CN113277934B - 一类具有mdr逆转活性的千金子二萜衍生物及其应用 - Google Patents

一类具有mdr逆转活性的千金子二萜衍生物及其应用 Download PDF

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CN113277934B
CN113277934B CN202110569775.0A CN202110569775A CN113277934B CN 113277934 B CN113277934 B CN 113277934B CN 202110569775 A CN202110569775 A CN 202110569775A CN 113277934 B CN113277934 B CN 113277934B
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焦威
向玲
邵华武
王飞
张中辉
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Abstract

本发明属于有机化学领域，具体涉及一类具有MDR逆转活性的千金子二萜衍生物及其应用。本发明从大戟植物千金子中提取得到新的千金子二萜衍生物，并对这些化合物进行结构改造，设计并合成了系列化合物，在MCF‑7/ADR细胞内逆转活性优于维拉帕米。其中，化合物17的逆转倍数高达460.32。

Description

一类具有MDR逆转活性的千金子二萜衍生物及其应用
技术领域
本发明属于有机化学领域，具体涉及一类具有MDR逆转活性的千金子二萜衍生物及其应用。
背景技术
多药耐药(MDR)是指对一种药物耐药的同时，伴有对其他结构和功能无关的药物也产生耐药的现象；MDR是恶性肿瘤化疗失败的主要原因之一。导致MDR发生的主要机制之一为P-gp的过表达。P-gp作为一个能量依赖的外排泵，可减少细胞内抗癌药物的积累，从而降低药物的疗效。因此，能够抑制P-gp活性的化合物可能具有逆转MDR的潜力。
在众多化合物中，大戟因子L1-L11被发现具有抗肿瘤、抗病毒、抗炎等活性，被视为P-gp介导的MDR的最有希望的逆转剂。但已公开的大戟因子及其衍生物，逆转效果并不显著。
发明内容
本发明的目的是提供一类具有MDR逆转活性的千金子二萜衍生物及其应用。
为实现上述发明目的，本发明所采用的技术方案是：一类千金子二萜衍生物，结构通式为：
Figure BDA0003082198360000011
其中，R1为H或benzoyl或m-nitrobenzoyl或p-Toluoyl或p-nitrobenzoyl或p-(trifluoromethyl)benzoyl或Ac，
R2为H或furoyl或m-nitrobenzoyl或cinnamoyl或benzoyl或p-(trifluoromethyl)benzoyl或Ac或p-Toluoyl或p-nitrobenzoyl，
R3为H或Ac或p-chlorophenyl或m-nitrobenzoyl或p-Tolylaceyl或cinnamoyl或1-naphthylacetyl或2-naphthoyl或benzoyl或furoyl或cinnamoyl或p-(trifluoromethyl)benzoyl或p-Toluoyl或p-nitrobenzoyl，
R4为H或Ac。
优选的，R1＝H，R2＝R3＝p-(trifluoromethyl)benzoyl，R4＝H。
优选的，R1＝H，R2＝R3＝furoyl，R4＝H。
优选的，R1＝R2＝H，R3＝1-naphthylacetyl，R4＝H。
优选的，R1＝R2＝H，R3＝2-naphthoyl，R4＝H。
优选的，化合物1：R1＝R2＝R3＝R4＝H；或；
化合物2：R1＝R2＝H，R3＝Ac，R4＝H；或；
化合物3：R1＝R2＝H，R3＝p-chlorophenyl，R4＝H；或；
化合物4：R1＝R2＝H，R3＝m-nitrobenzoyl，R4＝H；或；
化合物5：R1＝R2＝H，R3＝p-nitrobenzoyl，R4＝H；或；
化合物6：R1＝R2＝H，R3＝p-Tolylaceyl，R4＝H；或；
化合物7：R1＝R2＝H，R3＝cinnamoyl，R4＝H；或；
化合物9：R1＝benzoyl，R2＝R3＝H，R4＝H；或；
化合物11：R1＝R3＝benzoyl，R2＝H，R4＝H；或；
化合物12：R1＝R3＝m-nitrobenzoyl，R2＝H，R4＝H；或；
化合物14：R1＝H，R2＝R3＝m-nitrobenzoyl，R4＝H；或；
化合物15：R1＝H，R2＝R3＝cinnamoyl，R4＝H；或；
化合物16：R1＝H，R2＝R3＝benzoyl，R4＝H；或；
化合物18：R1＝R3＝benzoyl，R2＝R4＝Ac；或；
化合物19：R1＝R2＝R3＝p-Toluoyl，R4＝H；或；
化合物20：R1＝R2＝R3＝m-nitrobenzoyl，R4＝H；或；
化合物21：R1＝R2＝R3＝benzoyl，R4＝H；或；
化合物22：R1＝R2＝R3＝p-nitrobenzoyl，R4＝H；或；
化合物23：R1＝R2＝R3＝p-(trifluoromethyl)benzoyl，R4＝H；或；
化合物24：R1＝R2＝R3＝Ac，R4＝H；或；
化合物25：R2＝R3＝benzoyl，R1＝Ac，R4＝H；或；
相应的，所述千金子二萜衍生物在药物领域的应用。
优选的，在逆转MDR上的应用。
本发明具有以下有益效果：本发明从千金子中提取到了4种化合物：大戟因子化合物18、38、39、41。并对这四种化合物进行结构改造，设计并合成了化合物1～17，19～37、40，共37个衍生化合物，经核磁共振氢谱、质谱、x-ray单晶衍射等方法确定其结构，再对其进行药理活性研究，为寻找多药耐药逆转剂提供了有益的参考。
本发明得到了25个在MCF-7/ADR细胞内逆转活性优于维拉帕米的二萜化合物，且化合物17、29的逆转倍数分别为460.32、911.94，分别是VRP的229倍、454倍，远高于目前已知的此类化合物。
附图说明
图1为化合物8、化合物31分别在MCF-7/ADR细胞内的罗丹明积累变化示意图；
图2为MCF-7/ADR在化合物17、29、38不同浓度下处理2h后DOX积累，用Image J软件分析得到的荧光数据柱状图；
图3为化合物8、17、29、31和38P-gp表达的western blot实验结果示意图。
具体实施方式
本发明从大戟植物千金子中提取得到化合物18、38、39、41，并分别对这些化合物进行结构改造，设计并合成了化合物1～17、19～37、40。经核磁共振氢谱、质谱、x-ray单晶衍射等方法确定上述41个化合物的结构，再对其进行药理活性研究，最终筛选得到25个在MCF-7/ADR细胞内逆转活性优于维拉帕米的二萜化合物。其中，化合物17、29、38的逆转倍数分别为460.32、911.94、438.69。
化合物1～41的结构式如下所示：
Figure BDA0003082198360000031
其中，化合物1～25的各R基分别对应如下：
化合物1：R1＝R2＝R3＝R4＝H。
化合物2：R1＝R2＝H，R3＝Ac，R4＝H。
化合物3：R1＝R2＝H，R3＝p-chlorophenyl，R4＝H。
化合物4：R1＝R2＝H，R3＝m-nitrobenzoyl，R4＝H。
化合物5：R1＝R2＝H，R3＝p-nitrobenzoyl，R4＝H。
化合物6：R1＝R2＝H，R3＝p-Tolylaceyl，R4＝H。
化合物7：R1＝R2＝H，R3＝cinnamoyl，R4＝H。
化合物8：R1＝R2＝H，R3＝1-naphthylacetyl，R4＝H。
化合物9：R1＝benzoyl，R2＝R3＝H，R4＝H。
化合物10：R1＝R2＝H，R3＝2-naphthoyl，R4＝H。
化合物11：R1＝R3＝benzoyl，R2＝H，R4＝H。
化合物12：R1＝R3＝m-nitrobenzoyl，R2＝H，R4＝H。
化合物13：R1＝H，R2＝R3＝furoyl，R4＝H。
化合物14：R1＝H，R2＝R3＝m-nitrobenzoyl，R4＝H。
化合物15：R1＝H，R2＝R3＝cinnamoyl，R4＝H。
化合物16：R1＝H，R2＝R3＝benzoyl，R4＝H。
化合物17：R1＝H，R2＝R3＝p-(trifluoromethyl)benzoyl，R4＝H。
化合物18：R1＝R3＝benzoyl，R2＝R4＝Ac。
化合物19：R1＝R2＝R3＝p-Toluoyl，R4＝H。
化合物20：R1＝R2＝R3＝m-nitrobenzoyl，R4＝H。
化合物21：R1＝R2＝R3＝benzoyl，R4＝H。
化合物22：R1＝R2＝R3＝p-nitrobenzoyl，R4＝H。
化合物23：R1＝R2＝R3＝p-(trifluoromethyl)benzoyl，R4＝H。
化合物24：R1＝R2＝R3＝Ac，R4＝H。
化合物25：R2＝R3＝benzoyl，R1＝Ac，R4＝H。
2、
Figure BDA0003082198360000041
其中，化合物26～29、34的各R基分别对应如下：
化合物26：R1＝H，R2＝R3＝p-nitrobenzoyl，R4＝H。
化合物27：R1＝R2＝R3＝p-nitrobenzoyl，R4＝H。
化合物28：R1＝R3＝benzoyl，R2＝Ac，R4＝H。
化合物29：R1＝H，R2＝R3＝2-thiophenecarbonyl，R4＝H。
化合物34：R1＝R3＝benzoyl，R2＝Ac，R4＝Br。
3、
Figure BDA0003082198360000051
其中化合物30、31的各R基分别对应如下：
化合物30：R1＝R3＝benzoyl，R2＝R5＝Ac，R4＝H。
化合物31：R1＝R3＝benzoyl，R2＝Ac，R4＝R5＝H。
4、
Figure BDA0003082198360000052
其中，化合物32、33的各R基分别对应如下：
化合物32：R1＝R3＝benzoyl，R2＝R4＝R5＝Ac。
化合物33：R1＝R3＝benzoyl，R2＝Ac，R4＝R5＝propionyl。
5、
Figure BDA0003082198360000053
化合物35的各R基分别为：R1＝R3＝benzoyl，R2＝R4＝Ac。
6、
Figure BDA0003082198360000054
化合物36的各R基分别为：R1＝phenylacetyl，R2＝R3＝Ac。
7、
Figure BDA0003082198360000055
化合物37：R1＝H。化合物38：R1＝benzoyl。
8、
Figure BDA0003082198360000061
化合物39：R1＝cinamoyl，R2＝R3＝Ac。化合物40：R1＝R2＝R3＝H。
9、
Figure BDA0003082198360000062
化合物41：R1＝phenylacetyl，R2＝R3＝Ac。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例一：化合物1～41的制备和测定
1、化合物1、37、40的制备。在1％(m/v)NaOH/MeOH溶液体系中，分别以化合物18、38、39作为原料(化合物18对应化合物1，以此类推)，常温反应24h。反应后，用乙酸乙酯萃取，浓缩，色谱柱分离(石油醚与乙酸乙酯混合溶液洗脱)，得到目标产物。化合物1、37、40的核磁共振数据分别如下所示。
化合物1：HRMS-ESI-TOF：[M+Na]+373；mp 226-228℃；1H NMR(400MHz,Acetone)δ7.29(d,J＝11.6Hz,1H,H-12),5.07(s,1H,H-17α),4.96(d,J＝8.5Hz,1H,H-7),4.81(s,1H,17β),4.68(s,1H,OH-15),4.44(s,1H,H-5),4.37(s,1H,H-3),4.32(s,2H,OH-5,OH-7),4.06(s,1H,OH-3),3.07(dd,J＝13.2,8.8Hz,1H,H-1α),2.21(dd,J＝8.4,3.3Hz,1H,H-4),1.96–1.85(m,2H,H-2,H-8α),1.69–1.61(m,4H,H-1β,H-20),1.45(dd,J＝11.6,8.4Hz,1H,H-11),1.31–1.25(m,2H,H-8β,H-9),1.16(s,3H,H-19),1.12(s,3H,H-18),1.07(d,J＝6.8Hz,3H,H-16).
化合物37：HRMS-ESI-TOF：[M+Na]+357；1H NMR(400MHz,Acetone)δ7.87(d,J＝12.4Hz,1H,H-12),4.40(s,1H,OH-15),3.98-3.95(m,1H,H-3),3.83(d,J＝4.9Hz,1H,OH-3),3.44(d,J＝9.5Hz,1H,H-5),3.15(dd,J＝12.3,7.3Hz,1H,H-1α),2.02–1.98(m,1H,H-2),1.97-1.95(m,1H,H-7α),1.80(d,J＝1.0Hz,3H,H-20),1.76-1.70(m,1H,H-1β),1.67–1.64(m,1H,H-4),1.63-1.61(m,1H,H-11),1.49–1.41(m,1H,H-7β),1.28(dd,J＝9.6,3.3Hz,2H,H-8α,H-8β),1.24-1.22(m,1H,H-9),1.20(s,3H,H-18),1.13(s,3H,H-17),1.09(s,3H,H-19),1.05(d,J＝6.5Hz,3H,H-16).
化合物40：HRMS-ESI-TOF：[M+Na]+357；1H NMR(400MHz,Acetone)δ7.45(d,J＝12.9Hz,1H,H-12),5.88(d,J＝10.9Hz,1H,H-5),4.10(s,1H,OH-15),4.04(d,J＝5.2Hz,1H,OH-3),3.94–3.86(m,1H,H-3,H-17α),3.52(dd,J＝12.0,6.8Hz,1H,H-17β),3.39(dd,J＝6.8,4.2Hz,1H,OH-17),3.31(dd,J＝13.5,9.0Hz,1H,H-1α),2.35(dd,J＝10.9,3.2Hz,1H,H-4),2.31–2.26(m,1H,H-9),2.22-2.18(m,1H,H-7α),1.91-1.84(m,1H,H-2),1.76(d,J＝1.0Hz,3H,H-20),1.62–1.53(m,1H,H-7β),1.52–1.47(m,1H,H-8α),1.44(dd,J＝9.3,7.1Hz,1H,H-1β),1.29(s,1H,H-8β),1.17(s,3H,H-19),1.15-1.09(m,1H,H-11),1.07(d,J＝6.8Hz,3H,H-16),1.05(s,3H,H-18).
2、化合物2、24的制备方法：在DCM、BF3·Et2O体系中，使用化合物1与乙酸酐反应得到目标产物。制备方法为：在氩气保护下，往冰浴的CH2Cl2(5mL)溶液中依次加入化合物1(76mg,0.22mmol)、乙酸酐(21uL,0.22mmol)，BF3·Et2O(3uL,0.026mmol)。随后，将反应混合物加热至室温，搅拌10分钟，反应完毕后用CH2Cl2稀释反应液，加入饱和NaHCO3中和反应液。反应液用乙酸乙酯萃取三次，合并有机层并减压蒸发。最后柱层析法分离得到化合物2(产率58％，石油醚/乙酸乙酯＝4:1)和化合物24(产率30％，石油醚/乙酸乙酯＝15:1)。化合物2、24的核磁共振数据分别如下所示。
化合物2：HRMS-ESI-TOF：[M+Na]+415；1H NMR(400MHz,Acetone)δ6.33(d,J＝10.3Hz,1H,H-12),5.13(s,1H,H-17α),5.05(s,1H,H-17β),4.97(s,1H,H-7),4.68(s,1H,OH-15),4.49(s,2H,H-5,OH-5),4.25(s,2H,H-3,OH-3),2.75(dd,J＝13.5,8.8Hz,1H,H-1α),2.36(s,1H,H-4),2.00–1.92(m,1H,H-2),1.92–1.82(m,4H,H-8α,H-20),1.68(dd,J＝13.3,11.3Hz,1H,H-1β),1.58–1.42(m,2H,H-8β,H-11),1.30–1.22(m,1H,H-9),1.15(s,3H,H-19),1.13(s,3H,H-18),1.05(d,J＝6.7Hz,3H,H-16).7-OAc：2.04(s,3H).
化合物24：HRMS-ESI-TOF：[M+Na]+499；1H NMR(400MHz,Acetone)δ7.32(d,J＝11.3Hz,1H,H-12),6.08(d,J＝9.0Hz,1H,H-5),5.48(t,J＝3.6Hz,1H,H-3),5.29(s,1H,H-17α),5.21(dd,J＝8.4,3.0Hz,1H,H-7),5.08(s,1H,H-17β),4.49(s,1H,OH-15),3.01(dd,J＝13.5,8.8Hz,1H,H-1α),2.72(dd,J＝9.0,3.6Hz,1H,H-4),2.29-2.21(m,1H,H-2),2.12(dd,J＝9.3,5.8Hz,1H,H-8α),1.96–1.91(m,1H,H-8β),1.72(d,J＝1.0Hz,3H,H-20),1.64(dd,J＝13.6,11.4Hz,1H,H-1β),1.55(dd,J＝11.5,8.5Hz,1H,H-11),1.38-1.31(m,1H,H-9),1.17(s,3H,H-19),1.16(s,3H,H-18),0.93(d,J＝6.8Hz,3H,H-16).3-OAc：2.03(s,3H).5-OAc：1.94(s,3H).7-OAc：1.88(s,3H).
3、化合物3～8、10的制备方法：在EDCI、DMAP、DCM体系中，底物化合物1与相应的酸反应得到目标产物。制备方法为：在0℃的CH2Cl2(5mL)溶液中，依次加入化合物1(70mg,0.2mmol)、4-氯苯乙酸(39mg,0.23mmol)、EDCI(44mg,0.23mmol)和DMAP(4mg,0.23mmol)。将混合物在室温下搅拌6h。反应完毕后加水，乙酸乙酯萃取反应液三次，合并有机层并用无水Na2SO4干燥，减压蒸发，柱层析(石油醚/乙酸乙酯＝8:1)纯化得到化合物3，为白色无定形固体(产率46％)。其他条件相同，将4-氯苯乙酸改为间硝基苯甲酸，制备得化合物4，将4-氯苯乙酸改为对硝基苯甲酸，制备得化合物5，将4-氯苯乙酸改为对甲基苯乙酸，制备得化合物6，将4-氯苯乙酸改为肉桂酸，制备得化合物7，将4-氯苯乙酸改为1-萘乙酸，制备得化合物8，将4-氯苯乙酸改为2-萘甲酸，制备得化合物10。
化合物3：HRMS-ESI-TOF：[M+Na]+525；1H NMR(400MHz,Acetone)δ6.40(d,J＝10.4Hz,1H,H-12),5.11(s,1H,H-17α),5.05–5.00(m,1H,H-7),4.99(s,1H,H-17β),4.67(s,1H,OH-15),4.52(s,1H,H-5),4.47(d,J＝4.8Hz,1H,OH-5),4.25(s,1H,H-3),4.23(s,1H,OH-3),2.77(dd,J＝13.6,8.6Hz,1H,H-1α),2.36(t,J＝3.5Hz,1H,H-4),2.00-1.93(m,1H,H-2),1.93–1.88(m,1H,H-8α),1.86(s,3H,H-20),1.69(dd,J＝13.6,11.1Hz,1H,H-1β),1.63–1.53(m,1H,H-8β),1.47(dd,J＝10.5,8.9Hz,1H,H-11),1.23–1.17(m,1H,H-9),1.15(s,3H,H-19),1.13(s,3H,H-18),1.06(d,J＝6.8Hz,3H,H-16).7-4-Chlorophenylacetyl：7.41–7.32(m,4H),3.76(s,2H).
