Synthesis and allergenic potential of a 15-hydroperoxyabietic acid-like model: Trapping of radical intermediates

Article abstract of DOI:10.1021/tx9901433

To better understand the skin sensitization mechanism of 15-hydroperoxyabietic acid-like compounds, 2-(1'-hydroperoxy-1'-methylethyl)-4a-methyl-3,4,4a,5,6,7-hexahydronaphthalene 7 was synthesized. The allergenic activity of this compound was tested on mice using the local lymph node assay, and further evaluated on guinea pigs using the Freund's complete adjuvant test. Using these methods, hydroperoxide 7 was found to be a strong sensitizer. Radical-trapping experiments were carried out on this compound in the presence of the spin-trapping agent, 1,1,3,3-tetramethylisoindolin-2-yloxyl 17, using light to induce radicals. The formation of carbon-centered radicals derived from compound 7 by intramolecular cyclization of an oxygen-centered radical was observed. The reaction of these intermediates with 17 gave various adducts in a very low yield, but a 1/1 mixture of diastereomers 18a and 18b (0.1% yield) was isolated and characterized, together with compound 19 (0.03% yield). The results suggest that carbon-centered reactive radicals are formed from allylic hydroperoxides and that this could be the possible mechanism involved in allergic contact dermatitis to abietic acid.

Full text of DOI:10.1021/tx9901433

1028
Chem. Res. Toxicol. 2000, 13, 1028-1036
Syn th esis a n d Aller gen ic P oten tia l of a
15-Hyd r op er oxya bietic Acid -lik e Mod el: Tr a p p in g of
Ra d ica l In ter m ed ia tes
Vincent Mutterer,† Elena Gime´nez Arnau,† Ann-Therese Karlberg,‡ and
J ean-Pierre Lepoittevin*,†
Laboratoire de Dermatochimie associe´ au CNRS, Clinique Dermatologique,
Universite´ Louis Pasteur, CHU, F-67091 Strasbourg Cedex, France, and Dermatology Division,
National Institute for Working Life, S-11279 Stockholm, Sweden
Received August 3, 1999
To better understand the skin sensitization mechanism of 15-hydroperoxyabietic acid-like
compounds, 2-(1′-hydroperoxy-1′-methylethyl)-4a-methyl-3,4,4a,5,6,7-hexahydronaphthalene 7
was synthesized. The allergenic activity of this compound was tested on mice using the local
lymph node assay, and further evaluated on guinea pigs using the Freund’s complete adjuvant
test. Using these methods, hydroperoxide 7 was found to be a strong sensitizer. Radical-trapping
experiments were carried out on this compound in the presence of the spin-trapping agent,
1,1,3,3-tetramethylisoindolin-2-yloxyl 17, using light to induce radicals. The formation of carbon-
centered radicals derived from compound 7 by intramolecular cyclization of an oxygen-centered
radical was observed. The reaction of these intermediates with 17 gave various adducts in a
very low yield, but a 1/1 mixture of diastereomers 18a and 18b (0.1% yield) was isolated and
characterized, together with compound 19 (0.03% yield). The results suggest that carbon-
centered reactive radicals are formed from allylic hydroperoxides and that this could be the
possible mechanism involved in allergic contact dermatitis to abietic acid.
Ch a r t 1
In tr od u ction
Colophony (rosin) is a naturally occurring material
obtained from coniferous trees (Pinaceae family) either
from oleoresins, tapped from living trees, or by distillation
of tall oil, a byproduct of the paper pulp industry.
Colophony is a complex mixture of diterpene resinic acids
(85-95%) and their oxidation products (1), the major
constituents being abietic acid 1 and dehydroabietic acid
2 (Chart 1). Due to its stickiness and emulsifying and
insulating properties, colophony is a common ingredient
in many products, such as adhesives, printing ink, paper
size (used in a process to render paper hydrophobic),
paint, varnishes, soldering fluxes, cutting fluids, dental
products, and cosmetics (2, 3). Colophony is also known
to cause contact allergy (3, 4). Abietic acid 1, suspected
for many years of being the allergenic principle, is not
itself a sensitizer (5), but its autoxidation derivative in
air, the 15-hydroperoxyabietic acid 3, has been found to
be a major sensitizer and elicitant (6).
of immunocompetent cells, which is generally accepted
to occur through a nucleophile/electrophile mechanism
(7). However, the mechanism of interaction of hydroper-
oxides, derived from terpenes, with skin proteins does
not fit this model and is still unknown. In recent years,
mechanisms involving radicals have gained increased
prominence in the discussion of hapten-protein binding,
and our previous studies indicate that radical reactions
could be important in the case of haptens containing
hydroperoxide groups (8-10). Studies on the mechanisms
leading to the formation of radicals and their subsequent
reaction with proteins could give new insights in our
understanding of antigen formation.
To better understand the interaction of 15-hydroper-
oxyabietic acid-like compounds with proteins, we have
already reported the synthesis and allergenic properties
of a simplified model 4 (Figure 1) of 15-hydroperoxyabi-
etic acid 3, based on a single cyclohexene ring containing
a tertiary allylic hydroperoxide (8). The results confirmed
The first step of the allergic contact dermatitis (ACD)1
mechanism is thought to be the interaction of the
allergen, a low-molecular weight molecule, with proteins
* To whom correspondence should be addressed: Laboratoire
de Dermatochimie, Clinique Dermatologique, CHU, F-67091 Stras-
bourg Cedex, France. Fax: +33 3 88 14 04 47. E-mail: jplepoit@
chimie.u-strasbg.fr.
† Universite´ Louis Pasteur.
‡ National Institute for Working Life.
1 Abbreviations: ACD, allergic contact dermatitis; COSY, correlation
spectroscopy; FCA, Freund’s complete adjuvant; FCAT, Freund’s
complete adjuvant test; HMBC, heteronuclear multiple-bond correla-
tion; HSQC, heteronuclear single-quantum correlation; LLNA, local
lymph node assay; NOE, nuclear Overhauser effect; NOESY, nuclear
Overhauser effect spectroscopy; TTBP, tritertiobutylphenol; Fe3+-TPP,
Fe3+ tetraphenylporphine.
10.1021/tx9901433 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/21/2000

Radicals Derived from Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1029
Sch em e 1. Syn th etic P a th w a y to Hyd r op er oxid e 7
F igu r e 1. Formation of hydroxyepoxides 5 and 6 from allylic
hydroperoxide 4.
the high sensitizing potential of such tertiary and allylic
hydroperoxides, but ruled out a possible nucleophilic/
electrophilic route involving epoxides (5 and 6) as the
actual sensitizers derived from such hydroperoxides, as
both epoxides failed to induce a significant sensitization.
Reaction of 4 with NR-Ac-Cys-OMe and a catalytic
amount of FeCl3 led to the formation of a complex
mixture of products, the major product being the reduced
alcohol, but both epoxides 5 and 6 were also identified.
