CN105837650A - 一种甾体类化合物及其应用 - Google Patents
一种甾体类化合物及其应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域,具体为一种甾体类化合物及其抗肿瘤干细胞活性。本发明提供的一种甾体类化合物,其结构如式I所示:
Description
技术领域
本发明属于医药技术领域,涉及一种从珊瑚及珊瑚共附生真菌中分离得到的具有抑制肿瘤干细胞增殖活性的甾体类化合物。
背景技术
恶性肿瘤是严重威胁人类生命的疾病之一。对于多数恶性肿瘤患者而言,可采用化疗、放射疗法及生物免疫治疗等方法来杀死大部分肿瘤细胞,但是却无法从根本上治愈肿瘤。研究表明,恶性肿瘤发生发展及复发的主要原因在于肿瘤干细胞的存在(C TJordan,M L Guzman,M Noble.Cancer stem cells[J].N Engl J Med 2006,355,1253-1261.)。肿瘤干细胞通常仅占肿瘤细胞的不足1.5%,但这类细胞具有自我更新、多向分化及无限增殖的潜能,是肿瘤转移和复发的关键因素。因此,肿瘤干细胞一经发现立即受到强烈关注(D Bonnet,J E Dick.Human acute myeloid leukemia is organized as ahierarchy that originates from a primitive hematopoietic cell[J].Nat Med1997,3,730-737.),抗肿瘤干细胞活性物质研究迅速成为近年来抗肿瘤药物研究最为关注的热点领域。
甾体类化合物是广泛存在于生物体组织内的一类重要的天然有机化合物,尤其是在海洋中的分布广、含量高,随着海洋药物研究的不断深入,多年来已分离鉴定了多种甾体化合物,多具有抗肿瘤、抗炎、抗菌、抗病毒和酶抑制等生物活性。本课题组长期从事海洋天然活性成分的研究,最近我们从4种珊瑚:肉芝软珊瑚(Sarcophyton subviride)、紫柳珊瑚(Muriceopsis flavida)、小月柳珊瑚(Menella Kanisa)、侧扁软柳珊瑚(Subergorgiasuberosa)中分离鉴定了若干甾体化合物,活性测试部分化合物具有抗肿瘤和抗菌活性,但文献检索未发现这类甾体化合物具有抑制肿瘤干细胞增殖活性的报道。
发明内容
本发明的目的是提供一种甾体类化合物,它可应用于医学上抑制肿瘤干细胞的增殖活性。
为了实现上述目的,本发明采用的技术方案如下:
一种甾体类化合物,如下述化学通式所示:
其中,R1基团为H或OH或═O R2基团为H或Ac
R3基团为OH或H或OAc R4基团为OH或═O
R5基团为H或双键 R6基团为H或双键
R7选自以下结构(a~o)中的一个:
本发明的化合物有闭环和开环两种结构,分别为以下式Ia和式Ib:
本发明的部分优选化合物详见表1。
表1
本发明化合物的制备和结构鉴定方法如下:
1.制备化合物1~53
将经过预处理的珊瑚{肉芝软珊瑚(Sarcophyton subviride)、紫柳珊瑚(Muriceopsis flavida)、小月柳珊瑚(Menella Kanisa)、侧扁软柳珊瑚(Subergorgiasuberosa)}粉碎,用丙酮及甲醇超声提取数次,至提取液基本无色,浓缩后得到粗浸膏。将粗浸膏分散在等体积水中,用乙醚萃取数次,合并萃取液,减压回收乙醚至干,得乙醚层浸膏;或乙醚层浸膏再用90%甲醇-水溶液分散,正己烷萃取数次后,浓缩90%甲醇-水溶液得红褐色浸膏。将乙醚层浸膏或90%甲醇-水溶液浸膏用正相硅胶色谱(200~300目)分离,分别用不同的洗脱剂系统梯度洗脱,通过薄层板监测,根据馏分极性大小收集,将总浸膏合并成若干个部分。然后每部分用不同条件的洗脱剂进行Sephadex LH-20凝胶色谱分离、正反相硅胶柱层析,后经反相制备型HPLC纯化,最后得化合物1~53。
2.结构鉴定
化合物1:无色油状物;[α]D=-54(c 1.0,MeOH);m.p.260-263.5℃;ESIMS(m/z):437.6([M+H]+),C27H48O4。1H-NMR(CD3OD,400MHz):4.00(1H,m,H-3),3.94(1H,dd,1H,dd,J=11.5,4.8Hz,H-1),3.42(1H,br s,H-6),1.12(3H,s,Me-19),0.92(3H,d,J=6.4Hz,Me-21),0.87(3H,d,J=6.6Hz,Me-26),0.87(3H,d,J=6.6Hz,Me-27),0.71(3H,s,Me-18).
化合物2:白色无定形粉末;[α]D=-13.2(c 1.0,MeOH);m.p.267-269℃;ESIMS(m/z):450.4([M+H]+),C28H50O4;1H-NMR(CD3OD,400MHz):δH 4.00(1H,m,H-3),3.94(1H,dd,1H,dd,J=11.5,4.8Hz,H-1),3.42(1H,br s,H-6),1.12(3H,s,Me-19),0.92(3H,d,J=6.4Hz,Me-21),0.87(3H,d,J=6.8Hz,Me-28),0.80(3H,d,J=6.6Hz,Me-26),0.80(3H,d,J=6.6Hz,Me-27),0.70(3H,s,Me-18).
化合物3:无色晶体;m.p.279-281℃;1H-NMR(CD3OD,400MHz):δH 4.00(1H,m,H-3),3.94(1H,dd,1H,dd,J=11.5,4.8Hz,H-1),3.42(1H,br s,H-6),1.94(3H,s,-OAc),1.38(3H,d,J=7.0Hz,Me-26),1.38(3H,d,J=7.0Hz,Me-27),1.12(3H,s,Me-19),0.94(3H,d,J=6.8Hz,Me-21),0.88(3H,d,J=6.8Hz,Me-28),0.71(3H,s,Me-18).
化合物4:无色晶体;m.p.257-259℃;1H-NMR(CD3OD,400MHz):δH 4.00(1H,m,H-3),3.44(1H,br s,H-6),1.15(3H,s,Me-19),1.11(3H,d,J=5.6,Me-26),1.11(3H,d,J=5.6,Me-27),0.95(3H,d,J=6.8,Me-21),0.88(3H,d,J=6.8,Me-28),0.71(3H,s,Me-18).
化合物5:无色晶体;m.p.242-244℃;1H-NMR(CD3OD,400MHz):δH 4.09(1H,m,H-3),3.51(1H,br s,H-6),2.00(1H,s,-OAc),1.38(3H,s,Me-26),1.38(3H,s,Me-26),1.17(3H,s,Me-19),0.91(3H,d,J=6.8,Me-21),0.84(3H,d,J=6.8Hz,Me-28),0.67(3H,s,Me-18).
