CN106317196B - 咪唑并吡啶甲酰-k(k)-aa1-aa2-aa3-ak,其合成,活性及应用 - Google Patents
咪唑并吡啶甲酰-k(k)-aa1-aa2-aa3-ak,其合成,活性及应用 Download PDFInfo
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- CN106317196B CN106317196B CN201510352852.1A CN201510352852A CN106317196B CN 106317196 B CN106317196 B CN 106317196B CN 201510352852 A CN201510352852 A CN 201510352852A CN 106317196 B CN106317196 B CN 106317196B
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
技术领域
本发明涉及式I的4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys,涉及它们的制备方法,涉及它们的抗血栓活性,涉及它们的溶血栓活性以及涉及它们治疗缺血性中风的作用,因而本发明涉及它们在制备抗血栓药物,溶血栓药物以及缺血性中风药物中的应用。本发明属于生物医药领域。
背景技术
缺血性中风是一类较常见且危害严重的脑血管疾病,特点是发病率高、病死率高、致残率高和复发率高。目前临床治疗缺血性中风面临没有有效药物的现实,尤其中风面4h以上的患者非死即残。发明对中风面4h以上的患者有效的药物是临床的重要需求。发明人曾经公开式II的咪唑啉化合物在中风面24h的大鼠缺血性中风模型上,显示优秀疗效。即连续静脉注射6天式II的咪唑啉化合物,每天1次,首次剂量为5μmol/kg,后5次的剂量为2μmol/kg具有优秀疗效。式中aa1和aa2可为同时存在,aa1存在但aa2不存在,或同时不存在;当aa1和aa2同时存在时,aa1为R(Arg),且aa2为G(Gly),A(Ala)或Q(Gln);当aa1存在但aa2不存在时,aa1为R(Arg);aa3可为S(Ser),V(Val)或F(Phe)。由于式II的取代咪唑啉结构单元比较复杂需要简化。
发明人在经过3年实验研究,发现用4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰基代替式II的取代咪唑啉结构单元可以获得结构简单和疗效好意想不到的双重技术效果。按照这个发现,发明人提出了本发明。
发明内容
本发明的第一个内容是提供式I的4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys(当AA1=Gly,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Gly-Arg-Pro-Ala-Lys;当AA1=Ala,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Ala-Arg-Pro-Ala-Lys;当AA1不存在,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Arg-Pro-Ala-Lys;当AA1和AA2都不存在,AA3=Pro时AA1-AA2-AA3-Ala-Lys为Pro-Ala-Lys;当AA1,AA2和AA3都不存在时AA1-AA2-AA3-Ala-Lys为Ala-Lys)
本发明的第二个内容是提供式I的4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys(当AA1=Gly,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Gly-Arg-Pro-Ala-Lys;当AA1=Ala,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Ala-Arg-Pro-Ala-Lys;当AA1不存在,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Arg-Pro-Ala-Lys;当AA1和AA2都不存在,AA3=Pro时AA1-AA2-AA3-Ala-Lys为Pro-Ala-Lys;当AA1,AA2和AA3都不存在时AA1-AA2-AA3-Ala-Lys为Ala-Lys)的合成方法,该方法包括:
(1)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-Gly-Ala-Pro-Ala-Lys的合成方法:
(1-1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-羧酸;
(1-2)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl;
(1-3)在Pd/C存在下(6s)-3,5-二(Boc)-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc);
(1-4)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺(HOSu)缩合为Boc-Pro-OSu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(1-5)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(1-6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
(1-7)在DCC和HOBt存在下Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(1-8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(1-9)在DCC和HOBt存在下Boc-Gly在无水THF中与Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(1-10)在氯化氢-乙酸乙酯溶液中Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(1-11)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)在无水四氢呋喃中与HCl·Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(1-12)将步骤(1-11)所制备的化合物脱去保护基,得到式I所示的化合物。
(2)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-Ala-Arg-Pro-Ala-Lys的合成方法:
(2-1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-羧酸;
(2-2)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl;
(2-3)在Pd/C存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc);
(2-4)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺(HOSu)缩合为Boc-Pro-OSu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(2-5)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(2-6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro- Ala-Lys(Z)-OBzl;
