Metabolic activation of sulfur mustard leads to oxygen free radical formation.

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 µM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.

A plethora of carbene interconversions on the C(5)H(4)S energy surface: a computational study.

The potential energy surface for the reaction of atomic carbon with thiophene has been studied computationally. Intermediates which are energetically viable include the 2- and 3-thienylcarbenes 8 and 11, thiacyclohexa-3,5-dien-2-ylidene, 10, and thiacyclohexa-2,3,5-triene, 6. In accord with experimental data, 6 and 8 are in equilibrium. The lowest-energy pathway for rearrangement of 6 to 8, which is endothermic by 14.5 kcal/mol, involves ring opening to Z-2-penten-4-ynthial which then recloses to carbene 8. A 1,4 addition of C across the diene system in thiophene generates an ylid which rearranges with little or no barrier to cyclopentadienethione, the global minimum on this potential energy surface.

Discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase.

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (HNE) when the inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed specifically to afford a long residence time in the lung, it was not unexpected that inhibitors from this class of TFMKs were not active when administered orally. Upon evaluating a large number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were identified which demonstrated oral activity. Compounds were evaluated for their oral activity by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/Bw), the increase in red blood cells, and the increase in white blood cells induced by intratracheally administered HNE (100 micrograms/hamster). A number of tripeptidyl trifluoromethyl ketones containing neutral N-terminal groups displayed good oral activity, while those containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal 4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%, and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when administered either orally or intratracheally.

Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil elastase. 3. In vitro and in vivo potency of a series of peptidyl alpha-ketobenzoxazoles.

A series of peptidyl alpha-ketobenzoxazoles were synthesized and evaluated for their in vitro and in vivo inhibition of human neutrophil elastase (HNE). These compounds inhibit HNE by forming both a covalent bond between the ketone carbonyl carbon atom and the hydroxyl group of Ser-195 and a hydrogen bond between the benzoxazole nitrogen atom and His-57. Appending to the parent benzoxazole ring a variety of substituents which spanned a range of physicochemical properties had only a modest effect on in vitro potency (Ki = 3-0.4 nM). This apparent lack of a significant effect is believed to result from the fact that any increased ketone carbonyl activation by the ring substituent is counter balanced by a corresponding decrease in the hydrogen-bonding ability of the benzoxazole nitrogen atom. In contrast to the results in vitro, maximizing in vivo activity was critically dependent upon the choice of the benzoxazole ring substituent. Several substituted peptidyl alpha-ketobenzoxazoles effectively inhibited HNE-induced lung injury when administered intratracheally 24 h prior to the enzyme.

Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones.

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS)