Synthesis, trypanocidal activity and docking studies of novel quinoxaline-N-acylhydrazones, designed as cruzain inhibitors candidates

Article abstract of DOI:10.1016/j.bmc.2008.11.065

In this paper, we report the structural design, synthesis, trypanocidal activity and docking studies of novel quinoxaline-N-acylhydrazone (NAH) derivatives, planned as cruzain inhibitors candidates, a cysteine protease essential for the survival of Trypan

Full text of DOI:10.1016/j.bmc.2008.11.065

Bioorganic & Medicinal Chemistry 17 (2009) 641–652
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, trypanocidal activity and docking studies of novel
quinoxaline-N-acylhydrazones, designed as cruzain inhibitors candidates
Nelilma C. Romeiro a, Gabriela Aguirre b, Paola Hernández b, Mercedes González b, Hugo Cerecetto b,
Ignacio Aldana c, Silvia Pérez-Silanes c, Antonio Monge c, Eliezer J. Barreiro a, Lídia M. Lima a,
*
a Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro,
PO Box 68024, RJ 21944-970, Brazil
b Departamento de Química Orgánica, Facultad de Química-Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay
c Unidad en Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), Universidad de Navarra, c/Irunlarrea s/n, 31080 Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, we report the structural design, synthesis, trypanocidal activity and docking studies of
novel quinoxaline-N-acylhydrazone (NAH) derivatives, planned as cruzain inhibitors candidates, a cys-
teine protease essential for the survival of Trypanosoma cruzi within the host cell. The salicylaldehyde
N-acylhydrazones 7a and 8a presented IC50 values of the same magnitude order than the standard drug
nifurtimox (Nfx), when tested in vitro against epimastigote forms of Trypanosoma cruzi (Tulahuen 2
strain) and were non-toxic at the highest assayed doses rendering selectivity indexes (IC50 (macro-
Received 27 August 2008
Revised 20 November 2008
Accepted 24 November 2008
Available online 3 December 2008
Keywords:
Quinoxaline-N-acylhydrazone (NAH)
derivatives
phages)/IC50 (Trypanosoma cruzi)) of >25 for 7a and >20 for 8a, with IC50 values in macrophages >400 lM.
Ó 2008 Elsevier Ltd. All rights reserved.
Trypanosoma cruzi
Chagas disease
Docking
Cruzain
1. Introduction
tributed.6,7 Cruzain, the major cysteine protease in Trypanosoma
cruzi, is a lysosomal hydrolase with significant homology to
cathepsin L.8 It is expressed throughout the life cycle of Trypano-
soma cruzi and is known to be essential for the survival of the par-
asite within the host cell.8 Selective inhibitors of this protease
block the proliferation of both extracellular epimastigotes and
intracellular amastigotes, and arrest metacyclogenesis (transfor-
mation of epimastigotes to metacyclic trypomastigotes) in vitro.9
Therefore, cruzain represents an interesting target for the design
of potential trypanocidal drugs.9 The structures of current cruzain
Chagas’ disease is one of the most important medical problems
in South America. It is caused by the intracellular protozoan Try-
panosoma cruzi, which infects about 9–12 million people in Central
and South America.1
The World Bank estimated an annual loss of 2.74 million dis-
ability-adjusted life years, representing an economic loss to the en-
demic countries equivalent to US$ 6.5 billion per annum.2
The epidemiology of Chagas’ disease, first described in 1909,
still remains a challenge, given that the sylvatic transmission cycle
of the parasite occurs in a complex trophic network that includes
several mammalian species.3 The complexity of the epidemiology
is exemplified by the recently described new epidemiological fea-
tures expressed by outbreaks of human disease probably due to the
oral route described in Amazonia and Santa Catarina, a southern
area of the Atlantic Coastal Rain Forest, where no domiciliation
of triatomines had been reported up to now.4,5
inhibitors are diverse including a series of vinyl-sulfones (1),
a-
hydroxyketones (2), thiosemicarbazones (3) and 2-hydroxy-aryl-
N-acylhydrazone derivatives (4) (Chart 1).10–15
Recently, the N-acylhydrazone moiety (NAH, RCONHN@CHR)
has been described as an interesting privileged structure, being lar-
gely used in the design of new bioactive compounds with distinct
pharmacological profiles, such as anti-trypanosome (5, Chart 1 and
6, Chart 2).10,16–18
Cysteine proteases are proteolytic enzymes which depend on
the nucleophilic thiol group of a cysteine residue for their enzy-
matic activity and that are also functionally diverse and widely dis-
In this paper, we report the design, synthesis, trypanocidal
activity and docking studies of novel quinoxaline-N-acylhydrazone
(NAH) derivatives (7a–o and 8a, Chart 2), planned as cruzain inhib-
itors candidates. The design of these new derivatives was per-
formed by applying the classical bioisosterism strategy19 and
taking into consideration the role of the quinoxaline nucleus as
* Corresponding author. Tel.: +55 2125626503; fax: +55 2125626644.
E-mail address: lidia@pharma.ufrj.br (L.M. Lima).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.065

642
N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
O
O
O
H3C
N
N
H
OH
N
N
H
O
N
NH
H
S
O
O
O
(1)
O
N
(2)
OCH3
H3C
O
N
NAH
H
O
N
O
H
Cl
Cl
NH2
N
H
N
N
NAH
N
NO2
O
S
O
OH
(5)
(3)
OH
(4)
Chart 1.
imidazo[1,2-a]pyridine
quinoxaline ring
B
NAH
A
A
O
R
B
N
N
O
NAH
N
N
H
H
N
OH
N
N
N
H
H
OH
CH3
(6)
R= Ph (7a)
R=CH3 (8a)
classic bioisosterism
congenere series
Cl
Cl
Cl
F
Br
(e)
(g)
(b)
(f)
(c)
(d)
bioisosteric
replacement
OH
NO2
N(CH3)2
(k)
(j)
(h)
(i)
O
N
S
N
(o)
(l)
(n)
(m)
Chart 2.
an important biophore (scaffold), since some recently reported
derivatives with this chemotype have demonstrated interesting
trypanocidal activity.20 In addition, it also taken into account the
fact that the NAH subunit show an aza-vinylogue relationship with
the amide group present in peptides, as the primary sites of hydro-
lysis catalyzed by proteases, and that iminic double bond can work
like an hydrophobic anchor. The structural design of this new ser-
ies was accomplished by replacing the imidazo[1,2-a]pyridine nu-
cleus, present in the prototype (6), by a quinoxaline ring giving the
structures of derivatives 7a–o and 8a (Chart 2). The nature of the
para-substituent (F, Br, Cl, OH, N(CH3)2, NO2, iPr), present in the
phenyl group of subunit-B of the new compounds (7c–e, 7h–k),
was defined in order to introduce an important variation in Ham-
2. Results and discussion
2.1. Chemistry
The aforementioned compounds (7a–o and 8a) were prepared
according to synthetic process illustrated in Scheme 1. The synthe-
sis of the esters 10 and 11 was carried out by performing the con-
densation of benzofurazan oxide (14) obtained by previously
described methods,22 with ethyl acetoacetate or ethyl benzoylace-
tate, in the presence of potassium carbonate, using acetone as the
solvent, in a classical Beirut reaction. The synthesis of hydrazide
derivatives (12 and 13) was performed in good yield, exploring
the hydrazinolysis of the ethoxycarbonyl-group using hydrazine
hydrate in ethanol at reflux, in 72% and 63% yields, respectively.
Next, condensation of 12 and 13 with the appropriate aromatic
aldehydes yielded the new series of quinoxaline N-acylhydrazone
(7a–o, 8a) derivatives in excellent yields. Analysis of the 1H NMR
spectra of the new derivatives (7a–o, 8a) showed the presence of
mett0s
r
p values21 (ranging from ꢀ0.83 (N(CH3)2) to +0.78 (NO2)),
aiming at investigating any eventual electronic contribution of this
structural moiety in the trypanocidal activity. The hydrophobic
parameter
p
(ranging from ꢀ0.61 (4-OH) to +1.43 (4-iPr)) of the
aromatic substituents was also considered.21

