CN115403556B - 戊酮噻吩类化合物及其制备方法和在抗炎药物中的应用 - Google Patents
戊酮噻吩类化合物及其制备方法和在抗炎药物中的应用 Download PDFInfo
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Abstract
本发明公开了戊酮噻吩类化合物及其制备方法和在抗炎药物中的应用。本发明人通过对南极曲霉Aspergillus sp.SCSIO 05702液体发酵提取物分离纯化,从中获得了化合物Ochrathinols A和B((±)‑1和(±)‑2)。经结构分析,化合物1和2均为新化合物,具体结构如式(Ⅰ)所示。通过对化合物1和2的抗炎活性评价,发现化合物新化合物Ochrathinol A(1)对脂多糖(LPS)诱导小鼠巨噬细胞株RAW264.7产生的一氧化氮具有显著的抑制作用,同时抑制IL‑6和TNF‑α等促炎因子的mRNA表达,在相同浓度下,对RAW264.7细胞无细胞毒活性,可以作为抗炎药物开发的先导化合物。
Description
技术领域
本发明属于海洋天然产物化学领域,具体涉及戊酮噻吩类化合物及其制备方法和在制备抗炎药物中的应用。
背景技术
天然化合物是创新药物研究的重要源泉。统计表明,从1981年至2019年共计三十九年的时间里批准的小分子药物约有1602个,这些小分子药物中有三分之二可追溯到天然产物,或受天然产物启迪。目前,从南极等极端环境来源微生物中,获得骨架新颖的活性次生代谢产物已逐渐成为天然药物研究的重要方向之一。
发明内容
本发明的第一个目的是具有抗炎活性的戊酮噻吩类化合物Ochrathinols((±)-1和(±)-2)。
本发明的戊酮噻吩类化合物,其结构如式(Ⅰ)中的任一所示:
本发明的第二个目的是提供如式(Ⅰ)所示的化合物Ochrathinols A和B((±)-1和(±)-2)在制备抗炎药物中的应用。
本发明的第三个目的是提供南极曲霉Aspergillus sp.SCSIO 05702在制备上述戊酮噻吩类化合物的应用。
本发明的第四个目的是提供一种戊酮噻吩类化合物的制备方法,包括以下步骤:
制备南极曲霉Aspergillus sp.SCSIO 05702的发酵培养物;
将发酵培养物的发酵液和菌丝体分离,发酵液用乙酸乙酯萃取,合并乙酸乙酯萃取液,浓缩后得到浸膏A;菌丝体先用丙酮浸提,合并浸提液,浸提液回收丙酮后剩余水混合液用乙酸乙酯萃取,乙酸乙酯萃取液浓缩后得到浸膏B,将浸膏A和浸膏B合并,得到粗提物;粗提物经中压正相硅胶柱层析,二氯甲烷/甲醇从100:0梯度洗脱至0:100,收集二氯甲烷/甲醇体积比为92:8洗脱的馏分fr5;将该馏分进行葡聚糖凝胶Sephadex LH-20层析纯化处理,以二氯甲烷/甲醇体积比1:1作为流动相洗脱,收集含有210和300nm特征波长的吸收峰的馏分fr5-2,再经纯化得到戊酮噻吩类化合物。
优选,所述的发酵培养物是将南极曲霉Aspergillus sp.SCSIO 05702接种到发酵培养基中,培养获得发酵培养物;
所述的发酵培养基:每1000mL培养基是这样配制的:甘露醇20g、麦芽糖20g、葡萄糖 10g、谷氨酸钠10g、KH2PO4 0.5g、MgSO4·7H2O 0.3g、酵母浸膏3g、玉米干粉0.3g,然后溶于适量的水中,用水定容至1000mL。
优选,所述的培养是25℃,180rpm,培养制得种子液,将种子液以体积百分比5%的接种量接入到发酵培养基中,25℃,摇床培养35天,得到南极曲霉Aspergillus sp.SCSIO05702 的发酵产物。
优选,所述的纯化是以210和300nm波长做检测、采用4mL/min的流速,以乙腈:水(9:91, v/v)进行等梯度洗脱进行半制备高效液相分离,HPLC(YMC-pack ODS-A,10×250mm, 5μm),得到Ochrathinol A和Ochrathinol B。
