Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 0999 1039

Recently, some doubts have appeared on this widely accepted mechanism, because sulfenamides, but not N-hydroxyarylamines, were found upon addition of thiols to isolated semimercaptals (from nitrosobenzene and 4-chloronitrosobenzene reacted with 1-thioglycerol or 2-thioethanol)27,38. During the direct reaction of the nitrosoarenes with excess thiols, however, N-hydroxyarylamines were formed as the main products27,38. On the other hand, semimercaptals from 3- and 4-nitronitrosobenzene reacting with N- acetylcysteine methyl ester or GSH were reported to be thiolytically cleaved as expected29. These contrasting observations have remained without any well-founded explanation hitherto. Possibly, there exists an additional pathway, e.g. via radicals, leading from the nitrosoarene to the N-hydroxyarylamine, as discussed below in Section II.F.

3. Secondary products

Azoand azoxyarenes have been repeatedly observed during reactions of nitrosoarenes with thiols5 7,11,29,33,35,36,38 . The latter family presumably emerges from the interaction of the N-hydroxyarylamine with unreacted nitrosoarene, a reaction proceeding even in neutral solutions2,6,58. The formation of azoarenes may be due to condensation of the end-product arylamine with still unreacted nitrosoarene59.

D. The Sulfenamide Cation Descendants

1.The sulfinamide

a. Identification. In the reaction of a great variety of nitrosoarenes with alkanethiols, formation of a stable adduct has been repeatedly observed. This adduct was identified as N-

aryl-S-alkylsulfinamide (4) due to a lot of characteristics. Radioactive experiments26,35 as well as 1H-NMR spectra18,35,43 revealed a 1:1 adduct of nitrosoarene and thiol. N S bond

formation was indicated as ring substitution was excluded by 1H-NMR data and hydrolysis experiments: On acidification9,10,18,22,28,35,38,41,60,61 and alkalization11,24,33,43 the adduct

liberated the corresponding arylamine and the sulfinic acid18,33,60,62. In neutral aqueous solutions and in the presence of excess thiol, the adduct remained stable35,38. Only the glutathione sulfinamides of the heterocyclic 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole41 and 2-nitroso-6-methyldipyrido[1,2-a:30,20 -d]imidazole43 were observed to decay slowly at neutral conditions. Elemental analysis24,38 and FAB-MS spectra24,35,37,38,43,60 displayed the same mass as the respective semimercaptal, but no fragmentation corresponding to the loss of water was observed (see Table 1). IR spectra18,24,35,38,43 exhibited a broad intense

peak at ³ 1060 cm 1 indicative of a sulfoxide stretching vibration. Characteristic UV9,24,38,40,41,43,61 , 13C-NMR24,38 and 1H-NMR data35,43,63 are summarized in Table 1.

Among all products formed during reactions of nitrosoarenes with thiols, the sulfinamides display one special characteristic. Because of the stereostable pyramidal conformation at the sulfur atom, two enantiomeric forms are possible64 66. (Sulfenamides contain a stereogenic S N axis which, however, is stereolabile; therefore, enantiomers are not observable in many cases67. Most likely, this holds true for semimercaptals and N-hydroxyarylamines as well.) Therefore, nitrosoarenes reacting with optically active thiols as, for example, the physiological substrates L-cysteine or GSH (L-glutamyl-L- cysteinylglycine) will result in diastereomeric sulfinamides, provided the chirality of the thiol does not control the formation ratio24,38,61. In fact, doubling of NMR signals has been observed with sulfinamides containing chiral thiols24,35,38,43,61. Because of the relatively high inversion barrier of the stereogenic sulfur center65,66, the diastereomers are stable enough to allow isolation. Thus, distinct sulfinamides are eluted as double peaks,

