一类四环三萜酸衍生物及其制备方法与应用与流程

本发明属于医药技术领域，具体地说，涉及一种四环三萜酸衍生物及其制备方法与应用。

背景技术：

现有技术表明五味子有保肝、治疗肝炎的作用，已有文献报道五味子中甘五酸(sa)和安五酸(anwuweizicacid，aa)对hcv病毒抑制活性最好。sa和aa的结构如下：

初步作用机制研究结果表明，sa与现有的daas类药物作用靶点不同，它作用于hcv感染早期，即干扰hcv与细胞受体的结合，阻止hcv入侵细胞过程，从而将hcv“拒之门外”，属于病毒入侵抑制剂。

因此，在sa抑制hcv入侵细胞作用机制的研究基础上，以sa为先导物，可以对其进行结构改造，丰富化合物结构类型，以期寻找活性更好的具有阻滞hcv病毒入侵的先导化合物。

技术实现要素：

本发明的第一个目的是提供一种四环三萜酸衍生物。

本发明的第二个目的是提供一种所述四环三萜酸衍生物的制备方法。

本发明的第三个目的是提供一种所述四环三萜酸衍生物在制备抗hcv病毒药物中的应用。

为了实现上述目的，本发明采用的技术方案如下：

以五味子中的sa为原料对羰基或羧基进行结构修饰得到系列衍生物，然后测定抗hcv活性。

具体设计如下：

1、羰基修饰：

将sa羰基结构合成肟类衍生物，通过引入含氮杂原子；

将sa羰基还原为羟基。

2、双键修饰：

将sa双键还原，改变其刚性结构，增加其侧链柔性。

3、羧基修饰：

将sa的26位羧基进行酯化、酰胺化合成一系列衍生物。

本发明的第一个方面提供了一种四环三萜酸衍生物，结构如下所示：

r3为氢、烷基、环烷基、杂环基、取代或未取代芳基或杂芳基；

r4为氢、烷基、环烷基、杂环基、取代或未取代芳基或杂芳基；

r5为氢、烷基、杂环基、取代或未取代芳基或杂芳基。

较优选的，所述四环三萜酸衍生物中：

r3为氢、甲基、乙基、丙基、异丙基、丁基、叔丁基、戊基、

r4为氢、甲基、乙基、丙基、异丙基、丁基、叔丁基、戊基、

r5为氢、甲基、乙基、丙基、异丙基、丁基、叔丁基、戊基。

最优选的，所述四环三萜酸衍生物为以下结构中的一种：

本发明的第二个方面提供了一种所述四环三萜酸衍生物的制备方法，包括以下步骤：

将无水吡啶溶于乙醇中，然后加入摩尔比为1:(10～30)的sa和nh2oh·hcl，室温搅拌溶解反应，反应液加水淬灭，用盐酸调ph至中性，重结晶得到化合物1；

将摩尔比为1:(1.5～4)的sa和nabh4溶于无水甲醇中，超声反应，反应液加水淬灭，用盐酸调ph至中性，抽滤，烘干得到化合物2；

将sa和催化量的pd/c溶于无水乙醇中，在氢气催化条件下室温搅拌反应，反应完全后抽滤，减压浓缩，获得化合物3；

将0.07mmolsa溶于色谱纯甲醇、乙醇或丙醇中，向反应液滴加1ml浓盐酸或1滴浓硫酸，25～50℃搅拌反应，反应完全后制备获得化合物4a、4b或4c；

或，将摩尔比为1:(1.01～2):(1.01～2):(1.01～2)的sa、dcc、hobt和dmap溶于色谱纯甲醇、乙醇或丙醇中，室温搅拌溶解反应，反应完全后获得化合物4a、4b或4c；

将摩尔比为1:(1.01～3):(1.1～6):(1.1～4)的sa、pybop、diea和有机胺溶于无水二氯甲烷中，室温搅拌反应，获得化合物5a～5q；

将摩尔比为1:(1.01～3):(1.1～6):(1.1～4)的sa、pybop、diea和hobt溶于无水二氯甲烷中，室温搅拌反应，获得化合物sa-1；

取干燥的北五味子成熟果实，粉碎后用75％乙醇回流提取三次，提取液浓缩，得五味子乙醇粗提物浸膏，均匀分散于蒸馏水中，依次用石油醚、氯仿和正丁醇萃取；各有机相萃取液浓缩得石油醚部位浸膏、氯仿部位浸膏、正丁醇部位浸膏；将氯仿部位浸膏经反相硅胶柱层析、sephadexlh-20凝胶柱层析和c-18反相硅胶柱层析，得到ca。

所述有机胺选自甲胺、乙胺、丙胺、丁胺、异丙胺、环己胺、

本发明的第三个方面提供了一种所述四环三萜酸衍生物在制备抗hcv病毒药物中的应用。

所述抗hcv病毒药物为四环三萜酸衍生物及其组合物。

所述四环三萜酸衍生物及其组合物是指所述四环三萜酸衍生物单一成分作为药物或四环三萜酸衍生物与其他药学上可接受的成分构成组合物。

所述四环三萜酸衍生物与其他药学上可接受的成分构成组合物中，活性组分四环三萜酸衍生物的重量含量为0.1-99％。

本发明的第四个方面提供了一种预防hcv感染的抑制剂，是以所述四环三萜酸衍生物作为活性组分。

由于采用上述技术方案，本发明具有以下优点和有益效果：

本发明的四环三萜酸衍生物，初步的作用机制研究结果表明，其母环结构sa与现有的daas类药物作用靶点不同，作用于hcv病毒感染早期，即干扰hcv病毒与细胞受体的结合，阻止hcv病毒入侵细胞过程，从而将hcv病毒“拒之门外”，属于病毒入侵抑制剂。与目前临床上使用的抗hcv药物具有完全不同的作用机制。此外，还可以用作预防hcv感染的药物。

