一种双氢青蒿素衍生物、制备方法、药物组合物和其在制备抗肿瘤药物中的应用

1.本发明涉及医药技术领域，具体地说，涉及一种双氢青蒿素衍生物、制备方法、药物组合物和其在制备抗肿瘤药物中的应用。


背景技术：

2.癌症是继缺血性心脏病之后世界第二大死亡原因(897万人)，2060年后有可能成为世界第一大死亡原因(约1863万人)。据统计，2018年全球新增确诊病例1800万例，2020年新增病例还在持续上升。
3.双氢青蒿素是一种传统的一线抗疟疾的药物，近年来发现其除了具有抗疟疾的活性外还具有抗肿瘤活性。但是其发挥抗肿瘤作用的剂量远远高于其发挥抗疟疾的剂量，并且此类化合物的神经毒性问题一直是限制此类化合物应用的主要原因。开发有较高生物活性和较低副作用的双氢青蒿素类抗癌药物非常重要和迫切。
4.双氢青霉素衍生物的合成及抗癌活性研究，朴明男，延边大学硕士学位论文，2011年，公开了：在青蒿素的抗肿瘤作用机制研究中，认为最主要的机制为诱导细胞凋亡和亚铁离子作用过氧键基团，反应产生氧自由基损伤癌细胞膜性结构，但是确切的作用靶点和机制并不明确。
5.为了寻找比青蒿素抗肿瘤作用更强的药物，在保留过氧桥键的基础上，主要在双氢青蒿素10位碳上的-oh键引入一些亲水、亲脂基团进行结构修饰，合成了烷基醚、芳香醚、酯类、杂原子取代、二聚体化合物等青蒿素衍生物，并筛选出部分烷基醚、芳香醚类和部分双活性二聚体化合物比青蒿素具有更强的抗肿瘤活性。
6.线粒体是细胞内重要的亚细胞器，其与细胞的死亡密切相关。目前，靶向线粒体的药物研发已经成为该领域的研究热点。由于肿瘤细胞线粒体内膜电位比正常细胞更高，线粒体靶向基团tpp

可以依据线粒体膜电势的差异积聚在肿瘤细胞线粒体内，一般认为，靶向基团只会提高药物活性成分的靶向能力，对体外抗肿瘤细胞活性的提升并不会有明显影响。
7.synthesis and biological activities of novel mitochondria-targeted artemisinin ester derivatives，cangcang，xu，bioorg.med.chem.lett.39(2021)127912，发明人将双氢青蒿素和反应得到产物，制备路线如下所示。产物2a-2d对各肿瘤细胞的ic
50
值可以最低达到2.7μm，但是2d相比于dha，其抗肿瘤活性的提升并不明显。
8.

技术实现要素：

9.本发明的目的是针对现有技术中的不足，提供一类双氢青蒿素衍生物、制备方法、药物组合物和其在制备抗肿瘤药物中的应用，双氢青蒿素衍生物的抗肿瘤活性较高，其ic
50
值达到nm级别，具有开发为良好抗肿瘤药物的应用前景。
10.本发明提供一种双氢青蒿素衍生物，所述双氢青蒿素衍生物的结构为如下式ⅰ所示：
[0011][0012]
其中，所述n为3-12，r1为三苯基膦，r2为卤素，比如溴、氯、碘，优选为溴，其他卤素元素如氯、碘取代也具有类似的性能。
[0013]
具体的，所述双氢青蒿素衍生物为以下化合物之一：
[0014][0015]
本发明优选的化合物为d7-t和d8-t，最优选的为化合物d8-t。
[0016]
优选的，所述双氢青蒿素衍生物为式ⅰ化合物的各光学异构体、各晶型、药学上可接受的无机盐、有机盐或前药。
[0017]
本发明的盐可以由化合物中带正电荷的部分与具有相反电性的带负电荷形成；或者由化合物中带负电荷的部分与正电荷形成。
[0018]
所述的“结构式ⅰ的前药”通常指一种物质，当用适当的方法施用后，可在受试者体内进行代谢或化学反应而转变成结构式ⅰ的至少一种化合物或其盐。
[0019]
本发明的化合物可按照常规方法制备为药用盐的形式；包括其有机酸盐及无机酸盐：无机酸包括(但不限于)盐酸、硫酸、磷酸、二磷酸、氢溴酸、硝酸等，有机酸包括(但不限于)乙酸、马来酸、富马酸、酒石酸、琥珀酸、乳酸、对甲苯磺酸、水杨酸、草酸等。
[0020]
本发明提供一种双氢青蒿素衍生物的制备方法，步骤为，将双氢青蒿素和反应，n为3-12，r2为卤素，得到中间体，中间体的结构式如下：中间体和三苯基膦反应，得到双氢青蒿素衍生物。
[0021]
优选反应路线如下：
[0022][0023]
优选的，所述双氢青蒿素和反应的溶剂为二氯甲烷，催化剂为三氟化硼乙醚，优选在氮气保护下进行。
[0024]
优选的，中间体和三苯基膦反应的溶剂为乙氰，反应体系中还加入有碳酸盐，如碳酸钾或碳酸钠，其作为碱催化剂。
[0025]
本发明提供一种药物组合物，所述的药物组合物包括双氢青蒿素衍生物，还包括药学上可接受的赋形剂或载体。
[0026]
本发明提供一种双氢青蒿素衍生物在制备抗肿瘤药物中的用途。所述肿瘤为肺癌、食道癌、胃癌、肝癌、黑色素瘤、胰腺癌、肾癌、白血病、前列腺癌、膀胱癌、神经母细胞瘤、结肠癌、乳腺癌、子宫癌、卵巢癌或鼻咽癌。
[0027]
本发明的有益效果是，本发明的实验结果显示双氢青蒿素衍生物能显著抑制肿瘤细胞的增殖和迁移，ic
50
大大低于衍生化前的双氢青蒿素，其中以化合物d7-t、d8-t的活性最好，在j82、ovcar3和a549细胞中，本发明的双氢青蒿素衍生物活性是双氢青蒿素的2366、874和220倍，具有开发为良好抗肿瘤药物的应用前景。
附图说明
[0028]
图1为d3-t～d12-t在5种癌细胞株中的半数抑制浓度(ic
50
)。
[0029]
图2为d8-t和dha对人膀胱癌细胞系t24和j82的克隆形成能力抑制效果染色图。
[0030]
图3为d8-t和dha对人膀胱癌细胞系t24和j82的克隆形成能力抑制效果柱状图。
[0031]
图4为d8-t对人膀胱癌细胞系t24和j82迁移的抑制效果图。
[0032]
图5为dha对人膀胱癌细胞系t24和j82迁移的抑制效果图。
具体实施方式
[0033]
以下具体的实例用以详细说明本发明，但本发明绝非仅限于这些例子(除另有注明外，所用原料均有市售)。
[0034]
实施例1：中间体d3的制备
[0035][0036]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入3-溴-1-丙醇(0.325ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d3，产率12.93％。
[0037]
结构验证：
[0038]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.42(s,1h),4.80(d,j＝3.5hz,1h),4.00(ddd,j＝10.0,6.6,5.1hz,1h),3.49(q,j＝6.4hz,3h),2.64(qdd,j＝7.5,4.7,3.5hz,1h),2.37(ddd,j＝14.7,13.5,4.0hz,1h),2.15
–
1.99(m,3h),1.88(dddd,j＝13.7,6.8,4.0,3.1hz,1h),1.80
–
1.69(m,2h),1.63(dq,j＝13.2,3.4hz,1h),1.55
–
1.44(m,2h),1.44(s,3h),1.33(qp,j＝9.7,3.7,3.3hz,1h),1.28
–
1.21(m,2h),0.95(d,j＝6.4hz,3h),0.90(d,j＝7.4hz,3h).
13
cnmr(150mhz,chloroform-d)δ104.12,102.11,87.93,81.05,65.70,52.58,44.39,37.46,36.43,34.64,32.55,30.90,30.61,26.20,24.67,24.52,20.37,12.98.esi-ms m/z:c
18h29
bro
5 427.2(m na)

