CN1133048A - 由c-活性蛋白质片段衍生的寡肽 - Google Patents
由c-活性蛋白质片段衍生的寡肽 Download PDFInfo
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- CN1133048A CN1133048A CN94193726A CN94193726A CN1133048A CN 1133048 A CN1133048 A CN 1133048A CN 94193726 A CN94193726 A CN 94193726A CN 94193726 A CN94193726 A CN 94193726A CN 1133048 A CN1133048 A CN 1133048A
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Abstract
本发明涉及由C-活性蛋白质(以下简称CRP)片段衍生的寡肽,它们作为免疫调节剂以及在治疗心血管疾病和炎症方面的用途。
Description
本发明涉及由C-活性蛋白质(以下简称CRP)片段衍生的寡肽,它们作为免疫调节剂以及在治疗心血管疾病和炎症方面的用途。
CRP是一般具有非常低血液浓度的一种蛋白质,发炎后它可增至两千倍〔J.J.Morley和J.Kushner,Am.N.Y.Acad.Sci.,389,406-418(1989)〕。F.A.Robey等人在J.Biol.Chem.,262,No.15,7053-7057(1987)中公开了与某些吞噬作用激素非常相似的三种CRP四肽序列。化学合成的四肽有助于fagocytic白细胞和过氧化物的产生,并以似吞噬作用激素方式诱导单核细胞产生白细胞介素1。象吞噬作用激素一样,这三种CRP四肽被体内蛋白酶迅速代谢和灭活。
现在,令人惊奇地发现,所说CRP四肽片段的化学改性类似物被赋予免疫调节活性,而且可用于治疗心血管疾病和炎症,例如,治疗败血症休克。
因此,本发明涉及式(I)的寡肽及其与药物上可接受的酸或碱形成的盐,
A1-A2-A3-A4 (I)其中:A1为选自苏氨酸,亮氨酸,异亮氨酸,缬氨酸,肌氨酸,丙氨酸,甘氨酸和(C2-6)酰基一甘氨酸的氨基酸残基或不存在;A2为选自至少被一个(C1-6)烷基,苄基或(C2-6)酰基选择性Nα-取代的亮氨酸,异亮氨酸,缬氨酸,赖氨酸,鸟氨酸,和脯氨酸的氨基酸残基或不存在;A3为选自脯氨酸,亮氨酸,异亮氨酸和缬氨酸的氨基酸残基;A4为选自在C-端位选择性酰胺化的精氨酸,亮氨酸,谷氨酰胺的氨基酸残基,或是胍基丁胺残基,或不存在;条件是只能不存在A1,A2和A4其中一个;所说化合物进一步的特性在于上述氨基酸残基和胍基丁胺残基的侧链基团可以被选自(C1-6)烷基,苄基或(C2-6)酰基的一个或多个基团进一步取代,而且每个所说氨基酸残基可以在Cα上以D-或L-形式,或以可能的非对映体或对映体中的一种形式存在。
本发明优选的一组化合物是一些式(I)化合物及其与药物上可接受的酸或碱形成的盐,其中A1为选自甘氨酸,苏氨酸,亮氨酸,异亮氨酸,缬氨酸,肌氨酸,丙氨酸,(C2-6)酰基-甘氨酸的氨基酸残基或不存在;A2为选自被一个(C1-6)烷基,苄基或(C2-6)酰基Nα-取代的赖氨酸的氨基酸残基;A3为脯氨酸;A4为任意在C-端位酰胺化的谷氨酰胺,亮氨酸,精氨酸,或是胍基丁胺残基,或不存在;所说化合物进一步的特性在于所说氨基酸残基和胍基丁胺残基的侧链基团可以被选自(C1-6)烷基,苄基或(C2-6)酰基的一个或多个基团选择性取代,而且每个所说氨基酸残基可以在Cα上以D或L形式,或以可能的非对映体或对映体中的一种形式存在。
所说(C1-6)烷基指,例如,甲基,乙基,丙基,异丙基,丁基,仲丁基,叔丁基,正戊基,3-甲基-戊基,正己基和相应位置的异构体。所说(C2-6)酰基指,例如,甲酰基,乙酰基,丙酰基,丁酰基,戊酰基,己酰基和相应位置的异构体。
本发明另一个目的涉及式(I)的寡肽作为免疫调节剂和在治疗心血管疾病和炎症如败血症休克中的用途。
通式(I)的化合物可以用本领域普通技术人员熟知的肽合成方法,固相或溶液合成法制备〔例如,见Merrifield,R.B.,Biochemistry,3,1385(1964)〕。除非另有说明,氨基酸残基将用于Cα的L-构型中。
优选地,合成是在溶液中进行,以所选氨基酸为起始原料,通过逐步加成所需的氨基酸形成寡肽。当然,也可以先组合成二或三肽单元。尽管寡肽的合成可以从任何氨基酸开始,并且可以在N-端或C-端两个方向进行,但还是优选在N-端方向进行。这些氨基酸或,如果需要,预先合成的二或三肽可以直接使用或以通过酯化使羧基被保护,例如用叔丁基(tBu),或/和通过酰胺化使氨基被保护,例如用苄氧基羰基(Z),的相关衍生物形式使用,而且,在这种情况下,适宜选择保护侧链基团,例如用2,2,5,7,8-五甲基-苯并二氢吡喃-6-磺酰基(Pmc),叔丁氧羰基(Boc)或三氟乙酸(TFA)进行保护。这些保护反应可以用肽化学中普通技术人员熟悉的方法进行。
当然,上述保护的衍生物也可以是商品。但最好在与随后的氨基酸缩合之前除去α-氨基部分的保护基,例如,用中等强度的酸(如三氟乙酸)酸解,或用氢气或氢供体如甲酸或其盐,三乙基硅烷,碱中的肼等等催化氢解来实现。而且,最好根据用来去保护的氨基酸和其它因素(如果存在)选择在适当钯催化剂存在下进行。然后,与随后的氨基酸残基进行缩合,这些残基的不包括在所说反应中的部分被适当保护。
该缩合反应可以用几种已知方法中的一种来实现。具体地说,可用活性酯,例如,琥珀酰亚胺(Su),氟化物(F)或缩合剂如苯并三唑-1-基-氧-三-(二甲氨基)-磷鎓六氟磷酸酯(BOP),溴-三-吡咯烷鎓-磷鎓六氟磷酸酯(PyBroP),二环己基碳化二亚胺(DCC)等等,任选在催化剂如1-羟基-苯并三唑(HOBT),4-二甲基-氨基-吡啶(DMAP),三乙胺(TEA),N-甲基-吗啉,N-甲基-咪唑等存在下进行。
至于氨基酸的Nα-烷基衍生物,可以在低温下,在氰基硼氢化钠,选择性还原剂存在下,在极性溶剂,优选甲醇中,通过用适当的醛处理得到。
当需要C-端位是酰胺形式的式(I)寡肽时,带有这一部分的商品氨基酸可以用作起始原料,或可以用HOBT铵盐,或当A4是胍基丁胺残基时,用胍基丁胺本身来酰胺化C-端氨基酸。
其中A1和/或A2是酰化氨基酸残基的式(I)化合物可以在低温下,在催化剂如DMAP存在下,通过用适当酰基-酐处理得到。或者可以使用具有Nα-酰基-氨基酸残基的商品。
所得产物可以通过从适当溶剂中结晶纯化,或如果需要,用已知的色谱技术如反相色谱法和离子交换色谱法纯化。
下面将提供制备本发明一些改性寡肽的实施例。
氨基酸衍生物,保护片段和改性寡肽的HPLC分析是在下列实验条件下进行的:柱: Lichrosorb RP-18温度: 25℃(除非另有说明)流量: 1.5ml/分钟检测器: Jasco875-UV(230nm)洗脱剂A: 90%水,10%乙腈,0.1%三氟乙酸(TFA)洗脱剂B: 乙腈,0.1%TFA洗脱剂C: 水,0.1%TFA梯度: (I):从0—40%B于A中(20’)
至80%B于A中(10’)
(II):从0—50%A于C中(20’)至100%A
(3’),至40%B于A中(20’)。
除非另有说明,否则所有合成步骤均在室温下进行。
110℃用6MHCl水解22小时后,用Beckman SYSTEMGOLD氨基酸分析仪测定氨基酸的组成和比例。
