Studies on quinones. Part 39: Synthesis and leishmanicidal activity of acylchloroquinones and hydroquinones

Article abstract of DOI:10.1016/j.bmc.2005.04.041

Acylhydroquinone-based compounds are attractive targets for the design of new leishmanicidal drugs. We have previously described sesquiterpene quinones and hydroquinones series, which exhibit different degree of potency against Leishmania amazonensis. The present study details the preparation of acylchloroquinones and hydroquinones possessing lipophilic substituents and examines their in vitro activity against intracellular L. amazonensis amastigotes. The quinone or hydroquinone nucleus is essential for the activity of the members of the series. The lipophilicity of the cycloaliphatic systems in these members seems to attenuate the cytotoxical effect and increases the selectivity of those compounds containing the norbornene system.

Full text of DOI:10.1016/j.bmc.2005.04.041

Bioorganic & Medicinal Chemistry 13 (2005) 4153–4159
Studies on quinones. Part 39: Synthesis and leishmanicidal
activity of acylchloroquinones and hydroquinonesq
a
a
Jaime A. Valderrama,a, Carlos Zamorano, M. Florencia Gonzalez,
*
´
Eric Prinab and Alain Fournetc
a
´
´
Facultad de Quımica, Pontificia Universidad Catolica de Chile, Casilla 306, Santiago 22, Chile
´
b
Groupe de Recherche sur les Interactions Macrophage-Leishmania, Unite d’Immunophysiologie et Parasitisme Intracellulaire,
Institut Pasteur rue du Dr. Roux 75724, Paris Cedex, France
c
´
Institut de Recherche pour le Developpment (IRD), 213 rue La Fayette 75480, Paris Cedex, France
Received 3 March 2005; revised 15 April 2005; accepted 15 April 2005
Available online 3 May 2005
Abstract—Acylhydroquinone-based compounds are attractive targets for the design of new leishmanicidal drugs. We have previ-
ously described sesquiterpene quinones and hydroquinones series, which exhibit different degree of potency against Leishmania ama-
zonensis. The present study details the preparation of acylchloroquinones and hydroquinones possessing lipophilic substituents and
examines their in vitro activity against intracellular L. amazonensis amastigotes. The quinone or hydroquinone nucleus is essential
for the activity of the members of the series. The lipophilicity of the cycloaliphatic systems in these members seems to attenuate the
cytotoxical effect and increases the selectivity of those compounds containing the norbornene system.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tries. More recently the drug Miltefosine8,9 has been
introduced in chemotherapy of visceral leishmaniasis
with promising results. Due to the toxic side-effects
and variable efficacy of the drugs used in antileishmanial
chemotherapy, efforts are being made to find new and
inexpensive compounds as new leads.
Leishmaniasis is a widespread parasitic disease caused
by protozoan parasites of the genus Leishmania. The
disease is endemic in some tropical areas of the world
and in underdeveloped countries, directly affecting
about 2 million people annually, with approximately
350 million people who live at risk of contracting the
infection worldwide.2,3 Leishmaniasis is an opportunis-
tic infection that affects HIV infected individuals. This
co-infection involves the visceral form of leishmaniasis,
which is one of its most severe form.4 The drugs, which
have been used frequently in the treatment of the leish-
maniases are the pentavalent antimonials Pentostam
and Glucantime.5,6 AmBisome, a liposomal formulation
of amphotericin B,7 which overcomes the toxic limita-
tions of the parent drug, was developed for the treat-
ment of visceral leishmaniasis. However, because of its
cost, it is not suitable for use in underdeveloped coun-
Natural and synthetic naphthoquinones have been de-
scribed for many years for their antiprotozoal activity.10
In addition, quinones fused to a variety of heterocyclic
rings (e.g., furan, thiophene, pyridine, imidazole, and
pyran) were also described as antiprotozoal agents.11
Biological activities of quinones emerge due to their
ability to act as potent inhibitors of electron transport,
as uncouplers of oxidative phosphorylation, as DNA
intercalating agents, as bioreductive alkylating agents
and as producers of reactive oxygen radicals, by redox
cycling, under aerobic conditions. In all these cases,
the mechanisms require bioreduction of the quinone
nucleus as the first activating step.12–14
As part of our current research program toward the syn-
thesis of potential bioactive quinones,1,11,15–19 we have
reported the synthesis and in vitro leishmanicidal activ-
ity of sesquiterpene quinones and hydroquinones.1,15
Among the members of this family, compounds 1–3
Keywords: Quinones; Diels–Alder reaction; Enolization; Lipophilicity;
Leishmanicidal activity.
