Synthesis and trypanocidal activity of novel 2,4,5-triaryl-N- hydroxylimidazole derivatives

Article abstract of DOI:10.3390/molecules18033445

Herein, we report the design, synthesis and trypanocidal activity of some novel trisubstituted imidazole derivatives. These heterocyclic derivatives were structurally planned by exploring the concept of molecular hybridisation between two arylhydrazones derived from megazol, which has potent trypanocidal activity. The trypanocidal activity of these triarylimidazole derivatives was evaluated against infective trypomastigote forms of T. cruzi and the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-2,4'-biimidazol- 3'-ol showed moderate biological activity (IC₅₀ = 23.9 μM) when compared to benznidazole, a standard trypanocidal drug. These compounds did not present cytotoxic effects at concentrations near the trypanocidal IC₅₀, being considered a good starting point for the development of new anti-Chagas drug candidates.

Full text of DOI:10.3390/molecules18033445

Molecules 2013, 18, 3445-3457; doi:10.3390/molecules18033445
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Trypanocidal Activity of Novel
2,4,5-Triaryl-N-Hydroxylimidazole Derivatives
Ramon Borges da Silva 1,2, Vanessa Brandão Loback 1, Kelly Salomão 3,
Solange Lisboa de Castro 3, James L. Wardell 4, Solange M. S. V. Wardell 5,
Thadeu Estevam Moreira Maramaldo Costa 1, Carmen Penido 1,
Maria das Graças Muller de Oliveira Henriques 1, Samir Aquino Carvalho 1,
Edson Ferreira da Silva 1 and Carlos Alberto Manssour Fraga 2,6,
*
1
Instituto de Tecnologia em Fármacos — Farmanguinhos, Fundação Oswaldo Cruz, 21041-250,
Rio de Janeiro, RJ, Brazil; E-Mails: ramonpan@ig.com.br (R.B.S.);
nessaloback@gmail.com (V.B.L.); thadeucosta@far.fiocruz.br (T.E.M.M.C.);
cpenido@far.fiocruz.br (C.P.); gracahen@far.fiocruz.br (M.G.M.O.H.);
scarvalho@far.fiocruz.br (S.A.C.); edsonf@far.fiocruz.br (E.F.S.)
2
3
4
5
6
Programa de Pós-Graduação em Química, Instituto de Química,
Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro, RJ, Brazil
Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-900,
Rio de Janeiro, RJ, Brazil; E-Mails: ks@ioc.fiocruz.br (K.S.); solange@ioc.fiocruz.br (S.L.C.)
Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz, 21040-900,
Rio de Janeiro, RJ, Brazil; E-Mail: j.wardell@abdn.ac.uk
CHEMSOL, 1 Harcourt Road, Aberdeen AB15 5NY, Scotland, UK;
E-Mail: solangewardell@gmail.com
Programa de Pesquisa em Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, PO Box 68023, 21941-902, Rio de Janeiro, RJ, Brazil
* Author to whom correspondence should be addressed; E-Mail: cmfraga@ccsdecania.ufrj.br;
Tel.: +55-21-2562-6447; Fax: +55-21-2562-6478.
Received: 7 February 2013; in revised form: 25 February 2013 / Accepted: 8 March 2013 /
Published: 15 March 2013
Abstract: Herein, we report the design, synthesis and trypanocidal activity of some novel
trisubstituted imidazole derivatives. These heterocyclic derivatives were structurally
planned by exploring the concept of molecular hybridisation between two arylhydrazones
derived from megazol, which has potent trypanocidal activity. The trypanocidal activity of
these triarylimidazole derivatives was evaluated against infective trypomastigote forms of

Molecules 2013, 18
T. cruzi and the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-2,4'-biimidazol-
3446
3'-ol showed moderate biological activity (IC50 = 23.9 µM) when compared to
benznidazole, a standard trypanocidal drug. These compounds did not present cytotoxic
effects at concentrations near the trypanocidal IC50, being considered a good starting point
for the development of new anti-Chagas drug candidates.
Keywords: 2,4,5-triaryl-N-hydroxyimidazole; Trypanosoma cruzi; Chagas disease;
trypanocidal activity
1. Introduction
Chagas disease, also known as American trypanosomiasis or South American trypanosomiasis, is a
protozoan disease caused by the haemoflagellate parasite Trypanosoma cruzi [1]. It is a chronic and
debilitating parasitic infection that affects millions of people in Mexico, Central America, and South
America. Approximately 25% of the population of Latin America is at risk for acquiring the infection [2].
Currently, the available drug for the clinical treatment of Chagas disease is the nitroheterocyclic drug
benznidazole [3]. This drug is effective against the circulating form of the parasite (trypomastigotes) in
the acute phase of the disease, but its efficacy during the chronic stage is debatable [4].
Megazol [1-methyl-2-(5-amino-1,3,4-thiadiazole)-5-nitroimidazole] is a nitroheterocyclic derivative
shown to be highly active against T. cruzi in vitro and in vivo, including strains that are resistant to
benznidazole [5–7]; thus, it has become a core structure for the design of new drugs for the treatment
of Chagas disease. Megazol has been described as a scavenger of trypanothione, the cofactor for
trypanothione reductase [8,9]. Despite its noteworthy trypanocidal activity, megazol development was
discontinued due to reports of its in vitro mutagenic and genotoxic effects [10–12]. To circumvent this
undesirable profile, there have been numerous efforts to obtain megazol analogues [13–17].
The imidazole ring is commonly found in highly significant endogenous biomolecules including
biotin, the essential amino acid histidine and the autacoid histamine [18]. Several bioactive compounds
with this heterocyclic unit have valuable pharmacological properties such as antiparasitic [19],
antifungal [20], antimicrobial [21,22] and antidepressant [23] activity, among others. In this context,
2,4,5-triarylimidazole compounds have gained remarkable importance due to their widespread
biological activities and their applicability in synthetic organic chemistry. Moreover, N-hydroxyimidazoles
have been reported to possess fungicidal and bacteriostatic activities [24].
In our continuous effort to develop potent trypanocidal compounds, we decided to construct a new
class of 2,4,5-triaryl-N-hydroxyimidazole (TAI) derivatives 3–12 based on the molecular hybridisation [25]
of 1,3,4-thiadiazole prototypes 1 and 2 (Figure 1) [14]. In the design concept, the nitroimidazole
moiety (A) was preserved due to the pharmacophoric contribution of this group to the mechanism of
action against T. cruzi [14]. The 1,3,4-thiadiazole group (B1) of 1 and 2 was isosterically substituted
by an imidazole ring (B2) containing a hydroxyl group that mimics the proton donor/accepting
behaviour of the tautomeric N-H bond of the hydrazone group attached to B1 (Figure 1).

