Design, synthesis, and in vitro antiprotozoal, antimycobacterial activities of N-{2-[(7-chloroquinolin-4-yl)amino]ethyl}ureas

Article abstract of DOI:10.1016/j.bmc.2010.07.008

We have synthesized a new series of quinoline tripartite hybrids from chloroquine, ethambutol, and isoxyl drugs, using a short synthetic route. Compounds 1-8 were tested in vitro against five protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeb

Full text of DOI:10.1016/j.bmc.2010.07.008

Bioorganic & Medicinal Chemistry 18 (2010) 6398–6403
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and in vitro antiprotozoal, antimycobacterial activities
of N-{2-[(7-chloroquinolin-4-yl)amino]ethyl}ureas q
Carlos Nava-Zuazo a, Samuel Estrada-Soto a, Jorge Guerrero-Álvarez b, Ismael León-Rivera b,
Gloria María Molina-Salinas c, Salvador Said-Fernández c, Manuel Jesús Chan-Bacab d,
Roberto Cedillo-Rivera e, Rosa Moo-Puc e, Gumersindo Mirón-López f, Gabriel Navarrete-Vazquez a,
*
a Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
b Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
c División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, IMSS, Monterrey, Nuevo León 64720, Mexico
d Departamento de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, Campeche 24030, Mexico
e Unidad de Investigación Médica Yucatán, Unidad Médica de Alta Especialidad del Centro Médico Nacional Ignacio García Téllez, IMSS Mérida, Yucatán 97000, Mexico
f Facultad de Química, Universidad Autónoma de Yucatán, Merida, Yucatán 97000, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a new series of quinoline tripartite hybrids from chloroquine, ethambutol, and iso-
xyl drugs, using a short synthetic route. Compounds 1–8 were tested in vitro against five protozoa (Giar-
dia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Leishmania mexicana and Trypanosoma cruzi)
and Mycobacterium tuberculosis. N-(4-Butoxyphenyl)-N0-{2-[(7-chloroquinolin-4-yl)amino]ethyl}urea (6)
was the most active compound against all parasites tested. Compound 6 was 670 times more active than
metronidazole, against G. intestinalis. It was as active as pentamidine against L. mexicana, and it was two-
fold more potent than ethambutol and isoxyl versus M. tuberculosis. This compound could be considered
as a new broad spectrum antimicrobial agent.
Received 27 May 2010
Revised 30 June 2010
Accepted 5 July 2010
Available online 30 July 2010
Keywords:
Hybrid drugs
Quinolines
Giardia intestinalis
Leishmania mexicana
Chloroquine
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
line drugs: isoniazid, rifampicin, ethambutol, streptomycin, and
pyrazinamide.4
Protozoan diseases are a main problem in developing countries,
affecting hundreds of millions people and animals around the
world.1 Since protozoa are eukaryotic, they share many common
features with their mammalian host, making the development of
effective and selective drugs a hard job. The chemotherapy against
diseases such as giardiasis, amoebiasis, trichomoniasis, leishmani-
asis, and trypanosomiasis is limited by the existence of only a few
drugs in the market, most of which are of low efficacy, showing
toxic side effects, and frequently lead to the appearance of resistant
strains.2 This reflects the need to continue searching for new and
better antiprotozoal drugs.3
Isoxyl and phenylthiourea derivatives,5 have shown consider-
able antimycobacterial activity, however, the clinical use of isoxyl
was discontinued, apparently because of the poor bioavailability of
the highly non-polar product.6
Quinoline derivatives are a class of bioactive heterocycles
against Plasmodium falciparum and Leishmania mexicana.7–9 Qui-
nine, chloroquine (CQ), and mefloquine, are mainly used as anti-
malarial drugs.10 However, they possess moderate biological
activity against M. tuberculosis (MIC 25–100
tigations have reported the activity of some quinolines against
M. tuberculosis with MIC’s of 3.12–6.25
g/mL (Fig. 1).10–12
lg/mL). Recent inves-
l
On the other hand, tuberculosis (TB) is one of the most common
infectious diseases known by the mankind. Around 32% of the
world’s population is infected by Mycobacterium tuberculosis, the
main causal agent of TB. Problems in the chemotherapy of tubercu-
losis arise when patients develop bacterial resistance to the first-
Due to the antimalarial activity is known for this kind of
heterocycles, we have investigated the broad antiparasitic spec-
trum of quinolines against other protozoa and mycobacteria. As a
part of our search for basic information about the structural
requirements for new antiinfective molecules, we have synthe-
sized a series of novel chloroquine–ethambutol–isoxyl tripartite
hybrids 1–8 (Fig. 2), according to de Souza et al. approach.10 The
in vitro antiparasitic activity of these compounds on intestinal uni-
cellular parasites (Giardia intestinalis and Entamoeba histolytica), a
urogenital tract parasite (Trichomonas vaginalis) and kinetoplastid
q
Taken in part from the M. Pharm thesis of Carlos Nava Zuazo.
