Inhibition of nitric oxide synthase with pyrazole-1-carboxamidine and related compounds.
Guanidines, amidines, S-alkylisothioureas, and other compounds containing the amidine function (-C(=NH)NH2) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we report on the inhibition of the activity of NOS isoforms by compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines and related compounds. 1H-Pyrazole-1-carboxamidine HCl (PCA) inhibited the activity of purified inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) isoforms to a similar extent (IC50 = 0.2 microM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS [IC50 = 5 and 2.4 microM, respectively; cf. N(G)-methyl-L-arginine (NMA) IC50 = 6 microM]. Inhibition of purified iNOS by PCAs could be reversed completely by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug-free medium. This suggests a competitive mode of inhibition. PCA caused potent concentration-dependent inhibition of the acetylcholine-induced, endothelium-dependent relaxations of precontracted rat thoracic aorta (IC50 = 30 microM). 4-Methyl-PCA inhibited the relaxations only at > or = 300 microM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from lipopolysaccharide-treated rats. PCA and NMA, but not 4-methyl-PCA, caused marked increases in mean arterial pressure when administered i.v. to anesthetized rats. In conclusion, PCA and related compounds caused potent inhibition of NOS. Substitution of the pyrazole ring reduced potency, but improved selectivity towards iNOS as exemplified by 4-methyl-PCA.
Southan
G J
GJ
Intramural Research Support Program, SAIC-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702, U.S.A. gsouthan@mail.ncifcrf.gov
Gauld
D
D
Lubeskie
A
A
Zingarelli
B
B
Cuzzocrea
S
S
Salzman
A L
AL
Szabó
C
C
Wolff
D J
DJ
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D001794:Blood Pressure / Q000187:drug effects; D002417:Cattle; D002460:Cell Line; D004730:Endothelium, Vascular / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D011720:Pyrazoles / Q000008:administration & dosage / Q000138:chemical synthesis / Q000494:pharmacology*; D051381:Rats; D019323:omega-N-Methylarginine
D001120:Arginine; D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D011720:Pyrazoles; C092506:N(G)-monomethylarginine acetate; D019323:omega-N-Methylarginine; C107726:pyrazole-1-carboxamidine