CN103159827B - (1R,3S)-1-对硝基苯基-四氢-β-咔啉酰氨基酸、其合成、抗栓活性和应用 - Google Patents
(1R,3S)-1-对硝基苯基-四氢-β-咔啉酰氨基酸、其合成、抗栓活性和应用 Download PDFInfo
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- CN103159827B CN103159827B CN201110416859.7A CN201110416859A CN103159827B CN 103159827 B CN103159827 B CN 103159827B CN 201110416859 A CN201110416859 A CN 201110416859A CN 103159827 B CN103159827 B CN 103159827B
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Abstract
本发明公开了通式I的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸、公开了它们的合成、公开了它们的体外抗血小板聚集活性、进一步公开了它们在大鼠血栓模型上的体内抗血栓活性。因而本发明公开了(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸作为抗血栓剂的临床应用前景。
Description
发明领域
本发明涉及(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸、涉及它们的合成,涉及它们的体外抗血小板聚集活性、进一步涉及它们在大鼠血栓形成模型上的抗血栓活性。因而本发明涉及(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸作为抗血栓剂的临床应用前景。本发明属于生物医药领域。
背景技术
血栓性疾病是一种常见的心脑血管病,常表现为心肌梗死、缺血性脑梗死、静脉血栓栓塞等。每年每千人中有1~3人发生不同形式的血栓性疾病,严重影响人类健康。目前临床上常用的抗栓和溶栓药物都存在着这样或那样的缺点,如出血、肝损伤、胃黏膜损伤等毒副作用,寻找更加安全有效、毒副作用小的抗栓药物是药物研究研究的重要方向。
一般认为血栓的形成与多种因素导致的血小板聚集、粘附和释放有关。抗血小板药物成为了人们研究的热点。四氢-β-咔啉-3-羧酸具有抗血小板活性已被人们广泛认知,但水溶性和脂溶性都差使得它的生物利用度很低,一定的结构改造势在必行。在咔啉类抗血栓药物研究中,发明人揭示过用氨基酸修饰四氢咔啉可改善水溶性和提高生物利用度。发明人在多年的探索中发现往咔啉的1位引入对硝基苯基可获得活性更好的咔啉母核。依据这些发现,发明人提出了(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸、它们的合成,它们的体外抗血小板聚集活性及它们在大鼠血栓模型上的抗血栓活性的发明。
发明内容
本发明的第一个内容是制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸,该内容包括:
1)制备L-色氨酸甲酯;
2)L-色氨酸甲酯与对硝基苯甲醛经Pictet-Spengler缩合生成(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯;
3)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯皂化,生成(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸;
4)将(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸与氨基酸苄酯偶联制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯;
5)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯皂化制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸。
步骤1)的色氨酸甲酯的制备包括在甲醇溶液中滴加二氯亚砜,然后加入色氨酸。
步骤2)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯的制备包括在盐酸的甲醇溶液中色氨酸甲酯与对硝基苯甲醛进行Pictet-Spengler缩合后用柱色谱进行分离。
步骤3)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸的制备包括冰盐浴下向(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯四氢呋喃溶液先滴加2N氢氧化钠溶液至pH为12,反应完成之后再滴加2N的HCl溶液至pH为2。
