Synthesis, cytotoxicity, and anti-Trypanosoma cruzi activity of new chalcones

Article abstract of DOI:10.1021/jm800812k

Synthesis of a cytotoxic dihydrochalcone, first isolated from a traditional Amazonian medicinal plant Iryanthera juruensis Warb (Myristicaceae), followed by a comprehensive SAR analysis of saturated and unsaturated chalcone synthetic intermediates, led to the identification of analogues with selective and significant in vitro anti-Trypanosoma cruzi activity. Further SAR studies were undertaken with the synthesis of 21 new chalcones containing two allyloxy moieties that resulted in the discovery of 2′,4′-diallyloxy- 6′-methoxy chalcones with improved selectivity against this parasite at concentrations below 25 μM, four of which exhibited a selectivity index greater than 12.

Full text of DOI:10.1021/jm800812k

6230
J. Med. Chem. 2008, 51, 6230–6234
Synthesis, Cytotoxicity, and Anti-Trypanosoma cruzi Activity of New Chalcones
Jose´ C. Aponte,† Manuela Vera´stegui,§ Edith Ma´laga,§ Mirko Zimic,‡ Miguel Quiliano,‡ Abraham J. Vaisberg,‡
Robert H. Gilman,| and Gerald B. Hammond†,*
Department of Chemistry, UniVersity of LouisVille, LouisVille, Kentucky 40292, Laboratorios de InVestigacio´n y Desarrollo, Facultad de
Ciencias y Filosof´ıa, UniVersidad Peruana Cayetano Heredia, Lima, Peru´, Departamento de Microbiolog´ıa, Facultad de Ciencias y Filosof´ıa,
UniVersidad Peruana Cayetano Heredia, Lima, Peru´, and Department of International Health, Johns Hopkins UniVersity School of Public
Health, Baltimore, Maryland 21205
ReceiVed July 6, 2008
Synthesis of a cytotoxic dihydrochalcone, first isolated from a traditional Amazonian medicinal plant
Iryanthera juruensis Warb (Myristicaceae), followed by a comprehensive SAR analysis of saturated and
unsaturated chalcone synthetic intermediates, led to the identification of analogues with selective and
significant in vitro anti-Trypanosoma cruzi activity. Further SAR studies were undertaken with the synthesis
of 21 new chalcones containing two allyloxy moieties that resulted in the discovery of 2′,4′-diallyloxy-6′-
methoxy chalcones with improved selectivity against this parasite at concentrations below 25 µM, four of
which exhibited a selectivity index greater than 12.
Introduction
benznidazole, and nifurtimox; these drugs were developed for
veterinary use more than 30 years ago, and are effective only
in the acute phase of the disease. Their wider utilization has
been hindered by lack of compliance, low effectiveness, drug
resistance, high cost, and severe side effects.12,13 Clearly, a
search for new types of drugs with high selectivity, minimum
side effects, and low manufacture costs is urgently needed. The
economical, facile, and rapid synthesis of chalcones14 make them
attractive as potential drug candidates to fight some of the so-
called neglected diseases that affect the populations of many
countries in the Third World, chiefly among them leishmaniasis
and Chagas disease.
As part of our ongoing search for biologically active meta-
bolites from Peruvian medicinal plants,15 we recently reported
the isolation of two dihydrochalcones from the ethyl ether extract
of Iryanthera juruensis Warb (Myristicaceae); one of them, 2′,4′-
dihydroxy-4,6′-dimethoxydihydrochalcone, was found to be a
major cytotoxic metabolite when tested against a panel of cancer
cell lines.16 With the aim of confirming its structure, we
synthesized the natural product and three series of chalcones
used as intermediates in the synthesis, namely 2′,4′-dihydroxy-
6′-methoxychalcones (2′,4′-HC), 2′-hydroxy-4′,6′-dimethoxy-
chalcones (4′,6′-MC), and 2′,4′-diallyloxy-6′-methoxychalcones
(2′,4′-AC) (4-9 in Scheme 1). During this study, we screened
the in vitro antiproliferative and anti-T. cruzi activity of the
synthetic compounds (Tables 1 and 2). Motivated by the
promising anti-T. cruzi results found for the 2′,4′-AC series,
we synthesized and evaluated 19 new analogues, making
different substitutions on their B ring, to test whether their
selectivity could be further improved and their cytotoxicity
diminished (Table 3).
