A series of N-(2-amino-6-trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamides were designed combining previously identified pharmacophoric elements and evaluated as hTRPV1 antagonists. The SAR analysis indicated that specific hydrophobic interactions of the 2-amino substituents in the C-region of the ligand were critical for high hTRPV1binding potency. In particular, compound 49S was an excellent TRPV1 antagonist (Ki(CAP) = 0.2 nM; IC50(pH) = 6.3 nM) and was thus ca. 100- and 20-fold more potent, respectively, than the parent compounds 2 and 3 for capsaicin antagonism. Furthermore, it demonstrated strong analgesic activity in the rat neuropathic model superior to 2 with almost no side effects. Compound 49S antagonized capsaicin induced hypothermia in mice, but showed TRPV1-related hyperthermia. The basis for the high potency of 49S compared to 2 is suggested by docking analysis with our hTRPV1 homology model in which the 4-methylpiperidinyl group in the C-region of 49S made additional hydrophobic interactions with the hydrophobic region.

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1 Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH4 synthesis, N-acetyl-5-hydroxytryptamine (N-acetylserotonin, NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetized rat in vivo. 2 NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-y (IFNy)-stimulated RAW 264.7 macrophages and interleukin-lf (IL-lB)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis.3 Pretreatment of RAW 264.7 macrophages with the BH4 precursor, dihydrobiopterin (BH2, 0.1 mM)did not restore nitrite formation in the presence of NAS (1 mM).4 Intravenous administration of NAS (1 mg kg-' min-' for 30 min) in anaesthetized rats significantly reduced the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 Mg kg-') and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg-1).5 NAS pretreatment also attenuated iNOS activity in lung homogenates, as determined by the conversion of radiolabelled L-arginine to L-citrulline, and partially restored the constrictor effect of noradrenaline in aortic rings isolated from LPS-treated rats. Moreover, NAS significantly reduced the rise in the plasma concentration of tumour necrosis factor a (TNFa) in response to LPS. 6 These findings suggest that NAS inhibits the expression rather than the activity of iNOS in cultured macrophages and smooth muscle cells. This effect of NAS appears to be independent of the availability of BH4, but may be related to an attenuation of the release of TNFx following LPS administration, as shown in the anaesthetized rat. This mechanism may also account for the beneficial haemodynamic effect of NAS in our experimental model of endotoxaemia.

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