* Corresponding author. Tel./fax: +52 777 3297089.
E-mail address: gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.008

C. Nava-Zuazo et al. / Bioorg. Med. Chem. 18 (2010) 6398–6403
6399
NH2
Cl
HN
N
i
NH2
)
8
NH2
H
N
H
N
H2N
+
(
HN
N
Cl
Cl
N
9
11
O
10
O
F
OH
N
ii
O
Cl
N
R
O
C
H
N
H
MIC = 6.25 µg/mL
N
MIC = 3.12 µg/mL
12-19
R
HN
O
Figure 1. Antimycobacterial quinolines.10,11
Cl
N
1-8
parasites such as Trypanosoma cruzi and L. mexicana is reported in
this paper. Additionally, we also report the biocide effect against M.
tuberculosis.
Scheme 1. Reagents and conditions: (i) 6 equiv of 11, 80 °C; (ii) Et3N, CH3CN.
2. Results and discussion
quinolin-4-yl)ethane-1,2-diamine (9). The last compound was then
reacted with adequate isocyanate derivatives 12–19 under inert
atmosphere, triethylamine and acetonitrile as solvent (Scheme
1). Compounds were purified by recrystallization. The chemical
structures of the synthesized compounds were confirmed on the
basis of their spectral data.
2.1. Drug design of derivates 1–8
Compounds 1–8 were designed on the basis of the structure of
antiprotozoal drug chloroquine (CQ), and the antimycobacterial
drugs ethambutol (ETH) and isoxyl (ISO). The hybridization of
these three pharmacophores (7-chloroquinoline, ethylenediamine
spacer and phenylurea as thiourea bioisostere) leading the title
compounds (Fig. 2). As defined by Viegas-Junior et al. molecular
hybridization is a strategy of rational design of new ligands or pro-
totypes based on the recognition of pharmacophoric subunits in
the molecular structure of two or more known bioactive deriva-
tives which, through the adequate fusion of these, lead to the de-
sign of new hybrid architectures that maintain pre-selected
characteristics of the original templates.13 Recently, Saadeh et al.
have published a novel hybrid antiparasitic molecule, based on
the structures of metronidazole and chloroquine.14
In the nuclear magnetic resonance spectra (1H NMR; d ppm),
the signals of the respective protons of the compounds were veri-
fied on the basis of their chemical shifts, multiplicities and cou-
pling constants.
In compounds 3–8, the 4-substituted phenylurea region of the
1H NMR spectrum contained an A2B2 pattern signals ranging from
d 7.25–7.64 ppm, attributable to H-20 and H-60, whereas displace-
ments of H-30 and H-50 ranging from 6.77 to 8.14 ppm. We also
observed in all compounds a characteristic pattern for 7-chloro-
quinoline core: a doublet signal ranging in d 8.34–8.37 ppm, attrib-
utable to H-2, with ortho coupling constant (J = 5.2–5.4 Hz); a
doublet signal ranging from 6.52 to 6.60 ppm, assigned to H-3,
with Jo = 5.4–5.6 Hz. A third doublet signal in 8.15–8.37 ppm, with
Jo = 8.0–9.2 Hz, given to H-5. A doublet of doublets appears ranging
from 7.25 to 7.64 ppm, belongs to H-6 with ortho and meta cou-
pling constants, and the last doublet signal in 7.77–7.79 ppm,
attributable to H-8.
2.2. Chemistry
Compounds 1–8 were prepared starting from 4,7-dichloroquin-
oline (10). It was reacted with an excess of ethylenediamine (11),
through an addition-elimination mechanism to give N-(7-chloro-
N
HO
H
N
H
N
HN
H
N
N
H
S
O
O
Cl
N
HO
Ethambutol (ETH)
Isoxyl (ISO)
Chloroquine (CQ)
Tripartite hybridization
ISO bioisostere
H
ETH fragment
HN
H
N
N
O
R
Cl
N
CQ pharmacophore
Figure 2. Compounds used as lead and drug design of 1–8 through tripartite molecular hybridization.

6400
C. Nava-Zuazo et al. / Bioorg. Med. Chem. 18 (2010) 6398–6403
2.3. Biological activities
against M. tuberculosis strain H37Rv (ATCC 27294). Results revealed
that compounds 3 and 6 exhibited high mycobactericidal activity.
