Synthesis, biological evaluation and SAR of sulfonamide 4-methoxychalcone derivatives with potential antileishmanial activity

Article abstract of DOI:10.1016/j.ejmech.2008.04.016

Despite clinical importance of leishmaniasis, an infectious disease that affects 12 thousand million people in 88 countries, the treatment is still unsatisfactory due to its limited efficacy, cost expensive and undesirable side effects. Aiming to develop new antileishmanial lead compounds, we used a rational approach to synthesize a new set of sulfonamide 4-methoxychalcone derivatives (3a-3i) and evaluate the sulfonamide and methoxy moieties as promising adding-groups to chalcones. For that purpose we tested this new set against Leishmania braziliensis promastigotes and intracellular amastigotes and determined its cell toxicity profile. Interestingly all compounds presented a concentration-dependent antileishmanial profile and the benzylamino derivative (3i) showed a biological activity better than pentamidine. None of these compounds affected Trypanosoma cruzi epimastigotes, which suggests a specific antileishmanial profile. The structure-activity analysis of these sulfonamide 4-methoxychalcone derivatives pointed the molecular volume, the HOMO density concentrated in the chalcone moiety and the conformational structure of the compounds as important structural and stereoelectronic features for the antileishmanial activity. In addition, these compounds also fulfilled Lipinski rule of 5 and presented druglikeness similar to antileishmanial drugs. Altogether these results point the sulfonamide 4-methoxychalcone derivatives as potential lead compounds for designing new candidates for leishmaniasis treatment.

Full text of DOI:10.1016/j.ejmech.2008.04.016

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European Journal of Medicinal Chemistry 44 (2009) 755e763
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and SAR of sulfonamide
4-methoxychalcone derivatives with potential antileishmanial activity
Carla R. Andrighetti-Frohner a, Kely N. de Oliveira a, Daniela Gaspar-Silva c,
¨
Letıcia K. Pacheco , Antonio C. Joussef , Mario Steindel , Claudia M.O. Simoes ,
c
a
c
b
´
ˆ
´
´
Alessandra M.T. de Souza , Uiaran O. Magalhaes , Ilıdio F. Afonso ,
~
d,e
d
d
´
, Helena C. Castro
d,***
a,**
*
e,
Carlos R. Rodrigues
, Ricardo J. Nunes
a Departamento de Qu´ımica, Universidade Federal de Santa Catarina, UFSC,
Campus Universita´rio Trindade, CEP 88040 900 Floriano´polis, SC, Brazil
b Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Santa Catarina, UFSC,
Campus Universita´rio Trindade, CEP 88040 900 Floriano´polis, SC, Brazil
c Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, UFSC,
Campus Universita´rio Trindade, CEP 88040 900 Floriano´polis, SC, Brazil
d ModMolQSAR, Faculdade de Farma´cia, Universidade Federal do Rio de Janeiro, CEP 21941-590 Rio de Janeiro, RJ, Brazil
e LABioMol, Departamento de Biologia Celular e Molecular, IB/CEG, Universidade Federal Fluminense, CEP 24001-970 Nitero´i, RJ, Brazil
Received 18 February 2008; received in revised form 6 April 2008; accepted 15 April 2008
Available online 6 May 2008
Abstract
Despite clinical importance of leishmaniasis, an infectious disease that affects 12 thousand million people in 88 countries, the treatment is
still unsatisfactory due to its limited efficacy, cost expensive and undesirable side effects. Aiming to develop new antileishmanial lead com-
pounds, we used a rational approach to synthesize a new set of sulfonamide 4-methoxychalcone derivatives (3ae3i) and evaluate the sulfon-
amide and methoxy moieties as promising adding-groups to chalcones. For that purpose we tested this new set against Leishmania
braziliensis promastigotes and intracellular amastigotes and determined its cell toxicity profile. Interestingly all compounds presented a concen-
tration-dependent antileishmanial profile and the benzylamino derivative (3i) showed a biological activity better than pentamidine. None of these
compounds affected Trypanosoma cruzi epimastigotes, which suggests a specific antileishmanial profile. The structureeactivity analysis of these
sulfonamide 4-methoxychalcone derivatives pointed the molecular volume, the HOMO density concentrated in the chalcone moiety and the
conformational structure of the compounds as important structural and stereoelectronic features for the antileishmanial activity. In addition, these
compounds also fulfilled Lipinski rule of 5 and presented druglikeness similar to antileishmanial drugs. Altogether these results point the
sulfonamide 4-methoxychalcone derivatives as potential lead compounds for designing new candidates for leishmaniasis treatment.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Leishmaniasis; SAR; Sulfonamide 4-methoxychalcones; Antiparasite; Promastigote; Amastigote
* Corresponding author. Universidade Federal Fluminense, Instituto de Biol-
´
ogia, Departamento de Biologia Celular e Molecular, 24020-150 Niteroi, RJ,
1. Introduction
Brazil. Tel.: þ55 21 26292294; fax: þ55 21 26292284.