化合物4：HRMS-ESI-TOF：[M+Na]+522；1H NMR(400MHz,Acetone)δ6.63(d,J＝10.5Hz,1H,H-12),5.36(t,J＝5.5Hz,1H,H-7),5.19(s,2H,H-17α,H-17β),4.71(s,1H,OH-15),4.68(d,J＝4.8Hz,1H,H-5),4.52(d,J＝6.0Hz,1H,H-3),4.35–4.30(m,1H,OH-3),4.24(d,J＝3.7Hz,1H,OH-5),2.86–2.81(m,1H,H-1α),2.46(t,J＝4.0Hz,1H,H-4),2.23-2.16(m,1H,H-8α),1.96(s,1H,H-2),1.90(s,3H,H-20),1.87–1.77(m,1H,H-8β),1.72(dd,J＝13.5,11.1Hz,1H,H-1β),1.56(dd,J＝10.7,8.8Hz,1H,H-11),1.40(qd,J＝8.7,4.3Hz,1H,H-9),1.20(s,3H,H-19),1.16(s,3H,H-18),1.08(d,J＝6.8Hz,3H,H-16).7-m-nitrobenzoyl：8.87–8.82(m,1H),8.51(d,J＝8.6Hz,2H),7.86(t,J＝8.0Hz,1H).
化合物5：HRMS-ESI-TOF：[M+Na]+522；1H NMR(400MHz,Acetone)δ6.62(d,J＝10.5Hz,1H,H-12),5.35(t,J＝5.5Hz,1H,H-7),5.18(s,2H,H-17α,H-17β),4.70(s,1H,OH-15),4.67(d,J＝4.8Hz,1H,H-5),4.49(d,J＝6.0Hz,1H,H-3),4.35–4.30(m,1H,OH-3),4.23(d,J＝3.7Hz,1H,OH-5),2.86–2.81(m,1H,H-1α),2.46(t,J＝4.0Hz,1H,H-4),2.20-2.12(m,1H,H-8α),2.02-1.98(m,1H,H-2),1.90(s,3H,H-20),1.86–1.78(m,1H,H-8β),1.72(dd,J＝13.5,11.1Hz,1H,H-1β),1.56(dd,J＝10.7,8.8Hz,1H,H-11),1.42-1.36(m,1H,H-9),1.20(s,3H,H-19),1.16(s,3H,H-18),1.08(d,J＝6.8Hz,3H,H-16).7-p-nitrobenzoyl：8.42–8.33(m,4H).
化合物6：HRMS-ESI-TOF：[M+Na]+505；1H NMR(400MHz,Acetone)δ6.32(d,J＝10.4Hz,1H,H-12),5.10(s,1H,H-17α),5.02–4.98(m,1H,H-7),4.97(s,1H,H-17β),4.67(s,1H,OH-15),4.45(s,1H,OH-15),4.49(s,1H,H-5),4.24(s,2H,H-3,OH-3),2.76(dd,J＝13.6,8.7Hz,1H,H-1α),2.36(t,J＝3.4Hz,1H,H-4),2.01–1.92(m,1H,H-2),1.90–1.83(m,4H,H-8α,H-20),1.68(dd,J＝13.6,11.0Hz,1H,H-1β),1.58–1.49(m,1H,H-8β),1.46(dd,J＝10.5,8.9Hz,1H,H-11),1.23-1.18(m,1H,H-9),1.15(s,1H,H-19),1.12(s,1H,H-18),1.06(d,J＝6.8Hz,1H,H-16).7-p-Tolyacetyl：7.20(t,J＝7.4Hz,2H),7.16–7.09(t,J＝7.4Hz,2H),3.68(s,2H),2.30(s,3H).
化合物7：HRMS-ESI-TOF：[M+Na]+503；1H NMR(400MHz,Acetone)δ6.27(d,J＝10.4Hz,1H,H-12),5.19(s,1H,H-17α),5.12(br s,1H,H-7),5.10(s,1H,H-17β),4.70(s,1H,OH-15),4.60(d,J＝5.0Hz,1H,OH-5),4.53(s,1H,H-5),4.31-4.29(m,1H,OH-3),4.26(d,J＝3.6Hz,1H,H-3),2.82–2.73(m,1H,H-1α),2.46(t,J＝3.1Hz,1H,H-4),2.01-1.97(m,1H,H-2),1.95(s,1H,H-8α),1.94(s,3H,H-20),1.71(dd,J＝13.7,10.9Hz,1H,H-1β),1.60(dd,J＝12.8,2.5Hz,1H,H-8β),1.56–1.48(m,1H,H-11),1.35–1.27(m,1H,H-9),1.18(s,3H,H-19),1.14(s,3H,H-18),1.08(d,J＝6.8Hz,3H,H-16).7-O-cinnamoyl：7.75(d,J＝16.2Hz,1H),7.73–7.68(m,2H),7.46–7.42(m,3H),6.65(d,J＝16.0Hz,1H).
化合物8：HRMS-ESI-TOF：[M+Na]+541；1H NMR(400MHz,Acetone)δ6.26(d,J＝10.4Hz,1H,C-12),5.10(s,1H,H-17α),4.99(d,J＝3.4Hz,1H,H-7),4.97(s,1H,H-17β),4.64(s,1H,OH-15),4.22(br s,3H,H-3,H-5,OH-5),4.17(d,J＝3.5Hz,1H,OH-3),2.73(dd,J＝13.6,8.7Hz,1H,H-1α),2.35(s,1H,H-4),1.96(s,1H,H-2),1.84(s,3H,H-20),1.83–1.75(m,1H,H-8α),1.67(dd,J＝13.6,11.1Hz,1H,H-1β),1.53–1.43(m,1H,H-8β),1.41–1.34(m,1H,H-11),1.29(s,1H,H-9),1.12(s,3H,H-19),1.06(s,3H,H-18),1.04(d,J＝6.6Hz,3H,H-16).7-O-(1-naphthylacetyl)：8.08(d,J＝8.0Hz,1H),7.94(d,J＝8.5Hz,1H),7.87(d,J＝7.8Hz,1H),7.52(qd,J＝15.1,6.9Hz,4H),4.46(s,2H).
化合物10：HRMS-ESI-TOF：[M+Na]+527；1H NMR(400MHz,Acetone)δ6.38(d,J＝10.3Hz,1H,H-12),5.34-5.31(m,1H,H-7),5.23(s,1H,H-17α),5.20(s,1H,H-17β),4.73(s,1H,OH-15),4.64(s,1H,H-5),4.35(dd,J＝7.3,3.7Hz,1H,H-3),4.29(d,J＝3.8Hz,1H,OH-3),2.85–2.78(m,1H,H-1α),2.54(s,1H,H-4),2.20-2.14(m,1H,H-2),1.98(s,3H,H-20),1.78–1.67(m,2H,H-8α,H-11),1.57(dd,J＝10.4,9.0Hz,1H,H-1β),1.43-1.36(m,1H,H-8β),1.29(s,1H,H-9),1.22(s,3H,H-19),1.16(s,3H,H-18),1.10(d,J＝6.8Hz,1H,H-16).7-O-(2-naphthoyl)：8.76(s,1H),8.19–8.07(m,2H),8.05–7.99(m,2H),7.74–7.51(m,2H).
4、所述的化合物9、11～17、19～23、26～27和29的制备方法：在DMAP、Et3N体系中，底物化合物1与相应的酰氯、酸酐反应得到目标产物。化合物9和化合物21的制备方法：在冰浴下的CH2Cl2(10mL)溶液中依次加入化合物1(170mg,0.49mmol)、DMAP(7mg,0.06mmol)、Et3N(204μL,1.47mmol)和苯甲酰氯(226μL,1.96mmol)。将反应逐渐升温至室温，搅拌24h，反应完毕后柱层析进一步纯化，得到化合物9(石油醚/乙酸乙酯＝8:1，产率19％)和化合物21(石油醚/乙酸乙酯＝12:1，产率31％)，均为白色无定形固体。
参照化合物9的制备方法，其他条件相同的情况下，将苯甲酰氯替换为对甲基苯甲酰氯，反应得化合物19，将苯甲酰氯替换为间硝基苯甲酰氯，反应得化合物20，将苯甲酰氯替换为对硝基苯甲酰氯，反应同时得化合物22、化合物26和化合物27，将苯甲酰氯替换为对三氟甲基苯甲酰氯，反应得化合物23，将苯甲酰氯替换为噻吩甲酰氯，反应得化合物29。
化合物12和化合物14的制备方法为：在冰浴下的CH2Cl2(10mL)溶液中，依次加入化合物1(140mg,0.49mmol)、DMAP(7mg,0.06mmol)、Et3N(204μL,1.47mmol)和间硝基苯甲酰酐(113μL,0.98mmol)。反应液逐步升温至室温，搅拌反应12h，反应完毕后柱层析进一步纯化得到化合物12(石油醚/乙酸乙酯＝8:1，产率14％)和化合物14(石油醚/乙酸乙酯＝12:1，产率13％)。
参照化合物12的制备方法，其他条件相同的情况下，将间硝基苯甲酰酐替换为苯甲酸酐，反应得化合物11和化合物16，将间硝基苯甲酰酐替换为呋喃甲酰氯，反应得化合物13，将间硝基苯甲酰酐替换为肉桂酰氯，反应得化合物15，将间硝基苯甲酰酐替换为对三氟甲基苯甲酰氯，反应得化合物17。
化合物9：HRMS-ESI-TOF：[M+Na]+477；1H NMR(400MHz,Acetone)δ6.37(d,J＝10.4Hz,1H,H-12),5.26(dd,J＝6.8,3.1Hz,1H,H-7),5.21(s,1H,H-17α),5.14(s,1H,H-17β),4.71(s,1H,OH-15),4.60(br s,2H,H-5,OH-5),4.33(dd,J＝7.4,3.6Hz,1H,H-3),4.27(d,J＝3.8Hz,1H,OH-7),2.83–2.77(m,1H,H-1α),2.52–2.47(m,1H,H-4),2.14-2.07(m,1H,H-2),1.97-1.95(m,4H,H-8α,H-20),1.73(dd,J＝9.5,7.0Hz,1H,H-1β),1.70–1.63(m,1H,H-8β),1.55(dd,J＝10.4,8.9Hz,1H,H-11),1.38-1.32(m,1H,H-9),1.21(s,3H,H-19),1.15(s,3H,H-18),1.09(d,J＝6.8Hz,3H,H-16).3-O-benzoyl：8.12(d,J＝7.2Hz,2H),7.66(t,J＝7.4Hz,1H),7.54(t,J＝7.7Hz,1H).