N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
643
O
N
O
O
OCH2CH3
N
a)
O
N
O
R
N
R = Ph (
10
R = CH3 (11)
)
benzofurazan oxide (14)
b)
O
O
N
NH2
N
N
Ar
c)
N
H
N
H
H
N
R
N
R
R = Ph (
12
)
R = Ph (
7a-o)
R = CH3 (13)
R = CH3 (8a)
Ar=
OH
Cl
Cl
Cl
Br
F
(e)
(g)
(c)
(d)
(a)
(b)
(f)
N
S
O
N(CH3)2
OH
N
NO2
(h)
(j)
(k)
(o)
(i)
(n)
(l)
(m)
Scheme 1. Reagents and conditions: (a) ethyl acetoacetate or ethyl benzoylacetate; K2CO3; acetone; rt; 8 h; 32% (11) and 50% (10); (b) N2H4ꢁH2O; EtOH; reflux; overnight;
63% (13) and 72% (12); (c) Ar-CHO; EtOH; HCl (cat.); rt; 30 min.; 75–96% (7a–o, 8a).
two signals referring to the ylidenic hydrogen (N@CH), attributed
to a mixture of the E/Z-diastereomers. The assignment of (E) and
(Z) isomers was made in agreement with previous results disclosed
by Karabatsos and coworkers for the relative configuration of
hydrazones and related compounds.23,24 Moreover, we performed
a brief study of the relative energy of both possible diastereomers
terbinafine (Tbf) and ketoconazole (Ktz) as reference drugs.27,28
The results demonstrated that compounds 7a, 8a and 7o were
non-toxic at the highest assayed doses, rendering selectivity in-
Table 1
The in vitro trypanocidal activity of quinoxaline N-acylhydrazone derivatives (7a–o,
8a)
by molecular modeling, using the semiempirical AM1 method25
26
´
available in the PC SPARTAN 04 software, indicating a minor differ-
O
ence between the heat of formation values for the (E) and (Z) dia-
stereomers of all NAH-derivatives from this bioactive series (data
not shown).
N
N
Ar-W
N
H
H
N
R
2.2. Trypanocidal activities
Compound
W-Ar/R
PGIa(%)a,b,c
IC50 (l
M)c
NifurtimoxÒ
—
Ph/Ph
100
3
7.7
The primary screening data for all compounds (at 25 lM) are
n.d.d
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
15.9
n.d.
n.d.
n.d.
n.d.
n.d.
20.0
n.d.
shown in Table 1. All compounds were tested in vitro against epi-
mastigote forms of Trypanosoma cruzi, Tulahuen 2 strain. The new
quinoxaline N-acylhydrazones derivatives (7a–o, 8a) were incor-
LASSBio-1008 (7b)
LASSBio-1009 (7c)
LASSBio-1010 (7d)
LASSBio-1011 (7e)
LASSBio-1012 (7g)
LASSBio-1013 (7f)
LASSBio-1014 (7i)
LASSBio-1015 (7j)
LASSBio-1016 (7a)
LASSBio-1017 (7h)
LASSBio-1018 (7k)
LASSBio-1019 (7o)
LASSBio-1020 (7n)
LASSBio-1021 (7m)
LASSBio-1022 (8a)
LASSBio-1025 (7l)
4-F–Ph/Ph
4-Br–Ph/Ph
4-Cl–Ph/Ph
3-Cl–Ph/Ph
2-Cl–Ph/Ph
4-N(CH3)2–Ph/Ph
4-NO2–Ph/Ph
2-OH–Ph/Ph
4-OH–Ph/Ph
4-iPr–Ph/Ph
2-Thienyl/Ph
2-Furyl/Ph
2-Pyridinyl/Ph
2-OH–Ph/CH3
4-Pyridinyl/Ph
22
40
53
47
35
29
19
96
27
36
0
porated into the media at 25 lM and their ability to inhibit the par-
asite growth was evaluated in comparison to the standard drug
nifurtimox (Nfx).27 The percentage of growth inhibition was calcu-
lated and the IC50 concentration determined only for the com-
pounds with % inhibition superior to 80%.
The results shown in Table 1 demonstrate that seven, out of the
sixteen evaluated compounds, presented percentage of growth
inhibition superior to 30%, standing out the derivatives 7a and 8a
with inhibitions of 96% and 81%, respectively. These compounds
were selected and next the IC50 value determined. The quinoxaline
N-acylhydrazones presented IC50 values of the same magnitude or-
der than the standard drug Nfx. Also, unspecific cytotoxicity of the
most active derivatives 7a and 8a and one inactive compound (7o)
against mammalian cells was evaluated in vitro at 100, 200 and
0
0
81
0
a
Percentage of growth inhibition.
Inhibition of epimastigotes growth of Tulahuen 2 strain, doses = 25
The results are the means of three independent experiments with a SD less than
b
c
lM.
10% in all cases.
d
n.d.: Not determined.
400 lM, using J774 mice macrophages as the cellular model and

