Trypanosoma cruzi: Activities of lapachol and α- and β-lapachone derivatives against epimastigote and trypomastigote forms

Article abstract of DOI:10.1016/j.bmc.2007.10.038

Derivatives of natural quinones with biological activities, such as lapachol, α- and β-lapachones, have been synthesized and their trypanocidal activity evaluated in vitro in Trypanosoma cruzi cells. All tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity as compared with current trypanocidal drugs, nifurtimox and benznidazole. The results presented here show that the anti-T. cruzi activity of the α-lapachone derivatives can be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress through redox cycling or production of electrophilic metabolites are the potential biological mechanism of action for these synthetic quinones.

Full text of DOI:10.1016/j.bmc.2007.10.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 668–674
Trypanosoma cruzi: Activities of lapachol and a- and b-lapachone
derivatives against epimastigote and trypomastigote forms
a
a
a
Cristian Salas,a, Ricardo A. Tapia, Karina Ciudad, Veronica Armstrong,
Myriam Orellana,b Ulrike Kemmerling,b Jorge Ferreira,b
Juan Diego Mayab and Antonio Morellob
*
´
aDepartamento de Quı´mica Orga´nica, Facultad de Quı´mica, Pontificia Universidad Cato´lica de Chile,
Av. Vicun˜a Mackenna 4860, Macul, Santiago 6094411, Chile
bDepartamento de Farmacologı´a Molecular y Clı´nica, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Received 20 June 2007; revised 5 October 2007; accepted 15 October 2007
Available online 18 October 2007
Abstract—Derivatives of natural quinones with biological activities, such as lapachol, a- and b-lapachones, have been synthesized
and their trypanocidal activity evaluated in vitro in Trypanosoma cruzi cells. All tested compounds inhibited epimastigote growth
and trypomastigote viability. Several compounds showed similar or higher activity as compared with current trypanocidal drugs,
nifurtimox and benznidazole. The results presented here show that the anti-T. cruzi activity of the a-lapachone derivatives can
be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress
through redox cycling or production of electrophilic metabolites are the potential biological mechanism of action for these synthetic
quinones.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mastigote cultures at a concentration of 0.8 lg/mL.
This was one of the first natural product-derived mole-
cules that showed evidence of oxidative stress generated
in parasites.10–13 Interestingly, compounds derived from
b-lapachone and with an imidazole ring linked to the
naphthopyrane moiety showed enhanced activity com-
pared with 2.14 On the other hand, an oxyran derivative
of a-lapachone has been described as potent trypanoci-
dal agent.15
Chagas disease is one of the most important endemic
diseases caused by Trypanosoma cruzi, which affects
16–18 million people in large areas of Latin America.1,2
The drugs used for the treatment of this disease are nif-
urtimox, a nitrofuran derivative, and benznidazole, a
nitroimidazole derivate. Both drugs present severe side
effects. Furthermore, nifurtimox is no longer used in sev-
eral countries because of its toxicity.3–5 The need of
effective drugs, without adverse effects, has stimulated
the search for new compounds with potential clinical
utility.
In the search for new trypanocidal agents, in this work,
we evaluate the trypanocidal activity of lapachol, a- and
b-lapachone derivatives through inhibition of T. cruzi
epimastigote growth and trypomastigote viability. We
also investigate biological activity, including parasite
free radical production and respiration inhibition of
the parasite.
Many natural and synthetic naphthoquinones have been
tested against T. cruzi parasites as possible anti-chagasic
agents. Among natural naphthoquinones, lapachol (1),
b-lapachone (2), and its a-isomer (3) have demonstrated
trypanocidal activities.6–9 Compound 2 inhibited para-
site motility progressively and also the growth of epi-
Trypanocidal activity of all compounds was tested
against T. cruzi epimastigote growth. The quinones were
incorporated into the medium at different concentra-
tions. Growth inhibition until day 10 was evaluated
in comparison to control at day 5. Nifurtimox and
benznidazol were used as the reference trypanocidal
drugs. To establish the relative efficacy of the quinones
Keywords: Lapachol derivatives; a- and b-Lapachone derivatives;
Anti-trypanosomal activity; Redox cycling; Oxidative stress.
