz),8.21(1H,s),7.85(1H,dd,J＝1.31,1.37Hz),7.72-7–7.74(3H,m),7.36(1H,dd,J＝7.7,1.4Hz),4.46(1H,m),4.40(1H,m),3.26(1H,m),3.03(2H,m),2.06(1H,m),1.95(1H,m),1.52(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):167.9,156.0,154.8,137.7,133.0,131.1,130.2,129.9,129.6,126.6,123.8,100.3,47.4,46.5,46.5,32.2,31.5；HRMS(ESI)+calculated for C18H18F3N6O,[M+H]+:m/z 391.1489,found 391.1474.
化合物10,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.21(1H,s),8.19(1H,d,J＝7.7Hz),8.18(1H,s),7.72(1H,dd,J＝8.1,1.42Hz),7.47(1H,dd,J＝7.8,7.9Hz),7.36(1H,dd,J＝7.7,1.4Hz),4.51(1H,t,J＝14.3Hz),4.39(1H,m),3.20(1H,m),3.03(2H,m),2.10(1H,m),1.91(1H,m),1.98(2H,m),1.55(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.1,164.9,156.0,154.9,138.8,132.6,131.3,129.6,127.8,126.8,126.6,100.3,47.2,46.0,46.0,32.4,32.2；HRMS(ESI)+calculated for C17H17Cl2N6O,[M+H]+:m/z 391.0835,found 391.0870.
化合物11,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.38(1H,s),8.46(1H,s),8.23(1H,s),8.12(1H,s),8.04(1H,d,J＝7.5Hz),7.34(2H,d,J＝8.4Hz),7.02(2H,d,J＝8.4Hz),4.33(1H,m),4.14(1H,d,J＝13.3Hz),2.95(2H,t,J＝12.3Hz),2.22(3H,s),1.97(2H,d,J＝11.7Hz),1.50(1H,d,J＝11.3Hz),1.47(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.6,155.0,138.5,132.8,130.9,129.2,129.2,120.3,120.3,100.3,47.6,43.6,43.6,32.0,32.0,20.8；HRMS(ESI)+calculated for C18H22N7O,[M+H]+:m/z 352.1880,found 352.1882.
化合物12,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.39(1H,s),8.66(1H,s),8.23(1H,s),8.18(1H,s),8.15(1H,d,J＝7.3Hz),7.92(1H,s),7.04(2H,m),4.36(1H,m),4.16(1H,d,J＝13.3Hz),2.98(2H,t,J＝12.3Hz),2.17(6H,s),1.97(2H,d,J＝11.7Hz),1.52(1H,d,J＝11.3Hz),1.49(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.8,155.0,137.2,136.4,136.4,132.9,127.9,127.9,126.2,100.3,47.9,43.7,43.7,32.1,32.1,19.1,18.6；HRMS(ESI)+calculated for C18H22N7O,[M+H]+:m/z 366.2037,found366.2076.
化合物13,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.38(1H,s),8.41(1H,s),8.22(1H,s),8.14(1H,s),8.06(1H,d,J＝7.3Hz),7.35(2H,d,J＝8.9Hz),6.82(2H,d,J＝8.9Hz),4.33(1H,m),4.14(2H,d,J＝13.3Hz),3.69(3H,s),2.95(2H,t,J＝12.3Hz),1.97(2H,d,J＝11.7Hz),1.50(1H,d,J＝11.3Hz),1.47(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.7,155.0,154.9,134.1,132.7,122.0,122.0,113.9,113.9,100.3,55.6,47.6,43.5,43.5,32.0,32.0；HRMS(ESI)+calculated for C18H22N7O2,[M+H]+:m/z 368.1829,found 368.1813.
化合物14,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.37(1H,s),8.76(1H,s),8.23(1H,s),8.12(1H,s),8.04(1H,d,J＝7.4Hz),7.67(1H,d,J＝1.8Hz),7.41(1H,dd,J＝8.4,1.4Hz),7.25(1H,dd,J＝8.4,7.4Hz),6.97(1H,dd,J＝8.0,1.4Hz),4.35(1H,m),4.13(2H,d,J＝13.3Hz),2.99(2H,t,J＝12.3Hz),1.98(2H,d,J＝11.7Hz),1.50(1H,d,J＝11.3Hz),1.48(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.4,154.9,137.3,132.8,129.7,128.9,127.7,127.6,126.0,100.3,47.7,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C17H19ClN7O,[M+H]+:m/z 372.1334,found 3372.1361.
