Synthesis of the pterin carboxamides was achieved as shown

Synthesis of the pterin-7-carboxamides was achieved, as shown in , based on a previously reported method that utilizes DBU as a key additive to dissolve pterin in organic solvents and accelerate the reaction., With this method, the pterin-amino NG,NG-dimethyl-L-Arginine hydrochloride conjugates (–) were readily obtained by simply mixing pterin-7-carboxymethyl (7MCP) and unprotected amino acids in the presence of DBU in methanol. The ALR2 inhibitory activity of the synthesized pterin-7-carboxamides (–) was evaluated in vitro by measuring their inhibitory effects on the reduction of ,-glyceraldehyde with recombinant h-ALR2 in the presence of coenzyme NADPH as a reductant. When h-ALR2 reduces glyceraldehyde, coenzyme NADPH is oxidized to NADP. Therefore, the progress of the reduction reaction can be monitored spectrophotometrically by measuring the absorbance of NADPH at 340nm. This method has been proven reliable. The respective h-ALR2 inhibitory activities of – were reported as their IC values, which express the 50% inhibition concentration of the compounds in the bioreduction. We also measured the inhibitory activity of pterincarboxylic acids ( and ) and folic acid for comparison with that of epalrestat used as a positive standard. All of the IC values are compiled in . It was found that exhibited the highest inhibitory activity (IC=1.97μM). This value was almost comparable to that of sorbinil, a highly active ARI (IC∼2μM). Compounds and , which have a carboxy group directly attached to the pterin ring, yielded poor results. Compound was found to be insoluble in the assay medium. Compound showed some inhibition, with an IC=26.5μM, suggesting that a linker between the pterin ring and the carboxyl group is required. Folic acid exhibited weak but explicit inhibitory activity (IC=52.5μM). Owing to the synthetic difficulty, we have not constructed a library of 6-substitited pterin, but this result left the door open for further investigation of ALR2 inhibitory activity of 6-substituted pterins. A careful examination of the observed h-ALR2 inhibitory data for – reveals some structure–activity trends. First, compounds having -amino acid residues showed markedly higher inhibitory activity than the -counterparts (–), indicating that pterins conjugated with -amino acids are well suited for the polypeptide environment composed of -amino acids. Evaluation of the inhibitory activity of compounds having hydrophobic residues (–) showed that the activity apparently decreased with increasing size of the residues. To assess this observation quantitatively, the correlation between the pIC values and the physicochemical parameters presented in was examined by means of the multiple linear regression analysis. Here, π represents the hydrophobicity parameter for the amino acid residues, ν is the upsilon steric parameter, and L, B, and B are the STERIMOL length and maximum and minimum width parameters, respectively. The following equation was obtained from the analysis as a statistically significant model:Despite the limited number of samples (=10), the high coefficient of determination (=0.951) and low standard error (=0.163) revealed that a good correlation was obtained. The Fischer test proved this equation to be significant with 99% confidence interval (<0.01). It is obvious that the inhibitory activity can be explained predominantly with the steric parameters, B and B. The negative sign of the coefficients for these parameters denotes the decrease in activity with an increase in the bulkiness of the amino acid side chain. A plot of the experimental versus predicted inhibitory activity using this model proved the predictive potential of this model (). When the IC values of and are compared with those of and , respectively, it was found that replacing one hydrogen in the residues of Ala and Phe with a hydroxy group, giving Ser and Tyr, respectively, markedly decreases the activity. Perhaps the hydroxy groups allow additional hydrogen bonding that negatively influences the fitting of the substrate to the active site.