本发明的四环三萜酸衍生物，具有更好的抗hcv活性，大大的提高了此类化合物抗hcv的效果。

附图说明

图1是本发明实施例制备的化合物抗hcv的ic50值的柱状图。

图2是化合物3、5c、5e、5g、5h、5p、5q、sa-1、aa和ca的体外细胞毒性活性结果。

具体实施方式

为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。

本发明所用甘五酸、安五酸购于上海历鼎生物技术有限公司，并经进一步光谱检测确证了化合物结构。其它合成所用分析纯或光谱纯试剂均购上海泰坦科技股份有限公司。

实施例1

化合物1的合成：

将2.5ml95％乙醇和2.5ml无水吡啶依次加入50ml单口瓶中，然后依次加入sa(30mg，0.07mmol)和nh2oh·hcl(106mg，1.53mmol)，室温搅拌溶解，继续反应3.5h后，反应液加水淬灭，然后用2mol/l盐酸调ph至中性，析出白色固体，反应液继续放冷，进一步析出白色固体，抽滤，95％乙醇重结晶得20mg白色固体即化合物1，产率62.5％。m.p.90.0～92.4℃.ms(esi)m/z470.6[m+h]^+-.¹hnmr(300mhz,cdcl3)δ5.99(1h,t,j＝7.4hz,h-24),3.40(1h,d,3-oh),1.92(3h,s,ch3-27),1.11(3h,s,ch3-29),1.09(3h,s,ch3-28),0.96(3h,s,ch3-30),0.89(6h,d,j＝8.6hz,ch3-18,ch3-21),0.69(1h,d,j＝3.6hz,h-19β),0.48(1h,d,j＝4.1hz,h-19α).¹³cnmr(75mhz,cdcl3)δ171.7(c-26),166.6(c-3),143.5(c-24),125.0(c-25),50.2(c-17),47.5(c-5),47.3(c-14),45.9(c-8),43.8(c-13),41.5(c-4),34.3(c-1),34.2(c-20),34.0(c-22),31.5(c-15),31.3(c-12),27.9(c-19),26.5(c-7),25.2(c-16),24.9(c-11),24.7(c-10),24.4(c-2),24.4(c-23),22.2(c-29),20.4(c-6),19.8(c-9),19.3(c-27),19.2(c-30),18.0(c-28),16.9(c-21),16.5(c-18).

实施例2

化合物2的合成：

将5ml甲醇置于50ml单口瓶中，依次加入sa(148mg，0.33mmol)和nabh4(30.71mg，0.83mmol)，室温搅拌至溶解，超声反应2h，反应液加水淬灭，然后用2mol/l盐酸调ph至中性，析出白色固体，反应液继续放冷，进一步析出白色固体，抽滤，烘干得149mg浅白色固体即化合物2，产率100％。m.p.124.4～126.2℃.ms(esi)m/z457.4[m+h]⁺.¹hnmr(300mhz,cdcl3)δ6.08(1h,t,j＝7.3hz),3.28～3.22(1h,m,3-oh),1.92(s,3h,ch3-27),1.01～0.92(6h,m,ch3-28,ch3-29),0.90(6h,dd,ch3-18,ch3-30),0.82(3h,d,j＝5.2hz,ch3-21),0.56(1h,d,j＝4.0hz,h-19β),0.33(1h,d,j＝4.1hz,h-19α).¹³cnmr(75mhz,cdcl3)δ172.1(c-26),147.2(c-24),125.6(c-25),78.9(c-3),52.2(c-17),48.8(c-14),48.0(c-5),47.1(c-8),45.3(c-13),40.6(c-4),36.0(c-20),35.8(c-22),35.6(c-12),32.9(c-15),32.0(c-1),30.4(c-2),29.9(c-19),28.2(c-7),26.9(c-16),26.5(c-11),26.1(c-10),25.4(c-23),22.4(c-29),21.1(c-6),20.6(c-9),20.6(c-27),20.0(c-30),19.3(c-28),18.1(c-18),18.1(c-21).

实施例3

化合物3合成：

将3ml无水乙醇置于25ml单口瓶中，加入sa(51mg，0.11mmol)室温搅拌至溶解，称取50mg10％pd/c置于反应液中，在氢气催化条件下室温搅拌反应2.5d，反应液抽滤，滤液减压浓缩得灰白色固体，制备薄层[v(pe):v(ea)＝10:1]得到粗品，再用液相制备得到纯品，冷冻干燥得30mg白色固体即化合物3，产率60％。m.p.127.6～129.4℃.ms(esi)m/z457.4[m+h]⁺,479.3[m+na]⁺,455.4[m-h]^-.¹hnmr(300mhz,cdcl3)δ2.71(1h,td,j＝13.8,6.4hz,h-2β),1.19(3h,d,j＝6.8hz,ch3-27),1.10(3h,s,ch3-29),1.04(3h,s,ch3-28),0.99(3h,s,ch3-30),0.90(3h,s,ch3-18),0.86(3h,d,j＝6.3hz,ch3-21),0.78(1h,d,j＝3.8hz,h-19β),0.57(1h,d,j＝4.1hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.7(c-3),181.8(c-26),52.4(c-17),50.3(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.3(c-13),39.3(c-25),37.5(c-2),36.0(c-24),35.9(c-20),35.8(c-12),35.6(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.2(c-16),28.2(c-7),26.7(c-11),25.9(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.1(c-9),20.8(c-30)19.3(c-28),18.2(c-21),18.1(c-18),16.7(c-27).

实施例4

sa酯类衍生物合成，以化合物4a合成为例：

方法一：将3ml色谱纯甲醇置于25ml单口瓶中，加入sa(30mg，0.07mmol)室温搅拌至溶解，向反应液滴加1ml浓盐酸，40℃继续搅拌反应48h，减压蒸干甲醇，制备薄层[v(pe):v(ea)＝10:1]得10mg产物即化合物4a和10mg原料，产率48.5％。

