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CN111440172B - 丙烯酰胺类化合物及其应用 - Google Patents

丙烯酰胺类化合物及其应用 Download PDF

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CN111440172B
CN111440172B CN201910044871.6A CN201910044871A CN111440172B CN 111440172 B CN111440172 B CN 111440172B CN 201910044871 A CN201910044871 A CN 201910044871A CN 111440172 B CN111440172 B CN 111440172B
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丁克
李正球
国翠平
王鑫
徐嘉乾
胡俊
肖湘成
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Abstract

本发明涉及一种丙烯酰胺类化合物及其应用。所述丙烯酰胺类化合物具有如式(I)所示结构。该类分子探针化合物能够在活细胞内高选择性、高效率地标记其靶标蛋白，具有较高的灵敏度与可操作性，同时还可以作为一种工具分子探针，通过在活细胞体内竞争性的标记与成像实验筛选特定靶标的抗癌活性化合物。

Description

丙烯酰胺类化合物及其应用
技术领域
本发明涉及分子探针技术领域，特别是涉及丙烯酰胺类化合物及其应用。
背景技术
近年来，不可逆药物分子由于其持久的药效性质及良好的选择性得到广泛地应用，但其显著的毒副作用是该类药物分子的主要问题之一。
通过对药物分子构效关系进行分析，在不改变其药效母核的基础上引入生物正交反应基团，通过点击化学反应引入荧光基团或者生物素，可进行后续的蛋白分析鉴定工作。因此，利用分子探针探究该类药物分子的作用靶标，对药物分子的结构优化及临床指导用药具有重要的意义。
目前，尚无丙烯酰胺类的分子探针化合物的相关报道。
发明内容
基于此，有必要提供一种丙烯酰胺类化合物，该类化合物能够在活细胞内高选择性、高效率地标记其靶标蛋白，具有较高的灵敏度与可操作性，同时还可以作为一种工具分子探针，通过在活细胞体内竞争性的标记与成像实验筛选特定靶标的抗癌活性化合物。
一种如式(I)所示的丙烯酰胺类化合物：
Figure BDA0001948810530000011
其中，R0选自H或C1-C10烷基；
A包括如下基团中的一种或多种：
Figure BDA0001948810530000021
炔基、叠氮基、环烯基、C1-C10烷基；当A为C1-C10烷基时，与R0成环或不成环；
B包括如下基团中的一种或多种：
H、炔基、叠氮基、环烯基、
Figure BDA0001948810530000022
各基团之间的连接基团独立的选自单键、烷基、烷氧基、-NR0-、酰胺、
Figure BDA0001948810530000023
中的一种或多种；
各基团进一步被若干个R取代或不取代，R每次出现时，独立的选自卤素、C1-C5烷基、C1-C5烷氧基、NR0、
Figure BDA0001948810530000024
在其中一个实施例中，具有如式(I-1)、式(I-2)或式(I-3)所示结构：
Figure BDA0001948810530000025
其中A1、A2、A3的定义同A。
在其中一个实施例中，所述的丙烯酰胺类化合物，具有如下式所示结构：
Figure BDA0001948810530000031
在其中一个实施例中，B选自如下结构：
Figure BDA0001948810530000032
在其中一个实施例中，所述的丙烯酰胺类化合物，具有如下式所示结构：
Figure BDA0001948810530000033
其中，B选自如下结构：
Figure BDA0001948810530000034
在其中一个实施例中，R每次出现时，独立的选自F、Cl、C1-C2烷基、C1-C2烷氧基、羟基、N(CH3)2、
Figure BDA0001948810530000041
在其中一个实施例中，所述的丙烯酰胺类化合物，选自如下化合物：
Figure BDA0001948810530000042
其中，R1为
Figure BDA0001948810530000043
Figure BDA0001948810530000044
其中，R2为
Figure BDA0001948810530000045
Figure BDA0001948810530000046
Figure BDA0001948810530000051
本发明还提供所述的丙烯酰胺类化合物作为分子探针的应用。
在其中一个实施例中，所述丙烯酰胺类化合物作为分子探针在标记靶标蛋白中的应用。
在其中一个实施例中，所述靶标蛋白为EGFR。
在其中一个实施例中，所述丙烯酰胺类化合物作为分子探针在抗癌活性化合物筛选中的应用。
在其中一个实施例中，所述抗癌活性化合物为抑制BTK的活性化合物。
本发明还提供一种抗癌活性化合物的筛选方法，包括如下步骤：
(1)待细胞生长到对数生长期时，分到孔板中；
(2)将所述的丙烯酰胺类化合物和被筛选的抗癌活性化合物加入至所述孔板，进行孵育；
(3)于孵育后的细胞加入蛋白酶抑制剂的缓冲液进行裂解，超声，离心后取上清液；
(4)用蛋白标记试剂盒标定蛋白浓度；
(5)通过点击化学反应引入荧光基团，检测，即可。
与现有技术相比，本发明具有如下有益效果：
本发明提供的丙烯酰胺类化合物，其结构中含有α，β不饱和酰胺结构，能够被蛋白中的氨基、巯基等进攻，发生迈克尔加成反应，形成稳定的共价键。因此，该系列分子探针能够在活细胞内高选择性、高效率地标记其靶标蛋白，具有较高的灵敏度与可操作性，同时还可以作为一种工具分子探针，通过在活细胞体内竞争性的标记与成像实验筛选特定靶标的抗癌活性化合物。
附图说明
图1为分子探针AF-1标记靶蛋白EGFR的多功能激光扫描成像结果；
图2为分子探针AF-2标记靶蛋白EGFR的多功能激光扫描成像结果；
图3为分子探针IB-2/IB-3标记靶蛋白BTK多功能激光扫描成像结果；
图4为分子探针IB-3筛选靶向BTK的活性天然产物的多功能激光扫描成像结果。
具体实施方式
以下结合具体实施例对本发明的丙烯酰胺类化合物及其应用作进一步详细的说明。
