Design, synthesis and effect of triazole derivatives against some toxic activities of Bothrops jararaca venom

Article abstract of DOI:10.1007/s00044-020-02653-x

According to the World Health Organization, snakebite envenoming is a neglected disease that affects around 5.4 million people worldwide each year. In Brazil, in 2019 there were 29,000 cases of accidents, with 104 deaths. The genus Bothrops was responsibl

Full text of DOI:10.1007/s00044-020-02653-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02653-x
ORIGINAL RESEARCH
Design, synthesis and effect of triazole derivatives against some
toxic activities of Bothrops jararaca venom
Aldo R. da Silva1 Ana Cláudia R. da Silva1 Marcio Roberto H. Donza2 Gabriel Alves S. de Aquino2
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Carlos R. Kaiser2 Eladio F. Sanchez3 Sabrina B. Ferreira2 André L. Fuly
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Received: 9 July 2020 / Accepted: 8 October 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
According to the World Health Organization, snakebite envenoming is a neglected disease that affects around 5.4 million
people worldwide each year. In Brazil, in 2019 there were 29,000 cases of accidents, with 104 deaths. The genus Bothrops
was responsible for 90% of reported envenomations, mainly the species B. jararaca. The current therapy is performed with
an intravenous injection of antivenom, be it monovalent or polyvalent. However, this treatment has high manufacturing
costs, may induce side effects, and it does not effectively neutralize tissue necrosis. The latter issue may lead to deformity or
amputation of the affected limb. Therefore, new treatments are needed to aid or improve the efficacy of antivenoms. In this
study, nine triazole compounds (AM11–AM19) were chemically synthesized, characterized using infrared (IR) and nuclear
magnetic resonance (NMR) spectroscopy analyses, and tested against some in vitro (hemolysis, coagulation, and proteolysis)
and in vivo (hemorrhaging, lethal, and edema) activities of B. jararaca venom. Each compound was incubated with B.
jararaca venom (incubation protocol) or injected after the venom (treatment protocol), and then, biological assays were
performed. As a result, all the compounds inhibited the toxic activities of B. jararaca venom with different potencies in the
incubation protocol, while the compound AM13 inhibited hemorrhaging in the treatment protocol. In addition, the
compounds were devoid of toxicity, as shown through admetSAR analysis or in vitro cytotoxicity test. Thus, these
compounds may be an important tool for the development of antivenom molecules to improve serum therapy for recovering
patients envenomed by B. jararaca venom.
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Keywords Synthesis Triazole compounds Antivenom Snake Bothrops jararaca venom Neutralization
Introduction
However, the exact number of cases is unknown [2].
Besides deaths, 300,000 amputations and other permanent
disabilities occur due to snakebites. Thus, more efficient
treatments at impairing local effects of the venom should be
considered.
In Brazil, most cases of envenoming are caused by the
Bothrops genus (87%), followed by Crotalus (8%) and
Lachesis (2%) [3]. Within the Bothrops genus, the species
B. jararaca is responsible for the majority of accidents,
around 54%, and has a lethality index of 0.7% [4, 5]. B.
jararaca is a highly venomous pit viper, located in southern
Brazil, reaching a maximum length of 160 centimeters, with
typical envenomation symptoms including local hemorrha-
ging, inflammation, pain, massive tissue necrosis, bleeding,
renal and cardiac failure, and death [6, 7]. Besides death,
morbidities can occur as well.
Snakebite envenoming is a serious public health problem
worldwide, but with the highest incidence in the Americas.
According to the World Health Organization (WHO), sna-
kebite envenoming is a neglected disease, affecting 5.4
million people, with 100,000 deaths each year [1].
* André L. Fuly
andrefuly@id.uff.br
1
Department of Molecular and Cellular Biology, Institute of
Biology, Federal Fluminense University, Niterói, RJ 24020-141,
Brazil
2
Department of Organic Chemistry, Institute of Chemistry, Federal
University of Rio de Janeiro, Rio de Janeiro, RJ 21949-900, Brazil
Intravenous injection of antivenom is the official treat-
ment used to block toxic effects of venoms. In general,
antivenoms have been produced by hiperimmunization of
3
Center of Research and Development, Ezequiel Dias Foundation,
Belo Horizonte, MG 30510-010, Brazil

Medicinal Chemistry Research
horses, yielding polyvalent or monovalent products, which
are efficient to protect patients from death. However, anti-
venoms are unsatisfactory in blocking local effects, like
tissue necrosis, and this may lead to amputation or dis-
abilities of the affected limb [8, 9]. Antivenoms may induce
fever or anaphylactic reactions and their production and
manufacturing have high costs [10]. Thus, seeking for
alternative therapies that efficiently inhibit local effects and
have lower costs deserve more efforts. Natural products
derived from plants [11, 12] or seaweed [13] have been
tested, and some of them showed promising results. How-
ever, molecules derived from organic synthesis have not
been deeply investigated as antivenoms. Triazole com-
pounds belong to a five-membered ring family with two
carbons and three nitrogen atoms, and are widely studied as
a group of interest for developing active molecules against
diseases [14], including some reported by our group:
thrombosis [15, 16] or antivenom for the snakes Lachesis
muta or B. jararaca [17, 18]. Moreover, many biological
effects have been described elsewhere, like anti-malaria
[19], antifungal [20], anticancer [21], antiviral [22], and
antibacterial activity [23], and some of them have become
medicinal drugs, like fungicides [24].
Chemistry and synthetic compounds
Reagents specific for compound synthesis were used as pur-
chased, from Sigma-Aldrich or Bio-Grade, without further
purification. Column chromatography was performed with
F60 silica gel (Silicycle 40–65 μm). Analytical thin-layer
chromatography (TLC) was performed with silica gel plates
(Silicycle, TLC silica gel 60F-254), and the plots were
visualized under ultraviolet light or developed by immersion
in an ethanolic solution of vanillin. Yields refer to chroma-
tographically and spectroscopically homogeneous materials.
Melting points (mp) were obtained on a Fisatom apparatus
(430 D model). Infrared spectral data were recorded from KBr
pellets on a Thermo Scientific model Nicolet 6700-FTIR
spectrophotometer calibrated relative to the 1601.8 cm−1
absorbance of polystyrene. Elemental analysis was used to
ascertain purity > (95%) of all compounds for which biolo-
gical data were determined. CHN elemental analyses were
performed on a Perkin-Elmer 2400 CHN elemental analyzer.
NMR spectra were recorded on a Bruker AVHD 9.40 T
(400.13 MHz 1H e 100.61 MHz 13C) and AVIII 11,75 T
1
(500.13 MHz H e 125.76 MHz 13C) system in CDCl3 solu-
tions using tetramethylsilane as the internal reference standard
(0.0 ppm). Coupling constants (J) are reported in hertz and
refer to apparent peak multiplicities.
As part of our experiment and strategy to evaluate tria-
zole derivatives with antivenom potential, in this study, we
synthesized a novel family of 1,2,3-triazoles able to inhibit
coagulant, hemolytic, proteolytic, hemorrhagic, and ede-
matogenic activities of B. jararaca venom and we also
evaluated their predicted toxicity using the tool admetSAR.
General procedure for obtaining aromatic azides from
aromatic amines (2a-i)
An aqueous solution of 1.5 mmol of sodium nitrite (NaNO2)
in 2.5 mL of distilled water was slowly added under vigorous
stirring to an Erlenmeyer flask containing 1 mmol of aromatic
amine (1a-i) and 1 mL of 6 M hydrochloric acid solution
(50%) in an ice bath (maintaining temperature between
0–5 °C). Thereafter, stirring was continued at a low tem-
perature for 30 min. Subsequently, a 4 mmol solution of
sodium azide (NaN3) in 5 mL of distilled water was slowly
added while maintaining the temperature between 0–5 °C.