*
Corresponding author. Tel.: +56 2 6864427; fax: +56 2 6864744;
e-mail: cosalas@uc.cl
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.038

C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
Table 1. Effect of lapachol derivatives upon culture growth and
oxygen uptake in T. cruzi epimastigotes
669
O
O
O
a
Compound
ICk50
Respirationb Oxygen redox
cyclingc
Control
Lapachol (1)
— 33.28 (100)
31.3 0.01 16.21 (51.8)
2.2 + 0.15 (100)
1.8 0.30 (82)
i)
2
b-Lapachone (2) 0.21 0.01 32.52 (103.9) 2.0 0.40 (91)
a-Lapachone (3) 24.7 0.36 33.65 (107.5) 2.2 0.40 (100)
O
O
O
4
3.75 1.36 30.30 (96.8)
2.2 0.20 (100)
O
6
35.2 0.07 31.49 (100.6) 2.2 0.20 (100)
24.5 0.29 43.44 (138.8) 3.8 0.20 (173)
1.93 0.07 31.49 (100.6) 2.2 0.10 (100)
OH
7
ii)
8
10
O
26.2 1.14 29.86 (95.4)
36.3 0.15 44.60 (142.5) 3.7 0.15 (168)
2.1 0.20 (96)
11
12
3
>100 39.13 (125)
0.19 0.02 10.39 (33.2)
2.4 0.30 (109)
1.2 0.15 (55)
1
18
19
iii)
20.1 0.04 76.15 (243.3) 5.6 0.30 (254)
38.1 0.04 31.36 (100.2) 2.2 0.15 (100)
22.2 0.07 42.41 (135.5) 3.7 0.20 (168)
9.5 0.10 37.69 (120.4) 2.5 0.15 (113)
O
20
21
O
Nifurtimox
Benznidazole
20.6 0.10 18.09 (57.8)
1.8 0.10 (82)
O
a ICkc50, drug concentration needed to lower the growth constant (kc)
by 50%.
b Oxygen consumption expressed as nanoatoms-gram of oxygen/min/
OH
4
mg protein when drug concentration used was equal to ICk50
Numbers in parentheses correspond to percentage of control.
.
Scheme 1. Synthesis of a- and b-lapachone and derivative 4. Reagents
and conditions: (i) H2SO4, rt, 30 min, 80%; (ii) HCl, 50 ꢁC, 3 h, 70%;
(iii) CH2Cl2, rt, MCPBA, 24 h, (26%).
c Oxygen consumption expressed as nanoatoms-gram of oxygen/min/
mg protein when drug concentration used was equal to ICk50 and
20 mM KCN. Numbers in parentheses correspond to percentage of
control with KCN. The results are mean values of three different
experiments. See Section 4.
alcohol intermediate, in 64–75% yields (Scheme 2). In
the present study, the new a-lapachone derivative 10
was synthesized through Michael adduct 9,24 prepared
by reaction of 2-hydroxy-1,4-naphthoquinone 5 with
benzalacetone in refluxing quinoline for 3 h in 35% yield
(Scheme 1). Reduction of compound 9 with NaBH4 for
3 h in ethanol and further treatment with HCl under re-
flux for 2 more hours gave the pyranonaphthoquinone
10 in 59% yield as a mixture of diastereoisomers. On
the other hand, compounds 11, 12 were synthesized
from 5 according to our previously described proce-
dures.25,26 Finally, derivatives 18–21 were easily pre-
pared in a single step by a Diels–Alder reaction of the
pyranonaphthoquinone 13 and 1-substituted electron-
rich dienes of type 14–17, followed by aromatization
of the resulting adducts (Scheme 3).27–29
in vitro compared with the standard drugs, the ICk50
was determined. ICk50 is defined as the drug concentra-
tion needed to decrease the growth constant (k) by 50%.
The anti-trypanosomal activity of quinones has been
attributed to oxygen radical formation and conse-
quently strong oxidative stress.16–19 To evaluate this
possibility, oxygen uptake experiments with and without
cyanide were undertaken. Respiration is a composite va-
lue where oxygen uptake depends upon mitochondrial
reactions as well as extra-mitochondrial reactions such
as redox cycling.20 Thus, cyanide addition inhibits oxy-
gen consumption in the respiratory chain and allows
possible redox cycling induced by the drug to be ob-
served more clearly. The effect of quinones on T. cruzi
respiration was studied measuring oxygen consumption
by epimastigotes at different concentrations of these
compounds. The results are presented in Table 1.
2.2. Biological evaluation
Table 1 shows the effect of the quinones tested on the
´
growth of Tulahuen strain T. cruzi epimastigotes, at
ICk50 concentration. It is evident from these data that
all tested compounds inhibit parasite growth, the lapa-
chol-derived naphthoquinone 12 being the least active
of the series. However, those compounds with ICk50 val-
ues higher than 20 lM are equipotent when compared
with nifurtimox and benznidazole, and thus devoid of
pharmacological interest.
