Synthesis, protease inhibition, and antileishmanial activity of new benzoxazoles derived from acetophenone or benzophenone and synthetic precursors

Article abstract of DOI:10.1007/s00044-017-1824-y

Abstract: This work reports the synthesis, protease inhibition, and antileishmanial activity of ten benzoxazole derivatives, which were obtained in a three-step synthetic route from 4-hydroxy-acetophenone and 4-hydroxy-benzophenone. These benzoxazoles, the synthetic intermediates, and the starting ketones were evaluated for their inhibitory effect on the activity of cysteine (papain, rCPB2.8, and rCPB3.0) and serine (trypsin) proteases. All compounds showed significant values of IC₅₀ against these enzymes (in the range of 0.0086–0.7612 μM for papain and 0.0075–0.5032 μM for trypsin), being more active than the standard inhibitors (1.7821 and 7.2318 μM, for E64 and TLCK, respectively). Following, all compounds were evaluated in vitro for their leishmanicidal activity against promastigote form of Leishmania amazonensis. The most active compounds were further evaluated against amastigote form and for its toxicity against murine macrophages. The benzoxazole 4d, a benzophenone derivative, and the intermediate 4-hydroxy-3-nitroacetophenone 2b showed significant antileishmanial activity (IC₅₀ = 90.3 μM and IC₅₀ = 130.9 μM, respectively) with selectivity indexes (5.22 and 18.09, respectively) compared to or better than those of two established leishmanicidal drugs, pentamidine (0.58) and amphotericin B (5.31). Graphical Abstract: [InlineMediaObject not available: see fulltext.].

Full text of DOI:10.1007/s00044-017-1824-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1824-y
ORIGINAL RESEARCH
Synthesis, protease inhibition, and antileishmanial activity of new
benzoxazoles derived from acetophenone or benzophenone and
synthetic precursors
Laís R. S. Folquitto1 Priscila F. Nogueira1 Patrícia F. Espuri2 Vanessa S. Gontijo1
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Thiago B. de Souza1 Marcos J. Marques2 Diogo T. Carvalho3
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1
Wagner A. S. Júdice4 Danielle F. Dias
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Received: 23 September 2016 / Accepted: 28 February 2017
© Springer Science+Business Media New York 2017
Abstract This work reports the synthesis, protease inhibi-
tion, and antileishmanial activity of ten benzoxazole
derivatives, which were obtained in a three-step synthetic
route from 4-hydroxy-acetophenone and 4-hydroxy-
benzophenone. These benzoxazoles, the synthetic inter-
mediates, and the starting ketones were evaluated for their
inhibitory effect on the activity of cysteine (papain,
rCPB2.8, and rCPB3.0) and serine (trypsin) proteases. All
compounds showed significant values of IC50 against these
enzymes (in the range of 0.0086–0.7612 µM for papain and
0.0075–0.5032 µM for trypsin), being more active than the
standard inhibitors (1.7821 and 7.2318 µM, for E64 and
TLCK, respectively). Following, all compounds were
evaluated in vitro for their leishmanicidal activity against
promastigote form of Leishmania amazonensis. The most
active compounds were further evaluated against amastigote
form and for its toxicity against murine macrophages. The
benzoxazole 4d, a benzophenone derivative, and the inter-
mediate 4-hydroxy-3-nitroacetophenone 2b showed
significant antileishmanial activity (IC50 = 90.3 µM and
IC50 = 130.9 µM, respectively) with selectivity indexes
(5.22 and 18.09, respectively) compared to or better than
those of two established leishmanicidal drugs, pentamidine
(0.58) and amphotericin B (5.31).
Graphical Abstract
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Keywords Leishmania amazonensis
Benzophenone
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Acetophenone Benzoxazoles Proteases Antiproteolytic
activity
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1824-y) contains supplementary material,
which is available to authorized users.
* Danielle F. Dias
daniferreiradias@yahoo.com.br
Introduction
1
Instituto de Química—Universidade Federal de Alfenas, Alfenas,
MG 37130-001, Brazil
Leishmaniosis is a group of infectious diseases caused by
many protozoa of the genus Leishmania, which are trans-
mitted to humans and small mammals by more than 30
different species of phlebotomine sandflies (Phillips and
Stanley 2011). This disease can manifest as the less severe
cutaneous form or as the lethal visceral form (Eschenlauer
et al. 2009). According to the World Health Organization,
2
Instituto de Ciências Biomédicas—Universidade Federal de
Alfenas, Alfenas, MG, Brazil
3
Faculdade de Ciências Farmacêuticas—Universidade Federal de
Alfenas, Alfenas, MG, Brazil
4
Centro Interdisciplinar de Investigação Bioquímica, Universidade
de Mogi das Cruzes, UMC, Mogi das Cruzes, SP, Brazil

Med Chem Res
there is an estimation of approximately 1.3 million cases of
different clinical forms of leishmaniasis identified each year
(WHO 2016). Throughout the Brazilian territory, many
cases of both visceral and cutaneous leishmaniosis are
documented each year (WHO 2015).
There are about 25 drugs used to treat the clinical com-
plications associated to leishmaniosis, but few of them are
classified as antileishmania drugs for human use and the
most part is not administered orally (Singh and Sivakumar
2004). In some localities, the high consumption of these
drugs has caused resistance to treatment, which leads to an
urge need for the development of alternatives to treat these
diseases (Ready 2014).
Other studies indicated that some heterocycles could act
as potent protease inhibitors and showed interesting antil-
eishmanial activity (Loedige 2015; Sangshetti et al. 2015;
Steert et al. 2010). Among them, benzoxazoles have been
explored as bioactive agents in face of their wide biological
potential as antibacterial, antiparasitic, anti-inflammatory,
and anticancer agents (Radi et al. 2008). Tipparaju et al.
(2008) pointed out the promising activities of natural and
synthetic benzoxazoles against these parasites. In this work,
we describe the synthesis of substances containing the
benzoxazole ring fused to an acetophenone or benzophe-
none nucleus, their protease inhibitor potential and in vitro
antileishmanial activity. Benzoxazoles derived from acet-
ophenones or benzophenones can act as protease inhibitors
since the carbonyl carbon can act as an acceptor electro-
philic center and the benzoxazole nucleus can contribute to
the fixation of these compounds in the active site of the
enzymes (Martins et al. 2009; Steert et al. 2010).
Intense investigations in host-parasite interaction and
Leishmania virulence factors allowed the discovery of new
targets for drug design. There are several enzymes that are
essential for survival and proliferation of Leishmania, and
among them the proteases are well studied, mainly cysteine,
serine, aspartate, and metalloproteinases (Das et al. 2013).
Studies consistently show that in vitro and in vivo inhibition
of cysteine proteases leads to a significant loss of virulence
of parasite (Lima et al. 2013), proving to be very promising
targets in the development of antileishmanial drugs.
The mechanism of action is very similar for serine and
cysteine proteases (Powers et al. 2002). Each enzyme has a
specific amino acid residue in its active site, which is
important for the catalytic action. For cysteine proteases, the
mechanism of peptide hydrolysis of a substrate occurs by
the nucleophilic attack of a thiol group belonging to a
cysteine residue (Fricker 2010). However, serine proteases
use the hydroxyl group of this amino acid as nucleophile for
hydrolysis (Martins et al. 2009). After substrate accom-
modation, the sulfur or oxygen atoms of cysteine or serine,
respectively, attack the carbonyl carbon of the substrate
(Fricker 2010). A good protease inhibitor must mimic the
natural substrate and possess sites which may react with the
nucleophile of the corresponding amino acid residue. It is
known that the pseudopeptide inhibitors containing het-
erocycles and α-ketoeterocycles are quite relevant, since
they mimic this site from the initial attack (Steert et al.
2010).
Some reports on the bioactivities of natural benzophe-
nones and benzophenone analogs (Ayres et al. 2007; Costa
Júnior et al. 2013; Hay et al. 2008; Lenta et al. 2007; Pereira
et al. 2010), and even synthetic and semi-synthetic benzo-
phenones (Al-Kahraman et al. 2012; Maciel-Rezende et al.
