Trimethoxy-chalcone derivatives inhibit growth of Leishmania braziliensis: Synthesis, biological evaluation, molecular modeling and structure-activity relationship (SAR)

Article abstract of DOI:10.1016/j.bmc.2011.06.023

In this work we described the synthesis, the antileishmanial activity and the molecular modeling and structure-activity relationship (SAR) evaluations of a series of chalcone derivatives. Among these compounds, the methoxychalcones 2h, 2i, 2j, 2k and 2l showed significant antileishmanial activity (IC ₅₀ <10 μM). Interestingly 2i (IC₅₀ = 2.7 μM), 2j (IC₅₀ = 3.9 μM) and 2k (IC₅₀ = 4.6 μM) derivatives presented better antileishmanial activity than the control drug pentamidine (IC₅₀ = 6.0 μM). Our SAR study showed the importance of methoxy di-ortho substitution at phenyl ring A and the relationship between the frontier orbital HOMO coefficients distribution of these molecules and their activity. The most active compounds 2h, 2i, 2j, 2k, and 2l fulfilled the Lipinski rule-of-five which theoretically is important for good drug absorption and permeation through biological membranes. The potential profile of 2j (IC ₅₀ = 3.9 μM and CC₅₀ = 216 μM) pointed this chalcone derivative as a hit compound to be further explored in antileishmanial drug design.

Full text of DOI:10.1016/j.bmc.2011.06.023

Bioorganic & Medicinal Chemistry 19 (2011) 5046–5052
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Trimethoxy-chalcone derivatives inhibit growth of Leishmania braziliensis:
Synthesis, biological evaluation, molecular modeling and
structure–activity relationship (SAR)
Murilo Lamim Bello a,b, Louise Domeneghini Chiaradia c, Luiza Rosaria Sousa Dias b,
Letícia Kramer Pacheco d, Taisa Regina Stumpf c, Alessandra Mascarello c, Mário Steindel d,
a,
Rosendo Augusto Yunes c, Helena Carla Castro e, Ricardo José Nunes c, , Carlos Rangel Rodrigues
⇑
⇑
a Laboratório de Modelagem Molecular e QSAR (ModMolQSAR-3D), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
b Laboratório de Química Medicinal (LQMed) - Faculdade de Farmácia, Universidade Federal Fluminense, Niterói, RJ, Brazil
c Laboratório Estrutura e Atividade (LEAT), Departamento de Química, Universidade Federal de Santa Catarina, UFSC, Campus Trindade, 88040-900 Florianópolis, SC, Brazil
d Laboratório de Protozoologia, Departamento de Parasitologia, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil
e Laboratório de Antibióticos, Bioquímica e Modelagem Molecular (LABioMol), Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we described the synthesis, the antileishmanial activity and the molecular modeling and
structure–activity relationship (SAR) evaluations of a series of chalcone derivatives. Among these com-
pounds, the methoxychalcones 2h, 2i, 2j, 2k and 2l showed significant antileishmanial activity
Received 28 March 2011
Revised 3 June 2011
Accepted 8 June 2011
Available online 21 June 2011
(IC50 <10
l
M). Interestingly 2i (IC50 = 2.7
l
M), 2j (IC50 = 3.9
l
M) and 2k (IC50 = 4.6
l
M) derivatives pre-
M). Our SAR study
sented better antileishmanial activity than the control drug pentamidine (IC50 = 6.0
l
showed the importance of methoxy di-ortho substitution at phenyl ring A and the relationship between
the frontier orbital HOMO coefficients distribution of these molecules and their activity. The most active
compounds 2h, 2i, 2j, 2k, and 2l fulfilled the Lipinski rule-of-five which theoretically is important for
good drug absorption and permeation through biological membranes. The potential profile of 2j
Keywords:
Chalcones
Leishmania braziliensis
Antileishmanial activity
Molecular modeling
SAR
(IC50 = 3.9
lM and CC50 = 216 lM) pointed this chalcone derivative as a hit compound to be further
explored in antileishmanial drug design.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
occurs by the bite of the female sandfly of the Phlebotomus sp. and
in the new world by Lutzomyia sp.3,6,7
Neglected tropical diseases are an uncontrolled public health
problem in development countries. They currently threaten more
than one billion people and cause around 500.000 deaths every
year.1 Some of these diseases are caused by parasitic protozoan
