CN1030255C - 环形生长激素释放因子(grf)类似物的制备方法 - Google Patents
环形生长激素释放因子(grf)类似物的制备方法 Download PDFInfo
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Abstract
本发明公开了有关线性和环形生长激素释放因子类以物,以及将有效量的本发明的化合物施用于患者,刺激其生长激素释放的方法。
Description
动物的生长是由一联串生物调节分子调节的。下丘脑产生一种叫做生长激素释放因子(GRF)的物质,该物质再作用于垂体,引起生长激素释放。垂体是通过生长激素释放抑制因子和胰岛素生长因子(IGF)保持负反馈控制。已经发现,GRF极为活跃,能促进血液中生长激素以每毫升微克的水平释放。在大多数领域中,GRF可用于治疗目的,可取代生长激素的应用,如:可用来治疗由于异常生长激素的产生而引起的垂体性矮小症和糖尿病,加速伤口愈合,治疗烧伤或延缓衰老过程。
GRF的成功分离部分基于下述发现,即:与肢端肥大症有关的腺瘤异位产生大量的GRF。已经分离出三种形式的GRF,它们具有同源氨基酸顺序,但长度不同(44.40.37个氨基酸)。
具有44个氨基酸的酰胺化型GRF被认为是母体分子。已生产了多种合成类似物。它们包括多肽或其具有各种氨基酸取代物的类似物。所述多肽具有与最初分离的一种GRF或有生物活性的一段片段相同的氨基酸顺序。已专门设计了若干改变形式,以获得其生物性能比母体分子生物性能更为优越的合成类似物。因此,希望设计出具有如下特点的类似物,即:在效力、有效性和稳定性等方面均具有最高的生物活性。
至今为止,所有已知的GRF类似物均是线性构型的。一般而言,线性肽是具有挠性的分子,缺乏十分确定的构象。当线性肽中的每个氨基酸暴露在周围环境下时,它对酶和化学降解的敏感度更大。
所谓环形肽(内酰胺)是这样的一种肽,即:其酸性氨基酸(如Asp或Glu)的侧链羧基末端经过所产生的酰胺与碱性氨基酸(如Lys)的
侧链氨基末端连接。
环肽与其线性类似物相比,其生物性质通常可改变。环肽更具挠性,有十分确定的构形和内氨基酸残基,从而与周围环境隔离。这些不同之处反映在肽的生物性质中。由于环肽具密实结构,使其对化学和酶降解不太敏感,因此作用时间更长。被隔离的内氨基酸残基引起组织分布的改变,从而提高了环肽的生物利用率。此外,环肽的十分确定的构象,使其对靶受体具有更好的专一性,由此减少了产生不良生物副作用的可能性。就线性肽而言,具有中央和***受体的特定线性肽一般具有交叉活性,而具有受体的特定肽对另一种肽同样具有相当大的交叉活性。
本发明涉及具有上述特定氨基酸顺序的GRF线性类似物和环形类似物,包括其药物上可接受的盐。
本发明还涉及一种促进受治疗者生长激素释放的方法,该方法包括:将有效量的本发明化合物施用于受治疗者。
本说明书中所采用的符号和术语的定义如下:
1.环形肽:或内酰胺,指一种肽,由酸性氨基酸(如:Asp或Glu)的侧链羧基末端通过酰胺键(内酰胺)与碱性氨基酸(如:Lys)的侧链氨基末端连接。
2.8 12 指肽链中的第八个氨基酸“A”与链中的第
构。
3.GRF:指人体生长激素释放因子,具有氨基酸顺序的多肽,其氨基酸顺序为:
5
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-
10 15
Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-
20 25
Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-
30 35
Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-
40
Gly-Ala-Arg-Ala-Arg-Leu-NH2