实施例1
Figure BDA0001948810530000061
AF-1的合成方法：
Figure BDA0001948810530000062
AF-Br的合成参考文献(M.R.V.Finlay,M.Anderton,S.Ashton,etal.J.Med.Chem.2014,57,8249-8267)。
化合物AF-1的合成：
AF-Br50mg(0.095mmol,1equiv.)溶解在约5mLDMF中，在0℃加入化合物10.0317mL(0.38mmol,4equiv.)，加入K2CO326mg(0.19mmol,2equiv.)，KI27mg(0.19mmol,2equiv.)。反应温度升到40℃，反应3h。薄层色谱法(TLC)监测反应。反应完后，反应混合物滴加到水中，乙酸乙酯萃取。盐水洗，Na2SO4干燥，浓缩，柱层析分离(甲醇：乙酸乙酯＝1：10)。得到黄色固体AF-1 23mg，收率48％。1H NMR(300MHz,CDCl3)δ9.00(s,1H),8.56(s,1H),8.35(s,1H),8.10(s,1H),7.79(dd,J＝3.0,6.0Hz,1H),7.46(m,1H),7.09(s,1H),7.0(m,1H),6.96(m,1H),6.23(d,J＝15.0Hz,1H),5.10(s,1H),4.15(d,J＝12.0Hz,1H),4.02(m,2H),3.90(m,1H),3.37(s,2H),3.28(d,J＝6.0Hz，2H),2.43(m,1H),3.26(s,3H),2.29(t,J＝3.0Hz,1H),2.22(m,1H).13C NMR(75MHz,CDCl3)δ163.98,156.98,154.63,150.60,148.16,143.95,135.46,127.91,125.63,124.17,121.89,120.74,116.49,110.68,109.61,108.30,79.48,78.24,77.23,73.95,73.12,67.41,56.55,45.79,42.11,32.84.ESI-MS(m/z)[M+H]+calcd:510.2；Found:510.5.[M+2H]2+/2 calcd:255.6；Found:255.9.HR-MS(m/z)[M+H]+calcd:510.1703；Found:510.1666。
实施例2
AF-2的合成：
Figure BDA0001948810530000071
0℃下，1mLDMF中加入NaN3 6mg(0.09mmol,1equiv.)，加入AF-Br50mg(0.095mmol,1equiv.)。0℃反应30min，室温反应4h。加入水，乙酸乙酯萃取。NaSO4干燥。柱层析分离(甲醇/CH2Cl2＝3:50)，得到黄色固体37mg，收率80％。1H NMR(400MHz,CDCl3)δ9.10(s,1H),8.65(s,1H),8.13(s,1H),7.93(dd,J＝4.0,8.0Hz,1H),7.75(s,1H),7.54(m,1H),7.21(s,1H),7.14(t,J＝8.0Hz,1H),7.01(dt,J＝4.0 12.0Hz,1H),6.32(dt,J＝4.0,12.0Hz,1H),5.19(t,J＝4.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.14(d,J＝4.0Hz,2H),4.11(d,J＝4.0Hz,1H),4.05(m,1H),3.95(m,1H),2.45(m,1H),2.25(m,1H).13C NMR(100MHz,CDCl3)δ163.29,156.96,154.81,150.57,148.34,139.60,128.24,125.66,124.43,121.95,121.89,116.83,116.61,110.18,109.60,108.72,79.70,73.18,67.48,51.49,32.98,29.92.ESI-MS(m/z)[M+H]+calcd:484.1；Found:484.3.HR-MS(m/z)[M+H]+calcd:484.1295；Found:484.1290。
实施例3
AF-3的合成
Figure BDA0001948810530000081
化合物2的合成参考文献(Anon.IP.com Journal 2014，1-15)。
化合物4的合成
化合物2 55mg(0.1mmol,1equiv.)溶解在四氢呋喃中，0℃下搅拌，加入NaH16mg(0.4mmol,4equiv.)，混合溶液温度升到室温，加入化合物3 16mg(0.1mmol,1equiv.)溶解在1mL四氢呋喃中，室温搅拌反应。薄层色谱法(TLC)监测反应。反应完后，加入水稀释，乙酸乙酯萃取。柱层析分离(EA:PE＝1:20)得到纯化合物15mg，收率30％。1H NMR(400MHz,Chloroform-d)δ9.12(s,1H),8.66(s,1H),8.10(s,1H),7.94(dd,J＝6.6,2.7Hz,1H),7.88-7.68(m,2H),7.55(ddt,J＝6.1,4.2,2.9Hz,2H),7.16(t,J＝8.8Hz,1H),7.09-6.95(m,1H),6.18(d,J＝15.2Hz,1H),5.24-5.14(m,1H),4.85(s,1H),4.33(t,J＝6.7Hz,2H),4.19(d,J＝10.7Hz,1H),4.12-4.01(m,5H),3.98-3.92(m,2H),2.46(dt,J＝14.4,7.2Hz,2H),1.51(s,9H)。
化合物AF-3的合成
化合物4 10mg(0.018mmol)加入TFA和CH2Cl2的混合溶液(v:v＝1:3)，30min后旋掉溶剂，得到粗产品，溶解在DMF中，加入TCO-NHS 3.2mg(0.012mmol)，加入TFA0.001mL(0.006mmol)溶解在1mLDMSO中。室温搅拌1h。柱层析分离(DCM:MeOH＝50:1)，得到白色固体5mg，收45％。1H NMR(400MHz,Chloroform-d)δ9.12(s,1H),8.66(s,1H),8.10(s,1H),8.03-7.80(m,2H),7.56(dt,J＝9.0,3.3Hz,1H),7.16(t,J＝8.8Hz,1H),7.00(dt,J＝15.2,4.9Hz,1H),6.18(d,J＝15.2Hz,1H),5.66–5.45(m,2H),5.20(t,J＝5.4Hz,1H),4.91(s,1H),4.41(s,1H),4.22-4.03(m,5H),3.96(td,J＝8.6,5.1Hz,1H),2.52-2.22(m,6H),2.12-1.93(m,6H),1.77(dd,J＝15.3,5.7Hz,2H),1.65–1.57(m,1H)。