:429.2(m 2 na)

＝1:1。
[0039]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.34(s,1h),4.44(d,j＝9.2hz,1h),4.07(dt,j＝10.1,5.1hz,1h),3.64
–
3.47(m,3h),2.46
–
2.32(m,2h),2.21(ddq,j＝16.7,8.5,5.6hz,1h),2.09
–
1.96(m,2h),1.88(ddt,j＝13.6,6.8,3.5hz,1h),1.76(dq,j＝13.6,3.7hz,1h),1.72
–
1.66(m,1h),1.57
–
1.46(m,2h),1.43(s,3h),1.36
–
1.26(m,3h),1.04
–
0.98(m,1h),0.96(dd,j＝6.8,4.7hz,3h),0.89(d,j＝7.1hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.29,100.42,91.21,80.35,66.45,51.65,45.33,37.39,36.32,34.24,32.78,32.67,30.83,26.06,24.72,22.21,20.29,12.58.esi-ms m/z:c
18h29
bro
5 427.2(m na)

:429.2(m 2 na)

＝1:1。
[0040]
实施例2：中间体d4的制备
[0041][0042]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入4-溴-1-丁醇(0.328ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d4，产率10.09％。
[0043]
结构验证：
[0044]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.37(s,1h),4.77(d,j＝3.5hz,1h),3.88(dt,j＝9.8,6.2hz,1h),3.50
–
3.35(m,3h),2.69
–
2.57(m,1h),2.36(ddd,j＝14.7,13.5,4.0hz,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.97
–
1.84(m,3h),1.79
–
1.68(m,4h),1.63(dq,j＝13.3,3.4hz,1h),1.54
–
1.44(m,2h),1.43(s,3h),1.33(ttd,j＝12.4,6.3,4.0hz,1h),1.28
–
1.15(m,2h),0.95(d,j＝6.4hz,3h),0.90(d,j＝7.4hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.10,102.01,87.92,81.10,67.44,52.58,44.42,37.49,36.44,34.64,33.63,30.90,29.82,28.34,26.21,24.68,24.50,20.37,13.04.esi-ms m/z:c
19h31
bro
5 441.2(m na)

:443.2(m 2 na)

＝1:1。
[0045]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.44(s,1h),4.96(d,j＝5.2hz,1h),3.91(dt,j＝9.8,6.3hz,1h),3.51(dt,j＝9.9,6.4hz,1h),3.45(t,j＝6.8hz,2h),2.30(td,j＝14.0,3.9hz,1h),2.02(ddd,j＝14.5,4.8,3.0hz,1h),1.97(dq,j＝9.7,7.0hz,2h),1.90(ddq,j＝13.5,6.6,3.5hz,1h),1.76(p,j＝6.9hz,2h),1.72
–
1.61(m,3h),1.55
–
1.47(m,2h),1.47
–
1.42(m,2h),1.42(s,3h),1.18(d,j＝7.2hz,3h),0.99(td,j＝12.3,11.7,4.1hz,1h),0.94(d,j＝5.9hz,3h),0.93
–
0.86(m,1h).
13
c nmr(150mhz,chloroform-d)δ103.01,102.78,89.17,81.67,67.77,51.86,46.55,39.89,37.32,36.53,34.42,33.71,31.64,29.74,28.34,25.98,24.69,20.07,19.51.esi-ms m/z:c
19h31
bro
5 441.2(m na)

:443.2(m 2 na)

＝1:1。
[0046]
实施例3：中间体d5的制备
[0047][0048]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入5-溴-1-戊醇(0.438ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d5，产率19.72％。
[0049]
结构验证：
[0050]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.39(s,1h),4.77(d,j＝3.4hz,1h),3.85(dt,j＝9.8,6.3hz,1h),3.47
–
3.32(m,3h),2.68
–
2.52(m,1h),2.42
–
2.30(m,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.91
–
1.84(m,3h),1.81(dd,j＝13.9,3.6hz,1h),1.75(tt,j＝14.3,3.8hz,1h),1.66
–
1.55(m,4h),1.54
–
1.45(m,4h),1.43(s,3h),1.42
–
1.28(m,3h),1.28
–
1.16(m,1h),0.95(d,j＝6.4hz,3h),0.90(d,j＝7.8hz 3h).
13
c nmr(150mhz,chloroform-d)δ104.08,102.01,87.93,81.15,68.03,52.59,44.46,37.47,36.45,34.68,33.84,32.43,30.94,28.84,26.23,24.98,24.69,24.50,20.37,13.05.esi-ms m/z:c
20h33
bro
5 455.2(m na)

:457.2(m 2 na)

