Synthesis, antitubercular activity, and SAR study of N-substituted- phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides

Article abstract of DOI:10.1016/j.bmc.2011.07.035

Tuberculosis treatment remains a challenge that requires new antitubercular agents due to the emergence of multidrug-resistant Mycobacterium strains. This paper describes the synthesis, the antitubercular activity and the theoretical analysis of N-substit

Full text of DOI:10.1016/j.bmc.2011.07.035

Bioorganic & Medicinal Chemistry 19 (2011) 5605–5611
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antitubercular activity, and SAR study
of N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides
Alessandro K. Jordão a, Plínio C. Sathler b,e, Vitor F. Ferreira a, Vinícius R. Campos a, Maria C. B. V. de Souza a,
Helena C. Castro b,e, Andressa Lannes b,e, André Lourenco b,e, Carlos R. Rodrigues c, Murilo L. Bello c,
Maria C. S. Lourenco d, Guilherme S. L. Carvalho d, Maria C. B. Almeida a, Anna C. Cunha a,
⇑
a Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de Química, Outeiro de São João Baptista, 24020-141 Niterói, RJ, Brazil
b Universidade Federal Fluminense, LABioMol, Departamento de Biologia Celular e Molecular, Outeiro de São João Baptista, CEP 24020-141, Niterói, RJ, Brazil
c Universidade Federal do Rio de Janeiro, ModMolQSAR, Faculdade de Farmácia, Ilha do Fundão, CEP 21541-590, Rio de Janeiro, RJ, Brazil
d Fundação Oswaldo Cruz, Instituto de Pesquisa Evandro Chagas, Manguinhos, CEP 21040-030, Rio de Janeiro, RJ, Brazil
e Universidade Federal Fluminense, Hospital Universitário Antônio Pedro, Departamento de Patologia, Centro, CEP 24033-900, Niterói, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis treatment remains a challenge that requires new antitubercular agents due to the emergence
of multidrug-resistant Mycobacterium strains. This paper describes the synthesis, the antitubercular activ-
ity and the theoretical analysis of N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydra-
zides (8a–b, 8e–f, 8i–j and 8n–o) and new analogues (8c–d, 8g–h, 8l–m and 8p–q). These derivatives
were synthesized in good yields and some of them showed a promising antitubercular profile. Interest-
ingly the N-acylhydrazone (NAH) 8n was the most potent against the Mycobacterium tuberculosis H37Rv
Received 11 May 2011
Revised 11 July 2011
Accepted 19 July 2011
Available online 24 July 2011
Keywords:
strain (MIC = 2.5 lg/mL) similar to or better than the current drugs on the market. The theoretical struc-
1,2,3-Triazole derivatives
Diazocompounds
Carbohydrazides
ture–activity relationship study suggested that the presence of the furyl ring and the electronegative
group (NO2) as well as low lipophilicity and small volume group at R position are important structural fea-
tures for the antitubercular profile of these molecules. NMR spectra, IR spectra and elemental analyses of
these substances are reported.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Currently MDR-TB and XDR-TB are detected worldwide, includ-
ing in Eastern Europe and in Africa.12 XDR-TB is associated with a
Tuberculosis (TB) is an infection caused by Mycobacterium
tuberculosis with high mortality rates.1–3 Currently, tuberculosis
is endemic throughout the world, and HIV-associated TB, inade-
quate public health resources,2,4 extreme poverty and the develop-
ment of M. tuberculosis resistance to commonly used first-line
drugs (i.e., isoniazid and rifampin)6–11 are factors that contribute
to the disease resurgence.5
TB therapy is a multi-drug regimen over a long period of time.
