Mechanism of Fluorine-induced Adrenergic Selectivities[unreadable] [unreadable] The important and diverse biological functions of adrenergic agonists have made the search for compounds that selectively stimulate or inhibit the activity of the different types of adrenergic receptors an important area of medicinal chemistry. In the 1980s we synthesized 2-,5-, and 6-flouronorepinehprine (FNE) and 2-,5-, and 5-fluoroepineprhine (FEPI) and found that fluorine in the 2-position inhibited binding to alpha-adrenergic receptors and fluorine in the 6-position of inhibited binding to beta-adrenergic receptors. We now are using a combination of molecular modeling (docking experiments) and site specific mutagenesis to attempt to identify which amino acid residues on these receptors are responsible for fluorine-induced adrenergic selectivities.[unreadable] [unreadable] Preliminary computational studies of epinephrine binding to receptors alpha-1b and beta-2 show differences in composition of the binding pocket with the following pairs of residues occupying comparable spatial relationships with the ligands:[unreadable] [unreadable] Alpha-1b Cys129 Gly196 Val197 Leu314[unreadable] Beta-2 Val117 Asp192 Phe193 Asn293[unreadable] [unreadable] The next step consisted of using point mutations to switch these positions. [unreadable] [unreadable] N293L showed promising results: the affinity for 2FNE significantly decreased (more than 10 times) whereas the affinity for 6-FNE was significantly improved. Thus, replacement of the polar group of Asn - 293 with a non polar group from Leu caused a shift in selectivity.[unreadable] [unreadable] Further investigation by using a smaller non-polar group (Ala) or a larger non polar group (Phe) revealed that N293F made this mutant receptor 6-FNE (alpha)-selective (Ki = 6.91) relative to 2-FNe (Ki = 21.59). (The brta-2 wild type was originally much more selective for 2-FNE).[unreadable] [unreadable] To further investigate the binding pocket of the receptors, chimeras were prepared by replacing the 5th transmembrane helix of the b2 adrenergic receptors and mutants by the helix of a1b adrenergic receptor, since this helix seems to interact with the aromatic OH groups on the ligands. Binding data showed that the 5th transmembrane helix has little influence on the selectivity of the chimeras toward the ligands. The Ki values for 6-FNE of the mutants and chimeras were comparable.[unreadable] [unreadable] In the next stage of this iterative process, computations were carried out to determine the stable conformations of the ligands and several residues in the pocket of the beta-2 model in complexes with norepinephrine, 2F-norepinephrine, and 6F-norepinephrine, using Monte Carlo Multiple Minimum (MCMM) method as implemented in MacroModel 9.5 (Schrdinger, LLC). A total of 1000 search steps were performed using OPLS2005 force field. Energy minimization was employed with the Polak-Ribiere Conjugate Gradient (PRCG) procedure until convergence to the gradient threshold of 0.05 kJmol.[unreadable] [unreadable] We looked first at the native beta-2 receptor, incorporating the hydrogen bonds with key serine and aspartate residues. A key difference in the binding of 2-FNE and 6-FNE is seen in that the C-F bond is pointed into a hydrophobic pocket with 2-FNE and away from the hydrophobic pocket with 6-FNE.[unreadable] [unreadable] Similar calculations done with the N to F mutation showed that the binding pocket in this construct was constricted, causing fluorine in 6-FNE to flip and point into a hydrophobic region with aromatic residues. Although calculations with the N293A mutant have not been done, we assume that alanine is not large enough to cause a similar shift. [unreadable] [unreadable] Although speculative at this time, this could provide an explanation for the selectivity of the beta-2 receptor. According to the polar hydrophobic effect, placing the C-F bond of 6-fluoronorepinephrine into the hydrophobic pocket should cause fluorine to shed any residual water molecules, and event that will be attended by a substantial gain in entropy of binding. The fact that the C-F bond of 2FNE is already in this pocket in the WT receptor would be reflected in favorable binding of this analogue to the WT receptor.[unreadable] [unreadable] Fluorinated Indoles[unreadable] [unreadable] We had previously prepared 4-fluoro, 5-fluor-, 6-fluoro- and 4,6-difluoroserotonins and melatonins to study the effects of fluorine substitution on the biological properties of these important bioindoles. We now have expanded this inventory of fluorinated analogues by the synthesis of 7-fluoro- and 6,7-difluoroserotonin and melatonin by condensation of the appropriately substituted phenylhydrazine with 4-aminobutyraldehyyde diethyl acetal under acidic conditions.[unreadable] [unreadable] The affinities of the ring-fluorinated serotonins have been evaluated at 5HT1A, 5HT2A, 5HT2B, and 5HT5A receptors. Although there are individual differences in binding, the trends and orders of magnitude for the fluorinated compounds were comparable to serotonin. Thus, ring fluorination appears not to have dramatic effect such as seen with the adrenergic agonists, norepinephrine and epinephrine.[unreadable] [unreadable] A capsaicin fluorine scan.[unreadable] [unreadable] Capsaicin (8-methyl-non-6-enoic acid 4-hydroxy-3-methoxy-benzylamide) is the active component of hot chili peppers. Capsaicin binds to the vanilliod receptor subtype 1, an ion channel receptor that is also activated by heat, physical abrasion, and acidic pH. Drugs binding to this receptor have applications in pain alleviation. Interactions of aromatic rings with receptors and enzyme active sites can be probed by replacing each available hydrogen with a fluorine atom. The effect of this substitution on biological activity is then examined. This is now termed a fluorine scan. We have prepared the requisite carboxylic acid and this has been coupled to 5-fluoro- and 6-fluorovanillyl amine (prepared from the corresponding vanillins by reductive amination) to produce the mono-ring fluorinated analogues of capsaicin. To complete the series work is in progress to prepare the required 2-fluorovanillin from 2-aminovanillin using a photochemical Schiemann reaction. Following completion of the syntheses, effects of fluorine substitution on receptor binding will be studied.