CN112301063B - 基于微生物转化续随子二萜烷型衍生物的方法及其制药用途 - Google Patents
基于微生物转化续随子二萜烷型衍生物的方法及其制药用途 Download PDFInfo
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Abstract
本发明属生物药物技术领域,涉及续随子二萜烷型化合物的转化方法及用途,本发明利用已知微生物多型孢毛霉Mucor polymorphosporus,雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera对续随子二萜醇型化合物千金子甾醇及环氧续随子醇进行微生物转化,将类二萜的C‑6,17位环氧结构特异性的还原为双键,其中伞枝犁头霉Absidia corymbifera还在其C‑8位引入了酰基,制得相应的续随子二萜烷型衍生物。本发明为该类二萜化合物的结构修饰提供了新的方法。进一步,制得的续随子烷型二萜衍生物化合物用于制备抗肿瘤多药耐药性的药物或药物组合物。
Description
技术领域
本发明属生物药物技术领域,涉及续随子二萜烷型化合物的转化方法及用途,具体涉及利用已知微生物对千金子甾醇及环氧续随子醇进行微生物转化,制备具有抗肿瘤多药耐药活性的续随子二萜烷型衍生物。
背景技术
现有技术公开了续随子二萜烷型化合物具有较明显的抗肿瘤活性与抗肿瘤多药耐药性(MDR)。据构效关系研究表明,其母核上取代基的差异能明显改变该类化合物的抗肿瘤活性和MDR活性。且,该类化合物的抗肿瘤多药耐药活性是通过抑制细胞膜P-糖蛋白(P-gp)的过度表达实现的。
续随子二萜烷型化合物是从大戟科植物续随子Euphorbia lathyris L.中首次分离得到,迄今为止已有近30个该类二萜在千金子中发现;千金子甾醇(euphorbiasteroid)是从续随子Euphorbia lathyris中分离得到的一种具有一定抗肿瘤活性续随子烷型二萜醇酯,且该化合物与抗癌药物联用可有效克服肿瘤细胞的多药耐药性;但该化合物是否为发挥逆转MDR活性的最优结构,对其进行适当的结构修饰是否会影响其活性,已引起研究这关注,因此,为了发现活性更强的续随子二萜衍生物,特别是对其结构中低活性碳氢位点的结构修饰产物,对进一步研究该类化合物的构效关系有着重要的意义。
微生物转化是利用微生物生长过程中合成的特异酶完成特定生化反应。据报道,微生物在生长过程中可以合成多种酶,催化不同反应,例如氧化反应、还原反应、水解反应、脱氢反应、缩合反应和开环反应等,该类酶催化反应具有特异性和专属性,相对于化学反应,在底物的非活性碳氢位点上进行反应更高效环保。迄今为止尚未见有关千金子甾醇或其母核结构环氧续随子醇的微生物转化研究报道。
基于现有技术的基础与现状,本申请的发明人提供了基于微生物转化续随子二萜烷型衍生物的方法及其制药用途,并获得难以用传统化学合成制备的特异修饰位点的转化产物。
发明内容
本发明的目的是基于现有技术的基础与现状,提供续随子二萜烷型化合物非活性碳氢位点的结构改造方法,尤其是制备具有生理活性、难以通过传统化学法得到的续随子二萜烷型类化合物的微生物转化方法。
本发明使用多型孢毛霉Mucor polymorphosporus分别对千金子甾醇(euphorbiasteroid)和环氧续随子醇(epoxylathyrol)进行微生物转化,并使用雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera对千金子甾醇进行了微生物转化,获得以下结构通式的续随子烷型二萜衍生物:
