CN107129453B - 化合物、毒蕈碱m受体拮抗剂、组合物及应用 - Google Patents

化合物、毒蕈碱m受体拮抗剂、组合物及应用 Download PDF

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CN107129453B
CN107129453B CN201610105954.8A CN201610105954A CN107129453B CN 107129453 B CN107129453 B CN 107129453B CN 201610105954 A CN201610105954 A CN 201610105954A CN 107129453 B CN107129453 B CN 107129453B
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梁鑫淼
杜娜娜
刘艳芳
张秀莉
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Dalian Institute of Chemical Physics of CAS
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Abstract

本发明公开了一种茄科植物中分离的含饱和氮杂环的咖啡酸衍生物，或其药学可接受的盐，本发明提供的化合物为毒蕈碱M受体拮抗剂，可用于痉挛、心绞痛、胆绞痛、肾绞痛、内脏痉挛等疾病的治疗。本发明通过采用无标记细胞靶点药理学技术，量效关系研究显示该化合物剂量依赖性地拮抗乙酰胆碱引起的DMR响应信号，表明此化合物具有较强的毒蕈碱M受体拮抗活性。相对于已知的M受体拮抗剂阿托品、东莨菪碱等，本发明提供的化合物结构差别很大，预期可以改善对于肌肉腺体和神经系统的选择性。目前研究表明毒蕈碱M受体与痉挛、镇痛、镇静、精神分裂症等疾病相关，据此可为相关疾病提供作用靶点明确的高效新配体。