q For Part 38, see Ref. 1.
*
Corresponding author. Tel.: +56 2 6864432; fax: +56 2 6864744;
e-mail: jvalderr@puc.cl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.041

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J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
Figure 1.
(Fig. 1) showed promising activities against macro-
phages. These studies also revealed that the activity de-
pends on the acylquinone or acylhydroquinone core and
that the sesquiterpene fragment is not an essential
requirement but, in some cases, appears to cause a slight
increase of selectivity toward macrophages.
Scheme 1. Synthesis of 2-acyl-3-chloro-1,4-benzoquinones 7 and 11.
however, isolation of 11 was unsuccessful due to its
unstability even in dichloromethane solution.
Cycloaddition reaction of quinone 7 with cyclopentadi-
ene gave Diels–Alder adduct 12, which after column
chromatography on silica gel, was isolated as enoliza-
tion product 13. The synthesis of compound 13 was
achieved by cycloaddition of quinone 7 with cyclopenta-
diene followed by treatment of the crude with silica gel
in dichloromethane at room temperature. Under these
conditions, compound 13 was isolated in 80% total
yield.
These observations prompted us to investigate the
synthesis of new compounds containing the biologically
active acylhydroquinone or acylquinone framework
fused to lipophilic cycloaliphatic fragments. Here we
wish to report the synthesis and leishmanicidal activity
of acylchlorobenzoquinones, acylchlorohydroquinones
and cycloaliphatic fused derivatives. The in vitro biolo-
gical studies have been performed using intracellular
Leishmania amazonensis amastigotes stage in mouse
peritoneal macrophages and Miltefosine as the reference
drug.
The synthesis of compound 16 was performed by follow-
ing the procedure described for the preparation of com-
pound 13 (Scheme 2). Cycloaddition of
7 with
cyclohexa-1,3-diene followed by enolization of Diels–
Alder adduct 15, afforded compound 16 in 78% total
yield.
2. Results and discussion
2.1. Chemistry
The 1,4-dihydro-1,4-alkanonaphthalene-5,8-diones 14
and 17 were obtained in 97% and 89% yields, respec-
tively, by oxidation of compounds 13 and 16 with man-
ganese dioxide in dichloromethane.
On the basis of the results of Kayser et al.20 on the
increase of the leishmanicidal activity of naphthoqui-
nones by introducing an alicyclic ring at the quinone
nucleus, we designed 6-acyl-7-chloro-1,4-alkanonaph-
thalenes 13, 14, 16, and 17 for this study. For the
synthesis of the target compounds we took advantage
of a previous work on the Diels–Alder reaction of
2-acyl-3-chloro-1,4-benzoquinones with 1,3-butadienes.21
The requisite precursors 6, 7, 10, and 11 were prepared
by the route described in Scheme 1. Acetylbenzoqui-
none 5 was obtained from 4 and manganese dioxide
and then reacted with hydrogen chloride in dichloro-
methane to give addition compound 6 in 82% yield.
Further oxidation of 6 with manganese dioxide in
dichloromethane afforded 2-acetyl-3-chloro-1,4-benzo-
quinone 7 in 70% yield.
