Additional pathways of S-conjugate formation during the interaction of thiols with nitrosoarenes bearing pi-donating substituents.

During the well-established reaction pathways of nitrosoarenes interacting with thiols, a reactive N-(thiol-S-yl)-arylamine cation was implicated in the so-called rearrangement reaction which transforms the semimercaptal to the sulfinamide. In the case of nitrosoarenes with pi-donating substituents, this cationic transition state includes resonance structures bearing the positive charge in 2 and 4 position, thereby facilitating the attack of nucleophiles to the aromatic ring. Investigating the reaction products of 4-nitrosophenetol and reduced glutathione in chemical systems and human red cells, some glutatione S-conjugates were detected other than the already known sulfenamide and sulfinamide. Three of them were separated by HPLC and identified by mass spectroscopy, 1H-NMR, and UV-visible spectroscopy, by determination of pKa values and chemical behavior. The hitherto unknown conjugates are 4-ethoxy-2-(glutathione-S-yl)-aniline, N-(4-ethoxyphenyl)-N'-(glutathione-S-yl)-benzoquinonediimine, and 4-amino-4'-ethoxy-2-(glutathione-S-yl)-diphenylamine. In preliminary experiments, some of these conjugates were shown to be highly active in producing ferrihemoglobin. Considerations on the formation pathways of these metabolites lend further support to the electrophilic N-(glutathione-S-yl)arylamine cation as a reactive intermediate that may be implicated in nitrosoarene toxicity.


Introduction
Reactions of nitrosoarenes with cellular thiols are gaining increasing toxicologic interest (1). During the well-established reaction pathways leading to N-hydroxyarylamines, sulfinamides, and sulfenamides (2)(3)(4), reactive intermediates are formed, such as phenylnitroxide free radicals (5) and the metastable semimercaptals (6)(7)(8). Recently, an intermediate N-(thiol-S-yl)-arylamine cation was implicated during the so-called rearrangement reaction which transforms the semimercaptal to the sulfinamide (7,9). In the case of nitrosoarenes with i-donating substituents, this cationic transition state includes resonance structures bearing the positive charge in 2 and 4 position, thereby facilitating the attack of nucleo-philes to the aromatic ring (7). During the reaction of 4-nitrosophenetol (NOPt) with reduced glutathione (GSH) in chemical systems and human red cells, hitherto unknown glutathione S-conjugates were detected which confirm the intermediate occurrence of the electrophilic N-(glutathione-S-yl)-arylamine cation.
The corresponding ,-mercaptoethanol and t-butylmercaptan derivatives were syn-Environmental Health Perspectives thesized similarly at pH 11. t-Butylmercaptan reacted only during refluxing at 100°C for 17 hr. The products were purified by thin-layer chromatography on silica gel plates 60-F254, 0.25 mm thickness from E. Merck (Darmstadt, Germany), using chloroform/hexan mixtures as mobile phase.
'H-NMR spectra were recorded with an AM-400 MHz or an AM-500 MHz instrument from Bruker (Rheinstetten-Forchheim, Germany) with H20 set to 4.80 ppm. Electron impact mass spectra (EI-MS) and fast atom bombardmentmass spectra (FAB-MS) were recorded with  [u-ring-14C]-NOPt (5 mM) was added to GSH (in sodium phosphate buffer, 0.2 M, pH 7.4) and incubated for 0.5 hr at 37°C. Lipophilic products were removed by ether extraction. Samples were separated by HPLC on Novapak C18 with MeOH/sodium phosphate buffer (pH 7) gradient, detection at 220 to 320 nm (for structure see Figure 2). (A) 25 mM GSH; the hydrophilic metabolites amounted to 15% of total activity. (B) 1 mM GSH; the hydrophilic metabolites amounted to 5% of total activity. See Table 1 (14) with a LKB Rackbeta 1217 liquid scintillation counter. Human hemoglobin was obtained from outdated blood and purified by column chromatography as described (15). The ferrihemoglobin content was less than 5% throughout. Free SH groups (,B93 Cys) were alkylated with N-ethylmaleimide (16) and excess reagent removed by dialysis against phosphate buffer. Experiments on ferrihemoglobin formation by NOPt metabolites were carried out in sodium phosphate buffer (0.2 M, pH 7.4) at 37°C under free access of air. The volumes of added metabolite solutions were less than 3% throughout. Hemoglobin, ferrihemoglobin, and glutathione were determined as described previously (10).

Results and Discussion
During the HPLC separation of NOPt/ GSH incubates, some hydrophilic metabolites were detected other than the already known sulfenamide and sulfinamide ( Figure 1).

