Studies of Chemical Selectivity of Hapten, Reactivity, and Skin

Chem. Res. Toxicol. 2003, 16, 627-636

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Studies of Chemical Selectivity of Hapten, Reactivity, and Skin Sensitization Potency. 3. Synthesis and Studies on the Reactivity toward Model Nucleophiles of the 13C-Labeled Skin Sensitizers, 5-Chloro-2-methylisothiazol-3-one (MCI) and 2-Methylisothiazol-3-one (MI) Rubeń Alvarez-Sańchez,† David Basketter,‡ Camilla Pease,‡ and Jean-Pierre Lepoittevin*,† Laboratoire de Dermatochimie (UMR 7123), Universite´ Louis Pasteur, Clinique Dermatologique, CHU, Strasbourg, France, and SEAC, Unilever, Colworth, Sharnbrook, Bedford, United Kingdom Received November 18, 2002

The skin sensitizers, 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI), have been synthesized isotopically labeled with 13C at all carbon positions. The reactivity of 3-[13C]-, 4-[13C]-, and 5-[13C]MCI and MI toward a series of model nucleophiles for protein amino acid residues, i.e., butylamine, imidazole, sodium propanethiolate, and sodium phenoxide, was followed by 13C and 1H{13C} NMR spectroscopy. While MCI was found to react quantitatively with sodium propanethiolate and butylamine and significantly with imidazole and sodium phenoxide, MI reacted only with sodium propanethiolate. Reaction of MCI with nonthiol nucleophiles proceeded through an initial addition-elimination at position 5, leading to stable substitution adducts in the case of imidazole and sodium phenoxide. In the case of butylamine, the initial adduct was subjected to extra reactions at the sulfur atom through a cleavage of the S-N bond, leading to open adducts of the thioamide or amide type. Experiments carried out with N-acetyl-Cys, in excess or in deficiency, indicated that thiol nucleophiles reacted first at the sulfur atom through a cleavage of the S-N bond followed by extra nucleophilic reactions leading to open adducts of the mercaptothioester or mercaptoester type. Reaction of MCI with thiol nucleophiles gave products consistent with the formation of a reactive thioacyl chloride intermediate able to react with other nucleophiles present in the reaction medium. As a consequence, N-acetyl-Cys was found to be able to activate MCI toward NR-acetyl-Lys under physiological conditions to form adducts of the thioamide or amide type. Thus MCI, a strong sensitizer, and MI, a weak sensitizer, were found to react with different nucleophiles through different mechanisms. Although both MCI and MI can react with thiol nucleophiles, only MCI is capable of significantly reacting with amino nucleophiles of the Lys or His type. Moreover, MCI could be activated by a prior reaction with thiols.

Introduction

Chart 1. Chemical Structures of MCI (1), MI (2), and MDI (3)

The isothiazolone biocides developed by Rohm and Haas have gained considerable success due to their efficacy and biodegradability. They have been shown to exhibit a strong activity at relatively low concentrations against a broad spectrum of Gram+ and Gram- bacteria as well as on fungi and molds. These preservatives have therefore been used in many household cleaning products, personal care products, and also in some industrial products. Structural variations have been introduced on the nitrogen atom as well as in positions 4 and 5 (Chart 1) of the isothiazolone heterocycle to allow adjustment in solubility, stability, and modulation of activity in relation with the expected biocide activity. These biocides are formulated as aqueous solutions, in concentrations ranging from 1.5 to 15% of active ingredients associated

with magnesium and copper salts as stabilizers. The final use concentrations of active molecules usually range from 2 to 20 ppm (1). Among the biocides derived from isothiazolones, Kathon CG (cosmetic grade), based on two isothiazolones (Chart 1), namely, MCI (1)1 and MI (2), in aqueous solution has attracted attention due to cases of ACD. Isothiazolones were readily identified as significant skin sensitizers in predictive guinea pig tests (2), and this has

* To whom correspondence should be addressed. Tel: +33 388 350 664. Fax: +33 388 140 447. E-mail: [email protected]. † Universite ´ Louis Pasteur. ‡ SEAC, Unilever.

1 Abbreviations: ACD, allergic contact dermatitis; HSAB, hard and soft acids and bases; QSAR, quantitative structure activity relationships; MCI, 5-chloro-N-methyl-isothiazol-3-one; MDI, 4,5-dichloro-Nmethyl-isothiazol-3-one; MI, N-methyl-isothiazol-3-one.

10.1021/tx0256634 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/05/2003

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been confirmed as highly relevant to humans (3-5). Tests performed with pure components of Kathon CG, isolated by HPLC, have shown that the chlorinated derivative MCI was the stronger sensitizer while the nonchlorinated derivative MI was a much weaker sensitizer. Thus, when patch-tested in serial dilutions on patients sensitized to Kathon CG, MI gave positive reactions down to 100 ppm while MCI gave positive reactions down to 10 ppm (6) and this was further confirmed on guinea pig tests (7). Furthermore, an impurity present at less than 0.003%, namely, MDI (3), was identified and also shown to be a strong sensitizer (6, 8). MCI has been shown to give crossreactions in sensitized guinea pigs with MDI but not with MI, indicating an activation of two populations of T-cell clones. ACD is a common disease induced by the modification of skin proteins by haptens (9). Subsequent processing of these modified proteins by Langerhans cells, the main antigen-presenting cell of the epidermis, leads to the selective activation of T-lymphocytes with receptors able to bind antigenic hapten-modified peptides. The relation between the chemical reactivity of a molecule and its ability to induce a skin sensitization have been wellestablished and used as a base for the development of QSARs for ACD. For some years, we have been investigating the reactivity pattern of haptens toward nucleophilic residues of proteins to see if this could add some value to predictive models of skin sensitization. For example, we have shown recently that the difference in sensitization potential between two analogous sultones, hex-1-ene-1,3-sultone and hexane-1,3-sultone, could be explained in terms of selective reactivity toward Lys residues rather than in terms of general chemical reactivity (10, 11). Therefore, it was interesting to investigate the mechanisms by which MCI and MI were interacting with proteins to cause sensitization and to see if differences in mechanisms and/or in amino acid modifications could explain the observed difference in sensitizing potential. In this paper, we report the synthesis of 3-, 4-, and 5-[13C]MCI (1a-c), MI (2a-c), and MDI (3a-c), respectively. The reactivity of 1 and 2 toward model nucleophiles has been studied using 13C and 1H{13C} NMR spectroscopy. Results of these experiments are used to discuss the sensitizing properties of MCI and MI.

Materials and Methods Caution: Skin contact with isothiazolone derivatives must be avoided. Because these are sensitizing substances, they must be handled with care. Chemistry. 1H and 13C NMR spectra were recorded on a Bruker AC200-MHz or Bruker AC300-MHz spectrometer in CDCl3 unless otherwise specified. Chemical shifts are reported in ppm (δ) with respect to TMS, and CHCl3 was used as an internal standard (δ ) 7.26 ppm). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), and m (multiplet). Infrared spectra were recorded on a Perkin-Elmer FT-IR 1600 spectrometer, and the peaks were reported in reciprocal centimeters. Melting points were determined on a Buchi Tottoli 510 apparatus and are uncorrected. Dried solvents were freshly distilled before use. Methylene chloride was dried over P2O5 before distillation. All air or moisture sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. Chromatographic purifications were performed either on silica gel columns according to the flash chromatography technique or on alumina columns.