(2-7)在DCC和HOBt存在下Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(2-8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(2-9)在DCC和HOBt存在下Boc-Ala在无水THF中与Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(2-10)在氯化氢-乙酸乙酯溶液中Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(2-11)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)在无水四氢呋喃中与HCl·Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(2-12)将步骤(2-11)所制备的化合物脱去保护基,得到式I所示的化合物。
(3)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-Arg-Pro-Ala-Lys的合成方法:
(3-1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-羧酸;
(3-2)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl;
(3-3)在Pd/C存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc);
(3-4)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺(HOSu)缩合为Boc-Pro-OSu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(3-5)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(3-6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
(3-7)在DCC和HOBt存在下Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(3-8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(3-9)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)在无水四氢呋喃中与HCl·Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
(3-10)将步骤(3-9)所制备的化合物脱去保护基,得到式I所示的化合物。
(4)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-Pro-Ala-Lys的合成方法:
(4-1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-羧酸;
(4-2)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl;
(4-3)在Pd/C存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc);
(4-4)在二环己基碳二亚胺(DCC)存在下Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺(HOSu)缩合为Boc-Pro-OSu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
(4-5)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
(4-6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
(4-7)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)在无水四氢呋喃中与HCl·Pro-Ala-Lys(Z)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-Pro-Ala-Lys(Z)-OBzl;
(4-8)将步骤(4-7)所制备的化合物脱去保护基,得到式I所示的化合物。
(5)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-Ala-Lys的合成方法:
(5-1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-羧酸;
(5-2)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸 在无水四氢呋喃中与Lys(Boc)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl;
(5-3)在Pd/C存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc);
(5-4)在DCC和HOBt存在下Boc-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Ala-Lys(Z)-OBzl;
(5-5)在氯化氢-乙酸乙酯溶液中Boc-Ala-Lys(Z)-OBzl脱去Boc保护基生成Ala-Lys(Z)-OBzl;
(5-6)在DCC和HOBt存在下(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)在无水四氢呋喃中与HCl·Ala-Lys(Z)-OBzl缩合为(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-L-Lys(Boc)-Ala-Lys(Z)-OBzl;
(5-7)将步骤(5-6)所制备的化合物脱去保护基,得到式I所示的化合物。
其中所述HOBt是N-羟基苯并三氮唑的缩略术语,DCC是二环己基羰二亚胺的缩略术语,Boc为叔丁氧羰基的缩略术语,Pd/C为钯/碳。
本发明的第三个内容是评价式I的4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys抗血栓活性,溶血栓活性以及治疗缺血性中风的活性。
附图说明
图1(6s)-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys(当AA1=Gly,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Gly-Arg-Pro-Ala-Lys;当AA1=Ala,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Ala-Arg-Pro-Ala-Lys;当AA1不存在,AA2=Arg和AA3=Pro时AA1-AA2-AA3-Ala-Lys为Arg-Pro-Ala-Lys;当AA1和AA2都不存在,AA3=Pro时AA1-AA2-AA3-Ala-Lys为Pro-Ala-Lys;当AA1,AA2和AA3都不存在时AA1-AA2-AA3-Ala-Lys为Ala-Lys)的合成路线.(i)H2SO4,HCHO,60℃;(ii)1N NaOH,(Boc)2O;(iii)DCC,HOBt,NMM;(iv)H2,Pd/C;(v)4N氯化氢-乙酸乙酯溶液;(vi)TFA/TFSA;(vii)HOSu,DCC;(viii)TFA。
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(6s)-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸(1)
将10g(64.5mmol)L-His用80mL蒸馏水和20mL甲醛混合溶液溶解,然后往里滴加1mL浓H2SO4,60℃微波反应5小时,冷却到室温,往反应化合物中冰浴下滴加浓氨水调pH至7,有大量沉淀析出。过滤,得10.5g(97%)标题化合物,为无色固体。