The reaction was maintained at room temperature for the
necessary time until completion. Then, the mixture was
extracted with ethyl acetate, and the organic phase was
washed with saturated sodium bicarbonate solution and water
and dried with anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure to obtain aromatic azides
(1a-i). The residual crude product was used directly without
purification.
Materials and methods
Venom, antivenom, animals, and reagents
Lyophilized B. jararaca venom or antibothropic polyvalent
serum (antivenom) was provided by Fundação Ezequiel
Dias, Belo Horizonte, MG, Brazil, and stored at −20 °C
until the time for its use in assays. Snake venom collection
was conducted under the authorization of the Brazilian
National System for the Management of Genetic Heritage
and Associated Traditional Knowledge (SISGEN) (Process
number A39CD4E). Swiss mice (18–20 g) were obtained
from the Animal Care Laboratory of the Federal Fluminense
University (UFF), and housed under constant light and
temperature (24 1 °C) conditions. All in vivo experiments
were approved by the UFF Institutional Committee for
Ethics in Animal Experimentation (protocol number 847),
which is in accordance with the guidelines from the Bra-
zilian Committee for Animal Experimentation (COBEA).
Dimethylsulfoxide (DMSO) and azocasein were purchased
by Sigma-Aldrich (San Louis, Missouri, USA), and all the
organic solvents or reagents are of the best grade available.
1-azido-2-trifluoromethyl-4-chlorobenzene (1a)
Compound 1a was obtained as a brown liquid with 83%
yield. Rf = 0.60 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3059 (C-H sp²), 2100 (-N=N=N). 1H NMR
(CDCl3, 500.13 MHz) δ: 7.11 (d, 1H, J = 2.5 Hz, H-Ar),

Medicinal Chemistry Research
7.36 (d, 1H, J = 2.3 Hz, H-Ar), 7.65 (s, 1H, H-Ar).
13C NMR (CDCl3, 125.76 MHz) δ: 113.00 (C-Ar), 118.46
(C-Ar), 120.47 (C-Ar), 125.90 (C-Ar), 129.45 (C-Ar),
142.05 (C-Ar), 147.18 (q, J = 2.93 Hz, C-CF3), 163.38 (q,
J = 245.5 Hz, CF3).
ν(cm−1): 2117 and 2113 (-N=N=N), 1579 (C=C).
1H NMR (CDCl3, 500.13 MHz) δ: 7.06 (dd, 1H, J = 2.3 Hz
and J = 8.5 Hz, H-Ar), 7.15 (d, 1H, J = 2.3 Hz, H-Ar), 7.30
(d, 1H, J = 8.6 Hz, H-Ar). 13C NMR (CDCl3, 125.76 MHz)
δ: 119.99 (C-Ar), 123.66 (C-Ar), 125.99 (C-Ar), 131.69
(C-Ar), 133.68 (C-Ar), 138.60 (C-Ar).
1-azido-3-methylbenzene (1b)
1-azido-4-nitrobenzene (1g)
Compound 1b was obtained as a brown liquid with 60%
yield. Rf = 0.61 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3060 (C-H sp²), 2945 (C-H sp3), 2105
(-N=N=N). 1H NMR (CDCl3, 500.13 MHz) δ: 2.34 (s, 3H,
CH¬3), 6.80–6.84 (m, 2H, H-Ar), 6.5 (s, 1H, H-Ar), 7.19–
7.25 (m, 1H, H-Ar). 13C NMR (CDCl3, 125.76 MHz) δ:
21.52 (CH3), 116.32 (C-Ar), 119.79 (C-Ar), 125.95 (C-Ar),
129.74 (C-Ar), 140.08 (C-Ar).
Compound 1 g was obtained as a yellow solid with 90%
yield. Rf = 0.45 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3056 (C-H sp²), 2127 (-N=N=N), 1517 and 1351
(N=O). 1H NMR (CDCl3, 500.13 MHz) δ: 7.12 (d, 2H, J =
2.1 Hz, H-Ar), 8.25 (d, 2H, J = 2.3 Hz, H-Ar). 13C NMR
(CDCl3, 125.76 MHz) δ: 119.60 (C-Ar), 125.82 (C-Ar),
144.85 (C-Ar), 147.08 (C-Ar).
1-azido-2-methoxybenzene (1c)
1-azido-4-chlorobenzene (1 h)
Compound 1c was obtained as a brown liquid with 88%
yield. Rf = 0.69 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3066 (C-H sp²), 2940 (C-H sp3), 2113
(-N=N=N). 1H NMR (CDCl3, 500.13 MHz) δ: 3.87 (s, 3H,
OCH3), 6.89 (d, 1H, J = 8.0 Hz, H-Ar), 6.9 (t, 1H, J =
7.8 Hz, H-Ar), 7.01 (d, 1H, J = 7.8 Hz, H-Ar), 7.10 (t, 1H,
J = 7.9 Hz, H-Ar). 13C NMR (CDCl3, 125.76 MHz) δ:
56.07 (OCH3), 112.25 (C-Ar), 120.46 (C-Ar), 121.47
(C-Ar), 125.84 (C-Ar), 128.49 (C-Ar), 152.05 (C-Ar).
Compound 1 h was obtained as a brown liquid with 74%
yield. Rf = 0.61 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3054 (C-H sp²), 2130 (-N=N=N). 1H NMR
(CDCl3, 500.13 MHz) δ: 6.95 (d, 1H, J = 2.0 Hz, H-Ar),
7,30 (d, 2H, J = 2.1 Hz, H-Ar). 13C NMR (CDCl3,
125.76 MHz) δ: 120.48 (C-Ar), 130.05 (C-Ar), 130.44
(C-Ar), 138.88 (C-Ar).
1-azido-3-chlorobenzene (1i)
1-azido-4-hydroxybenzene (1d)
Compound 1i was obtained as a brown liquid with 44%
yield. Rf = 0.62 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 3068 (C-H sp²), 2103 (-N=N=N). 1H NMR
(CDCl3, 500.13 MHz) δ: 6.90 (d, 1H, J = 2.3 Hz, H-Ar),
7.02 (s, 1H, H-Ar), 7.12 (d, 1H, J = 2.2 Hz, H-Ar), 7.25 (t,
1H, J = 2.0 Hz and 2.1 Hz, H-Ar). 13C NMR (CDCl3,
125.76 MHz) δ: 117.45 (C-Ar), 119.53 (C-Ar), 125.27
(C-Ar), 130.85 (C-Ar), 135.65 (C-Ar), 141.68 (C-Ar).
Compound 1d was obtained as a brown liquid with 44%
yield. Rf = 0.36 (hexane/ethyl acetate: 7/3). IR (KBr pellet) ν
(cm−1): 3075 (C-H sp²), 2110 (-N=N=N). 1H NMR (CDCl3,
500.13 MHz) δ: 7.12 (d, 2H, J = 2.4 Hz, H-Ar), 7.25 (d, 2H,
J = 2.3 Hz, H-Ar).13C NMR (CDCl3, 125.76 MHz) δ: 120.60
(C-Ar), 126.82 (C-Ar), 145.85 (C-Ar), 145.90 (C-Ar).
1-azido-2-chlorobenzene (1e)
General method for preparing (1-phenyl-1H-1,2,3-triazol-4-
yl-)methanol compounds from aromatic azides (AM11-
AM19)
Compound 1e was obtained as a brown liquid with 89%
yield. Rf = 0.74 (hexane/ethyl acetate: 7/3). IR (KBr pellet)
ν(cm−1): 2135 and 2106 (-N=N=N), 1585 (C=C).
