α- and β-thujones (herbal medicines and food additives): Synthesis and analysis of hydroxy and dehydro metabolites

Article abstract of DOI:10.1021/jf001445+

Essential oils containing α- and β-thujones are important herbal medicines and food additives. The thujone diastereomers are rapidly metabolized convulsants acting as noncompetitive blockers of the γ-aminobutyric acid-gated chloride channel. Synthesis and analysis of the metabolites are essential steps in understanding their health effects. Oxidation of α- and β-thujones as their 2,3-enolates with oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) gave the corresponding (2R)-2-hydroxythujones assigned by ¹H and ¹³C NMR and X-ray crystallography, α-Thujone was converted to 4-hydroxy-α- and -β-thujones via the 3,4-enol acetate on oxidation with peracid and osmium tetroxide, respectively. Ozonation provided 7-hydroxy-α- and -β-thujones, and by dehydration provided the 7,8-dehydro compounds. 4,10-Dehydrothujone was prepared from sabinene via sabinol. The hydroxy and dehydro derivatives are readily identified and analyzed by GC/MS as the parent compounds and trimethylsilyl and methyloxime derivatives. A separate study established that all of these compounds are metabolites of α- and β-thujones.

Full text of DOI:10.1021/jf001445+

J. Agric. Food Chem. 2001, 49, 1915−1921
1915
r- a n d â-Th u jon es (Her ba l Med icin es a n d F ood Ad d itives):
Syn th esis a n d An a lysis of Hyd r oxy a n d Deh yd r o Meta bolites
Nilantha S. Sirisoma, Karin M. Ho¨ld, and J ohn E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and
Management, 115 Wellman Hall, University of California, Berkeley, California 94720-3112
Essential oils containing R- and â-thujones are important herbal medicines and food additives. The
thujone diastereomers are rapidly metabolized convulsants acting as noncompetitive blockers of
the γ-aminobutyric acid-gated chloride channel. Synthesis and analysis of the metabolites are
essential steps in understanding their health effects. Oxidation of R- and â-thujones as their 2,3-
enolates with oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) gave the corre-
sponding (2R)-2-hydroxythujones assigned by 1H and 13C NMR and X-ray crystallography. R-Thujone
was converted to 4-hydroxy-R- and -â-thujones via the 3,4-enol acetate on oxidation with peracid
and osmium tetroxide, respectively. Ozonation provided 7-hydroxy-R- and -â-thujones, and by
dehydration provided the 7,8-dehydro compounds. 4,10-Dehydrothujone was prepared from sabinene
via sabinol. The hydroxy and dehydro derivatives are readily identified and analyzed by GC/MS as
the parent compounds and trimethylsilyl and methyloxime derivatives. A separate study established
that all of these compounds are metabolites of R- and â-thujones.
Keyw or d s: R- and â-Thujones; oxidation reactions; metabolite synthesis; metabolite derivatization;
GC/ MS analysis
INTRODUCTION
R-Thujone (1R) and â-thujone (1â) are bicyclic mono-
terpenes which differ in the stereochemistry of the C-4
methyl group (1) (Figure 1). The isomer ratio depends
on the plant source, with high content of R-thujone in
cedarleaf oil (2) and â-thujone in wormwood oil (3). They
are also common constituents in herbal medicines,
essential oils, foods, flavorings, and beverages (2, 4, 5).
R-Thujone is perhaps best known as the active ingredi-
ent of the alcoholic beverage absinthe which was a very
popular European drink in the 1800s and gained
considerable notoriety as a preferred liqueur of artists
and writers (3, 6-10). Although absinthe became an
epidemic health problem, leading to a widespread ban
in the early 1900s (8), its use continues on a small scale
either legally or illicitly (11). The primary health
concern is from consumption in herbal medicines, foods,
and drinks, leading to the U.S. National Toxicology
Program recommending chronic toxicity studies on
thujone in rats and mice (4).
