Synthesis of chalcone analogues with increased antileishmanial activity

Article abstract of DOI:10.1016/j.bmc.2005.10.005

Eighteen analogues of an active natural chalcone were synthesized using xanthoxyline and some derivatives, and these analogues were tested for selective activity against both promastigotes and intracellular amastigotes of Leishmania amazonensis in vitro. Three analogues (10, 12, and 19) containing nitro, fluorine or bromine groups, respectively, displayed increased selective activity against the parasites as compared with the natural chalcone. The nitrosylated chalcone 10 was also tested intralesionally in infected mice and was found to be as effective as Pentostan reference drug at a dose 100 times higher than that of the chalcone in controlling both the lesion growth and the parasite burden.

Full text of DOI:10.1016/j.bmc.2005.10.005

Bioorganic & Medicinal Chemistry 14 (2006) 1538–1545
Synthesis of chalcone analogues with increased
antileishmanial activity
a
c
a
´
Paula Boeck, Camila Alves Bandeira Falcao, Paulo Cesar Leal,
˜
Rosendo Augusto Yunes,a Valdir Cechinel Filho,b Eduardo Caio Torres-Santosc
and Bartira Rossi-Bergmannc,*
aUniversidade Federal de Santa Catarina, Departamento de Quı´mica, Campus Trindade, CEP 88.040-90, Floriano´polis, SC, Brazil
b
ˆ
´
´
Nu´cleo de Investigac¸o˜es Quı´mico-Farmaceuticas (NIQFA/CCS), Universidade do Vale do Itajaı, CEP 88.303-202 Itajaı, SC, Brazil
cUniversidade Federal do Rio de Janeiro, Instituto de Biofı´sica Carlos Chagas Filho, Ilha do Funda˜o,
CEP 21.949-900 Rio de Janeiro, RJ, Brazil
Received 10 December 2004; revised 2 August 2005; accepted 4 October 2005
Available online 28 December 2005
Abstract—Eighteen analogues of an active natural chalcone were synthesized using xanthoxyline and some derivatives, and these
analogues were tested for selective activity against both promastigotes and intracellular amastigotes of Leishmania amazonensis
in vitro. Three analogues (10, 12, and 19) containing nitro, fluorine or bromine groups, respectively, displayed increased selective
activity against the parasites as compared with the natural chalcone. The nitrosylated chalcone 10 was also tested intralesionally
in infected mice and was found to be as effective as Pentostan reference drug at a dose 100 times higher than that of the chalcone
in controlling both the lesion growth and the parasite burden.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
present in a variety of plant species with a vast spectrum
of pharmacological activities including antibacterial,
antifungal, immunosuppressive, and antinociceptive
properties.8–10 One of the most studied antileishmanial
chalcones is licochalcone A isolated from the Glycyrrhi-
za spp. Chinese plant, which inhibits the parasite
enzyme fumarate reductase.11 However, licochalcone A
and some synthetic derivatives have also been shown
to affect human cell functioning by inhibiting lympho-
cyte proliferation and cytokine production.12
Leishmaniases are a group of diseases caused by differ-
ent species of the protozoan parasite Leishmania, which
affect more than 12 million people worldwide.1 The
clinical manifestations may range from single cutaneous
lesions to fatal visceral leishmaniasis. Conventional
chemotherapy relies on multiple parenteral injections
with pentavalent antimonials that are considerably toxic
and prone to induce resistance. Second-line drugs, such
as Amphotericin B and its lipid formulations, are either
too toxic or expensive for routine use in developing
countries. At the same time, the efficacy of Miltefosine
against cutaneous leishmaniasis remains to be ascer-
tained.2,3 These facts call for safer, cheaper, and more
effective new antileishmanial drugs.
We have previously reported the selective activity of the
20,60-dihydroxy-40-methoxychalcone (DMC) isolated
from the inflorescences of Piper aduncum against intra-
cellular amastigotes of Leishmania amazonensis13 and
the improvement of its therapeutic activity in mice by
encapsulation in biodegradable nanospheres.14 Aiming
at developing compounds with improved antileishma-
nial activity, we synthesized a similar active chalcone
(20-hydroxy-40,60-dimethoxychalcone, compound 2) by
using xanthoxyline, a natural compound present in the
leaves and stems of Sebastiana chottiana15 with reported
antibacterial,16 antifungal,17 and antioedematogenic18
properties. In this work, other 17 chalcone analogues
were then synthesized by base-catalyzed condensation
In the past decade, chalcones emerged as a new class of
antileishmanial agents.4–7 Chalcones are structurally
simple compounds of the flavonoid family and are
Keywords: Chalcone; Leishmania; Xanthoxyline; Cutaneous
leishmaniasis.
