Skin sensitization to eugenol and isoeugenol in mice: Possible metabolic pathways involving ortho-quinone and quinone methide intermediates

Article abstract of DOI:10.1021/tx960087v

With the aim of providing further mechanistic insights into the mode of action of eugenol (4-allyl-2-methoxyphenol) and isoeugenol (4-propenyl-2- methoxyphenol), we have synthesized two series of modified compounds which were tested in the mouse local lym

Full text of DOI:10.1021/tx960087v

Chem. Res. Toxicol. 1997, 10, 335-343
335
Sk in Sen sitiza tion to Eu gen ol a n d Isoeu gen ol in Mice:
P ossible Meta bolic P a th w a ys In volvin g or th o-Qu in on e
a n d Qu in on e Meth id e In ter m ed ia tes
Franck Bertrand,† David A. Basketter,‡ David W. Roberts,§ and
J ean-Pierre Lepoittevin*,†
Laboratoire de Dermatochimie associe´ au CNRS, Universite´ Louis Pasteur,
Clinique Dermatologique, CHU, F-67091 Strasbourg, France, Unilever Environmental Safety
Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, United Kingdom, and
Unilever Research Port Sunlight Laboratory, Quarry Road East Bebington, Wirral,
Merseyside L63 3J W, United Kingdom
Received J une 3, 1996X
With the aim of providing further mechanistic insights into the mode of action of eugenol
(4-allyl-2-methoxyphenol) and isoeugenol (4-propenyl-2-methoxyphenol), we have synthesized
two series of modified compounds which were tested in the mouse local lymph node assay for
their skin sensitizing potential. The replacement of the methoxy group by an isopropoxy group
led to a complete loss of sensitization for the eugenol derivative 6a , while no significant effect
was observed for the isoeugenol derivative 6b. In the eugenol series, when methyl groups
were present in the 3-, 5-, or 6-position a significant reduction in sensitization potential was
observed while in the isoeugenol series only methyl substitution in the 3- and 5-position had
a discernable effect. Introduction of three methyl groups on the aromatic ring of eugenol (3,5,6-
trimethyl-4-allyl-2-methoxyphenol, 7) and of a tert-butyl substituent at the γ-position of the
alkyl chain of isoeugenol (4-[3′,3′,3′-trimethylpropenyl]-2-methoxyphenol, 8) led to a strong
decrease of the sensitizing capacity. Our findings indicate that, at least in the mouse, eugenol
could sensitize via a demethylation pathway followed by oxidation to the o-quinone which could
act directly as a hapten even if we cannot exclude a reaction via its tautomeric p-quinone
methide. Isoeugenol, on the other hand, could act via a mechanism not involving demethylation
and for which the evidence is consistent with a direct oxidation to the p-quinone methide.
involving oxidation to the o-quinone, which acts as an
electrophilic Michael acceptor for protein nucleophiles.
These two mechanisms are shown in Figure 1. Studies
in the mouse have recently provided new data on the
sensitizing potential of eugenol and isoeugenol. In the
local lymph node assay it is found that both eugenol and
isoeugenol are potent skin sensitizers, although isoeu-
genol is still stronger than eugenol (6). Furthermore,
recent publications by Bolton et al. (7, 8) on the isomer-
ization of o-quinones to p-quinone methides lead us to
consider the possibility of modes of action additional to
the mechanisms outlined above.
In tr od u ction
The skin sensitization potential of eugenol 1 and
isoeugenol 2 (structures shown in Figure 1) continues to
be the subject of mechanistic studies (1-3). Although
the two compounds appear structurally very similar, they
show large differences in their sensitization properties
in guinea pig sensitization tests (1). In human predictive
studies, eugenol seems to be a weaker sensitizer than
isoeugenol (4), and this is borne out by clinical evidence
(5). Similarly, eugenol is a very weak sensitizer in the
guinea pig, whereas isoeugenol is a strong sensitizer. The
clinical evidence also suggests that eugenol and isoeu-
genol may not cross react since individuals often give
positive patch test reactions to one, but not both, of these
perfume chemicals. Furthermore, guinea pig cross-
challenge studies involving eugenol, isoeugenol, and
related compounds have provided some evidence that the
two compounds could act by different molecular mecha-
nisms (1). Two metabolic pathways are usually consid-
ered for skin sensitization to eugenol and isoeugenol. The
first one considers the formation of a phenolic radical
which can either haptenate proteins by reaction at the
unsubstituted ortho-position or form a reactive quinone
methide intermediate. The second one considers an in
vivo demethylation to form 4-alkyl catechols which can
then haptenate proteins by the “poison ivy mechanism”
With the aim of providing further mechanistic insights
into the modes of action of eugenol, isoeugenol, and
related compounds, we now describe the synthesis of two
series of compounds and sensitization studies in mice.
In these series of compounds the basic structures of
eugenol and isoeugenol were modified by introduction of
methyl groups in the 3-, 5-, and 6-positions of the
aromatic ring and also by replacement of the methoxy
group by the isopropoxy group (Figure 2). Futhermore,
to confirm our initial findings on skin sensitization to
eugenol and isoeugenol, additional compounds 7 and 8
were synthesized and tested.
Ma ter ia ls a n d Meth od s
Ca u tion ! Skin contact with eugenol and isoeugenol deriva-
tives must be avoided. As sensitizing substances, these com-
pounds must be handled with care.
Ch em istr y. 1H and 13C NMR spectra were recorded on a
* Corresponding author.
† Universite´ Louis Pasteur.
‡ Unilever Environmental Safety Laboratory.
§ Unilever Research Port Sunlight Laboratory.
X Abstract published in Advance ACS Abstracts, February 15, 1997.
Bruker AC200-MHz spectrometer in CDCl3 unless otherwise
S0893-228x(96)00087-2 CCC: $14.00 © 1997 American Chemical Society

336 Chem. Res. Toxicol., Vol. 10, No. 3, 1997
Bertrand et al.
F igu r e 1. Potential reaction pathways for the skin sensitization to eugenol 1 and isoeugenol 2.
F igu r e 2. Structures of eugenol and isoeugenol derivatives tested for their sensitizing potential in mice (LLNA).
specified. Chemical shifts are reported in ppm (δ) with respect
to TMS, and CHCl3 was used as internal standard (δ ) 7.27
ppm). Multiplicities are indicated by s (singlet), d (doublet), t
(triplet), and m (multiplet). Infrared spectra were obtained on
a Perkin-Elmer FT-IR 1600 spectrometer; peaks are reported
in reciprocal centimeters. Melting points were determined on
a Buchi Tottoli 510 apparatus and are uncorrected. Dried
solvents were freshly distilled before use. Tetrahydrofuran and
ethyl ether were distilled from sodium benzophenone. Trieth-
ylamine was distilled from powdered calcium hydride. Meth-
ylene 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 conducted on silica gel columns according to
the flash chromatography technique.
