Monoterpene hydroperoxides with trypanocidal activity from Chenopodium ambrosioides

Article abstract of DOI:10.1021/np010445g

Four monoterpene hydroperoxides were isolated from aerial parts of Chenopodium ambrosioides along with ascaridole (1), the anthelmintic principle of this plant, as anti-trypanosomal compounds. The structures of these monoterpenes were determined to be (-)

Full text of DOI:10.1021/np010445g

J . Nat. Prod. 2002, 65, 509-512
509
Mon oter p en e Hyd r op er oxid es w ith Tr yp a n ocid a l Activity fr om Ch en opod iu m
a m br osioid es
Fumiyuki Kiuchi,*,† Yoshiaki Itano,† Nahoko Uchiyama,† Gisho Honda,† Akiko Tsubouchi,‡
J unko Nakajima-Shimada,‡ and Takashi Aoki‡
Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida, Sakyo-ku, Kyoto, 606-8501, J apan, and
School of Medicine, J untendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, J apan
Received September 12, 2001
Four monoterpene hydroperoxides were isolated from aerial parts of Chenopodium ambrosioides along
with ascaridole (1), the anthelmintic principle of this plant, as anti-trypanosomal compounds. The
structures of these monoterpenes were determined to be (-)-(2S,4S)- and (-)-(2R,4S)-p-mentha-1(7),8-
dien-2-hydroperoxide (2a and 3a ) and (-)-(1R,4S)- and (-)-(1S,4S)-p-mentha-2,8-dien-1-hydroperoxide
(4a and 5a ) on the basis of spectroscopic methods and chemical correlations. In vitro trypanocidal activities
of ascaridole (1) and these hydroperoxides (2a -5a ) against epimastigotes of Trypanosoma cruzi were 23,
1.2, 1.6, 3.1, and 0.8 µΜ, respectively. Fresh leaves of C. ambrosioides also contained isomeric
hydroperoxides 6a and 7a , and the content ratio of 2a -7a suggested that these hydroperoxides were
formed through the singlet-oxygen oxidation of limonene.
Chenopodium ambrosioides L. var. antherminticum A.
Gray (Chenopodiaceae) is used to produce chenopodium oil,
which has been used as an anthelmintic for the treatment
of intestinal worms.1 In our search for anti-trypanosomal
compounds from natural sources, an AcOEt extract of
aerial parts of this plant showed potent trypanocidal
activity against the epimastigotes of Trypanosoma cruzi,
the etiologic agent of American trypanosomiasis (Chaga’s
disease).2 In this paper, we report the isolation and
characterization of four p-menthane-type monoterpene
hydroperoxides (2a -5a ) together with ascaridole (1), the
anthelmintic principle of chenopodium oil, as the trypano-
cidal constituents of this plant.
and 5.01 (each 1H, d, J ) 2 Hz) in 3a . Compounds 4a and
5a also showed similar spectra, with signals of an exocyclic
methylene and a cis-double bond: δ 4.74 and 4.76 (each
1H, br s), δ 5.62 (1H, br d, J ) 9.9 Hz), δ 5.82 (1H, dd, J )
9.9, 2.6 Hz) in 4a ; δ 4.68 and 4.79 (each 1H, br s), δ 5.70
(1H, dd, J ) 10.3, 2.0 Hz), δ 5.82 (1H, dd, J ) 10.3, 3.4
Hz) in 5a . Detailed analyses of their two-dimensional NMR
spectra (HMQC, HMBC, COSY) revealed that 2a and 3a
are epimers of p-mentha-1(7),8-dien-2-hydroperoxide at the
2-position. In compound 2a , H-2 (δ 4.45, d with fine
splittings, J ) 11.6 Hz) and H-4 (δ 2.21, 1H, tt, J ) 12.2,
3.2 Hz) are oriented axially, whereas in 3a , H-2 (δ 4.51, t,
J ) 2.9 Hz) and H-4 (δ 2.35, 1H, tt, J ) 12.9, 3.2 Hz) are
in equatorial and axial positions, respectively. Thus, the
structures of 2a and 3a were assigned as indicated.
