CN103145797B - [(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽,其合成和在医学中的应用 - Google Patents
[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽,其合成和在医学中的应用 Download PDFInfo
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Abstract
本发明涉及Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys及Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽及其合成方法和应用。通式I是本发明化合物的结构。式中n=1或2,AA选自Ser残基、Phe残基或Val残基。n=1时通式I为Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽。n=2时通式I为两个(3s)-1,2,3,4-四氢异喹啉-3-甲酰基分别连接到Lys的α-氨基和侧链氨基上,Lys的羧基再与RGD四肽,即Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽。本发明采用体外抗血小板聚集实验以及体内抗血栓活性实验(大鼠颈动静脉旁路插管模型)评价了本发明化合物的抗血小板聚集活性和抗血栓活性。试验结果表明,本发明化合物具有优异的抗血栓活性,以及具有自组装性能,可制备成抗血栓剂。
Description
技术领域
本发明涉及四氢异喹啉的衍生物,尤其涉及RGDS(F/V)修饰的四氢异喹啉衍生物及其合成方法,本发明还涉及它们作为抗血栓剂的应用,本发明属于生物医药领域。
背景技术
血管栓塞性疾病是危害人们生命健康的重要疾病。血栓形成是血管栓塞性疾病发病的最重要的病因。寻找抗血栓药物是新药研究的热点之一。1,2,3,4-四氢异喹啉-3-S-羧酸是广泛存在于中草药之中的一类重要生物碱,具有抗血小板聚集活性。发明人在抗血栓药物研究中发现用氨基酸对1,2,3,4-四氢异喹啉-3-羧酸进行修饰,可以改善1,2,3,4-四氢异喹啉-3-羧酸的生物利用度。发明人在抗血栓药物研究中还发现RGDS、RGDF和RGDV既具有抗栓活性,又具有良好的水溶性。用它们修饰杂环化合物,可以改善它们在体内的吸收、分布及提高生物活性。本发明用RGDS、RGDF和RGDV对1,2,3,4-四氢异喹啉-3-S-羧酸进行修饰,改善它在体内的吸收、分布及提高生物活性。按照我们的认识,含RGDS、RGDF和RGDV的杂环分子,在适当的条件下通过分子间非共价键相互作用自组装,形成纳米结构。借助纳米结构往往可以进一步可改善杂环在体内的转运、延缓在体内的降解速率和减少用药剂量。这是本发明的另一个出发点。根据发明人的这些认识背景,发明人提出了本发明。
发明内容
本发明依据血管栓塞性疾病是危害人们生命健康的重要疾病。血栓形成是血管栓塞性疾病发病的最重要的病因。寻找抗血栓药物是新药研究的热点之一。依据1,2,3,4-四氢异喹啉-3-S-羧酸是抗血小板聚集活性的天然物质、1,2,3,4-四氢异喹啉-3-S-羧酸是广泛存在于中草药之中的具有抗血小板聚集活性的重要生物碱。依据发明人在抗血栓药物研究中发现的用氨基酸对1,2,3,4-四氢异喹啉-3-羧酸进行修饰可以改善1,2,3,4-四氢异喹啉-3-羧酸的生物利用度。依据发明人在抗血栓药物研究中发现的RGDS、RGDF和RGDV既具有抗栓活性,又具有良好的水溶性。依据用它们修饰杂环化合物,可以改善它们在体内的吸收、分布及提高生物活性。依据发明人认识的含RGDS、RGDF和RGDV的杂环分子在适当的条件下通过分子间非共价键相互作用自组装,形成纳米结构。依据发明人认识的借助纳米结构往往可以进一步可改善杂环在体内的转运、延缓在体内的降解速率和减少用药剂量。本发明建立了制备Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA和Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA的方法、制备了3种Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA和3种Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA化合物、采用体内抗血栓活性实验(大鼠颈动静脉体外旁路插管模型)评价了它们作为抗血栓剂的应用。