4.[Leu27]GRF:指具有与GRF相应氨基酸顺序的多肽,其中甲硫氨酸残基在27位被亮氨酸残基取代。这类GRF类似物一般由GRF前面括号中的取代氨基酸表示。
5.GRF(1-29):指具有完整的开始29个氨基酸顺序的GRF肽片段。一般情况下,GRF后括号中的数字相当于具有原来GRF顺序的氨基酸位置,表示片段的N末端和C末端。
6.脱NH2Tyr′:指在位置1从酪氨酸残基脱除的氨基末端NH2。
7.OcHex:指环己基。
8.DIEA:指二异丙基乙胺。
9.DTE:指二硫乙烷。
图1表示本发明化合物促进鼠垂体细胞中生长激素释放的效果。将两种垂体(“pit”)腺(1∶4稀释)浸在过滤测定器的各滤片上;该测定器通常测试生物活性。采用下列专用缩写:
“rGH”表示重组长生激素,“[A15环8-12]GRF(1-29)-NH2”表示环8,12[Asp8,Ala15]-GRF(1-29)-NH2;“环(8-12)[D-Ala 2 Ala 15]GRF(1-29)-NH2”表示环8,12[D-Ala2,Asp8,Ala15]-GRF(1-29)-NH2,“环(8-12)[脱NH2Tyr 1 D-A2 A15](1-29)-NH2”表示环8,12[脱NH2Tyr′,D-Ala2,Asp8,Ala15]-GRF(1-29)-NH2以及“环(8-12)[脱NH2Tyr 1A 15](1-29)-NH2”表示环8,12[脱NH2Tyr1,Asp8,Ala15]-GRF(1-29)-NH2。
本发明包括下式所示的环肽及其药物上可接受的盐:
R1-R2-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-R3-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-R4-R5-R6-x
式中:
R1=Tyr,脱NH2-Tyr,Ac-Tyr,His,
N-甲基-L-Tyr
R2=Ala,D-Ala,N-甲基-D-Ala
R3=Gly,Ala,Leu,Val,Ile,Nle,NVal,β-Ala,
α-Aib
R4=Met,Leu,Nle,Ile
R5=Ser,Asn
R6=氨基酸顺序,选自
Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu或其片段,其中从羧基末端开始片段减少1-15个氨基酸
X=OH、NH2、N(R7)(R8),其中R7和R8=H或低级烷基
A=Asp,Glu,α-氨基庚二酸,α-氨基己二酸
B=Lys,Orn,二氨基丙酸、二氨基丁酸
其中A的侧链羧基末端通过酰胺键同B的侧链氨基末端连接。
较理想的是,式Ⅰ肽中的R1=Tyr、脱NH2Tyr、N-甲基-L-Tyr;R2=Ala、D-Ala;R3=Ala;X=NH2。
更为理想的是,上述式Ⅰ肽中的A=Asp;B=Lys(并且Asp的侧链羧基末端通过酰胺键与Lys的侧链氨基末端共价连接);R4=Met;R5=Ser;R6=Arg。
尤其理想的是,肽中的R1=Tyr,R2=Ala,R3=Ala,其结构式为:
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2.
同样特别理想的肽是其中R2=D-Ala的肽,其结构式为:
Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2.
同样理想的肽是式Ⅰ中R1=脱NH2Tyr的肽。
特别理想的是其中R2=Ala的一类肽,其结构式如下:
脱NH2-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2.
同样特别理想的肽是其中R2=D-Ala的一类肽,其结构式如下:
脱NH2-Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2.
