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A series of symmetrically bis-substituted imidazole analogs bearing at the N-1 and N-3 two biphenyl moieties ortho substituted either with tetrazole or carboxylate functional groups was designed based on docking studies and utilizing for the first time an extra hydrophobic binding cleft of AT1 receptor. The synthesized analogs were evaluated for their in vitro antagonistic activities (pA 2 values) and binding affinities (–logIC 50 values) to the Angiotensin II AT1 receptor. Among them, the potassium (–logIC 50 = 9.04) and the sodium (–logIC 50 = 8.54) salts of 4-butyl- N , N ′-bis{[2′-(2 H -tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide ( 12a and 12b , respectively) as well as its free acid 11 (–logIC 50 = 9.46) and the 4-butyl-2-hydroxymethyl- N , N ′-bis{[2′-(2 H -tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide ( 14 ) (–logIC 50 = 8.37, pA 2 = 8.58) showed high binding affinity to the AT1 receptor and high antagonistic activity (potency). The potency was similar or even superior to that of Losartan (–logIC 50 = 8.25, pA 2 = 8.25). On the contrary, 2-butyl- N , N ′-bis{[2′-[2 H -tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide ( 27 ) (–logIC 50 = 5.77) and 2-butyl-4-chloro-5-hydroxymethyl- N , N ′-bis{[2′-[2 H -tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide ( 30 ) (–logIC 50 = 6.38) displayed very low binding affinity indicating that the orientation of the n -butyl group is of primary importance. Docking studies of the representative highly active 12b clearly showed that this molecule has an extra hydrophobic binding feature compared to prototype drug Losartan and it fits to the extra hydrophobic cavity. These results may contribute to the discovery and development of a new class of biologically active molecules through bis-alkylation of the imidazole ring by a convenient and cost effective synthetic strategy.

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.