1H NMR (CDCl3, 500.13 MHz) δ: 7.06–7.10 (m, 1H, H-Ar),
7.18 (dd, 1H, J = 8.0 Hz, H-Ar), 7.28–7.31 (m, 1H, H-Ar),
7.38 (dd, 1H, J = 8.0 Hz, H-Ar). 13C NMR (CDCl3,
125.76 MHz) δ: 119.89 (C-Ar), 125.21 (C-Ar), 125.85
(C-Ar), 128.09 (C-Ar), 130.96 (C-Ar), 137.41 (C-Ar).
5 mmol of propargyl alcohol (3), 0.1 mmol of CuSO4.5H2O
and 0.2 mmol of sodium ascorbate were added to a solution
containing 1 mmol of the appropriate aromatic azide (2a-i)
in dichloromethane (1 mL) and water (1 mL). The resulting
suspension was maintained in reflux for 2 hr. After this
time, the reaction was quenched with NaHCO3 to neutralize
the solution. The mixtures were diluted with 5 mL of
dichloromethane and 5 mL of water. The organic phases
were separated, dried with anhydrous sodium sulfate, and
concentrated under reduced pressure, furnishing the 1,2,3-
triazole compounds, which were analyzed by 1H and
1-azido-2,5-dichlorobenzene (1f)
Compound 1 f was obtained as a brown liquid with 75%
yield. Rf = 0.56 (hexane/ethyl acetate: 7/3). IR (KBr pellet)

Medicinal Chemistry Research
13C NMR spectroscopy and IR spectroscopy. When isolated
as crude material, the compounds were subjected to pur-
ification prior to biological evaluation to ensure that no
residual metals or other organic impurities were present.
Filtration was performed in a flash column, using an elution
gradient of hexane/ethyl acetate.
(1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methanol (AM14)
Compound AM14 was obtained as a white powder with
65% yield. mp 125–128 °C; Rf = 0.20 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3350 (O-H), 3060 (C-H
sp2), 1241 (C-O). 1H NMR (CDCl3, 400.13 MHz) δ 4.60 (d,
2H, J = 5.7, CH2OH), 5.40 (s, 1H, OH), 7.60 (d, 2H, J =
9.0, H-Ar), 7.90 (d, 2H, J = 9.1, H-Ar), 8.50 (s, 1H,
CHtriazole). 13C NMR (CDCl3, 100.61 MHz) δ 54.61
(CH2OH), 121.00 (CHtriazole), 123.75 (2C-Ar), 130.97
(2C-Ar), 131.87 (Cq-Ar), 137.95 (Cq-Ar), 149.00 (Cq-
triazole). Anal. Calcd for C9H9N3O2: C, 56.54; H, 4.75;
N, 21.98. Found: C, 56.53; H, 4.79; N, 21.90.
(1-(2-trifluoromethyl-4-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methanol (AM11)
Compound AM11 was obtained as a white powder with
80% yield. mp 110–115 °C; Rf = 0.34 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3280 (O-H), 3120 (C-H
1
sp2), 1600 (C=C). H NMR (CDCl3, 500.13 MHz) δ 4.70
(s, 2H, CH2OH), 5.40 (s, 1H, OH), 7.30 (d, 1H, J = 3.9 Hz
H-Ar), 7.41 (d, 1H, J = 3.7 Hz H-Ar), 7.65 (s, 1H, H-Ar),
8.08 (s, 1H, CHtriazole). 13C NMR (CDCl3, 125.76 MHz) δ
53.5 (CH2OH), 120.3 (CHtriazole), 121.6 (C-Ar), 123.38
(q, J = 240.5 Hz, CF3), 124.9 (C-Ar), 130.0 (C-Ar), 131.6
(C-Ar), 132.3 (C-Ar), 134.6 (C-Ar), 144.2 (Cq-triazole).
Anal. Calcd for C10H7ClF3N3O: C, 43.26; H, 2.54;
N, 15.14. Found: C, 43.20; H, 2.50; N, 15.13.
(1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methanol (AM15)
Compound AM15 was obtained as a brown powder with
72% yield. mp 83–86 °C; Rf = 0.44 (hexane/ethyl acetate:
1/1). IR (KBr pellet) ν(cm−1): 3294 (O-H), 3134 (C-H sp2).
1H NMR (CDCl3, 500.13 MHz) δ 3.80 (s, 1H, OH), 4.91 (s,
2H, CH2OH), 7.42–7.48 (m, 2H, H-Ar), 7.56–7.60 (m, 2H,
H-Ar), 8.00 (s, 1H, CHtriazole). 13C NMR (CDCl3,
125.76 MHz) δ 56.38 (CH2OH), 124.15 (CHtriazole),
127.94 (C-Ar), 128.09 (C-Ar), 128.80 (C-Ar), 130.92 (C-
Ar), 130.99 (C-Ar), 134.97 (C-Ar), 147.74 (Cq-triazole).
Anal. Calcd for C9H8ClN3O: C, 51.57; H, 3.85; N, 20.05.
Found: C, 51.53; H, 3.89; N, 20.00.
(1-(2-methylphenyl)-1H-1,2,3-triazol-4-yl)methanol (AM12)
Compound AM12 was obtained as a white powder with 82%
yield. mp 58–59 °C; Rf = 0.54 (hexane/ethyl acetate: 1/1). IR
(KBr pellet) ν(cm−1): 3266 (O-H), 3112 (C-H sp2), 2935 (C-
H sp3), 1498 (C=C). 1H NMR (CDCl3, 500.13 MHz) δ 2.25
(s, 3H, CH3), 4.63 (d, 2H, J = 5.4, CH2OH), 5.45 (s, 1H,
(1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methanol
(AM16)
OH), 7.40–7.49 (m, 4H, H-Ar), 8.23 (s, 1H, CHtriazole). 13
C
NMR (CDCl3, 125.76 MHz) δ 17.8 (CH3), 55.5 (CH2OH),
124.6 (C-Ar), 126.2 (C-Ar), 127.3 (CHtriazole), 130.0 (C-Ar),
131.6 (C-Ar), 133.3 (C-Ar), 136.6 (C-Ar), 148.2 (Cq-tria-
zole). Anal. Calcd for C10H11N3O: C, 63.48; H, 5.81;
N, 22.21. Found: C, 63.40; H, 5.89; N, 22.20.
Compound AM16 was obtained as a white powder with
90% yield. mp 114–116 °C. Rf = 0.26 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3282 (O-H), 3140 (C-H
1
sp2), 1240 (C-O). H NMR (CDCl3, 400.13 MHz) δ 3.04
(s, 1H, OH), 4.92 (s, 2H, CH2OH), 7.44 (dd, 1H, J =
8.6 Hz, H-Ar), 7.52 (d, 1H, J = 8.7 Hz, H-Ar), 7.67–7.68
(m, 1H, H-Ar), 8.02 (s, 1H, CHtriazole). 13C NMR (CDCl3,
100.61 MHz) δ 56.61 (CH2OH), 123.88 (CHtriazole),
126.89 (C-Ar), 127.97 (C-Ar), 131.00 (C-Ar), 131.87
(C-Ar), 133.95 (C-Ar), 147.94 (Cq-triazole). Anal. Calcd
for C9H7Cl2N3O: C, 44.29; H, 2.89; N, 17.22. Found: C,
44.26; H, 2.89; N, 17.30.
(1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methanol
(AM13)
Compound AM13 was obtained as a white powder with
85% yield. mp 110–113 °C; Rf = 0.34 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3278 (O-H), 3124 (C-H
sp2), 2939 (C-H sp3), 1602 (C=C). 1H NMR (CDCl3,
500.13 MHz) δ 3.75 (s, 1H, OH), 3.86 (s, 3H, OCH3), 4.89
(s, 2H, CH2OH), 7.06–7.09 (m, 2H, H-Ar), 7.41 (t, 1H, J =
7.9 Hz H-Ar), 7.73 (d, 1H, J = 7.7 Hz, H-Ar), 8.11 (s, 1H,
CHtriazole).13C NMR (CDCl3, 125.76 MHz) δ 56.09
(OCH3), 56.50 (CH2OH), 112.42 (C-Ar), 121.37 (C-Ar),
124.24 (CHtriazole), 125.69 (C-Ar), 126.43 (C-Ar), 130.34
(C-Ar), 147.25 (Cq-triazole), 151.35 (C-Ar). Anal. Calcd
for C10H11N3O2: C, 58.53; H, 5.40; N, 20.48. Found:
C, 58.50; H, 5.42; N, 20.47.