Chalcones, or 1,3-diaryl-2-propen-1-ones, are prominent sec-
ondary metabolites precursors of flavonoids and isoflavonoids
in plants. Chemically, they consist of open-chain flavonoids in
which the two aromatic rings are joined by a three-carbon R,ꢀ-
unsaturated carbonyl system. The wide variety of pharmacologi-
cal activities reported for these compounds include anticancer,
antiinflammatory, immunomodulatory, antibacterial, and im-
munosuppressive, as well as antiprotozoan activity, including
trypanocidal, leishmanicidal, and antimalarial.1-7 Chagas’ dis-
ease or American trypanosomiasis, caused by the vector-borne
flagellate protozoan parasite Trypanosoma cruzi,a is an endemic
tropical disease that has infected 20 million people in Central
and South America and approximately between 50000 and
100000 people in the United States.8,9 Responsible for around
20000 deaths per year, Chagas’ disease manifests itself as
potentially fatal cardiopathy or dilations in the digestive tract.10
Its cycle starts when the reduviid bug feeds on blood and
releases an infectious metacyclic trypomastigote into the
bloodstream. Trypomastigotes penetrate various tissues, where
they differentiate by binary fission into amastigotes. Intracellular
amastigotes transform back into trypomastigotes bursting the
host cell and releasing the infectious form to the bloodstream,
giving rise to the clinical manifestations and amplifying the
infection.11 With no available vaccine, the only control interven-
tion is chemotherapy involving the use of nitroheterocycles,
* To whom correspondence should be addressed. Phone: (+1)502-852-
5998. Fax: (+1)502-852-3899. E-mail: gb.hammond@louisville.edu.
†
Department of Chemistry, University of Louisville.
Laboratorios de Investigacio´n y Desarrollo, Facultad de Ciencias y
‡
Filosof´ıa, Universidad Peruana Cayetano Heredia.
§
Departamento de Microbiolog´ıa, Facultad de Ciencias y Filosof´ıa,
Universidad Peruana Cayetano Heredia.
Chemistry
|
Department of International Health, Johns Hopkins University School
of Public Health.
The synthesis of these compounds started with the preparation
of different series of acetophenones, as summarized in Scheme
1. Initially, 2,4,6-trihydroxyacetophenone is transformed into
2,4-dihydroxy-6-methoxyacetophenone using dimethyl sulfate
as methylating agent. Then, the methyl group on the para-
methoxy position of the acetophenone is cleaved using AlCl3
to obtain 2,4-dihydroxy-6-methoxyaceptophenone,17 which is
protected using allyl bromide to give 2,4-allyloxy-6-methoxy-
a Abbreviations: T. cruzi, Trypanosoma cruzi; 2′,4′-HDC, 2′,4′-dihydroxy-
6′-methoxydihydrochalcones; 4′,6′-MDC, 2′-hydroxy-4′,6′-dimethoxydihy-
drochalcones; 2′,4′-HC, 2′,4′-dihydroxy-6′-methoxychalcones; 4′,6′-MC, 2′-
hydroxy-4′,6′-dimethoxychalcones; 2′,4′-AC, 2′,4′-diallyloxy-6′-methoxy-
chalcones; TPSA, topological polar surface area; %ABS, percentage of
absorption; n-ROTB, number of rotatable bonds; miLogP, logarithm of
compound partition coefficient between n-octanol and water; n-OHNH,
number of hydrogen bond donors; n-ON, number of hydrogen bond accep-
tors.
10.1021/jm800812k CCC: $40.75
2008 American Chemical Society
Published on Web 09/18/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6231
Scheme 1. Strategy for the Synthesis of Compounds 1-9a
Table 2. In Vitro anti-T. cruzi Activity of Compounds 1-9
IC50 (µM)a
compd
T. cruzi
VEROb
SIc
1
2
3
4
5
6
7
8
9
>25
>100
>100
>25
>25
>25
9.4
21.4
13.6
0.52
NDd
ND
ND
ND
ND
ND
25.5
99.9
73.5
80.1
2.7
4.7
5.4
Nifurtimox
154
a IC50: concentration that produces 50% inhibitory effect. b VERO, normal
African green monkey kidney epithelial cells. c SI: Selectivity index )
IC50,VERO/IC50,T. cruzi.
d Not determined.
