Synthesis, antiprotozoal activity and cytotoxicity in U-937 macrophages of triclosan–hydrazone hybrids

Article abstract of DOI:10.1007/s00044-017-2019-2

The synthesis and biological activities (cytotoxicity, leishmanicidal, and trypanocidal) of 11 triclosan–hydrazone hybrids are described herein. The structure of the products was elucidated by spectral data (NMR, IR) and mass spectrometric analyses. The s

Full text of DOI:10.1007/s00044-017-2019-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2019-2
ORIGINAL RESEARCH
Synthesis, antiprotozoal activity and cytotoxicity in U-937
macrophages of triclosan–hydrazone hybrids
Sebastian Vergara1 Miguel Carda2 Raül Agut2 Lina M. Yepes3 Iván D. Vélez3
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Sara M. Robledo3 Wilson Cardona Galeano1
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Received: 23 February 2017 / Accepted: 31 July 2017
© Springer Science+Business Media, LLC 2017
Abstract The synthesis and biological activities (cyto-
toxicity, leishmanicidal, and trypanocidal) of 11
triclosan–hydrazone hybrids are described herein. The
structure of the products was elucidated by spectral data
(NMR, IR) and mass spectrometric analyses. The synthe-
sized compounds were evaluated against amastigotes forms
of L. (V) panamensis, which is the most prevalent Leish-
mania species in Colombia, and against Trypanosoma
cruzi, which is the major pathogenic species to Chagas
disease in humans. In addition, the cytotoxic activity of the
synthesized compounds was evaluated against human U-
937 macrophages. Hydrazone hybrids were obtained as E-
synperiplanar and E-antiperiplanar conformers. Nine of
them were active against L. (V) panamensis (5a–5d, 5f–5j)
and eight of them against T. cruzi (5a, 5c, 5d, 5f–5j), with
EC50 values lower than 40 µM. The compounds 5c, 5e, and
5h exhibit the best selectivity index against both L. (V)
panamensis and T. cruzi, with values ranging from 5.90 to
16.55, thus showing potential as starting compounds for the
eventual development of drugs against these parasites.
The presence of hydroxy or methoxy groups in positions
2 and 4 of the aromatic ring of the benzylidene moiety
increases both activity and cytotoxicity. There is no clear
relationship between the antiprotozoal activity and the
methylation pattern of the hydroxy groups, since in some
cases methylation decreases the activity (5d vs. 5g) while in
other cases the activity is increased (5c vs. 5f and 5i vs. 5j).
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Keywords Leishmaniasis Chagas disease Trypanosoma
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cruzi Antiprotozoal activity Cytotoxicity Triclosan-
Hydrazone Hybrids
Introduction
Protozoal diseases are a cause of mortality in various
developing countries of tropical and subtropical regions.
For leishmania and Chagas-endemic countries these dis-
eases cause significant health problems affecting more than
one billion people worldwide (WHO 2002, 2013; Alvar
et al. 2012; Nouvellet et al. 2015). This situation is aggra-
vated by increasing treatment failures with available drugs
(Bhutta et al. 2014). Chagas disease (American trypanoso-
miasis) and leishmaniasis are parasitic diseases caused by
the parasitic protozoan Trypanosoma cruzi (T. cruzi) and
Leishmania species, respectively.
The Leishmaniasis involves a wide spectrum of clinical
manifestations in which L. (V) panamensis is one of the
most prevalent Leishmania species involved in human cases
of cutaneous leishmaniasis in Colombia (Alvar et al. 2012).
Chagas disease (also named American trypanosomiasis) is
produced by the protozoan parasite T. cruzi that is trans-
mitted to the mammalian host through the bite of triatomine
* Sara M. Robledo
sara.robledo@udea.edu.co
* Wilson Cardona Galeano
wilson.cardona1@udea.edu.co
1
Chemistry of Colombian Plants, Institute of Chemistry, Exact and
Natural Sciences School, University of Antioquia-UdeA, Calle 70
No. 52-21, Medellín AA 1226, Colombia
2
Department of Inorganic and Organic Chemistry, Jaume I
University, Castellón E-12071, Spain
3
PECET-Medical Research Institute, School of Medicine,
University of Antioquia-UdeA, Calle 70 No. 52-21, Medellín AA
1226, Colombia

Med Chem Res
Fig. 1 Compounds with antiprotozoal activity
bugs belonging to Triatoma, Rhodnius, and Panstrongylus
genus (Nouvellet et al. 2015). Current treatments for cuta-
neous leishmaniasis are based on pentavalent antimonials
(meglumine antimoniate and sodium stibogluconate). For
the treatment of Chagas disease nitroaromatic compounds
(benznidazole and nifurtimox) are usually employed.
However, these treatments are not without significant side
effects, particularly in patients undergoing high-dose and
long-term treatments. In addition, the development of drug
resistance has significantly increased the health problems
associated with these diseases (Chatelain and Ioset 2011;
Den Boer et al. 2011; Keenan and Chaplin 2015).
concentrations (EC50) of 13.1 and 4.7 µM, respectively
(Arango et al. 2012). Triclosan–chalcone (1c) and
triclosan–chromone (1d) hybrids showed no cytotoxicity
against U-937 cells but were active against L. panamensis
amastigotes (LC50 = > 326.7 µM, EC50 = 15.4 and 5.5 µM,
respectively) (Otero et al. 2014) (see Fig. 1). Quinoline–
hydrazone hybrid 1e showed activity against L. panamensis
and against T. cruzi with EC50 of 2.6 and 4.6 µM, respec-
tively (Coa et al. 2015). Some furoxanyl N-acylhydrazone
derivatives were evaluated in vitro against amastigote form
of L. panamensis and against T. cruzi. The compound 1f
exhibited excellent profile as anti-T. cruzi with an IC50
=
Triclosan is an uncompetitive inhibitor of purified enoyl-
acyl carrier protein reductase (ENR), which has demon-
strated in vitro inhibitory activity against Plasmodium fal-
ciparum (Kapoor et al. 2004; McLeod et al. 2001; Surolia
and Surolia 2001; Perozzo et al. 2002). A previous study
showed that triclosan has in vitro anti-leishmanial activity
against axenic amastigotes of L. panamensis with an
effective concentration (EC50) of 39 µM.
Further, hydrazones constitute an important type of
biologically active compounds (Rollas and Küçükgüzel
2007; Singh and Raghav 2011; Verma et al. 2014) with
high ability to elicit anti-leishmanicidal (Bernardino et al.
2006; Rando et al. 2008; Taha et al. 2014) and trypanocidal
activity (Carvalho et al. 2012; Porcal et al. 2008; Jorge et al.
2013; Massarico Serafim et al. 2014).
0.91 µM. On the other hand, compounds 1g and 1h showed
very good anti-Leishmania activity with IC50 = 1.3 and 1.7
µM, respectively. In addition, compounds 1g and 1h dis-
played higher selectivity than the reference drug Amph
(Hernández et al. 2013). Finally, antileishmanial activity of
several phenyl-linked oxadiazole–phenylhydrazone hybrids
was evaluated, compound 1i being the most potent antil-
eishmanial agent among this type of hybrids, displaying an
IC50 of 0.95 0.01 µM (Taha et al. 2017) (Fig. 1).
In the search for new therapeutic alternatives to treat
cutaneous leishmaniasis and Chagas disease a number of
triclosan–hydrazone hybrids have been designed and syn-
thesized. Their leishmanicidal and trypanocidal activities, as
well as their cytotoxicity in U-937 macrophages, have been
evaluated in vitro (Fig. 2).
