CHARACTERIZATION OF THE RADICAL TRAPPING ACTIVITY OF A NOVEL SERIES OF CYCLIC NITRONE SPIN TRAPS

alpha-Phenyl-tert-butyl nitrone (PEN) is a nitrone spin trap, which ha s shown efficacy in animal models of oxidative stress, including strok e, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them f or radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O-2) and hydroxyl radical ((OH)-O-.) trappin g activity as determined biochemically and using electron spin resonan ce (ESR) spectroscopy. All cyclic nitrones tested were much more poten t as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than P BN. An analysis of the analogs of MDL 101,002 revealed a direct correl ation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, in as m uch as the calculated octanol/water partition coefficient for MDL 101, 002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nit rones are inherently more effective radical traps than PBN in a membra ne system. The most active compound was a dichloro analog in the seven -membered ring series (MDL 104,342), which had an IC50 of 26 mu M, whi ch was 550-fold better than that of PBN. The cyclic nitrones were show n to trap (OH)-O-. with MDL 101,002 being 20-25 times more active than PBN as assessed using 2-deoxyribose and p-nitroso dimethylaniline as substrates, respectively. Trapping of (OH)-O-. by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, t he (OH)-O-. adduct of MDL 101,002 yielded a six-line spectrum with hyp erfine coupling constants distinct from that of PEN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less th an 1 min at physiologic pH. MDL 101,002 also trapped the O-2, radical to yield a six-line spectrum with coupling constants very distinct fro m that of the (OH)-O-. adduct. In mice, the cyclic nitrones ameliorate d the damaging effects of oxidative stress induced by ferrous iron inj ection into brain tissue. Similar protection was not afforded by the l ipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the i n vivo activity, the stability of the nitroxide adducts, and the abili ty to distinguish between trapping of (OH)-O-. and O-2 suggest the cyc lic nitrones to be ideal reagents for the study of oxidative cell inju ry.