2013; Román-Luque-Ortega et al. 2010) showed promising
in vitro antileishmanial activity. In addition, these com-
pounds were also quite active in inhibiting cysteine pro-
teases that were specific of the genus Leishmania (Almeida
et al. 2015). Moreover, compounds with the acetophenone
scaffold have also demonstrated potential antileishmanial
activity (Ansari et al. 2012).
Material and methods
Chemistry
Reagents and solvents were purchased as reagent grade and
were used without further purification. All melting point
determinations were measured on an Aaker PFM-II appa-
ratus and were not corrected. Infrared (IR) spectroscopy was
performed on a Thermo Scientific Nicolet-iS50 spectro-
meter. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AVANCE DRX 300 MHz spectro-
1
meter (300 MHz for H NMR and 75 MHz for 13C spectra)
or on a Bruker AVANCE DRX 400 MHz spectrometer
(400 MHz for 1H NMR and 100 MHz for 13C spectra) using
deuterated chloroform, methanol or dimethyl sulfoxide
(DMSO). The results are presented as chemical shifts (δ)
reported in parts per million (ppm) with reference to tetra-
methylsilane as internal standard. Coupling constants (J)
were reported in Hertz (Hz) and the following abbreviations
1
were used for the H multiplicities: singlet (s), doublet (d),
double doublet (dd), triplet (t), triple triplet (tt), and multi-
plet (m). The reactions were followed by thin-layer chro-
matography (TLC) on commercial silica gel 60 plates.
Column chromatography purifications were performed over
silica gel 60, 70–230 mesh (Merck). All the obtained sub-
stances are soluble in common organic solvents such as
ethyl acetate, chloroform, methylene chloride, and metha-
nol. High-resolution mass spectrometry (HRMS) were
acquired using UltrOTOFq (Bruker Daltonics) ESI-qTOF
mass spectrometer controlled by the software CLASS-VP
6.14 and the samples were solubilized in acetronitrilo. The
clogP and pKa values were obtained from the ChemDraw
Ultra 11.0 and Marvin Sketch 5.6.0.1, respectively.

Med Chem Res
The enzymes rCPB2.8 and rCPB3.0 were generously
gifted by Dr. Luiz Juliano (Department of Biophysics,
Federal University of São Paulo, Brazil), papain, trypsin, E-
64 (1-[[(Ltrans-epoxysuccinyl)-L-leucyl]amino]-4-guanidi-
nobutane), and fluorogenic substrate Z-FR-AMC (carbo-
129.9 (CH, C-6), 126.3 (CH, C-1), 120.7 (CH, C-2), 115.7
(CH, C-5), 26.4 (CH3, C-8). pKa = 5.4.
General procedure for the synthesis of 3a and 3b
(Belamy and Ou 1984)
benzyloxy-Phe-Arg-(7-amino-4-methylcoumarin)
were
commercially obtained from Sigma-Aldrich Sigma (St.
Louis, USA). Substrate hydrolyzes were monitored in a
F2500 Hitachi spectrofluorometer using λEx = 380 nm and
λEm = 460 nm as wavelengths and the enzymatic molar
concentrations were estimated by titration according to
kinetic parameters (Gontijo et al. 2015).
A mixture of 2a or 2b (1.03 mmol), SnC12.2H2O (5.14
mmol) and absolute ethanol (9 mL) was heated at 70 °C
under stirring. When the reaction was over, observed by
TLC (hexane/ethyl acetate, 8:2v/v), the clear solution was
cooled to room temperature and then poured into ice. The
pH was made slightly basic (pH 7–8) by addition of 10%
aqueous sodium bicarbonate and then the mixture was
extracted with ethyl acetate (3 × 30 mL). The organic phase
was dried over magnesium sulfate, filtered and the solvent
removed under reduced pressure. The obtained product was
used immediately in the next step, without further
purification.
General procedure for the synthesis of 2a and 2b
(Sudarma et al. 2014)
To
a
solution of 4-hydroxybenzophenone or 4-
hydroxyacetophenone (1.26 mmol) in dichloromethane
(15 mL), potassium hydrogen sulfate (6.82 mmol), sodium
nitrate (7.32 mmol), and 0.875 g of wet silica 50% p/p was
added. The slurry was was left under constant stirring at
room temperature until the completion of the reaction,
checked by TLC (hexane/ethyl acetate, 8:2v/v). Then the
mixture was filtered through silica, the solid was washed
with dichloromethane and the solvent removed under
reduced pressure. The products were then purified by
recrystallization.
3-amino-4-hydroxy-benzophenone (3a) (Jadhav et al. 2017)
97% yield, yellow oil; clogP = 2.34; IR (νmax cm–1): 3440,
3382, 3351, 3053, 1651, 1584, 1564, 1280, 1213. pKa =
9.0.
3-amino-4-hydroxy-acetophenone (3b) (Blum et al. 2003)
95% yield, brown oil; clogP = 0.75; IR (νmax cm–1): 3482,
3384, 3322, 3080, 1615, 1581, 1518, 1283, 1212. pKa =
9.3.
4-hydroxy-3-nitrobenzophenone (2a) (Cohen et al. 1984)
90% yield, yellow solid after recrystallization from water/
methanol (1:1v/v); M.p. 105–107 °C; clogP = 3.12; IR
(νmax cm–1): 3296, 3069, 1660, 1609, 1525, 1319. 1H NMR
(300 MHz, CDCl3) δ: 7.28 (1H, d, H-5, 3J = 8.7 Hz),
General procedure for the synthesis of 4a–h (Sener et al.
2000; Nakamura et al. 2013)
The appropriate acid chloride (1.02 mmol) was dissolved in
ethyl ether (5 mL) and this solution was added dropwise
during 30 min to a stirred ice-cooled mixture of the appro-
priate o-aminophenol 3a or 3b (1.02 mmol) and ethyl ether
(5 mL). The mixture was then stirred at room temperature
until the completion of the reaction, as showed by TLC
(hexane/ethyl acetate, 6:4v/v). Following, the mixture was
washed with water and extracted with ethyl acetate (3 × 30
mL). The organic phase was further washed with 10%
aqueous sodium bicarbonate (3 × 25 mL), followed by
water, and it was dried over anhydrous magnesium sulfate.
After filtration and removal of the solvent under reduced
pressure, the obtained product (1.02 mmol) and p-toluene-
sulfonic acid (3.06 mmol) were taken in toluene (25 mL)
and this suspension was allowed to heating at 110 °C under
stirring. When the reaction was complete, as recorded by
TLC (hexane/ethyl acetate, 7:3v/v), the solvent was
removed under reduced pressure and the crude product was
purified by column chromatography.
3
7.49–7.55 (2H, m, H-3′, H-5′), 7.64 (1H, tt, H-4′, J = 1.2
4
Hz, J = 7.5 Hz), 7.74–7.78 (2H, m, H-2′, H-6′), 8.12 (1H,
4
3
4
dd, H-6, J = 2.1 Hz, J = 8.7 Hz), 8.58 (1H, d, H-2, J =
2.1 Hz). 13C NMR (75 MHz, CDCl3) δ: 193.2 (C, C-7),
157.8 (C, C-4), 138.4 (CH, C-6, C, C-1′), 136.6 (C, C-3),
132.9 (CH, C-4′), 129.8 (C, C-1), 129.6 (CH, C-2′, CH, C-
6′), 128.6 (CH, C-3′, CH, C-5′), 127.8 (CH, C-2), 120.3
(CH, C-5). pKa = 5.1.
