Antiviral evaluation of N-amino-1,2,3-triazoles against Cantagalo virus replication in cell culture

Article abstract of DOI:10.1016/j.ejmech.2009.04.046

This paper describes the antiviral evaluation of new N-amino-1,2,3-triazole derivatives, 1-(substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters, 3 and 1-(4-substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic

Full text of DOI:10.1016/j.ejmech.2009.04.046

European Journal of Medicinal Chemistry 44 (2009) 3777–3783
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Antiviral evaluation of N-amino-1,2,3-triazoles against Cantagalo
virus replication in cell culture
Alessandro K. Jorda˜o a, Priscila P. Afonso b, Vitor F. Ferreira a, Maria C.B.V. de Souza a,
Maria C.B. Almeida a, Cristiana O. Beltrame b, Daniel P. Paiva b, Solange M.S.V. Wardell c,
,
,
*
James L. Wardell d e, Edward R.T. Tiekink f, Clarissa R. Damaso b, Anna C. Cunha a
a
´
ˆ
´
˜
˜
´
Universidade Federal Fluminense, Departamento de Quımica Organica, Instituto de Quıımica, Outeiro de Sao Joao Baptista, 24020-141 Niteroi, RJ, Brazil
Universidade Federal do Rio de Janeiro, Laboratorio de Biologia Molecular de Vırus, Instituto de Biofısica Carlos Chagas Filho, CCS, 21949-900 Rio de Janeiro, RJ, Brazil
b
´
´
´
c FAR-MANGUINHOS, Rua Sizenando Nabuco 100, Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil
d Department of Chemistry, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, UK
e Centro de Desenvolvimento Tecnolo´gico em Sau´de (CDTS), Fundaç˜ao Oswaldo Cruz (FIOCRUZ), Casa Amarela, Campus de Manguinhos, Av. Brasil 4365,
21040-900 Rio de Janeiro, RJ, Brazil
f Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78248-0698, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the antiviral evaluation of new N-amino-1,2,3-triazole derivatives, 1-(substituted-
phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters, 3 and 1-(4-substituted-phe-
nylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazides, 4, on Cantagalo virus replication.
1-(4-Fluoro-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazide, 4e, exhibited
a significant antiviral effect. Characterization of all compounds was confirmed by IR, 1H and 13C spec-
troscopies and elemental analysis. In addition, molecular structure of 4e was also reported.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 22 July 2008
Received in revised form
14 April 2009
Accepted 23 April 2009
Available online 8 May 2009
Keywords:
1,2,3-Triazole derivatives
Diazo compounds
Cantagalo virus
Anti-poxvirus agents
Molecular structure
X-ray crystallography
1. Introduction
It is a general consensus in the literature that VACV has no
natural host. Nevertheless, vaccinia-like viruses (VACV-like) have
The poxviruses present a complex morphology, a double-
stranded DNA genome and replication in the cytoplasm of the host
cells [1]. The family Poxviridae includes eight genera, four of which
are of greater importance to humans: Orthopoxvirus (variola,
vaccinia, cowpox and monkeypox viruses), Parapoxvirus
(orf, Milker’s nodules and pseudocowpox viruses), Yatapoxvirus
(yatapox and tanapox viruses) and Molluscipoxvirus (molluscum
contagiosum virus) [2]. The Orthopoxvirus is by far the most
studied genus, with species of particular importance clinically and
historically, especially variola virus (VARV) and vaccinia virus
(VACV). VARV is the causative agent of smallpox, a disease that
ravaged the human population until its eradication in 1977 by
a worldwide vaccination campaign with VACV [1,2].
been frequently isolated from dairy cattle and humans in different
states of Brazil for the last 7 years [3–7]. Cantagalo virus (CTGV) was
the first sample isolated during a poxvirus outbreak in 1999 in Rio
de Janeiro State, and it was characterized as a VACV strain [1].
In recent years, the potential use of VARV or another ortho-
poxvirus, such as monkeypox virus, as a biological weapon has
heightened our awareness as to our vulnerability to smallpox since
routine vaccination was discontinued in 1980 [8]. A few drugs
[9,10], that have approval for other uses, have been identified as
anti-poxvirus agents, and efforts have been intensified to discover
and develop potentially active compounds against smallpox and
other diseases caused by orthopoxviruses. As already mentioned,
outbreaks of CTGV infection and other VACV-like viruses have been
frequently notified all over Brazil, leading to major agricultural
losses, but no treatment is yet available [5] (Fig. 1).
