Synthesis, biological, and theoretical evaluations of new 1,2,3-triazoles against the hemolytic profile of the Lachesis muta snake venom

Article abstract of DOI:10.1016/j.bmc.2009.09.031

The current treatment used against envenomation by Lachesis muta venom still presents several side effects. This paper describes the synthesis, pharmacological and theoretical evaluations of new 1-arylsulfonylamino-5-methyl- 1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters (8a-f) tested against the hemolytic profile of the L muta snake venom. Their structures were elucidated by one- and two-dimensional NMR techniques (¹H, APT, HETCOR ¹J_CH and ¹¹J_CH,. n = 2, 3) and high-resolution electrospray ionization mass spectrometry. The series of triazole derivatives significantly neutralized the hemolysis induced by L. muta crude venom presenting a dose-dependent inhibitory profile (IC₅₀- 30-83 μM) with 1-(4'chlorophenylsulfonylamino)-5-methyl-1 H-[1,2,3 ]-triazole-4-carboxylic acid ethyl ester (8e) being the most potent compound. The theoretical evaluation revealed the correlation of the antiophidian profile with the coefficient distribution and density map of the Highest Occupied Molecular Orbitals (HOMO) of these molecules. The elucidation of this new series may help on designing new and more efficient antiophidian molecules.

Full text of DOI:10.1016/j.bmc.2009.09.031

Bioorganic & Medicinal Chemistry 17 (2009) 7429–7434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological, and theoretical evaluations of new 1,2,3-triazoles
against the hemolytic profile of the Lachesis muta snake venom
Vinícius R. Campos a, Paula A. Abreu b, Helena C. Castro b, Carlos R. Rodrigues c, Alessandro K. Jordão a,
Vitor F. Ferreira a, Maria C. B. V. de Souza a, Fernanda da C. Santos a, Laura A. Moura b, Thaisa S. Domingos b,
a,
Carla Carvalho b, Eládio F. Sanchez d, André L. Fuly b, , Anna C. Cunha
*
*
a Universidade Federal Fluminense, Departamento de Química Orgânica, Programa de Pós-Graduação em Química, Outeiro de São João Batista, s/n°, Niterói 24020-141,
Rio de Janeiro, Brazil
b Departamento de Biologia Celular e Molecular, Instituto de Biologia, Outeiro de São João Baptista, 24020-141 Universidade Federal Fluminense, Niterói, RJ, Brazil
c Laboratório de Modelagem Molecular e QSAR (ModMolQSAR), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, CEP 21941-590, RJ, Brazil
d Fundação Ezequiel Dias, Centro de Pesquisa e Desenvolvimento, Belo Horizonte, MG, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2009
Revised 16 September 2009
Accepted 17 September 2009
Available online 24 September 2009
The current treatment used against envenomation by Lachesis muta venom still presents several side
effects. This paper describes the synthesis, pharmacological and theoretical evaluations of new 1-aryl-
sulfonylamino-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters (8a–f) tested against the
hemolytic profile of the L. muta snake venom. Their structures were elucidated by one- and two-dimen-
sional NMR techniques (1H, APT, HETCOR 1JCH and nJCH, n = 2, 3) and high-resolution electrospray ioniza-
tion mass spectrometry. The series of triazole derivatives significantly neutralized the hemolysis induced
Keywords:
Triazoles
Diazo compounds
Snake venoms
Biological activities
Lachesis muta
by L. muta crude venom presenting a dose-dependent inhibitory profile (IC50 = 30ꢀ83
l
M) with 1-(40-
chlorophenylsulfonylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl ester (8e) being the
most potent compound. The theoretical evaluation revealed the correlation of the antiophidian profile
with the coefficient distribution and density map of the Highest Occupied Molecular Orbitals (HOMO)
of these molecules. The elucidation of this new series may help on designing new and more efficient
