Design, synthesis and antileishmanial in vitro activity of new series of chalcones-like compounds: A molecular hybridization approach

Article abstract of DOI:10.1016/j.bmc.2011.05.055

The chalcone-like series 1a-1g was efficiently synthesized from Morita-Baylis-Hillman reaction (52-74% yields). Compounds 1a-1g were designed by molecular hybridization based on the anti-inflammatory drug methyl salicylate (3) and the antileishmanial moie

Full text of DOI:10.1016/j.bmc.2011.05.055

Bioorganic & Medicinal Chemistry 19 (2011) 4250–4256
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antileishmanial in vitro activity of new series
of chalcones-like compounds: A molecular hybridization approach
Ticiano P. Barbosa a, Suervy C. O. Sousa a, Francianne M. Amorim b, Yara K. S. Rodrigues b,
Priscilla A. C. de Assis a, John P. A. Caldas c, Márcia R. Oliveira b,c, Mário L. A. A. Vasconcellos a,
⇑
a Laboratório de Síntese Orgânica Medicinal da Paraíba (LASOM-PB), Departamento de Química, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
b Departamento de Biologia Molecular, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
c Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The chalcone-like series 1a–1g was efficiently synthesized from Morita–Baylis–Hillman reaction
(52–74% yields). Compounds 1a–1g were designed by molecular hybridization based on the anti-inflam-
matory drug methyl salicylate (3) and the antileishmanial moiety of the Morita–Baylis–Hillman adducts
2a–2g. The 1a–1g compounds were much more actives than precursor series 2a–2g, for example,
Received 12 March 2011
Revised 21 May 2011
Accepted 26 May 2011
Available online 1 June 2011
IC50 = 7.65
compared to IC50 = 50.08
ues of compound 3 (228.49
(108.50 M on L. amazonensis and 118.83
Leishmania.
l
M on Leishmania amazonensis and 10.14
M on L. amazonensis and 82.29
M on L. amazonensis and 261.45
l
M on Leishmania chagasi (compound 1c) when
M on L. chagasi (compound 2c). The IC50 val-
M on L. chagasi) and acryloyl salicylate 4
l
l
Keywords:
l
l
Molecular hybridization
Morita–Baylis–Hillman adducts
Antileishmanial activity
Methyl salicylate
l
l
M on L. chagasi) were determined here, by the first time, on
Ó 2011 Elsevier Ltd. All rights reserved.
Acryloyl salicylate
1. Introduction
the primary etiologic agent of the diffuse cutaneous form of the
disease.9
Leishmaniasis affects almost 12 million people in nearly 90
countries, representing a worldwide public health problem. More
than 350 million people are currently at risk, and 2 million new cases
are registered each year. An estimated 51,000 annual deaths for
leishmaniasis have been reported.1–3 Leishmaniasis infection causes
many different clinical manifestations, such as cutaneous,4 mucocu-
taneous5 and a fatal infection of the liver and spleen (visceral
leishmaniasis, also known as kalazar).6,7 Human infection with
Leishmania chagasi, the protozoan causing South American visceral
leishmaniasis (VL), causes diverse sequelae ranging from subclinical
infection to progressive fatal disease.8 However, the cutaneous form
is the most common. Both the cutaneous and mucocutaneous forms
can cause severe disfigurement to patients, including ulcerative skin
lesions and the destruction of the mucous membranes of the nose,
mouth, and throat, leading to permanent disfigurement (diffuse
cutaneous form of the disease) and frequently social ostracization.
The most common species in the Americas and the most important
causative agent of cutaneous and mucocutaneous leishmaniasis in
Brazil is Leishmania braziliensis, while Leishmania amazonensis is
For the last 60 years, the treatment options for leishmaniasis
are limited and involve the administration pentavalent antimonial
family agents, represented by Sodium stibogluconate (PentostamÒ)
and Meglumine antimoniate (GlucantimeÒ). Second line drugs in-
clude pentamidine and Amphotericin B, but these drugs have not
experienced widespread use due to the severe toxicities and high
costs.10 Pentamidine present several side effects, including renal
and hepatic toxicity, pancreatitis, hypotension, dysglycemia, and
cardiac abnormalities.11 Amphotericin B is quite effective for vis-
ceral leishmaniasis being, however very expensive and do not ap-
pear to be suitable for treatment of non-visceral diseases.11 Up to
now, no vaccine approved for human use is available.12,13 There-
fore, there is an increasingly urgent need for the development of
new, inexpensive, effective and safe drugs for the treatment of
leishmaniasis and then, the discovery of new lead compounds for
this disease is a pressing concern for global health programs.
A promising route towards development of improved therapeu-
tic agents for diseases caused by human pathogens, such as para-
sitic protozoa, is the identification of key differences between the
metabolism of the host and the parasite, and the development of
inhibitors of parasite-specific enzymes.14 The mechanisms by
which different Leishmania species cause different pathologies
are largely unknown. In a pioneering paper on this subject, Baiocco
⇑
Corresponding author. Tel.: +55 083 3216 7589; fax: +55 083 3216 7333.
E-mail address: mlaav@quimica.ufpb.br (M.L.A.A. Vasconcellos).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.055

T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
4251
et al.15 by first time disclosed the molecular mechanism of action
OH
O
OH O
of antimonial drugs against the parasite Sb(III), which is coordi-
nated by the two redox-active catalytic cysteine residues (Cys52
and Cys57), one threonine residue (Thr335), and His4610 of the
2-fold symmetry related subunit in the dimer, strongly inhibits try-
panothione reductase (TR).