Among the known drugs, the most studied agent against
orthopoxviruses is the cidofovir, an acyclic nucleoside phosphonate
* Corresponding author. Tel.: þ55 21 2629 2148; fax: þ55 21 2629 2364.
E-mail address: annac@vm.uff.br (A.C. Cunha).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.046

3778
A.K. Jord˜ao et al. / European Journal of Medicinal Chemistry 44 (2009) 3777–3783
NH2
N
phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl
NH2
N
ester, 3e, and 1-(4-fluoro-phenylamino)-5-methyl-1H-[1,2,3]-tri-
azole-4-carboxylic acid hydrazide, 4e (Fig. 2). In designing these
compounds, a para-fluoro substituent was introduced into the N-
phenyl rings of the parent compounds 3a and 4a in order to
increase the structural similarity to 5.
N
N
O
O
O
O
O
O
P
2
17
O
O
O
3
P
15
HO
O
HO
OH
OH
2. Results and discussion
1,HDP-CDV
2,ODE-CDV
2.1. Chemistry
Fig. 1. Potential antiviral drugs against orthopoxvirus replication.
Compounds 3–4 were prepared according to the synthetic
pathways described in Scheme 1. Ethyl 2-diazoacetoacetate 6
prepared in a 70% yield by the method of Danheiser and co-workers
[38] was condensed with the desired phenylhydrazine (7a) or with
phenylhydrazine hydrochlorides (7b–e), giving the corresponding
diazo-hydrazone intermediates 8a–e, which underwent 1,5-elec-
trocyclization leading to the 4-carbethoxy-triazoles 3a–e (Scheme 1).
The 1,5-electrocyclization reaction using phenylhydrazine 7a
was carried out in MeOH/AcOH. Those using 7b–d in MeOH, and
that using 7e in MeOH/DMSO (4:1), as illustrated in Scheme 1. The
choice of solvent was determined by the solubilities of 7.
The yields of the reactions and the melting points of triazole
derivatives 3a–e are listed in Table 1. A good yield was obtained
from 7a, while compounds having moderate electron withdrawing
substituents, i.e. 7b, 7c and 7d, gave the corresponding 1,2,3-tri-
azoles 3b, 3c and 3d in reasonable yields. Compound 7e having
strong electron withdrawing group produced the triazole product
3e in low yield.
[9,11] approved for intravenous use in the treatment of cytomeg-
alovirus (CMV) retinitis in HIV-infected patients. However, its oral
bioavailability is the major limitation for use in a large-scale
emergency situation such as a smallpox outbreak [10,12]. The
alkoxyalkyl esters of cidofovir, such as hexadecyloxypropyl-cido-
fovir (1, HDP-CDV) and octadecyloxyethyl-cidofovir (2, ODE-CDV)
were designed and developed as potential oral drugs for the
prophylaxis and treatment of variola virus infection [13].
1,2,3-Triazoles have a wide range of applications as antimicro-
bial [14], cytostatic [15], anti-HIV [16], antineoplastic [17], and anti-
inflammatory agents [18] and as potassium channel activators [19].
Two general methods are available for the construction of 1,2,3-
triazole rings: Huisgen’s [1,3]-dipolar cycloaddition reactions [20],
in particular the copper(I)-catalyzed cycloaddition [21], and the
intramolecular 1,5-electrocyclization of
carbonyl compounds [22,23].
b-substituted-a-diazo-
Wolff [24], in 1912, reported the synthesis of ethyl 1-(phenyl-
amino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylate, 3a, from ethyl
2-diazoacetoacetate 6 and phenylhydrazine 7a (Scheme 1) and
then Shukla and co-workers [25] and Sezer and workgroup [26]
prepared other NH-triazoles by variation of this methodology.
Following our interest in triazoles [27–34] with potential phar-
macological activity, we have developed this methodology to
produce triazole derivatives 3a–d, from substituted phenyl-
hydrazines 7b–e and ethyl 2-diazoacetoacetate 6.
Finally, the 4-carbethoxy-triazole derivatives 3a–e were easily
converted into the corresponding acylhydrazides 4a–e in moder-
ated yields (Table 1), on treatment with hydrazine hydrate in
refluxing ethanol (Scheme 1).