In abietic acid derivatives, the presence of two conju-
gated double bonds will lead to the formation of allylic
radicals or epoxides which could have different reactivi-
ties. We therefore synthesized a bicyclic compound 7
(Scheme 1) to test a more realistic model containing both
B and C rings of 15-hydroperoxyabietic acid 3. In this
paper, we report the synthesis of this new bicyclic model
and a study of its sensitizing properties in mice using
the local lymph node assay (LLNA), further evaluated
on guinea pigs using the Freund’s complete adjuvant test
(FCAT). The rearrangement of the allylic hydroperoxide
7 was further investigated using a radical-trapping agent,
and the isolation and characterization of intermediates
are also reported.
pumps. The solutions were analyzed and compounds separated
on reversed phase columns [C18, Nucleosil (5 µm), Interchrom
(4.6 mm); C18, RSil (5 µm), Interchrom (10 mm)], the absorption
of the eluted material being monitored by an M686 tunable
absorbance detector at 254 nm. Mass spectrometry was per-
formed using a Finnigan model TSQ-700 instrument equipped
with a Digital DEC Station 5000 and with ICIS2 software.
Electron impact mass spectrometry was performed in the direct
probe electron impact mode, ionization being achieved with a
70 eV electron beam and the ion source temperature being
180 °C.
Ch em ica ls. Dried solvents were freshly distilled before use.
Tetrahydrofuran and diethyl ether were distilled from sodium/
benzophenone. Dichloromethane was dried over P2O5 before
distillation. All air- or moisture-sensitive reactions were per-
formed in flame-dried glassware under an atmosphere of dry
argon. Chromatographic purifications were carried out on silica
gel columns (Merck silica gel G 60, 0.040-0.063 mm) according
to the flash chromatography technique. Freund’s complete
adjuvant (FCA) was purchased from Difco (Detroit, MI). Olive
oil was purchased from Aldrich.
Ma ter ia ls a n d Meth od s
Ha za r d ou s Ma ter ia ls: Skin contact with hydroperoxides
must be avoided. Since these compounds are skin-sensitizing
substances, they must be handled with care.
Ma ter ia ls. 1H and 13C NMR spectra were recorded on Bruker
AC200 and AM500 MHz spectrometers in CDCl3 or in benzene-
d6. The chemical shifts are reported in parts per million (δ) and
are relative to that of CDCl3 at 7.25 ppm or to that of benzene-
d6 at 7.15 ppm. Multiplicities are indicated by s (singlet), d
(doublet), t (triplet), and m (multiplet). The two-dimensional
phase-sensitive 1H-1H COSY (correlation spectroscopy) spectra
were collected using a standard pulse sequence with a 254 ms
acquisition time, a sweep width of 12.25 ppm, 512 complete t1
data points, and 8 scans per increment. The two-dimensional
phase-sensitive 1H-1H NOESY (nuclear Overhauser effect
spectroscopy) spectra were collected using an 800 ms mixing
time and a 254 ms acquisition time, a sweep width of 12.25 ppm,
512 complete t1 data points, and 16 scans per increment. The
two-dimensional 1H-13C HSQC (heteronuclear single-quantum
1
correlation) spectra and H-13C HMBC (heteronuclear multiple-
bond correlation) spectra were collected using a standard pulse
sequence, with a 254 ms acquisition time, 512 complete t1 data
points, 16 (HSQC) or 32 (HMBC) scans per increment, and a
spectral width in the f1 dimension of 209.23 ppm. The data were
processed using XWINNMR software. Infrared spectra were
recorded on a Perkin-Elmer FT-IR 1600 spectrometer; the peaks
are reported in reciprocal centimeters. The HPLC analyses were
performed on a Waters Associates apparatus equipped with a
model M680 automated gradient controller and two M510
4a-Meth yl-2,3,4,4a,5,6,7,8-octah ydr on aph th alen -2-on e (8).
To a solution of previously distilled 2-methylcyclohexanone (112
g, 1.0 mol) in toluene (200 mL) were successively added methyl
vinyl ketone (100 mL, 1.22 mol) and sulfuric acid (0.8 mL, 97%).
The mixture was heated to reflux for 24 h, cooled to room
temperature, and poured into hexane (250 mL). The organic
solution was washed with an aqueous solution of potassium
hydroxide (4 × 150 mL, 5% w/w) and with brine (3 × 100 mL),
dried over MgSO4, filtered, and concentrated under reduced

1030 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Mutterer et al.
pressure to give an orange oil which was purified by double
distillation under reduced pressure to give 70.9 g (0.43 mol, 43%
yield) of 8 as a colorless oil: 1H NMR (CDCl3) δ 1.23 (s, 3H,
CH3), 1.24-1.47 (m, 2H, CH2), 1.61-1.95 (m, 6H, 3 × CH2),
2.25-2.60 (m, 4H, 2 × CH2), 5.72 (s, 1H, dCH); 13C NMR
(CDCl3) δ 21.8, 22.1, 27.2, 32.8, 34.1, 36.0, 38.1, 41.6, 124.2,
170.6, 199.7; IR (neat) ν 1670 (CdO), 2859 cm-1 (C-H).
methane (20%)] to give 6.52 g (34.6 mmol, 75% yield) of 12 as
a colorless oil (mixture of two isomers) together with (1-chloro-
1-methyl)ethyl phenyl sulfoxide 13 (0.91 g, 4.5 mmol, 10% yield).
Major isomer of compound 12: 1H NMR (CDCl3) δ 1.61 (d, 3H,
CH3, J ) 6.7 Hz), 4.51 (q, 1H, CH, J ) 6.7 Hz), 7.47-7.76 (m,
5H, dCH); 13C NMR (CDCl3) δ 17.8, 72.3, 125.6 (2C), 129.1 (2C),
132.1, 141.0. Minor isomer of compound 12: 1H NMR (CDCl3)
δ 1.74 (d, 3H, CH3, J ) 6.7 Hz), 4.70 (q, 1H, CH, J ) 6.7 Hz),
7.47-7.76 (m, 5H, dCH); 13C NMR (CDCl3) δ 17.4, 70.9, 125.9
(2C), 128.9 (2C), 132.1, 138.9; IR (neat) ν 751 (C-Cl), 1032 (Sd
O), 3006 cm-1 (C-H). Anal. Calcd for C8H9ClOS: C, 50.92; H,
4.80. Found: C, 51.01; H, 4.99. Compound 13: 1H NMR (CDCl3)
δ 1.55 (s, 3H, CH3), 1.87 (s, 3H, CH3), 7.46-7.60 (m, 3H, 2 ×
4a-Meth yl-1,2,3,4,4a,5,6,7-octah ydr on aph th alen -2-on e (9).
Ketone 8 (1.0 g, 6.1 mmol) was dissolved in freshly distilled
diglyme (30 mL), and 18-C-6 crown ether (160 mg, 0.6 mmol),
previously dried for 12 h under reduced pressure, was added.
The solution was cooled to 0 °C and potassium tert-butoxide (1.0
g, 7.9 mmol) added. The reaction mixture was allowed to warm
to room temperature and was then stirred for 1 h before a cold
aqueous acetic acid solution (33 mL, 57 mmol, 10% v/v) was
added and the solution stirred vigorously for 1 min. A saturated
aqueous solution of sodium hydrogen carbonate (140 mL, 114
mmol) was added, and then after 5 min, the aqueous phase was
extracted with pentane (8 × 50 mL). The combined organic
phases were washed with water (5 × 40 mL) and with brine (3
× 40 mL), dried over MgSO4, filtered, and concentrated under
reduced pressure. Pentane (5 mL) was added to the yellowish
oil that was produced, and then the solution was left at -30 °C
for 12 h. The crystals were filtered and washed with cold
pentane (2 × 5 mL, -30 °C), and recrystallization was repeated
twice. For each crystallization, the mother solution was con-
centrated under reduced pressure and the oil that was produced
crystallized again. Compound 9 (680 mg, 4.14 mmol, 68% yield)
was obtained as a colorless oil at room temperature: 1H NMR
(CDCl3) δ 1.23 (s, 3H, CH3), 1.33-1.97 (m, 8H, 4 × CH2), 2.31-
3.25 (m, 4H, 2 × CH2), 5.33-5.38 (m, 1H, dCH); 13C NMR
(CDCl3) δ 18.9, 23.9, 25.6, 34.1, 37.9, 38.3, 38.9, 48.6, 123.4,
138.0, 209.9; IR (neat) ν 1713 (CdO), 2933 cm-1 (C-H).