化合物6:白色固体粉末。HRESIMS(m/z):427.6303([M+Na]+),C26H44O3;(c0.050,Acetone);IR(film)νmax:3373,3026,2959,2928,2854,1600,1496,1453,1379,1302,1260,1040,966,749,699;UV(CHCl3)λmax nm(Abs):278(0.4411);1H-NMR和13C-NMR见表2。
化合物7:白色粉末状固体。HRESIMS(m/z):441.6371[(M+Na)+],C27H46O3;(c0.200,Acetone);IR(film)νmax:3388,3061,3022,2927,2854,1602,1455,1376,1041,966,748,699;UV(CHCl3)λmax nm(Abs):232(0.3947)。1H-NMR(400MHz,CD3OD)δ:4.03(1H,m,H-3),3.46(1H,brs,H-6),0.70(3H,s,H-18),1.16(3H,s,H-19),0.94(3H,d,J=6.6,H-21),1.60(3H,s,H-26),1.67(3H,s H-27),5.08(1H,t,J=7.0,14.2Hz,H-24);13C-NMR(100MHz,CD3OD)δ:33.5(t,C-1),31.6(t,C-2),68.4(d,C-3),41.4(t,C-4),76.9(s,C-5),76.6(d,C-6),35.3(t,C-7),31.6(d,C-8),46.6(d,C-9),39.3(s,C-10),22.3(t,C-11),41.4(t,C-12),44.0(s,C-13),57.4(d,C-14),25.2(t,C-15),29.3(t,C-16),57.6(d,C-17),12.6(q,C-18),17.5(q,C-19),36.8(d,C-20),19.4(q,C-21),37.3(t,C-22),25.8(t,C-23),126.3(d,C-24),131.6(s,C-25),26.2(q,C-26),18.1(q,C-27)。
化合物8:白色粉末。ESI-MS(m/z):418.5([M+H]+),C27H46O3。1H-NMR(400MHz,CD3OD)δ:4.02(1H,m,H-3),3.46(1H,brs,H-6),0.72(3H,s,H-18),1.16(3H,s,H-19),1.00(3H,d,J=6.6,H-21),0.87(3H,d,J=6.6,H-26),0.87(3H,d,J=6.6,H-27),5.21(1H,dd,J=8.2,15.2Hz,H-22),5.28(1H,dd,J=7.2,15.0Hz,H-23);13C-NMR(100MHz,CD3OD)δ:33.5(t,C-1),31.7(t,C-2),68.3(d,C-3),41.5(t,C-4),76.8(s,C-5),76.4(d,C-6),35.8(t,C-7),31.4(d,C-8),46.5(d,C-9),39.4(s,C-10),21.4(t,C-11),41.4(t,C-12),44.0(s,C-13),57.6(d,C-14),24.2(t,C-15),28.3(t,C-16),57.4(d,C-17),12.6(q,C-18),17.3(q,C-19),40.2(d,C-20),20.6(q,C-21),138.3(d,C-22),126.6(d,C-23),42.1(t,C-24),28.6(d,C-25),22.3(q,C-26),22.4(q,C-27)。
化合物9:白色粉末。ESI-MS(m/z):432.3([M+H]+),C28H48O3。1H-NMR(400MHz,CD3OD)δ:4.02(1H,m,H-3),3.46(1H,brs,H-6),0.71(3H,s,H-18),1.16(3H,s,H-19),0.96(3H,d,J=6.6,H-21),1.02(3H,d,J=6.8,H-26),1.02(3H,d,J=6.8,H-27),4.70(1H,s,H-28),4.60(1H,s,H-28);13C-NMR(100MHz,CD3OD)δ:33.5(t,C-1),31.7(t,C-2),68.3(d,C-3),41.5(t,C-4),76.8(s,C-5),76.4(d,C-6),35.8(t,C-7),31.4(d,C-8),46.5(d,C-9),39.4(s,C-10),21.4(t,C-11),39.9(t,C-12),44.0(s,C-13),57.1(d,C-14),24.2(t,C-15),28.3(t,C-16),57.1(d,C-17),12.6(q,C-18),17.1(q,C-19),36.4(d,C-20),18.6(q,C-21),35.3(d,C-22),31.9(d,C-23),156.6(t,C-24),34.5(d,C-25),22.1(q,C-26),22.3(q,C-27),117.7(q,C-28)。
化合物10:白色粉末。ESI-MS(m/z):432.3([M+H]+),C28H48O3。1H-NMR(400MHz,CD3OD)δ:4.02(1H,m,H-3),3.46(1H,brs,H-6),0.72(3H,s,H-18),1.16(3H,s,H-19),1.00(3H,d,J=6.6,H-21),0.85(3H,d,J=7.2,H-26),0.83(3H,d,J=7.2,H-27),0.92(3H,d,J=6.8Hz,H-28),5.16(1H,dd,J=8.2,15.2Hz,H-22),5.20(1H,dd,J=7.2,15.3Hz,H-23);13C-NMR(100MHz,CD3OD)δ:33.5(t,C-1),31.7(t,C-2),68.3(d,C-3),41.5(t,C-4),76.8(s,C-5),76.4(d,C-6),35.8(t,C-7),31.4(d,C-8),46.5(d,C-9),39.4(s,C-10),21.4(t,C-11),41.4(t,C-12),44.0(s,C-13),57.6(d,C-14),24.2(t,C-15),28.3(t,C-16),57.4(d,C-17),12.6(q,C-18),17.3(q,C-19),40.0(d,C-20),20.9(q,C-21),135.1(d,C-22),132.6(d,C-23),42.8(t,C-24),33.0(d,C-25),20.0(q,C-26),20.4(q,C-27),18.1(q,C-28)。
化合物11:白色粉末状固体;m.p.235-237℃(hexane);ESI-MS(m/z):420.4([M+H]+),C27H48O3;1H-NMR(400MHz,CD3OD)δ:4.02(1H,m,H-3),3.46(1H,brs,H-6),0.72(3H,s,H-18),1.16(3H,s,H-19),0.92(3H,d,J=6.6,H-21),0.87(3H,d,J=6.6,H-26),0.87(3H,d,J=6.6,H-27);13C-NMR(100MHz,CD3OD)δ:33.5(t,C-1),31.8(t,C-2),68.3(d,C-3),41.7(t,C-4),76.6(s,C-5),76.5(d,C-6),35.4(t,C-7),31.6(d,C-8),46.4(d,C-9),39.3(s,C-10),22.1(t,C-11),41.1(t,C-12),43.7(s,C-13),57.4(d,C-14),25.2(t,C-15),29.3(t,C-16),57.3(d,C-17),12.6(q,C-18),17.4(q,C-19),37.2(d,C-20),19.4(q,C-21),36.0(t,C-22),24.0(t,C-23),41.0(t,C-24),30.6(d,C-25),23.0(q,C-26),23.2(q,C-27)。
化合物12:白色粉末状固体;m.p.234-236℃(MeOH);ESI-MS(m/z):416.3([M+H]+),C27H44O3。1H-NMR(600MHz,CDCl3)δ:4.10(1H,m,H-3),3.64(1H,brs,H-6),5.37(1H,brs,H-7),5.24(1H,dd,J=8.4,15.0Hz,H-22),5.31(1H,dd,J=6.6,14.0Hz,H-23),0.62(3H,s,H-18),1.10(3H,s,H-19),1.04(3H,d,J=6.6,H-21),0.89(3H,d,J=7.2,H-26),0.88(3H,d,J=6.6,H-27);13C-NMR(125MHz,CDCl3)δ:31.0(t,C-1),32.9(t,C-2),67.7(d,C-3),39.4(t,C-4),73.6(s,C-5),76.0(d,C-6),117.6(d,C-7),143.9(s,C-8),43.4(d,C-9),37.1(s,C-10),22.0(t,C-11),39.3(t,C-12),43.9(s,C-13),54.7(d,C-14),22.9(t,C-15),27.7(t,C-16),56.0(d,C-17),12.0(q,C-18),18.8(q,C-19),39.7(d,C-20),20.6(q,C-21),137.7(d,C-22),126.6(d,C-23),41.8(t,C-24),28.5(d,C-25),22.3(q,C-26),22.3(q,C-27)。