with distinct morphological stages during development in both
vertebrate and invertebrate hosts.2–4
Leishmaniasis is an infectious disease caused by at least 20 dif-
ferent species of parasites of genus Leishmania. They are responsi-
ble for different clinic manifestations that classify this disease (e.g.,
cutaneous or visceral leishmaniasis).5 The life cycle of this parasite
includes two forms: (i) the flagellated promastigote, which colo-
nizes the gut of the sandfly vector and is the infective form, and
(ii) the obligatory intracellular amastigote in the mammalian mac-
rophage.3,5–7 The transmission of promastigote forms in old world
Despite the progress in basic knowledge on the parasite, current
therapy for leishmaniasis is still unsatisfactory, due to the limited
effectiveness, long term treatment, high cost and undesirable sec-
ondary effects like hepatic, heart, pancreas and kidney toxicity.8–10
Pentavalent antimonials (SbV) such as sodium stibogluconate
(PentostamÒ) and meglumine antimoniate (GlucantimeÒ) are the
first choice for treatment. These drugs only exist in their parenteral
forms and currently second-line compounds are necessary includ-
ing pentamidine (PentacarinatÒ) and amphotericin
B (AmBi-
someÒ).5,9–12 The development of drug resistance by the
pathogens, especially in HIV-Leishmania co-infected patients in-
creased this health problem.5,13
Flavonoids are an extensive group of compounds occurring in
plants. Chalcones are open chain flavonoids with two aromatic
rings linked by a carbonyl group and two
a,b-insatured carbon
atoms.14–16 Chalcones are natural-like compounds, and as such
they can show multitarget profile similar to other compounds de-
scribed in the literature.17 Thus natural and synthetic chalcones are
described in the literature with different pharmacological profiles,
⇑
Corresponding authors. Tel.: +55 48 37216844x236 (R.J.N.) (Synthesis); tel.: +55
21 22609192 (C.R.R.) (Molecular modeling).
E-mail addresses: nunes@qmc.ufsc.br (R.J. Nunes), rangelfarmacia@gmail.com
(C.R. Rodrigues).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.023

M. L. Bello et al. / Bioorg. Med. Chem. 19 (2011) 5046–5052
5047
mastigote forms. Since changes in the 3-propenone moiety causes
the lost of the activity, all modifications were performed in both
aromatic rings. On that purpose we focused on the structure–activ-
ity relationship (SAR) of these new chalcone analogues to deter-
mine structural and stereoeletronic features that can improve the
antileishmanial profile by introducing substituent with different
electronic and volume properties in both phenyl rings moiety of
the chalcone (2).
2. Results and discussion
2.1. Chemistry
The chalcone derivatives new set 2a–2t (Table 1) was prepared
by aldol condensation between aromatic acetophenones and corre-
sponding aldehydes in methanol, KOH (50% w/v), at room temper-
ature and magnetic agitation for 24 h (Fig. 2).16 After precipitation
and recrystallization, the purified chalcones were obtained in
yields from 36% to 97% as described in Section 4.
Figure 1. Sulfonamide 4-methoxychalcone (1) and chalcone set studied in this
work (2).
2.2. Biological results
such as anti-inflammatory,18 trypanocidal,19 antibacterial,20 antivi-
ral,21,22 antitumoral,15 antimalarial,23 and antileishmanial activi-
ties.24–27 Recently, we described the effects of a new set of
sulfonamide 4-methoxychalcones against L. braziliensis promasti-
gote forms.24 Compound 1 ( Fig. 1) presented the best profile
The compounds (2a–2t) were evaluated against L. braziliensis
promastigote forms and most of them showed some level of activ-
ity (Table 1). Among the active derivatives, the analogues 2h, 2i, 2j,
2k and 2l presented the best antileishmanial profile, with values
(IC50) smaller than 10
(IC50 = 3.94 M) and 2k (IC50 = 4.62
tive than standard antileishmanial agent pentamidine
(IC50 = 6.0 M), used as positive control in this study. Out of these
three antileishmanial analogues, compound 2j showed the lowest
lM. Among them, 2i (IC50 = 2.70
lM), 2j
l
l
M) derivatives were more ac-
against L. braziliensis promastigote forms (IC50 = 3.5 0.6 lM).