O
O
CO2CH3
O2N
O2N
1a
2a
Non-steroid anti-inflammatory drug NSAIDs are major drugs
used in the treatment of inflammation and pain in a wide variety
of disorders.16 NSAIDs constitute a diverse group of chemicals, cat-
egorized according to their chemical structures that share the same
therapeutic properties. Among the main compounds are aspirin and
salicylate, diclofenac and flurbiprofen. The best-known mechanism
of action of NSAIDs is the inhibition of prostaglandin synthesis sec-
ondary to their action on cyclooxygenases (COXs). However, data
have been accumulating through the years indicating that NSAIDs
also act on other targets to counteract pain17,18 Since anti-inflam-
matory activities of natural19 and non-natural20 salicylate deriva-
tives have been described and there is an important roles of COXs
and PGE2 during Leishmaniasis infection,21 the design of the new
hybrid salicylate compounds 1a–1g (Fig. 1) could be an attractive
strategy for discover new antileishmania substances.
OH
O
OH
O
O2N
O2N
O
O
CO2CH3
1b
2b
NO2 OH
O
NO2 OH O
O
O
CO2CH3
1c
2c
OH
O
O
OH
O
O
N
N
CO2CH3
In our continuing search for bioactive substances22–26 and in
connection with our efforts towards reactivity of Morita–
Baylis–Hillman reaction (MBHR) study (Scheme 1),27–30 our
research group described in 2006 the molluscicidal activities of
simple aromatic Morita–Baylis–Hillman adducts (MBHA) against
Biomphalaria glabrata (Say) snails, intermediate schistosomiasis
host.31 In sequence, some aromatic MBHA were presented as very
active compounds against the L. amazonensis (cutaneous and
mucocutaneous infections).32 Following that work, we published
the biological evaluation of aromatic MBHA against L. chagasi
(visceral infections) parasites,33 and in 2010, we have shown that
the MBHA 3-hydroxy-2-methylene-3-(4-nitrophenyl) propaneni-
trile, a high antileishmania compound, is also a highly active
compound against epimastigote and trypomastigote form of
Trypanosoma cruzi, the parasite that causes Chagas disease.34 In
the same year, we presented an improved synthesis for 16 MBHA
and their biological evaluation against L. amazonensis and L. chagasi
and we proposed, at the first time, a Structure–Activity-Relation-
ship (SAR) analysis for this class of new antiparasitic compounds.35
In this context, we present in this Letter the design, synthesis,
inhibitory activities on antipromastigote form of L. amazonensis
and L. chagasi of new congener hybrids 1a–1g (Fig. 1). The design
is based on the molecular hybridization36 between the antileish-
mania compounds 2a–2g presented by us in previous paper35
and the methyl salicylate (3) (Fig. 1) (e.g., MetedicÒ). It is important
to detach that the molecular hybridization is a relatively new con-
cept in drug design and development based on the combination of
pharmacophoric moieties of different bioactive substances to pro-
duce a new hybrid compound with improved bioactivity, when
compared to the parent compounds.36
1d
2d
OH
O
OH
OH
O
N
O
N
O
CO2CH3
1e
2e
OH
O
O
O
O
O
CO2CH3
Br
Br
1f
2f
OH
O
OH
O
O
CO2CH3
1g
2g
O
O
HO
CO2CH3
CO2CH3
4
3
Figure 1. Previous antileishmanial compounds 2a–2g,
chalcone-like compounds synthesized here 1a–1g, methyl salicylate 3, and a new
compound acryloyl salicylate 4.
a
new hybrid series of
Methylsalicylate (MS) is a naturally occurring compound that
is used as a major active ingredient of balms and liniments sup-
plied as topical analgesics. Despite the common use of MS as a
pain reliever, the underlying molecular mechanism is not fully
understood. Recently, Ohta et al. showed that the inhibitory ac-
tion of MS on transient receptor potential vanilloid subtype 1
(TRPV1) is one of the underlying mechanisms for its analgesic ef-
fects in vivo.37
In this Letter we also present, by the first time, the toxicity eval-
uation against promastigotes forms of L. amazonensis and L. chagasi
of methyl salicylate (3) and the acryloyl salicylate (4) (Fig. 1). All
molecular hybrids MBHA designed here are in accord with the Lipin-
ski’s rule of five38 and also with the recent Reynisson’s rule.39
We also should emphasize the structural similarity between
compounds 1a–1g designed here and the chalcones (Scheme 2)
O
OH
EWG
EWG
+
N
R
R
conditions
NR3 (promoter or catalyst)
N
DABCO
R= alkyl, aryl, H
EWG= CO2R, CN, COR, others
MBHA
Scheme 1. The Morita–Baylis–Hillman reaction (MBHR).
that exhibit various biological activities, such as antibacterial,
antifungal, antitumor, anti-inflammatory and antiparasitic
properties.40–42 Several chalcone-like compounds have been
recently synthesized and also exhibit various biological activities.43

4252
T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
O
C6
O
C6
C3
C6
C3
C6
NO2 O
O
O2N
O
N
O2N
O
OH
O
5a
5d
5h
5b
5c
O
O
a Chalcone
O
Chalcone-Like structures
O
N
Scheme 2. The structural similarities between compounds 1a–1g and chalcones.