2.2. Biology
The acylchlorobenzoquinones and hydroquinones were
tested in vitro against intracellular L. amazonensis
amastigotes stage in mouse peritoneal macrophages.
The results of the biological evaluation are indicated
in Table 1. The IC50 and TC50 of compounds 1–3, pre-
viously reported,15 and that of 1,4-naphthoquinone also
are included in Table 1 in order to compare their antilei-
shmanicidal activity and cytotoxic effects with those of
the new compounds.
Although almost all of the tested compounds showed
leishmanicidal activity, none was as effective as miltefo-
sine used here as the reference drug against L. amazon-
ensis intracellular amastigotes. All compounds showed
a weak selectivity (i.e., the ratio of between cytotoxic
and leishmanicidal effect).
Formylbenzoquinone 9 prepared from 8 and silver(II)
oxide was reacted with hydrogen chloride in dichloro-
methane to give addition product 10 in 69% yield. Prep-
aration of 2-formyl-3-chloro-1,4-benzoquinone 11 by
oxidation of 10 with silver(II) oxide was attempted;

J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
4155
Scheme 2. Synthesis of 6-acetyl-7-chloro-1,4-alkanonaphthalenes 13, 14, 16, and 17.
Table 1. Inhibitory concentrations IC50 and cytotoxicity TC50 of compounds 6, 7, 10, 11, 13, 14, 16, and 17 against Leishmania amazonensis
macrophages
Entry
Compound
Structure
IC50 (lM)
TC50 (lM)
Log Pa
1
1
9
25
6.76
2
3
4
5
2
3
6
7
16
18
50
51
8
7.76
7.68
0.96
3.08
13
6.3
6.4
6
7
10
13
55
27.2
1.40
4.55
18.7
9.4
(continued on next page)

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J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
Table 1 (continued)
Entry
Compound
Structure
IC50 (lM)
TC50 (lM)
Log Pa
8
9
14
18.7
9.4
5.07
16
17
35.5
8.9
8.9
4.73
5.54
10
>100
11
12
—
—
5.0
2.5
1.75
—
Miltefosine
3.12
12.5
a The values of lipophilicity (log pKa) were calculated using the Dixon method in the Spartan package.22
Comparison of quinones 7, 14, and 17 (entries 5, 8,
and 10) indicates that cytotoxicity decreases with
lipophilicity and a similar trend is observed for hydro-
quinones 6, 13, and 16 (entries 4, 7, and 9). Further-
more, comparison of compounds 1, 6, and 10 (entries
1, 4, and 6) shows that selectivity increases with the
lipophilicity.
3. Conclusion
In conclusion, the high yield synthesis of 6-acetyl-7-
chloro-5,8-dihydroxy-1,4-alkanonaphthalenes 13 and
17, and its corresponding quinones 14 and 16, were per-
formed from 2,5-dihydroxycetophenone 4 through the
sequence: (a) oxidation of 4 followed by conjugate addi-
tion of hydrogen chloride, (b) oxidation of the addition
product 6, (c) Diels–Alder reaction of 7 with cyclopenta-
diene and cyclohexa-1,3-diene, (d) enolization of the
Diels–Alder adducts 12 and 15, and (e) oxidation. The
compounds reported here exhibit weak to moderate
leishmanicidal activity and high toxicity compared with
Miltefosine. Regarding molecular structure, these in
vitro studies show that the antileishmanial activity is
determined by the presence of quinone or hydroquinone
groups and lipophilic fragments. The lipophilicity of the
cycloaliphatic fragments in these members seems to
attenuate the cytotoxical effects and, in the case of the
norbornene derivatives (13 and 14), increases the leish-
manicidal activity compared with their monocyclic
precursors 6 and 7.
It is noteworthy that cycloaliphatic substituted quinones
14 and 17 are less cytotoxic than 1,4-naphthoquinone
(entries 11 and 12) probably due to the high lipophilicity
of the former two. It seems possible that substituents on
14 and 17 play two roles in the parasitic activity: (i) by
lowering the oxidation potential of the quinone nucleus
with respect to 1,4-naphthoquinone and (ii) preventing
bioreductive alkylation reactions.
