Win N H, Ishikawa T, Saito N, Kato M, Yokokura H, Watanabe Y, Iida Y, Hidaka H
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Eur J Pharmacol. 1996 Mar 28;299(1-3):119-26. doi: 10.1016/0014-2999(95)00844-6.
HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.
合成了HF - 2035，即2 - [N - (2 - 氨基乙基)-N - (2,4,5 - 三氯苯磺酰基)]氨基 - N - (4 - 氯肉桂基)-N - 甲基苄胺，并研究了其对钙调蛋白依赖性酶的作用。HF - 2035抑制钙调蛋白激酶I、钙调蛋白激酶II和肌球蛋白轻链激酶，其IC50值分别为1.3微摩尔、1.6微摩尔和68微摩尔。HF - 2035还抑制重组大鼠神经元一氧化氮合酶（一种钙调蛋白依赖性酶）的活性，其Ki为0.78微摩尔。大鼠脑部分纯化的一氧化氮合酶也被HF - 2035抑制，IC50为3.2微摩尔。动力学分析表明，HF - 2035的这种抑制作用相对于钙调蛋白是竞争性的。我们在生物测定中使用有内皮和无内皮的兔颈动脉血管条，研究了HF - 2035对组成型一氧化氮合酶的影响。HF - 2035抑制乙酰胆碱和钙离子载体A23187（6S - [6α(2S*,3S*),8β(R*),9β,11α]-5 - (甲氨基)-2 - [[3,9,11 - 三甲基 - 8 - [1 - 甲基 - 2 - 氧代 - 2 - (1H - 吡咯 - 2 - 基) - 乙基]-1,7 - 二氧杂螺[5.5]十一烷 - 2 - 基]甲基]-4 - 苯并恶唑羧酸）诱导的有内皮血管条的舒张，ED50分别为1.5±0.5微摩尔和2.8±1微摩尔。然而，该化合物不抑制外源性一氧化氮供体N - 亚硝基 - N - 吗啉代氨基乙腈（SIN - 1A）诱导的无内皮血管条的舒张。W - 7（N - (6 - 氨基己基)-5 - 氯 - 1 - 萘磺酰胺）抑制乙酰胆碱诱导的舒张，ED50为46±7微摩尔，其效力比HF - 2035低30倍。HF - 2035不能抑制用大肠杆菌脂多糖处理的大鼠离体胸主动脉中诱导型一氧化氮合酶的活性。这些发现表明，HF - 2035是一种新型强效钙调蛋白拮抗剂，可作为母体化合物开发更具选择性的组成型一氧化氮合酶抑制剂。