N
Br
5f
5g
5e
2. Material and methods
2.1. Chemistry
Figure 2. The aromatic aldehydes 5a–5h used as electrophiles in this work.
Commercially available reagents were purchased from AldrichÒ
and AcrosÒ and used without further purification. The solvents
used in this study were purchased from TediaÒ, the acryloyl chlo-
ride was purchased from MerckÒ and methyl salicylate was pur-
chased from DynamicsÒ. All the new compounds were
characterized by 1H NMR, 13C NMR, FT-IR, and HR-MS, using a
NMR Varian Unitty Plus (300 MHz to 1H and 75 MHz for 13C), a
NMR Varian Unmrs (400 MHz 1H and 100 MHz for 13C) in CDCl3
using TMS as an internal standard. The Fourier Transform Infrared
Spectroscopy (FT-IR) spectra were obtained using a spectropho-
tometer IR-Prestige-21 (Shimadzu). HR-MS spectra were obtained
on a Shimadzu LC–MS-IT-TOF. Thin-layer chromatography was
performed on 0.25 mm plates for TLC silica gel Kieselgel 60 (What-
manÒ) and spots were visualized with short wavelength UV light
254 nm.
under reduced pressure. After purification by flash chromatogra-
phy using ethyl acetate/hexane (1:9; 2:8 or 3:7) as eluent, the
products were ready for in vitro bioevaluations.
2.1.2.1. Methyl-2-{2-[hydroxy(4-nitrophenyl)methyl])acryloyl-
oxy}benzoate (1a).
3478, 3001,2954, 1732, 1612, 1531, 1446, 1400, 1346, 972, 852,
825, 756, 732 cmꢁ1 1H NMR (300 MHz, CDCl3) d 33.76 (s, 3H),
Seventy-one % yield; yellow oil; IR (KBr):
;
4.06 (d, 1H, J = 4.0 Hz, CHOH), 5.79 (d, 1H, J = 3.6 Hz), 5.96 (s,
1H), 6.65 (s, 1H), 7.04 (dd, 1H, J = 0.8 Hz/8.0 Hz), 7.31 (ddd, 1H,
J = 1.0 Hz/7.8 Hz), 7.54 (ddd, 1H, J = 1.8 Hz/7.6 Hz/7.9 Hz), 7.63 (d,
2H, J = 8.8 Hz) 8.01 (dd, 1H, J = 1.8 Hz/7.8 Hz) 8.19 (d, 2H,
J = 8.8 Hz); 13C NMR (CDCl3, 50 MHz) d 52.3; 71.9, 122.4, 123.4
(2C), 123.6, 126.3; 127.5(2C), 129.5, 131.8, 134.1, 140.9, 147.2,
148.5, 145.0, 164.2, 164.7. HR-MS-Mass calculated: 380.0746
[M+23], found: 380.0701; C18H15NNaO7.
2.1.1. Synthesis of methyl salicylate acrylic ester (4)
In a 125 mL flask, 40 mmol (6.08 g) of methyl salicylate (3) was
dissolved in 20 mL of dichloromethane. Then, it was added
42 mmol of triethylamine. The resulting mixture was kept stirring
for 15 min in an ice bath, shortly after this time was dripped into
the flask a solution containing 42 mmol (3.5 mL) of acryloyl chlo-
ride in 10 mL of dichloromethane. The reaction mixture was kept
under agitation and product formation was accompanied by TLC
analysis using ethyl acetate/hexane (1:9 by volume). After 2 h of
reaction, the mixture was put into a separation funnel washed with
a 10% NH4Cl solution (30 mL) to remove the triethylammonium
salt and then preceded to the extraction with dichloromethane
(3 ꢀ 15 mL). The organic phase was then dried with anhydrous
Na2SO4 and the solvent was evaporated under reduced pressure
in rotaevaporatory. The reaction product was isolated by flash
chromatography column on silica gel, using hexane as eluent
(200 mL), and increasing slowly the polarity of eluent. The frac-
tions were collected, and the product was obtained in the form
of oil in 71% yield; yellow oil; IR (KBr): 2999, 2953, 1726, 1631,
2.1.2.2. Methyl 2-{2-[hydroxy(3-nitrophenyl)methyl])acryloyl-
oxy}benzoate (1b)
3020, 2951, 1728, 1608, 1531, 1435, 1350, 1300, 1083, 806, 759,
690 cmꢁ1 1H NMR (300 MHz, CDCl3) d 3.80 (s, 3H), 5.82 (d, 1H,
.
Seventy % yield; oil; IR (KBr): 3498, 3078,
;
J = 5.4 Hz), 5.97 (s, 1H), 6.69 (s, 1H), 7.07 (dd, 1H, J = 1.2/8.0 Hz),
7.33 (ddd, 1H, J = 1.2/7.6/7.8 Hz), 7.54 (t, 1H, J = 7.8/8.0 Hz), 7.57
(ddd, 1H, J = 1.8/2.0/7.8 Hz), 7.83 (d, 1H, J = 7.8 Hz), 8.04 (dd, 1H,
J = 1.6 Hz/7.8 Hz), 8.16 (ddd, 1H, J = 0.8/1.2/2.2/7.8 Hz), 8.35 (sl,
1H).; 13C NMR (CDCl3, 7 MHz) d 52.4, 72.1, 121.7, 122.6, 122.7,
123.7, 126.4, 129.3, 129.7, 131.9, 132.9, 134.1, 141.0, 143.4,
148.3, 150.1, 164.4, 164.8, HR-MS-Mass calculated: 380.0746
[M+23], found: 380.0692; C18H15NNaO7.
