Chem. Res. Toxicol. 1994, 7,130-133
130
Interactions of Allergenic Hydroperoxides with Proteins: A Radical Mechanism? Jean-Pierre Lepoittevin**tand Ann-Ther6se KarlbergI Laboratoire de Dermatochimie associ6 au CNRS, Uniuersite Louis Pasteur, Clinique Dermatologique, CHU, F-67091 Strasbourg, France, and Department of Occupational Dermatology, National Institute of Occupational Health, S-17184Solna, Sweden Receiued December 23,199P
l-(l-Hydroperoxy-l-methylethy1)cyolohexene was synthesized as a model compound for the study of the interaction of 15-hydroperoxyabieticacid-like terpenes with proteins. Two related epoxides, 1-(l-hydroxy-l-methylethyl)-2-oxabicyclo[4.1.0] heptane and 2,2-dimethyl-l-oxaspiro[2.5]octan-4-01, were also prepared as reference materials. Treatment of the hydroperoxide with FeCl3 and N'-Ac-Cys-OMe led to the formation of the corresponding alcohol and of both epoxides. The allergenic activity of these compounds was tested in guinea pigs using the Freund's complete adjuvant test. The hydroperoxide was found to be a strong sensitizer while both epoxides were found to be inactive a t the same doses. The generation of highly-reactive radicals in the epidermis could lead to the formation of antigenic structures, the first step of the allergic contact dermatitis mechanism.
Introduction Among naturally occurring allergenic substances, terpenes have a special status as they need to be air-oxidized to become reactive; however, their method of interacting with skin proteins is still unknown. The first investigations on allergenic terpenes took place in the 1950s with the study of turpentine (1)which was widely used in paints and varnishes. It wasshown at that time that the allergenic principles were several hydroperoxides derived from autoxidation of A3-carene. Today, allergic contact dermatitis (ACD)l to colophony, the distillation residue of oleoresin from Pinus palustris Mill. or other Pinus species, is one of the 10 most commonly encountered allergic reactions (2). It has been found that pure abietic acid, the main resinic acid of colophony, suspected for many years to be the allergenic principle, is not itself a sensitizer (31, but that several oxidation products are formed in air (4). Of these autoxidation derivatives, the lbhydroperoxyabietic acid 1 (Chart 1)is a major sensitizer and elicitant (5).
The first step of the ACD mechanism is thought to be the interaction of the allergen with proteic structures of immunocompetentcells (6),but the interaction mechanism of hydroperoxidesderived from terpenes with skin proteins is still unknown. As the synthesis of the l&hydroperoxyabietic acid 1 is difficult, we have synthesized a simplified model 2 based on a single cyclohexene ring in order to study such interactions. It is generally accepted that the main hapten-protein interaction is the formation of a covalent bond between the hapten and nucleophilic residues of proteins through a nucleophile/electrophilemechanism, although a radical mechanism has also been suggested (7). When treated with transition-metal catalysts such as FeCls or copper-
* Addreae correspondence to this author at Laboratoire de Dermatochimie, Clinique Dermatologique, CHU, F-67091 Strasbourg, France. Cliiique Dermatologigue, Univenrite Louis Pasteur. National Institute of Occupetional Health. 0 Abstract published in Advance ACS Abstracts, March 1, 1994. 1Abbreviations: allergic contactdermatitis,ACD m-chloroperbenzoic acid, m-CPBA: Freund's complete adjuvant test, FCAT. 0893-228x/94/2707-0130$04.50/0
Chart 1
3
4
(11)trifluoromethanesulfonate (8),allylic hydroperoxides are known to rearrange readily into a-dioxolanyl alkyl radicals that may react with molecular oxygen to give hydroxy epoxides. These epoxides which are highly substituted are not expected to be highly reactive. However, as is the case for limonene oxide (9) which is a skin sensitizer, they might be candidates for electrophilic structures able to react with nucleophilic residues of proteins. In order to test this hypothesis, both rearrangement epoxides 3 and 4 derived from the model hydroperoxide 2 have been synthesized and tested for their allergenic properties.
