Structure-activity relationships of fluorinated lysophosphatidic acid analogues

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions with seven-transmembrane G-protein-coupled receptors (GPCRs) and the nuclear hormone receptor PPARγ. To identify receptor-selective LPA analogues, we describe a series of fluorinated LPA analogues in which either the sn-1 or sn-2 hydroxyl group was replaced by a fluoro or fluoromethyl substituent. We also describe stabilized phosphonate analogues in which the bridging oxygen of the monophosphate was replaced by an α-monofluoromethylene (-CHF-) or α-difluoromethylene (-CF₂-) moiety. The sn-2- and sn-1-fluoro-LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. We first tested these LPA analogues on insect Sf9 cells induced to express human LPA₁, LPA₂, and LPA₃ receptors. While none of the analogues were found to be more potent than 1-oleoyl-LPA at LPA ₁ and LPA₂, several LPA analogues were potent LPA ₃-selective agonists. In contrast, 1-oleoyl-LPA had similar activity at all three receptors. The α-fluoromethylene phosphonate analogue 15 activated calcium release in LPA₃-transfected insect Sf9 cells at a concentration 100-fold lower than that of 1-oleoyl-LPA. This activation was enantioselective, with the (2S)-enantiomer showing 1000-fold more activity than the (2R)-enantiomer. Similar results were found for calcium release in HT-29 and OVCAR8 cells. Analogue 15 was also more effective than 1-oleoyl-LPA in activating MAPK and AKT in cells expressing high levels of LPA₃. The α-fluoromethylene phosphonate moiety greatly increased the half-life of 15 in cell culture. Thus, α-fluoromethylene LPA analogues are unique new phosphatase-resistant ligands that provide enantiospecific and receptor-specific biological readouts.