4-hydroxy-3-nitroacetophenone (2b) (Wang et al. 2010)
65% yield, yellow solid after recrystallization from water/
methanol (1:1v/v); M.p. 145–148 °C; clogP = 1.53; IR
(νmax cm–1): 3269, 3106, 2927, 1676, 1616, 1525, 1334. 1H
NMR (300 MHz, CDCl3) δ: 2.62 (3H, s, H-8) 7.24 (1H, d,
3
4
H-5, 3J = 9.0 Hz), 8.20 (1H, dd, H-6, J = 9.0 Hz, J = 2.1
Hz), 8.71 (1H, d, H-2, J = 2.1 Hz). 13C NMR (75 MHz,
4
CDCl3) δ: 194.8 (C, C-7), 158.4 (C, C-4), 136.7 (C, C-3),

Med Chem Res
(2-phenyl-5-benzoxazolyl)phenyl methanone (4a) (So and
Decaire 1998)
H-4). 13C NMR (100 MHz, CDCl3) δ: 195.9 (C, C-8), 163.7
(C, C-2), 153.5 (C, C-7a), 142.0 (C, C-3a), 138.5 (C, C-5′),
137.8 (C, C-5), 134.8 (C, C-12), 132.6 (CH, C-7a′), 130.2
(CH, C-4′ and CH, C-6′), 129.6 (CH, C-11 and CH, C-13),
129.2 (CH, C-10 and CH, C-14), 128.5 (C, C-9), 127.9
(CH, C-3a′ and CH, C-7′), 125.0 (CH, C-6), 122.9 (CH, C-
4), 110.8 (CH, C-7). pKa = 14.7. HRMS-ESI: m/z calcd. for
C20H12ClNO2 (M+Na)+: 356.0449; found: 356.0440;
calcd. for (M+H)+: 334.0634; found: 334.0619; calcd. for
(M + 2 + H)+: 336.0605; found: 336.0597.
71% yield, white solid after column chromatography
(hexane/ethyl acetate, 95:05v/v); M.p. 182–193 °C; clogP
= 4.84; IR (νmax cm–1): 3052, 1643, 1603, 1596, 1278,
1048. 1H NMR (400 MHz, CDCl3) δ: 7.50 (2H, t, H-3a′ and
3
H-7′, J = 8.0 Hz), 7.53–7.58 (3H, m, H-11, H-12 and H-
13), 7.61 (1H, tt, H-7a′, 3J = 7.6 Hz, 4J = 1.2 Hz), 7.69 (1H,
3
3
d, H-7, J = 8.4 Hz), 7.69 (1H, d, H-7, J = 8.4 Hz), 7.84
(2H, dd, H-4′ and H-6′, 3J = 8.2 Hz, 4J = 1.2 Hz), 7.94 (1H,
dd, H-6, 3J = 8.4 Hz, 4J = 1.6 Hz), 8.21 (1H, d, H4, 4J = 1.6
Hz), 8.28 (2H, dd, H-10 and H-14, 3J = 7.8 Hz, 4J = 1.2 Hz).
13C NMR (100 MHz, CDCl3) δ: 196.1 (C, C-8), 164.7 (C,
C-2), 153.5 (C, C-7a), 142.1 (C, C-3a), 137.9 (C, C-5′),
134.7 (C, C-5), 132.6 (CH, C-7a′), 132.2 (C, C-9), 130.2
(CH, C-4′ and CH, C-6′), 129.2 (CH, C-11 and CH, C-13),
128.5 (CH, C-12), 127.9 (CH, C-3a′ and CH, C-7′), 127.8
(CH, C-10 and CH, C-14), 126.8 (CH, C-6), 122.9 (CH, C-
4), 110.8 (CH, C-7). pKa = 14.7. HRMS-ESI: m/z calcd. for
[2-(4-methoxyphenyl)-5-benzoxazolyl]phenyl methanone
(4d)
33% yield, white solid after column chromatography
(hexane/ethyl acetate, 95:05v/v); M.p. 170–175 °C; clogP
= 4.86; IR (νmax cm–1): 3067, 2922, 1645, 1608, 1498,
1292, 1030. 1H NMR (400 MHz, CDCl3) δ: 3.90 (3H, s, H-
3
15), 7.04 (2H, d, H-11 and H-13, J = 9.2 Hz), 7.50 (2H, t,
C20H14NO2 (M+H)+: 300.1019; found: 300.1020.
H-3a′ and H-7′, 3J = 7.6 Hz), 7.59–7.62 (1H, m, H-7a′), 7.64
+
3
3
(1H, d, H-7, J = 8.4 Hz), 7.83 (2H, d, H-4′ and H-6′, J =
3
4
[2-(4-nitrophenyl)-5-benzoxazolyl]phenyl methanone (4b)
7,2 Hz), 7.90 (1H, dd, H-6, J = 8.4 Hz, J = 1.6 Hz), 8.16
(1H, d, H-4, 4J = 1.2 Hz), 8.21 (2H, d, H-10 and H-14, 3J =
8.8 Hz). 13C NMR (100 MHz, CDCl3) δ: 196.1 (C, C-8),
164. 8 (C, C-2), 162.9 (C, C-12), 153.5 (C, C-7a), 142.2 (C,
C-3a), 137.9 (C, C-5′), 134.5 (C, C-5), 132.5 (C, C-7a′),
130.2 (CH, C-4′ and CH, C-6′), 129.8 (CH, C-3a′ and CH,
C-7′), 128.5 (CH, C-6), 127.4 (C, C-9), 122.4 (CH, C-4),
119.2 (CH, C-10 and CH, C-14), 114.6 (CH, C-11 and CH,
C-13), 110.5 (CH, C-7), 55.7 (CH3, C-15). pKa = 14.7.
HRMS-ESI: m/z calcd. for C21H15NO3(M+Na)+:
352.0944; found: 352.0944.
40% yield, yellow solid after column chromatography
(hexane/ethyl acetate, 95:05v/v); M.p. 221–225 °C, with
decomposition; clogP = 4.60; IR (νmax cm–1): 3108, 1654,
1
1596, 1554, 1512, 1340, 1281, 1049. H NMR (300 MHz,
3
CDCl3) δ: 7.51 (2H, t, H-3a′ and H-7′, J = 7.2 Hz), 7.62
3
4
(1H, tt, H-7a′, J = 7.2 Hz, J = 1.5 Hz), 7.73 (1H, d, H-7,
3J = 8.4 Hz), 7.83 (2H, dd, H-4′ and H-6, 3J = 7.2 Hz, 4J =
1.5 Hz), 7.99 (1H, dd, H-6, J = 8.7 Hz, J = 1.5 Hz), 8.24
3
4
4
3
(1H, d, H-4, J = 1.2 Hz), 8.39 (2H, d, H-10 and H-14, J
= 9.0 Hz), 8.44 (2H, d, H-11 and H-13, J = 9.0 Hz). 13C
3
NMR (75 MHz, CDCl3) δ: 195.7 (C, C-8), 162.2 (C, C-2),
153.6 (C, C-7a), 149.8 (C, C-12), 141.8 (C, C-3a), 137.6 (C,
C-5′), 135.3 (C, C-9), 132.8 (C, C-5), 132.3 (CH, C-7a′),
130.2 (CH, C-10 and CH, C-14), 128.7 (CH, C4′, CH, C-6′,
CH, C-11, and CH, C-13), 128.5 (CH, C-3a′ and CH, C-6′),
124.4 (CH, C-6), 123.4 (CH, C-4), 111.1 (CH, C-7). pKa =
1-(2-phenyl-5-benzoxazolyl)ethanone (4e) (Ge et al. 2012)
21% yield, white solid after column chromatography
(hexane/ethyl acetate, 95:05v/v); M.p. 160–165 °C; clogP
= 3.24; IR (νmax cm–1): 3066, 2920, 1672, 1610, 1554,
1267, 1059. 1H NMR (400 MHz, CDCl3) δ: 2.69 (3H, s, H-
15), 7.52–7.58 (3H, m, H-11, H-12 and H-13), 7.63 (1H, d,
+
14.9. HRMS-ESI: m/z calcd. for C20H13N2O3 (M+H)+:
345.0870; found: 345.0870.
3
3
4
H-7, J = 8.4 Hz), 8.05 (1H, dd, H-6, J = 8.4 Hz, J = 1.6
3
4
[2-(4-chlorophenyl)-5-benzoxazolyl]phenyl methanone (4c)
Hz), 8.26 (2H, dd, H-10 and H-14, J = 8.0 Hz, J = 1.,2
Hz), 8.37 (1H, d, H-4, J = 1.6 Hz). 13C NMR (100 MHz,
4
60% yield, white solid after column chromatography
(hexane/ethyl acetate, 95:05v/v); M.p. 195–198 °C; clogP
= 5.56; IR (νmax cm–1): 3067, 1657, 1617, 1596, 1293,
1048, 1016. 1H NMR (400 MHz, CDCl3) δ: 7.48–7.54 (4H,
CDCl3) δ: 197.3 (C, C-8), 164.6 (C, C-2), 153.6 (C, C-7a),
142.2 (C, C-3a), 134.6 (C, C-5), 132.3 (C, C-9), 129.2 (CH,
C-11 and CH, C-13), 127.9 (C, C-12), 126.7 (CH, C-10 and
CH, C-14), 125.9 (CH, C-6), 121.1 (CH, C-4), 110.8 (CH,
C-7), 26.9 (CH3, C-15). pKa = 14.7. HRMS-ESI: m/z calcd.