antiophidian molecules.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
proteins, phospholipases A2 (sPLA2s) isoforms6–8 constitute the
major toxic components of the snake venoms. They exhibit a wide
The family Viperidae, represented by three main genera Crotalus
(rattlesnakes), Bothrops (lance-heads), and Lachesis (bushmaster, in
Brazil popularly known as surucucu), consists in the most important
group of snakes regarding public health issues since they are respon-
sible for most of the serious ophidian accidents reported not only in
Brazil but also in other Western countries.1–4 This latter genus com-
prises two subspecies: Lachesis muta muta, found in the Amazon
tropical forest, and Lachesis muta rhombeata, distributed in the
Atlantic forest of the eastern regions of Brazil.3,4
Envenomation by snakes of the family Lachesis usually causes
local tissue damage, including edema, hemorrhage, and myonecro-
sis as well as systemic disorders such as nausea, vomiting, diar-
rhea, hypotension and bradycardia, coagulation disturbs, and
renal malfunction.4 The venom of Lachesis muta contains several
components such as highly active enzymes, arginyl-ester hydrolase
and thrombin-like, hemorrhagic and neurotoxic substances, and
fibrinogenase and kininogenase substances.2,5 Among the bioactive
range of pharmacological effects, that is, pre- or postsynaptic
neurotoxicity, cardiotoxicity, myotoxicity, effects on platelet func-
tion, edema, hemolysis, anticoagulation, convulsion and hypoten-
sion, and their catalytic activity on cell membranes of specific
tissues suggests an important role in venom toxicity.9,10
Nowadays, the regular treatment for ophidic accidents is the
parentheral use of antiserum obtained from hyperimmunized
equines. However, this treatment presents drawbacks including
poor availability in distant regions, refrigerated storage need and
some allergic reactions. In addition, despite systemic and lethal ef-
fects are usually reversed or avoided, local tissue damages are not,
which lead to a high morbidity.11 Therefore, the search for new
natural and synthetic compounds that can prevent the toxins from
reaching the mammalian targets and acting as cytotoxic agents is
still relevant.12
Several natural compounds with activity against snake venoms
have been reported in the literature.13,14 For example, koninginins15
(1–2), rosmarinic acid16 (3), and pterocarpans12 (4) are snake venom
phospholipase A2 inhibitors isolated from Trichoderma koningii, Cor-
dia verbenacea, and cabenegrina A–I, respectively.
* Corresponding authors. Tel.: +55 21 26292148; fax: +55 21 26292135 (A.C.C.).
E-mail address: annac@vm.uff.br (A.C. Cunha).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.031

7430
V. R. Campos et al. / Bioorg. Med. Chem. 17 (2009) 7429–7434
Suramin (5) is a polysulfonated napthylurea that was originally
the new 1,2,3-triazole derivatives 8a–f (Scheme 1). The known sul-
fonylhydrazides 10a–f were prepared in excellent yields by adding
the corresponding arylsulfonyl chlorides 9a–f dissolved in THF to a
slightly excess of hydrazine hydrate solution 80%, according to the
procedure described in the literature28 (Scheme 1). The reaction
yields and the melting points of new triazole derivatives are listed
in Table 1.
developed for the treatment of African trypanosomiasis and oncho-
cerciasis.17–19 This charged compound also prevents muscle necro-
sis induced by some snake venoms, inhibiting their myotoxicity
and in vitro neuromuscular blocking activities of Lys49 phospho-
lipases A2 from Bothrops species. The benzoyl phenyl benzoate 6
is an effective inhibitor of phospholipase A2 and hyaluronidase en-
zymes in several snake families (e.g., Elapidae, Viperidae, and
Crotalidae).20 The imidazolic compound 7 exhibits a significant
7
PLA2 enzyme inhibitory activity against group II PLA2 (Fig. 1).
