CN106074561A - 羧甲司坦的药物组合物及其对白血病的治疗作用 - Google Patents
羧甲司坦的药物组合物及其对白血病的治疗作用 Download PDFInfo
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Abstract
本发明公开了羧甲司坦的药物组合物及其对白血病的治疗作用,本发明提供的羧甲司坦的药物组合物中含有羧甲司坦和一种从石韦的干燥叶中分离得到的结构新颖的天然产物化合物(Ⅰ),羧甲司坦和该天然产物单独作用时,对白血病具有一定的治疗作用;二者联合作用时,对白血病的治疗效果更为显著,可以开发成治疗白血病的药物,与现有技术相比具有突出的实质性特点和显著的进步。
Description
技术领域
本发明属于生物医药领域,涉及羧甲司坦的新用途,具体涉及一种羧甲司坦的药物组合物及其对白血病的治疗作用。
背景技术
羧甲司坦为S-(羧甲基)半胱氨酸,由半胱氨酸和氯乙酸在一定条件下反应制得。羧甲司坦是一种化痰止咳药,主要是在细胞水平影响支气管腺体的分泌,使低粘度唾液蛋白分泌增加,高粘度岩藻粘蛋白产生减少,使痰液稠度降低而易于咳出,临床用于慢性支气管炎、支气管哮喘等疾病引起的痰液粘稠、咯痰困难和痰阻气管等症。
迄今为止,尚未见羧甲司坦及其药物组合物与白血病的相关性报道。
发明内容
本发明的目的在于提供一种羧甲司坦的药物组合物,该药物组合物中含有羧甲司坦和一种从石韦叶中分离得到的结构新颖的天然产物,羧甲司坦和该天然产物可以协同发挥治疗白血病的作用,开发成治疗白血病的药物。
本发明的上述目的是通过下面的技术方案得以实现的:
一种具有下述结构式的化合物(Ⅰ),
一种羧甲司坦的药物组合物,包括羧甲司坦、如上所述的化合物(Ⅰ)和药学上可以接受的载体。
如上所述的化合物(Ⅰ)的制备方法,包含以下操作步骤:(a)将石韦的干燥叶粉碎,用70~80%乙醇热回流提取,合并提取液,浓缩至无醇味,依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇萃取物用大孔树脂除杂,先用15%乙醇洗脱6个柱体积,再用70%乙醇洗脱10个柱体积,收集70%洗脱液,减压浓缩得70%乙醇洗脱浓缩物;(c)步骤(b)中70%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为80:1、30:1、15:1和5:1的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分3用正相硅胶进一步分离,依次用体积比为25:1、15:1和2:1的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为72%的甲醇水溶液等度洗脱,收集8~14个柱体积洗脱液,洗脱液减压浓缩得到纯的化合物(Ⅰ)。
进一步地,步骤(a)中用75%乙醇热回流提取,合并提取液。
进一步地,所述大孔树脂为D101型大孔吸附树脂。
如上所述的化合物(Ⅰ)在制备治疗白血病的药物中的应用。
如上所述的羧甲司坦的药物组合物在制备治疗白血病的药物中的应用。
本发明的优点:
本发明提供的羧甲司坦的药物组合物中含有羧甲司坦和一种从石韦叶中分离得到的结构新颖的天然产物,羧甲司坦和该天然产物单独作用时,对白血病具有一定的治疗作用;二者联合作用时,对白血病的治疗效果更为显著,可以开发成治疗白血病的药物。本发明与现有技术相比具有突出的实质性特点和显著的进步。
具体实施方式
下面结合实施例进一步说明本发明的实质性内容,但并不以此限定本发明保护范围。尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
实施例1:化合物(Ⅰ)分离制备及结构确证