本发明目的之一是提供具有抗血栓活性的Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA和Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-AA(AA选自Ser、Phe和Val)其结构式为通式I的化合物Ia-f。
其中,AA选自丝氨酸残基、苯丙氨酸残基和缬氨酸残基;n=1时通式I为Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽。n=2时通式I为两个(3s)-1,2,3,4-四氢异喹啉-3-甲酰基分别连接到Lys的α-氨基和侧链氨基上,Lys的羧基再与RGD四肽,即Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽。
本发明目的之二是提供一种合成上述具有抗血栓活性的通式I的化合物Ia-f的方法,包括:
(1)制备1,2,3,4-四氢异喹啉-3-羧酸;
(2)制备N-Boc-1,2,3,4-四氢异喹啉-3-羧酸;
(3)将步骤(2)的产物分别与Tos·Lys(Boc)-OBzl和Tos·Lys(HCl)-OBzl缩合,得到Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-OBzl和Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-OBzl;
(4)将步骤(3)的产物与HCl·Arg(NO2)Gly-Asp(OBzl)AA-OBzl缩合,得到Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)Arg(NO2)GlyAsp(OBzl)AA-OBzl和Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)Arg(NO2)GlyAsp(OBzl)AA-OBzl。
(5)将步骤(3)的产物脱去保护基,得到通式I的化合物Ia-f
其中,AA选自Ser(Bzl)、Phe和Val;
本发明的第三个内容是评价通式通式I的Ia-f 6种抗血栓化合物的体内抗血栓活性,以及化合物的自组装性能。
附图说明
图1本发明通式I的化合物Ia-f的合成路线。试剂及反应条件:(i)浓HCl,甲醛,油浴90℃,7小时;(ii)(Boc)2O,二氧六环,2N NaOH,0℃;(iii)HOBt,DCC,NMM,无水THF,Tos·Lys(Boc)-OBzl/Tos·Lys(HCl)-OBzl;(iv)甲醇,2N NaOH,0℃;(v)HOBt,DCC,NMM,无水THF,HCl·R(NO2)GD(OBzl)AA-OBzl,在5a中AA=S,5b中AA=F,5c中AA=V;在8a中AA=S,8b中AA=F,8c中AA=V(vi)三氟醋酸,三氟甲磺酸,0℃。在Ia和Id中AA=Ser,Ib和Ie中AA=Phe,Ic和If中AA=Val。
图2代表性的纳米照片。
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11,2,3,4-四氢异喹啉-3-羧酸(1)的制备
往4.0g(24.2mmol)L-苯丙氨酸中先逐滴加入21.6mL甲醛,再逐滴加入36mL 35%浓盐酸,加热至90-100℃搅拌2两个小时苯丙氨酸完全溶解,反应2.5小时后,开始有白色沉淀生成,反应7小时,TLC(CHCl3/CH3OH=5/1)显示L-苯丙氨酸消失,抽滤得4.2g淡黄色固体,干燥,然后将所得淡黄色固体加入到86mL乙醇(80%)中80℃油浴加热至无色固体溶解,冷却至室温,慢慢滴加2mol/mL氢氧化钾溶液,有无色沉淀析出,过滤得4.17g(97.5%)标题化合物,为白色固体。ESI-MS(m/e):176[M-H]-.
实施例2N-Boc-1,2,3,4-四氢异喹啉-3-羧酸(2)的制备
冰浴下把2.49g(62.15mmol)氢氧化钠溶入62.2mL水,然后加入10g(56.49mmol)(3S)-1,2,3,4-四氢异喹啉-3-羧酸制成混悬液。用40mL四氢呋喃溶解14.8g(67.8mmol)(Boc)2O,滴加入混悬液中。反应室温搅拌24小时,反应过程中不断的抽除生成的CO2,当溶液变澄清,TLC(甲醇/氯仿∶1∶10)显示(3S)-1,2,3,4-四氢异喹啉-3-羧酸消失,停反应。除去溶剂,得到油状物用乙酸乙酯溶解,依次用5%KHSO4和饱和NaCl水溶液洗。有机层用无水硫酸钠干燥、过滤、滤液减压浓缩至干,经乙醚溶解做重结晶,得无色固体14.84g(95%)。ESI-MS(m/e):278.2[M+H]+.