Among compounds 7, 14, 17, and 6, 13, 16 those pos-
sessing the lipophilic norbornene system (13 and 14)
are the most active. It is noteworthy that compounds
13 and 14 show the highest leishmanicidal activity of
the series, which are similar to those exhibited by com-
pounds 2 and 3 (entries 2 and 3).
These observations could be interpreted by considering
that the in vitro leishmanicidal activity of the com-
pounds depends on the redox capability of the quinone
and hydroquinone nucleus and on the presence of the
lipophilic cycloaliphatic rings, which seemed to increase
the leishmanicidal effect and attenuate the cytotoxical
effect. These results are encouraging and further in vitro
experiments with acylquinones and hydroquinones must
be performed to understand the mechanism of drug toxi-
city in L. amazonensis.
4. Experimental
4.1. Chemical synthesis
All reagents were of commercial quality, reagent grade
and were used without further purification. Melting
points (mp) were determined on a Ko¨fler hot-stage
apparatus and are uncorrected. The IR spectra were re-
corded on a FT Bruker vector22-FT spectrophotometer
using KBr discs and the wave numbers are given in

J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
4157
1
cmꢀ1. H NMR spectra were measured on Bruker AM-
5-H), 2.48 (s, 3H, COCH3); 13C NMR (50 MHz,
CDCl3): d 207.0, 196.0, 183.0, 178.7, 143.1, 137.8,
136.3, 136.1.
200 and AM-400 in CDCl3. Chemical shifts are
expressed in ppm downfield relative to TMS (d scale)
and the coupling constants (J) are reported in hertz.
13C NMR spectra were acquired in deuteriochloroform
at 50 and 100 MHz on Bruker AM-200 and AM-400
spectrometers. The elemental analyses were performed
in a Fison SA, model EA-1108 apparatus. Silica gel
Merck 60 (70–230 mesh), and TLC aluminum foil 60
F254 were used for preparative column and analytical
TLC, respectively.
4.1.4. 6-Acetyl-7-chloro-5,8-dihydroxy-1,4-dihydro-1,4-
methanonaphthalene (13). A solution of quinone 7
(197 mg,
1.07 mmol),
cyclopentadiene
(100 mg,
1.5 mmol), and dichloromethane (10 mL) was left for
24 h at room temperature. Silica gel (3 g) was added to
the mixture and the suspension was stirred for 24 h at
room temperature. The suspension was filtered and the
solid was washed with dichloromethane. The filtrate
was evaporated to afford compound 13 ( 216 mg,
80%). Column chromatography of the crude, eluting
with dichloromethane gave pure 13 (145 mg, 54%) as a
pale yellow solid, mp 156–158 ꢁC; IR (KBr): mmax 3390
4.1.1. 2,5-Dihydroxy-6-chloroacetophenone (6). A sus-
pension of 2,5-dihydroxyacetophenone (4) (500 mg,
3.29 mmol), manganese dioxide (3.0 g, 34.5 mmol),
magnesium sulfate (500 mg), and dichloromethane
(50 mL) was vigorously stirred for 30 min at room tem-
perature. The mixture was filtered through Celite and
the solids were washed with dichloromethane (15 mL).
