CN115108935B - 白英中的生物碱类化合物及其制备方法和应用 - Google Patents
白英中的生物碱类化合物及其制备方法和应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
- C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/16—Preparation of optical isomers
- C07C231/20—Preparation of optical isomers by separation of optical isomers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/32—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D207/33—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms with substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D207/333—Radicals substituted by oxygen or sulfur atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
白英中的生物碱类化合物及其制备方法和应用,属于医药技术领域,具体涉及从白英(Solanum lyratum Thunb.)中提取到的八种生物碱类化合物,以及所述生物碱类化合物在制备神经退行性疾病药物中的应用。所述的生物碱类化合物的结构如下,并通过如下方法制备:白英用工业乙醇进行回流提取,提取液减压浓缩成浸膏,正丁醇萃取物采用硅胶减压柱、ODS开放色谱柱、HPLC等多种色谱学手段分离得到。所述化合物具有显著的体外神经细胞保护活性,具有进一步开发预防和治疗神经退行性疾病的医药新用途。
Description
技术领域
本发明属于医药技术领域,涉及白英中的生物碱类化合物及其制备方法和其在医药方面的用途,尤其涉及从植物白英全草中提取制备的生物碱类化合物及其在制备治疗神经退行性疾病药物方面的应用。
背景技术
神经退行性疾病是一种慢性进行性的中枢神经组织退行性病变和丢失而产生的疾病总称,主要病理表现为脑或脊髓神经元变性和丧失,而导致痴呆等认知障碍。其具体包括阿尔兹海默病(Alzheimer’s disease,AD)、帕金森氏病(Parkinson’s disease,PD)和亨廷顿氏病(Huntington’s disease,HD)等。由于这些疾病易受人口老龄化的影响,使得患者生活质量严重下降,增加了世界各地医疗系统的负担,所以从天然药物中制备具有神经保护活性的化合物具有重大的意义。
白英(Solanum lyratum Thunb.),属茄科(Solanaceae)茄属(Solanum),别名毛风藤、白毛藤、葫芦草、鬼目草等,是一种资源丰富的草质藤本植物。白英主要分布于安徽、浙江、江苏等江南地区。白英的化学成分有甾体类、生物碱类、倍半萜类、木脂素类和其他类化合物。现代药理学研究表明,其具有抗肿瘤、抗炎、保肝护肝、神经保护等药理作用,是一种具有极高药用价值和研究前景的天然药物。
本发明中涉及的八个生物碱类化合物,经实验证明,所述的化合物具有神经细胞保护活性,可应用于治疗神经退行性疾病。
发明内容
本发明的目的是提供八种从茄科茄属植物白英(Solanum lyratum Thunb.)中分离得到的生物碱类化合物,结构如下:
本发明的所述八个生物碱类化合物的制备方法,包括如下步骤:
(1)白英的干燥全草用70%-80%乙醇进行提取,提取液减压浓缩成浸膏;
(2)浸膏采用乙酸乙酯和正丁醇分别进行萃取并将所得流份经硅胶柱色谱,以二氯甲烷/甲醇系统100:1-10:1进行梯度洗脱,共收集到2个流份A、B;