Description

化合物、毒蕈碱M受体拮抗剂、组合物及应用
技术领域
本发明属于天然药物活性成分作用机制的发现及应用领域，涉及一种含饱和氮杂环的咖啡酸衍生物，及其在解痉药物中的作用机制及其用途。
背景技术
唐古特山莨菪Anisodus tanguticus为茄科山莨菪属的植物，是中国的特有植物，藏语称“唐川那保”。分布在中国大陆的甘肃、西藏、云南、青海等地，生长于海拔2,800米至4,200米的地区，一般生长在山坡及草坡阳处，目前尚未由人工引种栽培。作为传统的藏药，藏医用唐古特山莨菪及种子入药，具有麻醉镇痛的作用[1]。关于唐古特山莨菪的化学成分研究鲜有报道，多是托品烷类生物碱如莨菪碱、东莨菪碱、山莨菪碱和樟柳碱[2]，而唐古特山莨菪中一直以来广为人知的药用成分也是托品烷类生物碱，1960年祝寿河等用该药材中托品烷类生物碱改善微循环障碍，挽救了大量脑膜炎儿童的生命；山莨菪碱使暴发型流脑的病死率由过去的50％以上降至10％左右[3]。
痉挛是临床上常见的疾病，是指肌肉突然做不随意挛缩，俗称抽筋，会令患者突感剧痛，肌肉动作不协调，常见小腿、大腿、手、足、膀胱等痉挛。绞痛在医学上指痉挛性的剧烈疼痛并伴有闷塞的感觉，尤指胸部闷塞性疼痛(心绞痛)，临床常见内脏绞痛(腹绞痛)、心绞痛、肾绞痛、胆绞痛等。痉挛和绞痛多是由肌肉的不正常收缩引起[4]。
毒蕈碱M受体与腺体和平滑肌的收缩和舒张有关，M受体的拮抗剂有舒张平滑肌的作用，临床可用于解痉[5]。如临床用解痉药阿托品，为托品烷类生物碱，用药历史已久，其解痉作用机制即是M受体拮抗作用。然而阿托品由于对腺体、平滑肌、神经系统均有作用，因此在治疗疾病的同时有多种副作用，相对于此，不同结构的M受体拮抗剂的发现，尤其是本身是新化合物的，可能有更小的副作用。本专利即公开了一种新化合物，为含饱和氮杂环的咖啡酸衍生物，研究结果表明该化合物有很好的M受体拮抗活性，预示着可能作为一种副作用更小的解痉药物应用于临床。
我们应用高效液相色谱制备色谱系统，选用适合生物碱分离的反相模式[6]和离子交换模式[7,8]，对于唐古特山莨菪中的生物碱进行制备，得到一种新化合物，为含饱和氮杂环的咖啡酸衍生物。前期研究中我们采用无标记细胞靶点药理学技术建立了毒蕈碱M受体拮抗剂的筛选模型，并应用该模型筛选了唐古特山莨菪中的生物碱一维馏分对毒蕈碱M受体的拮抗活性。本发明用已经建立的M受体拮抗剂的筛选模型进行化合物的筛选，发现此新化合物对于M受体有很好的拮抗作用，而此类化合物还没有明确报道的作用靶点，这就为这类化合物的临床应用前景提供了一个新的开发方向。
参考文献：
[1]王环，沈建伟，张晓峰.山莨菪中4种生物碱含量与海拔关系的研究.中国中药杂志,2002,27(2):151-152
[2]张晓峰等.山莨菪植物体内4种莨菪烷类生物碱含量的变化.西北植物学报,2002,22(3):630-634
[3]祝寿河等.山莨菪碱在暴发型流脑综合治疗中的作用及机理的探讨.中国中西医结合研究会微循环专业委员会第一次学术会,1987.
[4]张翠平.浅析抗胆碱能药物在急性胆绞痛治疗中的作用.中国保健营养,2013,23(6):3277.
[5]史一鸣,钮因尧,陆阳.M受体及相关选择性药物研究进展.国际药学研究杂志,2009,36(5):355-361.
[6]Zhimou Guo et.al.Polar-copolymerized approach based on horizontalpolymerization on silica
surface for preparation of polar-modified stationary phases.Journalof Chromatography A,2010,1217:4555–4560.
[7]Long Z et.al.Selective separation and purification of highly polarbasic compounds using a silica-based strong cation exchange stationaryphase.Analytica Chimica Acta,2013,804:304-312.
[8]Long Z et.al.Two-dimensional strong cation exchange/positivelycharged reversed-phase liquid chromatography for alkaloid analysis andpurification.J.Sep.Sci,2013,36,3845–3852。
发明内容
本发明的目的在于提供一类含饱和氮杂环的咖啡酸衍生物的新化合物MMPVP，该化合物为如下结构所示的化合物、或其药学可接受的盐
其通式为
其中R1＝H,OH,OCH3；R2＝H,OH,OCH3；
本发明所述化合物药学上可接受的盐，选自但不限于：硫酸盐、盐酸盐、氢溴酸盐、磷酸盐、三氟乙酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、三氟甲磺酸盐、樟脑磺酸盐、甲酸盐、乙酸盐、丙酸盐、己酸盐、己二酸盐、酒石酸盐、柠檬酸盐、苯甲酸盐、富马酸盐、马来酸盐、乳酸盐、琥珀酸盐等。
本发明化合物MMPVP的制备方法是应用高效液相色谱制备色谱系统，选用适合生物碱分离的反相模式色谱柱XCharge C18和离子交换模式色谱柱XCharge SCX，对于唐古特山莨菪中的生物碱进行制备,得到此化合物。
本发明的另一目的是提供一类含有所示结构式及其类似化合物MMPVP的权利要求1-3任一化合物，或其药学可接受的盐的药物组合物，即以所示结构式的含饱和氮杂环的咖啡酸衍生物或其药学可接受的盐作为主要有效活性成分，还可加入一种或多种药物上可接受的辅料，以改善药物吸收效果或便于服用，如制成注射液、冻干粉针剂、注射微球、脂质体、片剂、胶囊剂、水剂、散剂、糊剂、喷雾剂、颗粒剂、软胶囊、滴丸剂、凝胶剂、贴片、膏剂等，其中优选注射液、冻干粉针剂、片剂和胶囊剂、贴片等；本发明所述的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、表面活性剂、稳定剂等，必要时还可以加入香味剂、色素和甜味剂。
本发明化合物使用方法包括给所述患者施用有效量的药物，可采用任何适当的给药方式施用这些治疗单体化合物或组合物，可以是但不限于经静脉注射、口服、直肠、舌下、粘膜、鼻、眼、肌肉、静脉内、经皮、脊椎、鞘内、关节内、动脉内、蛛网膜下、支气管、淋巴和子宫、皮下、皮肤表面、局部注射等方式给药。
本发明化合物为毒蕈碱M受体拮抗剂，可用于镇痛、解痉、镇静、解救有机磷中毒等疾病药物中，本发明的化合物不用于已有报道的托品烷类生物碱，且有良好的M受体拮抗活性，可能有更小的副作用，更好的作用效果。毒蕈碱M受体为G蛋白偶联受体，与痉挛、镇静、帕金森症、精神分裂症、抑郁症等相关，根据靶点与疾病的相关性，可以拓宽此化合物的临床应用范围。