本发明还包括上面特别提到的环肽的线性类似物。
可采用适宜的方法,如采用全部固相合成法、部分固相合成法、片段缩合或经典溶液合成等方法来合成肽。重组DNA技术也可用于仅含天然氨基酸残基的类似物。较为可取的是,按照Merrifield在J.Am.Chem.Soc.85,2149(1963)中所述固相肽合成法,制备本发明的肽。在氨基酸α-氨基末端被保护的情况下,进行合成。同样,用适宜的基团对带有不稳定侧链的三官能氨基酸进行保护,可防止肽集合过程中在不稳定侧链发生化学反应。有选择地脱除α-氨基保护基,以便随后在氨基末端进行偶联反应。脱除α-氨基保护基所用的反应条件不会导致侧链保护基脱去保护。
α-氨基保护基是分步合成肽的技术中公知的一类基团,包括酰基型保护基(如:甲酰基、三氟乙酰基、乙酰基),芳族尿烷型保护基(如:苄氧基羰基(Cbz)和取代的苄氧羰基)、脂族尿烷型保护基(如:叔丁氧基羰基(Boc)、异丙氧基羰基、环己氧基羰基)和烷基型保护基(如:苄基、三苯基甲基)。优选的保护基是Boc。Tyr侧链保护基包括四氢
吡喃基、叔丁基、三苯甲基、苄基、Cbz、4-Br-Cbz和2,6-二氯苄基。Tyr的优选侧链保护基是2,6-二氯苄基。Asp侧链保护基包括苄基、2,6-二氯苄基、甲基、乙基和环己基。Asp的优选侧链保护基是环己基。Thr和Ser的侧链保护基包括乙酰基、苯甲酰基、三苯甲基、四氢吡喃基、苄基、2,6-二氯苄基和Cbz。Thr和Ser的优选保护基是苄基。Arg侧链保护基包括硝基、甲苯磺酰基(Tos)、Cbz、金刚烷氧基羰基或Boc。Arg的优选保护基是Tos。可用Cbz、2-Cl-Cbz、Tos或Boc对Lys侧链氨基进行保护。2-Cl-Cbz是Lys的优选保护基。侧链保护基的选择基于以下因素,即:在偶联过程中侧链保护基保持完整,并且在脱去氨基末端保护基时或在偶联反应条件下不会***。在不改变所述肽的反应条件下,于最终肽的合成结束后,所述侧链保护基必须是可脱除的。
通常使α-氨基已保护的(侧链保护)氨基酸与适宜的固体载体连接,从待合成的肽的羧基末端进行固相合成。当氨基酸与氯甲基化或羟甲基树脂连接时,形成酯键,并且所得的靶肽在C-端带有游离羧基。另外,采用二苯甲基胺或对甲基二苯甲基胺树脂。在此情况下,形成酰胺键,所得的靶肽在C-端带有羧酰胺基。这些树脂是市售的,其制备方法如Stewart等人在“固相肽合成”(2nd Edition,Pierce Chemical Co.,Rockford,IL.1984)中所述。
C-末端氨基酸(在侧链用Tos保护,在α-氨基功能团用Boc保护)为Arg时,采用包括二环己基碳化二亚胺(DCC),N,N′-二异丙基碳化二亚胺或羰基二咪唑在内的各种激活剂,可将所述氨基酸与二苯甲基胺树脂连接。与树脂载体连接后,利用三氟乙酸(TFA)或HCl,在二噁烷中,于0℃至25℃,脱除α-氨基保护基。引入甲硫氨酸(Met)后,将二甲硫加到TFA中,以抑制可能产生的S-烷基化。脱去α-氨基保护基后,按所要求的规程,将余下的已保护氨基酸分步连接,以获得所需的肽顺
序。可用于该偶联反应的各种激活剂包括DCC、N,N-二异丙基碳化二亚胺、(苯并***-1-基-氧)[三(二甲基氨基)]六氟磷酸鏻(BOP)和DCC-羟基苯并***(HOBt)。过量(>2.5当量)地使用各保护氨基酸,通常在二甲基甲酰胺(DMF)、CH2Cl2或它们的混合物中进行偶联反应。根据Kaiser等人在Anal.Biochem.,34,595(1970)中所述,通过水合茚三酮反应,监测偶联反应每一步完成的程度。若偶联反应不完全,则重复进行偶联反应。可采用Vega 250一类的合成仪,应用生物***合成仪或其他市售设备,自动进行偶联反应。表1给出本发明的肽合成中一个典型的合成循环步骤。
表1 一个典型的合成循环步骤a
步骤 试剂 时间
1 1%DMS/CH2Cl21×1min
2 50%TFA/CH2Cl2+1%DMS(v/v) 1×1min
3 1%DMS/CH2Cl21×1min
4 50%TFA/CH2Cl2+1%DMS(v/v) 1×20min
5 CH2Cl23×1minb
6 10%DIEA/CH2Cl21×5min
7 CH2Cl22×1min
8 重复步骤6,7
9 MeOH 2×1min
10 CH2Cl23×1min
11a 2.5 eq.Boc-AA-COOH/CH2Cl25minC
b 2.5 eq.DCC/CH2Cl260min
c 1.5% DIEA/CH2Cl215min
12 重复步骤10,11
13 CH2Cl22×1min
14 MeOH 1×1min
15 CH2Cl21×1minb
a 测得洗涤及偶联反应用溶剂的体积达15-20ml/g树脂