Alvarez-Sa´ nchez et al. 2-Bromoethyl Acetate (5). To a mixture of acetic acid (1.01 g, 16.80 mmol, 1 equiv) was added PBr3 (0.8 mL, 8.25 mmol, 0.5 equiv) and then slowly bromine (3 mL, 58.43 mmol, 3.5 equiv). The reaction mixture was stirred at room temperature for 30 min, heated at 60 °C for 6 h, and then allowed to cool before the addition of anhydrous ethanol (1.8 mL, 30.85 mmol, 1.9 equiv). After the mixture was stirred for 15 h, the reaction was hydrolyzed by adding ethanol (5 mL) and water (15 mL). The mixture was extracted with ether (30 and then 15 mL), and combined organic layers were neutralized with a saturated solution of sodium hydrogen carbonate (2 × 15 mL) and then washed with a saturated solution of sodium thiosulfate to remove the excess of bromine. The mixture of organic solvents was then dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give an orange oil, which was purified by distillation (160 °C, 760 mm Hg). 2-Bromoethyl acetate (2.21 g, 13.21 mmol, 79%) was thus obtained as a colorless oil. 1H NMR (200 MHz, CDCl3): δ 1.28 (t, J ) 7.1 Hz, 3H, CH3), 3.81 (s, 2H, CH2Br), 4.22 (q, J ) 7.1 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl ): δ 14.0 (CH ), 25.9 (CH Br), 62.3 3 3 2 (OCH2), 167.2 (CO2Et). IR, CHCl3, ν (cm-1): 1285 (C-O), 1738 (CdO). 1-[13C]-2-Bromoethyl Acetate (5a). The same procedure as for the synthesis of 5 was used starting from [1-13C]acetic acid (1.04 g, 17.10 mmol) to give 5a (2.24 g, 14.53 mmol, 85% yield) as a colorless oil. 1H NMR (200 MHz, CDCl3): δ 1.29 (t, J ) 7.1 Hz, 3H, CH3), 3.82 (d, 2JHC ) 4.7 Hz, 2H, 13CH2Br), 4.23 (qd, J ) 7.1 Hz, 3JCH ) 3.2 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.0 (CH3), 25.9 (d, 1JCC ) 65.6 Hz, CH2Br), 62.3 (OCH2), 167.2 (13CO2Et). 2-[13C]-2-Bromoethyl Acetate (5b). The same procedure as for the synthesis of 5 was used starting from [2-13C]acetic acid (1.00 g, 16.38 mmol) to give 5b (1.93 g, 11.48 mmol, 70% yield) as a colorless oil. 1H NMR (200 MHz, CDCl3): δ 1.29 (t, J ) 7.0 Hz, 3H, CH3), 3.82(d, 1JHC ) 153.1 Hz, 2H, 13CH2Br), 4.23 (q, J ) 7.0 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.0 (CH3), 25.9 (13CH2Br), 62.3 (OCH2), 167.2 (d, 1JCC ) 65.6 Hz, CO2Et). (Ethoxycarbonylmethyl)triphenylphosphonium Bromide (6). To a solution of PPh3 (3.7 g, 13.97 mmol, 1.1 equiv) in ethyl acetate (12 mL) was slowly added a solution of ethyl bromoacetate 5 (2.21 g, 13.21 mmol, 1 equiv) in ethyl acetate (12 mL). The reaction was stirred at room temperature for 20 h, and the white precipitate was filtered off, washed with ethyl acetate (15 mL) and pentane (15 mL), and then dried under vacuum to give the phosphonium salt 6 (4.90 g, 11.42 mmol, 87% yield) as a white solid; mp 155-157 °C (lit. 159-161 °C). 1H NMR (200 MHz, CDCl ): δ 1.07 (t, J ) 7.1 Hz, 3H, CH ), 3 3 4.04 (q, J ) 7.1 Hz, 2H, OCH2), 5.62 (d, 2JHP ) 13.5 Hz, 2H, CH2P), 7.66-7.96 (m, 15H, ArH). 13C NMR (50 MHz, CDCl3): δ 13.6 (CH3), 33.2 (d, 1JCP ) 55.7 Hz, CH2P), 62.7 (OCH2), 117.8 (d, 1JCP ) 88.5 Hz, C1(Ar)), 130.2 (d, 3JCP ) 13.1 Hz, C3(Ar)), 133.9 (d, 2JCP ) 9.8 Hz, C2(Ar)), 135.1 (d, 4JCP ) 3.3 Hz, C4(Ar)), 164.6 (d, 2JCP ) 3.3 Hz, CO2Et). IR, CHCl3, ν (cm-1): 1733 (CdO). 2-[13C]-(Ethoxycarbonylmethyl)triphenylphosphonium Bromide (6a). The same procedure as for the synthesis of 6 was used starting from ethyl [1-13C]-2-bromoacetate (1.93 g, 11.48 mmol) to give the phosphonium 6a (4.33 g, 10.06 mmol, 88% yield) as a white solid. 1H NMR (200 MHz, CDCl3): δ 1.06 (t, J ) 7.1 Hz, 3H, CH3), 4.03 (dq, 3JHC ) 3.2 Hz, J ) 7.1 Hz, 2H, OCH2), 5.57 (dd, 2JHP ) 13.8 Hz, 2JHC ) 7.4 Hz, 2H, CH2P), 7.58-7.88 (m, 15H, ArH). 13C NMR (50 MHz, CDCl3): δ 13.7 (CH3), 33.1 (dd, 1JCP ) 55.7 Hz, 1JCC ) 59.0 Hz, CH2P), 62.7 (OCH2), 117.8 (d, 1JCP ) 88.5 Hz, C1(Ar)), 130.3 (d, 3JCP ) 13.2 Hz, C3(Ar)), 134.1 (d, 2JCP ) 11.5 Hz, C2(Ar)), 135.2 (d, 4JCP ) 3.3 Hz, C4(Ar)), 164.6 (d, 2JCP ) 3.3 Hz, 13CO2Et). 1-[13C]-(Ethoxycarbonylmethyl)triphenylphosphonium Bromide (6b). The same procedure as for the synthesis of 6 was used starting from ethyl-[2-13C]-2-bromoacetate (2.44 g, 14.53 mmol) to give the phosphonium 6b (5.19 g, 12.05 mmol, 83% yield) as a white solid. 1H NMR (200 MHz, CDCl3): δ 1.06 (t, J ) 7.1 Hz, 3H, CH3), 4.04 (q, J ) 7.1 Hz, 2H, OCH2), 5.62