*
Corresponding author. Tel.: +55 21 2260 6963; fax: +55 21 2280
8193; e-mail: bartira@biof.ufrj.br
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.005

P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
1539
of appropriate aldehyde and xanthoxyline or modified
xanthoxyline.
idineꢁ were used as reference drugs for antipromastigote
and antiamastigote activities, respectively. The toxicity
against murine macrophages (low IC50 values), as mea-
sured by the release of the cytoplasmic enzyme lactate
dehydrogenase, was also considered to be indicative of
unselective drug toxicity. The results showed that de-
spite its high activity against intracellular amastigotes
(IC50 = 4 lM), the natural chalcone DMC displayed a
slight toxicity against the host mammalian cell
(IC50 = 67.4 lM). These effects were similar to those
produced by Pentostanꢁ, in agreement with what was
previously found.13 The first compound synthesized in
this work was 2, and it presented similar activity against
intracellular parasites compared to DMC (1). This result
indicated that methoxy moiety at ring A in 2 is bioequiv-
alent to hydroxy group in DMC and for easiness of syn-
thesis, compound 2 served as a template for further
modifications. Xanthoxyline 1 and its derivative 2-bro-
moxanthoxyline 16 (Scheme 1) were both devoid of
antileishmanial activity (not shown), indicating that
their reported antibacterial16 and antifungal17 properties
were not extensive to the protozoan leishmania and that
the phenyl vinyl structure was important for the antile-
ishmanial activity. Almost all substitutions at the para
position of B ring reduced both the antileishmanial
and the antimacrophage toxicity (compounds 3, 5, 6,
7, and 11). It can be observed that not only electron-ac-
ceptor but also electron-donor substituents at position 4
reduced the antiamastigote activity relating to DMC,
while electron-acceptor substituents, such as the nitro
group at position 3 (10) and chlorine atom at position
2 (8), exhibited, practically, the same activity. This is
The chalcones were tested in vitro against L. amazonensis,
a causative agent of cutaneous leishmaniasis, using both
the promastigote forms found in the insect vector and
the intracellular amastigote forms found in mammalian
hosts. Cytotoxicity against murine macrophages was
evaluated for drug selectivity. One of the most active
chalcones was also tested for evaluation of efficacy in mice
with cutaneous leishmaniasis.
2. Results and discussion
2.1. Chalcones
The natural chalcone DMC was purified from Piper
aduncum as previously described.13 The 18 synthetic
chalcones were prepared as shown in Scheme 1. All
the starting materials were commercially obtained
(Merck), except for xanthoxyline (1) and 2-hydroxy-
3-bromo-4,6-dimethoxyacetophenone (16), which were
prepared as previously described.18 This method allowed
the preparation of a variety of, especially, B-ring-substi-
tuted compounds.
2.2. In vitro antileishmanial activity
Table 1 displays IC50 values of natural and synthetic
chalcones against promastigotes and intracellular
amastigotes of L. amazonensis. Pentostanꢁ and Pentam-
OH
O
OH
O
OH
O
R
a
d
H3CO
OCH3
H3CO
OCH3
O
H3CO
OCH3
15
O
02 R: H
1
03 R: 4-OCH3
04 R: 3,4-diCl *
05 R: 4-Cl
c
OH
O
b
06 R: 4-CH3
07 R: 4-NO2 *
08 R: 2-Cl *
OH
O
O
H3CO
OCH3
09 R: 2-COOH *
10 R: 3-NO2 *
11 R: 4-Br *
12 R: 4-F *
14 *
H3CO
OCH3
13 *
OH
O
OH
O
OH
O
R
Br
Br
e
a
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
1
16
17 R: 2-Cl *
18 R: H
19 R: 3-NO2 *
Scheme 1. Reagents: (a) CHO-Ph-X, NaOH/EtOH; (b) 2-naphthaldehyde, NaOH/EtOH; (c) 2-furaldehyde, NaOH/EtOH; (d) 3,4-(methylene-
dioxy)benzaldehyde; (e) Br2/ AcOH. *New compounds.

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P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
Table 1. In vitro antileishmanial activity
O
A
B
Chalcone
DMC
A
B
IC50 (lM)
Promastigotes
25.3 0.5
Amastigotes
Macrophages
67.3 0.8
OH
OH
OH
OH
OH
OH
4.0 0.4
8.4 0.3
27.9 0.1
3.8 0.2
>100
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
OH
2
3
4
5
6
21.4 0.2
72.6 0.6
11.9 0.8
>100
65.6 1.0
OCH3
OCH3
OCH3
OCH3
OCH3
>100
OCH3
Cl
Cl
8.0 0.0
>100
Cl
>100
>100
>100
CH3
OH
OH
OH
7
8
9
>100
>100
>100
NO2
H3CO
H3CO
H3CO
OCH3
OCH3
OCH3
Cl
5.1 0.3
0.8 0.5
3.6 0.3
28.5 0.3
54.2 1.5
7.6 0.8
COOH
OH
NO2
10
0.7 0.1
15.8 0.4
>100
H3CO
OCH3
OH
OH
OH
11
12
13
59.8 0.9
0.8 0.0
38.0 0.3
>100
>100
Br
F
H3CO
H3CO
H3CO
OCH3
OCH3
OCH3
4.3 0.8
3.4 0.5
>100
16.5 2.3
OH
14
5.0 0.9
3.4 0.4
64.0 0.0
O
H3CO
OCH3

P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
1541
Table 1 (continued)
Chalcone
A
B
IC50 (lM)
Promastigotes
Amastigotes
Macrophages
OH
OH
OH
OH
O
15
17
18
19
0.4 0.1
3.6 0.2
14.2 0.1
4.1 0.7
6.3 0.5
18.3 0.0
9.5 0.1
58.0 1.0
>100
O
H3CO
OCH3
Cl
Br
0.9 0.1
46.7 0.1
0.5 0.4
H3CO
OCH3
OCH3
OCH3
Br
H3CO
NO2
Br
H3CO
Pentostan
Pentamidine
nd
4.4 0.2
nd
72.0 1.0
nd
6.0 0.5
Arithmetic means SD (n = 3).