(3)流份B经胶柱色谱,以二氯甲烷/甲醇系统30:1-3:1进行梯度洗脱,共收集到2个流份B1、B2;
(4)流份B1经HP-20大孔吸附树脂,以乙醇/水系统10:90-90:10进行梯度洗脱,共收集到2个流份B1-1、B1-2;
(5)流份B1-1经ODS柱色谱,以乙醇/水系统20:80-70:30进行梯度洗脱,并通过薄层色谱和HPLC分析进一步得到2个流份B1-1-1、B1-1-2;
(6)所得流份经硅胶柱色谱以二氯甲烷/甲醇系统50:1-8:1进行梯度洗脱,得到4个流份1-4;
(7)利用半制备HPLC对流份1-4以乙腈/水系统进行洗脱,得到化合物(±)-1、化合物(±)-2、化合物(±)-3、化合物4和化合物5;采用手性色谱柱拆分得到化合物1a/1b、2a/2b和化合物3a/3b。
具体操作步骤如下:
利用半制备HPLC对流份1以乙腈/水系统31:69进行洗脱,得到了化合物(±)-2,对流份3以乙腈/水系统35:65进行洗脱,分离得到了化合物(±)-1和化合物(±)-3,对流份4以乙腈/水系统41:59进行洗脱,分离得到了化合物4和化合物5;利用Daicel Chiralpak IC手性色谱柱以正己烷/异丙醇系统1:1对化合物(±)-1和化合物(±)-2进行手性拆分得到化合物1a/1b和2a/2b;利用Daicel Chiralpak IA手性色谱柱以正己烷/异丙醇系统1:1对化合物(±)-3进行手性拆分得到化合物3a/3b。
所述的制备方法,其中,
所述步骤(1)中,提取为回流提取,提取2-3次,每次2-3小时。
使用的白英为茄科茄属植物白英(S.lyratum)。
上述制备方法所得化合物1a/1b、2a/2b、3a、4和5经过系统结构鉴定结果如下:
利用紫外光谱、高分辨质谱、一维和二维NMR技术对化合物1-5的平面结构进行鉴定。利用实测ECD和计算ECD方法,对拆分后的光学纯化合物1a/1b、2a/2b和3a的绝对构型进行确定(如图1-41)。
化合物1:黄色油状物(甲醇)。UV(MeOH)λmax(logε)319(3.57),289(3.49),232(3.58),219(3.58),204(3.84)nm;HR-ESIMS给出准分子离子峰m/z 396.1478[M+Na]+(calcd for C20H23NO6Na,396.1418),结合1H和13C NMR数据,推测其分子式为C20H23NO6,计算不饱和度为10。1H NMR(600MHz,DMSO-d6)和13C NMR(100MHz,DMSO-d6)谱:δH 7.11(1H,d,J=1.8Hz,H-2),6.97(1H,dd,J=8.2,1.8Hz,H-6),6.84(1H,d,J=1.8Hz,H-2′),6.78(1H,d,J=8.1Hz,H-5),6.76(1H,overlap,H-5′),6.71(1H,dd,J=8.1,1.8Hz,H-9′),δC 148.5(C-4),147.9(C-3),147.6(C-3′),146.2(C-4′),130.5(C-1′),126.3(C-1),121.6(C-6),119.5(C-6′),115.7(C-5′),115.2(C-5),110.7(C-2),110.5(C-2′)提示为两组具备ABX耦合系统的芳环波谱信号;δH 7.97(1H,t,J=5.7Hz)提示为氨基上的氢信号;δH 7.30(1H,d,J=15.7Hz,H-7),6.53(1H,d,J=15.7Hz,H-8),δC 139.0(C-7),118.9(C-8)提示为一组反式双键的波谱信号。在较高场区:给出了一组与氧相连的次甲基波谱信号δH 4.16(1H,dd,J=7.9,4.6Hz,H-7′),δC 81.7(C-7′);一组同碳偕偶的亚甲基波谱信号δH 3.39(1H,m,H-8′a),3.31(1H,m,H-8′b),δC 45.2(C-8′)。此外,还给出两组苯环上的甲氧基波谱信号δH 3.80(3H,s,3-OCH3),3.77(3H,s,3′-OCH3),δC 55.5×2(3/3′-OCH3);一组脂肪链上的甲氧基波谱信号δH 3.12(3H,s,7′-OCH3),δC 55.9(7′-OCH3)。