Labeled Isothiazolones (dd, 1JHC ) 134.6 Hz, 2JHP ) 13.5 Hz, 2H, 13CH2P), 7.64-7.93 (m, 15H, ArH). 13C NMR (50 MHz, CDCl3): δ 13.6 (CH3), 33.2 (d, 1JCP ) 55.7 Hz, 13CH2P), 62.7 (OCH2), 117.8 (d, 1JCP ) 88.5 Hz, C1(Ar)), 130.2 (d, 3JCP ) 13.1 Hz, C3(Ar)), 133.9 (d, 2JCP ) 9.8 Hz, C2(Ar)), 135.1 (d, 4JCP ) 3.3 Hz, C4(Ar)), 164.3 (dd, 2JCP ) 4.9 Hz, 1JCC ) 59.0 Hz, CO2Et). (Ethoxycarbonylmethylene)triphenylphosphorane (7). To phosphonium 6 (4.90 g, 11.42 mmol, 1 equiv) in dichloromethane (30 mL), a solution of sodium hydroxide (15 mL, 1 M, 15 mmol, 1.3 equiv) was slowly added. The biphasic mixture was stirred for 1 h, and the organic layer was recovered by decantation. The aqueous phase was further extracted with dichloromethane (2 × 20 mL), and combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a solid, which was purified by crystallization (150 mL, cyclohexane/heptane, 6/4). The phosphorane 7 (3.81 g, 10.95 mmol, 96% yield) was thus obtained as a yellowish solid; mp 124 °C (lit. 120-122 °C). 13C NMR (200 MHz, CDCl3): δ 14.8 (CH3), 30.1 (d, 1JCP ) 124.6 Hz, CH2P), 57.8 (OCH2), 128.0 (d, 1JCP ) 90.7 Hz, C1(Ar)), 128.6 (d, 3JCP ) 13.2 Hz, C3(Ar)), 131.8 (C4(Ar)), 132.9 (d, 2JCP ) 11.5 Hz, C2(Ar)), 172.2 (d, 2JCP ) 13.0 Hz, CO2Et). IR, CHCl3, ν (cm-1): 1610 (Cd O). 2-[13C]-(Ethoxycarbonylmethylene)triphenylphosphorane (7a). The same procedure as for the synthesis of 7 was used starting from 6a (5.19 g, 12.05 mmol) to give 7a (3.95 g, 11.31 mmol, 94% yield) as a yellowish solid. 13C NMR (50 MHz, CDCl3): δ 14.9 (CH3), 30.2 (q, 1JCP ) 124.5 Hz, 1JCC ) 87.5 Hz, CH2P), 57.8 (OCH2), 128,0 (d, 1JCP ) 90.7 Hz, C1(Ar)), 128.6 (d, 3J 2 CP ) 11.5 Hz, C3(Ar)), 131.9 (C4(Ar)), 132.9 (d, JCP ) 11.5 Hz, C2(Ar)), 172.2 (d, 2JCP ) 11.5 Hz, 13CO2Et). [1-13C]-(Ethoxycarbonylmethylene)triphenylphosphorane (7b). The same procedure as for the synthesis of 7 was used starting from 6b (4.33 g, 10.06 mmol) to give 7b (3.26 g, 9.35 mmol, 93% yield) as a yellowish solid. 13C NMR (50 MHz, CDCl3): δ 14.9 (CH3), 30.2 (d, 1JCP ) 124.5 Hz, 13CH2P), 57.9 (OCH2), 128.0 (d, 1JCP ) 90.7 Hz, C1(Ar)), 128.7 (d, 3JCP ) 11.5 Hz, C3(Ar)), 131.9 (C4(Ar)), 133.0 (d, 2JCP ) 9,8 Hz, C2(Ar)), 172.2 (dd, 2JCP ) 11.5 Hz, 1JCC ) 87.6 Hz, CO2Et). Ethyl-3-acetyl-sulfanyl-propionate (9). To a solution of phosphorane 7 (2.32 g, 6.66 mmol, 1 equiv) in dioxane (20 mL), an aqueous solution of formaldehyde (0.52 g, 37-40%, 6.67 mmol, 1 equiv) was added. The reaction mixture was stirred at room temperature for 20 min, heated at 50 °C for 24 h, and then allowed to cool to room temperature. Thioacetic acid (1 mL, 13.20 mmol, 2 equiv) was then added to the reaction mixture, which was stirred for 7 days and then concentrated under reduced pressure. Ether (25 mL) and water (25 mL) were then added, the aqueous layer was extracted with ether (3 × 25 mL), and combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The pink solid obtained was treated overnight with refluxing pentane (250 mL). The liquid phase was filtered and concentrated under reduced pressure to give a crude product, which was purified by column chromatography on silica (9.3/0.7 Hex/AcOEt) to give thioacetate 9 (0.98 mg, 5.57 mmol, 84% yield) as a reddish oil. 1H NMR (200 MHz, CDCl ): δ 1.23 (t, J ) 7.1 Hz, 3H, CH CH ), 3 2 3 2.30 (3H, CH3COS), 2.59 (t, J ) 6.9 Hz, 2H, CH2CO2), 3.08 (t, J ) 6.9 Hz, 2H, SCH2), 4.12 (q, J ) 7.1 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.1 (CH2CH3), 24.2 (SCH2), 30.5 (CH3COS), 34.4 (CH2CO2), 60.7 (OCH2), 171.6 (CO2Et), 195.4 (COS). IR, ν (cm-1): 1735 (CdO, ester), 1693 (CdO, thioester). Ethyl-1-[13C]-3-acetyl-sulfanyl-propionate (9a). The same procedure as for the synthesis of 9 was used starting from 7a (2.51 g, 7.18 mmol) to give 9a (83 mg, 4.68 mmol, 65% yield) as a reddish oil. 1H NMR (200 MHz, CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H, CH2CH3), 2.31 (3H, CH3COS), 2.60 (td, 2JHC ) 6.9 Hz, J ) 6.9 Hz, 2H, CH213CO2), 3.09 (td, J ) 6.9 Hz, 2H, 3JHC ) 4.9 Hz, SCH2), 4.13 (qd, J ) 7.1 Hz, 3JHC ) 3.2 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.2 (CH2CH3), 24.2 (SCH2), 30.5 (CH3COS), 34.4 (d, 1JCC ) 57.4 Hz, CH2CO2), 60.8 (OCH2), 171.6 (13CO2Et), 195.4 (COS).

Chem. Res. Toxicol., Vol. 16, No. 5, 2003 629 Ethyl-2-[13C]-3-acetyl-sulfanyl-propionate (9b). The same procedure as for the synthesis of 9 was used starting from 7b (3.26 g, 9.35 mmol) to give 9b (1.07 g, 6.04 mmol, 65% yield) as a reddish oil. 1H NMR (200 MHz, CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H, CH2CH3), 2.32 (3H, CH3COS), 2.60 (A part of an ABX system, 1JHC ) 129.6 Hz, J ) 7.1 Hz, 2H, 13CH2CO2), 3.10 (B part of an ABX system, J ) 7.1 Hz, 2JCH ) -3.5 Hz, 2H, SCH2), 4.13 (q, J ) 7.1 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.2 (CH2CH3), 24.2 (d, 1JCC ) 36.06 Hz, SCH2), 30.5 (CH3COS), 34.4 (13CH2CO2), 60.8 (OCH2), 171.7 (d, 1JCC ) 57.4 Hz, CO2Et), 195.4 (COS). Ethyl-3-[13C]-3-acetyl-sulfanyl-propionate (9c). The same procedure as for the synthesis of 9 was used starting from 7 and a 20% aqueous solution of [13C]-labeled formaldehyde (990 mg, 6.38 mmol) to give 9c (943 mg, 5.32 mmol, 83% yield) as a reddish oil. 1H NMR (200 MHz, CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H, CH2CH3), 2.31 (3H, CH3COS), 2.59 (B part of an ABX system, J ) 7.0 Hz, 2JCH ) -6 Hz, 2H, CH2CO2), 3.09 (A part of an ABX system, 1JHC ) 139.0 Hz, J ) 7.0 Hz, 2H, S13CH2), 4.13 (q, J ) 7.1 Hz, 2H, OCH2). 13C NMR (50 MHz, CDCl3): δ 14.2 (CH2CH3), 24.2 (S13CH2), 30.5 (CH3COS), 34.4 (d, 1JCC ) 38.8 Hz, CH2CO2), 60.8 (OCH2), 171.6 (CO2Et), 195.4 (COS). 3,3′-Dithiobis(N-methylpropionamide) (10). To thioacetate 9 (397 mg, 2.25 mmol, 1 equiv), an aqueous solution of methylamine (0.8 mL, 41%, 9,47 mmol, 4.2 equiv) was added at 0 °C and the reaction mixture was stirred at room temperature for 15 h. A solution of hydrogen peroxide (0.2 mL, 35%, 2.33 mmol, 1 equiv) was added at 0 °C, and brine (15 mL) was added before extraction with dichloromethane (6 × 20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a white solid. A crystallization from benzene (10 mL) gave the dithiopropionamide derivative 10 (190 mg, 0.80 mmol, 71% yield) as white crystals; mp 112 °C (lit. 105-108 °C). 1H NMR (200 MHz, CD3OD): δ 2.57 (t, J ) 7.1 Hz, 2H, CH2CON), 2.71 (3H, CH3), 2.93 (t, J ) 7.1 Hz, 2H, SCH2). 13C NMR (50 MHz, CD3OD): δ 26.4 (CH3), 35.1 (SCH2), 36.4 (CH2CON), 174.2 (CON). IR, CHCl3, ν (cm-1): 1637 (CdO); 3415, 3264 (N-H). 1,1′-[13C]-3,3′-Dithiobis(N-methylpropionamide) (10a). The same procedure as for the synthesis of 10 was used starting from 9a (309 mg, 1.74 mmol) to give 10a (145 mg, 0.61 mmol, 70% yield) as white crystals. 1H NMR (200 MHz, CD3OD): δ 2.57 (dt, 2JHC ) 6.9 Hz, J ) 6.9 Hz, 2H, CH213CON), 2.71 (d, 3J 3 HC ) 3.7 Hz, 3H, CH3), 2.91 (dt, JHC ) 4.2 Hz, J ) 7.1 Hz, 2H, SCH2). 13C NMR (50 MHz, CD3OD): δ 26.4 (NCH3), 35.1 (SCH2), 36.4 (d, 1JCC ) 49.0 Hz, CH213CON), 174.2 (13CON). 2,2′-[13C]-3,3′-Dithiobis(N-methylpropionamide) (10b). The same procedure as for the synthesis of 10 was used starting from 9b (478 mg, 2.70 mmol) to give 10b (238 mg, 1.00 mmol, 74% yield) as white crystals. 1H NMR (200 MHz, CD3OD): δ 2.58 (A part of an ABX system, 1JHC ) 136.0 Hz, J ) 7.1 Hz, 2H, 13CH2CON), 2.71 (3H, CH3), 2.92 (B part of an ABX system, J ) 7.1 Hz, 2JCH ) -3.5 Hz, 2H, SCH2). 13C NMR (50 MHz, CD3OD): δ 26.3 (NCH3), 35.1 (d, 1JCC ) 36.0 Hz, SCH2), 36.4 (13CH2CON), 174.2 (d, 1JCC ) 49.0 Hz, CON). 3,3′-[13C]-3,3′-Dithiobis(N-methylpropionamide) (10c). The same procedure as for the synthesis of 10 was used starting from 9c (363 mg, 2.05 mmol) to give 10c (172 mg, 0.73 mmol, 71% yield) as white crystals. 1H NMR (200 MHz, CD3OD): δ 2.56 (B part of an ABX system, J ) 7.1 Hz, 2JCH ) -6 Hz, 2H, CH2CON), 2.71 (3H, CH3), 2.91 (A part of an ABX system, 1JHC ) 144.4 Hz, J ) 7.1 Hz, 2H, S13CH2). 13C NMR (50 MHz, CD3OD): δ 26.3 (CH3), 35.1 (S13CH2), 36.4 (d, 1JCC ) 36.1 Hz, CH2CON), 174.2 (CON). Isothiazolones. To a suspension of 10 (169 mg, 0.71 mmol, 1 equiv) in dichoromethane (12 mL) was added, at -78 °C, sulfuryl chloride (541 mg, 4.00 mmol, 5.6 equiv). The reaction mixture was stirred at -78 °C for 1 h and then allowed to warm to room temperature and stirred for an additional period of 15 h. The suspension was taken up with methanol (20 mL), silica (0.4 g) was added, and solvents were removed under reduced pressure. The solid residue was purified by column chromatog-