Understanding the health effects of the thujone
diastereomers requires defining their mode of action and
metabolic fate. The acute toxicity of R-thujone can be
attributed to blocking the γ-aminobutyric acid (GABA)-
gated chloride channel (3). Metabolite synthesis and
analysis are essential steps in defining the metabolic
pathways of R-thujone and â-thujone and the biological
activities of their metabolites. Our recent studies of the
fate of R-thujone and â-thujone in vitro and in vivo led
to identification of the hydroxythujones and dehy-
drothujones shown in Figure 1 (3, 12). A large number
of isomeric hydroxythujones are involved with similar
GC characteristics and MS fragmentations. An impor-
F igu r e 1. Structures of R- and â-thujones and designations
for isomeric hydroxythujones and dehydrothujones reported
here and identified as metabolites.
tant step was to establish that all of the hydroxythu-
jones and dehydrothujones can be separated by GC
either underivatized or as their trimethylsilyl (TMS) or
methyloxime (MOX) derivatives. Synthesis and full
chromatographic and spectral characterization of these
candidate metabolites and their TMS and MOX deriva-
tives described here makes it possible to identify them
and ultimately to determine their health effects.
MATERIALS AND METHODS
Sa fety. The preparation of MoOPH involves hydrogen
peroxide and should be performed behind a safety shield to
minimize explosion hazards (13).
Ch em ica ls. R-Thujone (99% pure) was from Fluka (St.
Louis, MO) and sabinene (70% pure, from nutmeg essential
oil) was from R. C. Treatt and Co., Ltd. (Bury St. Edmunds,
Suffolk, U.K.). â-Thujone was isolated from wormwood oil
(3.2% R-thujone and 35% â-thujone) (3) (Lhasa Karnak,
Berkeley, CA) by chromatography of a 5-g sample on a silica
gel column using 1-5% ethyl acetate in hexane. The â-thujone
fractions contained an alkene impurity which was removed
by treatment with m-chloroperoxybenzoic acid (mCPBA) in
CHCl3, and chromatography using 3-5% ethyl acetate in
* To whom correspondence should be addressed. Tel: (510)
642-5424.Fax: (510)642-6497.E-mail: ectl@nature.berkeley.edu.
10.1021/jf001445+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/30/2001

1916 J. Agric. Food Chem., Vol. 49, No. 4, 2001
Sirisoma et al.

Thujone Metabolites: Synthesis and Analysis
J. Agric. Food Chem., Vol. 49, No. 4, 2001 1917
Ta ble 2. 13C NMR (75 MHz) Da ta (δ) in CDCl3
Carbon
1Ra
1â
2R
2â
3Rb
3â
4R
4â
5R
6
1
2
3
4
5
6
7
8
9
29.6
39.7
221.1
47.3
25.5
18.7
32.8
19.9
19.6
18.1
32.6
41.7
218.2
45.3
24.6
12.4
27.4
19.7
19.6
14.6
33.2
74.8
218.9
44.5
23.2
14.3
33.2
19.7
20.0
17.9
31.0
75.8
218.0
42.7
24.2
9.4
23.9
11.7
17.2
20.8
29.7
38.0
217.4
79.6
28.0
15.4
32.5
19.5
19.6
25.9
31.9
41.0
220.3
47.2
23.5
16.2
69.8
27.5
27.2
23.4
31.0
45.4
217.5
42.6
13.0
12.5
70.3
27.2
27.6
22.8
29.2
42.7
219.9
47.3
19.7
19.4
145.5
110.1
26.9
30.3
41.5
205.8
113.3
32.6
21.8
26.4
19.5
19.6
39.5
215.3
77.4
29.7
15.8
32.1
19.7
19.8
28.3
10
18.1
148.0
a
b
Assignment based on Bohlman et al. (16). Assignment by Alaoui et al. (15). Reported C-5 and C-7 chemical shifts interchanged.
