Trypanocidal terpenoids from Laurus nobilis L.

Article abstract of DOI:10.1248/cpb.50.1514

Trypanocidal constituents of dried leaves of Laurus nobilis L. (Lauraceae) were examined. Activity-guided fractionation of the methanol extract resulted in the isolation of two guaianolides, dehydrocostus lactone (1) and zaluzanin D (2), and a new p-menthane hydroperoxide, (1R,4S)-1-hydroperoxy-p-menth-2-en-8-ol acetate (3). The minimum lethal concentrations of these compounds against epimastigotes of Trypanosoma cruzi were 6.3, 2.5, and 1.4 μM, respectively.

Full text of DOI:10.1248/cpb.50.1514

1514
Notes
Chem. Pharm. Bull. 50(11) 1514—1516 (2002)
Vol. 50, No. 11
Trypanocidal Terpenoids from Laurus nobilis L.
a
a
Nahoko UCHIYAMA, Keiji MATSUNAGA, Fumiyuki KIUCHI, ,a Gisho HONDA,a Akiko TSUBOUCHI,b
*
Junko NAKAJIMA-SHIMADA,b and Takashi AOKI
b
a Graduate School of Pharmaceutical Sciences, Kyoto University; 46–29 Yoshida, Sakyo-ku, Kyoto 606–8501, Japan: and
b School of Medicine, Juntendo University; 2–1–1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan.
Received July 4, 2002; accepted August 10, 2002
Trypanocidal constituents of dried leaves of Laurus nobilis L. (Lauraceae) were examined. Activity-guided
fractionation of the methanol extract resulted in the isolation of two guaianolides, dehydrocostus lactone (1) and
zaluzanin D (2), and a new p-menthane hydroperoxide, (1R,4S)-1-hydroperoxy-p-menth-2-en-8-ol acetate (3). The
minimum lethal concentrations of these compounds against epimastigotes of Trypanosoma cruzi were 6.3, 2.5,
and 1.4 mM, respectively.
Key words Laurus nobilis; Trypanosoma cruzi; p-menthane hydroperoxide; guaianolide; Lauraceae
Trypanosoma cruzi, a parasitic protozoan of Zoomastigo- showed that 3 was a monoterpene with two oxygen-bearing
phoria, is the etiologic agent of Chagas’ disease which is carbons (dC 78.6, 84.4) and an acetoxy group (dC 22.5,
endemic in Central and South America.1) In our screening 170.6). The 1H-NMR spectrum showed the presence of three
work on medicinal plants used in Asian counties for trypano- singlet methyls [dH 1.34, 1.39, 1.44 (each 3H, s)], an acetoxy
cidal activity, extracts of the dried leaves of Laurus nobilis group [dH 2.00 (3H, s)], and a cis olefin [dH 5.65 (1H, ddd,
L. (Lauraceae) showed strong trypanocidal activity against Jꢀ10.0, 2.5, 1.5 Hz), 5.92 (1H, br dd, Jꢀ10.0, 1.5 Hz)]. The
epimastigotes of T. cruzi. Leaves of L. nobilis are used as a molecular formula C12H20O4, suggested by a weak molecular
spice known as laurel or bay leaf. They are also used as a ion at m/z 229 (MHꢁ) in CI-MS, together with the relatively
folk medicine for rheumatism and scabies.2) Although large chemical shift of the non-acetoxy oxygen-bearing car-
some biological activities such as inhibition of elevation bon (dC 78.6) indicated the presence of a hydroperoxy group.