In this work we synthesized novel chalcones (2) based on a 1,3-
diacetyl biphenyl nucleus without sulfonamide group (Fig. 1) and
evaluated their antileishmanial profile against L. braziliensis pro-
l
Table 1
Comparison of antileishmanial activity in promastigote forms (IC50), cytotoxicity profile in VERO cells (CC50) and selective index (SI) of chalcone derivatives (2a–2t)
R4
O
X
Y
A
B
R3
R1
Z
W
R2
Chalcone
R1
R2
R3
R4
X
Y
W
Z
IC50
(
lM)
CC50
(
lM)
SI
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
H
H
Cl
H
H
H
H
H
Cl
H
Cl
H
NO2
H
C10H8
C10H8
H
H
H
Cl
H
Cl
OCH3
H
H
Cl
H
Cl
H
H
Cl
H
H
Cl
H
OCH3
Cl
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
12.8
29.7
15.4
Nd
147.8 16
14.0
Nd
8.2
2.7
3.9
4.6
7.4
20.0
29.7
11.7
Nd
4
1
5
392
279
382
Nd
906
295
Nd
147
9
216
109
135
84
30.6
9.4
24.8
—
6.1
21.1
-
17.9
3.3
55.4
23.7
18.2
4.2
112.8
10.6
-
4
2
1
1
1
1
2
2
1
NO2
H
H
H
NO2
3350
124
Nd
Nd
Nd
*
**
2q
2r
2s
2t
Nd
Nd
Nd
194.1 30
6.0
-
-
-
1.8
Y-OCH2O-W***
H
Cl
H
H
H
H
H
N(CH3)2
H
Nd
341
H
Pentamidine
Amphotericine B
0.3
SI (Selectivity index) = CC50/IC50.
Nd = not detected at concentration of 50
l
M.
*
B aromatic ring exchange by 1-naphthyl ring.
B aromatic ring exchange by 2-naphthyl ring.
**
***
Methylenedioxy functional group as substituent at Y and W B aromatic ring positions.

5048
M. L. Bello et al. / Bioorg. Med. Chem. 19 (2011) 5046–5052
Figure 2. Synthesis of 2,4,5-trimethoxyacetophenone (3), 2,4,6-trimethoxyacetophenone (4) and xanthoxyline (5), and corresponding chalcones (2a–2t). (a) acetyl chloride,
AlCl3, dichloromethane, 10 °C, 1 h; (b) corresponding aldehyde, KOH 50% w/v, methanol, rt, 24 h; (c) (i) CH3CN, ZnCl2, ether, HCl (g), 0–5 °C; (ii) H2O, reflux; (d) Me2SO4, K2CO3,
acetone; (e) Me2SO4, NaOH/acetone, reflux, 24 h.
Figure 3. Electrostatic potential map and HOMO density encoded onto a van der Waals surface of chalcone derivatives (2a–2t), and structural alignment of most active
derivative (2i) and its isomer (2c) (inset). Molecular electrostatic potential in a 0.002 eV/au3 surface and energy range varying from deepest red to deepest blue.
toxicity level in Vero cells assays and the best selectivity index
against promastigote forms of L. braziliensis. Interestingly, 2j was
proach and theoretically evaluated the chalcone derivatives. Inter-
estingly, an overall look into the structure-activity relationship
data revealed that most chalcones analogues with methoxy substi-
tuent at different positions on chalcone phenyl ring A (R1–R4)
and other substituents at chalcone phenyl ring B (X, Y, W and X)
introduced diversified stereoelectronic effects and modulated the
antileishmanial profile against Leishmania braziliensis promastigote
form.
able to inhibit iNOS, an enzyme involved in inflammatory process,
16
with no toxicity on macrophages at IC50
.
2.3. Molecular modeling and structure–activity relationship
(SAR) studies
In order to provide useful guidelines for designing new more
potent antileishmanial agents, we used a molecular modeling ap-
The theoretical analysis of chlorine atoms substituent at phenyl
ring B (2h, 2i, 2j, 2k and 2l) pointed to an intricate relationship be-

M. L. Bello et al. / Bioorg. Med. Chem. 19 (2011) 5046–5052
5049
Table 2
Theoretical molecular electronic properties (EHOMO, ELUMO and dipole moment) and Lipinski parameters (volume, hydrogen bond donor and acceptor groups, c log P and molecular
weight (MW)) of chalcone derivatives 2a–2t
Chalcone
Dipole moment (debye)
Ehomo (eV)
Elumo (eV)
Lipinski rule of 5
HBA
Volume (Å3)
HBD
c log P
MW (Da)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2.64
2.96
2.10
2.15
3.07
3.27
1.85
3.61
2.23
2.49
3.59
3.28
3.66
2.62
6.76
3.63
2.87
2.96
3.52
1.98
ꢀ5.43
ꢀ5.51
ꢀ5.50
ꢀ5.45
ꢀ5.56
ꢀ5.39
ꢀ5.68
ꢀ5.91
ꢀ5.91
ꢀ5.87
ꢀ5.98
ꢀ5.90
ꢀ6.01
ꢀ5.90
ꢀ6.09
ꢀ5.62
ꢀ5.63
ꢀ5.53
ꢀ5.11
ꢀ5.91
ꢀ1.61
ꢀ1.78
ꢀ1.80
ꢀ1.67