Present assignments are based on HMQC (heteronuclear multiple quantum coherence).
hexane to obtain pure â-thujone (>99%). Following are the
sources for the other chemicals: N-methylmorpholine N-oxide,
mCPBA, and p-toluenesulfonic acid, Aldrich (Milwaukee, WI);
N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and
MOX reagent (2% solution of methoxyamine HCl in pyridine),
Pierce (Rockford, IL). p-Toluenesulfonic acid was dried over
P2O5 at 10 µm Hg. Oxodiperoxymolybdenum(pyridine)(hexa-
methylphosphoric triamide) (MoOPH) was prepared by the
procedure of Vedejs and Larsen (13).
saturated NaCl (10 mL) was added and the reaction mixture
was extracted with ether (3 × 20 mL). The combined extracts
were washed with 1N HCl (50 mL), dried over anhydrous
MgSO4, filtered, and concentrated to obtain a residue which
was chromatographed using 2-4% ethyl acetate in hexane to
obtain 2R (325 mg, 1.93 mmol, 58%). IR ν 3463, 2961, and
1750. The 2,4-dinitrophenylhydrazone was prepared and
recrystallized (hexane/ethyl acetate) to afford yellow crystals
(mp 109.5-110.5 °C).
An a lyses. 1H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were measured as CDCl3 solutions on a Bruker AM-
300 spectrometer using the residual CHCl3 as the standard.
Infrared (IR) spectra were recorded as neat samples on a
Perkin-Elmer 1600 series Fourier transform IR spectrometer
with bands reported as cm-1. GC/MS was performed on a
HP5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA)
coupled to a HP5971 Mass Selective Detector (Hewlett-
Packard) using positive chemical ionization. The GC was
equipped with a DB-5 fused silica gel capillary column (30 m,
0.25 mm i.d., 0.25 µm film thickness) (J &W Scientific, Folsom,
CA). The initial column temperature of 80 °C was programmed
at 5 °C/min up to 200 °C, followed by an increase at 20 °C/
min to 300 °C where it was maintained for 2 min. Helium was
used as the carrier gas at a flow rate of 0.8 mL/min. The
reagent gas was methane. Temperatures of the injection port
and detector were 250 and 280 °C, respectively. All column
chromatography was on silica gel.
Trimethylsilyl (TMS) derivatives were formed on reaction
of alcohols with MSTFA (30 min at 80 °C) and methyloxime
(MOX) derivatives were formed on coupling ketones with MOX
reagent (60 min at 80 °C) (12). The structure of one compound
(2R) was determined as its 2,4-dinitrophenylhydrazone deriva-
tive by X-ray crystallography. A yellow plate of dimensions
0.01 × 0.04 × 0.06 mm was the largest specimen that could
be found. Data were collected using a Siemens P4 diffractom-
eter equipped with a Siemens LT-2 low-temperature ap-
paratus, a cold stream of 130 K, and an ω scan of 2.2° with a
1.6° offset from the center for left and right background
measurements to a maximum 2θ of 112° (nickel filtered Cu
KR radiation from a Siemens rotating anode source). The
structure was solved by direct methods in the space group P21
and refined by full-matrix least-squares based on F2 (14). An
R1 value of 0.120 was obtained based on 408 parameters and
2516 observed data.
Preparation of 2â. â-Thujone was converted to 2â by the
same procedure as above for R-thujone to 2R in 61% yield. IR
ν 3446, 2963, and 1748.
P r epar ation of 4-Hydr oxyth u jon es (3r an d 3â) (Sch em e
2). Preparation of the 3,4-Enol Acetate. R-Thujone (185 mg,
1.22 mmol) in isopropenyl acetate (1.5 mL) was treated with
p-toluenesulfonic acid (18 mg, 0.105 mmol) and refluxed for 3
days. The reaction mixture was concentrated and chromato-
graphed using 1-4% ethyl acetate in hexane to obtain a
nonresolved mixture (220 mg, 93%) of 90% 3,4-enol acetate
and 10% 2,3-enol acetate by GC/MS analysis.