nd = not determined.
consistent with the presence of a steric effect at position
4. The fact that the fluorine atom at position 4 did not
reduce the activity may be attributed to the small size
of this atom. The lack of reduction in the activity exhib-
ited by compounds 13 and 15 that possess a carbon and
an oxygen atom, respectively, at position 4, can be
attributed to two factors: (i) both atoms are smaller than
the chloride atom, which is the smallest substituent that
promotes a reduction in activity at position 4, (ii) the
carbon and oxygen atoms are vertices of an aromatic
and pentacyclic ring, respectively, and thus their bond-
ing electrons are far from the place where the steric hin-
drance at position 4 is stronger.
mm
12
FU
6
Lesion sizes
Parasite loads
10
8
4
6
4
2
2
0
0
0
1
2
3
4
5
6
PBS
Pent Chalc10
weeks after infection
Figure 1. Effectiveness of chalcone 10 in the treatment of murine
cutaneous leishmaniasis. BALB/c mice were infected with Leishmania
amazonensis-GFP in the ear. From day 7 of infection they were treated
twice a week with chalcone 10 (m, 2 lg/dose), Pentostan (j, 200 lg/
dose) or 10 lL of vehicle alone (d, PBS + 1% DMSO) for 4 weeks. The
lesion sizes were measured at the indicated times and expressed in
millimeters (mm). The parasite loads in the lesion lysates were
measured on day 45 of infection and expressed as specific fluorescence
units (FU). Mean SD (n = 5).
It is interesting to note that while the NO2 group at po-
sition 4 renders the molecule inactive, the same group at
position 3 conserves the activity and (10, 19) confers in-
creased selectivity irrespective of the presence of a bro-
mine atom in the A ring. This finding indicates that
chalcones with a NO2 substituent at the meta position
of the B ring should be investigated for the development
of highly selective antileishmanial compounds.
2.3. In vivo antileishmanial activity
activity of compound 10 (Table 1), probably by promot-
ing increased permeation of the molecule through the
macrophage membrane. Experiments are underway to
evaluate whether that chalcone is more effective than 10
in treating cutaneous leishmaniasis.
One of the most potent analogues, chalcone 10, was tested
in mice infected with L. amazonensis. We found that int-
ralesional treatment with nitrosylated chalcone 10 effec-
tively controlled the growth of the lesions (Fig. 1). The
potency of 10 was much superior to that of Pentostanꢁ
reference drug, considering that the former in a dose
100 times smaller than that ofthe latter produced the same
protective effect. The reduction in lesion size was not due
to an antiinflammatory effect, since the number of para-
sites was also significantly reduced (right panel). Insertion
of bromine in ring A (19) enhances the antileishmanial
3. Conclusions
Altogether, those findings point to a significant increase
in the antileishmanial activity of the natural chalcone
DMC and 2 due to substitutions in their B ring with
fluorine or a nitro group at para and meta positions,

1542
P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
respectively. The resulting compounds 10, 12, and 19 are
non-toxic to the host cells and retain high antiparasite
activity, suggesting that they act on specific and critical
parasite targets. We have supporting evidence that these
three chalcones do not interfere with parasite fumarate
reductase activity even at supraoptimal concentra-
tions,19 which indicates that their mechanism of action
for Leishmania is different from that for other chalcones
such as licochalcone A.12 The potent selective activity
and simple synthesis of these chalcones suggest that they
are potential candidates for the development of new
antileishmanial drugs. This is supported by the finding
that local injections with small doses of compound 10
were more effective than Pentostanꢁ reference drug in
treating murine cutaneous leishmaniasis.
200 lm in thickness with several solvent systems of
different polarities. Compounds were visualized with
ultraviolet light (254 nm) and using ferric chloride
solution followed by heat as developing agent and
purified by recrystallization from ethyl ether and hex-
ane. H NMR spectra revealed that all the chalcones,
except for compounds 3, 7, and 15, were geometrically
pure and configured E (JHa–Hb = 15–16 Hz).18
1
4.2.1. Compound 2: 20-hydroxy-40,60-dimethoxychalcone.
1
IR (KBr) 1628 (C@O), 1586 (C@C) cmꢀ1. H NMR
(CDCl3)
d
14.34 (s, 1H, OH), 7.87 (d, 1H
J = 15.34 Hz, Hb), 7.76 (d, 1H, J = 15.34 Hz, Ha),
7.50 (d, 2H, J = 8.06 Hz, H2, H6), 7.20 (d, 2H,
J = 8.06 Hz, H3, H5), 6.10 (d, 1H, J = 2.36 Hz, H30),
5.96 (d, 1H, J = 2.36 Hz, H50), 3.91 (s, 3H, OCH3),
3.83 (s, 3H, OCH3). 13C NMR (CDCl3) d 193.40
(C@O), 169.03 (C20), 166.82 (C40), 163.18 (C60),
143.23 (Cb), 141.22 (C4), 133.50 (C10), 130.30 (C2,C6),
129.08 (C3,C5), 127.17 (C1), 107.04 (Ca), 94.48 (C30),
91.94 (C50), 56.53 (OCH3), 56.27 (OCH3). Anal. Calcd
for C17H16O4: C, 71.82; H, 5.67; found: C, 71.57; H,
5.68. Yield = 52%; mp = 85–86 ꢂC.
4. Experimental
4.1. Synthesis
The chalcones were synthesized as shown in Scheme 1.