化合物13:白色粉末状固体;m.p.244-246℃(MeOH);ESI-MS(m/z):431.6([M+H]+),C28H46O3。1H-NMR(600MHz,CDCl3)δ:4.10(1H,m,H-3),3.64(1H,brs,H-6),5.37(1H,brs,H-7),4.67(1H,s,H-22),4.73(1H,s,H-23),0.60(3H,s,H-18),1.10(3H,s,H-19),0.98(3H,d,J=6.2,H-21),1.036(3H,d,J=7.2,H-26),1.042(3H,d,J=7.2,H-27);13C-NMR(125MHz,CDCl3)δ:31.0(t,C-1),32.9(t,C-2),67.7(d,C-3),39.4(t,C-4),76.0(s,C-5),73.5(d,C-6),117.6(d,C-7),143.9(s,C-8),43.4(d,C-9),37.1(s,C-10),22.0(t,C-11),39.3(t,C-12),43.9(s,C-13),54.7(d,C-14),22.9(t,C-15),27.7(t,C-16),56.0(d,C-17),12.0(q,C-18),18.8(q,C-19),36.1(d,C-20),18.8(q,C-21),34.5(t,C-22),30.8(t,C-23),156.6(s,C-24),33.8(d,C-25),21.8(q,C-26),21.8(q,C-27),106.0(t,C-28)。
化合物14:白色粉末状固体;m.p.227-229℃(MeOH);ESI-MS(m/z):416.3([M+H]+),C27H46O3。1H-NMR(600MHz,CDCl3:CD3OD 1:1)δ:4.00(1H,m,H-3),3.57(1H,brs,H-6),5.30(1H,brs,H-7),0.62(3H,s,H-18),1.07(3H,s,H-19),0.95(3H,d,J=6.6,H-21),0.873(3H,d,J=6.6,H-26),0.877(3H,d,J=6.6,H-27);13C-NMR(125MHz,CDCl3)δ:31.0(t,C-1),32.9(t,C-2),67.7(d,C-3),39.4(t,C-4),73.6(s,C-5),76.0(d,C-6),117.6(d,C-7),143.9(s,C-8),43.4(d,C-9),37.1(s,C-10),22.0(t,C-11),39.3(t,C-12),43.9(s,C-13),54.7(d,C-14),22.9(t,C-15),27.7(t,C-16),56.0(d,C-17),12.0(q,C-18),18.8(q,C-19),35.2(d,C-20),18.6(q,C-21),36.0(t,C-22),23.8(t,C-23),39.4(t,C-24),28.1(d,C-25),22.5(q,C-26),22.8(q,C-27)。
化合物15:白色粉末状固体;m.p.215-217℃(MeOH);ESI-MS(m/z):403.3([M+H]+),C26H42O3。1H-NMR(600MHz,CDCl3)δ:4.10(1H,m,H-3),3.64(1H,brs,H-6),5.37(1H,brs,H-7),5.19(1H,dd,J=8.4,15.6Hz,H-22),5.29(1H,dd,J=8.4,15.0Hz,H-23),0.60(3H,s,H-18),1.10(3H,s,H-19),1.03(3H,d,J=6.6,H-21),0.96(3H,d,J=6.6,H-26),0.95(3H,d,J=7.2,H-27);13C-NMR(125MHz,CDCl3)δ:33.6(t,C-1),30.9(t,C-2),67.7(d,C-3),39.5(t,C-4),76.6(s,C-5),73.6(d,C-6),117.6(d,C-7),145.5(s,C-8),43.5(d,C-9),37.2(s,C-10),22.0(t,C-11),39.4(t,C-12),43.9(s,C-13),54.7(d,C-14),22.9(t,C-15),27.7(t,C-16),56.1(d,C-17),12.1(q,C-18),18.9(q,C-19),39.9(d,C-20),21.6(q,C-21),135.5(d,C-22),131.8(d,C-23),41.5(d,C-25),20.6(q,C-26),21.3(q,C-27)。
化合物16:白色粉末状固体;m.p.246-248℃(MeOH);ESI-MS(m/z):433.4([M+H]+),C28H48O3。1H-NMR(600MHz,CDCl3)δ:4.10(1H,m,H-3),3.64(1H,brs,H-6),5.37(1H,brs,H-7),0.60(3H,s,H-18),1.10(3H,s,H-19),0.94(3H,d,J=6.6,H-21),0.80(3H,d,J=7.2,H-26),0.82(3H,d,J=6.6,H-27),0.87(3H,d,J=6.5Hz,H-28);13C-NMR(125MHz,CDCl3)δ:33.6(t,C-1),30.9(t,C-2),67.7(d,C-3),39.5(t,C-4),76.6(s,C-5),73.6(d,C-6),117.6(d,C-7),145.5(s,C-8),43.5(d,C-9),37.2(s,C-10),22.0(t,C-11),39.4(t,C-12),43.9(s,C-13),54.7(d,C-14),22.9(t,C-15),27.7(t,C-16),56.1(d,C-17),12.1(q,C-18),18.9(q,C-19),36.5(d,C-20),18.9(q,C-21),33.5(t,C-22),31.5(t,C-23),40.5(d,C-24),31.1(d,C-25),18.4(q,C-26),20.8(q,C-27),17.6(q,C-28)。
化合物17:白的固体粉末;HRESIMS(m/z):469.3297([M+Na]+),C28H46O4;(c0.275,Acetone);IR(film)νmax:3454,2953,2941,2867,1716,1599,1496,1452,1382,1367,1281,1161,1037,968,699;UV(CHCl3)λmax nm(Abs):257(0.3642);1H-NMR和13C-NMR见表3。
化合物18:白色固体粉末;HRESIMS(m/z):483.2692([M+Na]+),C29H48O4;(c0.035,Acetone);IR(film)νmax:3369,2956,2927,1736,1664,1599,1458,1378,1260,1043,968,699;UV(CHCl3)λmax nm(Abs):240(0.8793);1H-NMR和13C-NMR见表3。
化合物19:白色固体粉末。ESI-MS(m/z):463.7([M+H]+),C29H50O4。1H-NMR(400MHz,CDCl3)δ:5.14(1H,m,H-3),3.53(1H,brs,H-6),0.68(3H,s,H-18),1.18(3H,s,H-19),0.90(3H,d,J=6.5,H-21),0.85(3H,d,J=6.6,H-26),0.85(3H,d,J=6.6,H-27),2.02(3H,s,H-29);13C-NMR(100MHz,CDCl3)δ:34.7(t,C-1),32.1(t,C-2),71.1(d,C-3),37.0(t,C-4),75.7(s,C-5),76.2(d,C-6),26.7(t,C-7),30.2(d,C-8),45.6(d,C-9),38.4(s,C-10),21.1(t,C-11),39.9(t,C-12),42.8(s,C-13),55.8(d,C-14),24.1(t,C-15),28.2(t,C-16),56.2(d,C-17),12.1(q,C-18),16.7(q,C-19),35.8(d,C-20),18.7(q,C-21),36.1(t,C-22),23.9(t,C-23),39.5(d,C-24),28.0(d,C-25),22.6(q,C-26),22.8(q,C-27),170.9(s,C-28),21.4(q,C-29)。