The failure of patients to complete or manage the therapy has
led to the emergence of multidrug-resistant TB (MDR-TB) and
extensively drug-resistant TB (XDR-TB). MDR-TB is defined as the
bacterial resistance to at least isoniazid and rifampicin, the most
potent antibiotics used against the disease2,6,8 whereas XDR-TB is
the resistance to at least rifampicin and isoniazid in addition to
any fluoroquinolone and to at least one of the three injectable
drugs commonly used in antitubercular treatment (capreomycin,
kanamycin and amikacin).6,13
higher mortality rate than MDR-TB due to the limited options for
effective treatment. This treatment deficiency is considered a seri-
ous public health threat, especially in populations already stricken
by HIV and therefore more susceptible to TB infection.6,13,14 Thus
these strains, particularly XDR-TB, seriously compromises TB con-
trol efforts.12
Currently, there is an overwhelming need to develop new struc-
tural classes of antitubercular agents that allow shorter and more
effective therapies.1,15,16 N-acylhydrazones (NAH) 1–317–20 and
several 1,2,3-triazole analogs 3–710,21–23 have been reported with
antitubercular activity (Fig. 1). The studies have showed that the
hydrazone group plays an important role for the antitubercular
profile of compounds 1–3.17–20 Simultaneously, compounds con-
taining 1,2,3-triazoles are among several heterocycles with broad
medicinal applications, such as dopamine D2 receptor ligands, for
treating schizophrenia,24 antiviral,25 antineoplastic,26 antiplate-
let,27 trypanocidal28 and antiophidic agents.29
In this work, we designed new derivatives exploring the intro-
duction of different aromatic rings (e.g., phenyl, bromothiophenyl,
furyl and nitrofuryl) attached to the imine group to verify the effect
of a pharmacophoric group at the C-4 position of the 1,2,3-triazole
⇑
Correspondence author. Tel.: +55 21 26292148; fax: +55 21 26292145.
E-mail address: annac@vm.uff.br (A.C. Cunha).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.035

5606
A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
O
Ar
N
N
N
H
H
N
Me
N
O
Ar
N
N
H
H
H
N
X
N
N
N
CF3
3a, Ar = 3-Methyl-2-thiophenyl
3b, Ar = 4-Cl-Ph
1a, X = N and Ar = 4-CF3C6H4
O
O
1b, X = C and Ar = 5-nitro-2-furfuryl
2
OBz
CF2
N
N
N
N
N
N
N
H3C
N
N
N
N
OCH3
O
OH
4
F
N
N
N
R1
5
OH
R1
R3
4a, R1= H and R2 =R3 = F
4b, R1 = R3 =H, R2 = F
4c, R1= R3= F and R2 = H
R2
7
R2
R3
6a, R1= R3 =H and R2 = OCH3
6b, R1 = R3 = Cl and R2 =H
Figure 1. Tuberculostatic agents 1–7.
ring. In rational design research area, halogens are known for
inductive, conjugative, steric, and/or electronic effects that can be
directly involved in the biological activity (i.e., antimicrobial).30
The replacement of a halogen atom at C–O or C–H centers espe-
cially in the medicinal chemistry area can produce marked results
and unexpected biological activity.30 Therefore we synthesized the
new bromo- and fluoro substituted analogues 8c–d, 8g–h, 8l–m
and 8p–q to evaluate the contribution of the steric and electronic
effects of these halogens for their antitubercular activity.
We also described the antitubercular profile against M. tubercu-
losis H37Rv and the structure–activity relationship (SAR) study
using a molecular modeling approach of the 4-acylhydrazone
derivatives27 8a–b, 8e–f, 8i–j, 8n–o and of the new analogs
N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbo-
hydrazides 8c–d, 8g–h, 8l–m and 8p–q (Fig. 2) to identify struc-
tural features important for the biological activity.
The NAH derivatives 8a–b, 8e–f and 8i–j as well as the novel
compounds 8c–d, 8g–h and 8l–m (Table 1) were prepared by the
condensation of compounds 10a–d with suitable aromatic alde-
hydes (Route I) in ethanol using hydrochloric acid as the catalyst
(Route I). The 5-nitrofuran derivatives 8n–q were obtained by
the condensation of compounds 10a–d with commercially avail-
able 5-nitro-2-furfurylidene diacetate in a 1:1 mixture of ethanol
and sulfuric acid (Route II).
