Comparative study of the trypanocidal activity of the methyl 1-nitrophenyl-1,2,3,4-9H-tetrahydro-β-carboline-3-carboxylate derivatives and benznidazole using theoretical calculations and cyclic voltammetry

Article abstract of DOI:10.1016/j.ejmech.2008.03.044

The cis and trans isomers of methyl 1-(m-nitro)phenyl and 1-(p-nitro)phenyl-1,2,3,4-tetrahydro-9H-β-carboline-3-carboxylates (compounds 3a,b, 4a and b) were synthesized and evaluated in vitro against epimastigote forms of Trypanosoma cruzi. Among all of the evaluated tetrahydro-β-carboline derivatives, the compound trans-methyl 1-(m-nitro)phenyl-1,2,3,4-9H-tetrahydro-β-carboline-3-carboxylate (3b) was found to exhibit significant trypanocidal activity (IC₅₀ = 22.2 μM). Theoretical studies of molecular conformations and electronic properties for the synthesized compounds and benznidazole, as well as, the cyclic voltammetric (CV) behaviors' determination were performed. A comparative study of the trypanocidal activity of the nitrophenyl-tetrahydro-β-carbolines derivatives and benznidazole, using the results of theoretical calculations and of the cyclic voltammetry experiments, is presented.

Full text of DOI:10.1016/j.ejmech.2008.03.044

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European Journal of Medicinal Chemistry 44 (2009) 1745e1750
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Short communication
Comparative study of the trypanocidal activity of the methyl
1-nitrophenyl-1,2,3,4-9H-tetrahydro-b-carboline-3-carboxylate derivatives
and benznidazole using theoretical calculations and cyclic voltammetry
a
Lilian T. Dusman Tonin , Valeria A. Barbosa , Cleverson Cassero Bocca ,
a
a
´
Erika R.F. Ramos , Celso V. Nakamura , Willian Ferreira da Costa , Ernani A. Basso ,
¨
b
b
a
a
´
b
Tania U. Nakamura , Maria Helena Sarragiotto
a,
*
ˆ
a Departamento de Qu´ımica, Universidade Estadual de Maringa´, Av. Colombo, 5790, CEP 87020-900, Maringa´, PR, Brazil
b Laborato´rio de Microbiologia Aplicada aos Produtos Naturais e Sinte´ticos, Departamento de Ana´lises Cl´ınicas, Universidade Estadual de Maringa´,
Av. Colombo, 5790, CEP 87020-900, Maringa´, PR, Brazil
Received 25 November 2007; received in revised form 17 March 2008; accepted 20 March 2008
Available online 10 April 2008
Abstract
The cis and trans isomers of methyl 1-(m-nitro)phenyl and 1-( p-nitro)phenyl-1,2,3,4-tetrahydro-9H-b-carboline-3-carboxylates (compounds
3a,b, 4a and b) were synthesized and evaluated in vitro against epimastigote forms of Trypanosoma cruzi. Among all of the evaluated tetrahydro-
b-carboline derivatives, the compound trans-methyl 1-(m-nitro)phenyl-1,2,3,4-9H-tetrahydro-b-carboline-3-carboxylate (3b) was found to
exhibit significant trypanocidal activity (IC50 ¼ 22.2 mM). Theoretical studies of molecular conformations and electronic properties for the syn-
thesized compounds and benznidazole, as well as, the cyclic voltammetric (CV) behaviors’ determination were performed. A comparative study
of the trypanocidal activity of the nitrophenyl-tetrahydro-b-carbolines derivatives and benznidazole, using the results of theoretical calculations
and of the cyclic voltammetry experiments, is presented.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Tetrahydro-b-carbolines; Trypanosoma cruzi; Cyclic voltammetry; Theoretical calculations
1. Introduction
serious side effects, such as peripheral neuropathy, anorexia,
vomiting, allergy, and cardiac and renal toxicities [3].
Natural and synthetic b-carbolines and tetrahydro-b-carbo-
lines alkaloids are well-known compounds that possess a vari-
ety of biological properties, such as anticonvulsive, ansiolytic,
sedative, antimicrobial, antithrombotic, anti-HIV, antiprolifer-
ative, insecticidal, and parasiticidal [4e6]. In the recent years,
this class of compounds has been tested in trypanocidal assays
against T. cruzi [7e9].
Trypanosomiases are a widespread group of parasitic dis-
eases that affect over several 100 millions of people, especially
in the tropical and subtropical countries [1]. The parasite Try-
panosoma cruzi is the causative agent of Chagas disease, an
endemic infection that affects mainly the Central and South
America, where 18 million people are infected and approxi-
mately 400,000 deaths per year [2].
Nifurtimox (nfx) and benznidazole (bnz), the drugs used in
the treatment of this pathology, are effective only during the
acute phase of the disease. Moreover, these compounds cause
The importance of the search for new effective chemother-
apy agents for the treatment of Chagas disease together with
the trypanocidal activity reported for tetrahydro-b-carboline
alkaloids, lead us to study this class of compounds.
In this work we report the in vitro trypanocidal activities of cis
and trans isomers of 1-(m-nitro)phenyl and 1-( p-nitro)phenyl-
1,2,3,4-tetrahydro-9H-b-carboline-3-carboxylates (compounds
* Corresponding author. Tel.: þ55 44 32613657; fax: þ55 44 32614125.
E-mail addresses: mhsarragiotto@uem.br, mhelena@wnet.com.br (M.H.
Sarragiotto).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.044