方法二：将2ml色谱纯甲醇置于25ml单口瓶中，依次加入sa(50mg，0.11mmol)、dcc(n,n'-二环己基碳酰亚胺)(26mg，0.13mmol)、hobt(1-羟基苯并三唑)(16mg，0.12mmol)和dmap(4-二甲氨基吡啶)(18mg，0.15mmol)，加料完毕室温搅拌至溶解，继续搅拌反应6h，减压蒸干甲醇，制备薄层[v(pe):v(ea)＝10:1]得到粗品，再用液相制备得到纯品，冷冻干燥得30mg白色固体即化合物4a，产率58.3％。m.p.117.6～120.4℃.ms(esi)m/z469.5[m+h]⁺.¹hnmr(300mhz,cdcl3)δ5.92(1h,t,j＝7.3hz,h-24),3.72(3h,s,h-1′),1.88(3h,s,ch3-27),1.08(3h,s,ch3-29),1.03(3h,s,ch3-28),0.98(3h,s,ch3-30),0.89(6h,s,ch3-18,ch3-21),0.77(1h,d,j＝3.8hz,h-19β),0.56(1h,d,j＝4.2hz,h-19α)。¹³c-nmr(75mhz,cdcl3)δ216.7(c-3),168.6(c-26),144.2(c-24),126.4(c-25),53.4(c-1′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.3(c-13),37.4(c-2),35.9(c-20),35.8(c-12),35.5(c-1),33.4(c-15),32.7(c-22),29.5(c-19),28.1(c-7),26.7(c-16),26.6(c-11),25.9(c-10),25.8(c-23),22.1(c-29),21.5(c-6),21.1(c-9),20.7(c-30),20.7(c-27),19.3(c-28),18.1(c-21),18.1(c-18).

实施例5

化合物4b的制备方法同实施例4，将实施例4中的方法二中甲醇替换为乙醇即可制备得到化合物4b。yield60.0％.m.p.127.6～129.4℃.ms(esi)m/z483.5[m+h]⁺.¹hnmr(300mhz,cdcl3)δ6.73(1h,t,j＝7.3hz,h-24),4.16(2h,q,j＝7.1hz,h-1′),1.81(3h,s,ch3-27),1.25～1.19(3h,m,ch3-2′),1.08(3h,s,ch3-29),1.02(3h,s,ch3-28),0.97(3h,s,ch3-30),0.89(6h,d,j＝6.4hz,ch3-18,ch3-21),0.76(1h,d,j＝3.8hz,h-19β),0.56(1h,d,j＝4.2hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.5(c-3),168.3(c-26),142.8(c-24),127.4(c-25),60.3(c-1′),52.1(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.3(c-13),37.4(c-2),35.9(c-20),35.5(c-1),34.9(c-12),33.4(c-15),32.7(c-22),29.5(c-19),28.1(c-7),26.6(c-16),25.9(c-11),25.8(c-23),25.6(c-10),22.1(c-29),21.5(c-6),21.0(c-9),20.7(c-27),20.7(c-30),19.2(c-28),18.1(c-18),18.1(c-21),14.3(c-2′).

实施例6

化合物4c的制备方法同实施例4，将实施例4中的方法一或方法二中甲醇替换为丙醇即可制备得到化合物4c。yield55.0％.m.p.109.5～110.4℃.ms(esi)m/z497.7[m+h]⁺.¹hnmr(300mhz,cdcl3)δ5.90(1h,t,j＝7.2hz,h-24),4.09(2h,t,j＝6.6hz,h-1′),1.88(3h,s,ch3-27),1.08(3h,s,ch3-29),1.03(s,3h,h-3′),1.01～0.92(6h,m,ch3-28、ch3-30),0.88(6h,d,j＝4.2hz,ch3-18,ch3-21),0.77(1h,d,j＝3.7hz,h-19β),0.56(1h,d,j＝4.1hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.6(c-3),168.3(c-26),143.6(c-24),126.8(c-25),65.7(c-1′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.3(c-13),37.4(c-2),35.9(c-20),35.9(c-12),35.5(c-1),33.4(c-15),32.7(c-22),29.5(c-19),21.5(c-6),28.1(c-7),26.7(c-11),26.6(c-16),25.9(c-23),25.8(c-10),22.1(c-29),22.0(c-2′),21.0(c-9),20.7(c-27),20.7(c-30),19.3(c-28),18.1(c-21),18.1(c-18),10.6(c-3′).

实施例7

sa酰胺类衍生物合成，以化合物5k合成为例：

将3ml无水二氯甲烷置于25ml单口瓶中，依次加入sa(30mg，0.07mmol)、pybop(40.1mg，0.08mmol)、diea(36.2mg，0.28mmol)和对氯苯胺(18.1mg，0.17mmol)搅拌至溶解，继续室温搅拌反应24h，减压蒸干溶剂，制备薄层[v(pe):v(ea)＝10:1]得到粗品，再用液相制备得到纯品，冷冻干燥得20mg白色固体即化合物5k，产率50.8％。m.p.118.6～120.9℃.ms(esi)m/z565.6[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.54(s,1h,ar-h),7.51(s,1h,ar-h),7.33(s,1h,ar-h),7.30(s,1h,ar-h),7.17(s,1h,nh),5.66(t,j＝7.4hz,1h,h-24),2.72(td,j＝13.9,6.5hz,1h,h-2β),2.00(s,3h,ch3-27),1.11(s,3h,ch3-29),1.06(s,3h,ch3-28),0.99(s,3h,ch3-30),0.88(d,j＝5.4hz,6h,ch3-18,ch3-21),0.79(d,j＝4.2hz,1h,h-19β),0.58(d,j＝4.2hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ215.6(c-3),167.1(c-26),135.3(c-24),134.6(c-ar),130.5(c-ar),128.1(c-ar),128.1(c-ar),128.1(c-25),120.0(c-ar),120.0(c-ar),51.1(c-17),49.2(c-4),47.7(c-14),47.4(c-5),46.9(c-8),44.3(c-13),36.5(c-2),35.2(c-20),34.9(c-12),34.5(c-1),32.4(c-15),31.7(c-22),28.5(c-19),27.2(c-7),25.7(c-16),25.6(c-11),24.9(c-10),24.8(c-23),21.2(c-29),20.5(c-6),20.0(c-9),19.8(c-30),19.6(c-27),18.1(c-28),17.2(c-21),17.1(c-18).