´
** Corresponding author. Departamento de Quımica, Universidade Federal de
Santa Catarina, UFSC, Campus Universitario Trindade, 88040 900 Florianop-
´
´
Disseminated leishmaniasis is an emerging infectious
disease, mostly due to Leishmania braziliensis. Importantly
the disease presents clinical and histopathological features
where L. braziliensis is responsible for both cutaneous
and mucocutaneous leishmaniasis in several Latin American
countries [1]. Therefore, currently these parasitic diseases
olis, SC, Brazil. Tel.: þ48 37216845x236.
*** Corresponding author. Universidade Federal do Rio de Janeiro, Faculdade
´
de Farmacia, ModMolQSAR, 24020-150 Rio de Janeiro, RJ, Brazil. Tel.: þ55
21 25626444.
E-mail addresses: rangel@pharma.ufrj.br (C.R. Rodrigues), nunesqmc@
ufsc.br (R.J. Nunes), hcastrorangel@yahoo.com.br (H.C. Castro).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.016

756
C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
cause significant morbidity and mortality, mainly in the
developing world [2].
cytotoxicity profile in mouse peritoneal macrophages was
evaluated, which allowed the determination of their selectivity
index. Finally, these biological effects were analyzed with the
Despite the progress in important fundamental knowledge
about L. braziliensis, the current chemotherapy for leishmani-
asis is still unsatisfactory due to the limited efficacy, long-term
treatment, cost expensive and undesirable side effects [3e5].
The development of drug resistance by the pathogens,
especially in HIVeLeishmania co-infected patients, has also
aggravated the health problem [6]. Thus, there is an urgent
need for the development of new, efficient and safe drugs
against leishmaniasis.
compounds’ different theoretical parameters (dipole, EHomo
E
,
Lumo, cLogP, molecular weight and volume, higher HOMO
coefficient orbital and density) calculated using a molecular
modeling approach in the structureeactivity relationship
(SAR) study [20].
2. Results and discussion
Natural and synthetic chalcones are described presenting
a wide range of potential pharmacological profiles [7], such
as anti-inflammatory [8], trypanocidal [9], antibacterial [10],
antiviral [11,12], antitumoral [13], antimalarial [14], and
antileishmanial [15e18] activities. Recently a series of substi-
tution-containing chalcone derivatives (1) have been reported
as antileishmanial agents (Scheme 1) [9]. In addition, sulfon-
amide analogues based on the herbicide oryzalin (2), presented
antiparasitic activity including an antileishmanial profile
(Scheme 1) [19].
In this work we used a rational approach and synthesized
a new set of sulfonamide 4-methoxychalcone derivatives
(3ae3i) to identify the sulfonamide and methoxy as promising
adding-groups to chalcones and develop new lead antiparasitic
compounds. In addition we focused on the structureeactivity
relationships (SAR) of these sulfonamide 4-methoxychalcones
series to determine structural and stereoelectronic features that
could lead to the antileishmanial profile (Scheme 1). For that
purpose we analyzed the role of groups with different
electronic and volume properties in the sulfonamide moiety
of the 4-methoxychalcones’ B ring.
2.1. Design and synthesis
The sulfonamide 4-methoxychalcone derivatives are pre-
pared as shown in Scheme 2. The 4-methoxychalcone (3)
was obtained by aldolic condensation of 4-methoxybenzalde-
hyde and acetophenone according to the general literature
procedure [19] (Scheme 2).
Treatment of 4-methoxychalcone (3) with chlorosulfonic
acid at room temperature for one week [21] afforded the
sulfonyl chloride derivative. Preparation of sulfonamides
3ae3i was accomplished by reaction of sulfonyl chloride de-
rivative with different amines in the presence of methanol at
room temperature. We characterized all the synthesized com-
pounds by spectroscopic data such as IR NMR and elemental
analysis [22]. The chalcones 3a, 3b and 3e were prepared as
previously described [22,23].
2.2. Biological evaluation
In this work we evaluated the inhibitory profile of the 4-
methoxychalcone (3) and sulfonamide 4-methoxychalcone
derivatives (3ae3i) against L. braziliensis promastigote forms
(Fig. 1A). Interestingly all the chalcones tested showed
a concentration-dependent inhibitory effect on the in vitro
Leishmania growth assays (Fig. 1A).
Interestingly, the addition of sulfonamide generated deriva-
tives with a better inhibitory profile against L. braziliensis than
the 4-methoxychalcone (3), except for the aniline substituted
sulfonamide 4-methoxychalcone (3e) (Fig. 1A and Table 1).
According to our results, most of the sulfonamide 4-methoxy-
chalcone derivatives showed a more potential inhibitory
To compare with other chalcone series described in the lit-
erature [9], the new 3-sulfonamide 4-methoxychalcone series
were evaluated for its antiparasitic activity against L. brazilien-
sis promastigotes and Trypanosoma cruzi epimastigotes. We
also tested the most active compound against L. braziliensis
intracellular amastigotes to evaluate its ability for decreasing
the parasite burden in infected host cells. In addition the
Scheme 1. Rational approach to design the sulfonamide 4-methoxychalcone
derivatives.