In recent years a promising strategy has emerged based on
hybrid molecules, which bear in their structures two distinct
pharmacophores having, for example, anti-protozoal, anti-
inflammatory, anti-fungal, or anti-cancer activity, thus
showing a dual mode of action (Keith et al. 2005; Meunier
2008). These hybrid molecules may display dual activity, but
do not necessarily act on the same biological target (Opsenica
et al. 2008; Roth et al. 2004; Walsh et al. 2007).
Materials and methods
Chemical synthesis
General remarks
Triclosan–quinoline hybrids with shorter methylene units
spacers (1a and 1b) have in vitro activity against intracel-
lular amastigotes of L. panamensis with effective
Microwave reactions were carried out in a CEM Discover
microwave reactor in sealed vessels (monowave, maximum
power 300 W, temperature control by IR sensor, fixed

Med Chem Res
Fig. 2 Design of triclosan–hydrazone hybrids as antiprotozoal agents
1
temperature). H and 13C NMR spectra were recorded on a
hexane–ethyl acetate (9:1 ratio) to obtain the ester 2 with
75% yield (0.016 mol, 5.9 g). Monitoring of the reaction
progress and product purification were carried out by TLC.
White solid, m.p. 58–60 °C; IR (cm−1): νmax max 2985
(C–H), 1745 (C=O), 1492 (C=CAr), 1215 ((C=O)–O), 829
Varian instrument operating at 500 and 125 MHz, respec-
tively. The signals of the deuterated solvent (CDCl3) were
used as reference (the singlet at δ = 7.27 ppm for H NMR
1
and the triplet centered at δ = 77.00 ppm for 13C NMR).
Carbon atom types (C, CH, CH2, CH3) were determined by
using the DEPT or APT pulse sequence. Signals were
assigned using two-dimensional heteronuclear correlations
(COSY and HSQC). High-resolution mass spectra were
recorded using electrospray ionization-mass spectrometry
(ESI-MS). A QTOF Premier instrument with an orthogonal
Z-spray–electrospray interface (Waters, Manchester, UK)
was used for operating in the W-mode. The drying and cone
gas was nitrogen-set to flow rates of 300 and 30 L/h,
respectively. Methanol sample solutions (ca. 1 × 10−5 M)
were directly introduced into the ESI spectrometer at a flow
rate of 10 µL/min. A capillary voltage of 3.5 kV was used in
the positive scan mode, and the cone voltage was set to Uc
= 10 V. For accurate mass measurements, a 2 mg/L stan-
dard solution of leucine enkephalin was introduced via the
lock spray needle at a cone voltage set to 85 V and a flow
rate of 30 μL/min. IR spectra were recorded on a Spectrum
RX I FT-IR system (Perkin-Elmer, Waltham, MA, USA) in
KBr disks. Silica gel 60 (0.063–0.200 mesh, Merck,
Whitehouse Station, NJ, USA) was used for column chro-
matography, and precoated silica gel plates (Merck 60 F254
0.2 mm) were used for thin layer chromatography (TLC).
1
(C–HAr), 723 (C–Cl). H-NMR (CDCl3, 300 MHz): δ 1.28
(CH3, t, J = 7.1 Hz), 4.23 (–OCH2–, q, J = 7.1 Hz), 4.66
(–OCH2C=O–, s), 6.79 (H3, d, J = 8.8 Hz), 6.91 (H6, d, J =
8.8 Hz), 6.94–7.00 (H4, H6, m), 7.14 (H5ʹ, dd, J = 8.8, 2.5
Hz), 7.44 (H3ʹ, d, J = 2.5 Hz); 13C-NMR (CDCl3, 75 MHz): δ
14.12 (CH3), 61.49 (–OCH2–), 66.29 (–OCH2–), 115.91
(C6), 118.77 (C3), 121.81 (C6ʹ), 122.54 (C4), 124.76 (C4ʹ),
127.82 (C2ʹ), 128.31 (C5ʹ), 130.19 (C5), 130.22 (C3ʹ), 143.61
(C2), 149.78 (C1), 151.90 (C1ʹ), 168.00 (C=O).
Synthetic procedure for 2-(5-chloro-2-(2,4-dichlorophe-
noxy)phenoxy)acetohydrazide Hydrazine
monohydrate
(3 mL of a 80% solution) was added to a solution of 2 (5 g,
0.013 mol) in ethanol (15 mL). The reaction mixture was
submitted to microwave irradiation and maintained under
reflux for 30 min. Then, the reaction mixture was poured on
ice and the resulting precipitate was filtered out, affording
the title compound 3 in 83% yield (3.90 g, 0.011 mmol).
White solid, m.p. 148–150 °C; IR (cm−1): νmax max 3298
(N–H), 1656 (C=O), 1473 (C=CAr), 1232 ((C=O)–N), 975
1
(C–HAr), 796 (C–Cl). H-NMR (CDCl3, 300 MHz): δ 4.55
(–OCH2C=O–, s), 6.86 (H3, d, J = 8.7 Hz), 6.99–7.08 (H4,
H6ʹ, m), 7.20 (H6, d, J = 2.0 Hz), 7.33 (H5ʹ, dd, J = 8.7, 2.3
Hz), 7.70 (H3ʹ, d, J = 2.3 Hz), 8.57 (NH), 9.10 (NH2);
13C-NMR (CDCl3, 75 MHz): δ 55.25 (–OCH2–), 115.75
(C6), 119.50 (C3), 122.24 (C6ʹ), 122.38 (C4), 123.98 (C4ʹ),
127.71 (C2ʹ), 128.94 (C5ʹ), 129.75 (C5), 130.24 (C3ʹ), 143.07
(C1), 150.50 (C2), 152.01 (C1ʹ), 166.62 (C=O).
Synthetic procedure for ethyl 2-(5-chloro-2-(2,4-dichloro-
phenoxy)phenoxy)acetate Triclosan 1 (6.0 g, 0.021 mol),
potassium hydroxide (1.7 g, 0.03 mol), and acetonitrile
(20 mL) were placed in a 50 mL flat-bottomed flask equipped
with a magnetic stirring bar. The mixture was stirred and
heated under microwave irradiation to reflux for a period of 5
min. Then, ethyl bromoacetate (2.4 mL, 3.67 g, 0.022 mol)
was added to the reaction mixture, which was then refluxed
for 30 min (200 W). The crude reaction mixture was con-
centrated on a rotatory evaporator, and the residue was pur-
ified by column chromatography over silica gel eluting with
Synthetic procedure for hydrazones
A triclosan–carbohydrazide 3 (0.5 g, 1.38 mmol) solution in
methanol (2 mL) was sonicated for 2 min and then

Med Chem Res
benzaldehyde 4 (1.38 mmol) and acetic acid (0.1 mL) were
added dropwise to the reaction mixture. Upon completion of
the reaction (5–10 min), the product was filtered, sequen-
tially washed with water (20 mL) and ethyl ether (5 mL),
dried in vacuo and recrystallized from ethanol, affording the
corresponding hydrazones in yields ranging from 42 to
93%.