化合物11：HRMS-ESI-TOF：[M+Na]+581；1H NMR(400MHz,Acetone)δ6.44(d,J＝10.0Hz,1H,H-12),5.86(t,J＝3.6Hz,1H,H-3),5.25(dd,J＝6.3,4.1Hz,1H,H-7),5.22(s,1H,H-17α),5.13(s,1H,H-17β),5.08(s,1H,OH-15),4.45(s,1H,OH-5),3.17(d,J＝3.2Hz,1H,H-5),2.95(dd,J＝14.2,9.0Hz,1H,H-1α),2.49(s,1H,H-4),2.09(dd,J＝7.1,3.4Hz,1H,H-2),2.04(s,3H,H-20),1.88(dd,J＝14.1,10.9Hz,1H,H-1β),1.69–1.60(m,1H,H-8α),1.56(dd,J＝10.3,8.9Hz,1H,H-11),1.40-1.33(m,1H,H-8β),1.29(s,1H,H-9),1.21(s,3H,H-19),1.16(s,3H,H-18),1.04(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.14(d,J＝7.2Hz,2H),7.67–7.56(m,1H),7.52(t,J＝7.6Hz,2H).7-O-benzoyl：8.04(d,J＝7.3Hz,2H),7.67–7.56(m,1H),7.40(t,J＝7.7Hz,2H).
化合物12：HRMS-ESI-TOF：[M+Na]+671；1H NMR(400MHz,Acetone)δ6.79(br s,1H,H-12),5.87(t,J＝3.5Hz,1H,H-3),5.36(t,J＝5.7Hz,1H,H-7),5.30(s,1H,H-17α),5.20(s,1H,H-17β),5.08(s,1H,OH-15),4.58(t,J＝5.0Hz,1H,OH-5),3.28(t,J＝4.8Hz,1H,H-5),3.02(dd,J＝13.9,8.9Hz,1H,H-1α),2.53-2.46(m,1H,H-4),2.21-2.15(m,1H,H-2),1.97(s,3H,H-20),1.90(dd,J＝13.9,11.2Hz,1H,H-1β),1.86–1.77(m,1H,H-8α),1.58(dd,J＝10.7,8.7Hz,1H,H-11),1.46–1.38(m,1H,H-8β),1.29(s,H,H-9),1.22(s,3H,H-19),1.17(s,3H,H-18),1.05(d,J＝6.8Hz,3H,H-16).3-m-nitrobenzoyl：8.88(t,J＝4Hz,1H),8.50(d,J＝1.9Hz,1H),8.41(dd,J＝8.3,2.2Hz,1H),7.84(t,J＝8.0Hz,1H).7-m-nitrobenzoyl：8.74–8.70(t,J＝4Hz,1H),8.48–8.46(t,J＝4Hz,1H),8.35(d,J＝7.7Hz,1H),7.64(t,J＝8.0Hz,1H).
化合物13：HRMS-ESI-TOF：[M+Na]+561；1H NMR(400MHz,Acetone)δ7.36(d,J＝11.2Hz,1H,H-12),6.37(d,J＝9.0Hz,1H,H-5),5.49(dd,J＝8.4,2.9Hz,1H,H-7),5.44(s,1H,H-17α),5.20(s,1H,H-17β),4.84(s,1H,OH-15),4.23–4.19(m,1H,H-3),3.95(d,J＝5.5Hz,1H,OH-3),3.10(dd,J＝13.9,9.8Hz,1H,H-1α),2.65(dd,J＝8.9,3.0Hz,1H,H-4),2.39-2.30(m,J＝15.2,12.2,8.5Hz,1H,H-8α),2.18–2.06(m,2H,H-2,H-1β),1.78(s,3H,H-20),1.68–1.58(m,2H,H-8β,H-11),1.49–1.40(m,1H,H-9),1.25(s,3H,H-19),1.20(s,3H,H-18),1.07(d,J＝6.8Hz,3H,H-16).5-O-furoyl：7.68(s,1H),7.04(d,J＝3.4Hz,1H),6.49(dd,J＝3.5,1.7Hz,1H).7-O-furoyl：7.51(s,1H),6.90(d,J＝3.5Hz,1H),6.40(dd,J＝3.5,1.7Hz,1H).
化合物14：HRMS-ESI-TOF：[M+Na]+671；1H NMR(400MHz,Acetone)δ7.51(t,J＝8.0Hz,1H,H-12),6.63(d,J＝9.8Hz,1H,H-5),5.68–5.58(m,2H,H-7,H-17α),5.39(s,1H,H-17β),4.85(s,1H,OH-15),4.21–4.14(br s,1H,H-3),3.85(d,J＝5.8Hz,1H,3-OH),3.13(dd,J＝12.7,10.9Hz,1H,H-1α),2.82–2.74(m,1H,H-4),2.55(dd,J＝24.7,12.1Hz,1H,H-8α),2.32(d,J＝17.0Hz,1H,H-8β),2.19–2.09(m,1H,H-2),1.77(s,3H,H-20),1.68(dd,J＝11.3,2.5Hz,1H,H-11),1.62(dd,J＝9.8,6.7Hz,1H,H-1β),1.56–1.45(m,1H,H-9),1.30(s,3H,H-19),1.23(s,3H,H-18),1.00(d,J＝6.8Hz,3H,H-16).5-m-nitrobenzoyl：8.30(d,J＝8.8Hz,2H),8.08(d,J＝8.8Hz,1H),7.51(t,J＝8.0Hz,1H),7-m-nitrobenzoyl：8.19(d,J＝8.2Hz,1H),8.13(d,J＝10.4Hz,1H),8.01(d,J＝8.8Hz,1H),7.44(t,J＝8.0Hz,1H).
化合物15：HRMS-ESI-TOF：[M+Na]+633；mp 216-218℃；1H NMR(400MHz,Acetone)δ6.33(d,J＝9.5Hz,1H,H-12),5.43(d,J＝9.1Hz,1H,H-7),5.40(s,1H,H-17α),5.14(s,1H,H-17β),4.79(s,1H,OH-15),4.18(s,1H,H-5),4.03(d,J＝4.8Hz,1H,H-3),3.12(dd,J＝13.8,9.8Hz,1H,H-1α),2.82(s,OH-3),2.60(dd,J＝9.5,2.7Hz,1H,H-4),2.34-2.24(m,1H,H-2),2.17–2.09(m,1H,H-8α),1.73(s,3H,H-20),1.66(dd,J＝10.6,7.1Hz,1H,H-1β),1.61(dd,J＝8.3,5.3Hz,1H,H-8β),1.45-1.39(m,2H,H-9,H-11),1.24(s,3H,H-19),1.21(s,3H,H-18),1.08(d,J＝6.8Hz,3H,H-16).5-O-cinnamoyl：7.69(d,J＝16.0Hz,1H),7.36(t,J＝7.7Hz,4H),7.27(t,J＝7.4Hz,1H),6.54(d,J＝16.0Hz,1H),7-O-cinnamoyl：7.56(d,J＝11.1Hz,4H),7.14(t,J＝7.6Hz,2H),6.28(d,J＝16.1Hz,1H).
化合物16：HRMS-ESI-TOF：[M+Na]+581；1H NMR(400MHz,Acetone)δ7.28(d,J＝11.1Hz,1H,H-12),6.38(d,J＝8.1Hz,1H,H-5),5.52(dd,J＝7.5,3.3Hz,1H,H-7),5.43(s,1H,H-17α),5.24(s,1H,H-17β),4.89(s,1H,OH-15),4.24-4.21(m,1H,H-3),3.88(d,J＝6.0Hz,1H,OH-3),3.09(dd,J＝14.0,9.9Hz,1H,H-1α),2.75(dd,J＝8.1,2.9Hz,1H,H-4),2.41-2.32(m,1H,H-8α),2.22–2.10(m,2H,H-2,H-8β),1.84(s,3H,H-20),1.70–1.59(m,1H,H-1β,H-9),1.47-1.42(m,1H,H-11),1.28(s,3H,H-19),1.21(s,3H,H-18),1.07(d,J＝6.8Hz,3H,H-16).5-O-benzoyl：7.88(d,J＝7.3Hz,2H),7.50(t,J＝7.4Hz,1H),7.35(t,J＝7.7Hz,2H).7-O-benzoyl：7.72(d,J＝7.3Hz,2H),7.40(t,J＝7.4Hz,1H),7.16(t,J＝7.8Hz,2H).
化合物17：HRMS-ESI-TOF：[M+Na]+717；1H NMR(400MHz,Acetone)δ7.50(s,1H,H-12),6.60(d,J＝9.7Hz,1H,H-5),5.63–5.59(m,1H,H-7),5.59(s,1H,H-17α),5.34(s,1H,H-17β),4.85(s,1H,OH-15),4.22-4.18(m,1H,H-3),3.89(d,J＝5.8Hz,1H,OH-3),3.23–3.10(m,1H,H-1α),2.75(dd,J＝9.7,2.9Hz,1H,H-4),2.59–2.47(m,1H,H-8α),2.30–2.22(m,1H,H-8β),2.18-2.10(m,1H,H-2),1.78(s,3H,H-20),1.70–1.59(m,2H,H-1β,H-11),1.54–1.45(m,1H,H-9),1.29(s,3H,H-19),1.22(s,3H,H-18),1.02(d,J＝6.8Hz,3H,H-16).5-O-p-(trifluoromethyl)benzoyl：7.92(d,J＝8.1Hz,2H),7.78(d,J＝8.1Hz,2H).7-O-p-(trifluoromethyl)benzoyl：7.57(d,J＝8.2Hz,2H),7.42(d,J＝8.2Hz,2H).
