Fluorine-containing benzothiazole as a novel trypanocidal agent: Design, in silico study, synthesis and activity evaluation

Article abstract of DOI:10.1007/s00044-015-1475-9

Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas disease. In the bloodstream of humans, this parasite is in the trypomastigote stage and can infect host cells. Its metabolism is dependent on the glycolysis pathway, and one enzyme important fo

Full text of DOI:10.1007/s00044-015-1475-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1475-9
ORIGINAL RESEARCH
Fluorine-containing benzothiazole as a novel trypanocidal agent:
design, in silico study, synthesis and activity evaluation
1
1
2
•
Roberto I. Cuevas-Hern a´ ndez
•
Jos e´ Correa-Basurto
•
C e´ sar A. Flores-Sandoval
3
4
4
•
Itzia I. Padilla-Mart ´ı nez
•
Benjam ´ı n Nogueda-Torres
•
Mar ´ı a de Lourdes Villa-Tanaca
1
1
1
Feliciano Tamay-Cach
•
Juan J. Nolasco-Fidencio
•
Jos e´ G. Trujillo-Ferrara
Received: 19 May 2015 / Accepted: 26 September 2015
Ó Springer Science+Business Media New York 2015
Abstract Trypanosoma cruzi (T. cruzi) is the etiologic
agent of Chagas disease. In the bloodstream of humans,
this parasite is in the trypomastigote stage and can infect
host cells. Its metabolism is dependent on the glycolysis
pathway, and one enzyme important for the optimal func-
tioning of this metabolic pathway is triosephosphate iso-
merase (TIM). The significant difference (48 %) between
the interfacial residues of TIMs from humans and try-
panosomes and the importance of these residues for the
stability of T. cruzi TIM (TcTIM) make this enzyme a
possible therapeutic target. In the present study, 204 ben-
zazole derivatives were designed as TcTIM inhibitors,
including some well-known ligands. Compounds were
analyzed with docking simulations and a QSAR study, and
their molecular physicochemical properties were calcu-
lated. The five compounds selected from in silico screening
were later synthesized and assayed in vitro for their
trypanocidal activity on a resistant strain of T. cruzi. All
compounds showed affinity for the aromatic cluster of the
TcTIM interface, with important electrostatic, hydrophobic
and p–p interactions. The benzothiazole derivatives had
better physicochemical attributes than the currently pre-
scribed drug, benznidazol. Although BT1and BT2 showed
toxicity problems, BT3 did not. The QSAR study indicates
that the inhibition of TcTIM improves when the com-
pounds (especially benzothiazoles) are substituted with
hyper-conjugated systems and there is a sulfur atom in their
structure. It was found with in vitro assays that compound
BT3 is a better trypanocidal agent than the currently used
drug on the market for the treatment of Chagas disease,
benznidazol.
Keywords Anti-trypanosomal drug Á
Docking simulation Á Fluorine-containing benzothiazole Á
T. cruzi Á Triosephosphate isomerase Á
Trypanocidal activity Á
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1475-9) contains supplementary
material, which is available to authorized users.
Quantitative structure–activity relationship
Feliciano Tamay-Cach
dr.felicianotamay@gmail.com
&
&
Roberto I. Cuevas-Hern a´ ndez
rcuevash@ipn.mx
Juan J. Nolasco-Fidencio
jesusprestige@hotmail.com
Jos e´ G. Trujillo-Ferrara
jtrujillo@ipn.mx
Jos e´ Correa-Basurto
corrjose@gmail.com
1
Departamento de Bioqu ´ı mica, Laboratorio de Modelado
molecular y Bioinform a´ tica, Escuela Superior de Medicina,
Instituto Polit e´ cnico Nacional, 11340 Mexico, D.F., Mexico
C e´ sar A. Flores-Sandoval
caflores@imp.mx
2
Instituto Mexicano del Petr o´ leo, 07730 Mexico, D.F., Mexico
Itzia I. Padilla-Mart ´ı nez
ipadillamar@ipn.mx
3
Unidad Profesional Interdisciplinaria de Biotecnolog ´ı a,
Instituto Polit e´ cnico Nacional, 07340 Mexico, D.F., Mexico
Benjam ´ı n Nogueda-Torres
bnogueda@hotmail.com
4
Departamento de Parasitolog ´ı a y Departamento de
Microbiolog ´ı a, Escuela Nacional de Ciencias Biol o´ gicas,
Instituto Polit e´ cnico Nacional, 11340 Mexico, D.F., Mexico
Mar ´ı a de Lourdes Villa-Tanaca
loudesvilla@hotmail.com
123

Med Chem Res
Introduction
related compounds were first evaluated with in silico tools
in order to establish a model structure. QSAR was assessed
with molecular modeling, physicochemical descriptors and
docking simulations. Possible toxicity was also analyzed
using Web databases. Taking into account this in silico
screening, a new molecule was designed with the features
needed to inhibit TcTIM, while another two were formed
in order to validate our model. In the end, five compounds
among the series were synthesized and tested in vitro for
possible trypanocidal activity.
Chagas disease is a type of trypanosomiasis caused by the
protozoan parasite Trypanosoma cruzi (T. cruzi). It con-
stitutes the greatest threat to public health in the parts of
Latin America that suffer from widespread poverty. T.
cruzi proliferates in communities where houses have dirt
floors and adobe walls. This parasite progressively invades
human cells and lyses them in order to multiply and spread,
and as a result causes chronic incurable complications such
as Chagasic cardiomyopathy as well as damage to the
gastrointestinal tract and peripheral neurological lesions
(
Rassi Jr. et al., 2010; Marin et al., 2007). An estimated 8
Materials and methods
Computational studies
million people are infected with T. cruzi in Latin America,
the Caribbean and the Southern United States. Due to the
increasing mobility of people in the world, this parasite has
now spread to other continents (WHO, 2015), and today
more than 100 million people are considered to be at risk
for this disease (OPS, 2006). A parasite related to T. cruzi
has been found in Africa (causing sleeping sickness) and
Asia (causing human Asian trypanosomiasis).