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raphy over silica (7/3 hex/AcOEt) to give a mixture of 1 and 3 (three fractions) and then (9.2/0.8 AcOEt/EtOH) to give 2. Column chromatography over alumina (hex/AcOEt 8/2) of the first fraction gave 3 (22 mg) while column chromatography over alumina (Gradient hex/AcOEt 8/2 f 7/3) of the second fraction gave 1 (62 mg) and 3 (12 mg). Column chromatography over alumina (hex/AcOEt 7/3) of the third fraction gave 1 (45 mg) while column chromatography over alumina (AcOEt/EtOH 9.2/ 0.8) of the fourth fraction gave 2 (34 mg). Thus, 1 (107 mg, 0.71 mmol, 50% yield), 2 (34 mg, 0.30 mmol, 21% yield), and 3 (34 mg, 0.18 mmol, 13% yield) were obtained as white solids. 5-Chloro-2-methyl-isothiazol-3-one (1). mp 52 °C (Litt. 54-55 °C). 1H NMR (200 MHz, CDCl3): δ 3.29 (3H, CH3), 6.28 (1H, CH). 13C NMR (50 MHz, CDCl3): δ 29.9 (CH3), 114.3 (CH), 145.3 (CCl), 166.9 (CON). IR, CHCl3, ν (cm-1): 1659 (CdO). EIMS (relative intensity): m/z 151.0 (M, 37Cl, 37.1), 149.0 (M, 35Cl, 100). 2-Methyl-isothiazol-3-one (2). mp 44 °C (lit. 48-50 °C). 1H NMR (200 MHz, CD3OD): δ 3.35 (3H, CH3), 6.22 (d, J ) 6.1 Hz, 1H, CHCON), 8.42 (d, J ) 6.1 Hz, 1H, SCH). 13C NMR (50 MHz, CD3OD): δ 31.2 (CH3), 114.9 (CHCON), 143.8 (SCH), 172.0 (CON). IR, CHCl3, ν (cm-1): 1648 (CdO). EIMS (relative intensity): m/z 115.1 (M, 100). 4,5-Dichloro-2-methyl-isothiazol-3-one (3). mp 116 °C (lit. 116-118 °C). 1H NMR (200 MHz, CDCl3): δ 3.35 (3H, CH3). 13C NMR (50 MHz, CDCl ): δ 31.4 (CH ), 115.1 (CClCON), 138.1 3 3 (CSCl), 162.0 (CON). IR, CHCl3, ν (cm-1): 1667 (CdO). EIMS (relative intensity): m/z 186.9 (M, 2 × 37Cl, 14.6), 184.9 (M, 35Cl + 37Cl, 71.3), 182.9 (M, 2 × 35Cl, 100). 3-[13C]Isothiazolones. The same procedure as for the synthesis of nonlabeled isothiazolones was used starting from amide 10a (145 mg, 0.61 mmol) to give 3-[13C]-5-chloro-2-methylisothiazol-3-one (1a; 113 mg, 0.75 mmol, 62% yield), 3-[13C]-2methyl-isothiazol-3-one (2a; 23 mg, 0.20 mmol, 16% yield), and 3-[13C]-4,5-dichloro-2-methyl-isothiazol-3-one (3a; 16 mg, 0.09 mmol, 7% yield) as white solids. 3-[13C]-5-Chloro-2-methyl-isothiazol-3-one (1a). 1H NMR (200 MHz, CDCl3): δ 3.29 (3H, CH3), 6.28 (1H, CH). 13C NMR (50 MHz, CDCl3): δ 30.0 (CH3), 114.5 (d, 1JCC ) 70.5 Hz, CH), 145.4 (d, 2JCC ) 8.2 Hz, CCl), 167.1 (13CON). EIMS (relative intensity): m/z 152.1 (M, 37Cl, 39.8), 150.1 (M, 35Cl, 100). 3-[13C]-2-Methyl-isothiazol-3-one (2a). 1H NMR (200 MHz, CDCl3): δ 3.35 (d, 3JCH ) 2.5 Hz, 3H, CH3), 6.26 (dd, J ) 6.1 Hz, 2JHC ) 6.1 Hz, 1H, CH13CON), 8.03 (dd, 3JHC ) 10.8 Hz, J ) 6.1 Hz, 1H, SCH). 13C NMR (50 MHz, CDCl3): δ 31.0 (d, 2JCC ) 34 Hz, CH3), 114.6 (d, 1JCC ) 69 Hz, CH13CON), 138.6 (SCH); 169.1 (13CON). EIMS (relative intensity): m/z 116.1 (M, 100). 3-[13C]-4,5-Dichloro-2-methyl-isothiazol-3-one (3a). 1H (200 MHz, CDCl3): δ 3.37 (d, 3JHC ) 2.72 Hz, 3H, CH3). 13C NMR (50 MHz, CDCl3): δ 31.4 (CH3), 115.0 (d, 1JCC ) 80.3 Hz, CClCON), 138.2 (d, 2JCC ) 13.1 Hz, CSCl), 162.0 (13CON). EIMS (relative intensity): m/z 188.1 (M, 2 × 37Cl, 15.3), 186.1 (M, 35Cl + 37Cl, 70.6), 184.1 (M, 2 × 35Cl, 100). 4-[13C]Isothiazolones. The same procedure as for the synthesis of nonlabeled isothiazolones was used starting from amide 10b (227 mg, 0.95 mmol) to give 4-[13C]-5-chloro-2-methylisothiazol-3-one (1b; 170 mg, 1.13 mmol, 59% yield), 4-[13C]-2methyl-isothiazol-3-one (2b; 43 mg, 0.37 mmol, 20% yield), and 4-[13C]-4,5-dichloro-2-methyl-isothiazol-3-one (3b; 15 mg, 0.09 mmol, 4% yield) as white solids. 4-[13C]-5-Chloro-2-methyl-isothiazol-3-one (1b). 1H NMR (CDCl3): δ 3.28 (3H, CH3), 6.27 (d, 1JHC ) 183.0 Hz, 1H, 13CH). 13C NMR (50 MHz, CDCl ): δ 29.7 (CH ), 114.2 (13CH), 145.2 3 3 (d, 1JCC ) 77.0 Hz, CCl), 166.7 (d, 1JCC ) 70.5 Hz, CON). EIMS (relative intensity): m/z 152.1 (M, 37Cl, 39.1), 150.1 (M, 35Cl, 100). 4-[13C]-2-Methyl-isothiazol-3-one (2b). 1H NMR (200 MHz, CDCl3): δ 3.35 (3H, CH3), 6.26 (dd, 1JHC ) 179.3 Hz, J ) 6.2 Hz, 1H, 13CHCON), 8.03 (dd, J ) 6.2 Hz, 2JHC ) 2.5 Hz, 1H, SCH). 13C NMR (50 MHz, CDCl3): δ 30.4 (CH3), 114.6 (13CHCON), 143.5 (d, 1JCC ) 65.6 Hz, SCH), 171.6 (d, 1JCC ) 68.4 Hz, CON). EIMS (relative intensity): m/z 116.2 (M, 100).