ESI-MS(m/z)167[M+H]+。
实施例2制备(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸(2)
冰浴下将1.67g(10mmol)(6s)-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸溶于5ml 2N氢氧化钠水溶液。往该反应液中加5.23g(24mmol)(Boc)2O与10mL二氧六环的溶液。室温搅拌,TLC(CH2Cl2∶MeOH=15∶1)监控反应原料点消失。反应完成后,过滤,滤液减压浓缩除去二氧六环。残留的水层用饱和KHSO4水溶液酸化至pH值至2,用乙酸乙酯萃取三次,合并乙酸乙酯层,并用少量水反洗,乙酸乙酯层用无水Na2SO4干燥,过滤,减压浓缩得到的淡黄色固体用乙酸乙酯浸泡磨洗,过滤,得1.55g(42%)标题化合物,为无色固体。ESI-MS(m/z)367[M+H]+。
实施例3制备(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-Lys-(Boc)-OBzl(3)
冰浴和搅拌下3.67g(10.0mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸,1.48g(11.0mmol)HOBt和2.47g(12.0mmol)DCC加50ml无水THF溶解,反应液活化30分钟。然后将3.91g(10.5mmol)Tos·Lys(Boc)-OBzl与50mL无水THF并用1.0mLNMM调节pH至9的悬浮液滴加到活化的反应液中。撤去冰浴,室温搅拌12小时,滤除二环己基脲(DCU)。滤液减压浓缩至干,残留物用乙酸乙酯溶解,再滤除DCU。滤液层依次用饱和NaHCO3溶液洗3次,饱和NaCl溶液洗3次,饱和KHSO4溶液洗3次,饱和NaCl溶液洗3次,饱和NaHCO3溶液洗3次,饱和NaCl溶液洗3次,乙酸乙酯层用无水Na2SO4干燥,过滤、滤液减压浓缩,残留物经柱层析(石油醚/丙酮体系8∶1-2∶1)纯化得3.97g(58%)标题化合物,为无色固体,ESI-MS(m/z)686[M+H]+。
实施例4制备(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)(4)
称取200mg(0.29mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)-OBzl于50mL茄形瓶中,用10mL甲醇溶解后,加入20mg Pd/C,先用真空水泵抽走反应瓶中的空气,然后通入氢气,如此反复三次,最后保持通氢气状态反应12小时,利用TLC(石油醚∶丙酮=3∶1)监测至原料斑点消失后,减压过滤,将滤液减压浓缩至干得160mg(92%)标题化合物,为无色固体,ESI/MS(m/e):596[M+H]+。
实施例5制备HCl·Pro-Ala-Lys(Z)-OBzl
5-1)制备Boc-Pro-Ala
冰浴下向1.075g(5.0mmol)Boc-Pro和20mL无水THF的溶液中加0.637g(5mmol)N-羟基琥珀酰亚胺(HOSu),并使完全溶解。往该溶液中加少量无水THF与1.236g(6.0mmol)二环己基羰二亚胺(DCC)的溶液。室温搅拌7小时,TLC(二氯甲烷∶甲醇=10∶1)监测Boc-Pro消失。滤除DCU,滤液减压浓缩除去THF。残留物先用乙酸乙酯溶解,然后依次用饱和NaHCO3水溶及饱和NaCl水溶液洗。乙酸乙酯层减压浓缩至干,残留物加入适量THF溶解。往该溶液中加已溶于少量水的0.489g(5.5mmol)Ala溶液。得到的反应液用NaHCO3固体调pH到9,室温反应12小时,减压浓缩除去THF,残留物加5mL水溶解,用饱和KHSO4水溶液调pH到2,用乙酸乙酯少量多次萃取,合并的乙酸乙酯层用饱和NaCl水溶液洗至中性,用无水硫酸钠干燥。过滤,滤液减压浓缩得1.41g(98%)标题化合物,为无色固体。ESI-MS(m/e):285[M-H]-。
5-2)制备Boc-Pro-Ala-Lys(Z)-OBzl
采用实施例3的方法从1.43g(5.0mmol)Boc-Pro-Ala和2.71g(5.0mmol)Lys(Z)-OBzl得3.09g(97%)标题化合物,为米黄色固体。ESI-MS(m/e):639[M+H]+。.
5-3)制备HCl·Pro-Ala-Lys(Z)-OBzl
将0.638g(1mmol)Boc-Pro-Ala-Lys(Z)-OBzl溶于10mL乙酸乙酯。冰浴下向得到的溶液中加15mL浓度为4N的氯化氢-乙酸乙酯溶液,TLC(二氯甲烷∶甲醇=1∶1)显示Boc-Pro-Ala-Lys(Z)-OBzl消失。反应混合液在室温下减压浓缩,残留物再用乙酸乙酯溶解并室温减压浓缩,如此反复3次,除净游离氯化氢。残留物用无水乙醚析晶,得0.516g(90%)标题化合物,直接用于下步反应。ESI-MS(m/e):539[M+H]+。
实施例6制备HCl·Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
采用实施例3的方法从798mg(2.5mmol)Boc-Arg(NO2)和1.322g(2.3mmol)HCl·Pro-Ala-Lys(Z)-OBzl得1.642g(78%)Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl,为无色固体。ESI-MS(m/z)864[M+Na]+。
采用实施例5-3的方法从2.099g(2.5mmol)Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得1.9g(98%)标题化合物,为无色固体。直接用于下步反应。ESI-MS(m/z)742[M+H]+。
实施例7制备Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
采用实施例3的方法从817mg(4.32mmol)Boc-Ala和3.36g(4.32mmol)HCl·Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得3.27g(83%)标题化合物,为无色固体。ESI-MS(m/e)911[M+H]+。
实施例8制备HCl·Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
采用实施例5-3的方法从2.241g(2.46mmol)Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得2.04g(98%)标题化合物,为无色固体。直接用于下步反应。ESI-MS(m/e)811[M+H]+。
实施例9制备Boc-Ala-Lys(Z)-OBzl
采用实施例3的方法从473mg(2.5mmol)Boc-Ala和936mg(2.3mmol)HCl·Lys(Z)-OBzl得1.204g(89%)标题化合物,为无色固体。ESI-MS(m/z)565[M+Na]+。
实施例10制备HCl·Ala-Lys(Z)-OBzl
采用实施例5-3的方法从1.354g(2.5mmol)Boc-Ala-Lys(Z)-OBzl得1.181g(99%)标题化合物,为无色固体,直接用于下步反应。ESI-MS(m/e)443[M+H]+。
实施例11制备Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
采用实施例3的方法从817mg(4.67mmol)Boc-Gly和3.62g(4.77mmol)HCl·Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得3.43g(82%)标题化合物,为无色固体。ESI-MS(m/e)897[M+H]+。
实施例12制备HCl·Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
