Novel inhibitors of NO and PGE2 production: pyrimidine analogues

Z Zídek1, P Kostecka1, E Kmonickova1, Z Janeba2, A Holý2, P Jansa2. 1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Department of Pharmacology, 142 20, Czech Republic, 2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610, Czech Republic

We have synthesized a series of original analogues of 2,4-diamino-6-hydroxypyrimidine (DAHP)* and investigated their potential to modulate the in vitro production of nitric oxide (NO) and prostaglandin E2 (PGE2) in immune-activated mouse (C57BL6) peritoneal cells (2 x 106/mL). High output NO production was activated by interferon-γ (5 ng/mL) plus lipopolysaccharide (LPS; 100 pg/mL). Concentration of nitrite in supernatants was determined by Griess reagent at the interval of 24 h of culture. Production of PGE2 was induced by LPS and assayed using ELISA after 16 h of culture. Effects of compounds were analysed in 3-5 identical experiments, all culture variants being run in duplicate. The data were statistically evaluated using the ANOVA and Bonferroni´s multiple comparison test.

None of the compounds had any stimulatory activity. In contrast, a number of them possessed the inhibitory mode of action on production of both NO and PGE2. The effects were closely bound to their structure. The most effective proved to be pyrimidines containing 2-(N,N-dimethylamino)methyleneamino group and pyrimidines containing 2-formamido group. Individual compounds (50 µM; n = 10) inhibited the formation of NO by 69.9-99.8% (SEM range: 0.17-4.81) and reduced the production of PGE2 by 8.0-91.9% (SEM range: 0.07-15.92). The IC 50 values ranged within the interval of 2-10 µM. Similar to DAHP, the pyrimidines containing 2-amino group remained without any influence on NO and PGE2 production.

The studied compounds have been found devoid of any signs of cytotoxicity. Possible alterations in the availability of fundamental factors required for undisturbed NO biosynthesis, i.e. L-arginine, a substrate for NO biosynthesis, and tetrahydobiopterin (BH4), a known cofactor of inducible NO synthase (iNOS), were ruled out as determinants of the prominent NO-inhibitory effects. Supplementation of culture medium with arginine and sepiapterin, a substituent of BH4, did not overcome the inhibitory effects of pyrimidine analogues. Using the arginase inhibitor N ω-hydroxy-nor-L-arginine, a participation of potentially enhanced activity of arginase, a competitive enzyme to iNOS, on reduction of NO production was excluded either. Whereas no effects on the expression of iNOS mRNA were observed, the synthesis of iNOS protein was inhibited by the action of the pyrimidine analogues. It may be presumed therefore that the mechanism of the NO-inhibitory effects of these compounds is mediated at the level of iNOS protein translation. Suppression of PGE2 production seems to be influenced by the interference with both transcription and translation mechanisms of the cyclooxygenase-2 expression.

Acknowledgements. The research was supported by grant no. CZ:GA ČR:303/12/0172 from the Grant Agency of the Czech Republic.

* Patent pending