m/z 481.0125 [M + H]+, calcd for C21H15Cl3N2O5:
481.0115.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(3,4-
dihydroxybenzylidene)acetohydrazide (5c) Yield 60%
(0.828 mmol, 398 mg); brown solid, m.p. 216–218 °C; IR
(cm−1): νmax max 3433 (Ar–OH), 3300 (N–H), 1658
(C=O), 1598 (C=N), 1475 (C=CAr), 1269 ((C=O)–N),
1
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(2-
hydroxybenzylidene)acetohydrazide (5a) Yield 91%
(1.256 mmol, 585 mg); white solid, m.p. 178–180 °C; IR
(cm−1): νmax max 3450 (Ar–OH), 3100 (N–H), 1680
(C=O), 1494 (C=N), 1409 (C=CAr), 1259 ((C=O)–N),
802 (C–HAr), 730 (C–Cl). H-NMR (DMSO-d6): δ 4.72,
5.20 (~1:1, –OCH2, s), 6.76, 6.77 (H5ʺ, d, J = 8.8 Hz),
6.85–6.94 (H3, H6ʹ, m), 7.00–7.10 (H4, H6ʺ m), 7.13, 7.19
(H6, d, J = 2.0 Hz), 7.26–7.35 (H5ʹ, H2ʺ, m), 7.68, 7.70 (H3ʹ,
d, J = 2.0 Hz), 7.81, 7.98 (~1:1, N=C–H, s), 9.27 (OH),
11.20, 11.32 (~1:1, NH). 1H (Acetone-d6): δ 5.28, 4.77
(~3:1 –OCH2, s), 7.92, 7.99 (~3:1, N=C–H, s), 10.31, 9.94
(~3:1, NH, s). 13C-NMR (DMSO-d6): δ 65.93, 67.32
(–OCH2), 113.14, 113.38, (C2ʺ), 115.12, 115.44 (C6),
115.82, 115.98 (C5ʺ), 119.09, 119.26 (C3), 120.59, 121.19
(C6ʺ), 121.66, 122.25 (C6ʹ), 122.75, 121.85 (C4), 123.66,
123.82 (C2ʹ), 125.80 (C5ʹ), 127.29, 127.53 (C4ʹ), 128.80,
128.92 (C1ʺ), 129.89, 129.96 (C5), 130.15, 130.24 (C3ʹ),
142.60, 142.90 (C2), 145.11, 145.53 (C3ʺ), 146.06, 146.16
(C4ʺ), 148.29, 148.58 (C1), 150.73, 151.15 (N=CH),
152.18, 152.37 (C1ʹ), 163.46, 168.30 (C=O). EIMS: m/z
481.0125 [M + H]+, calcd for C21H15Cl3N2O5: 481.0126.
1
829 (C–HAr), 754 (C–Cl). H-NMR (DMSO-d6): δ 4.78,
5.23 (~1:1, –OCH2, s), 6.82–6.94 (H3, H3ʺ, H5ʺ, H6ʹ, m),
7.01–7.10 (H4, H4ʺ, m), 7.19–7.31 (H6, H6ʹ, m), 7.34 (H5ʹ,
dd, J = 8.9, 2.0 Hz), 7.18–7.38 (H3ʹ, m), 8.28, 8.43 (~1:1,
N=C–H, s), 9.98, 10.92 (OH), 11.50, 11.71 (~1:1, NH). 1H
(Acetone-d6): δ 5.23, 4.79 (~2:1 –OCH2, s), 8.28, 8.42
(~2:1, N=C–H, s), 11.53, 11.75 (~2:1, NH, s). 13C-NMR
(DMSO-d6): δ 66.10, 67.34 (–OCH2), 115.58, 116.07 (C6),
116.54, 116.80, (C3ʺ), 119.08, 119.38 (C5ʺ), 121.68, 122.38
(C3), 122.62, 122.80 (C6ʹ), 123.68, 123.94 (C4), 127.06,
127.26 (C2ʹ), 127.28, 127.61 (C5ʹ), 128.74, 128.90 (C5),
129.55, 129.80 (C4ʹ), 129.93, 130.09 (C6ʺ), 130.11, 130.22
(C3ʹ), 131.65, 131.99 (C4ʺ), 142.07, 142.66 (C2), 148.08,
148.25 (C1), 150.63, 151.10 (N=CH), 152.13, 152.35 (C1ʹ),
156.83, 157.73 (C2ʺ), 163.83, 168.42 (C=O). EIMS: m/z
465.0176 [M + H]+, calcd for C21H15Cl3N2O4: 465.0172.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(2,4-
dihydroxybenzylidene)acetohydrazide (5d) Yield 64%
(0.883 mmol, 425 mg); light yellow solid, m.p. 222–226 °C;
IR (cm−1): νmax max 3448 (Ar–OH), 3344 (N–H), 1705
(C=O), 1496 (C=N), 1471 (C=CAr), 1251 ((C=O)–N),
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(2,3-
dihydroxybenzylidene)acetohydrazide (5b) Yield 52%
(0.718 mmol, 345 mg); yellow solid, m.p. 182–186 °C; IR
(cm−1): νmax max 3468 (Ar-OH), 3290 (N–H), 1680
(C=O), 1494 (C=N), 1475 (C=CAr), 1271 ((C=O)–N),
1
866 (C–HAr), 770 (C–Cl). H-NMR (DMSO-d6): δ 4.75,
5.18 (~1:1, –OCH2, s), 6.28–6.32 (H5ʺ, m), 6.35, 6.33 (H3ʺ,
d, J = 2.2 Hz), 6.87, 6.89 (H3, d, J = 8.9 Hz), 7.01–7.10
(H4, H6ʹ m), 7.20 (H6, d, J = 2.0 Hz), 7.27, 7.35 (H5ʹ, H6ʺ,
m), 7.67, 7.70 (H3ʹ, d, J = 2.4 Hz), 8.15, 8.28 (~1:1,
N=C–H, s), 11.07, 9.94 (OH), 11.31, 11.51 (~1:1, NH). 1H
(Acetone-d6): δ 5.23, 5.31 (~1:2 –OCH2, s), 8.16, 8.25
(~1:2, N=C–H, s), 11.39, 10.38 (~1:2, NH, s). 13C-NMR
(DMSO-d6): δ 66.89, 67.77 (–OCH2), 102.28, 102.52 (C3ʺ),
110.30, 111.41 (C5ʺ), 115.07, 115.56 (C1ʺ), 118.62, 118.89
(C6), 121.16, 121.87 (C3), 122.12, 122.30 (C6ʹ), 123.19,
123.45 (C4), 126.79, 127.13 (C2ʹ), 128.26, 128.41 (C5ʹ),
129.31, 129.43 (C4ʹ), 129.59, 129.62 (C5), 129.73, 129.76
(C3ʹ), 130.98, 131.00 (C6ʺ), 142.17, 142.62 (C2), 148.78
(C1), 150.16, 150.64 (N=CH), 152.64, 151.87 (C1ʹ),
158.01, 159.26 (C2ʺ), 160.45, 160.82 (C4ʺ), 162.87, 167.45
(C=O). EIMS: m/z 481.0125 [M + H]+, calcd for
C21H15Cl3N2O5: 481.0130.