Scheme 2. Synthetic approach used to obtain the sulfonamide 4-methoxychal-
cone derivatives.

C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
757
Fig. 1. Biological evaluation of 4-methoxychalcone (3) and sulfonamide 4-methoxychalcone derivatives (3ae3i). Inhibitory effects on Leishmania braziliensis in
vitro proliferation (A), cytotoxicity profile on mouse macrophages (B), selectivity index (SI) calculated as described by Ferreira et al. [24] (C) and the inhibition of
the amastigotes infection on macrophages by the most active compound (3i) (D).
activity (IC50 ¼ 3.5 ꢀ 0.6 to 8.6 ꢀ 0.4 mM) than that of 4-
methoxychalcone (3) (IC50 ¼ 16.6 ꢀ 1.6 mM) and other
chalcones without substituent groups such as those described
by Lunardi and co-workers [9] (IC50 ¼ 13.7e182.3 mM)
(Table 1). Although our compounds were less active than
amphotericin B (IC50 ¼ 0.3 ꢀ 0.02 mM), they were more
active than pentamidine isothionate (IC50 ¼ 19.6 mM) [25],
one current drug used for leishmaniasis treatment.
The addition of the aniline on the substituent group in the
compound 3e was the only one that negatively affected the in-
hibitory activity on the growth of L. braziliensis promastigotes
forms (IC50 ¼ 69 ꢀ 3.7 mM) compared to 3. Importantly, all
substitutions (electron-acceptor or electron-donor substituents)
at the sulfonamide moiety or at para-position of aniline
aromatic ring maintained the antileishmanial profile (com-
pounds 3ae3d and 3fe3h) suggesting the feasibility of new
interactions on these positions (Fig. 1 and Table 1).
The compound 3i showed the best profile against L. brazil-
iensis promastigotes (IC50 ¼ 3.5 ꢀ 0.6 mM). Apparently the
benzylamino group significantly contributes to this activity,
since this compound was about 20-fold more potent than the
compound 3e (IC50 ¼ 69.0 ꢀ 3.7 mM) (Fig. 1 and Table 1).
Notably the addition of the carbon spacer on the substituent
(3i) was enough to avoid the deleterious effect of the aniline
group (3e), probably allowing hydrophobic interactions with
the parasite target. We also carried out amastigotes assays
with the most active compound (3i) to evaluate its ability for
reducing the parasite load in infected host cells. In the
untreated controls we obtained an infection rate of 30.1%
(mean ¼ 2.9 parasites/infected cell). The results showed that
3i is able to reduce significantly the number of intracellular
amastigotes suggesting its potential as a lead antileishmanial
compound (Fig. 1D).
In this work we experimentally evaluated the cytotoxic
activity of 4-methoxychalcone (3) and sulfonamide 4-
methoxychalcone derivatives (3ae3i) against mouse perito-
neal macrophages. Our results showed CC50 values ranging
from 57.8 ꢀ 4.8 to 105.7 ꢀ 6.5 mM (Fig. 1B). Interestingly
the presence of the substituents of the sulfonamide group
seems to play a more important role for the antileishmanial
activity than for the cytotoxicity profile (Fig. 1 and Table 1).
The CC50 values were used to calculate the selectivity index
(SI) for these derivatives, which were higher than the non-
substituted 4-methoxychalcone, except for 3c and 3e. This
result showed the improvement of the selectivity index of
the derivatives compared to the leading compound (Fig. 1C).
Finally, we also tested the sulfonamide 4-methoxychalcone
derivatives’ effects against T. cruzi epimastigotes forms.
However, different from the chalcone series described by
Lunardi et al. that was active on both L. braziliensis
(21.9e182.3 mM) and T. cruzi strains (24.8e126.4 mM), this
new series showed no activity in concentrations up to 500 mM
(not shown). These data suggest that the sulfonamide 4-
methoxychalcone series target is probably different from that
of Lunardi series [9], due to its specificity for Leishmania. Pre-
vious studies showed that chalcone derivatives might act in the
parasite mitochondria by the inhibition of fumarate reductase,
succinate dehydrogenase, NADH dehydrogenase, or succinate-
and NADH-cytochrome c reductase activity [26,27]. However,
as these enzymes may be present in T. cruzi and the series
described herein does not affect this parasite, further investiga-
tion about its mechanism of action should be performed.