为了更加明了,下面实施例所用缩写的含意列表如下:BDHA-Cl: 苄基二甲基十六烷基氯化铵Boc: 叔丁氧羰基(Boc)20: 二-叔丁基-碳酸氢盐BOP: 苯并三唑-1-基-氧-三-(二甲氨基)-磷
鎓六氟磷酸酯BSA: N,O-双(三甲基甲硅烷基)-乙酰胺DCC: 二环己基碳化二亚胺DMAP: 4-二甲氨基-吡啶DMF: 二甲基甲酰胺F: 氟化物HOBT: 1-羟基-苯并三唑Pmc: 2,2,5,7,8-五甲基-苯并二氢吡喃-6-磺
酰基PyBroP: 溴-三-吡咯烷鎓-磷鎓六氟磷酸酯tBu: 叔丁基TBAF: 四丁基氟化铵TEA: 三乙胺TFA: 三氟乙酸Su: 琥珀酰亚胺Z: 苄氧羰基
实施例1H-Sar-Lys-Pro-Arg-OH·2AcOH
A)在50℃和60分钟内将8.64ml(36mmol)N,N-二甲基甲酰胺-二-叔丁基-乙缩醛加到6.9g(12mmol)Z-Arg(Pmc)-OH的60ml二氯乙烷浆液中。添加结束后,在50℃搅拌该反应混合物40分钟,然后加入20ml 5%碳酸氢钠水溶液。真空蒸发掉二氯乙烷并用100ml乙酸乙酯稀释水相。分离有机相,用5%碳酸氢钠和饱和氯化钠水溶液洗涤至中性。将有机相脱水并真空蒸发,粗产物在硅胶柱上纯化(洗脱剂:乙酸乙酯/正己烷6∶4),得到3.4gZ-Arg(Pmc)-OtBu(HPLC,梯度(I):R.t.32分钟;纯度99%)。B)将1.416g(21.44mmol)甲酸铵的3ml水和Pd海绵(约1g)加到3.385g(5.36mmol)A)化合物的80ml甲醇溶液中。在室温缓慢搅拌该反应混合物约2小时。滤除催化剂后真空蒸发溶剂,将剩余物溶解于乙酸乙酯并用5%碳酸氢钠水溶液和水洗涤至中性。将有机相脱水并真空蒸发,得到2.88gZ-Arg(Pmc)-OtBu·HCOOH(HPLC,梯度(I):R.t.23.40分钟;纯度99.3%)。C)将B)化合物溶解于30ml DMF/二氯甲烷(1∶1)。另外,将1.403g(5.63mmol)Z-Pro-OH溶解于20mlDMF/二氯甲烷(1∶1),并加入BOP(2.49g,5.63mmol),HOBT(0.76g,5.63mmol)和TEA(1.56ml,11.26mmol)。将两种溶液混合并搅拌所得反应混合物1小时。然后真空蒸发溶剂,剩余物溶解于乙酸乙酯,用5%碳酸氢钠和水洗涤至中性。将有机相脱水并真空蒸发,剩余物在乙醚中研制,得到3.71gZ-Pro-Arg(Pmc)-OtBu(HPLC,梯度(I):R.t.29.49分钟;纯度99.6%)。D)氮气下将350mgPd/C加到3.709g(5.08mmol)C)化合物的50ml甲醇溶液中,接着非常缓慢地加入4ml(24mmol)三乙基硅烷。2小时后,过滤反应混合物并真空蒸发溶剂,得到3.01gH-Pro-Arg(Pmc)-OtBu(HPLC,梯度(I):R.t.24.25分钟;纯度99.56%)。E)将D)化合物(2.375g,4mmol)溶解于20mlDMF/二氯甲烷(1∶1v/v)。再将Z-Lys(Boc)-OH(1.826g,4.8mmol)溶解于20ml同样的混合溶液中,然后加入BOP(2.12g,4.8mmol),HOBT(0.648g,4.8mmol)和TEA(1.33ml,9.6mmol)。将两种溶液混合,搅拌该反应混合物1小时,然后如C)所述进行处理。剩余物在乙醚中研制,得到3.64gZ-Lys(Boc)-Pro-Arg(Pmc)-OtBu。F)将1.321g(20mmol)甲酸铵的3ml水和约1g新鲜Pd海绵加到3.64g(3.8mmol)E)化合物的70ml甲醇溶液中。进行如B)所述过程,得到3.27gH-Lys(Boc)-Pro-Arg(Pmc)-OtBu。G)将Z-Sar-OH(0.196g,0.88mmol)溶解于4mlDMF/二氯甲烷(1∶1v/v),接着加入BOP(0.39g,0.88mmol),HOBT(0.119g,0.88mmol),TEA(0.24ml,1.76mmol)和溶解于4ml同样的混合溶液中的F)化合物(0.694g,0.8mmol)。将该溶液搅拌1小时,然后真空蒸发溶剂,剩余物溶解于乙酸乙酯,依次用5%碳酸氢钠,2.5%硫酸氢钾和水洗涤至中性。将有机相脱水并真空蒸发,剩余物在乙醚中研制,得到0.746gZ-Sar-Lys(Boc)-Pro-Arg(Pmc)-OtBu。H)将G)化合物(0.746g,0.726mmol)溶解于10ml95%TFA的水中。75分钟后用水稀释反应混合物并真空蒸发。将剩余物溶解于水,用乙醚洗涤并冷冻干燥。所得产物用反相顶替色谱纯化。将产物溶解于3mlTFA(0.1%v/v)水溶液中,并以0.5ml/分钟流速加到用TFA(0.1%v/v)水溶液预先平衡的VYDAC C18柱(250×10mm)上。用含有TFA(0.1%v/v)的50mMBDHA-Cl水溶液以0.5ml/min洗脱该柱。洗脱约1小时后,收集0.5ml级分直到顶替剂洗脱。用HPLC分析这些级分并将含有纯产物的级分合并,冷冻干燥,得到0.2gZ-Sar-Lys-Pro-Arg-OH(HPLC,梯度(I):R.t.11.04分钟;纯度>95%)。I)将H)化合物(0.2g,0.28mmol)溶解于85%甲酸(5ml)中并加入新鲜Pd海绵。温和搅拌反应混合物100分钟。滤除催化剂后,用水稀释反应混合物并冷冻干燥。产物在S-Sepharose F/F柱(16×200mm)上通过离子交换色谱纯化,300分钟内以3ml/分钟速率,用pH为5的乙酸铵以0.015M至0.15M梯度洗脱。用HPLC分析收集级分,并将含有纯产物的级分合并,多次冷冻干燥,得到0.1g标题产物。HPLC:梯度(II),R.t.7.30分钟;纯度>99%。FAB-MS:m/z-471amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;NH-Lys)8.06;d(1H;NH-Arg)7.17;m(1H;Cα-Pro)4.63;m(1H;Cα-Lys)4.32÷4.24;q(1H;Cα-Arg)3.79;m(1H;Cδ-Pro)3.70;s(2H;Cα-Sar)3.05;t(3H;Cα-Arg)3.03;t(2H;Cε-Lys)2.75;s(3H;CH3-Sar)2.24;m(4H;Cβ+γ-Pro)1.99÷1.85;s(6H;CH3COO-)1.81;m(10H;Cβ+γ-Arg;Cβ+γ+δ-Lys)1.72÷1.29.
实施例2H-(D)Ala-Lys-Pro-Arg-OH·2AcOH
以0.122g(0.55mmol)Z-(D)Ala-OH和0.434g(0.5mmol)实施例1F)化合物为起始原料,基本上按照实施例1G)-I)的方法进行,得到0.089g标题产物。HPLC:梯度(II),R.t.9.59分钟;纯度>99%。FAB-MS:m/z-471amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;NH-Lys)8.12;d(1H;NH-Arg)7.17;m(1H;Cα-Pro)4.60;m(1H;Cα-Lys)4.31÷4.25;q(1H;Cα-Arg)3.78;m(2H;Cδ-Pro)3.70;g(1H;Cα-Ala)3.31;t(2H;Cδ-Arg)3.03;t(2H;Cε-Lys)2.74;m(14H;Cβ+γ-Arg;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.09÷1.28;s(6H;CH3COO-)1.80;d(3H;CH3-Ala)1.12.