本发明人通过对南极曲霉Aspergillus sp.SCSIO 05702液体发酵提取物分离纯化,从中获得了化合物Ochrathinols A和B((±)-1和(±)-2)。经结构分析,化合物1和2均为新化合物,具体结构如式(Ⅰ)所示。通过对化合物1和2的抗炎活性评价,发现化合物新化合物Ochrathinol A (1)对脂多糖(LPS)诱导小鼠巨噬细胞株RAW264.7产生的一氧化氮具有显著的抑制作用,同时抑制IL-6和TNF-α等促炎因子的mRNA表达,在相同浓度下,对RAW264.7细胞无细胞毒活性,可以作为抗炎药物开发的先导化合物。
曲霉Aspergillus sp.SCSIO 05702于2015年1月6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),地址:北京市朝阳区北辰西路1号院3号中科院微生物研究所,保藏编号为:CGMCC No.10279。
附图说明
图1:戊酮噻吩类化合物1和2主要的1H-1H COSY,HMBC和NOESY信息;
图2:戊酮噻吩类化合物1和2的单晶X-Ray;
图3:戊酮噻吩类化合物1和2抑制NO释放活性;
图4:戊酮噻吩类化合物1和2抑制炎症因子活性。其中control代表空白对照,LPS代表脂多糖处理,LPS+(+)-1,LPS+(-)-1,LPS+(±)-1,LPS+(+)-2,LPS+(-)-2,LPS+(±) -2代表LPS+各化合物处理,A中每组柱形图从左至右依次为control,LPS,LPS+(+)-1,LPS+(-)-1,LPS+(±)-1,LPS+(+)-2,LPS+(-)-2,LPS+(±)-2;B中每组柱形图从左至右依次为control,LPS,LPS+(+)-1,LPS+(-)-1,LPS+(±)-1。
具体实施方式
以下结合实施例来进一步解释本发明,但实施例并不对本发明做任何形式的限定。
实施例1:化合物1和2的制备及结构鉴定
1、制备南极曲霉Aspergillus sp.SCSIO 05702的发酵培养物。
发酵培养基:每1000mL培养基是这样配制的:甘露醇20g、麦芽糖20g、葡萄糖10g、谷氨酸钠10g、KH2PO4 0.5g、MgSO4·7H2O 0.3g、酵母浸膏3g、玉米干粉0.3g,然后溶于适量的水中,用水定容至1000mL,121℃高温灭菌20min,备用。
将南极曲霉Aspergillus sp.SCSIO 05702接种到上述发酵培养基中,25℃,180rpm,培养制得种子液,将种子液以体积百分比5%的接种量接入到发酵培养基中,25℃,摇床培养35 天,得到南极曲霉Aspergillus sp.SCSIO 05702的发酵产物。
2、化合物1和2的分离纯化
上述发酵培养物,用大号布氏漏斗将发酵液和菌丝体分离,发酵液用乙酸乙酯萃取,合并乙酸乙酯萃取液,浓缩后得到浸膏A;菌丝体先用丙酮浸提,合并浸提液,浸提液回收丙酮后剩余水混合液用乙酸乙酯萃取,乙酸乙酯萃取液浓缩后得到浸膏B,将浸膏A和浸膏B合并,得到粗提物;粗提物经中压正相硅胶柱层析,二氯甲烷/甲醇从100:0梯度洗脱至0:100,梯度洗脱顺序得7个组分(fr1-fr7),收集二氯甲烷/甲醇体积比为92:8洗脱的馏分fr5;将该馏分进行葡聚糖凝胶Sephadex LH-20层析纯化处理,以二氯甲烷/甲醇体积比1:1作为流动相洗脱,获得5个馏分(fr5-1-fr5-5);馏分fr5-2(特征就是它含有210和300nm特征波长的吸收峰) 以210和300nm波长做检测、采用4mL/min的流速,以乙腈:水(9:91,v/v)进行等梯度洗脱进行半制备高效液相分离,HPLC(YMC-pack ODS-A,10×250mm,5μm),得到化合物2 (19.8mg,保留时间tR 25.8min)和化合物1(2.6mg,保留时间tR 31.0min)。
表1.化合物1和2的核磁数据(500MHz 1H and 125MHz 13C in CD3OD)
3、化合物1和2的结构鉴定
对获得的戊酮噻吩类化合物1和2进行核磁共振(NMR)、质谱(MS)、圆二色谱(CD)和单晶衍射(X-Ray)等数据测试,从而确定化合物的化学结构。