The stereochemistry of the imine double bond in this series was
determined for the understanding of the biological results. A de-
tailed analysis of the 1H NMR spectra of the derivatives 8a–q re-
vealed only one N–H signal for each compound, which was
attributed to the (E)-diastereomer, on the basis of a nuclear Over-
hauser (nOe) experiment. For example, irradiation of the N–H
hydrogen signal at 10.22 ppm of compound 8n resulted in a
NOE-enhancement of the N@CH hydrogen signal at 8.54 ppm
(14.4%) (Fig. 3). Despite the possibility of the formation of two dia-
stereoisomers, only the (E)-isomer was isolated from the reaction.
2. Results and discussion
2.1. Chemistry
2.2. In vitro and in silico analyses of the antitubercular profile
against Mycobacterium tuberculosis H37Rv – Structure–activity
relationship (SAR) study
The synthesis of the 4-acylhydrazone derivatives 8a–b, 8e–f,
8i–j, 8n–o, and of the new analogues 8c–d, 8g–h, 8l–m, 8p–q is
shown in Scheme 1. The carbohydrazides 10a–d were prepared
in moderated yields by the condensation of the corresponding 4-
carbethoxy-triazole derivatives 9a–d with hydrazine hydrate in
refluxing ethanol according to the method described in our previ-
ous report.27
Initially, we tested the compounds 8a–q against M. tuberculosis
H37Rv (ATCC 27294) at a screening concentration (100 lg/mL).
These data revealed an antitubercular profile only for the acylhyd-
razone derivatives 8e and 8i–q (Table 2). To analyze the impor-
tance of the carbohydrazide functional group at the C-4 position
Differents aromatic rings
Ar
H
O
N
R
N
H
N
8e
8f
, R = H and Ar = 5-bromo-2-thienyl
8a
, R = H and A r = Ph
, R = Cl and Ar = 5-bromo-2-thienyl
N
8b
, R = Cl and A r = Ph
Me
N
N
8g
, R = Br and Ar = 5-bromo-2-thienyl
8c
8d
, R = Br and A r = Ph
8
8h
, R = F and Ar = 5-bromo-2-thienyl
, R = F and A r = Ph
H
Ar =
8n
, R = H and Ar = 5-nitro-2-furfuryl
8i
8j
8l
, R = H and Ar = 2-furfuryl
, R = Cl and Ar = 2-furfuryl
, R = Br and Ar = 2-furfuryl
8o
, R = Cl and Ar = 5-nitro-2-furfuryl
8p
8q
, R = Br and Ar = 5-nitro-2-furfuryl
, R = F and Ar =5-nitro- 2-furfuryl
8m
, R = F and Ar = 2-furfuryl
R = H, Cl, Br and F
Figure 2. Strategy for the synthesis of compounds 8a–q.

A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
5607
O
ROUTE I
Ar
H
O
N
C
O
N
N
H
ArCHO/ HCl cat.
N
N
N
H
NH2
NH2NH2 80%
EtOH, reflux
N
N
N
O
Me
Me
N
N
8a
, R = H and A r = Ph
Me
N
N
EtOH
, R = H
8b
, R = Cl and A r = Ph
, R = Br and A r = Ph
Me
N
N
H
8c
8d
H
10a
10b
, R = F and A r = Ph
H
, R =Cl
R
9a, R = H
R
10c, R = Br
10d, R = F
8e
, R = H and Ar = 5-bromo-2-thienyl
9b, R = Cl
9c, R = Br
9d, R = F
R
8f, R = Cl and Ar = 5-bromo-2-thienyl
8g
, R = Br and Ar = 5-bromo-2-thienyl
ROUTE II
8h, R = F and Ar = 5-bromo-2-thienyl
OAc
OAc
8i, R = H and Ar = 2-furfuryl
8j, R = Cl and Ar = 2-furfuryl
EtOH/ H2SO4 50%
O
O2N
H
8l
, R = Br and Ar = 2-furfuryl
8m, R = F and Ar = 2-furfuryl
NO2
O
O
N
C
N
N
H
H
8n
, R = H
N
8o
Me
, R = Cl
N
N
8p
8q
, R = Br
, R = F
H
R
Scheme 1. Synthetic pathways used for the synthesis of compounds 8a–q.