2.1.2.3. Methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyl
oxy}benzoate (1c).
3078, 3001, 2954, 1732, 1612, 1531, 1446, 1350, 1300, 1087,
856, 736 cmꢁ1 1H NMR (400 MHz, CDCl3) d 3.85 (s, 3H), 5.66 (s,
Seventy-four % yield; oil; IR (KBr): 3479,
;
1H), 6.44 (s, 1H), 6.56 (s, 1H), 7.17 (dd, 1H, J = 0.8/1.2/8.0 Hz),
7.35 (ddd, 1H, J = 0.8/1.2/8.0 Hz), 7.50 (ddd, 1H, J = 1.2/1.6/
8.0 Hz), 7.59 (ddd, 1H, J = 1.6/8.0 Hz), 7.71 (ddd, 1H, J = 1.2/
7.6 Hz), 8.00 (d, 1H, J = 8.0 Hz), 8.03 (dd, 1H, J = 1.2/8.8 Hz), 8.05
(dd, 1H, J = 1.6/8.0 Hz); 13C NMR (CDCl3, 100 MHz) d 52.5, 67.7,
122.7, 124.0, 124.8, 126.3, 128.4, 128.7, 129.3, 132.0, 133.7,
134.1, 136.1, 141.2, 145.0, 150.4, 164.5, 165.1. HR-MS-Mass calcu-
lated: 380.0746 [M+23], found: 380.0675; C18H15NNaO7.
1606, 1452, 1404, 1300, 1205, 1153, 1083, 756 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 3.82 (s, 3H), 6.02 (dd, 1H, J = 1.8/10.2 Hz),
6.35 (dd, 1H, J = 10.2/17.2 Hz), 6.61 (dd, 1H, J = 1.8/17.2 Hz), 7.11
(dd, 1H, J = 1.0/8.0 Hz), 7,29 (dt, 1H, J = 1.2/7.6 Hz) 7.54 (dt, 1H,
J = 1.8/7.6 Hz/8.0 Hz) 8.00 (dd, 1H, J = 1.6/7.8 Hz); 13C NMR (CDCl3,
75 MHz) d 52.1; 123.2; 123.7; 126.0; 127.6; 131.7; 132.7; 133.8;
150.3; 164.6; 164.8. HR-MS-Mass calculated: 229.0477 [M+23],
found: 229.0417; C12H13NNa O6.
2.1.2.4. Methyl
2-{2-[hydroxy(pyridin-4-yl)methyl]acryloyl-
2.1.2. General protocols for the 1a–1g MBHA preparations
Reactions were carried out using the corresponding 5a–5h
(Fig. 2) aldehydes (1 mmol), the acrylate 4 (x mmol, Table 1),
solvent-free condition or 1.0 mL of protic/nonprotic solvent
(Table 1), 1 or 0.3 mmol of DABCO at 0 or 25 °C (Table 1) for
x min hꢁ1 dayꢁ1 (Table 1). After that, the reaction media was di-
rectly filtered through silica gel, using ethyl acetate/hexane (2:8;
3:7 or 4:6) as eluent and the reaction products were concentrated
oxy})benzoate (1d).
Sixty-seven % yield; oil; IR (KBr): 3113,
2951, 2850, 1728, 1708, 1600, 1454, 1296, 1199, 1130, 1056,
968. cmꢁ1 1H NMR (300 MHz, CDCl3) d 3.74 (s, 3H); 5.68 (s, 1H);
;
5.99 (s, 1H); 6.64 (s, 1H); 7.01 (dd, 1H, J = 1.0/8.2 Hz); 7.30 (ddd,
1H, J = 1.2/7.4/7.8 Hz); 7.38 (dd, 2H, J = 1.0/4.8 Hz); 7.53 (ddd, 1H,
J = 1.4/1.6/8.0 Hz); 8.01 (dd, 1H, J = 1.6/8.0 Hz); 8.52 (dd, 2H,
J = 1.4/4.4 Hz).; 13C NMR (CDCl3, 75 MHz) d 52.3, 71.7, 121.6 (2C),
122.7, 123.6, 126.3, 129.6, 131.9, 134.0, 140.9, 149.6 (2C), 150.0,

T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
4253
Table 1
Different experimental protocols and results in the preparation of 1a–1g compounds (see Scheme 6)
Entry
Compd
Solvent
DABCO (equiv)
Temp (°C)
Time
Yielda (%)
1
2
3
4
5
6
7
8
1a
1b
1c
1a
1a
1a
1b
1c
1a
1b
1c
1c
1d
1e
1f
Solvent-free condition
Solvent-free condition
Solvent-free condition
tBuOH/H2Ob
Methanol
Methyl salicylate
1
1
1
1
1
1
1
1
0.3
0.3
0.3
0.3
0.3
0.3
1
0
0
0
25
25
0
0
0
0
0
0
0
0
0
0
0
3 h
3 h
6 h
30 min
30 min
24 h
48 h
24 h
3 h
24
15
48
20 min
3 h
27
15
26
8c
0d
53
48
51
70e
61
0
Methyl salicylate
Methyl salicylate
9
Acryloyl salicylate (20 equiv)
Acryloyl salicylate (20 equiv)
Acryloyl salicylate (20 equiv)
Acryloyl Salicylate (20 equiv), methyl salicylate
CH2Cl2
CH2Cl2
Solvent-free condition
Acetonitrile
10
11
12
13
14
15
16
74e
67
60
52
65
4 h
24 h
1g
1
a
b
c
Purified yields.