H
ortho and Hpara), 7.70-7.79 (m, 2H, 2 × Hmeta); 13C NMR (CDCl3)
δ 24.1, 27.5, 84.3, 127.1 (2C), 128.5 (2C), 132.0, 139.0; IR (neat)
ν 751 (C-Cl), 1032 (SdO), 3058 cm-1 (C-H). Anal. Calcd for
C9H11ClOS: C, 53.32; H, 5.46. Found: C, 53.26; H, 5.35.
2-(1′,2-E p oxyet h yl-1′-p h en ylsu lfoxy)-4a -m et h yl-1,2,3,-
4a ,5,6,7-octa h yd r on a p h th a len e (14). To a solution of diiso-
propylamine (0.9 mL, 6.4 mmol) in anhydrous tetrahydrofuran
(20 mL) at -30 °C was added dropwise a solution of butyllithium
(4 mL, 6.4 mmol, 1.6 M solution in hexanes). The reaction
mixture was stirred at -30 °C for 45 min and then cooled to
-78 °C. A solution of chloroethyl phenyl sulfoxide 12 (1.21 g,
6.4 mmol) in anhydrous tetrahydrofuran (10 mL) was then
added, and the reaction mixture was stirred for 30 min. To this
solution was slowly added ketone 9 (0.87 g, 5.3 mmol) in
anhydrous tetrahydrofuran (10 mL). After 3 h at -78 °C, the
mixture was warmed to room temperature, treated with aqueous
sodium hydroxide (5.4 mL, 50% w/w), and stirred for 1 h.
Extraction with ether, followed by washing with water and
brine, drying, and solvent removal, gave a yellow oil which was
purified by silica gel column chromatography [hexane/AcOEt
(10%), dichloromethane (20%)] to give 1.56 g (4.93 mmol, 92%
yield) of the yellow oil 14 as a mixture of two isomers. Major
isomer (54%, 1H integration): 1H NMR (CDCl3) δ 1.15 (s, 3H,
CH3), 1.20-3.49 (m, 12H, 6 × CH2), 1.30 [s, 3H, C(S)(CH3)],
5.49-5.54 (m, 1H, dCH), 7.46-7.56 (m, 3H, 2 × Hortho and Hpara),
7.62-7.70 (m, 2H, 2 × Hmeta); 13C NMR (CDCl3) δ 7.7, 18.9, 23.5,
25.9, 27.0, 34.2, 36.3, 38.4, 38.9, 70.8, 79.4, 123.9, 125.2 (2C),
Ch lor om eth yl P h en yl Su lfoxid e (11). To a solution of
thioanisole 10 (20 mL, 0.17 mol) in dichloromethane (40 mL)
was added a solution of sulfuryl chloride (17 mL, 0.21 mol) in
dichloromethane (20 mL). After being stirred for 2 h under
reflux, the reaction mixture was cooled to 0 °C, and then water
(21 mL), silica (21 g, 1 g/mL H2O, Merck Geduran SI 60), and
a solution of sulfuryl chloride (17 mL, 0.21 mol) in dichloro-
methane (20 mL) were added dropwise. The reaction mixture
was allowed to warm to room temperature and then was stirred
for 2 h before potassium carbonate (21 g, 1 g/mL H2O) was added
and the solution was stirred for 30 min at room temperature.
The solution was filtered and concentrated under reduced
pressure to give a residue which was purified by silica gel
column chromatography (hexane/AcOEt, 30%) to give 24.8 g
(0.14 mol, 84% yield) of 11 as a yellowish oil: 1H NMR (acetone-
d6) δ 4.70 (AB system, 2H, CH2, J AB ) 11.0 Hz, ∆ν ) 40.1 Hz),
7.60-7.65 (m, 2H, 2 × Hmeta), 7.73-7.79 (m, 3H, 2 × Hortho and
1
128.8 (2C), 131.0, 138.1, 139.7. Minor isomer (46%, H integra-
tion): 1H NMR (CDCl3) δ 1.15 (s, 3H, CH3), 1.22-3.12 (m, 12H,
6 × CH2), 1.32 [s, 3H, C(S)(CH3)], 5.33-5.37 (m, 1H, dCH),
7.46-7.56 (m, 3H, 2 × Hortho and Hpara), 7.62-7.70 (m, 2H, 2 ×
H
meta); 13C NMR (CDCl3) δ 7.7, 18.9, 23.5, 25.9, 26.2, 34.2, 37.1,
38.5, 38.9, 70.8, 79.6, 123.7, 125.2 (2C), 128.8 (2C), 131.0, 138.1,
139.7; IR (CHCl3) ν 1040 (SdO), 2968 cm-1 (C-H). Anal. Calcd
for C19H24O2S: C, 72.11; H, 7.64. Found: C, 71.85; H, 7.77.
2-Ace t yl-4a -m e t h yl-3,4,4a ,5,6,7-h e xa h yd r on a p h t h a -
len e (15). A solution of compound 14 (400 mg, 1.26 mmol) in
freshly distilled toluene (10 mL) was treated with silica (1.0 g,
Merck, Geduran SI 60, 0.040-0.063 mm) and heated to reflux.
After 4 h, the reaction mixture was cooled to room temperature,
filtered, and concentrated under reduced pressure. The yellow
oil that was produced was purified by silica gel column chro-
matography (10% hexane/AcOEt) to give 0.18 g (0.96 mmol, 76%
yield) of 15 as a yellowish oil: 1H NMR (CDCl3) δ 0.95 (s, 3H,
CH3), 1.18-2.60 (m, 10H, 5 × CH2), 2.32 [s, 3H, C(O)CH3], 5.86-
5.88 (m, 1H, dCH-CH2), 6.92 (s, 1H, dCH-C); 13C NMR (CDCl3)
δ 18.2, 20.6, 23.4, 25.4, 26.5, 32.0, 36.3, 36.9, 133.2, 135.5, 139.3,
141.2, 199.3; IR (neat) ν 1660 (CdO), 2925 cm-1 (C-H). Anal.
Calcd for C13H18O: C, 82.05; H, 9.53. Found: C, 81.83; H, 9.32.
H
para); 13C NMR (CDCl3) δ 61.4, 124.9 (2C), 129.4 (2C), 132.2,
141.0; IR (neat) ν 690 (C-Cl), 1056 (SdO), 3006 cm-1 (C-H).
Ch lor oeth yl P h en yl Su lfoxid e (12). To a solution of
diisopropylamine (7.2 mL, 51 mmol) in anhydrous tetrahydro-
furan (100 mL) at -30 °C was added dropwise a solution of
butyllithium (31.2 mL, 49.2 mmol, 1.6 M solution in hexanes).