Dry hydrogen chloride was bubbled through the filtrate
for 5 min and the resulting solution was left overnight at
room temperature. Evaporation of the solvent left a
solid, which was purified by column chromatography
on silica gel (dichloromethane) to give pure 6 (497 mg,
82%); mp 96–98 ꢁC (lit.23 mp 94–96 ꢁC); IR (KBr): mmax
1
(OH), 1605 (COMe); H NMR (400 MHz, CDCl3): d
11.73 (s, 1H, C1-OH), 6.89 (dd, 1H, J = 5.2; 3.1 Hz, 6-
H or 7-H), 6.80 (dd, 1H, J = 5.2; 3.1 Hz, 7-H or 6-H),
5.44 (s, 1H, C4-OH), 4.27 (m, 1H, 5-H or 8-H), 4.23
(m, 1H, 8-H or 5-H), 2.77 (s, 3H, COMe), 2.29 (dt,
1H, J = 7.3; 1.5 Hz, 9-H or 9-H0), 2.23 (dt, 1H,
J = 7.3; 1.5 Hz, 9-H0 or 9-H); 13C NMR (100 MHz,
CDCl3): d 204.5, 151.2, 147.8, 144.0, 142.0, 140.6,
140.5, 117.8, 117.0, 70.6, 48.7, 47.3, 33.8. Anal. Calcd
for C13H11O3Cl: C, 62.29; H, 4.42. Found: C, 62.25;
H, 4.40.
1
3418 (OH), 1635 (C@O); H NMR (200 MHz, CDCl3):
d 11.91 (s, 1H, C2-OH), 7.19 (d, 1H, J = 9 Hz, 3-H or 4-
H), 6.92 (d, 1H, J = 9 Hz, 4-H or 3-H), 5.49 (s, 1H, C5-
OH), 2.83 (s, 3H, COCH3); 13C NMR (50 MHz,
CDCl3): d 204.0, 157.3, 144.9, 123.4, 118.9, 118.8,
117.9, 33.4.
4.1.5. 6-Acetyl-7-chloro-5,8-dihydroxy-1,4-dihydro-1,4-
ethanonaphthalene (16). A solution of quinone 7
(197 mg, 1.07 mmol), cyclohexa-1,3-diene (100 mg,
1.25 mmol), and dichloromethane (10 mL) was left for
24 h at room temperature. Silica gel (3 g) was added to
the mixture and the suspension was stirred for 24 h at
room temperature. The suspension was filtered and the
solid was washed with dichloromethane. Evaporation
of the solvent afforded crude compound 16 (221 mg,
78%); further column chromatography (dichlorometh-
ane) gave pure 16 (207 mg, 73%) as yellow crystals,
mp 150–152 ꢁC; IR (KBr): mmax 3404 (OH), 1624
4.1.2. 2,5-Dihydroxy-6-chlorobenzaldehyde (10). A sus-
pension of 2,5-dihydroxybenzaldehyde (8) (170 mg,
1.23 mmol), silver(II) oxide (510 mg, 2.2 mmol), magne-
sium sulfate (500 mg), and dichloromethane (35 mL)
was vigorously stirred for 30 min for 2 h. The mixture
was filtered and the solids were washed with dichloro-
methane (15 mL). Dry hydrogen chloride was bubbled
through the filtrate for 5 min and the resulting solution
was left overnight at room temperature. The solvent was
removed and the residue was chromatographed over sil-
ica gel (dichloromethane) to give pure 10 as a gold yel-
low solid (146 mg, 69%); mp 155–157 ꢁC; IR(KBr):
mmax 3271 (OH), 2773 and 2719 (C@O); 1H NMR
(200 MHz, CDCl3): d 11.50 (s, 1H, C2-OH), 10.35 (s,
1H, CHO), 7.26 (d, 1H, J = 9 Hz, 3-H or 4-H), 6.88
(d, 1H, J = 9 Hz, 4-H or 3-H), 5.41 (s, 1H, C5-OH);
13C NMR (50 MHz, CDCl3): d 194.7, 157.9, 144.5,
125.9, 120.6, 117.8, 115.8. Anal. Calcd for C7H5O3Cl:
C, 48.72; H, 2.92. Found: C, 48.87; H, 2.65.