Similarly, 4a and 5a were considered to be epimers of
p-mentha-2,8-dien-1-hydroperoxide at the 1-position. The
stereochemistry of these compounds was determined to be
1,4-cis for 4a and 1,4-trans for 5a , as described below.
These compounds have been reported as products of the
singlet-oxygen oxidation of limonene,3,4 and the negative
optical rotation values of these compounds indicated that
they have the same absolute configuration at the 4-position
as l-limonene.5 While these oxidation products of limonene
have been analyzed by GC-MS3 and characterized by IR
Resu lts a n d Discu ssion
Fresh aerial parts of C. ambrosioides were extracted
overnight with AcOEt at room temperature. The diethyl
ether-soluble fraction of this extract, which showed strong
in vitro trypanocidal activity, was separated by silica gel
column chromatography to give ascaridole (1), the anthel-
mintic principle of this plant, as a major constituent.
Although 1 showed strong trypanocidal activity, more
potent activity was found in compounds that appeared just
below the spot of 1 and gave a bright bluish-green color
with p-anisaldehyde/sulfuric acid reagent on TLC. These
compounds were separated by repeated column chroma-
tography to give 2a -5a .
Compounds 2a -5a were monoterpenes with two double
bonds and one oxygen function based on their 13C NMR
spectra. The chemical shifts (δ 79.4-85.5) of the oxygen-
bound carbon of these compounds suggested that the
oxygen function is a hydroperoxy group, which is consistent
with their molecular formula (C10H16O2 based on high-
resolution chemical ionization mass spectrum (HRCIMS))
and their relative instability. Compounds 2a and 3a
showed similar NMR spectra, with signals of two exocyclic
methylenes: δ 4.72 (2H, br s), δ 4.84 and 4.93 (each 1H, d,
J ) 1.5 Hz) in 2a ; δ 4.68 and 4.70 (each 1H, br s), δ 5.00
1
and 60 MHz H NMR,4 they have not been fully character-
ized by high-field NMR spectroscopy. Thus, the structures
of 2a -5a were confirmed by chemical means. Compounds
2a -5a were treated with PPh3 to give the corresponding
alcohols 2b-5b. The negative signs of the optical rotations
of these alcohols confirmed that the absolute configuration
at the 4-position was the same as that in l-limonene.5 The
13C NMR data of the alcohol obtained from 2a were
consistent with those of the known alcohol 2b,6 which has
a cis relationship between the hydroxy and isopropenyl
groups as assigned above. The alcohols derived from 4a
and 5a were further hydrogenated to give tetrahydro
derivatives 8 and 9, respectively. The 13C NMR spectra of
8 and 9 were in good agreement with those of 1,4-cis and
1,4-trans alcohols, respectively,6 which established the
stereochemistries of the alcohols 4b and 5b, and hence
those of the hydroperoxides 4a and 5a , as indicated in their
* To whom correspondence should be addressed. Tel: +81-75-753-4534.
Fax: +81-75-753-4591. E-mail: fkiuchi@pharm.kyoto-u.ac.jp.
† Kyoto University.
‡ J untendo University.
10.1021/np010445g CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/15/2002

510 J ournal of Natural Products, 2002, Vol. 65, No. 4
Kiuchi et al.
Ta ble 1. Anti-trypanosomal Activity of Compounds 1 and
3a -5a against T. cruzi in the HeLa Infection Assaya
structures. On the basis of these results, the structures of
the hydroperoxides isolated from C. ambrosioides were
concluded to be (-)-(2S,4S)-p-mentha-1(7),8-dien-2-hydro-
peroxide (2a ), (-)-(2R,4S)-p-mentha-1(7),8-dien-2-hydro-
peroxide (3a ), (-)-(1R,4S)-p-mentha-2,8-dien-1-hydroper-
oxide (4a ), and (-)-(1S,4S)-p-mentha-2,8-dien-1-hydro-
peroxide (5a ). Many hydroperoxides have been reported
from various plant sources,7 including trans-pinocarveyl-
hydroperoxide from C. ambrosioides.8 Although compounds
4a and 5a (absolute stereochemistry not reported) have
been reported from Citrus iyo,9 this is the first report of
isolation and full characterization of these p-menthane
hydroperoxides from natural sources.