All the starting materials were commercially obtained
(Merck), except for xanthoxyline (1) and 2-hydroxy-
3-bromo-4,6-dimethoxyacetophenone (16), which were
prepared as previously described.18 The substituted
chalcone derivatives 2–15 were prepared by stirring a
solution of xanthoxyline (1) (0.18 g; 0.92 mMol),
EtOH (15 mL), NaOH (0.1 g; 2.5 mMol, with a mini-
mum of H2O), and an appropriate aldehyde
(0.95 mMol) at room temperature for 1–23 h. The
substituted chalcone derivatives 17–19 were prepared
4.2.2. Compound 3: 4-methoxy-20-hydroxy-40,60-dimeth-
oxychalcone. IR (KBr) 1622 (C@O), 1580 (C@C) cmꢀ1
.
1H NMR (CDCl3) d 14.41 (s, 1H, OH), 7.80 (s, 2H, H2,
H3), 7.57 (d, 1H J = 8.65 Hz, Hb), 6.93 (d, 1H,
J = 8.65 Hz, Ha), 6.11 (d, 1H, J = 2.3 Hz, H30), 5.96
(d, 1H, J = 2.3 Hz, H50), 3.92 (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (CDCl3) d
192.55 (C@O), 168.33 (C20), 165.98 (C40), 162.41
(C60), 161.32 (C4), 142.44 (Cb), 130.07 (C10,C2,C6),
128.27 (C1), 125.07 (Ca), 114.31 (C3,C5), 93.76 (C30),
91.18 (C50), 55.79 (OCH3), 55.53 (OCH3), 55.35
(OCH3). Anal. Calcd for C18H18O5: C, 68.78; H, 5.77;
found: C, 68.53; H, 5.77. Yield = 32%; mp = 109–
110 ꢂC.
by stirring
a solution of compound 16 (0.2 g;
0.73 mMol), EtOH (15 mL), NaOH (0.1 g; 2.5 mMol,
with a minimum of H2O), and an appropriate alde-
hyde (0.95 mMol) at room temperature for 2–3 h.
All the crude products were isolated by acidification
of the cool diluted solution and recrystallized from
ethyl ether and hexane (chalcones 4, 7, 8, 9, 10, 11,
12, 13, 17, and 19 and 14 are new). Chalcone 2 was
previously described by Kimura;20 chalcones 5, 15,
16, and 18 have been described by Cechinel and
co-workers,18 whereas chalcones 03 and 06 were
described by Morio and co-workers.21
4.2.3. Compound 4: 3,4-dichloro-20-hydroxy-40,60-dimeth-
oxychalcone. Yellow solid; mp = 120–123 ꢂC; UV kmax
343 (3.80); IR (KBr) 1622 (C@O), 1586 (C@C) cmꢀ1
.
1H NMR (CDCl3) d 14.14 (s, 1H, OH), 7.85 (d, 1H,
J = 15.60 Hz, Hb), 7.62 (d, 1H, J = 15.60 Hz, Ha),
7.43–7.51 (m, 3H, H2, H3, H6), 6.14 (d, 1H,
J = 2.3 Hz, H30), 5.97 (d, 1H, J = 2.3 Hz, H50), 3.92 (s,
3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (CDCl3) d
191.96 (C@O), 168.47 (C40), 166.53 (C60), 162.45
(C20), 139.23 (Cb), 135.19 (C1), 133.75 (C3), 133.12
(C4), 132.04 (C5), 130.83(C2), 129.67 (Ca), 106.22
(C10), 93.81 (C30), 91.37 (C50), 55.94 (OCH3), 55.64
(OCH3). Anal. Calcd for C17H14Cl2O4: C, 57.81; H,
4.00; found: C, 57.62; H, 4.09. Yield = 47%.