Table 3. In vitro anti-T. cruzi Activity of Compounds 10-28
a Reagents and conditions: (a) K2CO3, (CH3)2SO4, (CH3)2CO, 65 °C,
6 h. (b) AlCl3, benzene, reflux, 1 h. (c) K2CO3, allyl bromide, DMF, rt,
overnight. (d) Claisen-Schmidt aldol condensation of an acetophenone with
an aromatic aldehyde, KOH, H2O, CH3OH, rt, 1-48 h. (e) K2CO3, catalytic
Pd(PPh3)4, MeOH, 60 °C, 1 h. (f) catalytic Pd/C 5%, H2 gas, 250 psi, EtOAc,
rt, 1.5 h.
Table 1. Cytotoxicity of Compounds 1-9
GI50 (µM) in indicated cell linea
compd 3T3 H460 DU145 MCF-7 M-14 HT-29 PC3 K562
1
2
3
4
5
6
7
8
9
>206 128.0 >206 65.5
109.2 >206 59.9 22.8
>197 >197 >197 >197 >197 >197 >197 >197
21.5
17.2
39.0
6.7
105.3 60.2
24.1 30.8
42.4
17.5
82.0
22.5
144.7 39.1 28.2
45.3 15.2 20.5
>190 >190 140.1 >190 >190 118.5 18.9
14.0
15.6
20.0
14.5
9.0
17.3
20.4
19.5
10.9
6.5
19.6
22.6
27.6
17.2
22.4
21.6
20.7
28.4
24.1
8.9
19.3 17.0
18.5 16.2
18.1 16.6
5.1
12.4
21.0
17.5
15.4
41.0
31.7
9.6
12.2
5FUb <1.54 4.1
14.6 16.7
a 3T3, BALB/3T3 clone A31 embryonic mouse fibroblast cells; H460,
human large cell lung cancer; DU145, human prostate carcinoma; MCF-7,
human breast adenocarcinoma; M-14, human melanoma; HT-29, human
colon adenocarcinoma; PC3, human prostate adenocarcinoma; K562, human
chronic myelogenous leukemia cells. b 5FU, 5-Fluoruracil.
acetophenone.18 Claisen-Schmidt aldol condensation of ac-
etophenone with the corresponding aromatic aldehyde in the
presence of aqueous KOH gives the chalcone product.2,3 After
a mild deprotection procedure to remove the allyl-protecting
groups, using Pd(PPh3)4 and K2CO3, the resulting 2′4′-dihy-
droxy-6-methoxy chalcones19 were finally reduced to produce
the corresponding dihydrochalcones.20
a IC50: concentration that produces 50% inhibitory effect. b VERO, normal
African green monkey kidney epithelial cells. c SI: Selectivity index ) IC50,
VERO/IC50, T. cruzi.
d Not determined.
Results
In the present study, three dihydrochalcones and six chalcones
were tested for their antiproliferative activity against a panel of
seven cancer cell lines and one nontumorogenic cell line (Table
1). Those initial nine compounds, plus 19 synthetic chalcones
analogues, were also evaluated for their in vitro anti-T. cruzi
activity. Their structures and bioactivities are presented in Tables
2 and 3. The cytotoxic assays showed that dihydrochalcones 1
and 2 were less active than their R,ꢀ unsaturated counterparts,
namely chalcones 6 and 4 respectively. Interestingly, dihydro-
chalcone 3 showed greater cytotoxicity than its corresponding
parent chalcone, 5. 2′,4′-HC series was more active than the
other two series of chalcones, 4′,6′-MC and 2′,4′-AC. However,
when assayed for in vitro anti-T. cruzi activity, only chalcones
7-9 showed activity. We were pleased to find a moderate degree

6232 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
Table 4. Physical Chemical Properties of Compounds 1-28a
n- n-
of selectivity for the two 2′,4′-diallyloxy chalcones when
compared with compound 7. Thus, we halted our anticancer
screening due to the lack of selectivity observed with the cell
lines assayed and instead directed our efforts to investigate the
more promising antitrypanosomal activity. As shown in Table
3, 16 of the new synthetic 8 and 9 derivatives (compounds
10-13, 15-23, 25, 16, and 28) showed activity against T. cruzi
at concentrations below 25 µM, and four of them exhibited a
selectivity index greater than 12.