化合物20:白色固体粉末;HRESIMS(m/z):455.3501([M+Na]+);C28H48O3;(c0.040,Acetone);IR(film)νmax:3374,3026,2929,2867,1600,1496,1454,1379,1262,1078,1032,754,699;UV(CHCl3)λmax nm(Abs):337(0.4653);1H-NMR和13C-NMR见表3。
化合物21:白色固体粉末。ESI-MS(m/z):435.3([M+H]+),C28H50O3。1H-NMR(400MHz,CDCl3)δ:3.82(1H,m,H-3),3.89(1H,brs,H-6),0.68(3H,s,H-18),1.18(3H,s,H-19),0.90(3H,d,J=6.5,H-21),0.86(3H,d,J=6.6,H-26),0.85(3H,d,J=6.6,H-27),3.15(3H,s,H-29);13C-NMR(100MHz,CDCl3)δ:34.4(t,C-1),31.2(t,C-2),67.7(d,C-3),33.5(t,C-4),78.9(s,C-5),70.1(d,C-6),32.2(t,C-7),30.2(d,C-8),44.7(d,C-9),39.4(s,C-10),21.1(t,C-11),39.9(t,C-12),42.8(s,C-13),55.9(d,C-14),24.1(t,C-15),28.3(t,C-16),56.3(d,C-17),12.2(q,C-18),17.7(q,C-19),35.8(d,C-20),18.7(q,C-21),36.2(t,C-22),23.9(t,C-23),39.5(d,C-24),28.0(d,C-25),22.6(q,C-26),22.8(q,C-27),48.1。
化合物22:白色固体粉末。HRESIMS(m/z):443.2857([M+Na]+),C26H44O4;(c0.085,Acetone);IR(film)νmax:3316,2953,2926,2851,1600,1496,1455,1379,1212,1098,1040,1009,960;UV(CHCl3)λmax nm(Abs):278(0.1204);1H-NMR和13C-NMR见表2。
化合物23:白色固体粉末。ESIMS(m/z):435.4([M+H]+),C27H46O4。1H-NMR(400MHz,CD3OD)δ:4.03(1H,m,H-3),3.44(1H,brs,H-6),3.95(1H,dd,J=4.9,11.5Hz,H-1),5.20(1H,dd,J=8.1,15.4Hz,H-22),5.27(1H,dd,J=6.5,13.5Hz,H-23),0.72(3H,s,H-18),1.13(3H,s,H-19),1.00(3H,d,J=7.2,H-21),0.87(3H,d,J=6.6,H-26),0.87(3H,d,J=6.6,H-27)。13C-NMR(100MHz,CD3OD)δ:74.3(d,C-1),43.1(t,C-2),65.9(d,C-3),41.5(t,C-4),77.4(s,C-5),77.0(d,C-6),35.1(t,C-7),35.1(d,C-8),47.3(d,C-9),44.8(s,C-10),25.5(t,C-11),41.6(t,C-12),42.4(s,C-13),57.6(d,C-14),24.9(t,C-15),29.7(t,C-16),57.6(d,C-17),12.7(q,C-18),10.0(q,C-19),41.8(d,C-20),21.3(q,C-21),127.3(d,C-22),139.5(d,C-23),43.2(t,C-24),25.7(d,C-25),19.9(q,C-26),19.9(q,C-27)。
化合物24:白色固体粉末。ESIMS(m/z):435.4([M+H]+),C28H50O4。1H-NMR(400MHz,CD3OD)δ:4.03(1H,m,H-3),3.44(1H,brs,H-6),3.95(1H,dd,J=4.9,11.5Hz,H-1),0.72(3H,s,H-18),1.13(3H,s,H-19),0.93(3H,d,J=6.6,H-21),0.87(3H,d,J=6.6,H-28),0.80(3H,d,J=6.6,H-26),0.82(3H,d,J=6.6,H-26)。13C-NMR(100MHz,CD3OD)δ:74.3(d,C-1),41.6(t,C-2),66.1(d,C-3),42.5(t,C-4),77.8(s,C-5),77.1(d,C-6),35.3(t,C-7),32.6(d,C-8),47.4(d,C-9),44.9(s,C-10),25.1(t,C-11),42.1(t,C-12),43.4(s,C-13),57.6(d,C-14),25.6(t,C-15),29.2(t,C-16),57.6(d,C-17),12.6(q,C-18),10.2(q,C-19),37.9(d,C-20),19.4(q,C-21),35.0(t,C-22),31.7(t,C-23),40.5(d,C-24),32.7(d,C-25),20.6(q,C-26),18.3(q,C-27),16.1(q,C-28)。
化合物25:白色无定形状粉末;m.p.205-207℃;(c 0.15,MeOH);IR(film)νmax3381,2925,2854,1459,1376,966cm-1;1H-NMR和13C-NMR数据见表4;HRESIMS m/z485.3032[M+Cl]-(calcd for C27H46O5Cl,485.3034)。
化合物26:白色无定形状粉末;m.p.235-237℃;(c 0.35,MeOH);IR(film)νmax3307,2924,2852,1459,1376,1005cm-1;1H-NMR和13C-NMR数据见表4;HRESIMS m/z433.3319[M-H]-(calcd for C27H45O4,433.3318)。
化合物28:1H-NMR(CDCl3,400MHz):δH 3.97(1H,m,H-3),2.79(1H,t,J=12.5Hz,H-7),2.15(1H,dd,J=12.5,4.6Hz,H-7),2.03(1H,ov,H-12),1.87(1H,ov,H-4),1.86(1H,ov,H-2),1.84(1H,m,H-16),1.82(1H,ov,H-9),1.80(1H,ov,H-4),1.70(2H,ov,H-8,H-1),1.54(1H,ov,H-1),1.50(1H,m,H-15),1.51(1H,ov,H-25),1.45(2H,ov,H-11,H-2),1.35(1H,m,H-20),1.34(1H,m,H-22),1.31(1H,m,H-23),1.30(1H,ov,H-14),1.28(1H,m,H-11),1.25(1H,m,H-16),1.23(1H,ov,H-12),1.12(2H,ov,H-17,H-23),1.06(2H,m,H-24),1.02(1H,m,H-15),1.01(1H,m,H-22),0.90(3H,d,6.6,H3-21),0.87(3H,d,6.6,H3-26),0.86(3H,d,6.6,H3-27),0.81(3H,s,H3-19),0.64(3H,s,H3-18)。13C-NMR(CDCl3,125MHz):δC 212.2(C,C-6),80.9(C,C-5),67.3(CH,C-3),56.4(CH,C-14),56.2(CH,C-17),44.6(CH,C-9),43.2(C,C-13),42.5(C,C-10),41.8(CH,C-7),39.6(CH2,C-24),39.5(CH2,C-12),37.3(CH,C-8),36.5(CH2,C-4),36.1(CH,C-22),35.7(CH,C-20),30.5(CH2,C-2),29.8(CH2,C-1),28.0(CH,C-25),28.1(CH2,C-16),23.9(CH2,C-15),23.9(CH,C-23),22.8(CH3,C-26),22.6(CH3,C-27),21.5(CH2,C-11),18.7(CH3,C-21),14.1(CH3,C-19),12.0(CH3,C-18)。