2.2. Antiviral activity on Cantagalo virus
The structural similarity of these compounds to ST-246 5 [35]
(Fig. 2) led us to investigate their antiviral potential as poxvirus
inhibitors. Compound 5 is an anti-poxvirus agent that exhibits
activity against vaccinia virus and cowpox virus with EC50 values of
The antiviral activity of the 4-carbethoxy-triazoles 3a–e and
acylhydrazides 4a–e against Cantagalo virus replication was eval-
uated in BSC-40 cells (Table 2).
The pharmacological studies indicated for each of the triazole
series with different substituents in the phenyl group distinct
effects on the observed inhibitory activity against virus replication.
Of the 10 compounds tested, three inhibited the formation of
<0.5 mM and has been reported as one of the most promising
prototypes to be used against smallpox [36,37]. Also, it has recently
proved to be effective in treating severe side effects of smallpox
vaccination with VACV [8].
viral plaques by more than 30% at 50 mM after 48 h post-infection
In addition to 3a–d and 4a–d, we decided to investigate the
pharmacological profile of two fluoro derivatives, 1-(4-fluoro-
and treatment. Compound 3c (R1 ¼4-Br) had higher activity than
the less lipophilic, unsubstituted compound 3a (R1 ¼ H). At a higher
O
O
NH2
N
O
Me
HN
N
N
N
O
Me
O
O
N
Me
N
N
See
+
Me
HN
Me
Me
O
3a, R1 = H, 73%
conditions
on the table
H
3b, R1 = 4-Cl, 62%
3c, R1 = 4-Br, 62%
3d, R1 = 2,5-Cl2, 56%
3e, R1 = 4-F, 40%
N2
7a
6
R1
8a-e
R1
or
NH2NH2 80%
EtOH, reflux
NH2.HCl
R1
7b, R1= 4-Cl
7c, R1 = 4-Br
7d, R1 = 2,5-Cl2
7e, R1 = 4-F
HN
Aromatic
Conditions
phenylhydrazines
O
MeOH/AcOH (4:1), rt
7a
N
N
N
H
Me
NH2
7b and 7c-d
7e
MeOH, rt
4a, R1 = H, 56%
N
4b, R1 = 4-Cl, 58%
4c, R1 = 4-Br, 62%
4d, R1 = 2,5 Cl2, 50%
4e, R1 = 4-F, 50%
MeOH/DMSO (4:1), rt
N
H
R1
Scheme 1. Synthetic pathways used for 3a–e and 4a–e.

A.K. Jord˜ao et al. / European Journal of Medicinal Chemistry 44 (2009) 3777–3783
3779
H
O
NHNH2
Me
O
OEt
Me
H
H
SIMPLIFICATION
MOLECULAR
O
O
H
N
H
N
N
N
N
H
N
N
N
N
N
H
3a, R1 =H
O
3b, R1 =4-Cl
3c, R1 =4-Br
3d, R1 =2,5-Cl2
3e, R1 =4-F
R1
CF3
R1
5
4a, R1 = H
4b, R1 = 4-Cl
4c, R1 = 4-Br
4d, R1 = 2,5 Cl2
4e, R1 = 4-F
Fig. 2. Structural analogy between ST-246 5 vs N-aminotriazoles 3 and 4.
dose level 100
m
M, 3c induced 84.62% inhibition of viral plaque
effect of these compounds on CTGV replication is still unknown, but
cidofovir has been the most studied anti-poxvirus drug evaluated
so far. Its EC50 values range from 10 to 46 mM, depending on the
VACV strain used [9]. Nevertheless, compound 5 reveals lower EC50
values compared with cidofovir [36].
formation. Unfortunately, this compound was toxic to the cells,
altering cell morphology and reducing cell viability to 52.6% (data
not shown) and so was not further studied. The dichloro derivative
3d had a greater activity than did the monochloro derivative 3b.
Except for compound 3e, the change from a 4-carbethoxy-triazole
3a–d to the corresponding acylhydrazides 4a–d resulted in
a decreasing inhibitory response on Cantagalo virus replication.