Molecules 2013, 18
Figure 1. Design concept of the new triaryl-N-hydroxyimidazole derivatives 3–12.
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2. Results and Discussion
2.1. Chemistry
The synthetic route used for the preparation of the title compounds 3–12 is outlined in Scheme 1.
1,2-Dimethyl-5-nitro-1H-imidazole (13) was converted into the corresponding phenylvinyl benzoate
14 in 92% yield through its base-catalysed condensation with benzoyl chloride [26]. Next, the
nitrosation of 14 furnished the key ketoxime intermediate 15 in 80% yield [26]. Finally, the desired
TAI derivatives 3–12 were obtained, in yields varying from 20–70%, after condensation of 15 with the
1
corresponding benzaldehydes in the presence of ammonium acetate [27]. The H- and 13C-NMR
spectra and mass spectra of the synthesised compounds 3–12 were consistent with the proposed
structures, which was corroborated by X-ray crystallography of the p-bromo-TAI derivative 6 (CCDC
928948 contains the supplementary crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html) as illustrated in Figure 2.
2.2. Trypanocidal Activity
The trypanocide profiles of the new TAI derivatives 3–12 was evaluated in vitro using bloodstream
trypomastigote forms of T. cruzi (Y strain) isolated from infected Swiss mice and are summarised in
Table 1.

Molecules 2013, 18
Scheme 1. Synthesis of the new triaryl-N-hydroxyimidazole derivatives 3–12.
3448
Figure 2. Atom arrangements for 2'-(4-bromophenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-
2,4'-biimidazol-3'-ol (6).

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Table 1. Physical and biological properties of 2,4,5-triaryl-N-hydroxyimidazole derivatives 3–12.
O2N
CH3
N
OH
R1
R3
N
N
R2
N
3-12
Molecular
Yield
M.P.
(°C)
log P
Cpn
R1
R2
R3
M.W.
IC50 (µM) a
Formula
C19H15N5O3
C19H14FN5O3
(%)
69
48
20
69
62
62
71
42
45
43
-
values b
3
4
H
H
H
F
H
H
361.35
379.38
217–218
248–249
249–250
218–219
361–362
218–219
108.2 ± 5.65
67.8 ± 8.39
34.6 ± 2.43
23.9 ± 4.88
241.6 ± 37.6
191.8 ± 25.3
2.92 ± 0.98
3.14 ± 1.02
3.69 ± 0.99
3.86 ± 1.02
2.88 ± 0.99
3.09 ± 0.99
5
H
Cl
H
C19H14ClN5O3 395.80
C19H14BrN5O3 440.25
6
H
Br
H
7
H
NO2
OCH3
OH
OH
OCH3
OH
-
H
C19H14N6O5
C20H17N5O4
C19H15N5O4
C20H15N5O5
C20H17FN5O5
C20H16N6O7
-
406.35
391.38
377.35
393.35
407.38
452.38
-
8
H
H
9
H
H
145–146 294.1 ± 27.98 2.56 ± 0.99
224–225 360.5 ± 24.67 2.34 ± 0.99
10
11
12
Bzn
OH
OH
OCH3
-
H
H
213–214
245–247
-
199.9 ± 2.02
241.8 ± 7.54
10.8 ± 0.4
2.53 ± 1.00
3.22 ± 1.01
0.91 ± 1.00
NO2
-
a
Mean ± standard deviation of at least four separate experiments, performed with trypomastigote forms of
T. cruzi; b Theoretical values calculated using the program ACDLABS.
The screening of the TAI derivatives 3–12 showed that compound 6, with a p-bromophenyl group
attached to the core imidazole ring, was the most active, with an IC50 = 23.9 µM, which is slightly
inferior to that displayed by the standard drug benznidazole (IC50 = 10.8 µM). The superior
trypanocidal profile of 6, compared with the other monosubstituted TAI analogues 3–5 and 7–9, could
be explained by the remarkable hydrophobic contribution of its bromo group (Hantzsch’s  = 0.86),
increasing the log P of this compound to 3.86 (Table 1). We did not find a clear correlation among the
stereoelectronic character of para-substituents in TAI compounds 3–9 and their trypanocidal activity.
In fact, the introduction of para-substituents from different halogens, independently of any
electron-withdrawing or electron-releasing properties, prominently reduced the trypanocidal profile
when compared with an unsubstituted TAI derivative 3. Nevertheless, we have found a good
correlation (r2 = 0.74) between theoretical log P value and the trypanocide profile of monosubstituted
TAI derivatives 3–9, indicating the importance of their relative lipophilicity for the displayed
biological activity (Figure 3).
Di- and tri-substituted TAI derivatives 10–12 have shown poor trypanocidal activity, distinct from
the corresponding brazilizone derivatives, e.g., 1 [14,15].
The cellular viability in the presence of most active TAI derivatives 4–6 was determined by
microplate Alamar Blue assay (Invitrogen) at three different concentrations, 100, 10 and 1 M. The
results displayed in Table 2 were expressed in percentage cell viability. Excepting the p-bromo TAI
derivative 6, that weakly reduced cell viability at 100 M, the other tested compounds were not
cytotoxic to the host cells at concentrations near the IC50 for trypanocidal action (Table 1).