6-Meth ylgu a ia col (9). A mixture of o-vanillin (5 g, 33
mmol), monohydrated hydrazine (16 mL, 330 mmol, 10 equiv),
and potassium carbonate (20 g, 145 mmol, 4.4 equiv) in
triethyleneglycol (100 mL) was heated under reflux for 1.5 h
and then at 200 °C for an additional 3 h. After it was cooled,
the mixture was diluted in ether (100 mL) and washed with
water (3 × 100 mL). Combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure to
give a crude phenol which was purified by column chromatog-
raphy over silica (AcOEt 20%, hexane) to give 4.09 g of 9 (29.7
mmol, 90% yield) as a yellow oil. 1H NMR (200 MHz, CDCl3):
δ 2.27 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 5.70 (s, 1H, OH), 6.70-
6.80 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3): δ 15.5, 56.0,
108.3, 119.2, 123.3, 124.0, 143.9, 146.3. IR (CHCl3): ν 3540
(OH). Anal. Calcd for C8H10O2: C, 69.54; H, 7.30. Found: C,
69.43; H, 7.53.
3-Meth ylgu a ia col (11). To a solution of 3-methylcatechol
(5.0 g, 40.3 mmol) in THF (200 mL) were added, at 0 °C, NaH
(2.9 g, 121 mmol, 3 equiv) and tert-butyldimethylsilyl chloride
(6.1 g, 40.5 mmol, 1 equiv). The reaction was stirred at room
temperature for 4 h, MeI (5 mL, 80.3 mmol, 2 equiv) was then
added, and the mixture was stirred for an additional 4 h before
hydrolysis with 10% HCl (50 mL) under reflux for 4 h. The
reaction was extracted with ether (2 × 200 mL), the combined
organic layers were dried over MgSO4, filtered, and concentrated
under reduced pressure, and the crude product was purified by
column chromatography over silica (AcOEt 10%, hexane) to give
3.9 g (28.2 mmol, 70% yield) of 11 as a yellow oil. 1H NMR
(200 MHz, CDCl3): δ 2.31 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.63
(s, 1H, OH), 6.60-7.00 (m, 3H, ArH). 13C NMR (50 MHz,
CDCl3): δ 15.8, 60.5, 113.4, 122.5, 124.6, 131.0, 145.6, 149.0.
IR (CHCl3): ν 3532 (OH). Anal. Calcd for C8H10O2: C, 69.54;
H, 7.30. Found: C, 69.17; H, 7.05.

(Iso)Eugenol Sensitization
Chem. Res. Toxicol., Vol. 10, No. 3, 1997 337
6-Meth yla llylgu a ia col (13). To a solution of 6-methylguai-
acol 7 (2.5 g, 18 mmol) in acetone (50 mL) were added potassium
carbonate (10.0 g, 72 mmol, 4 equiv) and allyl bromide (3.2 mL,
36 mmol, 2 equiv). The reaction mixture was stirred for 12 h,
diluted with hexane (50 mL), filtered, and concentrated under
reduced pressure. The crude product was purified by column
chromatography over silica (AcOEt 20%, hexane) to give 2.74 g
(15.4 mmol, 85% yield) of 13 as a colorless oil. 1H NMR (200
MHz, CDCl3): δ 2.28 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.48 (ddd,
dt-like, 2H, J Hâ-HR ) 6 Hz, J HR-Hγcis ) 2 Hz, J HR-Hγtrans ) 1 Hz,
HR), 5.22 (ddt, 1H, J Hâ-Hγtrans ) 10 Hz, J Hγtrans-Hγcis ) 2 Hz,
Hγtrans), 5.37 (ddt, 1H, J Hâ-Hγcis ) 17 Hz, Hγcis), 6.12 (ddt,
1H, Hâ), 6.70-7.00 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3):
δ 16.3, 55.8, 73.5, 110.1, 117.2, 122.9, 123.7, 132.3, 134.7, 146.3,
152.8. Anal. Calcd for C11H14O2: C, 74.13; H, 7.92. Found:
C, 73.73; H, 8.22.
Hz, Hγtrans), 5.40 (s, 1H, OH), 5.87 (ddt, 1H, Hâ), 6.46 (s, 1H,
H3), 6.91 (s, 1H, H6). 13C NMR (50 MHz, CDCl3): δ 18.7, 37.4,
56.2, 112.1, 115.4, 116.4, 129.1, 129.4, 137.1, 143.8, 144.7. IR
(CHCl3): ν 3536 (OH). Calcd for C11H14O2: C, 74.13; H, 7.92.
Found: C, 74.03; H, 8.24.
3-Meth yleu gen ol (5a ). To a solution of pentabromophenol
(11.0 g, 22.5 mmol, 4 equiv) in CH2Cl2 (500 mL) was added,
dropwise, a solution of trimethylaluminum (5.6 mL, 11.2 mmol,
2 equiv, 2 M). After 1 h at room temperature, the solution was
cooled down to -78 °C and a solution of 15 (1 g, 5.6 mmol) in
CH2Cl2 (100 mL) was added. The reaction mixture was stirred
at room temperature for 2 weeks, hydrolyzed with 5% HCl (20
mL), and extracted with CH2Cl2 (50 mL). Organic layers were
dried over MgSO4, filtered, and concentrated under vacuum, and
the residue was purified by column chromatography over silica
(AcOEt 10%, hexane) to give 560 mg (3.1 mmol, 56% yield) of
5a as a yellow oil. 1H NMR (200 MHz, CDCl3): δ 2.22 (s, 3H,
CH3), 3.29 (ddd, dt-like, 2H, J Hâ-HR ) 6 Hz, J HR-Hγcis ) 2 Hz,
J HR-Hγtrans ) 2 Hz, HR), 3.77 (s, 3H, OCH3), 4.97 (ddt, 1H,
J Hâ-Hγcis ) 16 Hz, J Hγtrans-Hγcis ) 2 Hz, Hγcis), 5.04 (ddt, 1H,
J Hâ-Hγtrans ) 10 Hz, Hγtrans), 5.53 (s, 1H, OH), 5.93 (ddt, 1H,
Hâ), 6.70-6.90 (m, 2H, ArH). 13C NMR (50 MHz, CDCl3): δ
11.7, 37.2, 60.5, 112.8, 115.2, 125.3, 129.4, 130.7, 136.8, 145.5,
147.1. IR (CHCl3): ν 3536 (OH). Anal. Calcd for C11H14O2:
C, 74.13; H, 7.92. Found: C, 73.99; H, 8.17.
5-Meth yla llylgu a ia col (14). Same procedure as for 13
starting from 5-methylguaiacol 10 (5.0 g, 36 mmol) to give 5.74
g (32.2 mmol, 89% yield) of 14 as a yellow oil. 1H NMR (200
MHz, CDCl3): δ 2.86 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.60 (ddd,
dt-like, 2H, J Hâ-HR ) 5 Hz, J HR-Hγcis ) 1 Hz, J HR-Hγtrans ) 1 Hz,
HR), 5.28 (ddt, 1H, J Hâ-Hγtrans ) 10 Hz, J Hγtrans-Hγcis ) 1 Hz,
Hγtrans), 5.41 (ddt, 1H, J Hâ-Hγcis ) 17 Hz, Hγcis), 6.10 (ddt,
1H, Hâ), 6.65-6.80 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3):
δ 21.0, 56.1, 69.9, 111.9, 114.9, 117.8, 121.4, 130.4, 131.6, 147.5,
147.9. Anal. Calcd for C11H14O2: C, 74.13; H, 7.92. Found:
C, 74.04; H, 8.24.
