CN103086972A - 吡唑-酰腙类化合物的制备及其在抗癌治疗中的应用 - Google Patents
吡唑-酰腙类化合物的制备及其在抗癌治疗中的应用 Download PDFInfo
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Abstract
一类吡唑-酰腙类衍生物1e-20e,其特征是它有如下通式:
Description
技术领域
本发明涉及吡唑-酰腙类化合物及其制备方法与抗癌治疗中的应用。
背景技术
端粒酶是一种核蛋白逆转录酶,可以合成染色体末端的端粒,使得因每次细胞分裂而逐渐缩短的端粒长度得以补偿,进而保证染色体的完整性。其活性在成体的大多数体细胞中被抑制,而在肿瘤细胞中被重新激活,保持端粒长度,从而使得肿瘤细胞无休止的增殖。近年有关端粒酶与肿瘤关系的研究表明,在肿瘤细胞中端粒酶还参与了对肿瘤细胞的凋亡和基因组稳定的调控过程。因此,抑制肿瘤细胞中端粒酶的活性将成为肿瘤治疗的重要途径和有效手段。
近年,X-射线衍射晶体学揭示了端粒酶活性部位由人类端粒酶RNA(hTR)和人类端粒酶逆转录酶(hTERT)催化亚基两个主要部分,以及许多端粒酶相关蛋白等组成,这将为端粒酶抑制剂提供重要作用靶点。
研究发现,许多含吡唑结构的端粒酶抑制剂表现出显著端粒酶抑制作用。例如,VER49009通过间接抑制端粒酶与相关蛋白的结合,从而导致端粒的去帽保护与细胞凋亡。另外,文献报道,酰胺基团作为主要结构骨架存在于多种端粒酶抑制性抗癌药物中。例如,BIBR1532是至今为止最有希望的hTERT特异性抑制剂,作为端粒酶非竞争性抑制剂,特异性地阻止DNA链的延伸。再者,由于腙的衍生物的特殊结构,尤其酰腙类衍生物,其表现出显著的配位性与配位多样性。一方面,酰腙基团可以与金属离子形成配合物,从而阻止由该金属离子催化的生理反应,或者促进药物在体内的吸收、转移、分配与代谢等;另一方面,酰腙基团可能与靶蛋白或DNA形成疏水相互作用和氢键,从而抑制细胞增殖。
正是基于以上多种基团的药效及其配伍研究,我们设计并合成了20种吡唑-酰腙类衍生物(15个化合物未经报道),并对其进行生物药效评价,推断这些新化合物可能表现出吡唑、酰胺和酰腙类化合物在抗癌作用中的协同性。我们测试了这批化合物的活性,并发现其对MCF7和B16-F10具有良好的抑制作用,且能有效抑制端粒酶活性从而阻止细胞周期,诱导细胞凋亡。
发明内容
本发明的目的在于提供一类新型吡唑-酰腙类化合物以及它们的制备方法与用途。
本发明的技术方案如下:
1.一类吡唑-酰腙类衍生物1e-20e,其特征是它有如下通式:
结构式中R1为:氟、氯、氢、甲氧基、甲基;
R2为:氢、甲基;
R3为:氯、溴、氢。
2.一种制备上述吡唑-酰腙类衍生物的方法,它由下列步骤组成:
步骤1.制备希夫碱(化合物a):
将乙酸钠(40mmol)加入到取代基苯乙酮(20mmol)与盐酸苯肼(25mmol)的乙醇溶液中,磁力搅拌,于50-60℃反应约3h,(TLC检测反应进行程度),产物以固体析出。反应结束后抽滤,并以无水乙醇冲洗固体物3次,干燥得取代基希夫碱(化合物a)。用无水乙醇将产物重结晶。
步骤2.制备吡唑-醛类衍生物(化合物b):
将步骤1所得产物加入到冰的DMF(10ml)溶液中,随后再逐滴加入POCl3(8ml),磁力搅拌,于50-60℃反应约6h(TLC检测反应进行程度);待反应结束,将反应体系倒入冰水中,并用氢氧化钠饱和溶液调节pH至中性,产物将以沉淀析出,真空抽滤后进行水洗,干燥,最后利用乙醇溶液进行重结晶,即得产物b。
步骤3.二氯亚砜成酯法制备水杨酸甲酯类衍生物(化合物c):
将水杨酸溶于甲醇中,放置于冰浴,向体系内逐滴加入SOCl2(约1.5eq),加毕,于冰浴中反应30-1h,然后放置于常温下,反应大概2-3h,转移到45℃,反应过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去甲醇及SOCl2,用乙酸乙酯与饱和NaHCO3萃取,旋转蒸发仪抽去萃取液,最后用乙醇重结晶,即得产物c。