化合物19：HRMS-ESI-TOF：[M+Na]+727；mp 222-224℃；1H NMR(400MHz,Acetone)δ6.64(d,J＝10.4Hz,1H,H-12),6.14(d,J＝6.5Hz,1H,H-7),5.85(t,J＝3.6Hz,1H,H-3),5.43(t,J＝4.9Hz,1H,H-5),5.37(s,1H,H-17α),5.29(s,1H,H-17β),4.79(s,1H,OH-15),3.09(dd,J＝6.2,3.7Hz,1H,H-4),2.99(dd,J＝14.0,8.9Hz,1H,H-1α),2.55-2.47(m,1H,H-2),2.09-2.06(m,1H,H-8α),2.04(s,3H,H-20),1.81(dd,J＝14.0,11.0Hz,1H,H-1β),1.68-1.62(m,1H,H-8β),1.52–1.44(m,1H,H-11),1.28(s,1H,H-9),1.26(s,3H,H-19),1.18(s,3H,H-18),0.96(d,J＝6.7Hz,3H,H-16).3-O-p-Toluoyl：7.87(d,J＝8.1Hz,2H),7.21(d,J＝8.0Hz,2H),2.39(s,3H).5-O-p-Toluoyl：7.65(d,J＝8.1Hz,2H),7.04(d,J＝8.0Hz,2H),2.30(s,3H).7-O-p-Toluoyl：7.35(d,J＝8.1Hz,2H),6.74(d,J＝8.0Hz,2H),2.20(s,3H).
化合物20：HRMS-ESI-TOF：[M+Na]+820；1H NMR(400MHz,Acetone)δ7.37(dt,J＝23.8,8.0Hz,1H,H-5).6.71(d,J＝9.3Hz,1H,H-12),5.85(t,J＝3.3Hz,1H,H-3),5.73(s,1H,H-17α),5.66(dd,J＝8.8,2.1Hz,1H,H-7),5.59(s,1H,H-17β),5.34(s,1H,OH-15),3.23(dd,J＝9.2,3.5Hz,1H,H-4),3.14(dd,J＝13.6,8.7Hz,1H,H-1α),2.59–2.48(m,1H,H-2),2.44-2.38(m,1H,H-8α),2.30–2.21(m,1H,H-8β),1.89(s,3H,H-20),1.88–1.81(m,1H,H-1β),1.70(dd,J＝11.5,8.6Hz,1H,H-11),1.59–1.50(m,1H,H-9),1.30(s,3H,H-19),1.23(s,3H,H-18),0.91(d,J＝6.8Hz,3H,H-16).3-m-nitrobenzoyl：8.56(s,1H),8.46(d,J＝8.2Hz,1H),8.27(d,J＝7.8Hz,1H),7.90(s,1H).5-m-nitrobenzoyl：8.19(s,1H),8.08(d,J＝8.2Hz,2H),7.94(d,J＝7.8Hz,1H).7-m-nitrobenzoyl：7.77(t,J＝8.0Hz,1H),7.70(d,J＝7.7Hz,1H),7.37(dt,J＝23.8,8.0Hz,2H).
化合物21：HRMS-ESI-TOF：[M+Na]+685；mp 192-194℃；1H NMR(400MHz,Acetone)δ6.75(d,J＝10.6Hz,1H,H-12),6.23(d,J＝6.4Hz,1H,H-5),5.89(t,J＝3.6Hz,1H,H-3),5.48(t,J＝5.0Hz,1H,H-7),5.42(s,1H,H-17α),5.34(s,1H,H-17β),4.89(s,1H,OH-15),3.13(dd,J＝6.3,3.7Hz,1H,H-4),3.03(dd,J＝13.9,8.9Hz,1H,H-1α),2.52(m,1H,H-2),2.16-2.09(m,2H,H-8α,H-8β),2.05(s,3H,H-20),1.85(dd,J＝13.9,11.1Hz,1H,H-1β),1.67(dd,J＝10.8,8.9Hz,1H,H-11),1.51(m,1H,H-9),1.28(s,3H,H-19),1.20(s,3H,H-18),0.98(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.01(d,J＝7.1Hz,2H),7.44(dt,J＝15.6,7.7Hz,3H).5-O-benzoyl：7.79(d,J＝7.2Hz,2H),7.31–7.21(m,3H).7-O-benzoyl：7.50(d,J＝7.1Hz,2H),7.59(t,J＝7.4Hz,1H),6.97(t,J＝7.8Hz,2H).
化合物22：HRMS-ESI-TOF：[M+Na]+820；1H NMR(400MHz,Acetone)δ7.26(br s,1H,H-12),6.62(d,J＝9.0Hz,1H,H-5),5.85(t,J＝3.3Hz,1H,H-3),5.70(s,1H,H-17α),5.63(dd,J＝8.3,2.6Hz,1H,H-7),5.56(s,1H,H-17β),5.23(s,1H,OH-15),3.21(dd,J＝9.1,3.5Hz,1H,H-4),3.11(dd,J＝13.6,8.7Hz,1H,H-1α),2.57-2.49(m,1H,H-2),2.42–2.29(m,1H,H-8α),2.27–2.18(m,1H,H-8β),1.91(s,3H,H-20),1.85(dd,J＝13.6,11.6Hz,1H,H-1β),1.70(dd,J＝11.5,8.6Hz,1H,H-11),1.58–1.49(m,1H,H-9),1.28(s,3H,H-19),1.22(s,3H,H-18),0.92(d,J＝6.7Hz,1H,H-16).3-p-nitrobenzoyl：8.27(d,J＝8.9Hz,2H),7.84(d,J＝8.9Hz,2H).5-p-nitrobenzoyl：8.13(d,J＝8.9Hz,2H),7.77(d,J＝8.9Hz,2H).7-p-nitrobenzoyl：7.90(d,J＝8.8Hz,2H),7.57(d,J＝8.8Hz,2H).
化合物23：HRMS-ESI-TOF：[M+Na]+889；1H NMR(400MHz,Acetone)δ7.50(br s,1H,H-12),6.55(d,J＝8.8Hz,1H,H-5),5.85(t,J＝3.4Hz,1H,H-3),5.65(s,1H,H-17α),5.60(dd,J＝8.0,2.9Hz,1H,H-7),5.52(s,1H,H-17β),5.15(s,1H,OH-15),3.19(dd,J＝8.9,3.6Hz,1H,H-4),3.09(dd,J＝13.7,8.7Hz,1H,H-1α),2.55-2.48(m,1H,H-2),2.39–2.27(m,1H,H-8α),2.21-2.15(m,1H,H-8β),1.93(s,3H,H-20),1.85(dd,J＝13.6,11.5Hz,1H,H-1β),1.69(dd,J＝11.4,8.6Hz,1H,H-11),1.59–1.49(m,1H,H-9),1.28(s,3H,H-19),1.21(s,3H,H-18),0.93(d,J＝6.7Hz,3H,H-16).3-O-p-(trifluoromethyl)benzoyl：7.79(d,J＝8.3Hz,2H),7.43(d,J＝8.3Hz,2H).5-O-p-(trifluoromethyl)benzoyl：7.74(d,J＝8.2Hz,2H),7.32(d,J＝8.3Hz,2H).7-O-p-(trifluoromethyl)benzoyl：8.10(d,J＝8.1Hz,2H),7.53(d,J＝8.2Hz,2H).
化合物26：HRMS-ESI-TOF：[M+Na]+671；1H NMR(600MHz,Acetone)δ6.12(d,J＝10.4Hz,1H,H-5),5.72(dd,J＝11.3,1.4Hz,1H,H-7),5.68(s,1H,H-17α),5.41(s,1H,H-17β),4.22(d,J＝9.8Hz,1H,H-12),3.95(d,J＝3.4Hz,1H,H-3),3.93-3.89(m,1H,OH-3),2.97(dd,J＝11.1,3.4Hz,1H,H-4),2.64(dd,J＝25.2,11.8Hz,1H,H-13),2.38(dd,J＝9.8,7.2Hz,1H,H-2),2.24(dd,J＝12.7,2.9Hz,1H,H-1α),1.98-1.93(m,1H,H-8α),1.89(dd,J＝12.3,6.7Hz,1H,H-8β),1.73(t,J＝12.7Hz,1H,H-1β),1.25(s,3H,H-18),1.16(d,J＝7.2Hz,3H,H-20),1.14(s,3H,H-19),1.01(d,J＝6.6Hz,3H,H-16),0.98-0.93(m,1H,H-9),0.88(d,J＝9.2Hz,1H,H-11).5-p-nitrobenzoyl：8.10(d,J＝8.9Hz,2H),8.01(d,J＝8.8Hz,2H).7-p-nitrobenzoyl：7.93(d,J＝8.8Hz,4H).
化合物27：HRMS-ESI-TOF：[M+Na]+820；1H NMR(400MHz,Acetone)δ6.33(d,J＝11.1Hz,1H,H-5),5.73(d,J＝12.8Hz,2H,H-17α,H-7),5.63(t,J＝3.1Hz,1H,H-3),5.53(s,1H,H-17β),4.40(d,J＝9.7Hz,1H,H-12),3.47(dd,J＝11.0,3.7Hz,1H,H-4),2.74-2.65(m,1H,H-13),2.53-2.45(m,1H,H-2),2.38–2.30(m,1H,H-8α),2.27(dd,J＝14.4,3.1Hz,1H,H-8β),2.19(dd,J＝13.1,7.0Hz,1H,H-1α),1.81(t,J＝13.1Hz,1H,H-1β),1.46(s,3H,H-20),1.37(s,1H,H-9),1.29(s,3H,H-19),1.20(s,3H,H-18),1.13–1.08(m,1H,H-11),0.93(d,J＝6.7Hz,3H,H-16).3-p-nitrobenzoyl：8.42(d,J＝8.8Hz,2H),8.36(d,J＝8.8Hz,2H).5-p-nitrobenzoyl：7.93(t,J＝8.5Hz,4H).7-p-nitrobenzoyl：8.12(d,J＝8.8Hz,2H),7.81(d,J＝8.8Hz,2H).