2-Isop r op oxy-4-a llylp h en ol (6a ). Same procedure as for
the synthesis of 3a starting from 16 (2.3 g, 10.4 mmol) to give
600 mg (3.1 mmol, 30% yield) of 6a as a colorless oil. 1H NMR
(200 MHz, CDCl3): δ 1.36 (d, 6H, J ) 6 Hz, Me2CH-O), 3.31
(bd, 2H, J HR-Hâ ) 7 Hz, HR), 4.58 (m, 1H, Me2CH-O), 5.05 (m,
1H, Hγtrans), 5.06 (m, 1H, Hγcis), 5.59 (s, 1H, OH), 5.96 (ddt,
1H, J Hâ-Hγcis ) 17 Hz, J Hâ-Hγtrans ) 9 Hz, Hâ), 6.60-7.00 (m,
3H, ArH). 13C NMR (50 MHz, CDCl3): δ 22.3, 40.0, 71.7, 114.1,
114.6, 115.5, 121.4, 131.8, 138.1, 144.7, 145.1. IR (CHCl3): ν
3536 (OH). Anal. Calcd for C12H16O2: C, 74.96; H, 8.39.
Found: C, 74.73; H, 8.68.
3-Meth yla llylgu a ia col (15). Same procedure as for 13
starting from 11 (2.5 g, 18 mmol) to give 3.16 g (17.7 mmol,
98% yield) of 15 as colorless oil. 1H NMR (200 MHz, CDCl3): δ
2.28 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.58 (ddd, dt-like, 2H,
J Hâ-HR ) 5 Hz, J HR-Hγcis ) 2 Hz, J HR-Hγtrans ) 1 Hz, HR), 5.28
(ddt, 1H, J Hâ-Hγtrans ) 10 Hz, J Hγtrans-Hγcis ) 2 Hz, Hγtrans), 5.43
(ddt, 1H, J Hâ-Hγcis ) 17 Hz, Hγcis), 6.09 (ddt, 1H, Hâ), 6.70-
7.00 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3): δ 15.9, 53.5,
69.5, 112.0, 117.2, 123.2, 123.6, 132.1, 133.6, 147.9, 151.8. Anal.
Calcd for C11H14O2: C, 74.13; H, 7.92. Found: C, 74.03; H, 8.19.
6-Meth ylisoeu gen ol (3b). To a solution of 6-methyleugenol
3a (2.5 g, 14 mmoles) in Me2SO (DMSO)1 (50 mL) was added
potassium tert-butanolate (3.2 g, 28.5 mmol, 2 equiv). The
reaction mixture was heated at 110 °C for 2 h, diluted with
water (50 mL), and extracted with ether (3 × 50 mL). Combined
organic layers were dried over MgSO4, filtered, and concentrated
under reduced pressure. The resulting crude oil was purified
by column chromatography over silica (AcOEt 20%, hexane) to
give 2.27 g (12.7 mmol, 91% yield) of 3b as a mixture of cis/
trans isomers (1/9). 1H NMR (200 MHz, CDCl3): δ 1.86 (dd,
3H, J Hâ-Hγ ) 6 Hz, J HR-Hγ ) 1 Hz, allyl-CH3), 2.24 (s, 3H, CH3),
3.89 (s, 3H, OCH3), 5.64 (s, 1H, OH), 6.11 (dd, 1H, J HR-Hâ ) 15
Hz, Hâ), 6.31 (dd, 1H, HR), 6.65-6.80 (m, 2H, ArH). 13C NMR
(50 MHz, CDCl3): δ 15.5, 18.4, 56.0, 105.7, 121.3, 123.0, 123.7,
129.6, 131.1, 143.1, 146.4. IR (CHCl3): ν 3540 (OH). Anal.
Calcd for C11H14O2: C, 74.13; H, 7.92. Found: C, 74.05; H, 8.26.
2-Isop r op oxya llylp h en ol (16). Same procedure as for the
synthesis of 13 starting from 2-isopropoxyphenol 12 (4 mL, 27
mmol) to give 5.2 g (27 mmol, quantitative yield) of 16 as a
colorless oil. 1H NMR (200 MHz, CDCl3): δ 1.36 (d, 6H, J ) 6
Hz, Me2CH-O), 4.51 (m, 1H, Me2CH-O), 4.59 (ddd, dt-like, 2H,
J HR-Hâ ) 5 Hz, J HR-Hγcis ) 2 Hz, J HR-Hγtrans ) 2 Hz, HR), 5.26
(m, 1H, Hγtrans), 5.51 (m, 1H, Hγcis), 6.08 (ddt, 1H, J Hâ-Hγcis
) 17 Hz, J Hâ-Hγtrans ) 10 Hz), 6.80-7.00 (m, 4H, ArH). 13C NMR
(50 MHz, CDCl3): δ 22.3, 70.1, 72.1, 115.3, 117.1, 118.0, 121.5,
121.8, 133.9, 148.2, 150.1. Anal. Calcd for C12H16O2: C, 74.96;
H, 8.39. Found: C, 74.88; H, 8.63.
6-Meth yleu gen ol (3a ). 6-Methylallylguaiacol 13 (2.0 g, 11.2
mmol) was stirred under argon at 180 °C for 4 h. The reaction
mixture was purified by column chromatography over silica
(AcOEt 20%, hexane) to give 1.52 g (8.5 mmol, 76% yield) of 3a
as a yellow oil. 1H NMR (200 MHz, CDCl3): δ 2.25 (s, 3H, CH3),
3.30 (bd, 2H, J HR-Hâ ) 7 Hz, HR), 3.89 (s, 3H, OCH3), 5.07 (m,
1H, Hγtrans), 5.11 (m, 1H, Hγcis), 5.59 (s, 1H, OH), 5.97 (ddt,
1H, J Hâ-Hγcis ) 17 Hz, J Hâ-Hγtrans ) 10 Hz, Hâ), 6.70-6.90 (m,
2H, ArH). 13C NMR (50 MHz, CDCl3): δ 15.5, 40.1, 56.0, 108.7,
115.4, 123.2, 123.7, 131.0, 138.1, 142.1, 146.3. IR (CHCl3): ν
3544 (OH). Anal. Calcd for C11H14O2: C, 74.13; H, 7.92.
Found: C, 73.93; H, 8.24.
5-Meth ylisoeu gen ol (4b). Same procedure as for 3b start-
ing from 4a (2.5 g, 14 mmol) to give 2.24 g (12.6 mmol, 90%
yield) of 4b as a cis/trans mixture (15/85). 1H NMR (200 MHz,
CDCl3): δ 1.89 (dd, 3H, J Hâ-Hγ ) 6 Hz, J HR-Hγ ) 2 Hz, allyl-
CH3), 2.24 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 5.48 (s, 1H, OH),
5.97 (dd, 1H, J HR-Hâ ) 16 Hz, Hâ), 6.52 (dd, 1H, HR), 6.70 (s,
1H, H6), 7.91 (s, 1H, H3). 13C NMR (50 MHz, CDCl3): δ 15.6,
19.0, 56.1, 108.1, 116.3, 124.7, 128.1, 128.8, 128.9, 144.6, 144.9.
IR (CHCl3): ν 3540 (OH). Anal. Calcd for C11H14O2: C, 74.13;
H, 7.92. Found: C, 74.31; H, 7.90.