b 开氏(Kaiser)水合茚三酮试验
c Boc-Arg(Tos)-OH用的DMF/CH2Cl2
将所要合成的肽全部集合后,在0℃下,使肽-树脂先后同TFA/二硫乙烷和诸如液态HF试剂反应1-2小时,用液态HF将肽从树脂上切下,除去所有的侧链保护基。
在固态载体上进行侧链至侧链的环化需要采用正交保护法。通过这种方法,可以有选择地将酸性氨基酸(如Asp)的侧链官能团同碱性氨基酸(如Lys)的侧链官能团分开。为此,Asp侧链可采用9-芴甲基OFm保护基,Lys侧链可采用9-芴甲氧基羰基(Fmoc)保护基。在这些情况下,于含哌啶的DMF中,可选择性地除去Boc保护的肽-树脂侧链保护基(OFm和Fmoc)。应用各种活化剂,包括DCC、DCC/HOBt或BOP,可在固体载体上实现环化。HF反应在上述经过环化的肽-树脂上进行。
可采用肽化学中众所周知的方法,将本发明的多肽纯化。可采用高压液相色谱(HPLC)法,提纯本发明的多肽,然而,还可利用其他公知的色谱法,如凝胶渗透法,离子交换法和分配色谱法或逆流分配法进行纯化。
本发明还包括如下方法,即:将有效量的式Ⅰ肽施用于受治疗者,以促进其体内生长激素的释放。
本发明的多肽具有生长激素释放的活性。本发明的药物组合物包括分散在药物上或兽用药物上可接受的液态或固态载体中的长度约为29至44个氨基酸的类似物或这些类似物的任何无毒性的盐。上述药物组合物可用于人体或兽类治疗或诊断药物。例如,它们可用来治疗与生长有关的一类疾病,如:由于产生异常的生长激素而引起的垂体性侏儒症和糖尿病。此外,它们可用来促进生长或提高饲养动物的进食率,以增加鲜肉,奶和蛋的产量。
本发明多肽的适宜施用剂量根据具体受治疗者而定,更具体地说,取决于其生长激素的缺乏程度和治疗的病情程度。根据与正常生长有关的已知的生长激素和本发明多肽的生长激素释放活性的循环水平,专业人员能够确定合适的剂量。具体地说,以受治疗者的体重计,可采用0.04μg/kg/天至20.0μg/kg/天的剂量,来促进生长激素的释放。用于促进牲畜生长活性的剂量显著高于用于治疗人体生长激素缺乏症(如垂体性侏儒症)的剂量(以受治疗者的每公斤体重计)。牲畜一般以0.4μg/kg/天至100μg/kg/天的剂量经皮下施用,便可促进垂体生长激素的释放。
本发明还提供了一种治疗以生长激素缺乏为特征以及与生长有关的一类疾病的方法。该方法包括施用有效量的本发明的类似物,促进生长激素产生,达到正常生长的水平。
生长激素的正常水平存在个体差异,就具体个人而言,生长激素循环水平在一天内变化也很大。据报道,成人生长激素的正常血清水平约在0~10毫微克/毫升内变化,儿童生长激素的正常血清水平大约在0~20毫微克/毫升内变化。
为利用上述类似物有效地治疗由垂体机能不良引起的侏儒症,在正常生长时期进行治疗。女性治疗期一般不要距始经期太晚。因此,根据个人的具体情况,女患者的治疗应在约12至16岁时进行。男性在***过后的较长一段时间里,仍有可能促进生长。因此,男患者的有效治疗期可延至18至19岁,在某些情况下,可延至25岁。
本发明还提供了一种加快动物生长速度的方法。即:施用有效量的类似物,促进生长激素以高于正常生长的水平释放。
可以以人体或兽用的药物组合物的形式施用本发明的多肽。所述的药物组合物可通过常规制剂技术制备。可采用适于口服、静脉内、皮下、肌内、腹膜内、鼻内或经皮用药的组合物形式。药用的适宜剂型中含大
约0.01至0.5mg的本发明化合物,可将其冻干并与无菌水或生理盐水再组合。应将该组合物维持在约pH5.0以下,以保持类似物的稳定性。还可将取自受治疗者的血清白蛋白(例如人的血清白蛋白和奶牛牛血清白蛋白等)与其他公知的佐药一起使用。
结合以下仅用于说明的实施例,描述本发明。
实施例1
环8,12[Asp8,Ala15]-GRF(1-29)-NH2的合成
Boc-Arg(Tos)-二苯甲基胺-树脂的制备
各用900mlCH2Cl2、MeOH、CH2Cl2、含25%Et3N的CH2Cl2(3倍)、CH2Cl2、MeOH和CH2Cl2洗涤二苯甲基胺-树脂(60g,0.7meq/g,42meq)。加入含BOC-Arg(Tos)-OH(35.95g,84mmol,2eq)的DMF(80ml)-CH2Cl2(600ml),将该混合物摇振5分钟,加入DCC(17.3g,84mmol,2eq),反应24小时。用DMF、CH2Cl2、MeOH和CH2Cl2对所得到的Boc-Arg(Tos)-BHA-树脂进行洗涤。对等分液的氨基酸进行分析,结果表明:取代程度为0.38mmol/g。于25℃,用300ml 50%的Ac2O吡啶将树脂乙酰化2小时,用CH2Cl2、MeOH、CH2Cl2洗涤,真空干燥,得到70g Boc-Arg(Tos)-BHA-树脂(取代程度:0.6meq/g)。
