Pyrazole-1- Carboxamidine and related compounds and novel inhibitors of nitric oxide synthase: Pharmacological characterization in cultured cells and vascular rings

Compounds containing the amidine function (-C(NH₂)=NH) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we investigated the effects of compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines on the activity of NOS isoforms. Pyrazole-1-carboxamidine (PCA) inhibited the activity of purified inducible NOS (iNOS). endothelial NOS and neuronal NOS isoforms to a similar extent (IC₅₀ = 0.2 mM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS (IC₅₀ = 5 and 2.4 mM, respectively, cf. N^G-methyl-L-arginine (NMA) IC₅₀ = 6 mM). Inhibition of purified iNOS by PCAs was reversed by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug free medium, suggesting a competitive mode of inhibition. PCA caused a potent dose-dependent inhibition of the acetylcholine-induced endothelium-dependent relaxations of precontracted rat thoracic aorta. 4-methyl-PCA inhibited the relaxations only at ≥ 300 μM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from LPS treated rats. PCA and NMA, but not 4-methyl-PCA, markedly increased blood pressure when administered to anaesthetized rats. Thus, PCA and related compounds are inhibitors of NOS. Substitution of the pyrazole ring reduces potency, but improves iNOS selectivity, as exemplified by 4-methyl-PCA.