The mixture was stirred at -30 °C for 45 min and then cooled
to -78 °C. Simultaneously, a solution of chloromethyl phenyl
sulfoxide 11 (8.0 g, 46 mmol) in anhydrous tetrahydrofuran (40
mL) was prepared, cooled to -78 °C, and then slowly added to
the first solution via a cannula. The colorless reaction mixture
turned orange; after being stirred for 45 min at -78 °C, methyl
iodide (8.5 mL, 137 mmol) was added dropwise. Stirring was
continued at -78 °C for 2 h, and then the temperature was
allowed to rise to -10 °C. The reaction was quenched with a
saturated ammonium chloride solution (60 mL), and the mixture
was stirred for 15 min and then poured into 40 mL of water.
The aqueous phase was extracted with ether (4 × 50 mL), and
then the combined organic phases were washed with brine (3
× 30 mL), dried over MgSO4, filtered, and concentrated under
reduced pressure to leave a residue that was purified by silica
gel column chromatography [hexane/AcOEt (20%), dichloro-
2-(1′-H yd r oxy-1′-m e t h yle t h yl)-4a -m e t h yl-3,4,4a ,5,6,7-
h exa h yd r on a p h th a len e (16). To a solution of ketone 15 (2.78
g, 14.6 mmol) and lithium bromide (1.3 g, 14.7 mmol) in dry
ether (80 mL) at -78 °C was added dropwise methyllithium (21
mL, 29.4 mmol, 1.4 M solution in ether). After 30 min at -78
°C, methanol (16 mL) was slowly added, and the reaction
mixture was allowed to warm to room temperature. Ether (50
mL) was then added and the solution filtered, dried over MgSO4,
and concentrated under reduced pressure. Purification of the
crude product by silica gel column chromatography (15% hexane/

Radicals Derived from Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1031
Ta ble 1. 1H a n d 13C NMR Da ta of Tr a p p in g P r od u cts
18a a n d 18ba
AcOEt) gave 2.77 g (13.4 mmol, 92% yield) of 16 as a colorless
oil: 1H NMR (CDCl3) δ 0.98 (s, 3H, CH3), 1.22-2.21 (m, 11H,
5 × CH2 and OH), 1.35 [s, 3H, C(OH)CH3], 1.36 [s, 3H, C(OH)-
CH3], 5.42-5.46 (m, 1H, dCH-CH2), 6.05 (s, 1H, dCH-C); 13C
NMR (CDCl3) δ 18.5, 22.1, 23.2, 25.8, 28.9, 29.0, 31.8, 37.3, 37.5,
72.9, 121.3, 123.8, 140.9, 142.9; IR (neat) ν 2872 (C-H), 3597
cm-1 (OH). Anal. Calcd for C14H22O: C, 81.49; H, 10 74. Found:
C, 81.52; H, 10.92.
2-(1′-Hydr oper oxy-1′-m eth yleth yl)-4a-m eth yl-3,4,4a,5,6,7-
h exa h yd r on a p h th a len e (7). To a solution of hydrogen per-
oxide (10 mL, 35%) and sulfuric acid (1 drop, 97%) was added
a solution of alcohol 16 (0.5 g, 2.4 mmol) in pentane (1.5 mL).
The reaction mixture was vigorously stirred for 4 h at 0 °C and
then extracted with dichloromethane (4 × 8 mL). The combined
organic layers were washed with brine (2 × 8 mL), dried over
MgSO4, filtered, and concentrated under reduced pressure to
give the crude hydroperoxide which was purified by silica gel
column chromatography [hexane/AcOEt (20%), dichloromethane
(20%)] to give 0.19 g (0.87 mmol, 36% yield) of 7 as a colorless
oil: 1H NMR (CDCl3) δ 0.84-2.24 (m, 10H, 5 × CH2), 1.01 (s,
3H, CH3), 1.25 [s, 3H, C(OOH)CH3], 1.31 [s, 3H, C(OOH)CH3],
5.39-5.42 (m, 1H, dCH-CH2), 6.08 (s, 1H, dCH-C), 6.87 (s, 1H,
OOH); 13C NMR (CDCl3) δ 18.9, 22.0, 23.4, 23.8, 24.4, 26.1, 32.1,
37.6, 37.7, 83.8, 124.2, 125.1, 139.6, 141.0; IR (neat) ν 2885
(C-H), 3425 cm-1 (OOH). Due to the instability of compound
7, no satisfactory elemental analysis was obtained.
18a
18b
position
1H [J (Hz)]
13C
1H [J (Hz)]
13C
1
2
3
4
4.57 (m)
83.4 4.25 (m)
68.5
86.4
67.8
19.0
46.6
1.52 (m)
18.9 1.57 (m)
47.5 1.26 (m), 2.24
(dd, 14.1, 2.9)
35.1
39.7 1.44 (m), 1.55 (m)
25.8 1.85 (m)
39.1 1.94 (m), 3.38
(ddd, 13.4, 4.9, 2.7)
121.7 5.23 (m)
140.2
1.57 (m), 2.40
(dd, 12.5, 3.8)
4a
5
6
34.5
40.0
25.8
34.0
1.44 (m), 1.54 (m)
1.85 (m)
2.30 (m), 2.60
(ddd, 14.2, 5.4, 2.9)
5.24 (m)
7
8
8a
9
2′
3′
121.2
141.3
26.2
60.9
20.9
20.8
68.1
68.5
146.2
Tr a p p in g Exp er im en t. The free radical-trapping agent,
1,1,3,3-tetramethylisoindolin-2-yloxyl 17, was synthesized from
N-benzylphthalimide by a previously described method (11) by
reaction with methylmagnesium bromide in refluxing toluene,
followed by hydrogenolysis and oxidation. Hydroperoxide 7 was
prepared from the tertiary alcohol 16 (1.0 g, 4.8 mmol) as
described above and used without further purification. The
crude product (281 mg) resulting from the hydroperoxidation
reaction was dissolved in degassed dichloromethane (20 mL).
The chemically stable nitroxide free radical 17 (721 mg, 3.8
mmol) was added to the solution and the stirred reaction
mixture irradiated for 3 weeks with a 150 W tungsten filament
lamp at a distance of 30 cm. The heat of the lamp induced
dichloromethane reflux, and the solution, initially yellow,
became dark orange. The mixture was concentrated under
reduced pressure and fractionated into polar and nonpolar
compounds by silica gel column chromatography (15% hexane/
Et2O). Further purification of the nonpolar fraction was carried
out using HPLC (C18 reversed phase, 25/75 H2O/CH3CN) to give
a nonseparable mixture of two diastereomers (18a and 18b) in
a 51/49 ratio (1.9 mg, 0.1% yield), together with compound 19
(0.6 mg, 0.03% yield). The polar fraction was found to contain
mainly the tertiary alcohol 16 and the R,â-unsaturated ketone
8, characterized by comparison with authentic samples.