化合物29:1H-NMR(CDCl3,400MHz):δH 5.27(1H,ddd,J=15.2,7.0,6.9Hz,H-23),5.20(1H,dd,J=15.2,8.2Hz,H-22),3.97(1H,m,H-3),2.70(1H,t,J=12.5Hz,H-7),2.13(1H,dd,J=12.5,4.6Hz,H-7),2.02(1H,m,H-20),1.99(1H,ov,H-12),1.88(1H,ov,H-4),1.86(1H,ov,H-2),1.84(1H,ov,H-9),1.83(2H,m,H-24),1.80(1H,ov,H-4),1.71(1H,ov,H-8),1.70(1H,ov,H-1),1.68(1H,m,H-16),1.56(1H,ov,H-25),1.54(1H,ov,H-1),1.47(1H,m,H-11),1.45(1H,ov,H-2),1.48(1H,m,H-15),1.29(1H,ov,H-14),1.28(1H,m,H-11),1.25(1H,m,H-16),1.23(1H,ov,H-12),1.17(1H,ov,H-17),1.02(1H,m,H-15),1.00(3H,d,6.6,H3-21),0.88(3H,d,6.6,H3-26),0.86(3H,d,6.6,H3-27),0.81(3H,s,H3-19),0.66(3H,s,H3-18)。13C-NMR(CDCl3,125MHz):δC 211.9(C,C-6),137.8(CH,C-22),126.5(CH,C-23),80.9(C,C-5),67.3(CH,C-3),56.5(CH,C-14),55.9(CH,C-17),44.6(CH,C-9),43.1(C,C-13),42.5(C,C-10),42.0(CH2,C-24),41.8(CH,C-7),40.1(CH,C-20),39.5(CH2,C-12),37.3(CH,C-8),36.5(CH2,C-4),30.4(CH2,C-2),29.8(CH2,C-1),28.6(CH,C-25),28.5(CH2,C-16),23.9(CH2,C-15),22.3(CH3,C-26),22.3(CH3,C-27),21.5(CH2,C-11),20.8(CH3,C-21),14.1(CH3,C-19),12.2(CH3,C-18)。
化合物30:白色无定形状粉末;m.p.248-250℃;(c 0.21,MeOH);IR(film)νmax3308,2923,2852,1698,1459,1376,1068cm-1;1H-NMR和13C-NMR数据见表5;HRESIMS m/z429.3372[M-H]-(calcd for C28H45O3,429.3369)。
化合物31:白色无定形状粉末;m.p.218-220℃;(c 0.20,MeOH);IR(film)νmax 3305,2953,2852,1708,1463,1381,963cm-1;1H-NMR和13C-NMR数据见表5;HRESIMS m/z 401.3058[M-H]-(calcd for C26H41O3,401.3056)。
化合物32:白色无定形状粉末;m.p.241-243℃;(c 0.08,MeOH);IR(film)νmax3315,2930,2854,1712,1459,1371,963cm-1;1H-NMR和13C-NMR数据见表5;HRESIMSm/z429.3373[M-H]-(calcd for C28H45O3,429.3369)。
化合物33:白色无定形状粉末;m.p.237-239℃;(c 0.15,MeOH);IR(film)νmax 3307,2926,2854,1698,1458,1370,970cm-1;1H-NMR和13C-NMR数据见表5;HRESIMS m/z 429.3366[M-H]-(calcd for C28H45O3,429.3369)。
化合物34:白色无定形状粉末;m.p.190-192℃;(c 0.15,MeOH);IR(film)νmax3371,2931,2868,1699,1467,1381cm-1;1H-NMR和13C-NMR数据见表4;HRESIMS m/z417.3366[M-H]-(calcd for C27H45O3,417.3369)。
化合物36:白色无定形状粉末;m.p.235-237℃;(c 0.20,MeOH);IR(film)νmax 3390,2927,2853,1698,1463,1377,1122,964cm-1;1H-NMR和13C-NMR数据见表6;HRESIMS m/z 431.3163[M-H]-(calcd for C27H43O4,431.3161)。
化合物37:白色无定形状粉末;m.p.192-194℃;(c 0.12,MeOH);IR(film)νmax 3377,2926,2854,1699,1463,1376cm-1;1H-NMR和13C-NMR数据见表6;HRESIMS m/z431.3158[M-H]-(calcd for C27H43O4,431.3161)。
化合物38:白色无定形状粉末;m.p.225-227℃;(c 0.09,MeOH);IR(film)νmax 3400,2929,2870,1698,1465,1379cm-1;1H-NMR和13C-NMR数据见表6;HRESIMS m/z445.3315[M-H]-(calcd for C28H45O4,445.3318)。
化合物39:白色无定形状粉末;m.p.237-239℃;(c 0.13,MeOH);IR(film)νmax 3390,2926,2853,1698,1456,1378,962cm-1;1H-NMR和13C-NMR数据见表6;HRESIMS m/z 417.3007[M-H]-(calcd for C26H41O4,417.3005)。
化合物41:白色无定形状粉末;m.p.204-206℃;(c 0.18,MeOH);IR(film)νmax 3365,2924,2851,1698,1456,1382,965cm-1;1H-NMR和13C-NMR数据见表7;HRESIMS m/z 431.3163[M-H]-(calcd for C27H43O4,431.3161)。
化合物42:白色无定形状粉末;m.p.192-194℃;(c 0.11,MeOH);IR(film)νmax 3334,2925,2853,1690,1466,1377cm-1;1H-NMR和13C-NMR数据见表7;HRESIMS m/z431.3159[M-H]-(calcd for C27H43O4,431.3161)。
化合物43:白色无定形状粉末;m.p.197-199℃;(c 0.12,MeOH);IR(film)νmax 3305,2923,2850,1689,1464,1378cm-1;1H-NMR和13C-NMR数据见表7;HRESIMS m/z445.3321[M-H]-(calcd for C28H45O4,445.3318)。
化合物44:白色针状结晶,(c 0.21,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.54(1H,s,H-7),4.24(2H,m,H2-11),4.17(1H,dd,J=10.0,1.8Hz,H-6),3.45(1H,m,H-3),3.25(1H,ov,H-14),2.23(1H,br d,J=11.6Hz,H-4),1.96(3H,s,-OAc),0.96(3H,d,J=6.8Hz,CH3-21),1.09(3H,s,CH3-19),0.66(3H,s,CH3-18),0.85(3H,s,CH3-27),0.84(3H,s,CH3-26)。
化合物45:白色针状结晶,(c 0.18,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.53(1H,d,J=1.8Hz,H-7),4.18(1H,dd,J=9.8,1.8Hz,H-6),3.69,(2H,m,H2-11),3.45(1H,m,H-3),3.25(1H,m,H-14),2.22(1H,br d,J=12.5Hz,H-4),2.05(1H,t,J=9.8Hz,H-17),1.27(3H,s,CH3-21),1.10(3H,s,CH3-19),0.84(3H,s,CH3-18),0.86(3H,d,J=6.6Hz,CH3-27),0.85(3H,d,J=6.6Hz,CH3-26);13C-NMR(100MHz,CDCl3)δ:206.2(C,C-9),149.9(CH,C-7),136.9(C,C-8),76.3(C,C-20),70.1(CH,C-3),69.7(CH,C-6),59.1(CH2,C-11),53.1(CH,C-17),47.3(CH,C-5),47.3(C,C-13),43.5(CH,C-14),42.1(C,C-10),40.9(CH2,C-22),40.0(CH2,C-12),40.0(CH2,C-24),33.2(CH2,C-4),31.3(CH2,C-1),30.7(CH2,C-2),30.5(CH,C-25),27.5(CH2,C-15),26.6(CH3,C-21),23.9(CH2,C-16),22.5(CH2,C-23),20.0(CH3,C-27),19.7(CH3,C-26),19.6(CH3,C-18),16.5(CH3,C-19)。