9:1 t-BuOH/water.
After 30 min a large amount of the acrylate 4 was transformed in the methyl salicylate (3).
After 30 min the acrylate 4 was converted on the methyl acrylate (transesterifications reaction).
Acryloyl salicylate (4) and methyl salicylate (3) excess could be easily recovery in high yields (>95%) by a simple filtration.
d
e
150.6, 164.3, 164.8. HR-MS-Mass calculated: 336.0848 [M+23],
found: 336.0814; C17H15NNaO5.
(sl, 3H), 5.94 (s, 1H), 6,54 (s, 1H), 7,10 (dd, 1H, J = 1.0 Hz/8,0 Hz),
7,17 (dd, 1H, J = 1.0 Hz/8,0 Hz), 7,32 (m, 1H), 7,56 (m, 1H), 8.05
(m, 1H); 13C NMR (CDCl3, 75 MHz) d 27.1, 32.8, 52.2 (2C), 123.2 e
123.4 (2C), 123.8 (2C), 126.0 (2C), 128.2, 131.7, 131.8, 133.81
(2C), 138.1, 150.6 (2C), 165.0 (2C), 165.3, 171.4 HR-MS-Mass calcu-
lated: 435.1056 [M+23], found: 435.0965; C22H20O8.
2.1.2.5. Methyl
2-{2-[hydroxy(pyridin-3-yl)methyl]acryloyl-
oxy})benzoate (1e).
Sixty % yield; oil; IR (KBr): 3151, 3055,
1728, 1608, 1454, 1431, 1203, 1053, 964, 732 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 3.68 (s, 3H); 5.74 (s, 1H); 6.20 (s, 1H); 6.67
(s, 1H); 7.00 (d, 1H, J = 8.0 Hz); 7.24 (d, 1H, J = 7.8 Hz,); 7.28 (ddd,
1H, J = 1,0/7.8 Hz); 7.51 (ddd 1H, J = 1,8/7.8 Hz); 7.81 (dd, 1H,
J = 7.8 Hz); 7.98 (dd, 1H, J = 1.6/7.8 Hz); 8.39 (d, 1H, J = 4.6 Hz);
8.56 (d, 1H, J = 1.8 Hz).; 13C NMR (CDCl3, 75 MHz) d 51.9, 69.9,
122.8, 123.3, 123.5, 126.0, 128.0, 131.6, 133.8, 134.9, 137.6,
141.5, 148.2 (2C), 149.9, 164.1, 164.7. HR-MS-Mass calculated:
336.0848 [M+23], found: 336.0791; C17H15NNaO5.
2.2. Biology33
Logarithmic phase promastigotes (1 ꢀ 106 parasites mLꢁ1) of
L. amazonensis (MHOM/IFLA/BR/67/PH8) and L. chagasi (MCAN/
BR/99/JP15) were incubated at 26 °C in Schneider’s Drosophila
medium supplemented with 20% of fetal bovine serum, streptomy-
cin (100 l
g mLꢁ1), and penicillin (100 U mLꢁ1), in the presence or
absence of different concentrations of new hybrids chalcones-like
1a–1g MBHA, methyl salicylate acrylic ester (4), methyl salicylate
(3) and methyl salicylate acrylic ester dimer 6 The reference drugs
GlucantimeÒ and Amphotericin BÒ were used as control. After 72 h,
parasites were collected, fixed in isotonic solution (10.5 g citric
acid, 7.0 g NaCl, 5.0 mL formalin and 1000 mL distilled water)
and examined under light microscopy. The inhibitory effect of
the compounds on cell growth was estimated by cell counting
using a Neubauer chamber. The IC50/72 h, concentration responsi-
ble for a 50% reduction in culture growth after 72-hour incubation
was determined by the probity regression model using the soft-
ware SPSS 8.0 for Windows. All experiments were performed in
triplicate and repeated at least three times. Statistical analysis of
data was done by means of one-way ANOVA. P values of 0.05 or
less were considered significant.
2.1.2.6. Methyl 2-{2-[(4-bromophenyl-hydroxy)methyl]acryloyl
oxy}benzoate (1f).
3078, 3001, 2951, 1716, 1604, 1485, 1435, 1404, 1300, 1041,
960, 844, 810, 756, 729 cmꢁ1 1H NMR (300 MHz, CDCl3) d 3.76
Fifty-two % yield; oil; IR (KBr): 3502,
;
(s, 3H); 5.68 (s, 1H); 5.92 (s, 1H); 6.61 (s, 1H); 7.05 (dd, 1H,
J = 1.2/8.0 Hz); 7.31 (1H) 7.33 (d, 2H, J = 8.6 Hz); 7.48 (d, 2H,
J = 8.0 Hz); 7.55 (ddd, 1H, J = 1.6/7.6 Hz); 8.02 (dd, 1H, J = 1.8/
7.8 Hz); 13C NMR (CDCl3, 75 MHz) d 52.3, 72.3, 121.7, 122.8,
123.7, 126.3, 128.5 (2C), 128.8, 131.4 (2C), 131.8, 134.1, 140.1,
141.6, 150.1, 164.6, 164.8. HR-MS-Mass calculated: 390.0103 [M],
found: 390.0138; C18H15BrO5.