新化合物1结构鉴定:无色晶体,高分辨质谱m/z 171.0474[M+H]+(calcd for171.0474)建议其分子式为C8H10O2S含有4个不饱和度,1H和13C NMR数据见表1,13C NMR结合DEPT-135 谱提示其8个碳原子信号包括:1个甲基,2个sp3杂化亚甲基,2个sp3杂化的次甲基,3个sp2杂化的季碳。二维核磁谱图1H-1H COSY(图1)确认了出CH2-6/CH(OH)-7和CH3-9/CH-3/CH2-2 两个独立的自选耦合体系。HMBC的相关信息(图1)(H2-6/H-7与C-4,C-5,C-8相关)进一步确定了包含a,b不饱和酮片段的环戊烯酮结构片段的存在。通过CH3-9和CH2-2跟C-4的BC相关信号,证明CH3-9/CH-3/CH2-2饱和碳链片段是连接在环戊烯酮的C-4的位置。结合高分辨质谱及CH2-2跟C-6’的BC相关信号,提示CH2-2与C-6’是通过硫原子相连形成五元环结构。进一步通过单晶X-Ray衍射证明上述推断的正确性(图2),同时也确定化合物1的相对构型为 3S*,7S*。同时单晶数据显示化合物1为单斜空间群P2(1)/c,表明1是以对映异构体的形式天然存在。最终通过手性柱拆分(FLM chiral-ND(2)-RH chiral column),将化合物1分别拆分为(+)-1 和(-)-1,确定其绝对构型分别为3R,7R和3S,7S。化合物1为崭新的结构,命名为Ochrathinol A。
新化合物2结构鉴定:无色晶体,高分辨质谱数据显示化合物2跟化合物1分子式完全一致, 1D NMR数据也几乎完全一致,1H和13C NMR数据见表1,它们仅在3位甲基处化学位移有较明显的差异,推测化合物2和1是差向异构体。进一步二维核磁数据COSY,HSQC和HMBC谱也证实了它们是差向异构体的推论。通过单晶X-Ray衍射证明也确定化合物2是以一对对映异构体的形式天然存在(图2)。同样通过手性柱拆分(FLM chiral-ND(2)-RH chiralcolumn),将化合物2分别拆分为(+)-2和(-)-2,确定其绝对构型分别为3S,7R和3R,7S。化合物2为崭新的结构,命名为Ochrathinol B。
化合物1和2的结构式如式(Ⅰ)所示:
实施例2:戊酮噻吩类化合物1和2的抗炎活性实验数据
采用Griess法检测戊酮噻吩类化合物对RAW264.7细胞NO的影响,ELISA试剂盒检测戊酮噻吩类化合物对RAW264.7细胞促炎因子水平的影响,RT-PCR检测巨噬细胞极化状态相关因子的基因表达水平。实验结果表明戊酮噻吩类化合物Ochrathinol A(1)能够有效抑制LPS 诱导的促炎细胞因子(IL-6和TNF-α)水平的趋势(图4A),同时也能有效抑制IL-6和TNF-α等促炎因子的mRNA表达(图4B)。这与戊酮噻吩类化合物Ochrathinol A(1)对NO释放的抑制作用是一致的(图3)。在相同浓度下,对RAW264.7细胞无细胞毒活性,可以做为抗炎药物开发的先导化合物,对我国海洋微生物药物资源的开发利用也具有重要意义。
Claims (8)
1.戊酮噻吩类化合物,其结构如式(Ⅰ)中的任一所示:
Ochrathinols A
式(Ⅰ)。
2.权利要求1所述的戊酮噻吩类化合物在制备抗炎药物中的应用。
3.一种抗炎药物,其特征在于,含有权利要求1所述的戊酮噻吩类化合物作为活性成分。
4.南极曲霉Aspergillus sp. SCSIO 05702 CGMCC 10279在制备权利要求1所述的戊酮噻吩类化合物中的应用。
5.一种权利要求1所述的戊酮噻吩类化合物的制备方法,其特征在于,包括以下步骤:
制备南极曲霉Aspergillus sp.SCSIO 05702 CGMCC 10279的发酵培养物;