of blood ethanol level in ethanol-loaded rat,3) inhibition of Comparison of 13C-NMR data with those of known monoter-
nitric oxide production in lipopolysaccharide-activated penes suggested that 3 had a p-menthane-type skeleton and
1
1
mouse peritoneal macrophages,4) induction of glutathione S- analyses of H–1H correlation spectroscopy (COSY), H-de-
transferase activity,5) and nematocidal activity6) were re- tected heteronuclear multiple quantum coherence (HMQC)
ported for the constituents of L. nobilis, nothing has been re- and heteronuclear multiple bond connectivity (HMBC) spec-
ported on anti-protozoan constituents of this plant. Here, we tra (Fig. 1) revealed the structure to be 1-hydroperoxy-p-
report the isolation and identification of trypanocidal con- menthan-2-en-8-ol acetate. Treatment of 3 with PPh3 gave an
stituents of L. nobilis.
acetoxy alcohol 4, which showed oxygen-bearing carbons at
dC 67.0 (C-1) and dC 84.4 (C-8). This confirmed that 3 had a
hydroperoxy and an acetoxy group at positions 1 and 8, re-
Results and Discussion
Dried leaves of Laurus nobilis were successively extracted spectively. Since nuclear Overhauser effect (NOE) experi-
with hexane, CHCl3 and MeOH, and each extract was tested ments did not provide conclusive stereochemical informa-
for anti-trypanosomal effect by the HeLa cell infection tion, the stereochemistry of 3 was determined by chemical
assay.7) As the CHCl3 and MeOH extracts inhibited infection correlations. Photo-oxidation11) of d-limonene followed by
and proliferation of T. cruzi, and the MeOH extract formed reduction with PPh3 gave (1S,4R)-p-mentha-2,8-dien-1-ol (5,
the major part of the extracts, the MeOH extract was further [a]Dꢀꢁ60.8°) and (1R,4R)-p-mentha-2,8-dien-1-ol (6,
fractionated under the guidance of in vitro trypanocidal ac- [a]Dꢀꢁ186.4°). Oxymercuration of 5 followed by treatment
tivity against epimastigotes of T. cruzi.8) The MeOH extract with NaBH4 gave (1S,4R)-p-mentha-2-en-1,8-diol (7,
1
was suspended in aqueous MeOH and fractionated into [a]Dꢀꢂ28.0°). The H- and 13C-NMR spectra of 7 were
hexane soluble, AcOEt soluble, and water soluble fractions. identical with those of the diol 8, which was derived from 3
Since the AcOEt soluble fraction showed the strongest activ- by alkaline hydrolysis followed by reduction with PPh3.12)
ity, it was fractionated by silica gel column chromatography This result established that the relative stereochemistry of 3
to give three trypanocidal compounds (1—3). These com- is identical with that of 7. However, the diols 7 and 8 show-
pounds were also isolated from the CHCl3 extracts.
ed different retention times on GC-MS analysis with chiral
Compounds 1 and 2 were obtained as colorless needles capillary column, revealing an enantiomeric relationship
from hexane, mp 58—60 °C, and as a colorless oil, respec- between them.13) Thus, the structure of 3 was concluded to
1
tively. The H- and 13C-NMR spectra indicated that both be (1R,4S)-1-hydroperoxy-p-menth-2-en-8-ol acetate.
A
compounds were guaiane-type sesquiterpene with three exo- sesquiterpene hydroperoxide, verlotorin, has been isolated
cyclic methylene moieties. Based on comparisons of the from L. nobilis.14) However, this is the first report of the iso-
spectral data with those reported, compounds 1 and 2 were lation of a monoterpene hydroperoxide from this plant.
identified with dehydrocostus lactone (1)9) and zaluzanin D
(2),10) respectively.
Compounds 1—3 showed strong trypanocidal activity
against epimastigotes of T. cruzi with minimum lethal con-
Compound 3 was obtained as colorless needles from pe- centrations (MLCs) of 6.3 mM (1), 2.5 mM (2), and 1.4 mM (3).
troleum ether, mp 87.5—88.5 °C. The 13C-NMR spectrum The activities were stronger than that of the anthelmintic
∗ To whom correspondence should be addressed. e-mail: fkiuchi@pharm.kyoto-u.ac.jp
© 2002 Pharmaceutical Society of Japan

November 2002
1515
Chart 1
Experimental
General Melting points were determined on a Yanagimoto micro melt-
ing point apparatus. Optical rotation ([a]D) was measured on a JASCO DIP-
370 polarimeter. 1H- and 13C-NMR spectra were measured on a JEOL JNM-
LA500 spectrometer with TMS as an internal standard and chemical shifts
were recorded in d ppm. Fuji Silysia BW-127ZH silica gel was used for col-
umn chromatography.
