Design, Synthesis, and Characterization of α‑Ketoheterocycles That Additionally Target the Cytosolic Port Cys269 of Fatty Acid Amide Hydrolase

A series of α-ketooxazoles incorporating electrophiles at the C5 position of the pyridyl ring of <b>2</b> (OL-135) and related compounds were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH) that additionally target the cytosolic port Cys269. From this series, a subset of the candidate inhibitors exhibited time-dependent FAAH inhibition and noncompetitive irreversible inactivation of the enzyme, consistent with the targeted Cys269 covalent alkylation or addition, and maintained or enhanced the intrinsic selectivity for FAAH versus other serine hydrolases. A preliminary in vivo assessment demonstrates that these inhibitors raise endogenous brain levels of anandamide and other FAAH substrates upon intraperitoneal (i.p.) administration to mice, with peak levels achieved within 1.5–3 h, and that the elevations of the signaling lipids were maintained >6 h, indicating that the inhibitors effectively reach and remain active in the brain, inhibiting FAAH for a sustained period

Potent Vinblastine C20′ Ureas Displaying Additionally Improved Activity Against a Vinblastine-Resistant Cancer Cell Line

A series of disubstituted C20′-urea derivatives of vinblastine were prepared from 20′-aminovinblastine that was made accessible through a unique Fe­(III)/NaBH₄-mediated alkene functionalization reaction of anhydrovinblastine. Three analogues were examined across a panel of 15 human tumor cell lines, displaying remarkably potent cell growth inhibition activity (avg. IC₅₀ = 200–300 pM), being 10–200-fold more potent than vinblastine (avg. IC₅₀ = 6.1 nM). Significantly, the analogues also display further improved activity against the vinblastine-resistant HCT116/VM46 cell line that bears the clinically relevant overexpression of Pgp, exhibiting IC₅₀ values on par with that of vinblastine against the sensitive HCT116 cell line, 100–200-fold greater than the activity of vinblastine against the resistant HCT116/VM46 cell line, and display a reduced 10–20-fold activity differential between the matched sensitive and resistant cell lines (vs 100-fold for vinblastine)

Rational Design of Fatty Acid Amide Hydrolase Inhibitors That Act by Covalently Bonding to Two Active Site Residues

The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (<b>3</b> and <b>4</b>) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of <b>2</b> (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of <b>3</b> bound to a humanized variant of rat FAAH revealed that <b>3</b> was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor <b>2</b>, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, <b>3</b> reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor <b>2</b>, providing effects that were unchanged over the 1–6 h time course monitored