A Brønsted plot for the sulfinamide rearrangement of N-(glutathion-S-yl)-N- hydroxyaniline revealed a slope of 0.6, indicating that the proton is not fully transferred to the hydroxy group in the transition state30. The developing positive charge is obviously highly delocalized, thereby enabling the hydroxide anion to leave without catalysis. In addition to the neighboring sulfur atom, the aromatic ring provides a large system for delocalization. A Hammett correlation for the sulfinamide rearrangement revealed a reasonable fit only for C constant and a reaction constant of C D 3.530. These findings once more corroborate a substantial build-up of a positive charge in the transition state and a significant stabilizing contribution of the (substituted) benzene ring. Accordingly, a couple of metabolites could be attributed to ring-localized nucleophilic addition reactions to the sulfenamide cation (see below). For comparison with the analogous Bamberger reaction of N-phenylhydroxylamine and N-imidazoylhydroxylamines the account of Kazanis and McClelland30 is recommended.

The significant inverse correlation of sulfinamide formation with thiol concentration18,22,24,25,28 30,33,35,36,38 has already been discussed in Section II.C.2. Accordingly, the rearrangement pathway from the semimercaptal to the sulfinamide is favored at low thiol concentrations at the expense of N-hydroxyarylamine formation (see equation 2). In the case of bulky thiols as t-butylthiol38 or Hb-SH40 the sulfinamide is the main product since reduction by a second thiol is sterically hindered.

The reaction of nitrosoarenes with alkanethiols may provide a new and simple synthetic route to N-aryl-S-alkylsulfinamides which has not been mentioned hitherto62. Nitrosoarenes are frequently accessible by simple redox reactions of the commercially available arylamines or nitroarenes2,71. High yields of the desired sulfinamide may be achieved by adjusting stoichiometry, pH and solvent polarity. With aryl thiols, however, this method may not be applicable because of the very sluggish reaction (see Table 2). Whether such a synthetic route can be extended to alkylnitroso compounds remains to be established.

2.Sulfenamide and arylamine

a. Formation and identification. During reaction of various electron-rich nitrosoarenes with physiological thiols, several indications of a further, metastable adduct arose which

liberated the corresponding arylamine on prolonged incubation33,35,72. Again, radioactive experiments26,35 and 1H-NMR data24,35,72 revealed a 1:1 adduct without ring substitution. MS spectra24,35,72 and elemental analysis24 indicated a product containing one oxygen atom less than the sulfinamide, and IR spectroscopy24,35,38 proved the sulfoxide structure to be absent. Correspondingly, 1H-NMR35,72, 13C-NMR24,38 and UV data24,38,72 indicated a lower electron withdrawal of the sulfenyl sulfur compared to the sulfinamide (see Table 1). According to these spectroscopic characteristics and the chemical behavior described below, this family was identified as N-aryl-S-alkylsulfenamides (6).

The instability of N-aryl-S-alkylsulfenamides observed in the reaction mixture was shown to be due to hydrolysis and a more rapid reaction with nucleophiles. In the presence of millimolar concentrations of GSH isolated sulfenamides yielded the arylamine and the thiol disulfide within a few minutes35,72.

(6)

Klehr argued against this thiolytic cleavage of sulfenamides, as he did not observe accelerated aniline formation from Ph NH SR in the presence of 1-thioglycerol24,38. However,

this negative result was probably due to the distinct higher pKa and, therefore, lower reactivity of 1-thioglycerol (see Table 2). In fact, thiolytic cleavage of aromatic sulfenamides does not always proceed spontaneously and requires proton catalysis73.

Moderate instability of various isolated N-aryl-S-alkylsulfenamides towards hydrolysis was observed24,35,72 with formation of one equivalent arylamine and 2/3 equivalent thiol disulfide38. This decomposition may be rationalized by protonation of the sulfenamide nitrogen atom and subsequent sulfenylation of a solvent molecule24,35,38.

The sulfenic acids formed thereby are known to be highly unstable and were presumed to disproportionate to 2/3 thiol disulfide equivalent and 1/3 sulfinic acid38,74 77.