Alvarez-Sa´ nchez et al. 4-[13C]-4,5-Dichloro-2-methyl-isothiazol-3-one (3b). 1H NMR (200 MHz, CDCl3): δ 3.37 (3H, CH3). 13C NMR (50 MHz, CDCl3): δ 30.8 (CH3), 114.4 (13CClCON), 138.1 (d, 1JCC ) 86.9 Hz, CSCl), 161.4 (d, 1JCC ) 82.0 Hz, CON). EIMS (relative intensity): m/z 188.1 (M, 2 × 37Cl, 15.9), 186.1 (M, 35Cl + 37Cl, 69.7), 184.1 (M, 2 × 35Cl, 100). 5-[13C]Isothiazolones. The same procedure as for the synthesis of nonlabeled isothiazolones was used starting from amide 10c (242 mg, 1.02 mmol) to give 5-[13C]-5-chloro-2-methylisothiazol-3-one (1c; 173 mg, 1.15 mmol, 57% yield), 5-[13C]-2methyl-isothiazol-3-one (2c; 43 mg, 0.37 mmol, 18% yield), and 5-[13C]-4,5-dichloro-2-methyl-isothiazol-3-one (3c; 44 mg, 0.24 mmol, 12% yield) as white solids. 5-[13C]-5-Chloro-2-methyl-isothiazol-3-one (1c). 1H NMR (200 MHz, CDCl3): δ 3.27 (3H, CH3), 6.26 (d, 2JHC ) 0.7, 1H, CH). 13C NMR (50 MHz, CDCl3): δ 30.1 (d, 3JCC ) 3.3 Hz, CH3), 114.7 (d, 1JCC ) 77.0 Hz, CH), 145.6 (13CCl), 167.3 (d, 2JCC ) 8,2 Hz, CON). EIMS (relative intensity): m/z 152.1 (M, 37Cl, 38.0), 150.1 (M, 35Cl, 100). 5-[13C]-2-Methyl-isothiazol-3-one (2c). 1H NMR (200 MHz, CD3OD): δ 3.35 (3H, CH3), 6.22 (dd, 2JHC ) 6.4 Hz, J ) 6.4 Hz, 1H, CHCON), 8.39 (dd, 1JHC ) 191.6 Hz, J ) 6.4 Hz, 1H, S13CH). 13C NMR (50 MHz, CD OD): δ 30.8 (CH ), 114.4 (d, 1J 3 3 CC ) 67.7 Hz, CHCON), 143.2 (S13CH), 171.6 (CON). EIMS (relative intensity): m/z 116.2 (M, 100). 5-[13C]-4,5-Dichloro-2-methyl-isothiazol-3-one (3c). 1H NMR (200 MHz, CDCl3): δ 3.36 (3H, CH3). 13C NMR (50 MHz, CDCl3): δ 31.3 (CH3), 114.8 (d, 1JCC ) 86.9 Hz, CClCON), 138.1 (13CSCl), 161.9 (d, 2JCC ) 13.1 Hz, CON). EIMS (relative intensity): m/z 188.1 (M, 2 × 37Cl, 14.3), 186.1 (M, 35Cl + 37Cl, 68.3), 184.1 (M, 2 × 35Cl, 100). Reaction of Isothiazolones 1 or 2 with Model Nucleophiles. Reactions were followed by 13C NMR, and products formed were characterized by 13C and 1H data obtained by heteronuclear 1H{13C} HSQC and HMBC NMR experiments. The combination of these different data allowed for a full assignment of carbon 13 and proton chemical shifts on the isothiazolone structure and in some case on the model nucleophile. Chemical shifts are reported in parts per million (δ) with respect to TMS (CH3CN was used as internal standard at δ ) 119.65) and are indicated in Schemes 2-5. Structure Assignment. Structures of the different adducts were assigned using a combination of {1H}-decoupled 13C NMR and 1H{13C} HSQC and HMBC experiments carried out on a Bruker AM400 or Bruker ARX500. The measured chemical shifts were compared with those calculated using additivity principle (ChemNMR) and NMR data derived from analogous compounds (ACD/CNMR 5.12 and ACD/HNMR 5.12). Reaction of MCI or MI with n-Butylamine. To butylamine (10 equiv) in a mixture of H2O and D2O (0.35 and 0.05 mL, respectively), isothiazolone 1 or 2 (1 mg) was added. The solution was filtered into an NMR tube, a trace of acetonitrile was added as an internal reference, and the reaction was followed by 13C NMR. Reaction of MCI or MI with Imidazole. To imidazole (10 equiv) in a mixture of H2O and D2O (0.35 and 0.05 mL, respectively), isothiazolone 1 or 2 (1 mg) was added. The solution was filtered into an NMR tube, a trace of acetonitrile was added as an internal reference, and the reaction was followed by 13C NMR. Reaction of MCI or MI with Sodium Phenolate. To sodium phenolate (10 equiv) in a mixture of H2O and D2O (0.35 and 0.05 mL, respectively), isothiazolone 1 or 2 (1 mg) was added. The solution was filtered into an NMR tube, a trace of acetonitrile was added as an internal reference, and the reaction was followed by 13C NMR. Reaction of MCI with Sodium Propanethiolate. A solution of sodium propanethiolate (0.3 mL, 66 µmol, 0.22 M, 10 equiv) prepared from n-propanethiol (169 mg, 2.2 mmol), sodium hydroxide (80 mg, 2.0 mmol), and H2O (10 mL) was added to