758
C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
Table 1
Comparison of the antileishmanial activity (IC50) of 4-methoxychalcone (3) and sulfonamide 4-methoxychalcone derivatives (3ae3i) with their theoretical
molecular electronic properties (dipole, EHomo, ELumo, cLog P, volume, molecular weight (MW)) and Lipinski profile, including the number of hydrogen bond
donor and acceptor groups (HDG and HAG, respectively)
C
R
IC50 (mM)
Dipole (Debye)
HOMO (eV)
LUMO (eV)
Lipinski rule of 5
3
Volume (A )
˚
HDG
HAG
clogP
MW
3
e
16.6 ꢀ 1.6
8.6 ꢀ 0.4
5.6 ꢀ 0.4
13.0 ꢀ 1.3
5.9 ꢀ 1.3
69.0 ꢀ 3.7
4.6 ꢀ 1.3
8.1 ꢀ 1.6
7.5 ꢀ 0.8
3.39
7.32
8.95
7.23
7.31
8.67
8.20
7.84
7.56
ꢁ8.43
ꢁ8.38
ꢁ8.40
ꢁ8.76
ꢁ8.59
ꢁ8.57
ꢁ8.19
ꢁ8.89
ꢁ8.27
2.01
2.04
2.09
0.95
1.87
1.87
2.09
0.951
2.06
263.80
346.07
382.91
371.83
379.89
391.84
405.13
418.27
419.38
0
0
0
0
0
1
1
1
1
2
5
5
5
6
5
5
5
6
3.63
2.61
3.29
2.93
2.21
4.04
4.60
5.16
3.91
238.29
345.42
373.47
371.46
387.46
393.46
427.91
462.35
423.49
3a
3b
3c
3d
3e
3f
3g
3h
3i
3.5 ꢀ 0.6
7.17
ꢁ8.59
1.87
410.52
1
5
4.11
407.49
2.3. Molecular modeling and SAR studies
To evaluate the electronic properties of the AM1 minimal
energy conformations, they were submitted to a Density
Functional Theory (DFT) calculation with a 6-31G* basis
set of the SPARTAN’06 package. The electronic properties
(HOMO and LUMO energy, HOMO density and dipole
moment) were calculated for all compounds. Theoretical
logP (clogP) was calculated at AM1 semi-empirical level
using the Villar method, included in Spartan [24,28e30].
In this study we compared the biological profile of this
new series with its structural features to establish a struc-
tureeactivity relationship. All calculations were performed
using SPARTAN’06 (Wavefunction Inc., Irvine, CA, 2000).
The structures were minimized and the equilibrium geometry
was obtained in vacuum using a semi-empirical AM1 module.

C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
759
An overall analysis of the activity of the compounds
showed that the addition of sulfonamide substituent increased
the activity of these compounds in comparison to compound 3.
Interestingly the increase of molecular volume, weight and
dipole moment of these derivatives seem to improve the activ-
ity compared to 3 (Table 1) except for 3e.
HOMO and LUMO (Highest and Lowest Unoccupied Molec-
ular Orbitals) energy values, HOMO and LUMO orbital
coefficients distribution, and molecular electrostatic potential
(MEP). Our results showed that sulfonamide 4-methoxychal-
cone derivatives led to different HOMO and LUMO energy
values, and MEPs that alone did not present any direct corre-
lation with the antileishmanial activity (Table 1 and Fig. 2).
In the effort to study the hydrophobic pattern, we calculated
the theoretical parameters related to the oral bioavailability,
according to Lipinski rule of 5 [31] (Table 1). Our results
revealed that the sulfonamide 4-methoxychalcone lipophilicity
(2.21 > cLogP < 4.60) is not greater than 5.0, which, accord-
ing to Lipinski, is an important feature for good drug absorp-
tion and permeation [32e34](Table 1).
The conformational analysis of these compounds inferred
that the enhancement of the biological activity is probably
due to the new interactions in a new plane of these molecules
caused by the addition of the sulfonamide group (Fig. 2). The
HOMO (Highest Occupied Molecular Orbital) density of
the most active compounds is concentrated in the center of
the chalcone moiety (the carbonyl group and unsaturated
linker between A and B rings e Scheme 1) thus suggesting
a role for these regions in the antileishmanial activity (Fig. 2).
The aniline 3e derivative presents a substituent that signif-
icantly compromised the biological activity, compared to 3,
and that reoriented the HOMO density to the substituent
(Fig. 2). Importantly, the addition of chlorine, an electron-
acceptor atom, in 3f restored the activity to a significant level.
However, the volume, the steric and electronic features are
probably important to the antileishmanial profile of this series
as the addition of two chlorines (3g) or a methoxy (3h) group,
which possess a bigger volume and different electrostatic
features, decreased the activity in more than 2-fold compared
to 3f. However, it should be considered that these substituents
still contributed for the activity as these derivatives were more
potent than 3e (Table 1).
Interestingly the addition of a spacer (C1) in the 3i struc-
ture, the most active compound, led to the best activity and
reoriented the HOMO density to the chalcone moiety, which
apparently contributed to the antileishmanial profile compared
to 3e. The most stable conformation of this derivative revealed
a pep stacking interaction between the benzyl and the chal-
cone ring, which may be important for the interaction with
the target and for avoiding the predicted prohibitive areas
(Figs. 2 and 3).
In addition the molecular volume and weight of derivatives
3
3
˚
˚
(346.07 A > MV < 419.38 A and 345.42 > MW < 462.35)
are similar to more than 80% of all Fluka traded drugs
(MW < 450 Da) and to that determined by Lipinski ‘‘Rule
of 5’’ [31e34]. In fact, according to our research, most of
the sulfonamide 4-methoxychalcone derivatives evaluated
(2.21 > Log P < 4.60, 345.42 Da > MW < 423.49 Da, hydro-
gen bond acceptors ¼2e5 and donors ¼ 0e1) fulfilled all
Lipinski rules, which states that most ‘‘drug-like’’ molecules
have log P ꢂ 5, molecular weight ꢂ 500, number of hydrogen
bond acceptors ꢂ 10, and donors ꢂ 5 (Table 1). These data
may suggest the potentiality of these derivatives as new candi-
date to antileishmanial agents.