The proposed mechanism of sulfenamide hydrolysis is consistent with various findings on the reactivity of sulfenamides. Thus, protonation of sulfenamides is widely suggested to occur at the nitrogen atom, both from theoretical calculations78 and from experimental results79 82. Accordingly, the reaction of sulfenamides with electrophiles involves the coordination of the electrophile with the nitrogen atom and subsequent nucleophilic attack on the sulfur atom83. This mechanism of hydrolysis could also explain the apparent high instability of the sulfenamide of 2-nitroso-6-methyldipyrido[1,2-a:30,20 - d]imidazole37,43 (see Section II.E): The electron-rich heterocyclic N-aryl substituent may drastically raise the pKa of the sulfenamide nitrogen atom as observed with donor substituted sulfenanilides80. Despite all these indications, the postulated reverse hydrolysis mechanism of proton-assisted cleavage of the thiol with liberation of a nitrenium ion35 may not be totally excluded. The sulfenamide N-(glutathion-S-yl)-2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine was observed to hydrolyze spontaneously with formation of the 5-hydroxyarylamine84. This reaction might be rationalized by addition of water to the highly resonance-stabilized nitrenium ion85.

b. Remarks on the formation mechanism. Klehr observed the formation of sulfenamides from isolated semimercaptals in the presence of excess thiol24,38. Thus, reduction of the semimercaptal does not only lead to N-hydroxyarylamines (see Section II.C), but also to the sulfenamides (Scheme 1). Formation of the latter product or the subsequent arylamine has been reported for a variety of electron-rich nitrosoarenes24,26,33,35,37 39,43,72, but has hardly been observed in the case of acceptor substituted nitrosoarenes at neutral conditions29,36,38. Nevertheless, in an acidic milieu formation of 3-nitroaniline was reported to be a main pathway during the reaction of 3-nitrosonitrobenzene with GSH29 (the probable intermediate sulfenamide will rapidly hydrolyze at the pH of reaction). These data suggest the sulfenamides to emerge from thiol-mediated reduction of the sulfenamide cation which is preferentially formed from electron-rich nitrosoarenes and/or at low pH (see Section II.D.1). Accordingly, high concentrations35,38 and low pKa33 of the thiol favored the sulfenamide-arylamine pathway at the expense of the sulfinamide route.

The occurrence of an intermediate N,N-bis(thiol-S-yl)-arylamine (full mercaptal) has been repeatedly surmised to be involved in the semimercaptal reduction25,29,33,37

Me

SCHEME 6. Charge distribution in two different sulfenamide cations (total charge densities as revealed by MNDO calculation; H. -U. Wagner in Reference 56)

Kazanis and McClelland, in their outstanding work30, have proposed a detailed mechanism of semimercaptal reduction (Scheme 5, pathway 2). Accordingly, a thiolate may add to the p-position of the sulfenamide cation, resulting in an unstable ipso-adduct. Attack of a second thiolate with elimination of thiol disulfide will restore aromaticity in the sulfenamide. This thiolytic cleavage of the ipso-adduct implicates a 1:1 stoichiometry in the formation of sulfenamide and thiol disulfide. However, the initial sulfenamide formation during the reaction of 4-nitrosophenetol with GSH (0.1 and 0.5 mM, respectively, pH 7.4, 37 °C, argon atmosphere) was accompanied by formation of only about 1/5 equivalent glutathione disulfide (GSSG)56. Therefore, an alternative route of hydrolytic cleavage of the ipso-adduct has been proposed (see Scheme 5)24,56, as already mentioned for sulfenamide decomposition. In summary, the ipso-adduct mechanism reflects plausibly the events occurring during semimercaptal reduction.

According to this mechanistic conception, -donor substituted nitrosoarenes exhibit a strong correlation between sulfenamide and arylamine yield, respectively, and the thiol concentration employed38,56. In the case of nitrosobenzene and 4-chloronitrosobenzene, however, the 1-thioglycerol proportion (1:2 and 1:25, respectively) had virtually no effect on the sulfinamide/sulfenamide ratio at pH 6 938. As to our understanding, this effect lacks any reasonable explanation.