分离方法:(a)将石韦的干燥叶(2kg)粉碎,用75%乙醇热回流提取(20L×3次),合并提取液,浓缩至无醇味(4L),依次用石油醚(4L×3次)、乙酸乙酯(4L×3次)和水饱和的正丁醇(4L×3次)萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇萃取物用D101型大孔树脂除杂,先用15%乙醇洗脱6个柱体积,再用70%乙醇洗脱10个柱体积,收集70%洗脱液,减压浓缩得70%乙醇洗脱浓缩物;(c)步骤(b)中70%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为80:1(8个柱体积)、30:1(8个柱体积)、15:1(8个柱体积)和5:1(10个柱体积)的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分3用正相硅胶进一步分离,依次用体积比为25:1(8个柱体积)、15:1(10个柱体积)和2:1(5个柱体积)的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为72%的甲醇水溶液等度洗脱,收集8~14个柱体积洗脱液,洗脱液减压浓缩得到化合物(Ⅰ)(280mg,HPLC归一化纯度大于98%)。
结构确证:淡黄色固体,HR-ESI-MS显示[M+H]+为m/z 561.3895,结合核磁特征可得分子式为C37H52O4,不饱和度为12。核磁共振氢谱数据δH(ppm,CDCl3,500MHz):H-1a(2.45,d,J=13.1Hz),H-1b(2.34,d,J=13.1Hz),H-4(2.28,q,J=6.6Hz),H-6a(1.41,m),H-6b(1.92,dt,J=13.1,3.0Hz),H-7a(1.57,m),H-7b(1.66,m),H-8(1.59,m),H-10(1.75,m),H-11a(1.31,td,J=13.8,3.3Hz),H-11b(2.06,d,J=13.8Hz),H-12a(1.39,m),H-12b(1.52,m),H-15a(1.38,m),H-15b(1.64,m),H-16a(1.41,m),H-16b(1.71,m),H-18(1.83,m),H-19a(1.63,m),H-19b(1.86,m),H-21a(1.18,m),H-21b(1.39,m),H-22a(1.28,m),H-22b(1.56,m),H-23(0.92,d,J=6.8Hz),H-24(0.83,s),H-25a(4.51,d,J=12.4Hz),H-25b(4.95,d,J=12.4Hz),H-26(1.07,s),H-27(1.17,s),H-28(1.14,s),H-29(1.09,s),H-30(1.16,s),H-3’,7’(7.98,br,d,J=7.6Hz),H-4’,6’(7.46,t,J=7.6Hz),H-5’(7.57,t,J=7.6Hz);核磁共振碳谱数据δC(ppm CDCl3,125MHz):36.2(CH2,1-C),198.6(C,2-C),187.5(C,3-C),50.4(CH,4-C),42.3(C,5-C),41.4(CH2,6-C),18.3(CH2,7-C),53.4(CH,8-C),41.9(C,9-C),60.8(CH,10-C),30.4(CH2,11-C),31.7(CH2,12-C),39.7(C,13-C),37.6(C,14-C),33.2(CH2,15-C),35.2(CH2,16-C),33.4(C,17-C),41.9(CH,18-C),37.0(CH2,19-C),30.1(C,20-C),34.7(CH2,21-C),36.4(CH2,22-C),7.3(CH3,23-C),14.6(CH3,24-C),62.7(CH2,25-C),20.8(CH3,26-C),18.1(CH3,27-C),33.5(CH3,28-C),28.8(CH3,29-C),25.3(CH3,30-C),166.7(C,1’-C),130.4(C,2’-C),129.3(CH,3’,7’-C),128.5(CH,4’,6’-C),133.6(CH,5’-C)。红外波谱中的1735cm-1吸收带表明结构中存在羰基,而1448cm-1与1523cm-1吸收带表明结构中存在苯环结构。