2.1.2.7. Methyl 2-{2-[hydroxy(naphthalen-2-yl)methy])acryloyl
loxy}benzoate (1g).
3055, 2951, 1732, 1604, 1485, 1446, 1300, 1087, 1041, 960, 856,
817, 752 cmꢁ1 1H NMR (300 MHz, CDCl3) d 3.69 (s, 3H); 5.90 (sl,
Sixty-five % yield; oil; IR (KBr): 3498,
3. Results and discussion
3.1. Chemistry
;
1H); 5.97 (s, 1H); 6.64 (s, 1H); 7.02 (dd, 1H, J = 0.8/8.0 Hz); 7.28
(ddd, 1H, J = 0.8/7.8 Hz); 7.50 (m, 4H); 7.84 (m, 3H); 7.95 (sl,
1H); 8.01 (dd, 1H, J = 1.6/7.8 Hz); 13C NMR (CDCl3, 75 MHz) d
52.3, 72.8, 123.0, 123.8, 124.8, 125.8, 126.0, 126.2, 126.2, 127.7,
128.1, 128.2, 128.8, 131.9, 133.1, 133.3, 133.9, 138.4, 142.0,
150.2, 164.8, 165.0 HR-MS-Mass calculated: 385,1052 [M+23],
found: 385.0976; C17H15NNaO5.
We chose as strategy the use of MBHR44 between the unpub-
lished acrylate 4 (Scheme 3) and the commercials aldehydes
5a–5h (Fig. 2) as key-step of the MBHA 1a–1g one-pot syntheses.
The synthesis of 4 was carried out in good yield reacting methyl
salicylate (3) and commercial acryloyl chloride on CH2Cl2 as sol-
vent at 0 °C for 2 h (71%, Scheme 3). The acrylate 4 is stable for
more than 30 days at low temperature (ꢁ4 °C/ꢁ20 °C) or stable
at room temperature by addition of BHT as antipolymerization.
However, a new acrylate 6 was formed after 48 h at room
2.1.2.8. Bis(2-(methoxycarbonyl)phenyl)-2-methylenepentane
dioate (25).
Fifty-one % yield; oil; IR (KBr): 2999, 2953,
1726, 1631, 1606, 1452, 1404, 1300, 1205, 1153, 1083, 756. cm
ꢁ1
;
1H NMR (300 MHz, CDCl3) d 2.96 (m, 4H), 3.84 (sl, 3H), 3.85

4254
T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
CO2CH3
OH
CO2CH3
DABCO
O
O
O
OH
(0.3 or 1 Equiv.)
TEA, CH2Cl2
0oC, 2h, 71%
O
Cl
+
+
O
Ar
O
Ar
O
O
Condition
(solvent, solvent-free,
temperature, rate)
O
O
O
O
3
4
(1-20 Equiv.)
,
5a-5e
5g, 5h
Scheme 3. Synthesis of acrylate 4.
4
1a-1g
Scheme 6. Morita–Baylis–Hillman reaction on the 1a–1g preparations (see Table
1).
O
O
48h, r.t.
solvent-free
O
O
O
2
0.3 equiv trying to minimize the dimers formation between the
acrylates. Satisfactorily, this experimental modification was effi-
cient and we could to prepare the 1a and 1b MBHA in good yields
(Table 1, entries 9 and 10). Curiously, MBHA 1c could not be pre-
pared using this methodology even after fifteen days of reaction
(Table 1, entry 11). Fortunately, in this case, the addition of methyl
salicylate (3) as protic solvent and using 20 equiv of 4 was efficient
for the MBHA 1c preparation in good yield (Table 1, entry 12). The
acrylate 4 could be easily recovery in high yields (>95%) by a sim-
ple filtration. These efficient results can be understood based on
the very recent Cantillo and Kappe work.49 They proven that the
use of phenols as a solvent or an addictive can improve rate and
yields on MBH reaction by change it slow determination rate
(RDS).49
For the preparation of the 1d and 1e pyridine MBHA derivatives,
reaction were carried out between the aldehydes pyridine-4-car-
boxaldehyde (5d) or pyridine-3-carboxaldehyde (5e) with the
acrylate 4, on dichloromethane as solvent. In these cases, the reac-
tions mixtures showed low solubilities in solvent-free conditions.
Dichloromethane also has the advantage being easily evaporated
and do not contained significant traces of water, which could
hydrolyze the MBHA. These experimental protocols produced 1d
and 1e in moderated or good yields (Table 1, entries 13 and 14).
Unlike of the expected, reaction between pyridine-2-carboxal-
dehyde (5f) and acrylate 4 produced several byproducts and the
corresponding chalcone-like compound could not be isolated (not
shown). Based on characteristics fragmentation obtained by
CGꢁMS, we speculate that principal byproducts is the indolizine
8 (m/z = 295, m/z = 264, m/z = 144 and m/z = 116, Scheme 7).
