CN102477069B - Pro-Ala-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3、及其合成和作为溶血栓剂的应用 - Google Patents
Pro-Ala-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3、及其合成和作为溶血栓剂的应用 Download PDFInfo
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Abstract
本发明公开了具有溶血栓活性的寡肽及其合成方法和用途。本发明提供了结构式为通式I所示的具有溶血栓活性的寡肽,式中n=6、8、10、12、14或16。体外和体内溶血栓活性试验表明,本发明通式I所示的寡肽化合物具有优秀的溶血栓活性,可作为溶血栓药物应用。
Description
技术领域
本发明涉及溶血栓寡肽,尤其涉及Pro-Ala-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3(n为6、8、10、12、14和16)及其合成方法,本发明进一步涉及它们在大鼠动静脉旁路插管模型上的溶血栓作用和应用,本发明属于溶血栓剂领域。
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,例如脂肪醇链修饰的多肽,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
本发明目的之一是提供具有溶血栓活性的寡肽。
本发明目的之二是提供一种合成上述具有溶血栓活性寡肽的方法。
本发明目的之三是将上述寡肽应用于制备溶血栓药物。
本发明上述目的是通过以下技术方案来实现的:
具有溶血栓活性的寡肽(结构式用通式I表示):
通式I
式中n=6、8、10、12、14或16。
一种合成上述具有溶血栓活性寡肽的方法,包括:
1)Boc-Pro与Ala-OBzl缩合为Boc-Pro-Ala-OBzl;
2)将Boc-Pro-Ala-OBzl氢解为Boc-Pro-Ala;
3)Boc-Pro-Ala与Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Boc)-OBzl;
4)将Boc-Pro-Ala-Lys(Boc)-OBzl氢解为Boc-Pro-Ala-Lys(Boc);
5)Boc-Asp与饱和脂肪醇缩合为Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3,其中n=6、8、10、12、14或16;
6)Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3脱去Boc生成Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
7)Boc-Pro-Ala-Lys(Boc)与Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
8)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)n-CH3]-OCH2(CH2)nCH3脱除Boc,即得。
为了达到更好的合成效果,步骤(1)中优选在DCC和HOBt存在下Boc-Pro在无水THF中与Ala-OBzl缩合为Boc-Pro-Ala-OBzl;步骤2)中优选在甲醇中将Boc-Pro-Ala-OBzl氢解为Boc-Pro-Ala;步骤3)中优选在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Boc)-OBzl;步骤4)中优选在甲醇中将Boc-Pro-Ala-Lys(Boc)-OBzl氢解为Boc-Pro-Ala-Lys(Boc);步骤5)中优选在DCC存在下Boc-Asp在无水THF中与饱和脂肪醇缩合为Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;步骤6)中优选在氯化氢-乙酸乙酯溶液中Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3脱去Boc生成Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;步骤7)中优选在DCC和HOBt存在下Boc-Pro-Ala-Lys(Boc)在无水THF中与Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;步骤8)中优选在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)n-CH3]-OCH2(CH2)nCH3脱除Boc。
体外和体内溶血栓活性试验表明,本发明通式I所示的寡肽化合物具有优秀的溶血栓活性活性,可作为溶血栓药物应用。
缩略术语
THF 四氢呋喃
HOBt N-羟基苯并三氮唑
DCC 二环己基羰二亚胺
TLC 薄层色谱
Boc 叔丁氧羰基
附图说明
图1本发明通式I化合物的结构式。
图2本发明通式I化合物的合成路线图.i)DCC、HOBt和NMM;ii)NaOH;iii)HCl/EA。
图3本发明化合物4b的透射电子显微镜照片。
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。
实施例1制备Boc-Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3](1a)
将3.0g(12.9mmol)Boc-Asp用20ml无水THF溶解。向得到的溶液中加入4.17g(30.9mmol)N-羟基苯并三氮唑(HOBT)。10分钟后,在冰浴下加入6.37g(30.9mmol)二环己基羰二亚胺(DCC)与25ml无水THF的溶液,得到反应液(I)。把4.02g(30.9mmol)脂肪醇链溶于10ml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,然后室温搅拌12h,TLC(石油醚/乙酸乙酯,15∶1)显示Boc-Asp消失。反应混合物过滤,滤除二环己基脲(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水溶液洗和饱和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,残留物经柱层析纯化,得到4.23g(72%)目标化合物,为无色粉末。ESI-MS(m/e):458[M+H]+.