在HMBC谱中,7′-OCH3(δH 3.12)与C-7′存在相关,H-7′与C-2′/C-6′存在相关,H-8′与C-9/C-1′存在相关;-NH(δH 7.97)与C-9存在相关,说明甲氧基连接在酰胺基团的C-7′位上,且酪胺基团与C-9位直接相连,由此确定了化合物1的平面结构。由于化合物的旋光值数值较小且在CD谱图中无明显的Cotton效应,因此推测该化合物可能为外消旋混合物,所以采用Daicel Chiralpak IC手性色谱柱在HPLC液相上进行进一步分离,得到了一对峰面积比约为1:1的对映异构体/>和 其绝对构型通过计算ECD确定的,化合物1b与计算的7′S构型吻合较好,从而确定化合物1a的绝对构型为7′R,而1b的绝对构型为7′S。
化合物2:白色粉末(甲醇)。UV(MeOH)λmax(logε)279(3.17),226(3.68),208(3.85)nm;HR-ESIMS给出准分子离子峰m/z 368.1467[M+Na]+(calcd for C19H23NO5Na,368.1468),其分子式为C19H23NO5,分子量为345,化合物2的不饱和度为9。1H NMR(600MHz,DMSO-d6)及13CNMR(150MHz,DMSO-d6)谱,δH 7.80(1H,t,J=5.6Hz,8-NH)归属为氨基的活泼氢信号;δH6.81(1H,d,J=1.8Hz,H-2′),6.73(1H,d,J=8.0Hz,H-5′),6.67(1H,dd,J=8.0,1.8Hz,H-6′),δC 147.5(C-3′),146.0(C-4′),132.1(C-1′),119.1(C-6′),115.1(C-5′),110.3(C-2′)归属为1个三元取代芳环的波谱信号;δH 6.93(2H,d,J=8.4Hz,H-2/6),6.65(2H,d,J=8.4Hz,H-3/5),δC155.6(C-4),129.6(C-1),129.5×2(C-2/6),115.1×2(C-3/5)归属为1个具有AA′BB′耦合的芳环波谱信号;δH 3.19(1H,m,H-8a),3.11(1H,m,H-8b),2.53(2H,overlap,H-7),2.53(1H,overlap,H-8′a),2.25(1H,dd,J=14.2,5.1Hz,H-8′b),δC 40.6(C-8),34.3(C-7)归属为2组亚甲基波谱信号;δH 4.44(1H,dd,J=8.7,5.1Hz,H-7′),δC79.9(C-7′)归属为1个连氧次甲基波谱信号;δH3.76(3H,s,3′-OCH3),3.05(3H,s,7′-OCH3),δC 55.8(7′-OCH3),55.5(3′-OCH3)分别归属为苯环和支链上的甲氧基波谱信号;除此之外,还包括1个sp2杂化酮羰基碳信号δC 169.3(C-9′)。
为了进一步确定化合物2的平面结构,我们进行了二维谱的测试。在HMBC谱中,H-2′/C-4′,C-6′,C-7′,H-5′/C-1′,C-3′,H-6′/C-2′,C-4′,C-7′,H-7′/C-2′,C-6′,H-8′/C-1′,δH 3.76(3′-OCH3)/C-3′,δH 3.05(7′-OCH3)/C-7′这一系列信号,提示该化合物存在1个分别在C-3′位和C-7′位被甲氧基取代的对乙基苯酚片段。H-7′/C-9′,H-8′/C-9′,H-8/C-9′这一系列信号,提示酪胺片段通过C-9′位的酮羰基与另一个片段相连。由此,化合物2的平面结构得以确定。由于化合物2的旋光值较弱且无明显的Cotton效应,所以推测为一对对映异构体,使用Daicel Chiralpak IC手性拆分柱进行分离,得到/> 和/>它们的绝对构型是通过计算ECD确定的,2b的实验CD谱中的Cotton效应与7′R构型的计算ECD谱中的Cotton效应有较高的吻合度,所以化合物2a和2b的绝对构型分别为7′S和7′R。