Unfortunately due to the existence of several byproducts of similar
polarities of 8, we cannot isolate pure indolizine 8 and than its
complete characterization was not done. Basavaiah et al. estab-
lished that several indolizines can be synthesized from MBHR.50
Several conditions (not shown) were investigated aiming to pre-
pare 8 efficiently, but without any success.
CO2CH3
CO2CH3
O
51%
CO2CH3
4
6
Dimerization
Scheme 4. Spontaneous dimerization of acrylate 4. Synthesis of acrylate 6.
temperature in solvent-free condition, from a spontaneous dimer-
ization reaction (Scheme 4).
Our first protocol aim to prepare 1a–1g MBHA was at low tem-
perature and on solvent-free condition.35 When the reaction be-
tween excess of acrylate 4 and aldehyde 5a was performed under
1 equiv of DABCO as promoter, the expected adduct 1a was ob-
tained in low yield (Table 1, entry 1) and the principal product of
reaction was the dioxanone 7 (Scheme 5), that can be understood
based on nonprotic mechanism proposed by McQuade.45 When
the reactions were carried out with aldehydes 5b and 5c (on the
same conditions) the 1b and 1c adducts were obtained in low
yields and over again the corresponding dioxanones were obtained
as principal products (Table 1, entries 2 and 3, see also Scheme 6).
Aiming to minimize the dioxanones formation, the MBHR with
aldehyde 5a was carried out in the presence of protic t-butanol/
water (9:1) as solvent, modifying the nonprotic to protic mecha-
nism, as proposed by Aggarwal.46 Both, protic and the nonprotic
mechanisms to the MBHR were recently corroborated by Coelho.47
As expected, there was a total disappearance of 7. Unfortunately,
due the salicylate moiety on MBHA 1a to be a good leaving group,
the adduct 1a was also obtained in low yield, and the methyl salic-
ylate (3) was the principal compound obtained of this reaction
(Table 1, entry 4). The use of anhydrous methanol as solvent
presented to be also inefficient producing the methyl acrylate from
a transesterification reaction (Table 1, entry 5). However, when the
methyl salicylate itself was used as protic solvent, the 1a, 1b, and
1c MBHA could be obtained in moderated yields (Table 1, entries
6, 7, and 8).
For the adduct 1f preparation (Fig. 1) was used solvent-free con-
dition at 0 °C (Table 1, entry 15, and 52% yield). Unfortunately, the
use of other protocols not improved yield on 1f preparations. Final-
ly, we prepared, in improved possible way, the MBHA 1g in mod-
erated yield (65% yields, Table 1, entry 16) by use acetonitrile as
solvent. Curiously, in this last case the solvent-free condition, prot-
ic and phenol addition as solvent not improved yields.
As reported by McQuade, excess of aldehydes under a pro-
longed reaction time leads to the formation of dioxanones like
7.48 Therefore, we realize to used a larger amount of acrylate 4
(20 equiv) aim to minimize the formation of dioxanones. Besides
that, in diluted reaction medium, the formation of dioxanones
would be less favored, after the proton transfer step (rate-
determining step, RDS). We also decrease the DABCO quantity to
NO2
DABCO
O
O
O
OH
(1Equiv.)
O
O
+
+
O
O
no solvent
O
0 ºC
O
O
O
O
NO2
NO2
NO2
1 mmol
2 mmol
27%
>50%
4
5a
1a
7
Scheme 5. Dioxanone 7 as the principal product of MBH reaction on solvent-free condition.

T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
4255
CO2CH3
N
O
O
8
O
CO2CH3
O
N
N
O
N
N
O
O
O
C8H7N
m/z: 116
C16H10NO3
m/z: 264
C17H13NO4
m/z: 295
C9H6NO
m/z: 144
Scheme 7. Characteristics fragmentation of 8.
from that occurs on compound 1a, the NO2 group in 1c is not com-
pletely conjugated with the aromatic moiety. This conformational
modification caused by the o-nitro group can change the interac-
tion between ligand–enzyme or/and change the reduction poten-
tial of these compounds modifying, for example, the redox
system TR/trypanothione that is vital for parasite survival within
the host cell.52–54 Even if, up to now, the correct biological mecha-
nism of action of these MBHA is not known, it is reasonable to
speculate for these different biological activities on 1a, 1b, and
1c compounds in this series.
Table 2
a,b
Antileishmanial in vitro activity of 3, 1a–1g, 4 and dimer 6 and GlucantimeÒ as
reference compound
Compd
IC50
g mLꢁ1) L.