number of amastigotes/
compound
infection rate (%)
infected cell
controlb
1
3a
4a
12.0 ( 0.9
8.5 ( 2.5
18.8 ( 3.8
22.8 ( 1.1
18.6 ( 6.0
19.1 ( 5.1
12.0 ( 4.8
10.9 ( 3.5*
4.4 ( 0.3*
1.5 ( 1.3
5a
0.13 ( 0.11*
7.0 ( 2.3*
allopurinol
a
The mean values ( SE (* p < 0.05 compared with control) at
b
1 µg/mL. Ethanol (10 µL).
The hydroperoxides 2a -5a showed stronger trypanocidal
activity than ascaridole (1). The minimum lethal concen-
trations (MLCs) of ascaridole (1) and hydroperoxides 2a -
5a against epimastigotes of Trypanosoma cruzi were 23,
1.2, 1.6, 3.1, and 0.8 µΜ, respectively. A few monoterpe-
noids, espintanol,12 piquerol A,13 and terpinen-4-ol,14 all of
which have OH function, have been reported to show anti-
trypanosomal activity. However, the alcohols 2b-5b, cor-
responding to the hydroperoxides 2a -5a , did not show
trypanocidal activity even at 400 µM, indicating that
hydroperoxy group is essential for the activity of this type
of compounds.
To evaluate the effect on the blood stream forms of the
parasite, the anti-trypanosomal effect of the hydroperoxides
3a -5a was also tested with the HeLa cell infection assay.15
Under this assay condition, these compounds were toxic
to HeLa cells at 10 µg/mL. At 1 µg/mL, 5a almost
completely inhibited the infection of HeLa cells by the
trypomastigotes, and 3a and 4a inhibited the infection by
63% and 88%, respectively (Table 1). However, they did
not inhibit the proliferation of amastigotes in infected cells.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were determined on a J ASCO DIP-370 polarimeter. 1H and
13C NMR spectra were measured on a J EOL J NM-LA500
spectrometer with tetramethylsilane as an internal standard,
and chemical shifts are given as δ values. Mass spectra were
measured on a J EOL J MS-HX/HX110A spectrometer. GC was
performed on a Hitachi G-5000 system equipped with an FID
detector. l-Limonene was a generous gift from Takasago
International Co. (-)-Carveol (a mixture of epimers 6b and
7b) was purchased from Kanto Chemical Co.
Extr a ction a n d Isola tion . The seeds of C. ambrosioides
L. var. antherminticum A. Gray were provided by the Kyoto
Medicinal Plant Garden of Takeda Chemical Industries Ltd.
The plants were cultivated, and a voucher specimen (No. 4212)
was deposited at the Experimental Station of Medicinal Plants,
Faculty of Pharmaceutical Sciences, Kyoto University. Fresh
aerial parts including immature seeds (10 kg) were extracted
with AcOEt at room temperature overnight, and the extract
was concentrated to dryness under reduced pressure. The
residue was suspended in water and extracted with diethyl
ether. The extract was dried over anhydrous sodium sulfate
and concentrated to dryness to give an ether-soluble fraction
(108 g). Some of this extract (21.0 g) was fractionated by silica
gel column chromatography (hexane-AcOEt, 20:1) to give
three fractions: fraction 1 (1.46 g), fraction 2 (4.93 g), and
fraction 3 (2.97 g), and the rest was eluted with MeOH to give
fraction 4. Minimum lethal concentrations (MLCs) of these
fractions were 12.5, 1.56, 0.1, and >6.25 µg/mL, respectively.