Labeled Isothiazolones

Chem. Res. Toxicol., Vol. 16, No. 5, 2003 631

Scheme 1. Synthetic Pathways to the Labeled Phosphorane 7 and Isothiazolones 1-3

1a-c (1 mg, 6.6 µmol) in CD3CN (0.2 mL). The solution was filtered into an NMR tube, and the reaction was followed by 13C NMR. Reaction of MI with Sodium Propanethiolate. A solution of sodium propanethiolate (0.3 mL, 66 µmol, 0.29 M, 10 equiv) prepared from n-propanethiol (221 mg, 2.9 mmol), sodium hydroxide (104 mg, 2.6 mmol), and H2O (10 mL) was added to 2a-c (1 mg, 8.7 µmol) in CD3CN (0.2 mL). The solution was filtered into an NMR tube, and the reaction was followed by 13C NMR. Reaction of MCI with an Excess of N-Acetyl-Cys. To a solution of N-acetyl-Cys (11 mg, 66 µmol, 10 equiv) in degassed phosphate buffer (0.25 mL, 0.1 M, pH 7.7) was added a solution of 1b-c (1 mg, 6.6 µmol) in CD3CN (0.25 mL). The solution was filtered into an NMR tube, and the reaction was followed by 13C NMR. Reaction of MCI with a Deficiency of N-Acetyl-Cys. A solution of 1b,c (12.8 mg, 85.7 µmol, 40 equiv) in degassed CD3CN (0.25 mL) was mixed with a solution of N-acetyl-Cys (0.35 mg, 2.1 µmol, 1 equiv) in phosphate buffer (0.25 mL, 0.1 M, pH 7.7). The solution was filtered into an NMR tube, and the reaction was followed by 13C NMR. Reaction of MCI and MI with N-Acetyl-Cys. A solution of MCI 1c (1.3 mg, 8.6 µmol, 1 equiv) and MI 2b (1 mg, 8.6 µmol, 1 equiv) in a mixture of PBS (0.25 mL, 0.1 M, pH 7.7) and CD3CN (0.25 mL) was filtered into an NMR tube. N-AcetylCys (1.4 mg, 8.6 µmol, 1 equiv) was added, and the reaction was followed by 13C NMR. Reaction of MCI with Nr-Acetyl-Lys in the presence of N-Acetyl-Cys. A solution of 1b (12.9 mg, 85.7 µmol, 30 equiv) in degassed CD3CN (0.25 mL) was mixted to a solution of NRacetyl-Lys (6 mg, 32 µmol, 10 equiv) and N-acetyl-Cys (0.5 mg, 3 µmol, 1 equiv) in phosphate buffer (0.25 mL, 0.1 M, pH 7.7). The solution was filtered into an NMR tube, and the reaction was followed by 13C NMR.

Results and Discussion Synthesis. To study reaction mechanisms of isothiazolone derivatives with model protein residues and to investigate amino acid specificity in relation to sensitization potential, access to isothiazolones derivatives 1-3 labeled at positions 3-5 was required. We therefore developed a synthesis of carbon 13-labeled N-methylisothiazolones based on two main sequences of reactions. The first sequence, common to the synthesis of 3-[13C]

and 4-[13C] derivatives (Scheme 1) starts with a HellVollhardt-Zelinsky reaction of 1-[13C]- or 2[-13C]acetic acid with PBr3 and Br2 followed by ethanolysis to give, in 78% average yields, 1-[13C]- or 2-[13C]-2-bromo ethyl acetates, 5a,b, respectively. Reaction of these two bromo derivatives with triphenylphosphine in ethyl acetate gave phosphonium salts 6a,b, respectively, which were converted into their stabilized phosphorus ylides 7a,b, respectively, by treatment with aqueous sodium hydroxide in methylene chloride. The second sequence of reaction, which is common to the synthesis of 3-[13C]-, 4-[13C], and 5-[13C]-N-methylisothiazolones, starts with a tandem Wittig-Michael reaction between ylides 7a,b, respectively, with formaldehyde or between ylide 7 and [13C]formaldehyde in conditions close to that used by Cappon et al. (12). Formed ethyl acrylates were immediately reacted in a one pot reaction with thioacetic acid to give thioacetates 9a-c, respectively, in 75% average yields. Reaction of these thioacetates with aqueous methylamine, followed by a subsequent one pot oxidation with hydrogen peroxide, led to the formation of dithiopropionamides 10a-c, respectively, in 71% average yields as white solids. Isothiazolones 1a-c, 2a-c, and 3a-c were then formed from 10a-c, respectively, following Lewis conditions (SO2Cl2 in methylene chloride) (13) optimized for the formation of 5-chloro-2-methyl-isothiazol-3-one 1a-c. The mixture of isothiazolones was purified by column chromatography over alumina to give 1a-c in about 60% yields, 2a-c in about 20% yields, and 3a-c in about 10% yields. Reaction of MCI with Model Nucleophiles. To investigate the chemical reactivity of MCI toward amino acids, carbon 13-labeled derivatives 1a-c were first reacted with model nucleophiles. Reactions were followed by 13C NMR, and products formed were characterized by 13C and 1H data obtained by heteronuclear 1H{13C} HSQC and HMBC NMR experiments. The combination of these different data allowed for a full assignment of carbon 13 and proton chemical shifts on the isothiazolone structure and in some cases on the model nucleophile.

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Scheme 2. Reaction of 1a-c with Imidazole, Butylamine, Sodium Phenoxide, and Sodium Propanethiolate

1. Reaction with Imidazole (Scheme 2). Imidazole used as a simple model for His was found to react quite slowly with MCI at room temperature in water. Thus, after 45 days of reaction, only the residual signal of 1ac, respectively, was observed together with a new peak at 169.6, 103.6, and 150.7 ppm, respectively. Heteronuclear 1H{13C} NMR experiments gave us access to the chemical shift of proton H-4 at 6.39 ppm and N-CH3 at 3.26 ppm. These NMR data are in agreement with the structure of derivative 11 that can be formed through an addition-elimination reaction at position 5 of MCI. The presence of imidazole at position 5 was further confirmed by HMBC experiments carried out on adduct 1c for which imidazole protons, coupled with the carbon 13 at position 5, were observed at 7.10, 7.37, and 8.00 ppm, respectively. 2. Reaction with Butylamine (Scheme 2). Reaction of MCI with butylamine is rapid as compared to the one of imidazole, and no starting material remained after 24 h. Following the reaction of 1b over time, the formation of a first intermediate 12 could be detected at 84.0 ppm; this later was transformed into three final products 1315 with chemical shifts at 111.5, 52.4, and 43.8 ppm, respectively. Similar experiments carried out on derivatives 1a,c, completed by 1H{13C} NMR HSQC and HMBC experiments, gave us access to the full set of 1H/13C chemical shifts for the three adducts with butylamine. The intermediate 12 could result from an additionelimination of butylamine at position 5, as already observed with imidazole. Proton chemical shifts at about 2.65 ppm for N-CH3 indicate then an opening of the isothiazolone ring with formation of a secondary amide for 13-15. Such a reaction resulting from an attack at the sulfur atom by an excess of nucleophile has already been reported (14) even if in our case the precise nature of this nucleophile is not known (excess of butyamine or hydroxyl ion). This process could lead to the formation of 13 and 14 that are in equilibrium under reaction conditions. Derivative 15 appeared to be the oxygenated

analogue of 14, and acidification of the reaction medium led to a slow conversion of 14 into 15, as expected. 3. Reaction with Sodium Phenoxide (Scheme 2). MCI was found to be nonreactive toward phenol, a model for Tyr, in water. Nevertheless, to obtain 13C/1H NMR reference values for further studies, we forced the reaction by using 10 equiv of sodium phenoxide instead of phenol. The reactivity of this nucleophile appeared to be intermediate between butylamine and imidazole with a complete disappearance of 1 after 15 days. Three signals increased in intensity during the reaction, and 1H{13C} NMR experiments starting from 1a-c gave us NMR data in accordance with structures of compounds 16-18. Chemical shifts of N-CH3 between 3.10 and 3.21 ppm indicated the presence of a cyclic isothiazolone while 13C chemical shifts at position 5, 174.8, 159.3, and 156.0, respectively, indicated the presence of either a C-O or a C-C bond. While the major adduct 16 could arise from a classical O-alkylation of isothiazolone 1 through an addition-elimination mechanism, minor adducts 17 and 18 could arise from a C-alkylation, at the either ortho or para position of the phenoxide ring. These structures were further confirmed by 1H{13C} HMBC experiments, carried out on isothiazolone 1c, with the detection of the aromatic protons coupled with the carbon 13 at position 5. 4. Reaction with Sodium Propanethiolate (Scheme 2). Propanethiol, usually used as model for Cys, was found to be only slightly soluble in the mixture of water and acetonitrile, and we therefore used 10 equiv of sodium propanethiolate to get adducts. Under these conditions, the reaction appeared to be very fast and only one adduct could be detected. Combined 13C and 1H NMR data, with a chemical shift of N-CH3 around 2.70 ppm, suggest an open structure of mercaptoacrylamide type 19. This structure was further confirmed by neutralization of the reaction medium, which led to the formation of a mercaptothioester 20 that was fully characterized in CDCl3. The formation of 19 could arise from either a