化合物15,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.39(1H,s),8.28(1H,s),8.23(1H,s),8.17(1H,s),8.13(1H,d,J＝7.2Hz),7.48(1H,dd,J＝8.1,1.4Hz),7.45(1H,dd,J＝8.1,1.4Hz),7.29(1H,td,J＝7.7,1.3Hz),7.14(1H,td,J＝7.7,1.3Hz),4.36(1H,m),4.14(2H,d,J＝13.3Hz),3.01(2H,t,J＝12.3Hz),1.98(2H,d,J＝11.7Hz),1.55(1H,d,J＝11.3Hz),1.52(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.4,154.9,137.3,132.8,128.2,128.2,128.1,126.3,124.8,100.3,47.7,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C21H22N7O,[M+H]+:m/z 372.1334,found 372.1361.
化合物16,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.38(1H,s),8.74(1H,s),8.22(1H,s),8.14(1H,s),8.08(1H,d,J＝7.3Hz),7.47(2H,d,J＝8.9Hz),7.39(2H,d,J＝8.9Hz),4.34(1H,m),4.15(2H,d,J＝13.3Hz),2.98(2H,t,J＝12.3Hz),1.97(2H,d,J＝11.7Hz),1.50(1H,d,J＝11.3Hz),1.45(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.9,155.2,140.6,132.7,131.5,131.5,121.9,121.9,113.5,100.3,53.8,47.5,43.4,43.4,32.0,32.0；HRMS(ESI)+calculated for C17H19BrN7O,[M+H]+:m/z416.0829,found 416.0804.
化合物17,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.39(1H,s),8.99(1H,s),8.24(1H,s),8.14(1H,s),8.08(1H,d,J＝6.8Hz),7.71(2H,d,J＝8.5Hz),7.59(2H,d,J＝8.5Hz),4.36(1H,m),4.18(1H,d,J＝13.3Hz),3.02(2H,t,J＝12.3Hz),2.00(2H,dd,J＝2.9,12.3Hz),1.52(1H,td,J＝12.4,4.0Hz),1.47(1H,td,J＝12.4,4.0Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.9,155.0,145.0,132.7,126.0,126.0,123.4,121.8,119.3,119.3,100.3,47.5,43.6,43.6,32.0,32.0；HRMS(ESI)+calculated forC18H19F3N7O,[M+H]+:m/z 406.1598,found 406.1608.
化合物18,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.39(1H,s),8.40(1H,s),8.23(1H,s),8.17(1H,s),8.14(1H,d,J＝7.2Hz),7.39(1H,dd,J＝8.1,1.4Hz),7.22(1H,d,J＝1.4Hz),7.01(1H,d,J＝8.1Hz),4.34(1H,m),4.12(2H,d,J＝13.3Hz),2.99(2H,t,J＝12.3Hz),1.98(2H,d,J＝11.7Hz),1.52(1H,d,J＝11.3Hz),1.49(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.8,155.2,154.8,154.7,143.4,132.9,130.9,130.6,119.2,100.3,46.6,43.5,43.5,31.9,31.9；HRMS(ESI)+calculated forC17H18F2N7O,[M+H]+:m/z 374.1535,found 374.1510.
化合物19,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.38(1H,s),8.87(1H,s),8.24(1H,s),8.13(1H,s),8.05(1H,d,J＝7.4Hz),7.47(2H,s),4.36(1H,m),4.14(2H,d,J＝13.3Hz),3.00(2H,t,J＝12.3Hz),1.99(2H,d,J＝11.7Hz),1.51(1H,d,J＝11.3Hz),1.48(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.4,154.9,137.3,132.8,128.2,128.2,128.1,126.3,124.8,100.3,47.7,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C17H18Cl2N7O,[M+H]+:m/z 406.0944,found 406.0992.
化合物20,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.38(1H,s),8.40(1H,s),8.22(1H,s),8.18(1H,s),8.16(1H,d,J＝7.7Hz),7.59(1H,d,J＝2.0Hz),7.50(1H,d,J＝8.6Hz),7.37(1H,dd,J＝8.6,2.0Hz),4.36(1H,m),4.13(2H,d,J＝13.3Hz),3.01(2H,t,J＝12.3Hz),1.97(2H,d,J＝11.7Hz),1.55(1H,d,J＝11.3Hz),1.54(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):180.9,156.9,156.1,155.1,142.8,133.2,132.7,130.4,121.6,119.2,118.1,100.3,47.5,43.5,43.5,32.0,32.0；HRMS(ESI)+calculated forC17H18Cl2N7O,[M+H]+:m/z 406.0944,found 406.0992.
化合物21,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.39(1H,s),9.40(1H,s),9.21(1H,s),8.26(1H,s),8.22(1H,s),8.16(1H,d,J＝7.2Hz),7.57(1H,s),4.36(1H,m),4.19(2H,d,J＝13.3Hz),3.11(2H,t,J＝12.3Hz),1.99(2H,d,J＝11.7Hz),1.53(1H,d,J＝11.3Hz),1.51(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.1,156.0,155.8,154.7,143.4,132.9,130.9,130.6,125.3,125.3,122.6,119.2,119.0,100.3,46.6,43.5,43.5,31.8,31.8；HRMS(ESI)+calculated for C19H18F6N7O,[M+H]+:m/z 474.1472,found474.1500.