化合物46:白色针状结晶,(c 0.44,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.59(1H,s,H-7),4.20(1H,dd,J=10.0,1.8Hz,H-6),3.74(1H,m,H2-11),3.60(1H,m,H2-11),3.48(1H,m,H-3),3.27(1H,ov,H-14),2.27(1H,br d,J=11.6Hz,H-4),1.01(3H,d,J=6.8Hz,CH3-21),1.15(3H,s,CH3-19),0.72(3H,s,CH3-18),0.89(3H,d,J=6.5Hz,CH3-27),0.89(3H,d,J=6.5Hz,CH3-26)。
化合物47:白色针状结晶,(c 0.27,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.58(1H,s,H-7),4.28(1H,dd,J=9.9,1.8Hz,H-6),3.88(1H,m,H-11),3.67(1H,m,H-11),3.63(1H,m,H-3),3.43(1H,t,J=10.1,9.6Hz,H-14),2.31(1H,br d,J=11.0Hz,H-4),1.14(3H,s,CH3-19),0.96(3H,d,J=6.8Hz,CH3-21),0.86(3H,d,J=7.0Hz,CH3-28),0.83(3H,d,J=6.6Hz,CH3-27),0.82(3H,d,J=6.6Hz,CH3-26),0.64(3H,s,CH3-18)。
化合物48:白色针状结晶,(c 0.77,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.54(1H,d,J=1.7Hz,H-7),5.32(2H,m,H-22,H-23),4.16(1H,dd,J=9.8,1.8Hz,H-6),3.71(1H,m,H-11),3.59(1H,m,H-11),3.45(1H,m,H-3),3.25(1H,ov,H-14),2.23(1H,ov,H-4),1.11(3H,s,CH3-19),1.04(3H,d,J=6.8Hz,CH3-21),0.85(3H,s,CH3-27),0.85(3H,s,CH3-26),0.69(3H,s,CH3-18);13C-NMR(100MHz,CDCl3)δ:206.2(C,C-9),149.4(CH,C-7),138.1(CH,C-22),137.3(C,C-8),128.6(CH,C-23),70.8(CH,C-3),69.8(CH,C-6),59.3(CH2,C-11),52.1(CH2,C-17),44.0(CH2,C-24),49.9(CH,C-5),47.2(C,C-13),46.1(C,C-10),43.2(CH,C-14),42.4(CH2,C-12),39.9(CH,C-20),33.7(CH2,C-4),33.2(CH2,C-1),31.3(CH2,C-2),29.8(CH,C-25),28.1(CH2,C-15),27.0(CH2,C-16),22.7(CH3,C-27),22.7(CH3,C-26),22.0(CH3,C-21),18.0(CH3,C-18),16.5(CH3,C-19)。
化合物49:白色针状结晶,(c 0.32,CH3OH)。NMR数据见表8。
化合物50:白色针状结晶,(c 0.66,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.55(1H,d,J=5.8,1.8Hz,H-7),5.24(2H,m,H-22,H-23),4.17(1H,br d,J=9.9Hz,H-6),3.72(1H,m,H-11),3.60(1H,m,H-11),3.45(1H,m,H-3),3.25(1H,ov,H-14),2.23(1H,brd,J=12.8Hz,H-4),1.11(3H,s,CH3-19),1.04(3H,d,J=6.8Hz,CH3-21),0.91(3H,d,J=6.8Hz,CH3-28),0.85(3H,d,J=7.0Hz,CH3-27),0.85(3H,d,J=7.0Hz,CH3-26),0.69(3H,s,CH3-18);13C-NMR(100MHz,CDCl3)δ:206.1(C,C-9),149.3(CH,C-7),137.4(C,C-8),136.2(CH,C-22),134.2(CH,C-23),70.8(CH,C-3),69.7(CH,C-6),59.2(CH2,C-11),51.7(CH,C-17),49.6(CH,C-5),47.2(C,C-13),46.1(C,C-10),44.4(CH2,C-24),43.8(CH,C-14),42.3(CH2,C-12),40.2(CH,C-20),33.7(CH2,C-4),33.2(CH2,C-1),31.3(CH2,C-2),29.1(CH,C-25),28.2(CH2,C-15),27.1(CH2,C-16),22.1(CH3,C-21),20.6(CH3,C-27),20.0(CH3,C-26),18.1(CH3,C-28),17.7(CH3,C-18),16.5(CH3,C-19)。
化合物51:白色针状结晶,(c 0.13,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.58(1H,d,J=1.5Hz,H-7),5.12(1H,br s,H-24),4.28(1H,br d,J=8.4Hz,H-6),3.88(1H,m,H-11),3.66(1H,m,H-11),3.62(1H,m,H-3),3.43(1H,t,J=9.8,10.3Hz,H-14),2.32(1H,m,H-4),1.14(3H,s,CH3-19),0.96(3H,d,J=6.8Hz,CH3-21),1.68(3H,s,CH3-27),1.68(3H,s,CH3-26),0.65(3H,s,CH3-18)。
化合物52:白色针状结晶,(c 0.12,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.55(1H,d,J=1.8Hz,H-7),4.71(1H,s H-28),4.64(1H,s,H-28),4.17(1H,dd,J=10.0,2.0Hz,H-6),3.71(1H,m,H-11),3.57(1H,m,H-11),3.45(1H,m,H-3),3.25(1H,ov,H-14),2.23(1H,ov,H-4),1.12(3H,s,CH3-19),1.02(3H,ov,CH3-21),1.01(3H,ov,CH3-27),1.00(3H,ov,CH3-26),0.69(3H,s CH3-18)。
化合物53:白色针状结晶,(c 0.16,CH3OH)。1H-NMR(400MHz,CD3OD)δ:6.58(1H,s,H-7),5.10(1H,m,H-28),4.28(1H,d,J=10.4Hz,H-6),3.87(1H,m,H-11),3.65(1H,m,H-11),3.59(1H,m,H-3),3.43(1H,t,J=10.3,9.8Hz,H-14),2.31(1H,m,H-4),1.59(3H,d,J=6.8Hz,CH3-29),1.14(3H,s,CH3-19),0.98(3H,d,J=6.8Hz,CH3-21),0.98(3H,s,CH3-27),0.97(3H,s,CH3-26),0.65(3H,s,CH3-18)。
表2.化合物6和22的核磁共振数据(in CD3OD)
表3.化合物17、18和20的核磁共振数据(in CDCl3)
表4.化合物25、26和34的核磁共振数据(a In CD3OD,b In CDCl3)
表5.化合物30、31、32和33的核磁共振数据(in CDCl3)
表6.化合物36、37、38和39的核磁共振数据(in CD3OD)
表7.化合物41、42和43的核磁共振数据(in CD3OD)
表8.化合物49的核磁共振数据(in CD3OD)
本发明还提供了上述的甾体类化合物在制备抗肿瘤药物中的应用。