Materials and Methods Caution: Skin contact with hydroperoxides must be avoided. As sensitizing substances, these compounds must be handled with care. Chemistry. 1H and '3C NMR spectra were recorded on a Bruker 200-MHz spectrometer in CDClS unless otherwise specified. Chemical shifts are reported in ppm (6) with respect to TMS, and CHC13 was wed as internal standard (6 = 7.27 ppm). Multiplicities are indicated by s (singlet),d (doublet),t (triplet), and m (multiplet). Infrared spectra were obtained on a PerkinElmer spectrometer;peaks are reported in reciprocal centimeters. Melting points were determined on a Buchi Tottoli 610 apparatus and are uncorrected. Dried solvents were freshly distilled before use. Tetrahydrofuran and ethyl ether were distilled from sodium benzophenone. Methylene chloride was dried over PZOSbefore distillation. All 0 1994 American Chemical Society
Chem. Res. Toricol., Vol. 7, No. 2, 1994 131
Communications air- or moisture-sensitive reactions were conducted in flamedried glassware under an atmosphere of dry argon. Chromatographic purifications were conducted on silica gel columns according to the flash chromatography technique. (A) 1-(1-Hydroxy-1-methylethy1)cyclohexene(6). To 1-acetyl-1-cyclohexene(15.5 g, 125 mmol) in THF (150 mL) was added, at -78 OC, MeLi (100mL, 140 mmol, 1.4 M in Et2O). After stirring at -78 OC for 2 h, the reaction mixture was allowed to warm up to room temperature. Aqueous NH&l (50 mL) was carefully added and the mixture extracted with ether (3 X 50 mL). Combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to lead to the crude alcohol, which was purified by column chromatogaphy on silica (10% AcOEt/ hexane) to yield 13.6 g (78% yield) of pure alcohol 6 as a colorless oil. lH NMR (CDC13) 6 1.29 (e, 3H, CH3), 1.30 (s, 3H, CH3), 1.48-1.64 (m, 5H, CH2 + OH), 2.02-2.06 (m, 4H, 2X =CCH2), 5.73 (dd, lH, J = 2.0 Hz, J = 3.6 Hz, =CH). '3C NMR (CDC13) 6 22.1,22.8,24.0,24.7,28.4(2C),72.4,118.3,143.5. IR (neat) v 3380 (OH), 1665 (C=C). Anal. Calcd for C9Hl60: C, 77.09; H, 11.50. Found: C, 77.12; H, 11.62. (B) 1- (1-Hydroperoxy - 1-methy lethy 1)cy clohexene (2). To a solution of H202 (50 mL, 35% in water) were added, at 0 "C, 1drop of 98% sulfuric acid and alcohol 6 (2 g, 14.28 mmol). The reaction mixture was vigourously stirred for 3 h at 0 O C and extracted with pentane (3 X 50 mL). Combined organic layers were dried over MgSO4, filtered, and evaporated under vacuum to give the crude hydroperoxide, which was purified by column chromatography (15% AcOEt/hexane) to give 1.8 g (81% yield) of the pure hydroperoxide 2 as a colorless oil. lH NMR (CDCl3) 6 1.32 (8, 6H, Me), 1.54-1.69 (m, 4H, CHz), 2.01-2.11 (m, 4H, CH2),5.77 (m,lH,=CH),7.32 (s,lH,OOH). 