2.3.1. In vitro antiprotozoal effects
Among others, these structures were found to be the most potent
The new tripartite hybrids 1–8 were tested in vitro as antiproto-
zoal agents. Biological assays results against the five protozoa tested
are summarized in Table 1. Comparison was made among new
compounds and the antiprotozoal drug of choice: metronidazole,
against G. intestinalis, E. histolytica, and T. vaginalis. In order to
compare bioactivities, chloroquine (CQ) and precursor 9 were also
tested. In vitro susceptibility assays were performed using a method
previously described.15,16
compounds with MIC’s of 4 and 2
6 was twofold more potent than reference drugs ethambutol and
isoxyl (MIC 4 g/mL), whereas compound 3 was as active as these
two antimycobacterial drugs (Table 2). Compounds 3 and 6 have a
high C log P value (4.60 and 5.16, respectively), leading us to the
hypothesis that an increase of lipophilicity could improve antimyco-
bacterial activity, in agreement with recent literature reports.18–20
lg/mL, respectively. Compound
l
In general, all the screened compounds showed high bioactivity
2.3.3. In vitro cytotoxic effect
(IC50 <1.8
lM) against G. intestinalis, being more potent than met-
The most active compound (6) was evaluated for its toxicity
ronidazole (IC50 = 5.36
lM). Compound 6, with a 4-butoxyphenyl
against mammalian VERO cell line, showing an CC50 of 5 lM. The
substituent, showed nanomolar potency (IC50 = 8 nM). It was
670-times more active than metronidazole, 50-fold more potent
than CQ, and 156-fold more active than precursor 9.
selectivity index (SI) of the compound, defined as the ratio of cyto-
toxicity to biological activity (SI = CC50 VERO cells/IC50 parasites)
was calculated. It is generally considered that biological efficacy is
not due to in vitro cytotoxicity when SI P10. Compound 6 showed
a nanomolar giardicidal effect, having a selectivity index of 625.
Against T. vaginalis, only compounds 3 and 6 showed high bio-
activity (<10
metronidazole. In comparison, CQ and precursor 9 had low poten-
cies, with IC50 >20 M.
lM). However, none of them were more active than
l
3. Conclusion
In vitro antiamoebic activity exhibited by compounds 1–3, 5,
and 6 was acceptable. All of them showed high bioactivity in the
range of 3.63–6.93 lM. Again, compound 6 was the most active
We have synthesized and screened the in vitro antiprotozoal
and antimycobacterial activities of new tripartite hybrids from
of the series against E. histolytica. It was three-times more active
than CQ, four-times more potent than precursor 9, but fourfold less
potent than metronidazole.
For L. mexicana, only compound 6 was as active as pentamidine
(second-line antileishmanial drug). The rest of compounds were
inactive against this kinetoplastid parasite.
Table 2
In vitro antimycobacterial activity of 1–8
Compd
R
MIC (lg/mL) M. tuberculosis H37Rv
1
2
3
4
5
6
7
8
Cyclohexyl
Ph
8
8
4
8
8
2
8
8
8
>8
4
4
In vitro antichagasic activity of compounds 3, 5–7 was moder-
ated (50
lM), whereas benznidazole (first-line antichagasic drug)
4-ClPh
4-FPh
4-EtOPh
4-BuOPh
4-NO2Ph
4-MeOPh
—
showed an IC50 = 34.38
l
M.
With these results, we could conclude that compounds 1–8 dis-
played a selective toxicity against G. intestinalis over the other uni-
cellular parasites tested.
9
CQ
—
—
2.3.2. In vitro antimycobacterial effect
Ethambutol
Isoxyl
Following the Microplate Alamar Blue Assay (MABA),17 tripartite
hybrids 1–8 were tested in vitro for their antimycobacterial activity
Table 1
In vitro antiparasitic activity of quinoline ureas (1–8)
H
N
H
N
HN
R
O
Cl
N
Compd
R
IC50 (lM)
G. intestinalis
T. vaginalis
E. histolytica
L. mexicana
T. cruzi
1
2
3
4
5
6
7
8
Cyclohexyl
Ph
1.647
1.171
0.267
0.927
0.339
0.008
1.748
0.595
1.253
0.401
5.360
—
66.668
45.974
9.589
23.212
30.128
8.444
12.026
21.738
22.986
30.000
0.290
—
6.933
5.370
6.866
17.664
5.934
3.636
10.563
10.678
14.597
9.579
0.770
—
>50
>50
50
>50
50
9.90
50
>50
>50
>50
—
>50
>50
50
>50
50
50
50
>50
>50
>50
—
4-ClPh
4-FPh
4-EtOPh
4-BuOPh
4-NO2Ph
4-MeOPh
—
—
—
—
—
9
CQ
Metronidazole
Pentamidine
Benznidazole
13.32
—
—
34.38
—
—
—

C. Nava-Zuazo et al. / Bioorg. Med. Chem. 18 (2010) 6398–6403
6401
pharmacophores 7-chloroquinoline (CQ), ethylenediamine spacer
4.3.2. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-
(ETH) and phenylurea as thiourea bioisostere (ISO). This study
demonstrated that the hybridization approach have generated a
new antiparasitic and mycobactericidal scaffold. The obtained re-
sults are very promising since many of the compounds showed
activity comparable with the current used antiprotozoal drugs
metronidazole and pentamidine, whereas compound 6 exhibited
even higher bioactivity, especially towards G. intestinalis, L. mexi-
cana, and M. tuberculosis. Further optimization and pharmacoki-
netic characterization of this series are in progress in our
laboratory.