Preparation of 3R. A solution of the above enol acetates (112
mg, 0.577 mmol) in CHCl3 (0.5 mL) was added to a solution of
mCPBA (75 mg) in CHCl3 (2.0 mL) at 0 °C. The mixture was
stirred at 0 °C for 0.5 h, then another portion of mCPBA (75
mg) was added, and the mixture was warmed to room
temperature with continued stirring for 3 h. The reaction
mixture was diluted with ethyl acetate (15 mL), then washed
in sequence with 5% NaHSO3 (20 mL), 5% NaHCO3 (20 mL),
and saturated NaCl (20 mL). The organic layer was dried over
anhydrous MgSO4, concentrated, and chromatographed using
1-6% ethyl acetate in hexane to obtain 4-acetoxy-R-thujone
(52 mg, 43%) with the remainder mostly R-thujone. 4-Acetoxy-
R-thujone (43 mg, 0.204 mmol) was dissolved in absolute
methanol (10 mL), and sodium methoxide (0.1 mmol in
methanol) was added at room temperature. Removal of the
acetate substituent was complete after 1.5 h. A few drops of
water were added, then most of solvent was evaporated under
vacuum. The residue was dissolved in ethyl acetate (15 mL)
and washed with water (20 mL) and saturated NaCl (20 mL).
The organic layer was dried over anhydrous MgSO4 and
concentrated to an oily residue which was chromatographed
using 20% ethyl acetate in hexane to obtain 3R as an oil (31
mg, 0.184 mmol, 90%). IR ν 3468, 2920, and 1748.
Preparation of 3â. A solution of the enol acetate isomers (100
mg, 0.514 mmol) in acetone (0.5 mL) was added to a mixture
containing OsO4 (10 mmol, added as a 2.5% solution in tert-
butyl alcohol) and N-methylmorpholine N-oxide monohydrate
(73 mg, 0.540 mmol) in water (1 mL) and acetone (2.2 mL) at
0 °C. The reaction mixture was stirred for 3 h at 0 °C and an
additional 6 h at room temperature. NaHSO3 (10 mg) was
added, the pH was adjusted to 7 with 1N H2SO4, and the
solvents were evaporated under vacuum. The residue was
dissolved in ethyl acetate (20 mL) and washed with water (20
mL) and saturated NaCl (20 mL); then the organic layer was
dried over anhydrous MgSO4, filtered, and concentrated. The
residue was chromatographed using 10-15% ethyl acetate in
hexane to obtain 3â as an oil (54 mg, 0.32 mmol, 62%). IR ν
3472, 2924, and 1751.
Hydroxythujones and dehydrothujones were synthesized as
described below and characterized by 1H NMR (Table 1),13
NMR (Table 2) and MS (Table 3).
C
P r epar ation of 2-Hydr oxyth u jon es (2r an d 2â) (Sch em e
1). Preparation of 2R. A 25-mL round-bottomed flask was
charged with THF (2 mL) and cooled to -78 °C. A solution of
n-butyllithium (1.6 M, 2.0 mL, 3.2 mmol) in hexane was added
followed by diisopropylamine (460 µL, 3.28 mmol). The reaction
mixture was stirred for 1 min and R-thujone (510 mg, 3.35
mmol) was added in THF (8 mL) over 0.5 h. The mixture was
stirred for 15 min at -78 °C, warmed to -25 °C, MoOPH (2.13
g, 5.09 mmol) was added as a solid over 1-2 min with
continued stirring at the same temperature for 0.5 h, and
finally quenched on addition of saturated aqueous NaHSO3
(10 mL). After the mixture was warmed to room temperature,

1918 J. Agric. Food Chem., Vol. 49, No. 4, 2001
Sirisoma et al.