采用实施例5-3的方法从2.241g(2.5mmol)Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得2.02g(97%)标题化合物,直接用于下步反应。ESI-MS(m/e)797[M+H]+。
实施例13制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5a)
采用实施例3的方法从819mg(1.38mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)和1.150g(1.38mmol)HCl·Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得680mg(36%)标题化合物,为无色固体产物。ESI-MS(m/e):1374[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.17-7.69(m,6H),7.35-7.20(m,9H),5.10(s,2H),5.00(s,2H),4.81(m,1H),4.51-4.21(m,5H),3.72-3.54(m,3H),3.42-3.35(m,2H),3.13(s,2H),2.98-2.92(m,3H),2.85-2.83(m,3H),1.83-1.78(m,2H),1.73-1.62(m,3H),1.57(s,9H),1.44(s,9H),1.37(s,9H),1.18-1.15(m,3H),1.12-1.07(m,3H)。
实施例14制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5b)
采用实施例3的方法从1.46g(2.46mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)和2.083g(2.46mmol)HCl·Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl得1.02g(30%)标题化合物,为无色固体产物。ESI-MS(m/e):1388[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.18(m,1H),8.07(s,1H),8.01-7.95(m,4H),7.35-7.20(m,9H),5.10(s,2H),5.00(s,2H),4.79(m,1H),4.45-4.18(m,6H),3.59-3.51(m,2H),3.15(s,2H),2.98-2.92(m,3H),2.86-2.84(m,2H),1.99-1.81(m,3H),1.72-1.62(m,3H),1.57(s,9H),1.44(s,9H),1.36(s,9H),1.30-1.10(m,11H)。
实施例15制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc) -Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5c)
采用实施例3的方法从819mg(1.38mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)和1.120g(1.44mmol)HCl·Arg(NO2)-Pro-Ala-Lys(Z)-OBzl,得526mg(29%)标题化合物,为无色固体。ESI-MS(m/e):1318[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.25-7.96(m,6H),7.35-7.20(m,8H),5.10(s,2H),5.00(s,2H),4.81(m,1H),4.61-4.07(m,5H),3.59-3.41(m,2H),3.23-2.94(m,3H),2.89-2.85(m,6H),2.73(s,3H),1.99-1.65(m,5H),1.57(s,9H),1.44(s,9H),1.37(s,9H),1.32-1.12(m,8H)。
实施例16制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Pro-Ala-Lys(Z)-OBzl(5d)
采用实施例3的方法从819mg(1.38mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)和834mg(1.45mmol)HCl·Pro-Ala-Lys(Z)-OBzl,得461mg(30%)标题化合物,为无色固体产物。ESI-MS(m/e):1116[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.22-8.09(m,2H),7.35-7.20(m,9H),5.10(s,2H),5.00(s,2H),4.79(m,1H),4.51-4.39(m,2H),4.28-4.22(m,3H),2.96-2.84(m,5H),1.74-1.62(m,4H),1.57(s,9H),1.44(s,9H),1.37(s,9H),1.29-1.15(m,7H)。
实施例17制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Ala-Lys(Z)-OBzl(5e)
采用实施例3的方法从819mg(1.38mmol)(6s)-3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-甲酰-L-Lys(Boc)和689mg(1.44mmol)HCl·Ala-Lys(Z)-OBzl,得631mg(45%)标题化合物,为无色固体。ESI-MS(m/e):1019[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.06(s,1H),7.39-7.28(m,9H),5.10(s,2H),5.00(s,2H),4.80(m,1H),4.44(m,1H),4.27-4.20(m,2H),2.99-2.84(m,5H),1.69(m,1H),1.57(s,9H),1.44(s,9H),1.37(s,9H),1.24-1.07(m,7H)。
实施例18制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7a)
冰浴下向100mg(0.073mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5a)中加入3mL TFA,搅拌30min后TLC(CH2Cl2∶MeOH=1∶1)显示原料点消失,停止反应。反应物用无水乙醚反复洗涤,减压浓缩至干,得化合物4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl。该产物继续采用实施例3的方法与31mg(0.08mmol)Boc-Lys(Z)得32mg(30%)标题化合物,为无色固体产物。ESI-MS(m/e):1437[M+H]+。
实施例19制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7b)
采用实施例18的方法从143mg(0.1mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5b),得化合物4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl。该产物继续采用实施例3的方法与43mg(0.11mmol)Boc-Lys(Z)得37mg(25%)标题化合物,为无色固体产物。ESI-MS(m/e):1451[M+H]+。
实施例20制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7c)
采用实施例18的方法从392mg(0.3mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(5c)得化合物4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl。该产物继续采用实施例3的方法与125mg(0.33mmol)Boc-Lys(Z)得110mg(27%)标题化合物,为无色固体。ESI-MS(m/e):1380[M+H]+。
