Although several ARIs have reached various phases

Although several ARIs have reached various phases of clinical experimentation however most of them have been withdrawn either due to an insufficient bioavailability, their poor efficacy or adverse side effects. Currently Epalrestat is in clinical use for the treatment of diabetic neuropathy. The side effects are due to a lack of selectivity over the aldehyde reductase (ALR1, EC 1.1.1.2), which is a cytosolic enzyme of the AKR superfamily and is closely related to ALR2, showing high structural homology (65% identity in their amino KU57788 sequences), substrate specificities and kinetic mechanisms. ALR1 plays a detoxification role in the efficient removal of toxic aldehydes such as hydroxynonenal (HNE), methylglyoxal and 3-deoxyglucosone arising from pathological conditions connected with oxidative stress, like diabetes.20, 21 ALR2 is a monomeric (α/β)8-barrel cytoplasmatic protein of 36kDa composed of 316 amino acid residues. The active site is located in the barrel core at the bottom of a large and deep hydrophobic pocket at the C-terminal of the barrel. The pyridine cofactor NADP+ is bound at the bottom of the cleft through a flexible loop named the “safety-belt” loop. Through a comparative analysis of the ALR2-inhibitor complex crystal structures it has been suggested that the ALR binding site splits into two parts with different flexibility properties. The first portion, mainly formed by the residues of the catalytic site (Trp20, Tyr48, Val47, His110, Trp49 and Trp111) and the flanking cofactor NADP+, is named the “anion binding pocket”. The second portion of the binding site is flexible and affects the segments Leu300 and Trp111 that are determinant for the appearance of a “specificity pocket”, also named the “induced cavity region”, which possesses hydrophobic features and shows a high degree of flexibility. This pocket can adopt different conformations depending on the size and nature of the bound ligand.25, 26 These features constitute important pharmacophoric requirements for the design of selective inhibitors binding to ALR2. In particular, among the members of the AKR superfamily the hydrophobic amino acid residues present in the specific pocket of the ALR2 active site are less conserved than the hydrophilic ones providing the structural basis for selectivity. The flexibility of the binding site of ALR2 permits accommodating a wide variety of small molecules with different shape and size. Although ALR2 inhibitors are structurally different, they have two common features: one or more aromatic moieties able to open the “specificity pocket” which possesses a high degree of induced-fit adaptations (useful to arrange structurally different ligands), and an acidic moiety which can interact with the “anionic binding pocket” by establishing ionic and/or hydrogen bond interactions with Tyr48 and His110. In previous papers from our laboratory we have studied several series of glycine (R=H, n=0) and β-alanine (R=H, n=1) derivatives (Fig. 3) as ARIs.27, 28, 29 In these studies, we have extensively examined the structure-activity relationships (SARs) within this class and explored the effects on inhibitory activity of aldose reductase by increasing both the distance between the aromatic portion and the carboxylic group and the nitrogen core, and by inserting a methylene spacer between them. Moreover, we have explored the effect obtained by shifting the phenyl ring away from the carbonyl group by inserting various spacers of different lengths and degrees of rigidity. The biological results indicate that the lengthened derivatives are in general less active with respect to the shorter ones. Thus, these distances between the pharmacophoric groups appeared to be a crucial element in determining the best spatial relationship between them within this structural class of compounds. In the present study we investigated three series of compounds starting from the structure of the most active compounds reported previously27, 28, 29 (R=H, Cl and R1=OMe, n=0, IC50=3.5μM and 6.3μM, respectively A and B of Fig. 4).