化合物22,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.28(1H,s),8.23(1H,s),8.17(1H,s),8.13(1H,d,J＝7.3Hz),7.48(1H,dd,J＝8.1,1.4Hz),7.45(1H,dd,J＝8.1,1.4Hz),7.29(1H,td,J＝7.7,1.3Hz),7.14(1H,td,J＝7.7,1.3Hz),4.36(1H,m),4.14(2H,d,J＝13.3Hz),3.01(2H,t,J＝12.3Hz),1.98(2H,d,J＝11.7Hz),1.55(1H,d,J＝11.3Hz),1.52(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.0,155.4,154.9,136.1,134.2,132.9,132.8,129.9,128.4,126.2,125.9,125.8,125.3,123.9,100.3,47.7,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C21H22N7O,[M+H]+:m/z 388.1880,found 388.1924.
化合物23,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),9.28(1H,s),8.24(1H,s),8.13(1H,s),8.07(1H,d,J＝7.8Hz),7.16(2H,d,J＝8.3Hz),7.11(2H,d,J＝8.3Hz),4.72(2H,d,J＝13.3Hz),4.46(1H,m),3.28(2H,t,J＝12.3Hz),2.28(3H,s),2.01(2H,d,J＝11.7Hz),1.60(1H,d,J＝11.3Hz),1.57(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):180.9,156.9,156.1,156.0,138.9,136.7,136.7,132.8,128.0,128.0,126.8,100.3,47.5,43.4,43.4,32.0,32.0,18.5；HRMS(ESI)+calculated for C18H22N7S,[M+H]+:m/z 368.1652,found 368.1645.
化合物24,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),8.94(1H,s),8.31(1H,s),8.25(1H,s),8.15(1H,s),8.11(1H,d,J＝7.5Hz),7.07(3H,s),4.79(2H,d,J＝13.3Hz),4.49(1H,m),3.31(2H,t,J＝12.3Hz),2.17(6H,s),2.03(2H,d,J＝11.7Hz),1.59(1H,d,J＝11.3Hz),1.56(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):180.3,156.0,155.2,154.8,136.6,132.9,129.7,129.2,129.1,128.6,127.7,100.3,47.5,43.6,43.6,31.9,31.9,18.4,17.2；HRMS(ESI)+calculated for C19H24N7S,[M+H]+:m/z 382.1808,found382.1881.
化合物25,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),9.24(1H,s),8.24(1H,s),8.15(1H,s),8.10(1H,d,J＝7.4Hz),7.17(2H,d,J＝8.8Hz),6.87(2H,d,J＝8.8Hz),4.74(2H,d,J＝13.3Hz),4.46(1H,m),3.74(3H,s),3.28(2H,t,J＝12.3Hz),2.02(2H,d,J＝11.7Hz),1.60(1H,d,J＝11.3Hz),1.57(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):181.8,156.9,156.1,156.0,134.5,132.8,127.8,127.8,113.6,113.6,124.8,100.3,47.7,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C18H22N7OS,[M+H]+:m/z384.1601,found 384.1615.
化合物26,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),9.26(1H,s),8.25(1H,s),8.14(1H,s),8.09(1H,d,J＝7.5Hz),7.49(1H,d,J＝7.9Hz),7.36–7.25(3H,m),4.76(2H,d,J＝13.3Hz),4.49(1H,m),3.33(2H,t,J＝12.3Hz),2.04(2H,d,J＝11.7Hz),1.62(1H,d,J＝11.3Hz),1.60(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):180.3,156.0,155.2,154.8,136.6,132.9,129.7,129.2,129.1,128.6,127.7,100.3,47.5,43.6,43.6,31.9,31.9,18.4,17.2；HRMS(ESI)+calculated for C17H19ClN7S,[M+H]+:m/z388.1106,found 388.1151.
化合物27,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),9.47(1H,s),8.25(1H,s),8.13(1H,s),8.08(1H,d,J＝7.6Hz),7.43(1H,dd,J＝1.5,1.4Hz),7.33(1H,dd,J＝8.0,7.7Hz),7.29(1H,dd,J＝7.7,1.5Hz),7.14(1H,dd,J＝8.0,1.4Hz),4.72(2H,d,J＝13.3Hz),4.48(1H,m),3.30(2H,t,J＝12.3Hz),2.05(2H,d,J＝11.7Hz),1.61(1H,d,J＝11.3Hz),1.58(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):181.3,156.9,156.1,156.0,143.2,132.8,132.5,130.0,124.9,124.2,123.7,100.3,47.8,47.2,47.2,31.8,31.8；HRMS(ESI)+calculated for C17H19ClN7S,[M+H]+:m/z 388.1106,found 388.1151.