4.2. Physico-chemical data of synthesized compounds
The purified chalcones were obtained in yields of
16–89%. Their structures were identified using infrared
spectroscopy (IR), nuclear magnetic resonance spec-
troscopy (NMR), and elementary analyses. Melting
points were determined with a Microquimica MG
APF-301 apparatus and are uncorrected. IR spectra
were recorded with a FT Perkin Elmer 16 PC spec-
trometer on KBr disks. NMR (1H and 13C NMR)
4.2.4. Compound 5: 4-chloro-20-hydroxy-40,60-dimethoxy-
1
spectra were recorded on
a
Brucker Ac-200
F
chalcone. IR (KBr) 1630 (C@O), 1566 (C@C) cmꢀ1. H
(200 MHz) with tetramethylsilane as an internal stan-
dard. Elementary analyses were obtained on a Perkin
Elmer 2400. Percentages of C and H were in agree-
ment with the product formula (within 0.4% of
theoretical values). The purity of the synthesized sub-
stances was analyzed by thin-layer chromatography
(TLC) using Merck silica pre-coated aluminum plates
NMR (CDCl3) d 14.23 (s, 1H, OH), 7.87 (d, 1H
J = 15.52 Hz, Hb), 7.71 (d, 1H, J = 15.52 Hz, Ha),
7.53 (d, 2H, J = 8.49 Hz, H3, H5), 7.37 (d, 2H,
J = 8.49 Hz, H2, H6), 6.11 (d, 1H, J = 2.26 Hz, H30),
5.96 (d, 1H, J = 2.26 Hz, H50), 3.91 (s, 3H, OCH3),
3.84 (s, 3H, OCH3). 13C NMR (CDCl3) d 192.28
(C@O), 168.40 (C20), 166.31 (C40), 162.42 (C60),

P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
1543
140.41 (Cb), 129.41 (C3, C5), 129.08 (C2, C6), 106 (Ca),
93.76 (C30), 91.26 (C50), 55.83 (OCH3), 55.57 (OCH3).
Anal. Calcd for C17H15ClO4: C, 64.06; H, 4.74; found:
C, 64.26; H, 4.69. Yield = 69%; mp = 173–175 ꢂC.
55.30 (OCH3). Anal. Calcd for C18H16O6: C, 65.85; H,
4.91; found: C, 65.64; H, 4.84. Yield = 44%.
4.2.9. Compound 10: 3-nitro-20-hydroxy-40,60-dimethoxy-
chalcone. Orange solid; mp = 171–172 ꢂC; UV kmax 335
1
4.2.5. Compound 6: 4-methyl-20-hydroxy-40,60-dimeth-
(3.74); IR (KBr) 1640 (C@O), 1580 (C@C) cmꢀ1. H
oxychalcone. IR (KBr) 1622 (C@O), 1586 (C@C)
NMR (CDCl3) d 14.09 (s, 1H, OH), 8.46 (s, 1H, H6),
8.22 (d, 1H, J = 7.75 Hz, H4), 7.98 (d, 1H,
J = 15.68 Hz, Hb), 7.84-7.88 (m, 2H, H3, H4), 7.74 (d,
1H, J = 15.68 Hz, Ha), 6.12 (s, 1H, H30), 5.98 (s, 1H,
H50), 3.94 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C
NMR (CDCl3) d 191.78 (C@O), 168.46 (C40), 166.45
(C60), 162.45 (C20), 148.71 (C3), 138.68 (Cb), 137.43
(C1), 134.01 (C6), 130.51 (C5), 129.78 (Ca), 123.99
(C4), 122.08 (C2), 106.20 (C10), 93.84 (C30), 91.37
(C50), 55.82 (OCH3), 55.56 (OCH3). Anal. Calcd for
C17H15NO6: C, 62.00; H, 4.59; N, 4.25; found: C,
62.08; H, 4.55; N, 4.26. Yield = 57%.
1
cmꢀ1. H NMR (CDCl3) d 14.34 (s, 1H, OH), 7.87 (d,
1H J = 15.34 Hz, Hb), 7.76 (d, 1H, J = 15.34 Hz, Ha),
7.5 (d, 2H, J = 8.06 Hz, H6), 7.2 (d, 2H, J = 8.06 Hz,
H3, H5), 6.1 (d, 1H, J = 2.36 Hz, H30), 5.96 (d, 1H,
J = 2.36 Hz, H50), 3.91 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3). 13C NMR (CDCl3) d 193.4 (C@O), 169.03
(C20), 166.82 (C40), 163.18 (C60), 143.23 (Cb), 141.22
(C4), 133.50 (C10), 130.30 (C2, C6), 129.08 (C3,C5),
127.17 (C1), 107.04 (Ca), 94.48 (C30), 91.94 (C50),
56.53 (OCH3), 56.27 (OCH3). Anal. Calcd for
C18H18O4: C, 72,47; H, 6.08; found: C, 72.31; H, 6.14.
Yield = 70%; mp = 125–126 ꢂC.
4.2.10. Compound 11: 4-bromo-20-hydroxy-40,60-dimeth-
4.2.6. Compound 7: 4-nitro-20-hydroxy-40,60-dimethoxy-
oxychalcone. Orange solid; mp = 150–151 ꢂC; UV kmax
chalcone. Orange solid; mp = 295–296 ꢂC; UV kmax 379
338 (4.08); IR (KBr) 1632 (C@O), 1588 (C@C) cmꢀ1
.
(3.80) R (KBr) 1664 (C@O), 1584 (C@C) cmꢀ1
.