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N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
dexes (IC50(macrophages)/IC50(Trypanosoma cruzi))29 of >25 for 7a
and >20 for 8a, with IC50(macrophages) > 400 M. (Table 2). This
toxicity parameter, along with the trypanocidal activity, revealed
these structures presented very close
case of LASSBio-1022 (8a), (Fig. 2), whose (E)-isomer has shown
a larger Gbind value difference in comparison to its (Z)-isomer
DGbind values, except in the
l
D
quinoxalines 7a and 8a as promising lead-compounds.
(ca. ꢀ7.00 kJ/mol, Fig. 2). Interestingly, the E/Z quinone adducts
Structurally, these compounds share as a common characteris-
tic the presence of an ortho-hydroxyphenyl group linked to the
hydrazone subunit. Salicylaldehyde N-acylhydrazones (or ortho-
hydroxyarylaldehydehydrazones) are described as inhibitors of
some cysteine proteases as in the case of Plasmodium falciparum
trophozoite cysteine protease (TCP) and Trypanosoma cruzi cruz-
ain.10 Aiming to rationalize the inhibitory activity profile of salicyl-
aldehyde N-acylhydrazones towards different proteases, Ifa and
coworkers proposed a possible theoretical mechanism of interac-
tion of compound 6 (Chart 2) with cysteine proteases.30 The pro-
posed mechanism involves the nucleophilic attack, through a
Michael addition mechanism, of the sulfur atom of a negatively
charged cysteine residue on a reactive ortho-quinonemethyde
intermediate, generated from the tautomeric equilibrium of
ortho-hydroxyarylaldehydehydrazone moiety.30
of LASSBio-1016 (7a) and LASSBio-1022 (8a) have the second most
favorable
DGbind values, close to the ones observed for the (E)-iso-
mers of these compounds (Fig. 2).
Figure 3 illustrates the top docking poses obtained for the ad-
ducts and (E)-isomers of LASSBio-1016 (7a) and LASSBio-1022
(8a) with amino acid residues in the active site of cruzain
(Fig. 3A–D).
From the docking experiments, the best complex of the quinone
adduct of LASSBio-1016 (Fig. 3A) with cruzain reveals that the car-
bon atom of the amide carbonyl group of LASSBio-1016 (7a) is
within 2.69 Å of the sulfur atom of Cys25, the key nucleophilic
amino acid residue in the active site of cruzain. The nitrogen atom
in position 1 of the quinoxaline ring also establishes a hydrogen
bond with His159; the oxygen atom of the hydrazide moiety
hydrogen bonds to Gln19 and to the –NH group of the backbone
of Cys25. One of the –NH groups of the molecule establishes a
hydrogen bond with the –SH group of Cys25.
2.3. Docking studies
The visual inspection of the top pose obtained for the (E)-isomer
of LASSBio-1016 (7a) (Fig. 3B) reveals that it is able to establish a
hydrogen bond with His159 via the nitrogen atom of the imine
group. Additional hydrogen bonds involve the –NH group and the
oxygen atom of the backbone of Asp158 and the 2-OH–Ph group
and the side chain of Gln19.
The docking analysis of the quinone adduct of LASSBio-1022 (8a)
and its (E)-isomer (Fig. 3C and D) reinforces the hydrogen bonding
pattern that had been observed for LASSBio-1016 (7a), its quinone
adduct and the (E)-isomer, since these structures interact with the
same amino acid residues (Fig. 3C and D). The putative binding
modes of these compounds are reinforced by the closer proximity
of the carbon atom of the amide pertaining to the acyl hydrazone
function of the quinone adduct and the sulfur atom of Cys25, with
a distance of 2.64 Å (Fig. 3C). Moreover, the b-keto carbon atom of
In an attempt to theoretically explain the difference found in
the trypanocidal activity of the new quinoxaline N-acylhydrazone
derivatives (7a–o, 8a), docking studies using the enzyme cruzain
were performed. The ortho-quinonemethyde-intermediates, the
tautomers of compounds 7a and 8a, were also considered in these
studies.
The molecular construction and docking analysis of quinoxaline
derivatives LASSBio 1008–1025 were performed as described in
Section 3. The binding modes of quinonemethyde-like tautomeric
forms of LASSBio-1016 (7a) and LASSBio-1022 (8a), denominated
15a and 16a (Fig. 1), respectively, were also evaluated by docking
into the active site of cruzain.
The docking analysis was carried out on the crystal structures of
Trypanosoma cruzi cysteine protease cruzain, with different bound
inhibitors,31–33 using the flexible docking software FlexE.34 Since
all compounds have been synthesized as Z/E diastereomeric mix-
tures, the modeling has been based on the isomers separately. In
this study, the proposed interaction modes of the quinoxaline
derivatives into the active site of cruzain were determined as the
highest scored conformations (best-fit ligand) among 30 confor-
mational and binding modes generated according to FlexX scoring
function, which correspond to the structure with the most favor-
the
a,b-unsaturated ketone in the quinone adduct is 4.44 Å away
from Cys25 (Fig. 3C). Interestingly, the carbon atom of the carbonyl
group of the (E)-isomer of LASSBio-1022 (8a) (Fig. 3D) is closer in
space to the sulfur atom of Cys25, with a distance of 2.09 Å, when
compared to the distance observed in the top pose obtained for the
(E)-isomer of LASSBio-1016 (7a), which is 2.69 Å (Fig. 3B).
Among compounds that bear lipophilic substituents in the phe-
nyl ring, such as LASSBio-1010 (7d) LASSBio-1011 (7e), LASSBio-
1012 (7g) and LASSBio-1013 (7f), the 4-Cl–Ph derivative 7e has
able free energy of binding, that is
DGbind (kJ/mol) (Fig. 2). DG val-
ues shown in this study have been based on MMFF94 charges.35
The investigation of the top poses obtained by docking with
FlexE has shown some interesting features, although this software
does not consider covalent binding, which would be the case of
interaction between Michael acceptors and cruzain. For instance,
there is no obvious preference for binding of Z and E isomers, since
shown a good theoretical DGbind (Fig. 2). Therefore, we have inves-
tigated the putative binding modes of its E/Z isomers with cruzain
(Fig. 4A and B). The visual inspection of the top pose obtained for
the (Z)-isomer of LASSBio-1011 (7e) and cruzain shows that this
derivative establishes hydrogen bonds with Gln19, His159 and
with Cys25. The observed interatomic distance between the carbon
atom of the amide carbonyl and the sulfur atom of Cys25 is 2.75 Å,
while the distance between the b-keto carbon atom of the
a,b-
Table 2
unsaturated ketone in the quinone adduct and the same sulfur
atom is 3.00 Å. No extra van der Waals interactions were observed
with the 4-Cl–Ph group (Fig. 4A).
Cytotoxicity of quinoxaline derivatives against J-774 mouse macrophages
Pcyt.a at 400 (
l
M)b
IC50,
SIc,d
b
Compound
macrophage
Also, the visual inspection of the top pose obtained for the (E)-
isomer of LASSBio-1011 (7e) and cruzain shows that this com-
pound interacts by hydrogen bonds to Gln156 and Leu157 via
the nitrogen atom in position 1 of the quinoxaline ring and the
nitrogen atoms of the hydrazide moiety. Worth of notice is the fact
that the putative Michael acceptors in this molecule are very dis-
tant from Cys25, in a range of 10–12 Å (Fig. 4B).
Interestingly, LASSBio-1017 (7h), a quinoxaline derivative that
bears a hydroxyl group in position 4 of the phenyl ring, showed a
low predicted bioactivity. Docking studies of this compound and
7a
7o
8a
Tbf
Ktz
14
33
12
100
100
>400
>400
>400
88.0
>25.2
—
>20.0
5.1
<5.0
e
<50.0
a
Pcyt = Percentage of cytotoxicity.
b
The results are the means of two different experiments with a SD less than 10%
in all cases.
c
SI: Selectivity index.
SI = IC50,macrophage/IC50,T2,epimastigote
Not determined.
d
e
.

N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
645
HO
H
HO
O
O
N
N
N
N
N
N
N
H
H
H
N
CH3
Ph
8a (LASSBio-1022)
7a (LASSBio-1016)
O
H
O
O
H
H
O
N
N
H
N
N
N
N
H
H
N
CH3
N
Ph
16a
15a
Figure 1. Possible Michael acceptor quinone adducts (15a and 16a), tautomers of LASSBio-1016 (7a) and LASSBio-1022 (8a).
0
-5
-10
-15
-20
-25
-30
-35
Z-isomer
E-isomer
Figure 2. In silico
DGbind values (kJ/mol) obtained from docking of the quinoxaline derivatives with cruzain.
cruzain showed that, opposite to what had been observed for the
other hydroxylated derivatives, LASSBio-1016 (7a) and LASSBio-
1022 (8a), the (Z)-isomer of LASSBio-1017 (7h) presented a differ-
ent binding pose in the enzyme active site (Fig. 4C). Most remark-
able is the fact that the carbon atom of the carbonyl pertaining to
the NAH moiety and the sulfur atom of Cys25 are 4.65 Å apart,
even though this derivative is able to establish hydrogen bonds
to Ser61 Gly66, Gln156 and with the backbone oxygen of the car-
bonyl group of Leu157, which may explain its lower bioactivity in
comparison to the other ortho-hydroxylated quinoxaline deriva-
tives, since the nucleophilic species is the sulfur atom from
Cys25. In addition, the distance between the carbon atom of the
imine group and the sulfur atom of Cys25 is 7.59 Å (Fig. 4C).
Finally, the visual inspection of the top pose obtained for the
(E)-isomer of LASSBio-1017 (7h) and cruzain shows that this com-
pound establishes hydrogen bonds to Gln156 and Leu157 via the
nitrogen atom in position 1 of the quinoxaline ring and the nitro-
gen atoms of the hydrazide moiety (Fig. 4D). Again, the putative
Michael acceptors in this molecule are very distant from Cys25,
in a range of 10–12 Å, which reinforces the possible inability of this
molecule to act as a Michael acceptor for the nucleophilic –SH
group of Cys25 (Fig. 4D).
The docking results presented herein have shown a reasonable
correlation with the structural design of the quinoxaline deriva-
tives towards cruzain and orientate our studies towards the molec-
ular mechanism of action of these substances against cysteine
proteases, enzymes that are the target of a wide range of therapeu-
tically active substances, including
a
few anti-chagasic
compounds.36
2.4. Lipinski’s rule of five
In addition to ligand–protein complex modeling, in vivo absorp-
tion capabilities of the designed molecules were tentatively as-

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N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
Figure 3. Top poses obtained by docking with FlexE of (A) the E isomer of the quinone adduct of LASSBio-1016 and (B) E isomer of LASSBio-1016; (C) the E isomer of the
quinone adduct of LASSBio-1022 and (D) E isomer of LASSBio-1022, with cruzain. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for
clarity.
sessed by means of theoretical calculations following Lipinski’s
rule of five,37 that predicts that a compound administered orally
will more likely have good absorption or permeation if it satisfies
the following criteria:
soma cruzi)) of >25 for 7a and >20 for 8a, with IC50 (macro-
phages) >400 M.
The docking results presented herein have shown a reason-
able correlation with the molecular design of the quinoxaline
l
derivatives towards cruzain, since the predicted in silico
DGbind
Hydrogen bond donors 6 5 (OH and NH groups)
Hydrogen bond acceptors 6 10 (N and O atoms)
Molecular weight < 500
values obtained through docking studies have pointed towards
LASSBio-1016 (7a) and LASSBio-1022 (8a) as the most promising
inhibitors, assuming that the molecular mechanism of these
compounds involves this target. Also, these studies suggest that
low bioactivity may be due to lack of adequate distances to
the sulfur atom of Cys25 of cruzain. Finally, the application of
parameters preconized by Lipinski’s rule of five showed that all
molecules have a potential for good in vivo absorption, since
the designed compounds had zero or less than two rule
violations.
Calculated logP (ClogP) < 5
This approach has been widely used as a filter in the decision-
making of which substances should be further developed in drug
design programs. The results of the calculations for the molecules
designed in this study show that all molecules have a potential for
good in vivo absorption, since the majority of the compounds, ex-
cept for LASSBio-1010 (7d) and LASSBio-1018 (7k), that did not
satisfy only one requisite, satisfied Lipinski’s rule of five with zero
violations (Table 3).
3. Experimental
3.1. Chemistry
2.5. Conclusions
The synthesized compounds were chemically characterized by
thin layer chromatography (TLC), infrared (IR), nuclear magnetic
resonance (1H NMR), mass spectra (MS) and elemental micro-
analysis (CHN). Alugram SIL G/UV254 (Layer: 0.2 mm) (Mache-
rey-Nagel GmbH & Co. KG. Postfach 101352. D-52313 Düren,
Germany) was used for TLC and Silica gel 60 (0.040–0.063 mm)
for column flash chromatography (Merck). The 1H NMR spectra
A
new series of trypanocidal quinoxaline-N-acylhydrazone
(NAH) derivatives were discovered, outstanding the salicylalde-
hyde N-acylhydrazones 7a and 8a with IC50 of the same magni-
tude order than the standard drug nifurtimox (Nfx). These
compounds were non-toxic at the highest assayed concentration
rendering selectivity indexes (IC50(macrophages)/IC50(Trypano-

