CN107698660B - 咪唑并吡啶-6-甲酰-Lys(Lys)-寡肽,其合成,活性和应用 - Google Patents
咪唑并吡啶-6-甲酰-Lys(Lys)-寡肽,其合成,活性和应用 Download PDFInfo
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Abstract
本发明公开了通式I的4,5,6,7‑四氢‑3H‑咪唑并[4,5‑c]吡啶‑6‑甲酰‑Lys(Lys)‑AA1‑AA2‑Asp‑AA3,公开了它们的制备方法,公开了它们的抗血栓活性以及抗炎活性,因而本发明公开了它们在制备抗血栓和抗炎药物中的应用。
Description
技术领域
本发明涉及4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3，涉及它们的制备方法,涉及它们的抗血栓活性以及抗炎活性,因而本发明涉及它们在制备抗血栓和抗炎药物中的应用。本发明属于生物医药领域。
背景技术
冠心病、脑卒中以及脉管炎等是非常常见的心脑血管疾病。随着人们生活水平的不断提高，心脑血管疾病的发病人数逐年递增。心脑血管疾病危险因素流行，我国心脑血管疾病的患病率持续上升。据资料统计，每5个成年人中就有1个心血管病患者。血栓形成是心脑血管疾病的重要病因。预防血栓形成仍然依赖药物治疗。临床常用的血栓类药物有阿司匹林、氯吡格雷和华法林等。它们都存在一些副作用，如出血和胃肠道刺激等。因此，寻找疗效好、副作用小的抗血栓药物是很有意义的。
近年来，发明人发现含有咪唑并四氢吡啶杂环的化合物是抗血栓活性的优秀结构。发明人曾经公开下式的4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-AA(式中AA为氨基酸残基)在10nmol/kgd的口服剂量下具有确切的抗血栓活性。不过，发明人对这个剂量并不满意。
经过3年实验探索，发明人发现用寡肽代替上式的AA不仅可以降低口服剂量，还可以获得额外的抗炎作用。按照这些发现，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通式I的4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3(式中AA1为Ala,AA2为Gly和AA3为Val时AA1-AA2-Asp-AA3为Ala-Gly-Asp-Val；式中AA1不存在AA2为Leu和AA3为Val时AA1-AA2-Asp-AA3为Leu-Asp-Val；式中AA1为Arg,AA2为Gly和AA3为Val时AA1-AA2-Asp-AA3为Arg-Gly-Asp-Val；式中AA1为Arg,AA2为Gly和AA3为Phe时AA1-AA2-Asp-AA3为Arg-Gly-Asp-Phe；式中AA1为Arg,AA2为Gly和AA3为Ser时AA1-AA2-Asp-AA3为Arg-Gly-Asp-Ser),公开了它们的制备方法,公开了它们的抗血栓活性以及抗炎活性,因而本发明公开了它们在制备抗血栓和抗炎药物中的应用。
本发明的第二个内容是提供通式I的4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3的合成方法，该方法包括：
(1)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Ala-Gly-Asp-Val的合成：
(1.1)L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(1.2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(1.3)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与Lys(Boc)-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl；
(1.4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)；
(1.5)Boc-Ala与甘氨酸苄酯偶联得到Boc-Ala-Gly-OBzl；
(1.6)Boc-Ala-Gly-OBzl在2N NaOH溶液中皂化成Boc-Ala-Gly；
(1.7)Boc-Asp(OBzl)与缬氨酸苄酯偶联得到Boc-Asp(OBzl)-Val-OBzl；
(1.8)Boc-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Asp(OBzl)-Val-OBzl；
(1.9)Boc-Ala-Gly与L-Asp(OBzl)-Val-OBzl偶联得到Boc-Ala-Gly-Asp(OBzl)-Val-OBzl；
(1.10)Boc-Ala-Gly-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Ala-Gly-Asp(OBzl)-Val-OBzl；
(1.11)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)与L-Ala-Gly-Asp(OBzl)-Val-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Ala-Gly-Asp(OBzl)-Val-OBzl；
(1.12)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Ala-Gly-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Ala-Gly-Asp(OBzl)-Val-OBzl；
(1.13)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Ala-Gly-Asp(OBzl)-Val-OBzl与Boc-Lys(Z)偶联得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Gly-Asp(OBzl)-Val-OBzl；
(1.14)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Gly-Asp(OBzl)-Val
-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Ala-Gly-Asp-Val。
(2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Leu-Asp-Val的合成：
(2.1)L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(2.2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(2.3)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与Lys(Boc)-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl；
(2.4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)；
