CN102477073B - 具有溶血栓活性的寡肽及其制备方法和应用 - Google Patents
具有溶血栓活性的寡肽及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及具有溶血栓活性的寡肽,属于生物医药领域。
背景技术
P6A(ARPAK)为纤维蛋白β链降解产物之一,具有溶血栓活性。在P6A的代谢研究中发现了代谢产物PAK。在大鼠动静脉旁路插管溶血栓模型上,PAK的溶血栓活性比母体P6A强。按照一般的认识,多肽在体内都会迅速降解。通过PAK的结构修饰延缓体内降解速率和提高溶血栓活性,是寡肽溶血栓药物研究的重要途径。
按照一般的认识,含多肽的两亲性分子,例如脂肪胺链修饰的多肽,在适当的条件下通过分子间非共价键相互作用可发生自组装,形成纳米结构。借助纳米结构可改善多肽在体内的输送、延缓多肽在体内的降解速率和提高多肽的体内活性。根据这些认识,发明人提出了本发明。
发明内容
本发明的目的在于提供具有溶血栓活性的寡肽,并通过动物实验评价了本发明寡肽的溶血栓活性,证明了本发明的寡肽具有优秀的溶血栓活性。
本发明是通过以下技术方案来实现的:
本发明的目的之一是提供以下结构的化合物,
其中,n为6、8、10、12、14或16。
本发明的目的之二是提供制备本发明化合物的方法,具体包括如下步骤:
1)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与N-羟基琥珀酰亚胺(HOSu)缩合为活泼酯中间体后,在THF与水的混合溶液中与Ala在弱碱性条件下生成Boc-Pro-Ala;
2)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl或Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl或Boc-Pro-Ala-Lys(Boc)-OBzl;
3)在甲醇中将步骤2)所得产物皂化为Boc-Pro-Ala-Lys(Z)或Boc-Pro-Ala-Lys(Boc);
4)在DCC存在下Boc-Lys(Boc)在无水THF中与饱和脂肪胺缩合为Boc-Lys(Boc)-NHCH2(CH2)nCH3;其中,n为6、8、10、12、14或16;
5)在氯化氢-乙酸乙酯溶液中Boc-Lys(Boc)-NHCH2(CH2)nCH3脱去Boc(叔丁氧羰基),生成Lys-NHCH2(CH2)nCH3;
6)在DCC和HOBt存在下,步骤3)所得产物在无水THF中与Lys-NHCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)nCH3或Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)nCH3;
7)当步骤6)的产物为Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)nCH3时,将步骤6)所得产物在氯化氢-乙酸乙酯溶液中脱除Boc生成Pro-Ala-Lys(Z)-Lys[Pro-Ala-Lys(Z)]-NHCH2(CH2)n-CH3;再将生成的Pro-Ala-Lys(Z)-Lys[Pro-Ala-Lys(Z)]-NHCH2(CH2)n-CH3在CH3OH、Pd/C和H2的条件下氢解,得到本发明化合物;
当步骤6)的产物为Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)nCH3时,将步骤6)的产物在氯化氢-乙酸乙酯溶液中脱除Boc,得到本发明化合物。
本发明的目的之三是提供一种药物组合物,该药物组合物含有治疗上有效剂量的本发明化合物,并含有一种或多种药学上可接受的赋型剂或者辅加剂。
本发明的目的之四是提供一种药物制剂,是将本发明化合物与药学上可接受的赋型剂或者辅加剂的混合物制成片剂、胶囊剂、粉剂、颗粒剂、锭剂或口服液。
本发明的化合物和本发明的药物组合物均可用于制备溶血栓药物。
本发明通过动物实验评价了本发明化合物的溶血栓活性,实验结果证明,本发明的寡肽具有优秀的溶血栓活性。
附图说明
图1为本发明化合物的结构通式;
图2为本发明化合物的合成路线;
图3为化合物4f的透射电子显微镜照片。
在图2中,i)DCC、HOBt和NMM;ii)NaOH;iii)HCl/EA;iv)Pd/C;H2。
具体实施方式
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。
在本发明的实施例中,所用的缩略术语表示以下含义:
THF 四氢呋喃
HOBt N-羟基苯并三氮唑
DCC 二环己基碳二亚胺
TLC 薄层色谱
Boc 叔丁氧羰基
实施例1制备Boc-Lys(Boc)-NHCH2(CH2)6CH3(1a)
将2.1g(6mmol)Boc-Lys(Boc)溶于20ml无水THF。向得到的溶液中加入0.81g(6mmol)N-羟基苯并三氮唑(HOBT)并完全溶解。10分钟后,在冰浴下加入1.2g(6mmol)二环己基二碳亚胺(DCC)与25ml无水THF的溶液,得到反应液(I)。将0.645g(5mmol)NH2CH2(CH2)6CH3溶于10m1无水THF并搅拌30分钟得到反应液(II)。冰浴下将反应液(II)加入反应液(I)中并室温搅拌12h,TLC(二氯甲烷/甲醇,20∶1)显示Boc-Lys(Boc)消失。反应混合物过滤除去生成的二环己基脲(DCU)。滤液减压浓缩除去THF,残留物用250ml乙酸乙酯溶解。得到的溶液依次用饱和NaHCO3水溶液洗、饱和NaCl水溶液洗、5%KHSO4水溶液洗及饱和NaCl水溶液洗。乙酸乙酯相用无水Na2SO4干燥、过滤、滤液减压浓缩至干,残留物柱层析纯化,得到1.83g(80%)目标化合物,为无色粉末。ESI-MS(m/e):458[M+H]+.