实施例2制备Boc-Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3](1b)
按照实施例1的方法由5.0g(21.5mmol)Boc-Asp和8.14g(51.5mmol)CH3(CH2)8CH2OH制得7.60g(69%)目标化合物,为无色粉末。ESI-MS(m/e):514[M+H]+.
实施例3制备Boc-Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3](1c)
按照实施例1的方法由5.0g(21.5mmol)Boc-Asp和9.58g(51.5mmol)CH3(CH2)10CH2OH制得7.94g(65%)目标化合物,为无色粉末。ESI-MS(m/e):570[M+H]+.
实施例4制备Boc-Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3](1d)
按照实施例1的方法由5.0g(21.5mmol)Boc-Asp和11.02g(51.5mmol)CH3(CH2)12CH2OH制得8.32g(62%)目标化合物,为无色粉末。ESI-MS(m/e):626[M+H]+.
实施例5制备Boc-Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3](1e)
按照实施例1的方法由3.0g(12.9mmol)Boc-Asp和7.48g(30.9mmol)CH3(CH2)14CH2OH制得5.16g(59%)目标化合物,为无色粉末。ESI-MS(m/e):682[M+H]+.
实施例6制备Boc-Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3](1f)
按照实施例1的方法由3.0g(12.9mmol)Boc-Asp和8.34g(30.9mmol)CH3(CH2)16CH2OH制得4.94g(52%)目标化合物,为无色粉末。ESI-MS(m/e):738[M+H]+.
实施例7制备Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3](2a)
将4.23g(9.26mmol)Boc-Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3]溶解在45ml 4N氯化氢-乙酸乙酯溶液中,室温搅拌1小时,TLC(石油醚/乙酸乙酯,15∶1)显示Boc-Asp(OC8H17)-OC8H17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3.39g(93%)目标化合物,为无色粉末。ESI-MS(m/e):358[M+H]+.
实施例8制备Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3](2b)
按照实施例7的方法5.96g(11.6mmol)Boc-Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3]制得4.96g(95%)目标化合物,为无色粉末。ESI-MS(m/e):414[M+H]+.
实施例9制备Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3](2c)
按照实施例7的方法6.28g(11.0mmol)Boc-Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3]制得5.24g(94%)目标化合物,为无色粉末。ESI-MS(m/e):470[M+H]+.
实施例10制备Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3](2d)
按照实施例7的方法6.32g(10.1mmol)Boc-Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3]制得5.39g(95%)目标化合物,为无色粉末。ESI-MS(m/e):526[M+H]+.
实施例11制备Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3](2e)
按照实施例7的方法5.18g(7.6mmol)Boc-Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3]制得4.27g(91%)目标化合物,为无色粉末。ESI-MS(m/e):582[M+H]+.
实施例12制备Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3](2f)
按照实施例7的方法4.94g(6.7mmol)Boc-Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3]制得4.06g(90%)目标化合物,为无色粉末。ESI-MS(m/e):638[M+H]+.
实施例13制备Boc-Pro-Ala-OBzl
按照实施例1的方法从3.0g(14.0mmol)Boc-Pro和5.88g(16.7mmol)Tos·Ala-Obzl制得到5.19g(99%)目标化合物,为黄色粉末。ESI-MS(m/e):377[M+H]+.
实施例14制备Boc-Pro-Ala
将5.19g(13.8mmol)Boc-Pro-Ala-OBzl用25ml甲醇溶解。往溶液中加入1.0gPd/C、通H2(0.02Mba)并室温搅拌至Boc-Pro-Ala-OBzl消失。滤除Pd/C、滤液减压浓缩至干。制得3.59g(91%)目标化合物,为黄色粉末。ESI-MS(m/e):285[M-H]-.
实施例15制备Boc-Pro-Ala-Lys(Boc)-OBzl
按照实施例1的方法由6.76g(23.6mmol)Boc-Pro-Ala和10g(19.7mmol)Tos·Lys(Boc)-OBzl制得6.78g(57%)目标化合物,为无色粉末。ESI-MS(m/e):605[M+H]+.
实施例16制备Boc-Pro-Ala-Lys(Boc)
按照实施例14的方法由6.78g(11.2mmol)Boc-Pro-Ala-Lys(Boc)-OBzl制得5.36g(93%)目标化合物,为无色粉末。ESI-MS(m/e):513[M-H]-.
