SPONTANEOUS REARRANGEMENT OF AMINOALKYLISOTHIOUREAS INTO MERCAPTOALKYLGUANIDINES, A NOVEL CLASS OF NITRIC-OXIDE SYNTHASE INHIBITORS WITH SELECTIVITY TOWARDS THE INDUCIBLE ISOFORM

1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas . Unlike most L-arginine based inhibitors, however, some guanidines an d S-alkylisothioureas, in particular aminoethylisothiourea (AETU), sho w selectivity towards the inducible isoform (iNOS) over the constituti ve isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitro gens, potently inhibit nitrite formation by immunostimulated J774 macr ophages (a model of iNOS activity) with EC(50) values ranging from 6-3 0 mu M (EC(50) values for N-G-methyl-L-arginine (L-NMA) and N-G-nitro- L-arginine were 159 and > 1000 mu M, respectively). The inhibitory eff ects of these aminoalkylisothioureas (AATUs) were attentuated by L-arg inine in the incubation medium, indicating that these agents may compe te with L-arginine for its binding site on NOS. 3 The above AATUs unde rgo chemical conversion in neutral or basic solution (pH 7 or above) a s indicated by (1) the disappearance of AATUs from solution as measure d by h.p.1.c., (2) the generation of free thiols not previously presen t and (3) the isolation of species (as picrate and flavianate salts) f rom neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were pr epared and shown to be potent inhibitors of iNOS activity with EC(50)s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intr amolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiolo gical pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than N-G -methyl-L-arginine and N-G-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrull ine in homogenates of bovine endothelial cells and by their presser ef fects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and m ercaptoethylguanidine (MEG), when given as infusions, gave slight decr eases in MAP in control rats. However, infusions of AETU or MEG to end otoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8 High doses of MEG (30-60 mg kg(-1)) c aused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanid inoethyldisulphide (GED), which caused pronounced, transient hypotensi ve responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AAT Us and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-amino thiazoline, in addition to MEG. This may account for the differences i n the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothio ureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibi tors of iNOS.