实施例3Ac-Gly-Lys-Pro-Arg-OH·AcOH
以Ac-Gly-OH(0.09g,0.77mmol)和实施例1F)化合物(0.607g,0.7mmol)为起始原料,按照实施例1G)-H)的方法进行,得到0.116g标题产物。HPLC:梯度(II),R.t.13.06分钟;纯度>99%。FAB-MS:m/z-499amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;NH-Lys)8.15;t(1H;NH-Gly)8.11;d(1H;NH-Arg)7.47;m(1H;Cα-Pro)4.59÷4.48;m(1H;Cα-Lys)4.33÷4.25;m(5H;Cα-Arg;Cα--Gly;Cδ-Pro)3.84÷3.59;t(2H;Cδ-Arg)3.01;t(2H;Cε-Lys)2.70;m(14H;Cβ+γ-Arg;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.03÷1.28;s(3H;CH3COO-)1.85;s(3H;CH3-CO-NH)1.72.
实施例4H-Gly-(Et)Lys-Pro-Arg-OH·2AcOH
A)将实施例1F)化合物(0.96g,1.1mmol)溶解于8ml甲醇中并加入0.071g(1.12mmol)氰基硼氢化钠。将反应混合物冷却至-15℃再加入0.062ml(1.12mmol)乙醛。60分钟后真空蒸发反应混合物并将剩余物溶解于水中,用HCl调至pH3。过滤沉淀并用pH3HCl洗涤,得到0.825g白色固体,其中70%为H-(Et)Lys(Boc)-Pro-Arg(Pmc)-OtBu,26%是二烷基化反应的副产物。B)将Z-Gly-OH(0.994g,4.75mmol)溶解于3mlDMF/二氯甲烷(4∶6v/v),然后连续加入PyBrop(2.21g,4.75mmol),二异丙基乙胺(2.4ml,14.25mmol)和溶解于6ml同样的混合溶液中的A)中所得固体。搅拌80分钟反应混合物。真空蒸发溶剂,将剩余物溶解于乙酸乙酯并依次用5%碳酸氢钠,2.5%硫酸氢钾和水洗涤至中性。将有机相脱水并真空蒸发,得到0.95g含有60%Z-Gly-(Et)Lys(Boc)-Arg(Pmc)-OtBu的混合物。C)用0.75gB)化合物作起始原料并按照实施例1H)方法进行,得到0.125gZ-Gly-(Et)Lys-Arg-OH。HPLC:梯度(I),R.t.12分钟;纯度94%。D)将C)化合物在CM-Sephadex C-25柱(16×200mm)上通过离子交换色谱纯化,在270分钟内以3ml/分钟速率,用pH6乙酸铵以0.02M至0.2M梯度洗脱。用HPLC分析收集的级分并将含有纯产物的级分合并,多次冷冻干燥,得到0.1gZ-Gly-(Et)Lys-Pro-Arg-OH·2AcOH(ITF1931)。HPLC:梯度(I),R.t.12分钟;纯度97.5%。FAB-MS:m/z-619amu[M+H]+。1H-NMR(200MHz;DMSO):t(1H;NH-Gly)7.46;m(5H;CH-aryl)7.37;d(1H;NH-Lys)7.24;m(1H;Cα-Lys)5.19;s(2H;Cα-Arg)5.05;m(1H;Cα-Pro)4.23;m(2H;Cα-Gly)3.92;m(1H;Cα-Arg)3.81;m(2H;Cδ-Pro)3.58;m(2H;CH2-Et)3.46;m(2H;Cδ-Arg)2.99;m(2H;Cε-Lys)2.65;m(4H;Cβ-and Cγ-Pro)2.03÷1.76;m(19H,Cβ-and Cγ-Lys and-Arg,and Cδ-Lys)1.69+1.19;s(6H,CH3COO-)1.66;t(3H,CH3-Et)1.04.E)将D)化合物按实施例1I)所述处理,得到0.089g标题产物。HPLC:柱温度:60℃;梯度(II),R.t.7.39分钟;纯度>99%。FAB-MS:m/z-485amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;Nα-Arg)7.12;m(0.8H;Cα-Pro)5.24;m(0.2H;Cα-Pro)4.43;m(1H;Cα-Lys)4.28+4.16;m(1H;Cα-Arg)3.81;m(1H;Cδ-Proe CH2-N)3.70÷3.16;s(2H;Cα-Gly)3.41;t(2H;Cδ-Arg)3.03;t(2H;Cε-Lys)2.75;m(4H;Cβ+γ-Pro)2.12÷1.75;s(6H;CH3COO-)1.79;m(10H;Cβ+ -Arg;Cβ+γ+δ-Lys)1.70÷1.14;t(2.4H;C*H3-CH2)0.98;t(0.6H;C*H3-CH2)0.89.
实施例5Ac-Lys-Pro-Arg-OH·TFA
A)将实施例1F)化合物(0.3g,0.34mmol)溶解于1ml二氯甲烷中。将该溶液冷却至-20℃并加入DMAP(0.048g,0.38mmol)和乙酸酐(0.035ml,0.38mmol)。30分钟后用5%碳酸氢钠水溶液和饱和氯化钠溶液洗涤上述溶液。有机相脱水并真空蒸发,得到0.28gAc-Lys(Boc)-Pro-Arg(Pmc)-OtBu。B)将A)化合物(0.28g,0.324mmol)按实施例1H)所述处理,得到0.085g标题产物。HPLC:梯度(II),R.t.13.64分钟;纯度>99%。FAB-MS:m/z-442amu[M+H]+1H-NMR(200MHz;DMSO):d(0.15H;NH-Lys)8.45;d(0.85H;NH-Lys)8.21;d(1H;NH-Arg)8.09;m(1H;Cα-Pro)4.55÷4.45;m(1H;Cα-Lys)4.43÷4.33;m(1H;Cα-Arg)4.21÷4.11;m(2H;Cδ-Pro)3.77÷3.47;q(2H;Cδ-Arg)3.13;m(2H;Cε-Lys)2.78;m(14H;Cβ+γ-Arg;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.18÷1.28;s(6H;C*H3COO- andC*H3-CO-NH)1.84.
实施例6H-Gly-(D)Lys-Pro-Arg-OH·2AcOH
A)以0.544g(1.43mmol)Z-(D)Lys(Boc)-OH和0.772g(1.3mmol)实施例1D)化合物为起始原料,按照实施例1G)的方法进行,得到1.2gZ-(D)Lys(Boc)-Pro-Arg(Pmc)-OtBu。B)将A)化合物(1.2g,1.29mmol)溶解于30ml甲醇中,然后加入0.325g(5.16mmol)甲酸铵的0.3ml水和约0.2g新鲜Pd海绵。2小时后滤除催化剂并真空蒸发溶剂。将剩余物溶解于50ml乙酸乙酯并用5%碳酸氢钠水溶液和水洗涤至中性。有机相脱水并真空蒸发,得到1.119gH-(D)Lys(Boc)-Pro-Arg(Pmc)-OtBu·HCOOH。C)用0.229g(1.43mmol)Z-Gly-OH和1.119g(1.29mmol)B)化合物作起始原料,按照实施例1G)-I)的方法进行,得到0.23g标题产物。HPLC:梯度(II),R.t.9.53分钟;纯度>99%。FAB-MS:m/z-457amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;NH-Lys)8.12;d(0.6H;NH-Arg)7.37;d(0.4H;NH-Arg)7.27;m(0.6H;Cα-Pro)4.80;m(0.4H;Cα-Pro)4.61;m(1H;Cα-Lys)4.34÷4.15;g(0.6H;Cα-Arg)3.95;t(0.4H;Cα-Arg)3.81;m(2H;Cδ-Pro)3.58÷3.35;s(0.4H;Cα-Gly)3.26;s(0.6H;Cα-Gly)3.11;t(2H;Cδ-Arg)3.05;m(2H;Cε-Lys)2.71;m(14H;Cβ+γ-Arg;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.11÷1.13;s(6H;CH3COO-)1.79.