For compound 4e, the electronic withdrawing effect of the
fluorine group and the hydrogen donor and acceptor functionalities
of the acylhydrazide moiety increased its biological activity. The
effects of 4-carbethoxy-triazole 3d and acylhydrazide 4e on the
yield of infectious virus particles were then analyzed after 24 h
post-infection and treatment. Both compounds inhibited virus
2.3. Molecular structure of 1-(4-fluoro-phenylamino)-5-methyl-
1H-[1,2,3]-triazole-4-carboxylic acid hydrazide 4e
A single crystal structure determination of 1-(4-fluoro-phenyl-
amino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazide 4e
was carried out at 120 K on a crystal grown by the slow evaporation
of an ethanolic solution. The structure was solved by direct methods
(SIR92) [39] and refined anisotropic displacement parameters, H
atoms in the riding model approximation and a weighting scheme
progeny production by more than 55% at 50 mM. Nevertheless,
using the neutral red assay to evaluate cellular toxicity, we
observed that 60% of the cells were viable when treated with the
w ¼ 1/[
s
2(Fo2) þ 0.078P2 þ 0.337P] where P ¼ (Fo2 þ 2Fc2)/3) with
SHELXL-97 [40] on F2. CCDC deposition number: 677104.
The atom connectivity is shown in Fig. 4. All bond lengths and
angles were in the expected ranges. The triazole ring exhibits
derivative 3d at 50 mM for 24 h (data not shown). At a similar
concentration, 4e generated 10% of non-viable cells after 24 h of
treatment (Fig. 3A) and inhibited the yield of infectious virus
particles by approximately 55% (Table 2 and Fig. 3B). Doses higher
than 100 mM were toxic to BSC-40 cells, although at this concen-
tration the virus yield was inhibited by nearly 80% with only 30% of
non-viable cells (Fig. 3B).
substantial delocalization of p-electron density as evidenced by its
planarity and pattern of bond distances; see figure caption. The
fluorophenyl ring adopts a conformation almost normal to the
triazole ring with the C10–O1–N5–N6 atoms essentially co-planar
with the attached triazole ring.
The accumulation of viral proteins during infection was also
inhibited in the presence of 4e (Fig. 3C). Western Blots, using anti-
bodies to detect virus structural proteins, revealed normal levels of
protein accumulation in infected cells treated with 0.5% DMSO
(control). Virus proteins were not detected in mock-infected cells, as
expected. Increasing concentrations of compound 4e gradually
reduced the accumulation of virus proteins, reaching 71.2% inhibition
3. Experimental protocols
Melting points were determined with a Fisher–Johns instru-
ment and are uncorrected. Infrared (IR) spectra were recorded on
Perkin–Elmer FT-IR, model 1600 senes spectrophotometer in KBr
pellets. NMR spectra, unless otherwise stated, were obtained in
deuterated Me2SO-d6 or CDCl3 using a Varian Unity Plus 300 MHz
at 100
mM, as determined by densitometric analysis of the blots
(Fig. 3D). The cellular protein
a-tubulin was detected as a control for
spectrometer. Chemical shifts (d) are expressed in ppm and the
coupling constant (J) in hertz. Column chromatography was
total protein loading and normalization for the densitometric analysis.
Compound 4e, therefore, revealed a great potential as a lead
structure that can be used in the development of new derivatives
with anti-Cantagalo virus activity. Cidofovir and compound 5 are
currently the most promising drugs against orthopoxviruses. The
Table 2
Antiviral activity of N-amino-1,2,3-triazole derivatives 3a–e and 4a–e on Cantagalo
virus replication.
Compound % Inhibition of virus plaque numbera,b % Inhibition of virus yielda,c
Table 1
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
15.15 ꢁ 4.75
11.00 ꢁ 3.9
34.41 ꢁ 3.16
47.16 ꢁ 6.41
9.42 ꢁ 4.53
6.10 ꢁ 3.08
3.79 ꢁ 7.15
8.40 ꢁ 5.5
ND
ND
ND
Yields and m.p. for N-amino-1,2,3-triazole derivatives 3 and 4.
Reagent
Product
R1
m.p. (ꢀC)
Yield (%)
67.51 ꢁ 4.84
7a
7b
7c
7d
7e
7a
7b
7c
7d
7e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
H
165–166
145–146
138–140
110
122
197
188–190
213–214
229–230
180–182
73
62
62
56
30
56
58
62
50
50
ND
4-Cl
4-Br
2,5-Cl2
4-F
ND
ND
ND
ND
9.71 ꢁ 2.8
H
48.26 ꢁ 5.25
55.7 ꢁ 3.6
4-Cl
4-Br
2,5-Cl2
4-F
ND: not determined.
a
The assay concentration was 50 mM.
Results observed after 48 h post-infection/treatment.