步骤4)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸与氨基酸苄酯偶联反应在DCC、HOBt和NMM存在下进行,其中氨基酸苄酯选自L-亮氨酸苄酯、L-酪氨酸苄酯、L-苯丙氨酸苄酯、L-丙氨酸苄酯、L-缬氨酸苄酯、L-色氨酸苄酯、L-天冬氨酸双苄酯、L-Nε-Boc-赖氨酸苄酯、L-蛋氨酸苄酯、L-丝氨酸苄酯、L-脯氨酸苄酯、甘氨酸苄酯或L-谷氨酰胺苄酯。与L-脯氨酸苄酯偶联时,先把(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸制备为N-叔丁氧羰基-(1S,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸,再与L-脯氨酸苄酯偶联,然后在氯化氢的乙酸乙酯溶液中脱去叔丁氧羰基。
步骤5)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯的四氢呋喃溶液先滴加2N氢氧化钠溶液至pH为12,反应完成之后再滴加2N的HCl溶液至pH为2得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸。本发明的第一个内容可以用图1的合成路线描述。
附图说明
图1.(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸的合成路线.i)甲醇和二氯亚砜;ii)对硝基苯甲醛和浓盐酸;iii)四氢呋喃和2N氢氧化钠溶液;iv)DCC,HOBt,四氢呋喃和NMM;v)四氢呋喃,2N氢氧化钠溶液和2NHCl溶液。4a和5a中AA为L-亮氨酸残基、4b和5b中AA为L-酪氨酸残基、4c和5c中AA为L-苯丙氨酸残基、4d和5d中AA为L-丙氨酸残基、4e和5e中AA为L-缬氨酸残基;4f和5f中AA为L-色氨酸残基、4g中AA为侧链苄酯保护的L-天冬氨酸残基、5g中AA为L-天冬氨酸残基、4h和5h中AA为L-赖氨酰基、4i和5i中AA为L-蛋氨酸残基、4j和5j中AA为L-丝氨酸残基、4k和5k中AA为L-脯氨酸残基、4l和5l中AA为甘氨酸残基;4m和5m中AA为L-谷氨酰胺残基。
本发明的第二个内容是评价(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸(5a-m)的抗血小板聚集活性。
本发明的第三个内容是评价(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸(5a-m)的抗血栓活性。
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
具体实施方式
实施例1制备L-色氨酸甲酯
冰-盐浴下,向150ml甲醇中边搅拌边滴加15ml二氯亚砜,半小时后加入L-色氨酸10.2g(50mmol),撤去冰盐浴,室温搅拌两天。TLC板显示原料基本消失后停止反应。减压去除甲醇。残留物用甲醇溶解并减压去除甲醇。该操作重复三次。残留物用乙醚溶解并减压去除乙醚。该操作重复三次。最后用甲醇/乙醚重结晶,经过两次重结晶共得到白色固体12.6g(99.0%)。Mp:218-220℃;(c=5.0,CH3OH);ESI+-MS(m/e):219[M+H]+.
实施例2制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯
在30ml甲醇中缓慢加入5ml浓盐酸,向稀盐酸溶液中加入2.55g(10mmol)L-色氨酸甲酯盐酸盐和1.66g(11mmol)对硝基苯甲醛,微波加热75℃反应两小时,TLC板显示基本反应完全。用浓氨水调pH值至6,过滤得到黄色固体。用石油醚/乙酸乙酯进行柱层析,得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯0.64g(18.2%)。Mp:197-198℃;ESI-MS(m/e):352[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.695(s,1H),8.206(d,J=8.7Hz,2H),7.573(d,J=8.4Hz,2H),7.477(d,J=7.8Hz,1H),7.261(d,J=7.8Hz,1H),7.086-6.965(m,2H),5.483(s,1H),3.817-3.779(m,1H),3.638(s,3H),3.100(dd,J=5.1Hz,J=15.3Hz,1H),2.943(dd,J=7.2Hz,J=14.7Hz,1H).
实施例3制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸
冰盐浴搅拌下用四氢呋喃溶解3.51g(10mmol)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯,滴加2N氢氧化钠溶液调节pH值至12,用TLC板检测反应。原料点基本消失后,用2N盐酸液调节pH值至5-6,过滤,滤渣用蒸馏水洗,干燥得到黄色固体3.1g(92%)。Mp:183-184℃;ESI-MS(m/e):338[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.883(s,1H),8.243(d,J=7.8Hz,2H),7.636(d,J=7.8Hz,2H),7.535(d,J=7.2Hz,1H),7.290(d,J=7.8Hz,1H),7.103-7.029(m,2H),5.805(s,1H),4.046-3.992(m,1H),3.284(d,J=12.3Hz,1H),3.099-3.025(m,1H).