实施例3Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-OBzl(3)的制备
取N-Boc-1,2,3,4-四氢异喹啉305mg,HOBt 178mg和DCC 272mg,冰浴搅拌下加无水THF溶解,30分钟后,将Tos·Lys(Boc)-OBzl 508mg用10mL干燥的THF悬浮,滴加0.5mLNMM调节反应液的PH值至8-9,然后滴加入反应液,撤去冰浴,室温搅拌12h,过滤,减压浓缩,乙酸乙酯溶解残余物,过滤,洗涤,乙酸乙酯层用无水Na2SO4干燥,过滤,减压浓缩得到标题化合物,白色固体产物576mg,产率为96.8%,ESI-MS(m/e):596[M+H]+.
实施例4Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)(4)的制备
将Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-OBzl 4.606g,溶解于30mL甲醇中,冰浴搅拌下,加入2N NaOH约8mL,反应1小时,用饱和KHSO4水溶液将反应液PH调至7,减压浓缩至干,向残余物中加入30mL蒸馏水,用饱和KHSO4水溶液将其PH调至2,用乙酸乙酯萃取(70mL×3)。将乙酸乙酯层合并,用饱和NaCl洗(50mL×3),用无水Na2SO4干燥过夜。过滤,减压浓缩至干,得到标题化合物3.89g,产率为99.5%,ESI-MS(m/e):504[M-H]-
实施例5Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (5a)的制备
将Nα[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)556mg,溶解于40mL干燥的THF中,加入HOBt 180mg,冰浴搅拌,向上述混合液中滴加20mL DCC 272mg的THF溶液。反应30分钟,将HCl·Arg(NO2)Gly-Asp(OBzl)Ser(Bzl)-OBzl 785mg,用无水THF溶解,滴加3-4mL NMM调节反应液的PH值至8-9,加入反应液,室温反应12h,过滤,减压浓缩,加少量的乙酸乙酯将残余物溶解,过滤,洗涤,用无水Na2SO4干燥,过滤,减压除去溶剂,得白色固体产物,经柱层析纯化得680mg,产率为55.1%。ESI-MS(m/e):1236.8[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.551-8.474(m,1H),8.294-8.271(d,J=6.9Hz,1H),8.158(s,1H),8.019-7.996(d,J=6.9Hz,1H),7.920(s,1H),7.688(s,1H),7.355-7.259(m,15H),7.158(s,4H),6.729(s,1H),5.767(s,1H),5.153-5.091(m,4H),4.857-4.206(m,10H),3.801-3.463(m,2H),3.093-2.566(m,8H),1.993(s,1H),1.942(s,1H),1.449(s,8H),1.361(s,18H),1.025(s,2H).
实施例6Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-A rg(NO2)-Gly-Asp(OBzl)-Ph e-OBzl(5b)的制备
按照实施例5的操作,得700mg,产率67.9%,ESI-MS(m/e):1206.9[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.414-8.392(d,J=6.6Hz,1H),8.231-8.208(d,J=6.6Hz,1H),8.120(s,1H),7.988(s,1H),7.915(s,1H),7.670(s,1H),7.393-7.163(m,19H),6.687(s,1H),5.111-4.998(m,4H),4.746-4.222(m,7H),3.698(s,2H),3.092-2.507(m,10H),1.646-1.446(m,8H),1.361(s,18H),1.116-1.014(m,2H).