Results observed after 24 h post-infection/treatment.
b
c

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A.K. Jord˜ao et al. / European Journal of Medicinal Chemistry 44 (2009) 3777–3783
Fig. 3. Cytotoxicity and antiviral activity of compound 4e. (A) Cytotoxicity assay: BSC-40 cells were treated with increasing concentrations of the derivative 4e for 24 h and
processed for the neutral red uptake assay. (B) Inhibition of virus progeny production: BSC-40 cells were infected with CTGV and treated with increasing concentrations of the
derivative 4e. After 24 h, the cells were harvested, and the formation of infectious virus particles was determined by plaque assay. (C) Accumulation of virus proteins: the cells were
infected and treated as above. The concentrations of compound 4e are indicated on the top of each lane. Samples were analyzed on SDS-PAGE followed by Western Blot using
specific anti-vaccinia virus antibodies. M ¼ mock-infected cells; C ¼ control cells treated with 0.5% DMSO. (D) The densitometric analysis of each lane of the Western Blot was
performed using tubulin to normalize protein load.
performed on silica gel flash from Acros. Reactions were routinely
monitored by thin layer chromatography (TLC) on silica gel pre-
coated F254 Merck plates. Microanalyses were performed using
a Perkin–Elmer Model 2400 instrument and all values were within
ꢁ0.4% of the calculated compositions.
substituted phenylhydrazine hydrochlorides 7b–d (1 mmol) or
a solution of the phenylhydrazine hydrochloride 7e (1 mmol) in
MeOH/DMSO (4:1, 10 mL) was added ethyl 2-diazoacetoacetate
(0.156 g, 1 mmol). The resulting mixture was stirred at room
temperature for 24 h. The crude reaction was then concentrated
under reduced pressure and the residue was column chromato-
graphed using silica gel and ethyl acetate/hexane (3:7) as eluent to
give the pure triazoles 3a–e.
The N-amino-1,2,3-triazole derivative 3a was prepared by
published procedure [24].
3.1. Chemistry
3.1.1.1. 5-Methyl-1-(phenylamino)-1H-[1,2,3]-triazole-4-carboxylic
acid ethyl ester 3a. Obtained in 73% yield as a yellow solid; m.p.
165–166 ꢀC; IR (KBr) nmax (cmꢂ1) 3211 (N–H); 1727 (C]O), 1218 (C–
3.1.1. General procedure for the preparation of the 4-carbethoxy-
triazole derivatives 3a–e
To a MeOH/acetic acid (5:1) (10 mL) solution of the phenyl-
hydrazine 7a (1 mmol) or a methanolic solution (10 mL) of the
O); 1H NMR (300 MHz; CDCl3):
d
1.45 (t, 3H, J ¼ 7.1, OCH2CH3), 2.57
(s, 3H, CH3), 4.46 (q, 2H, J ¼ 7.2, OCH2CH3), 6.50 (dd, 2H, J ¼ 8.5; 1.0,
Fig. 4. Atom arrangements for 4e. Probability ellipsoids are drawn at the 50% level. Hydrogen atoms are shown as spheres of arbitrary radius.

A.K. Jord˜ao et al. / European Journal of Medicinal Chemistry 44 (2009) 3777–3783
3781
H-20 and H-60), 7.01 (tt, 1H, J ¼ 7.3; 1.0, H-40), 7.21–7.27 (m, 2H, H-30
3.1.2.1. 5-Methyl-1-(phenylamino)-1H-[1,2,3]-triazole-4-carboxylic
acid hydrazide 4a. Obtained in 56% yield as a yellow solid; m.p.
210 ꢀC; IR (KBr) nmax (cmꢂ1) 3374, 3222 (N–H); 1631 (C]O); 1H NMR
and H-50), 7.63 (bs, 1H, N–H) ppm. 13C NMR (75 MHz; CDCl3):
d 8.6,
(CH3), 14.3 (–OCH2CH3), 61.1 (OCH2CH3) 113.7 (C-20 and C-60), 122.7
(C-40), 129.4 (C-30 and C-50), 135.2 (C-4 or C-5), 140.1 (C-4 or C-5),
145.0 (C-10), 161.3 (C]O). Anal. Calcd for C12H14N4O2: C, 58.53; H,
5.73; N, 22.75. Found: C, 57.9; H, 5.4; N, 22.4.