1746
L.T. Du¨sman Tonin et al. / European Journal of Medicinal Chemistry 44 (2009) 1745e1750
3a,b, 4a and b). In order to establish a possible relationship be-
tween electrochemical properties and trypanocidal activities we
performed electrochemical measurements by cyclic voltammetry
for the nitrophenyl-tetrahydro-b-carbolines synthesized. The
electronic distribution around the molecular surface, charge cen-
ters, electrostatic potential maps (EPMs), atomic charges, dipole
moments and Mulliken charges were also investigated for the
compounds synthesized and for benznidazole. A comparative
study of the trypanocidal activity of the nitrophenyl-tetrahydro-
b-carbolines derivatives and benznidazole, using the results of
theoretical calculations and of the cyclic voltammetry experi-
ments, was performed.
for protozoa). Compound 3b, which demonstrated better try-
panocidal activity, was also more selective against the para-
sites than the mammalian cells, with a selectivity index ratio
of SI 25.6 and 35.7 in LLC-MK2 and macrophages cells,
respectively.
2.3. Conformational analysis
The literature presents several studies correlating the trypa-
nocidal activity with structural parameters, as well as, with the
redox potential of the active compounds [15,16]. Thus, an in-
vestigation on these properties could be useful to understand
the action mechanism against the protozoan parasite. Based
on this prediction, we investigated the electronic distribution
around the molecular surface, charge centers, EPMs (electro-
static potential maps), atomic charges and dipole moments
for compounds 3a,b, 4a and b and benznidazole (Table 2).
The calculations were carried out on the most stable con-
former of each isomer. The conformer structures resulting
from the nitrogen inversion for cis and trans isomers were
optimized at 6-311 þ G(d) basis set. The spatial orientation
for the substituent groups was analyzed by the rotation of
NeC3eCeO and NeC1eC10eC20 dihedral angles. Benzni-
dazole was submitted to the same computational treatment,
in order to compare its conformational preference with the
tetrahydro-b-carboline derivatives synthesized.
2. Results and discussion
2.1. Chemistry
Methyl tetrahydro-b-carboline-3-carboxylates 3a,b, 4a and
b were prepared from the PicteteSpengler reaction of L-tryp-
tophan with an appropriated aldehyde [10] (Scheme 1). The
synthesis of these compounds was previously described in
the literature [11e13]. The compounds were characterized
by spectral data (1H and 13C NMR, MS and IV). The stereo-
chemistry of the cis and trans isomers was assigned on the ba-
sis of 13C NMR data [14].
2.2. Biology
Through the electrostatic potential maps showed in Fig. 1,
we can observe negative potential sites that include electro-
negative atoms and their neighborhoods and positive potential
sites, mainly around hydrogen atoms. The electronic density is
concentrated on nitro and carbonyl groups. In the EPM of
benznidazole (Fig. 2) we can observe the same electronic con-
centration on carbonyl and nitro groups. Table 2 contains the
electronic parameter values for all compounds and for
benznidazole.
The atomic charge values (Table 2) on the carbon atom of
carbonyl group for the nitrophenyl-tetrahydro-b-carboline de-
rivatives synthesized were all negatives and with the same
largeness, whereas for benznidazole was positive (þ0.158).
For the oxygen atom of the carbonyl group and for the nitro-
gen atom of the nitro group the values showed no significant
difference. The atomic charge values for the oxygen atoms
in the nitro group of the benznidazole was þ0.075 au; for
The compounds 3a,b, 4a and b were tested in vitro against
epimastigote forms of T. cruzi. Benznidazole was used as the
reference trypanocidal drug. The IC50 values for the trypano-
cidal activity of the tetrahydro-b-carbolines derivatives are
presented in Table 1. The results showed that among com-
pounds tested, trans-methyl 1-(m-nitro)phenyl-1,2,3,4-9H-tet-
rahydro-b-carboline-3-carboxylate (3b) was the most active
with IC50 of 22.2 mM. Moreover, the isomer trans-methyl
1-( p-nitro)phenyl-1,2,3,4-9H-tetrahydro-b-carboline-3-carboxyl-
ate (4b) was inactive (IC50 > 100 mM), indicating that the
activity depends on both stereochemistry at C-1 and position
of the nitro group in the aromatic ring. The toxicity for
LLC-MK2 and J774G8 macrophage cells and the activity
against the protozoa were compared by using the selectivity
index (SI) ratio (CC50 for LLC-MK2 or J774G8 cells/IC50
4
COOCH3
COOH
COOCH3
3
a
b
NH
R1
1
NH2
NH2
N
H
N
H
N
H
1
2
R2
3a: R1 = Hα; R2 = 3-NO2
3b: R1 = Hβ; R2 = 3-NO2
4a: R1 = Hα; R2 = 4-NO2
4b: R1 = Hβ; R2 = 4-NO2
Scheme 1. (a) H3COH, H2SO4, reflux, 12 h; (b) aldehyde (R1COH), CH2Cl2, TFA (2.2 mol equiv.), rt, 48 h.