实施例4制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰氨基酸苄酯(4a-m)
在50ml茄瓶里加入337mg(1.0mmol)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸,加入3ml无水四氢呋喃溶解,冰浴下搅拌,加入162mg(1.2mmol)HOBt,247mg(1.2mmol)DCC形成A液。在25ml茄瓶中加入1.05mmolL-氨基酸苄酯的四氢呋喃溶液,加入NMM调pH形成B液。半小时后向A液中加入B液,加入NMM调节pH至9。TLC板检测反应进度。反应完成后过滤除去二环己基脲(DCU),减压浓缩除去溶剂,残留物加乙酸乙酯溶解,分别用5%碳酸氢钠溶液,饱和氯化钠溶液,5%硫酸氢钾溶液,饱和氯化钠溶液,饱和碳酸氢钠溶液,饱和氯化钠溶液各洗3遍。乙酸乙酯层用无水硫酸钠干燥1小时,过滤,减压浓缩除去溶剂,得到粗产物。再通过柱色谱进行分离得到纯品。洗脱剂。石油醚∶乙酸乙酯5∶1-3∶1梯度洗脱。
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-亮氨酸苄酯(4a)
产量:216(40%)。Mp:65-66℃;ESI-MS(m/e):541[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.829(s,1H),8.253-8.191(m,2H),7.513(d,J=8.7Hz,,2H),7.429-7.278(m,7H),7.040(s,J=6.9Hz,,2H),5.120(s,2H),4.387-4.315(m,1H),3.538-3.491(m,1H),2.939(dd,J=4.8Hz,,J=15.3Hz,,1H),2.696(dd,J=9.0Hz,,J=15.0Hz,,1H),1.691-1.531(m,3H),0.885(d,J=5.7Hz,,3H),0.839(d,J=6.3Hz,,3H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-酪氨酸苄酯(4b)
产量:140mg(24%)。Mp:110-111℃;ESI-MS(m/e):591[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.836(s,1H),9.274(s,1H),8.195(d,J=8.7Hz,3H),7.479(d,J=8.7Hz,2H),7.431(d,J=7.5Hz,1H),7.361-7.277(m,7H),7.100-6.968(m,5H),5.276(s,1H),5.078(s,2H),4.473((dd,J=4.5Hz,J=14.1Hz,1H),3.458-3.414(m,1H),2.944-2.869(m,3H),2.745-2.665(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-苯丙氨酸苄酯(4c)
产量:254mg(44%)。Mp:69-70℃;ESI-MS(m/e):575[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.797(s,1H),8.309(d,J=7.5Hz,1H),8.194(d,J=8.7Hz,2H),7.486-7.185(m,1H),7.039(dt,J=7.2Hz,J=14.7Hz,1H),5.271(s,1H),5.088(s,2H),4.561(dd,J=7.8Hz,J=14.1Hz,1H),3.462-3.408(m,1H),3.120-2.950(m,2H),2.893(dd,J=4.5Hz,J=15.3Hz,1H),2.705(dd,J=9.0Hz,J=15.3Hz,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-丙氨酸苄酯(4d)
产量:302mg(61%)。Mp:88-89℃;ESI-MS(m/e):499[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.845(s,1H),8.312(d,J=7.2Hz,1H),8.204(d,J=8.7Hz,2H),7.513(d,J=8.7Hz,2H),7.434-7.286(m,7H),7.104-6.984(m,2H),5.369(s,1H),5.126(dd,J=12.6Hz,J=15.6Hz,2H),4.323(dq,J=7.2Hz,J=28.8Hz,1H),3.525-3.452(m,1H),2.954-2.891(m,1H),2.735-2.643(m,1H),1.322(d,J=7.2Hz,3H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-缬氨酸苄酯(4e)
