Conformationally constrained analogues of 2-arachidonoylglycerol

Abstract

Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol [meso-7 (AM5504), (+/-)-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of 2,3-dihydroxycyclohexanone followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (-)-8 (AM4435) were synthesized from (2S,3S)- and (2R,3R)-2,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and conformationally restricted analogues of 2-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for their affinities for CB1 and CB2 cannabinoid receptors. The observed biochemical differences between these ligands can help define the conformational requirements for 2-AG activity at each of the above endocannabinoid protein targets.

Figure 1

Endocannabinoids 2-arachidonoylglycerol (2-AG, 1) and N-arachidonoylethanolamine (AEA, 2).

Figure 2

Molecular models for (±)-5 (AM5501), (±)-6 (AM5502), meso-7 (AM5504), the enantiomers (−)-8 (AM4435) and (+)-8 (AM4434), and meso-9 (AM5505) were obtained on a Silicon Graphics Fuel workstation using Insight II (2000). The O-arachidonoyl groups were modeled in extended conformations^, but are not displayed. All structures were subject to molecular mechanics calculations using the steepest descent method for the first 1000 iterations, then the conjugate gradient method until the maximum derivative was less than 0.001 kcal/mol. Only for meso-7 (AM5504) was 2-O-arachidonoyl group found to be axial in the lowest energy conformation (see Figure 3).

Figure 3

Molecular models of both possible chair conformations of meso-7 (AM5504) were obtained on a Silicon Graphics Fuel workstation using Insight II (2000). The O-arachidonoyl groups were modeled in extended conformations as reported by Reggio, et al. for 2-AG and were constrained between C1 and C15. A restraint file for the cyclohexyl ring was also incorporated during the dynamics run in order to prevent possible isomerization and/or racemization at high temperature. The energy-minimized structures underwent constrained molecular dynamics performed by heating it to 1200 °K and recording 100 atomic coordinate trajectories every 10,000 iterations (1 fs per iteration). Next, each trajectory was subjected to simulated annealing followed by energy minimization using the steepest descent method for 100 iterations followed by conjugate gradient method until the maximum derivative was less than 0.001 kcal/mol. Two families of low-energy conformers were identified, and the conformer with the axial 2-O-arachidonoyl group (top) was found to be 2.0 kcal/mol lower in energy than the corresponding equatorial 2-O-arachidonoyl conformer (bottom).

Scheme 1

Syntheses of 1,2,3-cyclohexanetriol ester analogues of 2-AG.

Scheme 2

Chemoenzymatic syntheses of optically active starting 2,3-dihydroxycyclohexanones (−)-4 and (+)-4.