ꢀ1.93
ꢀ1.57
ꢀ1.41
ꢀ1.75
ꢀ1.78
ꢀ1.54
ꢀ1.92
ꢀ1.60
ꢀ2.54
ꢀ2.16
ꢀ2.70
ꢀ1.50
ꢀ1.71
ꢀ1.50
ꢀ1.25
ꢀ2.0
317.3
330.8
330.8
328.8
344.5
344.5
344.7
331.1
331.0
331.1
344.7
344.6
339.5
339.6
339.6
367.2
368.5
341.8
367.1
309.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.0
4.0
4.0
4.0
4.0
4.0
5.0
5.0
4.0
4.0
4.0
4.0
4.0
7.0
7.0
7.0
4.0
4.0
6.0
5.0
4.0
3.37
3.93
3.93
3.94
4.49
3.25
3.25
3.93
3.93
3.93
4.49
4.49
3.41
3.41
3.41
4.37
4.37
3.15
3.66
3.67
298.3
332.7
332.7
332.7
367.2
328.3
328.3
332.7
332.7
332.7
367.2
367.2
343.3
343.3
343.3
348,3
348.3
342.3
341.4
318.7
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
Figure 4. Theoretical toxicity evaluation (A), and the druglikeness and drugscore (B) values of the most active compounds (2h, 2i, 2j, 2k, and 2l) compared to the
antileishmanial drugs pentamidine and amphotericin B. The scale of toxicity risk ranges from low (0–1), medium (1–2) and high (2–3) calculated by using Osiris program.39
tween steric bulk and electronic properties of chlorine atom (Table 1).
Besides electronegative, halogens such as chlorine are also lipophilic,
which may allow the permeability through the cell memb rane.
Interestingly our data showed a higher activity profile for the
methoxy group substituent in the phenyl rings A and B compared
to the chlorine atom substitution probably due to the nature of the
methoxy position. In fact, the insertion of di-ortho- methoxy sub-
stituent at aromatic ring A (R1 and R4) imposes a structural rigidity
and reduces conformational freedom of the phenyl ring that appar-
ently improved the antileishmanial activity. The structural rigidity
imposed by methoxyl substituents at di-ortho- positions on the
aromatic ring is related to the volume of methoxy, which prevents
bond rotation due to the steric hindrance.
The comparison of methoxy group at the phenyl ring A in meta-
(R2) or di-ortho- (R1) (2c and 2i respectively) positions revealed an
improvement over seven orders of magnitude in the antileishma-
nial profile (Table 1). The structural alignment of these derivatives
showed a clear conformational difference (Fig. 3). Importantly the
torsion dihedral angles for all chalcone derivatives are defined as
the angle difference between the plane of the carbonyl group and
phenyl ring A. The derivatives with significant antileishmanial
activity have a larger variation of the torsion dihedral angles,
which are influenced by the methoxy group at ortho- position (R1
and R4). The compounds 2h and 2i have higher activity (Table 1)
and torsion dihedral angle (ꢀ64.01° and 63.12° respectively) com-
pared to 2b and 2c (ꢀ38.75° and ꢀ38.25° respectively). These data
pointed to the steric importance of the carbonyl group and the
phenyl ring A moiety to the antileishmanial activity.
The theoretical analysis of some electronic properties of these
derivatives, including HOMO and LUMO energies and distribution
and dipole moment values (Table 2) showed no clear direct correla-
tion with the antileishmanial activity. Nevertheless, the electro-
static potential map of the derivatives ( Fig. 3) revealed that the
electronic density is located on the carbonyl group. Derivatives that
presented better antileishmanial activity (2h, 2i, 2j, 2k and 2l) pos-
sess the electronic density HOMO at phenyl ring A, whereas 2p, 2q,
2r, and 2s with no antileishmanial activity presented the electronic
density HOMO at phenyl ring B far from carbonyl group (Fig. 3). A
similar result was observed for the series 1 synthesized by Fröhner
et al. since these derivatives also presented the electronic density
HOMO on the carbonyl group.24 Therefore our results reinforced
that the carbonyl group contributes not only with a steric perspec-
tive but also with an important electronic contribution to antileish-
manial activity on both chalcone derivatives series (1 and 2).