The structures of target compounds were fully optimized at
the B3LYP/6-31G(d) level, implemented in Gaussian 03
software (Frisch et al., 2003). Some QSAR descriptors
included in Hyperchem software were taken into account
from optimized structures. The training model was built
using stepwise subroutine multiple lineal regression (MLR)
The drugs currently available for treating this disease are
ineffective or have serious collateral effects. One strategy
for drug design is based on targeting the metabolism of T.
cruzi, which obtains its energy from the glycolysis path-
way. The key to this strategy is the identification of
enzymes that are distinct between T. cruzi and humans and
therefore can be targeted to selectively inhibit the parasite
without side effects for patients (Verlinde et al., 2001). One
such enzyme is triosephosphate isomerase (TIM), which
catalyzes the interconversion of dihydroxyacetone phos-
phate (DHAP) into R-glyceraldehyde-3-phosphate (G3P) in
parasites and humans (Knowles, 1991), leading to energy
from glycolysis.
(
(
Xu et al., 2012) of the SPSS for Windows version 13.0.
SPSS, 1999). The optimization of the linear models was
determined by considering the t-statistical models (with all
absolute values [2.0 accepted) as well as the overall F-
statistical test. We also performed Akaike’s Information
Criterion (AIC) (Akaike, 1974; Guha and Jurs, 2005).
Docking simulations were carried out by using Auto-
Dock 4.0.1 (Morris et al., 1998) and AutoDock tools 1.5.4
(Sanner, 1999). The specific conditions of interaction
between the ligands and the biological receptor (TcTIM)
were established by taking the crystallographic coordinates
of the Protein Data Bank. Before docking, water molecules
were removed from TcTIM and hydrogen atoms were
added to the polar atoms, considering a *7.4 pH value and
taking into account the Kollman charges for all atoms in
the receptor. The other parameters were left at their default
values. Finally, the whole protein was minimized in the
Nanoscale Molecular Dynamics (NAMD 2.6) program
using the CHARMM27 force field, and water molecules
were added using the Visual Molecular Dynamics (VMD
v.1.8.6) program (Phillips et al., 2005; Humphrey et al.,
TIM from T. cruzi (TcTIM) is a well-known dimeric
enzyme (Banner et al., 1975; Lolis and Petsko, 1990; Mal-
donado et al., 1998)whose crystal structure is available in the
protein data bank (PDB) with the code 1TCD (http://www.
rcsb.org/pdb/home/home.do). Several studies have shown
the structure–activity relationship of TIM (Zomosa et al.,
2
003; Olivares et al., 2007), as well as the significant dif-
ference of 48 % between theinterfacial residuesof TIM from
humans and parasites (Garza et al., 1998; Saab et al., 2011).
Recently, it has been demonstrated that a good strategy for
drug design is to seek inhibitors of the aromatic cluster of
residues present in the TcTIM dimer interface (as well as in
other TIMs from distinct parasite species) (Espinoza and
Trujillo, 2004, 2005, 2006; Espinoza et al., 2010; Alvarez
et al., 2010; Romo et al., 2011; Flores et al., 2013).
1996).
To identify binding modes, all the possible rotat-
able bonds, torsion angles, atomic partial charges, and
merge non-polar hydrogens of the ligands were assigned.
Binding modes were then docked on the interdimeric
˚
enzyme surface, using a grid box of 60 9 100 9 60 A, a
The aim of the present study was to rationally design a
series of 204 molecules as TcTIM inhibitors, which
included some well-known ligands. These structurally
˚
grid spacing of 0.375 A, and the following grid center:
X = 13.341, Y = 50.553 and Z = 55.738. The hybrid
Lamarckian Genetic Algorithm was used, with an initial
1
23

Med Chem Res
population of 100 randomly placed individuals and a
maximum number of energy evaluations of 107. All other
parameters were left at their default settings. The resulting
The appropriate 2-aminothiophenol was mixed with a
substituted benzaldehyde (both previously dissolved in
5 mL of DMSO anh.) and an equimolar amount of Na2-
S O . DMSO was then added to attain 30 mL of mixture,
docked orientations within a root-mean-square deviation of
˚
2
5
0
.5 A were clustered together, and the lowest energy
which was stirred at reflux at *120 °C for 40 min. The
desired compound was monitored by TLC analysis (using
Ethyl acetate as eluent). The mixture was cooled to room
temperature by adding cool water, and the resulting pre-
cipitate was collected by vacuum filtration. The filtrate was
then washed with an excess of water and left to dry. The
resulting powder was dissolved in CH Cl (70 mL), and
cluster returned by AutoDock for each compound was used
for further analysis. All ligand–enzyme complexes were
visualized by VMD v.1.8.6 and Discovery Studio 4.0
(
Dassault Syst e` mes, 2015) programs.
Validation parameters
2
2
the remaining traces of sodium metabisulfite were extrac-
ted with brine (70 mL) and solvent removed under vac-
uum. The resulting product was purified and recrystallized
in ethanol/water (1:3) with activated carbon to obtain
mostly white needles.