实施例21制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Pro-Ala-Lys(Z)-OBzl(7d)
采用实施例18的方法从113mg(0.1mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Pro-Ala-Lys(Z)-OBzl(5d)得化合物4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys-Pro-Ala-Lys(Z)-OBzl。该产物继续采用实施例3的方法与43mg(0.11mmol)Boc-Lys(Z)反应得29mg(24%)标题化合物,为无色固体产物。ESI-MS(m/e):1179[M+H]+。
实施例22制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Ala-Lys(Z)-OBzl(7e)
采用实施例18的方法从180mg(0.17mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Boc)-Ala-Lys(Z)-OBzl(5e)得化合物4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys-Ala-Lys(Z)-OBzl。该产物继续采用实施例3的方法与74mg(0.19mmol)Boc-Lys(Z)反应得57mg(30%)标题化合物,为无色固体产物。ESI-MS(m/e):1082[M+H]+。
实施例23制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys)-Gly-Arg-Pro-Ala-Lys(8a)
冰浴下向50mg(0.035mmol)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7a)中加3mL TFA和1mL TFMSA,搅拌30min后TLC(CH2Cl2∶MeOH=1∶1)显示原料点消失,停止反应。反应物用无水乙醚反复洗涤,减压浓缩至干,残留物用水溶解,用氨水调pH值至8。得到的溶液先用Sephadex G10除盐,然后用制备柱T3Prep OBDTM 5μm 30×150mm纯化。相应的馏分冻干得5mg(15%)标题化合物,为无色固体。Mp:150.9-152.8℃;IR(KBr):3272,2938,1659,1532,1448,1275,1173,1048,832,760,637,574cm-1;ESI-MS(m/e):934[M+H]+;1H-NMR(500MHz,DMSO-d6):δ/ppm=8.805(s,1H),8.740(s,1H),8.467(m,1H),8.257(m,1H),8.175(m,1H),8.159-8.145(m,3H),8.045(m,1H),7.995(m,1H),7.806(m,3H),7.736(m,2H),7.726(m,3H),4.504-4.474(m,2H),4.363-4.343(m,4H),4.276(m,1H),4.235(m,1H),4.126(m,1H),3.763-3.733(m,2H),3.670-3.622(m,2H),3.572(m,1H),3.369(m,1H),3.068-3.043(m,4H),2.840(m,1H),2.742-2.731(m,4H),1.877-1.829(m,2H),1.703-1.674(m,5H),1.579-1.495(m,10H),1.479-1.388(m,2H),1.388-1.344(m,7H),1.197-1.183(m,3H)。
实施例24制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys)-Ala-Arg-Pro-Ala-Lys(8b)
采用实施例23的方法从50mg(0.034mmol)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7b)得7mg(21%)标题化合物,为无色固体。Mp:137.1-138.8℃;IR(KBr):3272,2943,1659,1527,1448,1245,1174,1048,833,760,637,574cm-1;ESI-MS(m/e):948[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.656(s,1H),8.477(s,1H),8.122(s,2H),8.098(s,2H),8.039(m,1H),8.002(m,1H),7.772(m,3H),7.709(m,2H),7.658(m,3H),4.612-4.589(m,2H),4.454-4.368(m,4H),4.297(m,1H),4.233(m,1H),4.186-4.114(m,2H),3.762-3.694(m,2H),3.625(s,1H),3.542(m,1H),3.171-3.095(m,4H),2.754(m,5H),2.006-1.982(m,2H),1.951-1.854(m,3H),1.710-1.692(m,5H),1.535-1.428(m,10H),1.361-1.315(m,6H),1.230-1.188(m,6H)。
实施例25制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys)-Arg-Pro-Ala-Lys(8c)
采用实施例23的方法从50mg(0.036mmol)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl(7c)得7mg(22%)标题化合物,为无色固体。Mp:133.5-135.6℃;IR(KBr):3272,2943,1659,1527,1448,1245,1174,1048,833,760,637,574cm-1;ESI-MS(m/e):877[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.675(s,1H),8.511(s,1H),8.428(s,1H),8.260(m,1H),8.159(s,3H),8.075-8.030(m,2H),7.800(m,3H),7.750(m,3H),4.477-4.457(m,2H),4.370-4.350(m,4H),4.306-4.260(m,2H),4.085(m,1H),3.097(m,6H),2.842-2.751(m,6H),2.035(m,1H),1.954-1.858(m,4H),1.692-1.615(m,5H),1.615-1.439(m,10H),1.386-1.263(m,6H),1.229-1.205(m,3H)。
实施例26制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys)-Pro-Ala-Lys(8d)
采用实施例23的方法从50mg(0.04mmol)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基 -Lys(Lys(Z)-Boc)-Pro-Ala-Lys(Z)-OBzl(7d),得6mg(20%)标题化合物,为无色固体。Mp:137.1-139.8℃;IR(KBr):3058,2934,1660,1544,1531,1428,1178,1048,833,762,630,573cm-1;ESI-MS(m/e):721[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.780(m,1H),8.698(m,1H),8.437(m,1H),8.106(m,3H),8.043(m,1H),7.989(m,1H),7.703(m,3H),4.512-4.487(m,2H),4.399-4.386(m,3H),4.371(m,1H),4.271(m,1H),4.170(m,1H),3.685-3.633(m,2H),3.528(m,1H),3.371(s,1H),3.171-3.099(m,2H),2.840-2.753(m,5H),2.036(m,1H),1.940-1.887(m,3H),1.793-1.677(m,4H),1.625-1.530(m,7H),1.423-1.312(m,7H),1.257-1.238(m,3H)。
实施例27制备4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys)-Ala-Lys(8e)
采用实施例23的方法从50mg(0.046mmol)4,5,6,7-四氢-3H-咪唑[4,5-c]并吡啶-6-酰基-Lys(Lys(Z)-Boc)-Ala-Lys(Z)-OBzl(7e)得5mg(17%)标题化合物,为无色固体。Mp:119.4-121.8℃;IR(KBr):3047,2937,1659,1531,1428,1178,1048,835,763,639,557cm-1;ESI-MS(m/e):624[M+H]+;1H-NMR(300MHz,DMSO-d6):δ/ppm=8.743(m,1H),8.706(m,1H),8.487(m,1H),8.163(m,2H),8.139(m,2H),7.800(m,4H),4.374-4.264(m,5H),4.158-4.029(m,2H),3.693(s,1H),3.413(m,1H),3.101-3.080(m,2H),2.887-2.769(m,4H),1.725-1.688(m,4H),1.628-1.532(m,6H),1.480-1.332(m,8H),1.249-1.225(m,3H)。
实验例1评价化合物8a-e的抗血栓活性
将雄性SD大鼠(200±20g),随机分组,每组10只,饲养1天,停止喂食过夜。灌胃给予化合物8a-e的生理盐水溶液(剂量为1nmol/kg)或阿司匹林的生理盐水溶液(剂量为167μmol/kg)或生理盐水(剂量为10mL/kg)30min之后,大鼠用20%乌来糖的生理盐水溶液麻醉,之后手术。分离大鼠的右颈动脉和左颈静脉,将准确称重的丝线置于旁路插管,管的一端插入左静脉,另一端管插入右侧动脉并注射0.2mL肝素钠抗凝。使得血流从右侧动脉流经旁路插管进入左侧静脉,15min之后取出附有血栓的丝线称量,计算血液循环前后丝线的重量,得到的血栓重以均值±SD mg表示并代表抗血栓活性,作t检验。数据列入表1。结果表明口服1nmol/kg化合物8a-e能有效地抑制血栓形成。获得了意想不到的技术效果。
表1 1nmol/kg化合物8a-e的抗血栓活性
n=10;a)与生理盐水比p<0.01;b)与生理盐水比p<0.01,与阿司匹林相比p>0.05。
实验例2评价化合物8a-e的溶血栓活性
SD大鼠(雄性,200±20g)按1200mg/kg的剂量腹腔注射乌拉坦生理盐水溶液进行麻醉。麻醉大鼠后将其仰卧位固定,分离其右颈总动脉,近心端处夹住动脉夹,将近心端及远心端分别穿入手术线,远心端的手术线结扎,远心端插管,将动脉夹松开,取出约1mL动脉血,置于1mL离心管中。往垂直固定的橡胶管(长15mm,内径2.5mm,外径5.0mm,管底用胶塞密封,para膜封紧)内注入0.1mL大鼠动脉血,随后在管内迅速插入一支不锈钢材质的血栓的固定螺栓(血栓固定螺旋用直径为0.2mm的不锈钢丝绕成,螺旋部分长10mm,内含15个螺圈,螺圈的直径为1.0mm,托柄与螺旋相连,长约7.0mm,呈问号型)。血液凝固45min后,从玻璃管中小心取出被血栓包裹的血栓固定螺旋,精确称其重量。
旁路插管由三部分构成,中间段为长60.0mm,内径3.5mm的聚乙烯胶管;两端均为长100.0mm,内径1.0mm,外径2.0mm的相同的聚乙烯管,该管一端拉成尖管,长约10.0mm(用于插入大鼠颈动脉及静脉),外径为1.0mm,其另一端的外部套一段长为7.0mm,外径为3.5mm的聚乙烯管(用于插入中段的聚乙烯胶管内),3段管的内壁均需要硅烷化(1%的硅油乙醚溶液)。将血栓包裹的血栓固定螺旋置于中段聚乙烯胶管内,胶管的另外两端分别与两根聚乙烯的加粗端相套,保证在循环的过程中不会漏血。用注射器通过尖管端将管中注满肝素生理盐水溶液(50IU/kg),排除气泡,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,结扎远心端的血管,在暴露的左颈外静脉上剪一小口,将上述制备好的旁路管道尖管由小口插入左颈外静脉开口处,同时远离旁路管中段(含精确称量的血栓固定螺旋)内血栓固定螺旋。用注射器通过另一端的尖 管注入准确量的肝素钠的生理盐水溶液(50IU/kg),此时注射器不要撤离聚乙烯管,用动脉夹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,结扎远心端,在离动脉夹不远处将右颈总动脉剪一小口,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。旁路管道的两端均用4号手术缝线将动静脉固定。
用头皮针将生理盐水(3ml/kg)或尿激酶的生理盐水溶液(剂量为20000IU/kg)或化合物8a-e的生理盐水溶液(剂量为1nmol/kg)通过旁路管的中段(含精确称量的血栓固定螺旋),扎入远离血栓固定螺旋的近静脉端,松开动脉夹,使血流通过旁路管道从动脉流向静脉。将注射器中的溶液缓慢注入血液,通过血液循环,按静脉-心脏-动脉的顺序作用于螺旋的血栓上。血液循环1h之后,从旁路管道中取出固定血栓的螺旋,精确称量。计算每只大鼠旁路管道中固定血栓的螺旋血液循环前后血栓的重量差,以均值±SD mg表示并代表溶血栓活性,作t检验。数据列入表2。结果表明1nmol/kg化合物8a-e能有效地溶解形成的血栓。获得了意想不到的技术效果。
表2 1nmol/kg化合物8a-e的溶血栓活性
n=10;a)与生理盐水比p<0.01,与尿激酶比p>0.05。
实验例3评价化合物8a-e对缺血性中风大鼠的治疗作用
在雄性SD大鼠(体重300±20g)的颈部正中部竖直开约2cm长切口,沿胸锁乳突肌内侧缘分离出右颈总动脉、颈外动脉及颈内动脉。用无创动脉夹分别夹闭颈内动脉开口处和颈总动脉近心端,结扎颈外动脉的远心端,在颈外动脉剪一小口,松开颈总动脉近心端的动脉夹,取10μL血,之后再用无创动脉夹夹闭颈总动脉的近心端。将取得的10μL血放置在1mLEP管中常温放置30分钟使血液凝固,然后转移至-20℃冰箱中放置1小时,使血液凝块结实。大鼠用10%水合氯醛腹腔注射麻醉,剂量为400mg/kg。取出血液凝块,加入1mL生理盐水,用 钢铲把血液凝块捣成大小均一的细小血栓块,制备细小血栓的悬液并转移至1mL注射器内。松开颈总动脉近心端的动脉夹,将1mL血栓混悬液缓慢从大鼠颈外动脉向近心端经过颈内动脉注入大鼠的大脑,然后结扎颈外动脉近心端,打开颈内动脉和颈总动脉处得动脉夹,恢复血流。等待苏醒。大鼠苏醒24小时后按Zealonga方法评定神经功能缺损程度。0分表示无任何神经功能缺失体征、1分表示未损伤侧前肢不能伸展、2分表示向未损伤侧行走、3分表示向未损伤侧转圈成追尾状行走、4分表示意识障碍无自主行走、5分表示死亡。按照得分平均分组。各组大鼠经尾静脉每天注射1次化合物8a-e,剂量为1nmol/kg。连续注射6天,每天评分。结果列入表3-7。数据表明化合物8a连续治疗6天除可使1只脑缺血24小时的大鼠神经生物学评分为0分外,还可将11只脑缺血24小时神经生物学评分为2分、3分和4分的大鼠改善为1分。化合物8b连续治疗6天除可使1只脑缺血24小时的大鼠神经生物学评分为0分外,还可将8只脑缺血24小时神经生物学评分为2分、3分的大鼠改善为1分。化合物8c连续治疗6天除可使1只脑缺血24小时的大鼠神经生物学评分为2分外,还可将12只脑缺血24小时神经生物学评分为2分、3分的大鼠改善为1分。化合物8d连续治疗6天可使11只脑缺血24小时神经生物学评分为2分、3分和4分的大鼠全部改善为1分。化合物8e连续治疗6天可使8只脑缺血24小时神经生物学评分为1分、2分、3分的大鼠全部改善为1分。因为不像已经公开的化合物首次剂量需要5μmol/kg,后5次维持剂量需要2μmol/kg,化合物8a-e的6次剂量均为1nmol/kg。这样一来,首次剂量和维持剂量分别降低了5000倍和2000倍。