Ta ble 3. GC tR Va lu es a n d P ostu la ted MS F r a gm en ta tion P a tter n s for Th u jon es, Hyd r oxyth u jon es, a n d
Deh yd r oth u jon es a n d Th eir Tr im eth ylsilyl a n d Meth yloxim e Der iva tives
m/z values of the major protonated molecular ions or fragments
compound
tR (min)
and their relative abundance
Nonderivatizeda
[MH]+
[MH-H2O]+
135(100)
135(100)
133(43)
[MH-H2O-CO]+
[MH-H2O-C3H6]+
93(57)
93(45)
93(21)
93(26)
109(12)
109(87); 95(43)
109(59)
109(5)
109(14)
other
1R
1â
5R
5â
6
6.3
6.4
6.6
7.0
153(25)
153(10)
151(42)
151(42)
151(100)
169(3)
169(5)
169(4)
169(27)
123(4)
109(40);123(100)
109(61);123(100)
123(19)
133(66)
7.3
3R
3â
2R
2â
4R
4â
8.0
8.2
151(100)
151(100)
151(100)
151(100)
151(100)
151(100)
123(33)
123(24)
123(21)
123(58)
123(64)
123(62)
95(27)
8.2 (9.6)b
9.8 (8.4)
9.9
109(12)
109(23)
95(3);168(4)
168(3)
10.1
TMS derivativesa
[MH]+
241(15)
241(13)
241(6)
241(8)
241(2)
241(35)
[MH-CH4]+
225(52)
225(57)
225(81)
225(68)
[MH-TMSOH]+
151(100)
151(100)
151(100)
151(100)
151(100)
151(50)
other
3â
2â
2R
3R
4R
4â
11.0
11.2
11.3
12.1
12.9
13.1
109(38); 123(20)
123(23)
123(8)
109(57); 123(31)
109(6); 123(21)
123(22)
225(12)
225(100)
MOX derivatives
[MH]+
[MH-OCH3]+
150(33)
150(32)
148(53)
148(74)
148(31)
166(61)
166(54)
166(36)
166(64)
166(31)
[MH-H2O]+
[MH-H2O-OCH3]+
other
96(83)
96(100)
1R
1â
5R
5â
6
3R
2R
3â
2â
4R
8.3
8.7
182(100)
182(65)
180(100)
180(100)
180(100)
8.8 (8.4)b
9.4 (9.6)
9.8 (9.7)
10.8 (11.0)
10.9 (11.1)
11.0 (10.9)
12.0 (11.6)
12.4
107(100)
107(46)
71(64);124(37)
180(100)
180(100)
180(100)
180(100)
180(100)
148(28)
148(45)
148(26)
148(32)
148(37)
198(15)
71(36);124(36)
138(100)
112(17); 138(12);
150(43); 108(20)
150(13)
198(26)
198(35)
4â
12.5
198(18)
166(28)
180 (100)
148(31)
a
b
The corresponding data for thujol and neothujol under the identical conditions are given in the Supporting Information. Minor
peaks due to thermal decomposition of 2R and 2â and syn and anti geometrical isomers of MOX derivatives.
Sch em e 1a
a
(a) lithium diisopropylamide, -78 °C, THF; (b) MoOPH, THF.
P r ep a r a tion of 7-Hyd r oxyth u jon es (4r a n d 4â) a n d 7,8-
Deh yd r oth u jon es (5r a n d 5â) (Sch em e 3). Preparation of
4R and 4â. A solution of R-thujone (500 mg, 3.29 mmol) in dry
ethyl acetate (50 mL) was cooled to -25 °C. A steady stream
of ozone was passed though the solution for 6 h followed by a
stream of dry nitrogen for 30 min. The reaction mixture was
then warmed to room temperature and washed with water (50
mL) and saturated NaCl (20 mL). The organic layer was dried
over Na2SO4, filtered, and concentrated. The residue was
chromatographed using 20-25% ethyl acetate in hexane to
obtain unreacted R-thujone (108 mg, 0.71 mmol) and 4R (242
mg, 1.45 mmol, 44%). IR ν 3502, 2972, and 1733. 4R prepared
by this procedure was previously reported by Kutney and Chen
(17). The same method was used to convert â-thujone to 4â
(48%). IR ν 3608, 2981, and 1728.
apparatus. The reaction mixture was cooled to room temper-
ature, diluted with hexane (20 mL), and washed with water
followed by saturated NaHCO3 (20 mL each). The organic layer
was dried over Na2SO4, filtered, and chromatographed in
3-20% ethyl acetate in hexane to obtain 5R as an oil (34 mg,
0.226 mmol, 34%). The same procedure was used to convert
4â to 5â.