步骤4.制备酰肼类衍生物(化合物d):
将产物c溶解于乙醇中,逐滴加入80%水合肼,磁力搅拌,反应回流过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去溶剂直到沉淀析出,然后向其中加入水,静置,沉淀将大量析出,即得产物d。
步骤5.制备终产物:吡唑-酰腙类衍生物(化合物1e-20e):
将上述所得产物b和产物d溶解于无水乙醇中,磁力搅拌,回流反应约4h(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽净溶剂,然后用乙酸乙酯萃取,再用饱和NaCl连续洗涤,用无水Na2SO4干燥,抽滤,重结晶,即得终产物1e-20e。
本发明的吡唑-酰腙类衍生物具有抑制MCF-7和B16-F10细胞增殖的作用以及抑制端粒酶活性的作用。因此本发明的吡唑-酰腙类衍生物1e-20e可做潜在的抗肿瘤药物。
具体实施方式
实施例一:5-氯-N′-((3-(4-氟苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物1e)的制备
将乙酸钠(40mmol)加入到取代基苯乙酮(20mmol)与盐酸苯肼(25mmol)的乙醇溶液中,磁力搅拌,于50-60℃反应约3h,(TLC检测反应进行程度),产物以固体析出。反应结束后抽滤,并以无水乙醇冲洗固体物3次,干燥得取代基希夫碱(化合物a)。用无水乙醇将产物重结晶。取步骤1所得产物a,加入到冰的DMF(10ml)溶液中,随后再逐滴加入POCl3(8ml),磁力搅拌,于50-60℃反应约6h(TLC检测反应进行程度);待反应结束,将反应体系倒入冰水中,并用氢氧化钠饱和溶液调节pH至中性,产物将以沉淀析出,真空抽滤后进行水洗,干燥,最后利用乙醇溶液进行重结晶,即得产物b。
将水杨酸(1eq)溶于甲醇(>1eq)中,放置于冰浴,向体系内逐滴加入SOCl2(约1.5eq),加毕,于冰浴中反应30-1h,然后放置于常温下,反应大概2-3h,转移到45℃,反应过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去甲醇及SOCl2,用乙酸乙酯与饱和NaHCO3萃取,旋转蒸发仪抽去萃取液,最后用乙醇重结晶,即得产物c。取干燥产物c(1eq)溶解于乙醇中,逐滴加入80%水合肼(3eq),磁力搅拌,反应回流过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去溶剂直到沉淀析出,然后向其中加入水,静置,沉淀将大量析出,即得产物d。
将上述所得产物b(1eq)和产物d(1eq)溶解于无水乙醇中,磁力搅拌,回流反应约4h(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽净溶剂,然后用乙酸乙酯萃取,再用饱和NaCl连续洗涤,用无水Na2SO4干燥,抽滤,重结晶,即得终产物白色固体。Yield65%,mp:114-119℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);7.01(d,J=8.97Hz,1H);7.16-7.24(m,3H);7.62-7.67(m,3H);7.80(t,J=5.90Hz,3H);7.86-7.90(m,1H);8.27(s,1H);8.62(s,1H);9.53(s,1H);11.71(s,1H).ESI-MS:434.65(C23H17ClFN4O2[M+H]+).Anal.Calcd forC23H16ClFN4O2:C,63.53;H,3.71;N,12.88.Found:C,63.61;H,3.72;N,12.84.