N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
647
Figure 4. Top poses obtained by docking with FlexE of (A) and (B) LASSBio-1011 and (C) and (D) LASSBio-1017, Z and E isomers, respectively, with cruzain. Hydrogen bonds
are shown as green dashed lines. Only polar hydrogens are shown for clarity.
the frequencies are expressed in cmꢀ1. The mass spectra were
measured on an Agilent Technologies Model MSD/DS 5973N
Table 3
Compliance of compounds to computational parameters of bioavailability
(mod. G2577A) mass spectrometer with direct insertion probe
(DIP) (Waldbronn, Germany) and the ionization method was
electron impact (EI, 70 eV). Elemental microanalyses were ob-
tained on an Elemental Analyzer (Leco CHN-900, Tres Cantos,
Madrid, Spain) from vacuum-dried samples. The analytical re-
sults for C, H, and N, were within 0.4 of the theoretical values.
Compound
CLogP
Molecular
weight
Hydrogen
bond
donors
Hydrogen
bond
acceptors
Satisfies
the rule
of five?
LASSBio-1008 (7b)
LASSBio-1009 (7c)
LASSBio-1010 (7d)
LASSBio-1011 (7e)
LASSBio-1012 (7g)
LASSBio-1013 (7f)
LASSBio-1014 (7i)
LASSBio-1015 (7j)
LASSBio-1016 (7a)
LASSBio-1017 (7h)
LASSBio-1018 (7k)
LASSBio-1019 (7o)
LASSBio-1020 (7n)
LASSBio-1021 (7m)
LASSBio-1022 (8a)
LASSBio-1025 (7l)
4.24
4.38
5.10
4.95
4.95
4.35
4.70
3.98
4.77
4.14
5.67
3.89
3.41
2.96
3.29
2.76
352.391
370.381
431.287
386.836
386.836
386.836
395.459
397.388
368.390
368.390
394.471
358.419
342.353
353.379
306.320
353.379
1
1
1
1
1
1
1
1
2
2
1
1
1
1
2
1
4
4
4
4
4
4
5
6
5
5
4
4
5
5
5
5
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
3.1.1. General procedure for the preparation of 2-
(Ethoxycarbonyl)-3-substituted-quinoxaline 1,4-dioxide
derivatives (10–11)
7.34 mmol of the corresponding ester (ethyl acetoacetate or
ethyl benzoylacetate) and 9.55 mmol of potassium carbonate
were added to a solution of 7.34 mmol of benzofurazan oxide
in 50 mL of acetone. The suspension was stirred at room temper-
ature for 2 h. The quinoxaline 1,4-dioxide derivatives (10 and
11) were isolated by adding 50 mL of water, followed by extrac-
tion with dichlorometane (5 ꢂ 40 mL). The organic layer was
dried (Na2SO4), filtered, and evaporated to dryness. The residue
was purified by recrystallization from a mixture of methanol/
ether/n-hexane (2:4:4).
were recorded on
a
Bruker 400 Ultrashield instrument
(400 MHz), using TMS as the internal standard and with
DMSO-d6 and CDCl3 as the solvents; the chemical shifts are re-
ported in ppm (d) and coupling constants (J) values are given
in Hertz (Hz). Signal multiplicities are represented by: s (singlet),
d (doublet), t (triplet), q (quadruplet), dd (double doublet), hp
(septuplet) and m (multiplet). The IR spectra were performed
on a Thermo Nicolet Nexus FTIR (Madison, USA) in KBr pellets;
3.1.2. 2-(Ethoxycarbonyl)-3-phenylquinoxaline 1,4-dioxide
(10)
The derivative (10) was obtained by condensation of benzofura-
zan oxide with ethyl benzoylacetate, as a yellow powder in 50%
yield.

