Design and synthesis of new N-(5-Trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamides as possible antimalarial prototypes

Article abstract of DOI:10.3390/molecules16098083

A rational approach was used to synthesize a new set of 15 1H-1,2,4-triazol-3-yl benzenesulfonamide derivatives with the aim of developing new antimalarial lead compounds. These derivatives were prepared in yields between 50% and 62%, and their structures were elucidated using IR, ¹H-, ¹³C-, ¹⁹F-NMR, MS and elemental analysis. A docking study based on sulfonamides previously used against malaria identified trifluoromethyl-substituted derivatives to be the best lead compounds for new antimalarial drug development.

Full text of DOI:10.3390/molecules16098083

Molecules 2011, 16, 8083-8097; doi:10.3390/molecules16098083
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of New N-(5-Trifluoromethyl)-1H-1,2,4-
triazol-3-yl Benzenesulfonamides as Possible Antimalarial
Prototypes
Nubia Boechat *, Luiz C.S. Pinheiro, Osvaldo A. Santos-Filho and Isabor C. Silva
Departamento de Sintese Organica, Instituto de Tecnologia em Farmacos, Fundacao Oswaldo Cruz,
Manguinhos, CEP 21041-250, Rio de Janeiro, RJ, Brazil
* Author to whom correspondence should be addressed; E-Mail: boechat@far.fiocruz.br;
Tel.: +55-21-39772460; Fax: +55-21-25602518.
Received: 1 August 2011; in revised form: 9 September 2011 / Accepted: 16 September 2011 /
Published: 20 September 2011
Abstract: A rational approach was used to synthesize a new set of 15 1H-1,2,4-triazol-3-yl
benzenesulfonamide derivatives with the aim of developing new antimalarial lead
compounds. These derivatives were prepared in yields between 50% and 62%, and their
1
structures were elucidated using IR, H-, 13C-, 19F-NMR, MS and elemental analysis. A
docking study based on sulfonamides previously used against malaria identified
trifluoromethyl-substituted derivatives to be the best lead compounds for new antimalarial
drug development.
Keywords: malaria; sulfonamide; trifluoromethyl; dihydropteroate synthase; DHPS
1. Introduction
Malaria is a deadly mosquito-borne disease that afflicts as many as half a billion people; about
250 million malaria cases and nearly one million associated deaths have been reported in 106 countries
across Africa, Asia and Latin America [1]. Drug resistance, documented in Plasmodium falciparum,
P. malariae and P. vivax malaria species, has been one of the main obstacles in the fight against
malaria. Increasing resistance to antimalarial drugs has a number of implications for the eradication of
malaria. Inappropriate use of antimalarial drugs and the use of monotherapies or substandard and
counterfeit medicines are suggested to be the main contributors to widespread resistance in malaria
parasites [2].

Molecules 2011, 16
8084
Chemotherapeutic treatment of malaria can involve a number of compounds: quinine, chloroquine,
mefloquine, primaquine, amodiaquine, proguanil, pyrimethamine, sulfadoxine, artemisinin, halofantrine,
pyronaridine, piperaquine, and artemisinin derivatives, such as artemether, artesunate and
dihydroartemisinin. However, in the absence of an effective vaccine, and given the decreased
efficiency of classical medications against chloroquine-resistant strains, new antimalarial drugs that are
efficient, safe and synthetically economical are needed [3,4].
Organofluorine compounds have been broadly explored in medicinal chemistry and play a
continuing role as lead compounds for therapeutic applications. The introduction of a fluorine atom has
been an important strategy in the modification of chemical reactivity and physical and biological
properties of organic compounds. This strategy can critically influence pharmacodynamic and
pharmacokinetic properties, reinforce drug-receptor interactions, aid absorption and translocation
across lipid bilayers and induce conformational changes to block metabolism [5-11].
One of the most widespread fluorine-containing functional groups in bioactive molecules is the
trifluoromethyl moiety [12,13], a highly electronegative substituent that can exert significant electronic
influence on neighboring groups [9]. The trifluoromethyl group can also cause dramatic steric change,
as its van der Waals volume is comparable to that of an ethyl [12] or isopropyl [6] group, although
with a significantly different shape. It is among the most lipophilic groups known and is therefore
useful in improving the targeting of molecules to active sites [5,6]. Mefloquine, one of the most
important antimalarial agents, contains two trifluoromethyl groups [2]. Recent literature on compounds
that are active against malaria reports a large number of new molecules containing trifluoromethyl
groups [14-16].
Triazoles are important members of the azole class of antiparasitic drugs. They have been used to
treat cutaneous leishmaniasis and/or Chagas’ disease with variable rates of success [17,18], and they
possess antimalarial activity [19-23].
Sulfonamides are widely used in medicinal chemistry because of their low cost, low toxicity and
excellent biological activity. For example, sulfadoxine, sulfadiazine, and sulfalene are effective
malaria drugs that possess sulfonamide groups attached to a heterocyclic ring. Krungkrai and
coworkers reported a library of aromatic/heteroaromatic sulfonamides with diverse scaffolds and
assayed these compounds for the inhibition of carbonic anhydrase from Plasmodium falciparum
(pfCA) [24,25]. The literature describes several other sulfonamides with antimalarial activity [26-32].
Some of these studies involve the inhibition of folate metabolic enzymes that are crucial for the growth
of the malaria parasite. Some of these enzymes are absent in humans and are therefore potential targets
for malarial chemotherapy. However, parasitic resistance to currently used drugs has spread
worldwide, and the scientific community is researching alternatives.
A promising approach is the development of fixed-dose combinations of antimalarial agents, for
example, a pyrimethamine-sulfadoxine combination (Fansidar®), which has been widely used to treat
P. falciparum in Africa, where these parasites are resistant to chloroquine [33]. Pyrimethamine inhibits
the enzyme dihydrofolate reductase (DHFR) whereas sulfadoxine inhibits dihydropteroate synthase
(DHPS) [34-36].
The literature describes the synthesis and biological activity of some 1H-1,2,4-triazol-3-yl
benzenesulfonamides [37-39]. We sought to develop a potent and economical series of synthetic
1H-1,2,4-triazol-3-trifluoromethyl benzenesulfonamides as antimalarial agents. By using traditional

Molecules 2011, 16
8085
medicinal chemistry principles, we designed and synthesized a set of derivatives 13–27 as potential
antimalarial compounds (Scheme 1). Using bioisosteric principles, the heterocyclic ring of the
sulfonamides drugs (sulfadoxine, sulfadiazine, and sulfalene) was replaced by a triazole ring in the
designed compounds. Different substituents were incorporated to investigate the importance of the
group at position 5 on the triazole ring. The CF3 present in mefloquine was prioritized. We also varied
the group at position 4' of the aromatic ring to investigate its influence. The sulfonamide derivatives
were synthesized, nine new 13–17, 19, 20, 24, 25, and evaluated by molecular docking. Here, we
report the synthesis and modeling of 1H-1,2,4-triazol-3-yl benzenesulfonamides that potentially inhibit
P. falciparum DHPS.
Scheme 1. Rational approach to design the 1H-1,2,4-triazol-3-yl benzenesulfonamide
derivatives 13–27.
N
N
H
N
S
O
Heterocyclic
replacement
O
Sulfadiazine
HO
N
H
trifluoromethyl
group
H3CO
N
OCH3
H
N
N
N
HN
R'
H
N
CF3
N
S
O
R
O
CF3
N
S
O
O
Mefloquine
Sulfadoxine
(13-27)
R' = H, CH3, CF3
OCH3
N
H
R = H, CH3, F, Cl, Br
N
Sulfonamide
group
N
S
O
NH2
O
Sulfalene
1.1. Chemistry
The synthetic route for preparing the 1H-1,2,4-triazol-3-yl benzenesulfonamide derivatives 13–27
is illustrated in Scheme 2. The 3-amine-1H-1,2,4-triazoles 5–7 were prepared in excellent yield
(91%–99%) using aminoguanidine bicarbonate (1) to undergo condensation followed by cyclization
with the appropriate carboxylic acid 2–4.
To produce the target compounds, an equimolar mixture of 3-amino-1H-1,2,4-triazoles 5–7 and the
appropriate sulfonyl chloride derivative 8–12 was stirred in the presence of acetonitrile or DMF at
room temperature to produce the 1H-1,2,4-triazol-3-yl benzenesulfonamides 13–27. Products were
1
isolated in moderate yields (50%–62%) and characterized spectroscopically using IR, H-, 13C-,
19F-NMR, MS and elemental analysis.
1.2. Molecular Modeling and Docking Simulation
Each target molecule was modeled in silico, and energy minimization was performed over
1,000 steps using the steepest descent method, Gasteiger-Hückel charges, a dielectric constant of 80,
and the Tripos force field. The structures were further optimized using the conjugated gradient method.

Molecules 2011, 16
8086
Scheme 2. Synthetic approach used to obtain the 1H-1,2,4-triazol-3-yl benzenesulfonamide
derivatives 13–27.
H
N
O
HN
(i)
N
H2N
+
NH2
.H2CO3
R'
R'
OH
(2-4)
NH2
N
NH
(1)
(5-7)
O
S
Cl
R
O
HN
N
(ii)
H
(8-12)
R'
N
N
O2S
R
(13-27)
13: R' = CF3; R = F;
14: R' = CF3; R = Cl;
23: R' = H; R = F;
24: R' = H; R = Cl;
18: R' = CH3; R = F;
19: R' = CH3; R =Cl;
15
25
: R' = CF3; R = Br;
20
: R' = H; R = Br;
: R' = CH3; R = Br;
16: R' = CF3; R = CH3;
17: R' = CF3; R = H;
26: R' = H; R = CH3;
27: R' = H; R = H
21: R' = CH3; R = CH3;
22: R' = CH3; R = H;
Reagents and conditions: (i) toluene, reflux, 24 h; (ii) CH3CN or DMF, TEA, 0 oC, 6h.
A model of the active site of P. falciparum DHPS was constructed in silico from the crystal structure
of Escherichia coli DHPS (PDB entry: 1AJ0) [40], which includes a sulfonamide in its active site.
Ligand-enzyme docking simulations were performed with the molecular docking algorithm MolDock [41].
This software uses a heuristic search algorithm (termed “guided differential evolution”) that combines
differential evolution and a cavity-prediction algorithm. The docking scoring function is an extension
of the piecewise linear potential (PLP) [41]. After the ligands and the protein coordinates were
imported, all structural parameters, including bond type, hybridization, explicit hydrogens, charges,
and flexible torsions, were assigned using the automatic preparation function in Molegro Virtual
Docker software. For each compound, 100 docking runs were performed with an initial population of
150 individuals. After docking, each compound was energy minimized into the active site of the enzyme.
2. Results and Discussion
The preparation of 3-amino-1H-1,2,4-triazoles 5–7 was achieved through condensation followed by
cyclization of aminoguanidine bicarbonate (1) and the appropriate carboxylic acid 2–4. The triazoles
5–7 were obtained as white powders in excellent yields ranging from 91% to 99%. The 3-amino-1H-
1,2,4-triazoles 5–7 were used directly in the next step without purification. The chemical structures of
1
5–7 were confirmed by analyzing the crude product by H-, 13C- and 19F-NMR, as well IR. An IR
analysis showed an absorption band at 3456–3208 cm−1, attributed to the stretch vibrations of the NH
bond of the amino group.
The nucleophilic substitution reactions between 3-amino-1H-1,2,4-triazoles 5–7 and sulfonyl
chloride derivatives 8–12 were performed under magnetic stirring at room temperature in acetonitrile
or DMF, for derivatives 5–6 and 7, respectively, to prepare 1H-1,2,4-triazol-3-yl benzenesulfonamide
derivatives 13–27 in moderate yields (50%–62%). Derivatives 13–27 were identified using FT-IR; the

Molecules 2011, 16
8087
spectra lacked the previous diagnostic stretching vibration of the NH bond of the amino group and
1
included bands from the sulfonamide at 1,396–1,340 and 1,188–1,141 cm−1. In the H-NMR spectra,
the signals of the respective protons of the synthesized compounds were verified on the basis of their
chemical shifts, multiplicities and coupling constants. The 1H-NMR spectra showed double doublet or
multiplet signals corresponding to the aromatic hydrogens. The triazole protons were observed as a
singlet at 7.48–8.32 ppm for compounds 23–27, whereas compounds 18–22 showed another singlet at
1.96–2.00 ppm for the CH3 group. Compounds 13–17, which contain trifluoromethyl groups, were
19
13
confirmed mainly by characteristic F-NMR signals at −64.72 to −68.16 ppm. The C-NMR spectra
showed characteristic quartet signals at 118.42–118.45 ppm with J = 269 Hz, for the CF3 group, and a
quartet signal at 152.55–152.44 ppm with J = 39 Hz, for C-5. The p-F substituted compounds 13, 18,
and 23 showed C-F as a doublet signal at 165.66–166.21 ppm with J = 255 Hz, for the C4', a doublet
signal at 117.34–117.77 ppm with J = 23 Hz for C3' and C5', and a doublet signal at 130.83–131.38
ppm with J = 10 Hz, for C2' and C6'.
2.1. Molecular Modeling and Docking
P. falciparum DHPS is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin
pyrophosphokinase (PPPK) at the N-terminus of DHPS. No crystal structure is yet available for this
malarial enzyme, but its primary structure is available from proteome databases. We therefore used
GenBank to acquire and study the nucleotide sequence of P. falciparum DHPS (GenBank entry:
Q27865) [42]. A preliminary comparative model was constructed for the entire P. falciparum DHPS
protein. However, the overall score evaluation for this model was unsatisfactory; therefore, we
performed energy minimization with the AMBER force field [43]. The resulting optimized structure
still displayed a number of structural problems. This low resolution led us to discontinue our planned
structure-based design experiments using the model.
Since the active site residues of enzymes are well conserved through molecular evolution, we
decided to search for DHPS crystal structures from other organisms that could be used as templates for
modeling the active site of the P. falciparum enzyme. The malarial sequence (Q27865) was submitted
to the Basic Local Alignment Search Tool (BLAST) [44]. The crystal structures of DHPS from several
sources showed high local score similarity with the malarial enzyme, justifying their use in modeling
our active site. Because the Escherichia coli DHPS (PDB entry: 1AJ0) [40] was crystallized with a
sulfonamide bound to its active site, we used that structure as our main template.
Figure 1 shows a multiple sequence alignment of the top three matches for malarial DHPS. By
analyzing both the sequence alignment and the crystal structure of the bacterial enzyme (PDB entry:
1AJ0), it was possible to infer the active site of the malarial enzyme. Table 1 shows the active site
residues from the bacterial enzyme and the corresponding malarial residues. Notably, just three of the
15 active-site residues from E. coli enzyme differ from the P. falciparum enzyme active site. We
modified the appropriate residues for the E. coli DHPS and performed an energy minimization on each
mutated residue and their neighbor residues. A search for the best dihedral angles for the residues
involved ensured that structural clashes had been removed from the model.

Molecules 2011, 16
8088
Figure 1. Multiple sequence alignment of the malarial enzyme and the top-3 similar sequences.
Table 1. Active site residues of both E. coli and P. falciparum DHPS’s.
Active site residues of DHPS
N°.
E. coli
P. falciparum model
(PDB entry: 1AJ0)
from site-direct mutagenesis from 1AJ0
22
62
63
96
N
T
R
D
N
I
D
F
L
G
S
N
S
A
D
N
I
D
F
F
G
S
115
117
185
190
215
217
219
220
221
255
257
R
K
R
H
R
K
R
H
The docking score of the interactions between the active site of PfDHPS and each synthesized
molecule, as well as sulfadoxine, sulfadiazine and sulfalene, are shown in Table 2. Figures 2–4 show
specific receptor-ligand interactions in detail. There are a number of strong interactions between the

Molecules 2011, 16
8089
ligands and residues N22, S62, H257, and R255 that stabilize the intermolecular geometry. Another
interesting result from the docking calculation is the alignment of the triazole rings of all compounds.
Table 2. Docking score.
Ligand
Docking Score (kcal/mol)
−93.42
14
15
−91.03
16
−89.00
13
−88.89
20
−88.65
18
−87.73
19
−87.24
17
−85.98
25
−83.87
23
−83.85
21
sulfalene
sulfadoxine
22
−83.48
−78.78
−78.01
−76.55
26
−76.17
24
−76.15
27
−71.79
sulfadiazine
−70.03
Figure 2. Intermolecular hydrogen bonds between P. falciparum DHPS and the
synthesized ligands, sulfadoxine, sulfadiazine, and sulfalene.
N22
H257
S62
R255

Molecules 2011, 16
8090
Figure 3. Docking of sufadoxine into P. falciparum DHPS.
R255
S62
Figure 4. Docking of 14 into P. falciparum DHPS.
S62
According to the docking score results (Table 2), compounds 13–16 with CF3 groups on position 5 of
the triazole ring, but not compound 17, showed stronger interaction with the enzyme. The non-substituted
triazole analogs 23–27 exhibited the weakest intermolecular interactions with the enzyme.
All of the halogenated compounds 13–15, 18–20, 23–25, except compound 24, presented better
docking scores than did sulfalene and sulfadoxine (Table 2).

Molecules 2011, 16
8091
3. Experimental
3.1. General
The 1H-, 13C- and 19F-Nuclear Magnetic Resonance (NMR) spectra were obtained at 400.00, 100.00
and 376.00 MHz, respectively, on a Bruker Advance instrument, equipped with a 5 mm probe, or at
300.00, 75.0 and 188.00 MHz, respectively, on a Varian model Unity Plus spectrometer, using
tetramethylsilane as internal standard. The chemical shifts (δ) reported in ppm and the coupling
constants (J) in Hertz. Fourier transform infrared (FT-IR) absorption spectra were recorded on a
Shimadzu mode IR Prestige-21 spectrophotometer by reflectance in KBr. GC/MS employing a model
6890N gas chromatograph (Agilent, Palo Alto, CA, USA), equipped with a 7683B auto sampler and
coupled to a model MS 5973N single quadrupole mass spectrometer (Agilent). The GC is equipped
with a HP-5MS capillary column 30 m length, 0.25 mm diameter, 0.25 μm film thickness, in the
temperature program: 50 °C then increased to 300 °C at a rate oh 10 °C /min and kept for 10 min. the
helium flow rate was 0.5 mL/min. Melting points (m.p.) were determined with a Büchi model B-545
apparatus. TLC was carried out using silica gel F-254 Glass Plate (20 × 20 cm). All other reagents and
solvents used were analytical grade.
3.2. General Procedure for Preparing 3-Amine-1H-1,2,4-triazoles 5–7
A mixture of aminoguanidine bicarbonate (1) and the appropriate organic acid 2–4 (1 equivalent)
was stirred at room temperature to complete release of CO2 and then toluene (150 mL) was added. The
reaction mixture was heated to reflux with a Dean-Stark apparatus under magnetic stirring for 20 h.
The resulting white solid was then cooled to RT, filtered, washed with toluene, and dried. Compounds
5–7 were used without purification.
3-Amine-5-(trifluoromethyl)-1H-1,2,4-triazole (5). Yield: 91%. m.p. 200 °C. IR (KBr, cm−1): 3,441;
1
3,355 (ν NH); 1,654 (ν C=N); 1,580; 1,541 (δ NH); 1,347; 1,141; 780 (ν CF3). H-NMR (300 MHz,
DMSO-d6, δ in ppm): 12.24 (s; 1H; NH); 6.0 (s; 2H; NH2). 13C-NMR (75 MHz, DMSO-d6, δ in ppm):
19
148.7 (C-3); 140.8 (q; J = 36 Hz; C-5); 110.5 (q; J = 267 Hz; CF3). F-NMR (188 MHz, DMSO-d6,
δ in ppm): −64.79 (s, 3F, CF3). MS (70 eV) m/z (%): 152 (100); 133 (25); 104 (24); 69 (59).
3-Amine-5-methyl-1H-1,2,4-triazole (6). Yield: 99%. m.p. 265 °C. IR (KBr, cm−1): 3,456; 3,208 (ν NH);
1
1,672 (ν C=N); 1,554 (δ NH); 1,345 (ν CH3); 1,070–1,017 (ν C–N). H-NMR (300 MHz, DMSO-d6,
δ in ppm): 8.7–8.0 (s; 2H; NH2); 6.3–5.8 (s; 1H; NH); 2.13 (s; 3H; CH3). 13C-NMR (75 MHz, DMSO-d6,
δ in ppm): 159.9 (C-3); 154.9 (C-5); 13.2 (C-6). MS (70 eV) m/z (%): 98 (100); 57 (86).
3-Amine-1H-1,2,4-triazole (7). Yield: 93%. m.p. 142 °C. IR (KBr, cm−1): 3,413–3,332 (ν NH); 2,842
(ν C-H); 1,644 (ν C=N); 1,594 e 1,538 (δ N–H); 1,373–1,213 (δ C–N). 1H-NMR (300 MHz, DMSO-d6,
13
δ in ppm): 12.1 (s; 1H; NH); 7.5 (s; 1H; H5); 6.3–5.4 (s; 2H; NH2). C-NMR (75 MHz, DMSO-d6,
δ in ppm): 158.0 (C-3); 147.7 (C-5). MS (70 eV) m/z (%):84 (100); 57 (45).

Molecules 2011, 16
8092
3.3. General Procedure for Preparing 1H-1,2,4-Triazol-3-yl benzenesulfonamides 13–27
The appropriate sulfonyl chloride 8–12 (1.2 mmol) was treated with the respective 3-amine-1H-
1,2,4-triazole (1 mmol) in acetonitrile (for 5, 6) or DMF(for 7) at 0 °C. The mixture was reacted at
room temperature for 6 h and then poured into ice-water. The precipitate was collected by filtration,
washed with iced-acetonitrile, dried and recrystallized in ethanol.
4-Fluoro-N-(5-trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamide (13). Yield: 52%. m.p.
1
175–176 °C. IR (KBr, cm−1): 3,448; 3,176; 1,649; 1,384; 1,201; 1,141; 709. H-NMR (400 MHz,
13
DMSO-d6, δ in ppm): 8.21–7.60 (m, 5H, H–Ar); 7.95 (s, NH). C-NMR (100 MHz, DMSO-d6, δ in
ppm): 166.21 (d, J = 255 Hz, C4'); 157.72; 152.44 (q, J = 39 Hz, C-5); 131.62; 131.38 (d, J = 10 Hz,
C2', C6'); 118.45 (q, J = 270 Hz, CF3); 117.77 (d, J = 23 Hz, C3', C5'). 19F-NMR (376 MHz,
DMSO-d6, δ in ppm): −66.45 (s, 3F, CF3); −100.41 (s, 1F). MS m/z (%): 310; 159; 95 (100). Anal.
Calc. for: C9H6F4N4O2S (310.01) C, 34.84; H, 1.95; N, 18.06; found: C, 35.46; H, 1.21; N, 17.93.
4-Chloro-N-(5-trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamide (14). Yield: 51%. m.p.
203–205 °C. IR (KBr, cm−1): 3,475; 3,197; 1,643; 1,396; 1,149; 759. 1H-NMR (400 MHz, acetone-d6,
13
δ in ppm): 8.14 (d, J = 8 Hz, 2H, H3', H5'); 7.81 (d, J = 8 Hz, 2H, H2', H6'); 7.29 (s, NH). C-NMR
(100 MHz, DMSO-d6, δ in ppm): 157.78; 152.55 (q, J = 39 Hz, C-5); 141.27; 134.11; 130.50; 129.76;
118.42 (q, J = 269 Hz, CF3). 19F-NMR (376 MHz, DMSO-d6, δ in ppm): −68.16 (s, 3F, CF3). MS m/z
(%): 326; 175 (100); 111. Anal. Calc. for: C9H6ClF3N4O2S (325.98) C, 33.09; H, 1.85; N, 17.15;
found: C, 33.20; H, 1.81; N, 16.94.
4-Bromo-N-(5-trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamide (15). Yield: 50%. m.p.
1
194–196 °C. IR (KBr, cm−1): 3,473; 3,170; 1,643; 1,394; 1,186. H-NMR (400 MHz, DMSO-d6, δ in
13
ppm): 7.91 (d, J = 8.8 Hz, 2H, H3', H5'); 7.83 (d, J = 8.8 Hz, 2H, H2', H6'); 7.52 (s, NH). C-NMR
(100 MHz, DMSO-d6, δ in ppm): 157.77; 152.53 (q, J = 39 Hz, C-5); 134.53; 133.47; 132.87; 128.28;
118.42 (q, J = 269 Hz, CF3). 19F-NMR (376 MHz, DMSO-d6, δ in ppm): −64.72 (s, 3F, CF3). MS m/z
(%): 370; 219 (100); 155. Anal. Calc. for: C9H6BrF3N4O2S (369.93) C, 29.13; H, 1.63; N, 15.10;
found: C, 31.57; H, 1.39; N, 14.65.
4-Methyl-N-(5-trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamide (16). Yield: 53%. m.p.
1
170–172 °C. IR (KBr, cm−1): 3,448; 3,091; 1,591; 1,371; 1,174; 700. H-NMR (400 MHz, DMSO-d6,
δ in ppm): 7.48 (d, J = 10 Hz, 2H, H3', H5'); 7.13 (d, J = 10 Hz, 2H, H2', H6'); 2,28 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO-d6, δ in ppm): 157.11; 152.76 (q, J = 39 Hz, C-5); 140.78; 135.85;
130.91; 129.21; 118.63 (q, J = 269 Hz, C-9); 21.37. 19F-NMR (376 MHz, DMSO-d6, δ in ppm):
−66.39 (s, 3F, CF3). MS m/z (%): 306; 155; 91 (100). Anal. Calc. for: C10H9F3N4O2S (306.03) C,
39.22; H, 2.96; N, 18.29; found: C, 39.71; H, 2.88; N, 18.71.
N-(5-Trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamide (17). Yield: 53%. m.p. 133–135 °C.
1
IR (KBr, cm−1): 3,298; 2,775; 1,693; 1,591; 1,357; 1,176; 732. H-NMR (400 MHz, DMSO-d6, δ in
ppm): 8.08–7.73 (m, 5H, Ar); 7.92 (s, NH). 13C-NMR (100 MHz, DMSO-d6, δ in ppm): 157.79;
19
152.41 (q, J = 39 Hz, C-5); 136.02; 135.43; 130.26; 127.78; 118.45 (q, J = 269 Hz, C-9). F-NMR

Molecules 2011, 16
8093
(376 MHz, DMSO-d6, δ in ppm): −66.45 (s, 3F, CF3). MS m/z (%): 292; 141; 77 (100). Anal. Calc.
for: C9H7F3N4O2S (292.02) C, 36.99; H, 2.41; N, 19.17; found: C, 37.05; H, 2.25; N, 18.85.
4-Fluoro-N-(5-methyl-1H-1,2,4-triazol-3-yl) benzenesulfonamide (18). Yield: 57%. m.p. 281–283 °C.
IR (KBr, cm−1): 3,107; 1,593; 1,363; 1,188. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 8.05–7.11 (m,
13
4H, H–Ar); 7.24 (s, NH); 1.96 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6, δ in ppm): 165.66 (d,
J = 253 Hz, C4'); 161.55; 157.24; 132.74; 130.83 (d, J = 10 Hz, C2', C6'); 117.34 (d, J = 24 Hz, C3',
C5'); 13.90. 19F-NMR (376 MHz, DMSO-d6, δ in ppm): −101.57 (s, 1F). MS m/z (%): 256 (100); 95; 75.
Anal. Calc. for: C9H9FN4O2S (256.04) C, 42.18; H, 3.54; N, 21.86; found: C, 30.46; H, 4.54; N, 25.38.
4-Chloro-N-(5-methyl-1H-1,2,4-triazol-3-yl) benzenesulfonamide (19). Yield: 62%. m.p. 219–221 °C.
1
IR (KBr, cm−1): 3,468, 3,088; 1,637; 1,384; 1,159; 638. H-NMR (400 MHz, acetone-d6, δ in ppm):
8.04 (d, J = 8 Hz, 2H, H2', H6'); 7.75 (d, J = 8 Hz, 2H, H3', H5'); 6.71 (s, NH); 2.00 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO-d6, δ in ppm): 161.68; 157.31; 140.27; 135.21; 130.13; 129.35; 13.89.
MS m/z (%): 272; 111 (100); 75. Anal. Calc. for: C9H9ClN4O2S (272.01) C, 39.64; H, 3.33; N, 20.54;
found: C, 39.77; H, 3.28; N, 20.56.
4-Bromo-N-(5-methyl-1H-1,2,4-triazol-3-yl) benzenesulfonamide (20). Yield: 62%. m.p. 170–172 °C.
IR (KBr, cm−1): 3,468; 3,022; 1,683; 1,392; 1,188. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 7.94 (d,
J = 10 Hz, 2H, H3', H5'); 7.89 (d, J = 10 Hz, 2H, H2', H6'); 7.52 (s, NH); 2.00 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO-d6, δ in ppm): 160.18; 156.41; 147.55; 133.17; 130.66; 129.47; 13.56.
MS m/z (%): 318; 252; 219; 157; 75 (100). Anal. Calc. for: C9H9BrN4O2S (351,96) C, 34.08; H, 2.86;
N, 17.67; found: C, 34.05; H, 2.66; N, 17.56.
4-Methyl-N-(5-methyl-1H-1,2,4-triazol-3-yl) benzenesulfonamide (21). Yield: 60%. m.p. 207–209 °C.
IR (KBr, cm−1): 3,468; 3,089; 1,653; 1,363; 1,159. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 7.85 (d,
J = 8 Hz, 2H, H2', H6'); 7.49 (d, J = 8 Hz, 2H, H3', H5'); 7.23 (s, NH); 2.40 (s, 3H, CH3); 1.97 (s, 3H,
13
CH3). C-NMR (100 MHz, DMSO-d6, δ in ppm): 161.20; 157.20; 146.09; 133.62; 130.32; 127.45;
21.14; 13.89. MS m/z (%): 252; 188; 155; 91 (100). Anal. Calc. for: C10H12N4O2S (252.06) C, 47.61;
H, 4.79; N, 22.21; found: C, 48.01; H, 4.56; N, 22.08.
N-(5-Methyl-1H-1,2,4-triazol-3-yl) benzenesulfonamide (22). Yield: 59%. m.p. 167–168 °C. IR (KBr,
1
cm−1): 3,298; 3,005; 1,687; 1,361; 1,188. H-NMR (400 MHz, DMSO-d6, δ in ppm): 7.96–7.67 (m,
5H, H–Ar); 7.26 (s, NH); 1.98 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6, δ in ppm): 161.31;
157.22; 136.49; 135.12; 129.89; 127.36; 13.86. MS m/z (%): 238; 77(100). Anal. Calc. for:
C9H10N4O2S (238,05) C, 45.37; H, 4.23; N, 23.51; found: C, 45.41; H, 4.03; N, 23.36.
4-Fluoro-N-(1H-1,2,4-triazol-3-yl) benzenesulfonamide (23). Yield: 51%. m.p. 174–176 °C. IR (KBr,
cm−1): 3,471; 3,105; 1,649; 1,519; 1,384; 1,139. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 8.11–7.54
(m, 4H, H–Ar); 7.62 (s, 1H, H5); 7.40 (s, NH). 13C-NMR (100 MHz, DMSO-d6, δ in ppm): 165.69 (d,
J = 254 Hz, C4'); 156.89; 152.71; 132.52; 130.86 (d, J = 10 Hz, C2', C6'); 117.73 (d, J = 23 Hz, C3',
19
C5'). F-NMR (376 MHz, DMSO-d6, δ in ppm): −101.73 (s, 1F). MS m/z (%): 242; 159; 95 (100).
Anal. Calc. for: C8H7FN4O2S (242,02) C, 39.67; H, 2.91; N, 23.13; found: C, 39.77; H, 2.68; N, 22.95.

Molecules 2011, 16
8094
4-Chloro-N-(1H-1,2,4-triazol-3-yl) benzenesulfonamide (24). Yield: 51%. m.p. 200–202 °C. IR (KBr,
1
cm−1): NH 3,460; 3,097; 1,643; 1,386; 1,186; 621. H-NMR (400 MHz, acetone-d6, δ in ppm): 8.06
(d, J = 8 Hz, 2H, H3', H5'); 7.76 (d, J = 8 Hz, 2H, H2', H6'); 7.48 (s, 1H, H5); 6.82 (s, NH).
13C-NMR (100 MHz, acetone-d6, δ in ppm): 157.00; 152.88; 140.44; 135.02; 130.16; 129.40. MS m/z
(%): 258; 175; 111 (100). Anal. Calc. for: C8H7ClN4O2S (257.99) C, 37.14; H, 2.73; N, 21.66; found:
C, 37.35; H, 2.67; N, 21.62.
4-Bromo-N-(1H-1,2,4-triazol-3-yl) benzenesulfonamide (25). Yield: 55%. m.p. 205–207 °C. IR (KBr,
cm−1): 3,473; 3,093; 1,614; 1,386; 1,186. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 8.31 (s, 1H, H5);
7.99 (s, NH); 7.54 (d, J = 10 Hz, 2H, H3', H5'); 7.51 (d, J = 10 Hz, 2H, H2', H6'). 13C-NMR (100 MHz,
DMSO-d6, δ in ppm): 150.82; 147.23; 139.24; 130.76; 127.78; 121.90. MS m/z (%): 304; 238; 221;
155 (100); 75. Anal. Calc. for: C8H7BrN4O2S (301.94) C, 31.70; H, 2.33; N, 18.48; found: C, 29.57; H,
2.88; N, 18.19.
4-Methyl-N-(1H-1,2,4-triazol-3-yl) benzenesulfonamide (26). Yield: 50%. m.p. 197–199 °C. IR (KBr,
cm−1): 3,280; 3,035; 1,624; 1,340; 1,151. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 7.86 (d, J = 8 Hz,
2H, H2', H6'); 7.57 (s, 1H, H5); 7.49 (d, J = 8 Hz, 2H, H3', H5'); 7.32 (s, NH); 2.40 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO-d6, δ in ppm): 156.88; 152.47; 146.30; 133.42; 130.35; 127.48; 21.14.
MS m/z (%): 223; 149 (100). Anal. Calc. for: C9H10N4O2S (238,05) C, 45.37; H, 4.23; N, 23.51; found:
C, 45.40; H, 4.01; N, 23.53.
N-(1H-1,2,4-Triazol-3-yl) benzenesulfonamide (27). Yield: 57%. m.p. 163–165 °C. IR (KBr, cm−1):
3,278; 3,155; 2,775; 1,693; 1,369; 1,186. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 8.32 (s, 1H, H5);
13
8.05 (s, NH); 7.62–7.31 (m, 5H, H–Ar). C-NMR (100 MHz, DMSO-d6, δ in ppm): 150.79; 147.89;
139.21; 128.63; 127.74; 125.46. MS m/z (%): 223; 149 (100). Anal. Calc. for: C8H8N4O2S (224.03) C,
42.85; H, 3.60; N, 24.99; found: C, 41.51; H, 4.77; N, 21.96.
4. Conclusions
In this work, we designed and synthesized a set of 15 compounds analogs to sulfadoxine,
sulfadiazine, and sulfalene in order to identify their antiparasitic profiles, which could then be
correlated with their molecular properties. The 3-amine-1H-1,2,4-triazoles 2–4 were prepared in
good yield (91%–99%) from commercially available raw materials. The 1H-1,2,4-triazol-3-yl
benzenesulfonamide derivatives 13–27 were synthesized using compounds 2–4, which were already
prepared on a large scale by our research group. This simple methodology afforded the sulfonamide
derivatives using the appropriate sulfonyl chlorides. According to the docking calculations, compounds
with CF3 groups at position 5 of the triazole ring must be carefully considered when designing new
1H-1,2,4-triazol-3-yl benzenesulfonamide derivatives as potential inhibitors of PfDHPS. Moreover,
halogen groups at the 4' position increase the ligand-enzyme interaction. Overall, these results suggest
that N-(5-trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamides are potential lead compounds for
the development of new antimalarial drugs. The results reported herein should be useful in guiding
future efforts to discover compounds with increased antimalarial activity. Consequently, the reported
compounds are currently undergoing in vitro antimalarial tests.

Molecules 2011, 16
8095
Acknowledgements
The authors thank the Foundation for Research of the State of Rio de Janeiro (FAPERJ), National
Council of R&D (CNPq), Institute of Pharmaceutical Technology (Farmanguinhos) and Program of
Technological Development in Health Products of Oswaldo Cruz Foundation (PDTIS-FIOCRUZ) for
financial support.
References
1. World Health Organization (WHO). 10 Facts on Malaria. Available online:
http://www.who.int/features/factfiles/malaria/en/index.html/ (accessed on 10 March 2011).
2. World Health Organization (WHO). Global report on antimalarial drug efficacy and drug resistance:
2000–2010. Available online: http://www.who.int/malaria/en (accessed on 10 March 2011).
3. Kaur, K.; Jain, M.; Reddy, R.P.; Jain, R. Quinolines and structurally related heterocycles as
antimalarials. Eur. J. Med. Chem. 2010, 45, 3245-3264.
4. Biot, C.; Chibale, K. Novel approaches to antimalarial drug discovery. Infect. Disord.-Drug
Targets 2006, 6, 173-204.
5. Welch, J.T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry; John Wiley and Sons Ltd:
New York, NY, USA, 1991.
6. Ojima, I. Fluorine in Medicinal Chemistry and Chemical Biology; John Wiley and Sons Ltd:
Chichester, UK, 2009.
7. Elliott, A.J.M. Chemistry of Organic Fluorine Compounds. II. A Critical Review, ACS
Monograph 187; Hudlicky, M., Pavlath, A.E., Eds.; American Chemical Society: Washington,
DC, USA, 1995; pp. 1119-1125.
8. Kirk, K.L. Fluorination in medicinal chemistry: Methods, strategies, and recent developments.
Org. Process. Res. Dev. 2008, 12, 305-321.
9. Purser, S.; Moore, P.R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem.
Soc. Rev. 2008, 37, 320-330.
10. Smart, B.E. Fluorine substituent effects (on bioactivity). J. Fluorine Chem. 2001, 109, 3-11.
11. Park, B.K.; Kitteringham, N.R.; O’Neill, P.M. Metabolism of fluorine containing drugs. Ann. Rev.
Pharmacol. Toxicol. 2001, 41, 443-470.
12. Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: Looking beyond intuition.
Science 2007, 317, 1881-1886.
13. Boechat, N; Bastos, M.M. Trifluoromethylation of carbonyl compounds. Curr. Org. Synth. 2010,
7, 403-413.
14. Kgokong, J.L.; Smith, P.P.; Matsabisa, G.M. 1,2,4-Triazino-[5,6-b]indole derivatives: Effects of
the trifluoromethyl group on in vitro antimalarial activity. Bioorg. Med. Chem. 2005, 13, 2935-2942.
15. Jonet, A.; Dassonville-Klimpt, A.; Do Nascimento, S.; Leger, J-M.; Guillon, J. First enantioselective
synthesis of 4-aminoalcohol quinoline derivatives through a regioselective SN2 epoxide opening
mechanism. Tetrahedron: Asymmetry 2011, 22, 138-148.

