CN1158298C - 抗肿瘤化合物及其制备方法和制药用途 - Google Patents
抗肿瘤化合物及其制备方法和制药用途 Download PDFInfo
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Abstract
本发明涉及式A所示的化合物3β,5α,6β,7α-四羟基-8(9),22 Z-二烯麦角烷[ergosta-8(9),22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22Z),简称为化合物A]以及从海洋植物真菌2059(保藏号CCTCCM202030)的菌体中提取分离制备化合物A的方法和化合物A在制备抗肿瘤药物中的应用。
Description
技术领域
本发明涉及一种具有抗肿瘤活性的化合物3β,5α,6β,7α-四羟基-8(9),22 Z-二烯麦角烷[ergosta-8(9),22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22Z),以下简称为化合物A]及其制备方法和在制备抗肿瘤药物中的应用。
背景技术
自从青霉素(penicillin)引入了医学,就标志着真菌抗生素的时代已经开始。在过去的60年里,从陆地真菌发现了大量的结构独特的天然产物,其中很多已用作药物或作为生物医药发展的潜在工具。真菌的代谢产物作为药物的丰富来源,很多在临床上作为抗生素应用,大量的研究表明,真菌的代谢产物还有其它的药用价值,如抗肿瘤,治疗心血管疾病,免疫调节剂,酶抑制剂等。由于海洋环境的特殊性,海洋真菌具有独特的代谢途径和遗传背景,能提供陆生真菌无法提供的代谢产物。国际上已从海洋真菌中发现了一些结构独特的化合物,分别具有抗菌,抗病毒,抗肿瘤和心血管方面的活性。例如,1945年7月,Giuseppe Brotzu发现顶头孢霉(Cephalospoiun acremonium)有抑制革兰氏阳性细菌和革兰氏阴性细菌的生长的活性,后经分离得到多种抗菌素,即头孢菌素。五十多年过去了,头孢菌素在临床上仍广泛使用。从真菌筛选,到半合成结构改造,目前产品品种已达200多种,产量占世界上抗生素产量的60%以上,堪称后起之秀。还有其他的一些例子见Kerstin Liberra的文章《Marine fungi-a profileresource of biologically active natural products?》[Pharmazie 50(1995),H.9:583],国际上这方面的研究从八十年代以来呈加速发展的趋势。国内从海洋真菌中也发现一批新的活性物质(Lin YC et al Tetrahedron2000,Lin YC et al Tetrahedron Letters,2000,Lin YC et al J.Org.Chem.2001)。
有关四羟基甾醇的文献报道,目前还非常少。从陆地木腐菌Polyporus versicolor,真菌Agaricus blazei的子实体和海绵Spongia officinali[A.Migliuolo,et al.J.Nat.Prod.,1990,53,1414-1424]和中国苦红菇Russula rosacea中[王淮滨等,中草药,1994,25(7),342-343]分离到一种四羟基甾醇Ergosta-7,22-diene-3,5,6,9-tetraol(3β,5α,6β,9α)。T.Ishizuka等从真菌Grifola frondosa的子实体中,分离到18个甾醇类化合物,其中有两个四羟基甾醇Ergosta-8(9),22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22E)和Ergosta-8(14),22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22E)[T.Ishizuka,et al.Chem.Pharm.Bull.,1997,45(11),1756-1760]。根据文献报道,四羟基甾醇基本上都具有细胞毒活性,但三羟基甾醇很多并不具有细胞毒,羟基的存在和细胞毒性之间的关系,四羟基甾醇作用机理等现在都还不清楚,这些也都值得进一步的研究。
植物内源性真菌(endophytic fungi)生活在高等植物的组织中,对于内源性真菌的次级代谢物的研究还较为缺乏。据保守的估计内源性真菌的种类繁多,大约有1.5×106种,由于数量庞大,而且与其它生物之间紧密的生态关系,使这类真菌成为潜在的具有产生丰富的次级代谢物的来源。目前国内其它单位对生长于海洋环境的内源性真菌的次级代谢物的研究还未见报道。