Table 1
Yields and melting points of NAH derivatives 8c–d, 8g–h, 8l–m and 8p–q
R1
O
O
X
8l, R = Br, X = O and R1 = H
8m
N
C
N
N
H
N
C
, R = F, X = O and R1 = H
H
N
N
H
H
N
Me
N
N
N
8p, R = Br, X = O and R1 =NO2
Me
N
N
8q
, R = F, X = O and R1 = NO2
8c, R = Br
8d, R = F
H
8g
, R = Br, X = S and R1 = Br
H
8h
, R = F, X = S and R1 = Br
R
R
Compound
R
X
R1
Mp (°C)
Yield (%)
8c
8d
8g
8h
8l
8m
8p
8q
Br
F
Br
F
Br
F
Br
F
—
—
S
—
—
Br
Br
H
H
NO2
NO2
239–240
217–218
235–238
215–218
240–242
210–214
177–179
157–165
99
91
90
53
97
81
80
86
S
O
O
O
O
minimum inhibitory concentration (MIC) against M. tuberculosis
H37Rv using broth macrodilution assays (Table 2). Interestingly,
MIC assays of the triazole series 8a–q revealed the nitrofuran ana-
NO2
O
O
N C
N
N
H
log 8n as the most active compound (MIC = 2.5
antitubercular activity similar to clinical successful drugs (i.e., eth-
ambutol MIC = 2 g/ml) or those still in evaluation (i.e., isoxyl
MIC = 2.5
g/ml).31 The derivatives 8i, 8o and 8q also showed a
potential profile (MIC = 5.0 g/mL) as well as 8j, 8l and 8m
(MIC = 12.5–25 g/mL) comparable or better than some com-
pounds reported in the literature such as ethambutol derivative
SQ109 and pyrazinamide (MIC range in 5–64 g/mL and
12.5–25
g/mL respectively).2
lg/mL) with an
N
H
Me
N
14.4%
N
H
l
NOE
R
l
8n
Figure 3. (E)-configuration of N@C bond in the derivative 8n.
l
l
l
of the triazole ring, we also tested the compounds 10a–d used to
preparation of the NAH derivatives 8a–q. However no effect was
detected against M. tuberculosis H37Rv for any derivative evaluated
(not shown). This result suggests that the presence of the carbo-
hydrazide group at C-4 position of the triazole ring do not deter-
mine the biological activity per se.
Since the compounds 8a–d and 8f–h did not show any antitu-
lg/mL), only the active compounds
(8e and 8i–q) were further tested for determination of the
l
In this work we performed a structure–activity relationship
(SAR) analysis of the compounds 8a–q using a molecular modeling
approach. Overall, the structural analysis of the 3-D structure of
these derivatives revealed a conserved conformation for all acylhyd-
razone derivatives (Fig. 4A). This result pointed to other structural
parameters, instead of only the original conformation as important
to determine the antitubercular profile. Interestingly, the in vitro
and in silico analyses pointed the nature of the arylhydrazone
bercular activity (MIC >100

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A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
Table 2
Comparison of the antitubercular activity of compounds 8a–q (Minimal Inhibitory Concentration - MIC) against Mycobacterium tuberculosis H37Rv strain with the derivatives
electronic properties (Dipole moment, HOMO and LUMO energy and number of Hydrogen Bond Acceptor - HBA and Donor - HBD groups) and other structural and stereo features
(Molecular Volume, c log P, Molecular weight–Mwt, and Polar Surface Area–PSA). The most active compound of each subgroup is in bold.