化合物3a:白色粉末(甲醇)。UV(MeOH)λmax(logε)312(3.58),283(3.59),225(3.90),206(3.89)nm;HR-ESIMS给出准分子离子峰m/z 344.1493[M+H]+(calcd forC19H22NO5,344.1492),其分子式为C19H21NO5,计算化合物3的不饱和度为10。1H NMR(600MHz,DMSO-d6)和13C NMR(150MHz,DMSO-d6)谱显示:在低场区,给出氨基上的活泼质子信号δH8.14(1H,t,J=5.7Hz,8′-NH);一组顺式双键波谱信号δH 6.49(1H,d,J=13.0Hz,H-7),5.80(1H,d,J=13.0Hz,H-8),δC 137.3(C-7),120.7(C-8);一组具有ABX耦合系统的芳香波谱信号δH 7.71(1H,d,J=1.9Hz,H-2),7.12(1H,dd,J=8.2,1.9Hz,H-6),6.71(1H,d,J=8.2Hz,H-5),δC 147.5(C-4),146.8(C-3),126.7(C-1),124.4(C-6),114.8(C-5),114.5(C-2);一组具有AA′BB′耦合系统的芳环波谱信号δH 7.09(2H,d,J=8.5Hz,H-2′/6′),6.75(2H,d,J=8.5Hz,H-3′/5′),δC 157.1(C-4′),129.8(C-1′),128.0(C-2′),128.0(C-6′),115.1(C-3′),115.1(C-5′);一组磁不等价的亚甲基波谱信号δH 3.30(1H,overlap,H-8′a),3.24(1H,ddd,J=13.5,8.0,5.7Hz,H-8′b),δC 45.1(C-8′);一组连氧的次甲基波谱信号δH 4.15(1H,dd,J=8.0,4.6Hz,H-7′),δC 81.4(C-7′);两组甲氧基波谱信号δH 3.74(3H,s,3-OCH3),3.07(3H,s,7′-OCH3),δC 55.8(7′-OCH3),55.5(3-OCH3),其中一组偏高场区的信号为支链取代的甲氧基质子信号;δC 166.1(C-9)为羰基碳信号。
在二维谱中,可观察到H-2′/C-4′,C-6′,C-7′,H-3′/C-1′,C-5′,H-5′/C-1′,C-3′,H-6′/C-2′,C-4′,C-7′,H-7′/C-2′,C-6′,C-8′,δH 3.07(7′-OCH3)/C-7′一系列HMBC相关,以及δH 8.14(8′-NH)处氮上的氢与δH 3.30(H-8′)处的亚甲基,δH 3.30(H-8′)处的亚甲基与δH 4.15(H-7′)处的连氧次甲基一系列COSY相关,可推测出该化合物存在1个酪胺片段,且在C-7′位连有1个甲氧基。除此之外,还可观察到H-7/C-2,C-6,C-9,H-8/C-1,C-9,H-2/C-7一系列HMBC相关,可推测双键连接在苯环的C-1位上,且通过C-9位羰基与酪胺基团相连接。依据以上二维信息,确定了化合物(±)-3的平面结构。该化合物的实验旋光值较小且实测的CD曲线无明显的Cotton效应峰。推测该化合物以一对对映异构体的形式存在,由此使用Daicel Chiralpak IA手性色谱柱分离得到化合物的绝对构型通过计算ECD确定,化合物3a的实测ECD谱图与7′S构型的计算ECD谱图吻合度较高,所以确定化合物3a的绝对构型为7′S。
化合物1-3的核磁数据如下表所示:
表1化合物1-3在DMSO-d6中1H(600MHz)与13C NMR(150MHz)数据