2.2. Antihemolytic effect and structure–activity relationship
analysis
Literature has described different classes of 1,2,3-triazole com-
pounds with different pharmacological profiles. There are reports
of triazole analogs as antiplatelet agents,21 dopamine D2 receptor li-
gands for treating schizophrenia,22 tripanocidal,23 antimycobacteri-
al,24 and leishmanicidal diseases.25 1,2,3-Triazole has been elected
as a leadcompoundgroup for designing active molecules against dif-
ferent pathologies such as thrombosis, including recently by our
group.26 In this previous work we synthesized several 1,2,3-triazole
derivatives based on N0-[(40-bromophenyl)methylene)]-1-(p-chlo-
rophenyl)-1H-[1,2,3]-triazole-4-carbohydrazide and identified
them as significant platelet aggregation inhibitors.21,26
As part of an ongoing research program on the synthesis of new
biologically active triazoles and on the basis of our experience in
the field of the use of diazo compounds in organic synthesis, herein
we synthesized a novel family of 1-arylsulfonylamino-5-methyl-
1H-[1,2,3]-triazole-4-carboxylate derivatives, and evaluated their
ability to neutralize L. muta snake venom hemolytic activity and
their structure–activity relationship (SAR) by using a molecular
modeling approach.
The biological assay revealed that all triazole derivatives 8a–f
were able to completely inhibit the L. muta crude venom-induced
hemolysis at 130
dependent inhibitory profile with IC50 that ranges from 30 to
83 M (Fig. 2). Importantly, those compounds did not interfere
on the integrity of neither rabbit nor human erythrocytes when
lM. In fact, these compounds presented a dose-
l
tested alone, even up to 390
to such cells.
lM, inferring that they are not toxic
Since the new synthesized triazole derivatives presented this
promising antiophidian effect in a dose-dependent manner, we
performed a structure–activity relationship (SAR) analysis to corre-
late the stereoelectronic properties of the molecules with their
antihemolytic profile (Table 2).
The cross-correlation matrix showed that energy of HOMO,
electrostatic charge of the substituted carbon atom in phenyl ring,
lipophilicity and water solubility are the most correlated descrip-
tors to the experimental IC50 (Table 3). The most active compounds
8a, 8b, 8e, and 8f showed higher lipophilicity as well as lower
water solubility. In addition, low LUMO energies values are also
apparently related to these derivatives biological profile where
the most active compounds presented the lowest values for these
orbital energies (Table 2).
2. Results and discussion
2.1. Chemistry
The synthesis of new 1-arylsulfonylamino-5-methyl-1H-[1,2,3]-
triazole-4-carboxylic acid ethyl esters 8a–f is shown in Scheme 1.
The ethyl 2-diazoacetoacetate 12, prepared in 70% yield by the
method of Danheiser et al.,27 was condensed with arylsulfonylhyd-
razides 10a–f, giving the corresponding diazo-hydrazone interme-
diates 14a–f, which underwent 1,5-electrocyclization leading to
The analysis of HOMO density maps and coefficient distribution
showed a different distribution in the most active compounds with
a higher HOMO density concentrated in the triazole ring, whereas
in the less active derivatives, HOMO orbitals were concentrated in
the phenyl ring. Since the frontier orbitals are usually important for
ligand–target interaction, probably the location of HOMO in the
OH
O
R1
O
OH
R
HO
R
O
O
HO
R1
OH
OH
O
OH
R2
OH
H
O
1, Koninginin A
H
2a, R= OH, R1 = H and R2 = OH, Koninginin E
2b
O
O
HOOC
O
H
, R= OH, R1 = OH and R2 = H, Koninginin F
O
OH
3, Rosmarinic acid
R2
4a
, R =
, R1 = R2 = H
, R = R2 = H
OH
4b,
O
R1
=
N
NaO3S
NaO3S
H
Me
O
H
N
Me
SO3Na
O
Cl
N
O
H
COOEt
O
Cl
O
N
SO3Na
Cl
O
O
N
N
N
H
H
N
H
N
H
O
7
6
Cl
SO3Na
NaO3S
5, Suramin
Figure 1. Natural 1–4 and synthetic compounds 5–7 active against snakebites.