Some of fraction 2 (1.31 g) was purified by silica gel column
chromatography (hexane-AcOEt, 10:1) to give ascaridole (1,
1.13 g). Repeated column chromatography of fraction 3 on silica
gel (benzene-AcOEt, 98:2, CHCl3-AcOEt, 100:3) and a Lobar
column (LiChroprep Si-60B, Merck, benzene-AcOEt, 98:2)
gave compounds 2a (64 mg), 3a (151 mg), 4a (85 mg), and 5a
(67 mg).
It has been reported that the singlet-oxygen oxidation
of limonene produces six isomeric hydroperoxides, 2a -
7a .3,5,10 Therefore, we checked for the presence of the
isomeric hydroperoxides 6a and 7a in C. ambrosioides. The
peroxide fraction of the AcOEt extract was treated with
PPh3 to convert the hydroperoxides to alcohols and ana-
lyzed by GC. All of the six isomers were present: 2b
(23.8%), 3b (21.7%), 4b (35.7%), 5b (7.5%), 6b (3.1%), and
7b (8.2%). This ratio is very similar to that reported for
the products of the singlet-oxygen oxidation of limonene,3,10
which suggests that these hydroperoxides are formed by
the singlet-oxygen oxidation of l-limonene in C. ambrosio-
ides.
Pare´ et al. reported the isolation of the alcohol 2b as an
antifungal compound6 and Ahmed isolated several p-
menthane alcohols11 from C. ambrosioides. However, when
the fresh plants were extracted with AcOEt, the extract
did not contain any 2b-7b by TLC. However, when the
extract was heated, these alcohols appeared and their
amounts increased, accompanied by a decrease in the
amount of the hydroperoxides. Degradation of the hydro-
peroxides was evident when the fresh aerial part was
steam-distilled. The essential oil fraction obtained by
steam-distillation contained ascaridole as the major com-
ponent and a considerable amount of the alcohols. How-
ever, the hydroperoxides were not detectable by TLC.
Therefore, the p-menthane alcohols isolated from this plant
might be degradation products of the hydroperoxides 2a -
7a and ascaridole.

Monoterpene Hydroperoxides from Chenopodium
J ournal of Natural Products, 2002, Vol. 65, No. 4 511
1
Asca r id ole (1): pale yellow oil; [R]25 0° (c 3.0, CHCl3); H
triphenylphosphine as above to give the corresponding alcohols
3b (7.0 mg), 4b (11 mg), and 5b (28 mg), respectively.
(-)-(2S,4S)-p-Men th a -1(7),8-d ien -2-ol (2b): colorless oil;
[R]25D -16.4° (c 0.6, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.24
(2H, overlapped, H-3, 5), 1.65 (1H, br s, OH), 1.72 (3H, s, H-10),
1.83 (1H, d with fine splittings, J ) 12.5 Hz, H-5), 2.06 (1H, t
with fine splittings, J ) 13.5 Hz, H-6), 2.18 (2H, overlapped,
H-3, 4), 2.43 (1H, dt, J ) 13.5, 3.2 Hz, H-6), 4.45 (1H, d with
fine splittings, J ) 11.6 Hz, H-2), 4.71 (2H, br s, H-9), 4.79
(1H, d, J ) 1.5 Hz, H-7), 4.95 (1H, d, J ) 1.5 Hz, H-7); 13C
NMR (CDCl3, 125 MHz) δ 20.8 (q, C-10), 32.7 (t, C-5), 33.8 (t,
C-6), 42.1 (t, C-3), 44.1 (d, C-4), 72.2 (d, C-2), 103.9 (t, C-7),
109.1 (t, C-9), 148.6 (s, C-8), 151.2 (s, C-1).
D
NMR (CDCl3, 500 MHz) δ 0.95 (3H, s), 0.98 (3H, s), 1.34 (3H,
s), 1.48 (2H, m), 1.89 (1H, m), 1.99 (2H, m), 6.37 (1H, d, J )
8.5 Hz), 6.46 (1H, d, J ) 8.5 Hz); 13C NMR (CDCl3, 125 MHz)
δ 17.1, 17.2, 21.3, 25.5, 29.4, 32.1, 74.3, 79.7, 133.0, 136.3.