附图说明
图1.化合物的纯化制备过程色谱图；
图2.毒蕈碱M受体拮抗剂的DMR信号及M受体拮抗剂预处理后乙酰胆碱引起的DMR信号：
a.不同浓度的莨菪碱处理HT29细胞后引起的DMR信号；
b.不同浓度的莨菪碱预处理HT29细胞后，再加入乙酰胆碱(16μM)引起的DMR信号；
c.不同浓度的东莨菪碱处理HT29细胞后引起的DMR信号；
d.不同浓度的东莨菪碱预处理HT29细胞后，再加入乙酰胆碱(16μM)引起的DMR信号；
图3.不同剂量的MMPVP的DMR信号及MMPVP预处理后乙酰胆碱引起的DMR信号；
图4.化合物MMPVP预处剂量与16μM乙酰胆碱引起的DMR信号的量效关系曲线。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。下面以具体实施例的方式对本发明作进一步的说明。
本发明实施例中所使用的各种化学试剂如无特殊说明，均通过常规商业途径获得；乙酰胆碱购自百灵威公司；唐古特山莨菪药材由西北高原植物所青海所采，唐古特山莨菪中的生物碱化合物均由实验室自制；DMSO购买自Bio Basic公司；HBSS和McCoy’s 5A培养液购自Gibco公司；384孔生物感应器微型板购自Corning公司；HT29人结肠癌细胞从中国科学院典型培养物保藏委员会细胞库获得(中国，上海)。
实施例1唐古特山莨菪中生物碱化合物的纯化制备
1.实验仪器与试剂
唐古特山莨菪药材由西北高原植物所青海所采，经梅丽娟老师鉴定。色谱柱XCharge C18(4.6×250mm,7μm，华谱公司)，XCharge C18(100×280mm,7μm，华谱公司)，XCharge SCX(100×316mm,7μm，华谱公司)，XCharge C18(20×250mm,7μm，华谱公司)，无水硫酸钠，二水合磷酸二氢钠购自国药集团，乙醇胺，磷酸，甲酸购自百灵威，制备色谱级乙腈购自安徽时联。
2.化合物的纯化制备
提取，分两批提取,每批25kg，加80L乙醇浸泡后加热回流2h，分别提取三次，得到350L提取液。提取液浓缩至100L，用强阳离子交换的SCX SPE材料富集生物碱组分，1.5kg填料，每次上样浓缩液10L，甲醇10L洗涤非生物碱，250mM NH4ClO4/90％甲醇20L洗脱生物碱。生物碱洗脱液蒸干，重溶到6L的0.1％TFA/W，进行C18HCE的一维制备，采用ACN-Na2SO4/W体系，30min内ACN 5％-15％，收集保留时间20-21min的馏分F12(制备色谱图为图1a)，对其进行SCX的二维制备，ACN-30mM NaH2PO4-W,30min ACN 35％-50％，收集保留时间49-51min的色谱峰F12_15(制备色谱图为图1b)，进行C18HCE的2％ACN-0.1％FA/W三维制备，得到F12_15_P2。正离子ESI-MS给出的分子离子峰m/z：234.0([M+H]+，分子式C14H19NO2，分子量233.1)。核磁氢谱信息如下：1H-NMR(D2O，400MHz)δ:7.00(1H,s),6.88(1H,d),6.76(1H,d),6.67(1H,d),5.90(1H,dd),3.78(1H,m),3.73(3H,s),3.60(1H,m),3.05(1H,m),2.70(3H,s),2.25(2H,m),2.02(2H,m),1.86(1H,m)。鉴定为MMPVP。
实施例2毒蕈碱M受体拮抗剂的在Epic平台上的DMR信号特征
1.方法
1.1细胞培养
HT29人结肠癌细胞从中国科学院典型培养物保藏委员会细胞库获得(中国，上海)。HT29细胞用含有体积浓度10％的胎牛血清、50μg/ml氨苄青霉素、100μg/ml硫酸链霉素的McCoy’s 5A培养基(GIBCO,货号12800017，含D-Glucose 4500.0mg/L，添加NaHCO3 2.2g/L)，在37℃、体积含量5％CO2(空气)的培养箱中培养。
1.2毒蕈碱M受体拮抗剂的细胞活性实验方法
HT29细胞以2×104个/孔的密度接种到384孔生物感应器微型板中，置于37℃、体积含量5％CO2(空气)的培养箱中培养22h，检测前用HBSS缓冲液洗1次，再在每孔中加入30μL HBSS缓冲液，置于系统上平衡孵育1h。平衡后首先在系统上建立一个2min的基线，然后将莨菪碱、东莨菪碱10μL(每个化合物均有14个浓度点，最高工作浓度10μM，2倍逐级稀释，5μM，2.5μM，1.25μM等等)分别加入已接种HT29细胞的384孔生物感应器微型板中，在系统上监测60min，得化合物的DMR响应曲线。然后加入乙酰胆碱(16μM)继续监测60min，得乙酰胆碱DMR响应曲线。通过监测乙酰胆碱引起的DMR响应信号是否被预处理时加入的待测化合物所抑制来判断化合物对毒蕈碱M受体的拮抗活性。
1.3数据分析
DMR数据由Epic Imager软件(康宁公司，美国)记录，并经Imager Beta3.7软件(康宁公司，美国)处理得到。采用Microsoft Excel 2010和GraphPad Prism软件进行统计分析。所有的DMR信号都经空白校正得到。所有数据来自2次独立实验，每次3次重复。
2.结果
分别以不同浓度的毒蕈碱M受体拮抗剂莨菪碱或东莨菪碱处理HT29细胞后，两者都不引起DMR响应信号，见图2a、2c(不同浓度的拮抗剂对应的DMR曲线重合)，再次加入16μM乙酰胆碱(EC80)，乙酰胆碱引起的DMR响应信号被抑制，且拮抗剂的浓度越高抑制活性越强，呈剂量依赖性，见图2b、2d(拮抗剂浓度从10μM逐级稀释到1.22nM，所对应的DMR信号曲线从下到上排列)。由此得出，毒蕈碱M受体拮抗剂特征DMR谱为拮抗剂本身不引起DMR响应信号，拮抗乙酰胆碱引起的DMR响应信号。
实施例3MMPVP对毒蕈碱M受体拮抗活性
采用无标记细胞靶点药理学技术对活性筛选中M受体拮抗活性较强的化合物MMPVP进行量效关系考察。首先，将10μL待测化合物加入已接种HT29细胞的384孔生物感应器微型板中，在系统上监测60min，然后加入10μL乙酰胆碱(16μM)继续监测60min。以作用时间对化合物引起的DMR信号作图，如图3a(化合物浓度从200μM逐级稀释到48.8nM，对应的DMR曲线变化不大)；以作用时间对第二步加入乙酰胆碱后引起的DMR响应信号作图，结果如图3b(化合物浓度从200μM逐级稀释到48.8nM，所对应的DMR信号曲线从下到上排列)。从图中可以看出，MMPVP在HT29细胞上不引起DMR信号，而预处理剂量与再次加入乙酰胆碱引起的DMR响应信号呈剂量依赖性，即MMPVP浓度越高抑制活性越强。这与毒蕈碱M受体拮抗剂的特征相符合，由此推断，MMPVP为毒蕈碱M受体拮抗剂。
在第二步实验图中，选取化合物MMPVP各个浓度30min时间点的乙酰胆碱DMR信号值，以浓度对数为横坐标，DMR值为纵坐标，在prism软件中拟合拮抗活性曲线，得到化合物MMPVP的IC50值，见图4。