V. R. Campos et al. / Bioorg. Med. Chem. 17 (2009) 7429–7434
7431
SO2NHNH2
O
O
10
N
N
N
K2CO3 / CH2Cl2
O
N
N
N
O
O
O
O
O
Me
O
Me
R1
Me
OEt
Me
OEt
HN
Me
13a-f
HN
Me
N2
12
MeOH/AcOH
24 h, 25oC
H3C
S
N3
SO2
SO2
14a-f
11
O
R1
R1
O
O
SO2NHNH2
THF/ NH2NH2
81-92%
SO2Cl
N
10a, R1= H
10b, R1= Me
10c, R1= NH2
N
Me
N
8a,
R1= H
9
8b, R1= Me
8c, R1= NH2
8d, R1= OMe
8e, R1= Cl
N
R1
R1
10d
SO2
, R1= OMe
H
10e, R1= Cl
10f, R1= NO2
8f,
R1= NO2
R1
Scheme 1. Synthetic pathways used for 8a–f.
Table 1
biological activity observed (Table 2). As these substituents may be
interacting with the unknown target through noncovalent interac-
tion, apparently their nature is more important for the biological
activity than their influence on phenyl carbon to which is attached.
In this work, we submitted the new series of triazole derivatives
to the analysis29 of ‘Lipinski Rule of Five’ that indicates if a chemical
compound could be an orally active drug in humans. The ‘Rule of
Five’ states that a compound violating any two of the following rules
is likely to be poorly absorbed:29 (1) molecular weight less than
500 Da; (2) number of hydrogen bond donors (OH or NH groups)
equal to or less than 5; (3) number of hydrogen bond acceptors less
than 10; and (4) calculated clog P less than 5. Our results showed
that all compounds fulfilled this rule (molecular weight = 296.31–
341.30, clog P = 2.6–3.4, nHBA = 8–11 and nHBD = 1–3) pointing
for a good theoretical biodisponibility of this series (Table 2).
Yields and melting points (mp) of the 1-arylsulfonylamino-5-methyl-1H-[1,2,3]-
triazole-4-carboxylic acid ethyl esters 8a–f
Sulphonylhydrazides
1,2,3-Triazole
derivatives
R1
Mp (°C)
Yield (%)
10a
10b
10c
10d
10e
10f
8a
8b
8c
8d
8e
8f
H
151–152
140–141
200–203
173–175
155–157
195–197
60
61
55
60
60
63
Me
NH2
OMe
Cl
NO2
3. Conclusion
In summary, a novel family of 1-arylsulfonylamino-5-methyl-
1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters 8a–f has been
synthesized and evaluated for its ability to neutralize L. muta snake
venom’s hemolytic activity. All the compounds were able to neu-
tralize hemolytic property of venom. Based on literature,30,31 the
triazole derivatives could be possibly affecting the L. muta venom
phospholipase A2 that is involved in the hemolytic profile of this
snake venom. Since triazoles 8a–f presented a promising antihe-
molytic profile, these compounds may be useful as prototypes for
designing new antiophidian molecules to improve the current
treatment used for L. muta bites.
4. Experimental
Chemical reagents and all solvents used in this study were pur-
chased from Merck AG (Darmstadt, Germany) and VETEC LTDA
(Rio de Janeiro, Brazil). Melting points were determined with a
Fisher-Johns instrument and are uncorrected. Infrared (IR) spectra
were recorded on Perkin–Elmer FT-IR model 1600 senes spectro-
photometer, in KBr pellets. NMR spectra, unless otherwise stated,
were obtained in deuterated CDCl3 using a Varian Unity Plus
300 MHz spectrometer. Chemical shifts (d) are expressed in ppm
and the coupling constants (J) in hertz. High-resolution electro-
spray ionization mass spectrometry (HR-ESI-MS) was performed
in positive ion mode on a Waters-Micromass Q-Tof Micro instru-
ment. The progress of all reactions was monitored by TLC per-
formed on 2.0 cm ꢁ 6.0 cm aluminum sheets precoated with
Figure 2. Effect of 1,2,3-triazoles derivatives 8a–f on L. muta-induced hemolysis.
Different concentrations of sulfonamide derivatives (16–130 M) were pre-incu-
bated with L. muta snake venom (90 g/mL) for 30 min and then hemolytic test
performed. Data are expressed as mean SD of three individuals experiments
(n = 3).
l
l
triazole group may be contributing to a better biological profile
(Fig. 3).