实施例二:5-溴-N′-((3-(4-氟苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物2e)的制备
制备方法同实施例一。以3-溴水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield74%,mp:127-130℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);6.95(t,J=15H.72z,2H);7.11-7.28(m,2H);7.43(t,J=12.99Hz,1H);7.65-7.69(m,3H);7.87(d,J=5.49Hz,2H);8.27(s,1H);8.56(s,1H);8.69(s,1H);9.38(s,1H);10.06(s,1H).ESI-MS:479.34(C23H17BrFN4O2[M+H]+).Anal.Calcd for C23H16BrFN4O2:C,57.64;H,3.36;N,11.69.Found:C,57.87;H,3.37;N,11.66.
实施例三:N′-((3-(4-氟苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物3e)的制备
制备方法同实施例一。以水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield68%,mp:98-102℃.1H NMR(CDCl3,300MHz):3.73(d,J=6.39Hz,1H);6.88(t,J=15.36Hz,1H);7.06(d,J=8.58Hz,1H);7.13-7.24(m,2H);7.36(t,J=15.00Hz,1H);7.42-7.54(m,3H);7.60-7.65(m,2H);7.75-7.86(m,2H);8.24(s,1H);8.58(d,J=20.49Hz,1H);10.05(s,1H);11.80(s,1H).ESI-MS:400.14(C23H18FN4O2[M+H]+).Anal.Calcd for C23H17FN4O2:C,68.99;H,4.28;N,13.99.Found:C,68.71;H,4.29;N,14.03.
实施例四:N′-((3-(4-氟苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟基-3-甲苯酰肼(化合物4e)的制备
制备方法同实施例一。以3-甲基水杨酸代替例一中的5-氯水杨酸,得到淡黄色固体。Yield67%,mp:135-138℃.1H NMR(CDCl3,300MHz):2.28(s,3H);2.31(s,1H);6.80(t,J=15.36Hz,1H);7.16-7.24(m,1H);7.48-7.57(m,2H);7.63-7.67(m,1H);7.79(d,J=7.86Hz,4H);7.86-7.91(m,1H);8.22(s,1H);8.55(s,1H);8.66(s,1H);9.41(s,1H);12.12(s,1H);ESI-MS:414.24(C24H20FN4O2[M+H]+).Anal.Calcd for C24H19FN4O2:C,69.55;H,4.62;N,13.52.Found:C,69.82;H,4.63;N,13.55.
实施例五:5-氯-N′-((3-(4-氯苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物5e)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,得到淡黄色固体。Yield81%,mp:164-168℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);7.02(d,J=9.00Hz,1H);7.39(t,J=6.21Hz,3H);7.50(s,1H);7.63(d,J=8.22Hz,2H);7.79-7.87(m,4H);8.28(s,1H);8.67(s,1H);9.47(s,1H);11.71(s,1H).ESI-MS:451.69(C23H17Cl2N4O2[M+H]+).Anal.Calcd for C23H16Cl2N4O2:C,61.21;H,3.57;N,12.41.Found:C,60.96;H,3.58;N,12.45.
实施例六:5-溴-N′-((3-(4-氯苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物6e)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,以5-溴水杨酸代替例一中的5-氯水杨酸,得到黄色固体。Yield72%,mp:167-170℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);6.98(d,J=8.97Hz,2H);7.43-7.55(m,3H);7.65(d,J=8.61Hz,2H);7.80-7.87(m,2H);8.28(s,1H);8.56(s,2H);8.70(s,1H);10.07(s,1H);11.75(s,1H).ESI-MS:496.08(C23H17BrClN4O2[M+H]+).Anal.Calcdfor C23H16BrClN4O2:C,55.72;H,3.25;N,11.30.Found:C,55.94;H,3.24;N,11.26.