实施例4
IB-1的合成
Figure BDA0001948810530000101
化合物6的合成参考文献(J.Yang,Y.Liang,S.Jolita,et al.Chemistry-AEuropean Journal2014,20,3365-3375)。
化合物5 20.0mg(0.05mmol,1equiv.)溶解在约3mLDMF中，加入化合物66.2mg(0.05mmol,1equiv.)，EDCI 11.6mg(0.075mmol,1.5equiv.)，HOBT10.1mg(0.075mmol,1.5equiv.)和三乙胺0.014mL(0.10mmol,2equiv.)。室温搅拌反应过夜。薄层色谱法(TLC)监测反应。反应完后，滴加水淬灭。乙酸乙酯萃取，盐水洗，Na2SO4干燥，浓缩，柱层析分离(甲醇：二氯甲烷＝0.2：5)，得到浅黄色固体12.3mg，收率50％。1HNMR(300MHz,CDCl3)δ8.34(s,1H),7.62(d,J＝12.0Hz,2H),7.37(t,J＝6.0,9.0Hz,2H),7.14(t,J＝9.0Hz,3H),7.06(d,J＝6.0Hz,2H),6.53(m,1H),6.32(d,J＝3.0Hz,1H),5.66(s,1H),4.84(m,1H),4.13(d,J＝6.0Hz,1H),3.67(d,J＝6.0Hz,1H),3.31(m,1H),2.24(m,3H),2.11(s,3H),1.95(m,2H),1.69(m,1H).13CNMR(100MHz,CDCl3)δ166.07,158.53,157.84,156.35,156.18,155.79,154.25,143.83,129.97,127.75,124.05,119.54,119.30,119.14,115.54,101.24,98.59,53.62,52.70,49.87,46.16,45.72,42.13,35.96,31.91,30.38,29.26,27.22,22.46.ESI-MS(m/z)[M+H]+calcd:493.2；Foun d:493.5.HR-MS(m/z)[M+H]+calcd:493.2347；Found:493.2331。
实施例5
IB-2的合成
Figure BDA0001948810530000111
化合物7的合成参考文献(R.Y.Zhao,S.D.Wilhelm,C.Audette.J.Med.Chem.2011,54,3606-3623)。
化合物5 135.0mg(0.35mmol,1equiv.)溶解在约3mLDMF中，加入化合物745.0mg(0.35mmol,1equiv.)，EDCI 81.2mg(0.525mmol,1.5equiv.)，HOBt 70.9mg(0.525mmol,1.5equiv.)，三乙胺0.1mL(0.525mmol,1.5equiv.)。室温搅拌反应过夜。薄层色谱法(TLC)监测反应。反应完后，加入水，乙酸乙酯萃取，盐水洗，NaSO4干燥。柱层析分离(甲醇：二氯甲烷＝1：50)，得到浅黄色固体104mg，收率60％。1HNMR(400MHz,CDCl3)δ8.34(d,J＝21.2Hz,1H),7.68-7.59(m,2H),7.38(ddd,J＝8.5,7.3,2.1Hz,2H),7.20-7.11(m,3H),7.07(ddd,J＝8.6,2.2,1.1Hz,2H),6.29(ddt,J＝6.9,5.0,1.6Hz,1H),6.03–5.82(m,1H),5.20-5.06(m,1H),4.91-4.73(m,1H),4.54(dd,J＝10.2,6.5Hz,1H),4.10-3.98(m,1H),3.98-3.80(m,1H),3.70(dd,J＝13.2,10.6Hz,1H),3.48-3.09(m,2H),3.03(ddd,J＝16.1,6.5,1.8Hz,1H),2.85-2.72(m,1H),2.51-2.16(m,3H),2.03-1.87(m,1H),1.78-1.57(m,1H).ESI-MS(m/z)[M+H]+calcd:495.2；found:496.5.HR-MS(m/z)[M+H]+calcd:496.2204；Found:496.2185。
实施例6
IB-3的合成
Figure BDA0001948810530000112
化合物8的合成参考文献(E.Kim,K.S.Yang,R.H.Kohler,J.M.Dubach,H.Mikula,R.Weissleder.Bioconjug Chem.2015,26,1513-1518)。
化合物8 17.1mg(0.03mmol,1.5equiv.)溶解在约2mLTFA/DCM体积比为1:1的溶液中，室温搅拌30min，真空干燥，除去溶剂得到粗产品。粗产品溶解在约2mLDMF中，加入溶解在约2mLDMSO中的TCO-NHS5.3mg(0.02mmol,1equiv.)，三乙胺0.02mL(0.1mmol,5equiv.)。室温搅拌反应1h，薄层色谱法(TLC)监测反应。反应完后，加入水，乙酸乙酯萃取，盐水洗,柱层析分离(甲醇：二氯甲烷＝1：20)。得到浅黄色固体5.3mg收率16％。1HNMR(400MHz,CDCl3)δ8.37(d,J＝16.4Hz,1H),7.63(d,J＝8.2Hz,2H),7.40(dd,J＝8.5,7.4Hz,2H),7.21-7.13(m,3H),7.11-7.06(m,2H),6.75(s,1H),6.37(t,J＝18.0Hz,1H),5.88(d,J＝65.7Hz,1H),5.51(s,2H),4.80(d,J＝42.7Hz,2H),4.56(d,J＝12.9Hz,1H),4.34(d,J＝25.6Hz,1H),4.15(d,J＝11.6Hz,1H),3.92(d,J＝22.3Hz,3H),3.74(s,1H),3.35(t,J＝12.0Hz,1H),3.15(d,J＝14.8Hz,1H),2.87(s,1H),2.31(d,J＝30.9Hz,4H),2.04-1.81(m,12H).HR-MS(m/z)[M+H]+calcd:622.3136；Found:622.3126。
实施例7