实施例2制备Boc-Lys(Boc)-NHCH2(CH2)8CH3(1b)
按照实施例1的方法由1.903g(5.5mmol)Boc-Lys(Boc)和1.097g(6.94mmol)CH3(CH2)8CH2NH2制得1.997g(82%)目标化合物,为无色粉末。ESI-MS(m/e):486[M+H]+.
实施例3制备Boc-Lys(Boc)-NHCH2(CH2)10CH3(1c)
按照实施例1的方法由0.83g(2.4mmol)Boc-Lys(Boc)和0.37g(2mmol)CH3(CH2)10CH2NH2制得0.563g(55%)目标化合物,为无色粉末。ESI-MS(m/e):514[M+H]+.
实施例4制备Boc-Lys(Boc)-NHCH2(CH2)12CH3(1d)
按照实施例1的方法由0.83g(2.4mmol)Boc-Lys(Boc)和0.426g(2mmol)CH3(CH2)12CH2NH2制得0.541g(50%)目标化合物,为无色粉末。ESI-MS(m/e):543[M+H]+.
实施例5制备Boc-Lys(Boc)-NHCH2(CH2)14CH3(1e)
按照实施例1的方法由1.903g(5.5mmol)Boc-Lys(Boc)和1.679g(6.94mmol)CH3(CH2)14CH2NH2制得2.37g(76%)目标化合物,为无色粉末。ESI-MS(m/e):570[M+H]+.
实施例6制备Boc-Lys(Boc)-NHCH2(CH2)16CH3(1f)
按照实施例1的方法由2.1g(6mmol)Boc-Lys(Boc)和1.205g(5mmol)CH3(CH2)14CH2NH2制得1.61g(54%)目标化合物,为无色粉末。ESI-MS(m/e):598[M+H]+.
实施例7制备Lys-NHCH2(CH2)6CH3(2a)
将0.914g(2mmol)Boc-Lys(Boc)-NHCH2(CH2)6CH3溶于25ml 4N氯化氢-乙酸乙酯溶液中并室温搅拌1小时,TLC(二氯甲烷/甲醇,20∶1)显示Boc-Lys(Boc)-NHC8H17消失。减压浓缩除去乙酸乙酯,残留物反复加少量乙醚进行减压浓缩以除去氯化氢。最后加少量乙醚将残留物研磨成0.499g(97%)目标化合物,为无色粉末。ESI-MS(m/e):258[M+H]+.
实施例8制备Lys-NHCH2(CH2)8CH3(2b)
按照实施例7的方法由1.455g(3mmol)Boc-Lys(Boc)-NHCH2(CH2)8CH3制得0.97g(90%)目标化合物,为无色粉末。ESI-MS(m/e):286[M+H]+.
实施例9制备Lys-NHCH2(CH2)10CH3(2c)
按照实施例7的方法由1.539g(3mmol)Boc-Lys(Boc)-NHCH2(CH2)10CH3制得1.042g(90%)目标化合物,为无色粉末。ESI-MS(m/e):315[M+H]+.
实施例10制备Lys-NHCH2(CH2)12CH3(2d)
按照实施例7的方法由1.623g(3mmol)Boc-Lys(Boc)-NHCH2(CH2)12CH3制得1.130g(91%)目标化合物,为无色粉末。ESI-MS(m/e):343[M+H]+.
实施例11制备Lys-NHCH2(CH2)14CH3(2e)
按照实施例7的方法由2.0g(3.45mmol)Boc-Lys(Boc)-NHCH2(CH2)14CH3制得1.554g(97%)目标化合物,为无色粉末。ESI-MS(m/e):370[M+H]+.
实施例12制备Lys-NHCH2(CH2)16CH3(2f)
按照实施例7的方法由1.49g(2.5mmol)Boc-Lys(Boc)-NHCH2(CH2)16CH3制得0.89g(90%)目标化合物,为无色粉末。ESI-MS(m/e):398[M+H]+.