(2.5)Boc-Asp(OBzl)与缬氨酸苄酯偶联得到Boc-Asp(OBzl)-Val-OBzl；
(2.6)Boc-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Asp(OBzl)-Val-OBzl；
(2.7)Boc-Leu与L-Asp(OBzl)-Val-OBzl偶联得到Boc-Leu-Asp(OBzl)-Val-OBzl；
(2.8)Boc-Leu-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Leu-Asp(OBzl)-Val-OBzl；
(2.9)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)与L-Leu-Asp(OBzl)-Val-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Leu-Asp(OBzl)-Val-OBzl；
(2.10)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Leu-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Leu-Asp(OBzl)-Val-OBzl；
(2.11)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Ala-Gly-Asp(OBzl)-Val-OBzl与Boc-Lys(Z)偶联得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Leu-Asp(OBzl)-Val-OBzl；
(2.12)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Leu-Asp(OBzl)-Val-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Leu-Asp-Val。
(3)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg(NO2)-Gly-Asp-Val的合成：
(3.1)L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(3.2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(3.3)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与Lys(Boc)-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl；
(3.4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)；
(3.5)Boc-Arg(NO2)与甘氨酸苄酯偶联得到Boc-Arg(NO2)-Gly-OBzl；
(3.6)Boc-Arg(NO2)-Gly-OBzl在2N NaOH溶液中皂化成Boc-Arg(NO2)-Gly；
(3.7)Boc-Asp(OBzl)与缬氨酸苄酯偶联得到Boc-Asp(OBzl)-Val-OBzl；
(3.8)Boc-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Asp(OBzl)-Val-OBzl；
(3.9)Boc-Arg(NO2)-Gly与L-Asp(OBzl)-Val-OBzl偶联得到Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(3.10)Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(3.11)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)与L-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(3.12)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(3.13)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl与Boc-Lys(Z)偶联得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-
Gly-Asp(OBzl)-Val-OBzl；
(3.14)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-
Val-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Val。
(4)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg(NO2)-Gly-Asp-Phe的合成：
(4.1)L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(4.2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(4.3)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与Lys(Boc)-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl；
(4.4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)；
(4.5)Boc-Arg(NO2)与甘氨酸苄酯偶联得到Boc-Arg(NO2)-Gly-OBzl；
(4.6)Boc-Arg(NO2)-Gly-OBzl在2N NaOH溶液中皂化成Boc-Arg(NO2)-Gly；
(4.7)Boc-Asp(OBzl)与苯丙氨酸苄酯偶联得到Boc-Asp(OBzl)-Phe-OBzl；
(4.8)Boc-Asp(OBzl)-Phe-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Asp(OBzl)-Phe-OBzl；
(4.9)Boc-Arg(NO2)-Gly与L-Asp(OBzl)-Phe-OBzl偶联得到Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(4.10)Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(4.11)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)与L-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(4.12)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl在4N氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(4.13)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl与Boc-Lys(Z)偶联得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-