L.T. Du¨sman Tonin et al. / European Journal of Medicinal Chemistry 44 (2009) 1745e1750
1747
Table 1
IC50 values against T. cruzi and cathodic potential values for the tetrahydro-b-
carbolines 3a,b and 4a,b
In order to verify if there is correlation between the charge
on the carbon carrying the nitrophenyl groups and the trypano-
cidal activities of 3a,b and 4a,b the Mulliken charges on C-1
were calculated. Analyzing the data showed in Tables 1 and 2,
we can observe the same order for activity and Mulliken
charges values: 3b > 4a > 3a > 4b. Thus, the most active
compound for each series (m- or p-nitrophenyl) is also that
one presenting higher charge on C-1.
In summary, our results showed that the electrostatic poten-
tial and the atomic charges values on nitro group, as well as,
the Mulliken charges values on C-1, are useful parameters
for the analysis of trypanocidal activity of compounds 3a,b
and 4a,b.
Compound
Isomer C-1 substituent
IC50 (mM) Epc (V)
3a
cis
93.5
22.2
42.7
>100
ꢀ1.113
O2N
3b
trans
ꢀ1.098 (peak I)
ꢀ1.205 (peak II)
O2N
4a
cis
ꢀ1.096
ꢀ1.118
ꢀ1.044
O2N
2.4. Electrochemical measurements
Studies have demonstrated that the redox potential of the
nitro group has influence on the trypanocidal activity. The
main intermediate products responsible for the cytotoxic ac-
tion of the nitrocompounds are the nitro-radical anion (R-
NOꢁ2ꢀ) and hydroxylamine derivative (R-NHOH), which are
produced in the reduction of the nitro group [17,18].
Cyclic voltammograms and the values of the cathodic peak
potentials (Epc) of compounds 3a,b, 4a,b and benznidazole are
shown in Fig. 3 and Table 1, respectively. The voltammograms
of compounds 3a, 4a,b and benznidazole (Fig. 3) show only
one peak, which correspond to the nitro/nitro-radical anion
couple (R-NO2/R-NOꢁ2ꢀ). Our data for the benznidazole were
similar to those previously reported [19]. On the other hand,
the cyclic voltammogram for the most active compound 3b
shows two signals, the first one due to the nitro/nitro-radical
anion couple (peak I) and the second, due to the nitro-radical
anion reduction to form the hydroxylamine derivative (peak
II). The results presented in Table 1 show that the facility of
the nitro group reduction to nitro-radical anion for the active
compounds 3b (Epc ¼ ꢀ1.098 V for peak I) and 4a
(Epc ¼ ꢀ1.096 V) are very similar. These data led us to sug-
gest that the nitro-radical anion reduction to form the hydrox-
ylamine derivative (peak II) for 3b is responsible for its higher
activity in relation to 4a.
4b
trans
O2N
Benznidazole
the derivatives 3a,b, 4a and b the values were þ0.004,
þ0.003, ꢀ0.004 and ꢀ0.010 au, respectively.
The observed potential values are all positives for carbon
and negatives for oxygen atoms of the carbonyl group, and
negatives for the aromatic ring. A great difference was ob-
served for the electrostatic potential values of the nitro group.
The most active compounds (3b and 4a) presented more neg-
ative values for the electrostatic potential of the nitro group
when compared to those of the inactive compounds (3a and
4b). The potential values on the oxygen and nitrogen atoms
were both negatives for the actives compounds 3b and 4a, be-
ing ꢀ105.0 and ꢀ89.7 kcal/mol, for oxygen, and ꢀ18.0 and
ꢀ52.1 kcal/mol, for the nitrogen, respectively. The inactive
compounds (3a and 4b) exhibit minor potentials on the oxygen
atom (ꢀ80.3 and ꢀ36.4 kcal/mol) and positive potential
values on the nitrogen atom (þ5.30 and þ1.90 kcal/mol).
The potential values for the benznidazole were ꢀ23.8 and
ꢀ99.1 kcal/mol, for the nitrogen and oxygen atoms, respec-
tively. The difference in the activity between 3b and 4a is
probably due to the major atomic charge concentration in
the nitrogen atom of the nitro group, being ꢀ0.218 in 3b
and ꢀ0.204 in 4a.
From the Epc values for the first cathodic peak (Table 1) we
can observe the following order for the reduction: 3b > 3a and
4a > 4b. Therefore, the most active compound for each series
(m- or p-nitrophenyl) is also that one presenting higher easi-
ness of reduction.
Table 2
Electronic parameters, dipole moments and Mulliken charges at C-1 for 3a,b, 4a,b and benznidazole
Compounds
Electrostatic potential valuesa
Atomic chargesb
Dipolec
Mulliken charges at C-1
C]O (C/O)
þ79.0/ꢀ80.3
þ78.4/ꢀ76.0
þ78.4/ꢀ70.9
þ78.0/ꢀ74.0
þ18.8/ꢀ88.5
Ring
NO2 (N/O)
C]O (C/O)
NO2 (N/O)
3a
3b
ꢀ52.1
ꢀ37.0
ꢀ33.3
ꢀ36.4
ꢀ43.9
þ5.30/ꢀ80.3
ꢀ18.0/ꢀ105.0
ꢀ52.1/ꢀ89.7
þ1.90/ꢀ36.4
ꢀ23.8/ꢀ99.1
ꢀ0.183/ꢀ0.260
ꢀ0.163/ꢀ0.225
ꢀ0.203/ꢀ0.260
ꢀ0.198/ꢀ0.225
þ0.158/ꢀ0.292
ꢀ0.223/þ0.004
ꢀ0.218/þ0.003
ꢀ0.204/ꢀ0.004
ꢀ0.208/ꢀ0.010
ꢀ0.394/þ0.075
2.59
5.09
4.38
5.90
7.22
ꢀ0.5944
ꢀ0.8850
ꢀ0.6212
ꢀ0.3045
e
4a
4b
Benznidazole
a
In kcal/mol.
In au.
In Debye.
b
c

