Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors.

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC50 values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.