The electrostatic charge of the substituted carbon of phenyl ring
varied among the triazoles probably due to the nature of the sub-
stituents. However, these values were not directly correlated to the

7432
V. R. Campos et al. / Bioorg. Med. Chem. 17 (2009) 7429–7434
Table 2
Comparison of HOMO and LUMO energies (EHomo and ELumo), water solubility (log S), electrostatic charge of the substituted carbon of phenyl ring (Elect C), and Lipinski profile,
including lipophilicity (clog P), molecular weight (MW) and the number of hydrogen bond donor and acceptor groups (HBD and HBA, respectively) of the triazole derivatives
#
R1
IC50
(lM)
Energy (eV)
Elect C
Log S
Lipinski Rule of Five
cLog P
MW (Da)
nHBA
nHBD
HOMO
LUMO
8a
8b
8c
8d
8e
8f
H
41 4.1
44 5.8
63 3.4
83 5.5
30 0.57
44 4.5
ꢀ10.10
ꢀ10.05
ꢀ9.29
1.27
1.33
1.23
1.27
1.19
0.4
ꢀ0.047
0.547
0.329
0.665
0.235
0.038
ꢀ2.42
ꢀ2.76
ꢀ2.5
2.78
3.1
2.06
2.68
3.4
310.33
324.36
325.35
340.36
344.78
355.33
8
8
9
9
8
1
1
3
1
1
1
Me
NH2
OMe
Cl
ꢀ9.75
ꢀ2.44
ꢀ3.16
ꢀ2.88
ꢀ10.19
ꢀ10.36
NO2
2.65
11
Table 3
Cross-correlation matrix of the experimental biological activities (pIC50) for the triazole derivatives (8a–f) and the calculated parameters: EHomo and ELumo (HOMO and LUMO
energies, Ev), electrostatic charge of the substituted carbon of phenyl ring (Elect C), water solubility (log S),
l
(molecular dipole moment, Debye), MSA (molecular surface area, Å2),
MW (molecular weight, Da), clog P (calculated octanol/water partition coefficient), clog S (calculated water solubility), and nHBA (number of hydrogen bond acceptors, that is,
number of N and O atoms) and nHBD (number of hydrogen bond donors)
IC50
1.00
EHomo
ELumo
Elect C
Log S
cLog P
MW
nHBA
nHBD
l
MSA
PSA
IC50
EHomo
ELumo
Elect C
Log S
cLog P
MW
nHBA
nHBD
l
0.67
0.20
0.64
0.69
ꢀ0.61
0.02
0.21
0.31
0.49
0.48
0.17
1.00
0.50
0.45
1.00
0.49
0.36
1.00
0.16
0.02
0.59
1.00
ꢀ0.67
ꢀ0.62
ꢀ0.07
0.32
ꢀ0.72
ꢀ0.36
ꢀ0.15
0.85
0.14
1.00
0.17
ꢀ0.45
ꢀ0.77
ꢀ0.29
0.00
ꢀ0.70
ꢀ0.91
0.16
0.07
1.00
ꢀ0.22
0.68
ꢀ0.24
ꢀ0.72
0.78
1.00
0.07
0.06
1.00
0.46
0.78
0.90
0.56
0.60
ꢀ0.29
0.63
ꢀ0.66
1.00
MSA
PSA
ꢀ0.05
ꢀ0.39
ꢀ0.21
0.57
ꢀ0.19
ꢀ0.28
1.00
0.38
0.07
ꢀ0.83
ꢀ0.03
ꢀ0.63
0.52
0.94
0.38
ꢀ0.52
1.00
Figure 3. Coefficient distribution (up) and density maps (down) of the Highest Occupied Molecular Orbital (HOMO) of the compounds aligned according to the hemolysis IC50
potency (30–83 M). Blue and red areas in the density map indicate higher and lower HOMO energy values, respectively.
l
Silica Gel 60 (HF-254, E. Merck) to a thickness of 0.25 mm. The
developed chromatograms were viewed under ultraviolet light at
254 nm. E. Merck silica gel (60–200 mesh) was used for column
chromatography. Microanalyses were performed using a Perkin–
Elmer Model 2400 instrument and all values were within 0.4 of
the calculated compositions.
4.1. General procedure for the preparation of the 4-carbethoxy-
triazole derivatives 8a–f
To the sulfonylhydrazide solution (10a–f, 1 mmol) in MeOH/
acetic acid (5:1) (10 mL) was added ethyl 2-diazoacetoacetate
(0.156 g, 1 mmol). Stirring was kept, at room temperature, for

V. R. Campos et al. / Bioorg. Med. Chem. 17 (2009) 7429–7434
7433
24 h, and the resulting mixture was concentrated under reduced
4.1.5. 1-(40-Chlorophenylsulfonylamino)-5-methyl-1H-[1,2,3]-
pressure. The residue was purified by column chromatography
using silica gel and ethyl acetate:hexane (3:7) as eluent to give
the pure triazoles 8a–f.
triazole-4-carboxylic acid ethyl ester 8e
Obtained in 60% yield as a yellow solid; mp 155–157 °C; IR
(KBr) mmax (cmꢀ1) 3095 (N–H); 1700 (C@O), 1289 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.40 (t, 3H, J = 7.3, OCH2CH3), 2.61 (s, 3H,
CH3), 4.41 (q, 2H, J = 7.3, OCH2CH3), 7.41 (d, 2H, J = 8.8, H-20 and
H-60), 7.65 (d, 2H, J = 8.8, H-30 and H-50), 9.92 (br s, 1H, N–H)
ppm. 13C NMR (75 MHz, CDCl3) d: 9.1 (CH3), 14.2 (OCH2CH3),
61.5 (OCH2CH3), 129.4 (C-20 and C-60), 130.0 (C-30 and C-50),
134.7 (C-4 or C-5), 135.0 (C-10), 140.9 (C-4 or C-5), 141.0 (C-40),
160.9 (C@O) ppm. Anal. Calcd for C12H13ClN4O4S: C, 41.80; H,
3.80; N, 16.25. Found: C, 42.04; H, 4.09; N, 16.41. HRMS (ESI)
[M+Na]+ calcd for C12H13ClN4O4SNa 367.0238. Found: 367.0236.
4.1.1. 5-Methyl-1-(phenylsulfonylamino)-1H-[1,2,3]-triazole-4-
carboxylic acid ethyl ester 8a
Obtained in 60% yield as a yellow solid; mp 151–152 °C; IR
(KBr) mmax (cmꢀ1) 3099 (N–H); 1694 (C@O), 1292 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.40 (t, 3H, J = 7.1, OCH2CH3), 2.61 (s, 3H,
CH3), 4.42 (q, 2H, J = 7.1, OCH2CH3), 7.45–7.50 (m, 2H, H-30 and
H-50), 7.64 (tt, 1H, J = 7.5 and 1.2, H-40), 7.69–7.73 (m, 2H, H-20
and H-60), 9.38 (br s, 1H, N–H) ppm. 13C NMR (75 MHz, CDCl3) d:
9.1 (CH3), 14.3 (OCH2CH3), 61.3 (OCH2CH3), 128.5 (C-20 and C-60),
129.1 (C-30 and C-50), 134.2 (C-40), 134.6 (C-4 or C-5), 136.4
(C-10), 140.8 (C-4 or C-5), 160.8 (C@O) ppm. Anal. Calcd for
C12H14N4O4S: C, 46.44; H, 4.55; N, 18.05. Found: C, 46.48; H,
4.39; N, 17.09. HRMS (ESI) [M+Na]+ calcd for C12H14N4O4SNa
333.0627. Found: 333.0622.
4.1.6. 5-Methyl-1-(40-nitrophenylsulfonylamino)-1H-[1,2,3]-tri-
azole-4-carboxylic acid ethyl ester 8f
Obtained in 63% yield as a yellow solid; mp 195–197 °C; IR
(KBr) mmax (cmꢀ1) 3114 (N–H); 1719 (C@O), 1288 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.27 (t, 3H, J = 7.2, OCH2CH3), 2.53 (s, 3H,
CH3), 4.42 (q, 2H, J = 7.2, OCH2CH3), 8.0 (d, 2H, J = 8.9, H-20 and
H-60), 8.40 (d, 2H, J = 8.9, H-30 and H-50), 11.56 (br s, 1H, N–H)
ppm. 13C NMR (75 MHz, CDCl3) d: 9.0 (CH3), 14.0 (OCH2CH3), 60.6
(OCH2CH3), 124.6 (C-30 and C-50), 129.5 (C-20 and C-60), 134.3 (C-
4 or C-5), 140.0 (C-4 or C-5), 143.2 (C-10), 150.6 (C-40), 160.4
(C@O) ppm. Anal. Calcd for C12H13N5O6S: C, 40.56; H, 3.69; N,
19.71. Found: C, 40.63; H, 3.81; N, 19.63. HRMS (ESI) [M+Na]+ calcd
for C12H13N5O6SNa 378.0478. Found: 378.0492.
4.1.2. 5-Methyl-1-(40-methylphenylsulfonylamino)-1H-[1,2,3]-
triazole-4-carboxylic acid ethyl ester 8b
Obtained in 61% yield as a yellow solid; mp 140–141 °C; IR (KBr)
mmax (cmꢀ1) 3106 (N–H); 1697 (C@O); 1292 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.44 (t, 3H, J = 7.3, OCH2CH3), 2.43 (s, 3H,
CH3), 2.63 (s, 3H, CH3), 4.42 (q, 2H, J = 7.1, OCH2CH3), 7.29 (d, 2H,
J = 8.1, H-30 and H-50), 7.58 (d, 2H, J = 8.1, H-20 and H-60), 9.02 (br s,
1H, N–H) ppm. 13C NMR (75 MHz, CDCl3) d: 9.2 (CH3), 14.2
(OCH2CH3), 21.7, (CH3), 61.3 (OCH2CH3), 128.6 (C-20 and C-60),
130.0 (C-30 and C-50), 132.9 (C-10), 135.0 (C-4 or C-5), 140.8 (C-4 or
C-5), 145.9 (C-40), 160.9 (C@O) ppm. Anal. Calcd for C13H16N4O4S:
C, 48.14; H, 4.97; N, 17.27. Found: C, 48.03; H, 4.35; N, 16.63. HRMS
(ESI) [M+Na]+ calcd for C13H16N4O4SNa 347.0784. Found: 347.0785.
4.2. Antiophidic assays
4.2.1. Snake venom and antiserum
L. muta snake venom and anti-lachesis serum were provided
from Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil.
4.3. Antihemolytic activity
4.1.3. 1-(40-Aminophenylsulfonylamino)-5-methyl-1H-[1,2,3]-
triazole-4-carboxylic acid ethyl ester 8c
The hemolytic activity of L. muta venom was determined by the
indirect hemolytic test using rabbit erythrocytes and hen’s egg
yolk emulsion as substrate.31 The activity was performed in a
two-step reaction including (1) incubation of L. muta crude venom
with egg yolk emulsion and (2) measurement of the hemolytic
capacity of released lysolecithin by monitoring the hemoglobin at
A578 nm. The compounds were pre-incubated with L. muta crude
venom for 30 min at room temperature and then hemolytic activ-
ity was evaluated. The Inhibitory Concentration (IC50) was deter-
Obtained in 55% yield as a yellow solid; mp 200–203 °C; IR
(KBr) mmax (cmꢀ1) 2997 (N–H); 1699 (C@O), 1264 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.27 (t, 3H, J = 7.2, OCH2CH3), 2.53 (s, 3H,
CH3), 4.42 (q, 2H, J = 7.2, OCH2CH3), 7.29 (d, 2H, J = 8.7, H-20 and
H-60), 7.61 (d, 2H, J = 9.0, H-30 and H-50), 12.1 (br s, 2H, NH2),
6.40 (br s, 1H, N–H) ppm. 13C NMR (75 MHz, CDCl3) d: 9.0 (CH3),
9.6 (OCH2CH3), 60.6 (OCH2CH3), 112.7 (C-30 and C-50), 120.3
(C-10), 130.2 (C-20 and C-60), 134.1 (C-4 or C-5), 140.1 (C-4 or C-
5), 154.3 (C-40), 160.5 (C@O) ppm. Anal. Calcd for C12H15N5O4S:
C, 44.30; H, 4.65; N, 21.53. Found: C, 44.68; H, 5.07; N, 20.15. HRMS
(ESI) [M+Na]+ calcd for C12H15N5O4SNa 348.0736. Found:
348.0738.