发明内容
本发明的目的在于提供一种新的具有潜在药用价值的新化合物及其从海洋植物内生真菌提取分离的方法,以及该化合物在制备抗肿瘤药物中的用途。
本发明化合物A为下列结构式所示:
本发明的化合物A可以从海洋植物真菌,例如南海海洋红树林Castaniopsis fissa的内生真菌2059(以下称为海洋植物真菌2059)的菌体中提取分离而得到。
本发明所用的海洋植物真菌2059已保藏于中国典型培养物保藏中心(CCTCC,中国,武汉大学校内),保藏号为CCTCC №:M202030,保藏日为2002年8月3日。
本发明化合物A的制备方法包括以下步骤:
a.海洋植物真菌CCTCC M202030的种子培养:
选用PDA(potato dextro se agar)培养基,其组成为:马铃薯200g,葡萄糖20g,琼脂20g,水1L;制成试管斜面,挑取菌株接入斜面,25-28℃培养5-7天;
b.海洋植物真菌CCTCC M202030的发酵培养:
发酵培养基成分配比按重量比为:葡萄糖5-15,酵母提取物0.5-1.5,蛋白胨1-3,粗海盐30-50,水1000;将斜面中培养好的菌株挑入发酵培养基,于室温25-28℃静置1-2月;
c.将上述培养好的发酵液过滤,收集菌体,晾干;
d.菌体在室温下用甲醇浸泡,合并提取液,浓缩得到总提取物;
e.用乙酸乙酯溶解提取物,溶解物在硅胶柱中进行色谱分离,以石油醚-乙酸乙酯-甲醇梯度淋洗;
f.收集石油醚∶乙酸乙酯=1∶3得到的洗脱组分,进一步硅胶柱层析,以氯仿∶甲醇=7∶1为洗脱剂,纯化得到白色无晶形固体,即为所需的化合物A。
本发明的试验证明,化合物A能抑制肿瘤细胞株的生长,在以人肝癌细胞株和人大细胞肺癌细胞株为靶细胞的MTT还原法检测抗肿瘤活性试验中,化合物A的半数致死量IC50分别为8.445和5.03μg/ml。
表明化合物A可用于制备抗肿瘤药物。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1:化合物A的制备
a.海洋植物真菌CCTCC M202030的种子培养:
菌种以PDA为培养基,组成为:马铃薯200g,葡萄糖20g,琼脂20g,水1L。制成试管斜面,挑取菌株接入斜面,25-28℃培养7天后于4℃保存。
b海洋植物真菌CCTCC M202030的发酵培养:
发酵培养基组成为:葡萄糖10g,酵母提取物1g,蛋白胨2g,粗海盐40g,水IL;将斜面中培养好的菌株挑入发酵培养基,于室温25-28℃静置培养1个月;
c.将上述培养好的发酵液过滤,收集菌体,晾干;
d.菌体在室温下用甲醇浸泡7天,共浸提3次,合并提取液,旋转蒸发浓缩得到总提取物;
e.用乙酸乙酯溶解提取物,溶解物在硅胶柱中进行色谱分离,以石油醚-乙酸乙酯-甲醇梯度淋洗;
f.收集由石油醚-乙酸乙酯(1∶3)洗脱得到的组分,进一步硅胶柱层析,以氯仿-甲醇(7∶1)为洗脱剂,纯化得到白色无晶形固体化合物A。
化合物A的试验数据:
m.p.145~150℃。元素分析(w%,C28H46O4):C 75.54,H 10.22,N 0.000;计算值:C 75.34,H 10.31,,N 0.000;IRυ/cm-1(KBr):3423,2959,2872,1668,1634,1461,1378,1276,1158,1067,969,915,878,805,733。APCI-MS:429[M-H2O+H]+,411[M-2H2O+H]+,393[M-3H2O+H]+,375[M-4H2O+H]+,267,251,187,171,131,105等。化合物A的NMR实验数据如表1所示。
实施例2:MTT还原法检测化合物A抗肿瘤活性试验
1.材料:
1.1四脞盐(MTT):用0.01mol/L的磷酸盐缓冲液(PBS)溶解MTT〔3-(4,5-dimethythiazol-z-yl)2,5-diphenytetrazolium bromide,SIGMA〕终浓度5mg/ml,过滤除菌,分装后4℃避光保存。
1.2 MTT裂解液的配制:80g的十二烷基磺酸钠(SDS,华美生物工程公司)溶解在200ml的N-N-二甲基甲酰胺(北京化工厂)中,水浴加热助溶,加入200ml蒸馏水,用80%乙酸与1N盐酸(1∶1)混合调pH至4.7。
1.3细胞株选用:人正常细胞株(L-02),人肝癌细胞株(Bel-7402)和人大细胞肺癌细胞株(NCI4460)。
2.操作步骤:
a.单细胞悬液接种于96孔板(用RPMI-1640基础培养基将细胞稀释至3×104/ml,每孔加入200μl稀释好的细胞),37℃,5%CO2,饱和湿度下培养24小时;每组四个平行样;
b.去除培养基,取新配制培养基按系列浓度制备抗癌药物(化合物A)溶液,每孔200μl,培养48小时;
c.每孔加入2mg/ml的MTT20μl,孵育4小时;
d.吸出孔内培养液(尽量完全),加入DMSO液(150μl/孔),振荡10分钟,使结晶物充分溶解;
e.酶标仪检测各孔OD值,(λ=570nm);
f.绘制细胞活力曲线图,求出IC50值。
试验结果如表2所示。试验结果说明,化合物A具有较强的细胞毒,其毒性对正常细胞和癌细胞具有一定的选择性;化合物A可用于制备抗肿瘤药物。
表1化合物A的NMR数据(Acetone-d6,TMS,ppm)
碳位号 δC DEPT δH HMBC 1H-1HCOSY
1 31.1 CH2 1.54 1.13,1.57 1.73
1.73 1.54
2 31.9 CH2 1.57 1.85,3.76
1.85 1.57
3 68.6 CH 3.76 1.73,2.13 1.57,2.13
4 40.2 CH2 1.32 2.13
2.13 1.32,3.76
5 64.9 C 1.13,1.32,1.73
6 67.5 CH 4.20(brd,10.0Hz) 3.13 3.13,3.34
7 63.3 CH 3.13(d,2.5Hz) 4.20
8 128.2 C 2.22,3.13
9 134.3 C 1.13,1.94,2.02,2.22
10 39.0 C
11 24.4 CH2 1.94 1.38,2.01,2.02
2.02 1.38,1.94,2.01
12 36.8 CH2 1.38 0.64,1.94
2.01
13 42.8 C 0.64,1.22,2.18,2.22
14 50.8 CH 2.22 0.64,2.05 1.32
15 30.0 CH2 1.32 1.22 2.05,2.18
2.05 1.32
16 24.2 CH2 2.18 1.22,1.32
17 54.6 CH 1.22 0.64,1.05 1.32,2.10,2.18
18 11.7 CH3 0.64(s) 1.22,1.38
19 22.8 CH3 1.13(s)
20 41.3 CH 2.10 0.64,1.05,1.22 1.05,1.22,1.32
21 21.4 CH3 1.05(d,6.5Hz) 2.10
22 136.8 CH 5.24(d,6.0Hz) 1.05,1.88,2.10
23 132.6 CH 5.25(d,6.0Hz) 0.94,1.88,2.10
24 43.7 CH 1.88 0.84,0.86,0.94 0.94
25 18.1 CH3 0.94(d,7.0Hz) 1.88
26 33.8 CH 1.99 0.84,0.86,0.94
27 20.3 CH3 0.86(d,8.0Hz) 0.84 0.84
28 20.0 CH3 0.84(d,6.5Hz) 0.86 0.86
3-OH OH 3.68(d,5.0Hz)
5-OH OH 2.77
6-OH OH 3.34(d,10.0Hz)
7-OH OH 2.77
表2A的细胞毒性试验结果
试验细胞 正常人肝细胞株 人肝癌细胞株 人大细胞肺癌细胞株
L-02 Bel-7402 NCI4460
IC50(μg/ml) 13.621 8.445 5.03
Claims (3)
2.权利要求1所述化合物A的制备方法,其特征是该方法包括以下步骤:
a.海洋植物真菌CCTCC M202030的种子培养:
选用PDA培养基,其组成为:马铃薯200g,葡萄糖20g,琼脂20g,水1L;制成试管斜面,挑取菌株接入斜面,25-28℃培养5-7天;
b.海洋植物真菌CCTCC M202030的发酵培养:
发酵培养基成分配比按重量比为:葡萄糖5-15,酵母提取物0.5-1.5,蛋白胨1-3,粗海盐30-50,水1000;将斜面中培养好的菌株挑入发酵培养基,于室温25-28℃静置1-2月;
c.将上述培养好的发酵液过滤,收集菌体,晾干;
d.菌体在室温下用甲醇浸泡,合并提取液,浓缩得到总提取物;
e.用乙酸乙酯溶解提取物,溶解物在硅胶柱中进行色谱分离,以石油醚-乙酸乙酯-甲醇梯度淋洗;
f.收集石油醚∶乙酸乙酯=1∶3得到的洗脱组分,进一步硅胶柱层析,以氯仿∶甲醇=7∶1为洗脱剂,纯化得到白色无晶形固体,即为所需的化合物A。
3.权利要求1所述的化合物A用于制备抗肿瘤药物。
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