1748
L.T. Du¨sman Tonin et al. / European Journal of Medicinal Chemistry 44 (2009) 1745e1750
isomers of the tetrahydro-b-carbolines containing an m- or
p-nitrophenyl group at C-1. The results of the biological as-
says demonstrated that the isomer trans of the m-nitro-
phenyl-tetrahydro-b-carboline (compound 3b) was the most
active and less cytotoxic among all tested derivatives. Our re-
sults showed that the electronic potential on nitro group and
the Mulliken charges on C-1 are adequate parameters to ex-
plain the difference in the activity of the synthesized com-
pounds. Based on the theoretical calculations and on the
cyclic voltammograms data it was reasonable to conclude
that the difference in the trypanocidal activity of the com-
pounds 3a,b, 4a and b depends, mainly, on the easiness reduc-
tion of nitro group. The higher activity of 3b in relation to 4a
is probably due to the nitro-radical anion reduction to form the
hydroxylamine derivative, as observed in the voltammogram
of 3b.
3a
3b
4a
4. Experimental protocols
4.1. Biology
Epimastigote forms of T. cruzi Y strain were grown in liver
infusion tryptose (LIT) medium supplemented with 10% inac-
tivated fetal bovine serum (Gibco Invitrogen Corporation,
New York, USA) and assayed with synthetic chemical com-
pounds (5, 10, 50, and 100 mg/mL). The compounds were dis-
solved in 1% DMSO at the concentration which did not affect
the growth of the parasites. Next, 1 ꢂ 106 protozoa/mL was in-
troduced into 24-well plate each containing 1 mL of diluted
compounds. Cell growth was determined by counting the par-
asites with a Neubauer haemocytometer after incubation for
96 h at 28 ꢃC. Assays were performed in duplicate on separate
occasions.
4b
4.2. Computational methods
-120
0
120
Fig. 1. Calculated electrostatic potentials on the molecular surface for the most
stable conformers for the compounds 3a,b and 4a,b (colour ranges in kcal/
mol).
Molecular modeling studies were performed on the cis
and trans-methyl-1-(3-nitrophenyl)-1,2,3,4-tetrahydro-9H-b-
carboline-3-carboxylates 3a and b, cis and trans-methyl-1-
(4-nitrophenyl)-1,2,3,4-tetrahydro-9H-b-carboline-3-carboxylates
4a and b and the benznidazole, in order to calculate the stereo-
electronic properties of electronic surface density, including
electrostatic potential maps. A detailed conformational search
for each of the molecules was performed, using DFT methods,
to find out the minimum energy point, beginning with poten-
tial energy surface (PES) obtained through pre-determined di-
hedral turns (scan) and then, fully geometry optimization of
the minimum point. The PES was built using the semi-empir-
ical method (AM1); the geometry optimization was carried out
using the hybrid method B3LYP [20] along the 6-311 þ G(d)
basis set. In each case, frequency calculations were employed
to confirm the structures as minimum point. The electrostatic
potential maps (EPMs) were calculated at HF/6-31 G level of
theory and the maps were plotted using the MOLEKEL pack-
age [21]. The Gaussian 03 package [22] was used to perform
this set of calculations.
3. Conclusions
We reported the evaluation of the trypanocidal activity
against epimastigote forms of T. cruzi of the cis and trans
-100
0
115
Fig. 2. Calculated electrostatic potentials on the molecular surface for
benznidazole.

