CN116162094A - 蓬莱葛任碱型生物碱及其在制备抗肿瘤药物中的应用 - Google Patents
蓬莱葛任碱型生物碱及其在制备抗肿瘤药物中的应用 Download PDFInfo
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- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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Abstract
本发明公开了结构式如式Ⅰ、式Ⅱ所示的蓬莱葛任碱型生物碱：
其中R1～R4分别选自氢、羟基、甲氧基、乙氧基、乙酰基、烷基烃、卤素、三元氧环、甲醚基；其来源于离药蓬莱葛，本发明通过MTT法测定分离得到的生物碱对人胃癌细胞株、人肝癌细胞株、人结肠癌细胞株、和肺癌人类肺泡基底上皮细胞株的细胞毒活性，以顺铂为阳性对照，实验结果证实蓬莱葛任碱型生物碱具有抗肿瘤活性，且离药蓬莱葛植物原料易得，价格低廉，生物碱提取工艺简单；蓬莱葛任碱型生物碱在制备抗肿瘤药物中拥有良好的开发应用前景。
Description
技术领域
本发明属于药物制备技术领域，具体涉及一种蓬莱葛任碱型生物碱及其在制备抗肿瘤药物中的应用。
背景技术
近年来，癌症的发病率依然不断上升，它严重威胁着人类的生命和健康，因此抗肿瘤药物的开发受到了来自多方面的越来越多的关注。从植物中寻找天然抗肿瘤的活性物质或先导活性物质是开发新型抗肿瘤药物的重要途经。但是在研究开发新药的道路上仍然存在许多困难，一些天然源的抗肿瘤药物效果虽然很好，但由于其资源的限制性，限制了其使用范围，并且虽然有一些抗肿瘤药物已经应用于临床，但仍然存在一些缺陷，如毒副作用、耐药性及选择性差等问题，因此对于寻找低毒性、高活性的抗肿瘤药物是我们的首要任务。
研究发现，天然植物中的生物碱具有减少癌症的扩散，诱导癌细胞分化和凋亡，抑制癌细胞的增殖周期等生物活性。而且，生物碱在抗肿瘤活性方面具有耐受性好、疗效高、毒副作用小等优势；生物碱在抗肿瘤临床治疗方面也取得了良好的效果，其中，长春瑞宾、长春地辛、长春弗宁和脱水长春碱就是在长春碱类结构基础上半合成的抗癌药物。全面了解抗肿瘤天然生物碱及其作用机制，有利于合理开发利用其药物植物资源，为发现抗肿瘤药物的先导化合物和开发新的抗肿瘤药物提供科学、合理的依据。因而，从蓬莱葛属植物中寻找具有抗肿瘤作用的化合物，对于开发高效低毒的新型抗肿瘤药物具有很好的应用价值。本发明提供的蓬莱葛任碱型生物碱及其作为抗肿瘤药物尚未见报道。
发明内容
本发明提供了一类新化合物，即蓬莱葛任碱型生物碱，其分离自蓬莱葛属植物离药蓬莱葛(Gardneria distincta P.T.Li)，其化学结构式如式Ⅰ、式Ⅱ所示：
其中R1～R4分别选自氢、羟基、甲氧基、乙氧基、乙酰基、烷基烃、卤素、三元氧环、甲醚基。
当结构式Ⅰ中R1、R3、R4同时为甲氧基，R2为氢时，蓬蓬莱葛任碱型生物碱结构式如式Ⅲ所示：
式Ⅲ：
当结构式Ⅱ中R2、R3为甲氧基，R1、R4为羟基时，该化合物结构式Ⅳ如式所示：
式Ⅳ：
本发明另一目的是将上述蓬莱葛任碱型生物碱应用在制备抗肿瘤药物中。
本发明抗肿瘤药物中还可以加入一种或多种药物上可接受的辅料，所述辅料包括药学领域常规的填充剂、稀释剂、粘合剂、赋形剂、吸收促进剂、填充剂、表面活性剂和稳定剂等，必要时还可加入香味剂、色素和甜味剂等。
本发明所述应用除制成胶囊外，还可以制成丸剂、粉剂、片剂、粒剂、口服液和注射液等多种形式。