[Asp8(OFm)、Lys12(Fmoc),Ala15]-GRF(1-29)-
二苯甲基胺-树脂的制备
按表1给出的步骤,将Boc-Arg(Tos)-BHA-树脂(70g,0.6meq/g,42meq)脱去保护,并中和。三功能团氨基的保护如下:Boc-Arg(Tos)、Boc-Asp(OcHex)、Boc-Glu-(OBzl)、Boc-Lys(2Cl-Z)、Boc-Ser(Bzl)、Boc-Thr(Bzl)、Boc-Tyr-(2,6-Cl2Bzl)、Boc-Asp8(OFm)和Boc-Lys12(Fmoc)。所述步骤的例外情况是Boc-Gln-OH(24位和16位)的偶联以及紧接Gln(23位和15位)后的偶联,其中于DMF中采用3当量对称酐(双联)。在需要进行第三偶联的特殊例子中,采用各为2.5当量的Boc-氨
基酸、1-羧基苯并***和DCC,在DMF(120ml)中预活化,制备HOBt酯,然后过滤(脱除二环己基脲),用甲苯稀释至1.2l。偶联反应进行了2小时后,加入1.5%二异丙基乙胺,再反应15分钟。
除去一部分中间体[Ala15]-GRF(13-29)-BHA-树脂(1.88g,0.714mmol),如上所述,继续分步进行固相合成。导入Lys12(Fmoc)后,用0.6%DIEA/CH2Cl2代替步骤6。通过用1-羟基苯并***(6.6当量)和DCC(6当量)预活化,用Boc-Asn-OH(6当量)接肽。
环8,12[Asp8,Ala15]-GRF(1-29)-NH2的制备
在Boc-[Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(1-29)-BHA-树脂(2.72g,0.714mmol)结合后,用20%哌啶/DMF将树脂脱去保护20分钟,得到Boc-[Asp8,Ala15]-GRF(1-29)-BHA-树脂。在含Et3N(157μl,1.12mmol,2.8eq)的DMF(40ml)中,与BOP试剂(443mg,1.0mmol,2.5eq)反应4小时,从而将一部分树脂(1.40g,0.40mmol)环化。洗涤后,再环化两次,时间分别为11小时和3小时(Kaiser水合茚三酮阴性试验);并对该肽-树脂进行洗涤、干燥,于0℃,用含DTE(1ml/g树脂)的HF(约20ml)将肽链从树脂上切下来,时间为2小时。将HF蒸发后,用EtOAc洗涤,以TFA(6×5ml)萃取,蒸发,用醚研制。
环8,12[Asp8,Ala15]-GRF(1-29)-NH2的纯化和鉴定
将粗制产物(708mg)悬浮于20mlH2O(0.025%TFA)中,搅拌,离心,过滤,利用Synchropak RP-P柱(20×50cm)进行提纯[洗脱条件:洗脱液(A)H2O(0.025%TFA)-(B) ACN(0.025%TFA);线性梯度20~45%(B),120分钟;流速4ml/分]。每隔1分钟收集馏分。将馏分82-89汇集起来,冻干,得到84mg半纯产物。从馏分90-93得到副产物(33mg)。利用Nucleosil C18柱(1.0×50cm;5μ)将半纯物(84mg)再提纯[洗脱条件:洗脱液(A)H2O(0.1%TFA)-(B)ACN(0.1%TFA);线性梯度20-40%(B),120分钟;流速3ml/分]。每隔1分钟收集馏
分。将馏分120-121汇集起来,冻干,得到均匀产物(9mg),由馏份122-138得到42mg产物,其纯度高于97%。
hplc分析表明,该产物是均匀的。氨基酸分析(6M HCl,110°,24小时):
Asp,2.72;Thr,0.96;Ser,2.98;Glu,2.14;Ala,4.00;Val,
0.96;Met,0.97;Ile,1.89;Leu,4.28;Tyr,2.01;Phe,
0.98;Lys,2.06;Arg,3.04,
实施例2
环8,12[脱NH2Tyr1,D-Ala2,Asp8,Ala15]-
GRF(1-29)-NH2的合成