＝1:1。
[0051]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.44(s,1h),4.95(d,j＝5.0hz,1h),3.87(dt,j＝9.8,6.5hz,1h),3.48(dt,j＝9.8,6.5hz,1h),3.41(t,j＝6.8hz,2h),2.30(ddd,j＝14.6,13.4,3.9hz,1h),2.01(ddd,j＝14.6,4.8,3.1hz,1h),1.89(ddt,j＝13.9,9.7,5.3hz,3h),1.71(qd,j＝13.4,3.4hz,2h),1.63(ddt,j＝12.0,8.2,4.8hz,4h),1.57(dt,j＝13.3,3.5hz,2h),1.54
–
1.49(m,3h),1.41(s,3h),1.18(d,j＝7.3hz,3h),1.00
–
0.96(m,1h),0.94(d,j＝6.1hz,3h),0.90
–
0.85(m,1h).
13
c nmr(150mhz,chloroform-d)δ103.04,102.80,89.10,81.66,68.40,51.89,46.52,39.82,37.30,36.53,34.44,33.78,32.54,31.61,28.83,25.98,24.92,24.69,20.07,19.54.esi-ms m/z:c
20h33
bro
5 455.2(m na)

:457.2(m 2 na)

＝1:1。
[0052]
实施例4：中间产物d6的制备
[0053][0054]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入6-溴-1-己醇(0.471ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d6，产率13.10％。
[0055]
结构验证：
[0056]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.38(s,1h),4.77(d,j＝3.5hz,1h),3.83(dt,j＝9.7,6.5hz,1h),3.40(t,j＝6.8hz,2h),3.36(dd,j＝9.7,6.4hz,1h),2.61(qt,j＝7.4,4.1hz,1h),2.40
–
2.32(m,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.90
–
1.83(m,3h),1.80(dd,j＝13.7,3.6hz,1h),1.75(tq,j＝12.0,3.9hz,1h),1.66
–
1.59(m,1h),1.58(d,j＝4.2hz,2h),1.56
–
1.45(m,4h),1.44(s,3h),1.42
–
1.28(m,4h),1.24
–
1.16(m,1h),0.95(d,j＝6.3hz,3h),0.90(d,j＝7.3hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.07,101.99,87.92,81.15,68.24,52.61,44.48,37.51,36.46,34.68,33.80,32.73,30.95,29.49,27.89,26.24,25.46,24.70,24.49,20.39,13.05.esi-ms m/z:c21h35bro5 469.3(m na)

:471.3(m 2 na)

＝1:1。
[0057]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.33(s,1h),4.41(d,j＝9.2hz,1h),3.97(dt,j＝9.6,6.3hz,1h),3.47
–
3.35(m,3h),2.49
–
2.28(m,2h),2.02(ddd,j＝14.6,5.0,3.0hz,1h),1.87(h,j＝6.8hz,3h),1.76(dq,j＝13.5,3.8hz,1h),1.66(dddd,j＝33.0,15.3,7.8,4.7hz,2h),1.57
–
1.45(m,4h),1.44(s,4h),1.42
–
1.27(m,5h),1.01(td,j＝12.7,12.2,3.2hz,1h),0.95(d,j＝6.3hz,3h),0.88(d,j＝7.1hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.25,100.12,91.21,80.35,68.86,51.69,45.35,37.39,36.35,34.27,33.94,32.75,32.64,29.37,27.93,26.08,25.21,24.72,22.22,20.30,12.65.esi-ms m/z:c
21h35
bro
5 469.3(m na)

:471.3(m 2 na)

＝1:1。
[0058]
实施例5：中间产物d7的制备
[0059][0060]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入7-溴-1-庚醇(0.553ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d7，产率13.29％。
[0061]
结构验证：
[0062]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.38(s,1h),4.77(dd,j＝3.5,1.0hz,1h),3.82(dt,j＝9.6,6.6hz,1h),3.42
–
3.30(m,3h),2.67
–
2.51(m,1h),2.40
–
2.32(m,1h),2.07
–
1.99(m,1h),1.90
–
1.71(m,5h),1.63(dq,j＝13.1,3.3hz,1h),1.58
–
1.44(m,5h),1.44(s,3h),1.42
–
1.40(m,1h),1.39
–
1.29(m,6h),1.24
–
1.17(m,1h),0.95(d,j＝6.3hz,3h),0.90(d,j＝7.4hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.06,101.97,87.91,81.16,68.32,52.61,44.50,37.51,36.46,34.68,33.95,32.74,30.95,29.55,28.47,28.13,26.24,26.08,24.70,24.49,20.40,13.05.esi-ms m/z:c
22h37
bro
5 483.1724(m na)

:485.1708(m 2 na)

＝1:1。
[0063]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.33(s,1h),4.41(d,j＝9.2hz,1h),4.04
–
3.88(m,1h),3.45
–
3.29(m,3h),2.46
–
2.33(m,2h),2.02(ddd,j＝14.6,5.0,3.0hz,1h),1.86(tt,j＝14.5,7.0hz,3h),1.76(dq,j＝13.6,3.8hz,1h),1.68(dq,j＝13.3,3.4hz,1h),1.60
–
1.45(m,4h),1.44(s,3h),1.42
–
1.27(m,9h),1.04
–
0.97(m,1h),0.95(d,j＝6.3hz,3h),0.88(d,j＝7.1hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.25,100.12,91.21,80.36,69.02,51.69,45.35,37.39,36.35,34.27,34.01,32.76,32.64,29.45,28.54,28.12,26.08,25.83,24.73,22.23,20.30,12.65.esi-ms m/z:c
22h37
bro
5 483.1724(m na)

:485.1708(m 2 na)

＝1:1。
[0064]
实施例6：中间产物d8的制备
[0065][0066]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入8-溴-1-辛醇(0.617ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d8，产率19.07％。
[0067]
结构验证：
[0068]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.38(s,1h),4.76(d,j＝3.5hz,1h),3.83(ddt,j＝22.6,9.7,6.6hz,1h),3.39(td,j＝6.9,1.7hz,2h),3.35(dt,j＝9.7,6.5hz,1h),2.60(qt,j＝7.4,4.0hz,1h),2.36(td,j＝14.0,4.0hz,1h),2.02(ddd,j＝14.7,5.2,3.2hz,1h),1.84(ddd,j＝14.4,8.2,5.5hz,3h),1.81
–
1.67(m,2h),1.64(t,j＝3.6hz,1h),1.61
–
1.51(m,3h),1.51
–
1.44(m,2h),1.43(s,3h),1.32(dq,j＝8.7,5.6,4.5hz,9h),1.18(d,j＝7.2hz,1h),0.95(d,j＝6.3hz,3h),0.89(d,j＝7.3hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.04,101.95,87.91,81.16,68.39,52.61,44.50,37.50,36.46,34.70,33.98,32.80,30.95,29.62,29.13,28.72,28.09,26.24,26.15,24.70,24.48,20.40,13.05.esi-ms m/z:c
23h39
bro
5 497.3(m na)