Currently, there are many approaches that assess druglike-
ness of compounds based on topological descriptors, finger-
prints of molecular druglikeness, structural keys or other
properties as clogP and molecular weights [33]. In this work
we used the Osiris program (www.organic-chemistry.org/
prog/peo) for calculating the fragment-based druglikeness of
all compounds and other antileishmanial drugs including
pentamidine and glucantime (Fig. 3). Our theoretical data
showed that the most active compounds presented a druglike-
ness value higher than the compounds currently used in ther-
apy and evaluated in this work (Fig. 3). In this study we
also verified the drug score, which combines druglikeness,
We also evaluated some electronic properties of the sulfon-
amide 4-methoxychalcone chalcone derivatives including
Fig. 2. Minimal energy conformation (A), HOMO density encoded onto a van der Waals surface (B) and electrostatic potential map (C) of 4-methoxychalcone (3)
and sulfonamide 4-methoxychalcone derivatives (3ae3i). HOMO absolute density coefficient (isodensity 0.002 e/au3) mapped from deepest red (0.00) to deepest
blue (0.02). The chalcone moiety of all molecules is turned to the front in (A), (B) and (C). The position is slightly altered in (B) and (C) to allow a better view of
the parameters analyzed. (For interpretation of the references to color in figure legends, the reader is referred to the web version of this article.)

760
C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
Fig. 3. Structural alignment of the most (3i) and the less active (3e) derivatives of the sulfonamide 4-methoxychalcone series (A), and the druglikeness and drug
score values (B) and the theoretical toxicity evaluation (C) of the most active compounds (3b, 3d, 3f and 3i) compared to the non-substituted 4-methoxychalcone
and antileishmanial drugs. (A) The most (3i) and the less active (3e) compounds are represented in stick and ball colored by element (Grey ¼ carbon, white ¼ hy-
drogen, blue ¼ nitrogen, red ¼ oxygen and orange ¼ sulfur). The white color in the CPK structure shows the chalcone and sulfonamide parts in these compounds
(3i and 3e) whereas green represents the contributing region of the most active compound (3i) and purple indicates the prohibitive region of the less active de-
rivative (3e). (For interpretation of the references to color in figure legends, the reader is referred to the web version of this article.)
clogP, logS, molecular weight, and toxicity risks in one value
and that may be used to consider the compound overall poten-
tial to qualify for a drug. Our data showed that compound 3i
presented a close value to pentamidine and a good profile
based on the program values (Fig. 3) [24,28e30].
to antileishmanial drugs. These results point the sulfonamide
4-methoxychalcone derivatives as lead compounds for design-
ing new candidates for leishmaniasis treatment.
4. Experimental protocols
According to our theoretical toxicity evaluation of the
tumorigenic, irritant and reproductive profile of the sulfon-
amide 4-methoxychalcone derivatives (3ae3i), they show
a low profile for these toxicity effects similar to glucantime
and the non-substituted methoxychalcone (Fig. 3), except for
3h that presented a medium tumorigenic profile (not shown).
It is important to note that the toxicity predicted herein neither
is a fully reliable toxicity prediction nor guarantees that these
compounds are completely free of any toxic effect. However,
it reinforced the promising profile of these compounds for
further experimental investigation [24,28e30].
4.1. Chemistry
All reagents and solvents used were of analytical grade.
TLC was carried out using silica gel F-254 Glass Plate
(20 ꢃ 20 cm). The H NMR (200e400 MHz) and 13C NMR
1
(50e100 MHz) spectra were obtained from Bruker AC-200F
and Varian model Unity (Varian Oxford AS-400 spectrometer)
using tetramethylsilane as internal standard. The values of the
coupling constants (J ) are given in hertz. Fourier transform
infrared (FT-IR) absorption spectra were recorded in a Perkin-
eElmer 16PC spectrophotometer. The solid samples were
measured using potassium bromide pellets. Microanalyses
were carried out on a Carlo Erba EA 1110 instrument.
3. Conclusion
In conclusion, our data show that the combination of two
different pharmacophoric groups (i.e. chalcone and sulfon-
amide) enhanced the derivatives’ antileishmanial activity at
least 2-fold compared to 3 and other chalcones [9]. The sulfon-
amide 4-methoxychalcone derivatives exhibited a potential
antileishmanial activity in vitro where compound 3i presented
the highest activity against L. braziliensis. The structuree
function analysis revealed that volume, the HOMO density
concentrated in the chalcone moiety, and the conformational
structure of the compounds are important structural and
stereoelectronic features for the antileishmanial activity. The
theoretical study of these molecules also showed that they
fulfilled Lipinski rule of 5 and present druglikeness similar
4.1.1. General procedure for the preparation
of compounds 3ae3i
The sulfonyl chloride was treated with benzylamine in
methanol at 0 ꢄC. The mixture was reacted at room tempera-
ture for 2 h and then poured into iced-water. The precipitate
was collected by filtration, washed with iced-methanol, dried
and recrystallized in ethanol. Compound 3a: yield 43%, m.p.