3
m, H-11, H-13, H-3a′, H-7′), 7.61 (1H, tt, H-7a′, J = 7.2
4
3
5
+
Hz, J = 1.2 Hz), 7.66 (1H, dd, H-7, J = 8.4 Hz, J = 0.4
Hz), 7.82–7.84 (2H, m, H-4′ and H-6′), 7.94 (1H, dd, H-6,
for C15H12NO2 (M+H)+: 238.0863; found: 238.0870.
3J = 8.4 Hz, J = 1.6 Hz), 8.18–8.22 (3H, m, H-10, H-14,
4

Med Chem Res
1-[2-(4-nitrophenyl)-5-benzoxazolyl)] ethanone (4f) (Botija
10 and CH, C-14), 114.6 (CH, C-11 and CH, C-13), 110.5
et al. 1985)
(CH, C-7), 55.6 (CH3, C-16), 26.9 (CH3, C-15). pKa =
14.7. HRMS-ESI: m/z calcd. for C16H14NO3 (M+H)+:
+
20% yield, as yellow solid after column chromatography
(hexane/ethyl acetate 95:05); M.p. 244–248 °C with
decomposition; clogP = 3.00; IR (νmax cm–1): 3104, 2921,
268.0968; found: 268.0961.
General procedure for the synthesis of 4i–j (Temperini
et al. 2010; Trujillo-Ferrara et al. 2004)
1
1683, 1597, 1556, 1516, 1343, 1263, 1058. H NMR (400
MHz, CDCl3) δ: 2.71 (3H, s, H-15), 7.69 (1H, d, H-7, 3J =
8.4 Hz), 8.12 (1H, dd, H-6, 3J = 8.6 Hz, 4J = 1.6 Hz),
8.39–8.46 (5H, m, H-10, H-11, H-13, H-14, H-4). 13C
NMR (100 MHz, CDCl3) δ: 196.9 (C, C-8), 162.2 (C, C-2),
153.9 (C, C-7a), 149.8 (C, C-12), 142.3 (C, C-3a), 135.1 (C,
C-9), 132.3 (C, C-5), 128.8 (CH, C-10 and CH, C-14),
127.0 (CH, C-11 and CH, C-13), 124.4 (CH, C-6), 121.7
(CH, C-4), 111.2 (CH, C-7), 26.9 (CH3, C-15). pKa = 14.7.
A mixture of succinic anhydride (1.12 mmol) in ethyl ether
(5 mL) was added to a stirred solution of the o-aminophenol
3a or 3b (1.02 mmol) in ethyl ether (5 mL). The mixture
was kept under stirring at room temperature until the
completion of the reaction, as recorded by TLC (hexane/
ethyl acetate, 6:4v/v). Then, the solvent was removed under
reduced pressure and the product was purified by recrys-
tallization from water/methanol (1:1v/v). Next, a solution of
the obtained product (1.02 mmol) and acetic anhydride
(1.02 mmol) in tetrahydrofuran (THF) (17 mL) was allowed
to stir at 70 °C until the completion of the reaction, as
shown by TLC (hexane/ethyl acetate, 6:4v/v). After
removal of the solvent under reduced pressure, the pure
product was obtained after recrystallization from
isopropanol.
HRMS-ESI: m/z calcd. for C15H11N2O4 (M+H)+:
+
283.0713; found: 283.0721.
1-[2-(4-chlorophenyl)-5-benzoxazolyl)] ethanone (4g)
(Wynne et al. 2009)
10% yield, white solid after column chromatography
(hexane/ethyl acetate 95:05); M.p. 185–190 °C; clogP =
3.96; IR (νmax cm–1): 3066, 2921, 1671, 1621, 1606, 1262,
1043, 1008. 1H NMR (400 MHz, CDCl3) δ: 7.52 (2H, d, H-
3-(5-benzoyl-2-benzoxazolyl)propanoic acid (4i)
3
3
11 and H-13, J = 8.4 Hz), 7.63 (1H, d, H-7, J = 8.4 Hz),
3
4
8.06 (1H, dd, H-6, J = 8.6 Hz, J = 1.6 Hz), 8.19 (2H, d,
H-10 and H-14, 3J = 8.4 Hz), 8.36 (1H, d, H-4, 4J = 1.0 Hz).
13C NMR (100 MHz, CDCl3) δ: 197.1 (C, C-8), 163.7 (C,
C-2), 153.8 (C, C-7a), 142.4 (C, C-3a), 138.5 (C, C-5),
134.7 (C, C-12), 129.6 (CH, C-11 and CH, C-13), 129.2
(CH, C-10 and CH, C-14), 126.2 (C, C-9), 125.2 (CH, C-6),
121.1 (CH, C-4), 110.8 (CH, C-7), 26.9 (CH3, C-15). pKa
48% yield, beige solid; M.p. 192–196 °C; clogP = 4.60; IR
(νmax cm–1): 3483, 3066, 1761, 1712, 1651, 1597. 1H NMR
(300 MHz, DMSO-d6) δ: 2.76–2.91 (4H, m, H-9, H-10),
7.54–7.61 (3H, m, H-3a′, H-7′, H-7a′), 7.68–7.78 (3H, m,
4
H-7, H-4′, H-6′), 7.79 (1H, d, H-4, J = 2.1 Hz), 7.89 (1H,
dd, H-6, 3J = 8.4 Hz, 4J = 2.1 Hz). 13C NMR (75 MHz,
DMSO-d6) δ: 193.9 (C, C-8), 176.1 (C, C-11), 167.5 (C, C-
2), 149.2 (C, C-7a), 136.5 (C, C-3a), 134.4 (C, C-5’), 132.9
(C, C-5), 131.1 (CH, C-7a′), 130.9 (CH, C-4′, CH, C-6′),
129.5 (CH, C-3a′, CH, C-7′), 128.7 (CH, C-6), 125.0 (CH,
C-4), 124.2 (CH, C-7), 28.6 (CH2, C-9, CH2, C-10). pKa =
+
= 14.7. HRMS-ESI: m/z calcd. for C15H11ClNO2 (M
+H)+: 272.0473; found: 272.0475; calcd. for (M + 2 +
H)+: 274.0448; found: 274.0447.
4.1. HRMS-ESI: m/z calcd. for C17H14NO4 (M+H)+:
+
1-[2-(4-methoxyphenyl)-5-benzoxazolyl)] ethanone (4h)
(CAS Registry Number 1368597-94-7; source of
registration: Chemical Catalog, supplier: Otava
Chemicals)
296.0917; found: 296.0915.
3-(5-acetyl-2-benzoxazolyl)propanoic acid (4j)
15% yield, yellow solid; M.p. 196–200 °C; clogP = 3.01;
45% yield, white solid after column chromatography
(dichloromethane/hexane 7:3); M.p. 160–165 °C; clogP =
3.26; IR (νmax cm–1): 3067, 2920, 1672, 1607, 1581, 1252,
1
IR (νmax cm–1): 3073, 2955, 1759, 1700, 1682, 1601. H
NMR (300 MHz, CDCl3) δ: 2.58 (3H, s, H-12), 2.92 (4H, s,
1
1045. H NMR (400 MHz, CDCl3) δ: 2.68 (3H, s, H-15),
H-9, H-10), 7.46 (1H, d, H-7, 3J = 8.4 Hz), 7.87 (1H, d, H-
3
4
4
3
3.90 (3H, s, H-16), 7.04 (2H, d, H-11 and H-13, J = 8.8
4, J = 2.1 Hz), 8.03 (1H, dd, H-6, J = 2.1 Hz, J = 8.4
Hz). 13C NMR (75 MHz, CDCl3) δ: 195.8 (C, C-8), 175.1
(C, C-11), 167.3 (C, C-2), 149.5 (C, C-7a), 135.1 (C, C-3a),
130.1 (C, C-5), 129.4 (CH, C-6), 124.3 (CH, C-4), 124.2
(CH, C-7), 28.2 (CH2, C-9, CH2, C-10), 26.6 (CH3, C-12).