:499.3(m 2 na)

＝1:1。
[0069]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.32(s,1h),4.41(d,j＝9.2hz,1h),4.02
–
3.89(m,1h),3.40(t,j＝6.9hz,3h),2.48
–
2.31(m,2h),2.01(ddd,j＝14.6,5.0,3.0hz,1h),1.91
–
1.81(m,3h),1.75(dq,j＝13.6,3.9hz,1h),1.71
–
1.64(m,2h),1.63
–
1.45(m,5h),1.43(s,3h),1.42
–
1.36(m,2h),1.31(dd,j＝5.8,3.3hz,5h),1.27(d,j＝6.9hz,1h),1.00(td,j＝12.7,12.3,3.6hz,1h),0.95(d,j＝6.2hz,3h),0.93
–
0.89(m,1h),0.87(d,j＝7.1hz,3h).
13
cnmr(150mhz,chloroform-d)δ104.23,100.10,91.20,80.35,69.09,51.69,45.35,37.38,36.35,34.27,34.04,33.99,32.81,32.76,29.50,29.19,28.70,28.12,25.90,24.72,22.22,20.30,12.64.esi-ms m/z:c
23h39
bro
5 497.3(m na)

:499.3(m 2 na)

＝1:1。
[0070]
实施例7：中间产物d9的制备
[0071][0072]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入9-溴-1-壬醇(0.663ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d9，产率15.14％。
[0073]
结构验证：
[0074]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.39(s,1h),4.77(d,j＝3.5hz,1h),3.82(dt,j＝9.6,6.6hz,1h),3.49
–
3.33(m,3h),2.61(qdd,j＝7.4,4.6,3.5hz,1h),2.41
–
2.25(m,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.92
–
1.77(m,4h),1.74(dq,j＝14.3,4.0hz,1h),1.63(dq,j＝13.1,3.4hz,1h),1.57
–
1.45(m,4h),1.44(s,3h),1.42(d,j＝9.8hz,2h),1.38
–
1.27(m,10h),1.24
–
1.17(m,1h),0.95(d,j＝6.3hz,3h),0.89(d,j＝7.4hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.05,101.95,87.91,81.17,68.41,52.62,44.51,37.50,36.47,34.70,34.02,32.84,30.96,29.64,29.40,29.22,28.71,28.18,26.25,26.20,24.71,24.47,20.40,13.05.esi-ms m/z:c
24h41
bro
5 511.4(m na)

:513.3(m 2 na)

＝1:1。
[0075]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.33(s,1h),4.41(d,j＝9.2hz,1h),3.95(ddd,j＝9.6,7.0,5.8hz,1h),3.47
–
3.31(m,3h),2.49
–
2.30(m,2h),2.06
–
1.99(m,1h),1.95
–
1.87(m,1h),1.87
–
1.81(m,4h),1.77(tq,j＝13.6,3.8hz,1h),1.68(dq,j＝13.4,3.4hz,1h),1.58
–
1.46(m,4h),1.44(s,3h),1.41(q,j＝6.2,5.4hz,3h),1.31
–
1.29(m,6h),1.22
–
1.14(m,1h),1.08
–
0.98(m,2h),0.95(d,j＝6.3hz,3h),0.88(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.24,100.10,91.20,80.35,69.14,51.69,45.35,37.39,36.35,34.27,34.06,32.84,32.65,29.53,29.37,29.29,28.71,28.16,26.08,25.95,24.73,22.23,20.30,12.64.esi-ms m/z:c
24h41
bro
5 511.4(m na)

:513.3(m 2 na)

＝1:1。
[0076]
实施例8：中间产物d10的制备
[0077][0078]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入10-溴-1-葵醇(0.719ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d10，产率6.77％。
[0079]
结构验证：
[0080]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.41(s,1h),4.80(d,j＝3.6hz,1h),3.86(ddt,j＝21.0,9.6,6.6hz,1h),3.52
–
3.36(m,3h),2.63(qdd,j＝7.4,4.6,3.5hz,1h),2.36(dddd,j＝37.8,14.6,13.5,3.9hz,1h),2.05(dtd,j＝14.1,5.4,3.1hz,1h),1.95
–
1.80(m,4h),1.80
–
1.69(m,1h),1.65(dp,j＝12.8,2.9hz,1h),1.60
–
1.47(m,4h),1.46(d,j＝9.2hz,3h),1.43(d,j＝8.9hz,1h),1.32(d,j＝9.8hz,12h),1.27
–
1.18(m,2h),0.98(d,j＝6.2hz,3h),0.92(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.04,101.94,87.91,81.17,68.42,52.62,44.51,37.50,36.47,34.71,34.04,32.84,30.96,29.65,29.49,29.37,29.28,28.77,28.19,26.25,26.22,24.71,24.47,20.40,13.05.esi-ms m/z:c
25h43
bro
5 525.4(m na)

:527.4(m 2 na)

＝1:1。
[0081]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.33(s,1h),4.41(d,j＝9.2hz,1h),
3.95(ddd,j＝9.5,7.1,5.8hz,1h),3.47
–
3.27(m,3h),2.46
–
2.29(m,2h),2.02(ddd,j＝14.5,4.9,3.0hz,1h),1.91
–
1.81(m,3h),1.76(dq,j＝13.5,3.7hz,1h),1.68(dq,j＝13.4,3.4hz,1h),1.59
–
1.46(m,4h),1.44(s,3h),1.43
–
1.39(m,2h),1.37
–
1.26(m,13h),1.10(s,1h),1.05
–
0.98(m,1h),0.95(d,j＝6.3hz,3h),0.88(d,j＝7.8hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.24,100.10,91.20,80.36,69.18,51.69,45.36,37.39,36.36,34.28,34.08,32.85,32.65,29.71,29.55,29.46,29.37,28.75,28.18,26.08,25.98,24.73,22.23,20.30,12.64.esi-ms m/z:c
25h43
bro
5 525.4(m na)