1
810 (C–HAr), 732 (C–Cl). H-NMR (DMSO-d6): δ 4.79,
5.22 (~1:1, –OCH2, s), 6.67, 6.72 (H5ʺ, tapp, J = 7.8 Hz),
6.82, 6.85 (H3, d, J = 7.8 Hz), 6.88, 6.90 (H4ʺ, d, J = 8.8
Hz), 6.96, 7.12 (H6ʹ, d, J = 7.8 Hz), 7.03 (H6ʺ, dd, J = 8.6,
2.0), 7.05–7.09 (H4, m), 7.23, 7.30 (H6, d, J = 2.0 Hz),
7.28, 7.34 (H5ʹ, dd, J = 8.9, 2.3 Hz), 7.68, 7.70 (H3ʹ, d, J =
2.3 Hz), 8.28, 8.39 (~1:1, N=C–H, s), 9.22, 10.66 (OH),
11.49, 11.71 (~1:1, NH). 1H (Acetone-d6): δ 5.30, 4.86
(~2:1 –OCH2, s), 8.27, 8.35 (~2:1, N=C–H, s), 11.51,
11.39 (~2:1, NH, s). 13C-NMR (DMSO-d6): δ 65.39, 66.39
(–OCH2), 114.93, 115.42 (C6), 116.44, 116.83, (C4ʺ),
116.83, 117.24 (C1ʺ), 118.46, 118.51 (C3), 118.76, 118.95
(C5ʺ), 119.49, 120.23 (C6ʹ), 121.02, 121.73 (C6ʺ), 121.92,
122.12 (C4), 123.03, 123.31 (C2ʹ), 126.63, 126.97 (C5ʹ),
128.09, 128.25 (C4ʹ), 129.13, 129.26 (C3ʹ), 129.46, 129.58
(C5), 142.12, 142.48 (C2), 145.01 (C3ʺ), 145.37, 145.77
(N=CH), 148.37 (C2ʺ), 149.95, 150.44 (C1), 152.13, 152.35
(C1ʹ), 151.45, 151.69 (C2ʹ), 163.14, 167.68 (C=O). EIMS:
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(2,5-
dihydroxybenzylidene)acetohydrazide (5e) Yield 90%
(1.242 mmol, 597 mg); yellow solid, m.p. 220–224 °C; IR

Med Chem Res
(cm−1): νmax max 3325 (Ar–OH), 3167 (N–H), 1676
(C=O), 1492 (C=N), 1471 (C=CAr), 1255 ((C=O)–N),
6.42–6.53 (H3ʺ, H5ʺ, m), 6.88, 6.89 (H3, d, J = 8.9 Hz),
6.98–7.11 (H4, H6´ m), 7.21, 7.28 (H6, d, J = 2.0 Hz),7.30,
7.34 (H5´, dd, J = 8.8, 2.4 Hz), 7.42, 7.57 (H6ʺ, d, J = 8.6
Hz), 7.68, 7.70 (H3ʹ, d, J = 2.4 Hz), 8.19, 8.33 (~1:1,
N=C–H, s),10.15 (OH), 11.25, 11.41 (~1:1, NH). 1H
(Acetone-d6): δ 4.82, 5.25 (~3:1 –OCH2, s), 8.30, 8.20
(~3:1, N=C–H, s), 10.58, 10.64 (~3:1, NH, s). 13C-NMR
(DMSO-d6): δ 55.37, 55.62 (–OCH3, s), 66.00, 67.70
(–OCH2), 101.30, 101.55 (C3ʺ), 106.80, 106.99 (C5ʺ),
112.07, 113.36 (C1ʺ), 114.46, 115.95 (C6), 119.04, 119.32
(C3), 121.66, 122.34 (C6ʹ), 122.68, 122.87 (C4), 123.64,
123.89 (C4ʹ), 127.26, 127.59 (C2ʹ), 128.59, 128.79 (C5ʹ),
128.95, 129.22 (C5), 129.84, 129.96 (C3ʹ), 130.13, 130.25
(C6ʺ), 142.56, 143.02 (N=CH), 148.78, 148.86 (C1),
150.67, 151.14 (C2), 152.17, 152.37 (C1´), 158.41, 159.69
(C2ʺ), 162.36, 162.66 (C4ʺ), 163.56, 168.14 (C=O). EIMS:
m/z 495.0281 [M + H]+, calcd for C22H17Cl3N2O5:
495.0278.
1
910 (C–HAr), 785 (C–Cl). H-NMR (DMSO-d6): δ 4.77,
5.22 (~1:1, –OCH2, s), 6.67–6.74 (H3, H4ʺ, m), 6.89 (H3ʺ, d,
J = 8.8 Hz), 6.94–7.11 (H4, H6ʺ, H6´, m), 7.21, 7.29 (H6, d,
J = 2.0 Hz), 7.30, 7.34 (H5ʹ, dd, J = 8.9, 2.2 Hz), 7.68, 7.69
(H3ʹ, d, J = 2.2 Hz), 8.22, 8.35 (~1:1, N=C–H, s), 8.86,
8.94, 9.29, 10.07, (OH), 11.46, 11.59 (~1:1, NH). 1H
(Acetone-d6): δ 5.28, 4.84 (~1:2 –OCH2, s), 8.21, 8.28
(~1:2, N=C–H, s), 11.54, 10.62 (~1:2, NH, s). 13C-NMR
(DMSO-d6): δ 65.50, 66.86 (–OCH2), 111.39, 113.35 (C6ʺ),
115.00, 115.54 (C6), 116.91, 117.00 (C4ʺ), 118.64, 118.77
(C3ʺ), 118.88, 119.09 (C3), 120.12 (C1ʺ), 121.19, 121.85
(C6ʹ), 122.12, 122.28 (C4), 123.21, 123.44 (C2ʹ), 126.81,
127.12 (C5ʹ), 128.26, 128.40 (C4ʹ), 129.32, 129.44 (C5),
129.61, 129.72 (C3ʹ), 142.51, 142.60 (C2), 146.79, 147.10
(N=CH), 149.36, 149.84 (C5ʺ), 149.87, 150.04 (C1),
150.15, 150.61 (C1ʹ), 151.64, 151.86 (C2ʺ), 163.23, 167.83
(C=O). EIMS: m/z 481.0125 [M + H]+, calcd for for
C21H15Cl3N2O5: 481.0119.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-
(2,3,4-trihydroxybenzylidene)acetohydrazide (5h) Yield
82% (1.132 mmol, 562 mg); yellow solid, m.p. 184–186 °C;
IR (cm−1): νmax max 3462 (Ar–OH), 3310 (N–H), 1689
(C=O), 1494 (C=N), 1475 (C=CAr), 1265 ((C=O)–N),
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(4-
hydroxy-3-methoxybenzylidene) acetohydrazide (5f)
Yield 63% (0.870 mmol, 430 mg); white solid, m.p.
148–150 °C; IR (cm−1): νmax max 3431 (Ar–OH), 3084
(N–H), 1681 (C=O), 1496 (C=N), 1473 (C=CAr), 1251
1
804 (C–HAr), 715 (C–Cl). H-NMR (DMSO-d6): δ 4.76,
5.19 (~1:1, –OCH2, s), 6.36, 6.38 (H5ʺ, d, J = 8.6 Hz), 6.78,
6.87 (H3, d, J = 8.4 Hz), 6.89, 6.94 (H6ʺ, d, J = 8.6 Hz),
6.99–7.10 (H4, H6ʹ, m), 7.18–7.37 (H5ʹ, H6, m), 7.67, 7.70
(H3ʹ, d, J = 2.4 Hz), 8.13, 8.25 (~1:1, N=C–H, s), 8.45
9.32, 9.47, 11.06 (OH), 11.36, 11.56 (~1:1, NH). 1H
(Acetone-d6): δ 5.26, 4.82 (~1:3 –OCH2, s), 8.23, 8.15
(~1:3, N=C–H, s), 11.36, 10.36 (~1:3, NH, s). 13C-NMR
(DMSO-d6): δ 65.90, 67.28 (–OCH2), 107.62, 108.15 (C5ʺ),
111.11, 112.51 (C1ʺ), 115.51, 115.98 (C6), 119.09, 119.38
(C3), 121.46, 121.66 (C6ʹ), 122.36, 122.50 (C6ʺ), 122.63,
122.83 (C4), 123.65, 123.93 (C2ʹ), 127.27, 127.62 (C5ʹ),
128.78, 128.94 (C4ʹ), 129.82, 129.97 (C5), 130.13, 130.26
(C3ʹ), 133.12, 133.16 (C3ʺ), 142.57, 143.06 (C2), 144.43,
147.05 (N=CH), 147.85, 148.79 (C1), 149.34, 150.35 (C2ʺ),
150.65, 151.13 (C4ʺ), 152.12, 152.36 (C1ʹ), 163.43, 167.87
(C=O). EIMS: m/z 497.0074 [M + H]+, calcd for
C21H15Cl3N2O6: 497.0075.