实施例13制备Boc-Pro-Ala
将4.30g(20mmol)Boc-Pro用80ml无水THF溶解。向得到的溶液中加入2.53g(22mmol)N-羟基琥珀酰亚胺(HOSu)并完全溶解。10分钟后,在冰浴下加入4.944g(24mmol)二环己基碳二亚胺(DCC)与60ml无水THF的溶液,得到反应液。在室温下搅拌12h,TLC(石油醚/丙酮,5∶1)显示Boc-Pro消失。滤除二环己基脲(DCU),减压除去THF。残留物用250ml乙酸乙酯溶解,得到的溶液依次用饱和NaHCO3水溶液洗、饱和NaCl水溶液各洗三次,有机相减压浓缩至干。将有机相减压浓缩至干剩余物用60ml THF溶解,得到反应液(I)。将1.98g(22mmol)丙氨酸(Ala)用60ml水溶解,得到反应液(II)。搅拌下将反应液(II)加入反应液(I)中,用固体NaHCO3调混合液pH至8-9之间,室温下搅拌16h,反应过程中时时抽CO2气,并保持溶液pH至8-9之间,TLC(石油醚/丙酮,5∶1)显示原料点消失。反应后减压除去THF,残余液用饱和KHSO4水调溶液pH至2,加200ml乙酸乙酯萃取,有机层用饱和NaCl水溶液洗三次,分出有机相用无水Na2SO4干燥、过滤、滤液减压浓缩至干,得5.176g(90%)目标化合物,为白色固体粉末。ESI-MS(m/e):285[M-H]-.
实施例14制备Boc-Pro-Ala-Lys(Boc)-OBzl
按照实施例1的方法由3.146g(11mmol)Boc-Pro-Ala和2.966g(10mmol)Lys(Boc)-OBzl制得4.472g(85%)目标化合物,为无色粉末。ESI-MS(m/e):529[M+H]+.
实施例15制备Boc-Pro-Ala-Lys(Z)-OBzl
按照实施例1的方法由1.761g(6mmol)Boc-Pro-Ala和3.577g(6.6mmol)Lys(Z)-OBzl制得3.213g(84%)目标化合物,为无色粉末。ESI-MS(m/e):639[M+H]+.
实施例16制备Boc-Pro-Ala-Lys(Z)
将2.552g(4mmol)Boc-Pro-Ala-Lys(Z)-OBzl用20ml甲醇溶解,冰浴搅拌的下用2NNaOH调反应液pH至13,并使反应液保持在0℃,pH在13。2小时后,TLC(石油醚/丙酮,5∶1)检测显示Boc-Pro-Ala-Lys(Z)-OBzl消失。用饱和KHSO4水调反应后混合溶液pH至中性,减压除去甲醇后再用饱和KHSO4溶液调反应残余液pH至2,用200ml乙酸乙酯萃取,有机层用饱和NaCl水溶液洗至中性,分出有机相用无水Na2SO4干燥、过滤、滤液减压浓缩至干,得2.007g(91%)目标化合物,为无色粉末。ESI-MS(m/e):549[M-H]-.
实施例17制备Boc-Pro-Ala-Lys(Boc)
按照实施例16的方法由2.640g(4mmol)Boc-Pro-Ala-Lys(Boc)-OBzl制得2.397g(93%)目标化合物,为无色粉末。ESI-MS(m/e):515[M-H]-.
实施例18制备Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)6CH3(3a)
按照实施例1的方法由1.8g(3.3mmol)Boc-Pro-Ala-Lys(Z)和0.495g(1.5mmol)Lys-NHCH2(CH2)6CH3制得0.81g(41%)目标化合物,为无色粉末。Mp107℃;[α]D25=-47.5(c=0.50,甲醇);ESI-MS(m/e):1318[M+H]+;IR(KBr):3297,3073,2932,2865,1695,1643,1539,1452,1400,1251,1164,1127,1029,919,768,739,698,596.1H NMR(300MHz,DMSO-d6):δ/ppm=8.16-7.93(m,2H),7.93-7.58(m,5H),7.58-7.49(t,J=7.2Hz,J=7.8Hz,1H),7.47-7.38(t,J=7.8Hz,J=7.2Hz,1H),4.24-4.07(m,6H),3.08-2.91(m,4H),2.91-2.81(d,J=6.0Hz,4H),2.20-2.01(m,3H),1.87-1.70(m,6H),1.70-1.55(m,3H),1.45-1.28(m,42H),1.28-1.12(m,24H),0.86(t,J=5.7Hz,J=6.9Hz,3H).13C-NMR(75MHz,DMSO-d6):δ/ppm=172.73,172.52,172.21,171.51,155.98,154.34,153.73,127.75,124.93,119.58,110.06,79.26,78.85,77.76,59.75,52.92,49.17,48.55,47.19,46.96,38.89,32.33,31.75,31.31,30.29,29.68,29.46,29.42,29.15,28.58,28.46,26.73,24.31,18.85,18.27,17.75,14.65.Anal Calcd for C68H107N11O15:C,61.94;H,8.18;N,11.68;O,18.20.