2. Results and discussion
2.1. Chemistry
Lapachones 2 and 3 and its hydroxylic derivative 4 were
obtained from the commercially available lapachol (1)
by treatment with different acid conditions21 and
meta-chloroperbenzoic acid (MCPBA),22 respectively
(Scheme 1). Pyranonaphthoquinones 7–8 were prepared
from the easily available Michael adduct 623 by reduc-
tion with sodium borohydride followed by cyclization
in acid conditions, without isolation of the respective
The most active compound among lapachone deriva-
tives was pyranoquinolinequinone 18, an a-lapachone
derivative. This result is very interesting because nor-
mally a-lapachones have a weak trypanocidal activ-
ity,15,17,30 indicating that nitrogen substitution in the
aromatic ring increases the trypanocidal activity of these

670
C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
O
O
O
O
O
O
OH
i)
ii)
or
iii)
O
O
O
6
8
7
O
O
O
O
O
O
OH
OH
O
iv)
ii)
O
5
9
10
O
O
v)
O
O
11
O
vi)
OH
OH
O
12
Scheme 2. Synthesis of a- and b-lapachone derivatives 7–12. Reagents and conditions: (i) pyridine, MVK, reflux, 9 h, 90%; (ii) 1—EtOH, NaBH4,
reflux, 1 h; 2—H2SO4 20%, reflux, 2 h, 64% for 7 and X% for 10; (iii) 1—EtOH, NaBH4, reflux, 1 h; 2—HCl 10%, reflux, 3 h, 75%; (iv) quinoline,
benzalacetone, reflux, 3 h, 35%; (v) Ref. 21; (vi) Ref. 24.
O
O
N
O
ii)
N
O
N
NMe2
14
O
i)
O
O
OH
19
18
O
O
O
OCH3
OCH3
O
iii)
16
OH
13
O
20
OTMS
O
iv)
17
O
O
OAc
21
Scheme 3. Synthesis of a-lapachone derivatives 18–21. Reagents and conditions: (i) 1—CH2Cl2, 14, rt, 1,5 h; 2—SiO2, 10 min, Ag2O, rt, 5 h, 33%; (ii)
toluene, PTSA, reflux, 4 h, 68%; (iii) 1—MeOH, 15, rt, 1 h; 2—THF, NaH, 0 ꢁC, 30 min; 3—xilene, reflux, 45 min, 79%; (iv) 1—CH2Cl2, 17, rt, 24 h;
2—SiO2, chromatography, 90%.
compounds. A similar result was obtained in aza-naph-
thofuranoquinones, as previously reported.31 b-Lapach-
one derivatives 4 and 8 were less active than b-lapachone
2, indicating that a hydroxyl group at C-3 and one

C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
671
methyl group at C-2 diminish the activity compared
with b-lapachone.
urtimox and quinones 7, 11, 19, and 21 increase oxygen
uptake. Also, nifurtimox and quinones 7, 11, 19, and 21
present high oxygen redox cycling (Fig. 1), indicating
that reactive oxygen species such as superoxide anion
and hydroxyl radicals are generated, inducing oxidative
stress; (ii) a- and b-lapachone 2, and quinones 4, 6, 8,
and 20 do not inhibit cellular respiration neither pro-
duce redox cycling. The trypanocidal action of these
compounds is probably mediated by the production of
electrophilic metabolites, which bind to and inactivate
T. cruzi macromolecules. Figure 1 shows a proposal of
the metabolic pathways for a molecule with a p-quinone
nucleus in T. cruzi epimastigotes, including the biore-
ductive process, redox cycling, and generation of elec-
trophilic metabolites19; (iii) lapachol and compounds
10 and 18 diminish oxygen uptake and probably inhibit
cellular respiration.
Compound 18, an a-lapachone derivative, is the most
potent of this series, having an ICk50 lower than current
anti-chagasic drugs (50 times and 108 times lower than
nifurtimox and benznidazole, respectively). b-Lapach-
one 2 and its derivatives 8 and 4 showed ICk50 values
4–50 times lower than nifurtimox. Comparing com-
pounds 2 and 18 it is noticeable that p-quinone 18 shows
higher activity than b-lapachone 2,17,30 which is ob-
served for the first time. These results suggest that try-
panocidal activity may be increased in an o-quinone
system if the benzene ring is replaced by a pyridine
moiety.
The ability of these compounds to produce free radicals
in the parasite is a well-known mechanism for the try-
panocidal activity of quinones.16–19 To evaluate this
possibility, we conducted experiments of oxygen uptake
where cellular respiration was measured at the ICk50
concentrations. Quinone addition might produce an in-
crease (redox cycling), a decrease or no alteration in
oxygen uptake. The effect of the studied quinones upon
T. cruzi epimastigote cellular respiration at the ICk50
values can be seen in Table 1.