Plant Materials Dried leaves of Laurus nobilis were purchased from
Tochimoto Tenkaido.
Extraction and Isolation The dried leaves (225 g) were successively
extracted with hexane, CHCl3, MeOH (3 timesꢃ2 l) at room temperature
overnight. Each extract was concentrated to dryness to give hexane (3.6 g),
CHCl3 (5.8 g), and MeOH (53.4 g) extracts. The MeOH extract (53.4 g) was
suspended in 700 ml of aqueous MeOH and extracted with hexane
(250 mlꢃ3 times). The aqueous MeOH layer was concentrated to ca.
100 ml, diluted with water and extracted with AcOEt (250 mlꢃ3 times) to
give AcOEt soluble (13.5 g) and water soluble (34.3 g) fractions. The AcOEt
soluble fraction was applied to a silica gel column and eluted with
hexane–acetone 9 : 1 (fr. 1, 127 mg; fr. 2, 40 mg; fr. 3, 603 mg; fr. 4, 187 mg),
4 : 1 (fr. 5, 408 mg; fr. 6, 1.1 g; fr. 7, 330 mg), 7 : 3 (fr. 8, 646 mg; fr. 9, 2.4 g),
acetone (fr. 10, 3.8 g), and MeOH (fr. 11, 3.3 g), among which fractions 2, 3,
5, 6, and 8 showed trypanocidal activity at 25 mg/ml. Fractions 3, 5, and 6
were separately purified by silica gel column chromatography to give com-
pounds 1 (dehydrocostus lactone, 83 mg),9) 2 (zaluzanin D, 245 mg),10) and 3
(15 mg), respectively. Fraction 8 also gave zaluzanin D (2, 239 mg).
Compound 3: colorless needles from petroleum ether, mp 87.5—88.5 °C.
Fig. 1. Selected HMBC Correlations in 3
Table 1. Anti-trypanosomal Activity of Compounds 1—3 against T. cruzi
in the HeLa Infection Assay at 1 mg/ml
Inhibition of infection
(%)
Inhibition of proliferation
(%)
Compound
1
2
3
74
ꢂ8
98
75
38
83
42
Allopurinol
42
1
[a]Dꢀꢂ7.9° (cꢀ0.61, EtOH). H-NMR (CDCl3, 500 MHz) d: 1.34 (3H, s,
CH3-7), 1.39 (3H, s, CH3-9), 1.41 (1H, m, H-6), 1.44 (3H, s, CH3-10), 1.58
(2H, m, CH2-5), 2.00 (3H, s, Ac), 2.20 (1H, dtd, Jꢀ14.0, 3.5, 1.5 Hz, H-6),
2.80 (1H, m, H-4), 5.65 (1H, ddd, Jꢀ10.0, 2.5, 1.5 Hz, H-2), 5.92 (1H, br
dd, Jꢀ10.0, 1.5 Hz, H-3), 7.94 (1H, s, OOH). 13C-NMR (CDCl3, 125 MHz)
d: 20.1 (C-5), 22.5 (COCH3), 23.1 (C-10), 23.7 (C-9), 24.8 (C-7), 31.7 (C-
6), 44.4 (C-4), 78.6 (C-1), 84.4 (C-8), 129.9 (C-2), 133.7 (C-3), 170.6
(COCH3). CI-MS m/z: 229 (MHꢁ, 1), 211 (MHꢁ-18, 4), 195 (10), 153 (17),
151 (13), 135 (100).
Reduction of 3 with PPh3 Compound 3 (4.5 mg) dissolved in ether
(1 ml) was treated with PPh3 (8.5 mg) at room temperature for 20 min. The
mixture was concentrated to dryness and the residue was chromatographed
over silica gel to give an acetoxy alcohol 4 (3.4 mg).
monoterpene endoperoxide, ascaridole (MLCꢀ23 mM) and
were comparable to those of monoterpene hydroperoxides
isolated from Chenopodium ambrosioides.8) In the HeLa cell
infection assay, compound 3 very strongly inhibited the in-
fection of the trypomastigotes at 1 mg/ml (Table 1). Dehydro-
costus lactone (1) also significantly inhibited the infection at
1 mg/ml, whereas zaluzanin D (2) did not inhibit the infection
at the same concentration.
Several sesquiterpene lactones having an a,b-unsaturated
g-lactone moiety (15-deoxygoyazensolide,15) dehydroza-
luzanin C,16) lychnopholide17) naematolin,18) dehydroleuco-
dine19)) have been reported to show trypanocidal activity.
Brengio et al. reported that the effect of dehydroleucodine
was irreversible, but could be blocked by simultaneous addi-
tion of a thiol compound.19) This suggests that the trypanoci-
dal activity of this group of compounds relies on covalent
bond formation of the a,b-unsaturated g-lactone moiety with
nucleophiles which are essential for the life of the parasite.
Compound 4: Colorless oil. [a]Dꢀꢁ11.5° (cꢀ0.17, CHCl3). 1H-NMR
(CDCl3, 500 MHz) d: 1.29, 1.41, 1.46 (each 3H, s, CH3-7, 9, 10), 1.48 (1H,
m), 1.58 (1H, m), 1.68 (1H, m), 1.87 (1H, m), 2.00 (3H, s, Ac), 2.75 (1H,
m), 5.73 (2H, t-like, Jꢀ11 Hz, H-2, 3). 13C-NMR (CDCl3, 125 MHz) d: 20.3
(C-5), 22.6 (COCH3), 23.1 (C-10), 23.8 (C-9), 29.9 (C-7), 37.1 (C-6), 44.5
(C-4), 67.0 (C-1), 84.4 (C-8), 129.8 (C-2), 134.7 (C-3), 170.6 (COCH3).
Photo-oxidation and Reduction of d-Limonene A stirred solution of
d-limonene (2 g) and Rose Bengal (29 mg) in MeOH (29 ml) was irradiated
by a high pressure mercury lamp (Riko Kagaku, UV-100HA) under oxygen
atmosphere at 0 °C for 8.5 h. The mixture was concentrated to dryness, and
One possible target is the trypanothione (1,8-bis-glutathionyl the residue was dissolved in ether (30 ml) and treated with PPh3 (2.6 g) at
spermidine) redox system.20—22) Trypanosomes are known to
be sensitive to oxidative stresses. As a defense against these
stresses, they use trypanothione whose counterpart in mam-
room temperature for 1 h. The mixture was concentrated to dryness and the
residue was fractionated by repeated silica gel column chromatography with
hexane–AcOEt (4 : 1) to give (1S,4R)-p-mentha-2,8-dien-1-ol (5, 299 mg,
13.4%) and (1R,4R)-p-mentha-2,8-dien-1-ol (6, 53 mg, 5.3%).
malian cells is glutathione. a,b-Unsaturated g-lactones will
react with -SH groups of trypanothione and of enzymes in-
volved in trypanothione cycle,23) thus inactivating the de-
fense system and exposing the parasite to oxidative damage.
For hydroperoxide 3, reactive oxygen species derived from
the hydroperoxy moiety may be responsible for the try-
panocidal activity.