表3化合物8a连续治疗6天对脑缺血24小时大鼠神经生物学评分的影响
n=12
表4化合物8b连续治疗6天对脑缺血24小时大鼠神经生物学评分的影响
n=9
表5化合物8c连续治疗6天对脑缺血24小时大鼠神经生物学评分的影响
n=13
表6化合物8d连续治疗6天对脑缺血24小时大鼠神经生物学评分的影响
n=11
表7化合物8e连续治疗6天对脑缺血24小时大鼠神经生物学评分的影响
n=8。
Claims (9)
1.式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys,当AA1=Gly,AA2=Arg和AA3=Pro时,AA1-AA2-AA3-Ala-Lys为Gly-Arg-Pro-Ala-Lys;当AA1=Ala,AA2=Arg和AA3=Pro时,AA1-AA2-AA3-Ala-Lys为Ala-Arg-Pro-Ala-Lys;当AA1不存在,AA2=Arg和AA3=Pro时,AA1-AA2-AA3-Ala-Lys为Arg-Pro-Ala-Lys;当AA1和AA2都不存在,AA3=Pro时,AA1-AA2-AA3-Ala-Lys为Pro-Ala-Lys;当AA1,AA2和AA3都不存在时,AA1-AA2-AA3-Ala-Lys为Ala-Lys,
2.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的制备方法,其特征在于:其中4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Gly-Arg-Pro-Ala-Lys的合成方法,包括以下步骤:
1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸;
2)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys(Boc)-OBzl;
3)在Pd/C存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶6-甲酰-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成3,5-二-Boc-4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys(Boc);
4)在二环己基碳二亚胺存在下,Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺缩合为Boc-Pro-OSu,在NaHCO3存在下Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
5)在DCC和HOBt存在下Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
7)在DCC和HOBt存在下,Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
9)在DCC和HOBt存在下,Boc-Gly在无水THF中与Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
10)在氯化氢-乙酸乙酯溶液中Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
11)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys(Boc)在无水四氢呋喃中与Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys(Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
12)在TFA中3,5-二-Boc-4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys(Boc)-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
13)在DCC和HOBt存在下,Boc-Lys(Z)在无水四氢呋喃中与4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为4,5,6,7-四氢3H-咪唑并吡啶6-甲酰-Lys[Boc-Lys(Z)]-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
14)将步骤13)所制备的化合物脱去保护基,得到式I所示的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys。
3.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的制备方法,其特征在于:其中4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Ala-Arg-Pro-Ala-Lys的合成方法,包括以下步骤:
1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸;
2)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl;
3)在Pd/C存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc);
4)在二环己基碳二亚胺存在下,Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺缩合为Boc-Pro-OSu,在NaHCO3存在下,Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
5)在DCC和HOBt存在下,Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
7)在DCC和HOBt存在下,Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
9)在DCC和HOBt存在下,Boc-Ala在无水THF中与Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
10)在氯化氢-乙酸乙酯溶液中Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
11)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)在无水四氢呋喃中与Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
12)在TFA中3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
13)在DCC和HOBt存在下,Boc-Lys(Z)在无水四氢呋喃中与4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
14)将步骤13)所制备的化合物脱去保护基,得到式I所示的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Ala-Arg-Pro-Ala-Lys。
4.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的制备方法,其特征在于:其中4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的合成方法,包括以下步骤:
1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸;
2)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl;
3)在Pd/C存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc);