:527.4(m 2 na)

＝1:1。
[0082]
实施例9：中间产物d11的制备
[0083][0084]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入11-溴-1-十一醇(0.9g，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d11，产率12.14％。
[0085]
结构验证：
[0086]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.38(s,1h),4.77(d,j＝3.5hz,1h),3.81(dt,j＝9.6,6.6hz,1h),3.42
–
3.32(m,3h),2.68
–
2.56(m,1h),2.36(ddd,j＝14.6,13.5,4.0hz,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.92
–
1.80(m,4h),1.73(dq,j＝14.4,3.9hz,1h),1.64
–
1.59(m,1h),1.58
–
1.45(m,4h),1.43(s,3h),1.42
–
1.39(m,2h),1.35
–
1.23(m,15h),0.95(d,j＝6.3hz,3h),0.89(d,j＝7.4hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.05,101.94,87.91,81.18,68.44,52.61,44.51,37.49,36.47,34.70,34.06,32.84,30.96,29.71,29.65,29.56,29.47,29.44,29.31,28.79,28.19,26.24,24.70,24.47,20.40,13.05.esi-ms m/z:c
26h45
bro5539.2347(m na)

:541.2331(m 2 na)

＝1:1。
[0087]
实施例10：中间产物d12的制备
[0088][0089]
将dha(1g，3.5mmol)溶于200ml二氯甲烷中配成溶液，在室温条件下加入12-溴-1-十二醇(0.855ml，3.6mmol)，再加入4滴三氟化硼乙醚，n2保护，室温反应12h，tlc监测反应。反应结束，加入200ml饱和碳酸氢钠、二氯甲烷萃取三次，再加入无水碳酸钠干燥，过滤拌样，柱层析得中间产物d12，产率13.12％。
[0090]
结构验证：
[0091]
β-isomer；1h nmr(600mhz,chloroform-d)δ5.39(s,1h),4.77(d,j＝2.5hz,1h),3.82(dt,j＝9.6,6.6hz,1h),3.40(t,j＝6.9hz,2h),3.36(dt,j＝9.7,6.4hz,1h),2.65
–
2.55(m,1h),2.37(ddd,j＝14.6,13.5,4.0hz,1h),2.03(ddd,j＝14.6,5.0,3.0hz,1h),1.92
–
1.78(m,4h),1.74(dq,j＝14.2,3.8hz,1h),1.62(dq,j＝13.2,3.4hz,1h),1.56
–
1.44(m,4h),1.44(s,3h),1.43
–
1.39(m,2h),1.36
–
1.24(m,17h),0.95(d,j＝6.4hz,3h),0.90(d,j＝7.4hz,3h).
13
cnmr(150mhz,chloroform-d)δ104.04,101.94,87.92,81.18,68.45,52.62,44.52,37.49,36.47,34.71,34.07,32.85,30.97,29.66,29.60,29.55(
×
2),29.46,29.33,28.79,28.19,26.25(
×
2),24.71,24.47,20.40,13.05.esi-ms m/z:c
27h47
bro
5 553.4(m na)

:555.4(m 2 na)

＝1:1。
[0092]
α-isomer；1h nmr(600mhz,chloroform-d)δ5.33(s,1h),4.41(d,j＝9.2hz,1h),3.98
–
3.85(m,1h),3.44
–
3.30(m,3h),2.45
–
2.31(m,2h),2.02(ddd,j＝14.5,4.9,2.9hz,1h),1.90
–
1.82(m,3h),1.76(dq,j＝13.6,3.7hz,1h),1.68(dq,j＝13.4,3.5hz,1h),1.60
–
1.46(m,5h),1.44(s,3h),1.43
–
1.39(m,2h),1.38
–
1.27(m,13h),1.24
–
1.09(m,2h),1.03
–
0.97(m,1h),0.95(d,j＝6.3hz,3h),0.91(ddd,j＝9.3,5.3,2.6hz,1h),0.88(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ104.24,100.10,91.20,80.36,69.22,51.69,45.36,37.39,36.36,34.28,34.09,32.86,32.65,29.57(
×
2),29.54,29.52,29.44,29.42,28.78,28.19,26.08,26.00,24.73,22.23,20.30,12.64.esi-ms m/z:c
27h47
bro
5 553.4(m na)

:555.4(m 2 na)

＝1:1。
[0093]
实施例11：化合物d3-t的制备
[0094][0095]
将中间体d3(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d3-t，产率26.19％。
[0096]
结构验证：
[0097]
β-isomer；1h nmr(600mhz,chloroform-d)δ7.85
–
7.77(m,9h),7.69(td,j＝7.8,3.4hz,6h),5.26(s,1h),4.72(d,j＝3.5hz,1h),4.04
–
3.87(m,2h),3.82
–
3.67(m,2h),2.57(qt,j＝7.4,4.1hz,1h),2.32(td,j＝14.0,3.9hz,1h),1.99(ddd,j＝14.6,4.9,3.0hz,2h),1.96
–
1.90(m,2h),1.84(ddt,j＝13.5,6.7,3.5hz,1h),1.73
–
1.48(m,4h),1.44
–
1.38(m,2h),1.36(s,3h),1.19(td,j＝11.0,6.2hz,1h),0.92(d,j＝6.0hz,3h),0.83(d,j＝7.3hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.13(d,j＝3.0hz,ph-c),133.67(d,j＝9.0hz,ph-c),130.55(d,j＝12.0hz,ph-c),118.18(d,j＝85.5hz,ph-c),104.11,101.78,87.78,80.88,67.06,66.92,52.42,44.20,37.35,36.33,34.56,30.79,29.68,26.09,24.61(d,j＝7.5hz,ph-c),23.11(d,j＝3.0hz,ph-c),20.34,13.19.esi-ms m/z c
36h44
o5p

587.4(m-br)