1.18 (s)
25.9 1.51 (s)
62.2
22.7 1.13 (s)
21.3 1.23 (s)
67.3
1.30 (s)
1.63 (s)
4′
1′′
3′′
3′′a
4′′
5′′
6′′
7′′
7′′a
8′′
9′′
10′′
11′′
68.8
145.9
6.93 (ddd, 6.4, 2.3, 1.2) 121.8 6.97 (ddd, 6.4, 2.3, 1.2) 121.7
7.10 (dt, 6.3, 2.3)
7.10 (dt, 6.3, 2.3)
127.4 7.10 (dt, 6.3, 2.3)
127.1 7.10 (dt, 6.3, 2.3)
127.7
127.2
6.93 (ddd, 6.4, 2.3, 1.2) 121.9 6.97 (ddd, 6.4, 2.3, 1.2) 121.6
144.9
145.2
27.7
30.3
30.0
25.6
1.37 (s)
1.55 (s)
1.72 (s)
1.60 (s)
26.0 1.49 (s)
30.4 1.62 (s)
29.8 1.94 (s)
26.3 1.61 (s)
a
All NMR experiments were conducted in benzene-d6.
excised and pooled for each group, and a single-cell suspension
of lymph node cells was prepared. After washing and precipita-
tion with trichloroacetic acid, the extent of thymidine incorpora-
tion was determined by â-scintillation counting (13). Results are
expressed as the mean decompositions per minute per lymph
node for each experimental group and as the stimulation index
(SI), i.e., the test group value divided by the control group value.
Sen sit iza t ion E xp er im en t s in Gu in ea P igs. The sen-
sitization experiments were performed in outbred female
albino Dunkin-Hartley guinea pigs obtained from AB Sahlins
Fo¨rso¨ksdjursfarm (Malmo¨, Sweden). The animals were kept on
a standard diet from EWOS AB (So¨derta¨lje, Sweden), and their
average weight was 300-350 g. The experiments were carried
out according to the FCAT (14). Closed challenge was performed
according to previous experiments (15). For induction, the test
animals received intradermal injections in the upper back of
0.1 mL of the test substance hydroperoxide 7 (4.41%, w/w) in a
FCA/water (1/1) emulsion on days 0, 6, and 10, while the controls
received only the FCA/water emulsion. The induction concentra-
tion of 7 was chosen so that it was equimolar with respect to
15-hydroperoxyabietic acid in previous experiments (6). Chal-
lenge testing was performed on day 21. The test material in
olive oil (15 µL of each dose) was applied on the shaved flanks
for 24 h, using Finn chambers (alumina chambers, 8 mm i.d.).
Hydroperoxide 7 was tested at concentrations of 0.36, 1.0, and
3.1% (w/w), and the reactions were assessed 48 and 72 h after
application. The minimum criterion for a positive reaction was
a confluent erythema. A vehicle control with olive oil was applied
to all animals.
3,4,4a ,5,6,7-H e xa h yd r o-4a -m e t h yl-1H ,1R -(1′′,1′′,3′′,3′′-
tetr a m eth ylisoin d olin -2′′-yloxyl)-sp ir o-(n a p h th a len -2′,2′-
d im eth yl-2,2′-oxir a n e) (18a a n d 18b). 1H and 13C values are
listed in Table 1: EI-MS m/z 395.3 (C26H37O2N), 205.2 (C14H21O).
3,4,4a ,5,6,7-H e xa h yd r o-4a -m e t h yl-8H ,8R -(1′′,1′′,3′′,3′′-
tetr a m eth ylisoin d olin -2′′-yloxyl)-sp ir o-(n a p h th a len -2′,2′-
d im eth yl-2,2′-oxir a n e) (19). 1H and 13C values are listed in
Table 2: EI-MS m/z 395.3 (C26H37O2N), 205.2 (C14H21O).
Sen sitiza tion Exp er im en ts in Mice. The LLNA was
performed essentially as recommended by Kimber and Basketter
(12). Female mice (CBA/Ca strain, 6-10 weeks old), in groups
of four, received 25 µL of the test chemical dissolved in a 4/1
acetone/olive oil mixture on the dorsum of both ears for three
consecutive days. Test solutions were prepared fresh each day
and applied within 30 min. Compound 7 was tested in three
different concentrations: 1.4, 4.3, and 12.8% (w/w). Control mice
were treated with an equal volume of vehicle (4/1 acetone/olive
oil mixture) alone. Five days after the first treatment, all mice
were injected intravenously via the tail vein with 20 µCi of [3H]-
thymidine (specific activity of 2 Ci/mmol; Amersham Interna-
tional) in 250 µL of phosphate-buffered saline. After 5 h, the
mice were sacrified, the draining auricular lymph nodes were

1032 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Mutterer et al.
Ta ble 2. 1H a n d 13C NMR Da ta of Tr a p p in g P r od u ct 19a
Ta ble 3. Cell P r olifer a tion In d u ced by Hyd r op er oxid e 4
a n d 7 in th e LLNAa
[3H]thymidine
incorporation
(dpm/node)
concentrationb
(%, w/w)
stimulation
index
compd
control
1.0
3.0
9.0
control
1.4
325
962
5459
8033
503
3744
6234
11221
-
3.0
4
16.8
24.7
-
7.4
12.4
22.3
7
4.3
12.8
position
1H [J (Hz)]
13C
a
Groups of mice (n ) 4) received 25 µL of the test material
dissolved in vehicle (AOO) at the indicated concentrations, on the
dorsum of both ears daily for three consecutive days. Control
animals were treated in the same way with the vehicle alone. All
mice were injected intravenously 5 days after the first treatment,
with 250 µL of PBS containing 20 µCi of [3H]thymidine. Five hours
later, draining auricular lymph nodes were excised and pooled for
each group, and a single-cell suspension of lymph node cells was
prepared. The extent of thymidine incorporation was measured
by â-scintillation counting. The increase in the extent of thymidine
incorporation relative to vehicle-treated controls was derived for
each experimental group and recorded as a stimulation index.
b Compounds 4 and 7 were tested at the same molar concentration.
1
2
3
4
4a
5
6
7
8
8a
9
5.47 (m)
122.6
67.5
33.3
44.8
34.3
40.0
30.5
26.0
82.1
140.1
26.8
64.3
21.5
22.9
68.5
68.5
145.2
121.7
127.6
127.6
121.7
145.2
29.0
30.3
27.3
30.3
1.93 (m), 3.23 (ddd, 13.6, 5.0, 3.1)
1.90 (m), 1.96 (m)
1.41 (m), 1.56 (m)
1.55 (m), 1.76 (m)
1.88 (m), 1.96 (m)
4.08 (m)
1.46 (s)
2′
3′
4′
1.27 (s)
1.40 (s)
the use of 1-chloroethyl phenyl sulfoxide (12) as a synthon
for acylation, allowing the homologation of two carbon
atoms. The starting material for the synthesis of 12 was
thioanisole which was converted to chloromethyl phenyl
sulfoxide 11 using the method described by Hojo and
Masuda (21). Reaction of 11 with lithium diisopropyl-
amide in THF at -78 °C, followed by alkylation with
methyl iodide, gave the chloroethyl phenyl sulfoxide 12
in a 75% yield. Ketone 9 was treated at low temperatures
with the lithiated anion of sulfoxide 12, obtained at -78
°C after reaction with lithium diisopropylamide, to give
14 (92% yield) as a mixture of two isomers. Simple
treatment of 14 with silica in toluene under reflux readily
gave the desired R,â,γ,δ-unsaturated methyl ketone 15
(76% yield). Reaction of 15 with methyllithium (2 equiv)
and lithium bromide (1 equiv) at low temperatures
resulted in tertiary alcohol 16 in a 92% yield.
