The Role of Fluorine Atoms in a Fluorinated Prostaglandin Agonist

US, a phase III clinical study is currently underway. Hydrolyzed to its corresponding active acid form (AFP-172; 3) after topical application, compound 1 exhibits strong IOP reducing effects by increasing the drainage of aqueous humor through the uveoscleral outflow route. Compound 3 has a stronger binding affinity (Ki = 0.4 nm) [4] for the PGF2a receptor (FP-receptor) than its parent molecule, 2 (Ki = 129 nm), [5] suggesting that that the w-chain of compound 3 has an enhanced receptor-binding ability. The hydroxy group in the 15-position is critical for the pharmacological activity of 2, suggesting that the 15-F2 substituents in compound 3 may also play an essential role in receptor binding. However, the role of these fluorine atoms has not yet been elucidated because the FP-receptor is a G protein-coupled receptor (GPCR); the structural details known for this class of receptors are still limited to just several GPCRs. Additionally, the current understanding of interactions involving covalently bound fluorine is superficial. Recently, the role of covalently bound fluorine in small molecules and protein binding has become more recognized with increasing numbers of fluorinated ligand–protein complex structures. Therefore, we studied the binding mode of 3 using a computational approach to determine the role of the w-chain, in particular the 15-F2 substituents, in receptor binding. The apo FP-receptor was obtained by homology modeling and simulated annealing by AMBER (see Experimental Section), and compound 2 was placed among transmembrane (TM) domain 1, 2, 3 and 7 based on the previous PGD2 binding studies. Compound 2 was oriented to allow 1-COO and 11OH groups to bind the FP-receptor residues Arg 291 and His 81, respectively, which are indispensable for ligand binding. The binding mode predicted by molecular dynamics (MD) simulations was similar to that obtained in the PGD2 study; the 15-OH group forms hydrogen bonds with Asn 44NH and Asp 77O. A PGF2a analogue (4), [11] with reduced hydrogen bond donating ability, displayed fourto five-times weaker activity than compound 2, consistent with the simulated 15-OH binding mode. A marked loss of activity was observed for 15-deoxy-PGF2a (5), suggesting that the 15-F2 group of 3 plays a critical role in binding, analogous to the 15-OH of compound 2. Similar to 2, the binding geometry of 3 was obtained by MD simulations (2.6 ns) with distance constraints between the fluorine atoms and the neighboring Asn 44NH, Asp 77C, and Gln 297C (0–1 ns). The ligand retained its position after distance constraints were removed. Pro-S-F atom was closer to the polar Asn 44H2 atoms (2.54 and 2.58 ) [13] than pro-R-F atom, suggesting that it contributes more to ligand binding, as shown by the fluorine interaction energy estimate discussed below. This geometry did not appear to agree with the previous structure–activity relationship (SAR) studies, which proposed that (15 R)-mono-fluoro analogues of 2 had higher binding affinities than their (15 S)-isomers. However, several active PGF2a analogues with an inversed stereochemistry at carbon atom 15 15] may allow this geometry, since polar functional groups at the 15-position on the flexible w-chain are necessary for effective binding. A recent study suggested an inadequacy between molecular force field methods, except MMFF94, for estimating intermolecular interaction energies involving covalently bound fluorine atoms. Since the AMBER force field may display such propensity, a 3–receptor complex model structure was optimized using a fragment molecular orbital (FMO) method just after the 1 ns MD run. FMO allows typical amino acid units to be treated as CONHCR fragments in proteins and is also applicable to ligands (Figure 1). The two ligands obtained in the optimized model structures (see Experimental Section) overlapped well with one another. While no significant change was observed in compound 2, a small structural change about the phenoxy oxygen atom and its proximity was observed for compound 3 (Figure 2). For covalently bound fluorine atoms, characteristic “orthogonal interactions” have been reported between C F moieties Figure 1. Tafluprost and PGF2a. The dotted curves define the a-chain, cyclic, and w-chain fragments for the FMO calculation. In compound 3, R = pro-R-F and R = pro-S-F.