TPSA n- molecular
OHNH ON Lipinski’s
ID %ABS (Å2) ROTB weight miLogP donors acceptors violations
rule
1
2
3
4
5
6
7
8
<500
302.3
e5
3.26
<5
2
1
1
1
1
2
2
0
0
0
0
0
2
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
<10
5
5
6
5
6
5
6
5
6
4
4
5
5
6
6
7
4
4
4
4
4
7
6
4
5
5
5
5
e1
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
1
1
1
1
1
1
2
1
0
0
0
0
82.8 76.0
86.6 65.0
83.4 74.2
86.6 65.0
83.4 74.3
82.8 76.0
79.6 85.2
6
7
6
6
5
5
4
316.4
330.3
314.3
328.3
300.3
314.3
380.4
394.4
350.4
364.4
380.4
352.4
396.4
410.5
440.5
418.4
384.9
368.4
368.4
429.3
395.4
541.4
376.5
351.4
351.4
339.4
340.4
3.79
3.63
3.83
3.66
3.29
3.12
5.18
5.02
5.13
5.58
5.16
4.58
4.47
4.99
4.76
6.02
5.80
5.29
5.27
5.91
5.09
7.19
5.65
3.84
3.78
4.10
4.20
Discussion
90.4 54.0 11
87.2 63.2 10
9
The bioassay-guided fractionation of the ethyl ether extract
of I. juruensis Warb (Myristicaceae), a medicinal plant used
by the Aguaruna community living in the Peruvian rainforest,
led to the isolation of 2′,4′-dihydroxy-4,6′-dimethoxydihydro-
chalcone (1) as one of its main cytotoxic metabolites against a
panel of cancer cell lines.16 To synthesize the natural compound,
we prepared three series of chalcones (4′,6′-MC, 2′,4′-HC, and
2′,4′-AC), which were used as chemical intermediates. The
synthesis of this natural compound proved to be not an easy
task due to the presence of the dihydroxy phenolic groups. When
2,4-dihydroxy-6-methoxyacetophenone was subjected to a
Claisen-Schmidt condensation, no chalcone product was
obtained using regular conditions because of the low pKa (∼7)
of the resorcinol. Protection of these two positions in the
acetophenone was needed to proceed with the synthesis. We
chose the allyloxy group as a protecting agent, mainly because
it would be a natural-like moiety compared to silicon-based
protecting groups and that it would resist acidic pH when
compared to acetal protecting groups. Also, chalcones bearing
a benzyl ether protecting group have proved to be inactive as
antitumoral agents.3 In addition, the allyloxy moiety would
increase lipophilicity. To the best of our knowledge, this group
has not been used before as protecting group in the synthesis
of chalcones.
Extensive studies on the antimitotic and antiproliferative
effects of chalcones possessing hydroxyl and methoxyl groups
at the different positions of the A-ring and B-ring of chalcones
have been reported.2-5 Herein, we present, for the first time,
the antiproliferative effect of 2′,4′-HC and 2′,4′-AC chalcones.
In general, chalcones’ cytotoxicities were higher than their
dihydro counterparts lacking the R,ꢀ unsaturated carbonyl group,
which can react with nucleophiles such as glutathione (GSH).
Among chalcones, 2′,4′-HC chalcones (6, 7) were more active
than 4′,6′-MC (4, 5) and 2′,4′-AC chalcones (8, 9). The
antiproliferative activity of 2′,4′-HC chalcones is in agreement
with the results reported by Rao and co-workers,5 which showed
that the activity of 2′-hydroxychalcone derivatives was due to
an inhibition of cell cycle progression rather than a direct
cytotoxic effect, using the trypan blue exclusion test on Jurkat
and U937 cancer cells. The lower cytotoxic activity of 4′,6′-
MC and 2′,4′-AC chalcones may be explained by the substitu-
tions on the ortho position of the ring A, which could affect
the planarity of the molecule. Compound 8 is slightly less active
than its structurally related 4, which indicates that the cytotox-
icity decreases as the size of the ortho substituent on ring A
increases; however, it also demonstrates that some variations
are possible in the substitution pattern before the activity is lost.