3. Thio ether

Formation of some other products during reaction of the donor substituted 4- nitrosophenetol and N,N-dimethyl-4-nitrosoaniline with GSH has been implicated26,36. In fact, a stable glutathione conjugate was isolated from reaction mixtures of 4- nitrosophenetol and GSH in low yields68. FAB-MS analysis revealed the same molecular mass as the corresponding sulfenamide, but the UV spectrum was distinctly different, and acidic milieu did not decompose this compound. UV data, pKa value and 1H-NMR spectra indicated this product to be 4-ethoxy-2-(glutathion-S-yl)-aniline68. The formation of this adduct is consistent with the postulated intermediate occurrence of a resonancestabilized sulfenamide cation which is prone to nucleophilic ring addition of thiolate (see Scheme 1). The resulting 4-ethoxy-2,N-bis-(glutathion-S-yl)-aniline (5) has now been isolated and structurally confirmed by FAB-MS, 1H-NMR and chemical reactivity45. As already mentioned above, the sulfenamide group is sensitive to thiolytic and hydrolytic cleavage, yielding the stable arylamine thio ether (7). A similar mechanism was proposed

for the formation of the main product 2-amino-5-(glutathion-S-yl)-1-methylimidazole during reaction of 1-methyl-2-nitrosoimidazole with large excess of GSH57.

Formation of ring-substituted arylamine thio ethers occurs also by proton-catalyzed thermal rearrangement of the corresponding sulfenamides73,82,83. This alternative pathway may not completely be excluded in thio ether formation from nitrosoarenes, but it seems unlikely since these thio ethers were produced at neutral pH and low temperatures68. The discovery of the bis-conjugate additionally favors the pathway of nucleophilic ring addition of thiolate to the sulfenamide cation.

Hitherto, thio ether formation has clearly been proved only in the case of the - donor substituted 4-nitrosophenetol and the electron-rich 1-methyl-2-nitrosoimidazole. The low yields of this adduct (about 2% at 1:1- and about 10% at 1:5-stoichiometry for 4-nitrosophenetol reacting with GSH56) may be the reason for its rare discovery. However, other nitrosoarenes should yield this family, too. Semiempirical molecular orbital calculations (MNDO) indicate a similar positive charge at the o-position of the N- (methylthiol-S-yl)-aniline cation and -4-anisole cation as well (Scheme 6). Furthermore, formation of 1-(glutathion-S-yl)-2-naphthylamine was reported to occur in mixtures of 2-nitrosonaphthalene and GSH12.

4. N -Sulfenylquinonimines and resultant products

Formation of several colored products during reaction of nitrosoarenes with thiols has been repeatedly observed12,26,68. Two different orange-colored conjugates were found during HPLC separation of mixtures of 4-nitrosophenetol and GSH. The UV spectra were indicative of a quinoid structure, and further studies revealed these adducts to be a monocyclic and a bicyclic conjugate. In both cases the reactive quinoid structure gives rise to formation of secondary, stable end products.

a. Monocyclic products. An identical monocyclic conjugate was formed, both from 4- nitrosophenetol reacting with GSH as well as from 4-nitrosoanisole and 4-nitrosophenol, indicating an exchange of the p-substituent by an identical group. Accordingly, the signals of the p-substituent were lost in the 1H-NMR spectrum. Characteristically, 4 different aromatic signals, each splitted into a double doublet by o- and m-coupling, were observed. These findings are consistent with the structure of N-(glutathion-S-yl)-4- benzoquinonimine (Scheme 7, 9a) because the hindered rotation around the imino bond86 causes magnetic inequivalence of the quinoid protons. FAB-MS analysis corroborated the proposed structure. The isolated conjugate reacted with excess GSH with formation of various products. Mild reduction yielded the corresponding sulfenamide 12 followed by formation of 4-aminophenol (13) while addition to the quinoid system resulted in thio ethers of 4-aminophenol (14). However, these secondary products have not been observed directly in reaction mixtures of 4-nitrosophenetol and GSH, presumably because of the low yield of the quinonimine derivative (9a) (maximum 5% of theory)45.

