CN101879159B - 3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用 - Google Patents
3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明属制药领域,涉及3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用。本发明提供的3,5-二甲基补骨脂素的分子式为C13H10O3,分子量为214.22,具有广谱抗肿瘤活性,体外对多种人肿瘤细胞均有强烈的抑制效应,其抑制肝癌细胞HepG2增殖的机理是诱导癌细胞凋亡。因此其与药用敷料组合,可在制备抗肿瘤活性药物中应用,尤其是抗肝癌、抗乳腺癌、抗***、抗皮肤癌、抗鼻咽癌及抗结肠癌药物中的应用。本发明为上述癌症的治疗提供了更多选择,且3,5-二甲基补骨脂素对正常细胞的毒性小,有利于临床应用。
Description
技术领域
本发明涉及医药技术领域,具体涉及3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用。
背景技术
3,5-二甲基补骨脂素(简称DMFC),分子式为C13H10O3是补骨脂素衍生物,其合成方法见参考文献1,具有式(I)的结构,
迄今为止,没有文献报道3,5-二甲基补骨脂素在制备抗肿瘤药物中应用。恶性肿瘤是当今威胁人类身心健康最严重的疾病,死亡率高,目前尚无高效低毒的抗肿瘤药物报道,因此急需开发这类药物。
发明内容
本发明的目的是提供一种3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用。
3,5-二甲基补骨脂素(简称DMFC),具有式(I)的结构式:本发明所制备的3,5-二甲基补骨脂素药物,可在制备治疗肝癌、乳腺癌、子***、皮肤癌、鼻咽癌、结肠癌等药物中的应用。
本发明的3,5-二甲基补骨脂素可以和药学上允许的任意一种辅料或药物赋形剂制成药物,其制剂可以是药学上允许的任意一种剂型,包括但不限于液体制剂、颗粒剂、片剂、冲剂、软胶丸、软胶囊、滴丸剂或注射剂。
本发明提供的药物,其给药形式主要包括口服给药或注射给药。
实验证明本发明提供的3,5-二甲基补骨脂素,具有体外抑制肿瘤细胞增殖的作用,经四甲基偶氮唑盐(简称MTT法)检测,48小时处理肿瘤细胞后,对肝癌HepG2的半数抑制增值率IC50约为8.46±0.28μM,对乳腺癌MCF-7为33.5±4.95μM、对子***SIHA为68.5±9.19μM,对上皮癌A431为32.33±4.21μM、对鼻咽癌CNE2为105.5±8.91μM、对结肠癌CaCo2为124.5±7.62,并且3,5-二甲基补骨脂素杀死肝癌HepG2细胞的机理是诱导其产生凋亡,因此其与药用敷料组合,可在制备治疗恶性肿瘤药物中应用。
本发明的优点:本发明公开了3,5-二甲基补骨脂素在制备抗肿瘤药物中的应用,尤其是抗肝癌、抗乳腺癌、抗***、抗上皮癌、抗鼻咽癌及抗结肠癌药物中的应用,为上述癌症的治疗提供了更多选择,且3,5-二甲基补骨脂素对正常细胞的毒性小,有利于临床应用。
附图说明
图1为DMFC对肝癌HepG2细胞的细胞毒性试验。
图2为DMFC诱导肝癌HepG2细胞产生凋亡的DNA梯状条带凝胶电泳图。
图3为DMFC诱导肝癌HepG2细胞产生凋亡的AnnexinV-PI双染流式图。
图4为Caspase-8和Caspase-9抑制剂逆转DMFC诱导的HepG2细胞凋亡作用。
具体实施方式
以下将结合具体实施例和附图对本发明做进一步阐述,这些实例仅用于说明目的,而不用于限制本发明范围。
实施例一 3,5-二甲基补骨脂素(DMFC)的合成及鉴定[1]
(1)合成香豆素衍生物1
间苯二酚(3.96克,36毫摩尔)、乙酰乙酸乙酯(5.59克,43毫摩尔)和12M硫酸(60毫升)的混合物在室温下搅拌15小时,然后将该反应后的混合液倒入冰水(50毫升)中,沉淀物经过滤和水洗后,用甲醇重结晶得纯的香豆素衍生物1(5.70克,90%产率)。产物为白色粉末状固体,熔点:182-184℃。
(2)合成香豆素衍生物2
上述的香豆素衍生物1(5.46克,31毫摩尔)溶解于干燥的丙酮(75毫升)和二氯甲烷(75毫升),加入碳酸钾(8.55克,62毫摩尔)。搅拌下滴加氯代丙酮(5.7克,62毫摩尔),滴完后混合物搅拌回流24小时。反应结束后,混合液冷却、过滤,用丙酮和二氯甲烷洗涤滤渣各一次。滤液合并、旋干,用甲醇重结晶或柱层析(石油醚∶乙酸乙酯=1∶1作为洗脱液,硅胶为固定相)得纯的香豆素衍生物2(5.61克,78%产率)。产物为白色粉末状固体,熔点:147-149℃。
(3)合成补骨脂素衍生物(DMFC)I
将上述的香豆素衍生物2(5.33克,23毫摩尔)和1.0M氢氧化钠(140毫升)的混和液在氮气保护下回流24小时。反应混合液冷却后用2.0M盐酸酸化,沉淀物经过滤和水洗后,用甲醇重结晶或柱层析(石油醚∶乙酸乙酯=1∶2作为洗脱液,硅胶为固定相)得纯的补骨脂素衍生物I,根据氢核磁共振数据及熔点,对比参考文献[2]即知其为3,5-二甲基补骨脂素(4.04克,82%产率)。产物为白色粉末状固体,熔点:220-222℃;氢核磁共振(300MHz,CDCl3)δ(ppm):7.67(s,1H),7.46(s,1H),7.39(s,1H),6.25(s,1H),2.52(s,3H),2.29(s,3H)。
实施例二3,5-二甲基补骨脂素对人肿瘤细胞增殖抑制活性试验
实验方法:
肝癌细胞HepG2、乳腺癌细胞MCF-7、***细胞SIHA、上皮癌A431、鼻咽癌CNE2、结肠癌CaCo2均购自美国American Type Culture Collection。细胞培养在DMEM培养基(Invitrogene),添加5%小牛血清(Invitrogene),2mM谷氨酰胺,37℃,10%CO2培养箱。
四甲基偶氮唑盐(简称MTT法)检测药物对肿瘤细胞增殖的影响:选择对数生长期细胞,以7000/孔的密度接种于96孔板中,置37℃,10%培养箱中培养24小时后加入不同浓度的药物,每个浓度设复孔6个,分别培养24、48和72小时,吸出孔板中培养液,往每孔中加入浓度为1mg/ml的MTT 50μl,放入培养箱中继续培养3-4小时,取出加过MTT的96孔板,每孔加入150μl的DMSO,轻轻震荡孔板30min,待孔底部的结晶物完全溶解后,将孔板放入酶标仪中测波长在570nm的各孔的光吸收值,记录结果,实验至少重复三次,用IC50软件计算半数增殖抑制浓度IC50。
附图1a结果显示24、48和72小时的DMFC处理后,肝癌HepG2细胞的IC50分别为16.33±0.13μM for 24h,8.46±0.28μM for 48h and 5.97μM±0.55。可见DMFC对肝癌细胞HepG2细胞的细胞毒性表现为剂量和时间的依赖性,而对正常肝细胞MIHA的细胞毒性IC50为40.75±1.06μM(附图1b),较肝癌细胞小5倍,表明DMFC对正常细胞的毒性小,可以在临床上应用。
此外,DMFC处理48小时,对乳腺癌MCF-7的IC50为33.5±4.95μM、对***SIHA的IC50为68.5±9.19μM,对上皮癌A431的IC50为32.33±4.21μM、对鼻咽癌CNE2的IC50为105.5±8.9μM、对结肠癌的CaCo2的IC50为124.5±7.62表明DMFC可以在制备治疗上述恶性肿瘤中应用。
实施例三3,5-二甲基补骨脂素诱导肝癌细胞HepG2产生凋亡
(1)DNA梯状条带实验:肿瘤细胞HepG2经不同浓度的DMFC处理48h后收集细胞,加裂解液在37℃裂解过夜(5mM Tris-HCl,100mM EDTA,1%(w/v)SDS,and蛋白酶K),基因组DNA用酚/氯仿/异戊醇(25∶24∶1)抽提,70%酒精-20℃下沉淀,用RNA酶除去RNA后,加40μg DNA在1.2%琼脂糖凝胶上电泳并经凝胶成像仪拍照。
细胞凋亡最典型的特征之一,是细胞在接受药物作用后,DNA会被核酸酶在核小体连接处切断,产生180-200bp或其整数倍大小的DNA碎片,琼脂糖凝胶电泳时呈现有规则的梯状体条带,这是区分凋亡与坏死的重要标志。附图2结果显示琼脂糖凝胶电泳时呈现有规则的梯状体条带,DMFC以浓度0、8μM、16μM、32μM和64μM处理,可见DMFC可诱导HepG2产生典型的凋亡条带,并呈剂量依赖性。