phenylurea (2)
Recrystallized from ethanol. Yield: 77%, mp: 208–212 °C. 1H
NMR (200 MHz, DMSO) d: 8.34 (1H, d, Jo = 5.2, H2); 8.15 (1H, d,
Jo = 9.2, H5); 7.77 (1H, d, Jm = 2.0, H8); 7.42 (1H, dd, Jo = 9.0,
0
0
0
0
Jm = 1.8, H6); 7.34 (2H, m, Jo = 8, H2 –6 ); 7.19 (2H, t, Jo = 7.8, H3 –5
)
0
6.87 (1H, t, Jo = 7.6, H4 ); 6.57 (1H, d, Jo = 5.6, H3); 6.37 (1H, s,
NH); 3.71 (4H, s, H9,10) ppm. 13C NMR (50 MHz, DMSO) d: 151.72
(C-2), 150.75 (C-4), 150.27 (CO), 148.45 (C-8a), 139.90 (C-10),
133.59 (C-7), 128.62 (C-30–50), 126.99 (C-8), 124.26 (C-6), 117.95
(C-20–60), 117.16 (C-4a), 98.61 (C-3), 42.99 (C-9), 37.70 (C-
10) ppm; MS/FAB+: m/z 341 (M+H+). HRMS (FAB+): m/z 341.1172
[M+H]+ (Calcd for C18H18ClN4OH+ 341.1169).
4. Experimental
4.1. Instruments
4.3.3. N-(4-Chlorophenyl)-N0-{2-[(7-chloroquinolin-4-yl)amino]-
ethyl}urea (3)
Recrystallized from acetonitrile–methanol. Yield: 32%, mp:
228–230 °C 1H NMR (200 MHz, DMSO) d: 8.40 (1H, d, Jo = 5.4,
H2); 8.23 (1H, d, Jo = 9.2, H5); 7.79 (1H, d, Jm = 2.2, H8); 7.47 (1H,
Melting points were determined on an EZ-Melt MPA120 auto-
mated melting point apparatus from Stanford Research Systems
and are uncorrected. Reactions were monitored by TLC on
0.2 mm precoated Silica Gel 60 F254 plates (E. Merck). 1H NMR
spectra were recorded on a Varian INOVA 400 (400 MHz) and 13C
NMR (100 MHz) instruments, and Varian Mercury 200 instrument.
Chemical shifts are given in ppm relative to tetramethylsilane
(Me4Si, d = 0) in DMSO-d6; J values are given in Hz. The following
abbreviations are used: s, singlet; d, doublet; dd, doublet of dou-
blets; t, triplet; m, multiplet; br s, broad signal. MS and HRMS were
recorded on a JEOL JMS-700 spectrometer by Electron Impact or
Fast Atom Bombarded [(FAB+)]. The C log P values were obtained
using ACD/labs software v.4.5.
0
0
dd, Jo = 8.0, Jm = 2.2, H6); 7.43 (2H, m, Jo = 8.4, H2 –6 ); 7.27 (2H, t,
0
0
Jo = 9.2, H3 –5 ), 6.58 (1H, d, Jo = 5.4, H3); 6.37 (1H, s, NH); 3.37
(4H, s, H9,10) ppm. 13C NMR. (50 MHz, DMSO) d: 156.13 (C-2),
152. 55 (C-8a), 150.73 (CO), 149.64 (C-4), 140.00 (C-10), 134.06
(C-7), 129.09 (C-8), 128.18 (C-30–50), 126.00 (C-40), 124.78 (C-6),
124.54 (C-5) 119.43 (C-20–60), 118.05 (C-4a), 99.38 (C-3), 43.60
(C-9), 40.63 (C-10) ppm; MS/FAB+: m/z 375 (M+H+). HRMS
(FAB+): m/z 375.0800 [M+H]+ (Calcd for C18H17Cl2N4OH+
375.0779).
4.3.4. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-(4-fluoro-
phenyl)urea (4)
4.2. Synthesis of N-(7-chloroquinolin-4-yl)ethane-1,2-diamine
(9)
Recrystallized from ethanol. Yield: 50%, mp: 220–221 °C. 1H
NMR (500 MHz, DMSO) d: 8.40 (1H, d, Jo = 5.4, H2); 8.23 (1H, d,
Jo = 9.2, H5); 7.79 (1H, d, Jm = 2.2, H8); 7.47 (1H, dd, Jo = 9.2,
A mixture of 4,7-dichloroquinoline (3.0 g, 0.0151 mol) and eth-
ylenediamine (5.45 g, 6.08 mL, 0.0909 mol, 6 equiv), was stirred
under reflux for 3 h. After cooling to room temperature, the reac-
tion was verted under water–ice mixture. The precipitate formed
was filtered off, and the residue washed with cold water. Com-
pound was recrystallized from ethanol to give 3.13 g (94%), mp
143.5–145.4 °C (lit. 145–147 °C).10
0
0
Jm = 2.2, H6); 7.40 (2H, m, Jo = 9.0, H2 –6 ); 7.06 (2H, t, Jo = 9.2,
0
0
H
H
3 –5 ), 6.57 (1H, d, Jo = 5.4, H3); 6.37 (1H, s, NH); 3.29 (4H, s,
9,10) ppm. 13C NMR. (125 MHz, DMSO) d: 157.47 (J = 235.6, C–F);
156.28 (CO), 152.51 (C-2), 150.66 (C-4), 149.59 (C-8a), 137.29 (C-
10), 133.94 (C-7), 128.10 (C-8), 124.66 (C-6), 124.46 (C-5), 119.97
(J = 6.68, C-20–60), 117.95 (C-4a) 115.64 (J = 25.74, C-30–50), 99.25
(C-3), 40.36 (C-9), 39.70 (C-10) ppm. MS/FAB+: m/z 359 (M+H+).