Currently, many theoretical approaches such as the Lipinski rule
of five assess the physical-chemical theoretical parameters related
to the oral bioavailability (Table 2). Our results revealed that 2a–2t
derivatives lipophilicity (3.93 > c log P < 4.49) is not greater than
5.0, which, according to Lipinski, is an important feature for good
drug absorption and permeation.24,29–36
In addition the molecular volume and weight of the active deriv-
atives (331.1(Å3 > MV < 344.7 Å3 and 332.78 > MW < 367.22) are

5050
M. L. Bello et al. / Bioorg. Med. Chem. 19 (2011) 5046–5052
similar to the predicted by Lipinski ‘Rule of five’ (Table 2).33,36,37 In
fact, according to our research, all chalcone derivatives with antile-
ishmanial activity (2h, 2i, 2j, 2k, and 2l) (3.93 > c log P < 4.49,
332.78 Da > MW < 367.22 Da, hydrogen bond acceptors = 4 and do-
nors = 0) fulfilled all Lipinski rules, which states that most ‘drug-like’
molecules have log P 65, molecular weight 6500, number of hydro-
gen bond acceptors 610, and donors 65 (Table 1). These data may
suggest the potentiality of these derivatives as new candidates to
antileishmanial agents.
Currently, there are also many approaches that assess druglike-
ness of compounds based on topological descriptors, fingerprints of
molecular druglikeness, structural keys or other properties as
c log P and molecular weight.24,31,35,38 In this work we used the
Osiris software39 for calculating the fragment-based druglikeness
of all compounds also comparing with other antileishmanial drugs
including pentamidine and amphotericin B (Fig. 4). Our in silico re-
sults showed that the active compounds 2h, 2i, 2j, 2k, and 2l pre-
sented the druglikeness value better than pentamidine and
amphotericin B currently used in therapy (Fig. 4). We also analyzed
the drugscore, which evaluate druglikeness, c log P, cLogS, molecu-
lar weight, toxicity risks in one value and that may be used to con-
sider the compound overall potential as a drug.24,40–43 Our data
showed that all active compounds presented drugscore values
greater than the drugs pentamidine and amphotericin B (Fig. 4).
According to our theoretical toxicity evaluation of the tumor-
igenic, irritant, reproductive and mutagenic profiles of 2h, 2i, 2j,
2k, and 2l, all showed a low theoretical toxicity profile similar to
pentamidine and amphotericin B, except for 2h (Fig. 4). Far from
establishing that these compounds are completely free of any
toxicity effect, this theoretical result reinforced the promising
profile of these derivatives for further experimental investiga-
tion.24,38,40–43
to the reaction for total precipitation of the compounds, which
were then obtained by vacuum filtration and later recrystallized
in dichloromethane and hexane. All solvents used were of analyt-
ical grade. The purified chalcones were obtained in yields between
36% and 97%, and are soluble in dimethylsulfoxide, ethyl acetate,
acetone, chloroform and dichloromethane.
The chemical characterization of chalcones 2g–2t was previ-
ously described by our group16,44 whereas we described that of
2a–2f below. Chalcones 2b, 2c, 2d and 2e are new compounds
whereas chalcones 2a and 2f were previously described by Anja-
neyulu and col.45 The data of 2a and 2f are according with the lit-
erature. Melting points were determined with a Microquimica
MGAPF-301 apparatus and are uncorrected. Elementary analysis
was carried out using a CHNS EA 1110; percentages of C and H
agreed with the product formula (within 0.4% of theoretical val-
ues for C). IR spectra were recorded with Abb Bomen FTLA 2000
spectrometer on KBr disks. The 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were obtained from Varian Oxford AS-400 spec-
trometer, using tetramethylsilane as internal standard. The values
of the coupling constants (J) are given in Hertz. The delta value for
each methoxy group was determined as previously described using
sign integration (2,4,6-triOCH3 derivatives) and RMN based calcu-
lations (2,4,5-triOCH3 derivatives).46 The small difference in the
delta values between Ha and Hb can be explained by the presence
of methoxy groups at position ortho and para at A-ring, which re-
duces the withdrawing function of carbonyl group.