实施例七:N′-((3-(4-氯苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物7e)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,以水杨酸代替例一中的5-氯水杨酸,得到淡黄色固体。Yield67%,mp:123-125℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);6.91(t,J=13.71Hz,1H);7.07(d,J=8.58Hz,2H);7.46-7.54(m,3H);7.60(d,J=12.06Hz,2H);7.79-7.87(m,4H);8.25(s,1H);8.67(s,1H);9.46(s,1H);11.80(s,1H).ESI-MS:416.74(C23H18ClN4O2[M+H]+).Anal.Calcd for C23H17ClN4O2:C,66.27;H,4.11;N,13.44.Found:C,66.53;H,4.10;N,13.47.
实施例八:N′-((3-(4-氯苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-2-羟基-3-甲苯酰肼(化合物8e)的制备
制备方法同实施例一。以4-氯苯乙酮代替例一中的4-氟苯乙酮,以3-甲基水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield59%,mp:127-130℃.1H NMR(CDCl3,300MHz):2.29(s,3H);2.31(s,1H);6.80(t,J=15.36Hz,1H);7.33-7.39(m,3H);7.47(s,1H);7.61(t,J=8.61Hz,3H);7.77-7.83(m,3H);8.22(s,1H);8.55(s,1H);9.44(s,1H);12.11(s,1H).ESI-MS:430.68(C24H20ClN4O2[M+H]+).Anal.Calcd for C24H19ClN4O2:C,66.90;H,4.44;N,13.00.Found:C,66.63;H,4.43;N,13.04.
实施例九:5-氯-N′-((1,3-二苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物9e)的制备
制备方法同实施例一。以苯乙酮代替例一中的4-氟苯乙酮,得到白色固体。Yield72%,mp:132-139℃.1H NMR(CDCl3,300MHz):3.72-3.76(m,1H);6.99(d,J=5.31Hz,1H);7.37-7.39(m,3H);7.43-7.47(m,3H);7.71(d,J=39.54Hz,2H);7.79-7.84(m,3H);8.30(s,1H);8.61(s,1H);9.69(s,1H);11.32(s,1H).ESI-MS:416.94(C23H18ClN4O2[M+H]+).Anal.Calcd for C23H17ClN4O2:C,66.27;H,4.11;N,13.44.Found:C,66.54;H,4.12;N,13.47.
实施例十:5溴-N′-((1,3-二苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物10e)的制备
制备方法同实施例一。以苯乙酮代替例一中的4-氟苯乙酮,以5-溴水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield72%,mp:138-148℃.1H NMR(CDCl3,300MHz):3.71-3.78(m,1H);6.96(d,J=8.97Hz,1H);7.30(s,1H);7.37(d,J=7.50Hz,2H);7.50(t,J=15.36Hz,3H);7.72(s,2H);7.77(t,J=18.12Hz,3H);8.30(s,1H);8.62(s,1H);9.66(s,1H);11.80(s,1H).ESI-MS:461.23(C23H18BrN4O2[M+H]+).Anal.Calcd for C23H17BrN4O2:C,59.88;H,3.71;N,12.15.Found:C,59.64;H,3.72;N,12.19.
实施例十一:N′-((1,3-二苯基-1氢-吡唑-4-基)亚甲基)-2-羟苯酰肼(化合物11e)的制备
制备方法同实施例一。以苯乙酮代替例一中的4-氟苯乙酮,以水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield68%,mp:118-122℃.1H NMR(CDCl3,300MHz):3.71-3.76(m,1H);6.89(t,J=8.97Hz,1H);7.05(t,J=19.32Hz,1H);7.28(s,2H);7.35(t,J=8.79Hz,1H);7.45(t,J=7.89Hz,3H);7.64(d,J=4.29Hz,2H);7.77-7.84(m,3H);8.26(s,1H);8.63(s,1H);9.57(s,1H);11.83(s,1H).ESI-MS:382.19(C23H19N4O2[M+H]+).Anal.Calcd for C23H18N4O2:C,72.24;H,4.74;N,14.65.Found:C,72.53;H,4.75;N,14.62.