648
N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
1H NMR (CDCl3 ): d 1.08 (t, J = 7.2 Hz, OCH2CH3); 4.25 (q,
3.1.7. General procedure for the preparation of 3-phenyl (or 3-
methyl)-quinoxaline-2-arylidenehydrazides (7a–o, 8a)
J = 7.2 Hz, OCH2CH3); 7.53 (m, H30–H50); 7.61 (m, H20 and H60);
7.91 (m, H6 and H7); 8.65 (m, H5 and H8) ppm.
An equimolar amount of appropriates aromatic aldehydes was
added to a solution of hydrazide derivatives 12 or 13 (1.05 mmol)
in 20 mL of ethanol, in the presence of catalytic amount of hydro-
chloric acid. The reaction was stirred for 0.5–1.0 h, at reflux, and
the solvent was evaporated under reduced pressure. The colored
precipitate was collected by filtration, washed with cold water
and dried under vacuum to give the desired N-acylhydrazone
derivatives (7a–o and 8a), that were purified by recrystallization
in ethanol, yielding compounds 7a–o and 8a as a mixture of E
and Z stereoisomers. in good and excellent yields. The chemical
shifts of the different isomers were tentatively assigned according
to previously reported by Karabatsos and coworkers.23,24 When
mentioned isomer Z or E this means that most probably this signal
corresponds to the first mention isomer.
13C NMR (CDCl3): d 13.98 (OCH2CH3), 63.65 (OCH2CH3), 120.89
(C5), 121.08 (C8), 127.84 (C10), 129.16 (C30 and C50), 130.15 (C20
and C60), 131.26 (C40), 132.51 (C6), 132.53 (C7), 136.53 (C2),
137.73 (C10), 138.81 (C3), 140.08 (C9), 159.66 (CO2Et) ppm.
Ir (KBr): 2978 (ArC-H), 1746 (C@O), 1352 (N-oxide), 701 and
666 (mono-substituted phenyl) cmꢀ1
.
Mass: 310 (m/z, 100%), 294 (M+, 6%), 249 (M+, 51%), 221 (M+,
46%), 77 (M+, 46%).
Anal. Calcd for C17H14N2O4: C, 65.80; H, 4.52; N, 9.03. Found: C,
65.65; H, 4.57; N, 8.98.
3.1.3. 2-(Ethoxycarbonyl)-3-methylquinoxaline 1,4-dioxide
(11)
The derivative (11) was obtained by condensation of benzofura-
zan oxide with ethylacetoacetate as a yellow powder in yield 30%.
1H NMR (400 MHz, CDCl3) d: 1.52 (t, J = 7.1 Hz, OCH2CH3); 2.61 (s,
ArCH3); 4.60 (q, J = 7.1 Hz, OCH2CH3); 7.88 (m, H6 and H7); 8.62
(m, H5 and H8).
3.1.8. (E/Z)-N0-(2-Hydroxybenzylidene)-3-phenylquinoxaline-2-
hydrazide (7a)
Derivative 7a was obtained as a white solid by condensation of
12 with 2-hydroxybenzaldehyde in 77% yield.
13C NMR (100 MHz, CDCl3) d: 14.42 (OCH2CH3); 14.81 (ArCH3);
64.09 (OCH2CH3); 120.55 (C5); 120.77 (C8); 131.90 (C6); 132.97
(C7); 136.02 (C2); 137.31 (C9); 138.30 (C10); 139.34 (C3); 160.26
(CO2CH2CH3).
1H NMR (400 MHz, DMSO-d6): d 6.67 (dd, J = 7.2 Hz and 7.6 Hz,
H500, isomer Z or E), 6.74 (d, J = 8.0 Hz, H300, isomer Z or E); 6.93
(dd, J = 8.0 Hz and 8.4 Hz, H500, isomer E or Z), 6.94 (d, J = 8.0 Hz,
H300, isomer E or Z); 7.01 (d, J = 8.0 Hz, H600, isomer E or Z), 7.14
(dd, J = 8.4 Hz and 8.0 Hz, H400, isomer E or Z), 7.33 (dd, J = 7.2 Hz
and 8.0 Hz, H400, isomer Z or E), 7.53 (m, H30–50, isomer Z or E),
7.55 (m, H30–50, isomer E or Z), 7.61 (d, J = 7.6 Hz, H600, isomer Z
or E), 7.84 (m, H20 and H60, isomer Z and E), 7.97 (m, H6 and H7,
isomer Z and E), 8.16 (s, N@CH, isomer Z or E), 8.18 (m, H5 and
H8, isomer Z or E), 8.23 (m, H5 and H8, isomer E or Z), 8.60 (s,
N@CH, isomer E or Z), 9.80 (s, ArOH), 10.88 (ArOH), 12.18 (s, CONH,
isomer Z or E), 12.56 (s, CONH, isomer E or Z) ppm.
Ir (KBr): 1732 (C@O), 1334 (N-oxide), 1224 (C–O–C) cmꢀ1
.
Anal. Calcd for C12H12N2O2: C, 66.65; H, 5.59; N, 12.95. Found: C,
66.66; H, 5.59; N, 12.95.
3.1.4. General procedure for the preparation of 3-substituted
quinoxaline-2-hydrazide derivatives (12–13)
To a solution of 1.28 mmol of ester derivative 10 or 11 in 5 mL
of ethanol, was added 2.5 mL of hydrazine monohydrate. The reac-
tion mixture was maintained under reflux for 3 h, when TLC indi-
cated the end of the reaction. Then, the media was poured on ice
and the resulting precipitate was filtered out affording the title
compounds in 60–79% yields.
Mass: 368 (m/z, 17%), 248 (M+, 29%), 206 (M+, 71%), 191 (M+,
36%), 57 (M+, 100%).
Anal. Calcd for C22H16N4O2: C, 71.73; H, 4.38; N, 15.21. Found: C,
71.75; H, 4.37; N, 15.18.
3.1.5. 3-Phenylquinoxaline-2-hydrazide (12)
3.1.9. (E/Z)-N0-Benzylidene-3-phenylquinoxaline-2-
hydrazide(7b)
Derivative 7b was obtained as a white solid by condensation of
12 with benzaldehyde in 96% yield.
The derivative (12) was obtained as a white solid in 75% yield.
1H NMR (400 MHz, DMSO-d6): d 4.64 (br, CONHNH2); 7.53 (m,
H30–H50); 7.86 (m, H20 and H60); 7.90 (m, H6 and H7); 8.17 (m,
H5 and H8); 10.00 (br, CONHNH2) ppm.
1H NMR (400 MHz, DMSO-d6): d 7.20 (m, H200 and H600, isomer Z
or E), 7.26 (m, H300–H500, isomer Z or E); 7.55 (m, H30–50, isomer Z
and E), 7.54 (m, H200 and H600, isomer E or Z), 7.75 (m, H300 and
H500, isomer E or Z), 7.83 (m, H20 and H60, isomer Z and E), 7.85
(s, N@CH, isomer Z or E), 7.99 (m, H6 and H7, isomer Z and E),
8.18 (m, H5 and H8, isomer Z or E), 8.24 (m, H5 and H8, isomer E
or Z), 8.36 (s, N@CH, isomer E or Z), 12.16 (s, CONH, isomer Z or
E), 12.36 (s, CONH, isomer E or Z) ppm.
13C NMR (100 MHz, DMSO-d6): d 129.32 (C30 and C50), 129.52
(C20 and C60), 129.62 (C40), 129.85 (C6), 130.40 (C5), 131.65 (C7),
132.32 (C8), 138.17 (C10), 140.30 (C9), 141.61 (C2), 149.57 (C10),
151.58 (C3), 166.63 (CONHNH2) ppm.
Ir (KBr): 3241 (N–H), 3010 (C@H), 1703 (C@O), 768 (monosub-
stituted phenyl) cmꢀ1
.
Mass: 264 (m/z, 67%), 248 (M+, 70%), 205 (M+, 100%), 77 (M+,
59%).
Mass: 352 (m/z, 2%), 248 (M+, 47%), 206 (M+, 100%), 77 (M+,
15%).
Anal. Calcd for C22H16N4O: C, 74.98; H, 4.58; N, 15.90. Found: C,
74.97; H, 4.57; N, 15.88.
Anal. Calcd for C15H12N4O: C, 68.17; H, 4.58; N, 21.20. Found: C,
68.15; H, 4.57; N, 20.21.
3.1.6. 3-Methylquinoxaline-2-hydrazide (13)
The derivative (13) was obtained as a brown solid in 63% yield.
1H NMR (400 MHz, DMSO-d6 ): d 2.54 (ArCH3), 4.59 (br, CON-
HNH2); 7.88 (m, H6 and H7), 8.08 (m, H5 and H8); 9.95 (br,
CONHNH2) ppm.
´
3.1.10. (E/Z)-N-(4-Fluorobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7c)
Derivative 7c was obtained as a white solid by condensation of
12 with 4-fluorobenzaldehyde in 96% yield.
13C NMR (100 MHz, DMSO-d6 ): d 23.36 (ArCH3), 129.06 (C6),
129.75 (C5), 130.74 (C7), 132.01 (C8), 139.70 (C9), 142.35 (C2),
148.58 (C10), 151.98 (C3), 165.65 (CONHNH2) ppm.
Mass: 202 (m/z, 21%), 171 (M+, 5%), 143 (M+, 100%), 90 (M+,
23%).
1H NMR (400 MHz, DMSO-d6): d 7.11 (t, J = 8.8 Hz, H300 and H500,
isomer Z or E), 7.25 (t, J = 8.8 Hz, H200 and H600, isomer E or Z);
7.31 (d, J = 8.4 Hz, H300 and H500, isomer Z or E), 7.48 (m, H30–50, iso-
mer Z or E), 7.53 (m, H30–H50, isomer E or Z), 7.80 (m, H2’ and H60,
isomer Z and E; and H200 and H600, isomer E or Z), 7.85 (s, N@CH, iso-
mer Z or E), 7.98 (m, H6 and H7, isomer Z and E), 8.21 (m, H5 and
H8, isomer Z or E), 8.23 (m, H5 and H8, isomer E or Z), 8.36 (s,
Anal. Calcd for C10H10N4O: C, 59.40; H, 4.98; N, 27.71. Found: C,
59.39; H, 4.98; N, 27.68.