P r ep a r a tion of 4,10-d eh yd r oth u jon e (6) (Sch em e 4).
Sabinene (1 g) was dissolved in CH2Cl2 (3 mL), and SeO2 (6
mg, 0.054 mmol) was added followed by tert-butyl hydroper-
oxide (3 mL, 90% pure). The reaction mixture was stirred
overnight at room temperature, benzene (10 mL) was added
and most of the solvents were evaporated under reduced
pressure. Ether (20 mL) was added and the organic phase was
washed with 10% KOH (3 × 20 mL) and saturated NaCl (20
mL), dried over MgSO4, filtered, and concentrated. The oily
residue was chromatographed using 1-6% ethyl acetate in
hexane to obtain 622 mg impure sabinol. This material in CH2-
Preparation of 5R and 5â. A mixture of 4R (114 mg, 0.67
mmol) and pyridinium tosylate (28 mg, 0.11 mmol) in dry
benzene (14 mL) was refluxed for 2.5 h using a Dean-Stark

Thujone Metabolites: Synthesis and Analysis
J. Agric. Food Chem., Vol. 49, No. 4, 2001 1919
Sch em e 2a
a
(a) isopropenyl acetate, anhydrous p-toluenesulfonic acid, 110 °C; 2,3-enol acetate is a minor product; (b) mCPBA, CHCl3; (c)
NaOMe, MeOH; (d) OsO4, N-methylmorpholine N-oxide.
Sch em e 3a
totron with 1% ethyl acetate in hexane gave 320 mg of 6. IR
ν 2959, 1732, and 1637.
RESULTS AND DISCUSSION
Syn th esis a n d Ch a r a cter iza tion . The metabolites
of R-thujone and â-thujone have GC/MS features, di-
rectly and on derivatization, suggesting that in each
case they consist of a series of hydroxythujones and
dehydrothujones (12). Candidate metabolites were there-
fore synthesized for use as standards (Schemes 1-4).
a
(a) O3, -25 °C, EtOAc; (b) pyridinium tosylate or p-
toluenesulfonic acid.
2-H yd r oxyt h u jon es (2r a n d 2â): Syn t h esis
(Sch em e 1) a n d Ster eoch em ica l Assign m en t. The
lithium 2,3-enolates were prepared in situ from R-thu-
jone and â-thujone on treatment with lithium diisopro-
pylamide at -78 °C. R-Hydroxylation of the enolate with
MoOPH13 gave a single isomeric product in both cases
in good yield (58-61%). 1H NMR indicated retention of
configuration because H-4 in 2R is coupled only to the
C-10 methyl group but not to H-5, i.e., H-4 and H-5 are
orthogonal to each other. This is in contrast to 2â where
coupling between H-4 and H-5 gives rise to a multiplet
Sch em e 4a
a
(a) tert-butyl hydroperoxide, SeO2, CH2Cl2; (b) MnO2,
1
for H-4 (δ 2.96). H and 13C NMR established loss of
CH2Cl2.
one C-2 proton and long-range coupling (4J 2R-6R ) 2 Hz)
between H-2R and H-6R characteristic of planar zigzag
orientation consistent with the 2R and 2â assignments.