本发明采用MTT法检测了本发明化合物对人胃癌细胞株(MGC-803)、人肝癌细胞株(HepG-2)、人结肠癌细胞株(HCT-116)和肺癌人类肺泡基底上皮细胞株(A549)的抗肿瘤活性，实验结果显示生物碱1仅对人胃癌细胞株MGC-803具有较好抑制活性，生物碱2对肺癌人类肺泡基底上皮细胞株A549细胞显示出较好的抑制作用。
本发明优点和技术效果：
本发明从离药蓬莱葛中获得蓬莱葛任碱型生物碱，实验结果表明该2种生物碱化合物具有抑制肿瘤细胞增殖的活性，本发明化合物为研制抗肿瘤制剂提供了先导化合物，有利于植物药用资源的开发利用。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的采用常规市售试剂或按常规方法配置的试剂；
实施例1：蓬莱葛属生物碱的提取分离
取来源于云南腾冲的离药蓬莱葛样品风干粉碎，12kg粉碎物用85％甲醇加热60-80℃回流提取4次，料液比kg:L＝1:4，回流4h，回收溶剂，得到粗提物1.76kg，粗提物溶于水中，用质量浓度0.5％的盐酸调节pH为2-3，用乙酸乙酯萃取3次除去水溶性非碱后，酸水层用10％的氨水溶液调节pH至9-10，乙酸乙酯萃取4次，收集乙酸乙酯相，浓缩干燥后得总生物碱浸膏123g，用150g硅胶拌样，过1.5kg硅胶(100-200目)柱粗分，氯仿-甲醇作为流动相梯度洗脱(1:0、50:1、40:1、30:1、20:1、10:1、5:1、1:1、0:1)，TLC追踪检测合并含相同组分的洗脱液，得到5个分离段(Gd-Ⅰ～Gd-Ⅴ)；
Gd-Ⅲ(15g)过中压液相色谱，经甲醇-水(40％、50％、60％)洗脱，收集洗脱液浓缩得三个部分Gd-Ⅲ-a、Gd-Ⅲ-b和Gd-Ⅲ-c，其中生物碱1(35mg)从Gd-Ⅲ-b部分中结晶出来；
Gd-Ⅴ(12g)过中压液相色谱柱，用甲醇/水(40％、50％、60％)梯度洗脱得到三个组分Gd-Ⅴ-a、Gd-Ⅴ-b、Gd-Ⅴ-c，Gd-Ⅴ-a过硅胶柱，用石油醚-丙酮(体积比15:1、12:1、10:1)洗脱，得到Gd-Ⅴ-a-1、Gd-Ⅴ-a-2、Gd-Ⅴ-a-3，Gd-Ⅴ-a-2用制备型高效液相色谱(CH3CN-H2O，体积比45:55，TR＝10min)纯化分离，收集洗脱液浓缩得到生物碱2(9mg)。
生物碱1、2鉴定结果如下：
生物碱1：白色粉末；[α]25 D-319.78(c 0.09,MeOH)；UV(MeOH)λmax(logε)203(4.53),264(3.94),323(3.87)；IR(KBr)νmax 3434,2936,1581,1496,1268,1027cm-1；1D NMRdata(CDCl3)；HR-ESI-MS m/z 383.1960[M+H]+(calcd for C22H27N2O4 +,383.1965)。1H(400MHz)和13C(100MHz)NMR(CDCl3)数据见表1；以上数据结合2D NMR分析证实生物碱1为蓬莱葛任碱型生物碱，命名蓬莱葛碱B(Gardistine B)；
表1：蓬莱葛碱B(Gardistine B)的1H NMR和13C NMR数据
生物碱2：白色粉末；[α]24 D-99.63(c 0.09,MeOH)；UV(MeOH)λmax(logε)204(4.53),254(3.77),319(3.73)；IR(KBr)νmax 3437,1697,1491,1259cm-1；1D NMR data(DMSO-d6)；HR-ESI-MS m/z 403.1859[M+H]+(calcd for C21H27N2O6 +,403.1864)。1H(400MHz)和13C(100MHz)NMR(CDCl3)数据见表2。以上数据结合2D NMR分析证实生物碱2为蓬莱葛任碱型生物碱，命名蓬莱葛碱G(Gardistine G)；
