The newly synthesized compounds were evaluated for their

The newly synthesized compounds were evaluated for their FAAH inhibitory activity in vitro. In addition, molecular modeling, selectivity and reversibility behavior of the new class of FAAH inhibitors are presented in this article.
Results and discussion The potency of inhibition on the human recombinant (hr) enzyme FAAH for the newly synthesized compounds was evaluated; the relative IC50 values are given in Table 1, Table 2, Table 3, Table 4 [4-(5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-2-methylphenyl]-carbamic different phenylmethyl ester (CAY10499) and URB597 were used as the reference compounds. The most active compounds (22, 23, 25, 55 and 56) were further tested for their selectivity toward monoglyceride lipase MAGL (Table 5). For compound 23 the incubation studies were performed in order to evaluate the reversibility of FAAH inhibition. Thirty-nine pyrazole compounds were synthesized by varying the position of the phenylcarbamoyl chain on the pyrazole moiety (pyrazol-5-ylphenyl/pyrazol-3-ylphenyl carbamate), the position of carbamoyl chain on the phenyl ring (para-/meta-carbamoyl compounds), the presence of different functionalities (ester, carboxamide, carboxylic acid and alcohol) on the pyrazole nucleus, and the substitution of cyclohexyl with hexyl moiety. The SAR studies were extended by introduction of the electron-donating and electron-withdrawing groups on the aromatic substituent at the 1-position of the most active pyrazole compound 22 to afford compounds 23–28. As noted previously, we hypothesized that replacement of the benzene nucleus of the URB series with a pyrazolo nucleus would afford novel FAAH inhibitor compounds. A first set of fourteen compounds (20–33, Table 1) consisted in the 3-/4-(3-(ethoxycarbonyl)-1-substituted-pyrazol-5-yl)phenyl cyclohexylcarbamates were prepared. Among them, compounds 22 bearing a phenyl group at the N-1 position of pyrazole and the carbamoyl chain in the m-position of 5-phenyl ring was the most potent analogue tested (IC50 values of 11 nM). Introduction of a 4-chlorophenyl and 4-methylphenyl groups at N-1 of the pyrazolo moiety, as with compounds 23 and 25, resulted in a comparable activities (IC50 values of 17 and 29 nM, respectively) relative to the compound 22. In contrast, the 4-methoxy/-trifluoromethyl/-nitrophenyl analogues (24, 26 and 27, respectively) displayed a decrease of inhibitor activities for FAAH enzyme. These results imply that electronic effects on the N-phenyl moiety do not significantly control the inhibitor activity. Disubstitution of the aryl moiety at N-1 was also examined by the 2,4-dichlorophenyl derivative 28 which showed a significant decrease of activity for the FAAH enzyme in comparison with the corresponding unsubstituted or 4-chlorophenyl compounds (IC50 values of 123 nM vs 11 and 17 nM, respectively). Introduction of a benzyl moiety, as in compound 29, also exhibited a marked decrease of activity (7-folds) when compared with compound 22. We consequently investigated the impact of position of carbamoyl chain by moving it from the meta to para position of 5-phenyl moiety (compounds 30–33, Table 1). As shown by the biological assays, these new compounds displayed a loss of activity in comparison with the corresponding meta-carbamoyl compounds 20–23 except for analogue 30 bearing the N-methyl moiety that showed higher inhibitor activity than that of its analogue 20 (IC50 values of 81 nM vs 303 nM, respectively). These data confirm that the carbamoyl in meta-position of the phenyl ring greatly improves inhibitory potency of this class of molecules in analogy to the structure-activity profiles of URB series. Replacing the ethyl ester function in the 3-position of compounds 22–24 with a carboxamide moiety led to compounds 34–36. As shown in Table 2, the 3-carboxamide moiety reduced dramatically the inhibitor potency (34: IC50 = 169, 35: IC50 = 174, and 36: IC50 = 743 nM) relative to the 3-ethoxycarbonyl derivatives (22: IC50 = 11, 23: IC50 = 17, 24: IC50 = 103 nM). The 3-carboxamide analogues bearing an isopropyl (37), cyclohexyl (38) or diethyl (39) moieties also displayed lower potency at the FAAH enzyme when compared with the parent 3-carboxamide or 3-ethyl ester compounds (22 and 34, respectively). Within this series of 3-carboxamide-5-phenylpyrazoles, we also examined the effect of p-carbamoyl chain on inhibitor activity (compounds 40–42). These compounds showed comparable activities relative to the corresponding m-carbamoyl derivatives, except for the N-phenyl pyrazole 41 that showed higher potency than that of its analogue 34 (41: IC50 = 71, 34: IC50 = 169 nM).