产量:280mg(53%)。Mp:97-98℃;ESI-MS(m/e):527[M+H]+。1H-NMR(300MHz,DMSO-d6):δ/ppm=10.836(s,1H),8.208(d,J=8.7Hz,1H),8.099(d,J=8.1Hz,2H),7.520(d,J=8.4Hz,2H),7.439-7.282(m,7H),7.103-6.981(m,2H),5.370(s,1H),5.143(dd,J=12.6Hz,J=20.1Hz,2H),4.236(dd,J=6.0Hz,J=7.8Hz,2H),3.566-3.521(m,1H),3.442-3.391(m,1H),2.953(dd,J=4.5Hz,J=15.0Hz,2H),2.142-2.032(m,1H),0.882(dd,J=1.8Hz,J=6.6Hz,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-色氨酸苄酯(4f)
产量:190mg(31%)。Mp:86-87℃;ESI-MS(m/e):614[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.934(s,1H),10.810(s,1H),8.253(d,J=7.5Hz,1H),8.158(d,J=8.1Hz,1H),7.957(s,1H),7.510(d,J=7.8Hz,1H),7.446-7.373(m,3H),7.312-7.281(m,4H),7.205-6.944(m,7H),5.269(s,1H),4.638-4.574(m,1H),3.209-3.168(m,1H),2.957-2.890(m,2H),2.734-2.650(m,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-天冬氨酸双苄酯(4g)
产量:240mg(38%)。Mp:62-63℃;ESI-MS(m/e):633[M+H]+;1H-NMR(500MHz,DMSO-d6):δ/ppm=0.8379(s,1H),8.4945(d,J=8.1Hz,1H),8.1957(d,J=8.75Hz,2H),7.5091(d,J=8.75Hz,2H),7.428(d,J=7.75Hz,1H),7.3732-7.2901(m,11H),7.846(d,J=7.1Hz,1H),7.0316-7.002(m,1H),5.3674(d,J=4.95Hz,1H),5.1061-5.0519(m,4H),4.8092(dd,J=6.55Hz,J=14.3Hz,1H),3.4869-3.4581(m,1H),2.9789-2.8555(m,3H),2.7447-2.6971(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰赖氨酸苄酯(4h)
得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰赖氨酸(Boc)苄酯。在冰盐浴搅拌下向(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰脯氨酸(Boc)苄酯加入4N盐酸乙酸乙酯,反应结束后减压去除乙酸乙酯。残留物用乙酸乙酯溶解并减压去除乙酸乙酯。该操作重复三次。残留物用乙醚溶解并减压去除乙醚。该操作重复三次。得到154mg(24%)目标化合物,为泡状固体。Mp:162-163℃;ESI-MS(m/e):556[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=11.186(s,1H),8.275(d,J=8.4Hz,2H),8.103(s,1H),7.631(d,J=8.7Hz,1H),7.478-7.322(m,7H),7.191-7.069(m,2H),6.162(s,1H),5.159(s,1H),4.328-4.239(m,1H),3.734(m,1H),3.181(d,J=7.5Hz,1H),2.967(dd,J=10.5Hz,J=15.6Hz,1H),2.713(d,J=5.7Hz,2H),1.734(m,2H),1.559(m,2H),1.436-1.343(m,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-甲硫氨酸苄酯(4i)
产量:183mg(33%)。Mp:67-68℃;ESI-MS(m/e):559[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.834(s,1H),8.307(d,J=7.5Hz,1H),8.204(d,J=8.7Hz,2H),7.524(d,J=9.0Hz,2H),7.437-7.280(m,7H),7.101-6.981(m,2H),5.369(s,1H),5.136(dd,J=12.3Hz,J=16.2Hz,2H),4.502-4.431(m,1H),3.507-3.462(m,1H),2.948(dd,J=4.5Hz,J=15.3,1H),2.713(dd,J=9.0Hz,J=15.0Hz,1H),2.013-1.975(m,6H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-丝氨酸苄酯(4j)