化合物29：HRMS-ESI-TOF：[M+Na]+593；1H NMR(400MHz,Acetone)δ5.96(d,J＝10.9Hz,1H,H-5),5.63(d,J＝10.0Hz,1H,H-7),5.57(s,1H,H-17α),5.28(s,1H,H-17β),4.19(d,J＝9.8Hz,1H,H-12),4.10(d,J＝2.2Hz,1H,OH-3),3.91–3.83(m,1H,H-3),2.87(dd,J＝11.0,3.3Hz,1H,H-4),2.53(dd,J＝25.0,11.5Hz,1H,H-13),2.39-2.32(m,J＝14.3,7.2Hz,1H),2.13(d,J＝16.0Hz,1H,H-1α),2.02–1.92(m,1H,H-2),1.88(dd,J＝12.2,6.7Hz,1H,H-1β),1.73(t,J＝12.7Hz,1H,H-8α),1.29(s,2H,H-8β,H-9),1.22(s,3H,H-19),1.15(d,J＝7.2Hz,3H,H-20),1.13(s,3H,H-18),1.03(d,J＝6.6Hz,3H,H-16),0.95–0.88(m,1H,H-11).5-O-thiophenecarbonyl：7.71(d,J＝5.8Hz,1H),7.60(d,J＝3.7Hz,1H),7.04–7.00(m,1H).7-O-thiophenecarbonyl：7.56(d,J＝5.8Hz,1H),7.50(d,J＝3.7Hz,1H),6.92–6.85(m,1H).
5、所述的化合物25的制备方法：在甲醇溶液中，底物化合物18、三氯化铟、苯甲酰氯摩尔比为1：1：1，反应得到目标产物。
化合物25：HRMS-ESI-TOF：[M+Na]+623；1H NMR(400MHz,CDCl3)δ5.99(d,J＝10.1Hz,1H,H-12),5.83(t,J＝3.5Hz,1H,H-3),5.47(d,J＝4.6Hz,1H,H-5),5.30(s,1H,H-7),5.25(s,1H,H-17α),5.19(s,1H,H-17β),4.24(s,1H,OH-15),2.92–2.86(m,1H,H-4),2.82(dd,J＝14.7,9.6Hz,1H,H-1α),2.60–2.47(m,1H,H-2),2.13(s,3H,H-20),2.09–1.98(m,1H,H-8α),1.86(dd,J＝14.7,10.3Hz,1H,H-1β),1.76-1.66(m,1H,H-8β),1.46(dd,J＝20.3,10.4Hz,1H,H-11),1.33-1.28(m,1H,H-9),1.21(s,3H,H-19),1.14(s,3H,H-18),1.07(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.14(d,J＝8.4Hz,2H),7.60–7.56(m,1H),7.44(t,J＝7.6Hz,2H).5-OAc：1.31(s,3H).7-O-benzoyl：8.05(d,J＝8.4Hz,2H),7.56–7.52(m,1H),7.44(t,J＝7.6Hz,2H).
6、化合物28的制备方法：在甲醇溶液中，底物化合物18、三氯化铟、苯甲酰氯摩尔比为1：1：2，加热反应得到目标产物。
化合物28：HRMS-ESI-TOF：[M+Na]+623；mp 191-193℃；1H NMR(400MHz,Acetone)δ6.09(d,J＝10.9Hz,1H,H-5),5.66(d,J＝10.1Hz,1H,H-7),5.55(s,1H,H-17α),5.53(t,J＝4Hz,1H,H-3),5.23(s,1H,H-17β),4.32(d,J＝9.8Hz,1H,H-12),3.14(dd,J＝11.0,3.7Hz,1H,H-4),2.81(s,1H,H-13),2.69-2.60(m,1H,H-1α),2.46-2.39(m,1H,H-2),2.29-2.21(m,1H,H-8α),2.15–2.06(m,1H,H-8β),1.71(t,J＝13.0Hz,1H,H-1β),1.56(s,3H,H-20),1.29(s,1H,H-11),1.17(t,J＝3.5Hz,6H,H-18,H-19),1.02–0.94(m,1H,H-9),0.91(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.12(d,J＝8.4Hz,2H),7.65(t,J＝7.4Hz,1H),7.38(t,J＝7.8Hz,2H).5-OAc：1.41(s,3H).7-O-benzoyl：7.84(d,J＝7.2Hz,2H),7.57–7.48(m,3H).
7、所述的化合物30的制备方法：将原料化合物18(150mg,0.23mmol)溶解于20mL二氧六环溶剂中，加入过量NaBH4(0.5g)，室温搅拌反应6h,加入适量冰终止反应，用2N H2SO4调节反应液至中性。乙酸乙酯萃取三次，合并有机层浓缩并用柱层析法纯化，得化合物30(141mg,95％)，为白色无定形固体。
化合物30：HRMS-ESI-TOF：[M+Na]+667；1H NMR(400MHz,Acetone)δ5.86(s,1H,H-5),5.79(dd,J＝11.2,7.1Hz,2H,H-12,H-14),5.26(s,1H,H-17α),5.23(d,J＝7.8Hz,1H,H-7),5.01(s,1H,H-17β),4.89(s,1H,OH-15),3.73(s,1H,H-3),2.50(dd,J＝13.6,9.0Hz,1H,H-1α),2.45(d,J＝2.6Hz,1H,H-4),2.38(m,1H,H-8α),1.98(s,3H,H-20),1.97–1.87(m,3H,H-1β,H-2,H-8β),1.87–1.79(m,1H,H-9),1.30–1.22(m,1H,H-11),1.20(s,3H,H-19),1.11(s,3H,H-18),1.02(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.18–8.12(m,2H),7.68(t,J＝7.4Hz,1H),7.56(t,J＝7.7Hz,2H).5-OAc：1.49(s,3H).7-O-benzoyl：8.18–8.12(m,2H),7.62(t,J＝7.4Hz,1H),7.51(t,J＝7.6Hz,2H).14-OAc：2.09(s,3H).
8、所述的化合物31的制备方法：在氩气保护下，往冰浴的CH2Cl2(5mL)溶液中依次加入化合物30(142mg,0.22mmol)、乙酸酐(21uL,0.22mmol)，BF3·Et2O(3uL,0.026mmol)。随后，将反应混合物加热至室温，搅拌10分钟，反应完毕后用CH2Cl2稀释反应液，加入饱和NaHCO3中和反应液。反应液用乙酸乙酯萃取三次，合并有机层并减压蒸发。最后柱层析法分离得到化合物31(产率24％)。
化合物31：HRMS-ESI-TOF：[M+Na]+625.7132,found 623.2609；1H NMR(400MHz,Acetone)δ5.95(d,J＝11.2Hz,1H,H-5),5.67(s,1H,H-12),5.53(dd,J＝10.3,2.2Hz,1H,H-7),5.46–5.43(m,1H,H-3),5.42(s,1H,H-17α),5.36(s,1H,H-17β),5.06(s,1H,H-14),3.08(dd,J＝11.2,3.9Hz,1H,H-4),2.48(dd,J＝22.4,10.1Hz,1H,H-8α),2.21-2.16(m,1H,H-2),2.15–2.07(m,2H,H-1α,OH-15),2.02-1.97(m,2H,H-1β,H-8β),1.74(s,3H,H-20),1.30(s,3H,H-19),1.28(s,1H,OH-14),1.12(s,3H,H-18),0.87(d,J＝6.2Hz,3H,H-16),0.82-0.77(m,2H,H-9,H-11).3-O-benzoyl：8.12(d,J＝7.1Hz,2H),7.51(t,J＝6.8Hz,2H),7.34(t,J＝7.8Hz,1H).5-OAc：1.48(s,3H).7-O-benzoyl：7.85(d,J＝7.1Hz,2H),7.64(t,J＝6.9Hz,1H),751(t,J＝6.8Hz,1H),7.34(t,J＝7.8Hz,1H).
9、所述的化合物32、33的制备方法：在DCM、TMSOTf体系中，底物化合物18与相应酸酐反应得到目标产物。具体制备方法为：在氮气保护下，化合物18(100mg,0.16mmol)溶解于冰浴的干燥CH2Cl2(10mL)溶液中，依次加入乙酸酐(151uL,1.6mmol)，TMSOTf(一滴)。将反应混合物搅拌反应5分钟，反应完毕后用CH2Cl2稀释反应液，用饱和NaHCO3中和反应液。乙酸乙酯萃取三次，合并有机层，Na2SO4干燥，减压蒸发。最后柱层析纯化得到化合物32(18mg,产率18％)。其余条件相同，将乙酸酐替换为丙酸酐，反应得化合物33。
化合物32：HRMS-ESI-TOF：[M+Na]+725；1H NMR(400MHz,Acetone)δ5.85(t,J＝7.7Hz,3H,H-3,H-7,H-11),5.72(t,J＝9.5Hz,1H,H-12),5.61(d,J＝7.2Hz,1H,H-5),5.24(s,1H,H-17α),5.15(s,1H,H-17β),4.00(s,1H,H-4),3.48(s,1H,OH-15),2.49(dd,J＝14.3,5.8Hz,1H,H-1α),2.39(dd,J＝12.3,2.5Hz,1H,H-1β),2.32-2.27(m,1H,H-2),2.15–2.06(m,2H,H-8α,H-10),1.89(dd,J＝15.6,9.5Hz,1H,H-8β),1.82(s,3H,H-20),1.22(s,3H,H-19),1.17(s,3H,H-18),0.98(d,J＝6.2Hz,3H,H-16).3-O-benzoyl：8.07(d,J＝8.1Hz,2H),7.65(dd,J＝15.3,7.6Hz,1H),7.53(td,J＝7.7,2.0Hz,2H).5-OAc：1.95(s,3H).7-O-benzoyl：8.07(d,J＝8.1Hz,2H),7.65(dd,J＝15.3,7.6Hz,1H),7.53(td,J＝7.7,2.0Hz,2H).12-OAc：1.28(s,3H).14-OAc：2.16(s,3H).
化合物33：HRMS-ESI-TOF：[M+Na]+753；1H NMR(400MHz,Acetone)δ5.93–5.81(m,3H,H-3,H-7,H-11),5.73(t,J＝9.5Hz,1H,H-12),5.62(d,J＝7.3Hz,1H,H-5),5.24(s,1H,H-17α),5.15(s,1H,H-17β),4.00(s,1H,H-4),3.47(s,1H,OH-15),2.52-2.46(m,1H,H-1α),2.39(dd,J＝12.2,2.4Hz,1H,H-1β),2.35–2.29(m,1H,H-2),2.19–2.09(m,2H,H-8α,H-10),1.89(dd,J＝16.4,9.8Hz,1H,H-8β),1.83(s,3H,H-20),1.22(s,3H,H-19),1.17(t,7.6Hz,3H,H-18),0.98(d,J＝6.2Hz,3H,H-16).3-O-benzoyl：8.07(d,J＝8.1Hz,2H),7.65(dd,J＝15.3,7.6Hz,1H),7.53(m,2H).5-OAc：1.95(s,3H).7-O-benzoyl：8.07(d,J＝8.1Hz,2H),7.65(dd,J＝15.3,7.6Hz,1H),7.53(m,2H).12-O-propionyl：2.48(m,2H),1.17(t,7.6Hz,3H).14-O-propionyl：1.06(t,J＝7.6Hz,3H),2.26(dd,J＝15.1,7.5Hz,2H).
10、所述的化合物34的制备方法：在DCM溶液中，底物化合物25、溴化铜、乙酸酐摩尔比为1：1：10，常温反应得到目标产物。在氮气保护下，将化合物25(96mg,0.16mmol)溶解于冰浴的干燥CH2Cl2(10mL)溶剂中，依次加入乙酸酐(151uL,1.6mmol)，CuBr(36mg,0.16mmol)。室温搅拌反应12h，反应完毕后加水淬灭反应，乙酸乙酯萃取反应液三次，合并有机层，Na2SO4干燥，减压蒸发。最后柱层析纯化得到化合物38(22mg，石油醚/乙酸乙酯＝25：1),无色晶体，产率为20％。
化合物34：HRMS-ESI-TOF：[M+Na]+701；mp 213-215℃；1H NMR(400MHz,Acetone)δ6.84(s,1H,H-5),6.31(d,J＝10.8Hz,1H,H-12),5.83–5.76(m,1H,H-17α),5.71(d,J＝4.2Hz,1H,H-17β),5.03(s,1H,H-7),4.93(s,1H,H-3),3.70(dd,J＝11.2,5.0Hz,1H,H-4),2.98–2.88(m,1H,H-1α),2.80–2.71(m,1H,H-8α),2.24–2.15(m,1H,H-2),2.14–2.07(m,1H,H-1β),1.90(s,3H,H-19),1.88(s,3H,H-18),1.85–1.82(m,1H,H-8β),1.67(s,3H,H-20),1.19(s,1H,H-9),0.99-0.89(m,1H,H-11),0.86(d,J＝6.2Hz,3H,H-16).3-O-benzoyl：8.12(d,J＝7.4Hz,2H),7.54(dd,J＝16.4,8.2Hz,2H),7.37(t,J＝7.7Hz,1H).5-OAc：1.29(s,3H).7-O-benzoyl：7.96(d,J＝7.5Hz,2H),7.66(t,J＝7.3Hz,1H),7.54(dd,J＝16.4,8.2Hz,1H),7.37(t,J＝7.7Hz,1H).
11、所述的化合物35的制备方法：在DCM溶液中，底物化合物18、TMSOTf、乙酸酐摩尔比为10：1：1，反应得到目标产物。
化合物35：HRMS-ESI-TOF：[M+Na]+681；1H NMR(600MHz,Acetone)δ7.43(s,1H,H-12),5.78(t,J＝3.7Hz,1H,H-3),5.62(d,J＝11.7Hz,1H,H-5),5.41(s,1H,H-17α),5.32(dd,J＝11.2,2.6Hz,1H,H-7),5.04(s,1H,H-17β),3.30(s,1H,H-10),3.10(d,J＝10.4Hz,1H,H-9),3.05(dd,J＝11.7,4.2Hz,1H,H-4),2.49-2.41(m,1H,H-2),2.26-2.16(m,1H,H-1α),1.96(s,1H,H-8α),1.88–1.84(m,2H,H-1β,H-8β),1.74(t,J＝3.7Hz,3H,H-20),1.72(s,3H,H-18),1.65(s,3H,H-19),0.94(d,J＝6.6Hz,3H,H-16).3-O-benzoyl：8.48–8.40(m,2H),7.68–7.59(m,1H),7.57(dt,J＝15.1,4.3Hz,2H).5-OAc：1.90(s,3H).7-O-benzoyl：7.98(dd,J＝8.3,1.2Hz,2H),7.68–7.59(m,1H),7.47(t,J＝7.7Hz,2H).11-OAc：2.04(s,3H).
12、所述的化合物36的的制备方法：在DCM溶液中，底物化合物41、三氯化铟、苯甲酰氯摩尔比为1：1：2，反应得到目标产物。
化合物36：1H NMR(600MHz,Acetone)δ6.69(d,J＝11.3Hz,1H,H-12),6.27(s,1H,H-5),5.59(t,J＝4.4Hz,1H,H-3),3.94(d,J＝11.2Hz,1H,H-17α),3.90(d,J＝11.2Hz,1H,H-17β),3.84(d,J＝15.2Hz,1H,6-OH),3.03(dd,J＝11.7,5.3Hz,1H,H-1α),2.54(d,J＝5.3Hz,1H,H-7α),2.27–2.22(m,1H,H-8α),2.03(m,1H,H-2),1.96–1.86(m,2H,H-4,H-8β),1.84(s,3H,H-20),1.65(dd,J＝11.2,8.9Hz,1H,H-1β),1.51–1.46(m,1H,H-11),1.40–1.35(m,1H,H-7β),1.37(s,3H,H-19),1.20(s,3H,H-18),1.10(dd,J＝14.3,9.0Hz,1H,H-9),0.70(d,J＝6.7Hz,3H,H-16).3-O-phenylacetyl：7.36(d,J＝7.3Hz,2H),7.33(t,J＝7.5Hz,2H),7.26(t,J＝7.2Hz,1H),3.76(d,J＝16.1Hz,2H).5-OAc：2.01(s,3H).15-OAc：2.21(s,3H).
13、化合物18、38、39、41是从千金子中直接提取得到。提取方法为：用95％的乙醇/丙酮(体积比为5：1)在室温下对千金子粉末(5kg)进行浸泡，溶剂在真空下浓缩得到200g油状物。对油状物进行硅胶柱洗脱，得到3个粗馏分。在对馏分1进行进一步提纯，采用硅胶色谱法(按体积比，石油醚：乙酸乙酯＝15：1)得到化合物18(4.6g)，馏分2采用硅胶色谱法分离(按体积比，石油醚：乙酸乙酯＝12：1)得到化合物38(200mg)，化合物39(20mg)。馏分3采用硅胶色谱法(按体积比，石油醚：乙酸乙酯＝10：1)得到化合物41(20.7g)。
化合物18：HRMS-ESI-MS：665[M+Na]+；1H NMR(400MHz,Acetone)δ6.60(dd,J＝11.2,1.2Hz,1H,H-12),6.38(d,J＝7.9Hz,1H,H-5),5.76(t,J＝3.2Hz,1H,H-3),5.56(dd,J＝8.8,3.1Hz,1H,H-7),5.49(s,1H,H-17α),5.27(s,1H,H-17β),3.35(dd,J＝14.0,8.1Hz,1H,H-1α),3.05(dd,J＝7.8,3.4Hz,1H,H-4),2.43–2.30(m,1H,H-2,H-8α),2.23-2.17(m,1H,H-8β),1.83–1.75(m,1H,H-9),1.77(d,J＝1.1Hz,3H,H-20),1.62(dd,J＝11.2,8.5Hz,1H,H-1β),1.51–1.42(m,1H,H-11),1.26(s,3H,H-18),1.22(s,3H,H-19),0.91(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.10(dd,J＝8.3,1.3Hz,2H),7.66(t,J＝7.4Hz,1H),7.55(t,J＝7.6Hz,1H).5-OAc：1.30(s,3H).7-O-benzoyl：7.93(dd,J＝8.3,1.3Hz,2H),7.60(t,J＝7.4Hz,1H),7.46(t,J＝7.7Hz,1H).15-OAc：2.24(s,3H).
化合物38：HRMS-ESI-TOF：[M+Na]+461；1H NMR(400MHz,Acetone)δ7.88(d,J＝12.0Hz,1H,H-12),5.55(t,J＝3.6Hz,1H,H-3),5.20(s,1H,OH-15),3.43(d,J＝9.1Hz,1H,H-5),3.24(dd,J＝12.5,7.1Hz,1H,H-1α),2.00–1.95(m,1H,H-2),1.94–1.90(m,1H,H-7α),1.85(s,3H,H-20),1.79(d,J＝13.0Hz,1H,H-1β),1.76(dd,J＝9.2,4.4Hz,1H,H-4),1.65(dd,J＝11.5,7.9Hz,1H,H-11),1.63–1.53(m,1H,H-8α),1.35-1.27(m,3H,H-7β,H-8β,H-9),1.20(s,6H,H-17,H-18),1.08(s,3H,H-19),0.96(d,J＝6.7Hz,3H,H-16).3-O-benzoyl：8.16(d,J＝7.2Hz,2H),7.63(t,J＝7.4Hz,1H),7.52(t,J＝7.6Hz,2H).