化合物28,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.40(1H,s),9.86(1H,s),8.25(1H,s),8.13(1H,s),8.11(2H,s),8.08(1H,d,J＝7.6Hz),7.76(1H,s),4.76(2H,d,J＝13.3Hz),4.51(1H,m),3.31(2H,t,J＝12.3Hz),2.08(2H,d,J＝11.7Hz),1.64(1H,d,J＝11.3Hz),1.62(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):180.7,156.9,156.1,156.0,143.7,132.8,130.3,129.9,125.2,124.7,122.4,119.7,116.8,100.3,47.9,47.1,47.1,31.8,31.8；HRMS(ESI)+calculated for C19H18F6N7S,[M+H]+:m/z 490.1243,found490.1339.
化合物29,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.42(1H,s),9.56(1H,s),8.27(1H,s),8.17(1H,s),8.14(1H,d,J＝7.7Hz),7.79(1H,m),7.86(1H,m),7.84(1H,d,J＝8.2Hz),7.55–7.50(3H,m),7.32(1H,d,J＝7.2Hz),4.86(2H,d,J＝13.3Hz),4.53(1H,m),3.33(2H,t,J＝12.3Hz),2.09(2H,d,J＝11.7Hz),1.70(1H,d,J＝11.3Hz),1.67(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):182.7,156.1,156.0,154.9,137.9,134.3,132.8,131.2,128.4,126.9,126.5,126.3,126.2,126.0,124.1,100.3,47.7,47.4,47.4,31.9,31.9；HRMS(ESI)+calculated for C21H22N7S,[M+H]+:m/z 404.1652,found 404.1667.
实施例2 30-49的合成
Figure BDA0002410372550000141
流程2合成路线和条件(i)N,N-二异丙基乙胺，异丙醇，回流，4h；(ii)甲醇，HCl，室温，3h；(iii)酰氯衍生物，三乙胺，二氯甲烷，室温或者异氰酸酯衍生物(异硫氰酸酯衍生物)，三乙胺，氯仿，60℃。
中间体3b制备
将4-chloro-1H-pyrazolo[3,4-d]pyrimidine(30mmol)溶于异丙醇(60ml)中，加入DIEA(45mmol)和叔丁基4-氨基哌啶-1-羧酸盐(39mmol)，使混合物连续升温回流4小时。反应完成后，将残渣蒸发并加入50ml水，用三氯甲烷萃取。有机层用饱和的碳酸氢钠和盐水清洗，然后在无水硫酸钠上干燥。将溶剂在减压下除去后得粗品。采用硅胶色谱(二氯甲烷/甲醇30:1)对进行纯化，得到白色固体，收率89％。1H-NMR(400MHz,CDCl3),δ(ppm):8.50(1H,d,J＝5.5Hz),7.31(1H,m),6.88(1H,d,J＝5.5Hz),1.48(9H,s)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.9,156.0,154.7,137.0,132.6,100.3,84.1,45.4,45.4,31.8,31.8,33.1,33.1,33.1.
中间体4b合成
取250ml圆底烧瓶，加入90ml HCl气饱和甲醇，0℃，充分搅拌4小时，加入适量中间3b(20mmol)。实验完成后，过滤出中间产物4a为白色固体，直接用于下一步反应，不进行纯化，收率为91％。
化合物30-50合成通法
在二氯甲烷(10ml)中加入中间体4b(1mmol)和DIEA(1.1mmol)，室温滴加二氯甲烷(2ml)中酰氯衍生物或异氰酸酯衍生物(1.1eq)溶液3小时。用水、饱和的碳酸氢钠和盐水清洗混合物，然后在无水硫酸钠上干燥。减压脱除溶剂后，用硅胶闪蒸层析法纯化粗品，用二氯甲烷/甲醇(10-20％)洗脱，得率70-90％。
以下为化合物30-50的核磁检测结果。
化合物30,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.31(1H,s),8.28(1H,d,J＝7.9Hz),8.25(1H,s),7.77(2H,d,J＝8.2Hz),7.25(2H,d,J＝8.2Hz),4.70(2H,m),4.19(1H,m),3.35(2H,m),2.34(3H,s),1.96(2H,d,J＝13.4Hz),1.60(1H,d,J＝11.3Hz),1.57(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.9,156.7,156.1,155.2,141.4,134.1,132.2,129.2,129.2,127.8,127.8,99.7,46.8,44.4,44.4,31.6,31.6,21.4；HRMS(ESI)+calculated for C18H21N6O,[M+H]+:m/z 337.1771,found 337.1750.
化合物31,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.57(1H,d,J＝7.9Hz),8.30(1H,s),8.26(1H,s),8.24(1H,s),7.33–7.28(2H,m),7.24–7.19(2H,m),4.64(2H,m),4.16(1H,m),3.35(2H,m),2.33(3H,s),2.00(2H,dd,J＝13.4,2.4Hz),1.53(1H,d,J＝11.5Hz),1.50(1H,d,J＝11.5Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.9,156.6,156.1,155.2,137.8,135.3,134.1,130.7,129.6,127.4,125.9,99.7,46.5,44.4,44.4,31.6,31.6,19.7；HRMS(ESI)+calculated for C18H21N6O,[M+H]+:m/z 337.1771,found337.1750.
化合物32,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.55(1H,s),8.30(1H,s),8.23(1H,s),6.87(1H,s),6.84(1H,s),4.63(2H,m),4.18(1H,m),3.36(2H,m),2.22(6H,s),2.22(3H,s),1.99(2H,dd,J＝13.4,2.4Hz),1.49(1H,d,J＝11.5Hz),1.47(1H,d,J＝11.5Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.8,156.6,155.2,138.4,137.5,136.4,134.1,133.8,128.4,128.1,99.7,46.2,44.4,44.4,31.6,31.6,21.1,19.8,19.2；HRMS(ESI)+calculatedfor C20H25N6O,[M+H]+:m/z 365.2084,found 365.2061.
化合物33,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.31(1H,s),8.26(1H,s),8.18(1H,d,J＝7.8Hz),7.84(2H,d,J＝8.7Hz),6.98(2H,d,J＝8.7Hz),4.71(2H,m),4.18(1H,m),3.31(2H,m),1.97(2H,dd,J＝2.9,12.4Hz),1.59(1H,td,J＝12.4,4.0Hz),1.56(1H,td,J＝12.4,4.0Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.5,161.9,156.7,156.1,155.2,134.2,129.6,129.6,127.2,113.8,113.8,99.7,55.8,46.7,44.6,44.6,31.7,31.7；HRMS(ESI)+calculated for C18H21N6O2,[M+H]+:m/z 353.1721,found353.1717.
化合物34,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.39(1H,d,J＝7.8Hz),8.31(1H,s),8.26(1H,s),7.94(2H,d,J＝8.7Hz),7.28(2H,d,J＝8.7Hz),4.71(2H,m),4.20(1H,m),3.31(2H,m),1.98(2H,dd,J＝2.9,12.4Hz),1.59(1H,td,J＝12.4,4.0Hz),1.57(1H,td,J＝12.4,4.0Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.5,164.9,163.0,156.7,156.1,155.2,134.1,131.4,130.5,130.4,115.6,115.4,99.7,46.9,44.6,44.6,31.6,31.6；HRMS(ESI)+calculated for C17H18FN6O,[M+H]+:m/z 341.1521,found341.1535.
化合物35,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.39(1H,d,J＝7.8Hz),8.31(1H,s),8.26(1H,s),7.94(2H,d,J＝8.7Hz),7.28(2H,d,J＝8.7Hz),4.71(2H,m),4.20(1H,m),3.31(2H,m),1.97(2H,dd,J＝2.9,12.4Hz),1.59(1H,td,J＝12.4,4.0Hz),1.56(1H,td,J＝12.4,4.0Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.5,164.9,163.0,156.7,156.1,155.2,134.1,131.4,130.5,130.4,115.6,115.4,99.7,46.9,44.6,44.6,31.6,31.6；HRMS(ESI)+calculated for C17H18FN6O,[M+H]+:m/z 403.0720,found403.0707.
化合物36,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.64(1H,d,J＝7.7Hz),8.32(1H,s),8.26(1H,s),8.07(2H,d,J＝8.1Hz),7.84(2H,d,J＝8.1Hz),4.71(2H,m),4.22(1H,m),3.36(2H,m),2.00(2H,dd,J＝2.9,12.4Hz),1.63(1H,td,J＝12.4,2.8Hz),1.60(1H,td,J＝12.4,2.8Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):168.8,164.9,156.7,156.1,155.2,138.8,135.1,134.1,131.6,131.3,130.6,128.7,126.7,125.7,99.7,47.1,44.6,44.6,31.5,31.5；HRMS(ESI)+calculated for C18H18F3N6O,[M+H]+:m/z 391.1489,found 391.1474.
化合物37,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.57(1H,s),8.57(1H,d,J＝7.7Hz),8.32(1H,s),8.26(1H,s),8.19(1H,s),8.17(1H,d,J＝8.1Hz),7.91(1H,d,J＝7.8Hz),7.72(1H,d,J＝7.8Hz),4.72(2H,m),4.25(1H,m),3.35(2H,m),2.01(2H,m),1.59(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):164.5,156.8,156.1,155.3,135.8,134.1,131.9,130.4,129.7,129.4,128.2,125.8,124.3,99.7,47.1,44.6,44.6,31.5,31.5；HRMS(ESI)+calculated for C18H18F3N6O,[M+H]+:m/z 391.1489,found 391.1474.