5-Meth yleu gen ol (4a ). To a solution of 5-methylallylguai-
acol 12 (2.0 g, 11.2 mmol) in CH2Cl2 (20 mL) was added a
solution of diethylaluminum chloride (6.2 mL, 11.2 mmol, 1.8
M, 1 equiv). The reaction mixture was stirred for 15 min,
hydrolyzed with 5% HCl (10 mL), and extracted with CH2Cl2 (3
× 20 mL). Combined organic layers were dried over MgSO4,
filtered, and concentrated under vacuum to give a crude product
which was purified by column chromatography over silica
(AcOEt 10%, hexane) to give 840 mg (4.7 mmol, 42% yield) of
4a as a yellow oil. 1H NMR (200 MHz, C6D6) δ 2.01 (s, 3H, CH3),
3.15 (ddd, dt-like, 2H, J Hâ-HR ) 6 Hz, J HR-Hγcis ) 2 Hz, J HR-Hγtrans
) 2 Hz, HR), 3.20 (s, 3H, OCH3), 4.95 (ddt, 1H, J Hâ-Hγcis ) 17
Hz, J Hγtrans-Hγcis ) 2 Hz, Hγcis), 5.02 (ddt, 1H, J Hâ-Hγtrans ) 10
3-Met h ylisoeu gen ol (5b). Same procedure as for the
preparation of 3b starting from 5a (2.5 g, 14 mmol) to give 880
mg (4.9 mmol, 35% yield) as a mixture of cis/trans isomers (3/
17). 1H NMR (200 MHz, CDCl3): δ 1.88 (dd, 3H, J Hâ-Hγ ) 6
Hz, J HR-Hγ ) 2 Hz, allyl-CH3), 2.26 (s, 3H, CH3), 3.76 (s, 3H,
OCH3), 5.61 (s, 1H, OH), 5.98 (dd, 1H, J HR-Hâ ) 16 Hz, Hâ),
6.49 (dd, 1H, HR), 6.77 (d, 1H, J H5-H6 ) 8 Hz, H6), 7.09 (d, 1H,
1 Abbreviations: AAO, acetone-olive oil; DMSO, dimethyl sulfoxide;
LLNA, local lymph node assay; PBS, phosphate-buffered saline;
TBDMS, tert-butyldimethylsilyl.

338 Chem. Res. Toxicol., Vol. 10, No. 3, 1997
Bertrand et al.
H5). 13C NMR (50 MHz, CDCl3): δ 15.3, 18.7, 60.8, 113.0, 122.2,
122.6, 128.1, 128.5, 130.8, 145.3, 147.8. IR (CHCl3): ν 3532
(OH). Anal. Calcd for C11H14O2: C, 74.13; H, 7.92. Found: C,
73.83; H, 7.98.
reduced pressure. The crude residue was purified by chroma-
tography on silica (AcOEt 10%, hexane) to give 5.84 g (30 mmol,
76% yield) of 21 as a colorless oil. 1H NMR (200 MHz, CDCl3):
δ 2.12 (s, 3H, CH3), 2.23 (s, 6H, 2 CH3), 4.36 (bd, 2H, J HR-Hâ
)
6 Hz, HR), 5.25-5.40 (m, 1H, Hγtrans), 5.40-5.60 (m, 1H,
Hγcis), 5.62 (s, 1H, OH), 6.14 (ddt, 1H, J Hγcis-Hâ ) 17 Hz,
J Hγtrans-Hâ ) 10 Hz, Hâ), 6.68 (s, 1H, H4). 13C NMR (50 MHz,
CDCl3): δ 13.2, 15.2, 20.4, 74.5, 114.3, 118.3, 127.3, 129.4, 132.8,
133.8, 142.5, 146.3. IR (CHCl3): ν 3536 (OH). Anal. Calcd for
C12H16O2: C, 74.96 H, 8.39. Found: C, 74.62; H, 8.46.
2-Isop r op oxy-4-p r op en ylp h en ol (6b). Same procedure as
for the synthesis of 3b starting from 6a (2.5 g, 13 mmol) to give
2.23 g (11.6 mmol, 89% yield) of 6b as a yellow oil. 1H NMR
(200 MHz, CDCl3): δ 1.37 (d, 6H, J ) 6 Hz, Me2CH-O) 1.86
(dd, 3H, J Hâ-Hγ ) 6 Hz, J HR-Hγ ) 2 Hz, allyl-CH3), 4.60 (m, 1H,
Me2CH-O), 5.67 (s, 1H, OH), 6.05 (dd, 1H, J HR-Hâ ) 16 Hz, Hâ),
6.49 (dd, 1H, HR), 6.75-7.00 (m, 3H, ArH). 13C NMR (50 MHz,
CDCl3): δ 18.4, 22.2, 71.8, 111.0, 114.6, 119.4, 123.2, 130.6,
130.9, 144.7, 145.9. IR (CHCl3): ν 3536 (OH). Anal. Calcd for
C12H16O2: C, 74.96; H, 8.39. Found: C, 74.83; H, 8.61.
6-Meth oxya llylisop seu d ocu m en ol (22). To a solution of
6-hydroxyallylipsopseudocumenol 21 (5.0 g, 26 mmol) in acetone
(200 mL) were added potassium carbonate (14.3 g, 104 mmol, 4
equiv) and methyl iodide. The reaction mixture was stirred for
12 h, diluted with hexane (100 mL), and filtered, and the solid
residue was washed with hexane (10 mL). Combined organic
layers were concentrated under reduced pressure, and the crude
residue was purified by column chromatography over silica
(hexane) to give 5.15 g (25 mmol, 96% yield) of 22 as a colorless
liquid. 1H NMR (200 MHz, CDCl3): δ 2.17 (s, 3H, CH3), 2.28
(s, 3H, CH3), 2.31 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 4.47 (bd,
2H, J HR-Hâ ) 6 Hz, HR), 5.20-5.35 (m, 1H, Hγtrans), 5.35-
5.50 (m, 1H, Hγcis), 6.19 (ddt, 1H, J Hγcis-Hâ ) 17 Hz, J Hγtrans-Hâ
) 10 Hz, Hâ), 6.65 (s, 1H, H4). 13C NMR (50 MHz, CDCl3): δ
13.0, 15.4, 20.7, 55.9, 73.9, 111.8, 117.0, 127.8, 130.7, 131.6,
134.8, 144.4, 150.2. Anal. Calcd for C13H18O2: C, 75.68; H, 8.80.
Found: C, 75.42; H, 8.81.
6-Acetylisop seu d ocu m en ol (18). Acetyl isopseudocumenol
17 (13.0 g, 73 mmol) and aluminum chloride (10.0 g, 75 mmol,
1 equiv) were heated at 100 °C for 90 min. The reaction mixture
was hydrolyzed with crushed ice (50 g) and extracted with CH2-
Cl2 (3 × 50 mL). Combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure to
give a crude residue. Purification by column chromatography
over silica (AcOEt 5%, hexane) gave 11.0 g (62 mmol, 85% yield)
of 18 as a yellow solid, mp ) 32-34 °C. 1H NMR (200 MHz,
CDCl3): δ 2.20 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.26 (s, 3H, CH3),
2.60 (s, 3H, CH3-CO), 7.36 (s, 1H, H4), 12.55 (s, 1H, OH). 13C
NMR (50 MHz, CDCl3): δ 11.3, 16.7, 20.2, 26.5, 116.7, 125.2,
126.5, 128.1, 145.0, 158.9, 204.1. IR (CHCl3): ν 3400-3050
(OH), 1628 (CdO). Anal. Calcd for C11H14O2: C, 74.12; H, 7.92.