实施例19制备Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)8CH3(3b)
按照实施例1的方法由1.131g(2.2mmol)Boc-Pro-Ala-Lys(Boc)和0.358g(1mmol)Lys-NHCH2(CH2)8CH3制得0.259g(20%)目标化合物,为无色粉末。Mp 116℃;[α]D25=-56.9(c=0.70,甲醇);ESI-MS(m/e):1279[M+H]+;IR(KBr):3295,3084,2974,2931,2863,1691,1642,1535,1452,1398,1352,1248,1169,980,861,785,694.1H NMR(300MHz,DMSO-d6):δ/ppm=8.16-7.93(m,2H),7.93-7.58(m,5H),7.58-7.49(t,J=7.2Hz,J=7.8Hz,1H),7.47-7.38(t,J=7.8Hz,J=7.2Hz,1H),4.24-4.07(m,6H),3.08-2.91(m,4H),2.91-2.81(d,J=6.0Hz,4H),2.20-2.01(m,3H),1.87-1.70(m,6H),1.70-1.55(m,3H),1.45-1.28(m,42H),1.28-1.12(m,24H),0.86(t,J=5.7Hz,J=6.9Hz,3H).13C NMR(75MHz,DMSO-d6):δ/ppm=172.73,172.52,172.21,171.51,155.98,154.34,153.73,127.75,124.93,119.58,110.06,79.26,78.85,77.76,59.75,52.92,49.17,48.55,47.19,46.96,38.89,32.33,31.75,31.31,30.29,29.68,29.46,29.42,29.15,28.58,28.46,26.73,24.31,18.85,18.27,17.75,14.65.Anal Calcd for C64H115N11O15:C,60.12;H,9.07;N,12.05;O,18.77.
实施例20制备Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)10CH3(3c)
按照实施例1的方法由1.808g(3.3mmol)Boc-Pro-Ala-Lys(Z)和0.579g(1.5mmol)Lys-NHCH2(CH2)10CH3制得0.553g(35%)目标化合物,为无色固体粉末。Mp 99℃;[α]D 25=-56.5(c=0.67,甲醇);ESI-MS(m/e):1375[M+H]+;IR(KBr):3293,3076,2929,2860,1685,1640,1541,1453,1400,1253,1163,1131,1026,987,918,767,739,698.1H NMR(300MHz,DMSO-d6):δ/ppm=8.07-7.67(m,7H),7.34(s,9H),7.18(s,2H),5.00(s,4H),4.35-4.12(m,7H),2.98-2.94(m,8H),2.07(s,2H),1.77(s,7H),1.62(s,3H),1.39(s,14H),1.32(s,14H),1.23(s,29H),8.87-0.83(t,J=6.0Hz J=6.6Hz 3H).13C NMR(75MHz,CDCl3):δ/ppm=172.74,172.56,172.21,171.53,169.34,156.51,154.34,153.72,137.75,128.77,128.15,79.22,78.83,65.57,59.73,52.95,49.16,48.56,47.19,46.97,40.86,39.19,38.89,32.33,32.03,31.75,31.31,30.28,29.51,29.47,29.17,28.57,28.46,26.73.AnalCalcd for C72H115N11O15:C,62.90;H,8.43;N,11.21;O,17.46.
实施例21制备Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)12CH3(3d)
按照实施例1的方法由1.808g(3.3mmol)Boc-Pro-Ala-Lys(Z)和621mg(1.5mmol)Lys-NHCH2(CH2)12CH3制得672mg(32%)目标化合物,为无色粉末。Mp 107℃;[α]D 25=-57.7(c=0.58,甲醇);ESI-MS(m/e):1403[M+H]+;IR(KBr):3292,3076,2927,2857,2357,1698,1638,1543,1455,1399,1258,1162,1115,1028,1000,918,771,697,543.1H NMR(300MHz,DMSO-d6):δ/ppm=8.09-7.63(m,7H),7.33(s,10H),7.21(s,2H),4.99(s,4H),4.31-4.02(m,8H),3.35(s,2H),3.27(s,2H),2.98-2.94(m,8H),2.06(s,2H),1.99(s,1H),1.78-1.59(m,12H),1.38(m,10H),1.35(s,3H),1.32(s,12H),1.22(s,33H),8.87-0.83(t,J=6.0Hz J=6.9Hz 3H).13C NMR(75MHz,CDCl3):δ/ppm=171.95,171.86,171.65,171.55,170.89,170.30,65.29,64.84,59.13,52.82,49.19,49.09,46.10,41.66,40.85,40.57,39.46,39.18,38.89,36.32,32.00,31.75,30.10,29.46,29.41,29.16,28.49,26.88,25.78,25.72,24.03,22.97,22.54,18.43.Anal Calcd for C74H119N11O15:C,63.36;H,8.55;N,10.98;O,17.11.