实施例17制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3](3a)
按照实施例1的方法由1g(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.0g(2.54mmol)Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3]制得898mg(54%)目标化合物,为无色粉末。Mp 102℃;[α]D 25=-701(c=0.44,CH3OH);ESI-MS(m/e):854[M+H]+;IR(KBr):3285,3250,3242,3208,3071,2955,2922,2859,1740,1717,1692,1636,1526,1479,1454,1408,1366,1321,1273,1252,1221,1165,1136,1128,1096,1070,1011,777.1H NMR(300MHz,CDCl3):δ/ppm=7.44-7.32(m,1H),7.24-7.11(m,2H),7.02(s,1H),4.94(s,1H),4.81-4.77(m,1H),4.39(d,J=4.8Hz,2H),4.22(s,1H),4.08-3.97(m,4H),3.45-3.41(m,2H),3.04(d,J=5.1Hz,2H),2.98-2.75(m,2H),2.08(m,2H),1.97-1.86(m,3H),1.67-1.58(m,5H),1.42-1.14(m,44H),0.85(t,J=5.1Hz,J=6.9Hz,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.64,172.16,171.67,171.28,170.72,170.44,170.28,156.12,155.72,141.21,128.36,127.31,126.88,126.64,125.48,80.68,78.86,65.76,65.29,65.18,64.94,60.46,53.01,49.46,48.66,47.20,40.09,36.22,31.71,31.49,29.54,29.37,29.15,29.10,28.45,28.40,28.28,25.80,25.71,24.54,22.56,17.59,15.19,14.00.
实施例18制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3](3b)
按照实施例1的方法由1.0g(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.14g(2.54mmol)Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3]制得1.01g(57%)目标化合物,为无色粉末。Mp 94℃;[α]D 25=-541(c=0.58,CH3OH);ESI-MS(m/e):910[M+H]+;IR(KBr):3289,3277,3237,3063,3053,2953,2928,2857,1740,1684,1636,1526,1456,1402,1393,1366,1275,1248,1217,1165,1126,1099,988,914,864,692.1H NMR(300MHz,CDCl3):δ/ppm=7.35(s,1H),7.14-6.99(m,3H),4.93(s,1H),4.82-4.81(m,1H),4.41(d,J=4.5Hz,2H),4.24(s,1H),4.09-4.02(m,4H),3.44(s,2H),3.06(s,2H),2.95(d,J=15.9Hz,1H),2.81(d,J=9.9Hz,1H),2.10(m,2H),1.88(s,3H),1.59(s,6H),1.44-1.35(m,24H),1.25(s,29H),0.86-0.84(m,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.59,172.06,171.59,171.22,170.78,170.46,156.08,128.42,127.39,126.91,80.78,78.85,77.30,65.91,65.35,65.22,65.05,60.45,52.99,49.46,48.64,47.26,40.07,36.24,31.84,31.51,29.50,29.40,29.27,29.24,29.19,28.90,28.48,28.42,28.30,25.83,25.74,24.57,23.07,22.63,22.27,17.53,14.06.
实施例19制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3](3c)
按照实施例1的方法由1.0g(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.38g(2.72mmol)Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3]制得903mg(48%)目标化合物,为无色粉末。Mp 91℃;[α]D 25=-439(c=0.72,CH3OH);ESI-MS(m/e):966[M+H]+;IR(KBr):3970,3296,3277,3244,2957,2924,2855,1728,1684,1636,1530,1506,1456,1398,1395,1366,1275,1252,1206,1177,1119.1H NMR(300MHz,CDCl3):δ/ppm=7.25-7.10(m,2H),7.00-6.86(m,1H),4.94(s,1H),4.82-4.77(m,1H),4.41-4.39(m,2H),4.23(s,1H),4.09-4.00(m,4H),3.43(s,2H),3.04(d,J=4.8Hz,2H),2.95-2.90(m,1H),2.81-2.77(m,1H),2.08-2.06(m,2H),1.86(s,3H),1.67-1.57(m,5H),1.43-1.40(m,18H),1.34(d,J=6.9Hz,4H),1.23(s,35H),0.85(t,J=5.1Hz,J=6.9Hz,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.56,172.10,171.65,171.24,170.81,170.73,170.44,170.12,156.06,155.71,141.25,128.36,127.31,126.88,80.67,78.77,65.97,65.88,65.29,65.18,64.95,60.44,52.98,52.87,49.38,48.63,47.21,40.06,36.23,31.85,31.49,29.60,29.57,29.55,29.48,29.39,29.29,29.23,29.18,28.91,28.47,28.41,28.28,25.82,25.73,24.54,23.01,22.62,22.28,17.58,14.04.