4-Ethoxcy-2-(glutathione-S-yl)-aniline
This compound was obtained in optimized yield from incubates of NOPt with 10-fold excess GSH (pH 7.0, 20°C). Positive reactions with Echtblausalz B and with ninhydrin indicated a primary aromatic amine containing a glutathione residue. Its FAB' mass spectrum showed the molecular ion at m/z = 443 (M+1) and main fragments at m/z = 368 (M-Gly), 314 (M+2-Glu), and 168 (M-Glu-Ala-Gly), indicating a glutathionyl substituted phenetidine. The UV-visible spectrum (Table 1) differed distinctly from that of the sulfenamide N-(glutathione-S-yl)-4-ethoxyaniline (6,9) but agreed with that of 2-(thiol-S-yl)-4hydroxyaniline (17). The 1H-NMR spectrum showed aliphatic signals belonging to the ethoxy group and the glutathionyl substituent and aromatic signals of three protons indicating a 1,2,4-substituted aromatic ring. From the signal shifts in acidic solution it was derived that the ortho position to the amino group is substituted by glutathione (Table 2). This structure was confirmed by the distinctly lower pKa of GS-NH2Pt compared to the parent amine NH2Pt (Table 1). [It has been shown previously that the pKa of primary aromatic amino groups is considerably decreased by ortho-substitution with thiols but hardly by meta-substitution (17)].

4-Amino-4'-ethoxy-2-(glutathione-Syi)-diphenylamine
This metabolite from NOPt/GSH incubates was obtained in much higher yields when NH2EDPA was oxidized to the corresponding benzoquinone diimine and added to GSH. Positive reactions with Echtblausalz B and with ninhydrin pointed to a primary arylamine substituted with glutathione. The FAB' mass spectrum revealed the molecular ion at m/z = 534 Table 2. 1H-NMR data of 4-ethoxy-2-(glutathione-S-yl)-aniline (GS-NH2Pt) and 4-phenetidine (NH2Pt) in neutral (D20) and acidic solution (DCI, pD-1).  showed aliphatic signals belonging to the ethoxy and the glutathionyl group; besides the two proton pairs of the ethoxyring, the aromatic signals indicated three single protons, of which two were shifted to lower field during acidification (Table 3). Therefore, the position of the glutathione substituent was assumed to be meta referred to the primary amino group.
Accordingly, the pKal of this amino group was only slightly decreased upon glutathione substitution (Table 1). [A similar compound has been proposed to occur during the peroxidase-catalyzed oxidation of NH2Pt in the presence of GSH (19)].

N-(4-Ethoxyphenyl)-N'-(glutathione-S-yl)-4-benzoquinone diimine
The orange compound was immediately formed at maximal yields in incubates of NOPt with about 2.5-fold excess GSH (pH 7.4, 200C); further GSH addition resulted in discoloring. Interestingly, this NOPt metabolite was obtained in nearly quantitative yield during the reaction of 4ethoxy-4'-nitrosodiphenylamine with equivalent amounts of GSH at pH 10. Identification of the glutathione derivative by FAB' and El mass spectra was unsuccessful as the compound discolored instantly, probably by reduction. However, the ,3-mercaptoethanol and t-butylmercaptan derivatives revealed reasonable' mass spectra (in a hardly reducing 3-nitrobenzylalcohol matrix), indicating a benzoquinone diimine substituted with one ethoxyphenyl and one thiol group (Table 4). H-NMR spectra of the three derivatives showed the aliphatic signals of the ethoxy group and the respective thiol. The aromatic protons (relative intensity: 7-8 protons), however, revealed a highly complex splitting pattern resulting from the signal overlap of the ethoxyphenyl ring with two cis-transisomeric forms of the quinone diimine ring (J Sonnenbichler, personal communication, 1992). In order to clarify the position of the glutathionyl substitution in this metabolite, some chemical experiments were undertaken. Reduction of N-(4-ethoxyphenyl)-N'-(glutathione-S-yl)-4-benzoquinone diimine (GS-EPQDI) with GSH delivered 4amino-4'-ethoxy-2-(glutathione-S-yl)diphenylamine (GS-NH2EDPA) as one of the products, indicating that GS-EPQDI may be the corresponding benzoquinone diimine of GS-NH2EDPA. However, Volume 102, Supplement 6, October 1994 Table 4. Fast atom bombardment and electron impact mass spectra of the glutathione-, 1-mercaptoethanol-, and t-butylmercaptan-derivatives of V-(4-ethoxyphenyl)-N'-(thiol-S-yl)-4-benzoquinonediimine. when GS-NH2EDPA was oxidized with lead dioxide with concomitant color change to orange, no compound with the UV-visible spectrum of GS-EPQDI was observed; reduction with dithionite restored part of GS-NH2EDPA, indicating that the ring-substituted benzoquinone diimine had actually been formed. Therefore, GS-EPQDI was supposed to contain glutathione not at a ring position but at the second imino nitrogen. This presumption was confirmed as mild reduction of GS-EPQDI with ascorbate liberated glutathione, which was proved enzymically. In the reaction of GS-EPQDI with fmercaptoethanol, no GS-NH2EDPA was found, only the ,-mercaptoethanol derivative. These findings are in agreement with the proposed structure.