Molecules 2011, 16
8096
16. Kabri, Y.K.; Azas, N.; Dumetre, A.; Hutter, S.; Laget, M.; Verhaeghe, P.; Gellis, A.; Vanelle, P.
Original quinazoline derivatives displaying antiplasmodial properties. Eur. J. Med. Chem. 2010,
45, 616-622.
17. Dias, L.C.; Dessoy, M.A.; Silva, J.J.N.; Thiemann, O.H.; Oliva, G.; Andricopulo, A.D.
Quimioterapia da Doença de Chagas: Estado da Arte e Perspectivas no Desenvolvimento de
Novos Fármacos. Quim. Nova 2009, 32, 9, 2444-2457.
18. Al-Abdely, H.M.; Graybill, J.R.; Loebenberg, D.; Melby, P.C. Efficacy of the triazole SCH 56592
against Leishmania amazonensis and Leishmania donovani in experimental murine cutaneous and
visceral leishmaniases. Antimicrob. Agents Chemother. 1999, 43, 2910-2914.
19. Guantai, E.M.; Ncokazi, K.; Egan, T.J.; Gut, J.; Rosenthal, P.J.; Smith, P.J.; Chibale, K. Design,
synthesis and in vitro antimalarial evaluation of triazole-linked chalcone and dienone hybrid
compounds. Bioorg. Med. Chem. 2010, 18, 8243-8256.
20. Hans, R.H.; Gut, J.; Rosenthal, P.J.; Chibale, K. Comparison of the antiplasmodial and falcipain-2
inhibitory activity of β-amino alcohol thiolactone-chalcone and isatin-chalcone hybrids.
Bioorg. Med. Chem. Lett. 2010, 20, 2234-2237.
21. Vlahakis, J.Z.; Lazar, C.; Crandall, I.E.; Szarek, W.A. Anti-Plasmodium activity of imidazolium
and triazolium salts. Bioorg. Med. Chem. 2010, 18, 6184-6196.
22. Mishra, N.; Arora, P.; Kumar, B.; Mishra, L.C.; Bhattacharya, A.; Awasthi, S.K.; Bhasin, V.K.
Synthesis of novel substituted 1,3-diaryl propenone derivatives and their antimalarial activity
in vitro. Eur. J. Med. Chem. 2008, 43, 1530-1535.
23. D’hooghe, M.; Kenis, S.; Vervisch, K.; Lategan, C.; Smith, P.J.; Chibale, K.; De Kimpe, N.
Synthesis of 2-(aminomethyl)aziridines and their microwave-assisted ring opening to 1,2,3-
triaminopropanes as novel antimalarial pharmacophores. Eur. J. Med. Chem. 2011, 46, 579-587.
24. Krungkrai, J.; Krungkrai, S.R.; Supuran, C.T. Carbonic anhydrase inhibitors: Inhibition of
Plasmodium falciparum carbonic anhydrase with aromatic/heterocyclic sulfonamides—In vitro
and in vivo studies. Bioorg. Med. Chem. Lett. 2008, 18, 5466-5471.
25. Krungkrai, J.; Scozzafava, A.; Reungprapavut, S.; Krungkrai, S.R.; Rattanajak, R.;
Kamchonwongpaisan, S.; Supuran, C.T. Carbonic anhydrase inhibitors. Inhibition of Plasmodium
falciparum carbonic anhydrase with aromatic sulfonamides: Towards antimalarials with a novel
mechanism of action? Bioorg. Med. Chem. 2005, 13, 483-489.
26. Johann, L.; Pegraro, S.; Dormeyer, M.; Michael, L.; Aschenbrenner, A.; Karmer, B. Sulfonyl-
phenyl-ureido benzamidines: A novel structural class of potent antimalarial agents. Bioorg. Med.
Chem. Lett. 2004, 14, 1979-1982.
27. Domınguez, J.N.; Leon, C.; Rodrigues, J.; Domınguez, N.G.; Gut, J.; Rosenthal, P.J. Synthesis
and antimalarial activity of sulfonamide chalcone derivatives. Il Farmaco 2005, 60, 307-311.
28. Ryckebusch, A.; Deprez-Poulain, R.; Debreu-Fontaine, M.A.; Vandaele, R.; Mouray, E.;
Grellier, P.; Sergheraert, C. Parallel synthesis and anti-malarial activity of a sulfonamide library.
Bioorg. Med. Chem. Lett. 2002, 12, 2595-2598.
29. Posner, G.H.; Maxwell, J.P.; O'Dowd, H.; Krasavin, M.; Xie, S.; Shapiro, T.A. Antimalarial
sulfide, sulfone, and sulfonamide trioxanes. Bioorg. Med. Chem. 2000, 8, 1361-1370.
30. Parai, M.K.; Panda, G.; Srivastava, K.; Puri, S.K. Design, synthesis and antimalarial activity of
benzene and isoquinoline sulfonamide derivatives. Bioorg. Med. Chem. Lett. 2008, 18, 776-781.

Molecules 2011, 16
8097
31. Martyn, D.C.; Cortese, J.F.; Tyndall, E.; Dick, J.; Mazitschek, R.; Munoz, B.; Clardy, J.
Antiplasmodial activity of piperazine sulfonamides. Bioorg. Med. Chem. Lett. 2010, 20, 218-221.
32. Ekoue-Kovi, K.; Yearick, K.; Iwaniuk, D.P.; Natarajan, J.K.; Alumasa, J.; Dios, A.C.;
Roepe, P.D.; Wolf, C. Synthesis and antimalarial activity of new 4-amino-7-chloroquinolyl
amides, sulfonamides, ureas and thioureas. Bioorg. Med. Chem. 2009, 17, 270-283.
33. Kasekarn, W.; Sirawaraporn, R.; Chahomchuena, T.; Cowman, A.F.; Sirawaraporn, W. Molecular
characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate
synthase from Plasmodium falciparum. coli. Mol. Biochem. Parasitol. 2004, 137, 43-53.
34. Bzik, D.J.; Li, W.B.; Horii, T.; Inselburg, J. Molecular cloning and sequence analysis of the
Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl. Acad. Sci.
USA 1987, 84, 8360-8364.
35. Biswas, S.; Escalante, A.; Chaiyaroj, S.; Angkasekwinai, P.; Lal, A.A. Prevalence of point
mutations in the dihydrofolate re-ductase and dihydrofolate synthetase genes of Plasmodium
falciparum isolates from India and Thailand: A molecular epidemiologic study. Top. Med. Int.
Health 2000, 10, 737-743.
36. Triglia, T.; Cowman, A.F. Primary structure and expression of the dihydropteroate synthetase
gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 1994, 91, 7149-7153.
37. Paul, B.K.; Ghosh, S.N.; Bose, A.N.; Basu, U.P. Search for oral hypoglycemic agents. I. Synthesis
and biological activity of some 5-arylsulfonamido-1,2,4-triazoles. Indian J. Chem. 1968, 6, 618-620.
38. McColl, J.D.; Lee, C.F.; Hajdu, A. Effect of sulfonylurea derivatives in experimental ulcer
formation in the rat. Arch. Int. Pharm. Ther. 1963, 141, 181-189.
39. Joshi, K.C.; Singh, V.K. Potential organo-fluorine oral hypoglycemic agents. IV. Synthesis and
hypoglycemic activity of some 5-arylsulfonamido-1,2,4-triazoles. J. Prakt. Chem. 1971, 313,
169-173.
40. Achari, A.; Somers, D.O.; Champness, J.N.; Bryant, P.K.; Rosemond, J.; Stammers, D.K. Crystal
structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase. Nat. Struct. Biol.
1997, 4, 490-497.
41. Molegro Virtual Docker/4.0 Molegro ApS: Aarhus, Denmark, 2009.
42. Benson, D.A.; Karsch-Mizrachi, I.; Lipman, D.J.; Ostell, J.; Sayers, E.W. GenBank. Nucleic Acids
Res. 2009, 37 (Suppl. 1), D26-D31.
43. Cornell, W.D.; Cieplak, P.; Bayly, C.I.; Gould, I.R.; Merz, K.M., Jr; Ferguson, D.M.;
Spellmeyer, D.C.; Fox, T.; Caldwell, J.W.; Kollman, P.A. A second generation force field for the
simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 1995, 117,
5179-5197.
44. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search
tool. J. Mol. Biol. 1990, 215, 403-410.
Sample Availability: Not available
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).