(300 MHz, DMSO-d6) d: 2.40 (s, 3H, CH3), 4.46 (bs, 2H, NH–NH2),
6.44 (dd, 2H, J ¼ 8.5; 0.9, H-20 and H-60), 6.92 (tt, 1H, J ¼ 7.3; 0.9, H-
40), 7.22–7.27 (m, 2H, H-30 and H-50), 9.72 (bs, 1H, NH–NH2), 10.15
(bs, 1H; N–H). 13C NMR (75 MHz, DMSO-d6) : 7.9 (CH3), 112.7 (C-20
d
3.1.1.2. 1-(4-Chlorophenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid ethyl ester 3b. Obtained in 62% yield as a yellow
solid; m.p. 145–146 ꢀC; IR (KBr) nmax (cmꢂ1) 3215 (N–H); 1725
and C-60), 121.3 (C-40), 129.4 (C-30 and C-50), 136.4 (C-4 or C-5), 136.6
(C-4 or C-5), 146.2 (C-10), 160.0 (C]O). Anal. Calcd for C10H12N6O: C,
51.72; H, 5.21; N, 36.19. Found: C, 51.01; H, 5.12; N, 36.41.
(C]O), 1216 (C–O); 1H NMR (300 MHz; CDCl3):
d
1.44 (t, 3H, J ¼ 7.2,
OCH2CH3), 2.54 (s, 3H, CH3), 4,45 (q, 2H, J ¼ 7.2, OCH2CH3), 6.41 (d,
3.1.2.2. 1-(4-Chlorophenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid hydrazide 4b. Obtained in 58% yield as a white solid;
m.p. 190–191 ꢀC; IR (KBr) nmax (cmꢂ1) 3410, 3306 (N–H); 1670
2H, J ¼ 8.9, H-20 and H-60), 7.17 (d, 2H, J ¼ 8.9, H-30 and H-50), 7.99
(bs, 1H, N–H) ppm. 13C NMR (75 MHz; CDCl3):
d 8.6, (CH3), 14.2
(–OCH2CH3), 61.2 (OCH2CH3) 114.8 (C-20 and C-60), 127.6 (C-40),
129.3 (C-30 and C-50), 135.2 (C-4 or C-5), 140.1 (C-4 or C-5), 143.7 (C-
10), 161.0 (C]O). Anal. Calcd for C12H13ClN4O2: C, 51.34; H, 4.67; N,
19.96. Found: C, 52.00; H, 4.34; N, 19.13.
(C]O); 1H NMR (300 MHz, DMSO-d6)
d: 2.39 (s, 3H, CH3), 4.46
(bs, 2H, NH–NH2), 6.47 (d, 2H, J ¼ 8.8, H-20 and H-60), 7.29 (d, 2H,
J ¼ 8.8, H-30 and H-50), 9.74 (bs, 1H, NH–NH2), 10.31 (bs, 1H, N–H).
13C NMR (75 MHz, DMSO-d6) : 7.9 (CH3), 114.5 (C-20 e C-60), 125.0
d
(C-40), 129.2 (C-30 and C-50), 136.5 (C-4 or C-5), 136.6 (C-4 or C-5),
145.1 (C-10), 159.9 (C]O). Anal. Calcd for C10H11ClN6O: C, 45.04; H,
4.16; N, 31.51. Found: C, 45.12; H, 4.09; N, 31.18.
3.1.1.3. 1-(4-Bromophenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid ethyl ester 3c. Obtained in 62% yield as a yellow
solid; m.p. 138–140 ꢀC; IR (KBr) nmax (cmꢂ1) 3214 (N–H); 1724
(C]O), 1215 (C–O); 1H NMR (300 MHz; CDCl3):
d
1.44 (t, 3H, J ¼ 7.2,
3.1.2.3. 1-(4-Bromophenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid hydrazide 4c. Obtained in 62% yield as yellow solid;
m.p. 212 ꢀC; IR (KBr) nmax (cmꢂ1) 3317, 3259 (N–H); 1674 (C]O); 1H
OCH2CH3), 2.54 (s, 3H, CH3), 4.45 (q, 2H, J ¼ 7.2, OCH2CH3), 6.41
(d, 2H, J ¼ 8.9, H-20 and H-60), 7.17 (d, 2H, J ¼ 8.9, H-30 and H-50), 7.98
(bs, 1H, N–H) ppm. 13C NMR (75 MHz; CDCl3):