S-Conjugate Formation
The reaction pathways of nitrosoarenes with aliphatic thiols are known to be distinctly influenced by their aryl substituents. Recent results indicate that the initially formed semimercaptal undergoes N-O cleavage to give an intermediate sulfenamide cation (7,9). The NOPt metabolites presented here further support that the sulfenamide cation may play a central role in NOPt metabolism (compare Figure 2): a) The formation of N-hydroxy-4phenetidine from the semimercaptal was not observed hitherto (20,21). As generally deduced by Kazanis and McClelland (7), the i-donating ethoxy substituent should favor the N-O cleavage with formation of a sulfenamide cation rather than the nucleophilic substitution of GS-at the semimercaptal sulfur with formation of the corresponding hydroxylamine. Accordingly, the semimercaptal that has been detected with 1-thioglycerol (21) is rapidly converted to the resonance-stabilized sulfenamide cation.
b) The formation of appreciable amounts of sulfinamide was only observed when GSH reacted with excess NOPt (9) ( Figure 1B). At excess GSH, however, this pathway was not followed ( Figure 1A), suggesting that the positive charge is highly delocalized to the ring carbon atoms because of the strong i-donating ethoxy substituent.
c) The formation of GS-NH2Pt at excess GSH (Figure 1) again indicated the intermediate occurrence of a resonance-stabilized sulfenamide cation, which is prone to nucleophilic ring addition of GSH. The resulting 2,N-bis-(glutathione-S-yl)-4phenetidine may be rapidly reduced by GSH or hydrolyzed-as observed with other sulfenamides-to give GS-NH2Pt. d) Reaction of NOPt with excess GSH mainly led to the sulfenamide which decomposed to NH2Pt (9). According to the reaction sequences proposed by Kazanis and McClelland (7), addition of GSto the para position of the sulfenamide cation and subsequent reduction by further GSH or hydrolysis would yield the sulfenamide. Because the para position is thought to have the major partial charge in the sulfen-amide cation, the high yields of NH2Pt would be reasonably explained.
e) The formation of bicyclic metabolites supported the consideration that the para position of the sulfenamide cation may have the lowest electron density. Obviously not only GS-but also the less-nucleophilic NOPt metabolite NH2Pt undergoes ring addition to this position, delivering the Nsulfenylquinonediimine GS-EPQDI by elimination of EtOH. [This reaction mechanism would be consistent with the acidcatalyzed amination of p-nitrosophenol ethers (12), where the initial attack of a proton at the nitroso oxygen produces a positive charge at the para position of the ring.] Accordingly, the reaction of NOPt with GSH in the presence of authentic NH2Pt (1:1:1) resulted in immediate production of GS-EPQDI at increased yields. This reaction was shown to occur also with other arylamines but not with alkylamines.
f) Reduction of authentic GS-EPQDI with ascorbate resulted in discoloring and production of a metastable compoundprobably the 2e-reduction product N-(4ethoxyphenyl) -N'(glutathione-S-yl)phenylenediamine. This compound decomposed slowly (faster after acidification) to give the already known NOPt metabolite NH2EDPA (11). g) In incubates of NOPt with > 2.5 excess GSH, the orange color of GS-EPQDI disappeared after a few minutes. When authentic GS-EPQDI reacted with GSH, the ring-substituted GS-NH2EDPA was formed besides the reduction product NH2EDPA. The former pathway would be consistent with a reductive 1.4-Michael addition of GSH to the quinone diimine and subsequent hydrolysis or reduction of the ring substituted bicyclic sulfenamide.
Ferrihemoglobin-forming Activity of the NOPt Metabolites The Kiese cycle was found to play only a minor role during ferrihemoglobin formation by NOPt (10,22). Because in preliminary experiments the identified metabolites also were detected in NOPt-exposed human red cells, we investigated their activity in producing ferrihemoglobin. As shown in Table 5, the monocyclic metabolites hardly formed ferrihemoglobin in solutions of purified human hemoglobin. The bicyclic metabolites GS-EPQDI, NH2EDPA, and GS-NH 2EDPA, however, were highly active, producing many equivalents of ferrihemoglobin by hitherto unknown mechanisms.

Conclusion
The formation of ring-substituted glutathione S-conjugates during the reactions of NOPt with glutathione lend further support to the sulfenamide cation as reactive intermediate. This cation obviously reacted not only with GSH leading to monocyclic metabolites but also with sufficient nucleophilic arylamines producing various bicyclic metabolites. The latter were shown to be highly active in producing ferrihemoglobin, once more indicating that metabolic reactions with GSH should not be considered obligatory detoxication reactions. Despite its toxic action in erythrocytes, the stabilized electrophilic sulfenamide cation may play an important role in the in vivo toxicity of t-donor substituted nitrosoarenes.