Hz), 7.59 (1H, d, H-7, 3J = 8.4 Hz), 8.01 (1H, dd, H-6, 3J =
3
8.4 Hz, 4J = 1.6 Hz), 8.20 (2H, d, H-10 and H-14, J = 8.8
4
Hz), 8.32 (1H, d, H-4, J = 1.6 Hz). 13C NMR (100 MHz,
CDCl3) δ: 197.3 (C, C-8), 164.8 (C, C-2), 162.9 (C, C-12),
153.8 (C, C-7a), 142.7 (C, C-3a), 134.4 (C, C-5), 129.8
(CH, C-6), 125.5 (C, C-9), 120.6 (CH, C-4), 119.2 (CH, C-
+
pKa = 4.1. HRMS-ESI: m/z calcd. for C12H12NO4 (M
+H)+: 234.0761; found: 234.0763.

Med Chem Res
Biological assays
pentamidine (Sigma) and amphotericin B (Sigma) as the
reference drugs.
Antiproteolytic activity
Compounds 1a, 2b, and 4d were tested for their inhibitory
potential on papain, trypsin, rCPB2.8, and rCPB3.0 by
spectrofluorometric measurements. Enzyme inhibition
power data were stated as compound concentrations giving
50% decrease in enzyme activity (IC50 values), which had
been calculated by time-course dose–response curves using
inhibitor at different concentrations and the data analyzed in
the Grafit 5.0 software using the Eq. 1.
Leishmanicidal activity against amastigotes
Murine peritoneal macrophages were maintained in RPMI
1640 medium (Sigma, USA) supplemented with 10.0%
heat-inactivated fetal bovine serum at 37 °C in 5.0%
CO2 incubator. Cells were cultured in 24-well plates
chamber on the glass slides of 13 mm (Nunc, USA) in a
8 × 105 cells density per well and infected with late
log-phase promastigotes at a ratio of 10:1 (parasite/macro-
phage) and incubated at 37 °C in 5.0% CO2 incubator
for 24 h. Non-phagocytosed promastigotes were removed
by washing, and compounds 1a, 2a, 2b, and 4d solubilized
in DMSO (from 0.10 to 40.00 µg/mL) were administered at
the concentration of 0.6% v/v. After 72 h, chamber slides
were fixed in absolute methanol, stained with 10.0%
Giemsa and examined on an optical light microscope in oil
immersion. The percentage of infected cells per well was
calculated taking account at least 200 macrophages. The
ratio of inhibition (IC50 value) was calculated in compara-
tive to the control only with DMSO. All assays were per-
formed in triplicate on three different times using
pentamidine (Sigma) and amphotericin B (Sigma) as the
reference drugs.
Range
ꢀ
ꢁ
y ¼
s
ð1Þ
x
1 þ
IC50
where Range is the fitted uninhibited value, y is the enzyme
activity, x is the inhibitor concentration, and s is a slope
factor. The equation assumes that y falls with increasing x.
Inhibition reversibility assay enzymes were carried out in
100 mM sodium acetate buffer, pH 5.5, 20% glycerol (to
stabilize the enzymes), 5 mM EDTA, 5 mM dithiothreitol as
cysteine protease papain and cruzain activator, and the
enzyme solutions pre-activated by 5 min, at 37 °C. The
trypsin assays was carried out in 100 mM Trizma base HCl
buffer, pH 7.5, 10% glycerol (to stabilize the enzyme). The
substrate Z-FR-AMC was used as fluorogenic probe and the
hydrolysis of the substrate was followed in a spectro-
fluorometer Hitachi F2500 in the wavelength λEx = 360 nm
(excitation) and λEm = 480 nm (emission) as reference
inhibitors were used E-64 for papain, rCPB2.8 and rCPB3.0
and TLCK for trypsin (Gontijo et al. 2015).
Cytotoxicity evaluation
A suspension of 8 × 105 murine peritoneal macrophages, in
RPMI 1640 medium, supplemented with 10.0% heat-
inactivated fetal bovine serum and 1.0% penicillin (10000
UI/ml)/streptomycin (10 mg/mL) were added to each well in
96-well plates. The plates were incubated in a 5.0% CO2 air
mixture at 37 °C to adhesion of the cells. After 24 h, the
non-adherent cells were removed by washing with the
RPMI 1640 medium. Then, several concentrations of
compounds and reference drugs ranging from 3.91 to
500.00 µg/mL in DMSO at the final concentration of 0.6%
(v/v) were added to the wells containing the cells and the
plates were incubated for more 48 h. The non-adherent cells
were removed by washing with the RPMI 1640 medium.
Afterwards, the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazoliumbromide (MTT) was solubilized in
phosphate-buffered saline (5.0 µg/mL) as solvent. Ten
microliter of this solution was added to RPMI 1640 medium
in a final volume of 200.0 µL per well and incubated for 4 h
(Mosmann 1983). Then, the medium was removed and
100.0 µL of DMSO was added to each well and homo-
genized for 15 min. Next, the absorbance of each individual
well was calculated at 570 nm according to the following
Leishmanicidal activity against promastigotes
Promastigotes of L. amazonensis (MHOM/BR/71973/
M2269) were grown on 24-well plates in Schneider’s Dro-
sophila medium (Sigma, USA) supplemented with 10.0%
(v/v) heat-inactivated fetal bovine serum and 1.0% peni-
cillin (10,000 UI/mL)/streptomycin (10.0 mg/mL) (Sigma,
USA). Cells were harvested in the log phase, suspended in
fresh medium, counted in Neubauer’s chamber and adjusted
to a concentration of 1 × 106 cells/mL, using 24-wells
plates. The compounds 1a, 1b, 2a, 2b, 4a–j were added to
promastigote cultures (1 × 106 cells/mL) in the range of
0.10–160.00 µg/mL, solubilized in DMSO (0.6% v/v in all
wells) and incubated at 25 °C. After 72 h of incubation, the
surviving parasites were counted in a Neubauer’s chamber
and compared with controls, with just DMSO in con-
centration of 0.6% v/v, for the determination of 50.0%
inhibitory growth concentration (IC50). All tests were per-
formed in triplicate on three different times using

Med Chem Res
Scheme 1 Synthesis of the
benzoxazoles derived from
benzophenone or acetophenone
formula (OD represents optical density) using the Equation:
or 3b with the appropriate acyl chlorides (Nakamura et al.
2013) or succinic anhydride (Temperini et al. 2010), fol-
lowed by an intramolecular cyclization of the amide inter-
mediates in toluene and catalytic p-toluenesulfonic (4a–h)
or under heating in a THF/water mixture (4i–j). This
afforded the benzoxazoles in good yields and high purity
degree after column chromatography or recrystallization.
All the compounds were characterized by spectroscopic
ꢂ
ꢃ
ODcontrol ꢀ ODcompounds
ODcontrol ꢁ 100
Inhibition ¼
Each experiment was performed in triplicate on three
different occasions, and the percentage of viable cells was
calculated taking into account the cell culture control
(medium + cells + DMSO 0.6% v/v). The 50.0% cytotoxi-
city concentrations (CC50) were determined and the selec-
tivity index (SI) established by the ratio between the values
of CC50 and IC50 for promastigote and amastigote forms.
1
methods (IR, H, and 13C NMR and HRMS).
The analysis of NMR spectra of the proposed compounds
confirmed the success in obtaining the designed compounds.
1
In the H NMR spectra, in addition to the the signals of
benzophenone and acetophenone core, it was possible to note
the signals concerning to all substitution patterns of the
desired benzoxazole derivatives. In 13C NMR spectra, in
addition to the signals related to the different side chains of
these compounds it could be seen the signal of the azo-
methine carbon in the 162–167 ppm range which confirms
the ring closure. Other findings, as the maintenance of the
ketonic carbonyl (signals in 196.1–195.7 ppm range for
4a–d, 197.3–196.9 ppm range for 4e–h, 193.9 ppm for 4i
and 195.8 ppm for 4j) and the presence of three hydro-
genated aromatic carbons (signals in 7.64–8.34 ppm range)
corroborate the obtaining of final products.