实施例7H-Gly-Lys-Pro-Arg-NH2·3AcOH
A)将Z-Arg(Pmc)-OH(2.87g,5mmol)溶解于15mlDMF,然后加入0.837g(5.5mmol)HOBT铵盐并将反应混合物冷却至0℃。加入1.135g(5.5mmol)DCC后,将反应混合物升至室温并搅拌300分钟。滤除二环己基脲后,真空蒸发溶剂,将剩余物溶解于25ml乙酸乙酯并依次用5%碳酸氢钠水溶液和水洗涤至中性。有机相脱水,真空蒸发溶剂,剩余物从乙醚/正己烷中结晶,得到2.8gZ-Arg(Pmc)-NH2〔HPLC(梯度从20%到60%A中的B,20分钟):R.t.15.7分钟;纯度98%〕。B)氮气下将150mgPd/C加到1.045g(1.82mmol)A)化合物的50ml甲醇溶液中,接着非常缓慢地加入2ml(12mmol)三乙基硅烷。120分钟后,过滤反应混合物并真空蒸发溶剂,得到0.8gH-Arg(Pmc)-NH2〔HPLC(梯度从20%到60%A中的B,20分钟):R.t.8.6分钟;纯度98.5%〕。C)从Z-Pro-OH(0.499g,2mmol)和B)化合物(0.8g,1.82mmol)开始,按照实施例1G)所述方法进行,得到1.2gZ-Arg(Pmc)-NH2。将该化合物溶解于25ml甲醇中并加入100mg10%Pd/C,接着非常缓慢地加入1.52ml(9.1mmol)三乙基硅烷。130分钟后过滤反应混合物并真空蒸发溶剂,得到0.955gH-Pro-Arg(Pmc)-NH2。D)从0.745g(1.96mmol)Z-Lys(Boc)-OH和0.955g(1.78mmol)C)化合物开始,按照C)所述方法进行,得到1.19gH-Lys(Boc)-Pro-Arg(Pmc)-NH2。E)从0.385g(1.71mmol)Z-Gly-OH和1.19g(1.55mmol)D)化合物开始,按照实施例1G)-I)所述方法进行,得到粗产物,然后通过16×200mmCM-52纤维素柱进行离子交换色谱纯化,240分钟内以2ml/分钟速率,用pH7的乙酸铵以0.025M至0.30M的梯度洗脱。用HPLC分析收集的级分,合并纯的级分并冷冻干燥多次,得到0.283g标题产物。HPLC:梯度(II),R.t.4.7分钟;纯度=99.3%。FAB-MS:m/z-456amu[M+H].1H-NMR(200MHz;DMSO):d(0.2H;Nα-Arg)8.89;m(1.8H;Nα-Lys and Nα-Arg)8.16÷8.03;s(0.2H;NH2-Arg)7.77;s(0.8H;NH2-Arg)7.76;s(0.8H;NH2-Arg)7.07;s(0.2H;NH2-Arg)7.00;m(1H;Cα-Pro)4.61÷4.50;m(1H;Cα-Lys)4.47÷4.34;m(1H;Cα-Arg)4.26÷4.15;m(2H;Cδ-Pro)3.77÷3.54;s(2H;Cα-Gly)3.13;m(2H;Cδ-Arg)3.06;t(2H;Cε-Lys)2.67;m(14H;Cβ+γ-Arg;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.12÷1.26;s(9H;CH3COO-)1.77.
实施例8H-Gly-Lys-Pro-Agm:3AcOH
A)将Z-Lys-OH(0.84g,3mmol)溶解于6ml由3ml1M氢氧化钠和3ml二噁烷组成的溶液中,然后加入0.57ml(4.5mmol)乙基-硫羟-三氟乙酸酯。将反应混合物在40℃搅拌约7小时,然后真空蒸发溶剂并将剩余物溶解于40ml5%碳酸氢钠中。用乙酸乙酯洗涤水相并调至pH2,再用乙酸乙酯萃取。有机相脱水并真空蒸发,得到1gZ-Lys(TFA)-OH为油状物(HPLC,梯度(I):R.t.19.7分钟;纯度96%)。B)将H-Pro-OtBu·HCl(0.283g,1.364mmol)溶解于9mlDMF/二氯甲烷(1∶1)。再将A)化合物(0.565g,1.5mmol)溶解于9ml相同的混合溶液,然后加入BOP(0.66g,1.5mmol),HOBT(0.202g,1.5mmol)和TEA(0.63ml,4.5mmol)。将两种溶液合并后搅拌30分钟,然后按实施例1G)所述方法进行,得到0.725gZ-Lys-(TFA)-Pro-OtBu为油状物(HPLC,梯度(I):R.t.26.12分钟;纯度95.5%)。C)将80.26g(4.1mmol)甲酸铵的0.1ml水和约0.2g新鲜Pd海绵加到0.725g(1.36mmol)B)化合物的20ml甲醇溶液中。2小时后滤除催化剂并真空蒸发溶剂。将剩余物溶解于40ml乙酸乙酯并用5%碳酸氢钠水溶液和水洗涤至中性。有机相脱水并真空蒸发,得到0.5gH-Lys(TFA)-Pro-OtBu·HCOOH(HPLC,梯度(I):R.t.16.24分钟;纯度95%)。D)将C)化合物溶解于10mlDMF/二氯甲烷(1∶1)。再将Z-Gly-OH(0.291g,1.4mmol)溶解于10ml相同的混合溶液,然后加入BOP(0.615g,1.4mmol),HOBT(0.188g,1.4mmol)和TEA(0.39ml,2.8mmol)。将两种溶液合并后搅拌60分钟,然后按实施例1G)所述过程进行。有机相脱水并真空蒸发,得到的油状剩余物在硅胶柱上进行色谱分离(洗脱剂:乙酸乙酯/正己烷9∶1),得到0.588gZ-Gly-Lys(TFA)-Pro-OtBu(HPLC,梯度(I):R.t.24.51min.;纯度99.3%)。E)将D)化合物溶解于预先冷却至0℃的15ml37%HCl。在0℃8分钟后,用15ml水稀释反应混合物并真空蒸发。将剩余物溶解于水,用乙酸乙酯洗涤并冷冻干燥,得到0.531gZ-Gly-Lys(TFA)-Pro-OH(HPLC,梯度(I):R.t.17.69分钟;纯度95%)。F)将E)化合物(0.53g,1mmol)溶解于5ml乙酸乙酯,然后加入0.138g(1.2mmol)N-羟基琥珀酰亚胺并将反应混合物冷却至-20℃。加入0.248g(1.2mmol)DCC,将反应混合物置于室温90分钟,然后滤除沉淀并蒸发溶剂,得到0.625gZ-Gly-Lys(TFA)-Pro-OSu。将碳酸氢钠(0.115g,1.09mmol)溶解于22ml水中,接着加入胍基丁胺硫酸盐(0.498g,2.18mmol)和N-甲基-咪唑(0.087ml,1.09mmol)。在所得溶液中加入0.57g(0.91mmol)Z-Gly-Lys(TFA)-Pro-OSu,室温下搅拌反应混合物30分钟,然后将混合物调至pH3并用乙酸乙酯洗涤。冷冻干燥水相,得到0.58gZ-Gly-Lys(TFA)-Pro-Agm。G)将F)化合物溶解于35ml水中,加入1M氢氧化钠至pH12.7。30分钟后用1MHCl将pH调至7并冷冻干燥水相。将剩余物溶解于绝对乙醇,滤除盐并真空蒸发溶液。根据实施例1H)所述方法用反相顶替色谱纯化所得产物,得到0.251gZ-Gly-Lys-Pro-Agm(HPLC,梯度(I):R.t.10.77分钟;纯度97.5%)。H)基本上按实施例1I)所述方法处理G)化合物,得到0.229g标题化合物。HPLC:梯度(II),R.t.10.93分钟;纯度:98.5%。FAB-MS:m/z-413amu[M+H]+1H-NMR(200MHz;DMSO):t(0.2H;Nα-Agm)8.54;d(1H;Nα-Lys)8.10;t(0.8H;Nα-Agm)7.96;q(0.8H;Cα-Pro)4.55;q(0.2H;Cα-Pro)4.41;m(1H;Cα-Lys)4.28÷4.22;m(2H;Cδ-Pro)3.75÷3.34;m(6H;Cα-Gly;Cα+δ-Agm)3.21÷2.93;t(2H;Cε-Lys)2.67;;m(14H;Cβ+γ-Agm;Cβ+γ-Pro;Cβ+γ+δ-Lys)2.11÷1.25;s(9H;CH3COO-)1.76.