Table 2 shows the activity of b-lapachone derivatives
upon trypomastigote forms of the parasite. These forms
are found in mammalian blood and are the target for
anti-chagasic drugs. A viability test such as MTT reduc-
tion was carried out using drug concentrations equiva-
lent to ICk50 values for epimastigotes. Concentrations
used (Table 1) were in the range of 0.19–100 lM, and
the value of the MTT test ranged from 3 to 30 percent
compared with control. Derivatives 4, 8, and 18 showed
stronger activity against T. cruzi than nifurtimox or ben-
znidazole. These results indicate that mitochondrial
From our analysis of the results, three effects of the qui-
nones upon oxygen uptake can be distinguished: (i) nif-
Phosphate-pentose
pathway
Cytochrome
P450
NADP
NADPH
Reductase
FADRED
FADOX
O
O
O
O
O
O
R
R
Semiquinone Radical
Quinone
Oxidative damage
to DNA
O2
Protein and lipids
peroxidation
OH
O2
O
O2
+
2
Covalent bond to
nucleic acids
Fe+3
Superoxide
Dismutase
Enzyme
inactivation
Covalent bond to
proteins
(enzyme inactivation)
O2
Catalase
H O
+
H2O2
2
NADPH
2GSH
Glutathione
Peroxidase
Glutathione
Reductase
GS-SG
NADP+
2H2O
Figure 1. Schematic representation of p-quinone metabolism. Redox cycling and production of electrophilic metabolites.

672
C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
Table 2. Effect of lapachol derivatives upon T. cruzi trypomastigote
viabilitya
and the solvent was evaporated. The residue was puri-
fied by column chromatography on silica gel using
chloroform as eluent to afford the quinone 2 (40 mg,
80%), mp 153–154 ꢁC (lit.32 153–154 ꢁC). IR tmax
cmÀ1: 1700, 1645 (CO). 1H NMR (CDCl3): 1.40 (s,
6H, Me), 1.82 (t, 2H, J = 7.0 Hz, H-3), 2.57 (t, 2H,
J = 7.0 Hz, H-4), 7.60 (m, 2H, H-7 and H-8), 8.00
(m, 2H, H-6 and H-9). 13C (CDCl3): 16.2, 26.8 (2C),
31.6, 79.3, 112.3, 124.1, 128.6, 130.1, 130.7, 132.6,
134.8, 162.1, 178.6, 179.9.
Compound
Viability (% of control)
Control
Lapachol (1)
100
41.7
4.8
b-Lapachone (2)
a-Lapachone (3)
2.9
4
30.4
15.6
40.3
112.6
8.8
7
8
12
18
19
4.1.4. 3,4-Dihydro-2,2-dimethyl-2H-naphtho[2,3-b]pyran-
5,10-dione (3). A mixture of lapachol 1 (50 mg,
0.21 mmol) and 12 N hydrochloric acid (20 mL) was
stirred at 70 ꢁC for 3 h. After cooling to room tempera-
ture, ice water (20 mL) was added and the reaction mix-
ture was extracted with chloroform (2· 25 mL). The
combined organic layers were washed with a saturated
solution of sodium bicarbonate, water, dried over mag-
nesium sulfate, and the solvent was evaporated. The res-
idue was purified by column chromatography on silica
gel using chloroform as eluent to afford the quinone 3
(45 mg, 90%), mp 113–114 ꢁC (lit.32 115–116 ꢁC). IR
44.8
26.7
32.9
66.3
20
21
Nifurtimox
a Drug concentrations used were the ICk50 values from Table 1. See
Section 4.
reductase damage is very important in cytotoxicity
experiments.
1
3. Conclusion
tmax cmÀ1: 1682, 1615 (CO). H NMR (CDCl3): 1.44
(s, 6H, Me), 1.83 (t, 2H, J = 6.6 Hz, H-3), 2.63 (t, 2H,
J = 6.6 Hz, H-4), 7.66 (m, 2H, H-7 and H-8), 8.10 (m,
2H, H-6 and H-9). 13C (CDCl3):16.8, 26.5 (2C), 31.4,
78.2, 120.2, 126.0, 126.3, 131.2, 132.1, 132.9, 133.8,
154.6, 180.0, 184.4.
The most active compound against epimastigote and
trypomastigote forms of T. cruzi was the aza-a-lapachone
derivative 18. The results presented herein indicate that
compound 18 is a potential lead compound for the design
of new drugs for Chagas disease. This result may also be
important for the design of new b-lapachone derivatives
with a nitrogen isosteric modification on the aromatic
ring in order to improve the anti-T. cruzi activity.