实施例20制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3](3d)
按照实施例1的方法由1.0g(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.53g(2.72mmol)Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3]制得1.05g(52%)目标化合物,为无色粉末。Mp 80℃;[α]D 25=-626(c=0.50,CH3OH);ESI-MS(m/e):1022[M+H]+;IR(KBr):2955,2926,2855,1728,1684,1638,1404,1395,1366,1304,1169.1H NMR(300MHz,CDCl3):δ/ppm=7.05-6.99(m,2H),4.89-4.79(m,2H),4.47-4.40(m,2H),4.25(s,1H),4.13-4.05(m,4H),3.45(s,2H),3.08-3.06(m,2H),2.98-2.93(m,1H),2.85-2.75(m,1H),2.28-2.08(m,2H),1.86(s,3H),1.72-1.60(m,5H),1.45-1.36(m,22H),1.28-1.17(m,44H),0.85(t,J=5.7Hz,J=6.9Hz,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.57,172.04,171.54,171.19,170.86,170.78,170.45,170.40,170.03,156.13,156.07,80.77,78.85,66.04,65.92,65.35,60.49,53.00,52.89,49.41,48.65,48.51,47.23,40.13,36.25,36.14,32.29,31.89,31.49,29.65,29.63,29.58,29.51,29.41,29.32,29.25,29.21,28.83,28.50,28.43,28.30,25.85,25.76,24.56,23.09,22.65,22.26,17.54,14.07.
实施例21制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3](3e)
按照实施例1的方法由1.0g(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.57g(2.54mmol)Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3]制得966mg(46%)目标化合物,为无色粉末。Mp 74℃;[α]D 25=-907(c=0.34,CH3OH);ESI-MS(m/e):1078[M+H]+;IR(KBr):3304,3289,2955,2920,2851,1738,1688,1639,1537,1468,1456,1398,1393,1366,1339,1292,1279,1229,1171,1119.1H NMR(300MHz,CDCl3):δ/ppm=7.09-7.02(m,3H),4.92-4.84(m,2H),4.42(s,2H),4.26(s,1H),4.11-4.03(m,4H),3.46(s,2H),3.08(s,2H),2.97(d,J=16.2Hz,1H),2.83(d,J=14.4Hz,1H),2.12-1.89(m,5H),1.61(s,6H),1.45-1.37(m,25H),1.26(s,53H),0.87-0.86(m,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.68,172.15,171.57,171.26,170.88,170.80,170.45,170.39,170.27,156.16,155.86,141.40,128.42,126.68,125.67,117.89,110.75,80.81,78.92,65.95,65.38,65.25,60.51,53.06,49.50,48.68,48.55,47.28,40.10,36.25,31.90,31.45,29.68,29.64,29.59,29.52,29.38,29.33,29.27,29.22,28.93,28.50,28.43,28.31,25.85,25.76,24.59,22.66,17.51,14.08.
实施例22制备Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3](3f)
按照实施例1的方法由874mg(1.95mmol)Boc-Pro-Ala-Lys(Boc)和1.26g(1.87mmol)Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3]制得1131mg(59%)目标化合物,为无色粉末。Mp 88℃;[α]D 25=-526(c=0.60,CH3OH);ESI-MS(m/e):1134[M+H]+;IR(KBr):3306,3289,3233,2957,2928,2918,2853,1740,1690,1667,1636,1541,1533,1468,1456,1395,1366,1339,1277,1250,1227,1169,1138,1123,1076,772,721.1H NMR(300MHz,CDCl3):δ/ppm=7.06-6.98(m,3H),4.81(m,1H),4.41(m,2H),4.25(s,1H),4.10-4.02(m,4H),3.45(s,2H),3.06(m,2H),2.93(m,1H),2.83(m,1H),2.16-2.10(m,2H),1.99-1.88(m,4H),1.60(m,6H),1.45-1.42(m,20H),1.36(d,J=6.9Hz,4H),1.24(s,60H),0.86(t,J=5.1Hz,J=6.6Hz,6H).13C NMR(75MHz,CDCl3):δ/ppm=172.56,172.06,171.58,171.20,170.84,170.76,170.45,170.06,156.07,155.79,80.73,78.81,65.90,65.34,65.21,60.47,53.00,49.39,48.68,48.65,48.52,47.25,40.09,38.94,38.71,36.25,34.09,33.76,31.89,29.67,29.63,29.59,29.52,29.41,29.32,29.26,29.21,28.50,28.43,28.30,25.85,25.76,22.65,14.07.
