CN116410185A - 嗜氮酮类化合物及其制备方法和在制备神经退行性疾病药物中的应用 - Google Patents
嗜氮酮类化合物及其制备方法和在制备神经退行性疾病药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于海洋生物医药技术领域,具体涉及一种嗜氮酮类化合物及其制备方法和在抗神经退行性疾病药物中的应用。
背景技术
帕金森病(PD)是继阿尔茨海默病之后的第二大神经变性疾病,也是最常见的运动障碍疾病,我国65岁以上的老年人患病率已接近2%。PD的主要病理特征为黑质致密部多巴胺能神经元选择性变性,纹状体多巴胺水平降低和胞浆内路易小体的形成,最终导致静止性震颤、肌肉僵直、运动迟缓、姿势不稳等临床症状。研究发现线粒体功能障碍、氧化应激、谷氨酸毒性作用、蛋白质的错误折叠和铁的异常沉积等诸多因素都与PD的发病及进程密切相关,但直至目前PD的病因和发病机制及途径都未能完全弄清。随着社会人口的老年化加重,PD的发病率逐年上升,不仅严重影响患者日常生活,导致生存质量显著下降,随之而来的社会和经济负担也将进一步加剧,因此寻找治疗PD新型药物或新的治疗方法刻不容缓。
6-OHDA(6-羟基多巴损伤)是儿茶酚胺的羟基化衍生物,其结构与儿茶酚胺类似,是一种有效导致多巴胺神经元变性的神经毒剂,能高选择地引起交感神经肾上腺素能神经末梢急性溃变,是帕金森病发病机制研究及药物开发的经典模型。
发明内容
本发明提供一个对6-OHDA损伤的PC12细胞具有保护活性的嗜氮酮类化合物,其结构式如式(I)所示:
式(I)。
本发明另一目的在于提供上述化合物的制备方法。
为实现上述目的,本发明采用如下技术方案。
一种菌核青霉PenicilliumsclerotiorumUJNMF 0503,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No. 40172。
一种菌核青霉PenicilliumsclerotiorumUJNMF 0503及其发酵产物在生产治疗神经退行性疾病药物中的应用。
所述菌核青霉PenicilliumsclerotiorumUJNMF 0503培养后可产生发酵产物,所述发酵产物中含有对6-OHDA损伤的PC12细胞具有保护活性的物质,其发酵液可用于生产治疗神经退行性疾病药物或作为治疗神经退行性疾病的添加剂。
菌核青霉PenicilliumsclerotiorumUJNMF 0503产生的嗜氮酮类化合物,其结构如式(I)所示:
式(I)。
上述嗜氮酮类化合物的制备方法,包括以下步骤:
(1)菌核青霉PenicilliumsclerotiorumUJNMF 0503的发酵;
(2)从步骤(1)所得发酵物中经乙酸乙酯提取得到发酵提取物;
(3)步骤(2)中的提取物通过正相硅胶柱、快速中压层析柱、高效液相色谱等方法进行分离。
本发明提供一种含有步骤(2)中提取物,或式(I)所示化合物在制备治疗神经退行性疾病药物或药物中间体中的用途。
本发明提供一种含有步骤(2)中提取物,或式(I)所示化合物的治疗神经退行性疾病药物。所述抗神经退行性疾病药物还包括医学上可接受的辅料。所述治疗神经退行性疾病药物还可以包括其他有效成分,以增强神经保护效果。
本发明具有以下优点:
本发明的嗜氮酮类化合物可以通过PenicilliumsclerotiorumUJNMF 0503的发酵提取分离获得,具有神经保护活性,在制备抗神经退行性疾病药物方面具有应用潜力。
生物保藏信息
菌核青霉(Penicilliumsclerotiorum)UJNMF 0503,于2022年05月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为中国北京,北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏号:CGMCC No. 40172。