Found: C, 73.88; H, 7.64.
3,5,6-Tr im eth yleu gen ol (7). To a solution of 6-methoxyal-
lylisopseudocumenol 22 (3.0 g, 14.6 mmol) in dry toluene (50
mL) was added, dropwise, a solution of Et2AlCl in toluene (8.1
mL; 14.6 mmol; 1.8 M). The reaction was heated at 100 °C for
1 h, cooled down to 0 °C, and hydrolyzed with water (20 mL)
and then 10% HCl (50 mL). The mixture was extracted with
ether (3 × 50 mL), and combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure to
give a crude product. Purification by column chromatography
on silica (AcOEt 10%, hexane) gave 2.34 g (11.4 mmol, 78%
yield) of 7 as a yellow solid, mp ) 55-57 °C. 1H NMR (200
MHz, CDCl3): δ 2.12 (s, 3H, CH3), 2.16 (s, 3H, CH3), 2.22 (s,
3H, CH3), 3.46 (ddd, dt-like, 2H, J HR-Hâ ) 5 Hz, J HR-Hγcis ) 2
Hz, J HR-Hγtrans ) 2 Hz, HR), 3.74 (s, 3H, OCH3), 4.92 (ddt, 1H,
J Hγcis-Hâ ) 10 Hz, J Hγcis-Hγtrans ) 4 Hz, Hγcis), 5.04 (ddt, 1H,
J Hγtrans-Hâ ) 17 Hz, Hγtrans), 5.58 (s, 1H, OH), 5.97 (ddt, 1H,
Hâ). 13C NMR (50 MHz, CDCl3): δ 12.5, 15.7, 16.1, 31.3, 61.8,
115.3, 121.8, 127.1, 127.7, 132.2, 137.1, 143.5, 147.1. IR
(CHCl3): ν 3536 (OH). Anal. Calcd for C13H18O2: C, 75.68; H,
8.80. Found: C, 76.00; H, 8.90.
6-Acetyla llylisop seu d ocu m en ol (19). To a solution of
6-acetylisopseudocumenol 18 (10.0 g, 56 mmol) in acetone (200
mL) were added potassium carbonate (31 g, 224 mmol, 4 equiv)
and allylbromide (10 mL, 116 mmol, 2.1 equiv). The reaction
mixture was heated under reflux for 2 days, diluted with hexane
(200 mL), filtered, and concentrated under reduced pressure.
The crude oil was taken up in hexane (50 mL), filtered,
concentrated, and purified by chromatography over silica (AcO-
Et 5%, hexane) to give 11.8 g (54 mmol, 96% yield) of 19 as a
yellow oil. 1H NMR (200 MHz, CDCl3): δ 2.20 (s, 3H, CH3),
2.23 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.61 (s, 3H, CH3-CO), 4.25
(bd, 2H, J HR-Hâ ) 6 Hz, HR), 5.26 (dd, 1H, J Hγtrans-Hâ ) 10 Hz,
J Hγcis-Hγtrans ) 1 Hz, Hγtrans), 5.41 (dd, 1H, J Hγcis-Hâ ) 17 Hz,
Hγcis), 6.07 (ddt, 1H, HR), 7.35 (s, 1H, H4). 13C NMR (50 MHz,
CHCl3): δ 12.8, 16.4, 20.1, 30.5, 75.7, 117.6, 128.0, 130.6, 130.7,
132.2, 133.5, 141.5, 154.3, 200.7. IR (CHCl4) ν 1672 (CdO).
Anal. Calcd for C14H18O2: C, 77.02; H, 8.32. Found: C, 76.70;
H, 8.36.
9,9,9-Tr im eth ylisoeu gen ol (8). 4-Bromoguaiacol 23 (4 g,
20 mmol), palladium acetate (440 mg, 2 mmol, 0.1 equiv), tri-
o-tolylphosphine (600 mg, 2 mmol, 0.1 equiv), neohexene (26
mL, 0.2 mol, 10 equiv), and Et3N (11 mL, 80 mmol, 4 equiv)
were placed in a sealed tube which was heated at 110 °C for 3
days. The reaction mixture was cooled down and filtered on
Celite, and the organic layer was washed with 5% HCl, dried
over MgSO4, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography over
silica (AcOEt 30%, hexane) to give 3.45 g (16.7 mmol, 85% yield)
of 8 as a white solid, mp ) 63-64 °C. 1H NMR (200 MHz,
CDCl3): δ 1.12 (s, 9H, t-Bu), 3.92 (s, 3H, OCH3), 5.56 (s, 1H,
OH), 6.09 (d, 1H, J HR-Hâ ) 16 Hz, Hâ), 6.24 (d, 1H, HR), 6.80-
6.90 (m, 3H, ArH). 13C NMR (50 MHz, CDCl3): δ 29.7, 33.2,
55.9, 108.0, 114.4, 119.5, 124.4, 130.7, 139.7, 144.8, 146.6. IR
(CHCl3): ν 3544 (OH). Anal. Calcd for C13H18O2: C, 75.68; H,
8.80. Found: C, 75.29; H, 8.77.
6-Hyd r oxyeth yla llylip sop seu d ocu m en ol (20). To a solu-
tion of 6-acetyl allylisopseudocumenol 19 (10.0 g, 46 mmol) in
ether (200 mL) was added, by portions and at 0 °C, LiAlH4 (1.7
g, 45 mmol, 1 equiv). The reaction mixture was stirred at room
temperature for 30 min, cooled down to 0 °C, hydrolyzed with
water (20 mL), washed with 5% HCl (50 mL), and extracted
with ether (5 × 50 mL). Combined organic layers were dried
over MgSO4, filtered, and concentrated under reduced pressure
to give a crude product. Purification by column chromatography
on silica (AcOEt, 30% hexane) gave 9.5 g (43 mmol, 94% yield)
of 20 as a colorless oil. 1H NMR (200 MHz, CDCl3): δ 1.51 (d,
3H, CH3-CHOH), 2.18 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.28 (s,
3H, CH3), 2.65-2.80 (bs, 1H, OH), 4.30 (bd, 2H, J HR-Hâ ) 5 Hz,
HR), 5.17 (quint, 1H, CHOH), 5.30 (dd, 1H, J Hγtrans-Hâ ) 10 Hz,
J Hγcis-Hγtrans ) 1 Hz, Hγtrans), 5.47 (dd, 1H, J Hγcis-Hâ ) 17 Hz,
Hγcis), 6.13 (ddt, 1H, Hâ), 7.12 (s, 1H, H4). 13C NMR (50 MHz,
CHCl3): δ 13.1, 15.9, 20.5, 24.0, 65.0, 74.7, 117.3, 124.8, 129.5,
132.5, 133.9, 135.2, 135.9, 152.5. Anal. Calcd for C14H20O2: C,
76.31 H, 9.16. Found: C, 76.52; H, 9.19.
Mou se Loca l Lym p h Nod e Assa y (LLNA). This assay was
carried out as described (9) but with the exception that wherever
possible chemicals were tested at equimolar concentrations.