实施例22制备Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)14CH3(3e)
按照实施例1的方法,由1.131g(2.2mmol)Boc-Pro-Ala-Lys(Boc)和0.442g(1mmol)Lys-NHCH2(CH2)14CH3制得0.314g(21%)目标化合物,为无色粉末。Mp 111℃;[α]D 25=-58.7(c=0.70,甲醇);ESI-MS(m/e):1362[M+H]+;IR(KBr):3294,3083,2970,2928,2858,1691,1643,1535,1453,1398,1332,1249,1169,1119,984,927,862,773,689.1H NMR(300MHz,DMSO-d6):δ/ppm=8.15-7.92(m,2H),7.92-7.58(m,4H),7.58-7.36(m,1H),6.77-6.62(s,2H),4.39-4.05(m,6H),3.83-3.67(m,5H),3.60-3.15(m,7H),3.09-2.93(m,4H),2.93-2.81(m,5H),2.56(s,2H),2.15-1.99(m,2H),1.90-1.29(m,43H),1.29-1.13(m,31H),0.85(t,J=7.5Hz,J=6.9Hz,3H).13C NMR(75MHz,DMSO-d6):δ/ppm=172.73,172.52,172.22,171.50,155.97,154.35,153.75,128.28,127.52,124.82,119.52,110.12,79.25,78.85,77.75,65.59,59.78,54.65,52.97,49.21,48.58,46.96,45.26,38.91,32.32,31.73,30.27,29.68,29.17,29.13,28.48,26.74,24.28,23.60,23.12,22.98,18.57,18.32,18,19,15.02.Anal Calcd for C70H127N11O15:C,61.69;H,9.39;N,11.31;O,17.61.
实施例23制备Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)16CH3(3f)
按照实施例1的方法由2.4g(4.4mmol)Boc-Pro-Ala-Lys(Z)和0.94g(2mmol)Lys-NHCH2(CH2)16CH3制得0.961g(33%)目标化合物,为无色粉末。Mp 105℃;[α]D 25=-63.3(c=0.55,甲醇);ESI-MS(m/e):1458[M+H]+;IR(KBr):3300,3077,2926,2856,1696,1639,1543,1454,1400,1255,1164,1127,1013,702.1H NMR(300MHz,CDCl3):δ/ppm=7.42-7.24(m,15H),7.18-7.09(m,1H),5.17-5.00(m,4H),4.52-4.02(m,7H),3.58-3.32(m,5H),3.19(s,8H),2.42-2.1(m,1H),2.13-2.00(m,3H),1.97-1.82(m,6H),1.80-1.62(m,9H),1.62-1.32(m,38H),1.32-1.14(m,32H),0.88-0.84(d,J=5.1Hz,3H).13C NMR(75MHz,CDCl3):δ/ppm=172.09,156.12,78.80,65.41,53.50,52.70,52.23,40.27,31.88,29.65,29.61,29.57,29.49,29.31,29.21,28.44,28.32,28.29,25.78,24.60,22.64,14.08.Anal Calcd for C78H127N11O15:C,64.21;H,8.77;N,10.56;O,16.45.
实施例24制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)6CH3(4a)
将1.317g(1mmol)Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)6CH3用25ml甲醇溶解,加入0.066g Pd/C,一滴甲酸,通H2(0.02Mba),室温搅拌至原料点消失。滤除Pd/C、滤液减压浓缩至干。将残留物溶于25ml 4N氯化氢-乙酸乙酯溶液中并室温搅拌1小时,TLC(二氯甲烷/甲醇,10∶1)显示原料点消失。减压浓缩除去乙酸乙酯,残留物反复加少量乙醚进行减压浓缩以除去氯化氢。最后加少量乙醚将残留物研磨成0.679g(85%)目标化合物,为无色粉末。Mp 111℃;[α]D 25=-34.9(c=0.65,甲醇);ESI-MS(m/e):851[M+H]+;IR(KBr):3427,3263,3064,2934,1653,1546,1456,1382,1245,1160,1041,562.1H NMR(300MHz,MeOD):δ/ppm=8.75-8.13(d,J=5.7Hz,1H),8.13-7.86(m,1H),4.50-4.21(m,6H),3.49-3.28(m,14H),3.02-2.90(m,4H),2.61-2.40(m,2H),2.23-1.96(m,9H),1.81-1.61(m,8H),1.61-1.48(m,6H),1.48-1.37(m,9H),1.37-1.23(m,11H)0.96-0.82(d,J=6.9Hz,3H).13C NMR(75MHz,DMSO-d6):δ/ppm=172.12,171.83,171.65,171.52,168.29,59.10,53.00,49.18,46.06,32.36,32.00,31.74,31.44,30.08,29.46,29.14,26.79,23.98,23.07,22.53,21.51,18.37,18.31,14.40,11.34.Anal Calcd for C42H79N11O7:C,59.34;H,9.37;N,18.12;O,13.17.