13C-NMR、DEPT和HSQC谱中显示有37个碳信号,包括七个甲基,十一个亚甲基(一个连氧碳),四个次甲基,九个季碳(三个羰基碳)以及一组单取代苯环结构,以上功能结构再结合不饱和数表明该化合物为六环结构。1H-NMR谱结合HSQC谱显示一组单取代苯环质子信号δH 7.98(2H,br,d,J=7.6Hz)、7.46(2H,t,J=7.6Hz)、7.57(1H,t,J=7.6Hz),七个甲基质子信号δH 0.92(3H,d,J=6.8Hz)、0.83(3H,s)、1.07(3H,s)、1.17(3H,s)、1.14(3H,s)、1.09(3H,s)、1.16(3H,s),十一组亚甲基质子信号δH 2.45(1H,d,J=13.1Hz)与2.34(1H,d,J=13.1Hz)、1.41(1H,m)与1.92(1H,dt,J=13.1,3.0Hz)、1.57(1H,m)与1.66(1H,m)、1.31(1H,td,J=13.8,3.3Hz)与2.06(1H,td,J=13.8,3.3Hz)、1.39(1H,m)与1.52(1H,m)、1.38(1H,m)与1.64(1H,m)、1.41(1H,m)与1.71(1H,m)、1.63(1H,m)与1.86(1H,m)、1.18(1H,m)与1.39(1H,m)、1.28(1H,m)与1.56(1H,m)、5.51(1H,d,J=12.4Hz)与4.95(1H,d,J=12.4Hz),四个次甲基质子信号δH 2.28(1H,q,J=6.6Hz)、1.59(1H,m)、1.75(1H,m)、1.83(1H,m);由以上NMR数据可知该化合物为木栓烷型三萜类,并且结构中存在两个酮基以及一个羟甲基片段。根据HMBC谱中显示的H-3’,7’与C-1’相关信号以及C-1’、C-2’、C-3’、C-4’、C-5’、C-6’和C-7’的碳化学位移和1H-NMR数据可知该化合物含有苯甲酰基结构单元。通过分析HMBC谱中H2-25与C-1’相关信号以及C-25的化学位移可知苯甲酸是通过与C-25的羟甲基成酯形成苯甲酰基结构单元。此外,HMBC谱中,H2-1与C-2以及H3-23与C-3相关信号以及它们的碳化学位移数据可以确定C-2和C-3为酮基碳。综合氢谱、碳谱、HMBC谱和NOESY谱,以及文献关于相关类型核磁数据,可基本确定该化合物如下所示,立体构型进一步通过ECD试验确定,理论值与实验值基本一致。该化合物的化学结构和碳原子编号如下:
实施例2:治疗白血病作用
一、材料和仪器
K562细胞由中科院上海生物细胞所提供。化合物(Ⅰ)为按实施例1方法自制,HPLC归一化纯度大于98%。RPMI-1640干粉培养基购于Gibco公司。小牛血清购于杭州四季青生物工程材料研究所。青霉素、链霉素购于华北制药厂。Glutamine、MTT、DMSO购于AMRESCO。Annexinv-FITC细胞凋亡检测试剂盒购于南京凯基生物科技发展有限公司。MOPS,Rnasin、琼脂糖、嗅乙锭、溴酚兰华美生物工程公司华美生物工程公司。
CO2孵箱(美国Thermo公司),超净工作台(苏州净化设备厂),磁力搅拌器(上海南汇电讯器材厂),倒置显微镜(日本OLYMPUS公司),水平式离心机(上海安亭科学仪器厂),电子分析天平(德国Sartorius公司),自动酶标仪(德国Humareader公司),U-3010型紫外分光光度计(日本HITACHI公司),流式细胞仪(美国BECKMAN公司),Eppendorf Centrifuge5417R离心机(德国Eppendorf公司)。
二、试验方法
1、K562细胞的培养
将新购买的细胞株的培养瓶表面用75%酒精擦拭3遍。将细胞悬液吸入离心管,1000rpm离心10min。弃去上清,滴加含10%NBS的RPMI-1640完全培养基,轻轻吹打制备成细胞悬液。接种于培养瓶中,置于37℃,5%CO2培养箱中培养,每天换液传代。
2、实验分组
正常对照组:10%NBS的RPMI-1640;
羧甲司坦组:10%NBS的RPMI-1640+终浓度80mg/L的羧甲司坦;