Molecules 2013, 18
Figure 3. Correlation between the theoretical log P value of TAI derivatives 3–9 and their
3450
corresponding trypanocidal activity (see Table 1).
Table 2. Data of cytotoxic effects of 2,4,5-triaryl-N-hydroxyimidazole derivatives (4–6) on
murine macrophages cells 21 h after the treatment.
TAI
% Cell Viability/concentration (M)
Compound
100
97.6
93.1
78.7
10
1
4
5
6
98.9
97.6
97.6
94.2
95.7
97.3
3. Experimental
3.1. General Procedures
Melting points were determined on a Buchi apparatus and are uncorrected. Infrared spectra were
recorded on a Thermo Nicolet Nexus 670 spectrometer in potassium bromide pellets and frequencies
are expressed in cm−1. 1H-NMR spectra were recorded at room temperature on Bruker Avance 500 and
Bruker Avance 400 spectrometers operating at 500/125 and 400/100 MHz (1H/13C), respectively.
Chemical shifts are reported in ppm () downfield from tetramethylsilane, which was used as an
internal standard. Low resolution mass spectra (MS) were obtained by electron-spray ionisation in a
micromass ZQ 4000. Microanalysis data were obtained using a Perkin–Elmer 240 analyser, using a
Perkin–Elmer AD-4 balance. The progress of all reactions was monitored by TLC, which was
performed on 2.0 X 6.0 cm aluminium sheets that were precoated with silica gel 60 (HF-254, Merck)
to a thickness of 0.25 mm. The developed chromatograms were viewed under ultraviolet light
(254–265 nm).
3.2. Procedure for the Synthesis of (Z)-2-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1-phenylvinyl benzoate
(14) [26]
In a 250 mL flask, cooled in an ice bath, acetone (34 mL) and 1,2-dimethyl-5-nitroimidazole
(13, 10 g, 70.85 mmol) were added while stirring. Next, triethylamine (50 mL, 70.85 mmol) and then

