Next we were interested in replacing the ketobenzimidazole

Next, we were interested in replacing the ketobenzimidazole fragment of our inhibitors with a noncarbonyl bearing heterocycle. We reasoned that if we could replace the carbonyl while maintaining comparable FAAH inhibition, it would provide evidence for a noncovalent mechanism of action. Thus, we evaluated a number of simple 5,6-heteroycles. We realized success with the identification of 3-pyrrolo[2,3-]pyridine as a replacement for the ketobenzimidazole (see –, ). The synthesis of pyrrolopyridines – is summarized in . Condensation of -Boc-3-piperidinone with under basic conditions yielded . Hydrogenation and subsequent Boc removal gave . Finally, coupling with 2-Cl-4-[2-benzothiophene]pyrimidine followed by alkylation of the pyrrolopyridine nitrogen provided compounds and . Similarly, protection of as its -phenylsulfonyl-derivative, followed by methylation provided the 2-methylpyrrolopyridine . De-protection and elaboration as described above provided compounds –. As indicated by the SAR data in , the pyrrolopyridine-based FAAH inhibitors exhibited potencies comparable to the ketobenzimidazole-based inhibitors (cf. and with –), demonstrating that the carbonyl was not required for potent FAAH inhibition. Likewise, the potencies of the enantiomers of ( and ) showed that the activity resided in the -series, suggesting a binding mode similar to that of the ketobenzimidazoles. The X-ray co-crystal data obtained for compounds and bound in the rFAAH active site revealed an unexpected and unique binding mode. As shown in , both inhibitors adopted a binding mode in which the pyrimidine bridges F432 and M436 thus positioning the 4-aryl substituent in the solvent exposed portion of the membrane access channel (MAC) while the piperidine was positioned in the ABP. The aryl-pyrimidine makes van der Waals contact with several 12-O-tetradecanoyl phorbol-13-acetate residues including F432, M436, L433, W531, T488, and I491. Both the ketobenzimidazole (compound ) and pyrrolopyridine (compound ) fragments are perpendicular to the plane of the piperidine ring and directed down the cytosolic port. The ketobenzimidazole and pyrrolopyridine fragments make contact with the I238/G239 side of the oxy-anion hole, without engaging in a covalent interaction with S241. In fact, no specific polar interaction is apparent for either inhibitor. Overall, the binding mode displayed by both and is consistent with our SAR, noncovalent inhibition, and suggested that shape was the primary source of recognition of these inhibitors by FAAH. In moving forward we evaluated the in vivo pharmacokinetic (PK) parameters and CNS uptake of selected compounds (). As shown, compounds , and exhibited low clearance, displayed good absorption and reasonable mean residence times. Unfortunately, both and had very limited CNS exposure. We reasoned that the poor CNS penetration might be due in part to the highly lipophilic nature of these compounds (log=4.9–5.4). We had already demonstrated that we could decrease lipophilicity and improve potency by adding polarity to the ketobenzimidazole fragment, so next we turned to the aryl-pyrimidine subunit. Based on crystallographic data available at the time we suspected that adding a small polar group to the pyrimidine ring of compound would be well tolerated and was calculated to significantly reduce the compound′s lipophilicity. A NH (compound , log=4.3) group was found to be beneficial while larger groups were generally poorly tolerated. This modification, in combination with a p-fluorophenyl substituent on the pyrimidine ring led to the much less lipophilic (log=3.7). Compound was potent against rat and human FAAH in both our biochemical and whole cell assays and additionally had good permeability (35nM/s) and was not a substrate for P-gp (efflux ratio=1.1) resulting in improved CNS penetration. Correcting the total exposure of for the fraction unbound in rat plasma and brain tissue (), one calculates a brain/plasma ratio of 0.41 for compound , compared to a ratio of <0.05 for compound using the same formula.