(2)AnnexinV-PI双染实验:约1.5x105个对数生长期的肝癌HepG2细胞与DMFC共同孵育48h,,胰酶消化收集细胞,PBS清洗一次,加入500μl的AnnexinV-FITC结合缓冲液,含2.5μl的AnnexinV-FITC和0.5μl的PI缓冲液,然后避光孵育15min,用FACSCanto流式细胞仪(Becton Dickinson)分析结果。
细胞凋亡早期,细胞膜上的磷脂酰丝氨酸(PS)会从细胞膜内翻转到细胞膜外,AnnexinV可与其结合,而凋亡细胞的细胞膜仍然保持完整,染料PI不能进入细胞进行染色,因此通过AnnexinV-PI双染实验,可区分凋亡与坏死细胞。附图3a中HepG2细胞的5个不同DMFC浓度0、8μM、16μM、32μM和64μM处理。每张图的右下角百分数都表示该细胞在对应药物浓度下的凋亡百分率,而右上角百分数则代表相同条件下的死亡百分数。从图中可以看到,DMFC可以诱导HepG2细胞产生凋亡,并且这种作用呈现了剂量依赖性。图3b是图3a的量化分析图,图中清楚地表明随着DMFC浓度从0增至64μM,HepG2凋亡百分率上升至24%。
实施例四3,5-二甲基补骨脂素诱导肝癌细胞HepG2产生凋亡涉及的信号通路
(1)Caspase-8和Caspase-9抑制剂逆转DMFC诱导的HepG2细胞凋亡作用
在细胞凋亡的信号传导过程中,caspase(半胱氨酸水解酶)起着关键性的调节作用,因此它们的激活是凋亡的重要特征,两个主要的caspase家族成员caspase-8和caspase-9分别代表了两条凋亡信号通路即死亡受体通路和线粒体通的启动者。
约1.5x105个对数生长期的HepG2细胞分别与DMFC/caspase-8抑制剂(Z-IETD-FMK)或DMFC/caspase-9抑制剂(Z-LEHD-FMK)共同孵育48小时,胰酶消化收集细胞,PBS清洗一次,加入500μl的Annexin V-FITC结合缓冲液,含2.5μl的AnnexinV-FITC和0.5μl的PI缓冲液,然后避光孵育15min,用FACSCanto流式细胞仪(Becton Dickinson)分析结果。
附图4a中,HepG2细胞的对照、DMFC 32μM单独处理、DMFC加caspase-8抑制剂(8I)组合处理以及DMFC加caspase-9抑制剂(9I)组合处理。每张图的右下角百分数都表示该细胞在对应处理下的凋亡百分率。可以看出,DMFC 32μM作用于HepG2诱导细胞产生凋亡16.19%,而添加caspase-8和-9抑制剂,可以逆转这种凋亡的诱导,凋亡细胞百分比相应下调至8.67%,9.8%,附图4b是图4a的量化分析图。
可见,DMFC诱导HepG2细胞产生凋亡确实涉及了两条不同的通路,即细胞内(死亡受体)和细胞外(线粒体)通路。
综上实施例所述,本发明提供的3,5-二甲基补骨脂素(DMFC)对肝癌HepG2、乳腺癌MCF-7、***SIHA,上皮癌A431、鼻咽癌CNE2、结肠癌CaCo2均具有强烈的增殖抑制效果,DMFC杀死肝癌HepG2细胞的机理是通过细胞内(死亡受体)和细胞外(线粒体)两条通路诱导肿瘤细胞凋亡,且3,5-二甲基补骨脂素对正常细胞的毒性小,有利于临床应用。目前临床上缺乏高效低毒的药物,因此3,5-二甲基补骨脂素具有很好的制备抗肿瘤药物的应用前景。
本发明涉及的部分参考文献:
1.O.Gia,S.M.Magno,H.Gonazalez-Diaz,E.Quezada,L.Santana,E.Uniarte,L.D.Via,Bioorg.Med.Chem.2005,13,809.
2.Q.Shen,Q.Peng,J.Shao,X.Liu,Z.Huang,X.Pu,L.Ma,Y.M.Li,A.S.C.Chan,L.Gu,Synthesis and biological evaluation of functionalized coumarins asacetylcholinesterase inhibitors,European Journal of Medicinal Chemistry,2005,40(12),1307-1315.
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