实施例7Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(5c)的制备
按照实施例5的操作,得到650mg,产率56.1%,ESI-MS(m/e):1158.5[M+H]+;1180.9[M+Na]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.236(s,1H),8.186(s,1H),8.159(s,1H),7.983(s,1H),7.905(s,1H),7.779(s,1H),7.353(s,10H),7.162(s,4H),6.681(s,1H),.5.158-5.029(m,4H),4.795-4.750(m,1H),4.663-4.171(m,6H),3.770(s,2H),3.096-2.619(m,8H),2.303(s,1H),2.172(s,1H),2.121-1.990(m,1H),1.641-1.446(m,8H),1.338(s,18H),1.149-1.111(m,2H),0.854-0.832(d,J=6.6Hz,6H).
实施例8Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-Ser(Ia)的制备
取化合物5a 100mg,冰浴搅拌下加0.4mL三氟醋酸溶解,加入0.1mL三氟甲磺酸,反应2小时,加入大量乙醚搅拌,析出产品,然后静置,倾倒乙醚,再加乙醚搅拌,静置,倾倒,如此反复5次,抽干。加水溶解,用氨水调PH至7,经Sephadex G10填料柱子除盐,冻干,得目标产物48mg,产率82.8%,(c=0.5,CH3OH).mp 63.4-65.4℃,ESI-MS(m/e):721.3[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=9.876(s,1H),8.460(s,1H),8.434-8.425(m,1H),8.314-8.268(m,1H),7.976-7.951(m,1H),7.604-7.478(m,1H),7.160-6.964(m,4H),4.385-4.213(m,3H),3.869-3.534(m,8H),3.007-2.652(m,8H),1.902(s,2H),1.619-1.389(m,8H),1.148-1.033(m,2H).
实施例9Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-Phe(Ib)的制备
按照实施例8的操作,取化合物5b 100mg,制得50mg,产率73.5%,(c=0.5,CH3OH).mp 68.2-70.0℃,ESI-MS(m/e):781[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=9.534-9.404(m,1H),8.672-8.647(d,J=7.5Hz,1H),8.385-8.359(d,J=7.8Hz,1H),8.305-8.296(m,1H),8.028-7.807(m,1H),7.502-7.325(m,1H),7.205-7.171(m,8H),4.607-4.402(m,1H),4.241-4.166(m,2H),4.062(s,1H),3.842-2.706(m,15H),1.910(s,4H),1.910-1.150(m,10H).
实施例10Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-Val(Ic)的制备
按照实施例8的操作,取化合物5c 100mg,制得23mg,产率36.5%,(c=0.5,CH3OH).mp 66.5-67.8℃,ESI-MS(m/e):733.6[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=10.384(s,1H),9.041-9.014(d,J=8.1Hz,1H),8.660(s,1H),8.513-8.495(d,J=5.4Hz,1H),8.423-8.412(d,J=3.3Hz,1H),8.185-8.159(d,J=7.8Hz,1H),7.105-6.962(m,4H),4.414-4.191(m,3H),3.940-2.675(m,14H),1.903(s,4H),1.653-1.057(m,11H),0.785-0.763(d,J=6.6Hz,6H).
实施例11Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-OBzl(6)的制备
将N-Boc-1,2,3,4-四氢异喹啉727mg,HOBt 170mg和DCC 270mg混合,冰浴搅拌下,加入50mL的干燥THF溶液反应30分钟。将Tos·Lys(HCl)-OBzl 243mg用10mL干燥的THF悬浮,滴加0.5mL NMM调节反应液的PH值至8-9,然后滴加入反应液中,室温搅拌12h,过滤,减压浓缩,乙酸乙酯溶解残余物,再过滤,洗涤,用无水Na2SO4干燥,过滤,减压浓缩,经柱层析分纯,得到标题化合物,白色固体产物271mg,产率为65.5%。ESI-MS(m/e):755[M+H]+.