化合物4:黄色油状(甲醇)。UV(MeOH)λmax(logε)291(3.51),240(3.32),219(3.28)nm;HR-ESIMS给出准分子离子峰m/z 365.1462[M+Na]+(calcd for C19H22N2O4Na,365.1472),结合1H和13C NMR数据,推测其分子式为C19H22N2O4,计算不饱和度为10。1H NMR(600MHz,DMSO-d6)和13C NMR(150MHz,DMSO-d6)谱的低场区给出如下信号:δH 7.96(1H,t,J=5.7Hz,1′-NH)提示为氨基上的活泼氢信号;δH 9.50(1H,s)提示为醛基氢信号;δH 7.11(1H,d,J=1.8Hz,H-5),6.97(1H,dd,J=8.1,1.8Hz,H-9),6.78(1H,d,J=8.1Hz,H-8),δC148.2(C-7),147.8(C-6),126.4(C-4),121.5(C-9),115.6(C-8),110.7(C-5)提示为具有ABX耦合体系的芳环波谱信号;δH 7.29(1H,d,J=15.7Hz,H-3),6.40(1H,d,J=15.7Hz,H-2),δC 138.8(C-3),119.0(C-2)提示为1组反式烯烃波谱信号;δH 7.32(1H,m,H-8′),7.02(1H,dd,J=4.0,1.7Hz,H-6′),6.23(1H,dd,J=4.0,2.4Hz,H-7′),δC 132.2(C-8′),130.9(C-5′),124.5(C-6′),109.4(C-7′)提示为1个吡咯环上的波谱信号;δH 4.28(2H,t,J=7.2Hz,H-4′),3.14(2H,overlap,H-1′),1.66(2H,m,H-3′),1.36(2H,dt,J=14.6,7.2Hz,H-2′),δC 47.6(C-4′),38.2(C-1′),28.6(C-3′),26.3(C-2′)提示为4个亚甲基波谱信号;δH3.79(3H,s,6-OCH3),δC 55.5(6-OCH3)提示为苯环上的甲氧基波谱信号。
通过HMBC谱图信号H-4′/C-5′,H-6′/C-8′,C-9′,H-7′/C-5′,H-8′/C-5′提示了吡咯环与正丁基的连接方式,以及吡咯环被醛基的取代方式。H-3/C-1,C-5,C-9,H-2/C-4以上HMBC信号提示了双键所在的位置。综上,确定了化合物4的平面结构。
化合物5:黄色油状(甲醇)。UV(MeOH)λmax(logε)293(3.60),233(3.38)nm;HR-ESIMS给出准分子离子峰m/z 409.1742[M+Na]+(calcd for C21H26N2O5Na,409.1734),从而推测化合物5的分子量为386,结合1H和13C NMR数据确定其分子式为C21H26N2O5,计算其不饱和度为10。1H NMR(600MHz,DMSO-d6)和13C NMR(150MHz,DMSO-d6)谱的低场区给出如下信号:δH 8.04(1H,t,J=5.7Hz,1′-NH)归属为氨基上的活泼氢信号;δH 9.49(1H,s),δC 179.4(C-9′)归属为醛基波谱信号;δH 7.69(1H,d,J=1.9Hz,H-5),7.07(1H,dd,J=8.2,1.9Hz,H-9),6.70(1H,d,J=8.2Hz,H-8),δC 147.3(C-7),146.8(C-6),126.8(C-4),124.3(C-9),114.8(C-8),114.1(C-5)归属于1组具有ABX自旋体系的苯环信号;δH 6.48(1H,d,J=13.0Hz,H-3),5.76(1H,d,J=13.0Hz,H-2),δC 166.2(C-1),136.7(C-3),121.0(C-2)归属于α,β-不饱和酮信号,其中双键构型为顺式;δH 6.99(1H,d,J=3.9Hz,H-7′),6.27(1H,d,J=3.9Hz,H-6′),δC 138.9(C-5′),132.0(C-8′),123.7(C-7′),111.3(C-6′)归属于一个吡咯环中的两组双键波谱信号,其中δC138.9,132.0为季碳信号,δC 123.7,111.3为质子化碳信号。