140e144 ꢄC (lit. [22] 140e141 ꢄC). Compound 3b: yield
40%, m.p. 149e150 ꢄC. IR (KBr, cmꢁ1) nmax: 1659, 1595,
1
1323, 1217, 1144, 700. H NMR (200 MHz, CDCl3, ppm) d:
8.30 (s, 1H, Ar-H), 8.05 (d, 2H, Ar-H), 7.76 (d, J ¼ 15.7 Hz,
1H, olefinic H), 7.48 (m, 5H, Ar-H), 7.04 (d, 1H, Ar-H),

C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
761
3.98 (s, 3H, OCH3), 3.35 (q, 4H, CH2), 1.13 (t, 6H, CH3). 13
C
122.23, 120.26, 112.76, 56.83. Anal. Calcd for C22H17Cl2NO4
S: C, 57.15; H, 3.71; Cl, 15.34; N, 3.03; S, 6.94. Found: C,
56.93; H, 3.71; N, 3.09; S, 6.50%. Compound 3h: yield
60%, m.p. 183e185 ꢄC. IR (KBr, cmꢁ1) nmax: 3242, 1659,
NMR (50 MHz, CDCl3, ppm) d: 190.10, 157.99, 142.75,
137.97, 134.75, 132.83, 130.48, 129.88, 128.60, 128.46,
127.35, 121.46, 112.41, 56.13, 41.78, 14.24. Anal. Calcd for
C20H23NO4S: C, 64.32; H, 6.21; N, 3.75; S, 8.59. Found: C,
64.00; H, 6.11; N, 3.73; S, 8.03%. Compound 3c: yield
51%, m.p. 162e166 ꢄC. IR (KBr, cmꢁ1) nmax: 1661, 1594,
1
1595, 1154, 643. H NMR (200 MHz, DMSO-d6) d: 8.1 (d,
2H, Ar-H), 8.09 (d, 2H, Ar-H), 7.73 (d, J ¼ 15.7 Hz, 1H,
olefinic H), 7.69 (d, J ¼ 15.7 Hz, 1H, olefinic H), 7.50 (m,
3H, Ar-H), 7.02 (m, 5H, Ar-H), 4.09 (s, 3H, OCH3), 3.67 (s,
3H, OCH3). 13C NMR (50 MHz, DMSO-d6, ppm) d: 189.68,
158.52, 157.00, 143.10, 138.23, 135.83, 133.78, 131.44,
130.77, 129.44, 129.22, 127.76, 127.55, 122.05, 115.42,
114.87, 57.25, 55.76. Anal. Calcd for C23H21NO5S: C,
65.23; H, 5.00; N, 3.31; S, 7.57. Found: C, 65.58; H, 4.92; N,
3.41; S, 7.40%. Compound 3i: yield 42%, m.p. 167e169 ꢄC.
IR (KBr, cmꢁ1) nmax: 3210, 1655, 1591, 1162, 693. 1H
NMR (200 MHz, CDCl3, ppm) d: 8.25 (s, 1H, Ar-H), 8.04
(d, 2H, Ar-H), 7.73 (d, J ¼ 15.8 Hz, 1H, olefinic H) 7.63 (d,
1H, Ar-H), 7.42 (m, 4H, Ar-H), 7.14 (d, J ¼ 15.8 Hz, 1H,
olefinic H), 7.03 (m, 4H, Ar-H), 6.94 (d, 1H, Ar-H), 5.2 (t,
1H, NH), 4.14 (d, 2H, CH2), 4.09 (s, 3H, OCH3). 13C NMR
(50 MHz, CDCl3, ppm) d: 190.73, 157.99, 143.12, 138.63,
136.75, 135.96, 133.65, 129.99, 129.37, 129.21, 129.09,
128.57, 122.51, 113.10, 57.17, 48.43. Anal. Calcd for
C23H21NO4S: C, 67.79; H, 5.19; N, 3.44; S, 7.87. Found: C,
67.52; H, 5.11; N, 3.50; S, 7.62%.
1
1328, 1217, 1148, 701. H NMR (200 MHz, CDCl3, ppm) d:
8.3 (s, 1H, Ar-H), 8.01 (d, 2H, Ar-H), 7.81 (d, J ¼ 14.3 Hz,
1H, olefinic H), 7.77 (d, 1H, Ar-H), 7.47 (m, 3H, Ar-H and
1H, olefinic H), 7.08 (d, 1H, Ar-H), 3.99 (s, 3H, OCH3),
3.38 (t, 4H, NeCH2), 1.83 (q, 4H, CH2). 13C NMR
(50 MHz, CDCl3, ppm) d: 191.05, 157.36, 142.94, 137.97,
135.20, 133.13, 131.40, 128.90, 128.37, 128.11, 121.85,
112.76, 57.20, 48.05, 26.09. Anal. Calcd for C20H21NO4S:
C, 64.67; H, 5.70; N, 3.77; S, 8.63. Found: C, 64.22; H,
5.62; N, 3.83; S, 8.49%. Compound 3d: yield 38%, m.p.