经鉴定,所获得的续随子烷型二萜衍生物化合物为:
化合物1续随子醇:R1=R2=R3=R4=H
化合物2大戟因子L3:R1=Bz,R2=R4=Ac,R3=H
化合物3 8β-环丙甲酰氧基大戟因子L3:R1=Bz,R2=R4=Ac,R3=
本发明中所述的微生物转化法包括以下步骤:
(1)生产菌株为多型孢毛霉Mucor polymorphosporus,雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera,三种菌株在琼脂培养基上均生长良好,将生长在琼脂培养基上的三种菌丝分别划线接种于马铃薯固体培养基上,在20-28℃的恒温培养箱中培养3-7天,得到试管种;
(2)将试管种中的菌丝接种到250mL的三角瓶中,每瓶含有50mL的PDA培养基,培养温度为28℃,转速为120rpm,培养时间为3天,获得种子液;
(3)将种子液按2%体积比接入新鲜马铃薯培养基,28℃,120rpm条件下培养24-72小时后,加入转化底物千金子甾醇或环氧续随子醇,在28℃、120rpm的条件下转化培养7天;
本发明的一个实施例中,将多型孢毛霉Mucor polymorphosporus种子液按2%体积比接入PDA培养基,28℃培养48小时,再加入5mg/mL千金子甾醇或环氧续随子醇的乙醇溶液5mL;
(4)将发酵液用布氏漏斗减压过滤,获得发酵滤液和菌体,滤液按照1:1.5体积比用乙酸乙酯萃取3次,菌体剪碎,混悬于适量乙酸乙酯中,超声振荡提取10min后过滤,合并乙酸乙酯层,减压蒸干,发酵产物用半制备HPLC法分离得到发酵产物续随子醇(1)大戟因子L3(2)和8β-环丙甲酰氧基大戟因子L3(3);
其中,所述的液体培养基包括组分:200g马铃薯去皮,切成1立方厘米小丁,1L水微沸煎煮20分钟,随后用8层纱布趁热过滤,放凉后用水将滤液补齐至1L,加入20g葡萄糖,搅拌溶解;分装并经121℃高压灭菌20分钟后,放凉使用;
其中,所述的固体培养基包含组分:液体培养基加入1%琼脂,加热溶解并分装,121℃高压灭菌20分钟后,放凉使用。
本发明利用多型孢毛霉Mucor polymorphosporus、雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera对续随子二萜醇型化合物进行微生物转化,其中,利用多型孢毛霉Mucor polymorphosporus对千金子甾醇的C-6,17位环氧结构进行还原反应,制备得到其水解掉三个酯基的转化产物1;利用该菌对环氧续随子醇进行了微生物转化反应,同样制得到转化产物1;利用雅致小克银汉霉Cunninghamellaelegans对千金子甾醇进行了转化反应,制得其C-6,17位环氧的还原产物2;利用伞枝犁头霉Absidia corymbifera对千金子甾醇进行了转化反应,制得其C-8位发生环丙甲酰化的转化产物3。
本发明利用微生物对该类二萜的C-6,17位环氧结构进行了还原,伞枝犁头霉Absidia corymbifera还在其C-8位引入了酰基,丰富了续随子烷二萜醇的结构多样性。
本发明制得的续随子烷型化合物可制备抗肿瘤多药耐药性的药物或药物组合物,其中含有所述的化合物或其药学上可接受的溶剂及药学上可接受的载体。