(1S,4R)-p-Mentha-2,8-dien-1-ol (5): Colorless oil. [a]Dꢀꢁ60.8°
(cꢀ0.62, CHCl3) (lit., ꢁ67.3°).11) 1H-NMR (CDCl3, 500 MHz) d: 1.30 (3H,
s, CH3-7 ), 1.61 (2H, m, overlapped, H-5, H-6), 1.74 (3H, br s, CH3-10), 1.77
(1H, m, H-5), 1.85 (1H, m, H-6), 2.66 (1H, m, H-4), 4.75 (1H, br s, Ha-9),
4.78 (1H, quintet., Jꢀ1.5 Hz, Hb-9), 5.66 (1H, ddd, Jꢀ10.1, 2.2, 1.0 Hz, H-
3), 5.71 (1H, ddd, Jꢀ10.1, 2.2, 1.3 Hz, H-2). 13C-NMR (CDCl3, 125 MHz)
d: 20.8 (C-10), 24.9 (C-5), 29.4 (C-7), 36.7 (C-6), 43.4 (C-4), 67.4 (C-1),
110.6 (C-9), 132.1 (C-3), 133.9 (C-2), 148.1 (C-8).

1516
Vol. 50, No. 11
Graduate School of Pharmaceutical Sciences, Kyoto University, for allowing
us to use the NMR facilities, and Dr. N. Akimoto of the Faculty of Pharma-
ceutical Sciences, Kyoto University, for MS measurements. This work was
supported in part by a Grant-in-Aid for Scientific Research (No. 11672101)
from the Japan Society for the Promotion of Science.
(1R,4R)-p-Mentha-2,8-dien-1-ol (6): Colorless oil. [a]Dꢀꢁ186.4°
(cꢀ0.97, CHCl3) (lit., ꢁ163.8°).11) 1H-NMR (CDCl3, 500 MHz) d: 1.29
(3H, s, CH3-7), 1.56 (1H, m, Ha-5), 1.66 (1H, ddd, Jꢀ12.8, 9.5, 2.8 Hz, Ha-
6), 1.74 (3H, br s, CH3-10), 1.79 (1H, ddd, Jꢀ12.8, 8.9, 2.8 Hz, Hb-6), 1.90
(1H, m, Hb-5), 2.74 (1H, m, H-4), 4.67 (1H, br s, Ha-9), 4.78 (1H, quintet,
Jꢀ1.6 Hz, Hb-9), 5.61 (1H, dd, Jꢀ10.1, 3.4 Hz, H-3), 5.70 (1H, dd, Jꢀ10.1,
2.1 Hz, H-2). 13C-NMR (CDCl3, 125 MHz) d: 21.2 (C-10), 25.0 (C-5), 28.9
(C-7), 36.1 (C-6), 42.5 (C-4), 68.6 (C-1), 110.9 (C-9), 130.8 (C-3), 134.5
(C-2), 147.2 (C-8).
References and Notes
1) Bastien J. W., “The Kiss of Death, Chagas’ Disease in the Americas,”
The University of Utah Press, Salt Lake City, 1998.
2) Cuo J.-X. (ed.), “International Collation of Traditional and Fork Medi-
cine,” Vol. 4, World Scientific Publishing, Singapore, 1997, pp. 17—
18.
3) Matsuda H., Shimoda H., Uemura T., Yoshikawa M., Bioorg. Med.
Chem. Lett., 9, 2647—2652 (1999).
4) Matsuda H., Kagerura T., Toguchida I., Ueda H., Morikawa T.,
Yoshikawa M., Life Sci., 66, 2151—2157 (2000).
5) Wada K., Ueda N., Sawada H., Amemiya N., Haga M., Natural Medi-
cines, 51, 283—285 (1997).
Oxymercuration of (1S,4R)-p-Mentha-2,8-dien-1-ol (5) To a solution
of mercury (II) acetate in water (2 ml), THF (2 ml) and then a solution of 5
(64 mg) in THF (1 ml) were added, then the mixture was stirred at room tem-
perature for 10 min. To this mixture, 3 M NaOH (2 ml), and then 0.5 M
NaBH4 in 3 M NaOH (1 ml) were added and the mixture was stirred for
5 min. The mixture was saturated with NaCl and the THF layer was sepa-
rated, dried over anhydrous MgSO4, and concentrated to dryness. The
residue was purified by column chromatography on silica gel to give
(1S,4R)-p-mentha-2-en-1,8-diol (7, 14 mg, 19.6%).