158e158 ꢄC. IR (KBr, cmꢁ1) nmax: 1661, 1596, 1152, 1109,
699. 1H NMR (200 MHz, CDCl3, ppm) d: 8.23 (d, 1H,
Ar-H), 8.03 (d, 2H, Ar-H), 7.77 (d, J ¼ 15.7 Hz, 1H, olefinic
H), 7.74 (d, 1H, Ar-H), 7.45 (m, 3H, Ar-H and 1H, olefinic
H), 7.08 (d, 1H, Ar-H), 3.99 (s, 3H, OCH3), 3.74 (t, 4H,
OeCH2), 3.27 (t, 4H, NeCH2). 13C NMR (50 MHz, CDCl3,
ppm) d: 190.06, 158.25, 142.46, 137.95, 135.18, 132.94,
131.12, 128.66, 128.50, 127.72, 126.92, 121.81, 112.79,
66.71, 56.32, 46.01. Anal. Calcd for C20H21NO5S: C, 62.00;
H, 5.46; N, 3.62; S, 8.28. Found: C, 61.70; H, 5.37; N, 3.73;
S, 8.05%. Compound 3e: yield 47%, m.p. 200e202 ꢄC. IR
4.2. Biology
1
(KBr, cmꢁ1) nmax: 3329, 1661, 1597, 1158, 693. H NMR
4.2.1. Drugs
(200 MHz, DMSO-d6, ppm) d: 8.20 (s, 1H, Ar-H), 7.91 (d,
2H, Ar-H), 7.50 (m, 6H, Ar-H), 7.44 (d, J ¼ 15.7 Hz, 1H,
olefinic H), 7.02 (m, 5H, Ar-H), 4.10 (s, 3H, OCH3). 13C
NMR (50 MHz, DMSO-d6, ppm) d: 189.61, 157.49, 142.24,
137.53, 134.64, 132.50, 130.00, 128.65, 128.25, 127.24,
128.04, 126.93, 124.07, 120.17, 115.42, 56.05. Anal. Calcd
for C22H19NO4S: C, 67.16; H, 4.87; N, 3.56; S, 8.15. Found:
C, 66.82; H, 4.77; N, 3.50; S, 7.80%. Compound 3f: yield
51%, m.p. 205e207 ꢄC. IR (KBr, cmꢁ1) nmax: 3249, 1660,
1598, 1496, 1147, 689. 1H NMR (200 MHz, DMSO-d6,
ppm) d: 8.15 (d, 1H, Ar-H), 8.01 (d, 2H, Ar-H), 7.65 (m,
5H, Ar-H), 7.60 (d, J ¼ 14.0 Hz, 1H, olefinic H), 7.56 (d,
1H, Ar-H), 7.26 (d, J ¼ 14.0 Hz, 1H, olefinic H), 7.03 (d,
2H, Ar-H), 7.47 (s, 1H, Ar-H), 4.09 (s, 3H, OCH3). 13C
NMR (50 MHz, DMSO-d6, ppm) d: 190.05, 157.90, 142.42,
138.00, 135.88, 135.83, 133.22, 131.57, 130.41, 128.67,
128.92, 128.76, 128.53, 127.70, 122.96, 122.36, 112.88,
57.04. Anal. Calcd for C22H18ClNO4S: C, 61.75; H, 4.24;
Cl, 8.29; N, 3.27; S, 7.49. Found: C, 62.03; H, 4.22; N,
3.43; S, 7.42%. Compound 3g: yield 48%, m.p. 218e219 ꢄC.
All sulfonamide 4-methoxychalcone derivatives (3, 3ae3i)
were added into the cultures as a dimethyl sulfoxide (DMSO)
solution (50 mM). The final solvent (DMSO) concentrations
never exceeded 1% (v/v) and had no effect on the parasites’
proliferation or morphology.
4.2.2. Parasites and cell line
L. braziliensis (MHOM/BR/75/M-2904) promastigotes
were grown at 28 ꢄC in Schneider’s supplemented with 5%
of heat inactivated fetal bovine serum (FBS). T. cruzi (Y
strain) epimastigotes were grown at 28 ꢄC in LIT medium
supplemented with 10% of FBS. The J774.A1 macrophage
cell line was cultivated in RPMI 1640 (Gibco BRL) medium
supplemented with 2 g/L of sodium bicarbonate and 10% of
FBS without Hepes.
4.2.3. Antiparasitic assays
4.2.3.1. Promastigotes. For the parasite growth inhibition
assays, Leishmania promastigotes were harvested on the expo-
nential phase of growth and the concentration was adjusted to
10 ꢃ 106 parasites/ml in Schneider’s medium plus 5% FBS.