Gly-Asp(OBzl)-Phe-OBzl；
(4.14)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-
Phe-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Phe。
(5)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg(NO2)-Gly-Asp-Ser的合成：
(5.1)L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(5.2)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸；
(5.3)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与Lys(Boc)-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl；
(5.4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)；
(5.5)Boc-Arg(NO2)与甘氨酸苄酯偶联得到Boc-Arg(NO2)-Gly-OBzl；
(5.6)Boc-Arg(NO2)-Gly-OBzl在2N NaOH溶液中皂化成Boc-Arg(NO2)-Gly；
(5.7)Boc-Asp(OBzl)与丝氨酸苄酯偶联得到Boc-Asp(OBzl)-Ser-OBzl；
(5.8)Boc-Asp(OBzl)-Ser-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Asp(OBzl)-Ser-OBzl；
(5.9)Boc-Arg(NO2)-Gly与L-Asp(OBzl)-Ser-OBzl偶联得到Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl；
(5.10)Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl；
(5.11)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)与L-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl；
(5.12)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl在4N氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl；
(5.13)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl与Boc-Lys(Z)偶联得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-
Gly-Asp(OBzl)-Ser-OBzl；
(5.14)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Ser。
本发明的第三个内容是评价4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-peptide对SD大鼠的抗血栓作用。
本发明的第六个内容是评价4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Ala-Gly-Asp-Val和4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Ser对ICR小鼠炎症的抑制作用。
附图说明
图1.4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3，化合物为8e)的合成路线.i)HCHO,H2SO4,65℃；ii)(Boc)2O,4N NaOH；iii)二环己基碳二亚胺(DCC),1-羟基苯并三唑(HOBt),N-甲基吗啉(NMM),四氢呋喃(THF)；iv)2N NaOH,CH3OH；v)4N HCl/EA；vi)TFA/TFMSA。
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4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸(1)
冰浴和搅拌下向5g(32.3mmol)L-His与18mL水的溶液中缓慢滴加0.8mL H2SO4，使完全溶解。向该溶液中加6mL 40％甲醛水溶液。撤去冰浴，60℃反应8h，TLC(乙酸乙酯：水：冰醋酸＝4:1:1)监测原料点消失。将反应液冷却至室温，冰浴下用浓氨水调节pH至6，有大量无色固体析出。过滤。滤渣用水和丙酮洗涤，得4.52g(84％)标题化合物。ESI-MS(m/e):168[M+H]+。
实施例2制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸(2)
冰浴和搅拌下向5g(29.9mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸与40mL水的溶液中加40mL 1,4-二氧六环溶解的14.3g(65.7mmol)(Boc)2O，用4N NaOH溶液调节pH至8-9，室温反应24h，TLC(二氯甲烷：甲醇＝15:1)监测原料点消失。反应液用饱和KHSO4溶液调节pH至7，减压蒸出1,4-二氧六环后，用饱和KHSO4溶液调节pH至2。用乙酸乙酯萃取三次，酯层用饱和NaCl洗三次。用无水硫酸钠干燥30min，过滤。将滤液减压蒸干。加少量乙酸乙酯将其刚好溶解，静置。有固体析出，过滤。得1.2g(11％)标题化合物，为无色固体。ESI-MS(m/e):368[M+H]+。
实施例3制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl(3)
冰浴下，用20mL干燥的四氢呋喃(THF)将367mg(1mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-羧酸溶解。加入142mg(1.05mmol)HOBt以及258mg(1.25mmol)DCC，搅拌活化30min。用10mL干燥的THF将558mg(1.1mmol)Tos·Lys(Boc)-OBzl溶解，用NMM调节pH至8，将该溶液滴加至反应液中，最后用NMM调节反应液pH至8。室温反应5h，TLC(石油醚：丙酮＝3:1)显示原料消失。过滤除去二环己基脲(DCU)，减压蒸干反应液，剩余物用乙酸乙酯溶解后，依次用饱和NaHCO3溶液、饱和NaCl溶液、5％KHSO4溶液、饱和NaCl溶液、饱和NaHCO3溶液、饱和NaCl溶液各洗三次。乙酸乙酯层用无水Na2SO4干燥30min，过滤，滤液减压蒸干。所得黄色油状物经硅胶柱层析纯化(石油醚/丙酮为洗脱剂)，得248mg(36％)标题化合物，为无色固体。ESI-MS(m/e):686[M+H]+。