Scheme 3. Reaction of 2a-c with Sodium Propanethiolate

Scheme 4. Reaction of 1b-c, 2b-c, and an Equimolar Mixture of 1b/2c with N-Acetyl-Cys

classical addition-elimination at position 5 followed by a nucleophilic attack of propanethiolate at the sulfur atom or a prior attack of propanethiolate at the sulfur atom to form a disulfide intermediate, followed by a new addition of propanethiolate to give a reactive thioacyl chloride that could further react with water and thiolate. Reaction of MI with Model Nucleophiles. To investigate the chemical reactivity of MI toward amino acids, carbon 13-labeled derivatives 2a-c were first reacted with model nucleophiles. As for MCI, reactions were followed by 13C NMR and products formed were characterized by 13C and 1H data obtained by heteronuclear 1H{13C} HSQC and HMBC NMR experiments. 1. Reaction with Imidazole, Butylamine, and Sodium Phenoxide. The reactivity of MI toward amino nucleophiles and sodium phenoxide was found to be very different from the one of MCI as no reaction toward imidazole nor sodium phenoxide could be detected even after 50 days. MI was also found to be almost nonreactive toward butylamine with only the formation of many products in trace amounts. 2. Reaction with Sodium Propanethiolate (Scheme 3). By contrast with amino nucleophiles and sodium phenoxide, sodium propanethiolate was found to be very reactive toward MI. Initially, the formation of a major intermediate 23 and of a minor intermediate 24 was observed. Over time, 23 was slowly transformed into derivative 24 together with the formation of a minor adduct 25. Proton chemical shifts of N-CH3 ranging from 2.6 to 2.7 ppm were consistent, for all of these derivatives, with an open structure. Moreover, carbon 13 signals at around 120 and 157 ppm for positions C-4 and C-5, respectively, indicated for compounds 23 and 24 the presence of a double bond. NMR data and transformation of 23 into 24 over time are therefore in favor of a Z/E isomerization to form the most stable thioenolate probably through the hemithioacetal 25. At equilibrium, once 2a-c was consumed, a peak assigned to the hemithioacetal 25 with characteristic 1H chemical shift at 4.23 ppm for H-5 and 2.45/2.50 ppm for protons H-4 and H-4′ was seen. Reaction of MCI and MI with N-Acetyl-Cys. From experiments carried out with model nucleophiles, thiolates were found to be the most reactive nucleophiles toward both MCI and MI. Nevertheless, the very rapid reaction of isothiazolones with thiol residues never allowed us to identify intermediate products that could give information on the reaction mechanism. Thus, to better understand the reactivity of this nucleophile under more physiological conditions, we have reacted MCI or MI with a thiol soluble in a semiorganic reaction medium, namely, N-acetyl-Cys. As pointed out previously, the use of amino acids could introduce an extra complexity in the inter-

pretation of NMR spectra but this should be balanced by data and structures already obtained from experiments with propanethiolate. 1. Reaction of MCI with N-Acetyl-Cys (Scheme 4). Thus, in a first step, MCI was reacted with an excess (10 equiv) of N-acetyl-Cys, at pH 7.7, in a semiorganic medium of phosphate buffer and acetonitrile. As in the case of propanethiolate, MCI was found to react rapidly with N-acetyl-Cys to form an adduct 26 similar to 20 formed by protonation of 19, with characteristic chemical shifts of a mercaptothioester. Then, 26 was slowly converted into the thioester 27 together with 28, characteristic of an amide structure and probably resulting from the well-known intramolecular reaction of the R-amino function of the Cys following deacetylation (15). To further slow the reaction and thus avoid multiple thiol additions on MCI and/or on its reaction intermediates, we decided to react MCI with a deficiency of thiol. Thus, 1b,c were reacted with 0.03 equiv of N-acetyl-Cys in a semiorganic medium. After 6 h, the residual signal of MCI was still present together with one for a hydrolysis product of malonic nature (29) (46.0/3.01 ppm). The intensity of this signal then slowly increased over time. Interestingly, no signal corresponding to an additionelimination product at position 5 nor to 26 and 27 could be detected. These results are therefore more in favor of an initial attack of the thiol group at the sulfur atom through a cleavage of the sulfur-nitrogen bond to form a disulfide bridge. This intermediate disulfide could then give, through an intra- or intermolecular nucleophilic attack, a thioacyl chloride readily hydrolyzed into 29. 2. Reaction of MI with N-Acetyl-Cys (Scheme 4). The same reaction carried out on MI at pH 7.7, in a semiorganic medium, led to the formation of only one adduct assigned as the disulfide 30. This adduct confirmed the reaction hypothesis proposed for the addition of propanethiolate on MI and the high susceptibility of

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Alvarez-Sa´ nchez et al. Scheme 5. Reaction of 1b with Nr-Acetyl-Lys in the Presence of N-Acetyl-Cys

Figure 1. Reaction of an equimolar mixture of MCI (1c) and MI (2b) toward N-acetyl-Cys at pH 7.7.

MCI and MI to react at the sulfur atom with sulfur nucleophiles. 3. Competition of MCI and MI with N-Acetyl-Cys (Figure 1, Scheme 4). MCI and MI being used in mixture and both reacting rapidly with thiols, we have reacted an equimolecular mixture of these two isothiazolones labeled in 5 and 4, respectively, with 1 equiv of N-acetyl-Cys at pH 7.7 in a semiorganic medium. As expected, the reaction was fast and after 4 h MCI had almost been consumed with formation of the hydrolysis products 29 together with the thio acid 31. Over time, MCI was totally converted into 29 with consumption of 31. This further confirmed the hypothesis of an initial attack at the sulfur atom to form a thioacyl chloride, which is first hydrolyzed into the thio acid 31 and then into the acid 29. No adduct nor byproducts resulting from a reaction of N-acetyl-Cys with MI could be detected. 4. Activation of MCI by N-Acetyl-Cys toward other Nucleophiles (Figure 2, Scheme 5). Reactions

carried out with a deficiency of thiol were in favor of an initial attack at the sulfur atom through a cleavage of the S-N bond. A second attack of this disulfide by a nucleophile could result in the formation of a very reactive thioacyl chloride intermediate (16). The fact that an initial reaction with thiols could enhance the reactivity of MCI was already reported by Collier et al. (17) showing that the enzymatic activity of an alcohol dehydrogenase was decreased by MCI in the presence of dithiothreitol. Similar observations were also made by Ghosh (18) with an increased reactivity of amino groups toward isothiazolones in the presence of thiols. To investigate the activator effect of a catalytic amount of thiol, we have reacted MCI 1b with 0.3 equiv of NR-acetyl-Lys in the presence of 0.03 equiv of N-acetyl-Cys at pH 7.7 in a semiorganic medium. After 1 day, three new signals were detected, which were assigned to adduct 32 and 33 similar to adducts 14 and 15, respectively, together with the hydrolysis product 29. The reaction was then followed up to 3 days without any significant changes except a slight increase of the amount of hydrolysis product 29. The same reaction carried out in the absence of N-acetylCys and followed for 4 days showed only the slow formation of the hydrolysis product 29 without any trace of Lys adduct. This confirmed that in the presence of thiols, MCI was transformed to a very reactive intermediate able to react with amino groups of the Lys type. This activation was found to be rather specific for primary amino groups as the same reaction carried out with protected histidine or tyrosine showed no significant formation of adducts. Mechanistic Considerations (Scheme 6). MCI was found to be rather reactive with nucleophiles even if the behavior and level of reactivity were very different. Thus, with amino nucleophile of the imidazole type, the reaction was found to proceed through a rather clean substitution reaction at position 5 leading to the formation of a stable cyclic adduct and no further hydrolysis of this intermediate could be detected. A similar mechanism was observed in the presence of phenoxide with the formation of a cyclic adduct resulting from a substitution at position 5. Nucleophiles of the Lys type were also found to react

Figure 2. Reaction of 1b with NR-Ac-Lys in the presence of N-Ac-Cys.