(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methanol (AM17)
Compound AM17 was obtained as a white powder with
90% yield. mp 200–203 °C; Rf = 0.34 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3283 (O-H), 3138 (C-H
sp2), 1237 (C-O). 1H NMR (CDCl3, 400.13 MHz) δ 4.63 (s,
2H, CH2OH), 5.39 (s, 1H, OH), 8.23 (d, 1H, J = 9.1 Hz, H-
Ar), 8.44 (d, 1H, J = 9.1 Hz, H-Ar), 3.04 (s, 1H, H-10),
4.92 (s, 2H, H-9), 7.44 (dd, 1H, J = 8.6 Hz, H-Ar), 7.52 (d,

Medicinal Chemistry Research
1H, J = 8.7 Hz, H-Ar), 8.00 (s, 1H, CHtriazole). 13C NMR
(CDCl3, 100.61 MHz) δ 54.61 (CH2OH), 120.88 (CHtria-
zole), 120.95 (C-Ar), 124.90 (2C-Ar), 131.00 (2C-Ar),
145.87 (Cq-Ar), 149.95 (Cq-Ar), 147.94 (Cq-triazole).
Anal. Calcd for C9H8N4O3: C, 49.09; H, 3.66; N, 25.45.
Found: C, 49.10; H, 3.60; N, 25.43.
mixture was added to plasma, and coagulation was mon-
itored. Negative control groups were performed by incu-
bating each compound, saline or DMSO with plasma, in the
absence of venom.
Proteolytic activity of B. jararaca venom
(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methanol (AM18)
Proteolytic activity of B. jararaca venom was determined
according to Garcia et al. [25], using azocasein as a sub-
strate (0.2% w/v, in 20 mM Tris-HCl, 8 mM CaCl2, pH 8.8),
with minor modifications. Different concentrations of B.
jararaca venom (2–20 μg/mL) were incubated with azoca-
sein, and the amount of venom (μg/mL) that achieved
70–80% of maximum activity was considered to be 100%
proteolytic activity. This concentration of B. jararaca
venom (9 μg/mL), called the effective concentration (EC),
was incubated with 90 μg/mL of each compound
(AM11–AM19), saline or 0.9% DMSO (positive controls)
for 30 min at 25 °C. Finally, an aliquot of the mixture was
added to the reaction medium, and proteolytic activity was
evaluated. Negative control experiments were performed by
adding solely compounds, saline or DMSO to the reaction
medium, instead of the venom, while the positive control
was performed by incubating B. jararaca venom with saline
or DMSO.
Compound AM18 was obtained as a white powder with
90% yield. mp 144–146 °C; Rf = 0.46 (hexane/ethyl acet-
ate: 1/1). IR (KBr pellet) ν(cm−1): 3320 (O-H), 3070 (C-H
sp2), 1239 (C-O). 1H NMR (CDCl3, 400.13 MHz) δ 4.66 (d,
2H, J = 5.6, CH2OH), 5.37 (t, 1H, J = 5.6, OH), 7.66 (d,
2H, J = 9.1, H-Ar), 7.95 (d, 2H, J = 9.1, H-Ar), 8.71 (s, 1H,
CHtriazole). 13C NMR (CDCl3, 100.61 MHz) δ 55.61
(CH2OH), 122.00 (CHtriazole), 122.65 (2C-Ar), 129.97
(2C-Ar), 132.87 (Cq-Ar), 135.95 (Cq-Ar), 149.94 (Cq-
triazole). Anal. Calcd for C9H8ClN3O: C, 51.57; H, 3.85;
N, 20.05. Found: C, 51.50; H, 3.89; N, 20.02.
(1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methanol (AM19)
Compound AM19 was obtained as a white powder with
83% yield. mp 95–97 °C; Rf = 0.34 (hexane/ethyl acetate:
1/1). IR (KBr pellet) ν(cm−1): 3264 (O-H), 3090 (C-H sp2),
1236 (C-O). 1H NMR (CDCl3, 400.13 MHz) δ 4.61 (d, 2H,
J = 4.6, CH2OH), 5.35 (t, 1H, J = 5.6, OH), 7.55–7.58 (m,
1H, H-Ar), 7.64 (t, 1H, J = 8.0, H-Ar), 7.90–7.93 (m, 1H,
H-Ar), 8.03–8.07 (m, 1H, H-Ar), 8.71 (s, 1H, CHtriazole).
13C NMR (CDCl3, 100.61 MHz) δ 55.01 (CH2OH), 118.54
(CHtriazole), 119.65 (C-Ar), 122.00 (C-Ar), 128.32 (C-Ar),
130.97 (C-Ar), 133.87 (Cq-Ar), 137.95 (Cq-Ar), 149.04
(Cq-triazole Anal. Calcd for C9H8ClN3O: C, 51.57; H, 3.85;
N, 20.05. Found: C, 51.50; H, 3.80; N, 20.01.
Hemolytic activity of B. jararaca venom
The degree of hemolysis caused by B. jararaca venom was
determined by the indirect hemolytic test, using washed
human red blood cells and hen’s egg yolk emulsion as the
substrate [26]. After performing a concentration-response
curve of B. jararaca venom (1–30 μg/mL), the amount of
venom able to induce 100% hemolysis was called the
minimum indirect hemolytic concentration (MIHC). Then,
one MIHC of B. jararaca venom (10 μg/mL) was incubated
with each compound (100 μg/mL), saline or DMSO (posi-
tive controls) for 30 min at 25 °C, followed by the hemo-
lytic test. Negative control experiments were performed by
adding compounds or solvents to the reaction medium,
instead of venom.
Biological activities
Coagulating activity of B. jararaca venom
Coagulant activity of B. jararaca venom was determined
using a digital Amelung coagulometer, model KC4A
(Labcon, Germany). Plasma (obtained from the blood bank
of the UFF Hospital) was maintained for 1 min at 37 °C, and
then different concentrations of B. jararaca venom
(5–45 μg/mL) were added to the reaction medium and
plasma coagulation time was registered in seconds. The
amount of venom (μg/mL) capable of clotting plasma
around 60 sec was designated as the minimum coagulation
concentration (MCC). Then, this venom concentration
(25 μg/mL) was incubated for 30 min at 25 °C with saline or
0.9% v/v DMSO (positive controls) or with 250 μg/mL of
each compound (AM11–AM19). After incubation, each
Hemorrhagic activity of B. jararaca venom
Hemorrhagic lesions produced by B. jararaca venom were
quantified using a procedure described by Kondo et al. [27],
with modifications. B. jararaca venom was injected sub-
cutaneously (s.c.) into the abdominal skin of mice. Two
hours later, the animals were euthanized, and the abdominal
skin was removed, stretched, and inspected for visual
changes in its internal aspect in order to localize hemor-
rhagic spots. A minimum hemorrhagic dose (MHD) was
defined as the dose of venom (μg/mouse) able to produce a

Medicinal Chemistry Research
mice was recorded. Solvents, compounds or antivenom
were injected i.p. into mice in the absence of B. jararaca
venom. The number of dead or alive mice was counted after
6 h of experimentation. The total sample volume injected
into the mice was 100 μL.