化合物38,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.56(1H,s),8.31(1H,s),8.48(1H,d,J＝7.7Hz),8.31(1H,s),8.24(1H,s),7.50–7.36(4H,m),4.60(2H,m),4.16(1H,m),3.41(2H,t,J＝10.0Hz),2.01(2H,m),1.52(2H,m)；13C-NMR(100MHz,DMSO-d6),δ(ppm):166.1,156.6,156.1,155.2,137.6,134.1,131.1,130.3,129.9,129.2,127.5,99.7,46.6,44.3,44.3,31.5,31.5；HRMS(ESI)+calculated for C17H18ClN6O,[M+H]+:m/z 357.1225,found357.1195.
化合物39,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.56(1H,s),8.58(1H,d,J＝7.7Hz),8.30(1H,s),8.24(1H,s),7.70(1H,dd,J＝7.1,2.5Hz),7.44-7.38(2H,m),4.59(2H,m),4.15(1H,m),3.42(2H,m),2.01(2H,m),1.52(2H,m),；13C-NMR(100MHz,DMSO-d6),δ(ppm):165.4,156.6,156.1,155.2,139.9,134.1,132.4,131.4,128.9,128.5,127.7,99.7,46.7,44.1,44.1,31.4,31.4；HRMS(ESI)+calculated for C17H17Cl2N6O,[M+H]+:m/z391.0835,found 391.0870.
化合物40,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.28(1H,s),8.24(1H,s),8.22(1H,s),7.27(2H,d,J＝8.4Hz),7.02(2H,d,J＝8.4Hz),6.16(1H,d,J＝7.6Hz),4.55(2H,m),3.85(1H,m),3.41(2H,t,J＝11.3Hz),2.21(3H,s),1.99(2H,d,J＝13.4Hz),1.44(1H,d,J＝11.3Hz),1.42(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,155.1,138.3,134.2,130.3,129.5,129.5,118.3,118.3,99.7,46.5,44.3,44.3,32.3,32.3,20.7；HRMS(ESI)+calculated for C18H22N7O,[M+H]+:m/z352.1880,found 352.1882.
化合物41,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.28(1H,s),8.24(1H,s),7.44(1H,s),7.04–6.97(3H,m),6.18(1H,d,J＝7.6Hz),4.56(2H,m),3.83(1H,m),3.33(2H,t,J＝11.3Hz),2.16(6H,s),1.98(2H,d,J＝13.4Hz),1.45(1H,d,J＝11.3Hz),1.42(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.7,156.1,155.9,155.2,136.5,136.0,134.2,130.3,128.1,128.1,126.0,99.7,46.8,44.4,44.4,32.4,32.4,18.7,18.7；HRMS(ESI)+calculated for C19H24N7O,[M+H]+:m/z 366.2037,found 366.2076.
化合物42,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.35(1H,s),8.29(1H,s),8.24(1H,s),7.29(2H,d,J＝9.1Hz),6.81(2H,d,J＝9.1Hz),6.32(1H,d,J＝7.6Hz),4.51(2H,m),3.86(1H,m),3.69(3H,s),3.44(2H,t,J＝11.3Hz),1.98(2H,d,J＝13.4Hz),1.44(1H,d,J＝11.3Hz),1.42(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.0,155.3,154.7,154.4,134.1,133.5,119.8,119.8,114.3,114.3,99.7,55.6,46.4,44.3,44.3,32.3,32.3；HRMS(ESI)+calculated for C18H22N7O2,[M+H]+:m/z368.1829,found 368.1813.
化合物43,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.63(1H,s),8.29(1H,s),8.24(1H,s),7.38(1H,s),6.38(1H,d,J＝7.6Hz),4.54(2H,m),3.86(1H,m),3.43(2H,t,J＝11.3Hz),1.98(2H,d,J＝13.4Hz),1.45(1H,d,J＝11.3Hz),1.43(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.0,155.9,155.2,155.0,140.6,132.7,131.5,131.5,121.8,121.8,113.5,100.3,47.5,43.6,43.6,31.9,31.9；HRMS(ESI)+calculated forC17H19BrN7O,[M+H]+:m/z 416.0829,found 416.0804.
1-(1-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperidin-4-yl)-3-(4-(trifluoromethyl)phenyl)urea(8e)
化合物44,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.81(1H,s),8.29(1H,s),8.25(1H,s),7.61(1H,d,J＝9.1Hz),7.57(1H,d,J＝9.1Hz),6.40(1H,d,J＝7.6Hz)4.57(2H,m),3.89(1H,m),3.42(2H,t,J＝11.3Hz),2.00(2H,d,J＝13.4Hz),1.48(1H,d,J＝11.3Hz),1.45(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,154.6,144.6,134.2,126.4,126.4,123.8,117.7,117.7,99.7,46.6,44.2,44.2,31.1,31.1；HRMS(ESI)+calculated for C18H19F3N7O,[M+H]+:m/z 406.1598,found 406.1608.