。
[0098]
实施例12：化合物d4-t的制备
[0099][0100]
将中间体d4(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d4-t，产率21.09％。
[0101]
结构验证：
[0102]
α：β＝5:4；1h nmr(600mhz,chloroform-d)δ7.88
–
7.75(m,9h),7.72
–
7.66(m,6h),5.31/5.24(s,1h),4.66/4.44(d,j＝3.5hz and j＝9.2hz,1h),4.06
–
3.95/3.94
–
3.88(m,1h),3.82
–
3.74(m,3h),3.61
–
3.53/3.37
–
3.33(m,1h),2.79
–
2.59(m,1h),2.53(tdd,j＝10.0,6.7,3.3hz,1h),2.43
–
2.16(m,5h),2.04
–
1.93(m,3h),1.79
–
1.65(m,2h),1.58
–
1.44(m,3h),1.40/1.39(s,3h),0.93/0.91(d,j＝6.0hz,3h),0.75/0.69(d,j＝3hz,3h).
13
c nmr(150mhz,chloroform-d)δ134.97/134.92(d,j＝3.0hz,ph-c),133.81/133.72/133.69(d,j＝9.0hz,ph-c),130.48/130.46(d,j＝12.0hz,ph-c),118.39/118.33(d,j＝85.5hz,ph-c),104.07/103.99,101.88/99.84,91.20/87.80,80.96/80.59,67.53/66.66,59.90/59.52,52.46/51.76,45.41/45.31,38.15,37.36/37.28,36.38/36.27,34.51/34.19,32.96,31.24/30.77,26.16/25.70,24.65/24.42,22.69,20.37/20.28,13.01/12.63.esi-ms m/z c
37h46
o5p

601.4(m-br)

。
[0103]
实施例13：化合物d5-t的制备
[0104][0105]
将中间体d5(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d5-t，产率43.12％。
[0106]
结构验证：
[0107]
α：β＝1:5；1h nmr(600mhz,chloroform-d)δ7.85
–
7.82/7.68 7.63(m,6h),7.78
–
7.76/7.56
–
7.52(m,3h),7.71
–
7.70/7.48
–
7.43(m,6h),5.32/5.37(s,1h),4.67/4.88(d,j＝3.6hz and 5.4hz,1h),3.84
–
3.77(m,2h),3.72(dt,j＝9.7,6.4hz,2h),3.28(dt,j＝9.7,6.5hz,1h),2.61
–
2.50(m,1h),2.34(td,j＝14.0,4.0hz,2h),2.00(ddd,j＝14.5,4.9,3.1hz,2h),1.85(ddd,j＝10.1,6.4,3.2hz,2h),1.54
–
1.41(m,4h),1.39/1.40(s,3h),1.34
–
1.27(m,3h),1.22
–
1.12(m,3h),0.93(d,j＝6.3hz,3h),0.80(d,j＝7.3hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.00(d,j＝3.0hz,ph-c),133.77(d,j＝9.0hz,ph-c),133.70(d,j＝7.5hz,ph-c),132.09(d,j＝9.0hz,ph-c),131.97,131.96,130.51/128.51(d,j＝12.0hz,ph-c),118.43(d,j＝85.5hz,ph-c),104.04,101.98/102.15,87.90/88.30,
81.12/80.53,67.89,59.52,55.62/52.55,44.41,38.15,37.40,36.43,34.60,31.93/31.24,30.87,29.16,26.19,24.67,24.46,20.37,14.12/13.09.esi-ms m/zc
38h48
o5p

615.5(m-br)

。
[0108]
实施例14：化合物d6-t的制备
[0109][0110]
将中间体d6(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d6-t，产率25.52％
[0111]
结构验证：
[0112]
α：β＝1:1；1h nmr(600mhz,chloroform-d)δ7.85(dddd,j＝14.0,12.5,8.4,1.3hz,6h),7.78(tt,j＝7.4,1.6hz,3h),7.70(ddt,j＝11.0,7.4,3.3hz,6h),5.33/5.31(s,1h),4.70/4.38(d,j＝3.5hz and 9.2hz,1h),3.93
–
3.69(m,3h),3.38
–
3.24(m,1h),2.38
–
2.29(m,1h),2.04
–
1.95(m,1h),1.85(ddt,j＝13.9,7.0,3.6hz,1h),1.78
–
1.73(m,2h),1.71
–
1.66(m,3h),1.64
–
1.58(m,3h),1.56
–
1.42(m,5h),1.40/1.39(s,3h),1.37
–
1.25(m,4h),0.94/0.93(d,j＝6.6hz,3h),0.84/0.82(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ134.98/134.95(d,j＝3.0hz,ph-c),133.75/133.69(d,j＝9.0hz,ph-c),130.50/130.46(d,j＝12.0hz,ph-c),118.50/118.43(d,j＝85.5hz,ph-c),104.18/104.03,101.91/100.07,91.19/87.89,81.16/80.45,68.94/68.29,52.58/51.70,45.36/44.45,38.15,37.41/37.33,36.45/36.35,34.59/34.23,32.74,31.24/30.92,29.25/28.90,26.22/26.08,25.97/25.48,24.68/24.46,22.21,20.37/20.29,14.13/13.87,13.06/12.67.esi-ms m/z c
39h50
o5p

629.5(m-br)

。
[0113]
实施例15：化合物d7-t的制备
[0114][0115]
将中间体d7(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d7-t，产率25.52％。
[0116]
结构验证：
[0117]
α：β＝1:3；1h nmr(600mhz,chloroform-d)δ7.87
–
7.75(m,9h),7.70(td,j＝7.7,3.3hz,6h),5.34/5.31(s,1h),4.71/4.38(d,j＝3.6hz and 9.2hz,1h),3.90
–
3.65(m,3h),3.36
–
3.24(m,1h),2.38
–
2.27(m,2h),2.24
–
1.93(m,2h),1.84
–
1.82(m,2h),1.77
–
1.65(m,2h),1.54
–
1.43(m,4h),1.40/1.39(s,3h),1.33
–
1.13(m,10h),0.94/0.93(d,j＝6.0hz,
3h),0.84/0.81(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.02/134.98(d,j＝3.0hz,ph-c),133.71/133.68(d,j＝9.0hz,ph-c),130.51/130.48(d,j＝12.0hz,ph-c),118.40/118.44(d,j＝85.5hz,ph-c),104.21/104.04,101.94/100.13,91.18/87.90,81.19/80.44,69.14/68.38,52.59/51.69,45.35/44.48,37.42/37.34,36.45/36.34,34.62/34.23,32.71,30.92,30.26,30.16,29.54/29.35,29.04/28.80,26.23/26.07,25.82/25.50,24.68/24.44,22.46/22.21,20.39/20.29,13.06/12.66.esi-ms m/z c
40h52
o5p

643.5(m-br)