Under mechanistic aspects, discovery of the quinonimine derivative was not unexpected. The sulfenamide cation of 4-nitrosophenetol (3a) gives rise not only to ring addition of GSH but of other nucleophiles, too. Especially the p-position seems to be prone to nucleophilic attack since this aromatic carbon atom obtains additional positive charge by the I-effect of the adjacent oxygen atom as revealed by semiempirical molecular orbital calculations (see Scheme 6). The ethoxy group is known to be quite a good leaving group58. Thus, addition of H2O with concomitant loss of a proton may produce an unstable ipso-adduct which is prone to release either hydroxide or ethoxide, presumably with proton assistance (Scheme 7). A similar reaction pathway has already been described for the N-(thiophenol-S-yl)-4-anisidine cation87.

b. Bicyclic products. In analogy to the monocyclic quinonimine derivative, reaction of the main end-product 4-phenetidine (see Scheme 1) with the sulfenamide cation (3a) produces N-(40-ethoxyphenyl)-N0-(glutathion-S-yl)-4-benzoquinone diimine (Scheme 8, 10a). The corresponding 2-thioethanol and t-butylthiol derivatives

aromatic signals of 8 protons overlapped mutually68. Therefore, the pentafluorophenyl

another route was pursued: 4-nitrosophenetol was reacted with pentafluoroaniline under acid catalysis3,88, giving pentafluoro-40-nitrosodiphenylamine in low yield. Transformation with GSH delivered the desired benzoquinone diimine derivative 10b.) Chemical behavior of the 4-ethoxyphenyl (10a) and the pentafluorophenyl (10b) derivatives was similar, and FAB-MS analysis revealed the expected mass. The 1H-NMR spectrum exhibited 8 aromatic double doublets with a relative intensity of 0.5 proton

4-amino-40 -ethoxydiphenylamine (16) with intermediate occurrence of a metastable compound, probably the corresponding sulfenamide 15. Besides, ring addition of various thiols ultimately resulted in the formation of 4-amino-40-ethoxy-2-(thiol-S-yl)-diphenylamine (18), presumably via the corresponding sulfenamide 1768 (Scheme 8).

The quinone diimine 10a was discovered in incubates of 4-nitrosophenetol with about 2.5-fold excess of GSH. At higher GSH concentrations, this product was hardly formed because 4-nitrosophenetol and hence the sulfenamide cation (3a) had completely reacted before significant amounts of 4-phenetidine were formed (see Scheme 1)56. However, when GSH was slowly generated by an enzymic reaction in the presence of 4-nitrosophenetol, quinone diimine yields increased markedly56. Similarly, high yields of the quinone diimine 10a were obtained when authentic 4-phenetidine was present from the beginning in the mixtures of 4-nitrosophenetol and GSH. This path may provide a new and simple synthetic route for distinct N-sulfenylquinonimines which has not been mentioned hitherto86. In fact, a variety of N-sulfenylquinone diimine derivatives were obtained during reaction of 4-nitrosophenetol with other primary arylamines and other thiols. Because of the weaker nucleophilicity of acceptor substituted anilines and alkylamines, these amines presumably can hardly compete with the other nucleophiles for the sulfenamide cation56.

c. N-Sulfenylquinonimine formation from nitrosophenols and nitrosoanilines. Because of the ionizable proton of the aryl substituent, nitrosophenols and -anilines presumably will display a somewhat different metabolic pattern (Scheme 9). The semimercaptals of these -donor substituted nitrosoarenes will almost exclusively yield the sulfenamide cation which is prone to simple and rapid stabilization: Dissociation of the proton at the substituent oxygen and nitrogen results in the corresponding N-sulfenylquinone monoand

− H +

?sulfenamide arylamine thio ethers

SCHEME 9. Tentative pathways of nitrosophenols and nitrosoanilines reacting with thiols