IB-4的合成
Figure BDA0001948810530000121
化合物11的合成
化合物9 400mg(2mmol,1equiv.)溶解在约20mL二氯甲烷中，加入K2CO3552mg(4mmol,2equiv.)，60℃下搅拌反应1h。化合物10 355mg(2mmol,1equiv.)溶解在约5mL二氯甲烷中，滴加到反应液中。60℃反应20h。薄层色谱法(TLC)监测反应。反应完后，旋掉溶剂。柱层析分离，得到1123mg，收率80％。1HNMR(400MHz,DMSO-d6)δ6.87-6.70(m,2H),5.98(dt,J＝15.7,1.6Hz,1H),3.65(s,3H),3.18(dd,J＝7.5,3.7Hz,1H),3.08(dd,J＝5.9,1.7Hz,2H),2.72(dt,J＝11.8,3.7Hz,2H),1.95(td,J＝12.0,2.8Hz,2H),1.67(dd,J＝12.7,4.2Hz,2H),1.37(s,9H)。
化合物12的合成
化合物11 149mg(0.5mmol,1equiv.)溶解在约10mL甲醇中，0℃下慢慢加入NaOH2mL1M溶液，室温反应过夜。加水稀释，用1MHCl pH值调到3。乙酸乙酯萃取，盐水洗，Na2SO4干燥，浓缩，柱层析分离。得到化合物127mg，收率80％。1H NMR(400MH,Methanol-d4)δ6.86(dd,J＝15.2,7.3Hz,1H),6.20(d,J＝15.4Hz,1H),3.88(d,J＝6.9Hz,2H),3.59(s,1H),3.49-3.35(m,2H),3.17-3.01(m,2H),2.14-1.98(m,2H),1.85-1.68(m,2H),1.37(s,9H)。
化合物IB-4的合成
化合物12 19.6mg(0.03mmol,1.5equiv.)中加入2mLTFA:CH2Cl2(v:v＝1:3)，室温搅拌反应30min。旋蒸掉溶剂，粗产品溶解在约3mLDMF中。TCO-NHS5.3mg(0.02mmol,1equiv.)，加入三乙胺0.3mL(0.1mmol,5equiv.)。室温搅拌反应1h。薄层色谱法(TLC)监测反应。反应完后，加入水，乙酸乙酯萃取，NaSO4干燥。柱层析分离(甲醇:二氯甲烷＝1:50)。得到固体IB-4 2.8mg，收率20％。1H NMR(400MHz,DMSO-d6)δ8.25(s,1H),7.69-7.62(m,2H),7.48-7.40(m,2H),7.22-7.09(m,5H),6.65(s,1H),6.54(s,1H),5.57(ddd,J＝14.9,10.1,4.1Hz,1H),5.42(t,J＝13.5Hz,1H),4.69(d,J＝16.8Hz,1H),4.52(d,J＝12.6Hz,1H),4.25-4.00(m,3H),3.74(t,J＝11.3Hz,1H),2.21(d,J＝31.4Hz,4H),2.11(s,1H),2.01-1.78(m,6H),1.58(d,J＝24.2Hz,6H).HR-MS(m/z)[M+H]+ calcd:705.3871；Found:705.3866。
实施例8
IB-5的合成
Figure BDA0001948810530000141
化合物5 77mg(0.2mmol,1equiv.)，化合物1355mg(0.2mmol,1equiv.)溶解在约5mLDMF中，加入HATU91mg(0.2mmol,1equiv.)，加入三乙胺0.06mL(0.4mmol,2equiv.)。室温搅拌反应2h。薄层色谱法(TLC)监测反应。反应完后，反应液中加入水，乙酸乙酯萃取，2MHCl洗，水洗，5％NaHCO3洗，水洗。NaSO4干燥。柱层析分离(甲醇：二氯甲烷＝1：25)，得到白色固体36mg，收率23％。1H NMR(400MHz,DMSO-d6)δ9.98(d,J＝20.5Hz,1H),8.64(d,J＝14.6Hz,1H),8.19(d,J＝74.9Hz,1H),7.72–7.55(m,3H),7.44(qd,J＝7.4,2.1Hz,2H),7.24-7.05(m,6H),6.85-6.71(m,2H),6.67-6.47(m,2H),4.94-4.72(m,1H),4.66-4.31(m,1H),3.97-3.70(m,1H),3.57(dd,J＝13.0,10.4Hz,1H),3.15(dt,J＝38.6,13.1Hz,1H),2.85(ddd,J＝15.4,10.9,4.5Hz,1H),2.23(dq,J＝12.3,4.1Hz,1H),2.18-2.06(m,1H),1.94-1.79(m,1H),1.69(d,J＝13.5Hz,1H)。13CNMR(101MHz,DMSO-d6)δ166.66,163.80,160.27,158.69,158.60,158.25,157.59,156.77,156.15,155.95,154.43,152.08,143.70,136.77,136.51,130.60,130.58,130.49,129.74,129.69,126.93,126.83,125.62,124.25,121.17,119.44,119.42,119.40,114.14,111.84,102.43,97.86,52.77,52.45,50.33,46.13,44.92,30.33,30.17,24.63,23.75。
实施例9
IB-6的合成
Figure BDA0001948810530000151
化合物14的合成参考文献(G.H.Huan,G.H.Shi,H.Y.Sheng,etal.Angew.Chem.Int.2017,56,8686-8691)。
化合物IB-6的合成