实施例4制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)(4)
冰浴下，用20mL甲醇将685mg(1mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-OBzl溶解，用2N NaOH溶液调节pH至12，冰浴反应5h，TLC(石油醚：丙酮＝3:1)显示原料点消失。用饱和KHSO4溶液调节pH至7。减压蒸出甲醇，用饱和KHSO4溶液调节pH至2，用乙酸乙酯萃取3次，乙酸乙酯层用饱和NaCl溶液洗三次，用无水Na2SO4干燥30min，过滤，旋干滤液。得584mg(98％)标题化合物，为无色固体。ESI-MS(m/e):596[M+H]+。
实施例5制备Boc-Ala-Gly-OBzl
冰浴和搅拌下用30mL干燥的THF将945mg(5mmol)Boc-Ala溶解。依次加入708mg(5.25mmol)HOBt及1.291g(6.25mmol)DCC，活化30min。用干燥的THF将2.022g(6mmol)Tos·Gly-OBzl溶解后，用NMM调pH至8，然后将该溶液滴加至反应液中，最后用NMM调反应液pH至8。室温反应12小时，TLC(二氯甲烷：甲醇＝25:1)显示反应完毕后，将反应液减压蒸干，剩余油状物用乙酸乙酯溶解，过滤除去DCU，滤液依次用饱和NaHCO3溶液、饱和NaCl溶液、饱和KHSO4溶液、饱和NaCl溶液、饱和NaHCO3溶液、饱和NaCl溶液各洗三遍，乙酸乙酯层用无水Na2SO4干燥30min，过滤。滤液减压浓缩至干，将得到的黄色油状物经无水乙醚磨洗，得到1.471g(88％)标题化合物，为无色固体。ESI-MS(m/e):337[M+H]+。
实施例6制备Boc-Ala-Gly
冰浴下，用40mL甲醇将1.680g(5mmol)Boc-Ala-Gly-OBzl溶解，用2N NaOH溶液调节pH至12，冰浴反应5h，TLC(二氯甲烷：甲醇＝25:1)显示原料点消失。用饱和KHSO4溶液调节pH至7。减压蒸出甲醇，用饱和KHSO4溶液调节pH至2，用乙酸乙酯萃取三次，乙酸乙酯层用饱和NaCl溶液洗三次，用无水Na2SO4干燥30min，过滤。滤液减压浓缩至干。得1.11g(90％)标题化合物，为无色固体。ESI-MS(m/e):245[M+H]-。
实施例7制备Boc-Asp(OBzl)-Val-OBzl
按照实施例5的方法从1.615g(5mmol)Boc-Asp(OBzl)和2.369g(6mmol)Tos·Val-OBzl得到2.081g(81％)标题化合物，为无色固体。ESI-MS(m/e):513[M+H]+。
实施例8制备HCl·Asp(OBzl)-Val-OBzl
冰浴和搅拌下，用少量干燥的乙酸乙酯将1.024g(2mmol)Boc-Asp(OBzl)-Val-OBzl溶解。向该溶液中加20ml 4N氯化氢的乙酸乙酯溶液，冰浴下反应6小时，TLC(石油醚：丙酮＝3:1)显示反应完毕后，将反应液减压浓缩至干。向残余物中加干燥的乙酸乙酯，减压浓缩至干，重复两次。再向残余物中加无水乙醚，减压浓缩至干，重复三次。得0.751g(91％)标题化合物，为淡黄色固体。ESI-MS(m/e):413[M+H]+。
实施例9制备Boc-Ala-Gly-Asp(OBzl)-Val-OBzl
按照实施例5的方法从1.230g(5mmol)Boc-Ala-Gly和2.700g(6mmol)HCl·Asp(OBzl)-Val-OBzl得到2.650g(83％)标题化合物，为无色固体。ESI-MS(m/e):641[M+H]+。
实施例10制备HCl·Ala-Gly-Asp(OBzl)-Val-OBzl
按照实施例8的方法从1.280g(2mmol)Boc-Ala-Gly-Asp(OBzl)-Val-OBzl得到0.969g(90％)标题化合物，为淡黄色固体。ESI-MS(m/e):541[M+H]+。
实施例11制备Boc-Leu-Asp(OBzl)-Val-OBzl
按照实施例5的方法从1.155g(5mmol)Boc-Leu和2.700g(6mmol)HCl·Asp(OBzl)-Val-OBzl得到2.456g(79％)标题化合物，为无色固体。ESI-MS(m/e):626[M+H]+。
实施例12制备HCl·Leu-Asp(OBzl)-Val-OBzl
按照实施例8的方法从1.250g(2mmol)Boc-Leu-Asp(OBzl)-Val-OBzl得到0.950g(91％)标题化合物，为淡黄色固体。ESI-MS(m/e):526[M+H]+。
实施例13制备Boc-Arg(NO2)-Gly-OBzl
按照实施例5的方法从1.595g(5mmol)Boc-Arg(NO2)和2.022g(6mmol)Tos·Gly-OBzl得到1.866g(80％)标题化合物，为无色固体。ESI-MS(m/e):467[M+H]+。
实施例14制备Boc-Arg(NO2)-Gly
按照实施例6的方法从1.680g(5mmol)Boc-Arg(NO2)-Gly-OBzl得1.737g(92％)标题化合物，为无色固体。ESI-MS(m/e):375[M+H]-。
实施例15制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
按照实施例5的方法从1.880g(5mmol)Boc-Arg(NO2)-Gly和2.700g(6mmol)HCl·Asp(OBzl)-Val-OBzl得到2.903g(75％)标题化合物，为无色固体。ESI-MS(m/e):771[M+H]+。
实施例16制备HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
按照实施例8的方法从1.540g(2mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得到1.187g(89％)标题化合物，为淡黄色固体。ESI-MS(m/e):671[M+H]+。
实施例17制备Boc-Asp(OBzl)-Phe-OBzl
按照实施例5的方法从1.615g(5mmol)Boc-Asp(OBzl)和2.669g(6mmol)Tos·Phe-OBzl得2.500g(89％)标题化合物，为无色固体。ESI-MS(m/e):561[M+H]+。
实施例18制备HCl·Asp(OBzl)-Phe-OBzl
按照实施例8的方法从1.120g(2mmol)Boc-Asp(OBzl)-Phe-OBzl得0.802g(87％)标题化合物，为淡黄色固体。ESI-MS(m/e):461[M+H]+。
实施例19制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl
按照实施例5的方法从1.880g(5mmol)Boc-Arg(NO2)-Gly和3.014g(6mmol)HCl·Asp(OBzl)-Phe-OBzl得3.251g(80％)标题化合物，为无色固体。ESI-MS(m/e):819[M+H]+。
实施例20制备HCl·Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl
按照实施例8的方法从1.636g(2mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得1.301g(91％)标题化合物，为淡黄色固体。ESI-MS(m/e):719[M+H]+。