4.1.1. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-phenyl-2-propen-1-
one (2a)
Yellow solid; mp 112–114 °C; Yield: 71%; 1H NMR (CDCl3) d
3.90 (s, 3H, o-OCH3), 3.94 (s, 3H, m-OCH3), 3.97 (s, 3H, p-OCH3),
6.55 (s, 1H, H30), 7.40 (s, 1H, H60), 7.37–7.43 (m, 3H, H3, H4, H5),
7.60–7.64 (m, 2H, H2, H6), 7.68 (d, 1H, J = 16.0 Hz, H
a), 7.72 (d,
1H, J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 56.40 (o-OCH3), 56.58 (m-
3. Conclusion
OCH3), 57.02 (p-OCH3), 97.24 (C30), 113.39 (C60), 120.679 (C
a),
127.39 (C4), 128.54 (C2, C6), 129.06 (C3, C5), 130.17 (C10), 135.78
According to our results, the position of methoxy group in phe-
(C1), 142.10 (Cb), 143.63 (C50), 153.83 (C20), 155.09 (C40), 190.08
nyl ring A, in special at di-ortho- (R1 and R4) substituents and chlo-
(C@O); IR m
max/cmꢀ1 16450, 1217 (C@O), 1582 (C@C), 1265, 1031
rine atom at phenyl ring
antileishmanial activity. Among the chalcone series (2a–2t), 2j
derivative showed the most promising results including low IC50
B seems to be important to
(C–O), 2993, 2941, 2830, 1517, 1412, 1148, 980, 769 (Ar) (KBr);
Anal. Calcd for C18H18O4: C 72.47; H 6.08. Found: C 72.65; H 6.81.
against L. braziliensis (3.9
lM), good in silico and in vitro toxicolog-
4.1.2. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-(4-chlorophenyl)-2-
propen-1-one (2b)
ical profile (CC50 = 216 M) and with theoretical physical-chemical
l
indexes (druglikeness and drugscore) better than pentamidine and
amphotericin B. The theoretical study of 2j also showed that it ful-
filled Lipinski rule of five pointing it as a hit compound for further
exploring and to help on designing new candidates for leishmani-
asis treatment.
Light yellow solid; mp 129–130 °C; Yield: 70%; 1H NMR (CDCl3)
d 3.89 (s, 3H, o-OCH3), 3.91 (s, 3H, m-OCH3), 3.97 (s, 3H, p-OCH3),
6.35 (s, 1H, H30), 7.10 (d, 2H, J = 8.0 Hz, H2, H6), 7.13 (d, 2H,
J = 8.0 Hz, H3, H5), 7.46 (s, 1H, H60), 7.65 (d, 1H, J = 16.0 Hz, H
a),
7.73 (d, 1H, J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 56.31 (o-OCH3),
56.37 (m-OCH3), 56.85 (p-OCH3), 97.87 (C30), 112.53 (C60), 119.03
(C
139.43 (Cb), 143.16 (C50), 154.22 (C20), 155.08 (C40), 191.89
(C@O); IR
max/cmꢀ1 1660, 1217 (C@O), 1606 (C@C), 1275, 1026
a
), 128.26 (C2, C6), 129.54 (C3, C5, C10), 132.18 (C1, C4),
4. Materials and methods
4.1. Chemistry
m
(C–O), 2953, 2827, 1513, 1501, 1148, 821 (Ar) (KBr); Anal. Calcd
All reagents were obtained commercially, except for the ace-
tophenones 2,4,5-trimethoxyacetophenone (3), xanthoxyline (4)
and 2,4,6-trimethoxyacetophenone (5), which were prepared as
previously described, with yield of 81%, 70% and 84%, respec-
tively.16,25,28 All structures were confirmed by melting points and
by chemical identification data: infrared spectroscopy (IR), 1H
and 13C nuclear magnetic resonance spectroscopy (NMR) and ele-
mentary analysis. 1H NMR spectra revealed that all structures were
for C18H17ClO4: C 64.97; H 5.15. Found: C 64.98; H 5.91.
4.1.3. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-(3-chlorophenyl)-2-
propen-1-one (2c)
Yellow solid; mp 126–127 °C; Yield: 62%; 1H NMR (CDCl3) d
3.89 (s, 3H, o-OCH3), 3.93 (s, 3H, m-OCH3), 3.98 (s, 3H, p-OCH3),
6.34 (s, 1H, H30), 7.04–7.07 (m, 2H, H5, H6), 7.26 (s, 1H, H2),
7.33–7.35 (m, 1H, H4), 7.40 (s, 1H, H60), 7.64 (d, 1H, J = 16.0 Hz,
Ha
), 7.70 (d, 1H, J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 55.80 (o-
configured E (JH -Hb = 16.0 Hz).