Molecules 2013, 18
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benzoyl chloride (24.4 mL, 210.0 mmol, 3 equiv.) were added slowly. The temperature was adjusted to
20 °C, and acetone (46 mL) was then added. The reaction was stirred for 4 hours. Next, water (40 mL)
was added, and the suspension was filtered on a Buchner funnel and washed with acetone, affording 25 g
1
(92% yield) of 14. Light green solid; mp 245–275 °C; H-NMR (500 MHz, DMSO-d6) δ 4.05 (s, 3H,
5-NO2-imidazole-N-CH3), 7.28 (s, 1H, 5-NO2-imidazole-CH=C-), 7.49 (m, 3H, Ar(C3)H, Ar(C4)H and
Ar(C5)H), 7.61 (t, 2H, benzoate(C3)H and benzoate(C5)H), 7.75 (t, 1H, benzoate(C4)H), 7.83 (m, 2H,
Ar(C2)H and Ar(C6)H), 7.99 (s, 1H, 5-NO2-imidazole-(C4)H), 8.13 (d, 2H, benzoate(C2)H and
benzoate(C6)H): 13C-NMR (125 MHz, DMSO-d6) δ 33.16 (5-NO2-imidazole-N-CH3), 101.90
(5-NO2-imidazole-(C2)-CH=C-), 125.39 (benzoate(C3)H and benzoate(C5)H), 128.49 (benzoate(C1)),
128.97 (Ar(C3)H and Ar(C5)H), 130.00 (Ar(C2)H and Ar(C6)H), 130.29 (benzoate(C2)H and
benzoate(C6)H), 132.59 (Ar(C1)), 133.74 (5-NO2-imidazole-(C4)H and Ar(C4)H), 133.90
benzoate-(C4)H), 138.76 (5-NO2-imidazole-(C2)CH=C-), 146.44 (5-NO2-imidazole-(C5)NO2), 152.24
(5-NO2-imidazole-(C2)CH=C-), 163.71(Ar-COO-),: MS (ESI) m/z: 372.0 (M+.[+Na]) (100%). Anal.
Calcd. for C19H15N3O4: C: 65.32; H: 4.33; N: 12.03. Found: C: 65.31; H: 4.33; N: 12.03.
3.3. Procedure for the Synthesis of (1E)-1-(1-Methyl-5-nitro-1H-imidazol-2-yl)-2-phenylethane-1,2-
dione 1-oxime (15) [26]
In small portions, NaNO2 (3.82 g, 55.36 mmol) were added to a 250 mL flask that was seeded with
H2SO4 (56.6 mL) and cooled in an ice bath while stirring. After 30 min, glacial acetic acid (20.6 mL) and
then phenylvinyl benzoate 14 (10.0 g, 28.62 mmol) were added. The reaction was maintained at 65 °C
for 3 h and the temperature was then adjusted to 20 °C. Next, water (166 mL) was added, and this
mixture was subsequently filtered in a Buchner funnel. Compound 15 was suspended in ethanol and
filtered through a Buchner funnel, resulting in a yellow solid with a yield of 80%. mp 180–182 °C;
1H-NMR (500 MHz, DMSO-d6) δ 3.78 (s, 3H, 5-NO2-imidazole-N-CH3), 7.58 (m, 2H, Ar(C3)H and
Ar(C5)H), 7.71 (t, 1H, Ar(C4)H), 7.98 (m, 2H, Ar(C2)H and Ar(C6)H), 8.21 (s, 1H, 5-NO2-imidazole-
13
(C4)H), 13.85 (s, 1H, C=N-OH): C-NMR (125 MHz, DMSO-d6) δ 34.51 (5-NO2-imidazole-N-CH3),
128.47 (Ar(C3)H and Ar(C5)H), 130.16 (Ar(C2)H and Ar(C6)H), 132.24 (5-NO2-imidazole-(C4)H),
133.53 (Ar(C4)H), 135.79 (5-NO2-imidazole-(C5)NO2), 139.45 (5-NO2-imidazole-(C2)-C=N-OH),
142.46 (Ar(C1)), 146.04 (C=N-OH), 189.07 (C=O): MS (ESI) m/z: 273.1 (100%). Anal. Calcd. for
C12H10N4O4: C: 52.56; H: 3.68; N: 20.43. Found: C: 52.54; H: 3.68; N: 20.42.
3.4. General Procedure for the Synthesis of 2,4,5-Trisubstituted Imidazole Derivatives 3–12 [27]
In a 100 mL flask, ketoxime 15 (0.5 g, 1.82 mmol, 1 eq.), NH4OAc (0.850 g, 11.02 mmol, 6 eq.),
the corresponding benzaldehyde (1.82 mmol, 1 eq.) and ethanol (12 mL) were added while stirring.
The temperature was maintained at reflux for 5 h. The reaction was cooled 20 °C and then placed on
crushed ice. The precipitate was filtered on a Buchner funnel and washed with water. All new
2,4,5-triaryl-N-hydroxyimidazole derivatives 3–12 were purified by recrystallization from a 9:1
solution of ethanol/water.
1-Methyl-5-nitro-2',5'-diphenyl-1H,3'H-2,4'-biimidazol-3'-ol (3). Yellow solid; mp 217–218 °C; yield
1
69%; H-NMR (500 MHz, DMSO-d6) δ 3.72 (5-NO2-imidazole-N-CH3), 7.28 (t, 1H, Ar(C4)H), 7.34