实施例21制备Boc-Asp(OBzl)-Ser-OBzl
按照实施例5的方法从1.615g(5mmol)Boc-Asp(OBzl)和2.294g(6mmol)Tos·Ser-OBzl得1.940g(78％)标题化合物，为无色固体。ESI-MS(m/e):501[M+H]+。
实施例22制备HCl·Asp(OBzl)-Ser-OBzl
按照实施例8的方法从1g(2mmol)Boc-Asp(OBzl)-Ser-OBzl得1.669g(93％)标题化合物，为淡黄色固体。ESI-MS(m/e):401[M+H]+。
实施例23制备Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl
按照实施例5的方法从1.880g(5mmol)Boc-Arg(NO2)-Gly和2.621g(6mmol)HCl·Asp(OBzl)-Ser-OBzl得2.884g(76％)标题化合物，为无色固体。ESI-MS(m/e):759[M+H]+。
实施例24制备HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl
按照实施例8的方法从1.636g(2mmol)Boc-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得1.196g(91％)标题化合物，为淡黄色固体。ESI-MS(m/e):659[M+H]+。
实施例25制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Ala-Gly-Asp(OBzl)-Val-OBzl(5a)
按照实施例5的方法从893mg(1.5mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)和862mg HCl·Ala-Gly-Asp(OBzl)-Val-OBzl得1.22g(73％)标题化合物，为无色固体。ESI-MS(m/e):1118[M+H]+。
实施例26制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Leu-Asp(OBzl)-Val-OBzl(5b)
按照实施例5的方法从952mg(1.6mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)和906mg HCl·Leu-Asp(OBzl)-Val-OBzl得1.15g(65％)标题化合物，为无色固体。ESI-MS(m/e):1103[M+H]+。
实施例27制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(5c)
按照实施例5的方法从906mg(1.52mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)和1.06g HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得1.39g(73％)标题化合物，为无色固体。ESI-MS(m/e):1248[M+H]+。
实施例28制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl(5d)
按照实施例5的方法从595mg(1mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)和900mg HCl·Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得794mg(61％)标题化合物，为无色固体。ESI-MS(m/e):1296[M+H]+。
实施例29制备3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl(5e)
按照实施例5的方法从595mg(1.6mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)和834mg HCl·Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得721mg(58％)标题化合物，为无色固体。ESI-MS(m/e):1236[M+H]+。
实施例30制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Ala-Gly-Asp(OBzl)-Val-OBzl(6a)
按照实施例8的方法从100mg(0.09mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Ala-Gly-Asp(OBzl)-Val-OBzl得65.2mg(89％)标题化合物，为无色固体。ESI-MS(m/e):818[M+H]+。
实施例31制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Leu-Asp(OBzl)-Val-OBzl(6b)
按照实施例8的方法从100mg(0.09mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Leu-Asp(OBzl)-Val-OBzl得65.7mg(90％)标题化合物，为无色固体。ESI-MS(m/e):803[M+H]+。
实施例32制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(6c)
按照实施例8的方法从100mg(0.08mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得122.0mg(93％)标题化合物，为无色固体。ESI-MS(m/e):948[M+H]+。
实施例33制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl(6d)
按照实施例8的方法从100mg(0.08mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得70.6mg(92％)标题化合物，为无色固体。ESI-MS(m/e):996[M+H]+。
实施例34制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl(6e)
按照实施例8的方法从100mg(0.08mmol)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得68.6mg(91％)标题化合物，为无色固体。ESI-MS(m/e):936[M+H]+。
实施例35制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Gly-Asp(OBzl)-Val-OBzl(7a)