1
((C=O)–N), 806 (C–HAr), 780 (C–Cl). H-NMR (DMSO-
d6): δ 3.30, 3.80 (–OCH3, s), 4.73, 5.24 (~1:1, –OCH2, s),
6.81, 6.83 (H3, d, J = 8.5 Hz), 6.88 (H6ʹ, d, J = 8.7 Hz),
7.03 (H6ʺ, dd, 8.5, 2.0 Hz), 7.05–7.10 (H4, H5ʺ m), 7.19 (H6,
d, J = 2.0 Hz), 7.27 (H2ʺ, sapparent), 7.29, 7.34 (H5ʹ, dd, J =
8.7, 2.3 Hz), 7.68, 7.70 (H3ʹ, d, J = 2.3 Hz), 7.87, 8.05
(~1:1, N=C–H, s), 9.46, 9.52 (OH), 11.26, 11.41 (~1:1,
1
NH). H (Acetone-d6): δ 5.30, 4.78 (~2:1 –OCH2, s), 7.97,
8.12 (~2:1, N=C–H, s), 10.37, 10.10 (~2:1, NH, s). 13C-
NMR (DMSO-d6): δ 55.99 (–OCH3, s), 66.09, 67.25
(–OCH2), 119.46, 119.95 (C2ʺ), 115.44, 115.86 (C6),
119.05, 119.21 (C5ʺ), 121.61, 121.93 (C3), 122.24, 122.69
(C6ʺ), 121.76, 122.85 (C6ʹ), 123.65, 123.78 (C4), 125.74,
125.83 (C2ʹ), 127.28, 127.50 (C4ʹ, C5ʹ), 128.79, 128.91
(C1ʺ), 129.94, 130.15 (C3ʹ), 130.23, 131.07 (C5), 142.56,
142.86 (C2), 143.71, 144.78 (C3ʺ), 148.37, 148.46 (N=CH),
149.26, 149.57 (C1), 150.74, 151.16 (C4ʺ), 152.20, 152.36
(C1ʹ), 163.52, 168.50 (C=O). EIMS: m/z 495.0281 [M +
H]+, calcd for C22H17Cl3N2O5: 495.0290.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(3,4-
dihydroxy-5-methoxybenzylidene) acetohydrazide (5i)
Yield 78% (1.076 mmol, 550 mg); yellow solid, m.p.
106–110 °C; IR (cm−1): νmax max 3466 (Ar–OH), 3280
(N–H), 1659 (C=O), 1495 (C=N), 1475 (C=CAr), 1269
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(2-
hydroxy-4-methoxybenzylidene) acetohydrazide (5g)
Yield 80% (1.104 mmol, 547 mg); light yellow solid, m.p.
148–150 °C; IR (cm−1): νmax max 3400 (Ar–OH), 3250
(N–H), 1666 (C=O), 1496 (C=N), 1475 (C=CAr), 1274
1
((C=O)–N), 806 (C–HAr), 716 (C–Cl). H-NMR (DMSO-
d6): δ 3.31–3.79 (–OCH3, s), 4.73, 5.22 (~1:1, –OCH2, s),
6.76–6.82 (H3, m), 6.89 (H6ʹ, d, J = 8.8 Hz), 7.02, 7.04 (H2ʺ,
H6ʺ, d, J = 2 Hz), 7.07 (H4, dapparent, J = 9.5 Hz), 7.19, 7.27
(H6, d, J = 2.0 Hz), 7.29, 7.34 (H5ʹ, dd, J = 8.9, 2.5 Hz),
1
((C=O)–N), 806 (C–HAr), 712 (C–Cl). H-NMR (DMSO-
d6): δ 3.74, 3.76 (–OCH3, s), 4.76, 5.20 (~1:1, –OCH2, s),

Med Chem Res
7.69, 7.71 (H3ʹ, d, J = 2.5 Hz), 7.85, 8.03 (~1:1, N=C–H,
s), 8.58, 8.87, 8.93, 9.08 (OH), 11.36, 11.50 (~1:1, NH). 1H
(Acetone-d6): δ 5.29, 4.77 (~3:1 –OCH2, s), 7.91, 7.97
(~3:1, N=C–H, s), 10.37, 10.01 (~3:1, NH, s). 13C-NMR
(DMSO-d6): δ 56.31, 56.41 (–OCH3), 65.96, 67.43
(–OCH2), 102.99, 103.05 (C5ʺ), 108.52, 108.90 (C1ʺ),
115.41, 115.80 (C6), 119.08, 119.23 (C3), 121.62, 122.24
(C6ʹ), 122.80, 122.85 (C6ʺ), 123.66, 123.79 (C4), 124.62,
124.68 (C2ʹ), 127.29, 127.51 (C4ʹ), 128.79, 128.91 (C5ʹ),
129.90, 129.94 (C5), 130.16, 130.23 (C3ʹ), 137.01, 137.28
(C4ʺ), 142.57, 142.87 (C2) 145.14, 145.39 (C3ʺ), 146.28,
146.40 (N=CH), 148.89, 148.91 (C5ʺ), 150.73, 151.15 (C1),
152.19, 152.36 (C1ʹ), 163.39, 168.41 (C=O). EIMS: m/z
511.0230 [M + H]+, calcd for C22H17Cl3N2O6: 511.0152.
(C6), 119.05, 119.35 (C3), 121.69, 122.35 (C6ʹ), 122.63,
122.79 (C4), 123.91, 123.64 (C2ʹ), 127.26, 127.61 (C5ʹ),
128.77, 128.94 (C4ʹ), 129.82, 129.97 (C5), 130.11, 130.26
(C3ʹ), 142.52, 143.05 (N=CH), 144.59, 147.26 (C2), 150.07,
151.03 (C1), 152.13, 152.35 (C1ʹ), 159.67, 160.13 (C2ʺ, C6ʺ),
162.21, 163.09 (C4ʺ), 163.53, 167.12 (C=O). EIMS: m/z
497.0074 [M + H]+, calcd for C21H15Cl3N2O6: 497.0075.
Biological activity assays
The compounds were subjected to in vitro evaluation as
regards their cytotoxicity, leishmanicidal, and trypanocidal
activity against U-937 human cells and intracellular amas-
tigotes of L. (V) panamensis and T. cruzi, respectively.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-(4-
hydroxy-3,5-dimethoxybenzylidene) acetohydrazide (5j)
Yield 93% (1.283 mmol, 674 mg); white solid, m.p.