Molecules 2013, 18
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(t, 2H, Ar(C3)H and Ar(C5)H), 7.47 (t, 1H, Ar(C4’)H), 7.54 (t, 2H, Ar(C3’)H and Ar(C5’)H), 7.57 (d,
2H, Ar(C2)H and Ar(C6)H), 8.16 (d, 2H, Ar(C2’)H and Ar(C6’)H), 8.35 (s, 1H, 5-NO2-imidazole-
(C4)H), 12.48 (s, 1H, N-OH-imidazole-N-OH): 13C-NMR (125 MHz, DMSO-d6) δ 34.57 (5-NO2-
imidazole-N-CH3), 115.87 (N-OH-imidazole-C4), 125.89 (Ar(C2)H and Ar(C6)H), 127.39 (Ar(C2’)H
and Ar(C6’)H), 127.71 (Ar(C4)H), 128.42 (Ar(C1’)), 128.61 (Ar(C3)H and Ar(C5)H), 128.70 (Ar(C3’)H
and Ar(C5’)H), 129.33 (Ar(C4’)H), 132.75 (5-NO2-imidazole-(C4)H), 133.10 (N-OH-imidazole-C2),
137.56 (Ar(C1)), 139.97 (5-NO2-imidazole-(C2)), 141.28 (5-NO2-imidazole-(C5)NO2), 142.25
(N-OH-imidazole-C5): 13C-NMR DEPT (100 MHz, DMSO-d6)δ 34.57 (5-NO2-imidazole-N-CH3),
125.89 (Ar(C2)H and Ar(C6)H), 127.39 (Ar(C2’)H and Ar(C6’)H), 127.71 (Ar(C4)H), 128.61 (Ar(C3)H
and Ar(C5)H), 128.70 (Ar(C3’)H and Ar(C5’)H), 129.33 (Ar(C4’)H), 132.75 (5-NO2-imidazole-(C4)H):
IR (KBr) max cm−1: 3284 ( N-O-H), 3130–3064 ( C-H(aromatic), 1526–1470 ( C=C(aromatic),
1368 ( N=O2): MS (ESI) m/z: 360.5 (100%). Anal. Calcd. for C19H15N5O3: C: 63.15; H: 4.18; N:
19.38. Found: C: 63.17; H: 4.18; N: 19.40.
2'-(4-Fluorophenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (4). Yellow solid; mp
1
148–149 °C; yield 48%; H-NMR (400 MHz, DMSO-d6) δ 3.87 (5-NO2-imidazole-N-CH3), 7.27 (t,
1H, Ar(C4)H), 7.34 (t, 2H, Ar(C3’)H and Ar(C5’)H), 7.39 (d, 2H, Ar(C2’)H and Ar(C6’)H), 7.56 (d, 2H,
Ar(C2)H and Ar(C6)H), 8.19 (t, 2H, Ar(C3’)H and Ar(C5’)H), 8.34 (s, 1H, 5-NO2-imidazole-(C4)H),
12.49 (s, 1H, N-OH-imidazole-N-OH): 13C-NMR (100 MHz, DMSO-d6) δ 34.51 (5-NO2-imidazole-N-
CH3), 115.72 (d, J = 21.8 Hz, Ar(C3’)H and Ar(C5’)H), 124.95 (N-OH-imidazole-(C4)), 125.85
(Ar(C2)H and Ar(C6)H), 127.70 (Ar(C4)H), 128.57 (Ar(C3)H and Ar(C5)H), 129.63 (d, J = 8.7 Hz,
Ar(C2’)H and Ar(C6’)H), 132.70 (5-NO2-imidazole-(C4)H), 132.97 (N-OH-imidazole-(C2)), 137.49
(Ar(C1)), 139.95 (5-NO2-imidazole-(C2)), 141.17 (5-NO2-imidazole-(C5)NO2), 141.42 (N-OH-
imidazole-(C5)), 162.52 (d, J = 245.8 Hz, Ar(C4’)F): IR (KBr) max cm−1: 3284 ( N-O-H), 3130–3066
( C-H(aromatic), 1534–1468 ( C=C(aromatic), 1365 ( N=O2), 1225 ( C-F): MS (ESI) m/z: 378.2
(100%). Anal. Calcd. for C19H14FN5O3: C: 60.16; H: 3.72; N: 18.46. Found: C: 60.18; H: 3.72; N: 18.47.
2'-(4-Chlorophenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (5). green solid; mp
1
149–250 °C; yield 20%; H-NMR (500 MHz, DMSO-d6) δ 3.71 (5-NO2-imidazole-N-CH3), 7.27 (t,
1H, Ar(C4)H), 7.33 (t, 2H, Ar(C3)H and Ar(C5)H), 7.55 (d, 2H, J = 7.5 Hz, Ar(C2)H and Ar(C6)H),
7.60 (d, 2H, J = 7.5 Hz, Ar(C2’)H and Ar(C6’)H), 8.17 (d, 2H, J = 7.5 Hz, Ar(C3’)H and Ar(C5’)H),
13
8.33 (s, 1H, 5-NO2-imidazole-(C4)H), 12.58 (s, 1H, N-OH-imidazole-N-OH): C-NMR (125 MHz,
DMSO-d6) δ 34.54 (5-NO2-imidazole-N-CH3), 116.14 (N-OH-imidazole-(C4)), 125.86 (Ar(C2)H and
Ar(C6)H), 127.72 (Ar(C4)H), 128.58 (Ar(C3)H and Ar(C5)H), 128.77 (Ar(C2’)H and Ar(C6’)H), 128.86
(Ar(C3’)H and Ar(C5’)H), 132.72 (5-NO2-imidazole-(C4)H), 133.86 (N-OH-imidazole-(C2)), 137.60
(Ar(C1)), 139.94 (5-NO2-imidazole-(C2)), 141.10 (5-NO2-imidazole-(C5)NO2): IR (KBr) max cm−1:
3437 ( N-O-H), 3144–3058 ( C-H(aromatic), 1533–1470 ( C=C(aromatic), 1364 ( N=O2): MS
(ESI) m/z: 394.5 (100%). Anal. Calcd. for C19H14ClN5O3: C: 57.66; H: 3.57; N: 17.69. Found: C:
57.68; H: 3.57; N: 17.69.
2'-(4-Bromophenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (6). Yellow solid; mp
218–219 °C; yield 69%; 1H-NMR (500 MHz, DMSO-d6) δ 3.71 (s, 3H, 5-NO2-imidazole-N-CH3), 7.28