Results and discussion
Chemistry
The benzoxazoles derived from acetophenone and benzophe-
none were designed considering that the ketone carbonyl
would be the electrophilic group responsible for the nucleo-
philic attack by the amino acid residues of the proteases,
whereas the heterocycle core could assist increasing this effect.
Benzoxazoles 4a–j were synthesized following the steps
shown in Scheme 1. Briefly, this synthesis was initiated by
selective ortho aromatic nitration of 4-hydroxybenzo
phenone or 4-hydroxyacetophenone with potassium
hydrogen sulphate and sodium nitrate (Sudarma et al.
2014), followed by reduction by SnCl2.2H2O to obtain the
o-aminophenols 3a and 3b (Belamy and Ou 1984). Then,
the benzoxazole derivatives were prepared by reaction of 3a
Biological activity
The benzoxazoles 4a–j, 4-hydroxybenzophenone 1a, 4-
hydroxyacetophenone 1b, the nitro compounds 2a and 2b

Med Chem Res
and the reference protease inhibitors TLCK (N-α-tosyl-L-
lysinyl-chloromethylketone) and E64 (trans-epoxysuccinyl-
L-leucylamido(4-guanidino)butane) were next assayed to
determine their inhibitory potential against serine (trypsin)
and cysteine (papain, rCPB2.8, and rCPB3.0) proteases
(Table 1). As can be observed, all the compounds were very
active on both enzymes, but especially on papain. It is
noteworthy that all of them were more active (IC50 in the
range of 0.0086–0.7612 µM for papain, and 0.0075–0.5032
µM for trypsin) than the standard papain (E64—IC50
1.7821 µM) or trypsin (TLCK—IC50 7.2318 µM) reference
inhibitors. The benzophenone benzoxazoles were the most
potent inhibitors compounds against trypsin with IC50
values in the range of 0.0075–0.089 µM (except derivative
4a with an IC50 value of 0.3355 µM). The acetophenone
benzoxazoles 4e–h were much less active against trypsin
with IC50 values from 0.2865–0.523 µM and this may be
related to the absence of bulky phenyl group which might
be involved in favorable enzyme interactions, responsible
for the lower inhibition values seen in benzophenone series.
Considering papain, it was possible to notice that both
benzophenone and acetophenone derivatives showed inter-
esting inhibitory activities, but those carrying eletron
withdrawing groups (Cl or NO2) were the most active (4b,
IC50 0.0086 µM; 4c, IC50 0.0495 µM; 4f, IC50 0.0266 µM,
and 4g, IC50 0.0121 µM).
enzymes are also involved in several adaptation mechan-
isms for the survival of parasite, as the regulation in its
immune response, invasion and damage of tissues, differ-
entiation and dissemination of parasite and uptake of
essential nutrients (Coombs and Mottram 1997; Sajid and
McKerrow 2002; Aparicio et al. 2004). Proteases are also
key enzymes in the metabolism of proteins, or biologically
active peptides (Mótyán et al. 2013). Martins et al. (2009)
described three natural polyprenylated benzophenone deri-
vatives, which have shown potential inhibitory effect on
proteases. There is also another report about benzophenone
derivatives with inhibitory activity of cysteine (papain,
rCPB2.8, and rCPB3.0) and serine (trypsin) proteases
(Almeida et al. 2015). Besides, it was observed that dif-
ferent ketonic heterocycles show cysteine protease inhibi-
tors rCPB2.8 (Steert et al. 2010), wherein the authors
comment on the participation of heterocyclic framework in
providing greater inhibitor–enzyme interactions.
In face of these significant results in enzyme inhibition,
the most active compounds against papain (2a, 4b, 4g), as
well as their precursors (1a and 1b) were selected for eva-
luation against two specific strains of L. (L) mexicana
cysteine proteases, rCPB2.8, and rCPB3.0. According to
Pereira et al. (2011), cysteine proteases are expressed at
high levels in parasites of the genus Leishmania and
rCPB2.8 and rCPB3.0 are similar to papain. They represent
specific cysteine proteases from different species of Leish-
mania sp (Lanfranco et al 2008). Once again, it was pos-
sible to note excellent results for all compounds evaluated
Proteases have an essential role in the hydrolysis of
peptide bonds in parasites, such as trypanosomatids, leading
to peptides of variable sizes or even free amino acids. These
Table 1 Quantitative in vitro
Compounds
IC50 values (µM)a
inhibitory effect of synthetic
compounds on cysteine protease
papain and rCPB2.8 and
rCPB3.0 enzymes of L. (L)
mexicana and serine protease
trypsin
Trypsin
Papain
rCPB2.8
rCPB3.0
1a
0.5235 0.1502
0.2865 0.0038
0.3788 0.0525
0.5032 0.0641
0.3355 0.0489
0.0077 0.0013
0.0075 0.0018
0.0895 0.0003
0.4547 0.0196
0.3929 0.0304
0.4400 0.0354
0.4017 0.0159
0.3866 0.0156
0.4054 0.0422
7.2318 0.7185
–
0.4428 0.0294
0.0214 0.0029
0.0176 0.0062
0.4456 0.0432
0.1059 0.0038
0.0086 0.0003
0.0495 0.0036
0.7612 0.0554
0.0479 0.0079
0.0266 0.0022
0.0121 0.0080
0.0659 0.0023
0.030 0.0072
0.0212 0.0009
–
0.0115 0.0018
0.0146 0.0004
1b
0.0518 0.0012
0.0126 0.0006
2a
0.0131 0.0013
0.0227 0.0007
2b
–
–
4ª
–
–
4b
0.0103 0.0032
0.0291 0.0016
4c
–
–
4d
–
–
4e
–
–
4f
–
–
4g
0.0351 0.0092
0.0075 0.0015
4h
–
–
4i
–
–
4j
–
–
TLCKb
E64b
–
–
1.7821 0.1356
1.7821 0.1356
1.7821 0.1356
a Each IC50 value represents the mean standard deviation of triplicate determined by the software Grafit 5.0
b The TLCK and E64 substances were used as reference inhibitor for the corresponding proteases were
assayed

Med Chem Res
for inhibition of both enzymes (Table 1), with interesting
IC50 values found in the range 0.0103–0.0518 µM
against rCPB2.8 and 0.0075–0.0291 µM against rCPB3.0.
All compounds were about 34-folds more active than the
standard inhibitor of cysteine proteases E64 (IC50
1.7821 µM).
chloro-substituted benzoxazoles that were very active in
enzymatic tests did not have good activity in these tests. By
the other hand, the acetophenone analog 4h showed no
expressive activity and this may be, again, related to the
benzophenone nucleus in 4d. Although a SI greater than 10
is required to ensure good safety for a drug (Pires et al
2013), the two drugs used as reference did not reach this
value, which may explain the adverse effects related to
them. Although less active than amphotericin B and pen-
tamidine, the benzoxazole 4d showed a SI (5.22) compar-
able to that of amphotericin B (5.31) and nine-fold greater
than that of pentamidine (0.58). The two nitro derivatives
2a and 2b were also active at 158.4 and 130.9 µM,
respectively, against promastigotes forms, and for these two
compounds the methyl group of acetophenone nucleus
contributed to a better activity than the phenyl group in
benzophenone analog. Furthermore, derivative 2b showed a
SI of 18, considered ideal according to Pires et al (2013).
The more active compounds against promastigote forms
were then evaluated against amastigote intracellular forms.
Although benzoxazole 4d showed to be the most active
against the promastigote forms, it was less active on
amastigote forms and this low intracellular activity may be
related to its large molecular size, which may have restricted
its diffusion across the parasite cell membrane. Nitro deri-
vative 2b was more potent against amastigote forms (IC50
= 98.26 µM) compared to its action against promastigote
forms (IC50 = 130.9 µM) and we postulate that this differ-
ence in intracellular activity may be related to the formation
Thus, it could be confirmed that all evaluated compounds
exhibited excellent results on inhibition of cysteine pro-
teases papain, rCPB2.8 and rCPB3.0. As stated by Lima
and co-workers (2013), there are consistent studies showing
that a significant inhibition of cysteine protease can lead to
an expressive in vitro antileishmanial activity. Accordingly,
compounds 1a, 1b, 2a, 2b, and 4a–j were chosen for
in vitro antileishmanial evaluation against promastigote and
amastigote forms of L. (L) amazonensis. Unfortunately,
derivatives 4b, 4c, and 4f were not soluble in the culture
medium, so they could not be tested. The IC50 values of the
other compounds, pentamidine and amphotericin B are
listed in Table 2. The cytotoxic effects against murine
peritoneal macrophages (expressed as CC50 values) of the
most active compounds were also checked and were used to
determine their SI.