6) Kiuchi F., Nakamura N., Miyashita N., Nishizawa S., Tsuda Y., Kondo
K., Shoyakugaku Zasshi, 43, 279—287 (1989).
7) Nakajima-Shimada J., Hirota Y., Kaneda T., Aoki T., J. Protozool.
Res., 4, 10—17 (1994).
8) Kiuchi F., Itano Y., Uchiyama N., Honda G., Tsubouchi A., Nakajima-
Shimada J., Aoki T., J. Nat. Prod., 65, 509—512 (2002).
9) Taniguchi M., Kataoka T., Suzuki H., Uramoto M., Ando M., Arao K.,
Magae J., Nishimura T., Otake N., Nagai K., Biosci. Biotech.
Biochem., 59, 2064—2067 (1995).
10) Ando M., Kusaka H., Ohara H., Takase K., Yamaoka H., Yanagi Y., J.
Org. Chem., 54, 1952—1960 (1989).
11) Schenck G. O., Gollnick K., Buchwald G., Schroeter S., Ohloff G.,
Liebigs Ann. Chem., 674, 93—118 (1964).
12) The other possible diastereomer 9 was prepared from 6 (y. 33%) as de-
scribed for 7. The NMR spectra of 9 were different from those of 8.
Compound 9: colorless prisms (hexane–AcOEt), mp 110—112 °C. 1H-
NMR (CDCl3, 500 MHz) d: 1.17, 1.22 (each 3H, s, CH3-9,10), 1.29
(3H, s, CH3-7), 1.42 (1H, dt, Jꢀ13.5, 3.1 Hz, H-5), 1.65 (1H, td, Jꢀ13
2.8 Hz, H-6), 1.87 (1H, m, H-5), 1.94 (1H, m, H-6), 2.17 (1H, m, H-
4), 5.72 (1H, dt, Jꢀ10.7, 1.7 Hz, H-2), 5.75 (1H, dt, Jꢀ10.7, 1.3 Hz,
H-3). 13C-NMR (CDCl3, 125 MHz) d: 22.9 (C-5), 26.1 (C-9), 27.9 (C-
10), 28.2 (C-7), 38.2 (C-6), 46.9 (C-4), 69.5 (C-1), 72.6 (C-8), 127.8
(C-3), 136.5 (C-2).
13) Compound 8 showed positive optical rotation, although no reliable
[a]D value could be determined because of the scarcity of the com-
pound.
14) El-Feraly F. S., Benigni D. A., J. Nat. Prod., 43, 527—530 (1980).
15) Chiari E., de Oliveira A. B., Raslan D. S., Mesquita A. A. L., Tavares
K. G., Trans. Roy. Soc. Trop. Med. Hyg., 85, 372—374 (1991).
16) Fournet A., Muñoz V., Roblot F., Hocquemiller R., Cavé A., Gantier J.
C., Phytother. Res., 7, 111—115 (1993).
17) de Oliveria A. B., Saúde D. A., Perry K. S. P., Duarte D. S., Raslan D.
S., Boaventura M. A. D., Chiari E., Phytother. Res., 10, 292—295
(1996).
18) Inchaustil A., Yaluff G., Arias A. R., Torres S., Ferreria M. E.,
Nakayama H., Schinini A., Lorenzen K., Anke T., Fournet A., Phy-
tother. Res., 11, 193—197 (1997).
19) Brengio S. D., Belmonte S. A., Guerreiro E., Giordano O. S.,
Pietrobon E. O., Sosa M. A., J. Parasitol., 86, 407—412 (2000).
20) Fairlamb A. H., Cerami A., Annu. Rev. Microbiol., 46, 695—729
(1992).