产量:160mg(31%)。Mp:80-81℃;ESI-MS(m/e):515[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.851(s,1H),8.272~8.188(m,2H),7.933(d,J=8.4Hz,1H),7.572-7.490(m,1H),7.466-7.427(m,1H),7.364-7.286(m,6H),7.107-6.984(m2H),5.410(s,1H),5.143(s,2H),4.497-4.417(m,1H),3.827-3.774(m,1H),3.692(dd,J=4.2Hz,J=11.1Hz,1H),2.991(dd,J=4.5Hz,J=12.3Hz,1H),2.759-2.678(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-脯氨酸苄酯(4k)
冰浴下将337mg(1mmol)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉羧酸悬浮于含280mg(1.3mmol)(Boc)2O的四氢呋喃溶液中,然后加三乙胺调节pH8-9,TLC板检测反应,原料点消失后减压除去四氢呋喃,用乙酸乙酯溶液残留物,然后用5%硫酸氢钾溶液洗涤3次,饱和氯化钠溶液洗涤3次,无水硫酸钠干燥,过滤,减压浓缩得到N-Boc-(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉羧酸。然后在50ml茄瓶里加入437mg(1mmol)N-Boc-(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸,加入3ml无水四氢呋喃溶解,冰浴下搅拌,加入HOBt162mg(1.2mmol),DCC247mg(1.2mmol)形成A液。在25ml茄瓶中加入1.05mmol氨基酸苄酯,四氢呋喃或DMF溶解,加入NMM调pH形成B液。半小时后向A液中加入B液,加入NMM调节pH至8-9。TLC板检测反应进度。反应完成后过滤除去DCU,减压浓缩除去溶剂,加乙酸乙酯溶解,分别用5%碳酸氢钠溶液,饱和氯化钠溶液,5%硫酸氢钾溶液,饱和氯化钠溶液,饱和碳酸氢钠溶液,饱和氯化钠溶液各洗3遍。乙酸乙酯层用无水硫酸钠干燥1小时,过滤,减压浓缩除去溶剂,得到粗产物。再通过柱色谱进行分离得到纯品。洗脱剂:石油醚∶乙酸乙酯5∶1-3∶1梯度洗脱。在冰盐浴搅拌下向N-Boc-(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰脯氨酸苄酯加入4N盐酸乙酸乙酯,反应结束后减压去除乙酸乙酯。残留物用乙酸乙酯溶解并减压去除乙酸乙酯。该操作重复三次。残留物用乙醚溶解并减压去除乙醚。该操作重复三次。得到180mg(34%)目标化合物,为泡状固体。Mp:164-165℃;ESI-MS(m/e):547[M+Na]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.880(s,1H),8.245~8.187(m,3H),7.498(d,J=8.7Hz,2H),7.440(d,J=8.7Hz,1H),7.315~7.264(m,2H),7.106~6.931(m,3H),5.355(s,2H),5.116(dd,J=12.6Hz,J=18.6Hz,1H),4.378(dd,J=3.9Hz,J=8.7Hz,1H),4.03(dd,J=7.2Hz,J=14.4Hz,1H),3.631~3.603(m,1H),3.360(s,1H),2.889(s,1H),2.752-2.667(m,1H),2.193-2.083(m,1H),1.992(s,1H),1.885-1.712(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰甘氨酸苄酯(4l)
产量:122mg(25%)。Mp:79-80℃;ESI+-MS(m/e):485[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.835(s,1H),8.389(d,J=6.0Hz,1H),8.211(d,J=8.7Hz,2H),7.542(d,J=8.7Hz,2H),7.462(d,J=7.5Hz,1H),7.373-7.282(m,6H),7.104-6.979(m,1H),5.396(d,J=4.5Hz,1H),3.940(d,J=6.0Hz,2H),3.497-3.451(m,1H)2.979(dd,J=4.5Hz,J=15.0Hz,1H),2.814-2.763(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-谷氨酰胺苄酯(4m)
产量:115mg(18%)。Mp:193-194℃;ESI+-MS(m/e):556[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.850(s,1H),8.396(d,J=3.9Hz,1H),8.208(d,J=8.7Hz,2H),7.535(d,J=6.0Hz,2H),7.462-7.287(m,7H),7.046(dt,J=6.9Hz,J=14.7Hz,2H),5.380(s,1H),5.125(s,2H),4.307(dd,J=8.4Hz,J=12.6Hz,1H),3.495-3.454(m,1H),2.945(dd,J=4.5Hz,J=15.3Hz,1H),2.736-2.655(m,1H),2.159(d,J=7.5Hz,1H),2.064-1.952(m,1H),1.911-1.789(m,1H).