N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
649
N@CH, isomer E or Z), 12.17 (s, CONH, isomer Z or E), 12.38 (s,
CONH, isomer E or Z) ppm.
1H NMR (400 MHz, DMSO-d6): d 7.15 (d, J = 7.6 Hz, H400, isomer Z
or E), 7.22 (s, H600, isomer Z or E), 7.30 (dd, J = 7.6 Hz and 8.0 Hz,
H300, isomer Z or E); 7.35 (d, J = 8.0 Hz, H400, isomer Z or E), 7.49
(m, H30–50, isomer Z or E; H600, isomer E or Z), 7.53 (m, H30–H50, iso-
mer E or Z; H300, isomer E or Z), 7.72 (d, J = 8.0 Hz, H200, isomer Z or
E), 7.80 (m, H20 and H60, isomer Z or E), 7.82 (m, H20 and H60, isomer
E or Z, H200, isomer E or Z), 7.83 (s, N@CH, isomer Z or E), 7.98 (m,
H6 and H7, isomer Z and E), 8.20 (m, H5 and H8, isomer Z or E), 8.24
(m, H5 and H8, isomer E or Z), 8.37 (s, N@CH, isomer E or Z), 12.29
(s, CONH, isomer Z or E), 12.49 (s, CONH, isomer E or Z) ppm.
Mass: 386 (m/z, 2%), 248 (M+, 44%), 234 (M+, 21%), 205 (M+,
100%), 77 (M+, 17%).
Mass: 370 (m/z, 1%), 248 (M+, 51%), 205 (M+, 100%), 77 (M+, 13%).
Anal. Calcd for C22H15N4OF: C, 71.34; H, 4.08; N, 15.13. Found:
C, 71.35; H, 4.08; N, 15.15.
3.1.11. (E/Z)-N0-(4-Bromobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7d)
Derivative 7d was obtained as a white solid by condensation of
12 with 4-bromobenzaldehyde in 89% yield.
1H NMR (400 MHz, DMSO-d6): d 7.13 (d, J = 8.4 Hz, H200 and H600,
isomer Z or E), 7.47 (d, J = 8.4 Hz, H200 and H600, isomer E or Z); 7.48
(m, H30–50, isomer Z or E), 7.53 (m, H30–H50, isomer E or Z), 7.67 (d,
J = 8.4 Hz, H300 and H500, isomer Z or E), 7.70 (d, J = 8.4 Hz, H300 and
H500, isomer E or Z), 7.78 (m, H20 and H60, isomer Z or E), 7.82 (s,
N@CH, isomer Z or E), 7.83 (m, H20 and H60, isomer E or Z), 7.98
(m, H6 and H7, isomer Z and E), 8.19 (m, H5 and H8, isomer Z or
E), 8.23 (m, H5 and H8, isomer E or Z), 8.34 (s, N@CH, isomer E
or Z), 12.22 (s, CONH, isomer Z or E), 12.44 (s, CONH, isomer E or
Z) ppm.
Anal. Calcd for C22H15N4OCl: C, 68.31; H, 3.91; N, 14.48. Found:
C, 68.31; H, 3.90; N, 14.48.
3.1.15. (E/Z)-N0-(4-Hydroxybenzylidene)-3-phenylquinoxaline-
2-hydrazide (7h)
Derivative 7h was obtained as a white solid by condensation of
12 with 4-hydroxybenzaldehyde in 90% yield.
1H NMR (400 MHz, DMSO-d6): d 6.63 (d, J = 8.4 Hz, H300 and H500,
isomer Z or E), 6.84 (d, J = 8.4 Hz, H300 and H500, isomer E or Z); 7.02
(d, J = 8.4 Hz, H200 and H600, isomer Z or E), 7.45 (m, H30–50, isomer Z
or E), 7.51 (m, H30–H50, isomer E or Z), 7.59 (d, J = 8.4 Hz, H200 and
H600, isomer E or Z), 7.74 (s, N@CH, isomer Z or E), 7.79 (m, H20
and H60, isomer Z and E), 7.94 (m, H6 and H7, isomer Z and E),
8.15 (m, H5 and H8, isomer Z or E), 8.18 (m, H5 and H8, isomer E
or Z), 8.20 (s, N@CH, isomer E or Z), 11.92 (s, CONH, isomer Z or
E), 12.20 (s, CONH, isomer E or Z) ppm.
Mass: 431 (m/z, 2%), 248 (M+, 59%), 205 (M+, 100%), 77 (M+, 35%).
Anal. Calcd for C22H15N4OBr: C, 61.27; H, 3.51; N, 12.99. Found:
C, 61.30; H, 3.52; N, 12.96.
3.1.12. (E/Z)-N0-(4-Chlorobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7e)
Derivative 7e was obtained as a white solid by condensation of
12 with 4-chlorobenzaldehyde in 96% yield.
1H NMR (400 MHz, DMSO-d6): d 7.21 (d, J = 8.4 Hz, H300 and H500,
isomer Z or E), 7.34 (d, J = 8.4 Hz, H300 and H500, isomer E or Z); 7.48
(m, H30–50, isomer Z or E), 7.54 (m, H30–H50, isomer E or Z; H20 and
H60, isomer Z or E), 7.77 (d, J = 8.4 Hz, H200 and H600, isomer Z or E),
7.79 (m, H20 and H60, isomer E or Z), 7.82 (d, J = 8.4 Hz, H200 and H600,
isomer E or Z), 7.84 (s, N@CH, isomer Z or E), 7.99 (m, H6 and H7,
isomer Z and E), 8.19 (m, H5 and H8, isomer Z or E), 8.23 (m, H5 and
H8, isomer E or Z), 8.36 (s, N@CH, isomer E or Z), 12.22 (s, CONH,
isomer Z or E), 12.43 (s, CONH, isomer E or Z) ppm.
Mass: 386 (m/z, 2%), 248 (M+, 51%), 234 (M+, 23%), 205 (M+,
100%), 77 (M+, 10%).
Mass: 368 (m/z, 25%), 191 (M+, 51%), 108 (M+, 87%), 57 (M+,
100%).
Anal. Calcd for C22H16N4O2: C, 71.73; H, 4.38; N, 15.21. Found: C,
71.70; H, 4.40; N, 15.19.
3.1.16. (E/Z)-N0-(4-Dimethylaminobenzylidene)-3-phenylqui-
noxaline-2-hydrazide(7i)
Derivative 7i was obtained as a yellow solid by condensation of
12 with 4-dimethylaminobenzaldehyde in 84% yield.
1H NMR (400 MHz, DMSO-d6): d 2.85 (s, N(CH3)2, isomer Z or E),
2.97 (s, N(CH3)2, isomer E or Z), 6.54 (d, J = 8.8 Hz, H300 and H500, iso-
mer Z or E), 6.75 (d, J = 8.8 Hz, H300 and H500, isomer E or Z); 7.00 (d,
J = 8.8 Hz, H200 and H600, isomer Z or E), 7.48 (m, H30–50, isomer Z or
E), 7.53 (m, H30–H50, isomer E or Z), 7.55 (d, J = 8.4 Hz, H200 and H600,
isomer E or Z), 7.70 (s, N@CH, isomer Z or E), 7.81 (m, H20 and H60,
isomer Z or E), 7.84 (m, H20 and H60, isomer E or Z), 7.98 (m, H6 and
H7, isomer Z and E), 8.18 (m, H5 and H8, isomer Z or E), 8.19 (s,
N@CH, isomer E or Z), 8.22 (m, H5 and H8, isomer E or Z), 11.86
(s, CONH, isomer Z or E), 12.05 (s, CONH, isomer E or Z) ppm.
Mass: 395 (m/z, 67%), 248 (M+, 22%), 205 (M+, 87%), 146 (M+,
100%), 133 (M+, 87%), 77 (M+, 48%).
Anal. Calcd for C22H15N4OCl: C, 68.31; H, 3.91; N, 14.48. Found:
C, 68.30; H, 3.88; N, 14.46.
3.1.13. (E/Z)-N0-(2-Chlorobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7f)
Derivative 7f was obtained as a white solid by condensation of 4
with 2-chlorobenzaldehyde in 89% yield.
1H NMR (400 MHz, DMSO-d6): d 7.12 (d, J = 8.0 Hz, H300, isomer Z
or E), 7.20 (dd, J = 7.6 Hz and 7.2 Hz, H500, isomer Z or E); 7.31 (dd,
J = 8.0 Hz and 7.6 Hz, H400, isomer Z or E), 7.41 (d, J = 8.0 Hz, H300,
isomer E or Z), 7.45 (dd, J = 7.6 Hz and 8.0 Hz, H500, isomer E or
Z); 7.49 (m, H30–50, isomer Z or E), 7.55 (m, H30–H50, isomer E or
Z; H400, isomer E or Z), 7.84 (m, H20 and H60, isomer Z and E), 7.94
(d, J = 7.2 Hz, H600, isomer Z or E), 7.96 (m, H6 and H7, isomer Z
and E), 8.19 (d, J = 7.2 Hz, H600, isomer E or Z), 8.25 (s, N@CH, isomer
Z or E), 8.26 (m, H5 and H8, isomer Z and E), 8.79 (s, N@CH, isomer
E or Z), 12.34 (s, CONH, isomer Z or E), 12.64 (s, CONH, isomer E or
Z) ppm.
Anal. Calcd for C24H21N5O: C, 72.89; H, 5.35; N, 17.71. Found: C,
72.90; H, 5.35; N, 17.68.
3.1.17. (E/Z)-N0-(4-Nitrobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7j)
Derivative 7j was obtained as a yellow solid by condensation of
12 with 4-nitrobenzaldehyde in 93% yield.
1H NMR (400 MHz, DMSO-d6): d 7.46 (m, H30–50, isomer Z or E;
H200 and H600, isomer Z or E), 7.54 (m, H30–H50, isomer E or Z),
7.81 (m, H20 and H60, isomer Z or E), 7.83 (m, H20 and H60, isomer
E or Z); 7.97 (s, N@CH, isomer Z or E), 7.99 (m, H20 and H60, isomer E
and Z), 8.02 (d, J = 8.8 Hz, H200 and H600, isomer E or Z), 8.13 (d,
J = 8.8 Hz, H300 and H500, isomer Z or E), 8.20 (m, H5 and H8, isomer
Z or E), 8.24 (m, H5 and H8, isomer E or Z), 8.32 (d, J = 8.8 Hz, H300
and H500, isomer E or Z), 8.49 (s, N@CH, isomer E or Z), 12.46 (s,
CONH, isomer Z or E), 12.67 (s, CONH, isomer E or Z) ppm.
Mass: 397 (m/z, 2%), 248 (M+, 31%), 205 (M+, 100%), 77 (M+, 26%).
Mass: 386 (m/z, 1%), 248 (M+, 46%), 234 (M+, 20%), 206 (M+,
100%), 77 (M+, 19%).
Anal. Calcd for C22H15N4OCl: C, 68.31; H, 3.91; N, 14.48. Found:
C, 68.29; H, 3.89; N, 14.48.
3.1.14. (E/Z)-N0-(3-Chlorobenzylidene)-3-phenylquinoxaline-2-
hydrazide (7g)
Derivative 7g was obtained as a white solid by condensation of
12 with 3-chlorobenzaldehyde in quantitative yield.