化合物14 17mg(0.05mmol,1equiv.)溶解在2mL DMF中，加入HATU 23mg(0.06mmol,1.2equiv.)，加入TEA 0.02mL(0.1mmol,2equiv.)。混合液室温搅拌过夜，加入5mL 水，EA 萃取，饱和NaCl溶液洗，NaSO4干燥。旋干，得到粗产品。柱层析分离MeOH:CH2Cl21:30得到黄色固体10mg，收率29％。1H NMR(400MHz,DMSO-d6)δ10.06(d,J＝19.5Hz,1H),8.70(d,J＝12.7Hz,1H),8.19(d,J＝72.4Hz,1H),7.76–7.67(m,2H),7.65–7.59(m,1H),7.43(q,J＝8.1Hz,2H),7.25–6.95(m,8H),6.60(dd,J＝40.5,2.4Hz,2H),4.93(t,J＝2.3Hz,2H),4.89–4.73(m,1H),4.66–4.32(m,1H),3.97–3.71(m,1H),3.65(q,J＝2.2Hz,1H),3.58(dd,J＝13.1,10.4Hz,1H),3.26–3.05(m,1H),2.85(td,J＝12.9,3.0Hz,1H),2.24(dt,J＝15.6,8.4Hz,1H),2.18–2.07(m,1H),1.96–1.78(m,1H),1.78–1.61(m,1H).13C NMR(101MHz,DMSO-d6)δ166.72,166.63,163.95,159.18,158.69,158.60,158.05,157.59,157.54,156.76,156.72,156.15,155.95,154.43,151.64,143.85,143.71,136.94,136.70,130.59,130.57,130.48,129.55,129.50,128.38,128.31,125.89,125.78,125.59,124.25,124.23,122.41,119.44,119.41,113.78,102.08,97.87,79.31,79.09,56.54,52.78,52.46,50.33,46.12,44.94,30.32,30.17,24.65,23.75.HR-MS(m/z)[M+H]+calcd:682.2409；Found:682.2396。
实施例10
IB-7的合成
Figure BDA0001948810530000161
化合物15的合成参考文献(I.Bhattacharjee,N.Ghosh,et.al.Phys.Chem.Chem.Phys.2018,20,6060-6072)。
化合物15 33mg(0.1mmol,1equiv.)加入到5mLDMF中，加入HATU 46mg(0.12mmol,1.2equiv.)，加入中间体化合物5 39mg(0.1mmol,1equiv.)，加入EA 0.03mL(0.2mmol,2equiv.)。室温搅拌反应过夜，加入3mL水，EA萃取，饱和NaCl溶液洗，NaSO4干燥。浓缩，得到粗产品，柱层析分离(MeOH:CH2Cl2＝1:50)得到黄色固体39mg，收率56％。1H NMR(400MHz,DMSO-d6)δ9.87(d,J＝21.6Hz,1H),8.57(d,J＝18.5Hz,1H),8.20(d,J＝67.9Hz,1H),7.73–7.66(m,1H),7.65–7.59(m,1H),7.51–7.38(m,3H),7.20–7.09(m,5H),6.70(ddd,J＝9.0,2.5,1.4Hz,1H),6.62–6.48(m,3H),4.89(ddt,J＝15.1,10.2,4.3Hz,1H),4.78(tt,J＝11.1,4.4Hz,0H),4.61(dd,J＝12.5,4.4Hz,0H),4.37(dd,J＝10.2,6.7Hz,1H),3.91(dd,J＝13.1,4.2Hz,1H),3.75(d,J＝13.1Hz,0H),3.58(dd,J＝13.0,10.5Hz,1H),3.41(qd,J＝7.0,2.2Hz,4H),3.26–3.04(m,1H),2.83(td,J＝12.7,3.0Hz,1H),2.31–2.19(m,1H),2.19–2.09(m,1H),1.95–1.78(m,1H),1.76–1.60(m,1H).13C NMR(101MHz,DMSO-d6)δ166.84,166.75,163.51,158.69,158.61,157.58,157.51,156.77,156.15,155.97,154.44,153.20,149.75,143.70,136.33,136.06,130.59,130.56,130.49,129.46,129.41,128.39,128.34,128.07,125.77,124.25,124.21,119.43,119.37,118.87,118.84,109.96,107.93,107.88,97.87,97.07,52.77,52.45,50.33,46.14,44.94,44.47,30.35,30.20,29.50,24.62,23.75,12.79.HR-MS(m/z)[M+H]+ calcd:699.3038；Found:699.3026。
实施例11
IB-8的合成
Figure BDA0001948810530000171
化合物16的合成参考文献(S.Gurrapu,S.K.Jonnalagadda,M.A.Alam,etal.Bioorg.Med.Chem.Lett.2016,26,3282-3286)。
化合物17的合成
将化合物16 2.13g(10mmol,1equiv.)与硝基乙酸乙酯1.1ml(10mmol,1equiv.)溶解在5mL乙醇中，再加入L-脯氨酸345mg(3mmol,0.3equiv.)，在氩气保护下反应4小时，产物析出后过滤用乙醇洗涤，得到黄色产物18676.8mg，收率24％。1H NMR(400MHz,DMSO-d6)δ9.14(s,1H),7.87(d,J＝9.0Hz,1H),7.07(dd,J＝9.0,2.4Hz,1H),6.86(d,J＝2.4Hz,1H),4.45(d,J＝2.5Hz,4H),3.32(t,J＝2.4Hz,2H)。
化合物18合成
将SnCl2·2H2O 3.38g(15mmol,7.5equiv.)和20mL的37％HCl加入到50mL圆底烧瓶中。接下来，加入化合物564mg(2mmol,1equiv.)，并将所得溶液在室温下进一步搅拌6小时。然后，使用Na2CO3饱和溶液中和过量的酸，然后用乙酸乙酯萃取。用无水Na2SO4干燥有机层并蒸发至干。得到粗产物18，为棕色固体139mg，收率27％。1H NMR(400MHz,DMSO-d6)δ7.30(d,J＝8.6Hz,1H),6.93–6.76(m,2H),6.71(s,1H),5.27(s,2H),4.20(d,J＝2.5Hz,4H),3.26–3.13(m,2H)。
化合物19的合成
将化合物18 126(0.5mmol，1equiv.)溶解在丙酮中，再加入顺丁烯二酸酐59mg(0.6mmol,1.2equiv.)，反应搅拌过夜，产物析出，过滤，用丙酮润洗，得到橘黄色产物19100mg，收率57％。1H NMR(400MHz,DMSO-d6)δ12.99(s,1H),10.19(s,1H),8.62(s,1H),7.60(d,J＝8.8Hz,1H),6.96(dd,J＝8.8,2.5Hz,1H),6.85(d,J＝2.5Hz,1H),6.63(d,J＝12.1Hz,1H),6.41(d,J＝12.0Hz,1H),4.30(d,J＝2.5Hz,4H),3.23(t,J＝2.3Hz,2H)。
IB-8的合成
化合物5 11.6mg(0.03mmol,1equiv.)，化合物1810.5(0.03mmol,1equiv.)溶解在约2mLDMF中，加入HATU 13.7mg(0.036mmol,1.2equiv.)，加入三乙胺2滴。室温搅拌反应4h。薄层色谱法(TLC)监测反应。反应完后，反应液中加入水，乙酸乙酯萃取，2M HCl洗，5％Na2HCO3洗，水洗。Na2SO4干燥。柱层析分离(甲醇：二氯甲烷＝1：30)，得到黄色固体12.6mg，收率58％。1H NMR(400MHz,DMSO-d6)δ9.97(d,J＝20.5Hz,1H),8.65(d,J＝13.8Hz,1H),8.20(d,J＝71.3Hz,1H),7.73–7.67(m,1H),7.66–7.58(m,2H),7.44(td,J＝8.4,7.4Hz,2H),7.22–7.08(m,5H),6.95(dd,J＝8.7,2.5Hz,1H),6.85(t,J＝2.4Hz,1H),6.63(d,J＝3.0Hz,1H),6.53(d,J＝1.7Hz,1H),4.83(dddd,J＝42.0,11.3,6.5,4.2Hz,1H),4.61(dd,J＝12.6,4.4Hz,0H),4.38(d,J＝13.0Hz,1H),4.30(t,J＝2.2Hz,4H),3.91(dd,J＝13.0,4.2Hz,1H),3.75(d,J＝13.0Hz,0H),3.57(dd,J＝13.1,10.4Hz,1H),3.23(q,J＝2.3Hz,2H),3.20–3.06(m,1H),2.85(td,J＝12.8,3.2Hz,1H),2.23(tt,J＝12.0,3.9Hz,1H),2.13(dd,J＝12.8,4.2Hz,1H),1.95–1.78(m,1H),1.70(ddd,J＝12.8,8.6,4.4Hz,1H).13C NMR(101MHz,DMSO-d6)δ166.78,166.69,163.73,158.69,158.36,157.59,156.78,156.16,154.44,152.08,149.23,143.85,143.71,130.61,130.58,130.50,129.09,129.04,128.39,126.85,125.68,124.26,124.23,120.85,119.44,119.41,112.37,110.66,100.69,97.86,79.91,75.75,55.39。
实施例12
PO-1的合成
Figure BDA0001948810530000191
化合物20的合成参考文献(K.C.Bang,Y.H.Moon,Y.K.Chang,2013,WO2013051883)。
化合物22的合成
化合物21 149mg(0.5mol)加入到10mL甲醇中，加入2mL2NNaOH，混合物室温搅拌过夜。取2mg(0.1mmol,1equiv.)加入到2mLDMF中，加入HATU 46mg(0.12mmol,1.2equiv.)，TEA20mg(0.2mmol,2equiv.)，20(44mg,0.1mmol,1equiv.)，室温反应10h，加入水，EA萃取，饱和NaCL溶液洗，NaSO4干燥。浓缩，真空干燥的26mg，收率60％。1H NMR(400MHz,Chloroform-d)δ8.70(s,1H),8.46(t,J＝8.4Hz,1H),7.38-7.28(m,3H),7.23(s,1H),6.86(dt,J＝15.1,6.3Hz,1H),6.44(dd,J＝15.1,1.6Hz,1H),4.71(dt,J＝6.7,3.2Hz,1H),4.44(s,1H),4.01(s,3H),3.89(s,2H),3.72(d,J＝6.9Hz,1H),3.51(d,J＝35.5Hz,2H),3.13(dd,J＝6.2,1.5Hz,2H),2.83(d,J＝11.3Hz,2H),2.01-1.91(m,4H),1.63(s,5H),1.44(s,9H)。
PO-1的合成
化合物22 31.6mg(0.045mmol,1.5equiv.)加入到2mLTFA和6mLDCM的混合物溶液中，室温搅拌反应30min，溶剂旋干。加入2mLDMF，加入TCO-NHS 8.0mg(0.03mmol,1equiv.)，加入TEA。室温搅拌反应1h，加入水淬灭，EA萃取。柱层析分离(MeOH:DCM 1:25)得到纯化合物4.8mg，收率14％。1H NMR(400MHz,DMSO-d6)δ9.67(s,1H),8.40(s,1H),7.89(s,1H),7.60(d,J＝6.0Hz,2H),7.25(s,1H),6.95(d,J＝7.8Hz,1H),6.61(d,J＝4.3Hz,2H),5.57(ddd,J＝16.0,10.2,4.3Hz,1H),5.43(ddd,J＝15.9,10.9,3.5Hz,1H),4.78(t,J＝3.8Hz,1H),4.24–4.12(m,1H),3.95(s,3H),3.87(s,2H),3.53–3.41(m,2H),3.29–3.15(m,1H),3.07(d,J＝4.6Hz,2H),2.75(d,J＝11.0Hz,2H),2.26(td,J＝8.3,7.2,4.1Hz,3H),2.08–1.80(m,8H),1.73–1.64(m,4H),1.56(d,J＝5.5Hz,1H).13C NMR(101MHz,DMSO-d6)δ164.59,157.23,156.07,155.47,153.56,147.69,146.82,142.37,135.37,132.98,129.30,127.60,125.85,122.96,109.03,108.08,105.97,79.32,73.78,66.82,59.27,56.48,52.60,34.21,32.62,32.32,32.23,31.05.LC-MS(m/z)[M+H]+calcd:754.26；Found:755.45。
实施例13
JN-1的合成
Figure BDA0001948810530000211
化合物24的合成