实施例5制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰氨基酸(5a-m)
冰盐浴搅拌下在50ml茄瓶中称取0.1mmol(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯、加2ml无水THF溶解、滴加2N氢氧化钠溶液调节pH值至12、用TLC板检测反应。原料点基本消失后,用2N盐酸液调节pH值至2、过滤、滤渣用蒸馏水洗、干燥得到黄色固体。
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-亮氨酸(5a)
产量:40mg(90%)。ESI-MS(m/e):449[M-H]-;1H-NMR(300MHz,DMSO-d6):δ/ppm=10.97(s,1H),8.476(s,1H),8.245(d,J=8.1Hz,1H),7.548(dd,J=7.8Hz,J=20.7Hz,3H),7.399-7.316(m,J=8.1Hz,3H),7.206-7.033(m,2H),5.648(s,1H),4.276(dt,J=6.0Hz,J=13.2Hz,1H),3.791(d,J=6.0Hz,1H),3.198(dd,J=4.2Hz,J=15.3Hz,1H),2.88(dd,J=9.9Hz,J=15.3Hz,1H),0.911-0.750(m,6H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-酪氨酸(5b)
产量:43mg(87%)。ESI-MS(m/e):498[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=11.101(s,1H),9.454-9.356(m,1H),8.544-8.463(m,1H),8.378(d,J=8.4Hz,1H),8.249-8.189(m,1H),7.512(dd,J=8.4Hz,J=19.5Hz,2H),7.124-7.080(m,2H),7.030-6.990(m,2H),6.847(d,J=8.1Hz,1H),6.712(dd,J=8.4Hz,J=17.8Hz,2H),6.481(d,J=8.1Hz,1H),4.493(s,1H),4.363(m,1H),3.820-3.740(m,2H),3.148-3.103(m,2H),2.861-2.820(m,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-苯丙氨酸(5c)
产量:36mg(75%)。ESI-MS(m/e):483[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.153(s,1H),8.670(d,J=6.9Hz,1H),8.502-8.435(m,2H),8.371-8.346(m,1H),7.713-7.633(m,2H),7.325-7.254(m,8H),4.843(s,1H),4.441(m,1H),3.802-3.439(dd,J=6.0Hz,J=7.8Hz,2H),3.566-3.521(m,1H),3.442-3.391(m,1H),3.091-2.818(m,1H),1.812-1.578(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-丙氨酸(5d)
产量:35mg(86%)。ESI-MS(m/e):407[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.301(s,1H),8.939-8.841(m,2H),8.470(s,3H),7.778(d,J=8.4Hz,J=17.4Hz,1H),7.656-7.539(m,1H),7.314(d,J=3.6Hz,3H),4.623(dd,J=6.6Hz,J=13.5Hz,1H),4.495(s,1H),3.867-3.403(m,3H),1.527(s,3H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-缬氨酸(5e)
产量:36mg(84%)。ESI-MS(m/e):435[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=11.059(s,1H),8.427(d,J=8.1Hz,2H),7.595(d,J=7.8Hz,2H),7.464-7.319(m,4H),7.152-7.055(m,2H),5.801(s,1H),4.260(s,1H),4.052(s,1H),2.887-2.805(m,1H),2.112(t,J=6.3Hz,1H),0.998(s,1H),0.910(s,6H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-色氨酸(5f)
产量:40mg(77%)。ESI-MS(m/e):522[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=10.800(s,1H),8.496-8.453(m,1H),8.175(d,J=7.8Hz,1H),7.702-7.139(m,10H),7.096-6.926(m,4H),5.263-5.155(m,1H),4.497(d,J=8.1Hz,1H),3.807-3.745(m,2H),3.624-3.543(m,1H),3.225-3.157(m,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-天冬氨酸(5g)
产量:37mg(83%)。ESI-MS(m/e):451[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.140(s,1H),10.887(s,1H),8.505-8.411(m,2H),8.242(d,J=8.1Hz,1H),7.589-7.501(m,2H),7.334-7.321(m,2H),7.238-7.224(m,1H),7.155-7.008(m,2H),5.532(s,1H),4.520(s,3H),2.980(d,J=5.1Hz,1H),2.755-2.712(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-赖氨酸(5h)