实施例9H-Gly-Lys-Pro-OH:AcOH
A)将Z-Lys(Boc)-OH(1.487g,3.8mmol)溶解于15mlDMF/二氯甲烷(1∶1v/v)并依次加入BOP(1.72g,3.8mmol),HOBT(0.52g,3.8mmol),TEA(1.08ml,7.8mmol)和溶解于15ml相同的混合溶液中的H-Pro-OtBu(0.623g,3mmol)。搅拌1小时该溶液。真空蒸发溶剂,将剩余物溶解于乙酸乙酯,并依次用5%碳酸氢钠水溶液,2.5%硫酸氢钾水溶液和水洗涤至中性。有机相脱水并真空蒸发,剩余物在乙醚中研制,得到1.6gZ-Lys(Boc)-Pro-OtBu。B)将A)化合物(1.59g,2.9mmol)溶解于20ml甲醇中,并在所得溶液中加入甲酸铵(0.731g,11.6mmol)的0.6ml水溶液和新鲜Pd海绵(约0.5g)。2小时后滤除催化剂并真空蒸发溶剂。将剩余物溶解于50ml乙酸乙酯并用5%碳酸氢钠水溶液和水洗涤至中性。有机相脱水并真空蒸发,得到1.119gH-Lys(Boc)-Pro-Ot-Bu·HCOOH。C)将Z-Gly-OH(0.762g,3.64mmol)溶解于15mlDMF/二氯甲烷(1∶1v/v)并依次加入BOP(1.61g,3.64mmol),HOBT(0.491g,3.64mmol),TEA(1.01ml,7.28mmol;溶解于15ml相同的混合溶液中的H-Lys(Boc)-Pro-OtBu(1.119g,2.8mmol)。反应混合物用A)所述方法处理,得到1.16g Z-Gly-Lys(Boc)-Pro-OtBu。D)将C)化合物(0.746g,0.726mmol)溶解于20ml95%TFA水溶液。1小时后反应混合物用水稀释并真空蒸发。将剩余物溶解于水,用乙醚洗涤并冷冻干燥。所得产物通过反相顶替色谱纯化。将产物溶解于3ml含有TFA(0.1%v/v)的水溶液,并以0.5ml/分钟流速加到用TFA水溶液(0.1%v/v)预先平衡的VYDAC C18柱(16×200mm)上。用含有TFA(0.1%v/v)的50mMBDHA-Cl水溶液以0.5ml/分钟洗脱该柱。洗脱约1小时后,收集0.5ml级分直到顶替剂洗脱。用HPLC分析该级分,合并含有纯产物的级分并冷冻干燥,得到0.325gZ-Gly-Lys-Pro-OH。HPLC:梯度(I),R.t.11.48分钟;纯度>95%。E)将D)化合物(0.325g,0.59mmol)溶解于5ml85%甲酸中并加入新鲜Pd海绵。温和搅拌反应混合物约1小时。滤除催化剂后,用水稀释甲酸并冷冻干燥。产物用离子交换色谱法在S-SepharoseF/F柱(16×200mm)上纯化,在5小时内以3ml/分钟速率,用pH5的乙酸铵以0.015M至0.15M梯度洗脱。用HPLC分析收集的级分,将含有纯产物的级分合并,冷冻干燥多次,得到0.165g标题产物。HPLC:梯度(II),R.t.3.97分钟;纯度>99%。FAB-MS:m/z-301amu[M+H]+1H-NMR(200MHz;DMSO):d(1H;Nα-Lys)8.17;m(1H;Cα-Pro)4.60;m(1H;Cα-Lys)4.14;m(2H;Cδ-Pro)3.61;s(2H;Cα-Gly)3.12;t(2H;Cε-Lys)2.73;m(10H;Cβ+γ+δ-Lys;Cβ+γ-Pro)2.62÷1.40;s(3H;CH3COO-)1.88.
实施例10H-(Et)Lys-Pro-Arg-OH:2AcOH
A)将0.4g实施例4A)化合物在用氯仿/甲醇(9/1v/v)预先平衡的Lobar LiChroprep Si60,40-63μm(31×2.5cm)柱上通过硅胶色谱纯化。用相同的混合物以8ml/分钟速率洗脱该柱。合并含有纯产物的级分并真空蒸发溶剂,得到0.28gH-(Et)Lys(Boc)-Pro-Arg(Pmc)-OtBu。HPLC:梯度(I),R.t.28.5分钟;纯度>99%。B)将A)化合物(0.28g,0.33mmol)溶解于6ml95%TFA水溶液中。70分钟后,反应混合物用水稀释并真空蒸发。将剩余物溶解于水中,用乙醚洗涤并冷冻干燥。产物在CM-Sephadex C-25柱(16×200mm)上通过离子交换色谱纯化,在270分钟内以3ml/分钟速率,用pH6乙酸铵以0.02M至0.2M梯度洗脱。用HPLC分析收集级分,合并含有纯产物的级分并冷冻干燥多次,得到0.14g标题产物。HPLC:(柱温度:60℃)梯度(II),R.t.7.15分钟;纯度>99%。FAB-MS:m/z-428amu[M+H]+。1H-NMR(200MHz;DMSO):d(1H;NH-Arg)7.15;m(1H;Cα-Pro)4.30;q(1H;Cα-Arg)3.79;m(2H;Cδ-Pro)3.64;m(1H;Cα-Lys)3.41;m(2H;Cδ-Arg)3.02;m(2H;Cε-Lys)2,75;q(2H;CH2-Et)2.44;m(14H;Cβ-andCγ-Pro,Lys and-Arg,Cδ-Lys)2.11÷1.21;s(6H;CH3COO-)1,80;t(3H;CH3-Et)0,97.
实施例11(Et)2Lys-Pro-Arg-OH·2AcOH
A)将0.425g实施例4A)化合物溶解于7ml甲醇并加入0.063g(1mmol)氰基硼氢化钠。将反应混合物冷却至-15℃并加入0.155ml(2.5mmol)乙醛。90分钟后真空蒸发反应混合物并将剩余物在水中浆化,用HCl调至pH3。过滤得到的沉淀并用pH3盐酸洗涤,得到0.4g(Et)2Lys(Boc)-Pro-Arg(Pmc)-OtBu。HPLC:梯度(I),R.t.30.3分钟;纯度>98%。B)将上述A)化合物(0.4g,0.455mmol)按照实施例10B)的方法处理,得到0.14g标题产物。HPLC:(柱温度:60℃)梯度(II),R.t.11.08分钟;纯度>99%。FAB-MS:m/z-456amu[M+H]+。1H-NMR(200MHz,DMSO):d(1H;NH-Arg)7.08;m(1H;Cα-Pro)4.21;m(3H;Cα-Arg and Cδ-Pro)3.98÷3.65;m(1H;Cα-Lys)3.54;m(2H;Cδ-Arg)3.03;m(2H;Cε-Lys)2,75;(2H;CH2-Et)2.38;m(2H;CH2-Et)2.36;m(4H;Cβ-and Cγ-Pro)2.18+1.81;s(6H;CH3COO-)1.76;m(10H;Cβ-and Cγ-Lys and-Arg and Cδ-Lys)1.74÷1.33;t(6H;CH3-Et)0.96.