化合物23 60mg(0.11mmol,1equiv.)加入到3mLDMF中，加入4-N-叔丁氧羰基氨基哌啶27mg(0.13mmol,1.2equiv.)，加入K2CO3 31mg(0.22mmol,2equiv.)，40℃搅拌反应3h。加入水，EA萃取，饱和NaCl水洗，NaSO4干燥。柱层析分离，得到白色固体51mg，收率71％。1HNMR(400MHz,DMSO-d6)δ10.27(s,1H),10.23(s,1H),9.28(d,J＝2.2Hz,1H),8.99(s,1H),8.69(dd,J＝4.8,1.6Hz,1H),8.52(d,J＝5.1Hz,1H),8.49(dt,J＝8.0,2.0Hz,1H),8.14(t,J＝1.9Hz,1H),8.08(d,J＝2.1Hz,1H),7.91(dd,J＝8.0,2.0Hz,1H),7.64(d,J＝7.7Hz,1H),7.54(dd,J＝8.0,4.8Hz,1H),7.48(dd,J＝8.1,2.3Hz,2H),7.44(d,J＝5.4Hz,1H),7.22(d,J＝8.4Hz,1H),6.79–6.68(m,1H),6.27(d,J＝15.4Hz,1H),3.22(s,1H),3.11(d,J＝5.6Hz,2H),2.80(d,J＝11.1Hz,2H),2.23(s,3H),1.99(d,J＝11.6Hz,2H),1.70(d,J＝12.1Hz,2H),1.38(s,9H),1.27–1.23(m,2H)。
化合物JN-1的合成
化合物24 50mg(0.076mmol)加入到3mLDCM中，加入TFA 1mL，室温反应1h。旋干，加入水，EA萃取，浓缩，NaSO4干燥。取14mg(0.025mmol,1equiv.)，加入3mLDMF,加入TCO-NHS6.6mg(0.025mmol,1equiv.)，TEA 1滴。室温反应1h，加入水淬灭，EA萃取，饱和NaCl洗，NaSO4干燥，浓缩，柱层析分离得到8.2mg，收率46％。1H NMR(400MHz,DMSO-d6)δ10.27(s,1H),10.23(s,1H),9.28(dd,J＝2.3,0.8Hz,1H),8.99(s,1H),8.69(dd,J＝4.8,1.7Hz,1H),8.52(d,J＝5.1Hz,1H),8.51–8.47(m,1H),8.14(t,J＝1.9Hz,1H),8.07(d,J＝2.2Hz,1H),7.90(d,J＝8.1Hz,1H),7.63(dt,J＝7.8,1.2Hz,1H),7.54(ddd,J＝8.0,4.8,0.8Hz,1H),7.48(dd,J＝8.1,2.0Hz,2H),7.44(d,J＝5.3Hz,1H),7.22(d,J＝8.4Hz,1H),6.99(d,J＝7.9Hz,1H),6.78–6.73(m,1H),6.27(d,J＝15.4Hz,1H),5.57(dd,J＝10.5,5.6Hz,1H),5.47–5.41(m,1H),4.21(s,1H),3.10(d,J＝5.7Hz,1H),2.78(d,J＝11.2Hz,2H),2.30–2.26(m,2H),2.23(s,3H),2.00(d,J＝11.3Hz,2H),1.88(ddd,J＝22.5,10.9,7.4Hz,4H),1.70(d,J＝12.6Hz,2H),1.63–1.49(m,4H),1.40(s,4H).HR-MS(ESI)for C41H46N8O4[M+H]+calcd:715.360,Found:715.5400。
实施例14
HJ-1的合成
Figure BDA0001948810530000221
化合物25的合成参考文献(T.Xu,T.Peng,X.Ren,L.Zhang,L.Yu,J.Luo,Z.Zhang,Z.Tu,L.Tong,Z.Huang,X.Lu,M.Geng,H.Xie,J.Ding,K.Ding,Med.Chem.Commun.2015,6,1693-1697.)
HJ-1的合成
丙烯酰氯41.2μL(0.50mmol,1equiv.)溶解在约10mL DCM中，滴加化合物25 247mg(0.5mmol,1equiv.)，加入87.3μL(0.50mmol,1equiv.)。0℃搅拌反应1h。减压浓缩，柱层析分离(DCM:MeOH＝30:1)，得到黄色固体化合物178mg，收率65％。1H NMR(400MHz,DMSO)δ10.36(s,1H),8.81(s,1H),8.12(s,1H),7.88(d,J＝7.4Hz,1H),7.58(s,1H),7.51(t,J＝8.0Hz,1H),7.27(d,J＝8.9Hz,1H),6.98(d,J＝8.7Hz,1H),6.54(s,1H),6.44(dd,J＝16.9,10.1Hz,1H),6.33(s,1H),6.26(d,J＝17.0Hz,1H),5.77(d,J＝12.0Hz,1H),3.78(s,3H),3.34(s,2H),3.20(t,J＝2.3Hz,1H),3.05(s,4H),2.58(t,J＝4.7Hz,4H),2.46(s,3H).13CNMR(101MHz,DMSO)δ163.68,162.58,157.03,156.73,147.36,140.39,137.52,132.18,129.96,127.60,124.50,121.37,120.47,120.23,119.18,116.99,106.68,100.23,79.72,76.32,56.15,51.53,49.11,46.47,17.49.HR-MS(m/z)[M+H]+calcd:550.2449；Found:550.2524。
实施例15
FG-1及其中间体的合成
Figure BDA0001948810530000231
化合物26的合成参考文献(Y.Wang,Z.Chen,M.Dai,etal.Bioorg.Med.Chem.Lett.2017,27,2420-2423)。
化合物27的合成
化合物26 281mg(0.51mmol,1equiv.)溶解在约2mLTHF中，加入碳酸铯325mg(1.0mmol,2equiv.)，加入3-溴丙炔(0.76mmol,1.5equiv.)，温度加热到90℃，反应过夜。旋蒸除去溶剂，加入5mL水溶液，乙酸乙酯萃取。NaSO4干燥。柱层析分离，得到橘黄色油状物27207mg，两步反应收率76％。1H NMR(400MHz,Chloroform-d)δ8.19(s,2H),7.56(s,1H),7.36(d,J＝2.9Hz,1H),7.08(d,J＝2.9Hz,1H),6.63(s,1H),5.31(s,2H),3.95(s,6H),3.41(d,J＝2.5Hz,2H),3.34-3.28(m,4H),2.80-2.72(m,4H),2.32(t,J＝2.4Hz,1H),2.29(s,3H)。