21) Schirmer R. H., Müller J. G., Krauth-Siegel R. L., Angew. Chem. Int.
Ed. Engl., 34, 141—154 (1995).
(1S,4R)-p-Mentha-2-en-1,8-diol (7): Colorless oil. [a]Dꢀꢂ28.0° (cꢀ1.4,
CHCl3). 1H-NMR (CDCl3, 500 MHz) d: 1.19, 1.26 (each 3H, s, CH3-9, 10),
1.29 (3H, s, CH3-7), 1.53 (2H, m), 1.75 (1H, m), 1.88 (1H, m), 2.06 (1H, m,
H-4), 5.77 (1H, dt, Jꢀ10.1, 1.8 Hz, H-3), 5.88 (1H, dt, Jꢀ10.1, 1.8 Hz, H-
2). 13C-NMR (CDCl3, 125 MHz) d: 20.4 (C-5), 26.3 (C-9), 28.1 (C-10), 29.6
(C-7), 37.0 (C-6), 47.0 (C-4), 66.9 (C-1), 72.6 (C-8), 130.0 (C-3), 134.9 (C-
2).
Hydrolysis and Reduction of Compound 3 A mixture of compound 3
(0.5 mg) in MeOH (0.1 ml) and 10% NaOH (0.1 ml) was stirred at room
temperature for 1 h. The mixture was neutralized and applied to a silica gel
column, which was eluted with AcOEt to give an alcohol. The alcohol was
dissolved in ether (1 ml) and stirred with PPh3 (1.4 mg) at room temperature
for 40 min. The mixture was concentrated to dryness and the residue was pu-
rified by silica gel column chromatography with hexane : AcOEt (5 : 4) to
give the diol 8, whose 1H- and 13C-NMR spectra were identical with those of
(1S,4R)-p-mentha-2-en-1,8-diol (7).
GC-MS Analysis with Chiral Capillary Column GC-MS analysis was
performed on a Hitachi G-7000M/M-9000 system equipped with a Cy-
clodex-B chiral capillary column (0.25 mmꢃ30 m, J & W). GC conditions:
column temperature, starting at 40 °C, 10°/min to 100 °C, 5°/min to 130 °C,
3°/min to 160 °C, 20°/min to 220 °C; injector temperature, 100 °C; carrier
gas, He; flow rate, 1 ml/min. Retention time: the diol (8) derived from com-
pound 3, 20.68 min; compound 7, 20.92 min. MS conditions: mode, EI; ion-
ization voltage: 15 eV. The MS patterns of 7 and 8 were identical; m/z (%):
152 (MꢁꢂH2O, 1), 135 (13), 110 (33), 96 (25), 95 (98), 92 (18), 80 (100).
Trypanocidal Assay Trypanocidal assay was performed as described
previously.8) EtOH was used as a negative control. Ketoconazole and gentian
violet were used as positive controls. The MLCs of ketoconazole and gen-
tian violet under this assay condition were 50 mM and 6.3 mM, respectively.
HeLa Infection Assay The HeLa cell infection assay was performed as
described previously.7) A round coverslip was placed in each well of a 24-
well plate. Exponentially growing HeLa cells (5ꢃ103 cells/ml/well) were
added to each well, followed by incubation at 37 °C for 2 d in 5% CO2 in air.
The cells were then infected with T. cruzi trypomastigotes (1ꢂ2ꢃ104 para-
sites/well). A compound to be tested was dissolved in ethanol (10 ml) 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 contain-
ing more than one amastigote, and the mean number of amastigotes per in-
fected cell were determined by analyzing more than 200 host cells distrib-
uted in randomly chosen microscopic fields. Allopurinol was used as a posi-
tive control.
22) Sepúlveda-Boza S., Cassels B. K., Planta Med., 62, 98—105 (1996).
23) D’Silva C., Daunes S., Expert Opin. Investig. Drugs, 11, 217—231
(2002).
Acknowlegements The authors thank Professor K. Tomioka of the