。
[0118]
实施例16：化合物d8-t的制备
[0119][0120]
将中间体d8(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d8-t，产率22.36％。
[0121]
结构验证：
[0122]
α：β＝1:1；β-isomer；1h nmr(600mhz,chloroform-d)δ7.87
–
7.76(m,10h),7.72
–
7.67(m,5h),5.35/5.31(s,1h),4.72/4.38(d,j＝3.5and 9.3hz,1h),3.92
–
3.69(m,3h),3.63/3.58(t,j＝6.6hz,1h),3.38
–
3.26(m,2h),2.64
–
2.29(m,2h),2.03
–
1.96(m,2h),1.94
–
1.80(m,4h),1.80
–
1.65(m,3h),1.56
–
1.44(m,5h),1.41/1.40(s,1h),1.34
–
1.27(m,3h),1.21
–
1.13(m,3h),1.07
–
0.95(m,1h),0.94/0.93(d,j＝6.6hz,3h),0.91
–
0.87(m,1h),0.86/0.84(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.02/134.99(d,j＝3.0hz,ph-c),133.70/133.68(d,j＝9.0hz,ph-c),132.08(d,j＝9.0hz,ph-c),131.97(d,j＝3.0hz,ph-c),130.51/130.48(d,j＝12.0hz,ph-c),128.51(d,j＝12.0hz,ph-c),118.44/118.40(d,j＝85.5hz,ph-c),104.21/104.03,101.90/100.13,91.19/87.90,81.18/80.43,69.21/68.41,52.60/51.69,45.35/44.48,38.14,37.44/37.35,36.45/36.35,34.87/34.84,34.23,31.92,31.24/30.94,29.56/29.39,29.21/29.01,28.95/28.84,26.23/26.13,26.08/25.79,24.68/24.45,22.59/22.22,20.39/20.29,13.06/12.66.esi-ms m/z c
41h54
o5p

657.5(m-br)

。
[0123]
实施例17：化合物d9-t的制备
[0124][0125]
将中间体d9(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d9-t，产率17.71％。
[0126]
结构验证:
[0127]
α：β＝1:4；β-isomer；1h nmr(600mhz,chloroform-d)δ7.87(dddd,j＝12.5,6.7,3.2,1.3hz,6h),7.83
–
7.79(m,3h),7.76
–
7.69(m,6h),5.39/5.35(s,1h),4.77/4.42(d,j＝3.0hz and9.2hz,1h),3.99
–
3.61(m,4h),3.42
–
3.31(m,1h),2.44
–
2.30(m,1h),2.07
–
1.98(m,1h),1.95
–
1.68(m,7h),1.59
–
1.47(m,4h),1.46
–
1.38(m,4h),1.37
–
1.28(m,6h),1.24
–
1.16(m,5h),0.97/0.96(d,j＝6.6hz,3h),0.89/0.88(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ134.97(d,j＝3.0hz,ph-c),133.73/133.71(d,j＝10.5hz,ph-c),132.09(d,j＝9.0hz,ph-c),131.95(d,j＝3.0hz,ph-c),130.48/130.43(d,j＝13.5hz,ph-c),128.54/128.51(d,j＝12.0hz,ph-c),118.48(d,j＝85.5hz,ph-c),104.21/104.03,101.93/100.10,91.20/87.91,81.20/80.41,69.23/68.45,52.61/51.70,45.37/44.51,38.15,37.44/37.36,36.47/36.36,34.24,32.71,31.24/30.96,29.66,29.45/29.36,29.17,26.08,25.84,24.70,22.56,22.27/22.23,20.40/20.29,14.13/13.84,13.06/12.66.esi-ms m/z c
42h56
o5p

671.5(m-br)

。
[0128]
实施例18：化合物d10-t的制备
[0129][0130]
将中间体d10(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d10-t，产率20.86％。
[0131]
结构验证：
[0132]
α：β＝1:10；1h nmr(600mhz,chloroform-d)δ7.87
–
7.75(m,9h),7.70(td,j＝7.8,3.4hz,6h),5.36/5.31(s,1h),4.74/4.39(d,j＝3.0hz and 9.2hz,1h),3.94
–
3.56(m,4h),3.41
–
3.28(m,1h),2.41
–
2.28(m,2h),1.92
–
1.81(m,4h),1.79
–
1.66(m,3h),1.56
–
1.43(m,5h),1.41(s,3h),1.34
–
1.25(m,4h),1.22
–
1.12(m,8h),1.02
–
0.95(m,1h),0.94/0.93(d,j＝6.6hz,3h),0.86/0.85(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.03/130.54(d,j＝3.0hz,ph-c),134.67/132.08(d,j＝9.0hz,ph-c),130.52/130.49(d,j＝10.5hz,ph-c),128.52(d,j＝12.0hz,ph-c),118.43/118.40(d,j＝85.5hz,ph-c),104.22/104.03,100.14,91.19,80.42,69.29,52.60/51.69,45.35,37.36,36.35,34.24,32.70/32.62,29.51,29.36,29.26,29.11,29.02,26.07,25.90,24.70,24.45,22.61,22.22,20.40,20.29,13.05/12.65.esi-ms m/z c
43h58
o5p

685.5(m-br)

。
[0133]
实施例18：化合物d11-t的制备
[0134]
[0135]
将中间体d11(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d11-t，产率18.18％。
[0136]
结构验证：
[0137]
α：β＝1:4；1h nmr(600mhz,chloroform-d)δ7.87
–
7.80/7.67
–
7.62(m,6h),7.90
–
7.79/7.56
–
7.52(m,3h),7.70/7.45(ddd,j＝11.2,6.4,3.4hz,6h),5.37/5.32(s,1h),4.75/4.40(d,j＝3.0hz and 9.2hz,1h),3.96
–
3.68(m,3h),3.64
–
3.30(m,1h),2.43
–
2.32(m,2h),2.05
–
1.97(m,1h),1.94
–
1.78(m,5h),1.77
–
1.65(m,3h),1.58
–
1.44(m,4h),1.42/1.41(s,3h),1.40
–
1.37(m,1h),1.36
–
1.27(m,5h),1.22
–
1.14(m,8h),1.03
–
0.97(m,1h),0.95/0.92(d,j＝6.6hz,3h),0.86(d,j＝7.2hz,3h).
13
c nmr(150mhz,chloroform-d)δ135.00(d,j＝3.0hz,ph-c),133.71/133.69(d,j＝9.0hz,ph-c),132.09(d,j＝10.5hz,ph-c),131.97(d,j＝3.0hz,ph-c),130.49/130.47(d,j＝12.0hz,ph-c),128.51(d,j＝12.0hz,ph-c),118.47/118.44(d,j＝85.5hz,ph-c),104.22/104.03,101.92/100.13,91.20/87.91,81.19/80.41,69.30/68.46,52.61/51.69,45.36/44.51,37.46/37.37,36.46/36.35,34.68/34.25,31.24,29.52/29.49,29.41,29.37,29.31,29.21,29.15,29.09,26.07,25.93,24.71/24.46,22.92,22.64/22.60,22.22,20.40/20.29,13.06/12.65.esi-ms m/z c
44h60
o5p