Scheme 6. Reaction Mechanisms of MCI and MI toward Model Nucleophiles

through an initial substitution at position 5 but leading to an unstable intermediate readily opened through a nucleophilic attack at the sulfur atom and cleavage of the S-N bond to form adducts of thioamide and amide types. In contrast, the reactivity of MCI toward thiols was found to be much more complex. First, the reaction with an excess of thiol was found to be very fast leading to the formation of open structures of the mercaptothioester and mercaptoester type. These products could arise from two very different mechanisms. The first one, similar to that proposed for the reaction with primary amino groups, would start with an initial thiol substitution at position 5 followed by a nucleophilic attack at the sulfur atom and cleavage of the S-N bond. The second one would start with an initial nucleophilic attack at the sulfur atom with cleavage of the S-N bond and formation of a disufide bond. Reattack of this disulfide bond by a second equivalent of thiol would lead to a very reactive thioacyl chloride (16) that would either react with water to give hydrolysis products or with a third equivalent of thiol to form the observed adducts. Complementary reactions of MCI or mixtures of MCI and MI with a catalytic amount of thiol led to the formation of thio acid and acid hydrolysis products while the same reactions carried out in the presence of NRacetyl-Lys led to the formation of thioamide and amide adducts not formed under these conditions in the absence of thiol. This is strongly in favor of an initial attack of thiols at the sulfur atom and subsequent formation of a very reactive thioacyl chloride able to react with nucleophiles present in the medium. Skin Sensitization Potential. Three main parameters could explain the difference in sensitizing potential of MCI and MI. First, the difference in hydrophobicity could influence the skin penetration. Values of LogP ) 0.60 and -0.11 for MCI and MI, respectively, indicate that as expected MCI is more hydrophobic than MI and should therefore have a better skin penetration. Second, a difference in chemical reactivity toward a defined amino

acid allowed one molecule to modify proteins to a larger extent. To that respect, the only common reactive function was found to be thiol groups and competition experiments have shown that MCI was much more reactive than MI toward Cys, despite the fact that mechanisms and structures of formed adducts were very different. Third, a different reactivity pattern allowing one molecule to react with a key amino acid. Experiments carried out on model nucleophiles have shown that MCI was able to react with different chemical functions including amino groups while MCI was only able to react with thiol groups. The absence of cross-reactions between MCI and MI indicates the formation of two different subpopulations of T-lymphocytes and could fit with the second and third hypothesis. The fact that rather few free Cys are available on proteins and the observation that many strong sensitizers seem to be able to react with primary amino groups of the Lys type could give an advantage to the third hypothesis even if we do not have at the present time conclusive evidences. Further reactivity studies on proteins would be needed to further progress on this point.

Conclusions MCI and MI were found to have very different behaviors toward model nucleophiles. While MCI, which is a strong sensitizer, reacted easily and rapidly with most nucleophiles, MI, a weaker sensitizer, reacted only with thiols. Moreover, in case of competition under equimolar conditions, MCI was significantly more reactive toward thiols than MI. The difference in sensitizing potential could then be related to either an overall difference in chemical reactivity or a difference in reactivity toward specific peptidic residues. Thus, with respect to amino nucleophiles, His and Lys, which have been proposed in several studies to play a major role in the mechanism of skin sensitization, a significant reactivity of MCI is expected, while MI should be mostly inert. This difference in reactivity toward amino nucleophiles could even be reinforced by the very high reactivity of MCI toward thiol

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nucleophiles. Indeed, it is generally accepted that conjugation with GSH, the main source of free thiol groups in cells, would rather correspond to a detoxication process than to a toxication mechanism. While this scheme would apply to MI, which is reactive only with thiol nucleophiles and could therefore be detoxified, the reactivity of MCI toward thiols could paradoxically lead to an increase in reactivity by the formation of a very active thioacyl chloride intermediate able to react with amino nucleophiles.

Acknowledgment. We thank the Centre National de la Recherche Scientifique (CNRS-France) and Unilever Research for a fellowship to R.A.S.

References (1) Rossmore, H. W. (1979) Heterocyclic compounds as industrial biocides. Dev. Ind. Microbiol. 20, 41-71. (2) Chan, P. K., Baldwin, R. C., Parsons, R. D., Moss, J. N., Stiratelli, R., Smith, J. M., and Hayes, A. W. (1983) Kathon biocides: manifestation of delayed contact dermatitis in guinea pigs is dependent on the concentration for induction and challenge. J. Invest. Dermatol. 81, 409-411. (3) Maibach, H. I. (1985) Diagnostic patch test concentration for Kathon CG. Contact Dermatitis 13, 242-245. (4) Bjo¨rkner, B., Bruze, M., Dahlquist, I., Fregert, S., Gruvberger, B., and Persson, K. (1986) Contact allergy to the preservative Kathon CG. Contact Dermatitis 14, 85-90. (5) Fewings, J., and Menne´, T. (1999) An update for the risk assesment for methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) with focus on rinse-off products. Contact Dermatitis 41, 1-13. (6) Bruze, M., Dahlquist, I., and Gruvberger, B. (1989) Contact allergy to dichlorinated methylisothiazolinone. Contact Dermatitis 20, 219-220.

Alvarez-Sa´ nchez et al. (7) Bruze, M., Fregert, S., Gruvberger, B., and Persson, K. (1987) Contact allergy to the active ingredients of Kathon CG in the guinea pig. Acta Derm. Venereol. (Stockholm) 67, 315-320. (8) Bruze, M., Gruvberger, B., and Persson, K. (1987) Contact allergy to a contaminant in Kathon CG in guinea pigs. Dermatosen Beruf Umwelt 35, 165-168. (9) Lepoittevin, J. P., Basketter, D. A., Goossens, A., and Karlberg, A. T., Eds. (1997) Allergic Contact Dermatitis: The Molecular Basis, Springer, Berlin. (10) Meschkat, E., Barratt, M. D., and Lepoittevin, J. P. (2001) Studies of the chemical selectivity of hapten, reactivity, and skin sensitization potency. 1. Synthesis and studies on the reactivity toward model nucleophiles of the 13C-labeled skin sensitizers hex-1-eneand hexane-1,3-sultones. Chem. Res. Toxicol. 14, 110-117. (11) Meschkat, E., Barratt, M. D., and Lepoittevin, J. P. (2001) Studies of the chemical selectivity of hapten, reactivity, and skin sensitization potency. 2. NMR studies of the covalent binding of the 13C-labeled skin sensitizers 2-[13C]- and 3-[13C]hex-1-ene- and 3-[13C]hexane-1,3-sultones to human serum albumin. Chem. Res. Toxicol. 14, 118-126. (12) Cappon, J. J., Baart, G. A., Van der Walle, G. A. M., Raap, J., and Lugtenburg, J. (1991) Chemo-enzymatic synthesis of specifically stable isotope labeled L-glutamic acid and 2-oxoglutaric acid. Recl. Trav. Chim. Pays-Bas 110, 158-166. (13) Lewis, S. N., Miller, G. A., Hausman, M., and Szamborski, E. C. (1971) Isothiazoles I: 4-Isothiazolin-3-ones. A general synthesis from 3,3′-dithiopropionamides. J. Heterocycl. Chem. 8, 571-586. (14) Crow, W. D., and Leonard, N. J. (1965) 3-Isothiazolone-cis-3thiocyanoacrylamide equilibria. J. Org. Chem. 30, 2660-2665. (15) Martin, R. B., and Hedrick, R. I. (1962) Intramolecular S-O and S-N acetyl transfer reaction. J. Am. Chem. Soc. 85, 106-110, (16) Seybold, G. (1975) Aliphatische thiocarbonsaürechloride. Darstellung und eigenschaften. Angew. Chem. 87, 710-711. (17) Collier, P. J., Austin, P., and Gilbert, P. (1991) Isothiazolone biocides: enzyme-inhibition pro-drug. Int. J. Pharm. 74, 195201. (18) Ghosh, T. (1999) Reaction of isothiazolones with amines. Phosphor. Sulphur, Silicon Relat. Elem. 153, 367-368.

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Studies of Chemical Selectivity of Hapten, Reactivity, and Skin

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