One of the most classical methods for preparation of
tertiary allylic hydroperoxides is the quenching with
H2O2 of a carbocation generated from a hydroxyl function
under acidic conditions (22). Alcohol 16 was thus treated
with a solution of H2O2 (35% in water) under acidic
conditions (H2SO4) at 0 °C to give the hydroperoxide 7,
which has never been isolated in more than 40% yield
after purification on silica gel column. Several attempts
to purify this hydroperoxide on other chromatographic
supports gave no satisfactory results, due to the high
susceptibility of the molecule to â-scission reactions, re-
forming the ketone 8.
Sen sitiza tion Exp er im en ts Accor d in g to LLNA.
The LLNA was used as the initial screening test of the
allergenic activity of hydroperoxide 7 (Table 3). Hydro-
peroxide 7 fulfilled the criteria presented here for a
chemical to be classified as a sensitizer in the LLNA.
There was a dose-dependent increase in the level of
proliferation, and all the test concentrations induced a
g3-fold increase (SI value of g3) in isotope incorporation
compared with vehicle-treated controls (23). Sensitization
experiments were also carried out at the same molar
concentration on the previously reported monocyclic
hydroperoxide model 4 (8). Although the absolute values
are difficult to compare, it has been suggested that the
1′′
3′′
3′′a
4′′
5′′
6′′
7′′
7′′a
8′′
9′′
10′′
11′′
6.91 (ddd, 6.4, 2.3, 1.2)
7.10 (dt, 6.3, 2.3)
7.10 (dt, 6.3, 2.3)
6.91 (ddd, 6.4, 2.3, 1.2)
1.41 (s)
1.43 (s)
1.43 (s)
1.42 (s)
a
All NMR experiments were conducted in benzene-d6.
Resu lts a n d Discu ssion
Syn t h esis. The bicyclic model 7 was based on the
B and C cyclohexene rings of 15-hydroperoxyabietic
acid 3. However, we decided to introduce a methyl
group at the 4a position to avoid a possible isomerization
of the conjugated system into a cis conformation or
aromatization, and to facilitate further conformational
assignments. The synthesis of compound 7 is shown in
Scheme 1.
Ketone 8 was easily obtained, at a yield that was
relatively low (43%) but similar to that reported in the
literature, by a Robinson annulation reaction (16) be-
tween 2-methylcyclohexanone and methyl vinyl ketone
in acidic medium (17). The nonconjugated ketone 9 was
prepared according to a slight modification of the method
described by Ringold (18), whereas Ringold treatment
deconjugated the R,â-unsaturated ketones by treatment
with an excess of base (10 equiv of potassium tert-
butoxide in tert-butyl alcohol) at room temperature; the
yield of the reaction (55%) was improved (68%) by
replacing tert-butyl alcohol with diglyme and by using a
smaller amount of potassium tert-butoxide (1.3 equiv) and
a catalytic amount (0.1 equiv) of 18-C-6 crown ether.
To produce the R,â,γ,δ-unsaturated methyl ketone 15,
we were inspired by the method described by Reutrakul,
who suggested the use of chloromethyl phenyl sulfoxide
as a formylation and homologation synthon for obtaining
1-cyclohexenecarboxyaldehyde from cyclohexanone (19).
The method was improved by Taber (20), who proposed

Radicals Derived from Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1033
Ta ble 4. Sen sitiza tion Exp er im en ts in Gu in ea P igs Accor d in g to F CATa
3.10% hydroperoxide 7
1.00% hydroperoxide 7
0.36% hydroperoxide 7
guinea pigs
time (h)
(in olive oil)
(in olive oil)
(in olive oil)
olive oil
exposed groupb
(n ) 15)
48
72
10
12
<0.001
3
6
1
1
0
0
NSd/<0.01
NSd
p exp/coc control group
(n ) 15)
48
72
0
1
1
0
0
0
0
0
a
b
The number of positive animals at 48 and 72 h after challenge is given. All inductions were performed with 4.41% solutions in an
FCA/water emulsion. c According to Fischer’s exact test. NS, not significant.
d
Sch em e 2. F or m a tion of Ra d ica l-Tr a p p ed Com p ou n d s 18a , 18b, a n d 19 fr om Hyd r op er oxid e 7
minimal dose inducing a 3-fold increase in the level of
[3H]thymidine incorporation (the EC3 value) could be a
good indicator of the sensitizing potential (24). It there-
fore appears that the bicyclic hydroperoxide 7 could be a
slightly stronger sensitizer than the monocyclic hydro-
peroxide 4.
Sen sitiza tion Exp er im en ts Accor d in g to F CAT.
Experiments in guinea pigs were performed to further
study the sensitizing potential of hydroperoxide 7. The
results (Table 4) confirmed the LLNA findings, namely,
that with 12 of 15 positive animals at a challenge
concentration of 3.1% and 6 of 15 at a challenge concen-
tration of 1.0%, respectively, hydroperoxide 7 should be
considered a strong sensitizer. This is in agreement with
the sensitizing capacity seen for other hydroperoxides,
such as the hydroperoxide of abietic acid (6), and li-
monene hydroperoxide (10).
Ch em ica l-Tr a p p in g Exp er im en ts. The technique of
spin trapping was used to determine if a free radical
process could be involved in the mechanism of sensitiza-
tion to the tertiary allylic hydroperoxide 7. Radical-
trapping experiments, using tritertiobutylphenol (TTBP)
as the trapping agent and Fe3+ tetraphenylporphine
(Fe3+-TPP) as the radical inducer, have been reported in
the case of linalyl hydroperoxide (25) and polyunsatu-
rated fatty acid hydroperoxides (26). In both cases, the
reaction leads to the formation of alkoxy radical inter-
mediates, unstable species that rapidly decompose through
hydrogen abstraction or rearrange through intramolecu-
lar cyclization. Although reduction of hydroperoxides
with reduced metals, in particular Fe2+, is a common
method for alkoxy radical production, it has been shown
that photochemical cleavage of hydroperoxides is also an
efficient method for the same purpose (27-29). Photo-
chemical induction presents the advantage of being a
cleaner method that avoids the presence of metal con-
taminants. The formation of radical species of compound
7 was therefore investigated by photochemical homolytic
cleavage. The nitroxide free radical 17 was used as spin-
trapping agent, as it exhibits a chemical inertness quite
uncharacteristic of most other free radicals (30). More-
over, it is thermally stable, unreactive toward olefins,
symmetrical (so as to generate a minimum number of
isomers and NMR signals in the products), and a suitable
UV absorbing chromophore (the evolution of the reaction
was followed by thin-layer chromatography).
In the course of our investigations, hydroperoxide 7
was found to be quite unstable and decompose during
the purification process over silica gel, neutralized silica,
or even aluminum oxide. The trapping experiments were
therefore carried out directly on the crude product
resulting from the hydroperoxidation reaction of alcohol
16. After irradiation for 3 weeks in the presence of 17 in
degassed dichloromethane, there was no further change
in the reaction progress. The solvent was therefore
removed under reduced pressure, and the reaction com-
pounds were split into two fractions (polar and nonpolar).
The nonpolar fraction was further purified by semi-
preparative HPLC on a C18 reversed phase column. A
major peak was detected at 254 nm, corresponding to a
nonseparable mixture of two diastereomers, 18a and 18b.