Molecules 2013, 18
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(t, 1H, Ar(C4)H), 7.33 (t, 2H, Ar(C3)H and Ar(C5)H), 7.55 (d, 2H, J = 8.0 Hz, Ar(C2)H and Ar(C6)H),
7.75 (d, 2H, J = 8.0 Hz, Ar(C2’)H and Ar(C6’)H), 8.10 (d, 2H, J = 8.0 Hz, Ar(C3’)H and Ar(C5’)H),
13
8.34 (s, 1H, 5-NO2-imidazole-(C4)H), 12.51 (s, 1H, N-OH-imidazole-N-OH): C-NMR (125 MHz,
DMSO-d6) δ 34.54 (5-NO2-imidazole-N-CH3), 116.15 (N-OH-imidazole-(C4)), 122.68 (Ar(C4’)Br),
125.84 (Ar(C2)H and Ar(C6)H), 127.54 (Ar(C1’)), 127.75 (Ar(C4)H), 128.60 (Ar(C3)H and Ar(C5)H),
129.12 (Ar(C2’)H and Ar(C6’)H), 131.73 (Ar(C3’)H and Ar(C5’)H), 132.71 (5-NO2-imidazole-(C4)H),
132.89 (N-OH-imidazole-(C2)), 137.66 (Ar(C1)), 139.97 (5-NO2-imidazole-(C2)), 141.00
(5-NO2-imidazole-(C5)NO2), 141.15 (N-OH-imidazole-(C5)): IR (KBr) max cm−1: 3140–3056
( C-H(aromatic), 1533–1470 ( C=C(aromatic), 1364 ( N=O2), 828 ( C-N (Ar-NO2),: MS (ESI)
m/z: 440.3 (100%). Anal. Calcd. for C19H14BrN5O3: C: 51.83; H: 3.21; N: 15.91. Found: C: 51.85; H:
3.21; N: 15.91.
1-Methyl-5-nitro-2'-(4-nitrophenyl)-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (7). Yellow solid; mp
1
361–362 °C; yield 62%; H-NMR (500 MHz, DMSO-d6) δ 3.73 (s, 3H, 5-NO2-imidazole-N-CH3),
7.27 (t, 1H, Ar(C4)H), 7.33 (t, 2H, Ar(C3)H and Ar(C5)H), 7.57 (d, 2H, Ar(C2)H and Ar(C6)H), 7.80
(d, 1H, Ar(C2’)H, Ar(C3’)H, Ar(C5’)H, or Ar(C6’)H), 7.92 (t, 1H, Ar(C2’)H, Ar(C3’)H, Ar(C5’)H, or
Ar(C6’)H), 7.97 (t, 1H, Ar(C2’)H, Ar(C3’)H, Ar(C5’)H, or Ar(C6’)H), 8.15(d, 1H, Ar(C2’)H, Ar(C3’)H,
Ar(C5’)H, or Ar(C6’)H), 8.33 (s, 1H, 5-NO2-imidazole-(C4)H), 12.40 (s, 1H, N-OH-imidazole-N-OH):
13C-NMR (125 MHz, DMSO-d6) δ 34.39 (5-NO2-imidazole-N-CH3), 115.50 (N-OH-imidazole-(C4)),
124.63 (Ar(C3’)H and Ar(C5’)H), 125.94 (Ar(C2)H and Ar(C6)H), 127.78 (Ar(C4)H), 128.56 (Ar(C3)H
and Ar(C5)H), 131.15 (Ar(C2’)H and Ar(C6’)H), 132.60 (5-NO2-imidazole-(C4)H), 132.79 (N-OH-
imidazole-(C2)), 133.64 (Ar(C1)), 139.49 (N-OH-imidazole-(C5)), 140.05 (5-NO2-imidazole-(C2)NO2),
140.72 (5-NO2-imidazole-(C5)NO2), 148.48 (Ar(C4’)NO2): IR (KBr) max cm−1: 3120–3033 (
C-H(aromatic), 1524–1465 ( C=C(aromatic), 1349–1365 ( N=O2), 826 ( C-N (Ar-NO2),: MS (ESI)
m/z: 405.3 (100%). Anal. Calcd. for C19H14N6O5: C: 56.16; H: 3.47; N: 20.68. Found: C: 56.15; H:
3.47; N: 20.67.
2'-(4-Methoxyphenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (8). Yellow solid; mp
1
218–219 °C; yield 62%; H-NMR (400 MHz, DMSO-d6) δ 3.73 (s, 3H, 5-NO2-imidazole-N-CH3),
3.81 (s, 3H, Ar(C4’)OCH3), 7.08 (t, 1H, J = 8.4 Hz, Ar(C2’)H, Ar(C3’)H, Ar(C5’)H or Ar(C6’)H), 7.17
(d, 1H, J = 8.4 Hz, Ar(C2’)H, Ar(C3’)H, Ar(C5’)H or Ar(C6’)H), 7.25 (t, 1H, Ar(C4)H), 7.32 (t, 2H,
Ar(C2)H, Ar(C3)H, Ar(C5)H or Ar(C6)H), 7.51 (t, 2H, Ar(C3’)H and Ar(C5’)H), 7.56 (d, 2H, J = 7.2 Hz,
Ar(C2)H and Ar(C6)H), 8.34 (s, 1H, 5-NO2-imidazole-(C4)H), 11.72 (s, 1H, N-OH-imidazole-N-OH):
13C-NMR (100 MHz, DMSO-d6) δ 34.43 (5-NO2-imidazole-N-CH3), 55.72 (Ar(C4’)OCH3), 111.77 and
120.18 (Ar(C3’)H and Ar(C5’)H), 114.71 (N-OH-imidazole-(C4)), 117.77 (Ar(C1’)), 125.82 (Ar(C2)H
and Ar(C6)H), 127.38 (Ar(C4)H), 128.43 (Ar(C3)H and Ar(C5)H), 131.37 and 131.54 (Ar(C2’)H and
Ar(C6’)H), 132.63 (5-NO2-imidazole-(C4)H), 133.35 (N-OH-imidazole-(C2)), 137.48 (Ar(C1)), 139.85
(5-NO2-imidazole-(C2)), 141.58 (5-NO2-imidazole-(C5)NO2), 141.76 (N-OH-imidazole-(C5)), 157.70
(Ar(C4’)OCH3): IR (KBr) max cm−1: 3448 ( N-O-H), 3070–3054 ( C-H(aromatic), 1471–1488
( C=C(aromatic), 1363 ( N=O2), 825 ( C-N (Ar-NO2),: MS (ESI) m/z: 390.4 (100%). Anal. Calcd.
for C20H17N5O4: C: 56.16; H: 3.47; N: 20.68. Found: C: 56.15; H: 3.47; N: 20.67.