实施例十二:N′-(1,3-二苯基-1氢-吡唑-4-基)亚甲基)-2-羟基-3-甲苯酰肼(化合物12e)的制备
制备方法同实施例一。以苯乙酮代替例一中的4-氟苯乙酮,以3-甲基水杨酸代替例一中的5-氯水杨酸,得到淡黄色固体。Yield73%,mp:126-131℃.1H NMR(CDCl3,300MHz):2.27(s,3H);2.31(s,1H);6.79(t,J=9.15Hz,1H);7.32-7.38(m,3H);7.41-7.46(m,3H);7.66(d,J=4.11Hz,2H);7.80-7.85(m,3H);8.24(s,1H);8.68(s,1H);9.40(s,1H);12.13(s,1H).ESI-MS:396.13(C24H21N4O2[M+H]+).Anal.Calcd for C24H20N4O2:C,72.71;H,5.08;N,14.13.Found:C,72.42;H,5.10;N,13.17.
实施例十三:5-氯-2-羟基-N′-((3-(4-甲氧基苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物13e)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,得到淡黄色固体。Yield79%,mp:123-125℃.1H NMR(CDCl3,300MHz):2.31(s,3H);2.43(s,1H);6.10(d,J=8.97Hz,1H);7.27(s,1H);7.31-7.40(m,3H);7.46-7.53(m,4H);7.73(t,J=14.32Hz,2H);8.31(s,1H);8.59(s,1H);9.75(s,1H);10.11(s,1H).ESI-MS:446.54(C24H20ClN4O3[M+H]+).Anal.Calcd for C24H19ClN4O3:C,64.50;H,4.29;N,12.54.Found:C,64.76;H,4.30;N,12.50.
实施例十四:5-溴-2-羟基-N′-((3-(4-甲氧基苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物14e)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,以5-溴水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield83%,mp:111-118℃.1HNMR(CDCl3,300MHz):2.37(s,3H);2.41(s,1H);6.08(d,J=8.79Hz,1H);7.15(s,1H);7.25-7.34(m,3H);7.36-7.45(m,4H);7.73(t,J=13.31Hz,2H);8.11(s,1H);8.28(s,1H);9.34(s,1H);10.10(s,1H).ESI-MS:491.23(C24H20BrN4O3[M+H]+).Anal.Calcd for C24H19BrN4O3:C,58.67;H,3.90;N,11.40.Found:C,58.90;H,4.01;N,11.36
实施例十五:2-羟基-N′-((3-(4-甲氧基苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物15e)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,以水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield69%,mp:106-109℃.1H NMR(CDCl3,300MHz):2.29(s,3H);2.33(s,1H);6.81(t,J=9.15Hz,1H);7.33-7.39(m,3H);7.44-7.48(m,3H);7.67(d,J=4.21Hz,2H);7.81-7.86(m,3H);8.27(s,1H);8.69(s,1H);9.43(s,1H);12.12(s,1H).ESI-MS:412.68(C24H21N4O3[M+H]+).Anal.Calcd for C24H20N4O3:C,69.89;H,4.89;N,13.58.Found:C,70.17;H,4.91;N,13.54.
实施例十六:2-羟基-N′-((3-(4-甲氧基苯基)-1-苯基-1氢-吡唑-4-基)亚甲基)-3-甲苯酰肼(化合物16e)的制备
制备方法同实施例一。以4-甲氧基苯乙酮代替例一中的4-氟苯乙酮,以3-甲基水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield75%,mp:120-125℃.1HNMR(CDCl3,300MHz):2.39(s,3H);2.42(s,3H);2.43(s,1H);6.79(t,J=7.79Hz,1H);7.37(s,2H);7.50-7.65(m,4H);7.73(d,J=8.23Hz,1H);7.80(d,J=8.25Hz,3H);8.31(s,1H);8.53(s,1H);8.75(s,1H);10.12(s,1H).ESI-MS:426.13(C25H23N4O3[M+H]+).Anal.Calcd for C25H22N4O3:C,70.41;H,5.20;N,13.14.Found:C,70.69;H,5.19;N,13.11.