a
OCH3), 56.37 (m-OCH3), 56.90 (p-OCH3), 97.83 (C30), 112.94 (C60),
118.24 (C
), 126.31 (C6), 126.56 (C2), 128.13 (C4), 128.65 (C10),
129.36 (C5), 134.01 (C3), 140.19 (C1), 142.91 (Cb), 143.37 (C50),
153.94 (C20), 154.82 (C40), 190.93 (C@O); IR max/cmꢀ1 1661,
All chalcones were prepared by aldol condensation between
aromatic acetophenones and corresponding aldehydes, in metha-
nol, KOH (50% w/v), at room temperature and magnetic agitation
for 24 h.16 Distilled water and 10% hydrochloric acid were added
a
m

M. L. Bello et al. / Bioorg. Med. Chem. 19 (2011) 5046–5052
5051
1221 (C@O), 1609 (C@C), 1273, 1032 (C–O), 2934, 2838, 1517,
1472, 1409, 1158, 817 (Ar) (KBr); Anal. Calcd for C18H17ClO4: C
64.97; H 5.15. Found: C 64.80; H 5.87.
in Neubauer chamber and they were kept in Schneider medium
with 5% of BSF at 4 °C.
4.2.3. Antileishmanial and cytotoxic assays24
The assays were made in three independent experiments, in
plates of 96 wells. Initially we performed a screening in concen-
4.1.4. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-(2-chlorophenyl)-2-
propen-1-one (2d)
Yellow solid; mp 141–143 °C; Yield: 90%; 1H NMR (CDCl3) d 3.90
(s, 3H, o-OCH3), 3.92 (s, 3H, m-OCH3), 3.97 (s, 3H, p-OCH3), 6.54 (s,
1H, H30), 7.28–7.30 (m, 2H, H4, H5), 7.39 (s, 1H, H60), 7.41–7.44
trations of 500 and 50 lM of each chalcone derivative (20 lL), in
presence of 180
l
L of suspension of 5.0 ꢁ 106 promastigotes/mL
in Schneider0s medium supplemented with 5% of BSF. The plates
were incubated at 28 °C for 72 hours and visually evaluated in
inverted microscope to check the mobility of the protozoa. The
activity of the compounds was determined through the total
number counting of living protozoa in Neubauer chamber. The
protozoa were incubated also without chalcones, in presence of
1% of DMSO, as the negative control, and with pentamidine
and amphotericin B, for positive control. The compounds that
(m, 1H, H3), 7.60 (d, 1H, J = 16.0 Hz, Ha), 7.70–7.72 (m, 1H, H6),
8.08 (d, 1H, J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 56.15 (o-OCH3),
56.30 (m-OCH3), 56.74 (p-OCH3), 96.87 (C30), 113.13 (C60), 120.09
(C10), 126.94 (C
a
), 127.73 (C5), 129.63 (C6), 130.19 (C3), 130.58
(C4), 133.85 (C2), 135.33 (C1), 137.60 (Cb), 143.37 (C50), 153.74
(C20), 154.92 (C40), 189.59 (C@O); IR max/cmꢀ1 1648, 1217 (C@O),
m
1588 (C@C), 1267, 1024 (C–O), 1149 (C-Cl), 2935, 2834, 1616,
1517, 1483, 1408, 1358, 1198, 965, 819, 751 (Ar) (KBr); Anal. Calcd
for C18H17ClO4: C 64.97; H 5.15. Found: C 64.75; H 5.81.
inhibited the mobility of the protozoa with 50
to determine the IC50 values by evaluation in the concentrations
of 500, 100, 50, 25, 10, 5 and 1 M, in three independent exper-
lM were selected
l
4.1.5. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-(3,4-dichlorophen
yl)-2-propen-1-one (2e)
iments (like description above). The IC50 values were determined
by linear regression analysis, and represented the mean stan-
dard error of three independent experiments (GraphPad Soft-
ware). The cytotoxicity of the compounds was evaluated in
Vero cells, in the same conditions.24
Yellow solid; mp 136–137 °C; Yield: 43%; 1H NMR (CDCl3) d 3.90
(s, 3H, o-OCH3), 3.95 (s, 3H, m-OCH3), 3.98 (s, 3H, p-OCH3), 6.54 (s,
1H, H30), 7.46 (s, 1H, H60), 7.42 (d, 1H, J = 16.0 Hz, H
a), 7.46 (d, 1H,
J = 16.0 Hz, Hb), 7.59–7.62 (m, 2H, H5, H6), 7.68 (s, 1H, H2). 13C
NMR (CDCl3) d 56.44 (o-OCH3), 56.59 (m-OCH3), 57.04 (p-OCH3),
4.3. Molecular modeling methodology
97.02 (C30), 113.32 (C60), 120.17 (C
a), 127.58 (C6), 129.00 (C2),
129.88 (C10), 131.04 (C5), 133.31 (C4), 133.85 (C3), 135.97 (Cb),
138,99 (C1), 143.71 (C50), 152.92 (C20), 154.24 (C40), 190.35 (C@O);
Molecular modeling was performed using SPARTAN008 (Wave-
function Inc., Irvine, CA, 2000) and Osiris programs.24,39–43 Struc-
tures were minimized with mechanical molecular force field
MMFF and the equilibrium geometry was obtained in vacuum
using a semi-empirical RM1 module. In order to evaluate the elec-
tronic properties of the RM1 minimal energy conformations, they
were submitted to a single-point calculation using DFT (Density
Functional Theory) method with a 6-31G⁄ basis set of the SPAR-
TAN008 package. The three-dimensional isosurfaces of the molecu-
lar electrostatic potential maps (MEPs) at the van der Waals
contact surface represented electrostatic potentials superimposed
onto a surface of constant electron density (0.002 e/au3).24,40–43
The color-coded isosurface values provide an indication of the
overall molecular size and location of negative (red) or positive
(blue) electrostatic potentials.24,40–43 The electronic properties
(HOMO energy, HOMO orbital coefficients distribution, LUMO den-
sity, dipole moment and lipophilicity-c log P) were calculated for
all compounds.