HRMS (FAB+): m/z 359.1059 [M+H]+ (Calcd for C18H17ClFN4OH+
359.1075).
4.3. General method of synthesis of N-{2-[(7-chloroquinolin-4-
yl)amino]ethyl}-N0-arilureas (1–8)
4.3.5. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-(4-ethoxy-
phenyl)urea (5)
To a solution of N-(7-chloroquinolin-4-yl)ethane-1,2-diamine
(0.3 g, 0.0013 mol) in dry acetonitrile (5 mL), was added appro-
priate isocyanate (0.0014 mol, 1.1 equiv) in dry acetonitrile drop-
wise at 0 °C, and stirred at room temperature under nitrogen
atmosphere for 2–4 h. Solvent was removed in vacuo, and the
residue was suspended in water. The white precipitate was fil-
tered and dried. Crude compounds were purified by recrystalli-
zation.
Recrystallized from ethanol. Yield: 88%, mp: 213–216 °C. 1H
NMR (200 MHz, DMSO) d: 8.40 (1H, d, Jo = 5.2, H2); 8.37 (1H, d,
Jo = 8.0, H5); 7.79 (1H, d, Jm = 2.2, H8); 7.46 (1H, dd, Jo = 8.0,
0
0
Jm = 2.4, H6); 7.28 (2H, m, Jo = 8.8, H 2 –6 ); 6.79 (2H, t, Jo = 8.8,
0
0
H
3 –5 ), 6.57 (1H, d, Jo = 5.4, H3); 6.50 (1H, s, NH); 3.37 (4H, s, H
9,10), 3.93 (2H, c, CH2O), 1.28 (3H, t, CH3) ppm. 13C NMR (50 MHz,
DMSO) d: 155.91 (C-40), 153.18 (C-2), 151. 84 (C-4), 150.03 (CO),
148.93 (C-8a), 133.47 (C-7), 133.23 (C-10), 127.47 (C-8), 124.08
(C-6), 123.83 (C-20–60), 119.65 (C-30–50), 117.37 (C-4a) 114.37 (C-
5), 98.67 (C-3), 63.02 (OCH2), 43.19 (C-9), 40.34 (C-10), 14.76
(CH3) ppm; MS/FAB+: m/z 385 (M+H+). HRMS (FAB+): m/z
385.1415 [M+H]+ (Calcd for C20H22ClN4O2H+ 385.1431).
4.3.1. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-cyclohexy-
lurea (1)
Recrystallized from ethanol. Yield: 88%, mp: 210–212 °C. 1H
NMR (200 MHz, DMSO) d: 8.83 (1H, d, Jo = 5.2, H2); 8.16 (1H, d,
Jo = 8.8, H5); 7.78 (1H, d, Jm = 2.2, H8); 7.47 (1H, dd, Jo = 9.2,
Jm = 2.8, H6); 6.52 (1H, d, Jo = 5.6, H3); 5.98 (2H, s, NH); 3.32 (4H,
4.3.6. N-(4-Butoxyphenyl)-N0-{2-[(7-chloroquinolin-4-yl)amino]-
ethyl}urea (6)
0
0
0
0
s, H9,10); 1.67 (4H, m, H2 –6 ); 1.55 (4H, m, H4 ); 1.15 (4H, m, H3 –
0
5 ) ppm. 13C NMR (50 MHz, DMSO) d: 158.8 (CO), 152.5 (C-2),
Recrystallized from ethanol. Yield: 92%, mp: 202–204 °C. 1H
NMR (200 MHz, DMSO) d: 8.37 (1H, d, Jo = 5.4, H2); 8.19 (1H, d,
Jo = 9.0, H5); 7.79 (1H, d, Jm = 2.2, H8); 7.43 (1H, dd, Jo = 8.0,
150.8 (C-8a), 149.9 (C-4), 134.0 (C-7), 128.2 (C-8), 124.7 (C-6),
124.4 (C-5), 117.9 (C-4a), 99.3 (C-3), 48.6 (C-10), 44.6 (C-9), 40.6
(C-10), 33.9 (C-20-60), 25.2 (C-30–50), 20.02 (C-40) ppm; MS/FAB+:
m/z 347 (M+H+). HRMS (FAB+): m/z 347.1619 [M+H]+ (Calcd for
C18H24ClN4OH+ 347.1639).