A second peak was also detected corresponding to the
trapping adduct 19 (Scheme 2). Structures of the isolated
compounds were established by a combination of 1H and
13C NMR data, including 1H-13C correlation (HSQC and
HMBC) and 1H-1H correlation (COSY and NOESY)
experiments.
First, 1H NMR experiments were carried out in CDCl3,
but many overlapping or coincident signals, correspond-
ing to methyl groups, were found. We therefore decided
to continue the structure assignment in benzene-d6, as
it is known that transient complexation (stacking) be-
tween the studied compound and solvent molecules could,
in some cases, produce an enhanced resolution of all the

1034 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Mutterer et al.
Ta ble 5. HMBC Da ta Sh ow in g Lon g-Ra n ge 1H-13C
Cor r ela tion s of Ad d u cts 18a , 18b, a n d 19
13C position
1H
position
18a
18b
19
1
3
4
C-2, C-2′, C-4a
C-2, C-2′, C-4a
C-2′
C-8a, C-4a
C-4a, C-2, C-1
C-5, C-4a
C-8a, C-2, C-4a,
C-9
5
7
9
C-8a
C-8, C-8a
C-8a, C-4, C-5,
C-4a
C-8a, C-8,
C-8a, C-4, C-5,
C-4a
C-8a, C-4, C-5,
C-4a
3′
4′
C-2, C-2′
C-2, C-2′
C-2, C-2′
C-2, C-2′
C-2, C-2′
C-2, C-2′
4′′
5′′
6′′
7′′
C-3′′a, C-5′′, C-3′′ C-3′′a, C-5′′, C-3′′
C-4′′, C-6′′, C-3′′a C-4′′, C-6′′, C-3′′a
C-5′′, C-7′′, C-7′′a C-5′′, C-7′′, C-7′′a
C-6′′, C-7′′a, C-1′′ C-6′′, C-7′′a, C-1′′
C-7′′a, C-1′′, C-9′′ C-7′′a
C-3′′a, C-5′′, C-3′′
C-4′′, C-6′′, C-3′′a
C-5′′, C-7′′, C-7′′a
C-6′′, C-7′′a, C-1′′
8′′
F igu r e 2. Main NOE effects observed in compounds 18a , 18b,
and 19.
9′′
10′′
11′′
C-7′′a, C-8′′, C-1′′ C-7′′a
C-3′′a, C-11′′
C-3′′a, C-3′′
C-3′′a, C-11′′, C-3′′ C-3′′a, C-11′′
C-3′′a, C-10′′, C-3′′ C-3′′a, C-3′′, C-10′′
tion between the quaternary carbon at 141.3 ppm (C-8a)
and protons at 2.24 (H-4), 1.94 (H-7), and 1.51 ppm (H-
9). The methyl group at 1.51 ppm was also correlated
with carbons at 46.6 (C-4), 40.0 (C-5), and 34.5 ppm (C-
4a). The vinyl carbon atom at 121.2 ppm (C-8) was
correlated to the methylene group at 1.94 ppm (H-7), and
methyl protons at 1.30 (H-3′) and 1.63 ppm (H-4′) were
correlated with the quaternary carbons of the epoxide.
proton signals. In the case of the mixture of diastereo-
mers 18a and 18b, the high-field region of the spectra
was resolved into 14 singlet signals corresponding to 14
methyl groups, seven for each diastereomer, and for
compound 19, seven methyl signals were also clearly
visible. Benzene-d6 gave a better resolution of the spectra
and was therefore used for all subsequent NMR experi-
1
ments. The 500 MHz H NMR and 125 MHz 13C NMR
The stereochemistry determination was based on ob-
servations of nuclear Overhauser effects (NOE, Figure
2). A NOE between H-1 and the methyl group at C-9
allowed us to attribute the H-1 axial position for both
isomers. In the case of adduct 18b, NOE cross-peaks
between H-1 (4.25 ppm) and the methyl groups at C-3′
(1.13 ppm) and at C-4′ (1.23 ppm) confirmed the position
of the epoxide on the R side of the molecule. The absence
of these cross-peaks in adduct 18a is in favor of the
epoxide being on the â side of the molecule.
As it is shown in Table 1, the four methyl groups of
the 1,1,3,3-tetramethylisoindolin moiety are not chemi-
cally and, therefore, magnetically equivalent. This non-
equivalence must indicate a structure of the adducts in
which the 1,1,3,3-tetramethylisoindolin moiety is not free
to rotate rapidly. This is confirmed by the observation of
NOE cross-peaks between H-1 and the methyl groups at
C-8′′, C-9′′, C-10′′, and C-11′′ for adduct 18a and between
H-1 and the methyl groups at C-8′′ and C-9′′ for adduct
18b (Figure 2). The 1,1,3,3-tetramethylisoindolin moiety
must be placed therefore in a plane which is almost
perpendicular to the plane described by the bicycle moiety
of the adducts. Thus, all four methyl groups do not have
the same time-averaged chemical environment and do
not have the same resonance frequencies. Because of its
lone pair of electrons, the nitrogen atom of the 1,1,3,3-
tetramethylisoindolin moiety plays an important role that
exhibit hindrance to internal rotation. Hindered rotation
associated with the nitrogen atom could be the reason
for the loss of rapid rotation about the single bonds, and
explain the lack of equivalence of the four methyl groups.
data of the isolated adducts are listed in Tables 1 and 2.
Str u ctu r a l An a lysis of Com p ou n d s 18a a n d 18b.
The 500 MHz 1H NMR spectrum of the nonseparable
mixture 18a of 18b showed the disappearance of the
vinylic proton of the starting material 7 at 6.08 ppm. A
set of signals in the 6.90-7.20 ppm region corresponding
to the scavenger aromatic protons was present. The
spectrum also exhibited signals for only one vinyl proton
for each diastereomer at 5.24 and 5.23 ppm, respectively,
and for a proton in position R to an alkoxyamine at
4.57 and 4.25 ppm, respectively. In the 125 MHz 13C
NMR spectrum, the disappearance of signals at 139.6
ppm (dC) and at 83.8 ppm (C-OOH) and the appearance
of two new quaternary carbons at 68.5 and 62.2 ppm for
one isomer, and at 67.8 and 60.9 ppm for the other
isomer, were evidence for the formation of a 2,2′-spiro-
type epoxide. The HSQC experiments allowed us to
1
correlate proton and carbon nuclei via J (C, H) for each
isomer, and the skeleton of each adduct was determined
by long-range 1H-13C correlations via 2J (C, H) and
3J (C, H) (HMBC experiments, Table 5). Thus, protons
H-1 at 4.57 ppm (18a ) and at 4.25 ppm (18b) were
correlated via long-range coupling with the epoxide
quaternary carbons at 68.5 (C-2) and 62.2 ppm (C-2′), and
at 67.8 (C-2) and 60.9 ppm (C-2′), respectively. These
results confirmed the reaction between the spin-trapping
agent and the epoxy carbon-centered radical in position
1 for both isomers (Scheme 2). For the isomer 18a , the
8,8a-vinylic position was confirmed by long-range cor-
relations between the quaternary carbon at 140.2 ppm
(C-8a) and protons at 2.40 (H-4), 2.30 (H-7), and 1.18 ppm
(H-9). The methyl group at 1.18 ppm was also correlated
with carbons at 47.5 (C-4), 39.7 (C-5), and 35.1 ppm (C-
4a). The vinyl carbon atom at 121.7 ppm (C-8) was
correlated to the methylene group at 2.30 ppm (H-7).