实施例12Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(7)的制备
将化合物6(267mg,0.35mmol)溶解于10mL甲醇中,冰浴搅拌下,加入2N NaOH约4mL,冰浴搅拌1小时,TLC显示原料消失,用饱和KHSO4水溶液将反应液PH调至7,减压浓缩至干,向残余物中加入10mL蒸馏水,用饱和KHSO4水溶液将其PH调至2,用乙酸乙酯萃取(50mL×3)。将乙酸乙酯层合并,用饱和NaCl洗(50mL×3),用无水Na2SO4干燥过夜。过滤,减压浓缩至干,得到标题化合物230mg,产率为97.9%。ESI-MS(m/e):663[M-H]-.
实施例13Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl(8a)的制备
将Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(600mg,0.90mmol),HOBt(146mg,1.08mmol)和DCC(223mg,1.08mmol)混合,冰浴搅拌下,加入50mL的干燥THF溶液反应30分钟。将HCl·Arg(NO2)Gly-Asp(OBzl)Ser(Bzl)-OBzl(644mg,0.82mmol)用20mL干燥的THF悬浮,滴加0.5mL NMM调节反应液的PH值至8-9,然后滴加入反应液中,室温搅拌12h,过滤,减压浓缩,乙酸乙酯溶解残余物,再过滤,洗涤,用无水Na2SO4干燥,过滤,减压浓缩,经柱层析分纯,得到标题化合物,白色固体产物740mg,产率为64.7%。ESI-MS(m/e):1395.0[M+H]+;1417.6[M+Na]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.495(s,1H),8.316(s,1H),8.156(s,1H),8.031(s,1H),7.921(s,1H),7.762(s,1H),7.683(s,1H),7.352-7.260(m,15H),7.149(s,8H),4.859-4.264(m,13H),3.813-3.637(m,2H),3.083-2.512(m,10H),1.993(s,1H),1.942(s,1H),1.667(s,2H),1.441-1.008(m,26H).
实施例14Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl(8b)的制备
按照实施例13的操作,制得840mg,产率74.9%,ESI-MS(m/e):1365[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.413-7.934(m,7H),.7.348-7.146(m,19H),5.109-4.999(m,4H),4.748-4.067(m,10H),3.170-2.926(m,10H),2.757-2.687(m,2H),1.990(s,1H),1.915(s,1H),1.435-0.994(m,26H).
实施例15Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(8c)的制备
按照实施例13的操作,制得590mg,产率44.8%,ESI-MS(m/e):1317[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=8.495(s,1H),8.171(s,1H),8.079(s,1H),8.056(s,1H),7.953(s,1H),7.771(s,1H),7.695(s,1H),7.350(s,10H),7.141(s,8H),5.159-5.026(m,4H),4.771-4.174(m,9H),3.168-2.536(m,10H),2.122-2.009(m,1H),1.989(s,2H),1.656(s,2H),1.436-1.238(m,22H),1.176-0.999(m,4H),0.855-0.833(d,J=6.6Hz,6H).
实施例16Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-G ly-Asp-Ser(ld)的制备
取化合物8a 100mg,冰浴搅拌下加0.4mL三氟醋酸溶解,加入0.1mL三氟甲磺酸,反应2小时,加入大量乙醚搅拌,析出产品,然后静置,倾倒乙醚,再加乙醚搅拌,静置,倾倒,如此反复5次,抽干。加水溶解,用氨水调PH至7,经Sephadex G10填料柱子除盐,冻干,得目标产物48mg,产率76.2%,(c=0.5,CH3OH).mp 52.5-54.5℃,ESI-MS(m/e):880.7[M+H]+.1H-NMR(DMSO-d6,500MHz):δ/ppm=10.1131(s,1H),8.5759(s,1H),8.3952-8.2753(m,1H),8.1512-8.0968(m,1H),7.9686-7.8145(m,1H),7.4533-7.2457(m,2H),7.1706-7.0302(m,8H),4.4720-4.3231(m,3H),3.8845-3.8525(m,2H),3.3827(m,11H),3.1790-2.6153(m,11H),1.8924(s,3H),1.6670-1.1558(m,10H).