0
0
Jm = 1.4, H6); 7.25 (2H, m, Jo = 8.8, H 2 –6 ); 6.77 (2H, t, Jo = 8.8,
0
0
H
3 –5 ), 6.55 (1H, d, Jo = 5.4, H3); 6.25 (1H, s, NH); 3.30 (4H, s,

6402
C. Nava-Zuazo et al. / Bioorg. Med. Chem. 18 (2010) 6398–6403
H9,10), 3.84 (2H, t, CH2O), 1.60 (2H, c, CH2-200), 1.37 (2H, q, CH2-300),
performed in 96-well plates and all compounds were evaluated in
duplicate. Compounds were solubilized in DMSO and diluted in a
13
0.92 (3H, t, CH300) ppm. C NMR (50 MHz, DMSO) d: 156.55 (C-40),
154.02 (C-2), 152. 55 (C-4), 150.73 (CO), 149.64 (C-8a), 133.94 (C-
7), 133.94(C-10), 128.18 (C-8), 124.78 (C-6), 124.54 (C-5), 120.30
(C-20–60), 117.99 (C-4a) 115.08 (C-30–50), 99.32 (C-3), 67.85
(OCH2), 43.90 (C-9), 39.35 (C-10), 31.69 (CH2-200), 19.47 (CH2-300),
14.44 (CH3) ppm; MS/FAB+: m/z 413 (M+H+). HRMS (FAB+): m/z
413.1780 [M+H]+ (Calcd for C22H26ClN4O2H+ 413.1744).
liquid medium. A mixture of 100
100 L of culture medium containing 10,000 Leishmania prom-
astigotes or 20,000 T. cruzi epimastigotes was added to obtain con-
centrations of 10, 5, 2.5, 1.25 g/mL. Benznidazole (first-line
antichagasic drug) and pentamidine (second-line antileishmanial
drug) were used as positive controls. Cultures containing parasites
without compound solution were also included. The plate was
incubated at 26 °C for 72 h and the leishmanicidal and trypanocidal
activity of compounds were determined by direct count of para-
sites in a Neubauer chamber.22 The concentration required to inhi-
bit 50% of the parasites grow (IC50) was calculated by probit
analysis.
lL of compounds solution and
l
l
4.3.7. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-(4-nitro-
phenyl)urea (7)
Recrystallized from acetonitrile-methanol. Yield: 70%, mp: 223–
224 °C. 1H NMR (200 MHz, DMSO) d: 8.41 (1H, d, Jo = 5.4, H2); 8.23
0
0
(1H, d, Jo = 9.2, H5), 8.14 (2H, t, Jo = 8.8, H3 ,5 ) 7.79 (1H, d, Jm = 1.8,
H8); 7.46 (1H, dd, Jo = 8.8, Jm = 1.8, H6); 7.64 (2H, m, Jo = 8.8,
4.4.3. Anti-Mycobacterium tuberculosis assay17,18
0
0
H
2 –6 ), 6.67 (1H, s, NH); 6.60 (1H, d, Jo = 5.4, H3); 3.39 (4H, s,
H9,10) ppm. 13C NMR (50 MHz, DMSO) d: 154.71 (C-2), 154.02 (C-
4), 149. 98 (CO), 148.92 (C-8), 146.95 (C-10), 140.34 (C-40), 133.94
(C-7), 127.43 (C-8), 125.00 (C-20–60), 124.00 (C-6), 123.82 (C-5),
117.34 (C-4a) 116.82 (C-30–50), 98.63 (C-3), 41.26 (C-9), 40.31 (C-
10) ppm; MS/FAB+: m/z 386 (M+H+). HRMS (FAB+): m/z 386.1020
[M+H]+ (Calcd for C18H17ClN5O3H+ 386.1020).