实施例23制备Pro-Ala-Lys-Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3](4a)
按照实施例7的方法由580mg(0.68mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)6CH3]-[OCH2(CH2)6CH3]制得459mg(93%)目标化合物,为无色粉末。Mp120℃;[α]D 25=-725(c=0.44,CHCl3);ESI-MS(m/e):654[M+H]+;IR(KBr):3456,3426,3360,3237,3208,3046,2957,2928,2859,2735,1740,1655,1539,1460,1389,1368,1285,1213,1188,1053.1H NMR(300MHz,DMSO-d6):δ/ppm=10.23(s,1H),8.84(d,J=6.9Hz,1H),8.48-8.41(m,2H),8.12-8.09(m,3H),4.66-4.59(m,1H),4.40-4.33(m,1H),4.26-4.22(m,2H),4.02-3.95(m,4H),3.02(s,2H),2.81-2.66(m,4H),2.35-2.23(m,1H),1.91-1.87(m,3H),1.66-1.53(m,8H),1.25(s,25H),0.86(t,J=4.5Hz,J=6.9Hz,6H).13C NMR(75MHz,DMSO-d6):δ/ppm=171.93,171.82,170.90,170.31,168.26,65.27,64.80,59.03,52.53,49.10,48.91,46.02,38.85,36.28,32.61,31.74,31.67,30.12,29.04,28.48,26.85,25.78,25.68,23.99,22.52,22.46,22.28,18.30,14.37.C,62.94%;H,9.81%;N,10.49%;O,16.77%.
实施例24制备Pro-Ala-Lys-Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3](4b)
按照实施例7的方法由635mg(0.70mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)8CH3]-[OCH2(CH2)8CH3]制得503mg(92%)目标化合物,为无色粉末。Mp 120℃;[α]D 25=-554(c=0.58,CHCl3);ESI-MS(m/e):710[M+H]+;IR(KBr):3372,3227,3026,2955,2926,2907,2855,1744,1647,1533,1395,1173.1H NMR(300MHz,DMSO-d6):δ/ppm=10.22(s,1H),8.84(d,J=5.7Hz,1H),8.49(s,1H),8.41(d,J=7.8Hz,1H),8.11(d,J=6.9Hz,4H),4.66-4.59(m,1H),4.36(t,J=6.9Hz,J=13.8Hz,1H),4.26-4.22(m,2H),4.08-4.01(m,4H),3.21(s,2H),2.73(s,4H),2.32-2.23(m,1H),1.91-1.87(m,4H),1.57-1.53(m,7H),1.26(s,33H),0.85(t,J=4.5Hz,J=6.9Hz,6H).13CNMR(75MHz,DMSO-d6):δ/ppm=171.93,171.82,170.88,170.30,168.26,65.27,64.80,59.03,52.53,49.10,48.90,46.03,38.90,36.30,31.75,30.12,29.41,28.49,26.86,25.78,25.70,23.99,22.55,22.46,21.52,18.31,14.37.C,64.70%;H,10.16%;N,9.67%;O,15.47%.
