Recent findings of us suggest that compounds that

Recent findings of us suggest that compounds that inhibit both FAAH and TRPV1 may be more efficacious in pain relief than those targeting only one such protein. As continuation of our efforts to identify new molecules able to target simultaneously both FAAH and TRPV1 receptors, we hypothesized that the incorporation of a boronic arn 509 sale group (as the FAAH inhibiting moiety) into the pharmacophore model for a variety of TRPV1 antagonists (, ), could represent a simple strategy for the development of combined FAAH/TRPV1 blockers. The model can be generalized as a central hydrogen-bond acceptor/donor motif flanked by a lipophilic tail on one side and an aromatic group that incorporates a hydrogen-bond acceptor on the other side. Accordingly, 31 arylboronic acids inspired by this model were prepared and tested on FAAH and TRPV1 channel (compounds –, ). All the boronic acids were prepared as pinacol boronate esters and subsequent deprotection with sodium metaperiodate. In detail, amides , , and were synthesized by acylation of the corresponding aminobenzyl or anilino boronates with the appropriate carboxylic acids RCOH, using 1-hydroxybenzotriazole (HOBt)/-ethyl-′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as the carboxylate activator (), followed by cleavage of the boronates . The non-commercially available 4-(aminomethyl)-2-methoxyphenylboronic acid pinacol ester was prepared by a palladium-catalyzed cross-coupling reaction of diboron pinacol ester (Bpin) with the appropriate aryl triflate (). Amides and were obtained by reaction of the activated carboxylic acids with the appropriate amines RNH, followed by cleavage of the boronates (). Ureas and were prepared by reaction of the corresponding 4-aminobenzyl or anilino boronates with 4--butyl-phenylisocyanate followed by cleavage of the boronates (). The effects of arylboronic acids – on [C]anandamide hydrolysis by rat membranes (which express FAAH as the only AEA hydrolyzing enzyme) and on intracellular Ca elevation in HEK293 cells stably transfected with the human TRPV1 cDNA are shown in . The TRPV1 antagonist or desensitizing activity was assessed by adding the test compounds 5min before stimulation of cells with the reference agonist capsaicin. Data obtained for the two most potent boronic acids reported by Minkkilä et al. (compounds and ), compounds , , the selective TRPV1 antagonist SB-366791, and two ‘dual-target’ agents previously identified by us [-arachidonoylserotonin (AA-5-HT) and the piperazinyl carbamate OMDM-198] are also included in the table. The majority of arylboronic acids – showed, as expected, fairly good FAAH inhibitory activities (compounds ,–, –, –, –,–, ,), irrespective of the nature of the functional group X–Y connecting arylboronic acid and lipophilic moieties and of the presence or absence of a methylene bridge. The boronic acid functionality was essential for FAAH inhibitory activity, as demonstrated by the lack of activity of the analogs of , and . The choice of and as reference compounds was driven by the high, submicromolar modulating potency of the boronic acid on both FAAH and TRPV1. Notably, however, the movement of the B(OH) group away from the para-position resulted in a loss of activity (compare compounds and ). In their study, Minkkilä et al. found that para-substituted compounds were indeed more potent FAAH inhibitors than the meta-substituted ones, a result which was ascribed to the more steric tolerance near the enzyme’s catalytic site for the -substitution. A similar explanation may be invoked for the decrease in activity of the 2-methoxy-substituted compounds ,, as compared to their unsubstituted counterparts ,, . The choice of an ω-arylaliphatic chain as the lipophilic tail appeared to be beneficial for optimal activity on FAAH with a positive relationship with the chain length (compare compounds ,, and ; ,,, and ; and ; ,, and ). Conversely, the presence of an aliphatic chain (compounds ,) was detrimental for this activity. Boronic acids and were reported by Minkkilä et al. to inhibit FAAH in the low nanomolar range (IC=21 and 9.1nM, respectively), while the corresponding values recorded by us were one and three orders of magnitude higher (0.79 and 2.92μM, respectively). FAAH assays were however performed at different pH values (7.4 by Minkkilä et al., and FAAH optimal pH=9 by us) and it is well known that pharmacological properties of FAAH are highly pH-dependent. In particular, rat brain hydrolysis of [H]AEA shows an optimum around pH 8–9.