Claims (6)

1.一种化合物，其为含饱和氮杂环的咖啡酸衍生物，具有如下结构：
化合物为(E)-2-methoxy-5-(2-(1-methylpyrrolidin-2-yl)vinyl)phenol，简称MMPVP。
2.一种毒蕈碱M受体拮抗剂，其特征在于：所述毒蕈碱M受体拮抗剂为权利要求1所述化合物MMPVP及其药学上可接受的盐中的一种或两种以上。
3.根据权利要求2所述的毒蕈碱M受体拮抗剂，其特征在于：化合物药学可接受的盐选自硫酸盐、盐酸盐、氢溴酸盐、磷酸盐、三氟乙酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、三氟甲磺酸盐、樟脑磺酸盐、甲酸盐、乙酸盐、丙酸盐、己酸盐、己二酸盐、酒石酸盐、柠檬酸盐、苯甲酸盐、富马酸盐、马来酸盐、乳酸盐、琥珀酸盐中的一种或两种以上。
4.一种毒蕈碱M受体拮抗剂组合物，其特征在于：所述毒蕈碱M受体拮抗剂包括权利要求1所述化合物MMPVP及其MMPVP药学上可接受的盐中的一种或两种以上为活性成分，其中还可添加有药学上可接受的载体或赋形剂中的一种或两种以上。
5.根据权利要求4所述的毒蕈碱M受体拮抗剂组合物，其特征在于：化合物药学可接受的盐选自硫酸盐、盐酸盐、氢溴酸盐、磷酸盐、三氟乙酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、三氟甲磺酸盐、樟脑磺酸盐、甲酸盐、乙酸盐、丙酸盐、己酸盐、己二酸盐、酒石酸盐、柠檬酸盐、苯甲酸盐、富马酸盐、马来酸盐、乳酸盐、琥珀酸盐中的一种或两种以上。
6.一种含饱和氮杂环的咖啡酸衍生物在制备解痉药物或在制备毒蕈碱M受体拮抗活性药物中的应用，其特征在于：含饱和氮杂环的咖啡酸衍生物包括权利要求1所述化合物MMPVP及其所述化合物药学上可接受的盐中的一种或两种以上，其解痉作用机制为毒蕈碱M受体拮抗活性。
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