4.1.5. 3,4-Dihydro-3-hydroxy-2,2-dimethyl-2H-naphtho[1,2-
b]pyran-5,6-dione (4). To a solution of lapachol 1
(100 mg, 0.42 mmol) in dichloromethane (20 mL) was
added m-chloroperbenzoic acid (90 mg, 0.52 mmol)
and the mixture was stirred at room temperature for
24 h. The reaction mixture was washed with a saturated
solution of sodium bicarbonate, water, dried over mag-
nesium sulfate, and the solvent was evaporated. The res-
idue was purified by column chromatography on silica
gel using chloroform/ethyl acetate (2:1) as eluent to af-
ford quinone 4 (45 mg, 42%), mp 203–205 ꢁC (lit.33
204–206 ꢁC). IR tmax cmÀ1: 1700, 1630 (CO). 1H
NMR (CDCl3): 1.45 (s, 3H, Me), 1.53 (s, 3H, Me),
1.90 (s, 1H, OH), 2.64 (dd, 1H, J = 26 and 5.7 Hz, H-
4), 2.82 (dd, 1H, J = 26 and 5.3 Hz, H-4), 3.91 (dd,
1H, J = 5.7 and 5.3 Hz, H-3), 7.75 (m, 4H). 13C
(CDCl3): 21.0, 22.6, 23.2, 69.0, 79.7, 110.0, 124.3,
128.8, 130.1, 131.0, 132.0, 135.0, 161.2, 178.6, 179.4.
4. Experimental
4.1. Chemistry
4.1.1. General remarks. Melting points were determined
with a Meltemp apparatus and are not corrected. IR
spectra were recorded on a Bruker Model Vector 22
1
spectrophotometer using KBr discs. H and 13C NMR
spectra were obtained on Bruker ACP-200 and AM-
400 instruments, using tetramethylsilane as internal ref-
erence. Column chromatography was performed on sil-
ica gel Merck 60 (70–230 mesh). High-resolution mass
spectrum was obtained using a Thermo Finnigan Model
MAT95XP instrument.
4.1.6. 2-Hydroxy-3-(3-oxobutyl)-1,4-naphthoquinone (6).
To a solution of 2-hydroxy-1,4-naphthoquinone 5
(1.0 g, 5.74 mmol) in a pyridine/tert-butanol mixture
(1:10, 25 mL) was added 3-buten-2-one (1.0 mL) and
the mixture was heated at reflux for 12 h. The reaction
mixture was cooled, acidified with 1:1 hydrochloric acid,
and evaporated under reduced pressure. The residue was
washed with water and dried to yield quinone 5 (1.35 g,
96%), mp 149–150 ꢁC (lit.23 149–145.5 ꢁC). IR tmax
4.1.2. Synthetic procedures. Compounds 11–1325,26 and
18–2125–29 were prepared according to our previously
described procedures and their spectroscopic data have
been already reported.
4.1.3. 3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-
5,6-dione (2). A mixture of lapachol
1 (50 mg,
0.21 mmol) and concentrated sulfuric acid (20 mL)
was stirred at room temperature for 0.5 h. The reac-
tion mixture was poured into ice and extracted with
chloroform (2· 25 mL). The combined organic layers
were washed with a saturated solution of sodium
bicarbonate, water, dried over magnesium sulfate,
1
cmÀ1: 1712, 1660, 1650 (CO). H NMR (CDCl3): 2.19
(s, 3H, Me), 2.80 (m, 4H), 7.65 (s, 1H, OH), 7.70 (m,
2H, H-6 and H-7), 8.09 (m, 2H, H-5 and H-8). 13C
(CDCl3): 17.9, 29.6, 41.7, 122.8, 126.2, 126.8, 129.5,
132.8, 133.1, 134.9, 153.5, 181.1, 184.6, 208.2.

C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
673
4.1.7. 3,4-Dihydro-2-methyl-2H-naphtho[2,3-b]pyran-5,10-
dione (7). To a solution of 5 (50 mg, 0.21 mmol) in eth-
anol (10 mL) was added sodium borohydride (50 mg,
1.56 mmol) and the mixture was heated at reflux for
1 h. After cooling to room temperature, aqueous sulfu-
ric acid (20%, 30 mL) was added dropwise and the mix-
ture was heated at reflux for 2 h. The reaction mixture
was cooled and extracted with dichloromethane (2·
25 mL). The combined organic layers were washed with
a saturated solution of sodium bicarbonate, water, dried
over magnesium sulfate, and the solvent was evapo-
rated. The residue was purified by column chromatogra-
phy on silica gel using chloroform as eluent to afford the
quinone 7 (30 mg, 64%), mp 121–122 ꢁC (lit.32 122.5 ꢁC).
