Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrilline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The mast potent nNOS inhibitor among these compounds is L-Arg(NO2)-L-Dbu-NH2 (1) (K-i = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than I. Both of the results demonstrate the importance of the cl-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.