[0016] 实施例1化合物闭花木酮Cleistanone的制备
[0017] 化合物闭花木酮Cleistanone(I)的制备方法参照Van Trinh Thi Thanh等人发表的文献(Van Trinh Thi Thanh et al.,2011.Cleistanone:A Triterpenoid from Cleistanthus indochinensis with a New Carbon Skeleton.Volume 2011,Issue 22,pages 4108–4111,August 2011)的方法。
[0018]
[0019] 实施例2闭花木酮Cleistanone的O-溴乙基衍生物(II)的合成
[0020] 将化合物I(440mg,1.00mmol)溶于10mL苯,向溶液中加入四丁基溴化铵(TBAB)(0.04g),1,2-二溴乙烷(3.760g,20.00mmol)和6mL的50%氢氧化钠溶液。混合物在25摄氏度搅拌24h。24h之后将反应液倒入冰水中,立即用二氯甲烷萃取两次,合并有机相溶液。然后对有机相溶液依次用水和饱和食盐水洗涤3次,再用无水硫酸钠干燥,最后减压浓缩去除溶剂得到产物粗品。产物粗品用硅胶柱层析纯化(流动相为:石油醚/丙酮=100:1,v/v),收集黄色集中洗脱带即得到化合物II的黄色固体(344mg,63%)。
[0021] 1H NMR(500MHz,DMSO-d6)δ5.04(s,1H),4.82(s,1H),3.94(d,J=26.5Hz,1H),3.87(d,J=26.5Hz,2H),3.57(s,2H),2.40(d,J=14.0Hz,1H),2.39(d,J=14.0Hz,1H),
2.27(s,1H),2.21(s,1H),2.15(s,1H),1.82(s,1H),1.62(s,2H),1.57(d,J=3.3Hz,1H),
1.54(d,J=3.3Hz,1H),1.50(d,J=1.2Hz,1H),1.47(d,J=1.2Hz,1H),1.39(d,J=15.3Hz,
2H),1.34(d,J=15.3Hz,1H),1.26(dd,J=32.6,13.7Hz,4H),1.13(d,J=18.0Hz,2H),1.05(s,6H),0.98(s,1H),0.88(s,12H),0.78(s,3H),0.74(s,1H)。
[0022] 13C NMR(125MHz,DMSO-d6)δ216.59(s),154.50(s),105.23(s),74.63(s),69.85(s),59.71(s),52.55(s),51.21(s),47.92(s),44.10(s),42.25(s),41.73(s),40.64(s),40.16(s),38.88(s),38.65(s),37.21(s),36.23(s),33.34(d,J=1.1Hz),32.96(s),29.91(s),27.18(s),26.03(s),24.23(s),23.96(s),20.77(s),18.48(s),17.98(s),16.93(s)。
[0023] HRMS(ESI)m/z[M+H]+calcd for C32H52BrO2:547.3151;found 547.3159.[0024]
[0025] 实施例3闭花木酮Cleistanone的O-(1H-四氮唑基)乙基衍生物(III)的合成[0026] 将化合物II(273mg,0.5mmol)溶于15mL乙腈当中,向其中加入无水碳酸钾(345mg,2.5mmol),碘化钾(84mg,0.5mmol)和1H-四氮唑(1401mg,20mmol),混合物加热回流10h。反应结束后将反应液倒入冰水中,用等量二氯甲烷萃取三次,合并有机相。依次用水和饱和食盐水洗涤合并之后的有机相,再用无水硫酸钠干燥,减压浓缩去除溶剂得到产物粗品。因为互变异构作用,在反应条件下会生成1H-四氮唑基和2H-四氮唑基两种取代产物。产物粗品用硅胶柱层析纯化(流动相为:石油醚/丙酮=100:1,v/v),收集黄色集中洗脱带,再将洗脱带浓缩,用硅胶柱层析纯化(流动相为:石油醚/丙酮=100:0.5,v/v),依次收集两个淡黄色的洗脱带,浓缩前1个洗脱带即得到化合物III的淡黄色固体(56.5mg,21%)。
[0027] 1H NMR(500MHz,DMSO-d6)δ10.10(s,1H),4.63(s,1H),4.53(s,1H),4.33(d,J=4.7Hz,2H),4.26(s,1H),3.88(s,2H),2.37(d,J=3.0Hz,2H),2.26(d,J=13.0Hz,2H),2.20(s,1H),1.89(s,2H),1.81(s,1H),1.65(d,J=15.0Hz,3H),1.56(s,2H),1.54–1.47(m,3H),
1.42(s,1H),1.30(dd,J=26.0,22.6Hz,3H),1.22(s,1H),1.04(s,6H),0.96(s,12H),0.87(d,J=5.4Hz,4H),0.63(s,1H).
[0028] 13C NMR(125MHz,DMSO-d6)δ216.58(s),154.48(s),145.25(s),105.20(s),74.64(s),65.63(s),59.74(s),52.54(s),51.19(s),47.89(s),47.02(s),44.11(s),42.27(s),41.76(s),40.63(s),40.14(s),38.85(s),38.66(s),37.23(s),36.26(s),33.33(s),32.94(s),29.88(s),27.19(s),26.05(s),24.26(s),23.95(s),20.75(s),18.45(s),17.99(s),
16.95(s).
[0029] HRMS(ESI):m/z[M+H]+calcd for C33H53N4O2:537.4169;found:537.4161。
[0030]
[0031] 实施例4体外抗肿瘤活性筛选
[0032] 筛选细胞株为HepG2(人肝癌细胞),PANC-1(人胰腺癌),MCF-7(人乳腺癌细胞),SW620(人结肠癌细胞),A549(人肺癌细胞),HGC-27(人胃癌细胞)。
[0033] 实验方法:
[0034] 取对数生长期状态良好的细胞,胰蛋白酶消化,制成5×104细胞/mL的悬液。将细胞悬液移入96孔培养板,每孔100μL,置37℃、5%CO2条件下培养24h。
[0035] 将受试化合物III用DMSO配制成一定浓度的母液,再用RPMI1640培养基将衍生物