One of the most commonly applied internal validation
techniques is the leave-one-out cross-validation (LOO-
CV). The measurements connected to internal validation by
2
LOO-CV (q ) are defined as follows:
cv
ꢀ
ꢁ
P
2
n
obs
predcv
yi À yi
Chemical characterization
i¼1
2
cv
q
¼ 1 À
ð1Þ
Pn
2
obs
obs
i
ðyi À yꢀ Þ
i¼1
The reactions were monitored by TLC and all synthesized
1
compounds were characterized by
where y oi bs is the experimental (observed) value of the
predcv
property for the ith compound; yi
13
H, C NMR
is the predicted value
(300 MHz) spectra on a Jeol GSX-300 spectrometer using
DMSO-d6 as solvent and TMS as internal reference.
for the temporary excluded (cross-validated) ith
obs
compound; yꢀ i is the mean experimental value of the
Chemical shift values (d ) are in parts per million (ppm),
ax
property in the training set; and n is the number of
compounds in the training set. Cross-validation provides a
reasonable approximation of the ability of QSAR to predict
the activity values of new compounds. However, external
validation gives the ultimate proof of the true predictability
of a model. In this sense, to achieve a better external
and coupling constants (J values) are in Hertz (Hz). ESI–
MS were recorded on a Bruker micrOTOF-Q II. Infrared
spectra (IR) were obtained with a MIDAC M2000 FT-IR
spectrophotometer using KBr pellets. The uncorrected
melting points were obtained in open-ended capillary tubes
in an Electrothermal 9300 digital apparatus.
predictive potential of the model, a modified r2
was
mðtestÞ
2
-phenyl-1,3-benzothiazole
14.62 mmol) and benzaldehyde (14.96 mmol) were reac-
ted to give a compound as white needles, 77 % yield,
(BT1) 2-aminothiophenol
introduced with the following equation (Roy and Roy,
008; Roy et al., 2009):
(
2
ꢂ
where r is the squared correlation coefficient between
ꢃ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
-
1
2
2
2
2
0
mp = 95–96 °C; IR (KBr, cm ) m: 3064 (C-HAr), 1432
r
¼ r  1 À r À r
ð2Þ
mðtestÞ
1
(C=N), 684 (C-S); H NMR: d 7.49 (H-7), 7.56 (H-12,
2
H-13, H-14, H-8), 8.06 (H-6), 8.08 (H-11, H15), 8.1 (H-9);
1
3
2
0
C NMR: d 167.99 (C-2), 154.23 (C-4), 135.12 (C-5),
33.52 (C-10), 132.13.42 (C-13), 130.10 (C-12, C-14),
observed and predicted values, and r is the squared cor-
1
relation coefficient between the observed and predicted
values with the intercept set at zero. The value of r2
127.88 (C-11, C-15), 127.37 (C-7), 126.26 (C-8), 123.58
?
C-6), 123.06 (C-9). m/z (ESI) 212.05 [M ].
mðtestÞ
(
should be greater than 0.5 to be acceptable. Moreover, this
value may be used for selection of the best predictive
models among comparable models.
4
-(1,3-benzothiazol-2-yl)benzoic acid (BT2) 2-Aminoth-
iophenol (2.6 mmol) and 4-Formylbenzoic acid (2.8 mmol)
were reacted to give a compound as a white powder,
-
1
General procedure for synthesis of benzothiazole
and benzoxazole derivatives
6
3 % yield, mp = 332 °C; IR (KBr, cm ) m: 3059 (C-
1
HAr), 1684 (COOH), 1425 (C=N), 689 (C-S); H NMR:
d 7.49 (H-7, dd, J = 11.8), 7.55 (H-8, dd, J = 12), 8.07
Five compounds were synthesized, including three ben-
zothiazoles (BT1, BT2 and BT3) obtained by modifying
the reaction conditions described elsewhere (2011)
(
H-6, s), 8.10 (H-12, H15, s), 8.18 (H-11, H-14, s), 8.20
1
3
(H-9, s); C NMR: d 167.36 (C-16), 166.85 (C-2),
1
1
1
2
54.18 (C-4), 137.01 (C-5), 135.42 (C-10), 133.81 (C-13),
30.96 (C-11, C-15), 128.0 (C-12, C-14), 127.58 (C-8),
26.67 (C-7), 123.87 (C-6), 123.22 (C-9); m/z (ESI)
(Scheme 1) and two benzoxazoles (Cfad3 and Cfad5,
according to a previously described method) (Trujillo
?
et al., 2004).
54.0341 [M ].
123

Med Chem Res
Scheme 1 Synthesis pathway
for benzothiazole derivatives
O
1
2
1
2
R
R
NH2
SH
R
R
N
S
Na S O
2
2
5
3
+
R
DMSO
~120 °C
3
R
1
2
3
R = R = R = H
1
2
3
R = R = H, R = COOH
1
2
3
R = CF
3
, R = H, R = COOH
4
-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl]benzoic acid
the peak of parasitemia, bloodstream trypomastigotes
were obtained by cardiac puncture from male NIH mice.
(
BT3) 2-Amino-4-(trifluoromethyl)benzenethiol (7.31
mmol) and 4-Formylbenzoic acid (7.48 mmol) were reac-
ted to give a compound as a white flaky crystal, 51 %
5
195 lL of blood with 2 9 10 trypomastigotes was
placed in each well of sterile 96-well plates. Five
microliters of one of the concentrations of compounds
(Cfad5, Cfad3, BT1, BT2 or BT3) was added to obtain
a final concentration (5, 10, 50 or 100 lmol/mL). The
final concentration of DMSO in the culture medium
-
1
yield, mp = 258–260 °C; IR (KBr, cm ) m: 3068 (C-
HAr), 1686 (COOH), 1141 (C-F ), 691 (C-S); H NMR: d
1
3
7
.81 (H-7, d, J = 8.4), 8.12 (H-11, H-15 J = 8.4), 8.23
(
H-12, H-14 J = 8.4), 8.45 (H-6, d, J = 7.8), 8.45 (H-9,
1
3
s), 13.35 (H-17); C NMR: d 167.36 (C-17), 166.85 (C-2),
remained below 1 %. A solution of DMSO/H O (1:99)
2
1
1
1
3
54.18 (C-4), 137.01 (C-10), 135.42 (C-5), 133.81 (C-13),
30.96 (C-11, C-15), 128.0 (C-12, C-14), 127.58 (C-8),
26.67 (C-7), 123.87 (C-6), 123.22 (C-9), m/z (ESI)
was used as the negative control and 12.5 lmol/mL
crystal violet as the positive control (100 % lysis). The
plates were incubated at 4 °C for 24 h. Bloodstream
trypomastigotes were counted in a Neubauer chamber.