13CNMR(CDC13) 6 22.0, 22.7, 23.5 (2C), 23.6, 25.0, 84.1, 122.4, 139.8. IR (neat) v 3420 (OOH), 1660 (C=C). Anal. Calcd for CgH16O2: C, 69.19; H, 10.32. Found: C, 68.98; H, 10.49. (C) l-(l-Hydroxy-l-methylethyl)-2-oxabicyclo[4.1.0]heptane (3). To alcohol 6 (1g, 7.14 mmol) in CHzClz was added at 0 OC m-chloroperbenzoicacid (2.48 g, 7.2 mmol, 50% in water). After 1h at room temperature, the mixture was filtered and the precipitate washed with CH2C12. The organic layers were washed with a saturated solution of NaHCO3 (3 X 25 mL) and then with water (25 mL), dried over MgS04, filtered, and evaporated under reduced pressure to give the crude epoxide, which was purified by column chromatography (20% AcOEt/hexane) to give 1.04 g (94% yield) of 3 as a colorless oil. lH NMR (CDCl3) 6 1.22 (s, 3H, CH3), 1.24 (e, 3H, CH3), 1.17-1.52 (m, H, CH2), 1.72-1.83 (m, H, CH2), 1.94-2.04 (m, 2H, CHz), 3.37 (dd, lH, J = 1.6 Hz, J = 3.3Hz). '3CNMR(CDC13) 619.1,20.6,24.4,24.6,25.3,27.7,55.3, 64.9,69.8. IR (neat) v 3480 (OH). Anal. Calcd for CsHl602: C, 69.19; H, 10.32. Found: C, 69.34; H, 10.43. (D)2-(l-Hydroxy-l-methylethyl)cyclohexanone(8). To 2-acetylcyclohexanone(18.2 g, 130 mmol) in THF (200 mL) was added, at -78 OC, a solution of MeMgCl (130 mL, 260 mmol, 2 M in THF). After 3 h a t room temperature, the reaction mixture was carefully hydrolyzed with a saturated solution of NH4Cl(100 mL) and extracted with ether (3 X 50 mL). Combined organic layers were dried over MgS04and filtered and solvents removed under reduced pressure to give the crude alcohol, which was purified by column chromatography (20% AcOEt/hexane) to give 14.6 g (72% yield) of 8 as a colorless oil. lH NMR (CDC13) 6 1.21 (8, 6H, Me), 1.42-1.74 (m, 3H, CHz), 1.86-1.98 (m, lH, CHz),2.0-2.20 (m, 2H, CHz),2.26-2.46 (m, 3H, C(O)CH2 C(0)CH), 3.94 (8, lH, OH). 13CNMR (CDCl3) 6 25.0,25.4,27.5,28.0, 29.5,43.1,59.4,71.1,215.3. IR (neat) v 3520 (OH), 1700 (C=O). Anal. Calcd for C9H16O2: C, 69.19; H, 10.32. Found: C, 69.23; H, 10.47. (E)2-Isopropylidenecyclohexanone(9). To alcohol 8 (1g, 6.32 mmol) in Et20 (25 mL) was added, drop by drop and at 0 OC, &SO4 (0.5 mL, 98%). The reaction mixture was stirred until completion of the reaction. The reaction mixture was washed witha solution of Na2C03, dried over MgS04,and filtered and solvent removed under vacuum to give 9, which was purified by column chromatography (10% AcOEt/hexane) to give 0.59 g
+
Scheme 1. Synthetic P a t h w a y to Hydroperoxide 2 a n d Epoxide 3
MeLi (1.2,eq)
fl
THF, - 7 8 T
\ 6
5
\(yY
m-CPBA CH2C12,O"C
3
Scheme 2. Synthetic P a t h w a y t o Epoxide 4
1
8
/
,
9
LiAIHd
m-CPBA 4
CH2C12,O°C 4
10