除去一部分中间体[Ala15]-GRF(13-29)-BHA-树脂(5.1g,1.63mmol),按表1的步骤,继续进行分步固相合成。加入Lys12(Fmoc)后,用0.6%DIEA/CH2Cl2代替步骤6。继续进行合成,得到6.4g Boc-[Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(3-29)-BHA-树脂。将1.0g(0.255mmol)所得树脂与Boc-D-Ala-OH进行一个循环的固相合成,得到Boc-[D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(2-29)-BHA-树脂。将0.5所得树脂与脱NH2Tyr-OH进行最后一个循环的固相合成,得到[脱NH2Tyr1,D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(1-29)-BHA-树脂;用20%哌啶/DMF使所得树脂脱去保护20分钟。在含DIEA(0.3ml,2.2mmol)的DMF(20ml)中,用BOP试剂(170mg,0.384mmol,3eq)环化2小时。用新鲜的BOP试剂重复进行环化(Kaiser水合茚三酮阴性试验),(如上所述),将肽洗涤,干燥,于0℃下,用HF(~10ml),将肽从树脂上切下来,时间为2小时。蒸发HF,随后用EtOAc洗涤,用TFA萃取,蒸发,用醚研制,得到229mg粗制品。如上所述,用制备性hplc法对环8,12[Asp8,Ala15]-GRF(1-29)-NH2进行纯化。
实施例3
环8,12[脱NH2Tyr1,Asp8,Ala15]-GRF(1-29)-NH2的合成
将1.0g(0.255mmol)部分的Boc-[Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(3-29)-BHA-树脂先后与Boc-L-Ala-OH和脱NH2Tyr-OH进行两轮循环的固相合成,得到[脱NH2Tyr1,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(1-29)-BHA-树脂;用20%哌啶/DMF使所得树脂脱去保护20分钟,在含DIEA(0.3ml,2.2mmol)的DMF(20ml)中,用BOP试剂(170mg,0.384mmol,3eq)环化2小时。用新鲜的BOP试剂重复进行环化,Kaiser水合茚三酮阴性试验),并对肽进行洗涤,干燥,于0℃下用HF(~10ml)将肽从树脂上切下,所用时间为2小时。蒸发HF,随后用EtOAc洗涤,以TFA萃取,蒸发,用醚研制,得到450mg粗制产物。如前所述,经制备性hplc法,将环8,12[Asp8,Ala15]-GRF(1-29)-NH2纯化。
实施例4
环8,12[D-Ala2,Asp8,Ala15]-GRF(1-29)-NH2的合成
将0.5g取自例2的Boc-[D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(2-29)-BHA-树脂与Boc-Tyr[2,6-Cl2Bzl]-OH进行最终轮循环的固相合成,得到Boc-[D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(1-29)-BHA-树脂。用20%哌啶/DMF使所得树脂脱去保护20分钟,并用BOP试剂(170mg,0.384mmol,3eq)在含DIEA(0.3ml,2.2mmol)的DMF(20ml中环化2小时。(如上所述)用新鲜BOP试剂重复进行环化(开氏水合茚三酮阴性试验),并对肽进行洗涤,干燥,于0℃下,历时2小时,用HF(~10ml)将肽链从树脂上切下来。蒸发HF,继之以EtOAc洗涤,用TFA萃取,蒸发,用醚研制,得到198mg粗制产物。如上所述,经制备性hplc法,对环8,12[Asp8,Ala15]-GRF(1-29)-NH2进行纯化。
实施例5
环8,12[N-甲基-Tyr1,D-Ala2,Asp8,
Ala15]-GRF(1-29)-NH2的合成
将0.5gBoc-[D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(2-29)BHA-树脂(取自上例)与Boc-N-甲基-L-Tyr-(2,6-Cl2Bzl)-OH进行最终轮循环的固相合成,得到Boc-[N-甲基-L-Tyr′(2,6-Cl Bzl),D-Ala2,Asp8(OFm),Lys12(Fmoc),Ala15]-GRF(1-29)-BHA-树脂。用20%哌啶/DMF使所得树脂去保护20分钟,并用BOP试剂(170mg,0.384mmol,3eq)在含DIEA(0.103ml,0.765mmol,6eq)的DMF(5ml)中环化2小时。采用新鲜BOP试剂再进行两次环化(水合茚三酮阴性试验),并对肽进行洗涤,干燥,于0℃下用HF(~10ml)将肽从树脂上切下来(如上所述),时间为2小时。蒸发HF,继之以EtOAc洗涤,用TFA萃取,蒸发,用醚研制,得到219mg粗制产物。如上所述,经用hplc法,对环8,12[Asp8,Ala15]-GRF(1-29)-NH2进行纯化。