实施例8

化合物5a的制备方法同实施例7，将实施例7中对氯苯胺替换为甲胺即可制备得到化合物5a。yield61％.m.p.105.5～107.0℃.ms(esi)m/z468.7[m+h]⁺,490.7[m+na]⁺.¹hnmr(300mhz,cdcl3)δ5.45(1h,t,j＝7.2hz,h-24),2.82(3h,d,j＝4.8hz,ch3),1.82(3h,s,ch3-27),1.03(3h,s,ch3-29),0.98(3h,s,ch3-28),0.92(3h,s,ch3-30),0.81(6h,d,j＝7.9hz,ch3-18,ch3-21),0.72(1h,d,j＝3.9hz,h-19β),0.51(1h,d,j＝4.1hz,h-19α).¹³cnmr(75mhz,cdcl3)δ215.5(c-3),169.7(c-26),133.0(c-24),130.4(c-25),51.0(c-17),49.1(c-4),47.6(c-14),47.3(c-5),46.7(c-8),44.2(c-13),36.3(c-2),35.1(c-20),34.7(c-12),34.4(c-1),32.3(c-15),31.6(c-22),28.4(c-19),27.0(c-7),25.5(c-16),25.2(c-11),24.9(c-10),24.7(c-1′),24.7(c-23),21.0(c-29),20.3(c-6),19.9(c-9),19.8(c-30),16.9(c-18),17.0(c-21),19.6(c-27),18.1(c-28).

实施例9

化合物5b的制备方法同实施例7，将实施例7中对氯苯胺替换为乙胺即可制备得到化合物5b。yield62％.m.p.106.5～107.4℃.ms(esi)m/z482.8[m+h]⁺,505.0[m+na]⁺.¹hnmr(300mhz,cdcl3)δ5.52～5.44(1h,t,h-24),3.44～3.30(1h,m,h-1′),2.71(1h,td,j＝13.8,6.4hz,h-1′),1.89(3h,s,me-27),1.26(3h,s,me-29),1.19(3h,t,j＝7.2hz,me-2′),1.10(3h,s,me-28),1.02(6h,d,j＝16.8hz,me-18,me-30),0.90(3h,s,me-21),0.79(1h,d,j＝3.6hz,h-19β),0.58(1h,d,j＝4.0hz,h-19α).

实施例10

化合物5c的制备方法同实施例7，将实施例7中对氯苯胺替换为丙胺即可制备得到化合物5c。yield58％.m.p.106.3～107.4℃.ms(esi)m/z496.7[m+h]⁺.¹hnmr(600mhz,cdcl3)δ5.48(td,j＝7.5,1.4hz,1h,h-24),5.45(s,1h,nh),3.42～3.31(m,2h,ch2),2.71(td,j＝13.9,6.4hz,1h,h-2β),2.33～2.28(m,1h,h-2α),1.89(d,j＝1.1hz,3h,ch3-27),1.59～1.60(d,1h,h-17α),1.19(t,j＝7.3hz,3h,ch3),1.10(t,j＝7.3hz,3h,ch3-29),1.05(s,3h,ch3-28),0.99(s,3h,ch3-30),0.90(s,3h,ch3-18),0.88(s,3h,ch3-21),0.79(d,j＝4.1hz,1h,h-19β),0.58(d,j＝4.3hz,1h,h-19α).¹³cnmr(150mhz,cdcl3)δ215.5(c-3),169.1(c-26),132.5(c-24),130.8(c-25),51.1(c-17),49.2(c-4),47.7(c-14),47.4(c-5),46.8(c-8),44.3(c-13),36.4(c-2),35.2(c-20),34.9(c-12),34.5(c-1),33.1(c-1′),32.4(c-15),31.7(c-22),28.6(c-19),28.5(c-2′),27.1(c-7),25.6(c-16),25.3(c-11),24.9(c-23),24.8(c-10),21.1(c-29),20.4(c-6),20.0(c-9),19.8(c-30),19.7(c-27),18.2(c-28),17.1(c-21),17.0(c-18),13.9(c-3′).

实施例11

化合物5d的制备方法同实施例7，将实施例7中对氯苯胺替换为丁胺即可制备得到化合物5d。yield70％.m.p.104.5～106.0℃.ms(esi)m/z510.8[m+h]⁺,532.9[m+na]⁺.¹hnmr(300mhz,cdcl3)δ5.48(1h,t,j＝7.1hz,j＝6.7hz,h-24),3.39～3.29(1h,td,h-1′),2.72(1h,td,j＝13.8,6.4hz,h-1′),1.94～1.87(3h,m,ch3-27),1.39(3h,dd,j＝15.3,7.2hz,ch3-4′),1.11(3h,s,ch3-29),1.05(3h,s,ch3-28),0.99(3h,d,j＝7.3hz,ch3-30),0.90(6h,dd,j＝8.2,6.1hz,ch3-18,ch3-21),0.79(1h,d,j＝4.0hz,h-19β),0.59(1h,d,j＝4.2hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.6(c-3),170.3(c-26),146.3(c-24),125.9(c-25),52.1(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.3(c-13),35.5(c-1),39.0(c-1′),37.4(c-2),36.2(c-20),35.9(c-12),33.4(c-15),32.7(c-22),31.8(c-2′),29.7(c-19),28.1(c-7),26.7(c-16),26.4(c-11),25.9(c-23),25.8(c-10),22.1(c-29),21.5(c-6),21.0(c-9),20.9(c-30),20.7(c-3′),20.1(c-27),19.3(c-28),18.1(c-21),18.0(c-18),13.7(c-4′).

实施例12

化合物5e的制备方法同实施例7，将实施例7中对氯苯胺替换为异丙胺即可制备得到化合物5e。yield55％.m.p.108.4～112.5℃.ms(esi)m/z496.8[m+h]⁺.¹hnmr(300mhz,cdcl3)δ5.43(1h,t,j＝7.6hz,h-24),4.20～4.06(1h,m,h-2′),2.69(td,j＝13.8,6.4hz,1h,h-2β),1.86(3h,s,ch3-27),1.19～1.17(6h,d,(ch3)2),1.09～1.02(6h,m,ch3-28,ch3-29),0.97(3h,s,ch3-30),0.92～0.84(6h,m,ch3-18,ch3-21),0.77(1h,d,j＝3.9hz,h-19β),0.56(1h,d,j＝4.2hz,h-19α).¹³cnmr(75mhz,cdcl3)δ215.6(c-3),168.4(c-26),131.9(c-24),131.2(c-25),51.1(c-17),49.2(c-4),47.7(c-14),47.4(c-5),46.8(c-8),44.3(c-13),40.1(ch),36.4(c-2),35.2(c-20),34.9(c-12),34.5(c-1),32.4(c-15),31.7(c-22),28.5(c-19),27.1(c-7),25.6(c-16),25.2(c-11),24.9(c-23),24.8(c-10),21.8(ch3,ch3),21.1(c-29),20.4(c-6),20.0(c-9),19.8(c-30),19.7(c-27),18.2(c-28),17.1(c-21),17.0(c-18).