The trypanocidal effect was determined by comparing
the remaining trypomastigotes in each concentration with
the negative control group. Stock solutions (10 lmol/mL)
of each test compound and reference drugs were pre-
pared in DMSO, and subsequent dilutions were made
with sterile distilled water. Each assay was performed in
triplicate.
?
22.0214 [M ].
(
2E)-3-(1,3-benzoxazol-2-yl)prop-2-enoic acid (Cfad5)
Brown crystals, 70 % yield, mp = 227–229 °C; IR (KBr,
-
1
1
cm ): m 1710 (COOH), 1527 (C=N); H NMR: d 6.92 (H-
1
1, d, Jtrans = 15.8), 7.45 (H-10, d, Jtrans = 15.9), 7.77 (H-
7
, d, J = 8.6), 7.52 (H-6, d, J = 8.6), 7.44 (H-5, d,
o
o
1
3
Jo = 8.6) 7.84 (H-4, br); C NMR: d 166.0 (C-12), 159.9
C-2), 150.1 (C-8), 141.4 (C-9), 129.3 (C-11), 128.2 (C-
0), 126.9 (C-6), 125.2 (C-5), 120.5 (C-4), 111.1 (C-7); m/
(
1
?
z (ESI) 190.0411 [M ].
Results and discussion
(
2E)-3-(6-methyl-1,3-benzoxazol-2-yl)prop-2-enoic
acid
(
Cfad3) Light brown powder, 42 % yield, mp =
-
Molecular modeling and docking studies
1
2
38–239 °C; IR (KBr, cm ) m: 1710 (COOH), 1544
1
(
C=N); H NMR: d 2.44 (H-4, s), 6.83 (H-11, d,
Jtrans = 15.9), 7.37 (H-10, d, Jtrans = 15.9), 7.67 (H-7, d,
Two hundred and four potential ligands were designed
based on the lead compound, benzazole, with the aim of
exploring different electronic effects. Derivatives were
formed by substituting the benzazole ring at position C6 or
C7 with functional groups commonly found in organic
molecules. These compounds were divided into three
families according to the expected electronic effects. In the
benzazole ring there were compounds with N–NH (benz-
imidazoles), N–S (benzothiazoles) and N–O (benzoxa-
zoles). The electronic effects were evaluated with the
Hammett constants (rq,m) in order to establish whether a
given derivative is electron withdrawing or donating
(Hansch et al., 1991). These derivatives were also substi-
tuted at the C2 position to elongate the chain (including
unsaturated systems in cis and trans configurations) in
order to observe lipophilic and steric effects (Table 1). All
1
3
J = 8.0), 7.23 (H-5, d, J = 8.0), 7.5 (H-4, br); C NMR:
o
o
d 166.5 (C-12), 129.1 (C-10), 128.7 (C-11), 159.8 (C-2),
50.8 (C-9), 137.8 (C-8), 120.3 (C-7), 127.0 (C-6), 139.7
1
?
C-5), 111.5 (C-4); m/z (ESI) 204.0594 [M ].
(
In vitro susceptibility assays
For this study, a T. cruzi strain, INC-5, was isolated from
chronic Chagas patients of an endemic area of Mexico. The
T. cruzi stock strains were routinely maintained in tri-
atomine bugs (Meccus longipennis) and periodically pas-
sed through albino mice.
The methodology employed was carried out according
to the protocol reported elsewhere (D ´ı az et al., 2011). At
1
23

Med Chem Res
Table 1 Chemical structure of benzazole derivatives
Heteroatom in the azole ring (X = Family)
Substituent at C2 (Y = series)
Aromatic ring substituent at C6 or C7
1
R
2
a
r
R
A=NH (benzimidazole)
bad = –(CH
2
)
3
CO
2
H
1 = H
= H
3 = H
NH
OH
2
-0.66
-0.37
-0.17
2
fad = –(CH)
2
CO
2
H
CH
3
(
E) configuration
4
= H
CH
H
2
CH
3
-0.15
B=S (benzothiazole)
C=O (benzoxazole)
mad = –(CH)
2
CO
2
H
5 = H
0.00
(
Z) configuration
6
= H
F
0.06
0.23
pad = –(CH
2
)
2
CO
2
H
7 = H
Cl
8
9
1
1
1
1
1
1
1
1
= H
COOH
0.45
= H
NO
H
2
0.78
0 = NH
1 = OH
2
-0.16
0.12
H
2 = CH
3 = CH
4 = F
3
2
H
-0.07
-0.07
0.34
CH
3
H
H
5 = Cl
H
0.37
6 = COOH
7 = NO
H
0.37
2
H
0.71
Each derivative is formed from the lead compound and involves a substitution at position 1 with heteroatom NH (A), S (B) or O (C) to give a
total of three benzazole families (benzimidazole, benzothiazole and benzoxazole). A substitution was then made at the C2 position with the
fragments derived from butanoic acid (bad), propionic acid (pad), fumaric acid (fad) and maleic acid (mad) to give a total of four series for each
of the three families, resulting in 12 series. For each series, there was a substitution at position C6 or C7 to give a total of 17 combinations,
resulting in a total of 204 combinations. For example, compound Bfad12 corresponds to family B (benzothiazole), the fad series (a fumaric acid
1
2
moiety at the C2 position) and 12 corresponds to a substitution at R = CH
a
3
and R = H
p m
r or r = Hammett constants
compounds were geometrically optimized at the B3LYP/6-
Molecular physicochemical properties
3
1G(d) level.