IR m
max/cmꢀ1 1654, 1222 (C@O), 1591 (C@C), 1268, 1025 (C–O),
2941, 2838, 1524, 1476, 1413, 1354, 1147, 969, 821 (Ar) (KBr); Anal.
Calcd for C18H16Cl2O4: C 58.87; H 4.39. Found: C 58.68; H 5.00.
4.1.6. (2E)-1-(20,40,50-Trimethoxyphenyl)-3-(3-methoxyphenyl)-
2-propen-1-one (2f)
Yellow solid; mp 97–99 °C; Yield: 52%; 1H NMR (CDCl3) d 3.85
(s, 3H, o-OCH3), 3.90 (s, 3H, m-OCH3), 3.93 (s, 3H, p-OCH3), 3.97
(s, 3H, OCH3), 6.55 (s, 1H, H30), 6.94 (d, 1H, J = 8.0 Hz, H4), 7.14
(s, 1H, H2), 7.22 (d, 1H, J = 8.0 Hz, H6), 7.32 (t, 1H, J = 8.0 Hz, H5),
7.39 (s, 1H, H60), 7.61 (d, 1H, J = 16.0 Hz, H
a), 7.68 (d, 1H,
J = 16.0 Hz, Hb). 13C NMR (CDCl3) d 55.49 (o-OCH3), 56.34 (m-
OCH3), 56.53 (p-OCH3), 57.00 (OCH3), 97.21 (C30), 113.37 (C60),
11.71 (C2), 115.77 (C4), 120.62 (C6), 121.15 (C
130.05 (C5), 137.19 (C1), 141.97 (Cb), 143.62 (C50), 153.86 (C20),
155.10 (C40), 160.03 (C3), 190.07 (C@O); IR max/cmꢀ1 1646, 1219
a
), 127.73 (C10),
m
(C@O), 1586 (C@C), 1266, 1026 (C–O), 2938, 2834, 1517, 1480,
1409, 1144, 847, 792 (Ar) (KBr); Anal. Calcd for C19H20O5: C
69.50; H 6.14. Found: C 69.82; H 6.41.
Acknowledgments
This study was supported by grants and fellowships from
CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-
nológico), FAPERJ (Fundação de Amparo à Pesquisa do Estado do
Rio de Janeiro) and CAPES (Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior), Brasil. The group wishes to thank
the Central de Análises (Departamento de Química, UFSC) for
equipments to chemical analysis and Patricky Santos, Cid Felipe
Alves de Mattos Medeiros and Juliana Novais for their technical
assistance.
4.2. Biology
4.2.1. Chemicals
Serum was purchased from GIBCO (Grand Island, NY) and all
other reagents were purchased from Sigma Chemical Co. All chal-
cones (2a–2t) were solubilized in DMSO 0.3 M and kept at 4 °C.
The final concentration of DMSO into the cultures never exceeded
1% (v/v) and had no effect on the parasites proliferation or
morphology.
References and notes
4.2.2. Parasites and cell line
L. braziliensis (MHOM/BR/75/M-2904) promastigotes were ob-
tained in the exponential stage of growth in Schneider0s medium
at 28 °C supplemented with 5% of BSF (Bovine Serum Fetal) and
washed two times in PBS (Phosphate Buffer Saline) pH 7.2, at
4 °C, at 1,500ꢁg/10 min. The number of protozoa was determined
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