实施例十七:5-氯-2-羟基-N′-((1-苯基-3-(对-甲苯基)-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物17e)的制备
制备方法同实施例一。以4-甲基苯乙酮代替例一中的4-氟苯乙酮,得到白色固体。Yield79%,mp:137-140℃.1H NMR(CDCl3,300MHz):2.40(s,3H);2.45(s,1H);6.10(d,J=8.97Hz,1H);7.25(s,1H);7.32-7.41(m,3H);7.48-7.55(m,4H);7.75(t,J=21.96Hz,2H);8.29(s,1H);8.58(s,1H);9.72(s,1H);10.01(s,1H).ESI-MS:430.94(C24H20ClN4O2[M+H]+).Anal.Calcd for C24H19ClN4O2:C,66.90;H,4.44;N,13.00.Found:C,67.12;H,4.46;N,12.96.
实施例十八:5-溴-2-羟基-N′-((1-苯基-3-(对-甲苯基)-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物18e)的制备
制备方法同实施例一。以4-甲基苯乙酮代替例一中的4-氟苯乙酮,以5-溴水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield77%,mp:145-151℃.1H NMR(CDCl3,300MHz):2.38(s,3H);2.43(s,1H);6.08(d,J=8.97Hz,1H);7.15(s,1H);7.24-7.31(m,3H);7.38-7.45(m,4H);7.75(t,J=14.32Hz,2H);8.14(s,1H);8.29(s,1H);9.37(s,1H);10.11(s,1H).ESI-MS:475.13(C24H20BrN4O2[M+H]+).Anal.Calcd for C24H19BrN4O2:C,60.64;H,4.03;N,11.79.Found:C,60.88;H,3.74;N,11.82.
实施例十九:2-羟基-N′-((1-苯基-3-(对-甲苯基)-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物19e)的制备
制备方法同实施例一。以4-甲基苯乙酮代替例一中的4-氟苯乙酮,以水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield75%,mp:132-137℃.1H NMR(CDCl3,300MHz):2.37(s,3H);2.45(s,1H);6.90(t,J=14.46Hz,1H);7.06(d,J=8.43Hz,2H);7.33(d,J=7.14Hz,2H);7.48-7.58(m,2H);7.74(d,J=8.07Hz,3H);7.82(t,J=7.50Hz,2H);8.24(s,1H);8.55(s,1H);8.71(s,1H);10.07(s,1H).ESI-MS:396.58(C24H21N4O2[M+H]+).Anal.Calcd for C24H20N4O2:C,72.71;H,5.08;N,14.13.Found:C,73.00;H,5.10;N,14.18.
实施例二十:2-羟基-3-甲基-N′-((1-苯基-3-(对-甲苯基)-1氢-吡唑-4-基)亚甲基)-苯基酰肼(化合物20e)的制备
制备方法同实施例一。以4-甲基苯乙酮代替例一中的4-氟苯乙酮,以3-甲基水杨酸代替例一中的5-氯水杨酸,得到白色固体。Yield66%,mp:139-143℃.1HNMR(CDCl3,300MHz):2.37(s,3H);2.44(s,3H);2.45(s,1H);6.80(t,J=15.36Hz,1H);7.34(s,2H);7.53-7.62(m,4H);7.74(d,J=8.10Hz,1H);7.82(d,J=8.25Hz,3H);8.24(s,1H);8.55(s,1H);8.73(s,1H);10.07(s,1H).ESI-MS:410.21(C25H23N4O2[M+H]+).Anal.Calcd for C25H22N4O2:C,73.16;H,5.38;N,13.67.Found:C,73.45;H,5.40;N,13.63.