650
N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
Anal. Calcd for C22H15N5O3: C, 66.49; H, 3.80; N, 17.62. Found: C,
66.50; H, 3.82; N, 17.65.
isomer Z or E), 6.98 (d, J = 3.6 Hz, H300, isomer Z or E), 7.49 (m, H30–
H50, isomer Z or E), 7.53 (m, H30–H50, isomer E or Z), 7.62 (d,
J = 4.4 Hz, H500, isomer E or Z), 7.76 (s, N@CH, isomer Z or E), 7.78
(m, H20 and H60, isomer Z and E), 7.83 (m, H20 and H60, isomer E
and Z), 7.91 (d, J = 3.6 Hz, H300, isomer E or Z), 7.97 (m, H6 and
H7, isomer Z and E), 8.21 (m, H5 and H8, isomer Z or E), 8.23 (m,
H5 and H8, isomer E or Z), 8.24 (s, N@CH, isomer E or Z), 12.10
(s, CONH, isomer Z or E), 12.34 (s, CONH, isomer E or Z) ppm.
Mass: 342 (m/z, 1%), 248 (M+, 44%), 205 (M+, 100%), 77 (M+,
29%).
3.1.18. (E/Z)-N0-(4-Isopropylbenzylidene)-3-phenylquinoxaline-
2-hydrazide (7k)
Derivative 7k was obtained as a white solid by condensation of
12 with 4-isopropylbenzaldehyde in 84% yield.
1H NMR (400 MHz, DMSO-d6): d 1.11 (d, J = 6.8 Hz, RCH(CH3)2,
isomer Z and E), 1.22 (d, J = 6.8 Hz, RCH(CH3)2, isomer E and Z),
2.80 (hp, J = 6.8 Hz, RCH(CH3)2, isomer
Z and E), 2.93 (hp,
J = 6.8 Hz, RCH(CH3)2, isomer E and Z), 7.11 (d, J = 8.4 Hz, H300 and
H500, isomer Z or E), 7.14 (d, J = 8.4 Hz, H200 and H600, isomer Z or
E); 7.35 (d, J = 8.0 Hz, H300 and H500, isomer E or Z), 7.49 (m, H30–
50, isomer Z or E), 7.54 (m, H30–H50, isomer E or Z), 7.67 (d,
J = 8.0 Hz, H200 and H600, isomer E or Z), 7.82 (m, H20 and H60, isomer
Z and E), 7.83 (s, N@CH, isomer Z or E), 7.98 (m, H6 and H7, isomer
Z and E), 8.19 (m, H5 and H8, isomer Z or E), 8.23 (m, H5 and H8,
isomer E or Z), 8.32 (s, N@CH, isomer E or Z), 12.11 (s, CONH, iso-
mer Z or E), 12.29 (s, CONH, isomer E or Z) ppm.
Anal. Calcd for C20H14N4O2: C, 70.17; H, 4.12; N, 16.37. Found: C,
70.19; H, 4.10; N, 16.38.
3.1.22. (E/Z)-N0-(2-Thenylidene)-3-phenylquinoxaline-2-
hydrazide (7o)
Derivative 7o was obtained as a beige solid by condensation of
12 with thiophene-2-carboxaldehyde in 92% yield.
1H NMR (400 MHz, DMSO-d6): d 6.97 (t, J = 4.0 Hz, H400, isomer Z
or E), 7.16 (t, J = 4.0 Hz, H400, isomer E or Z), 7.23 (d, J = 3.2 Hz, H500,
isomer Z or E), 7.41 (d, J = 4.8 Hz, H300, isomer Z or E), 7.48 (m, H30–
H50, isomer Z or E), 7.50 (d, J = 3.2 Hz, H500, isomer E or Z), 7.53 (m,
H30–H50, isomer E or Z), 7.71 (d, J = 4.8 Hz, H300, isomer E or Z), 7.81
(m, H20 and H60, isomer Z and E), 7.94 (m, H6 and H7, isomer Z or
E), 7.96 (m, H6 and H7, isomer E or Z), 7.98 (s, N@CH, isomer Z or E),
8.19 (m, H5 and H8, isomer Z or E), 8.22 (m, H5 and H8, isomer E or
Z), 8.57 (s, N@CH, isomer E or Z), 12.11 (s, CONH, isomer Z or E),
12.34 (s, CONH, isomer E or Z) ppm.
Mass: 394 (m/z, 1%), 239 (M+, 57%), 206 (M+, 100%), 77 (M+, 34%).
Anal. Calcd for C25H22N4O: C, 76.12; H, 5.62; N, 14.20. Found: C,
76.13; H, 5.65; N, 14.23.
3.1.19. (E/Z)-N0-(Pyridin-4-ylmethylene)-3-phenylquinoxaline-
2-hydrazide (7l)
Derivative 7l was obtained as a white solid by condensation of
12 with pyridine-4-carboxaldehyde in 90% yield.
1H NMR (400 MHz, DMSO-d6): d 7.11 (d, J = 5.6 Hz, H200 and H600,
isomer Z or E), 7.47 (m, H30–50, isomer Z or E), 7.52 (m, H30–H50, iso-
mer E or Z), 7.69 (d, J = 5.6 Hz, H200 and H600, isomer E or Z), 7.78 (m,
H20 and H60, isomer Z or E), 7.82 (m, H20 and H60, isomer E or Z), 7.83
(s, N@CH, isomer Z or E), 8.0 (m, H6 and H7, isomer Z and E), 8.20 (m,
H5 and H8, isomer Z or E), 8.23 (m, H5 and H8, isomer E or Z), 8.37 (s,
N@CH, isomer E or Z), 8.45 (d, J = 5.6 Hz, H300 and H500, isomer Z or E),
8.66 (d, J = 5.6 Hz, H300 and H500, isomer E or Z), 12.65 (br, CONH, iso-
mer Z or E), 12.68 (br, CONH, isomer E or Z) ppm.
Mass: 358 (m/z, 2%), 248 (M+, 19%), 206 (M+, 100%), 77 (M+,
23%).
Anal. Calcd for C20H14N4OS: C, 67.02; H, 3.94; N, 15.63. Found:
C, 67.03; H, 3.95; N, 15.65.
3.1.23. (E/Z)-N0-(2-Hydroxybenzylidene)-3-methylquinoxaline-
2-hydrazide (8a)
Derivative 8a was obtained as a yellow solid by condensation of
13 with 2-hydroxybenzaldehyde in 90% yield.
Mass: 353 (m/z, 1%), 248 (M+, 29%), 234 (M+, 37%), 206 (M+,
1H NMR (400 MHz, DMSO-d6): d 2.72 (s, ArCH3, isomer Z or E),
2.89 (s, ArCH3, isomer E or Z), 6.68 (dd, J = 7.2 Hz and 7.6 Hz, H500,
isomer Z or E), 6.75 (d, J = 8.0 Hz, H300, isomer Z or E); 6.94 (dd,
J = 7.2 Hz and 6.8 Hz, H500, isomer E or Z), 6.95 (d, J = 8.8 Hz, H300,
isomer E or Z); 7.05 (d, J = 7.6 Hz, H600, isomer Z or E), 7.14 (dd,
J = 7.2 Hz and 7.6 Hz, H400, isomer Z or E), 7.32 (dd, J = 7.2 Hz and
8.8 Hz, H400, isomer E or Z), 7.58 (d, J = 6.8 Hz, H600, isomer E or Z),
7.93 (m, H6 and H7, isomer Z or E), 8.08 (d, J = 7.6 Hz, H5, isomer
Z and E), 8.18 (d, J = 7.6 Hz, H8, isomer Z and E), 8.4 (s, N@CH, iso-
mer Z or E), 8.74 (s, N@CH, isomer E or Z), 11.07 (s, ArOH, isomer Z
or E), 11.16 (ArOH, isomer E or Z), 12.39 (s, CONH, isomer Z or E),
12.51 (s, CONH, isomer E or Z) ppm.
100%), 77 (M+, 17%).
Anal. Calcd for C21H15N5O: C, 71.38; H, 4.28; N, 19.82. Found: C,
71.35; H, 4.31; N, 19.78.
3.1.20. (E/Z)-N0-(Pyridin-2-ylmethylene)-3-phenylquinoxaline-
2-hydrazide (7m)
Derivative 7m was obtained as a beige solid by condensation of
12 with pyridine-2-carboxaldehyde in 75% yield.
1H NMR (400 MHz, DMSO-d6): d 7.10 (d, J = 7.6 Hz, H600, isomer Z
or E), 7.28 (dd, J = 6.4 Hz and 6.0 Hz, H400, isomer Z or E), 7.48 (m,
H30–50, isomer Z or E; H600, isomer E or Z), 7.54 (m, H30–H50, isomer
E or Z), 7.65 (dd, J = 7.6 Hz and 8.0 Hz, H400, isomer E or Z), 7.78 (m,
H20 and H60, isomer Z or E), 7.82 (m, H20 and H60, isomer E or Z),
7.90 (s, N@CH, isomer Z or E), 7.92 (m, H500, isomer Z and E), 7.99
(m, H6 and H7, isomer Z and E), 8.20 (d, J = 4.0 Hz, H300, isomer Z
or E), 8.22 (m, H5 and H8, isomer Z and E), 8.41 (s, N@CH, isomer
E or Z), 8.47 (d, J = 4.0 Hz, H300, isomer E or Z), 12.40 (s, CONH, iso-
mer Z or E), 12.70 (s, CONH, isomer E or Z) ppm.
Anal. Calcd for C17H14N4O2: C, 66.66; H, 4.61; N, 18.29. Found: C,
66.67; H, 4.59; N, 18.32.
3.2. Trypanocidal activities
Epimastigote forms of Trypanosoma cruzi, Tulahuen 2 strain,
were grown in axenic BHI-Tryptose media complemented with
5% fetal bovine serum. The compounds dissolved in DMSO were
Mass: 353 (m/z, 1%), 248 (M+, 34%), 234 (M+, 29%), 206 (M+,
100%), 77 (M+, 15%).
incorporated into the media at 25 lM and their ability to inhibit
Anal. Calcd for C21H15N5O: C, 71.38; H, 4.28; N, 19.82. Found: C,
71.37; H, 4.28; N, 19.80.
the parasite growth was evaluated in comparison to the control
(no drug added). No effect on epimastigotes growth was observed
by the presence of up to 1% DMSO in the culture media. Nifurtimox
(Nfx) was used as the reference trypanocidal drug. Growth of the
parasite was followed by measuring the increase in absorbance
at 600 nm, which had previously been shown to be proportional
to the number of cells present. The percentage of inhibition was
calculated as follows: % = {1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢂ 100, where
Ap is A600 of the culture containing the drug at day 5; A0p is A600 of
3.1.21. (E/Z)-N0-(Furfurylidene)-3-phenylquinoxaline-2-
hydrazide (7n)
Derivative 7n was obtained as a beige solid by condensation of
12 with 2-furaldehyde in 89% yield.
1H NMR (400 MHz, DMSO-d6): d 6.47 (t, J = 4.0 Hz, H400, isomer Z
or E), 6.66 (t, J = 4.0 Hz, H400, isomer E or Z), 6.67 (d, J = 4.4 Hz, H500,

