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A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.

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.

In the literature, the introduction of fluorine into bioactive molecules has been known to enhance the biological activity relative to the parent molecule. Described in this article is the synthesis of 4R-fluoro-L-NIL (12) and 4,4-difluoro-L-NIL (23) as part of our iNOS program. Both 12 and 23 were found to be selective iNOS inhibitors as shown in Table 2 below. Secondarily, methodology to synthesize orthogonally protected 4-fluoro-L-lysine and 4,4-difluoro-L-lysine has been developed.

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