实施例13

化合物5f的制备方法同实施例7，将实施例7中对氯苯胺替换为环己胺即可制备得到化合物5f。yield64％.m.p.125.3～127.7℃.ms(esi)m/z536.9[m+h]⁺.¹hnmr(300mhz,cdcl3)δ5.45(1h,t,j＝7.0hz,h-24),5.33(1h,d,j＝8.8hz,nh),3.87(1h,dd,j＝11.5,7.5hz,h-1′),2.71(td,j＝13.3,6.0hz,1h,h-2β),1.88(3h,s,h-27),1.10(3h,s,ch3-29),1.04(3h,s,ch3-28),0.99(3h,s,ch3-30),0.88(6h,d,j＝8.2hz,ch3-21,ch3-18),0.78(1h,d,j＝4.0hz,h-19β),0.58(1h,d,j＝4.2hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.6(c-3),169.4(c-26),132.9(c-24),132.3(c-25),52.1(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),47.9(c-1′),45.3(c-13),37.4(c-2),36.2(c-22),35.9(c-20),35.5(c-1),33.4(c-15),33.3(c-2′),33.2(c-6′),32.7(c-12),29.5(c-19),28.1(c-16),26.7(c-11),26.3(c-7),25.9(c-10),25.8(c-23),25.5(c-4′),24.8(c-3′),24.8(c-5′),22.1(c-29),21.5(c-6),21.0(c-9),20.9(c-30),20.7(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例14

化合物5g的制备方法同实施例7，将实施例7中的方法对氯苯胺替换为噻吩-2-甲胺即可制备得到化合物5g。yield62％.m.p.108.4～113.0℃.ms(esi)m/z550.6[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.25(d,j＝6.1hz,1h,h-5′),7.01(d,j＝2.8hz,1h,h-3′),6.99～6.94(m,1h,h-4′),5.85～5.77(m,1h,nh),5.54(t,j＝6.7hz,1h,h-24),4.74～4.67(m,2h,ch2),2.81～2.65(m,1h,h-2β),2.34(d,j＝14.0hz,1h,h-2α),1.92(s,3h,ch3-27),1.12(s,3h,ch3-29),1.06(s,3h,ch3-28),0.99(s,3h,ch3-30),0.90(s,3h,ch3-18),0.85(d,j＝6.4hz,3h,ch3-21),0.80(d,j＝4.5hz,1h,h-19β),0.59(d,j＝4.3hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ216.6(c-3),169.7(c-26),140.89(c-2′),134.7(c-24),131.1(c-25),126.9(c-4′),126.1(c-3′),125.2(c-5′),52.1(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.3(c-13),38.1(ch2),37.4(c-2),36.2(c-20),36.0(c-12),35.5(c-1),33.4(c-15),32.7(c-22),29.5(c-19),28.1(c-7),26.7(c-11),26.4(c-16),25.9(c-10),25.8(c-23),22.1(c-29),21.5(c-6),21.0(c-9),20.8(c-30),20.7(c-27),19.3(c-28),18.1(c-21),18.1(c-18).

实施例15

化合物5h的制备方法同实施例7，将实施例7中对氯苯胺替换为呋喃-2-甲胺即可制备得到化合物5h。yield60％.m.p.118.9～120.5℃.ms(esi)m/z534.6[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.35(s,1h,h-5′),6.36～6.29(m,1h,h-4′),6.25(d,j＝3.0hz,1h,h-3′),5.78(s,1h,nh),5.52(t,j＝7.3hz,1h,h-24),4.50(dd,j＝5.4,2.5hz,2h,ch2),2.71(td,j＝13.8,6.4hz,1h,h-2β),1.89(s,3h,ch3-27),1.09(s,3h,ch3-29),1.04(s,3h,ch3-28),0.97(s,3h,ch3-30),0.89(s,3h,ch3-18),0.83(d,j＝6.3hz,3h,ch3-21),0.78(d,j＝4.0hz,1h,h-19β),0.57(d,j＝4.2hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ215.6(c-3),168.8(c-26),150.2(c-2′),141.1(c-5′),133.6(c-24),130.1(c-25),109.4(c-4′),106.5(c-3′),51.1(c-17),49.2(c-4),47.7(c-14),47.4(c-5),46.8(c-8),44.3(c-13),36.4(ch2),35.2(c-2),35.2(c-20),34.9(c-12),34.5(c-1),32.4(c-15),31.7(c-22),28.5(c-19),27.1(c-7),25.6(c-16),25.4(c-11),24.9(c-23),24.84(c-10),21.1(c-29),20.4(c-6),20.0(c-9),19.8(c-30),19.7(c-27),18.2(c-28),17.0(c-21),17.0(c-18).

实施例16

化合物5i的制备方法同实施例7，将实施例7中对氯苯胺替换为苯胺即可制备得到化合物5i。yield66％.m.p.128.3～130.3℃.ms(esi)m/z530.7[m+h]⁺.¹hnmr(600mhz,cdcl3)δ7.56(d,j＝7.6hz,2h,h-2′,6′),7.34(t,j＝7.9hz,2h,h-3′,5′),7.21(s,1h,nh),7.12(t,j＝7.4hz,1h,h-4′),5.61(t,j＝6.9hz,1h,h-24),2.70(td,j＝13.8,6.4hz,1h,h-2β),2.30(ddd,j＝14.0,4.1,2.7hz,1h,h-2α),1.99(s,3h,ch3-27),1.09(s,3h,ch3-29),1.04(s,3h,ch3-28),0.98(s,3h,ch3-30),0.87(d,j＝4.7hz,6h,ch3-18,ch3-21),0.78(d,j＝4.1hz,1h,h-19β),0.56(d,j＝4.3hz,1h,h-19α).¹³cnmr(150mhz,cdcl3)δ216.6(c-3),168.3(c-26),137.7(c-24),134.9(c-1′),132.0(c-25),129.1(c-3′),129.1(c-5′),124.4(c-4′),119.9(c-2′),119.9(c-6′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.4(c-13),37.5(c-2),36.2(c-20),35.9(c-12),35.5(c-1),33.4(c-15),32.8(c-22),29.5(c-19),28.2(c-7),26.7(c-16),26.6(c-11),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.1(c-9),20.8(c-30),20.8(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例17