The stereochemistry of 2R at C-2 was firmly assigned
by X-ray crystallography of the 2,4-dinitrophenylhydra-
zone derivative (Figure 2). The unit cell contained four
molecules of compound and one of water. Thus, 2-hy-
droxythujones from R-thujone and â-thujone are as-
Cl2 (16 mL) was added to a suspension of MnO2 (4.5 g, 51.8
mmol) in CH2Cl2 (20 mL). The reaction was complete after 0.5
h based on TLC on silica gel. The reaction mixture was diluted
with CH2Cl2 (50 mL) and filtered though a pad of Celite. The
filtrate was concentrated and chromatographed using 1-4%
ethyl acetate in hexane to afford 610 mg of product as a
mixture. Further purification on silica gel using a Chroma-

1920 J. Agric. Food Chem., Vol. 49, No. 4, 2001
Sirisoma et al.
hydroxy substituent is at C-7 and the double bond is at
the 7,8-position.
4,10-Deh yd r ot h u jon e (Sa bin on e, 6): Syn t h esis
(Sch em e 4) a n d Ch a r a cter iza tion . Sabinene was
oxidized to the allylic alcohol (sabinol) with tert-butyl
hydroperoxide and SeO2 (utilizing the general method
of Umbreit and Sharpless, 23) and then to 6 with MnO2.
Klinck et al. (24) used SeO2 to convert sabinol to
sabinone. The double bond is at the 4,10-position based
1
on H and 13C NMR, and UV absorbance on TLC.
GC/MS An a lysis of Th u jon es, Hyd r oxyth u jon es,
a n d Deh yd r oth u jon es. Standard analytical methods
and derivatization of alcohol and ketone functionalities
(Scheme 5) to TMS and MOX derivatives, respectively,
were applied to R-thujone and â-thujone and their
hydroxy and dehydro derivatives. Retention time (tR)
values and MS data are given in Table 3 and the
fragmentation patterns are illustrated in the Supporting
Information using 4R as an example. With the non-
derivatized hydroxy compounds the protonated molec-
ular ion (m/z 169) is very small or absent because of
the loss of H2O, and characteristic ions are [MH-H2O]+
m/z 151, [MH-H2O-CO]+ m/z 123, and [MH-H2O-
C3H6]+ m/z 109. The fragmentation patterns of 3R and
3â show higher relative abundance of m/z 109 than do
2R, 2â, 4R, or 4â, suggesting a facile loss of H2O and
C3H6. This difference is useful in recognizing individual
metabolites in the poorly resolved 3R plus 2R peak (12).
The [MH-CO]+ m/z 123 fragment is characteristic for
the dehydrothujones.
GC/MS An a lysis of TMS a n d MOX Der iva tives
of Th u jon es, Hyd r oxyth u jon es, a n d Deh yd r oth u -
jon es. With the hydroxythujone TMS derivatives, the
protonated molecular ion m/z 241 is still small, and
[MH-CH4]+ m/z 225 and [MH-TMSOH]+ m/z 151 are
common fragments. In the MOX derivatives the proto-
nated molecular ion for R-thujone and â-thujone is m/z
182 versus m/z 180 for the dehydrothujones and [MH-
OCH3]+ is m/z 150 and 148, respectively. With the
hydroxythujone MOX derivatives the protonated mo-
lecular ion m/z 198 is more stabilized and [MH-H2O]+
m/z 180, [MH-OCH3-H]+ m/z 166, [MH-OCH3-OH]+
m/z 150, and [MH-OCH3-OH-C3H6]+ m/z 108 are the
most common fragments.
F igu r e 2. Computer-generated perspective drawing of 2R as
the 2,4-dinitrophenylhydrazone derivative based on X-ray
crystal analysis. Crystallographic data are available as deposi-
tion number 153724 at the Cambridge Crystallographic Data
Centre. Copies of the data can be obtained free of charge from
CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax (+44)
1223-336-033; e-mail deposit@ccdc.cam.ac.uk.
signed as (2R)-2-hydroxy-R-thujone (2R) and (2R)-2-
hydroxy-â-thujone (2â), respectively.