Scheme 1 Synthetic route for the preparation of 1,2,3-Triazoles
Toxicity of compounds
hemorrhagic halo of 10 millimeters (mm), which was 16 μg/
mouse. The effect of compounds (AM11–AM19) on B.
jararaca venom-induced hemorrhaging was analyzed
through two protocols, incubation and treatment. In the
incubation protocol, compounds or solvents (saline or
DMSO) were incubated with 2 MHD of B. jararaca venom
(32 μg/mouse) for 30 min at 25 °C. Then, an aliquot of the
mixture was injected s.c. into mice, and the hemorrhage
halo of the venom was measured and compared to that
produced in the absence of the venom. In the treatment
protocol, B. jararaca venom was injected s.c. into mice, and
after 10 min, compounds or solvents were administered s.c.
at the same site as venom injection. Instead of compounds,
after injecting the venom, 100 μL of antivenom or saline
were injected intravenously (i.v.), as well. Negative control
experiments were performed by injecting solely solvents or
compounds s.c. or injecting antivenom i.v., instead of
venom. The total volume of sample injection into the mice
was 100 μL.
Prediction of pharmacological and toxicological properties
by admetSAR
The ADMET parameters of the nine compounds
(AM11–AM19) were determined using the AdmetSAR
online tool (http://lmmd.ecust.edu.cn/admetsar2) [29].
Cytotoxicity
Toxicity of the compounds (AM11–AM19) was evaluated
by the hemocompatibility test, according to Bauer et al.
[30], with modifications. All the compounds (600 μg/mL) or
saline (negative control) were incubated with a 13 % (v/v)
of red blood cell suspension for 3 h at 37 °C. After, samples
were centrifuged for 3 min at 1800 × g and lysis of cells was
detected by measuring hemoglobin at A578 nm, using a
Microplate Reader (SpectraMax, Model M4, Molecular
Devices, California, USA). 100 % of hemolysis (positive
control) was achieved by adding Triton X-100 (1%, v/v) or
water to the red blood cell suspension.
Edematogenic activity of B. jararaca venom
Statistical analysis
Edema-inducing activity of B. jararaca venom was deter-
mined according to Vishwanath et al. [28]. Groups of mice
received a single s.c. sub plantar injection of B. jararaca
venom into the right paw, and the left one received an
injection of saline or DMSO. Then, 1 h after injection,
edema was evaluated and expressed as the percent increase
in the weight of the right paw compared to the left one.
After that, B. jararaca venom (16 μg/mouse) was mixed
with 160 μg/mouse of compounds (AM11–AM19) or sol-
vents for 30 min at 25 °C, and then, the mixture was
injected s.c. into animals, and edema was evaluated, as
described. Negative control mice received a single injection
of either compounds or solvents, instead of venom. The
total sample volume injected into the mice was 50 μL.
Results are expressed as means standard deviation (SD) of
number of mice or experiments. Statistical significance of
differences among experimental groups was evaluated using
the ANOVA test. *p values < 0.05 were considered
significant.
Results
Chemistry
1,2,3-triazoles substituted with a methanol at position 4
were reported by Boechat et al. [29] and the methodology
described in the paper was followed in this study (Scheme 1).
The first step involved the preparation of aromatic azides
from aromatic amines and sodium nitrite, which were
appropriately combined to form diazonium salts; then, with
the addition of an aqueous sodium azide solution, aromatic
azides were obtained in good yields, ranging from 44–90%.
With the azides properly characterized, it was possible to
obtain the desired triazoles through a 1,3-dipolar cycload-
dition reaction between propargyl alcohol and aromatic
Lethal activity of B. jararaca venom
An intraperitoneal (i.p.) injection of B. jararaca venom
(130 μg/mouse) mixed with solvents (saline or DMSO) into
mice killed them in 60 min. This dose of B. jararaca venom
was incubated with each compound (AM11–AM19) or
antivenom for 30 min at 25 °C, and then, an aliquot of
mixture was injected i.p. into mice, and survival time of the

Medicinal Chemistry Research
*
azides catalyzed by Cu (I). Copper sulfate (CuSO4) and
sodium ascorbate guided region selectivity [31]. For the
formation of triazole, it is necessary that Cu(I) species be
present as a catalyst; however, the system used uses Cu (II),
so sodium ascorbate has the function of reducing this Cu(II)
ion to Cu(I), generating these ions in situ. The first stage of
the triazole formation mechanism consists of the π com-
plexation between copper and the terminal alkyne, which
reduces the pKa value of the alkyne, favoring the depro-
tonation of the species without the need for a strong base in
the reaction medium, enabling the formation of copper
acetylide. This acetylide then forms a complex with azide,
which favors the formation of a metallocycle, since copper
increases the electrophilicity of the azide’s terminal nitrogen
and the nucleophilicity of the β-vinylidene carbon. This
complex leads to the formation of copper triazolyl, which is
protonated, leading to obtaining the final products and
regenerating the Cu (I) ion. It was observed that the use of
dichloromethane and heating at 40 °C, replacing tert-
butanol at room temperature proved to be more efficient
in terms of reaction time and low formation of by-products
[29]. Triazole compounds were obtained as white solids
with yields ranging between 65–90%.
100
50
0
*
*
*
Saline DMSO AM 11 AM12 AM 13 AM 14 AM 15 AM 16 AM 17 AM 18 AM 19
B. jararaca venom
+
Fig. 1 Effect of compounds on B. jararaca venom-induced
coagulation
the mixture was added to plasma, and coagulant activity was
evaluated. As seen in Fig. 1, the compounds AM12, AM14,
AM15, and AM16 impaired coagulation induced by B. jar-
araca venom, when compared to the positive group (B. jar-
araca + saline or B. jararaca + DMSO). The other
compounds, AM11, AM13, AM17, and AM19, did not
inhibit coagulation (Fig. 1). None of the compounds (250 μg/
mL) or DMSO alone (at 0.9%, final concentration) induced
plasma coagulation nor interfered with the coagulation test.
B. jararaca venom (25 μg/mL) was incubated for 30 min
at 25 °C with 250 μg/mL of compounds (AM11–AM19),
saline or DMSO (0.9%, final concentration). Then, an ali-
quot of the mixture was added to plasma and coagulation
time was measured, as described in the methods section.
Results express the means SD of three individual experi-
ments (n = 3). *p < 0.05 in relation to positive controls (B.
jararaca venom + saline or B. jararaca venom + DMSO).
The chemical structures of the azides and triazoles
(AM11–AM19) were confirmed by analyzing the crude pro-
1
duct using FTIR, NMR of H and 13C. Analysis showed a
strong absorption band around 2100 cm−1, referring to the
stretch vibrations of N3 at IR of azides and the absence of
stretching vibrations of the azide group and the presence
of bands related to axial deformation of the OH bond at IR of
triazoles. In the 1H NMR spectrum, the signals of the
respective protons of the synthesized compounds were ver-
ified based on their chemical shifts, multiplicities, and cou-
pling constants. The characteristic proton signal for
identifying the triazolic nucleus can be observed as a singlet
around 8 ppm. All data obtained and analyzed were consistent
with data from the literature for this family of compounds.
Furthermore, all the compounds (AM11–AM19) were
tested against some in vitro or in vivo toxic activities of B.
jararaca venom, followed by theoretical toxicity using the
tool admetSAR. The compounds (AM11–AM19), at a
concentration of 600 μg/mL did not lyze red blood cells
evaluate by the hemocompability test (data not shown).
Thus, compounds were not cytotoxic.