Although the antiprotozoal activity of chalcones have been
proven against leishmaniasis and malaria,1,6,21 their activity
against T. cruzi have been scarcely reported.7 The use of an in
vitro assay using a T. cruzi strain with an enzyme insert
represents a major advance because the method allows for high
throughput screening for assessing multiple chemical agents for
10 93.5 44.8 10
11 93.5 44.8 10
12 90.4 54.0 11
13 82.8 76.0
9
14 83.4 74.2 11
15 87.2 63.2 12
16 84.0 72.5 13
17 93.5 44.8 11
18 93.5 44.8 10
19 93.5 44.8 10
20 93.5 44.8 10
21 93.5 44.8 10
22 77.9 90.6 11
23 87.2 63.2 16
24 93.5 44.8 11
25 89.1 57.7 10
26 89.1 57.7 10
27 88.1 60.6 10
28 89.0 57.9 10
a %ABS, percentage of absorption; TPSA, topological polar surface area;
n-ROTB, number of rotatable bonds; miLogP, logarithm of compound
partition coefficient between n-octanol and water; n-OHNH, number of
hydrogen bond donors; n-ON, number of hydrogen bond acceptors.
their anti T. cruzi efficacy.22,23 Our laboratory is now making
new inserts for other strains because there is significant strain-
to-strain variation in sensitivity. In addition, it is essential to
have toxicity assays performed because highly toxic compounds
are not useful for treatment of this disease. Drug candidates
that have high efficacy and low toxicity will need further testing
in animal models. In the present study, the anti-T. cruzi activity
of 2′,4′-AC is reported for the first time. Although active, it
can be observed that 2′,4′-AC bearing electron withdrawing
groups on ring B (17-23, 25, 26) are highly toxic to VERO
cells. On the other hand, 2′,4′-AC having electron donating
groups on ring B (11, 12, 15, 16, 28) showed selectivity indexes
greater than 7. As can be seen, when comparing compounds 1
and 24, the addition of a double bond on the R-ꢀ unsaturated
bridge results on the loss of the activity. The high activity of
pyridinium chalcones (25, 26) could not be maintained by the
pyrrole analogue 27; however, when ring B was replaced by
furan (28), we obtained the highest selectivity index of the series.
Although the selectivity indexes of our synthetic compounds
did not match the positive control (Nifurtimox), it is known
that this drug is only efficient during the acute phase of the
disease and is also responsible for severe side effects such as
anorexia, vomiting, and peripheral polyneuropathy, among
others.12 Chalcones are known to inhibit mitochondrial Leish-
mania parasite proteins such fumarate reductase, succinate
dehydrogenase, NADH dehydrogenase, and NADH-cytochrome
c reductase.21
A computational study for prediction of ADME properties
of all molecules was performed and is presented in Table 4.
Topological polar surface area (TPSA) is a good indicator of
drug absorbance in the intestines, Caco-2 monolayers penetra-
tion, and blood-brain barrier crossing.25 TPSA was used to

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6233
Synthesis of Chalcones and Dihydrochalcones. To prepare
chalcones, the corresponding acetophenone (1.2 mmol), aromatic
aldehyde (1.4 mmol), KOH (1.5 g, 26.7 mmol), H2O (1.5 mL),
and CH3OH (3.0 mL) were stirred at rt for 1 to 48 h. Deionized
water (50 mL) was added, and the solution was extracted with
EtOAc (2 × 30 mL) and the organic layer was dried over MgSO4
and evaporated. The crude extract was subjected to column
chromatography using hexanes:EtOAc gradient (10:1 to 1:1).2 To
obtain 2,4-dihydroxy-6-methoxychalcone, the appropriate 2,4-
diallyloxy-6-methoxychalcone (0.25 mmol) and Pd(Ph3P)4 (1
mmol%) were dissolved in CH3OH (3 mL); after 1 min of
sonication, K2CO3 (6 equiv) was added to the mixture and flushed
with argon gas for 3 min. The solution was stirred for 1 h at 60
°C, and then it was poured over a solution of HCl (2N, 20 mL).
