A straightforward synthetic pathway was
A straightforward synthetic pathway was adopted to synthesize the target compounds as outlined in . The starting chloromethylquinazolinones (–) were synthesized from anthranilic AS-605240 in two steps following reported procedures., , , The first step involves chloroacetylation of anthranilic acid using chloroacetyl chloride in dry benzene under reflux conditions. In the second step, cyclization to the desired chloromethylquinazolinones was achieved by direct reaction of the -chloroacetyl anthranilic acid derivatives with the appropriate anilines in presence of phosphorous oxychloride as a condensing agent in dry toluene. Subsequently, 4-hydroxybenzaldehyde or vanillin was alkylated with the chloromethylquinazolinones (–) in refluxing acetonitrile under the basic conditions of potassium carbonate and in the presence of potassium iodide to afford the aldehydes (–) in good yields as previously reported by us. The title rhodanineacetic acids (–) were obtained in fair yields via Knoevenagel-type condensation of the aldehydes with rhodanine-3-acetic acid in refluxing acetic acid using β-alanine as a condensing agent. On the other hand, compounds – were obtained by condensation of the appropriate aldehyde with rhodanine in the presence of sodium acetate. Structures of all the target compounds were fully characterized by means of H and C NMR spectroscopy and their purity were satisfactorily confirmed by elemental analysis. All the novel synthesized compounds were tested for their aldose reductase inhibitory activity following standard protocols., , , The obtained IC values are reported in . Our investigation started by synthesizing and testing four compounds of the benzylidene series, namely –. Their functional evaluation indicated that, moving from the hit (IC 60.9nM), the presence of the electron-withdrawing fluoro atom on the 3-phenyl ring of the quinazolinone core, as in (IC 55.6nM), was tolerated while the presence of the bulkier bromo atom, as in (IC 711nM), resulted in an almost 10-fold decrease of activity. Similarly, compound (IC 574nM), having an electron-donating 3-methyl substituent, turned out to be less active than the unsubstituted parent compound, . We than examined the effect on ALR-2 activity by inserting a 3-methoxy group on the central benzylidene ring. The hit compound of the novel series, namely (IC 49.7nM) turned out to be slightly more active than the unsubstituted counterpart (IC 60.9nM). On this basis, additional compounds of the 3-methoxybenzylidene series were synthesized and tested, bearing different electron-withdrawing and electron-donating substituents on the distal phenyl ring of the quinazolinone core. The observed functional data indicated that, regardless of both the type of the substituent and its position on the ring, peripheral substituents have a negative impact on ALR-2 activity. Actually, compared to the unsubstituted hit (IC 49.7nM), all the substituted compounds (–) showed IC values in the low micromolar range, as shown in . To test the importance of the presence of an acetic acid moiety, a series of representative rhodanines – was synthesized as well, and tested for their ALR-2 activity. All the compounds of the novel series displayed no or weak activity against the target enzyme, as shown in . To propose a structural binding mode of the rhodanine-3-acetic acid derivatives when bound to ALR2, docking experiments were carried out on the newly synthesized compounds starting from the most active, . It has been clearly proven that ALR2 can adopt different binding site conformations, depending on the structural characteristics of an inhibitor. To combat any computational bias, the target compound was docked into the binding pocket of the human ALR2/NADP-IDD594 complex (PDB code: ) using the automated Autodock Vina plugin package in UCSF chimera., Visual inspection of the docked pose with the best binding affinity (−11.05kcal/mol) (), revealed to bind favorably into the polar anion-binding pocket. Upon assessment of the residue-ligand interaction network, stabilizing hydrogen bond interactions were illustrated with residue Tyr48 and strong electrostatic interactions between and the nicotinamide moiety of the NADP cofactor. The presence of the rhodanine ring is also shown to well orient the planar and aromatic quinazolinone fragments into the “specificity pocket”, thus creating favorable hydrophobic bonds with Leu300.