实施例二十一:吡唑-酰腙类化合物对肿瘤的抑制活性的研究
采用MTT[3-(4,5)-双甲基-2-噻唑-(2,5)-苯基溴化四氮唑蓝]法来测定吡唑-酰腙类化合物对MCF7、B16-F10癌细胞及对端粒酶的半抑制浓度,即IC50;采用TRAP-PCR-ELISA法来检测化合物1e-20e在体外对端粒酶活性的影响。
(1)培养液(每升)的配制:①悬浮细胞:RPMI-1640培养粉一袋(10.4g),新生牛血清100ml,青霉素溶液(20万U/ml)0.5ml,链霉素溶液(20万U/ml)0.5ml,加三蒸水溶解后,用5.6%的NaHCO3溶液调PH值至7.2-7.4,最后定容至1000ml。过滤灭菌。②贴壁细胞:同上,再加入NaHCO32.00g,HEPES2.38g。
(2)D-Hanks缓冲液(每升)的配制:NaCl8.00g,KCl0.40g,Na2HPO4·12H2O0.06g,KH2PO40.06g,NaHCO30.35g。高压灭菌。
(3)胰蛋白酶液的配制:利用D-Hanks缓冲液配成浓度为0.5%胰蛋白酶液。过滤除菌。
(4)实验药液的配制:将测试样品用少量的三蒸水溶解配成储备液,一般按实验最高浓度的10倍配制储备液。根据化合物溶解性不同,可用三蒸水直接溶解,或用少量DMSO助溶,再加三蒸水溶解。DMSO在培养液中的浓度不宜过大,加药后的每孔细胞悬液中DMSO的终浓度一般不超过0.05%-0.1%。储备液保存于-20℃冰箱中备用。
(5)细胞培养:为贴壁生长细胞,常规培养于DMEM或者RPMI-1640培养液内(含10%小牛血清),置37℃、5%CO2培养箱中培养,每隔3-4天传代一次。传代时先弃去原培养液,再用D-Hanks缓冲液洗涤;然后用0.5%胰蛋白酶消化30秒左右,加入少量新鲜培养液终止消化;吹打,使贴壁细胞从培养瓶壁上脱落下来;移取适量至新鲜培养瓶中,再补充新鲜培养液至原体积(培养液体积约为培养瓶容量的1/10)。
(6)细胞孵育:取对数生长期的上述肿瘤细胞,调细胞悬液浓度为2×104个/ml。在96孔培养板中每孔加细胞悬液100μl,置37℃,5%CO2培养箱中培养24h。培养24h后,分别按设计加入药液。
(7)加药:将测试药液按照最终浓度的浓度梯度分别加入到各个孔中,每个浓度设6个平行孔。实验分为药物试验组(分别加入不同浓度的测试药)、对照组(只加培养液和细胞,不加测试药)和空白组(只加培养液,不加细胞和测试药)。将加药后的96孔板置于37℃,5%CO2培养箱中培养48h。
(8)存活细胞的测定:在培养了48h后的96孔板中,每孔加MTT40μl(用40μl PBS配成2.5mg/ml的MTT)。在37℃放置4h后,移去上清液。每孔加100μl提取液(10%SDS-5%异丁醇-0.01M HCl)。37℃孵育过夜,最后,利用自动酶标仪在570nm波长处检测各孔的光密度(OD值)。
抑制率的计算:细胞生长的抑制率按照下列公式计算:
生长抑制率=(1-存活率)×100%=[1-(OD实验-OD空白)/(OD对照-OD空白)]×100%(OD实验表示测试药物组的平均光密度,OD对照表示对照组的平均光密度,OD空白表示对照组的平均光密度)。
半数抑制浓度(IC50)定义为当50%的肿瘤细胞存活时的药物浓度。根据测定的光密度(OD值),制作细胞生长抑制率的标准曲线,在标准曲线上求得其对应的药物浓度。
测得的IC50见表2所示
(9)化合物1e-20e对端粒酶活性的影响的测定
使用端粒酶检测试剂盒进行TRAP-PCR-ELISA试验以测定测定吡唑-酰腙类化合物1e-20e对端粒酶的半抑制浓度,即IC50。将2μL的MCF-7细胞提取液与48μL TRAP反应液混合后,于94℃,PCR预变性去活120s,随后进行35个循环,每个PCR循环包括94℃变性30s,50℃退火30s,72℃延伸90s。取20μL PCR产物与DIG(异羟基洋地黄毒甙元)标记的端粒特异性检测探针杂交。接着通过生物素标记的引物将PCR产物固定在抗生物素蛋白链菌素覆盖的微量滴定板上。利用过氧化物酶结合的DIG抗体检测被固定的DNA片段,最后,利用自动酶标仪在490nm波长处检测微量板上样品的光密度(OD值)。