优选的,上述的甾体类化合物在制备抗肝癌药物中的应用。
进一步地,本发明提供了一种药物组合物,含有所述甾体类化合物作为活性成份和药学上可接受的载体。
本发明采用CCK-8法(Cell Counting Kit-8,Ishiyama M,Miyazono Y,SasamotoK,et al.A highly water-soluble disulfonated tetrazolium salt as a chromogenicindicator for NADH as well as cell viability[J].Talanta,1997,44(7):1299-1305.)对化合物1~53进行了体外抑制人肝癌Huh7干细胞增殖活性试验,试验结果表明,部分化合物对人肝癌Huh7干细胞增殖表现出了不同程度的抑制活性,可用于制备新型抗肝癌药物。
本发明对于制备新型抗肿瘤药物具有重要的参考价值,对开发利用中国的海洋药用生物资源具有重要意义。
具体实施方式
下面结合实施例对本发明作进一步详细的说明,但本发明的实施不仅限于此。
实施例1:化合物1~5的制备
取肉芝软珊瑚洗净剪碎,丙酮超声提取7次,再用甲醇超声提取3次,至提取液基本无色,合并丙酮及甲醇提取液,减压浓缩得粗浸膏,用等体积水分散,乙醚萃取数次,合并萃取液,减压回收乙醚至干,得乙醚层浸膏110g,乙醚层浸膏再用90%甲醇-水溶液分散,正己烷萃取5次后,浓缩90%甲醇-水溶液得红褐色浸膏10g。将90%甲醇-水溶液的10g粗浸膏,以二氯甲烷/乙醇梯度洗脱(100:1~1:1),经正相硅胶柱层析分离,薄层板监测,收集合并得到16个部分Fr.A~Fr.P。
Fr.O经过Sephandex LH-20凝胶柱色谱层析,流动相为甲醇:氯仿=1:1,根据薄层板监测,收集合并得到组分Fr.O-7,再经过反相硅胶柱(ODS)、RP-HPLC纯化,以86%甲醇-水溶液为流动相,示差检测器检测,收集化合物1和2。
Fr.N经过Sephandex LH-20凝胶柱色谱层析,流动相为甲醇:氯仿=1:1,薄层板监测,收集合并得到组分Fr.N-3,再经过反相硅胶柱(ODS)、RP-HPLC纯化,以79%甲醇-水溶液为流动相,示差检测器检测,收集化合物3。
Fr.M经过Sephandex LH-20凝胶柱色谱层析,流动相为甲醇:氯仿=1:1,薄层板监测,收集合并得到组分Fr.M-4,再经过反相硅胶柱(ODS)、RP-HPLC纯化,以84%甲醇-水溶液为流动相,示差检测器检测,收集化合物4。
Fr.L经过Sephandex LH-20凝胶柱色谱层析,流动相为甲醇:氯仿=1:1,薄层板监测,收集合并得到组分Fr.L-1,再经过反相硅胶柱(ODS)、RP-HPLC纯化,以96%甲醇-水溶液为流动相,示差检测器检测,收集化合物5。
实施例2:化合物6~24的制备
取新鲜紫柳珊瑚洗净剪碎,用重量为新鲜紫柳珊瑚重量的5~10倍的丙酮(丙酮中加入少量水)超声提取至提取液无色,减压回收丙酮至无丙酮味,用等体积水分散,乙醚萃取数次,合并萃取液,减压回收乙醚至干,得总浸膏。将总浸膏用正相硅胶色谱(200~300目)分离,用石油醚/乙酸乙酯系统梯度洗脱,通过薄层板监测,根据馏分极性大小收集,将总浸膏合并成16个部分。
Fr.12部分加入适量氯仿,过滤得滤渣,滤渣经过Sephandex LH-20凝胶柱色谱(流动相:甲醇:氯仿=1:1)洗脱,根据薄层板检测,收集合并得到Fr.12-1~Fr.12-4;Fr.12-3经过RP-HPLC纯化,以85%甲醇-水溶液为流动相,示差检测器检测,收集化合物6、7、8、9、10、11;Fr.12-2经过正相硅胶柱(流动相CHCl3:CH3OH=35:1)、RP-HPLC纯化,以85%甲醇-水溶液为流动相,示差检测器检测,收集化合物12、13、14、15、16;
Fr.6部分经过Sephandex LH-20凝胶柱色谱(流动相:石油醚:甲醇:氯仿=2:1:1)洗脱,根据薄层板检测,收集合并得到Fr.6-1~Fr.6-6;Fr.6-5,经正相硅胶柱(流动相:石油醚:丙酮=4:1)分离,薄层板检测,收集合并得到Fr.6-5-1~Fr.6-5-4;Fr.6-5-2经过RP-HPLC纯化,以90%甲醇-水溶液为流动相,示差检测器检测,收集化合物20、21;Fr.6-5-3经过RP-HPLC纯化,以95%甲醇-水溶液为流动相,示差检测器检测,收集化合物17、18、19;
Fr.13部分经正相硅胶柱分离,由氯仿:甲醇梯度洗脱,薄层板检测,收集合并得到Fr.13-1~Fr.13-4;Fr.13-3加入适量氯仿,过滤得滤渣,滤渣经过Sephandex LH-20凝胶柱色谱(流动相:甲醇:氯仿=1:1)洗脱,薄层板检测,收集合并得到Fr.13-3-1~Fr.13-3-3;Fr.13-3-2经过RP-HPLC纯化,以85%甲醇-水溶液为流动相,示差检测器检测,收集化合物22、23、24。
实施例3:化合物25~43的制备
取新鲜小月柳珊瑚洗净剪碎,用重量为新鲜小月柳珊瑚重量的5~10倍的丙酮(丙酮中加入少量水)超声提取至提取液无色,减压回收丙酮至无丙酮味,用等体积水分散,乙醚萃取数次,合并萃取液,减压回收乙醚至干,得总浸膏。将总浸膏用正相硅胶色谱(200~300目)分离,用石油醚/乙酸乙酯系统梯度洗脱,通过薄层板监测,根据馏分极性大小收集,将总浸膏合并成21个部分。
Fr.15部分经过Sephandex LH-20凝胶柱色谱(流动相:甲醇:氯仿=1:2)洗脱,薄层板检测,收集合并得Fr.15-1~Fr.15-8。Fr.15-7部分再经过正相硅胶色谱(二氯甲烷:甲醇=30:1)分离,薄层板监测,合并得到Fr.15-7-1~Fr.15-7-6。Fr.15-7-3通过半制备型RP-HPLC纯化,以90%甲醇-水溶液为流动相,示差检测器检测,收集化合物28、29、30、31、32、33;
Fr.19部分经过Sephandex LH-20凝胶柱色谱(流动相:甲醇:氯仿=1:2)洗脱,薄层板检测,收集合并得Fr.19-1~Fr.19-5。Fr.19-4部分再经过正相硅胶色谱(二氯甲烷:甲醇=20:1)分离,薄层板监测,收集合并得到Fr.19-4-1~Fr.19-4-19。Fr.19-4-7用半制备型RP-HPLC纯化,以95%甲醇-水溶液为流动相,示差检测器检测,收集化合物34;Fr.19-4-16用半制备型RP-HPLC纯化,以95%甲醇-水溶液为流动相,示差检测器检测,收集得到Fr.19-4-16-1~Fr.19-4-16-9。Fr.19-4-16-1用半制备型RP-HPLC纯化(流动相,90%的甲醇-水),紫外检测器检测,收集化合物25;Fr.19-4-16-4用半制备型RP-HPLC纯化(流动相,84%的甲醇-水),示差检测器检测,收集化合物35、40。Fr.19-4-16-3用半制备型RP-HPLC纯化(流动相,87%的甲醇-水),紫外检测器检测,收集得到Fr.19-4-16-3-a~Fr.19-4-16-3-i;Fr.19-4-16-3-d浓缩得到化合物39;Fr.19-4-16-3-g用半制备型RP-HPLC纯化(流动相,90%的乙腈-水),紫外检测器检测,收集化合物37和42;Fr.19-4-16-3-h用半制备型RP-HPLC纯化(流动相,78%的乙腈-水),紫外检测器检测,收集化合物36和41;Fr.19-4-16-3-i用半制备型RP-HPLC纯化(流动相,93%的乙腈-水),紫外检测器检测,收集化合物38和43。
Fr.20部分经过Sephandex LH-20凝胶柱色谱(流动相:甲醇:氯仿=1:2)洗脱,薄层板检测,收集合并得Fr.20-1~Fr.20-6。Fr.20-3部分再经过正相硅胶色谱(二氯甲烷:甲醇=20:1、15:1、10:1)洗脱,薄层板监测,收集合并得到Fr.20-3-1~Fr.20-3-9。Fr.20-3-8通过半制备型RP-HPLC纯化,以90%甲醇-水溶液为流动相,示差检测器检测,收集化合物26、27。
实施例4:化合物44~53的制备
取新鲜侧扁软柳珊瑚(Subergorgia suberosa)洗净剪碎,用重量为新鲜侧扁软柳珊瑚重量的5~10倍的丙酮超声提取至提取液无色,减压回收丙酮至无丙酮味,用等体积水分散,乙醚萃取数次,合并萃取液,减压回收乙醚至干,得总浸膏。总浸膏经过SephandexLH-20凝胶柱色谱(流动相:二氯甲烷:甲醇=1:1)洗脱,薄层板检测,收集合并得到Fr.A和Fr.B两部分。
Fr.A部分经过反相硅胶柱色谱,用甲醇/水梯度洗脱,薄层板监测,合并得到Fr.A1~Fr.A6。Fr.A4用半制备型RP-HPLC纯化,以70%乙腈-水溶液为流动相,示差检测器检测,收集化合物47和53;Fr.A5用半制备型RP-HPLC纯化,以77%乙腈-水溶液为流动相,示差检测器检测,收集得到化合物51;Fr.A3部分经正相硅胶柱层析,石油醚/丙酮体系梯度洗脱,薄层板监测,合并得到Fr.A2-4,Fr.A2-4用半制备型RP-HPLC纯化(流动相,80%的甲醇-水),示差检测器检测,收集化合物44、45、46、48、49、50和52。