化合物41：1H NMR(400MHz,Acetone)δ6.69(dd,J＝11.4,1.1Hz,1H,H-12),6.31(d,J＝9.0Hz,1H,H-5),5.38(t,J＝3.1Hz,1H,H-3),3.24(dd,J＝14.0,8.2Hz,1H,H-17α),2.55(d,J＝3.6Hz,1H,H-7α),2.19(dd,J＝3.4,2.1Hz,1H,H-17β),2.13–2.09(m,1H,H-2),2.08(d,J＝3.6Hz,1H,H-8α),2.05(m,2H,H-8β,H-4),1.85(dd,J＝9.0,3.4Hz,1H,H-7β),1.80(d,J＝1.1Hz,3H,H-20),1.74(d,J＝13.3Hz,1H,H-1α),1.62(dd,J＝11.4,8.2Hz,1H,H-11),1.38(dd,J＝13.9,12.3Hz,1H,H-1β),1.25(s,3H,H-19),1.22(s,3H,H-18),1.00–0.91(m,1H,H-9),0.64(d,J＝6.7Hz,3H,H-16).3-O-phenylacetyl：7.34(s,2H),7.33(s,2H),7.27(m,1H),3.70(d,J＝15.1Hz,1H),3.58(d,J＝15.1Hz,1H).5-OAc：1.99(s,3H).15-OAc：2.15(s,3H).
实施例二：化合物1～41的生物体外活性试验
1、千金子二萜衍生物的细胞毒性检测。具体操作：按照1×110个/孔限定数量，将MCF-7、MCF-7/ADR细胞接种于96孔板，每孔加入100μL含10％胎牛血清的MEM培养基培养15h，至细胞增长至覆盖孔面积的70％，依次加入最终浓度梯度为1μM、5μM、20μM、50μM、100μM的1～41号化合物，化合物皆用DMSO溶解，给药体积为1μL。设置终浓度梯度为0.01μM、0.1μM、1μM、5μM、10μM的多柔比星作为阳性对照，化合物DMSO溶解，给药体积为1μL。设置DMSO为阴性对照，给药体积为1μL。每组设置3个孔作为平行样。37℃培养48h后，抽去原培养基，用无血清MEM培养基按将CCK-8溶液稀释10倍，每孔加入100μL稀释液，37℃孵育1h，之后在450nm测定吸光度。
数据分析：将各孔细胞吸光度相对于DMSO组吸光度的相对百分比作为抑制率，然后将浓度梯度取10的对数，将其作为横坐标，将抑制率作为纵坐标，求得回归方程后带入50％抑制率计算IC50，同时根据每组内数据测得其平均值和方差，每组数据独立重复三次。41个化合物对MCF-7和MCF-7/ADR细胞的细胞毒(IC50)见表1。
表1各化合物对MCF-7和MCF-7/ADR细胞的细胞毒
Figure BDA0003082198360000241
2、千金子二萜衍生物逆转耐药能力检测。具体操作：按照1×110个/孔限定数量，将MCF-7/ADR细胞接种于96孔板，每孔加入100μL含10％胎牛血清的MEM培养基培养15h，至细胞增长至覆盖孔面积的70％，加入最终浓度梯度为10μM的1～41号化合物，以终浓度为10μM的维拉帕米(VRP)作为阳性对照。化合物皆用DMSO溶解，给药体积为1μL，37℃下孵育2h。之后加入终浓度梯度为0.01μM、0.1μM、1μM、5μM、10μM的多柔比星，化合物DMSO溶解，给药体积为1μL。设置DMSO为阴性对照，给药体积为1μL。每组设置3个孔作为平行样。37℃培养48h后，抽去原培养基，用无血清MEM培养基按将CCK-8溶液稀释10倍，每孔加入100μL稀释液，37℃孵育1h，之后在450nm测定吸光度。
数据分析：将各孔细胞吸光度相对于DMSO组吸光度的相对百分比作为抑制率，然后将浓度梯度取10的对数，将其作为横坐标，将抑制率作为纵坐标，求得回归方程后带入50％抑制率计算加入1～41号化合物后对于多柔比星的EC50，同时根据每组内数据测得其平均值和方差，每组数据独立重复三次，将耐药株(MCF-7/ADR)对于多柔比星的IC50平均值与加入1～41号化合物的EC50作比值，求出其逆转倍数。逆转倍数＝EC50(DOX)/EC50(sample+DOX)。
41个化合物对于MCF-7/ADR细胞的逆转活性数据见表2。
表2各千金子二萜衍生物在MCF-7/ADR细胞上的逆转效应
Figure BDA0003082198360000251
Figure BDA0003082198360000261
从表2可以看出，有25个化合物逆转活性都优于VRP。其中，化合物17、29、38、39活性最佳，逆转倍数分别是维拉帕米(VRP)的229倍、454倍、218倍和71倍。
实施例三：胞内聚集实验
本实施例提供了化合物8、31进行罗丹明123(Rh123)胞内聚集实验检测。具体操作：将MCF-7/ADR和MCF-7细胞以2×106个/孔的密度接种到24孔板中，在37℃下孵育16h，用逆转耐药筛选出来的化合物8和化合物31(浓度分别为2、10和20μM)预处理1h，采用终浓度为20μM的维拉帕米作为阳性对照。之后将细胞与终浓度为5μM的Rh123在37℃避光孵育1h。用PBS洗涤细胞两次，重悬于PBS缓冲液中，并通过流式细胞仪进行分析。实验结果如图1、表3所示。图1为化合物8、化合物31分别在MCF-7/ADR细胞内的罗丹明积累变化示意图。
表3化合物8、31抑制P-gp介导的罗丹明外排实验结果展示表
Figure BDA0003082198360000262
化合物8、31在MCF-7/ADR细胞内均可抑制Rh123的外排，并且化合物8、31抑制P-gp的外排能力有明显的浓度依耐性。化合物31随着浓度的增加抑制能力加强，浓度为80μM时基本完全逆转耐药性。化合物8在浓度为20μM抑制P-gp的外排的能力最佳，增加浓度逆转能力反而下降。
实施例四：对多柔比星(DOX)的积累实验
将MCF-7/ADR细胞以1×107个/孔的密度接种到6孔板盖玻片中，在37℃下孵育16h，用逆转耐药筛选出来的化合物17、29和38(浓度分别为2、10和20μM)预处理2h，再用DOX处理2h，终浓度为20μM的维拉帕米作为阳性对照。之后用4％多聚甲醛固定30min，终浓度为5μg/ml的dapi染液(4',6-二脒基-2-苯基吲哚)在于37℃避光孵育1h。用PBS洗涤细胞两次，取出盖玻片，用分片液封于载玻片上，通过荧光显微镜400倍下观察分析。实验结果图2、表4所示。图2为MCF-7/ADR在化合物17、29、38不同浓度下处理2h后DOX积累，用Image J软件分析得到的荧光数据柱状图。图2从左往右依次为：化合物17、化合物29、化合物38的结果展示图。
表4化合物17、29、38抑制P-gp介导的DOX外排实验结果展示表
Figure BDA0003082198360000271
化合物17、29、38均可以抑制MCF-7/ADR细胞内P-gp介导的DOX的外排且具有浓度依赖性。化合物17、29、38均有助于细胞内DOX的积累；化合物29在浓度为10μM时，抑制DOX外排能力优于维拉帕米，在浓度为10μM时，抑制DOX外排能力是维拉帕米的2.1倍。
实施例五：对p-gp表达的western blot实验检测
将MCF-7/ADR细胞以1×107个/孔的密度接种到6孔板中，在37℃下孵育16h，用逆转耐药筛选出来的化合物8、17、29、31和38(浓度分别为2、10和20μM)预处理1h，采用终浓度为20μM的维拉帕米作为阳性对照。PBS洗涤细胞两次，加入Ripa裂解细胞15min，13000r离心细胞20min，总蛋白浓度用BCA试剂盒进行定量检测。之后在8SDS-PAGE凝胶中将蛋白质分离，然后在100V的恒定电压下在4℃下电转移到硝酸纤维素膜上。将硝酸纤维素膜用5％牛血清白蛋白封闭2h。然后在4℃下与抗P-gp或抗GAPDH抗体(一抗)孵育过夜，GAPDH用作加载对照。用TBST(1x Tris缓冲盐水，0.1％Tween 20)洗涤3次后加入山羊抗兔IgG二抗，在室温下孵育2h。再次用TBST洗涤3次后，用超敏ECL化学发光底物检测结合的抗体复合物。结果如图3所示。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形、变型、修改、替换，均应落入本发明权利要求书确定的保护范围内。

Claims (2)

1.一类千金子二萜衍生物，其特征在于：结构通式为：
Figure DEST_PATH_IMAGE001
，
其中，
R1=H，R2=R3=对三氟甲基苯甲酰基，R4=H；或；
R1=H，R2=R3=呋喃甲酰基，R4=H；或；
R1=R2=H，R3=1-萘乙酰基，R4=H；或
R1=R2=H，R3=2-萘基，R4=H；或；
R1=R2=R3=R4=H；或；
R1=R2=H，R3=乙酰基，R4=H；或；
R1=R2=H，R3=对氯苯基，R4=H；或；
R1=R2=H，R3=间硝基苯甲酰基，R4=H；或；
R1=R2=H，R3=对硝基苯甲酰基，R4=H；或；
R1=R2=H，R3=对甲基苯乙酰基，R4=H；或；
R1=R2=H，R3=肉桂酰基，R4=H；或；
R1=苯甲酰基，R2=R3=H，R4=H；或；
R1=R3=苯甲酰基，R2=H，R4=H；或；
R1=R3=间硝基苯甲酰基，R2=H，R4=H；或；
R1=H，R2=R3=间硝基苯甲酰基，R4=H；或；
R1=H，R2=R3=肉桂酰基，R4=H；或；
R1=H，R2=R3=苯甲酰基，R4=H；或；
R1=R3=苯甲酰基，R2=R4=乙酰基；或；
R1=R2=R3=对甲基苯甲酰基，R4=H；或；
R1=R2=R3=间硝基苯甲酰基，R4=H；或；
R1=R2=R3=苯甲酰基，R4=H；或；
R1=R2=R3=对硝基苯甲酰基，R4=H；或；
R1=R2=R3=对-（三氟甲基）苯甲酰基，R4=H；或；
R1=R2=R3=乙酰基，R4=H；或；
R2=R3=苯甲酰基，R1=乙酰基，R4=H。
2.权利要求1所述千金子二萜衍生物在药物制备领域的应用，其特征在于：在逆转多药耐药上的应用。
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