Compounds 2a, 2b, 4d, and the benzophenone 1a were
active against promastigote forms with IC50 values of 158.4,
130.9, 90.3, and 190.9 µM, respectively (Table 2). Ben-
zoxazole 4d, which bears a methoxy group in the benzox-
azole ring, was twice as active than benzophenone 1a. In
fact, it was the only benzoxazole which showed significant
activity against promastigote form. Curiously, those nitro or
Table 2 Antipromastigote,
Compounds
Promastigotesa IC50
in µM/(SI)
,
Amastigotesa IC50
in µM/(SI)
,
Macrophagesb CC50
,
in µM
antiamastigote, and cytotoxicity
activities for benzoxazoles and
their precursors
1a
190.9 2.5/(2.85)
> 1176.5
19.93 2.02/(27.49)
547.9 17.5
1b
nd
nd
2a
158.4 77.4
> 164.61
nd
2b
130.9 0.55/(18.09)
98.26 1.10/(24.1)
2368.3 317.8
4a
> 535.1
nd
nd
nd
4b
nd
nd
4c
nd
nd
nd
4d
90.3 2.7/(5.22)
> 675.1
nd
> 121.45
471.1 64.5
4e
nd
nd
4f
nd
nd
4g
> 589.3
> 599.3
> 542.4
> 686.7
19.47 0.08/(0.58)
nd
nd
4h
nd
nd
4i
nd
nd
4j
nd
nd
Pentamidine
18.82 (0.6)
6.15 (4.4)
11.23 1.3
27 2.1
Amphotericin B 5.09 0.39/(5.31)
a Each IC50 value represents the mean standard deviation of triplicate determined by the software Grafit 5.0
b Cytotoxicity concentration for 50% of macrophages
nd not determined, SI selectivity index

Med Chem Res
Ansari M, Emami S, Khalilzadeh MA, Maghsoodlou MT, Foroumadi
A, Faramarzi MA, Samadi N, Ardestani SK (2012) Replacement
of the methylene of dihydrochalcones with oxygen: synthesis and
biological evaluation of 2-phenoxyacetophenones. Chem Biol
Drug Des 80:591–597
Aparicio IM, Scharfstein J, Lima AP (2004) A new cruzipain-mediated
pathway of human cell invasion by Trypanosoma cruzi requires
trypomastigote membranes. Infect Immun 72:5892–902
Ayres DC, Marcucci MC, Giorgio S (2007) Effects of Brazilian pro-
polis on Leishmania amazonensis. Memórias do Instituto
Oswaldo Cruz 102:215–220
of a more active metabolite after its penetration in the
infected cell. The SI value of 2b was 24, indicating again
the high selective toxicity of this derivative for the parasite.
The commercial benzophenone 1a was active against pro-
mastigote forms at 190.9 µM and was also more active
against amastigote forms with IC50 value comparable to that
of pentamidine (19.93 µM), which can also indicate a pos-
sible intracellular metabolism leading to a more active
substance. The selectivity index of 1a was higher than 27,
showing a low toxicity for this compound.
Belamy FD, Ou K (1984) Selective reduction of aromatic nitro com-
pounds with stannous chloride in non acidic and non aqueous
medium. Tetrahedron Lett 25:839–842
Blum G, Gazit A, Levitzki A (2003) Development of new insulin-like
growth factor-1 receptor kinase inhibitors using catechol mimics.
J Bio Chem 278:40442–40454
Conclusion
Botija MM, Gomez VP, Sanchez FA, Torres TC (1985) Procedure for
the preparation of 2-(2-arylbenzoxazol-5-yl) propionic acids. ES
Patent 1984-529620, filled 10 Feb 1984, issued 16 Aug 1985.
Cohen BJ, Karoly-Hafeli H, Patchornik A (1984) Active esters of
polymer-bound 4-hydroxy-3-nitrobenzophenone as useful acy-
lating reagents. Application to peptide synthesis. J Org Chem
49:922–924
Coombs GH, Mottram JC (1997) Parasite proteinases and amino acid
metabolism: possibilities for chemotherapeutic exploitation.
Parasitology 1997(114 Suppl):S61–80
Costa Júnior JS, Almeida AAC, Ferraz ABF, Rossatto RR, Silva TG,
Silva PBN, Militão GCG, Citó AMGL, Santana LCLR, Carvalho
FAA, Freitas RM (2013) Cytotoxic and leishmanicidal properties
of garcinielliptone FC, a prenylated benzophenone from Platonia
insignis. Nat Prod Res 27:470–474
Das P, Alam MN, Paik D, Karmakar K, De T, Chakrabort T (2013)
Protease inhibitors in potencial drug development for leishma-
niasis. Indian J Biochem Biophys 50:363–376
Ten benzoxazole derivatives, five of which are new com-
pounds (4b, 4c, 4d, 4i, 4j), were obtained of from a three-
step synthetic route from acetophenone and benzophenone
under mild conditions. These compounds and some of their
precursors showed good results when evaluated as inhibi-
tors of papain, rCPB2.8, and rCPB3.0 proteases. Benzo-
phenone benzoxazoles which bear electron withdrawing
groups were the best inhibitors of proteases. However, their
potential against L. amazonensis promastigote forms were
not significant, wherein benzoxazole 4d was the most active
with IC50 of 90.3 µM and SI value 9-fold greater than that of
pentamidine. In the in vitro evaluation with amastigote
forms of L. amazonensis compounds 1a, 2b and, again, 4d,
exhibited satisfactory activities and very low cytotoxicity
when compared to standards drugs. In view of the results,
we assume that these compounds are good candidates to be
exploited in further studies of chemical modifications in
order to improve their potential and check in vivo activity as
leishmanicidal drug candidates.
Eschenlauer SC, Faria MS, Morrison LS, Bland N, Ribeiro-Gomes FL,
Dos Reis GA, Coombs GH, Lima AP, Mottram JC (2009)
Influence of parasite encoded inhibitors of serine peptidases in
early infection of macrophages with Leishmania major. Cell
Microbiol 11:106–120
Fricker SP (2010) Cysteine proteases as targets for metal-based drugs.
Metallomics 2:366–377
Ge W, Jun Z, Min Z, Peng H, Weiping S (2012) Palladium-catalyzed
direct arylation of benzoxazoles with unactivated simple arenes.
Chem Commun 48:8964–8966
Gontijo VS, Espuri PF, Alves RB, Camargos LF, Santos FV, Judice
WAS, Marques MJ, Freitas RP (2015) Leishmanicidal, anti-
proteolytic, and mutagenic evaluation of alkyltriazoles and
alkylphosphocholines. Eur J Med Chem 101:24–33
Acknowledgements This work was supported by Grants from
Universal Project (Fapemig, Brazil, APQ-01209-13). The authors are
also grateful for the fellowships granted by CAPES, CNPq,
FAPEMIG.
Compliance with ethical standards
Hay AE, Merza J, Landreau A, Litaudon M, Pagniez F, Le Pape P,
Richomme P (2008) Antileishmanial polyphenols from Garcinia
vieillardii. Fitoterapia 79:42–46
Conflict of interest The authors declare that they have no competing
interests.