按照实施例5的方法从380mg(1mmol)Boc-Lys(Z)和898mg(1.1mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Ala-Gly-Asp(OBzl)-Val-OBzl得630mg(53％)标题化合物，为无色固体。ESI-MS(m/e):1180[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝11.730(s,1H),8.249-8.147(m,4H),7.945(d,J＝8.1Hz,1H),7.767(t,J＝8.1Hz,1H),7.425(s,1H),7.384-7.262(m,15H),6.774(d,J＝8.1Hz,1H),5.154-5.020(m,4H),4.992(s,2H),4.767(m,1H),4.329-4.246(m,2H),4.175(dd,J＝7.8Hz,6.3Hz,1H),3.810(m,1H),3.706(d,J＝3.3Hz,4H),3.449(dd,J＝8.7Hz,4.8Hz,1H),3.034-2.889(m,4H),2.792-2.550(m,4H),2.055(m,1H),1.682-1.055(m,24H),0.841(d,J＝6.6Hz,6H)。
实施例36制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Leu-Asp(OBzl)-Val-OBzl(7b)
按照实施例5的方法从103mg(0.27mmol)Boc-Lys(Z)和217mg(0.30mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Leu-Asp(OBzl)-Val-OBzl得198mg(63％)标题化合物，为无色固体。ESI-MS(m/e):1165[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝7.975(t,1H),7.731(t,1H),7.426-7.216(m,14H),5.159-5.057(m,4H),4.999(s,1H),4.701(dd,J＝13.8Hz,8.1Hz,1H),4.341-4.246(m,1H),4.202(dd,J＝7.8Hz,6.0Hz,1H),3.920-3.653(m,3H),3.510-3.421(m,2H),3.055-2.953(m,4H),2.817-2.714(m,1H),2.664-2.581(m,1H),2.151-2.002(m,1H),1.631-1.238(m,32H),0.870-0.821(m,12H)。
实施例37制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(7c)
按照实施例5的方法从91mg(0.24mmol)Boc-Lys(Z)和234mg(0.26mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得168mg(54％)标题化合物，为无色固体。ESI-MS(m/e):1310[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝8.313(d,J＝7.8Hz,1H),8.233-8.153(m,3H),7.809(s,1H),7.501(s,1H),7.385-7.244(m,15H),5.152-5.023(m,4H),4.990(s,2H),4.768(dd,J＝13.8Hz,8.4Hz,1H),4.297-4.221(m,2H),4.171(dd,J＝7.8Hz,6.3Hz,1H),3.807(s,2H),3.722(d,J＝5.1Hz,2H),3.241(s,2H),2.956(m,4H),2.776-2.705(m,1H),2.622-2.543(m,1H),2.060(m,1H),1.688-1.109(m,31H),0.840(d,J＝5.7Hz,6H)。
实施例38制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl(7d)
按照实施例5的方法从88mg(0.23mmol)Boc-Lys(Z)和230mg(0.25mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得168mg(45％)标题化合物，为无色固体。ESI-MS(m/e):1358[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝9.723(s,1H),8.624(s,1H),8.454(d,J＝7.5Hz,1H),8.265(d,J＝7.8Hz,1H),8.148(d,J＝4.8Hz,1H),7.792(s,1H),7.363-7.169(m,20H),7.023(s,1H),6.751(d,J＝8.1Hz,1H),5.112-4.992(m,6H),4.724(dd,J＝13.5Hz,8.1Hz,1H),4.492(dd,J＝7.8Hz,J＝14.7Hz,1H),4.333-4.306(m,2H),4.223-4.041(m,2H),3.816(dd,J＝13.8Hz,9.0Hz,1H),3.721(d,J＝5.1Hz,1H),3.155(s,3H),3.073-2.921(m,6H),2.837-2.794(m,1H),2.746-2.532(m,2H),1.700-1.236(m,27H)。
实施例39制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl(7e)
按照实施例5的方法从92mg(0.24mmol)Boc-Lys(Z)和227mg(0.26mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得126mg(62％)标题化合物，为无色固体。ESI-MS(m/e):1298[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝8.245(t,1H),8.161(d,J＝7.5Hz,1H),8.005(d,J＝7.2Hz,1H),7.781(t,J＝5.1Hz,1H),7.428-7.245(m,15H),6.781(d,J＝8.1Hz,1H),5.202-5.062(m,4H),4.991(s,2H),4.790(dd,J＝13.2Hz,8.1Hz,1H),4.368(dd,J＝4.5Hz,J＝12Hz,1H),4.282(m,1H),3.816-3.660(m,6H),3.550(m,1H),3.148(s,2H),3.051-2.890(m,4H),2.806-2.543(m,4H),1.699-1.233(m,28H),0.933-0.829(m,6H)。
实施例40制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Ala-Gly-Asp-Val(8a)
冰浴和搅拌下，向60mg(0.05mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Ala-Gly-Asp(OBzl)-Val-OBzl中滴加1.8mL三氟乙酸，使其完全溶解。滴加0.6mL三氟甲磺酸，溶液变为橘黄色。加干燥管冰浴反应25min，加入大量乙醚，有无色固体析出。离心后倾去上清液，固体再加乙醚，如此反复磨洗三次。用少量水将所得固体溶解，用稀释的氨水调节pH至8，再用稀释的冰醋酸调节pH至7。经Sephadex G10柱子除盐，冷冻干燥，得21mg(54％)标题化合物，为无色固体。Mp:170.9-171.8℃.
