Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase

Abstract

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions, and b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed this rearranged scaffold significantly reduces off-target binding.

Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2-Aminoquinoline Inhibitors

“…17 Unfortunately, 2 was selective for rat nNOS (rnNOS) over human nNOS (hnNOS), displayed low selectivity for human nNOS over human eNOS (heNOS), caused toxic side effects in rats, and was extremely promiscuous in CNS counterscreens. The second-generation, 18 rearranged phenyl ether 4 (optimized from lead 3 ), preserved the potency and selectivity of 1 and 2 while drastically decreasing the off-target binding, but this compound had significantly decreased Caco-2 permeability, low human nNOS activity, and similarly low selectivity for hnNOS over heNOS.…”

“…23 Van der Waals contact between inhibitors and this residue has been implicated in improved n/e selectivity, as this residue is replaced by a smaller valine in eNOS isoforms. 18 Similarly, compound 17 combines a 4-substitution pattern (as in 4 ) with the 5-cyano group.…”

Nitric oxide synthase and structure-based inhibitor design

“…Most recently the 2-aminoquinoline group (Fig. 9) was chosen to replace 2-aminopyridine [73,74]. Compounds were first screened with in vitro inhibitory assays and optimized via structural approaches against both rat and human enzymes.…”

“…Compounds were first screened with in vitro inhibitory assays and optimized via structural approaches against both rat and human enzymes. 20 has a K i = 58 nM for nNOS and exhibits 216-fold selectivity over eNOS [74]. The oral bioavailability of the best candidates were further tested using Caco-2 cell permeability assays [75] to approximate the permeability of the gut epithelium.…”

“…The oral bioavailability of the best candidates were further tested using Caco-2 cell permeability assays [75] to approximate the permeability of the gut epithelium. For nNOS specific inhibitors attention was also paid [74] as to whether compounds showed any off-target binding to other receptors in the central nerve system (CNS) via t he Psychoactive Drug Screening Program (PDSP) [76]. One of the more recent studies has focused on imidazole inhibitors that coordinate the heme iron [77].…”