The target amides of ibuprofen were synthesised according to the Schemes 1–3. The benzylamides were obtained by coupling ibuprofen (1) with substituted benzylamines (2) in the presence of EDC and HOBt in MeCN solution. This synthetic pathway was found to be clean and high yielding.

The benzylamides 3–16, along with the reference compound Ibu-AM5, were evaluated for their ability to inhibit FAAH. The inhibition assays were performed using 0.5 μM [³H]AEA as substrate and rat brain homogenates as the enzyme source. For comparison, the % inhibition produced by URB597 (which shows time-dependent inhibition) using this assay in our laboratory but following a 60 min preincubation, was 8, 38, 98 and 100 at concentrations of 0.1, 1, 10 and 100 nM, respectively³⁵. The results of these primary assays are shown in Table 1. The replacement of the Ibu-AM5 2-methylpyridine moiety with a benzyl moiety to give benzylamides 3–16 led to a reduction in inhibitory activity. In general, benzylamide derivatives showed IC₅₀ values ranging 18–51 µM with the exception of 11, and 15. Compounds 11 and 15 showed FAAH inhibitory activity with IC₅₀ values of 4.1 and 4.4 µM, respectively (Table 1). As shown in Table 1, the position and the kind of substituent on benzyl moiety affect the inhibitory activity. The presence of 4-substituent is not favourable for the inhibitory activity. Conversely, the presence of substituents at 2-position improved the activity as compared with the unsubstituted amide 3, as showed by the 2-chloro (11) and 2,5-dichloro (15) derivatives. Moreover, the moving of chlorine atom from 5-position of amide 15 to 4-position to give the analogue 14 led to a reduction in activity (IC₅₀ 21 µM). While the moving of a chlorine atom from 5-position to 6-position (amide 16) led to a complete loss of activity.

We also studied the binding of the newly designed compounds in the active site of FAAH by using the Induced fit protocol (IFD) implemented in Maestro (Schrödinger), that takes into consideration not only ligand flexibility, but also protein rearrangements upon ligand binding. Calculations were carried out in the monomer A of the rat FAAH (rFAAH) (PDB ID: 3QK5). Taking into considerations previous results on stereoselectivity of Ibu-AM5, demonstrating (S)-IbuAM5 being 10-fold more active than (R)-Ibu-AM5²³, we performed and discussed docking calculations on the (S)-enantiomers of selected compounds in the series. However, for the sale of completeness, docking results for the (R)-enantiomers were reported as Supplementary information. A preliminary validation of the computational protocol was performed on (S)-Ibu-AM5 and QK5 ligand. Results highlighted that the pose with the lowest Glide-emodel and IFDScore reproduced closely the binding mode of (S)-Ibu-AM5 and QK5 (RMSD: 1.34 Å and 0.58 Å respectively; (Supplementary Figure S1). We have compared the binding mode of amides 3, 11 and 15. Best poses of compounds 3 (Figure 2(a)), 11 and 15 (Figure 2(b)) were positioned within the ACB channel with the benzylamide moiety oriented toward the membrane, and the isobutyl moiety pointing toward the catalytic triad, but differed for the position along the ACB channel, being 3 slightly shifted toward the catalytic triad. The more active compounds of the series, 11 and 15 showed high similarity to the binding mode predicted for Ibu-AM5 (Supplementary Figure S2). All the compounds engaged one hydrogen bond (H-bond) interaction of the carbonyl with the Thr488 side chain, but differed for the positioning of the rest of the molecule (Figure 2). In particular, the isobutyl well matching the hydrophobic region formed by Ile491, Phe244, Ile238, and Leu192 in the case of 3, or Phe381, Ala377, Phe432, and Leu380 in the case of amide 11, and Leu192, Ile491, and Leu404 in the case of amide 15. The binding mode adopted by amides 11 and 15 positioned the substituted benzyl ring in a similar position within the hydrophobic region at the gorge of MA channel and appeared favoured with respect to amide 3, since they showed an additional H-bond interaction with Trp531 (amide 11) or Asp403 (amide 15).