(c＝0.11,CH3OH).IR(KBr):3259,3049,2931,2871,1647,1518,1455,1387,1231,1012,995,955,925,651,626,605；ESI-MS(m/e):766[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝8.756(s,1H),8.479(s,1H),8.384(d,J＝6.9Hz,1H),8.094-7.901(m,3H),7.433(s,1H),4.624-4.577(m,1H),4.341-4.277(m,3H),3.921(q,J＝4.8Hz,1H),3.817-3.802(m,1H),3.767-3.612(m,6H),3.580(s,1H),3.455(dd,J＝8.7Hz,4.8Hz,1H),3.169-3.036(m,2H),2.847-2.572(m,5H),2.447-2.261(m,1H),2.033(m,1H),1.686-1.205(m,15H),0.796(d,J＝6.6Hz,1H)。
实施例41制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Leu-Asp-Val(8b)
按照实施例40的方法从58mg(0.05mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Leu-Asp(OBzl)-Val-OBzl得18.7mg(51％)标题化合物，为无色固体。Mp 203.0-203.8℃.[α]20 D＝-63.543(c＝0.11,CH3OH).IR(KBr):3255,2961,2924,1645,1532,1393,1251,1200,1178,1132,1016,956,834,799,720,652,627,570；ESI-MS(m/e):751[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝8.394-8.272(m,1H),8.103(m,1H),7.443(s,1H),4.506(q,1H),4.347-4.330(m,3H),3.896(m,2H),6.773-3.450(m,6H),3.079(s,1H),2.473-2.583(m,4H),2.498-2.385(m,1H),2.012(m,1H),1.551-1.322(m,17H),0.878-0.791(m,12H)。
实施例42制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Val(8c)
按照实施例40的方法从52mg(0.04mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl得18.1mg(57％)标题化合物，为无色固体。Mp 202.7-203.9℃.
(c＝0.12,CH3OH).IR(KBr):3197,3058,2936,1660,1650,1533,1472,1395,1251,1201,1179,1132,1033,955,918,834,799,720,650,627,602；ESI-MS(m/e):851[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝10.359(s,1H),9.263(d,J＝8.1Hz,1H),8.760(s,1H),8.601(d,J＝7.8Hz,1H),8.256(d,J＝7.8Hz,1H),7.997(s,1H),7.417(s,1H),6.969(d,J＝8.1Hz,1H),4.330-4.258(m,3H),3.952(dd,J＝16.8Hz,5.7Hz,1H),3.780(q,1H),3.685(d,J＝7.5Hz,1H),3.433(dd,J＝12Hz,4.8Hz,1H),3.191-3.107(m,2H),3.058-3.021(m,2H),2.898(s,1H),2.730-2.701(m,3H),2.590-2.538(m,2H),2.244-2.174(m,1H),2.023-1.942(m,1H),1.712-1.290(m,22H),0.755(d,J＝5.7Hz,6H)。
实施例43制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Phe(8d)
按照实施例40的方法从54mg(0.04mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl得19.7mg(55％)标题化合物，为无色固体。Mp 178.1-179.2℃.
(c＝0.12,CH3OH).IR(KBr):3202,3047,2893,1890,1651,1470,1436,1177,840,801,724,601,569；ESI-MS(m/e):899[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝10.130(s,1H),9.056(d,J＝7.2Hz,1H),8.581(s,1H),8.267(d,J＝7.2Hz,1H),8.192(s,1H),8.025(d,J＝7.8Hz,1H),7.434(s,1H),7.150(m,7H),4.349(m,1H),4.259(m,2H),4.028(dd,J＝12.3Hz,5.4Hz,1H),3.708(s,1H),3.454(m,1H),3.130(m,1H),2.978(m,1H),2.715(m,4H),2.270(m,1H),1.976(m,1H),1.653-1.188(m,14H)。
实施例44制备4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys(Lys)-Arg-Gly-Asp-Ser(8e)
按照实施例40的方法从62mg(0.05mmol)4,5,6,7-四氢-3H-咪唑并[4,5-c]吡啶-6-甲酰-Lys[Boc-Lys(Z)]-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl得18.9mg(47％)标题化合物，为无色固体。Mp 155.2-156.7℃.