在高场区,δH 4.44(2H,s,H-10′),4.24(2H,m,H-4′),3.11(2H,dd,J=13.1,5.7Hz,H-1′),1.63(2H,dd,J=15.4,7.6Hz,H-3′),1.42(2H,dd,J=15.0,7.4Hz,H-2′),δC 64.7(C-10′),44.8(C-4′),38.2(C-1′),28.6(C-3′),26.3(C-2′)为五组脂肪族亚甲基信号;δH3.72(3H,s,6-OCH3),δC 55.4(6-OCH3)为苯环上的甲氧基信号;δH3.25(3H,s,10′-OCH3),δC57.3(10′-OCH3)为支链上的甲氧基信号。
为了进一步确定其平面结构,进行了HSQC、HMBC以及COSY谱图的测试。在COSY和HMBC谱中,观察到H-6′/C-8′,H-7′/C-5′,C-9′,H-10′/C-6′,δH 3.25(10′-OCH3)/C-5′这些信号,说明该化合物存在1个1,2,5-三取代吡咯环的存在,且C-8′位被醛基取代,C-5′位被含氧亚甲基取代。H-2/C-4,H-3/C-1,C-5,C-9,H-5/C-3的HMBC相关,说明α,β-不饱和酮片段连接在苯环的C-4位。H-1′/H-2′,H-2′/H-3′,H-3′/H-4′,H-1′/(δH 8.04)1′-NH具有COSY相关,H-1′/C-1,C-3′,C-4′/C-2′,H-4′/C-5′,C-8′的HMBC相关,说明化合物中存在1个正丁基片段,且一侧通过1′-NH与顺式双键相连,另一侧取代了-NH与吡咯片段直接相连。由此确定了化合物5的平面结构。
化合物4-5的核磁数据如下表所示:
表2化合物4-5在DMSO-d6中1H(600MHz)与13C NMR(150MHz)数据
对本发明所述七个新化合物的H2O2诱导的人SH-SY5Y神经细胞损伤的神经保护活性进行了考察,在给药浓度为50μM的条件下,化合物1a和1b显示出了与阳性药Trolox相当的体外神经保护活性,与H2O2诱导的模型组相比,化合物1a和1b使细胞生存率上升了30%。在给药浓度为25μM和50μM的条件下,化合物3a显示了良好的体外神经保护活性。除此之外,在给药浓度为12.5μM,25μM和50μM的条件下,化合物5显示了良好的体外神经保护活性。因此本发明所述的生物碱类化合物具有进一步开发预防和治疗神经退行性疾病的前景。
一种药物组合物,包含所述八个生物碱类化合物和药学上可接受的载体和赋形剂。
所述八个生物碱类化合物或所述药物组合物在制备治疗神经退行性疾病药物方面的应用。
一种白英提取物,包含所述八个生物碱类化合物,应用于制备治疗神经退行性疾病药物中。
本发明的优点在于,所述化合物均为新化合物,且均为立体构型确定的光学纯化合物。同时部分化合物具有显著的体外神经细胞保护活性,具有进一步开发预防和治疗神经退行性疾病的医药新用途。
附图说明
图1为化合物1的UV谱;
图2为化合物1的HRESIMS谱;
图3为化合物1的1H NMR(600MHz,DMSO-d6)谱;
图4为化合物1的13C NMR(150MHz,DMSO-d6)谱;
图5为化合物1的HSQC(600MHz,DMSO-d6)谱;
图6为化合物1的HMBC(600MHz,DMSO-d6)谱;
图7为化合物1的1H-1H COSY(600MHz,DMSO-d6)谱;
图8为化合物1的手性拆分色谱图;
图9为化合物1的实测与计算ECD谱图;
图10为化合物2的UV谱;
图11为化合物2的HRESIMS谱;
图12为化合物2的1H NMR(600MHz,DMSO-d6)谱;
图13为化合物2的13C NMR(150MHz,DMSO-d6)谱;
图14为化合物2的HSQC(600MHz,DMSO-d6)谱;
图15为化合物2的HMBC(600MHz,DMSO-d6)谱;