M. tuberculosis H37Rv (ATTC 27294) was used in the present
study, which is sensitive to all five first-line antituberculosis drugs
(STR, INH, RIF, ETH and PYR). Mycobacteria was cultured at 37 °C
and 5% CO2 atmosphere in Middlebrook 7H9 broth supplemented
with 0.2% glycerol and 10% OADC enrichment (oleic acid–
albumin–dextrose–catalase) until the logarithmic phase of growth
was reached. The inoculum for the assay was prepared by diluting
a logarithmically growing culture to match the McFarland 1 tur-
bidity standard and then further diluting this to 1:50 with Middle-
brook 7H9 broth to obtain a concentration of 6 ꢀ 106 colony
forming units/mL. The working suspension was prepared just be-
fore inoculation. The antibacterial activity of compounds against
M. tuberculosis was tested using the modified MABA. The concen-
4.3.8. N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}-N0-(4-meth-
oxyphenyl)urea (8)
Recrystallized from acetonitrile–ethanol. Yield: 76%, mp: 216–
219 °C. 1H NMR (200 MHz, DMSO) d: 8.42 (1H, d, Jo = 5.4, H2);
8.22 (1H, d, Jo = 9.2, H5), 7.79 (1H, d, Jm = 2.2), 7.79 (1H, d,
Jm = 1.8, H8), 7.46 (1H, dd, Jo = 8.8, Jm = 2.2, H6); 7.29 (2H, m,
Jo = 9.2, H2 –6 ), 6.81 (2H, t, Jo = 8.8, H3 –5 ), 6.28 (1H, s, NH); 6.58
(1H, d, Jo = 5.4, H3); 3.39 (4H, s, H9,10) ppm. 13C NMR (50 MHz,
DMSO) d: 155.91 (C-40), 153.97 (C-2), 151. 84 (C-4), 150.09 (CO),
148.93 (C-8), 133.35 (C-7), 133.35 (C-10), 127.47 (C-8), 124.08 (C-
6), 123.83 (C-5), 119.65 (C-20–60), 117.35 (C-4a), 113.37 (C-30–50),
98.67 (C-3), 55.08 (CH3O) 43.19 (C-9), 40.71 (C-10) ppm; MS/
FAB+: m/z 371 (M+H+). HRMS (FAB+): m/z 371.1273 [M+H]+ (Calcd
for C19H20ClN4O2H+ 371.1275).
0
0
0
0
trations for compounds ranged from 8.000 lg/mL to 0.016 lg/mL.
ETH was included as positive internal standard. All evaluations
were carried out in triplicate.
4.4.4. Cytotoxicity on VERO cell line
The cytotoxicity assay was performed as reported previously,16
where 1.5 ꢀ 104 viable cells from the VERO cell line were seeded in
a 96-well plate and incubated for 24–48 h. VERO cells were grown
in DMEM media supplemented with 10% (v/v) fetal bovine serum
with 100 UI/mL penicillin and 100 mg/mL streptomycin and main-
tained at 37 °C in a 5% CO2 atmosphere with 95% humidity. When
cells reached >80% confluence, the medium was replaced and the
cells were treated with the compounds at 6.25, 12.5, 25, 50, and
4.4. Biological assays
4.4.1. In vitro giardicidal, amebicidal and trichomonicidal assay
G. intestinalis strain IMSS:0696:1 was cultured in TYI-S-33 mod-
ified medium, supplemented with 10% calf serum and bovine bile.
T. vaginalis strain GT3 and E. histolytica HM1-IMSS were cultured in
TYI-S-33 medium, supplemented with 10% bovine serum. In vitro
susceptibility assays were performed using a method previously
described.15,16 Briefly: 4 ꢀ 104 trophozoites of Giardia intestinalis
or T. vaginalis or E. histolytica were incubated for 48 h at 37 °C with
increasing concentrations of synthesized compounds, chloroquine,
9, and metronidazole. As the negative control, trophozoites were
incubated in culture medium with DMSO used in the experiments.
After the incubation, trophozoites were washed and subcultured
for another 48 h in fresh medium alone. At the end of this period,
trophozoites were counted and the 50% inhibitory concentration
(IC50) was calculated by probit analysis. Experiments were carried
out in triplicate and repeated at least twice.
100
lg/mL dissolved in DMSO at a maximum concentration of
0.05%. After 72 h of incubation, 10
lL of a 0.005% 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution
(5 mg/mL) was added to each well and incubated at 37 °C for 4 h.
The medium was removed and the formazan, a product generated
by the activity of dehydrogenases in cells, was dissolved in acidi-
fied isopropanol (0.4 N HCl). The amount of MTT-formazan is di-
rectly proportional to the number of living cells and was
determined by measuring the optical density (OD) at 540 nm using
a Bio-assay reader. Metronidazole was used as a positive control,
whereas untreated cells were used as negative controls. The con-
centration of the compound that killed 50% of the cells (CC50
)
was calculated by GraphPad Prism 4 software. All determinations
were performed in triplicate.16
4.4.2. In vitro antileishmanial and trypanocidal assay21
Acknowledgments
The growth inhibition test was performed on promastigotes of
L. mexicana (MHOM/MX/ISETGS; clinical strain originally isolated
from a patient with diffuse cutaneous leishmaniasis) and epim-
astigotes of T. cruzi (MHOM/MX/1994/Ninoa; clinical strain origi-
nally isolated from a patient with the disease in acute phase).
Parasites were cultivated at 26 °C in Schneider’s drosophila
medium, supplemented with 10% fetal bovine serum, penicillin
This work was supported in part by internal funds from Facul-
tad de Farmacia, UAEM. We are grateful to Victoria Labastida from
CIQ, UAEM for the determination of mass spectra. G.M.M.S. wishes
to thank Centro de Investigación Biomédica del Noreste, I.M.S.S. to
support the evaluation of antimycobacterial activity of compounds
in this collaborative work. We are in debt to Johnatan Cauich Chan
(100 IU/mL) and streptomycin (100 lg/mL). Biological assays were

C. Nava-Zuazo et al. / Bioorg. Med. Chem. 18 (2010) 6398–6403
6403
10. de Souza, V. N. M.; Pais, C. K.; Kaiser, R. C.; Peralta, A. M.; de L Ferreira, M.;
Lourenço, C. S. M. Bioorg. Med. Chem. 2009, 17, 1474.
from the UADY, for his technical assistance during antiprotozoal
susceptibility assays.