The compounds solubilized in DMSO were diluted to appropri-
ate concentrations ranging from 100 to 1.6 mM in culture
medium. One hundred microliters of the parasite suspension
was added to 96-well plates and incubated at 28 ꢄC for 72 h in
the presence of different compounds’ concentrations. Ampho-
tericin B (ranging from 31.25 to 1000 nM) was used as positive
1
IR (KBr, cmꢁ1) nmax: 3238, 1652, 1590, 1495, 1160, 654. H
NMR (200 MHz, CDCl3, ppm) d: 8.15 (s, 1H, Ar-H), 8.01 (d,
2H, Ar-H), 7.78 (d, J ¼ 14.8 Hz, 1H, olefinic H), 7.67 (s, 1H,
Ar-H), 7.55 (m, 4H, Ar-H), 7.01 (dd, 1H, Ar-H), 7.09 (d,
J ¼ 14.8 Hz, 1H, olefinic H), 7.24 (d, 1H, Ar-H), 4.09 (s,
3H, OCH3). 13C NMR (50 MHz, CDCl3, ppm) d: 190.04,
157.35, 142.09, 137.86, 136.03, 135.79, 133.00, 130.92,
130.14, 129.14, 128.68, 128.53, 128.16, 126.69, 122.78,

762
C.R. Andrighetti-Fro¨hner et al. / European Journal of Medicinal Chemistry 44 (2009) 755e763
control and DMSO 1% was used as negative controls. Three
experiments were carried out in triplicate, and the number of
live parasites was determined by counting in Neubauer chamber.
The 50% inhibitory concentrations (IC50) were determined by
linear regression analysis, and represented the mean ꢀ standard
error of three independent experiments (GraphPad Software,
San Diego, CA). The IC50 for pentamidine isothionate was
obtained from Lima et al. [25].
T. cruzi epimastigotes were harvested at the exponential
phase of growth and the concentration adjusted to 5 ꢃ 106 par-
asites/ml in LIT medium plus 10% FBS. One hundred micro-
liters of the parasite suspension was added to 96-well plates
and incubated at 28 ꢄC for 72 h in the presence of different
compounds’ concentrations (ranging from 100 to 1.6 mM).
Benzonidazole (10 mM) was used as positive control and
DMSO 1% was used as negative control. Experiments were
carried out in triplicate, and the number of live parasites was
determined by counting in Neubauer chamber.
(http://www.organic-chemistry.org/prog/peo/druglikeness.html)
as described elsewhere [19,24,28e30]. Structures were mini-
mized and the equilibrium geometry was obtained in vacuum
using a semi-empirical AM1 module. In order to evaluate the
electronic properties of the AM1 minimal energy conforma-
tions, they were submitted to a single-point calculation using
DFT (Density Functional Theory) method with a 6-31G* basis
set of the SPARTAN’06 package. The three-dimensional iso-
surfaces of the molecular electrostatic potential maps
(MEPs) at the van der Waals contact surface represented elec-
trostatic potentials superimposed onto a surface of constant
electron density (0.002 e/au3). They were generated in a range
from ꢁ65 to þ23 kcal/mol. These color-coded isosurface
values provide an indication of the overall molecular size
and location of negative (red) or positive (blue) electrostatic
potentials. The electronic properties (HOMO energy, HOMO
orbital coefficients distribution, LUMO density, dipole mo-
ment, dipole moment vector and lipophilicity-cLogP) were
calculated for all compounds. Hydrogen bond acceptor and
donor, molecular weight and volume and the theoretical toxic-
ity properties were calculated in the Osiris Property Explorer
(http://www.organic-chemistry.org/) and in the in silico
screening program (http://www.molinspiration.com/cgi-bin/
properties).
4.2.3.2. Amastigotes. L. braziliensis axenic amastigotes were
obtained by differentiation of promastigotes from the station-
ary phase in Schneider medium plus 20% FBS pH 6.3 at 34 ꢄC
for 72 h in 25 cm2 tissue culture flasks [34]. The J774.A1 cells
were removed by scrapping and incubated at 34 ꢄC with
axenic amastigotes at a 10:1 parasite:cell ratio in RPMI
medium supplemented with 20% FBS and maintained under
gentle shaking overnight. One hundred microliters of the cell
suspension was seeded on 13 mm glass coverslips in 24-well
plates, and incubated with different 3i concentrations (1e100
mM) for 48 h at 34 ꢄC/5% CO2. After that, coverslips were
air-dried, methanol fixed, Giemsa stained. The rate of cell
infection as well as the number of amastigotes per cell was
microscopically evaluated (1000ꢃ) by counting 200 random
cells in at least of two independent experiments in duplicate.
The percentage of inhibition (PI) was calculated according
to Guru et al. and values higher than 25% were considered
significative. Amphotericin B was used as positive control,
and DMSO 1% as negative control.
Acknowledgements
The authors thank Conselho Nacional de Desenvolvimento
´
´
Cientıfico e Tecnologico/CNPq (Brazil), Fundac¸ao de Amparo
a Pesquisa do Estado do Rio de Janeiro (FAPERJ), CAPES
~
`
and Universidade Federal Fluminense (UFF) for the financial
support and scientist fellowships.
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