将发酵培养物的发酵液和菌丝体分离,发酵液用乙酸乙酯萃取,合并乙酸乙酯萃取液,浓缩后得到浸膏A;菌丝体先用丙酮浸提,合并浸提液,浸提液回收丙酮后剩余水混合液用乙酸乙酯萃取,乙酸乙酯萃取液浓缩后得到浸膏B,将浸膏A和浸膏B合并,得到粗提物;粗提物经中压正相硅胶柱层析,二氯甲烷/甲醇从100:0梯度洗脱至0:100,收集二氯甲烷/甲醇体积比为92:8洗脱的馏分fr5;将该馏分进行葡聚糖凝胶Sephadex LH-20层析纯化处理,以二氯甲烷/甲醇体积比1:1作为流动相洗脱,收集含有210和300nm特征波长的吸收峰的馏分fr5-2,再经纯化得到戊酮噻吩类化合物。
6.根据权利要求5所述的制备方法,其特征在于,所述的发酵培养物是将南极曲霉Aspergillus sp.SCSIO 05702 CGMCC 10279接种到发酵培养基中,培养获得发酵培养物;
所述的发酵培养基:每1000 mL培养基是这样配制的:甘露醇 20 g、麦芽糖20 g、葡萄糖10 g、谷氨酸钠10 g、KH2PO4 0.5 g、MgSO4·7H2O 0.3 g、酵母浸膏 3 g、玉米干粉 0.3 g,然后溶于适量的水中,用水定容至1000 mL。
7.根据权利要求6所述的制备方法,其特征在于,所述的培养是25 °C,180 rpm,培养制得种子液,将种子液以体积百分比5%的接种量接入到发酵培养基中,25 °C,摇床培养35天,得到南极曲霉Aspergillus sp.SCSIO 05702 CGMCC 10279的发酵产物。
8.根据权利要求5所述的制备方法,其特征在于,所述的纯化是以210 和 300 nm波长做检测、采用4 mL/min的流速,以乙腈:水9:91, v/v进行等度洗脱进行半制备高效液相分离,HPLC YMC-pack ODS-A, 10 × 250 mm, 5µm,得到Ochrathinol A。
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CN104860959A (zh) * | 2015-05-13 | 2015-08-26 | 中国科学院南海海洋研究所 | 一种α-吡喃酮混源萜及其制备方法和应用 |
CN106278877A (zh) * | 2016-07-20 | 2017-01-04 | 中国科学院南海海洋研究所 | 一类新颖结构倍半萜化合物及其在制备抗炎药物中的应用 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012070582A1 (ja) * | 2010-11-24 | 2012-05-31 | 住友化学株式会社 | 共役系化合物、並びにこれを用いた有機薄膜及び有機薄膜素子 |
CN104860959A (zh) * | 2015-05-13 | 2015-08-26 | 中国科学院南海海洋研究所 | 一种α-吡喃酮混源萜及其制备方法和应用 |
CN106278877A (zh) * | 2016-07-20 | 2017-01-04 | 中国科学院南海海洋研究所 | 一类新颖结构倍半萜化合物及其在制备抗炎药物中的应用 |
Non-Patent Citations (3)
Title |
---|
Junfeng Wang, et al..Antiviral Merosesquiterpenoids Produced by the Antarctic Fungus Aspergillus ochraceopetaliformis SCSIO 05702.《J. Nat. Prod.》.2015,第79卷59-65. * |
Junfeng Wang,et al..Ochracenes A−I, Humulane-Derived Sesquiterpenoids from the Antarctic Fungus Aspergillus ochraceopetaliformis.《J. Nat. Prod.》.2017,第80卷1725-1733. * |
于豪冰,等.极地生物活性次级代谢产物研究进展.《中国海洋药物》.2022,第41卷(第1期),70-85. * |
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