11. Upadhayaya, R. S.; Kulkarni, G. M.; Vasireddy, N. R.; Vandavasi, J. K.; Dixit, S. S.;
Sharma, V.; Chattopadhyaya, J. Bioorg. Med. Chem. 2009, 17, 4681.
12. Candéa, A. L. P.; Ferreira, M. L.; Pais, K. C.; Cardoso, L. N. F.; Kaiser, C. R.;
Henriques, M. G. M. O.; Lourenço, M. C. S.; Bezerra, F. A. F. M.; de Souza, M. V. N.
Bioorg. Med. Chem. 2009, 19, 6272.
13. Viegas-Junior, C.; Danuello, A.; Da Silva Bolzani, V.; Barreiro, E. J.; Fraga, C. A. M.
Curr. Med. Chem. 2007, 14, 1829.
14. Saadeh, H. A.; Mosleh, I. M.; Mubarak, M. S. Molecules 2009, 14, 1483.
15. Cedillo-Rivera, R.; Chávez, B.; González-Robles, A.; Tapia, A.; Yépez-Mulia, L. J.
Eukaryot. Microbiol. 2002, 49, 201.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.008. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
16. Hernández-Núñez, E.; Tlahuext, H.; Moo-Puc, R.; Torres-Gómez, H.; Reyes-
Martínez, R.; Cedillo-Rivera, R.; Nava-Zuazo, C.; Navarrete-Vazquez, G. Eur. J.
Med. Chem. 2009, 44, 2975.
References and notes
17. Molina-Salinas, G. M.; Ramos-Guerra, M. C.; Vargas-Villarreal, J.; Mata-
Cárdenas, B. D.; Becerril-Montes, P.; Said-Fernández, S. Arch. Med. Res. 2006,
37, 45.
18. Navarrete-Vázquez, G.; Molina-Salinas, G. M.; Duarte-Fajardo, Z. V.; Vargas-
Villarreal, J.; Estrada-Soto, S.; González-Salazar, F.; Hernández-Núñez, E.; Said-
Fernández, S. Bioorg. Med. Chem. 2007, 15, 5502.
1. Pink, R.; Hudson, A.; Marie-Annick, M.; Bendig, M. Nat. Rev. Drug Disc. 2005, 4,
727.
2. Argüello-García, R.; Cruz-Soto, M.; Romero-Montoya, L.; Ortega-Pierres, G.
Infect. Genet. Evol. 2009, 9, 1057.
3. Soares, M. B. Curr. Drug Targets 2009, 10, 176.
4. Sriram, D.; Yogeeswari, P.; Madhu, K. Bioorg. Med. Chem. Lett. 2005, 15, 4502.
5. Liav, A.; Angala, S. K.; Brennan, P. J.; Jackson, M. Bioorg. Med. Chem. Lett. 2008,
18, 2649.
19. Biava, M.; Porretta, C. G.; Poce, G.; De Logu, A.; Saddi, M.; Meleddu, R.; Manetti,
F.; De Rossi, E.; Botta, M. J. Med. Chem. 2008, 51, 3644.
20. Biava, M.; Porretta, C. G.; Poce, G.; De Logu, A.; Maleddu, R.; De Rossi, E.;
Manetti, F.; Botta, M. Eur. J. Med. Chem. 2009, 44, 4734.
21. Chan-Bacab, M. J.; Hernández-Núñez, E.; Navarrete-Vazquez, G. J. Antimicrob.
Chemother. 2009, 63, 1292.
22. Encarnacion-Dimayuga, R.; Murillo-Álvarez, J. I.; Christophersen, C.; Chan-
Bacab, M.; García Reiriz, M. L.; Zacchino, S. Nat. Prod. Commun. 2006, 1,
541.
6. Bhowruth, V.; Brown, A. K.; Reynolds, R. C.; Coxon, G. D.; Mackay, S. P.;
Minnikina, D. E.; Besra, G. S. Bioorg. Med. Chem. Lett. 2006, 16, 4743.
7. Ekoue, K. K.; Yearick, K.; Iwaniuk, D. P.; Natarajan, J. K.; Alumasa, J.; de Dios, A.
C.; Roepe, P. D.; Wolf, C. Bioorg. Med. Chem. 2009, 17, 270.
8. Kumar, A.; Srivastava, K.; Kumar, S. R.; Puri, S. K.; Chauhan, P. M. S. Bioorg. Med.
Chem. Lett. 2008, 18, 6530.
9. Faruk, K. M. O.; Levi, S. M.; Tekwani, L. B.; Wilson, H. N.; Borne, F. R. Bioorg. Med.
Chem. 2007, 15, 3919.