1
(COMe); H NMR (400 MHz, CDCl3): d 12.25 (s, 1H,
C1-OH); 6.54 (m, 1H, 6-H or 7-H), 6.46 (m, 1H, 7-H
or 6-H), 5.47 (s, 1H, C4-H), 4.53 (m, 1H, 5-H or 8-H),
4.48 (m, 1H, 8-H or 5-H), 2.79 (s, 3H, COMe), 1.54
(m, 2H, 9-H and 10-H), 1.46 (m, 2H, 9-H0 and 10-H0);
13C NMR (50 MHz, CDCl3): d 204.2, 151.4, 140.3,
139.6, 135.6, 133.8, 133.3, 116.0, 115.0, 34.6, 33.4,
32.9, 24.7, 24.6. Anal. Calcd for C14H13O3Cl: C, 63.52;
H, 4.95. Found: C, 63.68; H, 4.85.
4.1.6. 6-Acetyl-7-chloro-1,4-dihydro-1,4-methanonaphtha-
lene-5,8-dione (14).
A suspension of 13 (100 mg,
4.1.3. 2-Acetyl-3-chloro-1,4-benzoquinone (7). A suspen-
sion of 6 (150 mg, 0.8 mmol), manganese dioxide
(730 mg, 8.4 mmol), magnesium sulfate (500 mg) and
dichloromethane (15 mL) was vigorously stirred for
30 min at room temperature. After the usual work-up,
and column chromatography over silica gel (dichloro-
methane), compound 7 was obtained as an orange solid
(104 mg, 70%); mp 110–114 ꢁC (lit.23 mp 110–112 ꢁC);
IR (KBr): mmax 1717 (COMe), 1679, 1655 (C@O qui-
none); 1H NMR (200 MHz, CDCl3): d 6.98 (d, 1H,
J = 10 Hz, 5-H or 6-H), 6.84 (d, 1H, J = 10 Hz, 6-H or
0.4 mmol), manganese dioxide (367 mg, 4.2 mmol),
magnesium sulfate (0.5 g), and dichloromethane
(10 mL) was stirred for 30 min at room temperature.
The suspension was filtered, the solids were washed with
dichloromethane, and the solvent was removed under
vacuum to give crude quinone 14. Column chromato-
graphy of the crude afforded pure 14 (96 mg, 97%) as
an orange solid, mp 145–147 ꢁC. IR (KBr): mmax 1722
(COMe), 1672, 1648 (C@O quinone); 1H NMR
(400 MHz, CDCl3): d 6.89 (m, 2H, 6-H and 7-H), 4.18
(m, 1H, 5-H or 8-H), 4.12 (m, 1H, 8-H or 5-H), 2.46

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J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
(s, 3H, COCH3), 2.39 (dt, 1H, J = 7.3; 5.0 Hz, H-9 or
H0-9), 2.32 (dt, 1H, J = 7.3; 1.5 Hz, 9-H0 or 9-H); 13C
NMR (50 MHz, CDCl3): d 197.0, 179.7, 175.4, 161.3,
160.7, 142.4, 141.9, 137.0, 74.1, 49.2, 48.7, 31.0, 29.7.
Anal. Calcd for C14H13O3Cl: C, 62.79; H, 3.65. Found:
C, 62.41; H, 3.62.
Thirty-hours after drug addition, infected cultures were
examined using an inverted phase contrast Zeiss micro-
scope (magnification of 400). Toxic effects in the macro-
phages were evidenced by the change in morphological
features, that is, loss of refringency, vacuolation of cyto-
plasm or loss of cytoplasmic material.
4.1.7. 6-Acetyl-7-chloro-1,4-dihydro-1,4-ethanonaphtha-
lene-5,8-dione (17). A suspension of 16 (150 mg,
0.57 mmol), manganese dioxide (520 mg, 6.0 mmol),
magnesium sulfate (500 mg), and dichloromethane
(15 mL) was stirred for 30 min at room temperature.