实施例12H-Gly-(Et)Lys-Pro-Leu-OH·AcOH
A)将实施例9A)的化合物(5.33g,10mmol)在Pd/C的存在下溶解在130ml绝对乙醇中。在30分钟内滴加乙醛(0.745ml,12mmol)的20ml绝对乙醇,接着在60分钟内滴加三乙基硅烷(9.56ml,60mmol)。75分钟后,滤除催化剂并真空蒸发溶剂。将油状残余物溶解在50ml无水乙酸乙酯中,并加入3ml用HCl饱和的乙酸乙酯。过滤所得沉淀并真空干燥,得到4.25gH-(Et)Lys(Boc)-Pro-OtBu.HCl。HPLC:梯度(I)R.t.19.83分钟;纯度>98%。B)将Z-Gly-OH(3.78g,18mmol)溶解在12mlDMF和67ml二氯甲烷的混合物中。将上述溶液冷却至-15℃并加入DCC(1.86g,9.03mmol)。15分钟后过滤反应混合物并加到A)化合物(2.8g,6.02mmol)的40ml二氯甲烷中。加入N-甲基-吗啉(0.66g,6.02mmol)后将反应混合物保持在35℃60分钟。真空蒸发溶剂,将残余物溶解在乙酸乙酯中并依次用5%的碳酸氢钠水溶液和2.5%的硫酸氢钾水溶液洗涤。将有机相脱水并真空蒸发,得到3.7gZ-Gly-(Et)Lys(Boc)-Pro-OtBu。HPLC:梯度(I)R.t.28.60分钟;纯度:94%。C)将B)化合物(3.7g,6mmol)溶解在15ml95%TEA的水中。15分钟后将反应混合物慢慢倒入乙酸乙酯中,过滤所得沉淀并真空干燥,得到2.9gZ-Gly-Lys-Pro-OH。HPLC:梯度(I)R.t.14.93分钟;纯度:94%。D)将C)化合物(1g,1.73mmol)溶解于6.92ml0.5M二恶烷/氢氧化钠水溶液(1/1v/v)。将该溶液冷却至0℃并加入(Boc)20(0.415g,1.903mmol)。将pH12的反应混合物保持在室温45分钟。真空蒸发溶剂,将剩余物溶解于水并用乙醚洗涤。水相酸化至pH3并用30ml乙酸乙酯萃取。有机相脱水并真空蒸发,得到0.96gZ-Gly-(Et)Lys(Boc)-Pro-OH。HPLC:梯度(I)R.t.23.04分钟;纯度:92%。E)将D)化合物(0.281g,0.5mmol)溶解于4mlDMF/二氯甲烷(1∶1)并加入BOP(0.221g,0.5mmol),HOBT(0.067g,0.5mmol),TEA(0.219ml,1.57mmol)和H-Leu-OtBu·HCl(0.117g,0.525mmol)。使用实施例1C)方法,得到0.365gZ-Gly-(Et)Lys(Boc)-Pro-Leu-OtBu。HPLC:梯度(I)R.t.29.86分钟;纯度>99%。F)以E)化合物(0.365g,0.5mmol)为起始原料,基本上按照实施例1H)-I)所述方法进行,得到0.155g标题产物。HPLC:梯度(I)R.t.6.3分钟;纯度:98%。FAB-MS:m/z-442amu[M+H]+.1H-NMR(200MHz;DMSO):d(1H;NH-Leu)6.93;m(1H;Cα-Lys)5.23;m(1H;Cα-Pro)4.15;m(1H;Cα-Leu)3.84;m(6H;Cα-Gly,Cδ-Pro and CH2-Et)3.72÷3.03;m(2H;Cε-Lys)2.75;m(4H;Cβ-and Cγ-Pro)2,17÷1,77;s(3H;CH3COO-)1,85;m(9H;Cβ-and Cγ-Lys and-Leu,Cδ-Lys)1.68÷1.05;t(3H;CH3-Et)0.96;d(3H;CH3-Leu)0,88;d(3H;CH3-Leu)0,86.
实施例13H-Gly-(Et)Lys-Pro-Agm·3AcOH
A)以实施例12D)化合物(0.425g,0.75mmol),N-羟基琥珀酰亚胺(0.103g,0.9mmol)和DCC(0.186g,0.9mmol)为起始原料,依次加入碳酸钠(0.095g,0.9mmol),胍基丁胺硫酸盐(0.411g,1.8mmol)和N-甲基-咪唑(0.072g,0.9mmol),接着进行实施例8F)过程,得到0.5gZ-Gly(Et)Lys(Boc)-Pro-Agm。HPLC:梯度(I)R.t.20.6分钟;纯度:85%。B)从A)化合物(0.5g,0.74mmol)开始,按照实施例1H)-I)所述方法进行,得到0.169g标题产物。HPLC:(柱温度:60℃);梯度(II)R.t.13.87分钟;纯度>99%。FAB-MS:m/z-441amu[M+H]+。1H-NMR(200MHz;DMSO):t(1H;NH-Agm)7.98;m(1H;Cα-Lys)5.26;m(1H;Cα-Pro)4.15;m(10H;Cα-Gly,Cα-Agm,Cδ-Pro,Cδ-Agm and CH2-Et)3.65÷2.91;m(2H;Cε--Lys)2.66;m(14H;Cβ-and Cγ-Lys,-Pro and-Agm,and Cδ-Lys)2,18÷1,16;s(9H;CH3COO-)1,76;t(3H;CH3-Et)0.99.
实施例14H-Gly-(Et)Lys-Pro-OH·2AcOH
从实施例12C)化合物(0.2g,0.347mmol)开始,按照实施例1I)所述方法进行,得到0.085g标题产物。HPLC:(柱温度:60℃);梯度(II)R.t.9.32分钟;纯度>99%。FAB-MS:m/z-329amu[M+H]+。1H-NMR(200MHz;DMSO):m(1H;Cα-Lys)5.16;m(1H;Cα-Pro)4.05;m(6H;Cα-Gly and Cδ-Pro and CH2-Et)3.87÷2.92;m(2H;Cε-Lys)2.67;m(4H;Cβ-and Cγ-Pro)2.10÷1.65;s(6H;CH3COO-)1,78;m(6H;Cβ+γ+δ-Lys)1,59÷1.24;t(1.3H;CH3-Et)1.06;t(1.7H;CH3-Et)1,02.
实施例15H-Leu-(H)Lys-Pro-Arg-OH·2AcOH
A)氮气下将实施例12A)化合物(0.732g,1.5mmol)溶解于8ml乙腈,然后加入0.733ml(3mmol)BSA,1.6g(6mmol)Z-Leu-F(L.A.Carpino,E.M.E.Mansour,D.Sadat-Aalaee,J.Org.Chem.,1991,56,2611-2614)和溶解于2ml乙腈的0.094g(0.3mmol)TBAF。室温搅拌反应混合物240分钟。真空蒸发溶剂,剩余物溶解于乙酸乙酯,用5%碳酸氢钠和2.5%硫酸氢钾洗涤至中性。有机相脱水并真空蒸发,剩余物用Lobar LiChroprep Si60柱,40-63mm,(44×3.7cm),通过硅胶色谱纯化,硅胶柱预先在己烷/乙酸乙酯(7/3v/v)中平衡。该柱用同样的混合物以16ml/分钟洗脱。收集含有纯产物的级分并真空蒸发溶剂,得到0.5gZ-Leu-(Et)Lys(Boc)-Pro-OtBu。HPLC:梯度(I)R.t.31.5分钟;纯度:94%。B)将A)化合物(0.5g,0.74mmol)用实施例12C)-D)所述方法处理,得到0.43gZ-Leu-(Et)Lys(Boc)-Pro-OH。HPLC:梯度(I)R.t.31.5分钟;纯度>98%。C)将B)化合物(0.43g,0.695mmol)溶解于9ml1,2-二甲氧基乙烷并加入N-羟基琥珀酰亚胺(0.128g,1.112mmol),冷却至-20℃后再加入DCC(0.215g,1.042mmol)。15分钟后过滤反应混合物,在所得溶液中加入溶解于21ml0.15MDMF/KCl水溶液(2/1v/v)的H-Arg-OH。室温搅拌反应混合物110分钟。真空蒸发溶剂,将剩余物多次溶解于绝对乙醇,过滤,得到0.455gZ-Leu-(Et)Lys(Boc)-Pro-Arg-OH。HPLC:梯度(I)R.t.24.38分钟;纯度:84%。D)C)化合物用实施例1H)所述方法处理,得到Z-Leu-(Et)Lys-Pro-Arg-OH·TFA(ITF1929)。HPLC:梯度(I)R.t.17.63分钟;纯度:98%。FAB-MS:m/z-676amu[M+H]+.1H-NMR(200MHz;DMSO):t(1H;NHε-Arg)7.77;d(1H;NH-Leu)7.68;m(5H;CH-aryl)7.37;d(1H;NHα-Arg)7.34;m(1H;Cα-Lys)5.15;s(2H;CH2-z)5.03;m(1H;Cα-Leu)4.42;m(1H;Cα-Pro)4.26;m(1H;Cα-Arg)3,99;m(4H;Cδ-Pro and CH2-Et)3.68÷3.18;m(2H;Cδ-Arg)3.11;m(2H;CHε-Lys)2,74;m(17H;Cβ and Cγ-Leu,-Lys,-Pro and-Arg,and Cδ-Lys)2,18÷1,19;t(3H;CH3-Et)1,12;d(6H;CH3-Leu)0,89.E)D)化合物(0.341g,0.44mmol)用实施例1I)所述方法处理,得到0.06g标题产物。HPLC:梯度(II)R.t.29.13分钟;纯度:97%。FAB-MS:m/z-541amu[M+H]+.1H-NMR(200MHz;DMSO):d(1H;NH-Arg)7.10;m(1H;Cα--Lys)5.20;m(1H;Cα-Pro)4.19;m(1H;Cα-Arg)3.81;m(5H;Cα-Lys,CH2-Et and Cδ-Pro)3.66÷3,10;m(2H;Cδ-Arg)3.03;m(2H;Cε-Lys)2.74;m(17H;CβandCγ-Leu,-Lys,-Pro and-Arg,and Cδ-Lys)2.10÷1,18;s(6H;CH3COO-)1,77;t(3H;CH3-Et)1.03;d(3H;CH3-Leu)0,89;d(3H;CH3-Leu)0,87.