化合物(Ⅰ)组:10%NBS的RPMI-1640+终浓度80mg/L化合物(Ⅰ);
羧甲司坦与化合物(Ⅰ)组合物组:10%NBS的RPMI-1640+终浓度40mg/L的羧甲司坦+终浓度40mg/L化合物(Ⅰ)。
3、MTT法检测对K562细胞增殖的影响
取生长状态良好的细胞,用含10%NBS的RPMI-1640培养液将细胞配制成细胞悬液,以5×103细胞/孔接种于无菌96孔培养板中。(2)依实验分组,每组加入不同浓度的药物50μL,设无细胞孔为空白对照组,每组设8个复孔,将96孔培养板放入CO2培养箱中培养;(3)24、48和72h后取出96孔培养板,每孔加入20μL MTT(5mg/mL),放入CO2培养箱孵育4h,即有紫蓝色结晶物生成,结晶物的生成量与细胞数成正比。(4)每孔加入酸化的SDS 100μL放入CO2培养箱过夜。(5)用酶标仪测定OD,比色时以空白孔调零。
4、统计学处理
实验组和对照组均重复三次操作,结果用(x±s表示,P<0.05示有显著性差异。组间比较采用单因素方差分析。所有数据用SPSS 18.0统计软件进行统计学分析。
三、结果及结论
MTT法检测对K562细胞的增殖抑制作用
药物作用K562细胞24h、48h、72h后,用酶标仪检测OD490显示:与正常对照组比,作用24h后羧甲司坦组和化合物(Ⅰ)组对细胞K562细胞抑制作用不明显(P>0.05,P>0.05),羧甲司坦与化合物(Ⅰ)组合物组对K562细胞有抑制作用(P<0.05);与正常对照组比,作用48h和72h后,羧甲司坦组和化合物(Ⅰ)组对细胞K562细胞有抑制作用(P<0.05,P<0.05),羧甲司坦与化合物(Ⅰ)组合物组可显著抑制K562细胞(P<0.01)。
表1各组作用K562细胞后的MTT值(x±s,n=8)
结论:羧甲司坦与化合物(Ⅰ)组合物可显著抑制K562细胞的增殖,抑制作用强于羧甲司坦或化合物(Ⅰ)单独作用于K562细胞。羧甲司坦与化合物(Ⅰ)之间存在协同作用,可以开发成治疗白血病的药物。
上述实施例的作用在于说明本发明的实质性内容,但并不以此限定本发明的保护范围。本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和保护范围。
Claims (7)
1.一种具有下述结构式的化合物(Ⅰ),
2.一种羧甲司坦的药物组合物,其特征在于:包括羧甲司坦、如权利要求1所述的化合物(Ⅰ)和药学上可以接受的载体。
3.权利要求1所述的化合物(Ⅰ)的制备方法,其特征在于,包含以下操作步骤:(a)将石韦的干燥叶粉碎,用70~80%乙醇热回流提取,合并提取液,浓缩至无醇味,依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇萃取物用大孔树脂除杂,先用15%乙醇洗脱6个柱体积,再用70%乙醇洗脱10个柱体积,收集70%洗脱液,减压浓缩得70%乙醇洗脱浓缩物;(c)步骤(b)中70%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为80:1、30:1、15:1和5:1的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分3用正相硅胶进一步分离,依次用体积比为25:1、15:1和2:1的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为72%的甲醇水溶液等度洗脱,收集8~14个柱体积洗脱液,洗脱液减压浓缩得到纯的化合物(Ⅰ)。
4.根据权利要求3所述的化合物(Ⅰ)的制备方法,其特征在于:步骤(a)中用75%乙醇热回流提取,合并提取液。
5.根据权利要求3所述的化合物(Ⅰ)的制备方法,其特征在于:所述大孔树脂为D101型大孔吸附树脂。
6.权利要求1所述的化合物(Ⅰ)在制备治疗白血病的药物中的应用。
7.权利要求2所述的羧甲司坦的药物组合物在制备治疗白血病的药物中的应用。
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