Briefly, groups of mice (n ) 4) received 25 µL of the test chemical
dissolved in vehicle (acetone-olive oil 4:1) in the concentrations
indicated in Tables 1-3, on the dorsum of both ears daily for 3
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 phosphate-
6-Hyd r oxya llylip sop seu d ocu m en ol (21). To a solution of
6-hydroxyethyl allylipsopseudocumenol 20 (8.8 g, 40 mmol) in
THF (100 mL) were added a solution of H2O2 (50 mL, 35%
aqueous) and p-toluenesulfonic acid (760 mg, 4 mmol, 0.1 equiv).
The reaction mixture was stirred at room temperature for 3 days
and extracted with hexane (3 × 100 mL), and combined organic
layers dried over MgSO4, filtered, and concentrated under

(Iso)Eugenol Sensitization
Chem. Res. Toxicol., Vol. 10, No. 3, 1997 339
Ta ble 1. Cell P r olifer a tion In d u ced by Eu gen ol 1 a n d
Ta ble 2. Cell P r olifer a tion In d u ced by Isoeu gen ol 2 a n d
Der iva tives 3a -6a in th e Loca l Lym p h Nod e Assa ya
Der iva tives 3b-6b in th e Loca l Lym p h Nod e Assa ya
[3H]thymidine
[3H]thymidine
concentration incorporation stimulation
concentration incorporation stimulation
chemical
isoeugenol 2
(w/w %)
(dpm/node)
index
chemical
eugenol 1
(w/w %)
(dpm/node)
index
control
416
3545
4990
6859
659
3879
7341
10329
655
3514
3379
4559
659
1435
2822
3963
-
control
380
899
2091
6100
655
1263
3182
5436
713
1927
3477
3035
713
1037
1621
4578
655
1160
1185
1442
-
2.4
5.5
16.1
-
1.9
4.9
8.3
-
2.7
4.9
4.3
-
1.5
2.3
6.4
-
2.5 (0.15 M)
5 (0.30 M)
10 (0.61 M)
control
2.5 (0.14 M)
5.5 (0.31 M)
11 (0.62 M)
control
2.5 (0.14 M)
5.5 (0.31 M)
11 (0.62 M)
control
2.5 (0.14 M)
5.5 (0.31 M)
11 (0.62 M)
control
8.5
10 (0.61 M)
25 (1.52 M)
50 (3.05 M)
12.1
16.5
-
6-methylisoeugenol 3b
5-methylisoeugenol 4b
3-methylisoeugenol 5b
compound 6b
6-methyleugenol 3a control
11 (0.62 M)
5.9
11.1
15.7
-
5.4
5.2
7.0
-
2.2
27 (1.52 M)
54 (3.03 M)
5-methyleugenol 4a control
11 (0.62 M)
27 (1.52 M)
54 (3.03 M)
3-methyleugenol 5a control
4.3
6.0
-
11 (0.62 M)
27 (1.52 M)
54 (3.03 M)
control
12 (0.62 M)
29 (1.51 M)
59 (3.07 M)
659/449*
1333*
2574*
7320/4823*
7657
0.6 (31 mM)
1.2 (62 mM)
3 (0.16 M)
6 (0.31 M)
12 (0.62 M)
3.0*
5.7*
11.1/10.7*
11.6
11.9
compound 6a
1.8
1.8
2.2
7869
a
Groups of mice (n ) 4) received 25 µL of the test chemical
dissolved in vehicle (AOO) in the concentrations indicated, on the
dorsum of both ears daily for 3 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. 5 h later,
draining auricular lymph nodes were excised and pooled for each
group and a single-cell suspension of lymph node cells was
prepared. The thymidine incorporation was measured by â-scin-
tillation counting. The increase in thymidine incorporation rela-
tive to vehicle-treated controls was derived for each experimental
group and recorded as a stimulation index.
a
Groups of mice (n ) 4) received 25 µL of the test chemical
dissolved in vehicle (AOO) in the concentrations indicated, on the
dorsum of both ears daily for 3 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. 5 h later,
draining auricular lymph nodes were excised and pooled for each
group and a single-cell suspension of lymph node cells was
prepared. The thymidine incorporation was measured by â-scin-
tillation counting. The increase in thymidine incorporation rela-
tive to vehicle-treated controls was derived for each experimental
group and recorded as a stimulation index. Two sets of experi-
ments were carried out with compound 6b, and data marked with
asterisks correspond to the same set.
buffered saline (PBS) containing 20 µCi of [3H]thymidine. 5 h
later, draining auricular lymph nodes were excised and pooled
for each group, and a single-cell suspension of lymph node cells
was prepared. The thymidine incorporation was measured by
â-scintillation counting. The increase in thymidine incorpora-
tion relative to vehicle-treated controls was derived for each
experimental group and recorded as a stimulation index.
Ta ble 3. Cell P r olifer a tion In d u ced by Eu gen ol
Der iva tive 7 a n d Isoeu gen ol Der iva tive 8 in th e Loca l
Lym p h Nod e Assa ya
[3H]thymidine
concentration incorporation stimulation
chemical
(w/w %)
(dpm/node)
index
Resu lts
eugenol derivative 7
control
612
1018
1313
865
-
12.6 (0.61 M)
31.4 (1.52 M)
62.8 (3.05 M)
1.9
2.1
1.4
-
Ch em istr y. The synthesis of eugenol derivatives 3a -
6a was based on the thermal or catalyzed Claisen
transposition of appropriately functionalized allylphenol
compounds 13-16. The further transformation of eu-
genol into isoeugenol derivatives 3b-6b was achieved
by potassium tert-butanolate treatment in DMSO.
Sta r tin g Ma ter ia ls. The 6-methylguaiacol 9 was
prepared, in one step and with 90% yield, from o-vanillin
using a Wolf-Kischner reduction of the aldehydic func-
tion (10). The 3-methylcatechol was transformed into
3-methylguaiacol 11 through a one-pot selective protec-
tion of the phenol function at position 1 with tert-
butyldimethylsilyl chloride (NaH, THF) (11) followed by
the methylation of the second phenolic function with MeI
and potassium carbonate and the subsequent hydrolysis
of the silyl protective group (11) under acidic conditions
(10% HCl, reflux). The 3-methylguaiacol 11 was thus
obtained with an overall yield of 70%. The 5-methylguai-
acol 10 and the 2-isopropylcatechol 12 were commercially
available.
isoeugenol derivative 8 control
6.3 (0.30 M)
612
1984
2850
4903
3.2
4.7
8.0
12.6 (0.61 M)
31.4 (1.52 M)
a
Groups of mice (n ) 4) received 25 µL of the test chemical
dissolved in vehicle (AOO) in the concentrations indicated, on the
dorsum of both ears daily for 3 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. 5 h later,
draining auricular lymph nodes were excised and pooled for each
group and a single-cell suspension of lymph node cells was
prepared. The thymidine incorporation was measured by â-scin-
tillation counting. The increase in thymidine incorporation rela-
tive to vehicle-treated controls was derived for each experimental
group and recorded as a stimulation index.
thermal condition when both o-positions were function-
alized and under aluminum catalyze when one o-position
was free. It is known in the literature that aluminum
derivatives are able to catalyze Claisen transposition (12,
13) by chelating the O-allyl oxygen and that voluminous
groups will orientate the allyl function and promote the
transposition at the substituted ortho-position, leading
then to the para-transposition product (12). Thus com-
pound 13 gave the eugenol derivative 3a with 76% yield
Syn th esis of Eu gen ol a n d Isoeu gen ol Der iva tives.