实施例16H-Gly-(isoBu)Lys-Pro-Arg-OH·2AcOH
A)将实施例9B)化合物(2.38g,5.95mmol)溶解于34ml甲醇并加入20.4ml乙酸。将反应混合物冷却至-20℃,滴加异丁醛(1.36ml,14.87mmol)后加入氰基硼氢化钠(0.748g,11.9mmol)。室温搅拌反应混合物100分钟。真空蒸发溶剂,剩余物溶解于乙酸乙酯,用5%碳酸钠,pH2.5的HCl和水洗涤至中性。有机相脱水并真空蒸发,得到2.65gH-(isoBu)Lys(Boc)-Pro-OtBu。HPLC:梯度(I)R.t.22.85分钟;纯度:97%。B)以A)化合物(2.2g,4.8mmol)和Z-Gly-F(4.2g,20mmol)为起始原料,基本上按实施例15A)-D)操作,得到0.2g标题产物。HPLC:梯度(II)R.t.12.58分钟;纯度>98%。FAB-MS:m/z-513amu[M+H]+。1H-NMR(200MHz;DMSO):d(1H;NH-Arg)7.09;m(1H;Cα-Lys)5.25;m(0.3H;Cα-Pro)4.43;m(0.7H;Cα-Pro)4.16;m(3H;Cα-Arg and Cδ-Pro)3.83÷3.56;m(2H;Cα-Gly)3.38;m(4H;CH2-iBu and Cδ-Arg)3.09÷2.93;m(2H;Cε-Lys)2.75;m(15H;Cβ-and Cγ-Lys,-Pro and-Arg,Cδ-Lys and CH-iBu)2.13÷1.16;s(6H;CH3COO-)1.79;d(2.1H;CH3-iBu)0,81;d(3H;CH3-iBu)0,76;d(0,9H;CH3-iBu)0,69.
实施例17H-Gly-(isoBu)Lys-Pro-OH·AcOH
从实施例16A)得到的化合物(0.455g,1mmol)和Z-Gly氟化物(0.84g,4mmol)开始,基本上按实施例15A)和10B)操作,得到0.172g标题产物。HPLC:(柱温度:60℃);梯度(II)R.t.23.48分钟;纯度>98%。FAB-MS:m/z-357amu[M+H]+。1H-NMR(200MHz;DMSO):m(1H;Cα-Lys)5.13;m(0.3H;Cα-Pro)4.30;m(0.7H;Cα-Pro)4.00;m(4H;Cα-Glyand Cδ-Pro)3.68÷3.31;m(2H;CH2-iBu)3.03;m(2H;C-Lys)2.81÷2.61;m(11H;Cβ and Cγ-Lys and-Pro,Cδ-Lys and CH-iBu)2.05÷1,24;s(3H;CH3COO-)1,86;d(2.1H;CH3-iBu)0.82;d(3H;CH3-iBu)0,77;d(0.9H;CH3-iBu)0,69.
实施例18H-Gly-(Bzl)Lys-Pro-Arg-OH·2AcOH
从实施例9B)化合物(1.36g,3mmol)和苯甲醛(0.546ml,5.4mmol)开始,基本上按实施例16A)-B)方法进行,得到0.395g标题产物。HPLC:梯度(II)R.t.27.85分钟;纯度>99%。FAB-MS:m/z-546amu[M+H]+.1H-NMR(200MHz;DMSO):m(6H;CH-aryl and NH-Arg)7.39÷6,97;m(0.7H;Cα-Lys)5.42;m(0.3H;Cα-Lys)4.93;m(2H;Cα-Pro and Cα-Arg)4.65÷4.42;m(4H;Cα-Gly and Cδ-Pro)4.00÷3.43;m(2H;CH2-aryl)3.30;m(2H;Cδ-Arg)3.03;m(2H;Cε-Lys)2.75;m(14H;Cβ-and Cγ-Lys,-Pro and-Arg,and Cδ-Lys)2,29÷1,14;s(6H;CH3COO-)1,79.
实施例19H-Gly-Pro-Pro-Arg-OH·AcOH
A)以H-Pro-OtBu·HCl(0.315g,1.5mmol)和Z-Pro-OH(0.397g,1.59mmol)为起始原料,基本上按实施例1C)-D)方法进行,得到0.4gH-Pro-Pro-OtBu。HPLC:梯度(I)R.t.11.38分钟;纯度:93%。B)以Z-Gly-OH(0.333g,1.59mmol)和A)化合物(0.4g,1.5mmol)为起始原料,基本上按实施例1G)-H)方法进行,得到0.6gZ-Gly-Pro-Pro-OH。HPLC:梯度(I)R.t.14.81分钟;纯度>98%。C)基本上按实施例15C)-D)所述方法处理B)化合物,得到0.045g标题产物。HPLC:(柱温度:60℃);梯度(II)R.t.12.06分钟;纯度>98%。FAB-MS:m/z-426amu[M+H]+.1H-NMR(200MHz;DMSO):d(1H;NH-Arg)7.34;m(0.2H;Cα-Pro)4.73;m(0.8H;Cα-Pro)4.61;m(0.2H;Cα-Pro)4.39;m(0.8H;Cα-Pro)4.33;m(7H;Cα-Arg and-Gly,and 2Cδ-Pro)3.83÷3.26;m(2H;Cδ-Arg)3.00;m(12H;Cβ-and Cγ-Pro and -Arg)2,22÷1,26;s(3H;CH3COO-)1,83.