The aqueous solution was extracted with EtOAc (2 × 20 mL), and
the organic phase was dried over MgSO4 and evaporated. The
orange residue was purified by CC using hexanes:EtOAc step
gradients (6:1 to 1:2).19 2,4-Dihydroxy-6-methoxydihydrochalcones
were obtained by mixing the appropriate chalcone (1.5 mmol) with
Pd/C 5% (0.1 equiv) in EtOAc (10 mL) and stirring the solution in
a Parr flask under 250 psi of H2 gas at rt for 1.5 h. After this, the
solvent was evaporated and the residue purified by CC using
hexanes:EtOAc step gradients (20:1 to 4:1).20
calculate the percentage of absorption (%ABS) according to the
equation: %ABS ) 109 - 0.345 × TPSA, as reported by Zhao
et al.26 In addition, the number of rotatable bonds (n-ROTB)
and Lipinski’s rule of five27 were also calculated. From all these
parameters, it can be observed that although the oral bioavail-
ability of compounds with selectivity indexes greater than 12
(11, 16, 23, 28) could be affected (n-ROTB ranged from 10 to
16), they exhibited a great %ABS ranging from 84 to 94%.
Furthermore, 11, 16, and 28 violate one or none of Lipinski’s
parameters, making them potentially promising agents for anti-
trypanosomal therapy.
Conclusion
The screening of synthetic intermediates against a panel of
cancer cell lines and T. cruzi parasite during the synthesis of a
natural product led to the identification of new chalcones with
antiprotozoan activity and low toxicity. These series of com-
pounds showed not only promising drug-like properties but they
were also easy and economical to prepare, important charac-
teristics to support further in vivo studies and development for
possible use in developing countries, where the cost of drug
therapies is a major factor.
Cell Growth Inhibition Bioassay. The cytotoxicity of com-
pounds was assayed using previously described methodology.16
The Parasites. Trypanosoma cruzi (Tulahuen C4) transfected
with ꢀ-galactosidase (Lac Z) gene was obtained from Instituto de
Investigaciones Cientificas Avanzadas y Servicios de Alta Tecno-
log´ıasPanama´ (AIP). This strain permits high throughput screening
of drugs using a colorimetric enzyme assay. Drugs that inhibit the
growth of T. cruzi (Tulahuen C4) will have no or little color, while
those that do not inhibit growth will permit the strain to grow as
determined by a purple color change.22-24 The strain was main-
tained in monolayer VERO cells (African Green Monkey kidney
epithelial cells) in complete RPMI 1640 medium without phenol
red (Sigma company, St. Louis MO), supplemented with 10% heat
inactivated fetal bovine serum. All cultures and assays are conducted
at 37 °C under an atmosphere of 5% CO2/95% air mixture.
In Vitro Anti-T. cruzi Activity. The antitrypanosome activity
of chalcones and dihydrochalcones was evaluated by the colori-
metric method based on the reduction of the substrate chlorophenol
red ꢀ-D-galactopyranoside (CPRG) by ꢀ-galactosidase resulting
from the expression of the gene for T. cruzi Tulahuen C4.25 The
assay was realized in 96 well plates containing monolayer VERO
cells, which were infected with 5 × 104 trypomastigotes (Tulahuen
C4) per well; 24 h later, 10 µg/mL of each compound were added
and incubated at 37 °C. After 120 h, 25 µL of 900 µM CPRG
substrate (Roche) solution were added to each well to see the
antitrypanoside activity of the compound. Then they were incubated
at 37 °C for 4-5 h until color developed. The compounds that had
antitrypanoside activity (<50% growing inhibition) passed through
a second test for determining the inhibitory concentration for 50%
growth of the parasites (IC50). These compounds were evaluated
at 10, 2, 0.4, 0.8, and 0.16 µg/mL. Each compound and concentra-
tion was made in duplicate. The intensity of color resulting from
the cleavage of CPRG by ꢀ-galactosidase was measured at 570
nm using a VersaMax Micro microplate reader. The IC50 of the
compound were calculated by logarithmic regression of the OD
values obtained compared with the untreated control. All active
compounds also went through an evaluation of the cytotoxicity
using Thiozol Blue (MTT; 3-[4.5 dimethylthiazol-2-y1]-2,5-di-
phenyl tetrazolium bromide) (Aldrich, St. Louis MO). This is
specially important because compounds that may inhibit the parasite
may also be highly toxic and thus not useful as future drug
candidates. This reaction was measured at 570 nm using a
VersaMax Micro microplate reader. Nifurtimox (Bayer) was used
as positive control at concentrations of 0.1, 1, and 10 µg/mL.