Effect of compounds against of B. jararaca venom-
induced proteolysis
B. jararaca venom incubated with saline or DMSO (positive
controls) hydrolyzed azocasein in a concentration-dependent
manner, and reads of 0.2 at absorbance of 420 nm was called
the effective concentration (EC). One EC of B. jararaca
venom (9 μg/mL) was incubated for 30 min at 25 °C with
compounds (90 μg/mL), and then, the mixture was added to
the reaction medium, proteolytic activity was evaluated, and
result is shown as inhibition of proteolysis (Fig. 2). The
compounds AM15 (32%) or AM19 (53%) had the highest
values of inhibition against the proteolytic activity of B. jar-
araca venom; while the compounds AM14, AM16, and
AM18 achieved the lowest, below 20% (Fig. 2). The com-
pounds AM11, AM12, AM13, and AM17 inhibited
approximately 20% (Fig. 2).
Effect of the compounds against of B. jararaca
venom-induced plasma coagulation
B. jararaca venom (9 μg/mL) was incubated with 90 μg/
mL of each compound (AM11–AM19) for 30 min at 25 °C.
After that, the mixture was added to the reaction medium,
proteolytic activity was evaluated, and inhibition of pro-
teolysis was determined. Results express the means SD of
three individual experiments (n = 3).
The concentration of B. jararaca venom capable of coagu-
lating plasma in around 60 s was designated as the minimum
coagulant concentration (MCC). One MCC of B. jararaca
venom (25 μg/mL) was incubated with the compounds
(250 μg/mL) or solvents for 30 min at 25 °C. After incubation,

Medicinal Chemistry Research
100
80
60
40
20
0
venom (32 μg/mouse). Then, an aliquot of the mixture was
injected s.c. into mice, and hemorrhaging was analyzed, as
described. This approach was the incubation protocol. As seen
in Fig. 4a, the compounds AM11 (11%), AM13 (22%), AM18
(4%), and AM19 (17%) inhibited the hemorrhagic activity of
B. jararaca venom. Thus, the treatment protocol was employed
using compounds AM11, AM13, AM18, and AM19. B. jar-
araca venom (32 μg/mouse) was injected s.c. into mice, and
after 10 min, each of these compounds (300 μg/mouse) was
injected s.c. (at the same site as venom injection), followed by
an intravenous (i.v.) injection of commercial antivenom. As
seen in Fig. 4b, the compound AM13 inhibited 32% of B.
jararaca venom-induced hemorrhaging, while the other com-
pounds did not protect the mice from hemorrhaging (Fig. 4b).
The inhibitory percentage of AM13 (20%) in the absence of
antivenom was similar in the incubation (Fig. 4a) and treatment
protocols (Fig. 4b). On the other hand, inhibition of AM13 was
enhanced to 32 % in the presence of antivenom (Fig. 4b).
Moreover, administration of antivenom i.v. after administration
of B. jararaca venom did not inhibit hemorrhage as well as a
cocktail using the derivatives AM11, AM13, AM18, and
AM19 did not enhance inhibitory percentage (data not shown).
Injection of solvents (saline or DMSO), antivenom or the
compounds alone did not induce hemorrhaging in mice,
regardless of the protocol employed.
AM 11 AM12 AM 13 AM 14 AM 15 AM 16 AM 17 AM 18 AM 19
B. jararaca venom +
Fig. 2 Inhibition by compounds of proteolysis induced by B. jararaca
venom
100
80
60
40
20
0
AM 11 AM12 AM 13 AM 14 AM 15 AM 16 AM 17 AM 18 AM 19
B. jararaca venom +
Fig. 3 Inhibition by compounds of B. jararaca venom-induced
hemolysis
Effect of compounds against B. jararaca venom-
induced hemolysis
Effect of compounds against B. jararaca venom-
induced edema
Incubation of B. jararaca venom with saline or DMSO
(1%, final concentration) lysed a suspension of red blood
cells, with a similar CMHI of 10 μg/mL. Then, this venom
concentration was incubated with the compounds (100 μg/mL)
for 30 min at 25 °C. After that, an aliquot of the mixture was
removed and added to the reaction medium, and a hemo-
lytic activity test was performed (Fig. 3). The compounds
AM11, AM12, AM14, and AM15 inhibited ~30% of
B. jararaca venom-induced hemolytic activity, while the
others inhibited less than 15% of hemolysis (Fig. 3). None
of the compounds alone lysed red blood cells.
B. jararaca venom (10 μg/mL) was incubated with
100 μg/mL of compounds (AM11–AM19) for 30 min at
25 °C. After that, the mixture was added to red blood cells,
hemolytic activity was evaluated, and inhibition of hemo-
lysis was determined. Results express the means SD of
three individual experiments (n = 3).
The effect of compounds on edema caused by B. jararaca
venom was investigated. B. jararaca venom (16 μg/mouse)
was incubated for 30 min at 25 °C with 160 μg/mouse of each
compound (AM11–AM19), saline or DMSO (0.9% v/v, final
concentration), and then the mixture was injected s.c. into the
paw of the animals and edema was analyzed. All of the
compounds inhibited the edematogenic activity from B. jar-
araca venom with similar inhibitory percentages: AM11
(48%), AM12 (37%), AM13 (44%), AM14 (47%), AM15
(44%), AM16 (39%), AM17 (39%), AM18 (39%), and
AM19 (45%) (Fig. 5). None of the compounds induced
edema in the mice.
B. jararaca venom (16 μg/mouse) was incubated with
160 μg/mouse of compounds (AM11–AM19) for 30 min at
25 °C. Then, an aliquot of the mixture was injected into the
mice, edema was evaluated as described, and inhibition of
edema was determined. Results are expressed as the means
SD of three individual experiments (n = 3).
Effect of compounds against of B. jararaca venom-
induced hemorrhaging
Effect of compounds against lethality of B. jararaca
venom
A single injection of B. jararaca venom (16 μg/mouse) into
mice produced a hemorrhagic halo of 10 mm which represents
one MHD. Compounds (320 μg/mouse), saline or DMSO were
incubated for 30 min at 25 °C with 2 MHD of B. jararaca
Intraperitoneal (i.p.) injection of B. jararaca venom
(130 μg/mouse) incubated with saline or DMSO (0.9% v/v,

Medicinal Chemistry Research
Treatment
Incubation
40
30
20
10
0
40
30
20
10
0
A
B
*
AM11 AM12 AM13 AM14 AM15 AM16 AM17 AM18 AM19
AM11 AM13 AM18 AM19 AM11 AM13 AM18 AM19
antivenom +
B. jararaca venom +
saline +
B. jararaca venom +
Fig. 4 Inhibition by compounds on hemorrhaging from B. jararaca
venom. a Incubation protocol: B. jararaca venom (32 μg/mouse) was
incubated with 320 μg/mouse of each compound (AM11–AM19) for
30 min at 25 °C. Then, the mixture was injected s.c. into mice, and
hemorrhaging was analyzed. b Treatment protocol: B. jararaca venom
(32 μg/mouse) was administered s.c. into mice, and 10 min later,
320 μg/mouse of the compounds AM11, AM13, AM18, and AM19
were injected s.c., followed by i.v. injection of antivenom (black
columns) or saline (white columns). After 2 h of B. jararaca venom
injection, hemorrhaging was analyzed, as described. Results are shown
as inhibition of hemorrhage, and expressed as the means SD of three
individual experiments (n = 3). *p < 0.05 in relation to B. jararaca
venom + antivenom + AM13
Table 1 Effect of compounds on lethality from B. jararaca venom
100
80
60
40
20
Groups
Survival time (min)
B. jararaca + saline
B. jararaca + DMSO
B. jararaca + AM11
B. jararaca + AM12
B. jararaca + AM13
B. jararaca + AM14
B. jararaca + AM15
B. jararaca + AM16
B. jararaca + AM17
B. jararaca + AM18
B. jararaca + AM19
B. jararaca + antivenom
60
61
6
8
100 11*
69
68
5
5
0
AM 11 AM12 AM 13 AM 14 AM 15 AM 16 AM 17 AM 18 AM 19
57 20
B. jararaca venom +
76 19
154 11*
Fig. 5 Inhibition by compounds on edema from B. jararaca venom
66
73 10
71
360 10*
3
final concentration) killed the mice in 60 min (Table 1).