1H
1H NMR (CDCl3) d 14.21 (s, 1H, OH), 7.87 (d, 1H,
J = 15.51 Hz, Hb), 7.68 (d, 1H, J = 15.51 Hz, Ha),
7.47–7.51 (m, 4H, H2, H3, H5, H6), 6.11 (s, 1H,
H30), 5.96 (s, 1H, H50), 3.91 (s, 3H, OCH3), 3.84 (s,
3H, OCH3). 13C NMR (CDCl3) d 193 (C@O), 169.13
(C40), 167.07 (C60), 163.15 (C20), 141.47 (Cb), 135.21
(C1), 132.79 (C3, C5), 130.36 (C2, C6), 128.34 (Ca),
124.88 (C4), 106.99 (C10), 94.1 (C30), 92.03 (C50),
56.57 (OCH3), 56.30 (OCH3). Anal. Calcd for C17H15
BrO4: C, 56.22; H, 4.16; found: C, 56.04; H, 4.21.
Yield = 38%.
NMR (CDCl3) d 8.26 (d, 1H, J = 8.85 Hz, Hb), 7.98
(d, 1H, J = 8.85 Hz, Ha), 7.25 (s, 2H, H3, H5), 6.75 (s,
2H, H2, H6), 6.42 (s, 1H, H30), 6.17 (s, 1H, H50), 3.97
(s, 3H, OCH3), 3.93 (s, 3H, OCH3). 13C NMR (CDCl3)
d 192.21 (C@O), 172 (COOH), 169.51 (C40), 163.08
(C60), 160.05 (C20), 149.55 (C4), 147.22 (C1), 139.05
(Cb), 131.24 (C2, C6), 123.82 (Ca), 122.44 (C3, C5),
107.21 (C10), 94.41 (C30), 89,52 (C50), 56.62 (OCH3),
56.54 (OCH3). Anal. Calcd for C17H15NO6: C, 62.00;
H, 4.59; N, 4.25; found: C, 61.98; H, 4.56; N, 4.23.
Yield = 16%.
4.2.11. Compound 12: 4-fluoro-20-hydroxy-40,60-dimeth-
4.2.7. Compound 8: 2-chloro-20-hydroxy-40,60-dimethoxy-
oxychalcone. Yellow solid; mp = 140–141 ꢂC; UV kmax
chalcone. Yellow solid; mp = 136-137 ꢂC; UV kmax 336
339 (4.00); IR (KBr) 1632 (C@O), 1572 (C@C) cmꢀ1
.
1
(3.68); IR (KBr) 1630 (C@O), 1556 (C@C) cmꢀ1. H
1H NMR (CDCl3) d 13.72 (s, 1H, OH), 7.01–7.42 (m,
6H, Hb, Ha, H2, H3, H5, H6), 6.08 (d, 1H,
J = 2.22 Hz, H30), 6.91 (d, 1H, J = 2.22 Hz, H50), 3.83
(s, 3H, OCH3), 3.79 (s, 3H, OCH3). 13C NMR (CDCl3)
d 193.05 (C@O), 169.08 (C40), 166.93 (C60), 163.12
(C20), 141.69 (Cb), 132.48 (C1), 130.73(C2, C6), 128.12
(Ca), 116.43 (C3, C5), 106.91 (C10), 94.45 (C30), 91.94
(C50), 56.52 (OCH3), 56.26 (OCH3). Anal. Calcd for
C17H15FO4: C, 67.54; H, 5.00; found: C, 67.50; H,
5.04. Yield = 87%.
NMR (CDCl3) d 14.25 (s, 1H, OH), 8.19 (d, 1H,
J = 15.67 Hz, Hb), 7.91 (d, 1H, J = 15.67 Hz, Ha),
7.30–7.75 (m, 4H, H3, H4, H5, H6), 6.15 (d, 1H,
J = 2.36 Hz, H30), 6.0 (d, 1H, J = 2.36 Hz, H50), 3.88
(s, 3H, OCH3), 3.94 (s, 3H, OCH3). 13C NMR
(CDCl3) d 193.01 (C@O), 169.14 (C40), 167.08 (C60),
163.17 (C20), 138.55 (Cb), 136.05 (C1), 134.53 (C2),
131.35 (C4), 130.94 (C3), 130.73 (C6), 128.49 (C5),
127.65(Ca), 106 (C 10), 94.49 (C30), 91.99 (C50),
56.56 (OCH3), 56.30 (OCH3). Anal. Calcd for
C17H15ClO4: C, 64.06; H, 4.74; found: C, 63.87; H,
4.80. Yield = 89%.
4.2.12. Compound 13: 1-(20-Hydroxy-40, 60-dimethoxy-
phenyl)-3-(2-naphthyl)prop-2-en-1-one. Yellow solid,
mp = 110–112 ꢂC; UV kmax 336 (4.05); IR (KBr) 1638
4.2.8. Compound 9: 2-[-3-(20-Hydroxy-40,60-dimethoxy-
phenyl)-3-oxoprop- 1-enyl]benzoic acid. Yellow solid;
mp = 160–161 ꢂC; UV kmax 291 (3.82) IR (KBr) 1640
(C@O), 1560 (C@C) cmꢀ1. 1H NMR (CDCl3) d 13.99 (s,
1H, OH), 8.44 (d, 1H, J = 15.49 Hz, Hb), 7.81 (d, 1H,
J = 15.49 Hz, Ha), 7.96 (d, 1H, J = 7.87 Hz, H3), 7.47-
7.81 (m, 3H, H4, H5, H6), 6.12 (d, 1H, J = 2.39 Hz, H30),
6.09 (d, 1H, J = 2.38 Hz, H50), 3.93 (s, 3H, OCH3), 3.84
(s, 3H, OCH3). 13C NMR (CDCl3) d 199.66 (C@O),
167.15 (C60, C40), 162.43 (C20), 149.67 (Cb), 133.94 (C1),
128.93 (C3, C5), 125.50 (C2), 125.04 (C4, C6), 122.43
(Ca), 106 (C10), 93.57 (C30), 90.60 (C50), 55.44 (OCH3),
(C@O), 1586 (C@C) cmꢀ1 1H NMR (CDCl3) d
.