实施例25制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)8CH3(4b)
按照实施例7的方法由0.2g(0.16mmol)Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)8CH3制得0.155g(97%)目标化合物,为白色固体粉末。Mp 98℃;[α]D 25=-41.2(c=0.70,甲醇);ESI-MS(m/e):879[M+H]+;IR(KBr):3411,3260,3064,2928,2860,1653,1548,1456,1379,1307,1247,1160,1045,589.1H-NMR(300MHz,DMSO-d6):δ/ppm=8.90-8.81(d,J=6.6Hz,1H),8.61-8.45(m,1H),8.22-7.85(m,3H),7.75-7.64(d,J=8.4Hz,1H),7.59-7.51(t,J=7.2Hz,J=8.1Hz,1H),7.47-7.38(t,J=7.8Hz,J=7.5Hz,1H),4.42-4.28(m,2H),4.28-4.17(m,6H),3.65-3.32(m,14H),3.29-3.12(m,5H),3.12-2.90(m,4H),2.80-1.68(m,4H),2.39-2.25(m,2H),1.97-1.80(m,7H),1.75-1.46(m,9H),1.46-1.12(m,28H),0.86(t,J=5.1Hz,J=6.3Hz,3H).13C-NMR(75MHz,DMSO-d6):δ/ppm=172.33,172.14,171.84,171.53,168.30,128.42,127.62,124.85,119.50,110.25,59.11,53.08,49.29,46.04,38.90,32.35,31.98,31.73,31.43,30.09,29.44,29.12,26.77,23.98,23.07,22.52,21.52,18.33,14.62.Anal Calcd for C44H83N11O7:C,60.18;H,9.53;N,17.54;O,12.75.
实施例26制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)10CH3(4c)
按照实施例24的方法由0.653g(0.5mmol)Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)10CH3制得0.374g(78%)目标化合物,为白色固体粉末。Mp 104℃;[α]D 25=-13.4(c=0.55,甲醇);ESI-MS(m/e):907[M+H]+,IR(KBr):3439,3264,3065,2927,2857,1654,1548,1457,1381,1249,1164,1037,641,470.1H-NMR(300MHz,DMSO-d6):δ/ppm=10.12(s,1H),8.86-8.84(d,J=6.9Hz 2H),8.51(s,1H),8.25-7.89(m,8H),4.38-4.14(m,6H),3.21(s,4H),3.09-2.99(m,5H),2.74(s,3H),2.42-2.24(m,2H),1.91-1.79(m,6H),1.70-1.55(m,9H),1.45-1.05(m,32H),0.87-0.83(tJ=5.7Hz J=6.6Hz 3H).13C-NMR(75MHz,CDCl3):δ/ppm=172.13,171.84,171.65,171.55,168.29,59.10,53.00,49.18,46.06,32.36,32.00,31.74,31.44,30,08,29.46,29.14,26.79,23.98,23.07,22.53,21.51,18.37,18.31,14.40.Anal Calcd for C46H87N11O7:C,60.96;H,9.68;N,17.00;O,12.36.
实施例27制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)12CH3(4d)
按照实施例24的方法由0.668g(0.5mmol)Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)12CH3。制得0.337g(72%)目标化合物,为白色固体粉末。Mp 97℃;[α]D 25=-31.1(c=0.60,甲醇);ESI-MS(m/e):935[M+H]+;IR(KBr):3423,3255,3062,2926,2856,2361,1654,1547,1457,1381,1307,1247,1156,1041,992,947,652.1H-NMR(300MHz,DMSO-d6):δ/ppm=10.22(s,1H),8.90-8.88(d,J=6.3Hz,2H),8.56(s,1H),8.30-7.89(m,7H),4.24-4.02(m,6H),3.20(s,4H),3.15-2.96(m,4H),2.41-2.29(m,2H),1.97-1.86(m,8H),1.64-1.45(m,10H),1.35-1.13(m,37H),0.84-0.81(t,J=5.4Hz,J=6.6Hz,3H).13C-NMR(75MHz,CDCl3):δ/ppm=172.39,171.90,171.59,168.28,59.04,52.94,49.27,46.03,40.71,39.04,38.79,34.58,31.72,30.10,29.48,29.45,29.13,26.77,23.98,22.52,21.53,18.19,14.38.Anal Calcd forC48H91N11O7:C,61.71;H,9.82;N,16.49;O,11.99.
实施例28制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)14CH3(4e)
按照实施例7的方法由200mg(0.17mmol)Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)14CH3制得129mg(91%)目标化合物,为白色固体粉末。Mp 101℃;[α]D 25=-34.5(c=0.70,甲醇);ESI-MS(m/e):962[M+H]+;IR(KBr):3411,3293,3070,2920,2851,1643,1551,1465,1377,1307,1286,715,673.1H-NMR(300MHz,DMSO-d6):δ/ppm=10.11(m,2H),8.90-8.79(d,J=6.9Hz,2H),8.57-8.43(m,2H),8.22-7.17(m,9H),7.58-7.36(m,1H),4.42-4.10(m,6H),4.19-4.13(m,4H),4.13-3.89(m,1H),3.82-3.71(m,1H),3.27-3.12(m,4H),3.12-2.90(m,4H),2.73(s,4H),2.39-2.23(m,2H),2.02-1.77(m,6H),1.77-1.43(m,8H),1.43-1.13(m,34H),0.86(t,J=6.0Hz,J=6.3Hz,3H).13C-NMR(75MHz,DMSO-d6):δ/ppm=172.53,172.37,172.13,171.84,171.65,171.54,168.79,168.28,128.42,127.69,124.90,119.53,110.20,63.68,59.07,52.96,52.75,49.20,46.06,42.74,40.84,40.56,40.29,40.01,39.73,39.45,39.17,38.89,32.35,31.99,31.74,31.43,30.10,29.50,29.19,29.15,26.18,24.00,23.49,22.89,21.63,18.59,18.47,14.25.Anal Calcd for C50H95N11O7:C,62.40;H,9.95;N,16.01;O,11.64.