This dose of B. jararaca venom was incubated with 320 μg/
mouse of compounds (AM11–AM19) or antivenom for
30 min at 25 °C; then, the mixture was injected i.p., and
survival time of the mice was observed and compared to the
positive control groups (B. jararaca + saline or B. jararaca
+ DMSO). As seen in Table 1, the compounds AM11 and
AM16 prolonged survival time of the mice. Moreover,
antivenom fully protected mice from B. jararaca venom-
induced lethality (Table 1). The maximal period of obser-
vation of mice was 360 min, and i.p. injection of com-
pounds or antivenom alone did not kill the mice (data not
shown).
B. jararaca venom (130 μg/mouse) was incubated for
30 min at 25 °C with saline, DMSO, 320 μg/mouse of
compounds (AM11–AM19) or with antivenom. After
incubation, the mixture was injected i.p. into mice, and
survival time of the mice was recorded. Results are
expressed as the means SD of two individual experiments
(n = 6). *p < 0.05 when compared to B. jararaca + DMSO
or to B. jararaca + saline (positive controls). Maximal
observation of the mice was 360 min.
6
Theoretical studies of toxicity and bioavailability
In silico analysis of the compounds (AM11–AM19) was
investigated using the software ADMET, and the results
are shown in Table 2. Some parameters, such as
absorption, distribution, metabolism, excretion, and
toxicity were investigated (Table 2). Upon analysis of
the results through this software, we observed that the
compounds interfered with the following absorption
parameters: Blood-Brain Barrier, Human Intestinal
Absorption, and Caco-2 Permeability; but did not inter-
fere with P-glycoprotein Substrate and P-glycoprotein
Inhibitor. In the parameters of distribution and metabo-
lism, all the compounds acted on CYP450 1A2 Inhibitor,
and only derivative AM11 acted on CYP450 3A4 Sub-
strate. The compounds did not act on the CYP450 2C9

Medicinal Chemistry Research
Table 2 Pharmacokinetic and toxicological studies of the compounds AM11–AM19 using the tool AdmetSAR
Model
Lead compound
Absorption
AM11
AM12
AM13
AM14
AM15
AM16
AM17
AM18
AM19
Blood-brain barrier (BBB)
Human intestinal absorption (HIA)
Caco-2 permeability
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
P-glycoprotein substrate
P-glycoprotein inhibitor
Distribution and metabolism
CYP450 2C9 substrate
CYP450 2D6 substrate
CYP450 3A4 substrate
CYP450 1A2 inhibitor
CYP450 2C9 inhibitor
CYP450 2D6 inhibitor
CYP450 2C19 inhibitor
CYP450 3A4 inhibitor
CYP inhibitory promiscuity
Excretion and toxicity
Human Ether-a-go-go-related gene inhibition
AMES toxicity
No
No
No
No
No
No
No
No
No
–
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
–
–
+
–
–
–
–
L
–
+
+
–
–
–
–
L
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Carcinogen
–
–
–
–
–
–
–
–
–
Fish toxicity
+
–
–
–
+
–
+
–
+
–
–
+
–
+
–
Honey bee toxicity
–
–
–
Biodegradation
–
–
–
–
–
–
–
–
–
Acute oral toxicity
III
–
III
–
III
–
III
–
III
–
III
–
III
–
III
–
III
–
Carcinogen (three-class)
DMET predicted profile (regression)
Absorption
Water solubility (LogS)
Plasma protein binding (PPB) (100%)
Acute oral toxicity Kg/mol
Tetrahymena pyriformis (plGC50, μg/L)
−3.270 −1.429 −1.659 −1.860 −2.628 −2.628 −1.804 −2.445 −2.445
0.746
3.21
0.725
2.49
0.564
2.16
0.551
1.51
0.729
2.10
0.560
2.40
0.493
1.54
0.681
2.40
0.804
2.30
1.202
0.241
0.220
0.212
0.552
0.784
0.057
0.521
0.454
+ or − means positive or negative in silico toxicity profile, respectively, L means low, and acute oral toxicity III means slightly toxic and a slight
irritant
Substrate, CYP450 2D6 Substrate, CYP450 2C9 Inhi-
bitor, CYP450 2D6 Inhibitor, CYP450 2C19 Inhibitor,
CYP450 3A4 Inhibitor nor CYP Inhibitory Promiscuity.
According to the excretion and toxicity parameters, the
compounds did not act on Human Ether-a-go-go-Related
Gene Inhibition, AMES Toxicity, Carcinogens, Honey
Bee Toxicity, Biodegradation or Carcinogenicity. The
compounds showed level III Acute Oral Toxicity which
indicates slight toxicity and a slight irritant. On the other
hand, the compounds AM11, AM14, AM15, AM16,
AM18, and AM19 showed toxicity against fish, but not
to honey bee. Thus, the compounds are favorable to
become drugs according to their absorption parameters.
Discussion
Snakebite envenoming by the species B. jararaca induces a
range spectrum of clinical alterations in victims. On the
other hand, it is worth saying that snake venoms have
intriguing compositions and actions in victims; they may
induce toxic effects in victims or they can be used to
develop medicines and save lives. A good example of this
positive effect of venom is the drug known as angiotensin
converting enzyme inhibitor (ACEI). ACEI was developed
from a peptide isolated from B. jararaca venom and is
currently used to treat hypertension and some types of heart
failure [32].

Medicinal Chemistry Research
Antivenom is the official treatment for snakebites
worldwide, and in Brazil, it is produced by three major
public research centers: Vital Brazil Institute (Niterói, RJ),
Butantan Institute (São Paulo, SP), and the Ezequiel Dias
Foundation (Belo Horizonte, MG). The earliest studies
related to antivenom therapy were carried out at the end of
the 1800s [33], but only in 1901 did Vital Brazil develop
the first antivenom [34]. Antivenom binds to venom and
neutralizes its toxic effects. Thus, it is effective for pre-
venting death; however, it has some drawbacks. To date,
manufacturing processes of antivenoms have not been
improved to overcome these limitations. Therefore, due to
these limitations, new treatments to aid conventional anti-
venoms are being investigated. Natural products derived
from plants or seaweed have been used by citizens or
healers to treat many diseases, since ancient civilization.
However, in some cases, such products were chemically
modified to enhance efficacy, earn faster profits, diminish
side effects or attend to market demand [35, 36]. The
rational planning of compounds with lower costs of pro-
duction and side effects are an important challenge in
medicinal chemistry, and, because of that, the number of
new compounds has increased dramatically [37].
Among strategies to synthesize new compounds to
become drugs, the target-oriented synthesis strategy is
relevant. A synthetic compound can be used either to dis-
cover a target protein or as a modulating ligand. In this case,
it is quite common to screen collections of molecules with
low molecular weights [37]. 1,2,3-triazoles or 1,2,4-tria-
zoles triazole derivatives are widely used in medicine for
drug development because of their structure and low toxi-
city [18, 20, 38, 39, 41, 42].
Thus, here, we synthesized a novel family of 1,2,3-tria-
zoles able to inhibit some in vitro or in vivo activities of
B. jararaca venom. The 1,2,3-triazoles substituted with
methanol at position 4 were first described elsewhere, and
we followed the methodology described in that paper [29].
The first step involved the preparation of aromatic azides
from aromatic amines and sodium nitrite which were
appropriately combined to form diazonium salts; then, with
the addition of the aqueous sodium azide solution, aromatic
azides were obtained in good yields, ranging from 44–90%.