14.35 (s, 1H, OH), 7.47–7.99 (m, 9H, Hb, Ha, H2,
H4, H5, H6, H7, H9, H10), 6.13 (d, 1H, J = 2.26 Hz,
H30), 5.99 (d, 1H, J = 2.26 Hz, H50), 3.95 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3). 13C NMR (CDCl3) d
193.25 (C@O), 169.14 (C40), 166.93 (C60), 163.20
(C20), 143.17 (Ca), 134.86 (C2), 134.11 (C9), 133.77
(C10), 129.29 (C4, C8), 128.44 (C1, C5), 127.83 (C7),
127.35 (C6), 124.41 (C3, Ca), 107.07 (C10), 94.50
(C30), 91.99 (C50), 56.30, 56.60 (OCH3). Anal. Calcd
for C21H18O4: C, 75.43; H, 5.43; found: C, 75.52; H,
5.47. Yield = 30%.

1544
P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
4.2.13. Compound 14: 3-(2-furyl)-1-(20-hydroxy-40,60-
dimethoxyphenyl)prop-2-en-1-one. Yellow solid;
Calcd for C17H14BrNO: C, 50.02; H, 3.46; N, 3.43;
found: C, 50.10; H, 3.44; N, 3.40. Yield = 71%.
mp = 92–94 ꢂC; UV kmax 363 (3.42); IR (KBr) 1626
(C@O), 1586 (C@C) cmꢀ1. H NMR (CDCl3) d 7.79
1
4.3. Biological assays
(d, 1H, J = 15.37 Hz, Hb), 7.57 (d, 1H, J = 15.37 Hz,
Ha), 7.51 (s, 1H, H2), 6.67 (d, 1H, J = 3.31 Hz, H4),
6.50 (t, 1H, J = 1.68 Hz, H3), 3.91 (s, 3H, OCH3), 3.83
(s, 3H, OCH3). 13C NMR (CDCl3) d 192.74 (C@O),
169.09 (C40), 166.85 (C60), 163.21 (C20), 152.93 (C2),
145.32 (Cb, C5), 129.63 (Ca), 116.11 (C4), 113.20
(C3), 106.99 (C10), 94.91 (C30), 91.88 (C50), 56.45
(OCH3), 56.24 (OCH3). Anal. Calcd for C15H14O5: C,
65.69; H, 5.1; found: C, 65.76; H, 5.18. Yield = 52%.
4.3.1. In vitro antileishmanial activity. The antileishma-
nial activities of the test compounds were determined
in vitro against both the insect promastigote forms
and the intramacrophage amastigote forms of L. ama-
zonensis (Josefa strain) transfected with the green
fluorescence protein (GFP).22 For antipromastigote
activity, fluorescent promastigotes were plated in tripli-
cate at 105 parasites/ well with varying concentrations
of test compounds (0, 1, 10, and 100 lM) in a final
volume of 200 lL of medium containing 5% serum
and 1% hybri-max dimethyl sulfoxide (DMSO, Sigma).
After 72 h at 27 ꢂC, the fluorescence intensity of the
cultures was measured using a plate-reader fluorometer
(Fluoroskan) set at 435 nm excitation/538 nm emission.
For antiamastigote activity, 106 mouse peritoneal
macrophages were infected with 5 · 106 fluorescent
promastigotes for 4 h at 37 ꢂC, washed, and cultured
for a further 72 h in 500 lL with the test compounds
in 1% DMSO. Controls were 1% DMSO alone. The
fluorescence intensity of the cell monolayers was mea-
sured for each drug concentration as for promasti-
gotes. Maximum and minimum inhibitory activities
were fluorescence units of uninfected macrophages
and infected cells without drugs, respectively. The
IC50 (the concentration required to induce half of the
maximum activity) values were calculated by linear
regression of the mean fluorescence intensities. Two
reference drugs were used: Pentostanꢁ (sodium stibo-
gluconate, GlaxoSmithKline: antiamastigote activity)
and Pentamidineꢁ (pentamidine isothionate, Aventis:
antipromastigote activity), since Pentostanꢁ is not ac-
tive against promastigotes.