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2'-(4-Hydroxyphenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (9). Yellow solid; mp
145–146 °C; yield 71%; 1H-NMR (400 MHz, DMSO-d6) δ 3.70 (s, 3H, 5-NO2-imidazole-N-CH3), 6.90
(d, 2H, J = 9.2 Hz, Ar(C3’)H and Ar(C5’)H), 7.25 (t, 1H, Ar(C4)H), 7.32 (t, 2H, Ar(C3)H and Ar(C5)H),
7.53 (d, 2H, J = 7.6 Hz, Ar(C2)H and Ar(C6)H), 7.97 (d, 2H, J = 8.4 Hz, Ar(C2’)H and Ar(C6’)H), 8.36
(s, 1H, 5-NO2-imidazole-(C4)H), 9.89 (s, 1H, Ar(C4’)OH), 12.21 (s, 1H, N-OH-imidazole-N-OH):
13C-NMR (100 MHz, DMSO-d6) δ 34.52 (5-NO2-imidazole-N-CH3), 115.09 (N-OH-imidazole-(C4)),
115.42 (Ar(C3’)H and Ar(C5’)H), 119.40 (Ar(C1’)), 125.79 (Ar(C2)H and Ar(C6)H), 127.51 (Ar(C4)H),
128.54 (Ar(C3)H and Ar(C5)H), 129.04 (Ar(C2’)H and Ar(C6’)H), 132.75 (5-NO2-imidazole-(C4)H),
133.26 (N-OH-imidazole-(C2)), 137.13 (Ar(C1)), 139.87 (5-NO2-imidazole-(C2)), 141.56 (5-NO2-
imidazole-(C5)NO2), 142.74 (N-OH-imidazole-(C5)), 158.50 (Ar(C4’)OH): IR (KBr) max cm−1: 3123–3062
( C-H(aromatic), 1527–1465 ( C=C(aromatic), 1283 ( N=O2), 827 ( C-N (Ar-NO2),: MS (ESI)
m/z: 376.3 (100%). Anal. Calcd. for C19H15N5O4: C: 60.47; H: 4.01; N: 18.56. Found: C: 60.49; H:
4.01; N: 18.56.
4-(3'-Hydroxy-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-2'-yl)benzene-1,2-diol (10). Yellow
solid; mp 224–225 °C; yield 42%; 1H-NMR (500 MHz, DMSO-d6) δ 3.70 (s, 3H, 5-NO2-imidazole-N-
CH3), 6.85 (d, 1H, J = 8.0 Hz, Ar(C5’)H), 7.25 (t, 1H, Ar(C4)H), 7.32 (t, 2H, Ar(C3)H and Ar(C5)H),
7.49 (dd, 1H, J = 8.5 Hz, Ar(C6’)H), 7.53 (d, 2H, Ar(C2)H and Ar(C6)H), 7.62 (d, 1H, Ar(C2’)H), 8.32
(s, 1H, 5-NO2-imidazole-(C4)H), 9.23 (s, 1H, Ar(C3’)OH or Ar(C4’)OH), 9.33 (s, 1H, Ar(C3’)OH or
Ar(C4’)OH), 12.16 (s, 1H, N-OH- midazole-N-OH): 13C-NMR (125 MHz, DMSO-d6) δ 34.54 (5-NO2-
imidazole-N-CH3), 114.87 (Ar(C2’)H), 115.09 (N-OH-imidazole-(C4)), 115.63 (Ar(C5’)H), 119.17
(Ar(C6’)H), 119.69 (Ar(C1’)), 125.80 (Ar(C2)H and Ar(C6)H), 127.49 (Ar(C4)H), 128.54 (Ar(C3)H and
Ar(C5)H), 132.77 (5-NO2-imidazole-(C4)H), 133.31 (N-OH-imidazole-(C2)), 137.05 (Ar(C1)), 139.86
(5-NO2-imidazole-(C2)), 141.61 (5-NO2-imidazole-(C5)NO2), 142.69 (N-OH-imidazole-(C5)), 145.20
Ar(C3’)OH), 146.82 Ar(C4’)OH): IR (KBr) max cm−1: 3367 ( O-H), 3124–3066 ( C-H(aromatic),
1528–1468 ( C=C(aromatic), 1365 ( N=O2), 827 ( C-N (Ar-NO2),: MS (ESI) m/z: 392.2 (100%).
Anal. Calcd. for C19H15N5O5: C: 58.01; H: 3.84; N: 17.80. Found: C: 57.09; H: 3.84; N: 17.79.
2'-(3-Hydroxy-4-methoxyphenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (11). Gray
solid; mp 213 °C; yield 45%; 1H-NMR (400 MHz, DMSO-d6) δ 3.70 (s, 3H, 5-NO2-imidazole-N-CH3),
3.82 (s, 3H, Ar(C4’)OCH3), 7.05 (d, 1H, Ar(C2)H, Ar(C5)H or Ar(C6)H), 7.26 (t, 1H, Ar(C4)H), 7.33 (t,
2H, Ar(C3)H and Ar(C5)H), 7.53 (d, 2H, Ar(C2)H and Ar(C6)H), 7.61 (d, 1H, Ar(C2)H, Ar(C5)H or
Ar(C6)H), 8.32 (s, 1H, 5-NO2-imidazole-(C4)H), 9.28 (s, 1H, N-OH-imidazole-N-OH): 13C-NMR
(125 MHz, DMSO-d6) δ 34.46 (5-NO2-imidazole-N-CH3), 56.41 (Ar(C4’)OCH3), 113.63 (Ar(C2’)H,
114.73 (N-OH-imidazole-(C4)), 116.10 (Ar(C6’)H, 118.21 (Ar(C1’), 125.91 (Ar(C2)H and Ar(C6)H),
127.46 (Ar(C4)), 128.48 (Ar(C3)H and Ar(C5)H), 132.70 (5-NO2-imidazole-(C4)H), 137.11 (Ar(C1)),
133.15 (N-OH-imidazole-(C2)), 136.53 (Ar(C5’)NO2, 137.02 (Ar(C1)), 139.74 (5-NO2-imidazole-(C2)),
140.09 (Ar(C4’)OH, 141.59 (5-NO2-imidazole-(C5)NO2), 144.67 (N-OH-imidazole-(C5)), 149.82
(Ar(C3’)OCH3: IR (KBr) max cm−1: 3368 ( N-O-H), 3130–3056 ( C-H(aromatic), 1533–1503
( C=C(aromatic), 1369 ( N=O2), 827 ( C-N (Ar-NO2),: MS (ESI) m/z: 406.4 (100%). Anal. Calcd.
for C20H17N5O5: C: 58.97; H: 4.21; N: 17.19. Found: C: 58.98; H: 4.21; N: 17.19.