图16为化合物2的1H-1H COSY(600MHz,DMSO-d6)谱;
图17为化合物2的手性拆分色谱图;
图18为化合物2的实测与计算ECD谱图;
图19为化合物3a的UV谱;
图20为化合物3a的HRESIMS谱;
图21为化合物3a的1H NMR(600MHz,DMSO-d6)谱;
图22为化合物3a的13C NMR(150MHz,DMSO-d6)谱;
图23为化合物3a的HSQC(600MHz,DMSO-d6)谱;
图24为化合物3a的HMBC(600MHz,DMSO-d6)谱;
图25为化合物3a的1H-1H COSY(600MHz,DMSO-d6)谱;
图26为化合物3a的手性拆分色谱图;
图27为化合物3a的实测与计算ECD谱图;
图28为化合物4的UV谱;
图29为化合物4的HRESIMS谱;
图30为化合物4的1H NMR(600MHz,DMSO-d6)谱;
图31为化合物4的13C NMR(150MHz,DMSO-d6)谱;
图32为化合物4的HSQC(600MHz,DMSO-d6)谱;
图33为化合物4的HMBC(600MHz,DMSO-d6)谱;
图34为化合物4的1H-1H COSY(600MHz,DMSO-d6)谱;
图35为化合物5的UV谱;
图36为化合物5的HRESIMS谱;
图37为化合物5的1H NMR(600MHz,DMSO-d6)谱;
图38为化合物5的13C NMR(150MHz,DMSO-d6)谱;
图39为化合物5的HSQC(600MHz,DMSO-d6)谱;
图40为化合物5的HMBC(600MHz,DMSO-d6)谱;
图41为化合物5的1H-1H COSY(600MHz,DMSO-d6)谱。
具体实施方式
下面所列实施例有助于本领域技术人员更好地理解本发明,但不以任何方式限制本发明。
实施例1
白英中的生物碱类化合物1-5的制备方法,具体操作包括如下步骤:
(1)采用浓度为70%的工业乙醇对白英(S.lyratum)的40kg干燥全草加热回流提取3次,每次3小时,提取液减压浓缩后得到2.5kg浸膏。
(2)将浸膏用水混悬后,分别用18L乙酸乙酯和20L正丁醇萃取多次,依次得到293.0g乙酸乙酯层萃取物和1114.0g正丁醇层萃取物。使用二氯甲烷/甲醇系统100:1,50:1,30:1,15:1,10:1(v/v)进行梯度洗脱,共收集到2个流份A、B。
(3)流份B经胶柱色谱,以二氯甲烷/甲醇系统30:1,20:1,15:1,10:1,8:1,3:1(v/v)进行梯度洗脱,共收集到2个流份B1、B2。
(4)流份B1经HP-20大孔吸附树脂,以乙醇/水系统10:90,50:50,90:10(v/v)进行梯度洗脱,共收集到2个流份B1-1、B1-2。
(5)流份B1-1经ODS柱色谱,以乙醇/水系统20:80,40:60,60:40,70:30(v/v)进行梯度洗脱,并通过薄层色谱和HPLC分析进一步得到了共收集到2个流份B1-1-1、B1-1-2。
(6)所得流份经硅胶柱色谱以二氯甲烷/甲醇系统50:1,30:1,20:1,15:1,10:1,8:1(v/v)进行梯度洗脱,得到4个流份1-4。
(7)利用半制备HPLC对流份1以乙腈/水系统31:69进行洗脱,得到了化合物(±)-2,对流份3以乙腈/水系统35:65进行洗脱,分离得到了化合物(±)-1和化合物(±)-3,对对流份4以乙腈/水系统41:59进行洗脱,分离得到了化合物4和化合物5。利用DaicelChiralpak IC手性色谱柱以正己烷/异丙醇系统1:1对化合物(±)-1和化合物(±)-2进行手性拆分得到化合物1a/1b和2a/2b。利用Daicel Chiralpak IA手性色谱柱以正己烷/异丙醇系统1:1对化合物(±)-3进行手性拆分得到化合物3a/3b。
实施例2
化合物1-5在体外对H2O2诱导的人SH-SY5Y神经细胞损伤保护作用的活性考察。