Phenols 9-12 were allylated with 2 equiv of allyl bromide
in refluxing acetone with an excess of K2CO3 as base with
yields ranging from 85% to 98% (Scheme 1). Claisen
transpositions of allyl derivatives were performed under

340 Chem. Res. Toxicol., Vol. 10, No. 3, 1997
Bertrand et al.
Sch em e 1. Syn th etic P a th w a y to Eu gen ol 3a -6a a n d Isoeu gen ol 3b-6b Der iva tives
Sch em e 2. Syn th etic P a th w a y to th e Tr im eth yl Eu gen ol Der iva tive 7
after treatment at 180 °C for 4 h, but a similar treatment
Oxidation of the alcohol 20 with H2O2 under acidic
conditions (15) (p-TsOH) gave with 76% yield the phenol
21 which was subsequently methylated under classical
conditions. The Claisen transposition of 22 under ther-
mal conditions gave mainly decomposition products,
while treatment at room temperature with Et2AlCl gave
the ortho-transposition intermediate with 91% yield. We
thus decided to combine thermal and catalytic conditions.
As a result, the treatment of 22 in refluxing toluene with
1 equiv of Et2AlCl gave the expected eugenol derivative
7 with 78% yield.
applied to compounds 14-16 gave mainly the formation
of the expected ortho-substituted products. The use of
aluminum derivatives allowed us to decrease both reac-
tion time and temperature and to increase the para/ortho
ratio to a reasonable level. Thus the Claisen transposi-
tion of 14 and 15 catalyzed respectively with Et2AlCl and
methylaluminum bis(pentabromophenol) (MAP) gave
derivatives 4a and 5a with 42% and 56% yield, respec-
tively. The lowest yield (30%) was obtained for the
transposition of the isopropyl derivative 16 into 6a .
The 9,9,9-trimethylisoeugenol 8 was prepared in one
step (Scheme 3) from neohexene and 4-bromoguaiacol 23
using a classical Heck-type reaction with palladium
acetate (16). Only the trans isomer was formed (85%
yield).
Isoeugenol derivatives 3b-6b were prepared in high
yields from 3a -6a by treatment with 2 equiv of potas-
sium tert-butanolate in DMSO at 110 °C for 2 h, and
trans isomers were predominantly formed.
The 3,5,6-trimethyl eugenol 7 was synthesized from
6-acetyl-isopseudocumenol 17 (Scheme 2). A Fries rear-
rangement (14) of 17 with AlCl3 gave the phenol deriva-
tive 18 (85% yield) which was allylated under classical
conditions. The oxidation of 19 with H2O2, under either
basic or acidic conditions, gave poor results so we decided
to reduce the carbonyl function with LiAlH4 in ether.
LLNA. The mouse local lymph node assay results on
the eugenol and isoeugenol derivatives are shown in
Tables 1-3. The following points should be noted.
Eu gen ol Der iva tives. In contrast to the situation in
guinea pigs, a significant degree of sensitization to
eugenol was induced in the mouse, i.e., a dose dependent

(Iso)Eugenol Sensitization
Sch em e 3. Syn th esis of th e Isoeu gen ol
Chem. Res. Toxicol., Vol. 10, No. 3, 1997 341
in skin sensitization was reported by Landsteiner and
J acobs in the 1930s (17). Although the biological mech-
anism of sensitization was not at that time understood
in any detail, they had already come to the view that
sensitization to chemicals involved covalent bonding to
protein. Their results on aromatic halides and pseudoha-
lides showed a complete correspondence between ability
to react with aniline, chosen as a model for protein and
ability to sensitize. Further evidence for a relationship
between reactivity and ability to sensitize came from a
study of the guinea pig sensitization potential of R-sub-
stituted p-nitrotoluenes, XCH2C6H4NO2, reported in 1984.
The compounds with X ) H or OH did not react with
n-butylamine, used as a model for protein nucleophiles,
and were non-sensitizing, whereas the compounds with
X ) F, Cl, Br, I, and p-toluenesulfonyl all reacted as
electrophiles with butylamine, the X group being dis-
placed in an SN2 reaction, and were all sensitizers (18).
More recently, developments on quantitative structure-
activity relationships have shown that the ability of a
chemical to induce a skin sensitization reaction was a
function of three parameters: the dose, the partition
coefficient, and the rate constant toward a model nucleo-
phile such as n-butylamine (19-21).
With the development of the local lymph node assay
(9) it is now possible to apply conventional QSAR ap-
proaches in skin sensitization studies. The main reasons
for this are that the LLNA uses a single dose per test
group and then the extend of sensitization induced by
this dose is quantified via measurement of the cellular
proliferation induced in the lymph nodes draining the site
of chemical application. The data are presented as a
ratio, T/C, which compares the proliferation for each test
group to that from a vehicle-treated control group and is
taken as a quantifier of the degree of sensitization
induced.
Der iva tive 8
increase in proliferation and at least one test concentra-
tion inducing a 3-fold or greater increase (SI value g 3)
in isotope incorporation compared with vehicle-treated
controls (Table 1). However, it should be noted that in
the LLNA testing, higher concentrations were used for
eugenol (10-50%) and its derivatives than for isoeugenol
(2.5-10%) and its derivatives; taking this into account,
it can be inferred that isoeugenol is actually a stronger
sensitizer than eugenol in the mouse, but the difference
is less than in the guinea pig.
The replacement of the methoxy group in eugenol by
the isopropoxy group (compound 6a ) led to a complete
loss of sensitization potential, and when a methyl group
was present in the 3-position (compound 5a ) a significant
degree of sensitization was induced only at the highest
concentration of 54%. Introduction of methyl substitu-
ents in the 5- or 6-position (compounds 4a and 3a ,
respectively) also led to similar decreases in the sensitiz-
ing potential, while introduction of three methyl groups
on the aromatic ring (compound 7) led to a complete loss
of sensitization potential.
Isoeu gen ol Der iva tives. All isoeugenol derivatives
fulfill the criteria for a chemical to be classified as a
sensitizer in the LLNA. The sensitization potential of
isoeugenol in the mouse was not substantially affected
when the methoxy group was replaced by the isopropoxy
group (compound 6b). Methyl substitution in the 6-posi-
tion of isoeugenol (3b) had no discernible effect on the
sensitization potential, whereas methyl substitution in
the 3- and 5-positions of isoeugenol (5b and 4b, respec-
tively) led to a reduction in sensitization potential.
Introduction of a tert-butyl substituent at the γ-position
of the alkyl chain (compound 8) resulted in a strong
decrease of the sensitizing capacity.
Within each seriesseugenol and isoeugenol derivativess
compounds have been tested at the same molar concen-
tration and have little differences in their partition
coefficients. The difference in skin sensitization is
therefore strongly related to their differences in chemical
reactivity toward epidermal proteins.