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2'-(3-Hydroxy-4-methoxy-5-nitrophenyl)-1-methyl-5-nitro-5'-phenyl-1H,3'H-2,4'-biimidazol-3'-ol (12).
1
Red solid; mp 245–247 °C; yield 43%; H-NMR (400 MHz, DMSO-d6) δ 3.69 (s, 3H, 5-NO2-
imidazole-N-CH3), 3.90 (s, 3H, Ar(C3’)OCH3), 7.25 (t, 1H, Ar(C4’)OH), 7.32 (t, 2H, Ar(C3)H and
Ar(C5)H), 7.54 (d, 2H, Ar(C2)H and Ar(C6)H), 7.89 (s, 1H, 5-NO2-imidazole-(C4)H), 8.26 (d, 2H,
13
Ar(C2’)H and Ar(C6’)H): C-NMR (100 MHz, DMSO-d6) δ 34.43 (5-NO2-imidazole-N-CH3), 55.41
(Ar(C3’)OCH3), 125.90 (Ar(C2)H and Ar(C6)H), 127.46 (Ar(C4)H), 128.44 (Ar(C3)H and Ar(C5)H),
132.70 (5-NO2-imidazole-(C4)H), 137.15 (Ar(C1)), 139.74 (5-NO2-imidazole-(C2)), 140.09 (5-NO2-
imidazole-(C5)NO2), 141.59 (Ar(C1’)), 149.82 (Ar(C3’)OCH3): IR (KBr) max cm−1: 3530 ( O-H),
1555–1542 ( C=C(aromatic), 1365 ( N=O2), 827 ( C-N (Ar-NO2),: MS (ESI) m/z: 451.4 (100%).
Anal. Calcd. for C20H16N6O7: C: 53.10; H: 3.56; N: 18.58. Found: C: 52.98; H: 3.57; N: 18.55.
3.5. Activity against Bloodstream Trypomastigote Forms [28]
Stock solutions of NAH derivatives were prepared in DMSO. Bloodstream trypomastigote forms of
T. cruzi (Y strain) were isolated from infected Swiss mice and re-suspended with Dulbecco’s modified
Eagle medium plus 10% foetal calf serum (DMES) to a parasite concentration of 10 × 106 cells/mL.
This suspension (100 L) was added to the same volume of each NAH derivative, previously prepared
at twice the desired concentrations in DMES. Stock solutions of the NAH derivatives were prepared in
dimethylsulfoxide (DMSO), and they were assayed in the range of 0.5 to 2000 g/mL. The final
concentration of DMSO never exceeded 0.5%, thus, it had no deleterious effect on the parasites [29].
3.6. Cytotoxic Effect on Murine Macrophages
Cellular viability in the presence and absence of TAI derivatives (4–6) was determined using
Alamar Blue assay (Invitrogen). The macrophage cell line J774 was seeded into black clear flat-bottomed
96-well plates in a density of 2.5 × 106 cells/well. After 1 h of incubation in controlled atmosphere (5%
CO2, 37 °C),cells received fresh medium with or without Tween 20 (3%), DMSO (0.5%) and the TAI
compounds (1 to 100 M) in a quadruplicate assay. After 21 h of incubation in the previous
conditions, 20 L of Alamar Blue solution was added to each well and after 3 h, fluorescence was
measured using SpectraMax M5/M5e microplate reader (Molecular Devices; λexc = 555 nm, λem = 585 nm).
4. Conclusions
We have described herein a novel structural triarylheterocyclic template capable of displaying a
significant antitrypanosomal profile in vitro. Among these N-hydroxytriarylimidazole (TAI)
derivatives, we were able to identify the derivative 2'-(4-bromophenyl)-1-methyl-5'-phenyl-1H,3'H-
2,4'-biimidazol-3'-ol (6), which showed moderate trypanocidal activity (IC50 = 23.9 µM) when
compared to benznidazole, which is used as the standard drug. This compound did not present
cytotoxic effects at concentrations near the IC50, being considered a good starting point in the
development of new anti-Chagas drug candidates.

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Acknowledgments
The authors thank CAPES (BR), CNPq (BR), FAPERJ (BR) and FIOCRUZ (BR) for financial
support and fellowships.
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Sample Availability: Samples of the compounds are available from the authors.
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