实施例29制备Pro-Ala-Lys-Lys(Pro-Ala-Lys)-NHCH2(CH2)16CH3(4f)
按照实施例24的方法由0.729g(0.5mmol)Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)16CH3制得0.236g(69%)目标化合物,为无色固体粉末。Mp 107℃;[α]D 25=-45.3(c=0.69,甲醇);ESI-MS(m/e):991[M+H]+;IR(KBr):3423,3262,3062,2925,2855,1651,1547,1458,1380,1311,1247,1160,665.1H-NMR(300MHz,DMSO-d6):δ/ppm=8.91-8.78(d,J=6.6Hz,2H),8.33-7.80(m,5H),4.42-4.11(m,6H),3.29-3.10(m,4H),3.10-2.92(m,4H),2.83-2.66(m,4H),2.39-2.24(m,2H),1.99-1.72(m,7H),1.72-1.48(m,10H),1.48-1.12(m,44H),0.90-0.79(t,J=5.1Hz,J=6.6Hz,3H).13C-NMR(75MHz,DMSO-d6):δ/ppm=172.16,172.03,171.85,171.55,170.66,169.59,168.47,168.35,129.57,59.07,52.91,50.50,49.37,49.22,49.10,46.04,38.87,38.17,31.75,30.13,29.33,29.16,26.96,26.87,26.78,24.04,23.03,22.54,18.61,18.40,18.30,14.38.Anal Calcd forC52H99N11O7:C,63.06;H,10.08;N,15.56;O,11.31.
实验例14a-f的溶血栓活性
将200-220g雄性SD大鼠用20%乌拉坦溶液(6ml/kg,i.p.)进行麻醉。麻醉大鼠仰卧位固定,分离右颈总动脉,于近心端夹动脉夹,近心端和远心端分别穿入手术线,将远心端的手术线于皮毛用止血钳夹紧,在远心端插管,松开动脉夹,放出约1ml动脉血并装在1ml的EP管中。往垂直固定的玻璃管(长15mm,内径2.5mm,外径5.0mm,管底用胶塞密封)中注入0.1ml大鼠动脉血液,往管内迅速插入一支不锈钢质料的血栓固定螺栓。该血栓固定螺旋用直径为0.2mm的不锈钢丝绕成,螺旋部分长12mm,含15个螺圈,螺圈的直径为1.0mm,托柄与螺旋相连,长7.0mm,呈问号型。血液凝固15min后,打开玻璃管底部的胶塞,用镊子固定血栓固定螺旋的托柄,从玻璃管中取出被血栓包裹的血栓固定螺旋,精确称重。
旁路插管由3段构成,中段为聚乙烯胶管,长60mm,内径3.5mm,两端为相同的聚乙烯管,长100mm,内径1mm,外径2mm,该管的一端拉成尖管(用于插入大鼠颈动脉或静脉),外径1mm,另一端的外部套一段长7mm,外径3.5mm的聚乙烯管(加粗,用于插入中段的聚乙烯胶管内)。3段管的内壁均硅烷化。将血栓包裹的血栓固定螺旋放入中段聚乙烯胶管内,胶管的两端分别与两根聚乙烯的加粗端相套。用注射器通过尖管端将管中注满肝素生理盐水溶液(50IU/kg)备用。
分离大鼠的左颈外静脉,近心端和远心端分别穿入手术线,在暴露的左颈外静脉上小心地剪一斜口,将上面制备好的旁路管道的尖管由斜口插入左颈外静脉开口的近心端,同时远离旁路管中段(含精确称重的血栓固定螺旋)内血栓固定螺旋的托柄。用注射器通过另一端的尖管推入准确量的肝素生理盐水(50IU/kg),此时注射器不撤离聚乙烯管,用止血钳夹住注射器与聚乙烯管之间的软管。在右颈总动脉的近心端用动脉夹止血,在离动脉夹不远处将右颈总动脉小心地剪一斜口。从聚乙烯管的尖部拔出注射器,将聚乙烯管的尖部插入动脉斜口的近心端。旁路管道的两端都用4号手术缝线与动静脉固定。
用头皮针将生理盐水(3ml/kg),尿激酶的生理盐水溶液(20000IU/kg)或不同浓度化合物的生理盐水溶液通过旁路管的中段(含精确称重的血栓固定螺旋),刺入远离血栓固定螺旋的近静脉处,打开动脉夹,使血流通过旁路管道从动脉流向静脉,此即大鼠动静脉旁路溶栓模型,缓慢将注射器中的液体注入到血液中(约6min),使生理盐水(空白对照),尿激酶(阳性对照)或本发明的化合物通过血液循环,按静脉-心脏-动脉的顺序作用到血栓上。从开始注射时计时,1h后从旁路管道中取出血栓固定螺旋,精确称重。计算每只大鼠旁路管道中血栓固定螺旋给药前后的质量差,统计并评价化合物的体内溶栓活性。结果见表1。
表11nmol/kg 4a-f对大鼠血栓减重的影响