4-H yd r oxyt h u jon es (3r a n d 3â): Syn t h esis
(Sch em e 2) a n d St er eoch em ica l Assign m en t . 3R
and 3â were prepared from the 3,4-enol acetate, ob-
tained in high yield (93%) as a 9:1 mixture with the 2,3-
enol acetate on refluxing R-thujone in isopropenyl
acetate with p-toluenesulfonic acid using the general
procedure of House and Thompson (18). Two different
oxidants were used to prepare the final target com-
pounds. In the first procedure the enol acetate was
treated with mCPBA to form the 3,4-epoxy acetate in
situ which quickly undergoes intramolecular rearrange-
ment with C4-O bond dissociation to generate 4-ace-
toxy-R-thujone with inversion of configuration (analo-
gous cases have been reported, 19-21). The alternative
pathway involving C3-O bond dissociation to generate
4-acetoxy-â-thujone and ultimately 3â was not detected.
4-Acetoxy-R-thujone was treated with 0.5 equiv of
sodium methoxide to obtain 3R in 90% yield. In the
second approach the 3,4-enol acetate was oxidized with
OsO4 using a general method described by McCormick
et al. (22) to obtain 3â in 62% yield. Product stereo-
chemistry was assigned by two-dimensional nuclear
Overhauser effect spectroscopy (NOESY) which indi-
cated that the C-10 methyl group and H-6â are closer
in space in the product from OsO4 oxidation (3â) than
in the one from mCPBA oxidation (3R) (see Supporting
Information).
In conclusion, all of the major metabolites (Figure 1)
(3, 12) have been synthesized as reported here (Schemes
1-5). Procedures to analyze them as metabolites are
provided which differentiate each of the isomeric thu-
jones, dehydrothujones, and hydroxythujones. Many of
them have been tested for toxicity to insects and as
blockers of the GABA-gated chloride channel with
activities less than those of R-thujone and â-thujone,
i.e., the metabolites are detoxification products (12).
The methods of synthesis, derivatization, and analysis
developed here are applicable to other monoter-
7-Hyd r oxyth u jon es (4r a n d 4â) a n d 7,8-Deh y-
d r oth u jon es (5r a n d 5â): Syn th esis (Sch em e 3)
a n d Ch a r a cter iza tion . Ozonation of R-thujone (17)
and â-thujone provided 4R and 4â, respectively in 44-
48% yield. Treatment of these tertiary alcohols with
pyridinium tosylate or p-toluenesulfonic acid afforded
the corresponding dehydro derivatives (34% yield for
1
5R). H and 13C NMR established that the introduced
Sch em e 5a
a
(a) MSTFA; (b) MeONH2, pyridine.

Thujone Metabolites: Synthesis and Analysis
J. Agric. Food Chem., Vol. 49, No. 4, 2001 1921
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ABBREVIATIONS USED
mCPBA, m-choroperbenzoic acid; MoOPH, oxodiper-
oxymolybdenum(pyridine)(hexamethylphosphoric tria-
mide); MOX, methyloxime reagent or derivative; MST-
FA,
N-methyl-N-(trimethylsilyl)trifluoroacetamide;
NOESY, nuclear Overhauser effect spectroscopy; TMS,
trimethylsilyl substituent or derivative.
ACKNOWLEDGMENT
We thank our laboratory colleagues Shinzo Kagabu,
David Lee, Gary Quistad, Yoffi Segall, and Nanjing
Zhang for helpful suggestions. Crystallography was
done by Marilyn Olmstead and Håkon Hope (Depart-
ment of Chemistry, University of California, Davis), and
1H NMR NOESY experiments were conducted by Corey
Liu and Ted Barnum (Department of Chemistry, Uni-
versity of California, Berkeley).
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two-dimensional NOESY of 3â. Figure S2 giving mass spectra
of 4R and its TMS and MOX derivatives using positive
chemical ionization and postulated fragmentation pathways.
Table S1 giving GC tr values and proposed MS fragmentation
patterns for thujol and neothujol and their TMS derivatives.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Received for review December 4, 2000. Revised manuscript
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