(-)-(2S,4S)-p-Men th a -1(7),8-d ien -2-h yd r op er oxid e (2a ):
colorless oil; [R]20D -18.7° (c 4.7, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 1.27 (1H, qd, J ) 12.8, 4 Hz, H-5), 1.30 (1H, q, J )
11.9 Hz, H-3), 1.72 (3H, s, H-10), 1.82 (1H, d with fine
splittings, J ) 12.8 Hz, H-5), 2.08 (1H, t with fine splittings,
J ) 13.4 Hz, H-6), 2.21 (1H, tt, J ) 12.2, 3.2 Hz, H-4), 2.27
(1H, d with fine splittings, J ) 11.6 Hz, H-3), 2.43 (1H, ddd,
J ) 13.7, 4, 2.8 Hz, H-6), 4.45 (1H, d with fine splittings, J )
11.6 Hz, H-2), 4.72 (2H, br s, H-9), 4.84 (1H, d, J ) 1.5 Hz,
H-7), 4.93 (1H, d, J ) 1.5 Hz, H-7), 8.10 (1H, s, OOH); 13C
NMR (CDCl3, 125 MHz) δ 20.7 (q, C-10), 32.7 (t, C-5), 34.1 (t,
C-6), 37.0 (t, C-3), 43.9 (d, C-4), 85.2 (d, C-2), 105.3 (t, C-7),
109.4 (t, C-9), 146.7 (s, C-1), 148.4 (s, C-8); CIMS (isobutane)
m/z 169 [M + H+] (51), 151 (100), 135 (89); HRCIMS m/z
169.1219 (calcd for C10H17O2, 169.1228).
(-)-(2R,4S)-p-Men th a -1(7),8-d ien -2-ol (3b): colorless oil;
[R]25D -60.5° (c 0.7, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.30
(1H, qd, J ) 12.4, 4 Hz, H-5), 1.48 (1H, br s, OH), 1.53 (1H,
ddd, J ) 13.7, 12.2, 3.1 Hz, H-3), 1.73 (3H, s, H-10), 1.85 (1H,
d with fine splittings, J ) 12.4 Hz, H-5), 1.99 (1H, dq, J )
13.7, 3.1 Hz, H-3), 2.21 (1H, dt, J ) 13.7, 3.5 Hz, H-6), 2.48
(1H, t with fine splittings, J ) 13.4 Hz, H-6), 2.54 (1H, tt, J )
12.2, 3.2 Hz, H-4), 4.37 (1H, br s, H-2), 4.71 (2H, br s, H-9),
(-)-(2R,4S)-p-Men th a -1(7),8-d ien -2-h yd r oper oxid e (3a ):
colorless oil; [R]20D -78.1° (c 2.5, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 1.28 (1H, qd, J ) 13, 4.1 Hz, H-5), 1.54 (1H, td, J )
13.6, 3.3 Hz, H-3), 1.70 (3H, s, H-10), 1.86 (1H, d with fine
splittings, J ) 11.9 Hz, H-5), 2.16 (1H, dq, J ) 14.3, 3 Hz,
H-3), 2.29 (1H, dt, J ) 13.4, 3.2 Hz, H-6), 2.35 (1H, tt, J )
12.9, 3.2 Hz, H-4), 2.38 (1H, t with fine splittings, J ) 13.4
Hz, H-6), 4.51 (1H, t, J ) 2.9 Hz, H-2), 4.68 and 4.70 (each
1H, br s, H-9), 5.00 and 5.01 (each 1H, br s, H-7), 7.64 (1H, br
s, OOH); 13C NMR (CDCl3, 125 MHz) δ 20.7 (q, C-10), 30.4 (t,
C-6), 32.4 (t, C-5), 35.5 (t, C-3), 38.6 (d, C-4), 85.5 (d, C-2),
4.77 (1H, t, J ) 1.8 Hz, H-7), 4.86 (1H, t, J ) 1.5 Hz, H-7); 13
C
NMR (CDCl3, 125 MHz) δ 21.0 (q, C-10), 29.9 (t, C-6), 32.6 (t,
C-5), 38.1 (d, C-4), 39.0 (t, C-3), 72.4 (d, C-2), 108.9 (t, C-9),
109.9 (t, C-7), 149.4 (s, C-8), 149.8 (s, C-1).
(-)-(1R, 4S)-p-Men th a -2,8-d ien -1-ol (4b): colorless oil;
[R]D -68.1° (c 0.54, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.30
(3H, s, Me-7), 1.61 (2H, m, overlapped, H-5, H-6), 1.74 (3H, br
s, Me-10), 1.77 (1H, m, H-5), 1.85 (1H, m, H-6), 2.66 (1H, m,
H-4), 4.75 (1H, br s, H-9), 4.78 (1H, quint, J ) 1.5 Hz, H-9),
5.66 (1H, ddd, J ) 10.1, 2.2, 1.0 Hz, H-3), 5.71 (1H, ddd, J )
10.1, 2.2, 1.3 Hz, H-2); 13C NMR (CDCl3, 125 MHz) δ 20.9 (q,
C-10), 24.9 (t, C-5), 29.4 (q, C-7), 36.7 (t, C-6), 43.4 (d, C-4),
67.5 (s, C-1), 110.6 (t, C-9), 132.2 (d, C-3), 133.9 (d, C-2), 148.1
(s, C-8).
(-)-(1S, 4S)-p-Men th a -2,8-d ien -1-ol (5b): colorless oil; [R]D
-150.8° (c 1.07, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.29
(3H, s, Me-7), 1.56 (1H, m, H-5), 1.66 (1H, ddd, J ) 12.8, 9.5,
2.8 Hz, H-6), 1.74 (3H, br s, Me-10), 1.79 (1H, ddd, J ) 12.8,
8.9, 2.8 Hz, H-6), 1.90 (1H, m, H-5), 2.74 (1H, m, H-4), 4.67
(1H, br s, H-9), 4.78 (1H, quint, J ) 1.6 Hz, H-9), 5.61 (1H,
dd, J ) 10.1, 3.4 Hz, H-3), 5.70 (1H, dd, J ) 10.1, 2.1 Hz, H-2);
13C NMR (CDCl3, 125 MHz) δ 21.2 (q, C-10), 25.0 (t, C-5), 28.9
(q, C-7), 36.1 (t, C-6), 42.5 (d, C-4), 68.6 (s, C-1), 110.9 (t, C-9),
130.8 (d, C-3), 134.4 (d, C-2), 147.2 (s, C-8).
Hyd r ogen a tion of th e Alcoh ols 4b a n d 5b. The alcohols
4b and 5b were dissolved in AcOEt and hydrogenated in the
presence of 5% Pd-C at room temperature for 6 h to give the
corresponding saturated p-menthane alcohols 8 and 9, respec-
tively. 13C NMR in CDCl3: compound 8, δ 69.2 (C-1), 38.9 (C-
2,6), 25.0 (C-3,5), 43.4 (C-4), 31.3 (C-7), 32.6 (C-8), 19.9 (C-
9,10); compound 9, δ 71.1 (C-1), 40.3 (C-2,6), 27.2 (C-3,5), 43.4
(C-4), 25.6 (C-7), 32.3 (C-8), 20.0 (C-9,10).
GC An a lysis of th e Hyd r op er oxid e F r a ction of AcOEt
Extr a ct. Fresh leaves of C. ambrosioides were extracted with
AcOEt at room temperature for 3 h. The extract was dried
over anhydrous sodium sulfate and concentrated under re-
duced pressure. The residue was fractionated with a short
silica gel column with 4:1 hexane-AcOEt to give a peroxide
fraction. The peroxide fraction was dissolved in ether and
treated with PPh3 at room temperature for 5 min. The mixture
was concentrated and fractionated by a short silica gel column
(6:1 hexane-AcOEt then 3:1) to give the alcohol fraction, which
was analyzed by GC. The GC conditions were as follows:
column, TC-WAX (60 m × 0.25 mm, GL Sciences, J apan);
carrier gas, helium; injector temperature, 210 °C; temperature
program, 70 °C (4 min), 10 °C/min to 120 °C, 2 °C/min to
160 °C, 10 °C/min to 210 °C (10 min); detector temperature,
210 °C.
1
109.0 (t, C-9), 114.0 (t, C-7), 145.2 (s, C-1), 149.0 (s, C-8). H
NMR (C6D6, 500 MHz) δ 1.14 (1H, qd, J ) 13.0, 4.0 Hz, H-5),
1.33 (1H, td, J ) 13.6, 3.4 Hz, H-3), 1.56 (3H, s, H-10), 1.70
(1H, m, H-5), 2.03 (1H, dt, J ) 13.4, 3.1 Hz, H-6), 2.16 (1H,
ddd, J ) 14.0, 6.1, 3.1 Hz, H-3), 2.31 (1H, t with fine splittings,
J ) 13.7 Hz, H-6), 2.42 (1H, tt, J ) 12.5, 2.8 Hz, H-4), 4.32
(1H, t, J ) 2.7 Hz, H-2), 4.71 and 4.73 (each 1H, m, H-9), 4.81
and 4.85 (each 1H, t, J ) 2.1 Hz, H-7), 7.12 (1H, br s, OOH);
13C NMR (C6D6, 125 MHz) 20.8 (q, C-10), 30.7 (t, C-6), 32.8 (t,
C-5), 35.8 (t, C-3), 39.0 (d, C-4), 85.2 (d, C-2), 109.3 (t, C-9),
113.3 (t, C-7), 145.9 (s, C-1), 149.2 (s, C-8); CIMS (isobutane)
m/z 169 (M + H+, 17), 167 (38), 151 (100), 135 (47); HRCIMS
m/z 169.1224 [M + H+] (calcd for C10H17O2, 169.1228).
(-)-(1R,4S)-p-Men th a -2,8-d ien -1-h yd r op er oxid e (4a ):
colorless oil; [R]20D -49.6° (c 0.9, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 1.32 (3H, s, H-7), 1.43 (1H, m, H-6), 1.68 (2H, m, H-5),
1.71 (3H, s, H-10), 2.14 (1H, dt, J ) 13.8, 4.3 Hz, H-6), 2.66
(1H, br t, J ) 7.3 Hz, H-4), 4.74 (1H, br s, H-9), 4.76 (1H, br
s, H-9), 5.62 (1H, br d, J ) 9.9 Hz, H-2), 5.82 (1H, dd, J ) 9.9,
2.6 Hz, H-3), 7.29 (1H, br s, OOH); 13C NMR (CDCl3, 125 MHz)
δ 20.8 (q, C-10), 24.5 (t, C-5), 24.6 (t, C-7), 31.0 (t, C-6), 43.5
(d, C-4), 79.4 (s, C-1), 111.0 (t, C-9), 129.3 (d, C-2), 135.9 (d,
C-3), 147.7 (s, C-8); CIMS (isobutane) m/z 169 (M + H+, 23),
167 (43), 151 (51), 135 (100); HRCIMS m/z 169.1231 [M + H+]
(calcd for C10H17O2, 169.1228).
(-)-(1S,4S)-p-Men th a-2,8-dien -1-h ydr oper oxide (5a): col-
orless oil; [R]20 -164.2° (c 1.0, CHCl3); H NMR (CDCl3, 500
1
D
MHz) δ 1.32 (3H, s, H-7), 1.53 (1H, m, H-5), 1.63 (1H, m, H-6),
1.74 (3H, s, H-10), 1.94 (1H, m, H-5), 2.01 (1H, m, H-6), 2.77
(1H, m, H-4), 4.68 (1H, br s, H-9), 4.79 (1H, br s, H-9), 5.70
(1H, dd, J ) 10.3, 2.0 Hz, H-2), 5.82 (1H, dd, J ) 10.3, 3.4 Hz,
H-3), 7.35 (1H, br s, OOH); 13C NMR (CDCl3, 125 MHz) δ 21.2
(q, C-10), 24.3 (q, C-7), 24.8 (t, C-5), 30.0 (t, C-6), 42.6 (d, C-4),
80.8 (s, C-1), 111.0 (t, C-9), 130.0 (d, C-2), 134.6 (d, C-3), 147.2
(s, C-8); CIMS (isobutane) m/z 169 (M + H+, 13), 167 (14), 151
(26), 135 (100); HRCIMS m/z 169.1237 [M + H+] (calcd for
C
10H17O2, 169.1228).