166–170 °C; IR (cm−1): νmax max 3383 (Ar–OH), 3224
(N–H), 1662 (C=O), 1494 (C=N), 1480 (C=CAr), 1269
In vitro cytotoxicity
The cytotoxic activity of the compounds was assessed based
on the viability of the human promonocytic cell line U-937
(ATCC CRL-1593.2TM) evaluated by the MTT (3-(4,5-
1
((C=O)–N), 803 (C–HAr), 712 (C–Cl). H-NMR (DMSO-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
d6): δ 3.79, 3.80 (–OCH3, s), 4.75, 5.26 (~1:1, –OCH2, s),
6.86, 6.88 (H3, d, J = 8.9 Hz), 6.96 (H6ʹ, d, J=8.6 Hz), 7.01,
7.04 (H2´´, H6´´, d, J = 2.0 Hz), 7.07 (H4, d, J = 8.9 Hz),
7.19, 7.27 (H6, d, J = 8.9, 2.5 Hz), 7.29, 7.34 (H5ʹ, dd, J =
8.9, 2.5 Hz), 7.69, 7.71 (H3ʹ, d, J = 2.5 Hz), 7.85, 8.03
(~1:1, N=C–H, s), 8.87, 8.43 (OH), 11.36, 11.50 (~1:1,
assay following the methodology described elsewhere
(Pulido et al. 2012). Briefly, cells grown in tissue flasks were
harvested and washed with phosphate buffered saline (PBS)
by centrifuging. Cells were counted and adjusted at 1 × 106
cells/mL of RPMI-1640 supplemented with complete 10%
fetal bovine serum (FBS) and 1% antibiotics (100 U/mL
penicillin and 0.1 mg/mL streptomycin). One hundred µL
was dispensed into each well of a 96-well cell-culture plate
and then 100 mL of RPMI-1640 and the corresponding
concentrations of the compounds were added, starting at
200 µg/mL in duplicate. Plates were incubated at 37 °C, 5%
CO2 for 72 h in the presence of compounds. The effect of
compounds was determined by measuring the activity of the
mitochondrial dehydrogenase by adding 10 µL/well of MTT
solution (0.5 mg/mL) and incubated at 37 °C for 3 h. The
reaction was stopped by adding 100 µL/well of 50% iso-
propanol solution with 10% sodium dodecyl sulfate and 30
min incubation. The cell viability was determined based on
the quantity of formazan produced according to the intensity
of color (absorbance) registered as optical densities (O.D.)
obtained at 570 nm in a spectrophotometer (Varioskan™
Flash Multimode Reader—Thermo Scientific, USA). Cells
cultured in the absence of compounds were used as control
of viability (100% viability), while amphotericin B (AmB)
was used as non-cytotoxic and cytotoxic drug control,
respectively. Assays were conducted in two independent
runs with three replicates per each concentration tested.
1
NH). H (Acetone-d6): δ 5.30, 4.78 (~3:1 –OCH2, s), 7.96,
8.03 (~3:1, N=C–H, s), 10.39, 9.98 (~3:1, NH, s). 13C-
NMR (DMSO-d6): δ 56.45, 66.07 (–OCH2), 105.00, 105.13
(C2ʺ, C6ʺ), 115.00, 115.43 (C6), 116.53, 116.59 (C3),
119.05, 119.18 (C6ʹ), 120.98, 121.60 (C4), 122.74, 122.85
(C4ʹ), 123.66, 123.78 (C2ʹ), 124.58, 124.67 (C1ʺ), 127.29,
127.49 (C5ʹ), 128.79, 128.91 (C5), 129.93, 130.14 (C3ʹ),
137.39, 138.20 (C4ʺ), 138.50, 139.15 (C2), 142.55, 142.84
(C1), 144.84, 145.02 (C3ʺ, C5ʺ), 148.52, 148.74 (N=CH),
151.15, 152.36 (C1ʹ), 161.11, 162.88 (C=O). EIMS: m/z
525.0387 [M + H]+, calcd for C23H19Cl3N2O6: 525.0376.
(E)-2-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)-Nʹ-
(2,4,6-trihydroxybenzylidene)acetohydrazide (5k) Yield
42% (0.580 mmol, 288 mg); light yellow solid, m.p.
210–214 °C; IR (cm−1): νmax max 3425 (Ar–OH), 3310
(N–H), 1687 (C=O), 1495 (C=N), 1473 (C=CAr), 1259
1
((C=O)–N), 827 (C–HAr), 702 (C–Cl). H-NMR (DMSO-
d6): δ 4.74, 5.14 (~1:1, –OCH2, s), 5.83 (H3ʺ, H5ʺ, m), 6.87
(H3, H6ʹ, d, J = 8.9 Hz), 6.99–7.09 (H4, m), 7.25–7.30 (H6,
m), 7.34 (H5ʹ, dd, J = 8.9, 2.5 Hz), 7.69, 7.68 (H3ʹ, d, J =
2.5 Hz), 8.35, 8.55 (~1:1, N=C–H, s), 10.92, 10.25, 9.85
1
(OH), 11.38, 11.58 (~1:1, NH). H (Acetone-d6): δ 5.22,
In vitro anti-leishmanial activity
4.80 (~1:4 –OCH2, s), 8.54, 8.63 (~1:4, N=C–H, s), 10.24,
10.37 (~1:4, NH, s). 13C-NMR (DMSO-d6): δ 56.48, 67.24
(–OCH2), 94.80 (3ʺ,5ʺ), 99.14, 99.25 (1ʺ), 116.01, 116.08
The activity of the compounds was evaluated on intracel-
lular amastigotes of L. (V) panamensis transfected with the

Med Chem Res
green fluorescent protein gene (MHOM/CO/87/UA140-
EGFP) (Taylor et al. 2011). The effect of each compound
was determined according to the inhibition of the infection
evidenced by the decrease of the infected cells and parasite
inside the cells. Briefly, U-937 human cells at a con-
centration of 3 × 105 cells/mL in RPMI 1640 and 0.1 μg/mL
of phorbol-12-myristate-13-acetate (PMA) were dispensed
into each well of a 24-well cell culture plate and then
infected with 5 days old promastigotes in a 15:1 parasites
per cell ratio. Plates were incubated at 34 °C, 5% CO2 for 3
h and cells were washed two times with PBS to eliminate
internalized parasites. One mL of fresh RPMI 1640 supple-
mented with 10% FBS and 1% antibiotics was added into
each well, cells were incubated again to guarantee multi-
plication of intracellular parasites. After 24 h of infection,
culture medium was replaced by fresh culture medium
containing each compound at four-fold dilutions
(100–25–6.25 and 1.56 μg/mL) and plates were incubated at
37 °C, 5% CO2. After 72 h, inhibition of the infection was
determined. Shortly, cells were removed from the bottom
plate with a trypsin/EDTA (250 mg) solution, recovered
cells were centrifuged at 1100 rpm for 10 min at 4 °C, the
supernatant was discarded and cells were washed with 1 mL
of cold PBS by centrifuging at 1100 rpm for 10 min at 4 °C.
The supernatant was discarded and cells were suspended in
500 μL of PBS and analyzed by flow cytometry (FC
500MPL, Cytomics, Brea, CA, USA). All determinations
for each compounds and standard drugs were carried out in
triplicate, in two independent experiments (Pulido et al.
2012). Activity of the tested compounds was carried out in
parallel with infection progress in culture medium alone and
in culture medium with AmB as antileishmanial drugs.
spectrophotometer (Varioskan™ Flash Multimode Reader
—Thermo Scientific, USA) and intensity of color (absor-
bance) was registered as O.D. Infected cells exposed to
benznidazol (BNZ) were used as control for anti-
trypanosomal activity while infected cells incubated in
culture medium alone were used as control for infection.