实施例17Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-Phe(le)的制备
按照实施例16的操作,取化合物8b 100mg,制得47mg,产率65.3%,(c=0.5,CH3OH).mp 50.9-52.7℃,ESI-MS(m/e):940.6[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=9.579(s,1H),8.711-8.643(m,1H),8.492-8.466(d,J=7.8Hz,1H),8.363(s,1H),8.260-8.234(d,J=7.8Hz,1H),8.199-8.140(m,1H),7.433-7.275(m,1H),7.212-7.141(m,13H),4.376-4.273(m,4H),4.026-3.594(m,8H),3.167-2.770(m,12H),1.713-1.238(m,10H).
实施例18Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-Arg-Gly-Asp-Val(If)的制备
按照实施例16的操作,取化合物8c 100mg,制得54mg,产率76.1%,(c=0.5,CH3OH).mp 48.6-51.3℃,ESI-MS(m/e):892.6[M+H]+.1H-NMR(DMSO-d6,300MHz):δ/ppm=9.813(s,1H),8.861-8.833(d,J=8.4Hz,1H),8.425-8.332(m,1H),8.259-8.221(m,1H),7.439-7.251(m,2H),7.163(s,8H),4.390-4.263(m,4H),4.098-3.569(m,8H),3.168-2.824(m,10H),2.001-1.911(m,3H),1.670-1.298(m,11H),0.816-0.794(d,J=6.6Hz,6H).
实验例1Ia-f的体内抗血栓活性测定(大鼠颈动静脉体外旁路插管模型,活性用血栓湿重表示)
1)实验动物
SD雄性大鼠,体重200-220g,由北京维通利华实验动物技术有限公司提供。
2)实验材料
聚乙烯管(外径1.6mm及1.3mm两种)、肝素(50IU/mL)、乌拉坦(20%)、生理盐水。
3)测定方法与结果
以生理盐水(NS,3mL/kg)为空白对照,以阿司匹林(30mg/kg)作阳性对照。阿司匹林、Ia-f(10nmol/kg)在临用前配成生理盐水溶液。另配四氢异喹啉(100nmol/kg)的生理盐水溶液、RGDS的生理盐水溶液(100nmol/kg)、RGDF的生理盐水溶液(100nmol/kg)和RGDV的生理盐水溶液。将雄性SD大鼠(体重200-220g)灌胃给予生理盐水、阿司匹林的生理盐水溶液、四氢异喹啉的生理盐水溶液、RGDS的生理盐水溶液、RGDF的生理盐水溶液、RGDV的生理盐水溶液和Ia-f的生理盐水溶液,30分钟后,腹腔注射20%乌拉坦溶液进行麻醉,分离大鼠的右颈动脉和左颈静脉。在聚乙烯管的中段放入事先称重的6cm长的丝线,以肝素生理盐水(50IU/kg)充满聚乙烯管,将一端插入左颈静脉,用注射器从另一端加入定量肝素钠抗凝(50IU/mL,1mL/kg),然后插入右颈动脉。血流从右颈动脉流经聚乙烯管流入左颈静脉,15min后中断血流,取出丝线称重,总重量减去丝线重量即为血栓湿重。统计各组的血栓湿重的均值和标准差并作t检验,结果列入表1。表1的数据说明,在10nmol/kg剂量下Ia-f化合物具有良好的抗血栓活性。
表1Ia-f化合物体内抗血栓活性
n=10;a)与生理盐水组,四氢异喹啉组及RGDS组比p<0.01;b)与生理盐水组,四氢异喹啉组及RGDF组比p<0.01;b)与四氢异喹啉组比p<0.01;c)与生理盐水组,四氢异喹啉组及RGDV组比p<0.01;与生理盐水组相比P<0.01,与RGDS组比p<0.01;d)与生理盐水组相比P<0.01,与RGDF组比p<0.01;e)与生理盐水组相比P<0.01,与RGDV组比p<0.01。血栓湿重用表示。
实验例2Ic化合物的剂量依赖实验
按照实验例1的方法,选择活性较强的Ic化合物测定10nmol/kg、1nmol/kg和0.1nmol/kg三种剂量下的活性,结果见表2。表2的数据表明,Ic显示剂量依赖性的抗血栓活性。
表2Ic化合物体内抗血栓的量效关系
n=10,a)与生理盐水组、1nmol/kg Ic组及0.1nmol/kg Ic组比p<0.01;b)与与生理盐水组及0.1nmol/kg Ic组比p<0.01.血栓湿重用表示.