本发明提供了二萜化合物的结构修饰新的方法。本发明利用已知微生物多型孢毛霉Mucor polymorphosporus,雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera对续随子二萜醇型化合物千金子甾醇及环氧续随子醇进行微生物转化,将类二萜的C-6,17位环氧结构特异性的还原为双键,其中伞枝犁头霉Absidiacorymbifera还在其C-8位引入了酰基,制得相应的续随子二萜烷型衍生物。进一步,制得的续随子烷型二萜衍生物化合物用于制备抗肿瘤多药耐药性的药物或药物组合物;所述的药物组合物,含有制得的化合物或其药学上可接受的溶剂化物及药学上可接受的载体。
具体实施方式
实施例1制备续随子醇(1)
采用两步活化法活化菌种多型孢毛霉Mucor polymorphosporus,获得的种子液以2%体积比接种于装有250mL的PDA培养基、体积为1L的三角瓶中,28℃,120rpm条件下摇瓶培养48h,每瓶活化后的菌液加入5mg/mL千金子甾醇或环氧续随子醇的乙醇溶液,终浓度为0.1mg/mL。同条件培养7天后,发酵液抽滤获得发酵滤液和菌体,滤液按照1:1.5体积比用乙酸乙酯萃取3次,菌体剪碎,混悬于适量乙酸乙酯中,超声振荡提取10min后过滤,合并乙酸乙酯层,减压蒸干,得到发酵液提取物;
发酵液提取物用适量甲醇溶解,过滤后用HPLC制备,流动相条件如下:甲醇-水(40%-80%)40min梯度洗脱,得到化合物1,产率约为6%;化合物1的NMR结构鉴定数据如表1所示。
实施例2制备大戟因子L3(2)
采用两步活化法活化菌种雅致小克银汉霉Cunninghamella elegans,获得的种子液以2%体积比接种于装有250mL的PDA培养基、体积为1L的三角瓶中,28℃,120rpm条件下摇瓶培养48h,每瓶活化后的菌液加入5mg/mL千金子甾醇的乙醇溶液,终浓度为0.1mg/mL,同条件培养7天后,发酵液抽滤获得发酵滤液和菌体,滤液按照1:1.5体积比用乙酸乙酯萃取3次,菌体剪碎,混悬于适量乙酸乙酯中,超声振荡提取10min后过滤,合并乙酸乙酯层,减压蒸干,得到发酵液提取物;
发酵液提取物用适量甲醇溶解,过滤后用HPLC制备,流动相条件如下:甲醇-水(40%-80%)40min梯度洗脱,得到化合物2,产率约为5%;化合物2的NMR结构鉴定数据如表1所示。
实施例3制备8β-环丙甲酰氧基大戟因子L3(3)
采用两步活化法活化菌种伞枝犁头霉Absidia corymbifera,获得的种子液以2%体积比接种于装有250mL的PDA培养基、体积为1L三角瓶中,28℃,120rpm条件下摇瓶培养48h,每瓶活化后的菌液加入5mg/mL千金子甾醇的乙醇溶液,终浓度为0.1mg/mL,同条件培养7天后,发酵液抽滤获得发酵滤液和菌体,滤液按照1:1.5体积比用乙酸乙酯萃取3次,菌体剪碎,混悬于适量乙酸乙酯中,超声振荡提取10min后过滤,合并乙酸乙酯层,减压蒸干,得到发酵液提取物;
发酵液提取物用适量甲醇溶解,过滤后用HPLC制备,流动相条件如下:甲醇-水(40%-80%)40min梯度洗脱,得到化合物3,产率约为4%;化合物3的NMR结构鉴定数据如表1所示。
表1.转化产物续随子醇(1)、大戟因子L3(2)和8β-环丙甲酰氧基大戟因子L3(3)的NMR数据(600MHz,CD3OD).
