Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties

Abstract

Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using representative FAAH crystal structures revealed that compounds containing a novel piperidinyl thiazole isoxazoline core fit within the ligand binding domains. New potential FAAH inhibitors were designed and synthesized incorporating urea, carbamate, alkyldione and thiourea reactive centers as potential pharmacophores. A small library of candidate compounds (75) was then screened against human FAAH leading to the identification of new carbamate and urea based inhibitors (Ki=pM and nM, respectively). Representative carbamate and urea based chemotypes displayed slow, time dependent inhibition kinetics leading to enzyme inactivation which was slowly reversible. However, evidence indicated that features of the mechanism of inactivation differ between the two pharmacophore types. Selected compounds were also evaluated for analgesic activity in the mouse-tail flick test.

Keywords: Analgesia; Fatty acid amide hydrolase; Inactivation; Inhibition; Piperidinyl thiazole isoxazoline.

Figure 1

Oxathiapiprolin fungicide; Piperidine and piperazine aryl urea FAAH inhibitors (PF-750, JNJ-1661010); α-ketooxazole and carbamate FAAH inhibitors (OL-135, URB597).

Figure 2

Docking of compound 1a (green carbon backbone) to FAAH structure 2WAP.

Figure 3

A, Time dependent loss of FAAH activity in the presence of increasing concentrations of compound 1l. Baseline (open circles), 10nM (dark circles), 1 nM (open squares), 0.1 nM (dark squares), and the uninhibited reaction (open triangles). The solid lines represent the computer fit of the data using the apparent inactivation constant (k_i’) determined at each concentration of 1l. B, computer fit of the apparent k_i’ values for compound 2b to determine k_inact as described in Experimental procedures.

Figure 4

Recovery of FAAH activity (relative to controls) through exhaustive dialysis after inactivation by various compounds (a) compound 1a; (b) JNJ-1661010; (c) compound 2c; (d) compound 2d as described in Experimental procedures. Bars: light gray, 24 hrs; hash marks, 48 hrs; dark gray, 144 hrs; and black, range of control activity over the time course. Structures are given in Table 1 and Fig 1.

Figure 5

in vivo efficacies of novel FAAH inhibitors (Table1) in the mouse tail flick test. A–F shows tail flick latencies in the 52° C tail flick test following intraperitoneal injection of doses of novel FAAH inhibitors G shows summary data for all compounds where compound 1b shows the best in vivo potency. * p < 0.05, ** p < 0.01, *** p < 0.001 two way ANOVA with Bonferroni posthoc test.