N. C. Romeiro et al. / Bioorg. Med. Chem. 17 (2009) 641–652
651
the culture containing the drug right after addition of the inocula
(day 0); Ac is A600 of the culture in the absence of any drug (control)
at day 5; A0c is A600 in the absence of the drug at day 0. The IC50
(50% inhibitory concentration) was determined only for com-
pounds presenting high trypanocidal activity at the screening con-
indicators, etc.), the location of hydrogen atoms at hetero atoms,
and the definition of the active site atoms. The assignment of
hydrogen positions has been made on the basis of default rules ex-
cept for the definition of the hydrogen positions inside the histi-
dine side-chain. The side-chains of lysine, arginine and the
carboxylate groups of aspartic and glutamic acid have been mod-
eled in their ionized states. Water molecules contained in the
PDB file have been removed.
centration (25 l
M).27
3.3. Cytotoxicity to mice macrophages
J-774 mouse macrophages were seeded (100.000 cells/well) in
3.5. Lipinski’s rule of five
96 well flat bottom microplates (Nunclon) with 200 lL of RPMI
1640 medium supplemented with 20% heat inactivated foetal
calf serum. Cells were allowed to attach for 48 h in a humidi-
fied 5% CO2/95% air atmosphere at 37 °C. Then, cells were ex-
posed to derivatives 7a and 8a compounds (100, 200, and
CLOGP, the log of the octanol/water partition coefficient, was
calculated with the Clogp software41,42; Molecular Weight calcula-
tions have been performed with the MOLPROP facility, all available
in SYBYL 7.3.38
400 lM) for 48 h. Afterwards, the cells were washed with PBS
and incubated (37 °C) with 3-[4,5-dimethylthiazol-2-yl]-2,5-di-
Acknowledgements
phenyl tetrazolium bromide (MTT; Sigma) 0.4 mg/mL for 3 h.
Then, formazan was dissolved with DMSO (180
l
L) and optical
The authors wish to thank financial support from IM-INOFAR
(CNPq-BR #420.015/05-1), PROSUL (CNPq-BR # 490.600/2007-8),
FAPERJ (BR), and the ‘Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior’ (CAPES; BR) for the fellowship (to L.M.L.;
BEX0520/04-7) received.
densities were measured. Each concentration was assayed three
times and six growth controls were used in each test. Cytotox-
icity percentages (%C) were determined as follows:
%C = [100 ꢀ (ODd ꢀ ODdm)/(ODc ꢀ ODcm)] ꢂ 100, where ODd is
the mean of OD595 of wells with macrophages and different
concentrations of the compounds; ODdm is the mean of
OD595 of wells with different compounds concentration in the
medium; ODc is the growth control and ODcm is the mean of
OD595 of wells with medium only.28,29
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3.4.1. General procedures
All calculations have been performed on a PC running under Li-
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tions were performed using SYBYL 7.3.38
3.4.2. Preparation of the ligands
The preparation of the ligands for FLEXE was performed as for
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generated using the program Sketcher, available in SYBYL version
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´
25
point charges were assigned to the ligands.
3.4.3. Selection of protein crystal structures
Ligand-bound crystallographic structures of cruzain are avail-
able in the Protein Data Bank.40 In this study, 1EWL, 1F2C, 1ME3,
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recognition site of the ensemble has been defined as the collection
of residues within 10.0 Å of the bound inhibitor and comprised the
union of all ligands of the ensemble. All atoms located less than
10.0 Å from any ligand atom were considered. 1ME4 was used as
a reference structure for the united protein preparation. All carbox-
ylic acid and amino groups were modeled in their ionized forms.
Proteins were prepared for the docking studies using the Biopoly-
mer module of SYBYL 7.3. Amber7 FF9938 charges were attributed to
the protein atoms.
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process of analysis and correction of geometry parameters. For
each structure, the description of an ensemble contains the defini-
tion of the protein atoms (via chain identifiers and hetero groups),
the resolution of ambiguities in the PDB file (alternate location

652
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