Work up of the mixture followed by column chromato-
graphy afforded pure quinone 17 (134 mg, 89%) as yel-
low crystals, mp 144–146 ꢁC; IR (KBr): mmax 1724
(COMe), 1673, 1646 (C@O quinone); 1H NMR (400
MHz, CDCl3): d 6.41 (m, 2H, 6-H and 7-H), 4.43 (m,
1H, 5-H or 8-H), 4.34 (m, 1H, 8-H or 5-H), 2.46 (s,
3H, COCH3), 1.53 (m, 2H, 9-H and 10-H), 1.38 (m,
2H, 9-H0 and 10-H0); 13C NMR (100 MHz, CDCl3): d
197.2, 179.9, 175.8, 150.0, 148.9, 142.5, 137.2, 134.0,
133.9, 35.0, 34.4, 31.3, 25.0, 24.9. Anal. Calcd for:
C14H13O3Cl: C, 64.01; H, 4.22. Found: C, 63.94; H, 4.08.
Leishmanicidal effects of drugs are easily detectable
looking at the regression of parasitophorous vacuoles
and the overall decrease in parasite number. For some
drugs, a
achieved.
complete clearance of amastigotes was
Acknowledgements
Financial support by FONDECYT (Grant 1020885) is
gratefully acknowledged.
References and notes
´
1. Valderrama, J. A.; Benites, J.; Cortes, M.; Pessoa-Mah-
ana, H.; Prina, E.; Fournet, A. Bioorg. Med. Chem. 2003,
11, 4713.
2. Croft, S. L.; Coombs, G. H. Trends Parasitol. 2003, 19,
502.
3. Desjeux, P. Clin. Dermatol. 1966, 14, 417.
4. Molina, R.; Gradoni, L.; Alvar, J. Ann. Trop. Med.
Parasitol. 2003, 97, 29.
5. Croft, S. L.; Yardley, V. Curr. Pharm. Des. 2002, 8,
319.
6. Roberts, W. L.; McMurray, W. J.; Rainey, P. M.
Antimicrob. Agents Chemother. 1998, 42, 1076.
7. Adler-Moore, J.; Proffit, R. T. J. Antimicrob. Chemother.
2002, 49, 21.
8. Unger, C.; Maniera, T.; Kaufmann-Kolle, P.; Eibl, H.
Drugs Today 1998, 34, 133.
9. Escobar, P.; Yardley, V.; Croft, S. L. Antimicrob. Agents
Chemother. 2001, 45, 1872.
10. For recent reviews on natural products with leishmani-
4.1.8. In vitro activity against intracellular L. amazonen-
sis amastigotes. Female BALB/c mice aged 2–4 months
were obtained from the breeding center of the Pasteur
Institute. L. amazonensis strain LV79 (MPRO/BR/
1972/M1841) was propagated in BALB/c mice. L. ama-
zonensis amastigotes were isolated from lesions and
purified as described earlier.24 Bone marrow plugs from
tibias and femurs of BALB/c mice were suspended in
RPMI 1640 medium (Seromed) supplemented with
10% heat-inactivated fetal calf serum (FCS, Dutscher,
Brumath, France), 50 mg/mL of streptomycin, 50 IU/
mL of penicillin (culture medium) and with 15% L-929
fibroblast-conditioned medium. Cells were then distrib-
uted in bacteriologic Petri dishes (Greiner, Germany)
and were incubated at 37 ꢁC in a 5% CO2 atmosphere.
Five days later, adherent macrophages were washed
with DulbeccoÕs phosphate buffered solution (PBS)
and taken off by treatment for 20 min at 37 ꢁC with
Ca2+ and Mg2+-free DulbeccoÕs PBS containing 2 mg/
mL of glucose. Recovered macrophages were suspended
in culture medium and they were then deposited in flat-
bottom 96-well plates (Tanner, Switzerland) at a density
of 4 · 104 cells/well. Twenty-four hours after replating,
macrophages were infected at a multiplicity of five
amastigotes per host cell and were incubated at 34 ꢁC,
which is the permissive temperature for the survival
and multiplication of LV79 strain amastigotes. In most
instances, more than 95% of the macrophages were
found to be infected.
