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

Abstract

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, Caco-2 assay also revealed improved membrane permeability over previous compounds.

Improvement of Cell Permeability of Human Neuronal Nitric Oxide Synthase Inhibitors Using Potent and Selective 2-Aminopyridine-Based Scaffolds with a Fluorobenzene Linker

“…22, 24 Intermediate 27 , containing a cyanophenyl linker, was synthesized from bromophenyl precursor 22d by treatment with CuCN in DMF at 150 °C. Sonogashira coupling was then performed on 27 to install the amine tails.…”

“…Using a benzonitrile ring as the middle aromatic linker had some success in the 2-aminoquinoline- 22 and the 2-aminopyridine- 24 containing inhibitors in the past, but it showed the opposite effects with compounds 10 or 14 here. We were able to obtain the structures of rnNOS- 10 (Figure 5A) and hnNOS- 10 (Figure 5B).…”

“…The crystal growth conditions were as described previously. 22 Fresh crystals were first passed stepwise through cryoprotectant solutions and then soaked with 5–10 mM inhibitor for 3–4 h at 4 °C before being flash cooled with liquid nitrogen and stored until data collection. The presence of an acetate ion near the heme active site in bovine eNOS caused interference in the binding mode of some inhibitors.…”

“…17 These inhibitors, however, still suffer from low membrane permeability and poor ability to cross the BBB as a result of their high basicity and/or polarity. In the past few years our group has pursued various medicinal chemistry approaches to improve the pharmacokinetic values of nNOS inhibitors, such as incorporating intramolecular hydrogen bonds ( 1 ), 18 converting to prodrugs ( 2 ), 19 modulating the amine basicity ( 3 ), 20 and replacing an essential pharmacophore, such as replacement of the 2-aminopyridine with a 2-imidazolylpyrimidine ( 4 ) 21 or 2-aminoquinoline ( 5 ) 22 (Figure 1). Although some pharmacokinetic improvement has been achieved, these approaches can result in a diminution in activity, especially for human nNOS, the ultimate target for neurodegenerative diseases, and in the selectivity over human eNOS.…”

Overview of Recent Strategic Advances in Medicinal Chemistry

“…Water-mediated interactions, especially hydrogen bonds, play key roles in drug binding. 236,237 Careful examination of these water molecules and their energetics can contribute to successful drug design, as exemplified by neuronal nitric oxide synthase inhibitors, nonpeptidic urea HIV protease inhibitors and benzoxaborole non-nucleoside polymerase inhibitors of HCV. [238][239][240] Recently, several structural and computational studies to explore water-binding pockets have been reported.…”