Simplified 2-aminoquinoline-based scaffold for potent and selective neuronal nitric oxide synthase inhibition

Abstract

Since high levels of nitric oxide (NO) are implicated in neurodegenerative disorders, inhibition of the neuronal isoform of nitric oxide synthase (nNOS) and reduction of NO levels are therapeutically desirable. Nonetheless, many nNOS inhibitors mimic l-arginine and are poorly bioavailable. 2-Aminoquinoline-based scaffolds were designed with the hope that they could (a) mimic aminopyridines as potent, isoform-selective arginine isosteres and (b) possess chemical properties more conducive to oral bioavailability and CNS penetration. A series of these compounds was synthesized and assayed against purified nNOS enzymes, endothelial NOS (eNOS), and inducible NOS (iNOS). Several compounds built on a 7-substituted 2-aminoquinoline core are potent and isoform-selective; X-ray crystallography indicates that aminoquinolines exert inhibitory effects by mimicking substrate interactions with the conserved active site glutamate residue. The most potent and selective compounds, 7 and 15, were tested in a Caco-2 assay and showed good permeability and low efflux, suggesting high potential for oral bioavailability.

Figure 1

Representative nNOS inhibitors discussed in this study.

Figure 2

Inhibitor design strategy and 2-aminoquinolines synthesized in this study.

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Scheme 6

Figure 3

Active site structures of lead 5 bound to rat nNOS (a) and bovine eNOS (b). The omit F_o – F_c density map for the inhibitor is shown at the 2.5 σ contour level. Major hydrogen bonds are shown as dashed lines. In each panel, the four residues that line a hydrophobic pocket are highlighted by a dot surface representation. While residues in chain A of nNOS (a) and eNOS (b) are colored green and cyan, respectively, the residue from chain B (second monomer in the homodimeric structure) is distinguished by a different color. The same color scheme is used in the other figures as well. Figures were prepared with PyMol (www.pymol.org).

Figure 4

(a) Active site structure of 6 bound to nNOS. The omit F_o – F_c density map for the inhibitor is shown at the 2.5 σ contour level. The fluorophenethyl tail is partially disordered with weaker density. (b) Overlay of 5 (cyan) and 6 (yellow) in nNOS. The different tilt angles of the aminoquinoline ring relative to the heme plane is in part related to whether hydrogen bonds (dashed lines) from the heme propionates to the linker amine are present (compound 5) or absent (compound 6).

Figure 5

Active site structure of 7 (a) or 9 (b) bound to nNOS. The omit F_o – F_c density map for the inhibitor is shown at the 2.5 σ contour level. The fluorophenethyl tail of 7 shows weaker density, indicative of disordering. Major hydrogen bonds are shown as dashed lines.

Figure 6

Active site structure of 8 (a) or 15 (b) bound to nNOS. The omit F_o – F_c density map for the inhibitor is shown at the 2.5 σ contour level. The chlorophenethyl tail of 15 is partially disordered with weaker density. Major hydrogen bonds are shown as dashed lines.

Figure 7

Active site structure of 7 bound to eNOS. The omit F_o – F_c density map for the inhibitor is shown at the 2.5 σ contour level. The fluorophenethyl tail of 7 shows weaker density indicative of partial disordering. Major hydrogen bonds are shown as dashed lines.