d
8.6 (CH3), 14.2
NMR(300 MHz, DMSO-d6) d: 2.40 (s, 3H, CH3), 4.45 (bs, 2H, NH–
(OCH2CH3), 61.3 (OCH2CH3), 115.0 (C-40), 115.2 (C-20 and C-60), 132.2
(C-30 and C-50), 135.2 (C-4 or C-5), 140.2 (C-4 or C-5), 144.2 (C-10),
161.1 (C]O). Anal. Calcd for C12H13BrN4O2: C, 44.33; H, 4.03; N,
17.23. Found: C, 44.06; H, 4.09; N, 16.98.
NH2), 6.43 (d, 2H, J ¼ 9.0, H-20 and H-60), 7.41 (d, 2H, J ¼ 9.0, H-30
and H-50), 9.63 (bs, 1H, NH–NH2), 10.25 (bs, 1H, N–H). 13C NMR
(75 MHz, DMSO-d6) d
: 7.7 (CH3), 112.5 (C-40), 114.8 (C-20 and C-60),
131.9 (C-30 and C-50), 136.4 (C-4 or C-5), 136.5 (C-4 or C-5), 145.4 (C-
10), 159.8 (C]O). Anal. Calcd for C10H11BrN6O: C, 38.60; H, 3.56; N,
27.01. Found: C, 38.33; H, 3.61; N, 26.92.
3.1.1.4. 1-(2,5-Dichlorophenylamino)-5-methyl-1H-[1,2,3]-triazole-
4-carboxylic acid ethyl ester 3d. Obtained in 56% yield as a yellow
solid; m.p. 110 ꢀC; IR (KBr) nmax (cmꢂ1) 3308 (N–H); 1716 (C]O),
3.1.2.4. 1-(2,5-Dichlorophenylamino)-5-methyl-1H-[1,2,3]-triazole-
4-carboxylic acid hydrazide 4d. Obtained in 50% yield as a yellow
solid; m.p. 193 ꢀC; IR (KBr) nmax (cmꢂ1) 3304, 3225 (N–H); 1635
1202 (C–O); 1H NMR (300 MHz; CDCl3):
d
1.47 (t, 3H, J ¼ 7.2,
OCH2CH3), 2.60 (s, 3H, CH3), 4.48 (q, 2H, J ¼ 7.2, OCH2CH3), 5.99
(d, 1H, J ¼ 2.3, H-60), 6.96 (dd, 1H, J ¼ 8.4; 2.3, H-40), 7.31 (d, 1H,
J ¼ 8.4, H-30), 7.80 (bs, 1H, N–H) ppm. 13C NMR (75 MHz; CDCl3):
(C]O); 1H NMR (300 MHz, DMSO-d6)
d: 2.41 (s, 3H, CH3), 4.48 (bs,
2H, NH–NH2), 5.98 (d, 1H, J ¼ 2.4, H-60), 7.03 (dd, 1H, J ¼ 8.5; 2.4, H-
d
8.6, (CH3), 14.3 (OCH2CH3), 61.3 (OCH2CH3), 113.6 (C-60), 117.8 (C-
40), 7.51 (d, 1H, J ¼ 8.5, H-30), 9.78 (bs, 1H, NH–NH2), 10.26 (bs, 1H,N–
50), 123.3 (C-40), 130.6 (C-30), 134.0 (C-20), 135.4 (C-4 or C-5), 140.4
(C-4 or C-5), 141.9 (C-10), 161.0 (C]O). Anal. Calcd for
C12H12Cl2N4O2: C, 45.73; H, 3.84; N, 17.78. Found: C, 45.81; H,
3.72; N, 18.06.
H).13C NMR (75 MHz, DMSO-d6) : 7.9 (CH3),113.0 (C-60),116.7 (C-50),
d
122.0 (C-40), 131.3 (C-30), 133.0 (C-20),136.6 (C-4 or C-5), 137.0 (C-4 or
C-5), 143.0 (C-10), 160.0 (C]O). Anal. Calcd for C10H10Cl2N6O: C,
39.89; H, 3.35; N, 27.91. Found: C, 39.00; H, 3.77; N, 27.80.