The target compounds were docked on the interface of the
Although docking simulations provide a powerful tool for
selecting molecules with a certain structure, it is necessary
to consider other theoretical tools to estimate pharmaco-
logical activity. By using programs available on line,
important pharmacological properties can be taken into
account, including absorption, bioavailability, permeabil-
ity, blood–brain barrier penetration, metabolism and
excretion. All of these properties should be taken into
account to achieve greater affinity for the biological
receptor, lower toxicity and better scores according to
two monomers of the enzyme. According to the in silico results,
compound Bbad9 of the family of benzothiazoles showed the
greatest affinity for TcTIM, with a DG = -5.25 kcal/mol. The
binding energies for ligand–enzyme complexes for each of the
families of benzazoles are shown in Tables 2, 3 and 4. The
compounds with the highest affinity for TcTIM are those
containing electron-withdrawing groups on the benzazole ring.
These results are consistent with the high electron density
present in the interfacial region of TcTIM.
123

Med Chem Res
Table 2 DG values in Kcal/mol for the benzimidazole derivatives
Compound
DG
Compound
DG
Compound
DG
Compound
DG
Abad1
Abad2
Abad3
Abad4
Abad5
Abad6
Abad7
Abad8
Abad9
Abad10
Abad11
Abad12
Abad13
Abad14
Abad15
Abad16
Abad17
-4.80
-4.74
-4.40
-4.55
-4.26
-4.01
-4.62
-4.83
-5.18
-4.64
-4.61
-4.52
-4.53
-4.15
-4.46
-4.65
-4.86
Amad1
Amad2
Amad3
Amad4
Amad5
Amad6
Amad7
Amad8
Amad9
Amad10
Amad11
Amad12
Amad13
Amad14
Amad15
Amad16
Amad17
-4.19
-4.50
-3.77
-4.45
-3.62
-3.40
-3.96
-4.30
-4.64
-4.67
-4.01
-4.28
-4.32
-4.11
-3.84
-3.91
-4.28
Afad1
Afad2
Afad3
Afad4
Afad5
Afad6
Afad7
Afad8
Afad9
Afad10
Afad11
Afad12
Afad13
Afad14
Afad15
Afad16
Afad17
-4.24
-4.11
-3.97
-3.89
-3.80
-3.75
-4.00
-3.50
-3.73
-4.15
-4.13
-3.99
-3.9
Apad1
Apad2
Apad3
Apad4
Apad5
Apad6
Apad7
Apad8
Apad9
Apad10
Apad11
Apad12
Apad13
Apad14
Apad15
Apad16
Apad17
-4.10
-4.25
-4.61
-4.59
-3.74
-3.63
-4.68
-3.77
-4.22
-4.00
-4.44
-4.06
-4.72
-3.65
-4.11
-4.00
-4.05
-3.74
-4.02
-3.65
-3.84
Table 3 DG values in Kcal/mol for the benzothiazole derivatives
Compound
DG
Compound
DG
Compound
DG
Compound
DG
Bbad1
Bbad2
Bbad3
Bbad4
Bbad5
Bbad6
Bbad7
Bbad8
Bbad9
Bbad10
Bbad11
Bbad12
Bbad13
Bbad14
Bbad15
Bbad16
Bbad17
-5.01
-5.00
-4.58
-4.58
-4.44
-4.31
-4.61
-4.7
Bmad1
Bmad2
Bmad3
Bmad4
Bmad5
Bmad6
Bmad7
Bmad8
Bmad9
Bmad10
Bmad11
Bmad12
Bmad13
Bmad14
Bmad15
Bmad16
Bmad17
-4.64
-4.42
-4.10
-4.12
-3.91
-3.86
-4.1
Bfad1
Bfad2
Bfad3
Bfad4
Bfad5
Bfad6
Bfad7
Bfad8
Bfad9
Bfad10
Bfad11
Bfad12
Bfad13
Bfad14
Bfad15
Bfad16
Bfad17
-4.58
-4.27
-4.32
-4.29
-4.15
-4.11
-4.37
-3.78
-3.91
-4.52
-4.48
-4.29
-4.28
-4.57
-5.00
-3.77
-4.2
Bpad1
Bpad2
Bpad3
Bpad4
Bpad5
Bpad6
Bpad7
Bpad8
Bpad9
Bpad10
Bpad11
Bpad12
Bpad13
Bpad14
Bpad15
Bpad16
Bpad17
-4.75
-4.53
-4.6
-4.64
-4.36
-4.33
-4.68
-4.16
-4.38
-4.8
-4.71
-4.82
-4.54
-4.33
-4.12
-4.28
-3.9
-5.25
-5.07
-4.82
-4.66
-4.67
-4.35
-4.7
-4.7
-4.64
-4.65
-4.27
-4.75
-4.68
-4.99
-4.17
-4.88
-4.69
-4.94
-5.23
Lipinski’s Rules (Lipinski et al., 2001) and the contribution
of Veber et al., (2002).