With the azides properly characterized, it was possible to
obtain the desired triazoles through a 1,3-dipolar cycload-
dition reaction between propargyl alcohol and aromatic
azides catalyzed by Cu (I). Copper sulfate (CuSO4) and
sodium ascorbate guided the regioselectivity. It was
observed that the use of dichloromethane and heating at
40 °C, replacing tert-butanol at room temperature, proved to
be more efficient in terms of reaction time (change from 3 h
to 30 min) and low formation of by-products [29]. Only for
compounds AM11 and AM14, was purification with flash
column filtration necessary to remove by-products.
The chemical structures of the azides and triazoles were
confirmed by analyzing the product using FTIR, NMR of
1H, and 13C. Analysis showed a strong absorption band
around 2100 cm−1, referring to the stretch vibrations of N3
at IR of azides and the absence of stretching vibrations of
the azide group and the presence of bands related to axial
1
deformation of the OH bond at IR of triazoles. In the H
NMR spectrum, the signals of the respective protons of the
synthesized compounds were verified based on their che-
mical shifts, multiplicities, and coupling constants. The
characteristic proton signal for identifying the triazolic
nucleus can be observed as a singlet around 8 ppm. All data
obtained and analyzed were consistent with the literature for
this family of compounds.
The new 1,2,3-triazole compounds inhibited the proteo-
lytic, coagulant, hemolytic, edematogenic, lethal, and
hemorrhagic activity of B. jararaca venom. These assays
cover most of the active enzymes (phospholipase A2
(PLA2), snake venom metalloproteinases (SVMPs), and
snake venom serine proteases (SVSPs)) of this species as
well as the other venomous snakes. These enzymes are
responsible for most of the toxic symptoms of envenoma-
tion, but it is very difficult to correlate one enzyme with a
specific symptom or effect observed in victims, because
snake venoms are a complex mixture of active proteins.
SVMPs comprise a large group of zinc-dependent enzymes
with a domain enriched with histidine residues that induce
tissue damage, inflammation or disorders in the coagulation
cascade [43] and impair regeneration of the affected skeletal
muscle [44]. SVSPs exhibit multiple toxic activities, such as
the induction of plasma coagulation, hemorrhaging, and
effects on platelet aggregation, while the PLA2 enzymes
may induce edema, hemolysis, and tissue necrosis [45, 46].
Therefore, the neutralization of these enzymes is extremely
important in order to treat patients and help them recover
faster. As has been described in the literature, commercial
antivenom fails to fully inhibit these enzymes [47], even
following the standard guidelines recommended by autho-
rities, which is performed by incubating commercial anti-
venom with venoms (incubation protocol). On the other
hand, antivenom efficiently inhibited B. jararaca venom
lethality but failed to inhibit hemorrhaging [18]. Thus, if
antivenom is mixed with venom, rather than administered
after the venom, neutralization is more effective. Indeed, the
time of administration of antivenom should also be taken
into consideration, and some studies postulate that anti-
venom should be given around 60 min after a snakebite to
avoid death; however, the local effects of venoms are not
inhibited [45].
The literature has shown that the experimental strategy of
potency tests is performed by incubating antivenom with
venoms (incubation protocol). However, this approach does
not reflect how a snake accident really occurs, since a

Medicinal Chemistry Research
venomous snake injects venom into victims, and later,
victims receive antivenom intravenously. Here, all the
1,2,3-triazoles inhibited toxic activities of B. jararaca
venom through the incubation protocol, but only the com-
pound AM13 inhibited hemorrhaging in the treatment
protocol. This is a promising result and may turn, mainly
compound AM13, into a candidate molecule for neu-
tralization of B. jararaca-induced hemorrhaging, since its
envenomation is one of the most toxic of the Bothrops
species. Moreover, hemorrhaging is responsible for ampu-
tation or morbidities of victims.
In silico studies are an important virtual tool capable of
analyzing theoretical toxicity, enabling the screening of
molecules more efficiently and quickly, reducing the costs
of drug development, reducing the use of animals, eluci-
dating mechanisms of action or determining safety or side
effects to humans or the environment. The software
ADMET (absorption, distribution, metabolism, excretion,
and toxicity) is of vital importance in drug discovery
studies through the analysis of some parameters, such as
water solubility, sites of cytochrome P450 (CYP) meta-
bolism or metabolites, mutagenicity, toxicity, ability to
cross the blood-brain barrier (BBB), and gastrointestinal
absorption [48]. However, like any other technique, there
are limitations with this process [49]. Some programs use
the structure-activity relationship to preview the physical-
chemical structure of a molecule for possible biological
functions.
ADMET properties of the lead compounds were cal-
culated; admetSAR was used to predict some chemical
properties in order to investigate 1,2,3-triazole derivatives
(AM11–AM19) as potential molecules for the develop-
ment of antivenoms. According to the parameters in Table 2
(Caco-2 permeability, HIA or P-glycoprotein), the com-
pounds AM11–AM19 have good penetrability across the
blood-brain barrier and can be easily absorbed by the
human intestine, due to their chemical structure and non-
polarity. This is in accordance with the chemical structure
of triazoles [50]. High absorption of these compounds
(AM11–AM19) meant that they can be given orally to
patients, resulting in some advantages (they can be self-
administered without suffering pain or discomfort and they
can have relatively lower prices) or disadvantages (patient
must be conscious, molecules suffer first-pass effect, may
produce irritation to gastric mucosa or may be destroyed by
the action of digestive enzymes or extreme pH values).
According to ADMET distribution and metabolism, the
compounds (AM11–AM19) do not suffer liver metabolism
or renal clearance, and this fact may facilitate the desired
effect as antivenoms, or indeed, maintain an effective con-
centration of them in the plasma longer. On the other hand,
one negative consequence of circulating longer is their non-
specific interaction with plasma proteins, which means the
possibility of increasing side effects on any tissues. How-
ever, the compounds have low acute oral toxicity (below 3)
and low percentage (below 1%) of unbound to plasma
proteins (PPB parameter) that reduce even more, appear-
ance of undesirable effects. Moreover, LogS values above
-4 of the compounds indicate good water solubility and
distribution through human body that in turn is facilitated
by their low molecular weight (below 450 Kda). The
compounds (AM11–AM19) inhibited some of the enzymes
of cytochrome P450, the superfamily that is responsible for
metabolized drugs or xenobiotics [49]. The inhibitory action
of triazoles on the enzymatic activity of cytochrome P450
has been well established [49]. All the compounds
(AM11–AM19) are devoid of toxicity or mutagenicity,
since they did not inhibit the isoform CYP1A2 that is
responsible for metabolizing polycyclic aromatic hydro-
carbons (PAHs) to activate carcinogenic products. More-
over, these compounds fell into category III of toxicity
which means they produce mild toxicity or slight irritation.
According to WHO [51], toxicity of a chemical compound
is classified into four categories (I–IV); category I has the
lowest toxicity and category 4, the highest. Another result
that reinforces the lack of toxicity of the compounds was the
inability of them to lyse red blood cells, evaluated by the
in vitro hemocompatibility test that is regularly used to test
drugs or any molecule that enter into circulating blood.
Therefore, according to favorable data, 1,2,3-triazole
derivatives, especially AM13, may be a good molecule
candidate for use as antivenom, because it did neutralize
hemorrhagic, edematogenic, and lethal activities of B.
jararaca venom and it is devoid of toxicity. Moreover, a
simple and easy method to synthesize such derivatives and
reinforce the interest to study the mechanism of action and
develop pharmaceutical formulations using these
derivatives.
Conclusion
Thus, these new compounds derived from 1,2,3-triazoles
could be used as adjuvants of commercial antivenom to
enhance the effectiveness of neutralizing the local effects of
B. jararaca venom. However, an in-depth scientific inves-
tigation is imperative to evaluate other approaches for
deriving these therapeutically effective compounds against
symptoms of snakebites.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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Bothrops jararaca and Lachesis muta snakes. Biomed Res.
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