表2：蓬莱葛碱G(Gardistine G)的1H NMR和13C NMR数据
上述生物碱1、2均为新颖结构的天然产物，属于蓬莱葛任碱型生物碱。
实施例2：生物碱1、2对四种肿瘤细胞株的抗肿瘤活性
(1)材料
DMEM培养液(美国Gibco公司)、胎牛血清(美国Gibco公司)、RPMI1640培养液(美国Gibco公司)、磷酸盐缓冲液(上海beyotime公司)、1％青霉素-链霉素(美国HyClone公司)、DMSO(美国Sigma公司)、MTT(美国Sigma公司)，本发明生物碱及顺铂(Cisplatin)均用DMSO配制。
(2)分别称取生物碱1和生物碱2，加入DMSO溶解，上样前用DMEM不完全培养液稀释至所需浓度(20μM)，并使得上样后的DMSO终浓度不超过0.1％；
(3)本实施例中采用的四种癌症细胞：人胃癌细胞株(MGC-803)、人肝癌细胞株(HepG-2)、人结肠癌细胞株(HCT-116)和肺癌人类肺泡基底上皮细胞株(A549)；以顺铂(Cisplatin)为阳性对照，用含10％胎牛血清、1％青霉素-链霉素含量的培养液(DMEM或者RMPI1640)配成细胞悬浮液，以每孔浓度为1×105个/mL细胞接种到96孔板，每孔体积120μL，于5％CO2、37℃恒温培养箱中培养24小时后吸出里面的培养基后加入待测生物碱溶液，固定浓度40μM初筛(IC50＞40μM未被标记，抗肿瘤效果比较差)，在该浓度对肿瘤细胞生长抑制在50％附近的生物碱设3个浓度进入梯度复筛，每孔终体积120μL。
加药(顺铂及样品)后继续置于37℃，5％CO2培养箱培养；
(4)MTT法测定生物碱1和生物碱2对四种肿瘤细胞株的毒性作用
37℃培养24h后，吸弃孔内培养上清液，每孔加MTT溶液120μL，继续孵育4小时，终止培养，吸弃孔内培养上清液，每孔加120μL的DMSO(分析纯)，用微型振荡器震荡5min，待各孔中的结晶充分溶解后，在570nm条件下，用酶标仪测出OD值，记录结果，计算出化合物的IC50值。
(5)数据处理
实验数据OD值采用“平均数±标准差”来表示，数理统计及方差分析工作采用SPSS完成，实验结果见表3；
表3：生物碱1和生物碱2对四种细胞的细胞毒性
结果显示，生物碱1仅对人胃癌细胞株MGC-803具有较好抑制活性，其IC50值为11.71μM，抗肿瘤活性均优于阳性对照；生物碱1和生物碱2对肝癌肿瘤细胞株HepG-2和人结肠癌细胞株HCT-116没有抑制作活性(IC50＞40μM)；生物碱2对肺癌人类肺泡基底上皮细胞株A549细胞显示出较好的抑制作用，其IC50值为1.20μM，活性优于阳性对照；生物碱1和生物碱2对肿瘤细胞株的杀伤作用具有较好的选择性，可进一步研究以便于开发新的抗肿瘤药物。
Claims (3)
1.结构式如式Ⅰ、式Ⅱ所示的蓬莱葛任碱型生物碱：
式Ⅰ：
式Ⅱ：
其中，R1～R4分别选自氢、羟基、甲氧基、乙氧基、乙酰基、烷基烃、卤素、三元氧环、甲醚基。
2.根据权利要求1所述的蓬莱葛任碱型生物碱，其特征在于，其为如下任一结构：
3.权利要求1或2所述的蓬莱葛任碱型生物碱在制备抗肿瘤药物中的应用。
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