TcTIM (Alvarez et al., 2010; Flores et al., 2013). The
descriptors obtained with molinspiration cheminformatics
(2015) and OSIRIS Property Explorer (Organic Chemistry
Portal, 2015) were the number of hydrogen bond acceptors
(Nhba) and hydrogen bond donors (Nhbd), the number of
These molecular physicochemical properties were cal-
culated for all ligands under study, particularly for the six
compounds reported elsewhere as possible inhibitors of
1
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Med Chem Res
Table 4 DG values in Kcal/mol for the benzoxazole derivatives
Compound
DG
Compound
DG
Compound
DG
Compound
DG
Cbad1
-4.7
Cmad1
Cmad2
Cmad3
Cmad4
Cmad5
Cmad6
Cmad7
Cmad8
Cmad9
Cmad10
Cmad11
Cmad12
Cmad13
Cmad14
Cmad15
Cmad16
Cmad17
-4.71
-4.5
Cfad1
Cfad2
Cfad3
Cfad4
Cfad5
Cfad6
Cfad7
Cfad8
Cfad9
Cfad10
Cfad11
Cfad12
Cfad13
Cfad14
Cfad15
Cfad16
Cfad17
-4.38
-4.26
-4.03
-4.06
-3.92
-3.83
-4.08
-3.59
-4.01
-4.26
-4.14
-4.11
-4.06
-3.88
-4.16
-3.56
-3.75
Cpad1
Cpad2
Cpad3
Cpad4
Cpad5
Cpad6
Cpad7
Cpad8
Cpad9
Cpad10
Cpad11
Cpad12
Cpad13
Cpad14
Cpad15
Cpad16
Cpad17
-4.46
-4.44
-4.35
-4.42
-4.1
Cbad2
Cbad3
Cbad4
Cbad5
Cbad6
Cbad7
Cbad8
Cbad9
Cbad10
Cbad11
Cbad12
Cbad13
Cbad14
Cbad15
Cbad16
Cbad17
-4.6
-4.38
-4.37
-4.18
-4.11
-4.42
-4.70
-4.98
-4.79
-4.6
-4.26
-4.14
-3.83
-3.8
-4.02
-4.46
-4.39
-4.72
-4.47
-4.34
-4.34
-4.38
-4.43
-4.43
-4.28
-4.62
-4.22
-4.61
-4.23
-4.9
-4.94
-4.13
-4.22
-3.83
-4.21
-4.67
-4.91
-4.41
-4.41
-4.07
-4.43
-4.62
-5.03
2
Table 5 r
obtained from MLR of DGcalc as a function of descriptors considered for each benzazole family
mðtestÞ
Considering
Benzimidazole
Benzothiazole
Benzoxazole
2
2
2
All series (n = 68)
bad series (n = 17)
mad series (n = 17)
fad series (n = 17)
pad series (n = 17)
r
r
r
r
r
¼ 0:2342
¼ 0:7445
¼ 0:8501
¼ 0:9968
¼ 0:7705
r
r
r
r
r
¼ 0:1948
¼ 0:8646
¼ 0:9734
¼ 0:9929
¼ 0:8529
r
r
r
r
r
¼ 0:3021
¼ 0:9968
¼ 0:9968
¼ 0:9430
¼ 0:9771
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
mðtestÞ
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
mðtestÞ
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
m2 ðtestÞ
mðtestÞ
rotatable bonds (N ), polar surface area (PSA), molecular
B
screening. It is necessary to take several other factors into
account, including molecular geometry, lipophilicity and
electronic density. These parameters were evaluated for the
test compounds of the present study and compared to the
values of the compounds RefI, RefII, RefIII, RefIV, Cfad3 and
Cfad5 reported in the literature (86.67, 66.67, 70.0, 66.67,
83.33 and 83.33 %, respectively) (Flores et al., 2013).
Besides the relatively low score, compared to Bfad12, most
of the compounds taken from the literature had fragments in
their structure that act as irritants, and thus are potentially
mutagenic and tumorigenic, and/or lead to reproductive
malformations. For all compounds, the aforementioned
parameters are shown in the Supplementary information.
weight (MW), the partition coefficient (log P) and the
solubility coefficient (log S).
The values obtained for these physicochemical proper-
ties of the ligands show that the best candidate is com-
pound Bfad12, a benzothiazole derivative with a score of
9
3.33 %, which can be compared to Bbad9 with a score of
6.67 %. In the case of compound Bfad12, all of the car-
6
2
3
bons are sp , with the exception of an sp methyl group.
Additionally, the hydrocarbon chain at the C2 position is
shorter (3 carbons) and is an a, b-unsaturated system that
confers rigidity and extension of the conjugation, reducing
its degrees of freedom. In the case of Bbad9, all of the
2
carbons in the benzazole moiety are sp without exception.
3
At the C2 position, this compound has a chain with sp ,
QSAR study
which gives rise to greater freedom.