IC50
amazonensis
(
l
M) L. IC50
IC50 (lM)
L. chagasi
(
l
(l
g mLꢁ1
)
amazonensis
L. chagasi
3
1a
1b
1c
1d
1e
1f
1g
34.73
4.05
8.16
2.73
7.46
9.75
8.58
3.26
22.35
9.25
>4000
0.11
228.49
11.34
22.86
7.65
23.83
31.15
22.00
9.00
108.50
22.45
Variable
0.12
39.74
20.48
14.51
3.62
10.44
12.11
16.78
14.98
24.48
20.22
>4000
0.64
261.45
57.34
40.64
10.14
33.35
38.69
43.03
41.38
118.83
49.08
Variable
0.69
Further, the position 4 or 3 of the nitrogen atom in the pyridine
rings into the corresponding 1d and 1e compounds did not signif-
icantly alter these bioactivities (Table 2). The same tendency was
4
6
observed in the previous studied 2a–2g series,35 (IC50 = 488.98
lM
GlucantimeÒ
Amphotericin BÒ
on L. amazonensis; IC50 = 494.81
l
M on L. chagasi to the compound
2d and IC50 = 448.50
l
M on L. amazonensis; IC50 = 350.57 lM on L.
chagasi to the compound 2e).35 However, the 1d and 1e (Table 2)
were almost 20 times more active than the corresponding com-
pounds 2d and 2e.
a
IC50 values obtained from a minimum of three separate experiments performed
in triplicate are shown.
b
The reference drugs GlucantimeÒ and Amphotericin BÒ were used in this study
and in vitro activities.
The antileishmania activity of compound 1f is similar than 1d
and 1e (Table 2), differently that determinate to 2f,35 which is al-
most 5 times more active than the corresponding compounds 2d
and 2e in the 2a–2g series (shown in the previous paragraph).
It should be noted that the dimer 6 (Scheme 4), which also is an
analogues of chalcones, has a significant activity against the L.
amazonensis and L. chagasi, unlike the corresponding monomer 4.
This remark suggests once again the importance of the presence
of two the aromatic groups in the 1a–1g series, one of the
3.2. Biology
Our biological data are described in Table 2 and a few comments
about these results deserve be made. Initially, the IC50 evaluation of
methyl salicylate (3) and acryloyl salicylate (4) on L. amazonensis
and L. chagasi have shown a moderated antipromastigote activities
(Table 2) when compared to the designed new compounds 1a–1g.
Secondly, we can note that the new hybrids 1a–1g have higher tox-
icity on L. amazonensis than on L. chagasi. Additionally, the leish-
manicidal activities of compounds 2a–2g (IC50 = 50.08–731.37
lM
on L. amazonensis and IC50 = 52.92–724.59 M on L. chagasi) pre-
l
sented in our previous article.33 are much smaller than the hybrids
1a–1g of this new series of chalcones-like compounds (Table 2).
This experimental observation confirmed the importance of the
salicylate moiety in 1a–1g, to increase the leishmanicidal activity
of this new series, may be also a consequence of anti-COXs activity,
the best known mechanism of action to salicylate deriva-
tives.17,18,21,51 Thus, as idealized by us, the molecular hybridization
is critical to increase the leishmanicidal activity.
We can also notice in Table 2 that the ortho nitro compound 1c
presents the most leishmanicidal activity on both L. amazonensis
and L. chagasi in the 1a–1g series. We recently described that the
presence of the ortho nitro group also increases antileishmania
activity being the compound 2c (Fig. 1) the most active in the
2a–2g series.35 We can identify on geometric shapes of the 1a
and 1c calculated conformationals minima (Fig. 3) that, different
Figure 3. Conformationals minima of 1a and 1c, calculated by B3LYP/6-311++G
(d,p) as calculation level from GAUSSIAN 09 (versions for Linux).

4256
T. P. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 4250–4256
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lipophilic into this new series (calculated from Pharma-algo-
rithmsÒ, Log P = 4.54 0.51),55 lipophilicity it could be an impor-
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In this work, we successfully applied the current mechanistic
knowledge of MBHR, to improve synthetic results.45–49 Our data
also are useful to corroborate these mechanistic proposals for this
fine reaction.
Moreover, in this work, we idealized, synthesized and show
with success one more example of molecular hybridization, a very
important strategy in Medicinal Chemistry.36
Seven new chalcone-like compounds 1a–1g were designed
using molecular hybridization strategy, efficiently synthesized,
completely characterized and bioevaluated as a new series of
in vitro leishmanicides. Amphotericin
B presented a higher
in vitro activity compared with compounds 1a–1g and 2a–2g.
However, Amphotericin B is well-known for its severe and poten-
tially lethal side effects and very high cost to production.56 In addi-
tion, preparation of 1a–1g have advantages in terms of yield and
operational simplicity.
Finally, all these new original series of compounds 1a–1g were
active against the promastigotes form of L. amazonensis and
L. chagasi and much more active than the previous bioevaluated
series 2a–2g.35 This prospective work needed to study the real
mechanism of action of these new compounds. Then, the study
of toxicities in macrophages and the biological mechanisms of
action of these new compounds will now be investigated.
35. Junior, C. G. L.; de Assis, P. A. C.; Silva, F. P. L.; Sousa, S. C. O.; de Andrade, N. G.;
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1997, 23, 3.
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Acknowledgments
This work is supported by the CNPq and CAPES. The authors
thank Professor Alexandre José da Silva Góes (Antibiotic Depart-
ment/UFPE) by HR-MS, NMR (1H and 13C) analysis on the Chemis-
try Department of Federal University of Pernambuco (Recife City,
Pernanbuco, Brazil) where they could gently be made.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.055.
43. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Cara, C. L.; Cruz-Lopez, O.; Preti, D.;
Tolomeo, M.; Grimaudo, S.; Di Cristina, A.; Zonta, N.; Balzarini, J.; Brancale, A.;
Sarkar, T.; Hamel, E. Bioorg. Med. Chem. 2008, 16, 5367.
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