L.T. Du¨sman Tonin et al. / European Journal of Medicinal Chemistry 44 (2009) 1745e1750
1749
1.0x10-6
5.0x10-7
0.0
2.0x10-7
1.0x10-7
0.0
-5.0x10-7
-1.0x10-6
-1.0x10-7
-2.0x10-7
-3.0x10-7
-4.0x10-7
3a
3b
-1.5x10-6
-2.0x10-6
I
II
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
-1.6
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
E/V
E/V
1.0x10-7
2.0x10-7
1.0x10-7
5.0x10-8
0.0
0.0
-1.0x10-7
-2.0x10-7
-5.0x10-8
-1.0x10-7
-1.5x10-7
-2.0x10-7
-2.5x10-7
-3.0x10-7
-4.0x10-7
-5.0x10-7
4a
4b
-1.4
-1.2
-1.0
-0.8
-0.6
-1.6
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
E/V
E/V
6.0x10-7
3.0x10-7
0.0
-3.0x10-7
-6.0x10-7
-9.0x10-7
-1.2x10-6
benznidazole
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
E/V
Fig. 3. Cyclic voltamograms at 1 mM in DMF of the compounds 3a,b, 4a,b and benznidazole.
4.3. Cyclic voltammetry
working electrode, a glassy carbon as counter-electrode and
a calomel electrode as the reference electrode. The scan rate
used in all experiments was 0.100 V/s. Each sample was pre-
pared at a concentration of 1.0 mM in a 10 mL of a 0.10 M
supporting electrolyte solution of tetrabutylammonium nitrate
(Aldrich) in DMF. For all experiments, the solutions were de-
gassed with nitrogen for 5 min prior to analysis.
Cyclic voltammetry (CV) was performed using an Eco
Chemie Autolab potentiostat/galvanostat equipped with
a 663 VA stand Metrohm. All experiments were carried out us-
ing an electrochemical cell of 20 mL with a three-electrode
system. A drop mercury electrode (DME) was used as the

1750
L.T. Du¨sman Tonin et al. / European Journal of Medicinal Chemistry 44 (2009) 1745e1750
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4.4.1. General procedure for compounds 3a,b, 4a and b
Methyl tetrahydro-b-carboline-3-carboxylates 3a,b, 4a and
b were prepared using the PicteteSpengler reaction, accord-
ingly to the procedure previously reported [10].
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mixture of L-tryptophan methyl ester (480 mg,
2.2 mmol), 3-nitrobenzaldehyde or 4-nitrobenzaldehyde
(2.2 mmol) and trifluoroacetic acid (4.8 mmol) in CH2Cl2
(20 mL) was stirred at room temperature for 48 h followed
by evaporation of the solvent. Treatment of the crude product
with 10% Na2CO3, extraction with EtOAc (3 ꢂ 20 mL), dread-
ing of organic layer under anhydrous Na2SO4, filtration and
solvent evaporation afforded a residue which was purified on
chromatographic column (silica flash; hexane-EtOAc 20%)
to give the pure cis and trans products.
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for fellowship to Lilian T. Dusman Tonin.
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