IR tmax cmÀ1: 1675, 1650 (CO). 1H NMR (CDCl3): 1.50
(d, 3H, J = 6.4 Hz, Me), 1.85 (m, 2H, H-3), 2.6 (m, 2H,
H-4), 4.31 (m, 1H, H-2), 7.66 (m, 2H, H-7 and H-8),
8.06 (m, 2H, H-6 and H-9). 13C (CDCl3): 18.3, 20.5,
27.2, 74.4, 121.2, 126.0, 126.3, 131.0, 132.0, 133.0,
133.9, 155.4, 179.8, 184.3.
dure for the preparation of 7, yielding pure product 10
(28 mg, 59%), mp 210–211 ꢁC. IR tmax cmÀ1: 1682,
1652 (CO). H NMR (CDCl3): 1.38 (d, 3H, J = 6.3 Hz,
Me), 2.05 (2H, m, CH2), 4.34 (2H, m, H-2 and H-4),
7.36 (5H, m, arom H), 7.65 (2H, m, arom H), 8.06
(2H, m, arom H). 13C (CDCl3): 114.6, 117.4, 124.5,
126.3, 126.9, 126.7, 127.8, 128.6, 128.8, 130.4, 131.0,
132.2, 135.0, 143.6, 163.9, 178.1,179.6. HRMS calcd
for C20H16O3 304.10995. Found: 304.10922.
1
4.2. Biology
4.2.1. Epimastigote culture and growth inhibition assays.
´
Trypanosoma cruzi epimastigotes, Tulahuen strain, were
cultured, at an initial density of 3 · 106 parasites/mL, at
28 ꢁC in monophasic Diamond’s culture medium, sup-
plemented with 4 lM Hemin, bovine fetal calf serum
at a final concentration of 5% v/v, and sodium penicillin
100 mg/mL and streptomycin 100 mg/mL. The assayed
drugs were dissolved in 1% dimethylsulfoxide (DMSO).
All measurements were carried out in triplicate. Epimas-
tigote growth was followed daily by nephelometry for 10
days. The nephelometry measurements were directly
proportional to the number of parasites in suspension.
No toxic effect was attributable to DMSO at the final
concentration of 1%. Since parasite growth is exponen-
tial, the data were analyzed according to a first order
equation and the respective growth constant (k), corre-
sponding to the slope, was calculated for controls and
parasites treated with different drug concentrations.
The ICk50 is defined as the drug concentration needed
to lower the k by 50%.
4.1.8. 3,4-Dihydro-2-methyl-2H-naphtho[1,2-b]pyran-5,6-
dione (8). To a solution of 6 (50 mg, 0.21 mmol) in eth-
anol (10 mL) was added sodium borohydride (50 mg,
1.56 mmol) and the mixture was heated at reflux for
1 h. After cooling to room temperature the reaction mix-
ture was diluted with water (30 mL), acidified with 10%
HCl, and extracted with dichloromethane (2· 20 mL).
To the combined organic phase, dried with anhydrous
magnesium sulfate, were added five drops of boron tri-
fluoride-etherate and heated at reflux for 1 h. After
evaporation of the solvent, the residue was purified by
column chromatography on silica gel using chloroform
as eluent to afford quinone 8 (35 mg, 75%), mp 163–
164 ꢁC (lit.25,26 164 ꢁC). IR tmax cmÀ1: 1670, 1645
(CO). 1H NMR (CDCl3): 1.54 (d, 3H, J = 6.3 Hz,
Me), 1.84 (m, 2H, H-2), 2.60 (m, 2H, H-4), 4.38 (m,
1H, H-4), 7.55 (m, 2H, H-8 and H-9), 7.80 (m, 1H, H-
10), 8.06 (m, 1H, H-7). 13C (CDCl3): 18.0, 20.7, 27.7,
75.3, 113.7, 124.0, 128.7, 130.0, 130.7, 132.3, 134.6,
162.9, 178.7, 180.0.
4.2.2. Trypomastigotes. VERO cells were infected with
MF strain T. cruzi metacyclic trypomastigotes from
15 days old epimastigote cultures.34 Subsequently, the
trypomastigotes harvested from this culture were used
to reinfect further VERO cell cultures at 1 · 106 para-
sites per 25 cm2 density. VERO cell cultures infected
with trypomastigotes were incubated at 37 ꢁC in
humidified air and 5% CO2 for 5–7 days. After that
time, the culture medium was collected, centrifuged at
3000g for 5 min, and the trypomastigote-containing
pellet was resuspended in RPMI supplemented with fe-
tal bovine serum 5% and penicillin–streptomycin at a
final density of 1 · 107 parasites/mL. 210 · 106 trypom-
astigotes are equivalent to 1 mg of protein or 12 mg of
wet weight.
4.1.9. 2-Hydroxy-3-(1-phenyl-3-oxobutyl)-1,4-naphtho-
quinone (9). To a solution of 2-hydroxy-1,4-naphthoqui-
none 5 (1.0 g, 5.74 mmol) in quinoline (25 mL) was
added 4-phenyl-3-buten-2-one (1.0 g, 6.85 mmol) and
the mixture was heated at reflux for 3 h. The solvent
was removed at reduced pressure and the residue was
purified by column chromatography on silica gel using
chloroform as eluent to afford quinone 9 (0.65 g, 35%),
mp 143–144ꢁC (lit.24 143–144 ꢁC). IR tmax cmÀ1: 1712,
4.2.3. Oxygen uptake and redox cycling. Epimastigotes
were harvested on the 4th or 5th day of culture by
centrifugation at 500g for 10 min, followed by wash-
ing and re-suspension in 0.05 M sodium phosphate
buffer (0.107 M NaCl; 0.05 M KH2PO4; pH 7.4). Res-
piration measurements were carried out polarographi-
cally with a Clark 5331 electrode (Yellow Spring
Instruments) in a 53 YSI model oxygraph. The cham-
ber volume was 1.0 mL and the temperature 28 ꢁC.