化合物45,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.76(1H,s),8.29(1H,s),8.24(1H,s),7.67(1H,dd,J＝1.9,1.8Hz),7.24(1H,dd,J＝8.1,7.6Hz),7.20(1H,dddd,J＝8.1,7.6,1.9,1.8Hz),6.93(1H,ddd,J＝7.6,1.9,1.8Hz),6.46(1H,d,J＝7.6Hz),4.54(2H,m),3.87(1H,m),3.43(2H,t,J＝11.3Hz),1.99(2H,d,J＝13.4Hz),1.47(1H,d,J＝11.3Hz),1.44(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,154.8,142.5,134.2,133.6,130.7,121.1,117.4,115.4,99.7,46.5,44.2,44.2,32.1,32.1；HRMS(ESI)+calculated for C17H19ClN7O,[M+H]+:m/z 372.1334,found 372.1361.
化合物46,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.30(1H,s),8.24(1H,s),8.26(1H,s),8.18(1H,dd,J＝8.4,1.5Hz),7.96(1H,s),7.39(1H,dd,J＝8.0,1.5Hz),7.24(1H,td,J＝8.0,1.4Hz),7.16(1H,d,J＝7.3Hz),6.95(1H,td,J＝7.6,1.5Hz),4.51(2H,m),3.88(1H,m),3.52(2H,t,J＝11.3Hz),2.03(2H,d,J＝13.4Hz),1.46(1H,d,J＝11.3Hz),1.43(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):157.4,156.0,155.6,154.8,143.5,132.9,131.6,128.3,128.2,128.2,128.1,100.3,46.5,44.2,44.2,31.9,31.9；HRMS(ESI)+calculated for C17H19ClN7O,[M+H]+:m/z 372.1334,found 372.1361.
化合物47,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),9.27(1H,s),8.88(1H,s),8.29(1H,s),8.24(1H,s),7.84(1H,d,J＝2.0Hz),7.45(1H,d,J＝8.9Hz),7.26(1H,dd,J＝8.9,2.0Hz),6.52(1H,d,J＝7.6Hz),4.54(2H,m),3.88(1H,m),3.42(2H,t,J＝11.3Hz),1.98(2H,d,J＝13.4Hz),1.47(1H,d,J＝11.3Hz),1.45(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,154.7,141.2,134.2,131.4,130.9,122.6,119.1,118.2,99.7,46.5,44.2,44.2,32.1,32.1；HRMS(ESI)+calculated for C17H18Cl2N7O,[M+H]+:m/z 406.0944,found 406.0905.
化合物48,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.53(1H,s),8.33(1H,s),8.26(1H,s),8.24(1H,s),8.08(1H,dd,J＝8.4,1.5Hz),7.23(1H,d,J＝1.5Hz),7.01–6.90(2H,m),6.95(1H,td,J＝7.6,1.5Hz),4.48(2H,m),3.87(1H,m),3.50(2H,t,J＝11.3Hz),1.99(2H,d,J＝13.4Hz),1.45(1H,d,J＝11.3Hz),1.43(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,155.2,154.8,153.4,134.0,125.3,121.8,111.2,104.0,99.7,46.4,43.7,43.7,32.1,32.1；HRMS(ESI)+calculated for C17H18F2N7O,[M+H]+:m/z374.1535,found 374.1594.
化合物49,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.54(1H,s),9.27(1H,s),8.30(1H,s),8.25(1H,s),8.09(2H,s),7.55(1H,s),6.65(1H,d,J＝7.6Hz),4.59(2H,m),3.91(1H,m),3.40(2H,t,J＝11.3Hz),2.00(2H,d,J＝13.4Hz),1.52(1H,d,J＝11.3Hz),1.49(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.2,154.6,142.9,134.2,131.2,130.9,127.9,125.2,122.5,117.8,113.9,99.7,46.9,44.4,44.4,32.1,32.1；HRMS(ESI)+calculated for C19H18F6N7O,[M+H]+:m/z 474.1472,found 474.1500.
化合物50,1H-NMR(400MHz,DMSO-d6),δ(ppm):13.55(1H,s),8.62(1H,s),8.32(1H,s),8.26(1H,s),8.16(1H,d,J＝8.2Hz),8.04(1H,d,J＝7.1Hz),7.88(1H,dd,J＝7.1,2.0Hz),7.56–7.48(3H,m),7.43(1H,dd,J＝7.9,7.8Hz)6.94(1H,d,J＝7.6Hz),4.54(2H,m),3.94(1H,m),3.51(2H,t,J＝11.3Hz),2.05(2H,d,J＝13.4Hz),1.51(1H,d,J＝11.3Hz),1.49(1H,d,J＝11.3Hz)；13C-NMR(100MHz,DMSO-d6),δ(ppm):156.6,156.1,155.5,155.3,135.6,134.2,134.1,128.8,126.4,126.2,125.8,125.8,122.4,122.0,116.6,99.7,46.5,44.1,44.1,32.3,32.3；HRMS(ESI)+calculated for C21H22N7O,[M+H]+:m/z 388.1880,found 388.1924.