Non-specific absorbance was corrected by subtracting the
O.D. from the blank. Determinations were done by triplicate
in at least two independent experiments (Buckner et al.
1996; Insuasty et al. 2015).
Statistical analysis
Cytotoxicity was determined according to viability and
mortality percentages obtained for each isolated experiment
(compounds, amphotericin B, Benznidazole, and culture
medium alone). The results were expressed as 50 lethal
concentrations (LC50) that corresponds to the concentration
necessary to eliminate 50% of cells and calculated by Probit
analysis (Finney 1978). Percentage of viability was calcu-
lated by Eq. 1, where the O.D. of control corresponds to
100% of viability. In turn, mortality percentage corresponds
to 100%–%viability:
% Viability ¼ ðO:D Exposed cellsÞ
=ðO:D unexposed cellsÞ Â 100
The degree of toxicity was graded according to the LC50
value using the following scale: high cytotoxicity: LC50
ð1Þ
<
200 μM; moderate cytotoxicity: LC50 > 200–<400 μM and
potential non-cytotoxicity: LC50 > 400 μM.
Anti-leishmanial activity was determined according to
the percentage of infection (amount of parasites in infected
cells) obtained for each experimental condition by flow
cytometry. The percentage of infected cells was determined
as the number of positive events by double fluorescence
(green for parasites and red for cells) using dotplot analysis.
On the other hand, the amount of parasites in the infected
cells was determined by analysis of mean fluorescence
intensity (MFI) in fluorescent parasites (Pulido et al. 2012).
The parasite inhibition was calculated by Eq. 2, where the
MFI of control corresponds to 100% of parasites. In turn,
inhibition percentage corresponds to 100%–%Parasites.
Results of anti-leishmanial activity were expressed as EC50
determined by the Probit method (Finney 1978):
In vitro anti-trypanosomal activity
Compounds were tested on intracellular amastigotes of T.
cruzi, Tulahuen strain transfected with β-galactosidase gene
(donated by Dr. F. S. Buckner, University of Washington)
(Buckner et al. 1996). The activity was determined
according to the ability of the compounds to reduce the
infection of U-937 cells by T. cruzi. In this case, 100 μL of
U-937 human cells at a concentration of 2.5 × 105 cells/mL
in RPMI-1640, 10% SFB, and 0.1 μg/mL of PMA were
placed in each well of 96-well plates and then infected with
phase growth epimastigotes in 5:1 (parasites per cell) ratio
and incubated at 34 °C, 5% CO2. After 24 h of incubation
four-fold dilutions of each compound (100–25–6.25 and
1.56 mg/mL) were added to infected cells. After 72 h of
incubation, the effect of all compounds on viability of
intracellular amastigotes was determined by measuring
the β-galactosidase activity by spectrophotometry adding
100 μM CPRG and 0.1% nonidet P-40 to each well. After 3
h of incubation, plates were read at 570 nm in a
% Parasite ¼ ðMFI Exposed parasitesÞ
=ðMFI unexposed parasitesÞ Â 100
ð2Þ
Similarly, anti-trypanosomal activity was determined
according to the amount of parasites in infected cells
obtained for each experimental condition by colorimetry.

Med Chem Res
The parasite inhibition was calculated by Eq. 3, where the
O.D. of unexposed parasites corresponds to 100% of
parasites. In turn, percentage of inhibition corresponds to
100%–% Parasites. Results of anti-trypanosomal activity
were also expressed as EC50 determined by the Probit
method (Finney 1978):
signals: C(=O)–NH– (11.50, 11.71 ppm), –N=CH (8.28,
8.43 ppm), and Ar–O–CH2– (4.78, 5.23 ppm). 13C-NMR
spectra showed signals at 150.63 and 151.10 ppm due to the
presence of CH=N, signals at 163.83 and 168.42 ppm
corresponding to C=O group and signals to 66.10 and
67.34 due to Ar–O–CH2−, which indicated that these
molecules are present in two stereoisomeric forms (e.g.,
geometric or conformational isomers) (see Fig. 3).
% Parasite ¼ ðO:D Exposed parasitesÞ
=ðO:Dunexposed parasitesÞ Â 100
ð3Þ
Integration values of these signals showed a ~1:1 ratio
between the two stereoisomers when compounds were dis-
solved in DMSO-d6. However, a ~2:1 ratio was obtained when
1H-NMR spectra of hydrazones were taken in acetone-d6. This
result shows the presence of an equilibrium system that is
dependent on the polarity of the solvent (Rahman et al. 2005).
NOESY experiment showed coupling between C(=O)–NH–
and –N=CH, and between –N=CH and Ar–O–CH2−
hydrogens. These couplings can be explained for the presence
of E-antiperiplanar isomer, which is depicted in Fig. 3 (Basilio
et al. 2013). Therefore, the other constituent of the isomeric
mixture must be the E-synperiplanar isomer (see Fig. 3).
The anti-leishmanial and anti-trypanosomal activities
were graded according to the EC50 value using the fol-
lowing scale: high activity: EC50 < 40 μM, moderate
activity: EC50 > 40–<80 μM; and potential non-activity:
EC50 > 80 μM.
The selectivity index (SI), was calculated by dividing the
cytotoxic activity and the anti-leishmanial or anti-
trypanosomal activity using the following formula: SI =
LC50/EC50.
Biological activities
Results and discussion
Chemistry
The effect of hydrazones on cell growth and viability was
assessed in human macrophages (U-937 cells) (Pulido et al.
2012), which are the host cells for L. (V) panamensis and T.
cruzi parasites. On the other hand, the antiparasite activity of
these compounds was tested on intracellular amastigotes of L.
(V.) panamensis (Taylor et al. 2011) and T. cruzi (Buckner
et al. 1996; Insuasty et al. 2015) according to the ability of
these compounds to reduce the amount of parasite inside
infected macrophages. Results are summarized in Table 1.
All synthetic hydrazones, with the exception of 5c, 5e,
and 5h, were highly cytotoxic to U-937 cells showing LC50
< 200.0 μM (Table 1). Compounds 5c, 5e, and 5h showed
no cytotoxicity (LC50 > 400 μM). In turn, amphotericin B
and benznidazole showed high and moderate and no cyto-
toxicity, respectively.
The anti-leishmanial and anti-trypanosomal activities
were measured by determining the effective concentration
50 (EC50) that corresponds to the concentration of drug that
gives the half-maximal reduction of the amount of intra-
cellular parasites (Table 1). Dose–response relationship
showed that compounds 5a–5d, 5f–5j, and triclosan were
active against intracellular amastigotes of L. (V) panamensis
with EC50 < 40 μM. The most active hybrid compounds
were 5a, 5d, 5g, 5f, 5j, and 5i with an EC50 of >1.29, 1.64,
2.36, 6.88, 9.30, and 11.92 µM, respectively, followed by
5b, 5c, triclosan and 5h with EC50 of 24.70, 25.08, 38.61,
and 39.24 µM, respectively. Compounds 5k and 5e showed
moderate activity with a EC50 > 40 µM. As expected, the
anti-leishmanial drug amphotericin B showed activity with
low EC50 values.
Microwave-assisted Williamson etherification of triclosan 1
with ethyl bromoacetate gave rise to ester 2 in 75% yield
(Otero et al. 2014). Nucleophilic reaction of hydrazine
hydrate on compound 2 gave rise to acylhydrazide 3 in 83%
yield. Coupling compound 3 with a number of aldehydes in
alcoholic medium provided hydrazones 5a–6h in 42–93%
yields (Coa et al. 2015). This synthetic strategy involves
ultrasound-assisted or microwave-assisted reactions which
allows to achieve the compounds with shorter reaction times
than the conventional heating methods. In addition, the
products were obtained in very good to excellent yields and
without appreciable by-product formation (Scheme 1).