实验例3本发明化合物的自组装性能评价
1)本发明的Ia-f 6种化合物配置成10-4M、10-5M、10-6M浓度的水溶液,在激光散射粒度仪上观察25℃时的粒径大小,结果见表3。数据表明,本发明它们在水溶液中在具有自组装成为纳米球的性能。
表3 25℃的粒径测定结果
2)按照透射电镜的测定要求,配制了本发明的Ia-f 6种化合物的浓度为10-8、10-10、10-12mg/mL的水溶液,分别置于铜网上烘干后测定本发明的化合物的自主装性能。结果表明,它们在透射电镜下均可观察到纳米球、纳米棒、纳米块等不同自主装纳米形态。代表性的纳米照片见图2.
以上所述仅为本发明的较佳实施例,对本发明而言仅仅是说明性的,而非限制性的。本专业技术人员理解,在本发明权利要求所限定的精神和范围内可对其进行许多改变,修改,甚至等效,但都将落入本发明的保护范围内。
Claims (3)
1.通式I的化合物
n=1或2,AA选自Ser残基、Phe残基或Val残基,n=1时通式I为Nα-[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽,n=2时通式I为两个(3s)-1,2,3,4-四氢异喹啉-3-甲酰基分别连接到Lys的α-氨基和侧链氨基上,Lys的羧基再与RGD四肽连接,即Nα,Nω-双[(3s)-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys修饰的RGD四肽。
2.一种制备权利要求1所述化合物的方法,其特征在于,
(1)将L-苯丙氨酸在浓盐酸催化下与甲醛进行Pictet-Spengler缩合,得到1,2,3,4-四氢异喹啉-3-羧酸;
(2)将1,2,3,4-四氢异喹啉-3-羧酸在碱性条件下进行Boc保护得到N-Boc-1,2,3,4-四氢异喹啉-3-羧酸;
(3)将N-Boc-1,2,3,4-四氢异喹啉-3-羧酸与Tos·Lys(Boc)-OBzl缩合,得到Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-OBzl;
(4)将Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)-OBzl在NaOH水溶液存在下脱去苄酯保护基,得到Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc);
(5)将Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)与HCl·Arg(NO2)Gly-Asp(OBzl)AA-OBzl缩合,得到Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)Arg(NO2)GlyAsp(OBzl)AA-Obzl;
(6)将Nα-[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys(Boc)Arg(NO2)GlyAsp(OBzl)AA-OBzl在三氟醋酸和三氟甲磺酸的作用下,脱除保护基,得到通式I的化合物;
(7)将N-Boc-1,2,3,4-四氢异喹啉-3-羧酸与Tos·Lys(HCl)-OBzl缩合,得到Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-OBzl;
(8)将Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys-OBzl在NaOH水溶液存在下脱去苄酯保护基,得到Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys;
(9)将Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-Lys与HCI·Arg(NO2)Gly-Asp(OBzl)AA-OBzl缩合,得到Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-LysArg(NO2)GlyAsp(OBzl)AA-OBzl;
(10)将Nα,Nω-双[(3s)-N’-Boc-1,2,3,4-四氢异喹啉-3-甲酰基]-LysArg(NO2)GlyAsp(OBzl)AA-OBzl在三氟醋酸和三氟甲磺酸的作用下,脱除保护基,得到通式I的化合物。
3.权利要求1所述通式I的化合物在制备抗血栓药物中的应用。
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