mined as the concentration of compound (lM) able to inhibit
50% of hemolysis caused by snake venom.
4.4. Statistical analysis
Results are expressed as means SD obtained with the indi-
cated number of antihemolytic assays performed. The statistical
significance of differences among experimental groups was evalu-
ated using ANOVA test. P value of <0.05 was considered statisti-
cally significant.
4.1.4. 5-Methyl-1-(40-methoxiphenylsulfonylamino)-1H-[1,2,3]-
triazole-4-carboxylic acid ethyl ester 8d
Obtained in 60% yield as a yellow solid; mp 173–175 °C; IR
(KBr) mmax (cmꢀ1) 3109 (N–H); 1699 (C@O), 1264 (C–O); 1H NMR
(300.00 MHz, CDCl3) d: 1.40 (t, 3H, J = 7.2, OCH2CH3), 2.65 (s, 3H,
CH3), 3.87 (s, 3H, OCH3), 4.41 (q, 2H, J = 7.2, OCH2CH3), 6.93 (d,
2H, J = 8.9, H-30 and H-50), 7.64 (d, 2H, J = 8.9, H-20 and H-60), 9.70
(br s, 1H, N–H) ppm. 13C NMR (75 MHz, CDCl3) d: 9.2 (CH3), 14.2
(OCH2CH3), 55.7 (OCH3), 61.3 (OCH2CH3), 114.5 (C-30 and C-50),
127.3 (C-10), 130.9 (C-20 and C-60), 135.0 (C-4 or C-5), 140.8 (C-4
or C-5), 160.9 (C@O), 164.3 (C-40) ppm. Anal. Calcd for
C13H16N4O5S: C, 45.88; H, 4.74; N, 16.46. Found: C, 45.71; H,
4.96; N, 17.29. HRMS (ESI) [M+Na]+ calcd for C13H16N4O5SNa
363.0733. Found: 363.0724.
4.5. Molecular modeling
4.5.1. Structure–activity relationship (SAR) evaluation
The non-substituted derivative 5-methyl-1-(phenylsulfonyla-
mino)-1H-[1,2,3]-triazole-4-carboxylic acid ethyl ester 8a was sub-
mitted to the default systematic conformational analysis
procedure, available in the SPARTAN’06 software package (Wavefunc-
tion Inc. Irvine, CA, 2000), using the MMFF94 force field,32 and the

7434
V. R. Campos et al. / Bioorg. Med. Chem. 17 (2009) 7429–7434
10. White, J. Toxicon 2005, 45, 951.
most stable conformer was used to construct the other derivatives
8b–f. In order to evaluate the stereoelectronic properties, all
structures were submitted to a full geometry optimization process,
using the Recife Model 1 (RM1) semi-empirical Hamiltonian,33 and,
subsequently, to a single-point energy ab initio calculation, using
Hartree–fock method at 6–311+G level available in SPARTAN’06.
Then, some electronic properties, such as HOMO (Highest Occu-
pied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular
Orbital) energy and orbital coefficients distribution, molecular di-
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34
**
pole moment (l), and molecular electrostatic potential (MEP)
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Acknowledgments
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The authors are grateful to FAPERJ, CAPES, CNPq, and the Inter-
national Foundation for Science (IFS-Grant F/4571-1) for financial
support and fellowships and would like to thank Universidade Fed-
eral do Rio de Janeiro, Departamento de Química Orgânica, LA-
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obtaining the mass spectra data.
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