Sk in Sen sitiza tion to Eu gen ol a n d Its Der iva -
tives. In contrast to the situation in guinea pigs, a
significant degree of sensitization to eugenol was induced
in the mouse, i.e., a dose dependent increase in prolifera-
tion and at least one test concentration inducing a 3-fold
or greater increase (SI value g 3) in isotope incorporation
compared to vehicle-treated controls. The replacement
of the methoxy group in eugenol by the isopropoxy group
(compound 6a ) led to a complete loss of sensitization,
indicating that this derivative is no longer metabolized
into a reactive intermediate. This would suggest that a
demethylation step occurs as part of the eugenol sensi-
tization pathway (Figure 3). Previously it was assumed
that demethylation is followed by oxidation to the o-
quinone, which reacts as a Michael acceptor with protein
to form antigen. Several studies on the reactivity of
o-quinones (22, 23) have demonstrated that attacks of
nucleophiles were following the hard and soft acids and
bases (HSAB) theory (24), i.e., 1,4 Michael addition for
oxo and amino groups and 1,6 Michael addition for thio
groups. This has been further confirmed by Bolton et al.
on the o-quinone derived from hydroxychavicol with a
major addition of glutathione in the 6-position (7).
Previous studies on the sensitizing potential of 3-n-
alkylcatechols, which are believed to be active through
Discu ssion
The skin sensitization reaction to a chemical is a
multistep process which involves T-cells of the immune
system. During the induction phase, the chemical pen-
etrates into the epidermis, beneath the stratum corneum,
and there it binds to protein, thus modifying the protein’s
structure. The modified protein is then processed by
epidermal antigen presenting cells and is presented, in
a form which can be recognized as antigenic, to uncom-
mitted T-cells. Those T-cells whose receptors match the
modified protein are stimulated to multiply, producing
expanded clones of circulating T-cells capable of recogniz-
ing the modified protein.
It follows from the above simple description of the
biological mechanism that for a chemical to be a sensi-
tizer it must have the ability to bind, either directly or
after appropriate biochemical transformation, to protein
so that a non-self-antigen can be produced.
Since it is the chemical which is the driving force, it is
reasonable to consider that it is possible to relate chemi-
cal structure with the propensity to behave as a skin
sensitizer. One of the earliest structure-activity studies

342 Chem. Res. Toxicol., Vol. 10, No. 3, 1997
Bertrand et al.
The effects of methyl substitution on the sensitization
potential of isoeugenol in the LLNA should provide a good
test of the oxidation-quinone methide proposal. On the
basis of the findings of Bolton et al. we can assume that
if this proposal is correct, conjugation of the quinone
methide to protein to produce antigen occurs by nucleo-
philic attack at the R-carbon atom. This reaction should
be susceptible to steric retardation by methyl groups in
the 3- or 5-positions but not in the 6-position. In complete
agreement with this reasoning, 6-methylisoeugenol 3b
is very similar in sensitization potential to isoeugenol
while 3-methylisoeugenol 5b and 5-methylisoeugenol 4b
(Table 2) are significantly weaker sensitizers.
It is also important to note that the tert-butyl deriva-
tive 8 which cannot be oxidized directly to a quinone
methide has a very low sensitizing potential comparable
to the one of eugenol 1. It could be assumed that when
the direct oxidation into a p-quinone methide is not
available, the more classical demethylation-o-quinone
pathway is still available.
F igu r e 3. Proposed metabolic pathway for the sensitizing
properties of eugenol 1.
p-Qu in on e Meth id e vs o-Qu in on e P a th w a y. In
two recent papers (7, 8) Bolton and co-workers have
demonstrated a bioactivation pathway for the hepato-
carcinogen safrole (1-allyl-3,4-methylenedioxybenzene)
involving initial O-dealkylation of the methylene dioxy
ring to form the catechol hydroxychavicol, oxidation of
hydroxychavicol to the o-quinone, and tautomerization
of the o-quinone to the p-quinone methide. As discussed
by Bolton et al. p-quinone methides are much more
electrophilic than their o-quinone tautomers and could
account in the toxicity of such compounds. Bolton et al.
studied the reactions with the nucleophile glutathione
on oxidation intermediates generated from hydroxychavi-
col using either mushroom tyrosinase, silver oxide, or rat
liver microsomes (cytochrome P450). The o-quinone
generated by oxidation with tyrosinase in the presence
of glutathione led to the formation of adducts on the
aromatic ring, mainly in the C-6 position while the
quinone methide produced by reaction with silver oxide
was found to give, under kinetic control, the product
resulting from attack of the nucleophile at the R-position,
while under thermodynamic control, the product of
γ-attack was obtained. Treatment of safrole or hydroxy-
chavicol with rat liver microsomes in the presence of
tritiated glutathione gave mainly adducts on the aromatic
ring resulting from a reaction with the o-quinone inter-
mediate.
When this paper was at an advanced stage of prepara-
tion, we received information in a private communication
from Bolton of unpublished work in which isoeugenol
oxidation was studied. Consistent with our hypothesis,
Bolton and Leslie found that isoeugenol is more readily
oxidized than eugenol. However, in experiments in which
glutathione was added to trap the expected quinone
methide, only minimal amounts of the expected quinone
methide adducts were detected. One should keep in mind
that the balance of metabolic activities in the skin does
not necessarily reflect that found in the liver and that
the biological mechanism of skin sensitization which
involves the covalent modification of epidermal proteins
mainly through amino groups is far from the detoxifica-
tion process involving glutathione.
F igu r e 4. Proposed metabolic pathway for the sensitizing
properties of isoeugenol 2.
the formation of an o-quinone intermediate, have high-
lighted the fact that 1,4 addition positions, and thus
amino nucleophiles were largely responsible for the
allergenic activity (25-27). If the same theory applied
to eugenol derivatives, introduction of a methyl group at
the 5-position (compound 4a ) should have a pronounced
effect on the sensitizing potential, while methylation at
the 6-position should be less important. This is indeed
what was observed with a decreased SI of 8.3 at 3.05 M
for compound 3a and a SI of only 4.3 for compound 4a at
the same concentration (the SI at 3.05 M for eugenol 1
is 16.1).
For the present we do not consider the data to be
conclusive, but two additional pieces of evidence tend to
favor the involvement of o-quinone. When a methyl
group was present in the 3-position (compound 5a ) a
significant degree of sensitization was induced only at
the highest concentration of 54% (we could attribute this
to a steric effect hindering attack on the methoxy group
in demethylation) while the presence of three methyl
substituents on the aromatic ring (compound 7) com-
pletely abolished the sensitizing potential even at the
highest concentration (3.05 M).
Sk in Sen sitiza tion to Isoeu gen ol a n d Its Der iva -
tives. In contrast to the situation with eugenol deriva-
tives, the sensitization potential of isoeugenol in the
mouse was not substantially affected when the methoxy
group was replaced by the isopropoxy group (compound
6b). This suggests that a demethylation step is probably
not necessary for the formation of reactive metabolites
and the findings of Bolton et al. (7, 8) prompt us to
consider direct oxidation of isoeugenol to a methoxy
substituted p-quinone methide as the isoeugenol sensi-
tization pathway (Figure 4).
Con clu sion
To summarize, our findings indicate that, in the mouse,
eugenol seems to sensitize via a demethylation pathway

(Iso)Eugenol Sensitization
Chem. Res. Toxicol., Vol. 10, No. 3, 1997 343
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