The structures and stereochemistry of all hydrazones
have been established by a combined study of IR, ESI-MS,
1H-NMR, 13C-NMR, COSY, and NOESY spectra. IR
spectra exhibited characteristic absorption peaks corre-
sponding to Ar–OH (3325–3468 cm−1), N–H (3100–3290
cm−1), C=O (1658–1705 cm−1), C=N (1492–1598 cm−1),
C=CAr (1409–1480 cm−1), (C=O)–N (1251–1274 cm−1),
C–HAr (802–910 cm−1), and C–Cl (702–780 cm−1). ESI-
MS spectra showed characteristic [M + 1]+ peaks corre-
sponding to their molecular weights. The assignments of all
the signals to individual H-atoms or C-atoms have been
performed on the basis of typical δ-values and J-constants.
1
The H-NMR and 13C-NMR spectra of these compounds
dissolved in DMSO-d6 showed double signals. For exam-
ple, for compounds 5a we have the following H-NMR
1

Med Chem Res
Scheme 1 Synthetic pathway to triclosan–hydrazone hybrids
On the other hand, compounds 5d, 5a, 5f, 5g, and 5i
were highly active against intracellular amastigotes of T.
cruzi with EC50 of 2.28, 2.36, >5.04, 2.95, and 11.31 µM,
respectively, followed by compounds 5j, 5c, 5h, and tri-
closan with an EC50 of 25.11, 31.31, 37.11, and 48.97 µM,
respectively. In this case, benznidazole showed activity with
an EC50 of 40.3 µM.
In general, the anti-leishmanial and anti-trypanosomal
activity of the hydrazones were higher than their cytotoxi-
city. Thus, the SI values calculated for these compounds
were >1. Compounds 5c, 5e, and 5h showed the best SI with
values from 5.90 to 16.55 (Table 1). Amphotericin B has
very high SI values. Benznidazole exhibited a SI of 17.0.
Although several hybrid compounds showed better activity
than benznidazole, the SI of these compounds is affected by
their cytotoxicity. These results suggest that biological
activity of the triclosan–hydrazone hybrids is selective,
being more active against T. cruzi parasites than U-937 cells.
On a structure–activity relationship basis, it is worth
noting the synergistic effect of the parent subunits in the
hybrids in comparison with the unlinked cases. For exam-
ple, triclosan is by itself less potent than their hybrids 5a,
5d, 5f, 5g, 5i, and 5j. The presence of hydroxy or methoxy
groups in positions 2 and 4 on the benzylidene moiety
increases both the activity and cytotoxicity (5d and 5g vs.
5b, 5c, and 5e), which could be explained by a better
molecular recognition ability towards target bioreceptors.
There is not a clear relationship between the antiprotozoal
activity and the methylation of the hydroxy groups, since in
some cases methylation decreases the activity (5d vs. 5g)
while in other cases an increase in activity is measured (5c
vs. 5f and 5i vs. 5j). Furthermore, increasing the number of
hydroxy groups decreases the activity (5a vs. 5b and 5h; 5a
vs. 5d and 5k). The biological activity of these compounds
could be explained by their action as iron chelators (Wal-
court et al. 2004; Coa et al. 2015) or/and as alkylating

Med Chem Res
Fig. 3 Triclosan–hydrazone
hybrids stereoisomers
Table 1 In vitro cytotoxicity
and antiprotozoal activity of
triclosan–hydrazone hybrids
Compound
Cytotoxicity
Anti-leishmanial activity
Anti-trypanosomal activity
LC50 (Mean SEM)
EC50 (Mean SEM) SI
EC50 (Mean SEM) SI
[µM]
[µM]
[µM]
5a
2.79 0.65
91.96 9.38
>415.19
>1.29
<2
3.72
2.36 0.13
1.18
5b
24.70 1.54
25.08 0.44
1.64 0.27
70.33 12.48
6.88 0.36
2.36 0.12
39.24 2.18
11.92 0.82
9.30 0.52
45.61 6.19
38.61 2.38
0.054 0.011
NA
47.81 4.24
1.92
5c
>16.55 31.31 1.27
1.31 2.28 0.21
>5.90 58.48 6.85
>13.26
0.95
5d
2.08 0.13
>415.19
5e
>7.10
<2.16
1.83
5f
10.89 0.81
5.45 0.20
>401.84
1.58
2.28
>5.04
5g
2.95 0.49
5h
>10.24 37.11 6.43
>10.83
3.20
5i
36.15 5.10
23.78 5.15
62.08 1.61
193.41 32.81
3.03
2.55
1.36
5.01
842
11.31 0.27
25.11 4.31
42.39 6.73
48.97 4.21
NA
5j
0.95
5k
1.46
Triclosan
3.95
Amphotericin B 45.60 2.16
Benznidazole 687.8 16.14
Data represent mean value standard deviation
LC50 lethal concentration 50 in μM, EC50 effective concentration 50 in μM, SI selectivity index = LC50
EC50, NA not applicable
NA
NA
40.3 6.92
17.0
/
agents (Michael acceptor) (Cardona et al. 2014). Studies
in vitro have shown that chelating agents are able to inhibit
parasite growth and proliferation by deprivation of iron,
which is an essential nutrient for cell growth and division
(Richardson et al. 1995). An electrophilic-conjugated sys-
tem could be generated from o-hydroxybenzylidene-N-
acylhydrazone framework due to the ability of this system
to be converted into an electrophilic quinone methide
intermediate through a pericyclic rearrangement (Ifa et al.
2000). The generation of such a system would allow con-
jugate addition of nucleophilic amino acid residues such as
those found in Leishmania cysteine proteases (Mottram
et al. 2004).
triclosan–hydrazone hybrids are reported. Hydrazones were
obtained as two E-synperiplanar and E-antiperiplanar con-
formers. Nine of them were active against L. (V) pana-
mensis (5a–5d, 5f–5j) and eight of them were active against
T. cruzi (5a, 5c, 5d, 5f–5j), with EC50 values lower than 40
µM. Compounds 5c, 5e, and 5h showed the best SI against
both L. panamensis and T. cruzi, with values between 5.90
and 16.55. These results suggest that these compounds
have potential as templates for drug development against
protozoal diseases. The presence of hydroxy or methoxy
groups in positions
2 and 4 on the benzylidene
moiety increases both activity and cytotoxicity. There is no
clear relationship between the antiprotozoal activity and the
methylation pattern of the hydroxy groups, since in some
cases methylation decreases the activity (5d vs. 5g),
while in other cases the activity is increased (5c vs. 5f and
5i vs. 5j). The mechanism of action of these compounds
needs to be adressed and will be the objective of further
studies.
Conclusions
The synthesis, cytotoxicity, and activity against L. (V)
panamensis and T. cruzi amastigotes of 11

Med Chem Res
Acknowledgements The authors thank COLCIENCIAS (contract
no. 0333-2013, code: 111556933423) for financial support.
caffeine-based chalcones, pyrazolines and pyrazolo[3-4-b][1-4]
diazepines as potential antimalarial, antitrypanosomal and antil-
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Jorge SD, Palace-Berl F, Mesquita Pasqualoto KF, Ishii M, Ferreira
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Compliance with ethical standards
Conflict of interests The authors declare that they have no com-
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