化合物5j的制备方法同实施例7，将实施例7中对氯苯胺替换为对氟苯胺即可制备得到化合物5j。yield60％.m.p.118.9～121.5℃.ms(esi)m/z548.7[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.50～7.53(d,j＝4.5hz,j＝4.9hz,2h,ar-h),7.13(s,1h,nh),7.03(t,j＝8.6hz,2h,ar-h),5.63(t,j＝7.5hz,1h,h-24),2.71(td,j＝13.9,6.5hz,1h,h-2β),1.99(s,3h,ch3-27),1.09(s,3h,ch3-29),1.04(s,3h,ch3-28),0.98(s,3h,ch3-30),0.94～0.80(m,6h,ch3-18,ch3-21),0.78(d,j＝3.8hz,1h,h-19β),0.57(d,j＝4.2hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ216.6(c-3),168.2(c-26),135.4(c-ar),135.4(c-ar),133.7(c-24),131.7(c-25),121.7(c-ar),121.6(c-ar),115.9(c-ar),115.6(c-ar),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.4(c-13),37.5(c-2),36.2(c-20),35.9(c-12,35.5(c-1),33.4(c-15),32.88(c-22),29.6(c-19),28.2(c-7),26.7(c-16),26.6(c-11),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.0(c-9),20.8(c-30),20.8(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例18

化合物5l的制备方法同实施例7，将实施例7中对氯苯胺替换为间氯苯胺即可制备得到化合物5l。yield58％.m.p.108.9～110.5℃.ms(esi)m/z563.2[m-h]^-.¹hnmr(300mhz,cdcl3)δ7.70(s,1h,h-2′),7.41(d,j＝9.1hz,1h,h-6′),7.25～7.30(d,1h,h-5′),7.16～7.13(d,1h,h-4′),7.10(s,1h,nh),5.67(t,j＝7.6hz,1h,h-24),2.81～2.64(m,1h,h-2β),2.01(s,3h,ch3-27),1.11(s,3h,ch3-29),1.06(s,3h,ch3-28),0.99(s,3h,ch3-30),0.89(t,j＝2.9hz,6h,ch3-18,ch3-21),0.79(s,1h,h-19β),0.59(d,j＝4.2hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ215.6(c-3),166.8(c-26),134.8(c-24),133.7(c-ar),130.5(c-ar),129.0(c-ar),123.4(c-25),118.9(c-ar),116.7(c-ar),112.9(c-ar),51.1(c-17),49.2(c-4),47.7(c-14),47.4(c-5),46.9(c-8),44.3(c-13),36.5(c-2),35.2(c-20),34.9(c-12),34.9(c-1),32.4(c-15),31.7(c-22),28.5(c-19),27.2(c-7),25.7(c-16),25.6(c-11),24.9(c-23),24.8(c-10),21.2(c-29),20.5(c-6),20.0(c-9),19.8(c-27),19.8(c-30),18.2(c-28),17.2(c-21),17.1(c-18).

实施例19

化合物5m的制备方法同实施例7，将实施例7中对氯苯胺替换为对溴苯胺即可制备得到化合物5m。yield64％.m.p.109.8～112.6℃.ms(esi)m/z610.3[m+h]⁺；608.3[m-h]^-.¹hnmr(300mhz,cdcl3)δ7.45(s,3h,ar-h),7.19～7.10(m,1h,ar-h),5.64(t,j＝8.8hz,1h,h-24),2.79～2.63(m,1h,h-2β),1.99(s,3h,ch3-27),1.10(s,3h,ch3-29),1.04(s,3h,ch3-28),0.98(s,3h,ch3-30),0.86(d,j＝5.7hz,6h,ch3-18,ch3-21),0.78(d,j＝4.2hz,1h,h-19β),0.57(d,j＝4.1hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ216.7(c-3),160.8(c-26),136.8(c-24),135.7(c-1′),132.0(c-3′、c-4′、c-5′、c-25),121.4(c-2′、c-6′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.8(c-8),45.4(c-13),37.5(c-2),36.2(c-20),35.9(c-12),35.5(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.2(c-7),26.8(c-16),26.6(c-11),26.0(c-10),25.9(c-23),22.2(c-29),21.5(c-6),21.0(c-9),20.8(c-30),20.8(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例20

化合物5n的制备方法同实施例7，将实施例7中对氯苯胺替换为对甲氧基苯胺即可制备得到化合物5n。yield60％.m.p.108.3～110.5℃.ms(esi)m/z560.6[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.46(d,j＝8.9hz,2h,h-2′,h-6′),7.06(s,1h，nh),6.88(d,j＝8.8hz,2h，h-3′,h-5′),5.60(t,j＝6.7hz,1h,h-24),3.80(s,3h,och3),2.71(td,j＝13.8,6.4hz,1h,h-2β),1.99(s,3h,ch3-27),1.09(s,3h,ch3-29),1.04(s,3h,ch3-28),0.98(s,3h,ch3-30),0.87(d,j＝4.6hz,6h,ch3-18,ch3-21),0.78(d,j＝3.9hz,1h,h-19β),0.57(d,j＝4.3hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ216.7(c-3),168.1(c-26),156.5(c-4′),134.7(c-24),132.0(c-1′),130.8(c-25),121.7(c-2′),121.7(c-6′),114.2(c-3′),114.2(c-3′),55.5(-och3),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.4(c-13),37.5(c-2),36.3(c-20),35.9(c-12),35.5(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.2(c-7),26.7(c-16),26.7(c-11),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.1(c-9),20.9(c-30),20.8(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例21