产量:33mg(71%)。ESI-MS(m/e):464[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=11.250(s,1H),8.571-8.477(m,1H),8.233-8.188(m,4H),7.630-7.529(m,2H),7.297(m,2H),7.132-6.993(m,2H),6.172(s,1H),4.473(s,2H),3.150-3.032(m,2H),2.717(s,3H),1.902-1.211(m,6H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-蛋氨酸(5i)
产量:37mg(80%)。ESI-MS(m/e):467[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=11.248(s,1H),8.496-8.443(m,2H),8.267(d,J=6.9Hz,1H),7.653-7.545(m,3H),7.387-7.314(m,2H),7.148-7.069(m,2H),4.758-4.715(m,1H),4.481-4.364(m,2H),3.085-3.038(m,2H),2.759-2.709(m,1H),2.214(m,1H),2.044(d,J=15.0Hz,4H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-丝氨酸(5j)
产量:33mg(79%)。ESI-MS(m/e):423[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.183(s,1H),8.969(s,1H),8.798(d,J=8.1Hz,1H),8.520-8.471(m,2H),8.398(d,J=8.4Hz,1H),7.981(d,J=8.4Hz,1H),7.798-7.707(m,1H),7.646(d,J=7.5Hz,1H),7.568-7.518(m,1H),7.437-7.309(m,2H),4.619(d,J=3.9Hz,1H),3.975-3.912(m,1H),3.853-3.816(m,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-脯氨酸(5k)
产量:32mg(73%)。ESI-MS(m/e):434[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=11.135(s,1H),8.245-8.108(m,4H),7.622-7.537(m,2H),7.311(s,1H),7.125-7.057(m,1H),6.043(s,1H),4.300-4.191(m,1H),2.724(s,2H),2.075-1,928(m,1H),1.681-1.583(m,4H),1.413(m,2H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-甘氨酸(5l)
产量:35mg(88%)。ESI-MS(m/e):393[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.091(s,1H),9.058(d,J=6.0Hz,1H),8.926(s,1H),8.493-8.433(m,6H),7.718(d,J=8.1Hz,1H),7.654-7.604(m,1H),7.339(d,J=7.5Hz,2H),4.114(d,J=5.7Hz,4H),3.587(d,J=6.0Hz,1H).
(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰-L-谷氨酰胺(5m)
产量:35mg(76%)。ESI-MS(m/e):464[M-H]-。1H-NMR(300MHz,DMSO-d6):δ/ppm=12.107(s,1H),10.878(s,1H),8.513-8.427(m,2H),8.231(d,J=8.1Hz,1H),7.735-7.620(m,1H),7.563-7.485(m,1H),7.411-7.292(m,2H),7.114-7.012(m,2H),5.483(s,1H),4.580-4.552(m,1H),4.310-4.204(m,1H),3.309-2.946(m,1H),2.788-2.736(m,1H),2.235-2.137(m,3H),1.968-1.918(m,1H),1.844-1.748(m,2H).
实施例6体外抗血小板聚集活性评价
猪颈动脉取血,用3.8%枸橼酸钠(V枸橼酸钠∶V全血=1∶9)抗凝。1000g/min离心10分钟得富血小板血浆(PRP),再以3000g/min离心10分钟,得贫血小板血浆(PPP)。以胶原(ADP)、血小板活化因子(PAF)、凝血酶(TH)和花生四烯酸(AA)为诱导剂诱导血小板聚集。母核3和(1S,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-甲酰氨基酸(5a-m)均用生理盐水溶解。每个数据平行测6次。表1数据说明母核以及13个氨基酸修饰的目标化合物均对ADP诱导的血小板聚集的没有抑制作用,表2数据说明对PAF诱导的血小板聚集没有抑制作用,表3数据说明对TH诱导的血小板聚集具有抑制作用,表4数据说明对AA诱导的血小板聚集具有抑制作用。通过数据还可以看到本发明经过氨基酸修饰后的一些目标化合物抑制TH和AA诱导的血小板聚集作用比母核抑制作用增强。
表15a-m在100μM浓度下对ADP诱导的血小板聚集作用的影响
表25a-m在100μM浓度下对PAF诱导的血小板聚集作用的影响
表35a-m在100μM浓度下对TH诱导的血小板聚集作用的影响
表45a-m在100μM浓度下对AA诱导的血小板聚集作用的影响
实施例75a-m的抗血栓活性评价
实验前将化合物(母核3和5a-m)配成0.33nmol/l生理盐水溶液(加入少量的吐温80润湿助溶),用于体内的剂量为1.0nmol/kg。阳性药Asprin配成10g/l生理盐水溶液,即55.5mmol/l的浓度,用于体内的剂量为167μmol/kg。空白对照为生理盐水,抗凝剂为肝素钠2.4mg/ml生理盐水溶液。将雄性SD大鼠随机分组,每组12只大鼠。化合物(5a-m)、母核3、空白和阳性药共分为15组,以(3ml/kg)给大鼠灌胃,30分钟后,用乌拉坦(20g/100ml,7ml/kg),麻醉后,分离右颈动脉和左颈静脉。把一根6cm长的事先精密称重的丝线放在聚乙烯管中,将插管充满肝素钠的生理盐水溶液(50IU/ml)后,一端插入左侧静脉,从一端加入定量肝素钠抗凝,然后插入右侧动脉。血流从右侧动脉流经聚乙烯管流入左侧静脉,15分钟后取出丝线并记录血栓湿重。数据列入表3。
表35a-m对SD雄性大鼠血栓形成的影响
n=10,血栓湿重用表示;a)与生理盐水比p<0.01.