SH-SY5Y细胞处于对数生长期,密度为1.2×104个/孔,接种于96孔板上,在37℃和5%CO2的培养箱中培养12小时,直至细胞贴壁。加药组加入不同浓度的化合物(12.5,25,50μM),对照组与模型组加入等体积的空白培养基预处理1小时,模型组与加药组加入180μM的H2O2于培养箱中继续培养作用4小时以达到细胞的损伤率约为50%,每组设3个复孔。每孔加入20μL浓度为5mg/mL的MTT溶液于培养箱中孵育4小时后,吸去上清液,每孔加入150μL的DMSO于微量振荡器上振荡5分钟以完全溶解形成的甲瓒结晶。最后用酶标仪(λ=490nm)测每孔吸光度值(A),计算存活率。
存活率(%)=[A490(加药组)-A490(对照组)]/[A490(对照组)-A490(模型组)]×100%。
结果显示:在给药浓度为50μM的条件下,化合物1a和1b显示出了与阳性药Trolox相当的体外神经保护活性,与H2O2诱导的模型组相比,化合物1a和1b使细胞生存率上升了30%。在给药浓度为25μM和50μM的条件下,化合物3a显示了良好的体外神经保护活性。除此之外,在给药浓度为12.5μM,25μM和50μM的条件下,化合物5显示了良好的体外神经保护活性。
表3给药后细胞存活百分比
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Claims (9)
1.白英中的生物碱类化合物,其特征在于,所述化合物结构如下:
所述的白英中的生物碱类化合物,由茄科茄属植物白英(Solanum lyratum Thunb.)中分离得到。
2.一种权利要求1所述白英中的生物碱类化合物的制备方法,其特征在于,包括如下步骤:
(1)白英的干燥全草用70%-80%乙醇进行提取,提取液减压浓缩成浸膏;
(2)浸膏采用乙酸乙酯和正丁醇分别进行萃取并将所得流份经硅胶柱色谱,以二氯甲烷/甲醇系统100:1-10:1进行梯度洗脱,共收集到2个流份A、B;
(3)流份B经胶柱色谱,以二氯甲烷/甲醇系统30:1-3:1进行梯度洗脱,共收集到2个流份B1、B2;
(4)流份B1经HP-20大孔吸附树脂,以乙醇/水系统10:90-90:10进行梯度洗脱,共收集到2个流份B1-1、B1-2;
(5)流份B1-1经ODS柱色谱,以乙醇/水系统20:80-70:30进行梯度洗脱,并通过薄层色谱和HPLC分析进一步得到2个流份B1-1-1、B1-1-2;
(6)所得流份经硅胶柱色谱以二氯甲烷/甲醇系统50:1-8:1进行梯度洗脱,得到4个流份1-4;
(7)利用半制备HPLC对流份1-4以乙腈/水系统进行洗脱,得到化合物(±)-2、化合物4和化合物5;采用手性色谱柱拆分得到化合物2a/2b。
3.根据权利要求2所述的白英中的生物碱类化合物的制备方法,其特征在于,所述步骤(1)中,提取为回流提取,提取2-3次,每次2-3小时。
4.根据权利要求2所述的白英中的生物碱类化合物的制备方法,其特征在于,使用的白英为茄科茄属植物白英(S.lyratum)。
5.根据权利要求2所述的白英中的生物碱类化合物的制备方法,其特征在于,所述步骤(7)包括如下步骤:
利用半制备HPLC对流份1以乙腈/水系统31:69进行洗脱,得到了化合物(±)-2,对流份4以乙腈/水系统41:59进行洗脱,分离得到了化合物4和化合物5;利用Daicel ChiralpakIC手性色谱柱以正己烷/异丙醇系统1:1对化合物(±)-2进行手性拆分得到化合物2a/2b。
6.一种药物组合物,其特征在于,包含权利要求1所述白英中的生物碱类化合物和药学上可接受的载体和赋形剂。
7.一种白英提取物,其特征在于,包含权利要求1所述白英中的生物碱类化合物,应用于制备治疗神经退行性疾病药物中。
8.权利要求1所述白英中的生物碱类化合物在制备治疗神经退行性疾病药物方面的应用。
9.权利要求6所述药物组合物在制备治疗神经退行性疾病药物方面的应用。
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