Negative control was comprised of a media containing 0.1%
DMSO.
Experimental Section
1H and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, using CDCl3 or CDCl3/CD3OD as a solvent on a
Varian Inova 500. The chemical shifts are reported in ppm values
relative to CHCl3 (7.27 ppm for H NMR and 77.0 ppm for 13C
1
NMR). Coupling constants (J) are reported in hertz (Hz). Melting
points were measured on a Thomas-Hoover capillary melting point
apparatus and are uncorrected. All air and/or moisture sensitive
reactions were carried out under argon atmosphere. Elemental
analysis was performed at Atlantic Microlab, Inc., Norcross, GA.
Column chromatography (CC) was carried out over Silicycle silica
gel (230-400 mesh). Reactions and fractions obtained from CC
were monitored on Merck silica gel 60 F254 aluminum sheets. TLC
spots were visualized by inspection of plates under UV light (254
and 365 nm) and after submersion in 5% sulfuric acid or in 4%
phosphomolybdic acid and heating (110 °C). All commercial
reagents were obtained either from Aldrich, Acros, or Alfa Aesar
and used without any further purification. 3,5-Bisallyloxy-4-
bromobenzaldehyde was available from our laboratory.18
Synthesis of Acetophenones. To a refluxing solution of 2,4,6-
trihydroxyacetophenone-monohydrate (10 g, 53.7 mmol) and K2CO3
(15 g, 108.7 mmol) in acetone (150 mL), (CH3)2SO4 was added at
three-hour intervals (3 × 3.5 mL, 40.0 mmol). The solution was
filtered and the solvent was evaporated to afford 2,4-dimethoxy-
6-hydroxyacetophenone as a yellow solid (98%). To obtain 2,4-
dihydroxy-6-methoxyacetophenone, anhydrous AlCl3 (11.0 g, 82.5
mmol) and 2,4-dimethoxy-6-hydroxyacetophenone (11.0 g, 56.1
mmol) were suspended in chlorobenzene (133 mL) and heated at
reflux for 1 h. After cooling and evaporating of the solvent, an
ice-cold H2O-HCl (1:1, 290 mL) solution was added to the residue
and sonicated until the white precipitate seemed homogeneous. The
solution was filtered, and the solid was dissolved in EtOAc (200
mL) and extracted with an aqueous solution of NaOH (10%, 3 ×
200 mL). The aqueous portions were mixed and neutralized with
HCl (conc, 40 mL) to finally be extracted with EtOAc (2 × 250
mL) and recrystallized from the same solvent (44.1%).18 2,4-
Diallyloxy-6-methoxyacetophenone was prepared by mixing 2,4-
dihydroxy-6-methoxyacetophenone (5.8 g, 10.0 mmol), K2CO3
(21.8 g, 50.0 mmol), and allylbromide (11.0 mL, 40 mmol) in DMF
(100 mL). After stirring for 18 h, the mixture was dissolved in
deionized water (100 mL) and extracted with diethyl ether (3 ×
75 mL). The organic layers were pooled and extracted with
deionized water (3 × 50 mL). Finally, the organic phases were
combined and dried to be subjected to column chromatography
using hexanes:EtOAc step gradient (40:1 to 5:1, colorless oil,
85.0%).18
Physicochemical Parameters Calculation. Absorption (%ABS)
was calculated by: %ABS ) 109 - (0.345 × TPSA).26 Polar

6234 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
surface area (TPSA),24 miLogP, number of rotable bonds, and
violations of Lipinski’s rule of five27 were calculated using
Molinspiration online property calculation toolkit,28 according to
previously reported literature.25-28
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W. C. Efficient technique for screening drugs for activity against
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Acknowledgment. We are grateful to the U.S. Department
of Defense Prostate Cancer Research Program (PCRP) of the
Office of the Congressionally Directed Medical Research
Programs (CDMRP) (grant no. W81XWH-07-1-0299 to G.B.H.)
and the Infectious Diseases Training Program in Peru no. 5D43
TW006581 (R.H.G.) for their financial support.
Supporting Information Available: Melting points, isolated
yields, spectral data (1H and 13C NMR), elemental analysis, and
different LogP calculations of compounds 1-28. This material is
available free of charge via the Internet at http://pubs.acs.org.
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