Claims (9)
1.通式I的化合物,式中AA为L-亮氨酸残基、L-酪氨酸残基、L-苯丙氨酸残基、L-丙氨酸残基、L-缬氨酸残基、L-色氨酸残基、侧链苄酯保护的L-天冬氨酸残基、L-赖氨酸残基、L-蛋氨酸残基、L-丝氨酸残基、L-脯氨酸残基、甘氨酸残基或L-谷氨酰胺残基。
2.制备权利要求1的化合物的方法,该方法包括:
1)制备L-色氨酸甲酯;
2)L-色氨酸甲酯与对硝基苯甲醛经Pictet-Spengler缩合生成(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯;
3)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯皂化,生成(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸;
4)将(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸与氨基酸苄酯偶联制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯;
5)(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯皂化制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸。
3.按照权利要求2所述的方法,其特征在于步骤1)中所述的色氨酸甲酯的制备方法包括在甲醇溶液中滴加二氯亚砜,然后加入色氨酸。
4.按照权利要求2所述的方法,其特征在于步骤2)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯的制备方法包括在盐酸的甲醇溶液中色氨酸甲酯与对硝基苯甲醛进行Pictet-Spengler缩合后用柱色谱进行分离,得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯。
5.按照权利要求2所述的方法,其特征在于步骤3)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸的制备方法包括冰盐浴下向(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸甲酯四氢呋喃溶液先滴加2N氢氧化钠溶液至pH为12,反应完成之后再滴加2N的HCl溶液至pH为5-6得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸。
6.按照权利要求2所述的方法,其特征在于步骤4)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸与氨基酸苄酯偶联制备(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯,反应在二环己基羰二亚胺(DCC),N-羟基苯并三氮唑(HOBt)和N-甲基吗啉(NMM)存在下进行,其中氨基酸苄酯选自L-亮氨酸苄酯、L-酪氨酸苄酯、L-苯丙氨酸苄酯、L-丙氨酸苄酯、L-缬氨酸苄酯、L-色氨酸苄酯、L-天冬氨酸双苄酯、L-Nε-Boc-赖氨酸苄酯、L-蛋氨酸苄酯、L-丝氨酸苄酯、L-脯氨酸苄酯、甘氨酸苄酯或L-谷氨酰胺苄酯。
7.按照权利要求6所述的方法,其特征在于:先把(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸制备为N-叔丁氧羰基-(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉-3-羧酸,再与L-脯氨酸苄酯偶联,然后在氯化氢的乙酸乙酯溶液中脱去叔丁氧羰基。
8.按照权利要求2所述的方法,其特征在于步骤5)的(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸苄酯的四氢呋喃溶液先滴加2N氢氧化钠溶液至pH为12,反应完成之后再滴加2N的HCl溶液至pH为2得到(1R,3S)-1-对硝基苯基-1,2,3,4-四氢-β-咔啉酰氨基酸。
9.权利要求1的化合物在制备抗血栓药物中的应用。
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