化合物5o的制备方法同实施例7，将实施例7中对氯苯胺替换为对硝基苯胺即可制备得到化合物5o。yield60％.m.p.112.6～114.9℃.ms(esi)m/z592.8[m+h2o]^-.¹hnmr(300mhz,cdcl3)δ7.51(dd,j＝8.7,4.8hz,2h,ar-h),7.14(s,1h,nh),7.03(t,j＝8.7hz,2h,ar-h),5.63(t,j＝6.9hz,1h,h-24),2.71(td,j＝13.8,6.4hz,1h,h-2β),1.99(s,3h,ch3-27),1.08(d,j＝7.8hz,3h,ch3-29),1.05(d,j＝6.2hz,3h,ch3-28),0.98(s,3h,ch3-30),0.86(d,j＝4.1hz,6h,ch3-18,ch3-21),0.78(d,j＝4.0hz,1h,h-19β),0.57(d,j＝4.2hz,1h,h-19α).¹³c-nmr(75mhz,cdcl3)δ216.6(c-3),168.2(c-26),147.1(c-24),135.4(c-ar),133.7(c-ar),131.7(c-25),121.7(c-ar),121.6(c-ar),115.9(c-ar),115.6(c-ar),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.4(c-13),37.5(c-2),36.2(c-20),35.9(c-12),35.5(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.2(c-7),26.7(c-16),26.6(c-11),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.0(c-9),20.8(c-30),20.8(c-27),19.3(c-28),18.2(c-21),18.1(c-18).

实施例22

化合物5p的制备方法同实施例7，将实施例7中对氯苯胺替换为对氯苄胺即可制备得到化合物5p。yield63％.m.p.112.4～115.5℃.ms(esi)m/z578.3[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.31～7.23(4h,d,j＝8.6hz,ar-h),5.76(1h,t,j＝6.0hz,h-24),5.54(1h,t,j＝7.1hz,nh),4.48(2h,t,j＝5.3hz,ch2),1.91(3h,s,ch3-27),1.10(3h,s,ch3-29),1.05(3h,s,ch3-28),0.97(3h,s,ch3-30),0.88(3h,s,ch3-18),0.82(3h,d,j＝6.4hz,ch3-21),0.79(1h,d,j＝4.1hz,h-19β),0.58(1h,d,j＝4.3hz,h-19α).¹³cnmr(75mhz,cdcl3)δ216.7(c-3),170.0(c-26),136.9(c-24),134.7(c-1′),133.4(c-25),131.2(c-4′),129.3(c-2′,c-6′),128.9(c-3′,c-5′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.3(c-13),42.8(ch2),37.5(c-2),36.2(c-20),36.0(c-12),35.5(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.1(c-7),26.7(c-16),26.5(c-11),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.1(c-9),20.9(c-30),20.8(c-27),19.3(c-28),18.1(c-18),18.1(c-21).

实施例23

化合物5q的制备方法同实施例7，将实施例7中对氯苯胺替换为2,4-二氟苄胺即可制备得到化合物5q。yield62％.m.p.121.3～124.5℃.ms(esi)m/z580.7[m+h]⁺.¹hnmr(300mhz,cdcl3)δ7.39(dd,j＝15.0,8.3hz,1h,h-3′),6.89～6.84(dd,1h,ar-h),6.81～6.77(dd,1h,ar-h),5.84(s,1h,-nh),5.52(t,j＝7.0hz,1h,h-24),4.51(d,j＝5.9hz,2h,ch2),2.71(td,j＝13.8,6.4hz,1h,h-2β),1.89(s,3h,ch3-27),1.10(s,3h,ch3-29),1.04(s,3h,ch3-28),0.97(s,3h,ch3-30),0.88(s,3h,ch3-18),0.79(d,j＝6.4hz,4h,me-21,h-19β),0.58(d,j＝4.2hz,1h,h-19α).¹³cnmr(75mhz,cdcl3)δ216.7(c-3),170.0(c-26),134.7(c-2′),134.7(c-4′),131.5(c-24),131.2(c-6′),121.5(c-25),111.6(c-1′),111.3(c-5′),103.9(c-3′),52.2(c-17),50.2(c-4),48.7(c-14),48.4(c-5),47.9(c-8),45.3(c-13),37.5(c-2),36.9(ch2),36.2(c-12),36.0(c-20),35.5(c-1),33.4(c-15),32.8(c-22),29.6(c-19),28.1(c-7),26.7(c-11),26.4(c-16),26.0(c-23),25.9(c-10),22.2(c-29),21.5(c-6),21.1(c-9),20.8(c-30),20.8(c-27),19.3(c-28),18.1(c-21),18.0(c-18).

实施例24

化合物sa-1的制备

将3ml无水二氯甲烷置于25ml单口瓶中，依次加入sa(30mg，0.07mmol)、pybop(40.1mg，0.08mmol)、diea(36.2mg，0.28mmol)、0.08mmolhobt搅拌至溶解，继续室温搅拌反应24h，减压蒸干溶剂，制备薄层[v(pe):v(ea)＝10:1]得到粗品，再用液相制备得到纯品，冷冻干燥得20mg白色固体即化合物sa-1，产率76.3％。ms(esi)m/z572.4[m+h]⁺。¹hnmr(600mhz,cdcl3)：δ0.57、0.77,(h-19),0.87(h-18),0.89(h-21),0.98(h-30),1.04(h-28),1.09(h-29),1.61(h-17),2.22(h-27),6.44(h-24),7.42(h-7’),7.46(h-8’),7.55(h-6’),8.08(h-9’).¹³cnmr(150mhz,cdcl3)δ216.7(c-23),163.0(c-26),153.4(c-24),143.6(c-4′),128.7(c-3′),128.6(c-6′),124.8(c-8′),121.5(c-25),120.6(c-9′),108.3(c-7′),52.1(c-17),29.6(c-19),22.2(c-28),20.1(c-29),20.1(c-27),19.3(c-21),18.1(c-30),18.1(c-18).

实施例25

化合物coccinicacid(ca)的制备

北五味子干燥果实，产地辽宁。

取干燥的北五味子(schisandrachinensis(turcz.)baill，)成熟果实20kg(购于湖州心良药业有限公司)，粉碎后用75％乙醇回流提取三次，每次200l溶剂。提取液浓缩，得五味子乙醇粗提物浸膏13kg(产率65％)，均匀分散于150l蒸馏水中，依次用石油醚(5l×3)、氯仿(5l×3)和正丁醇萃取(5l×3)；各有机相萃取液浓缩得石油醚部位浸膏(50g，产率0.25％)、氯仿部位浸膏(620g，产率3.1％)、正丁醇部位浸膏(2218g，产率11.09％)。将氯仿部位浸膏(620g)经反相硅胶柱层析、sephadexlh-20凝胶柱层析和c-18反相硅胶柱层析，得到coccinicacid(ca)(1.0g)。

ca:m.p.154～156℃.ms(esi)m/z[m+h]⁺455.35；477.33[m+na]⁺；453.33[m-h]^-.

