The FAAH inhibitor activity of

The FAAH inhibitor activity of the 3-(5-(ethoxycarbonyl)-1-phenyl-1H-pyrazol-3-yl)phenyl cyclohexylcarbamates was investigated by preparation of compounds (43–52, Table 3). These new compounds displayed an important loss of activity in comparison with the corresponding regioisomers 5-phenyl compounds (20–33), revealing that the activity is quite sensitive to the modifications at the pyrazolo nucleus. These results also highlighted that position of carbamoyl moiety on the 3-phenyl ring (meta-,43–47 or para-,48–52) did not significantly influence the inhibitor potency for the FAAH enzyme. Recognizing that a significant enhancement in inhibitor potency had been seen only with the ethyl 1,5-diphenylpyrazole-3-carboxylate scaffold, the subsequent adopted strategy was to prepare analogues by the introduction of structural modifications on positions 3, and 5 of the pyrazolo template of compound 22 to define the correct structural requirements for inhibitor activity. This led to the synthesis of compounds 53–58 (Table 4). The m-hexylcarbamate 53 was prepared by replacing the cyclohexyl moiety with a noncyclic, yet lipophilic chain such as hexyl, which persuaded an 8-fold decreased activity toward FAAH enzyme compared to that of the analogue 22. Transformation of the ester functionality of 22 into the carboxylic N6-methyladenosine (compound 54) resulted in a dramatic loss of activity (IC50 = 2274 nM). The two 3-methyl and 3-propyl esters 55 and 56 (IC50 = 18 and 27 nM, respectively) showed interesting potencies similar to that observed for 22 (IC50 = 11 nM). The latter bearing the hexyl carbamoyl chain (compound 57) displayed lower inhibitor activity. Interestingly, the introduction of a hydroxymethyl group at the C-3 resulted in a significant loss of activity for the FAAH (58: IC50 = 462 nM) in comparison with that of the corresponding compound 22, confirming that the potency of this class of molecules is quite sensitive to variations at C-3 of the pyrazolo nucleus.
Conclusions In this study we have designed and synthesized a new series of pyrazole phenylcyclohexylcarbamates as FAAH inhibitors. The best activity values were obtained with compounds 22, 23, 25, 55 and 56 (IC50 = 11, 17, 29, 18, and 27 nM, respectively) bearing the N-phenyl moiety and 3-alkyloxycarbonyl functionality on the pyrazole nucleus. Compounds containing carboxamide moieties on the 3 position of pyrazole ring resulted in a decreased potency for FAAH enzyme. The most active compounds showed a marked FAAH selectivity, greater than 1000-fold, versus MAGL. The effect of preincubation on the inhibitory ability of compound 23 was evaluated suggesting that its binding to FAAH is reversible. Taken together, these data could be an apposite starting point for the optimization of novel FAAH reversible inhibitors.
Experimental section
In vitro assays, determination of FAAH activity The IC50 values for compounds were generated in 96-well microtiter plates. The FAAH reaction was conducted at room temperature at a final volume of 200 μL in 125 mM Tris buffer, pH 9.0, containing 1 mM EDTA. A total of 150 μL of AMC arachidonoyl amide 13.3 μM (final concentration = 10 μM) was added to 10 μL of DMSO containing the appropriate amount of compound. The reaction was initiated by the addition of 40 μL of FAAH (0.9 μg/well) in such a way that the assay was linear over 30 min. The final concentration of the analyzed compounds ranged for from 20 to 0.00002 μM. After the reaction had proceeded for 30 min, fluorescence values were then measured by using a VictorX3 PerkinElmer instrument at an excitation wavelength of 340 nm and an emission of 460 nm. Two reactions were also run: one reaction containing no compounds and the second one containing neither inhibitor nor enzyme. IC50 values were derived from experimental data using the sigmoidal dose−response fitting of GraphPad Prism software. To remove possible false positive results, for each compound concentration a blank analysis was carried out, and the final fluorescence results were obtained detracting the fluorescence produced by the presence of all the components except FAAH in the same conditions.