虽然结合优选实施例描述了本发明,但这并不意味着要将本发明的范围限制于所述特定形式,相反,意在复盖权利要求所定义的本发明的精神和范围内可能包含的变换、改进和等同物。
Claims (12)
1、一种制备下式所示的肽及其药物上可接受的盐的方法:
R1-R2-Asp-Ala-Ile-Phe-Thr-A-Ser-Tyr-Arg-B-Val-Leu-R3-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-R4-R5-R6-X
式中:
R1=Tyr,脱NH2-Tyr,
N-甲基-L-Tyr
R2=Ala,D-Ala,
R3=Ala,
R4=Met,
R5=Ser,
R6=氨基酸顺序,选自
Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Cly-Ala-Arg-Ala-Arg-Leu或其片段,其中片段从羧基末端开始减少1至15个氨基酸,
X=OH、NH2,
A=Asp,
B=Lys,
A的侧链羧基末端通过酰胺键与B的侧链氨基末端连结,该方法的特征在于:将已由固相肽合成法合成的、侧链已适当保护的树脂结合的相应氨基酸顺序的多肽同液体HF反应,需要时将这样的肽转化成药物上可接受的盐。
2、按权利要求1的方法,其中R′=Tyr、脱NH2-Tyr、N-甲基-L-Tyr,R2=Ala、D-Ala,R3=Ala,X=NH2。
3、按权利要求2的方法,其中A=Asp,B=Lys,并且Asp的侧链羧基末端经酰胺键与Lys的侧链氨基末端共价结合,R4=Met,R5=Ser,R6=Arg。
4、按权利要求3的方法,其中R1=Tyr。
5、按权利要求4的方法,其中R2=Ala,所述化合物的结构式如下:
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2。
6、按权利要求4的方法,其中R2=D-Ala,所述化合物的结构式如下:
Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2。
7、按权利要求3的方法,其中R1=脱NH2-Tyr。
8、按权利要求7的方法,其中R2=Ala,所述化合物的结构式如下:
脱
NH2-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asp-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2。
10、按权利要求3的方法,其中R1=N-甲基-L-Tyr。
11、按权利要求10的方法,其中R2=Ala,所述化合物的结构式如下:
N-甲 基
-L-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asp-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2。
12、按权利要求10的方法,其中R2=D-Ala,所述化合物的结构式如下:
N-甲 基 -L-Tyr-D-Ala-Asp-Ala-Ile-Phe-Thr-
-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2。
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DE (1) | DE3850015T2 (zh) |
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US5043322A (en) * | 1988-07-22 | 1991-08-27 | The Salk Institute For Biological Studies | Cyclic GRF analogs |
US5084442A (en) * | 1988-09-06 | 1992-01-28 | Hoffmann-La Roche Inc. | Cyclic growth hormone releasing factor analogs and method for the manufacture thereof |
US5756458A (en) * | 1989-06-16 | 1998-05-26 | Pharmacia & Upjohn Company | Stabilized potent GRF analogs |
AU625787B2 (en) * | 1989-06-16 | 1992-07-16 | Pharmacia & Upjohn Company | Stabilized, potent grf analogs |