附图说明
图1为式(I)所示化合物主要的1H-1H COSY、HMBC相关信息;
图2为式(I)所示化合物主要的NOESY相关信息;
图3为式(I)所示化合物的ECD谱图;
图4为PenicilliumsclerotiorumUJNMF 0503发酵提取物以及式(I)所示化合物对6-OHDA损伤下PC12细胞的存活率的影响。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。实施例中的实验方法,如无特别说明,均采用本领域常规技术,实验试剂均为商业购买。
实施例1 发酵提取物的制备
种子培养基的配制方法:土豆浸出液200 mL,葡萄糖20 g,海盐30 g,用水定容到1L。将培养基装入20个500 mL的三角烧瓶中,每瓶约150 mL,在121℃高压蒸汽灭菌25分钟,备用。
PDB发酵培养基配置方法:土豆浸出液200 mL,葡萄糖20 g,海盐30 g,用水定容到1 L。置于1 L三角瓶中,每瓶350mL液体培养基,共150瓶。121℃高压蒸汽灭菌25分钟,备用。
用无菌竹签挑取适量的真菌PenicilliumsclerotiorumUJNMF 0503菌种接种入种子培养基中,28℃摇床(160 rpm)培养3天得到种子液,然后用移液枪接种10 mL种子液到1L的装有PDB培养基的三角烧瓶中,于28℃静置培养30天后,收取发酵物。
将发酵物用纱布进行过滤分为菌体和菌液,菌体用95%乙醇浸泡,浸取液回收乙醇后剩余水相,再用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯提取物A,菌液用等量乙酸乙酯萃取三次,减压浓缩得到乙酸乙酯提取物B,A、B合并得到总提取物。
实施例2 嗜氮酮类化合物的制备
按照实施例1的方法获得70 L液体培养基,将发酵物用纱布进行过滤分为菌体和菌液,菌体用95%乙醇浸泡、浸取液回收乙醇后剩余水相再用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯提取物A,菌液用等量乙酸乙酯萃取三次,减压浓缩得到乙酸乙酯提取物B,A、B合并得到总提取物200 g。乙酸乙酯提取物用大孔树脂D101吸附材料进行柱层析,以乙醇/水作为洗脱剂,体积比30%、50%、75%、90%进行洗脱,回收洗脱溶剂,获得4个馏分(Fr.1-Fr.4)。
Fr.2进一步以二氯甲烷-甲醇作为洗脱剂进行正相硅胶(200-300目)柱层析,根据薄层层析情况合并各个流份,回收洗脱溶剂,获得8个馏分(Fr.2-1-Fr.2-8)。Fr.2.4通过中压RP-C18柱层析以梯度(甲醇/水25:75-100:0)进行洗脱,得到6个馏分(Fr.2.4.1-Fr.2.4.6),Fr.2.4.3通过Sephadex LH-20(甲醇),得到2个馏分(Fr.2.4.3.1-Fr.2.4.3.2),Fr.2.4.3.2通过Rp C-18半制备高效液相色谱(甲醇/水,v/v 55:45,3mL/min)于tR=17.8 min分离获得一纯品化合物(2.0 mg),化合物为淡黄色胶状物,易溶于氯仿、甲醇、DMSO,难溶于水,旋光度值为[α]27 D+173.0 (c 0.15, MeOH)。
对分离获得的化合物进行高分辨质谱(HR-ESIMS)、1H NMR、13C NMR、2D1H-1H COSY、HSQC、HMBC分析,确定了平面结构,再结合NOESY信号与ECD谱图分别确定了化合物的相对构型和绝对构型。化合物的1H和13C NMR数据见表1,主要的1H-1H COSY和HMBC相关信息见图1,主要的NOESY信号见图2,ECD谱图见图3。
表1 化合物在CDCl3中的1H和13CNMR数据
化合物结构鉴定:
黄色胶状,高分辨质谱(HRESIMS)在m/z处给出准分子离子峰385.1417([M + H]+,计算385.1412),结合NMR波谱数据,推测化合物的分子式为C19H25ClO6,不饱和度为7。NMR数据分析发现该化合物与文献报道的化合物peniaphilone A类似,但是C-3位侧链不同。1H-1HCOSY谱中H-13与H-12、H2-14、H3-17分别相关,H2-14与H-15相关,HMBC谱中H2-15与C-11的相关,H3-16与C-10/C-11/C-12相关,结合C11-C17的NMR数据,推测该化合物含有C-11连有二甲基四氢呋喃片段,进一步结合H-16与C-10,H-9与C-3/C-4/C-11的HMBC相关以及H-9与H-10的1H-1H COSY相关,推测出C-3位的侧链结构如图所示。
NOESY谱图中,H-8a与H3-18的相关表明H-8a与H3-18处于平面的同侧且处于直立位,进一步通过J 8,8a(3.1 Hz) 推测H-8与H-8a位于平面同侧;J 12,13(10.3 Hz) 说明H-12与H-13处于四氢吡喃环的反式直立位,H-12与H3-16的NOESY相关说明两者处于平面同侧;另外,该比较该化合物与已知化合物peniaphilone A的ECD谱图,推测该化合物的构型如式(I)所示。这是首次报道嗜氮酮类化合物在C-3位具有四氢吡喃环结构。
化合物的结构如下:
式(I)。
实施例3 式(I)所示化合物的神经保护活性测试
采用PC12细胞(鼠源肾上腺嗜铬细胞瘤)测定式(I)所示化合物的的神经保护活性。以6-OHDA作为神经毒剂诱导神经细胞损伤,刺激细胞死亡,实验中使用MTT法确定细胞存活率,检测化合物在6-OHDA作用下是否具有神经保护活性。将细胞按照1.5×105/mL(100 μL孔)的密度接种到96孔板中,37℃、5% CO2恒温培养24小时,设置空白对照组(DMSO)、对照组(6-OHDA)、实验组(6-OHDA+50 μM发酵提取物或化合物),分别预处理细胞6小时后,再加入适量6-OHDA刺激细胞13小时,最后加入MTT在培养箱中避光4小时,读取490nm处的吸光度(OD值),计算PC12细胞的存活率。
实验结果显示,在测试浓度为50 μM时,PenicilliumsclerotiorumUJNMF 0503发酵提取物以及式(I)所示化合物处理后,PC12细胞的存活率相较于对照组均有提升,表明PenicilliumsclerotiorumUJNMF 0503发酵提取物以及式(I)所示化合物可以减少6-OHDA诱导的细胞损伤,具有一定的神经保护作用,具有抗神经退行性疾病药物开发的潜力。
Claims (6)
2.一种如权利要求1所述的化合物的制备方法,其特征在于,包括以下步骤:
(1)将菌核青霉(Penicillium sclerotiorum)UJNMF 0503发酵,获得发酵物;
(2)将步骤(1)所得发酵物经乙酸乙酯提取得到发酵提取物;
(3)将步骤(2)得到的发酵提取物通过正相硅胶柱、快速中压层析柱、高效液相色谱分离纯化获得;
所述菌核青霉(Penicillium sclerotiorum)UJNMF 0503,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No. 40172。
3.一种如权利要求1所述的嗜氮酮类化合物、如权利要求2中步骤(2)所述的发酵提取物在制备治疗神经退行性疾病药物或药物中间体中的应用。
4.一种治疗神经退行性疾病的药物,其特征在于,包含有效量的作为活性成分的如权利要求1所述的嗜氮酮类化合物;或,
包含有效量的作为活性成分的权利要求2所述的步骤(2)所述的菌核青霉(Penicillium sclerotiorum) UJNMF 0503的发酵提取物。
5.根据权利要求4所述的药物,其特征在于,还包括医学上可接受的辅料以及其他有效成分。
6.如权利要求3所述的应用、如权利要求4或5所述的药物,其特征在于,所述神经退行性疾病为帕金森病。
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