699.5(m-br)

。
[0138]
实施例19：化合物d12-t的制备
[0139][0140]
将中间体d12(1eq)，三苯基磷(2eq)，碳酸钾70mg溶于5-10ml乙腈中，n2保护，60℃回流24h。tlc监测，待反应结束后过滤，旋干样品，柱层析得化合物d12-t，产率28.93％
[0141]
结构验证：
[0142]
α：β＝1:10；1h nmr(600mhz,chloroform-d)δ7.83/7.65(ddt,j＝12.5,7.0,1.3hz,6h),7.80
–
7.76/7.55
–
7.52(m,3h),7.73
–
7.67/7.47
–
7.44(m,6h),5.32/5.37(s,1h),4.40/4.75(d,j＝9.2hz and 3.6hz,1h),3.93(ddd,j＝9.6,7.1,5.8hz,1h),3.83
–
3.65(m,2h),3.41
–
3.32(m,1h),2.37(dddd,j＝17.4,14.5,10.2,4.3hz,2h),2.05
–
1.93(m,1h),1.86(ddt,j＝13.7,6.8,3.5hz,1h),1.74(dt,j＝13.6,3.8hz,2h),1.68(dq,j＝13.3,3.4hz,1h),1.56
–
1.43(m,4h),1.42
–
1.39(m,4h),1.35
–
1.25(m,7h),1.23
–
1.15(m,11h),1.10(s,2h),1.03
–
0.96(m,1h),0.94(d,j＝6.2hz,3h),0.89
–
0.82(m,3h).
13
c nmr(150mhz,chloroform-d)δ134.98/131.96(d,j＝3.0hz,ph-c),133.70/132.09(d,j＝9.0hz,ph-c),130.48/130.47(d,j＝12.0hz,ph-c),128.51(d,j＝12.0hz,ph-c),118.42/118.41(d,j＝85.5hz,ph-c),104.22/104.03,101.93/100.13,91.20,80.40,69.30,51.69,45.36,38.15,37.37,36.35,34.25,32.69,31.24,30.38(d,j＝15.0hz),29.55,29.48,29.45,29.37,29.18,29.11,26.07,25.97,24.71,22.92,22.62(d,j＝4.5hz),22.22,20.30,12.65.esi-ms m/z c
45h62
o5p

713.6(m-br)

。
[0143]
实验例1：药物ic
50
值测定
[0144]
一、实验材料
[0145]
人膀胱癌细胞系j82购自上海兴泊生物科技有限公司；
[0146]
人膀胱癌细胞系t24购自上海兴泊生物科技有限公司；
[0147]
人肺癌细胞系a549购自上海兴泊生物科技有限公司；
[0148]
人卵巢癌细胞系ovcar3购自武汉细胞所；
[0149]
人卵巢癌细胞系skov3来自湘雅医院。
[0150]
mtt购自solarbio公司。
[0151]
二、实验方法及结果
[0152]
取处于对数期的人膀胱癌细胞(j82、t24)、人卵巢癌细胞(ovcar3、skov3)和人肺癌细胞a549分别按照每孔5000个细胞进行种板，将接种完的96孔培养板放置于培养箱中过夜，用不同浓度的化合物d3-t、化合物d4-t、化合物d5-t、化合物d6-t、化合物d7-t、化合物d8-t、化合物d9-t、化合物d10-t、化合物d11-t、化合物d12-t处理96孔板，处理完成后将96孔培养板均放回培养箱中培养72小时，然后用mtt比色法检测细胞生存率，并分别计算ic
50
值，实验结果如表1所示。
[0153]
表1 d3-t～d12-t对5株肿瘤细胞的半数抑制浓度(ic
50
)
[0154][0155]
实验例2：细胞克隆试验
[0156]
一、实验材料
[0157]
10％的福尔马林购自国药集团化学试剂有限公司；
[0158]
结晶紫染液购自sigma-aldrich公司。
[0159]
二、实验方法及结果
[0160]
取处于对数生长期的人膀胱癌细胞系j82、t24，分别按照每孔1000个细胞接种于24孔培养板，将接种完的培养板均放置在培养箱中过夜，使细胞贴壁；人膀胱癌细胞系j82、t24分别给予0nm、6.25nm、12.5nm、25nm、50nm的药物(药物为d8-t和dha)处理；处理完后将培养板均放回培养箱中继续培养一周；待细胞融合至80％后，倒掉培养液，用pbs洗一遍，10％的福尔马林固定1个小时，用并500μl结晶紫染液染色一个小时，最后回收结晶紫染液；之后，分别将24孔板流水冲洗干净，并将24孔板倒置放在滤纸上，干燥后用相机拍摄图像并用酶标仪(biotek，synergy htx，vermont，usa)进行定量分析，实验结果如图2-3所示。
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实验例3：细胞划痕试验
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取处于对数生长期的人膀胱癌细胞系j82分别按照每孔3
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105个细胞接种于12孔培养板，将接种完的培养板均放置在培养箱中使细胞融合至70％-80％。用marker笔在12孔板背后用直尺在中间划一个十字作为固定监测点，使前后观察时位置固定。用10μl枪头比着直尺背后的横线划痕。pbs洗细胞一次，去除划下的细胞，加入无血清培养基。然后膀胱癌细胞系j82分别给予12.5nm、25nm、50nm的药物(药物为d8-t和dha)处理，放入37度5％co2培养箱培养。分别于0，24，48小时用显微镜(leica，dfc450c，wetzlar，germany)拍摄图像。实验结果如图4-5所示。由图可以看出，化合物对人膀胱癌细胞系t24、人膀胱癌细胞系umuc3、人肺癌细胞系a549的迁移抑制效果明显优于双氢青蒿素。