本发明化合物表现出具有免疫调节,心血管和抗炎活性。特别是,它们表现出可用作治疗败血症休克治疗剂。该活性可以通过下列药理试验确定。
BALB/C雌鼠,重量20-22g,以1mg/鼠用LPS(脂多糖血清型O127:B8-Sigma)的0.5ml生理溶液腹膜内接种。然后将它们分成组,每组10只,LPS施用20和120分钟后每组都用同样的本发明代表性化合物以62.5μg/鼠腹膜内接种,有一组只用LPS处理作为对照。
对这些动物至少监视4天。结果示于下列表中。表实施例 存活百分比(对照组) 3.5
2 23
4 40
5 23
14 38
19 23
本发明化合物的心血管活性通过试验进行了研究,试验的目的在于评价麻醉后的大鼠左冠状动脉闭塞引起局部缺血后的心脏保护活性,基本上按C.Clerk等人在.Journ.Exp.Methods,3,357,1980中所述进行。众所周知,冠状动脉闭塞造成许多改变ECG-模型的情况,例如包括缺氧,心律失常,最后实验动物死亡。
本试验中,雄性Charles River大鼠被分成12只一组,用戊巴比妥(65mg/kgi.p.)麻醉并与心电图仪连接连续监测ECG。随后,对动物实行胸廓切开手术,心包膜切开之后,用缝合线在其起点附近缠绕左冠状动脉。恢复10分钟后,如果心电图没有变化,给动物静脉注射预定剂量的溶解于或悬浮于盐水中的本发明化合物。对照组动物仅静脉注射盐水。5分钟后结扎左冠状动脉并保持结扎30分钟。
在代表性实验中,即使剂量为0.4μg/kg也能观察到心室性心博过速(VT),心室纤维性颤动(VF)和死亡率的明显降低。特别是发现实施例4的化合物当以3μg/kg给药时,发生在结扎后头18分钟的死亡率比对照组降低约80%,而VT和VF降低约50%。
本发明的目的还在于使用该新寡肽作为免疫调节剂及治疗心血管疾病和发炎病理疾病。涉及与所说用途有关的实用方面还包括它们的药物组合物。这些药物组合物的实例有片剂,糖衣和膜衣片剂,糖浆和小瓶制剂,后者适于口服和肌肉或静脉内给药。这些组合物或只含活性物质或含有活性物质与常规药物上可接受的载体及赋形剂组合。
活性物质的剂量可以在很宽的范围内变化,这取决于所用化合物根据治疗需要是一天给药一次还是多次。
Claims (10)
1.式(I)的寡肽及其与药物上可接受的酸或碱形成的盐,
A1-A2-A3-A4 (I)其中:A1为选自甘氨酸,苏氨酸,亮氨酸,异亮氨酸,缬氨酸,肌氨酸,丙氨酸,(C2-6)酰基-甘氨酸的氨基酸残基或不存在;A2为选自至少被一个(C1-6)烷基,苄基或(C2-6)酰基选择性Nα-取代的亮氨酸,异亮氨酸,缬氨酸,赖氨酸,鸟氨酸的氨基酸残基,或是脯氨酸残基或不存在;A3为选自脯氨酸,亮氨酸,异亮氨酸和缬氨酸的氨基酸残基;A4为选自任意在C-端位选择性酰胺化的精氨酸,亮氨酸和谷氨酰胺的氨基酸残基,或是胍基丁胺残基,或不存在;条件是只能不存在A1,A2和A4其中一个;所说化合物进一步的特性在于上述氨基酸残基和胍基丁胺残基的侧链基团可以被选自(C1-6)烷基,苄基或(C2-6)酰基的一个或多个取代基选择性取代;而且每个所说氨基酸残基可在带有侧链的碳原子上以D-或L-形式存在,或以可能的非对映体或对映体中的一种形式存在。
2.权利要求1定义的寡肽,其中A1为选自甘氨酸,肌氨酸,丙氨酸,(C2-6)酰基-甘氨酸的氨基酸残基或不存在;A2为氨基酸残基,选自赖氨酸和被一个(C1-6)烷基,苄基或(C2-6)酰基选择性Nα-取代的赖氨酸;A3为脯氨酸;A4为精氨酸,在C-端位选择性酰胺化的精氨酸,或胍基丁胺残基;所说化合物进一步的特性在于所说氨基酸残基和胍基丁胺残基的侧链基团可以被选自(C1-6)烷基,苄基或(C2-6)酰基的一个或多个取代基选择性取代;每个所说氨基酸残基可在Cα上以D或L形式存在,或以可能的非对映体或对映体中的一种形式存在;及其与药物上可接受的酸或碱形成的盐。
3.权利要求1定义的寡肽,其中A1甘氨酸,A2是Nα-乙基-赖氨酸,A3是脯氨酸及A4是精氨酸,及其与药物上可接受的酸或碱形成的盐。
4.权利要求1定义的寡肽,其中A1不存在,A2是Nα-乙酰基-赖氨酸,A3是脯氨酸及A4是精氨酸,及其与药物上可接受的酸或碱形成的盐。
5.权利要求1定义的寡肽,其中A1是甘氨酸,A2是Nα-乙基-赖氨酸,A3是脯氨酸及A4不存在,及其与药物上可接受的酸或碱形成的盐。
6.权利要求1-5的化合物,作为治疗剂。
7.根据权利要求6的化合物,作为心血管治疗剂。
8.根据权利要求6的化合物,作为消炎剂。
9.根据权利要求6的化合物,作为治疗败血症休克的治疗剂。
10.药物组合物,含有权利要求1的化合物和药物上可接受的赋形剂。
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1994
- 1994-05-16 PT PT94916966T patent/PT723552E/pt unknown
- 1994-05-16 CA CA002173939A patent/CA2173939C/en not_active Expired - Fee Related
- 1994-05-16 JP JP50541495A patent/JP3221881B2/ja not_active Expired - Fee Related
- 1994-05-16 CN CN94193726A patent/CN1041524C/zh not_active Expired - Fee Related
- 1994-05-16 AU AU68441/94A patent/AU684511B2/en not_active Ceased
- 1994-05-16 DK DK94916966T patent/DK0723552T3/da active
- 1994-05-16 HU HU9600934A patent/HUT75538A/hu unknown
- 1994-05-16 US US08/624,405 patent/US6057295A/en not_active Expired - Fee Related
- 1994-05-16 AT AT94916966T patent/ATE226591T1/de not_active IP Right Cessation
- 1994-05-16 WO PCT/EP1994/001574 patent/WO1995010531A1/en active IP Right Grant
- 1994-05-16 DE DE69431603T patent/DE69431603T2/de not_active Expired - Fee Related
- 1994-05-16 ES ES94916966T patent/ES2185652T3/es not_active Expired - Lifetime
- 1994-05-16 EP EP94916966A patent/EP0723552B1/en not_active Expired - Lifetime
- 1994-05-16 KR KR1019960701879A patent/KR100363408B1/ko not_active IP Right Cessation
- 1994-05-24 ZA ZA943610A patent/ZA943610B/xx unknown
- 1994-05-24 IL IL10976194A patent/IL109761A0/xx unknown
-
1996
- 1996-04-11 NO NO961423A patent/NO961423L/no unknown
- 1996-04-11 FI FI961584A patent/FI961584A/fi not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
HU9600934D0 (en) | 1996-08-28 |
AU6844194A (en) | 1995-05-04 |
NO961423L (no) | 1996-06-10 |
WO1995010531A1 (en) | 1995-04-20 |
DE69431603T2 (de) | 2003-03-13 |
JPH09503200A (ja) | 1997-03-31 |
EP0723552A1 (en) | 1996-07-31 |
CN1041524C (zh) | 1999-01-06 |
DK0723552T3 (da) | 2003-02-24 |
US6057295A (en) | 2000-05-02 |
FI961584A0 (fi) | 1996-04-11 |
HUT75538A (en) | 1997-05-28 |
IT1271486B (it) | 1997-05-28 |
FI961584A (fi) | 1996-04-11 |
CA2173939C (en) | 2002-08-27 |
NO961423D0 (no) | 1996-04-11 |
ITMI932154A0 (it) | 1993-10-12 |
CA2173939A1 (en) | 1995-04-20 |
DE69431603D1 (de) | 2002-11-28 |
IL109761A0 (en) | 1994-08-26 |
ZA943610B (en) | 1995-01-25 |
ATE226591T1 (de) | 2002-11-15 |
PT723552E (pt) | 2003-02-28 |
EP0723552B1 (en) | 2002-10-23 |
KR100363408B1 (ko) | 2003-02-11 |
ITMI932154A1 (it) | 1995-04-12 |
JP3221881B2 (ja) | 2001-10-22 |
ES2185652T3 (es) | 2003-05-01 |
AU684511B2 (en) | 1997-12-18 |
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