The amount of parasites used was equivalent to
1 mg of protein. The drugs were added at epimasti-
gote ICk50 values. To evaluate drug-induced oxygen
redox cycling, mitochondrial respiration was inhibited
with 20 mM KCN.
1
1660, 1650 (CO). H NMR (CDCl3): 2.16 (s, 3H, Me),
3.23 (dd, 1H, J = 18 and 8 Hz, CH2), 3.75 (dd, 1H,
J = 18 and 10 Hz, CH2), 4.96 (dd, 1H, J = 10 and
6 Hz, CH), 7.36 (m, 5H), 7.64 (s, 1H, OH), 7.69 (m,
2H, H-6 and H-7), 8.06 (m, 2H, H-5 and H-8). 13C
(CDCl3): 30.0, 36.4, 46.3, 124.6, 125.6, 126.1, 126.8,
127.0, 127.7, 128.6 (2C), 129.2 (2C), 132.9, 135.1,
141.7, 152.6, 181.7, 184.3, 207.1.
4.1.10. 3,4-Dihydro-2-methyl-4-phenyl-2H-naphtho[2,3-
b]pyran-5,10-dione (10). Quinone 10 was prepared from
compound 9 (50 mg, 0.16 mmol) according to the proce-

674
C. Salas et al. / Bioorg. Med. Chem. 16 (2008) 668–674
13. Boveris, A.; Stoppani, A. O.; Docampo, R.; Cruz, F. S.
Comp. Biochem. Physiol. C 1978, 61 C, 327–329.
14. De Moura, K. C.; Salomao, K.; Menna-Barreto, R. F.;
Emery, F. S.; Pinto Mdo, C.; Pinto, A. V.; de Castro, S. L.
Eur. J. Med. Chem. 2004, 39, 639–645.
15. (a) Ferreira, V. F.; Jorqueira, A.; Souza, A. M.; da Silva,
M. N.; de Souza, M. C.; Gouvea, R. M.; Rodrigues, C. R.;
Pinto, A. V.; Castro, H. C.; Santos, D. O.; Araujo, H. P.;
Bourguignon, S. C. Bioorg. Med. Chem. 2006, 14, 5459–
5466; (b) Jorqueira, A.; Gouvea, R. M.; Ferreira, V. F.; da
Silva, M. N.; de Souza, M. C.; Zuma, A. A.; Cavalcanti,
D. F.; Araujo, H. P.; Santos, D. O.; Bourguignon, S. C.
Parasitol. Res. 2006, 99, 429–433.
16. Teixeira, M. J.; de Almeida, Y. M.; Viana, J. R.; Holanda
Filha, J. G.; Rodrigues, T. P.; Prata, J. R., Jr.; Coelho, I.
C.; Rao, V. S.; Pompeu, M. M. Phytother. Res. 2001, 15,
44–48.
17. Lopes, J. N.; Cruz, F. S.; Docampo, R.; Vasconcellos, M.
E.; Sampaio, M. C.; Pinto, A. V.; Gilbert, B. Ann. Trop.
Med. Parasitol. 1978, 72, 523–531.
4.2.4. Trypomastigote viability assay. Viability assays
were performed using the MTT reduction method as de-
scribed previously.35 1 · 107 trypomastigotes were incu-
bated in FBS-RPMI culture medium at 37 ꢁC during
24 h with or without the drugs studied. An aliquot of
the parasite suspension was extracted and incubated in
a 96-flat bottom well plate and MTT was added at
0.5 mg/mL final concentration, incubated at 28 ꢁC dur-
ing 4 h and then made soluble with 10 % SDS–0.1 mM
HCl and incubated overnight. Formazan formation
was measured at 570 nm with reference wavelength at
690 nm in a multiwell reader (Labsystems Mult-
oskanMS, Finland).
Acknowledgments
This research was supported by FONDECYT (Research
Grants 1020874 and 1061072), Proyecto Anillo ACT 29
CONICYT/PBCT, and DIPUC (Proyecto de Inicio 22
PI/2005).
18. Amarante-Mendes, G. P.; Green, D. R. Braz. J. Med.
Biol. Res. 1999, 32, 1053–1061.