a)n=10,血栓湿重用均值±SD mg表示;b)与生理盐水组相比,p<0.01。
实验例2剂量对4e体内溶血栓活性的影响
按照实验例1的实验方法,选取溶栓效果最好的4e考察1nmol/kg、0.01nmol/kg和0.001nmol/kg三个剂量下的溶栓活性。结果如表2所示。结果表明,4e的溶栓作用显示剂量依赖性。
表2
表2为4e的剂量对大鼠血栓减重的影响a,其中,a表示样本数,即所用大鼠的个数,a)n=10,血栓减重用均值±SD mg表示;b)与生理盐水及0.01nmol/kg 4e组比,p<0.01;c)与生理盐水及0.001nmol/kg 4e组比,p<0.01;d)与生理盐水相比,p>0.05。
实验例24a-f的纳米球
1)水溶液中4a-f纳米球的粒径
在激光散射粒度仪上观察了25℃和37℃时4a-f在10-4M、10-5M和10-6M水溶液中形成的纳米球的粒径。结果如表3所示,结果表明,4a-f在水溶液中可组装为纳米球,25℃时粒径为132至593nm,37℃时粒径为122至803nm。
表3
2)4a-f的纳米球的形态
将4a-f配成浓度为1×10-12mg/ml的水溶液,然后将此溶液滴在铜网上,挥发干溶剂后在JEM-1230透射电子显微镜下观察纳米球的形态。测定表明,4a-f形成规则的纳米球。4f的透射电子显微镜照片作为代表,用图3描述。
Claims (6)
2.一种制备权利要求1所述的化合物的方法,其特征在于,包括如下步骤:
1)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与N-羟基琥珀酰亚胺(HOSu)缩合为活泼酯中间体后,在THF与水的混合溶液中与Ala在弱碱性条件下生成Boc-Pro-Ala;
2)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl或Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl或Boc-Pro-Ala-Lys(Boc)-OBzl;
3)在甲醇中将步骤2)所得产物皂化为Boc-Pro-Ala-Lys(Z)或
Boc-Pro-Ala-Lys(Boc);
4)在DCC存在下Boc-Lys(Boc)在无水THF中与饱和脂肪胺缩合为
Boc-Lys(Boc)-NHCH2(CH2)nCH3;其中,n为6、8、10、12、14或16;
5)在氯化氢-乙酸乙酯溶液中Boc-Lys(Boc)-NHCH2(CH2)nCH3脱去Boc(叔丁氧羰基),生成Lys-NHCH2(CH2)nCH3;
6)在DCC和HOBt存在下,步骤3)所得产物在无水THF中与Lys-NHCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)nCH3或Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)nCH3;
7)当步骤6)的产物为Boc-Pro-Ala-Lys(Z)-Lys[Boc-Pro-Ala-Lys(Z)]-NHCH2(CH2)nCH3时,将步骤6)所得产物在氯化氢-乙酸乙酯溶液中脱除Boc生成Pro-Ala-Lys(Z)-Lys[Pro-Ala-Lys(Z)]-NHCH2(CH2)n-CH3;再将生成的Pro-Ala-Lys(Z)-Lys[Pro-Ala-Lys(Z)]-NHCH2(CH2)n-CH3在CH3OH、Pd/C和H2的条件下氢解,得到权利要求1所述化合物;
当步骤6)的产物为Boc-Pro-Ala-Lys(Boc)-Lys[Boc-Pro-Ala-Lys(Boc)]-NHCH2(CH2)nCH3时,将步骤6)的产物在氯化氢-乙酸乙酯溶液中脱除Boc,得到权利要求1所述化合物。
3.一种药物组合物,其特征在于,含有治疗上有效剂量的权利要求1所述化合物,并含有一种或多种药学上可接受的赋型剂或者辅加剂。
4.一种药物制剂,其特征在于,是将权利要求1所述化合物与药学上可接受的赋型剂或者辅加剂的混合物制成片剂、胶囊剂、粉剂、颗粒剂、锭剂或口服液。
5.权利要求1所述化合物在制备溶血栓药物中的应用。
6.权利要求3所述药物组合物在制备溶血栓药物中的应用。
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CN101190941A (zh) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | 具有溶血栓活性的多肽、其制备方法及应用 |
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