With the aim to increase the hydrophilicity of the amides and to reduce the flexibility of the amide chain a new series of arylpiperazinoamides was designed. As indicated in Scheme 2, amides 18–27 were prepared by condensation between Ibuprofen (1) and aryl-substituted piperazines (17) using the EDC method. In general, all the arylpiperazinoamides displayed FAAH inhibitory activity better than benzylamide series. As shown in Table 2 the N-phenylpiperazinoamide 18 displayed inhibitory activity comparable to benzylamides 11 and 15. The introduction of 4-methoxy group on the phenyl ring (amide 23) did not change the activity, while the displacement of the methoxy group into the 3-position (amide 24) led to a drop in activity. The replacement of 4-methoxy group with fluorine atom produced a reduction in activity (compound 22, IC₅₀ 17 μM). In contrast, the introduction in the same position of chlorine atom to give compound 20 improved ten-fold the inhibitory activity. Moving the chlorine from 4- to 3-position (amide 19) did not affect the activity. However, the 3,4-dichlorophenyl substituted amide 21 that, contrary to expectation, showed inhibitory activity an order of magnitude lower potency than analogue 20. The substitution of 3-chlorine of amide 19 with the methyl group to afford compound 25 caused a clear reduction in activity. The introduction of the second methyl group in 2-position (amide 26) restored the activity, while the displacement of 3-methyl into 2-position produced loss of activity (amide 27).

Induced-Fit docking at the enzyme active site revealed that all the arylpiperazinoamide derivatives adopted a different binding mode when compared to benzylamides or to the lead Ibu-AM5, binding deeper in the ACB, establishing, in most cases, direct interactions with at least one residue of the catalytic triad through the carbonyl of the amide, and the arylpiperazinoamide moiety entering the cytosolic port (CP). In order to clarify the SARs of this interesting novel series of ibuprofen derivatives, we analysed the binding mode of 19, 20, 21, 25, and 26. Figure 3(a) represents the binding mode of amide 19, showing two H-bonds with the catalytic triad residues Ser241 and Ser217, and a clear interaction of the chlorine with the NH of Cys269. Accordingly, the introduction of a second chlorine in 4-position, would clash with the side chain of Val270, thus forcing the 3,4-dichlorophenyl ring of compound 21 to move toward Phe381 (Figure 3(a)). This movement also induces a rearrangement of the isobutylphenyl moiety determining the disruption of the hydrophobic network that could account for the 10-fold loss of activity. Interestingly compound 20, which showed the same FAAH inhibitory activity of analogue 19, showed the same position as the isobutylphenyl moiety, albeit the 4-chlorophenyl ring assumed an intermediate conformation with respect to amides 19 and 21. We also studied the binding of amides 25, and 26 differing for the number and the position of methyl groups on the piperazine phenyl ring (Figure 3(b)). Docking results did not allow to give a clear explanation of how these subtle structural differences gave rise to the observed striking differences in activity. The two compounds indeed occupied the same binding pocket, and all established a H-bond with Ser241. Nevertheless, the introduction of the 2-methyl forced the phenyl ring to assume a perpendicular conformation with respect to the piperazine ring as an effect of steric hindrance with piperazine ring hydrogens. This led to different binding modes for 2-methylphenyl (compound 26) and 3-methylphenyl analogue (25), that resulted in a different ability in engaging T-shape π-π stacking interactions with Phe381, only observed for 2-methyl derivative (Figure 3(b)). Albeit docking results gave interesting suggestions about some aspects of SAR of the benzyl- and the piperazine phenyl derivatives, the lack of correlation between IFDscores (Data not shown) and IC₅₀ suggests some caution in interpreting the results.

As further modification, a methylene bridge was inserted between the piperazine and the aromatic ring. The amides 30–34 were prepared by condensation of ibuprofen (1) with N-BOC-piperazine (29), using the same EDC method described above. After BOC-deprotection, a benzyl group was added by reductive alkylation, treating the intermediate 6 with the suitable substituted arylaldehyde in presence of NaHCO₃, NaBHAc₃ in CH₂Cl₂ solution (Scheme 3). Unfortunately, this modification afforded the poor active analogues 30–34 (Table 3). This loss of activity could be attributed to the protonation of the nitrogen atom of the piperazine ring that is unfavourable in the hydrophobic environment of the FAAH channels. The induced fit docking of 30 and 32 confirmed this hypothesis, yielding binding modes characterised by the loss of interactions with Ser241, and by a closed conformation (Supplementary Figure S3), due largely to an intramolecular cation-π interaction, thus explaining the loss in the inhibition activity.

In conclusion, the present study has further explored the pharmacophore of Ibu-AM5 with respect to its interaction with FAAH. Although the benzyl- and the piperazinoamides were logical areas to explore, neither set of derivatives improved upon the inhibitory potency of Ibu-AM5. Nevertheless, the arylpiperazinoamide derivatives showed a binding mode involving residues from the cytosolic port, which has been poorly explored as potential binding site of FAAH inhibitors and could, therefore, be considered interesting leads for the design of novel and more potent FAAH inhibitors.