(c＝0.14,CH3OH).IR(KBr):3066,2938,1660,1533,1434,1246,1183,1134,1030,927,838,799,761,722,639,601,577；ESI-MS(m/e):839[M+H]+.1H-NMR(300Hz,DMSO-d6):δ/ppm＝10.120(s,1H),8.758(s,1H),8.646(d,J＝8.4Hz,1H),8.351(d,J＝7.2Hz,1H),8.232(d,J＝6.3Hz,1H),7.963(d,J＝8.1Hz,1H),7.451(s,1H),7.133(d,J＝6.6Hz,1H),7.043(s,1H),4.332(m,2H),4.238(s,1H),4.007(m,2H),3.454(m,1H),3.854(dd,J＝12.0Hz,5.4Hz,1H),3.516(m,4H),3.468(dd,J＝9.3Hz,5.1Hz,1H),3.219(m,4H),2.990(m,2H),2.747(m,4H),2.595(m,2H),2.264(m,1H),2.015(m,1H),1.466(m,17H)。
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12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＝12；a)与生理盐水比p<0.01；b)与生理盐水比p<0.05；c)与生理盐水比p<0.01,与阿司匹林比p>0.05。
实验例2评价化合物8a和8c的抗炎活性:
1)实验方法
20±2g ICR雄性小鼠随机分为空白对照组、阳性用药组及给药组，小鼠使用前静息1天，操作间保持室内温度22℃，每组小鼠12只。一次给药30分钟后往小白鼠的左耳外廓涂二甲苯(0.03mL)，2小时后将小白鼠颈椎脱臼处死。将小鼠的左，右耳剪下，用直径7mm的打孔器在两耳的相同位置，取圆形耳片，分别称重，求出两圆耳片的重量差作为肿胀度。肿胀度＝左耳原片重量-右耳原片重量。
2)给药方法和剂量
给药方式为灌胃给药。空白对照：生理盐水，给药剂量为0.2mL/20g；阳性对照：阿司匹林，给药剂量为1.11mmol/kg；本发明化合物的给药剂量为1nmol/kg。
3)统计方法
本实验数据统计均采用t检验和方差分析，肿胀度以
表示。
4)实验结果
从表2结果可以看出，化合物治疗组的鼠耳肿胀度与生理盐水组相比具有显著性差异，表明化合物在发挥体内抗血栓作用的剂量下，还具有抗炎作用。根据表1的数据演绎，其它化合物也有相应的技术效果。
表2 1nmol/kg化合物8a和8c的抗炎活性
n＝12；a)与生理盐水相比p<0.01；b)与生理盐水相比p<0.01,与阿司匹林比p>0.05。
Claims (4)
1.通式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3,
式中AA1-AA2-Asp-AA3为Ala-Gly-Asp-Val。
2.权利要求1所述的通式I的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3的制备方法，AA1-AA2-Asp-AA3为Ala-Gly-Asp-Val，该方法包括：
(1) L-组氨酸在稀硫酸催化下与甲醛进行Pictet-Spengler缩合生成4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸；
(2) 4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸转化为3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸；
(3) 3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-羧酸与Nα-Boc-Lys-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Nα-Boc-Lys-OBzl；
(4)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Nα-Boc-Lys-OBzl在2N NaOH溶液中皂化成3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Nα-Boc-Lys；
(5)Boc-Ala与甘氨酸苄酯偶联得到Boc-Ala-Gly-OBzl；
(6)Boc-Ala-Gly-OBzl在2N NaOH溶液中皂化成Boc-Ala-Gly；
(7)Boc-β-羧基-OBzl-Asp与缬氨酸苄酯偶联得到Boc-β-羧基-OBzl-Asp-Val-OBzl；
(8)Boc-β-羧基-OBzl-Asp-Val-OBzl在4N 氯化氢的乙酸乙酯溶液中得到L-β-羧基-OBzl-Asp-Val-OBzl；
(9)Boc-Ala-Gly与L-β-羧基-OBzl-Asp-Val-OBzl偶联得到Boc-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl；
(10)Boc-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl在4N氯化氢的乙酸乙酯溶液中得到L-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl；
(11)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Nα-Boc-Lys与L-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl偶联得到3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Boc-Lys -Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl；
(12)3,5-二-Boc-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Boc-Lys-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl在4N 氯化氢的乙酸乙酯溶液中得到4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl;
(13)4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl与Boc- -Lys(Z)偶联得到Nα-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Boc-Lys-(Z-Lys)-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl；
(14)Nα-4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Boc-Lys-(Z-Lys)-Ala-Gly-β-羧基-OBzl-Asp-Val-OBzl在三氟醋酸/三氟甲磺酸中得到4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰- Lys(Lys)-Ala-Gly-Asp-Val。
3.权利要求1所述的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3在制备抗血栓药物中的应用。
4.权利要求1所述的4,5,6,7-四氢-3H-咪唑并吡啶-6-甲酰-Lys(Lys)-AA1-AA2-Asp-AA3在制备抗炎药物中的应用。
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