Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies

Abstract

N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region.

Keywords: Acetamidine; inhibitor; molecular docking; nitric oxide synthases; selective; synthesis.

Figure 1

Structures of 1400W and its benzyl derivatives 1 and 2.

Figure 2

Target molecule structures.

Scheme 1. Synthesis of 9–13

Reagents and conditions: (a) 1400W, iBuOCOCl, NMM, DMF_dry, N₂, 0 °C to r.t., 24 h; (b) 4 N HCl, dioxane, r.t., 24 h.

Scheme 2. Synthesis of 14

Reagents and conditions: (a) 1400W, TEA, DMAP, DMF_dry, N₂, 0 °C, 6 h; (b) 4 N HCl, dioxane, r.t., 24 h.

Figure 3

Effects of compound 10 on ACh-mediated NO-dependent vasodilation. Mesenteric vascular arteries (MVA) isolated from SD rats were contracted to ∼80% of maximal vasoconstriction with 10 μM NA. (A) Dose–response curves for ACh-induced vasorelaxation (0.01–3 μM/30 s) were obtained in MVA under control conditions and after pretreatment with 1400W (1 μM) and subsequent treatment with l-NAME (100 μM). (B–F) Dose–response curves for ACh-mediated vasorelaxation were obtained in MVA under control conditions, and after pretreatment with increasing concentrations of compound 10 and subsequent treatment with l-NAME. Results are mean ± SEM of duplicates from four independent experiments for each condition. Asterisks refer to significant differences found between indicated curves assessed by two way ANOVA for repeated measures. *p < 0.05; **p < 0.01 vs l-NAME (panels A–C) or CTRL (panels D–F) curves, respectively.

Figure 4

Effects of compound 10 on NA-induced vasoconstriction. (A) Aortas were isolated from SD rats injected with vehicle or LPS. Samples were homogenized and subjected to immunoblotting with iNOS, eNOS or β-actin antibodies. Representative blot shows overexpression of iNOS from vessels of LPS-treated, but not of vehicle-treated animals. (B) Dose–response curves for NA-induced vasoconstriction (10 nM–10 μM) were obtained in MVA under basal conditions (CTRL), after pretreatment with compound 10, and after subsequent treatment with l-NAME and 1400W. Maximal vasoconstriction to individual NA doses (peak effect) was measured as perfusion pressure value and expressed in mmHg. (C) Duration of vasoconstriction obtained with individual NA doses (10 nM–10 μM) was calculated as area under the curve (AUC) and expressed as perfusion pressure (mmHg) × time (sec). Values were compared among curves obtained under CTRL conditions, pretreatment with compound 10, and subsequent pretreatment with l-NAME and 1400W. Results are mean ± SEM of duplicates from four independent experiments repeated for each condition. Symbols refer to significant differences between NA doses under treatment with compound 10 vs CTRL (*p < 0.05), vs l-NAME (°p < 0.05), and 1400W (^†p < 0.05), respectively.

Figure 5

Best ranking pose of 14 in (left) iNOS and (right) eNOS binding sites. The H-bonds between ligand and receptor are represented by yellow dotted lines; in gray the side chains involved in H bonds with ligand, and in orange the cofactor heme.