´
cidal activity, see: (a) Chan-Bacab, M. J.; Pen˜a-Rodrıguez,
L. M. Nat. Prod. Rep. 2001, 18, 674; (b) Fournet, A.;
Mun˜oz, V. Curr. Top. Med. Chem. 2002, 2, 1215.
´
´
11. Tapia, R. A.; Alegrıa, L.; Pessoa, C. D.; Salas, C.; Cortes,
M. J.; Valderrama, J. A.; Sarciron, M.-E.; Pautet, F.;
Walchshofer, N.; Fillon, H. Bioorg. Med. Chem. 2003, 11,
2175, and references cited therein.
12. Abreu, F. C. de; Ferraz, P. A. de L.; Goulart, M. O. F. J.
Brazil. Chem. Soc. 2002, 13, 19.
13. Monks, T. J.; Hanzlik, P.; Cohen, G. M.; Ross, D.;
Graham, D. G. Toxicol. Appl. Pharm. 1992, 112, 2.
14. Goulart, M. O. F.; Freitas, L. R.; Tonholo, J.; de Abreu,
F. C.; Raslan, D. S.; Starling, S.; Zani, C. L.; Oliveira, A.
B.; Chiari, E. Bioorg. Med. Chem. Lett. 1997, 7, 2042, and
references cited therein.
´
15. Valderrama, J. A.; Benites, J.; Cortes, M.; Pessoa-Mah-
For all drugs, stock solutions were prepared in DMSO
at a concentration of 500 lg/mL. Twofold serial dilu-
tions were made from 250 lg/mL in culture medium
supplemented with 0.5% DMSO final. Twenty-four
hours after infection, freshly prepared drugs were added
to the infected cultures in triplicate. The first final drug
concentration was 25 lg/mL and the final DMSO con-
centration 0.1%. This DMSO concentration was proven
to have no effect on control cultures.
ana, D.; Prina, E.; Fournet, A. Tetrahedron 2002, 58,
881.
16. Valderrama, J. A.; Astudillo, C.; Tapia, R. A.; Prina, E.;
Estrabaud, E.; Mahieux, R.; Fournet, A. Chem. Pharm.
Bull. 2002, 50, 1215.
17. Pessoa-Mahana, C. D.; Valderrama, J. A.; Olmos, M. G.;
Espinoza, O. A.; Pessoa-Mahana, H.; Rojas de Aria, A.;
Nakayama, H.; Torres, S.; Miret, J. Heterocycl. Commun.
2002, 8, 135.

J. A. Valderrama et al. / Bioorg. Med. Chem. 13 (2005) 4153–4159
4159
´
21. Farin˜a, F.; Valderrama, J. A. An. Quım 1995, 91,
230.
22. Spartan version 5.1.3. Wavefunction Inc., Von Karman
18. Valderrama, J. A.; Pessoa-Mahana, D.; Tapia, R.; Rojas
de Arias, A.; Nakayama, H.; Torres, S.; Miret, J.;
Ferreira, M. E. Tetrahedron 2001, 57, 8653.
19. Valderrama, J. A.; Fournet, A.; Valderrama, C.; Bastias,
S.; Astudillo, C.; Rojas de Arias, A.; Inchausti, A.; Yaluff,
G. Chem. Pharm. Bull. 1999, 47, 1221.
´
Ave. 370, 18401 Irvine, CA, 1999.
´
23. Farin˜a, F.; Valderrama, J. An. Quım. 1974, 70,
258.
20. Kayser, O.; Kiderlen, A. F.; Laatsch, H.; Croft, S. L. Acta
Tropica 2000, 77, 307.
24. Antoine, J. C.; Jouanne, C.; Ryter, A. Parasitology 1989,
99, 1.