实施例5:本发明的甾体化合物的抗肿瘤干细胞活性
一、实验方法
采用CCK-8法对所述甾体化合物进行肿瘤干细胞增殖抑制试验。
1.实验用细胞株:human Huh7Stem cell(人肝癌干细胞)。实验用细胞株购自中国科学院细胞库。
2.实验试剂、耗材和仪器:
DMEM/F12培养液(Corning);ITS(Life technologies);B27(Life technologie);bFGF(Life technologie);EGF(Life technologie);EDTA胰酶(Invitrigen);DMSO(sigma);青霉素链霉素(Hyclone)、CCK-8(日本同仁);超低吸附10cm培养皿和96孔培养板(Corning);移液管(Corning);CO2孵箱(SANYO);酶标仪(Thermo Multiskan MK3)。
3.实验用药:
部分实施例1~4制备的甾体化合物,
阳性对照药:阿霉素(Adriamycin),大连美仑生物科技有限公司。
4.细胞培养
Huh7干细胞培养:人肝癌细胞(Huh7stemcell)在含有DMEM/F12培养液50ml、B27(1ml)、ITS(500μL)、bFGF(10μl,100μg/ml)、EGF(2μl,10μg/ml)、青霉素链霉素450μl配成的培养液里,37℃、5%CO2条件下培养,每天补充新鲜干细胞培养液,始终保持培养液微红。约第四天开始传第一代,以后每三天传一代,等传至第四代以后,干细胞纯度可达90%左右,可用于试验。
将细胞液完全吸入15ml离心管内,静置约20-30min,成球的肝癌肿瘤干细胞会慢慢沉积于离心管底部,小心吸去上层液,用0.25%的EDTA胰酶消化,直至液体澄清。终止消化,放入离心机内在1200R的条件离心5min,小心吸去上层液。用培养液混悬细胞,计数铺板。将细胞浓度调至106个/ml种于96孔板中,每孔100μl,于18~24h后进行实验。
5.细胞活力检测实验
Huh7干细胞活力检测实验:于实验前以105个细胞/100μl·孔的细胞浓度接种96孔板。37℃、5%CO2条件下培养24h后,每孔分别加100μl药物浓度为50μmol/L的培养液,设立两个重复组、DMSO对照组和阿霉素(10μg/ml)阳性对照组。给药后,37℃、5%CO2条件下孵育48h。每孔加入20μl CCK-8试剂,37℃、5%CO2条件下孵育2h。用酶标仪在450nm条件下检测OD值。
二、实验结果
通过CCK-8法测定部分实施例1~4制备的甾体类化合物体外抑制肿瘤干细胞增殖活性,部分实施例1~4制备的甾体类化合物表现出不同程度的抑制活性,其肿瘤干细胞抑制率见表9所示,其他化合物无抑制活性。
表9部分实施例1~4制备的甾体化合物的人肝癌Huh7干细胞增殖抑制试验(IC50μM)
由表9的数据可以看出,化合物1、2、8、11、23、50对人肝癌Huh7干细胞具有抑制活性,可用于制备新型抗肿瘤药物。
本发明对于制备新型抗肿瘤药物具有重要的参考价值,对开发利用中国的海洋药用生物资源具有重要意义。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
Claims (6)
1.一种甾体类化合物,其化学结构如式I所示:
式I中,R1基团为H或OH或═O R2基团为H或Ac
R3基团为OH或H或OAc R4基团为OH或═O
R5基团为H或双键 R6基团为H或双键
R7选自以下a~o中的任一个结构:
2.根据权利要求1所述的甾体类化合物,其特征在于,所述的甾体类化合物为
胆甾-1β,3β,5α,6β-四醇、
24S-胆甾-1β,3β,5α,6β-四醇、
24S-胆甾-1β,3β,5α,6β-四醇-25-单乙酸酯、
24S-胆甾-3β,5α,6β,25-四醇、
24S-胆甾-3β,5α,6β-三醇-25-单乙酸酯、
(22E)-22-烯-24-降胆甾-3β,5α,6β-三醇、
24(25)-烯-胆甾-3β,5α,6β-三醇、
(22E)-22-烯-胆甾-3β,5α,6β-三醇、
24(28)-烯-麦角甾-3β,5α,6β-三醇、
(22E)-22-烯-麦角甾-3β,5α,6β-三醇、
(22E)-22-烯-24-胆甾-3β,5α,6β-三醇、
(22E)-7,22-二烯-胆甾-3β,5α,6β-三醇、
7(8),24(28)-二烯-麦角甾-3β,5α,6β-三醇、
7-烯-胆甾-3β,5α,6β-三醇、
(22E)-7,22-二烯-24-降胆甾-3β,5α,6β-三醇、
(22E)-7,22-二烯-麦角甾-3β,5α,6β-三醇、
(22E)-22-烯-24-降胆甾-3β,5α,6β-三醇-3β-醋酸酯、
(22E)-22-烯-胆甾-3β,5α,6β-三醇-3β-醋酸酯、
胆甾-3β,5α,6β-三醇-3β-醋酸盐、
(22E)-22-烯-5α-甲氧基-胆甾-3β,6β-二醇、
5α-甲氧基-胆甾-3β,6β-二醇、
22-烯-24-降胆甾-1β,3β,5α,6β-四醇、
22-烯-胆甾-1β,3β,5α,6β-四醇、
麦角甾-1β,3β,5α,6β-四醇、
(23E)-23-烯-胆甾-25-过氧-3β,5α,6β-三醇、
24(25)-烯-胆甾-1β,3β,5α,6β-四醇、
24(28)-烯-麦角甾-1β,3β,5α,6β-四醇、
胆甾-3β,5α-二醇-6-酮、
(22E)-22-烯-胆甾-3β,5α-二醇-6-酮、
24(28)-烯-麦角甾-3β,5α-二醇-6-酮、
(22E)-22-烯-24-降胆甾-3β,5α-二醇-6-酮、
(24S,22E)-22-烯-麦角甾-3β,5α-二醇-6-酮、
(24R,22E)-22-烯-麦角甾-3β,5α-二醇-6-酮、
胆甾-1β,3β-二醇-6-酮、
胆甾-1β,3β,5α-三醇-6-酮、
(22E)-22-烯-胆甾-1β,3β,5α-三醇-6-酮、
24(25)-烯-胆甾-1β,3β,5α-三醇-6-酮、
24(28)-烯-麦角甾-1β,3β,5α-三醇-6-酮、
(22E)-22-烯-24-降胆甾-1β,3β,5α-三醇-6-酮、
胆甾-3β,5α,6β-三醇-1-酮、
(22E)-22-烯-胆甾-3β,5α,6β-三醇-1-酮、
24(25)-烯-胆甾-3β,5α,6β-三醇-1-酮、
24(28)-烯-麦角甾-3β,5α,6β-三醇-1-酮、
11-acetoxy-3β,6α-dihydroxy-9,11-seco-5α-cholest-7-en-9-one、subergorgol B、
3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7-ene-9-one、Sarcomilasterol、
subergorgol I、
24-nor-3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7,22(E)-dien-9-one、(24R)-methyl-3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7,22E-diene-9-one、subergorgolH、
3β,6α,11-trihydroxy-24-methylene-9,11-seco-5α-cholest-7-en-9-one,或subergorgol J。
3.根据权利要求2所述的甾体类化合物的制备方法,其特征在于,所述的制备方法如下:
将经过预处理的珊瑚粉碎,用丙酮及甲醇超声提取数次,至提取液基本无色,浓缩后得到粗浸膏;将粗浸膏分散在等体积水中,用乙醚萃取数次,合并萃取液,减压回收乙醚至干,得乙醚层浸膏;或乙醚层浸膏再用90%甲醇-水溶液分散,正己烷萃取数次后,浓缩90%甲醇-水溶液得红褐色浸膏;将乙醚层浸膏或90%甲醇-水溶液浸膏用正相硅胶色谱分离,分别用不同的洗脱剂系统梯度洗脱,通过薄层板监测,根据馏分极性大小收集,将总浸膏合并成若干个部分;然后每部分用不同条件的洗脱剂进行Sephadex LH-20凝胶色谱分离、正反相硅胶柱层析,后经反相制备型HPLC纯化,即得。
4.如权利要求1或2所述的甾体类化合物在制备抗肿瘤药物中的应用。
5.根据权利要求4所述的甾体类化合物在制备抗肿瘤药物中的应用,其特征在于,所述的肿瘤为肝癌。
6.一种药物组合物,其特征在于:所述的药物组合物包括如权利要求1或2所述的甾体类化合物作为活性成份和药学上可接受的载体。
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