实施例25制备Pro-Ala-Lys-Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3](4c)
按照实施例7的方法由345mg(0.36mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)10CH3]-[OCH2(CH2)10CH3]制得270mg(90%)目标化合物,为无色粉末。Mp 121℃;[α]D 25=-394(c=0.72,CHCl3);ESI-MS(m/e):766[M+H]+;IR(KBr):3401,3296,3223,3044,2955,2924,2853,2731,1740,1647,1547,1533,1464,1395,1288,1217,1173.1H NMR(300MHz,DMSO-d6):δ/ppm=8.80(d,J=7.2Hz,1H),8.41(d,J=7.8Hz,1H),8.14-8.04(m,3H),4.69-4.59(m,1H),4.38-4.31(m,1H),4.26-4.19(m,2H),4.02-4.00(m,4H),3.20-3.18(m,2H),2.74-2.72(m,4H),2.30-2.28(m,1H),1.86(s,3H),1.54(s,9H),1.25(s,41H),0.86(t,J=5.4Hz,J=6.6Hz,6H).13C NMR(75MHz,DMSO-d6):δ/ppm=171.93,171.80,170.88,170.30,168.27,65.28,64.81,59.07,52.51,49.06,48.89,46.07,38.88,36.32,31.76,30.09,29.47,29.41,29.17,29.13,28.49,26.90,25.78,25.71,23.98,22.54,22.46,18.30,14.37.C,66.20%;H,10.47%;N,8.98%;O,14.36%.
实施例26制备Pro-Ala-Lys-Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3](4d)
按照实施例7的方法由185mg(0.18mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)12CH3]-[OCH2(CH2)12CH3]制得152mg(94%)目标化合物,为无色粉末。Mp 109℃;[α]D 25=-580(c=0.50,CHCl3);ESI-MS(m/e):822[M+H]+;IR(KBr):3944,3433,3298,3229,2922,2855,2749,1742,1647,1545,1462,1387,1344,1292,1221,1169,1067,1003,723.1H NMR(300MHz,DMSO-d6):δ/ppm=10.07(s,1H),8.39(d,J=7.8Hz,1H),8.12(d,J=7.8Hz,1H),4.66-4.62(m,1H),4.36-4.31(m,1H),4.26-4.18(m,2H),4.02-3.99(m,4H),3.24-3.17(m,2H),2.74-2.72(m,4H),2.34-2.29(m,1H),1.91-1.85(m,3H),1.68-1.52(m,9H),1.27-1.23(m,49H),0.86(t,J=5.7Hz,J=6.6Hz,6H).13CNMR(75MHz,DMSO-d6):δ/ppm=172.22,171.96,171.83,170.85,170.40,170.29,168.19,168.12,65.27,64.80,58.98,52.52,52.41,49.08,48.97,48.85,48.73,45.99,38.74,36.32,31.77,30.06,29.55,29.49,29.44,29.18,28.50,28.47,26.80,26.23,25.80,25.73,23.95,22.88,22.55,22.46,18.28,14.36.C,67.20%;H,10.67%;N,8.52%;O,13.62%.
实施例27制备Pro-Ala-Lys-Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3](4e)
按照实施例7的方法由265mg(0.25mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)14CH3]-[OCH2(CH2)14CH3]制得206mg(88%)目标化合物,为无色粉末。Mp 104℃;[α]D 25=-1116(c=0.27,CHCl3);ESI-MS(m/e):878[M+H]+;IR(KBr):3300,3219,2957,2920,2851,2758,2654,2575,1740,1643,1547,1468,1395,1371,1341,1292,1225,1169,1070.1H NMR(300MHz,DMSO-d6):δ/ppm=10.11(s,1H),8.39(d,J=7.2Hz,1H),8.14(d,J=5.4Hz,1H),4.62(s,1H),4.35(d,J=6.6Hz,1H),4.25(s,2H),3.98-3.97(m,4H),3.22-3.19(m,2H),2.73(s,4H),2.32-2.30(m,1H),1.87(m,3H),1.53(s,8H),1.22(s,55H),0.85(m,6H).13C NMR(75MHz,DMSO-d6):δ/ppm=171.84,171.74,171.67,170.78,170.24,168.21,168.11,65.30,64.80,59.12,52.63,52.48,49.11,48.96,48.86,46.03,38.78,36.38,36.26,31.86,31.71,29.99,29.66,29.45,29.42,29.37,29.10,28.47,26.77,25.77,25.70,23.90,22.59,22.48,18.23,18.00,14.42,14.29,14.11.C,68.37%;H,10.90%;N,7.97%;O,12.75%.