试验例1化合物1～50的PAK1激酶抑制活性及抗增殖活性
本实验的目的是检测本发明的化合物对体外PAK1抑制活性(表1)。
表1化合物1～50激酶激动活性及抗肿瘤抑制活性
Figure BDA0002410372550000191
Figure BDA0002410372550000201
实验结果表明，本发明的化合物大多对PAK1具有抑制活性，其中，化合物46、化合物49显示出纳摩尔级的抑制活性。
试验例2化合物49对PAK1-3的选择性实验
为了进一步研究化合物49对PAK家族激酶的选择性，我们进行了酶活选择性测试，结果如表2所示。
表2化合物49对PAK1-3的激酶选择性.
Figure BDA0002410372550000211
a Each compound was tested in triplicate； the data are presented asthe mean ± SD.
试验例3化合物49抗增殖活性评价
为了检测49对肿瘤细胞的增殖抑制活性，我们利用MTT法测试了化合物49对MDA-MB-231细胞的抗增殖活性，结果显示化合物49能够显著的抑制肿瘤细胞的增殖，其IC50为3.48μM，结果见图1。
试验例4化合物49的诱导肿瘤细胞凋亡
为了检测化合物49是否能够诱导肿瘤细胞凋亡，我们使用Hoechst 33258对不同浓度的化合物49处理后的MDA-MB-231细胞进行荧光染色，结果显示随着化合物49浓度的增加，荧光染色亮度增强，证明化合物49诱导了MDA-MB-231细胞DNA片段化，即凋亡的发生(见图2)。随后，应用Annexin V-PI双染的流式细胞术检测了不同浓度化合物49处理后MDA-MB-231细胞的凋亡率，结果表明随着化合物49浓度升高，凋亡率增加(见图3)。
试验例5化合物49的抑制肿瘤细胞转移
为了检测化合物49是否能够抑制肿瘤细胞迁移，我们使用Transwell实验对不同浓度的化合物49处理后的MDA-MB-231细胞进行检测，结果显示化合物49可以剂量依赖的抑制MDA-MB-231细胞转移，结果见图4。

Claims (10)

1.通式Ⅰ-IV所示的化合物或其药学上可接受的盐：
Figure FDA0002410372540000011
其中，
R1为氢、C1-C6烷基、C1-C6烷氧基、卤素、硝基、氨基、卤代C1-C6烷基、6-10元芳基或杂芳基，所述的芳基或杂芳基可以被一个或多个R2取代，R2为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基；R1、R2为单取代或多取代。
2.根据权利要求1所述的化合物或其药学上可接受的盐，其特征在于：
R1为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基、苯基、萘基，所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C6烷基、C1-C6烷氧基、卤素、卤代C1-C6烷基；R1、R2为单取代或多取代；
X为O或S。
3.根据权利要求1或2所述的化合物或其药学上可接受的盐，其特征在于：
式(I)中：
R1为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O；
式(II)中：
R1为苯基、萘基；所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O或S；
式(III)中：
R1为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O；
式(IV)中：
R1为苯基、萘基；所述的苯基或萘基可以被一个或多个R2取代，R2为氢、C1-C4烷基、C1-C4烷氧基、卤素、卤代C1-C4烷基；X为O。
4.如下的化合物或其药学上可接受的盐：
Figure FDA0002410372540000021
Figure FDA0002410372540000031
Figure FDA0002410372540000041
5.权利要求1-4任何一项所述的化合物或其药学上可接受的盐的制备方法，其特征在于，
Figure FDA0002410372540000042
Figure FDA0002410372540000051
其中，R1如权利要求1所述。
6.药物组合物，包含权利要求1-4任一项所述的化合物或其药学上可接受的盐和药学上可接受的载体。
7.权利要求1-4任何一项所述的化合物或其药学上可接受的盐或权利要求6所述的药物组合物在制备PAK抑制剂中的应用。
8.权利要求1-4任何一项所述的化合物或其药学上可接受的盐或权利要求6所述的药物组合物在制备PAK1抑制剂中的应用。
9.权利要求1-4任何一项所述的化合物或其药学上可接受的盐或权利要求6所述的药物组合物在制备抗肿瘤药物中的应用。
10.权利要求9所述的应用，其特征在于，所述的肿瘤为肺癌、肝癌、乳腺癌、肾癌。
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