FG-1的合成
化合物27 207mg(0.35mmol,1equiv.)溶解在约5mL异丙醇：DCM＝2:1的混合溶液中，加入SnCl2 267mg(1.4mmol,4equiv.)。加热回流反应9h。薄层色谱法(TLC)监测反应。反应完后，旋掉溶剂，加入5mL水。NaOH调节pH值到14，DCM萃取，盐水洗，NaSO4干燥。柱层析分离，得到橘红色固体14 3mg，收率73％。1H NMR(400MHz,DMSO-d6)δ8.13(s,2H),7.82(s,1H),6.99(s,1H),6.16(d,J＝2.7Hz,1H),6.08(d,J＝2.6Hz,1H),5.21(s,2H),4.51(s,2H),3.94(s,6H),3.32(d,J＝3.4Hz,2H),3.19(t,J＝2.4Hz,1H),3.06(dd,J＝6.2,3.9Hz,4H),2.58(t,J＝5.0Hz,4H),1.95(s,3H)。
中间体化合物14 3mg(0.26mmol,1equiv.)溶解在2mLDCM中，加入DIPEA 0.08mL(0.53mmol,2equiv.)，0℃，氩气保护下，缓慢滴加丙烯酰氯21mg(0.23mmol,0.9equiv.)。0℃下反应0.5h。薄层色谱法(TLC)监测反应。反应完后，加入2mL水淬灭反应，DCM萃取。盐水洗，NaSO4干燥，柱层析分离。得终产物FG-1 79mg，收率50％。1H NMR(400MHz,Chloroform-d)δ8.17(s,2H),8.10(s,1H),7.91(s,1H),6.69(t,J＝8.1Hz,1H),6.61(d,J＝2.7Hz,1H),6.35(dd,J＝16.9,1.4Hz,1H),6.25–6.09(m,2H),5.70(dd,J＝10.2,1.4Hz,1H),5.13(t,J＝1.6Hz,2H),3.90(s,6H),3.38(d,J＝2.4Hz,2H),3.31(t,J＝5.0Hz,4H),2.73(t,J＝5.0Hz,4H),2.30(t,J＝2.4Hz,1H),2.17(s,3H).LC-MS(m/z)[M+H]+calcd:578.25；Found:579.29。
实施例16
本实施例为丙烯酰胺类的分子探针化合物在活细胞内标记靶标蛋白实验，以AF-1/AF-2为例。
在37℃下，5％CO2下培养人肺癌细胞A431，到对数生长期将细胞均匀地分到6孔板。待24h贴壁后，其中3个孔加入不同浓度的分子探针0.1、1、5μM，另外3个孔中加入分子探针0.1、1、5μM和5倍抑制剂。在细胞培养箱中孵育5h。去除培养基，用磷酸缓冲液(PBS)洗涤两次。然后，用DPBS细胞裂解液将细胞裂解，其中DPBS中加入蛋白酶和磷酸酶1片/10mL及3％的两性表面活性剂3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐。用BCA蛋白定量试剂盒进行定量蛋白浓度至1毫克每毫升。取一定量的细胞裂解液，加入点击化学试剂[THPTA(0.1mmol),TCEP(1mmol),CuSO4(1mmol)]及荧光染料TARMA-Azide(10μM)，进行点击化学反应，室温反应2h。随后，加入冰冻的丙酮溶液将蛋白变性析出并离心去除有机溶剂，得到的蛋白固体加入上样缓冲液，并通过聚丙烯酰胺凝胶电泳进行分离。最后，通过多功能激光扫描成像仪Typhoon FLA 9500测试。如图1和2所示，分子探针AF-1、AF-2能够标记其靶蛋白EGFR。
实施例17
通过在活细胞内竞争性标记实验利用分子探针来筛选靶向BTK的活性化合，以IB-2/IB-3为例。
在37℃下，5％CO2下培养人肺癌细胞弥漫大B细胞淋巴瘤细胞Toledo，到对数生长期将细胞均匀地分到6孔板。待24h贴壁后，其中4个孔加入不同浓度的分子探针1、10、10、100μM，第5个孔加入100μM的分子探针以及5μM被筛选的化合物16，第6个孔加入DMSO作为阴性对照。在细胞培养箱中孵育5h。去除培养基，用磷酸缓冲液(PBS)洗涤两次。然后，用DPBS细胞裂解液将细胞裂解，其中DPBS中加入蛋白酶和磷酸酶1片/10mL及3％的两性表面活性剂3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐。用BCA蛋白定量试剂盒进行定量蛋白浓度至1毫克每毫升。取一定量的细胞裂解液，加入点击化学试剂荧光染料Tz-Cy5(10μM)，进行点击化学反应，室温反应2h。随后，加入冰冻的丙酮溶液将蛋白变性析出并离心去除有机溶剂，得到的蛋白固体加入上样缓冲液，并通过聚丙烯酰胺凝胶电泳进行分离。最后，通过多功能激光扫描成像仪Typhoon FLA 9500获取实验结果，如图3所示。实验结果表明，竞争性实验结果是化合物16在分子探针70kD的条带消失，化合物16有可能是靶向BTK的活性化合物，同时说明利用分子探针IB-2、IB-3可以筛选靶向BTK的活性化合物。
另外，利用分子探针IB-3筛选靶向BTK的活性天然产物(化合物1-173)的多功能激光扫描成像结果见图4(a)-(g)，其中在IB-3 70kD的条带消失说明该化合物可能是靶向BTK的活性化合物，未消失说明该化合物不是靶向BTK的活性化合物。由此说明分子探针IB-3具有较为优异的靶向BTK的筛选能力。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (4)

1.具有如下式所示结构的丙烯酰胺类化合物：
Figure FDA0003316621150000011
2.权利要求1所述的丙烯酰胺类化合物在制备分子探针中的应用。
3.根据权利要求2所述的应用，其特征在于，所述分子探针用于标记靶标蛋白，或，所述分子探针用于筛选抗癌活性化合物。
4.一种抗癌活性化合物的筛选方法，其特征在于，包括如下步骤：
(1)待细胞生长到对数生长期时，分到孔板中；
(2)将权利要求1所述的丙烯酰胺类化合物和被筛选的抗癌活性化合物加入至所述孔板，进行孵育；
(3)于孵育后的细胞加入蛋白酶抑制剂的缓冲液进行裂解，超声，离心后取上清液；
(4)用蛋白标记试剂盒标定蛋白浓度；
(5)通过点击化学反应引入荧光基团，检测，即可。
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