¹hnmr(600mhz,cdcl3)δ6.09(t,j＝8.0hz,1h,h-24),5.29(d,j＝6.1hz,1h,h-11),2.75～2.69(ddd,j＝15.4,13.3,6.3hz,1h,h-2β),1.92(s,3h,ch3-27),1.23(s,3h,ch3-19),1.07(s,3h,ch3-29),1.07(s,3h,ch3-28),0.91(s,3h,ch3-18),0.75(s,3h,ch3-30),0.68(s,3h,ch3-21).¹³cnmr(150mhz,cdcl3)δ217.2(c-3),173.0(c-26),147.2(c-24),147.1(c-9),125.8(c-25),116.3(c-11),53.4(c-5),50.9(c-17),47.7(c-4),46.7(c-14),44.3(c-13),41.9(c-8),39.1(c-10),37.2(c-12),36.7(c-1),36.0(c-20),35.9(c-22),34.9(c-2),33.9(c-15),28.0(c-16),27.7(c-23),26.9(c-6),25.6(c-29),22.6(c-7),22.0(c-28),21.8(c-19),20.5(c-27),18.4(c-30),18.2(c-18),14.4(c-21).

光谱数据与文献(zhanghj,tangt,hoangvd,etal.naturalanti-hivagents.partiv.anti-hivconstituentsfromvaticacinerea1[j].journalofnaturalproducts,2003,66(2):263-268.)比较，与coccinicacid基本一致，可以确证为coccinicacid。

目标化合物体外抗hcvcc活性测试

一.实验药物、试剂及材料

所用试剂购自sigma公司。

1.细胞系huh7，人肝癌细胞株(详见：yimintong,yongzhezhu,xueshanxia,yuanliu,etal.tupaiacd81,sr-bi,claudin-1,andoccludinsupporthepatitiscvirusinfection,journalofvirology,2011；85(6):2793-2802；jinzhong,pablogastaminza,guofengcheng,etal.robusthepatitiscvirusinfectioninvitro，pnas,2005；102(26):9294-9299)。

2.细胞培养液配制，含10％胎牛血清、0.03％谷氨酰胺、非必需氨基酸、氨苄青霉素和链霉素100u/ml，调ph至7.4。

3.细胞消化液配制，含0.25％胰蛋白酶，用磷酸缓冲液配制。

4.hcvcc：细胞培养的感染性丙型肝炎病毒(详见：yimintong,yongzhezhu,xueshanxia,yuanliu,etal.tupaiacd81,sr-bi,claudin-1,andoccludinsupporthepatitiscvirusinfection,journalofvirology,mar.2011；85(6):2793-2802；jinzhong,pablogastaminza,guofengcheng,etal.robusthepatitiscvirusinfectioninvitro,pnas,2005；102(26):9294-9299)。

二、实验方法

(一)hcvcc的制备

1.病毒扩增

hcvcc病毒(丙肝病毒)来源：日本重症肝炎1型(japanesefulminanthepatitistype1,jfh-1)基因的质粒是由日本东京国立传染性研究所的wakita友情提供，并用来表达得到jfh-1hcvcc。

jfh-1嵌合hcvcc(10⁵ffu/ml)，取50μl感染接种于24孔板的huh7.5细胞，次日换液，随后根据细胞生长密度传代培养，观察细胞生长状态，待病毒快速增殖导致的细胞病变效应(cytopathiceffect，cpe)出现后，收集第7-20天的培养上清，取0.1ml用于病毒滴度测定，其余8000rpm离心5min弃细胞碎片后分装保存于-70℃备用。

2.病毒滴定

以空白huh7.5(1×10⁴cells/孔)、12h，弃培养上清，每孔加入100μl经10倍梯度稀释的hcvcc上清液，共孵育5h，换新鲜dmem全培养液继续培养72h，行免疫荧光法检测hcv阳性细胞，一抗用1:100稀释的hcv抗体阳性病人血清，二抗为1:100稀释的fitc标记羊抗人igg。在荧光显微镜下观察发光细胞，并记录最后一个可观察到绿色荧光阳性细胞的孔内绿色荧光阳性细胞数及相应的稀释梯度，计算出focusformingunit/ml(ffu/ml)数值，以此代表hcvcc滴度。

(二)hcvcc感染性的检测

取处于对数生长期的huh7细胞，调整细胞浓度为1×10⁵个/ml，取100μl种96孔板；培养24h后加入待测化合物及hcvcc，化合物按0、10、25、50、100μg/ml浓度稀释，37℃培养4h后去除化合物及hcvcc混液，换培养基继续培养；48h后进行免疫荧光检测，在荧光显微镜下读取各孔hcvcc阳性克隆数。

本发明实施例制备的化合物体外抗hcvcc活性结果如表1所示，图1是本发明实施例制备的化合物抗hcv的ic50值的柱状图，从表1和图1中可以看出，大部分表现出有较好的抗hcv活性，其中3、5c、5e、5g、5h、5p、5q和sa-1共8个衍生物及aa和ca活性均优于甘五酸，并且化合物3、5c、5e、5h、5q和sa-1抗hcv活性约为甘五酸的3倍或更高，其中sa-1的ic50达7.80μg/ml；5b、5f和5i三个衍生物活性弱于sa。

进一步采用cck-8法检测化合物对人肝癌huh7细胞毒性，实验结果显示(图2)，具有较好抗hcv活性的化合物3、5c、5e、5g、5h、5p、5q、sa-1和ca的细胞毒性(cc50)均低于甘五酸sa，具有较低的细胞毒性，说明这些化合物比sa具有更好的抗hcv活性和安全性。图2是化合物3、5c、5e、5g、5h、5p、5q、sa-1和ca的体外细胞毒性活性结果。

表1

以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专利的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。