Selective heterocyclic amidine inhibitors of human inducible nitric oxide synthase.

The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC(50)'s) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500x selectivity versus hec-NOS.

2-Iminohomopiperidinium salts as selective inhibitors of inducible nitric oxide synthase (iNOS).

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release has been implicated in the pathology of these diseases. A series of monosubstituted iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and 7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50 ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase in plasma nitrite/nitrate levels in a rat LPS model.

Substituted 2-iminopiperidines as inhibitors of human nitric oxide synthase isoforms.

A series of analogues of 2-iminopiperidine have been prepared and shown to be potent inhibitors of the human nitric oxide synthase (NOS) isoforms. Methyl substitutions on the 4-position (3) or 4- and 6-positions (8) afforded the most potent analogues. These compounds exhibited IC50 values of 0.1 and 0.08 microM, respectively, for hiNOS inhibition. Substitution with cyclohexylmethyl at the 6-position (13) afforded an inhibitor that showed the best selectivity for hiNOS versus heNOS (heNOS IC50/hiNOS IC50 = 64). Following oral administration, inhibitors were found to decrease serum nitrite/nitrate levels in an in vivo rat endotoxin assay. This series of 2-iminopiperidines were prepared via the described synthetic methodologies. The effect of ring substitutions on potency and selectivity for this class of cyclic amidines as NOS inhibitors is described.

Inhibitors of human nitric oxide synthase isoforms with the carbamidine moiety as a common structural element.

Identification of potent and selective inhibitors of inducible nitric oxide synthase (NOS) is of great interest because of their therapeutic potential for treatment of diseases mediated by excess production of nitric oxide. We present here a comparison of potency and selectivity for amino acid and nonamino acid based compounds as inhibitors of human inducible, human endothelial constitutive and human neuronal constitutive NOS isoforms. In addition, a novel series of substituted amidines has been identified as NOS inhibitors. 2-Methylthioacetamidine and 2-thienylcarbamidine were the most potent of the series examined with IC50 values of 3.9 and 2.9 microM for human neuronal constitutive NOS. Cyclopropylcarbamidine and 2-thienylcarbamidine were the most potent inhibitors for human inducible NOS with IC50 values of 5.2 and 6.5 microM, respectively. These substituted amidines represent a new class of NOS inhibitors and provide a foundation for potential therapeutic agents.

2-Iminopiperidine and other 2-iminoazaheterocycles as potent inhibitors of human nitric oxide synthase isoforms.

A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were the most potent inhibitors, with IC50 values of 1.0 and 2.0 microM, respectively, for human inducible nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS inhibitors and the foundation for potential therapeutic agents.

Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase.

Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10-12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl-L-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl-L-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl-L-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase.

L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase.

L-N6-(1-Iminoethyl)lysine (L-NIL) has been synthesized and is shown to be both a potent and selective inhibitor of mouse inducible nitric oxide synthase (miNOS). L-NIL has an IC50 of 3.3 microM for miNOS compared to an IC50 of 92 microM for rat brain constitutive NOS indicating that L-NIL is 28-fold more selective for inducible NOS. L-N5-(1-Iminoethyl)ornithine (L-NIO), which differs from L-NIL by having one less methylene group, has very similar potency for inducible NOS, but lacks selectivity. DL-N7-(1-Iminoethyl)homolysine was also synthesized and found to be substantially less potent than L-NIL or L-NIO, with intermediate selectivity for inducible NOS. These data suggest that L-NIL may be useful as a selective inhibitor of inducible NOS for determining the role of this enzyme in disease models.