Jadhav AG, Shinde SS, Lanke SK, Sekar N (2017) Benzophenone
based fluorophore for selective detection of Sn2+ ion: Experi-
mental and theoretical study. Spectrochim Acta A Mol Biomol
Spectrosc 174:291–296
References
Lanfranco MA, Loayza-Muro R, Clark D, Núnez R, Zavaleta AI,
Jimenez M, Meldal M, Coombs GH, Mottram JC, Izidoro M,
Juliano MA, Juliano L, Arévalo J (2008) Expression and sub-
strate specificity of a recombinant cysteine proteinase B of
Leishmania braziliensis. Mol Biochem Parasitol 161:91–100
Lenta BN, Vonthron-Sénécheau C, Weniger B, Devkota KP, Ngou-
payo J, Kaiser M, Naz Q, Choudhary MI, Tsamo E, Sewald N
(2007) Leishmanicidal and Cholinesterase Inhibiting Activities of
Phenolic Compounds from Allanblackia monticola and Sym-
phonia globulifera. Molecules 12:1548–1557
Al-Kahraman YMSA, Yasinzai M, Singh GS (2012) Evaluation of
some classical hydrazones of ketones and 1,2-diketones as
antileishmanial, antibacterial and antifungal. Agents Arch Pharm
Res 35:1009–1013
Almeida L, Alves KF, Maciel-Rezende CM, Jesus LOP, Pires FR,
Viegas Junior C, Izidoro MA, Júdice WAS, Santos MH, Marques
MJ (2015) Benzophenone derivatives as cysteine protease inhi-
bitors and biological activity against Leishmania (L.) amazo-
nensis amastigote. Biomed Pharmacother 75:93–99

Med Chem Res
Lima APCA, Reis FCG, Costa TFR (2013) Cysteine peptidase inhibi-
tors in trypanosomatid parasites. Curr Med Chem 20:3152–3173
Loedige M (2015) Design and synthesis of novel antileishmanial
compounds. Int J Med Chem 2015:1–21
Maciel-Rezende CM, Almeida L, Costa EDM, Pires FR, Alves KF,
Viegas Junior C, Dias DF, Doriguetto AC, Marques MJ, Santos
MH (2013) Synthesis and biological evaluation against Leish-
mania amazonensis of a series of alkyl-substituted benzophe-
nones. Bioorg Med Chem 21:3114–3119
Martins FT, Assis DM, Santos MH, Camps I, Veloso MP, Juliano MA,
Alves LC, Doriguetto AC (2009) Natural polyprenylated benzo-
phenones inhibiting cysteine and serine proteases. Eur J Med
Chem 44:1230–1239
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxic assays. J
Immunol Methods 65:55–63
Sajid M, McKerrow JH (2002) Cysteine proteases of parasitic
organisms. Mol Biochem Parasitol 120:1–21
Sangshetti JN, Khan FAK, Kulkarni AA, Aroteb R, Patil RH (2015)
Antileishmanial drug discovery: comprehensive review of the last
10 years. RSC Advances 5:32376–32415
Sener EA, Bingol KK, Oren I, Arpaci OT, Yalçin I, Altanlar N (2000)
Synthesis and microbiological activity of some N-(o-hydro-
xyphenyl)benzamides and phenylacetamides as the possible
metabolites of antimicrobial active benzoxazoles: part II. IlFar-
maco 55:469–476
Singh S, Sivakumar R (2004) Challenges and new discoveries in the
treatment of leishmaniasis. J Infect Chemother 10:307–315
So H, Decaire R (1998) 2-Arylbenzothiazoles and 2-Arylbenzoxazoles
from α,α,α-Trihalomethyl Aromatic Compounds. Synth Commun
28:4123–4135
Steert K, Berg M, Mottram JC, Westrop GD, Coombs GH, Cos P,
Maes L, Joossens J, Van der Veken P, Haemers A, Augustyns K
(2010) α-Ketoheterocycles as inhibitors of Leishmania mexicana
cysteine protease CPB. Chem Med Chem 5:1734–1748
Sudarma IM, Wazni N, Wildawaty N, Yuanita E, Suana IW (2014) An
Efficient Method on Nitration of Eugenol Using NH4NO3 and
KHSO4. Asian J Chem 26:173–175
Mótyán JA, Tóth F, Tőzsér J (2013) Research applications of pro-
teolytic enzymes in molecular biology. Biomolecules 3:923–942
Nakamura H, Yasui Y, Ban HS (2013) Synthesis and biological
evaluation of ortho-carborane containing benzoxazole as an
inhibitor of hypoxia inducible factor (HIF)-1 transcriptional
activity. J Organomet Chem 747:189–194
Pereira BA, Silva FS, Rebello KM, Marín-Villa M, Traub-Cseko YM,
Andrade TC, Bertho ÁL, Caffarena ER, Alves CR (2011) In
silico predicted epitopes from the COOH-terminal extension of
cysteine protease B inducing distinct imune responses during
Leishmania (Leishmania) amazonensis experimental murine
infection. BMC Immunol 12:1471–2172
Temperini A, Terlizzi R, Testaferri L, Tiecco M (2010) Additive-Free
Chemoselective Acylation of Amines. Synth Commun
40:295–302
Tipparaju SK, Joyasawal S, Pieroni M, Kaiser M, Brun R, Kozikowski
AP (2008) In pursuit f natural products leads: Synthesis and
biological evaluation of 2-[3-hydroxy-2-[(3-hydroxypyridine-2-
Pereira IO, Marques MJ, Pavan ALR, Codonho BS, Barbieri CL, Beijo
LA, Doriguetto AC, D’Martin EC, Santos MH (2010) Leishma-
nicidal activity of benzophenones and extracts from Garcinia
brasiliensis Mart fruits Phytomedicine 17:339–345
carbonyl)amino]phenyl]benzoxazole-4-carboxylic
acid
(A-
33853) and its analogues: discovery of N-(2-benzoxazol-2-
ylphenyl)benzamidines as novel antileishmanial chemotypes. J
Med Chem 51:7344–7347
Phillips MA, Stanley Jr SL (2011) Chemotherapy of protozoal infec-
tions: amebiasis, giardiasis, trichomoniasis, trypanosomiasis,
leishmaniasis, and other protozoal infections. In: Brunton LL,
Chabner BA, Knollmann BC (eds) Goodman & Gilman’s The
Pharmacological Basis of Therapeutics. 12th ed. McGraw-Hill,
New York, NY
Pires CM, Rodrigues SD, Bristot D, Gaeta HH, Toyama DO, Farias
WRL, Toyama MH (2013) Evaluation of macroalgae sulfated
polysaccharides on the Leishmania (L.) amazonensis promasti-
gote. Mar Drugs 11:934–943
Powers JC, Asgian JL, Ekici ÖD, James KE (2002) Irreversible
Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem
Rev 102:4639−4750
Radi M, Saletti S, Bota M (2008) A one-pot, two-step microwave-
assisted synthesis of highly functionalized benzoxazoles using
solid-supported reagents (SSRs). Tetrahedron Lett 49:4464–4466
Ready PD (2014) Epidemiology of visceral leishmaniasis. Clin Epi-
demiol 6:147–154
Trujillo-Ferrara JG, Padilla-Martínez II, Martínez-Martínez FJ, Höpfl
H, Farfan-García N, García-Báez EV (2004) The E and Z isomers
of 3-(benzoxazol-2-yl)prop-2-enoic acid. Acta Cryst C60:
o723–o726
Wang S, Yan J, Wang J, Chen J, Zhang T, Zhao Y, Xue M (2010)
Synthesis of some 5-phenylisoxazole-3-carboxylic acid deriva-
tives as potent xanthine oxidase inhibitors. Eur J Med Chem
45:2663–2670
World Health Organization (2015) Leishmaniasis. Fact sheet No. 375.
http://www.who.int/mediacentre/factsheets/fs375/en/. Accessed
15 Jan 2016
World Health Organization (2016) WHO to implement online epide-
miological surveillance for Leishmaniasis. http://www.who.int/
neglected_diseases/news/WHO_implement_epidemiological_
surveillance_leishmaniasis/en/. Accessed 8 Sept 2016.
Wynne GM, Wren SP, Johnson PD, Price PD, De Moor O, Nugent
GS, Storer RP, Pye RJ, Dorgan CR (2009) Preparation of ben-
zoxazoles, benzimidazoles, benzothiazoles, indoles and their
analogs for the treatment of muscular dystrophy and cachexia.
UK Patent WO 2009-019504, filled 1 Aug 2008, issued 12 Feb
2009
Román-Luque-Ortega J, Reuther P, Rivas L, Dardonville C (2010)
New benziphenone-derived bisphosphonium salts as leishmani-
cidal leads targeting mitochondria. throughv inhibition of
respiratory complex II. J Med Chem 53:1788–1798