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-cit rilline 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 r eported 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 ma st 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 (>150 0-fold) with excellent selectivity over iNOS (190-fold). Here we describe t he design and synthesis of a series of peptidomimetic analogues of this dip eptide as potential selective inhibitors of nNOS. The biochemical evaluatio n of these compounds also revealed the binding requirements of the dipeptid e inhibitors with NOS. Incorporation of protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decrease s in the inhibitory potency of nNOS. Masking the NH group of the peptide bo nd (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNO S 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 include d converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which reve aled less restricted binding requirements for the C-terminus of the dipepti de. Further optimization should be possible when we learn more about the bi nding requirements at the active sites of NOSs.