Con ver sion of Hyd r op er oxid es to Alcoh ols. Compound
2a (7.5 mg) and triphenylphosphine (15.3 mg) were dissolved
in ether (1 mL) and stirred at room temperature for 1 h. The
solvent was removed under reduced pressure, and the residue
was purified by silica gel column chromatography to give the
corresponding alcohol 2b (6 mg). Each of the hydroperoxides
3a (8.3 mg), 4a (50 mg), and 5a (50 mg) was treated with
Tr yp a n ocid a l Assa y. Epimastigotes of T. cruzi (Tulahuen
strain) were kept in GIT medium (Wako) supplemented with
hemin (12.4 µM, Wako). The epimastigotes in GIT medium
(10 µL) were incubated with a test sample dissolved in EtOH
(5 µL) and autoclaved saline (185 µL). After 24 h of incubation,
the movement of epimastigotes was observed under a micro-

512 J ournal of Natural Products, 2002, Vol. 65, No. 4
Kiuchi et al.
Su p p or t in g In for m a t ion Ava ila b le: Gas chromatogram of
the alcohols prepared from the hydroperoxide fraction of the AcOEt
extract. This material is available free of charge via the Internet at
http://pubs.acs.org.
scope (× 100). Each assay was performed in duplicate. Gentian
violet, which is used to disinfect trypanosomes from transfu-
sion blood in Latin America, is used as a positive control. MLC
of gentian violet under this assay condition was 6.3 µM.
HeLa In fection Assa y. The HeLa cell infection assay was
performed as described previously.15 In brief, a round coverslip
was placed in each well of a 24-well plate. Exponentially
growing HeLa cells (5 × 103 cells/mL/well) were added to each
well, followed by incubation at 37 °C for 2 days in 5% CO2 in
air. The cells were then infected with T. cruzi trypomastigotes
(1.2 × 104 parasites/well). A compound to be tested was
dissolved in ethanol (10 µL) and added immediately after
infection. HeLa cells attached to the coverslip were fixed and
stained with Diff-Quik (Kokusai Shiyaku) at day 4 after
infection. The coverslip was then transferred upside down to
a slide glass, and the cells were finally embedded in HSR
solution (Kokusai Shiyaku) for observation under a microscope.
The percentage of infected host cells, i.e., those containing
more than one amastigote, and the mean number of amastig-
otes per infected cell were determined by analyzing more than
200 host cells distributed in randomly chosen microscopic
fields. Allopurinol was used as a positive control.
Refer en ces a n d Notes
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(11) Ahmed, A. A. J . Nat. Prod. 2000, 63, 989-991.
(12) Hocquemiller, T.; Cortes, D.; Arango, G. J .; Myint, S. H.; Cave´, A.;
Angelo, A.; Munzo, V.; Fournet, A. J . Nat Prod. 1991, 54, 450-452.
(13) Castro, C.; J imenez, M.; La Parra, M. G. Planta Med. 1992, 58, 281-
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Res. 1994, 4, 10-17.
Ack n ow led gm en t. We are grateful to the Kyoto Medicinal
Plant Garden of Takeda Chemical Industries Ltd. for providing
seeds of C. ambrosioides. We also thank Professor K. Tomioka
of the Graduate School of Pharmaceutical Sciences, Kyoto
University, for allowing us to use the NMR facilities and Dr.
N. Akimoto of the Faculty of Pharmaceutical Sciences, Kyoto
University, for MS measurements. This work was supported
in part by Grants-in-Aid for Scientific Research (Nos. 11672101
and 12557199) from the J apan Society for the Promotion of
Science.
NP010445G