These results demonstrate that molecular modeling and
docking simulations are not sufficient for molecular
The purpose of applying a QSAR method is to establish
models that can improve our conception of molecular
123

Med Chem Res
Fig. 1 Graph for the benzimidazole derivatives: a considering the
four series together (n = 68), and b considering each individual series
(n = 17). For the benzothiazole derivatives: c considering the four
series together and d considering each individual series. For the
benzoxazole derivatives: e considering the four series together and
f considering each individual series. The abscissa axis represents
DG calculated from physicochemical parameters, and the ordinate
axis represents DG obtained by docking simulation
recognition based on behavioral data. Using physico-
chemical descriptors through a QSAR study in order to
explain the molecular recognition obtained by docking
simulations is a strategy that has established correlations
with high predictive power in other studies (Correa et al.,
2014; Zekri et al., 2009). This strategy has also been
1
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Med Chem Res
Table 6 Molecular physicochemical properties for BT1, BT2 and BT3 ligands
Compound
BT 1
BT2
BT3
DG (Kcal/mol)
MW (g/mol)
Log P
-4.35
211.0
4.45
-3.18
12.89
1
-5.08
255.29
3.97
-3.19
50.191
2
-4.93
323.29
4.73
Log S
-3.97
PSA
50.19
N
N
N
B
3
3
1
0
0
0
0
hba
hbd
1
3
0
1
M
T
0
0
Medium
Medium
0
Medium
Medium
0
I
RE
DG enzyme-ligand binding energy, Log P partition coefficient, Log S solubility coefficient, PSA polar surface area, MW molecular weight, Nhba
number of hydrogen bond acceptors, Nhbd number of hydrogen bond donors, N
I irritant, RE reproduction effects
B
number of rotatable bonds, M mutagenic, T tumorigenic,
employed to explain biological activity and mechanisms of
action, or to modulate receptor–ligand molecular recogni-
tion (Zhang et al., 2013; Voss et al., 2014). For this pur-
pose, quantum parameters were obtained from DFT
calculations at the B3LYP/6-31G(d) level. The parameters
taken into account for the QSAR study were the energy of
the frontier molecular orbitals (EHOMO and ELUMO), the
difference in energy between orbitals (gap), dipolar
For the benzimidazole derivatives, the r2
value is
mðtestÞ
acceptable when considering each series (bad, mad, fad or
pad) separately. The most precise correlation was found
with the fad series, which had an r2
value of 0.9968.
mðtestÞ
However, when an entire family is considered (68 com-
pounds), the r2
values have no correlation. This is
mðtestÞ
particularly true when substituting a,b-unsaturated moi-
eties in trans configuration at C2, which decreases freedom
and gives rise to an extension of the conjugation. Addi-
tionally, in these compounds the methyl group increases
lipophilicity. This information suggests that docking stud-
ies are insufficient for selecting the best compound and that
other physical–chemical properties must be taken into
account to improve the results (Fig. 1a, b).
momentum (l), molecular volume (V ), electronegativity
M
(
v), absolute hardness (g) and hydrophilicity (x). Also
considered was the electrostatic potential (EP) for nitrogen
N), sulfur (S) or oxygen (O) substituents, or the nitrogen
(
atom with a double bond. The atomic charges of the
N=C - X fragment (X = N, S or O) were obtained by
employing the Merz-Singh-Kollman population analysis.
For benzothiazole derivatives, there was a very low
correlation when including the whole family (all four ser-
Other descriptors considered were Nhba, Nhbd, N , PSA,
B
MW, log P, log S, molar refractivity (MR), polarizability
ies) (r2
¼ 0:1948). The best correlation was found with
mðtestÞ
2
(a) and hydration energy (EHydration). Finally, each family
(benzimidazole, benzothiazole and benzoxazole) includes
the sulfur substituent, where the r
value for the bad
mðtestÞ
series was 0.8656, for the mad series 0.9734, and for the
pad series 0.8529 (Fig. 1c, d).
four series of 17 compounds with differences in the chain
length and the configuration of the double bond at the C2
substituent: bad, mad, fad and pad. To carry out the QSAR
for each of the four series of the three families, the 17
compounds were separately analyzed by multiple linear
regression (MLR), for a total of 204 compounds (see
Table 5). The data for these parameters and the equations
are included in supplementary information.
For the benzoxazole family, the r2
value showed the
mðtestÞ
best correlation independently of whether the C2 sub-
stituent was saturated or unsaturated, or whether or not the
configuration had a double bond Fig. 1e, f).
The in silico results suggest greater affinity between
TcTIM and the proposed ligands whose substituent at the
2
C2 position has an sp carbon. Additionally, substituting
123

Med Chem Res
Table 7 The interactions involved in the ligand–enzyme complex, formed by the ligands BT1, BT2, BT3 and Bfad12 and the amino acid
residues of chain A or B of TIM
Bfad12
BT1
BT2
BT3
DG
-4.29
-4.35
-5.08
-4.93
A:Ile69
•
•
•
•
•
•
•
•
•
•
A:Thr70
A:Arg71
A:Phe75
A:Tyr103
A:Gly104
A:Glu105
A:Ile109
A:Lys113
B:Tyr102
B:Tyr103
B:Gly104
1
1
1
•
•
2
2
2
•
•
•
2
2
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
2
2
2
2
•
•
•
•
•
2
2
•
2
•
2
•
•
1
The binding energy (DG) is in Kcal/mol. The point (•) indicates that there is interaction with the amino acid residue; superscript one ( ) indicates
2
that the amino acid has hydrogen bond interactions with the ligand; superscript two ( ) indicates that the amino acid has p–p interactions with the
ligand
the NH fragment in position 1 with a sulfur atom gave
fitting correlations for the series with unsaturated systems.
However, in the case of the benzoxazoles, it was not pos-
sible to see any difference between any of the four series.
the new lead compound and added a trifluoromethyl group
(an electron-withdrawing group, r = 0.43) at C6 instead
m
of the methyl group of Bfad12 in order to obtain a better
log P (Table 6). Thus BT3 is notably more lipophilic than
BT1 and BT2, enabling it to pass through lipidic mem-
branes to reach T. cruzi, which is an intracellular parasite.
Organic compounds that contain fluoride groups have good
physicochemical properties in regard to biological activity
(Pursor et al., 2008; Filler and Saha, 2009), improving
lipophilicity and increasing the velocity of diffusion
through cellular membranes (Bhavanarushi et al., 2014).
Hence, BT3 is an isostere of the Bfad12 compound and
contains an unsaturated system at the C2 position.
Despite the good correlation in r2
values, their affinity
mðtestÞ
energies for TcTIM were lower than those of the other two
families. The physicochemical properties evaluated herein
indicate a high probability that the benzothiazole deriva-
tives could be effective drugs for treatment of T. cruzi.