实施例4大戟因子L3(2)的抗肿瘤多药耐药活性测试
采用斑马鱼模型,研究了千金子甾醇和转化产物2对P-gp的抑制作用。在显微镜下挑选发育健康且阶段一致的受精后6小时的野生型AB品系斑马鱼于六孔板中,每孔随机挑选30尾,每孔容量为3mL,分别给予不同浓度(3.125、6.25、12.5、25和50μM)的千金子甾醇和转化产物2;同时设置阳性对照组环孢菌素A(20μM)和正常对照组,置于28℃培养箱中培养18小时后,各实验组斑马鱼用5μM罗丹明B染色,染色后每组随机取10尾斑马鱼进行拍照,统计分析斑马鱼中罗丹明B的荧光信号强度(F),计算千金子甾醇和化合物2对P-gp的抑制率,并计算其抑制P-gp的IC50值;
给药浓度为3.125、6.25、12.5、25和50μM的千金子甾醇的斑马鱼荧光强度分别为1666875、1906829、2072213、2282093和2710139像素,对P-gp的抑制作用分别为23%、40%、52%、68%和96%。与正常对照组比较,3.125μM浓度组无显著性差异(P>0.05),其余浓度组均有显著性差异(P<0.001),说明千金子甾醇对斑马鱼P-gp具有明显的抑制作用,且呈剂量依赖性,其抑制斑马鱼P-gp的IC50为34.97μM;
给药浓度为3.125、6.25、12.5、25和50μM的化合物2的斑马鱼荧光强度分别为1924398、2180128、2354745、2764628和2989520像素,对P-gp的抑制作用分别为42%、60%、73%、103%和116%。与正常对照组比较,所有浓度组均有显著性差异(P<0.001),说明转化产物2对斑马鱼P-gp具有明显的抑制作用,且呈剂量依赖性,其抑制斑马鱼P-gp的IC50为15.50μM;
转化产物大戟因子L3(2)与底物千金子甾醇抑制肿瘤多药耐药相关蛋白P-gp的IC50分别为15.50μM和34.97μM。表明该转化产物2中被还原的C-6,17位环氧结构,能明显提高底物千金子甾醇的抗肿瘤多药耐药活性。
Claims (4)
1.基于微生物转化续随子二萜烷型衍生物的方法,其特征在于:用多型孢毛霉Mucorpolymorphosporus分别对千金子甾醇(euphorbiasteroid)或环氧续随子醇(epoxylathyrol)进行微生物转化;使用雅致小克银汉霉Cunninghamella elegans或伞枝犁头霉Absidia corymbifera对千金子甾醇(Euphorbiasteroid)进行了微生物转化,获得下式的续随子二萜烷型化合物:
其中,
化合物1 续随子醇:R1=R2=R3=R4=H
化合物2 大戟因子L3:R1=Bz,R2=R4=Ac,R3=H
化合物3 8β-环丙甲酰氧基大戟因子L3:R1=Bz,R2=R4=Ac,
所述方法包括步骤:
(1)将生长在琼脂培养基上的多型孢毛霉Mucor polymorphosporus、雅致小克银汉霉Cunninghamella elegans和伞枝犁头霉Absidia corymbifera三种菌丝分别划线接种于马铃薯固体培养基上,在20-28℃的恒温培养箱中培养3-7天,得试管种;
(2)将试管种中的菌丝接种到250mL的三角瓶中,每瓶含有PDA培养基,培养后获得种子液;
(3)将种子液接入新鲜马铃薯培养基,培养后,加入转化底物千金子甾醇或环氧续随子醇的乙醇溶液,转化培养;
(4)将发酵液过滤,获得发酵滤液和菌体,滤液用乙酸乙酯萃取,菌体剪碎,混悬于适量乙酸乙酯中,超声振荡提取后过滤,合并乙酸乙酯层,减压蒸干,发酵产物用半制备HPLC法分离得到发酵产物;
所述的方法中,以多型孢毛霉Mucor polymorphosporus作为微生物,以千金子甾醇或环氧续随子醇为底物进行转化,得到化合物1;以雅致小克银汉霉Cunninghamella elegans作为微生物,以千金子甾醇为底物进行转化,得到化合物2;以伞枝犁头霉Absidiacorymbifera作为微生物,以千金子甾醇为底物进行转化,得到化合物3。
2.按权利要求1所述的方法,其特征在于,所述步骤(2)中,所述的三角瓶中,含有50mL的PDA培养基,培养温度为28℃,转速为120rpm,培养时间为3天。
3.按权利要求1所述的方法,其特征在于,所述步骤(3)中,将种子液按2%体积比接入新鲜马铃薯培养基,28℃,120rpm条件下培养24-72小时后,加入转化底物千金子甾醇或环氧续随子醇的乙醇溶液,在28℃、120rpm的条件下转化培养7天。
4.按权利要求1所述的方法,其特征在于,所述步骤(4)中,所述的滤液按1:1.5体积比用乙酸乙酯萃取3次,超声振荡提取10min后过滤。
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