实施例28制备Pro-Ala-Lys-Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3](4f)
按照实施例7的方法由530mg(0.47mmol)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)16CH3]-[OCH2(CH2)16CH3]制得438mg(93%)目标化合物,为无色粉末。Mp 90℃;[α]D 25=-1760(c=0.01,CHCl3);ESI-MS(m/e):934[M+H]+;IR(KBr):3329,3275,3229,3048,2922,2855,1740,1663,1545,1458,1240,1173.1H NMR(300MHz,DMSO-d6):δ/ppm=8.75(d,J=7.2Hz,1H),8.26(d,J=7.8Hz,1H),8.03-8.11(d,J=7.8Hz,1H),4.64(t,J=5.7Hz,J=6.0Hz,1H),4.38-4.33(m,1H),4.26-4.20(m,2H),4.04-3.95(m,4H),2.74(m,4H),2.34-2.28(m,1H),1.92-1.86(m,3H),1.56(m,7H),1.37-1.25(m,68H),0.86(t,J=6.0Hz,J=6.6Hz,6H).13C NMR(75MHz,DMSO-d6):δ/ppm=171.85,171.65,170.78,170.25,168.24,168.12,65.57,65.31,64.81,59.75,59.15,59.06,52.61,52.47,52.38,52.28,49.21,49.03,48.86,46.38,46.30,46.19,46.07,38.95,36.46,31.86,31.71,29.98,29.44,29.09,28.51,28.48,26.80,25.78,25.71,23.90,22.48,22.41,18.26,15.73,14.29.C,69.41%;H,11.11%;N,7.49%;O,11.99%.
试验例1本发明通式I化合物的体内溶血栓活性评价试验
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1nmol/kg本发明化合物4a-f(实施例23-28所制备)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。结果表明,本发明化合物4a-f具有优秀的体内溶栓活性(表1)。
表1 1nmol/kg 4a-f对大鼠血栓减重的影响a
a)n=12,血栓减重用均值±SD mg表示;b)与生理盐水组比,p<0.01。
试验例2剂量对本发明化合物4d体内溶血栓活性的影响
按照试验例1的实验方法,选取溶栓效果最好的4d(本发明实施例26所制备)考察1nmol/kg、0.01nmol/kg和0.0001nmol/kg三个剂量下的溶栓活性。结果表明,4d的溶栓作用显示剂量依赖性(表2)。
表24d的剂量对大鼠血栓减重的影响a
a)n=10,血栓减重用均值±SD mg表示;b)与生理盐水及0.01nmol/kg 4d组比,p<0.01;c)与生理盐水及0.0001nmol/kg 4d组比,p<0.01;d)与生理盐水相比,p>0.05。
试验例3本发明通式I化合物的体外溶血栓活性评价试验
1)血栓形成装置的制作
将内径4mm,外径5.5mm,长度18mm的一段玻璃管安放在一个塑料易拆卸底座上,玻璃管和塑料底座的接缝处用一段乳胶管密封。玻璃管中放置一不锈钢丝螺旋,螺旋直径1mm,长度20mm,包括一端的2mm长的挂钩,血液即凝固在不锈钢螺旋的周围,称重时可将血栓挂起,在孵育时可以将血栓挂在反应瓶的溶液中,不碰到四壁,以免损伤血栓。
2)反应瓶的制作
用带橡胶塞的10ml西林瓶,在橡胶塞上穿一根不锈钢丝,在瓶中的一端弯成钩,血栓挂在钩上,悬浮于瓶内待测化合物溶液中,不锈钢丝可以在橡胶塞上上下移动,调节血栓在溶液中的高度,使其刚好浸在待测溶液中。体内环境的模拟:按照大鼠平均体重估计每只大鼠有13ml血液,如果是大鼠体内血栓模型,可能约8ml的血液可接触到血栓,故反应瓶中加入8ml待测溶液,在37℃恒温摇床孵育。
3)血栓的制备
将350-400g雄性SD大鼠用20%乌拉坦(6ml/kg,i.p.),麻醉,仰卧固定,分离右颈总动脉,动脉夹夹闭近心端,动脉夹上方插入30mm长聚乙烯管,每次放出约3-4ml血,大约可放2-3次,用硅烷化的5ml注射器立即将放出的血液逐个注入制备血栓用的玻璃管中,马上将不锈钢螺旋放入。静置40min使血栓形成,之后将玻璃管小心从底座上取下,用细针将血栓四周和玻璃管内壁分开,取出血栓挂在反应瓶的橡胶塞上,反应瓶中加入8ml蒸馏水,将血栓悬挂在水中静置1小时,除掉血栓表面浮血。1小时后,用滤纸吸去血栓表面的水分,逐个精确称重。
4)测定4a-f体外溶栓活性:
在每个反应瓶中重新注入4a-f(10nM)的生理盐水溶液,以生理盐水作为空白对照,尿激酶(100IU/ml)作为阳性对照,再将血栓悬挂于待测化合物的溶液中,37℃恒温摇床70rpm孵育2小时。孵育结束后,用滤纸吸取表面水再逐个精确称重,计算血栓在加入待测溶液前后的重量差,统计评价化合物体外溶栓活性。结果表明,4a-f有优秀的体外溶栓活性(表3)。
表310nM 4a-f体外溶栓活性a
a)n=6,血栓减重用均值±SD mg表示;b)与生理盐水组比,p<0.01。
实验例4浓度对对本发明化合物4b体外溶血栓活性的影响
按照实验例3的实验方法,选取溶栓效果最好的4b考察10nM,1nM,0.1nM和0.01nM四个浓度下的溶栓活性。结果表明,4b的体外溶栓作用显示浓度依赖性(表4)。
表24b的浓度对体外溶栓活性的影响a
a)n=6,血栓减重用均值±SD mg表示;b)与生理盐水及1nM 4b组比,p<0.01;c)与生理盐水及0.1nM 4b组比,p<0.01;d)与生理盐水及0.01nM 4b组比,p<0.01。
试验例5本发明通式I化合物的纳米球试验
1)水溶液中4a-f纳米球的粒径
先测得4a-f在水溶液中的临界胶束浓度为1×10-12mg/ml。然后在Nano-ZS90纳米粒度测定仪上测定4a-f在1×10-12mg/ml的粒径。结果表明,4a-f在水溶液中可组装为纳米球,粒径为182至343nm(表5)。
表54a-f在水溶液中组装的纳米球的粒径
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。4b的透射电子显微镜照片作为代表(图3)。
Claims (3)
1.具有溶血栓活性的寡肽,其结构式为通式I所示:
Pro-Ala-Lys-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
其中,n=6、8、10、12、14或16。
2.一种合成权利要求1所述寡肽的方法,包括以下步骤:
1)Boc-Pro与Ala-OBzl缩合为Boc-Pro-Ala-OBzl;
2)将Boc-Pro-Ala-OBzl氢解为Boc-Pro-Ala;
3)Boc-Pro-Ala与Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Boc)-OBzl;
4)将Boc-Pro-Ala-Lys(Boc)-OBzl氢解为Boc-Pro-Ala-Lys(Boc);
5)Boc-Asp与饱和脂肪醇缩合为
Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3,其中n=6、8、10、12、14或16;
6)Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3脱去Boc生成
Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
7)Boc-Pro-Ala-Lys(Boc)与Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
8)Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)n-CH3]-OCH2(CH2)nCH3脱除Boc,即得;
其中,步骤(1)中在DCC和HOBt存在下,Boc-Pro在无水THF中与Ala-OBzl缩合为Boc-Pro-Ala-OBzl;
步骤2)中在甲醇中将Boc-Pro-Ala-OBzl氢解为Boc-Pro-Ala;
步骤3)中在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Boc)-OBzl缩合为Boc-Pro-Ala-Lys(Boc)-OBzl;
步骤4)中在甲醇中将Boc-Pro-Ala-Lys(Boc)-OBzl氢解为
Boc-Pro-Ala-Lys(Boc);
步骤5)中在DCC存在下Boc-Asp在无水THF中与饱和脂肪醇缩合为
Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
步骤6)中在氯化氢-乙酸乙酯溶液中
Boc-Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3脱去Boc生成
Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
步骤7)中在DCC和HOBt存在下Boc-Pro-Ala-Lys(Boc)在无水THF中与 Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3缩合为Boc-Pro-Ala-Lys(Boc)-
Asp[OCH2(CH2)nCH3]-OCH2(CH2)nCH3;
步骤8)中在氯化氢-乙酸乙酯溶液中
Boc-Pro-Ala-Lys(Boc)-Asp[OCH2(CH2)n-CH3]-OCH2(CH2)nCH3脱除Boc。
3.权利要求1所述的寡肽在制备溶血栓药物中的用途。
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