4.2.14. Compound 15: 3,4-methylenedioxy-20-hydroxy-
40,60-dimethoxychalcone. IR (KBr) 1625 (C@O), 1592
1
(C@C) cmꢀ1. H NMR (CDCl3) d 14.36 (s, 1H, OH),
7.73 (s, 3H, H2, H5, H6), 7.09 (d, 1H J = 8.1 Hz, Hb),
6.83 (d, 1H, J = 8.1 Hz, Ha), 6.10 (d, 1H, J = 2.26 Hz,
H30), 6.07 (s, 2H, O-CH2-O), 5.96 (d, 1H, J = 2.26 Hz,
H50), 3.91 (s, 3H, OCH3), 3.83 (s, 3H, OCH3) 13C
NMR (CDCl3) d 193 (C@O), 169.07 (C20), 166.78
(C40), 163.12 (C60), 143.11 (Cb), 107.28 (Ca), 102.21
(CH2), 94.47 (C30), 91.92 (C50), 56.55 (OCH3), 56.26
(OCH3). Anal. Calcd for C18H16O6: C, 65.85; H, 4.91;
found: C, 65.82; H, 4.89. Yield = 32%; mp = 153–
154 ꢂC.
4.2.15. Compound 17: 2-chloro-30-bromo-20-hydroxy-
40,60-dimethoxychalcone. IR (KBr) 1624 (C@O), 1556
1
(C@C) cmꢀ1. H NMR (CDCl3) d 14.67 (s, 1H, OH),
8.17 (d, 1H J = 15.58 Hz, Hb), 7.81 (d, 1H,
J = 15.58 Hz, Ha), 7.28–7.70 (m, 4H, H3, H4, H5,
H6), 6.05 (s, 1H, H50), 3.98 (s, 6H, OCH3) 13C NMR
(CDCl3) d 193.25 (C@O), 163.92 (C20), 163 (C40),
162.9 (C60), 139.49 (Cb), 106 (Ca), 92.80 (C30), 87.93
(C50), 57.08 (OCH3), 56.85 (OCH3). Anal. Calcd for
C17H14 BrClO4: C, 51.35; H, 3.55; found: C, 51.45; H,
3.58. Yield = 47%; mp = 210–212 ꢂC.
4.3.2. Cytotoxicity against macrophages. For cytotoxici-
ty against mammalian cells, adherent mouse peritoneal
macrophages were cultured for 48 h at 37 ꢂC with vary-
ing concentrations of the test compounds. The release of
the cytoplasmic enzyme lactate dehydrogenase (LDH)
into the culture medium was measured using an assay
kit (Doles Reagentes, Brazil). Maximum and minimum
release values were cells cultured with 2% Triton
X-100 or 1% DMSO, respectively. The IC50 values were
calculated by linear regression analysis.
4.2.16. Compound 18: 30-bromo-20-hydroxy-40,60-dimeth-
oxychalcone. IR (KBr) 1628 (C@O), 1558 (C@C) cmꢀ1
.
1H NMR (CDCl3) d 14.83 (s, 1H, OH), 7.85 (d, 1H
J = 15.74 Hz, Hb), 7.76 (d, 1H, J = 15.74 Hz, Ha),
7.38–7.60 (m, 5H, H2, H3, H4, H5, H6), 6.02 (s, 1H,
H50), 3.98 (s, 3H, OCH3), 3.96 (s, 3H, OCH3) 13C
NMR (CDCl3) d 193.43 (C@O), 163.84 (C20), 162.94
(C40), 162.63 (C60), 143.97 (Cb), 135.94 (C10), 131.0
(C1), 107.53 (Ca), 92.58 (C30), 87.81 (C50), 57 (OCH3),
56.76 (OCH3). Anal. Calcd for C17H15 BrO4: C, 56.22;
H, 4.16; found: C, 56.05; H, 4.14. Yield = 40%;
mp = 178–180 ꢂC.
4.3.3. In vivo antileishmanial activity. BALB/c mice were
infected with L. amazonensis-GFP in the ear. From day
7 of infection they were locally injected twice a
week during 4 weeks with compound 10 (2 lg/dose),
Pentostan (200 lg/dose) or 10 lL vehicle alone (phos-
phate-buffered saline—PBS—plus 0.5% DMSO). The
lesion sizes were measured at the indicated times with
a dial caliper and expressed in millimeters. The parasite
loads in the individual lesions were measured on day 45
of infection as described previously23 and expressed as
arbitrary fluorescence units after deduction of the back-
ground fluorescence intensity of the contralateral unin-
fected ears (1.6 0.2 units).
4.2.17. Compound 19: 3-nitro-30-bromo-20-hydroxy-40,60-
dimethoxychalcone. Orange solid; mp = 264 ꢂC; UV kmax
1
288 (3.25)IR (KBr) 1632 (C@O), 1558 (C@C) cmꢀ1. H
NMR (CDCl3) d 14.55 (s, 1H, OH), 8.48-7.60 (m, 6H,
Hb, Ha, H2, H3, H4, H6), 6.10 (s, 1H, H50), 4.03 (s,
3H, OCH3), 4.01 (s, 3H, OCH3). 13C NMR (CDCl3) d
199.99 (C@O), 163.14 (C40), 163.09 (C60), 162.39
(C20), 148.30 (C3), 140.20 (Cb), 134.85 (C1), 131.00
(C6), 130.40 (C5), 124.81 (Ca), 107.02 (C10), 92.12
(C30), 87.16 (C50), 56.76 (OCH3), 56.18 (OCH3). Anal.

P. Boeck et al. / Bioorg. Med. Chem. 14 (2006) 1538–1545
1545
12. Barfod, L.; Kemp, K.; Hansen, M.; Kharazmi, A. Int.
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