Compounds proposed for synthesis and biological
study
The three compounds with the best score did not have an
excellent DG value, indicating a less than desirable ligand–
receptor affinity. However, these three compounds were all
from the benzothiazole family. Therefore, we took the one
with the best DG value as the lead compound to form three
new compounds. This lead compound was a benzothiazole
with the fad substituent at C2 and the methyl group at C6.
To form BT1 (2-phenyl-1,3-benzothiazole), we added a
benzyl group at C2 because the best 3 compounds all had
Of these three compounds, BT1 and BT2 had problems
with toxicity (based on the toxicological database for
several different drugs). BT3, however, was acceptable in
respect to this parameter. This clearly shows that the tri-
fluoromethyl group is particularly important for better
effects. Additionally, the DG is much better for BT3 than
BT1 or BT2. On the other hand, molecular recognition data
obtained from docking simulations show that the affinity
for TcTIM increases with BT3 as opposed to its isostere,
Bfad12. It seems that this is due to the incorporation of one
more aromatic ring at the C2 position, which interacts with
the aromatic cluster residues present in the TcTIM dimer
interface. This idea is in agreement with a previous report
(Espinoza and Trujillo, 2005). The consensus binding site
found for Bfad12, BT1, BT2 and BT3 corresponds to
the following amino acid residues of chain A or B of
TIM: A:Ile69, A:Thr70, A:Arg71, A:Phe75, A:Tyr103,
2
an sp geometry for all the carbons. To form BT2 (4-(1,3-
benzothiazol-2-yl)benzoic acid), we started with BT1 as the
new lead compound and added a carboxylic group in the
2
para position of the benzyl group in order to extend the sp
hybridization carbons, because all of the recognition
between the two chains (A and B) of TIM is by p–p
interactions. To form BT3 (4-[5-(trifluoromethyl)-1,3-
benzothiazol-2-yl]benzoic acid), we started with BT2 as
1
23

Med Chem Res
Fig. 2 Binding mode on TcTIM obtained by docking simulation. a–
d Stereoview of the ligand binding sites located at the TcTIM
interface. e–h Schematic representation of the binding site for Bfad12
(e), BT1 (f), BT2 (g) and BT3 (h). Dotted lines represent hydrogen
˚
bond interactions and distances are in A
A:Gly104, A:Glu105, A:Ile109, A:Lys113, B:Tyr102,
B:Tyr103 and B:Gly104. The interactions involved for
each complex are summarized in Table 7 and shown in
Fig. 2. Hence, BT1, BT2 and BT3 were synthesized,
evaluated in vitro with T. cruzi and compared with the
results found with Cfad3 and Cfad5, previously reported to
123

Med Chem Res
Fig. 3 Trypanocidal activity of compounds Cfad5, Cfad3, BT1, BT2, BT3 and Benznidazol (BZN), based on the percentage of lysis of the INC-
5
strain of T. cruzi that was isolated from the blood of Chagas patients
be trypanocides. Cfad3 and Cfad5 were compared with
benznidazol, but did not prove to have better trypanocidal
activity (Flores et al., 2013).
replacement of CH by CF improved the interaction with
3 3
the receptor as well as the lipophilicity of the compound,
thus allowing BT3 to diffuse more rapidly through cellular
membranes and reach its pharmacological target. Finally,
BT3 proved to be a better trypanocide than BT1 and BT2,
in agreement with predictions, and also better than the
currently used drug on the market for the treatment of
Chagas disease, benznidazol. In general, BT3 fulfills the
requirements proposed by Lipinski and Veber for com-
pounds to be considered as new drugs. BT3 should cer-
tainly be of interest for further testing in animal models
and/or clinical trials, especially when considering that there
is currently no effective treatment for Chagas disease.
Trypanocidal activity
After synthesis and characterization, BT1, BT2 and BT3
were tested in vitro for their biological activity against the
INC-5 strain of T. cruzi, isolated from the blood of patients
with chronic Chagas disease. The concentrations of the test
compounds were 5, 10, 50 and 100 lg/mL, and the results
showed that all the compounds have trypanocidal activity.
However, only compound BT3, beginning at the concen-
tration of 50 lg/mL, proved to have greater trypanocidal
activity than the reference compound benznidazol (BZN)
Acknowledgments We are grateful to the Consejo Nacional de
Ciencia y Tecnolog ´ı a (CONACyT), Comisi o´ n de Operaci o´ n y
Fomento de Actividades Acad e´ micas del Instituto Polit e´ cnico
Nacional (COFAA-IPN) for financial support and CNMN-IPN for
experimental support. We thank Bruce Allan Larsen for proofreading
this manuscript and Ariana Lucas for her contribution in the style of
the figures.
(
72.3 % versus 58.9 %; Fig. 3).
Conclusion
Regarding the biological activity of benzoxazoles Cfad3
and Cfad5, the in vitro and in silico results are in agree-
ment with each other and with previous reports, thus val-
Compliance with ethical standards
Conflict of interest The authors confirm that the article content has
no conflict of interest.
idating the current model. The r2
values of multiple
mðtestÞ
linear regression for the QSAR studies found an accept-
able correlation for the benzothiazoles, BT1, BT2 and
BT3, supporting the idea that the substitution at C2 with a
unsaturated system tends to favor molecular recognition
with TcTIM. For BT3, the substitution at the C2 position
with an aromatic ring increased affinity for the cluster of
aromatic residues present at the interface of the dimeric
monomers of TcTIM. The data obtained from the docking
studies show that affinity for TcTIM is greater with BT3
than with its isostere Bfad12 and that the isosteric
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