3.1.1.5. 1-(4-Fluoro-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid ethyl ester 3e. Obtained in 30% yield as a yellow
solid; m.p. 122 ꢀC; IR (KBr) nmax (cmꢂ1) 3264 (N–H); 1706 (C]O),
3.1.2.5. 1-(4-Fluoro-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carboxylic acid hydrazide 4e. Obtained in 50% yield as yellow solid;
m.p. 180–182 ꢀC; IR (KBr) nmax (cmꢂ1) 3321, 3218 (N–H); 1638
1208 (C–O); 1H NMR (300 MHz; CDCl3/Me4Si):
d
1.42 (t, 3H, J ¼ 7.1,
(C]O); 1H NMR (300 MHz, DMSO-d6)
d: 2.41 (s, 3H, CH3), 4,45 (bs,
OCH2CH3), 2.54 (s, 3H, CH3), 4.44 (q, 2H, J ¼ 7.1, OCH2CH3), 6.48 (d,
2H, NH–NH2), 6.49 (d, 2H, 9.0; 4.4, H-20 and H-60), 7.09 (dd, 2H,
2H, J ¼ 9.0; 4.2, H-20 and H-60), 6.91 (d, 2H, J ¼ 9.0; 8.3, H-30 and H-
J ¼ 9.0; 8.9, H-30 and H-50), 9.68 (bs, 1H, NH–NH2), 10.11 (bs, 1H, N–
50). 13C NMR (75 MHz; CDCl3/Me4Si):
d
8.6, (CH3), 14.2 (OCH2CH3),
H). 13C NMR (75 MHz, DMSO-d6)
d
: 7.9 (CH3), 114.6 (d, J ¼ 8.2, C-20
61.2 (OCH2CH3), 115.4 (d, J ¼ 8.0, C-20 and C-60), 116.0 (d, J ¼ 23, C-30
and C-50), 135.1 (C-4 or C-5),139.9 (C-4 or C-5),141.2 (C-10), 158.6 (d,
J ¼ 240, C-40), 161.2 (C]O). Anal. Calcd for C12H13FN4O2: C, 54.54; H,
4.96; N, 21.20. Found: C, 54.01; H, 4.97; N, 20.48.
and C-60), 116.0 (d, 2H, J ¼ 22.7, C-30 and C-50), 136.5 (C-4 or C-5),
136.6 (C-4 or C-5), 142.7 (d, J ¼ 2.6, C-10), 157.2 (d, 2H, J ¼ 235, C-40),
160.0 (C]O). Anal. Calcd for C10H11FN6O: C, 48.00; H, 4.43; N, 33.58.
Found: C, 47.96; H, 4.46; N, 33.29.
3.1.2. General procedure for the preparation of 4-carbohydrazide-
3.2. Biological evaluation
1,2,3-triazole derivatives 4a–e
A
solution of the appropriate ethyl ester derivative
3
3.2.1. Analysis of virus plaque formation and virus yield
(1.00 mmol), hydrazine monohydrate (1.00 mL of 80%) and catalytic
amount of 37% (w/v) hydrochloric acid in ethanol (5 mL) was
refluxed for 1–2 h. The reaction mixture was then concentrated
under reduced pressure and the resulting solid was collected by
filtration, washed with cold water and dried under vacuum to give
the desired hydrazide-triazoles 4.
Confluent BSC-40 cells (1 ꢃ106 cells/plate) were infected with
200 particle forming units (PFU) of CTGV per plate. After a 120-min
adsorption period, the inoculum was removed, and the cells were
incubated at 37 ꢀC with fresh medium containing 0.1% DMSO
(control cells) or 50
mM of a specified derivative. The plates were
incubated at 37 ꢀC for 48 h when the monolayers were stained

3782
A.K. Jord˜ao et al. / European Journal of Medicinal Chemistry 44 (2009) 3777–3783
with 0.1% crystal violet in 10% formaldehyde. The number of viral
plaques was determined by visual inspection. The cells were
infected with 5000 PFU of CTGV and, after the adsorption period,
the monolayers were treated with the specified concentration of
the derivative, as indicated in the figure legend. After 24 h of
infection/treatment, the cells were collected in PBS for yield
determination by plaque assay [41]. All measurements of virus
production are expressed as average of three experiments per-
formed in duplicates.
Compound 4e is a promising lead structure for the development
of new derivatives with anti-Cantagalo virus activity. We are
currently working with these compounds to increase their activi-
ties, in particular by introducing structural changes at position 4 of
the triazole ring.
Acknowledgements
This work was supported by the Brazilian agencies FAPERJ and
CNPq, and the International Foundation for Science. Fellowships by
CAPES, CNPq, CNPq-PIBIC and FAPERJ are gratefully acknowledged.
The authors thank the EPSRC X-Ray Crystallographic Service, at the
University of Southampton, England for the data collection.
3.2.2. Cytotoxicity assay
The neutral red assay was previously described [21], and it is
based on the incorporation of the supravital dye neutral red into
living cells. Confluent BSC-40 cells (96-well plate) were incubated
with several concentrations of the compounds for 24 h at 37 ꢀC. The
control was medium with 0.5% DMSO. Neutral red was added to
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