19. Ollinger, K.; Kagedal, K. Subcell Biochem. 2002, 36, 151–
170.
20. Morello, A.; Pavani, M.; Garbarino, J. A.; Chamy, M. C.;
Frey, C.; Mancilla, J.; Guerrero, A.; Repetto, Y.; Ferreira,
J. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endo-
crinol. 1995, 112, 119–128.
References and notes
1. Fournet, A.; Munoz, V. Curr. Top. Med. Chem. 2002, 2,
1215–1237.
2. Coura, J. R.; Castro, S. L. d. Mem. Inst. Oswaldo Cruz
2002, 97, 3–24.
3. Zahoor, A.; Lafleur, M. V.; Knight, R. C.; Loman, H.;
Edwards, D. I. Biochem. Pharmacol. 1987, 36, 3299–3304.
4. Croft, S. L.; Barrett, M. P.; Urbina, J. A. Trends Parasitol.
2005, 21, 508–512.
5. Castro, J. A.; de Mecca, M. M.; Bartel, L. C. Hum. Exp.
Toxicol. 2006, 25, 471–479.
6. Maya, J. D.; Morello, A.; Repetto, Y.; Tellez, R.;
Rodriguez, A.; Zelada, U.; Puebla, P.; Caballero, E.;
Medarde, M.; Nunez-Vergara, L. J.; Squella, J. A.; Bonta,
M.; Bollo, S.; San Feliciano, A. Comp. Biochem. Physiol.
C Toxicol. Pharmacol. 2000, 125, 103–109.
21. Hooker, S. C. J. Am. Chem. Soc. 1936, 58, 1190–1197.
22. Sun, J. S.; Geiser, A. H.; Frydman, B. Tetrahedron Lett.
1998, 39, 8221–8224.
23. Rafart, H. V. J.; Vega, J. C. An. Quim. 1976, 72, 804–808.
24. Zaugg, H. E. J. Am. Chem. Soc. 1949, 71, 1890–1891.
25. Saitz, C.; Valderrama, J.; Tapia, R. Synth. Commun. 1990,
20, 3108–3114.
26. Cassis, R.; Tapia, R.; Valderrama, J. An. Quim. 1977, 72,
1512–1515.
27. Zuloaga, F.; Tapia, R.; Quintanar, C. J. Chem. Soc.,
Perkin Trans. 2 1995, 939–943.
28. Tapia, R.; Valderrama, J.; Quintanar, C. Heterocycles
1994, 38, 1797–1802.
29. Saitz, C.; Valderrama, J.; Tapia, R. Synth. Commun. 1992,
22, 955–960.
7. Maya, J. D.; Bollo, S.; Nunez-Vergara, L. J.; Squella, J.
A.; Repetto, Y.; Morello, A.; Perie, J.; Chauviere, G.
Biochem. Pharmacol. 2003, 65, 999–1006.
8. Zani, C. L.; Chiari, E.; Krettli, A. U.; Murta, S. M.;
Cunningham, M. L.; Fairlamb, A. H.; Romanha, A. J.
Bioorg. Med. Chem. 1997, 5, 2185–2192.
9. Zani, C. L.; Fairlamb, A. H. Mem. Inst. Oswaldo Cruz
2003, 98, 565–568.
10. Boveris, A.; Docampo, R.; Turrens, J. F.; Stoppani, A. O.
Rev. Assoc. Argent Microbiol. 1977, 9, 54–61.
11. Docampo, R.; Lopes, J. N.; Cruz, F. S.; Souza, W. Exp.
Parasitol. 1977, 42, 142–149.
30. Goulart, M. O. F.; Zani, C. L.; Tonholo, J.; Freitas, L.
R.; deAbreu, F. C.; Oliveira, A. B.; Raslan, D. S.;
Starling, S.; Chiari, E. Bioorg. Med. Chem. Lett. 1997,
7, 2043–2048.
31. Tapia, R. A.; Salas, C.; Morello, A.; Maya, J. D.; Toro-
Labbe, A. Bioorg. Med. Chem. 2004, 12, 2451–2458.
32. Fieser, L. F. J. Am. Chem. Soc 1929, 49, 857–864.
33. Mock, J. M. S. T.; Rotchie, E.; Taylor, W. C. Aust. J.
Chem. 1973, 26, 1121–1130.
34. Contreras, V. T.; Salles, J. M.; Thomas, N.; Morel, C. M.;
Goldenberg, S. Mol. Biochem. Parasitol. 1985, 16, 315–
327.
12. Boveris, A.; Docampo, R.; Turrens, J. F.; Stoppani, A. O.
Biochem. J. 1978, 175, 431–439.
35. Muelas-Serrano, S.; Nogal-Ruiz, J. J.; Gomez-Barrio, A.
Parasitol. Res. 2000, 86, 999–1002.