Methyl protons at 1.30 (H-3′) and 1.63 ppm (H-4′) were
correlated with the quaternary carbons of the epoxide.
Similarly, in the case of isomer 18b, there was a correla-
Moreover, it is well-known that NMR measurements
conducted in different solvents can lead to different
results when there are specific interactions between the
solvent and the reference substance or sample. Especially
in steroid chemistry, specific solvent effects have been
systematically studied and used to advantage. Particu-
larly valuable for this purpose is benzene because of its

Radicals Derived from Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1035
high magnetic anisotropy and its tendency to form
specific complexes with the solute. If benzene is used
instead of chloroform, the proton resonance signals of
individual methyl groups in steroids can often be dif-
ferentiated (31). This property has been applied for the
resolution of all methyl group signals in adducts 18a and
18b. The transient stacking between the studied com-
pounds and solvent (benzene-d6) molecules leads to 14
singlet signals corresponding to 14 methyl groups, seven
for each diastereomer.
compound resulting from hydrogen abstraction by the
alkoxy or the carbon-centered radical intermediates were
detected.
It is now accepted that the first step of the mechanism
leading to the development of a contact allergic reaction
to a chemical is the formation of a covalent bond between
the allergen or hapten and the side chains of proteins.
Most known allergens are therefore low-molecular weight
electrophiles or molecules that are metabolized to elec-
trophiles that are able to react, in proteins, with the side
chains of amino acids containing electron-rich or nucleo-
philic groups (7). In this respect, lysine and cysteine are
those most often cited, but other amino acids containing
nucleophilic heteroatoms, such as histidine, methionine,
and tyrosine, could be involved in the skin sensitization
mechanism. Studies performed in the past few years have
shown for example that methyl alkanesulfonates, which
are lipophilic methylating agents, almost exclusively
modify histidine and methionine residues in proteins (32),
while alk-2-ene-γ-sultones, which have been shown to be
particularly strong skin sensitizers with sensitization
potential down to levels of approximately 1 ppm, react
mainly with tyrosine residues at physiological pH. In
this respect, the formation of radical intermediates
could lead to the modification of other amino acids or to
the formation of carbon-carbon bonds, which are not
usually considered in the mechanism of ACD. Several
examples of sensitizing allylic hydroperoxides mainly
derived from terpenes by autoxidation processes are
known (6, 10, 33), but the mechanism by which these
molecules react with proteins to make covalent bonds is
not clear. Allylic hydroperoxides from fatty acids are
known to form carbon-centered epoxyallylic radicals (26).
In the case of the model molecule 7, the formation of an
oxygen-centered radical also led to the formation of a
carbon-centered allylic epoxy radical and its associated
mesomeric form. Both radical intermediates could be
candidates for the modification of lateral chains of amino
acids such as tyrosine or histidine.
The electron impact mass spectrum supported the
structure with a molecular ion detected at m/z 395.3.
Str u ctu r a l An a lysis of Com p ou n d 19. The confor-
mation of adduct 19 was assigned on the basis of the
1
same arguments used for adducts 18a and 18b. H-13C
long-range correlations (Table 5) were consistent with a
reaction between the spin-trapping agent and an epoxy
carbon-centered radical in position 8 (Scheme 2). The
methyl group at 1.46 ppm (H-9) was correlated via long-
range coupling with carbons at 44.8 (C-4), 40.0 (C-5),
140.1 (C-8a), and 34.3 ppm (C-4a). The proton at 1.93
ppm (H-3) was correlated via long-range coupling with
carbons at 122.6 (C-1), 67.5 (C-2), and 34.3 ppm (C-4a).
Methyl protons at 1.27 (H-3′) and 1.40 ppm (H-4′) were
correlated with the quaternary carbons of the epoxide at
67.5 (C-2) and 64.3 ppm (C-2′). These results were
supported by NOE experiments (Figure 2) as the proton
at 4.08 ppm (H-8) exhibited important NOE cross-peaks
with protons at 1.88 (H-7) and 1.96 ppm (H-7), and with
the methyl groups of the radical scavenger at 1.41 (H-
8′′), 1.42 (H-11′′), and 1.43 ppm (H-9′′ and H10′′). The
vinylic proton at 5.47 ppm (H-1) exhibited significant
NOE cross-peaks with the methyl group of the epoxide
at 1.27 ppm (H-3′) and with a methyl group at 1.43 ppm
(H-9′′ and H-10′′). The epoxide methyl group at 1.40 ppm
(H-4′) exhibited a NOE signal with the proton at 1.93
ppm (H-3). All NOE cross-peaks suggested the position
of the epoxide on the â side of the molecule. The same
explanations that were given for the lack of magnetic
equivalence of the 1,1,3,3-tetramethylisondolin four meth-
yl groups in the case of adducts 18a and 18b can be
applied for adduct 19.
The rather low yield of trapping products obtained in
organic solvents provides little information about the
probability of a radical intermediate being an important
intermediate in vivo, but at least suggests that such
intermediates can be formed. The relevance of such
intermediates to the mechanism of sensitization to allylic
hydroperoxides will mainly depend on the environment
of the reaction, modification of skin proteins, which is
still not well-known. Nevertheless, the existence of this
intermediates could give new insight into the mechanism
of hapten-protein interactions.
The electron impact mass spectrum supported the
structure with a molecular ion detected at m/z 395.3.
Mech a n istic In ter p r eta tion s. The irradiation of
hydroperoxide 7 in the presence of the radical scavenger,
1,1,3,3-tetramethylisoindolin-2-yloxyl 17, led to the for-
mation of an alkoxy radical intermediate which was then
potentially capable of generating carbon-centered radicals
that are able to react with 17 (Scheme 2). Initially, the
alkoxy radical underwent immediate cyclization to an R
epoxy radical, which was trapped by 17 to give an
approximately 1/1 mixture of two diastereomers, 18a and
18b. As the only difference between the isomers was the
stereochemistry in position 2, we can conclude that the
alkoxy radical cyclization can occur on both the R and â
sides of the molecule without any preference. The R epoxy
radical can then react with the spin-trapping agent, and
the equatorial position is preferred for evident steric
reasons, leading to adducts 18a and 18b. The mesomeric
form of this carbon-centered radical, an epoxyallylic
radical, can also be trapped by 17, in an equatorial
position as well to give only isolated isomer 19 (Scheme
2). The fact that other isomers, formed during the
photochemical process at an extremely low yield, could
not be isolated and characterized cannot be excluded. No
Con clu sion s
The results of our experiments, obtained using a
combination of chemical-trapping and in vivo experimen-
tal sensitization techniques, favor the formation of
carbon-centered reactive radicals as intermediates in the
skin sensitization to hydroperoxide 7. We have demon-
strated that the radical rearrangement of this conjugated
allylic hydroperoxide model of 15-hydroperoxyabietic acid
is probable. The fact that hydroperoxide 7 radicals are
trapped with very low yields does not prove that they
are able to form adducts with skin proteins in vivo, but
allows us to think that the in situ generation of such
reactive radicals in the epidermis may be possible and
could lead to the formation of antigenic structures, the
first step of the ACD mechanism.

1036 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
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Ack n ow led gm en t. We truly thank the Swedish
Council for Work Life Research for financial support of
this research program and Dr. J ohan Montelius (National
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