(68% yield) of a colorless oil. lH NMR (CDCl3) 6 1.73 (m, 2H, CH2), 1.77 (s,3H, CHs), 1.84 (m, 2H, CHz), 1.98 (s,3H, CHs), 2.40 (m triplet-like, 2H, CH2), 2.48 (m triplet-like, 2H, CH2). l3C NMR (CDC13) 6 21.6, 22.6, 24.2 (2C), 29.4, 42.1, 132.1, 141.4, 203.8. IR (neat) v 1685 (C=O), 1615 (C=C). Anal. Calcd for CgH14O: C, 78.21; H, 10.21. Found: C, 78.34; H, 10.06. (F) 2-Isopropylidenecyclohexanol(10). To the ketone 9 (1 g, 7.14 mmol) in THF (25 mL) was added, at 0 OC, LiAlH4 (270.9 mg, 7.14 mmol). The reaction mixture was stirred at 0 OC for 2 h and hydrolyzed with a saturated solution of NH4Cl. The crude alcohol was purified by column chromatography (20% AcOEt/hexane) to give 0.83 g (83% yield) of 10 as a white solid mp 58-59 OC. 1H NMR (CDCl3) 6 1.07-1.16 (m, lH, CH), 1.291.42 (m, 2H, CHz), 1.60 (s,3H, CH3), 1.62-1.70 (m, 2H, CH2), 1.67 (s, 3H, CH3), 1.78-1.85 (m, lH, CH), 2.03 (t, lH, J = 13.7 Hz, CHC=),2.38(d,lH,J= 13.8 Hz,CHC=),4.80 (s,lH,CHOH). 13CNMR (CDCls) 6 19.4, 19.9, 20.1,24.8, 26.9, 33.9,66.2, 124.7, 132.8. IR (CHC13) v 3420 (OH). Anal. Calcd for CsHlsO: C, 77.09; H, 11.50. Found C, 76.84; H, 11.32. (G)2,2-Dimethyl-l-oxaspiro[2.5]octan-4-ol(4).To alcene 10 (1g, 7.14 mmol) in CH2Cl2 was added, at 0 OC, m-CPBA (2.48 g, 7.2 mmol, 50% in water). After 1h at room temperature, the mixture was filtered and the precipitate washed with CH2C12. The organic layers were washed with a saturated solution of NaHC03 (3 X 25 mL) and then with water (25 mL), dried over MgSO4, filtered, and evaporated under reduced pressure to give the crude epoxide,which was purified by column chromatography (20% AcOEt/hexane) to give 1.00 g (91% yield) of 3 as a colorless oil. 1H NMR (CDC13) 6 1.35 (8, 3H, CHs), 1.36 (e, 3H, CH3), 1.22-1.51 (m, 3H), 1.71-1.81 (m, 3H), 1.94-2.00 (m, lH), 2.122.23 (m, 2H), 3.82 (d, lH, J = 2.3 Hz). lSCNMR (CDCls)6 18.7, 20.2,20.4,24.6,24.9,31.9,62.9,68.3,68.7.IR (neat) v 3450 (OH). Anal. Calcd for CsHleO2: C, 69.19; H, 10.32. Found: C, 69.17; H, 10.43. (H) Reaction of Compound 2 Catalyzed with FeCla. To hydroperoxide 2 (37.44 mg, 0.24 mmol) and Na-Ac-Cys-OMe (86.73 mg, 0.49 mmol) in degassed THF (20 mL) was added a solution of FeC13 (20 pL, 0.1 M in ether). The reaction mixture was followed either by TLC and GC (SE30 column, 130 "C). Compounds were identified by coinjection of an aliquot of pure material. Biology. The sensitization experiments were performed in female outbred Dunkin-Hartley albino guinea pigs according to the modified Freund's complete adjuvant test (FCAT) (IO).
132 Chem. Res. Toxicol., Vol. 7, No. 2, 1994
Communications
Table 1. Sensitization Exwriments in Guinea Pigs According to FCAT (Freund's ComDlete Adjuvant Test). challenee material (% in olive oil)
~~~~
euinea Dies exposedb hydroperoxide 2 (n = 12) ~~~
p exp/coc epoxide 3 (n = 12) P explco
time (h)
48
72
12 12