测得的IC50见表2所示
Compound 1e-20e的通式
表1.吡唑-酰腙类化合物Compound 1e-20e的结构式
表2.Compound 1e-20e对MCF7、B16-F10细胞增值和端粒酶的抑制作用
a对肿瘤细胞生长的抑制;
b对端粒酶活性的抑制。
Claims (4)
2.一种制备上述吡唑-酰腙类衍生物的方法,它由下列步骤组成:
步骤1.制备希夫碱(化合物a):
将乙酸钠(40mmol)加入到取代基苯乙酮(20mmol)与盐酸苯肼(25mmol)的乙醇溶液中,磁力搅拌,于50-60℃反应约3h,(TLC检测反应进行程度),产物以固体析出。反应结束后抽滤,并以无水乙醇冲洗固体物3次,干燥得取代基希夫碱(化合物a)。用无水乙醇将产物重结晶。
步骤2.制备吡唑-醛类衍生物(化合物b):
将步骤1所得产物加入到冰的DMF(10ml)溶液中,随后再逐滴加入POC13(8ml),磁力搅拌,于50-60℃反应约6h(TLC检测反应进行程度);待反应结束,将反应体系倒入冰水中,并用氢氧化钠饱和溶液调节pH至中性,产物将以沉淀析出,真空抽滤后进行水洗,干燥,最后利用乙醇溶液进行重结晶,即得产物b。
步骤3.二氯亚砜成酯法制备水杨酸甲酯类衍生物(化合物c):
将水杨酸溶于甲醇中,放置于冰浴,向体系内逐滴加入SOCl2(约1.5eq),加毕,于冰浴中反应30-1h,然后放置于常温下,反应大概2-3h,转移到45℃,反应过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去甲醇及SOCl2,用乙酸乙酯与饱和NaHCO3萃取,旋转蒸发仪抽去萃取液,最后用乙醇重结晶,即得产物c。
步骤4.制备酰肼类衍生物(化合物d):
将产物c溶解于乙醇中,逐滴加入80%水合肼,磁力搅拌,反应回流过夜(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽去溶剂直到沉淀析出,然后向其中加入水,静置,沉淀将大量析出,即得产物d。
步骤5.制备终产物:吡唑-酰腙类衍生物(化合物1e-20e):
将上述所得产物b和产物d溶解于无水乙醇中,磁力搅拌,回流反应约4h(TLC检测反应进行程度);待反应结束,用旋转蒸发仪抽净溶剂,然后用乙酸乙酯萃取,再用饱和NaCl连续洗涤,用无水Na2SO4干燥,抽滤,重结晶,即得终产物1e-20e。
3.权利要求2所述的吡唑-酰腙类衍生物的制备方法。
4.权利要求1所述的吡唑-酰腙类衍生物在抗肿瘤药物治疗中的应用。
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CN107089950A (zh) * | 2017-06-16 | 2017-08-25 | 河南师范大学 | 一种由非环酮腙合成4‑酰基吡唑类化合物的方法 |
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CN107089950B (zh) * | 2017-06-16 | 2019-12-24 | 河南师范大学 | 一种由非环酮腙合成4-酰基吡唑类化合物的方法 |
CN110128343A (zh) * | 2019-06-18 | 2019-08-16 | 四川省人民医院 | 一种酰肼类化合物 |
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