4)在二环己基碳二亚胺存在下,Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺缩合为Boc-Pro-OSu,在NaHCO3存在下,Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
5)在DCC和HOBt存在下,Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
7)在DCC和HOBt存在下,Boc-Arg(NO2)在无水THF中与Pro-Ala-Lys(Z)-OBzl缩合为Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
8)在氯化氢-乙酸乙酯溶液中Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
9)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)在无水四氢呋喃中与Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
10)在TFA中3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
11)在DCC和HOBt存在下,Boc-Lys(Z)在无水四氢呋喃中与4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl缩合为4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl;
12)将步骤11)所制备的化合物脱去保护基,得到式I所示的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Arg-Pro-Ala-Lys。
5.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的制备方法,其特征在于:其中4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的合成方法,包括以下步骤:
1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸;
2)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl;
3)在Pd/C存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc);
4)在二环己基碳二亚胺存在下,Boc-Pro在无水THF中与和N-羟基琥珀酰亚胺缩合为Boc-Pro-OSu,在NaHCO3存在下,Boc-Pro-OSu与Ala反应生成Boc-Pro-Ala;
5)在DCC和HOBt存在下,Boc-Pro-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Pro-Ala-Lys(Z)-OBzl;
6)在氯化氢-乙酸乙酯溶液中Boc-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成Pro-Ala-Lys(Z)-OBzl;
7)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)在无水四氢呋喃中与Pro-Ala-Lys(Z)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Pro-Ala-Lys(Z)-OBzl;
8)在TFA中3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Pro-Ala-Lys(Z)-OBzl脱去Boc保护基生成4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Pro-Ala-Lys(Z)-OBzl;
9)在DCC和HOBt存在下,Boc-Lys(Z)在无水四氢呋喃中与4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Pro-Ala-Lys(Z)-OBzl缩合为4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Pro-Ala-Lys(Z)-OBzl;
10)将步骤9)所制备的化合物脱去保护基,得到式I所示的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Pro-Ala-Lys。
6.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的制备方法,其特征在于:其中4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys的合成方法,包括以下步骤:
1)在稀硫酸存在下,甲醛与L-组氨酸在60℃下反应,生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸;
2)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸在无水四氢呋喃中与Lys(Boc)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl;
3)在Pd/C存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-OBzl在甲醇溶液中脱去OBzl生成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc);
4)在DCC和HOBt存在下,Boc-Ala在无水THF中与Lys(Z)-OBzl缩合为Boc-Ala-Lys(Z)-OBzl;
5)在氯化氢-乙酸乙酯溶液中Boc-Ala-Lys(Z)-OBzl脱去Boc保护基生成Ala-Lys(Z)-OBzl;
6)在DCC和HOBt存在下,3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)在无水四氢呋喃中与Ala-Lys(Z)-OBzl缩合为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Ala-Lys(Z)-OBzl;
7)在TFA中3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Boc)-Ala-Lys(Z)-OBzl脱去Boc保护基生成4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Lys(Z)-OBzl;
8)在DCC和HOBt存在下Boc-Lys(Z)在无水四氢呋喃中与4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Lys(Z)-OBzl缩合为4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Lys(Z)-OBzl;
9)将步骤8)所制备的化合物脱去保护基,得到式I所示的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-Ala-Lys。
7.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys在制备抗血栓药物中的应用。
8.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys在制备溶血栓药物中的应用。
9.权利要求1式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-AA3-Ala-Lys在制备治疗缺血性中风药物中的应用。
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