AU4415796A (en) * | 1994-12-07 | 1996-06-26 | Bionebraska, Inc. | Production of c-terminal amidated peptides from recombinant protein constructs |
US8361964B2 (en) * | 2011-04-21 | 2013-01-29 | Theratechnologies Inc. | Growth hormone releasing factor (GRF) analogs and uses thereof |
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US4528190A (en) * | 1983-10-25 | 1985-07-09 | The Salk Institute For Biological Studies | GRF Analogs IV |
CA1271600A (en) * | 1985-01-07 | 1990-07-10 | David Howard Coy | Growth hormone-releasing peptides and method of treating mammals therewith |
US5002931A (en) * | 1987-05-22 | 1991-03-26 | The Salk Institute For Biological Studies | GRF analogs VII |
-
1988
- 1988-09-13 ES ES88114948T patent/ES2054754T3/es not_active Expired - Lifetime
- 1988-09-13 AT AT88114948T patent/ATE106895T1/de active
- 1988-09-13 EP EP88114948A patent/EP0307860B1/en not_active Expired - Lifetime
- 1988-09-13 DE DE3850015T patent/DE3850015T2/de not_active Expired - Fee Related
- 1988-09-14 ZA ZA886860A patent/ZA886860B/xx unknown
- 1988-09-14 NZ NZ226178A patent/NZ226178A/en unknown
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- 1988-09-16 IE IE283588A patent/IE64494B1/en not_active IP Right Cessation
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EP0307860A3 (en) | 1990-07-11 |
EP0307860A2 (en) | 1989-03-22 |
ZA886860B (en) | 1989-05-30 |
IE64494B1 (en) | 1995-08-09 |
PT88532B (pt) | 1992-11-30 |
DE3850015D1 (de) | 1994-07-14 |
AU609878B2 (en) | 1991-05-09 |
JP2715472B2 (ja) | 1998-02-18 |
ATE106895T1 (de) | 1994-06-15 |
PH25715A (en) | 1991-09-18 |
EP0307860B1 (en) | 1994-06-08 |
PT88532A (pt) | 1988-10-01 |
DE3850015T2 (de) | 1994-10-20 |
IE882835L (en) | 1989-03-18 |
DK519088A (da) | 1989-03-19 |
ES2054754T3 (es) | 1994-08-16 |
DK519088D0 (da) | 1988-09-16 |
AU2234688A (en) | 1989-03-23 |
NZ226178A (en) | 1991-06-25 |
CN1032014A (zh) | 1989-03-29 |
JPH01129000A (ja) | 1989-05-22 |
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