NG-nitro-L-arginine-containing peptidomimetics as selective neuronal nitric oxide synthase inhibitors. Structure-based design, synthesis, and biological evaluation and approaches toward enhanced CNS activity

Nitric oxide (NO) is a crucial signaling molecule in a diverse array of physiological events. Endogenous NO synthesis is carried out by a family of enzymes known as nitric oxide synthase (NOS). The distribution of different isoforms of NOS is usually related to their distinct physiological functions: neuronal NOS to neurotransmission, endothelial NOS to vascular homeostasis, and inducible NOS to immune response. As a result of the broad physiological role of NO, disordered generation of NO is associated with various pathological states. Hence, isoform-selective regulation of the localized excess production of NO has been targeted as a potential means of treating various diseases.;As highly selective neuronal nitric oxide synthase (nNOS) inhibitors, NG-nitro-L-arginine-containing dipeptidomimetics have been developed. X-ray crystal structures of complexes of nNOS with nNOS-selective dipeptidomimetic inhibitors led to the discovery of a conserved structural water molecule that forms a hydrogen bond between the active site heme propionates and the inhibitors. Based on this observation, we hypothesized that the attachment of a hydrogen bond donor group to the inhibitors might enable the inhibitors to displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues, which have either an N-hydroxyl or N-amino donor group, were designed and synthesized. X-ray crystal structures of nNOS with those inhibitors bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety. In vitro activity assay results indicated that the addition of a hydroxyl group slightly increased the potency against the neuronal isoform over the parent compound.;As an approach toward improved in vivo activity of the N G-nitro-L-arginine-containing dipeptidomimetic inhibitors, physico-chemical properties of the inhibitors were modified by two strategies; (1) masking a polar amine functionality with a pro-moiety, (2) rational modifications of the inhibitors for higher lipophilicity while maintaining the inhibitory activity. In the former strategy, (pivaloyloxy)ethyl carbamate prodrugs were designed and synthesized. The latter approach provided an aminopyrrolidine-sidechain dipeptidomimetic inhibitor with slightly increased in vitro potency over the parent compound.