#
R
X
R1
MIC (l
g/ml)
ELUMO (eV)
EHOMO (eV)
Dipole (debye)
Volume (Å3)
c log P
Mwt (amu)
HBA
HBD
R
⁄
PSA (Å2)
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
Cl
Br
F
H
Cl
Br
F
H
Cl
Br
F
H
Cl
Br
F
—
—
—
—
S
S
S
S
O
O
O
O
O
O
O
O
—
—
—
—
Br
Br
Br
Br
H
H
H
H
NO2
NO2
NO2
NO2
>100
>100
>100
>100
12.5
>100
>100
>100
5
12.5
25.5
12.5
2.5
2.41
2.33
2.32
2.36
1.86
1.80
1.79
1.82
2.29
2.21
2.20
2.24
0.98
0.93
0.93
0.95
ꢀ8.26
ꢀ8.32
ꢀ8.32
ꢀ8.3
3.61
2.88
2.88
2.97
5.38
4.27
4.19
4.49
3.43
2.55
2.55
2.64
10.01
8.88
8.78
8.90i
326.69
340.05
345.00
331.41
331.93
345.28
350.24
336.66
305.89
319.24
324.20
310.61
327.61
340.96
345.90
332.34
4.07
4.68
4.77
4.13
4.88
5.49
5.57
4.93
3.13
3.79
3.87
3.23
4.08
4.14
4.78
3.58
320.4
354.8
399.3
338.3
405.3
439.7
484.2
423.3
310.3
344.8
389.2
328.3
355.3
389.8
434.2
373.3
7
7
7
7
8
8
8
8
8
8
8
8
11
11
11
11
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
9
9
9
9
73.9
73.7
73.7
73.9
73.8
73.7
73.7
73.8
81.7
81.6
81.6
81.7
121.6
121.5
121.5
121.6
ꢀ8.02
ꢀ8.08
ꢀ8.08
ꢀ8.06
ꢀ7.71
ꢀ7.77
ꢀ7.77
ꢀ7.76
ꢀ8.61
ꢀ8.67
ꢀ8.68
ꢀ8.65
10
10
10
10
10
10
10
10
13
13
13
13
8j
8l
8m
8n
8o
8p
8q
5
6.25
5
Figure 4. Structural comparison of the N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (8a–q). (A) Superimposition of all compounds 3-D-
structure revealing their similar conformation. (B) Comparison of the Electrostatic Potential Maps (MEP) of the most active derivative of each subgroup substituted with
phenyl (8a), 5-bromo-2-thienyl (8e), furyl (8i) or nitrofuryl (8n) ring at C-2 (boxed). The analysis considering the biological activity (MIC in lg/mL) showed the less diffused
and more oriented charge distribution related to the best antitubercular profile. All compounds are at same orientation, negative and positive charges are represented by red
and blue respectively and range (-70 – 90 kJ/mol). (C) Comparison of the halogen substitution of 8n subgroup showing the volume of each halogen that negatively affects the
biological activity (insets).
moiety as important to improve the biological activity (Table 2 and
Fig. 4). In fact, both replacements of the bromothiophenyl ring (e.g.,
8e–h) by a furyl or a nitrofuryl moiety (e.g., 8i–m and 8n–q, respec-
tively) enhanced the antitubercular activity. According to the elec-
trostatic potential map analysis, these substitutions led to a
singular electrostatic potential distribution that specifically ori-
ented the positive and negative charges and probably allowed more
interactions with the bacterial target (Fig. 4B).
In Drug design, halogen atoms are used to improve penetration
through lipid membranes and tissues. They may also present a sig-
nificant reactivity depending on the structure of the molecule. In
this work, the introduction of a para-halogen substituent in the
N-phenyl ring of 8e (e.g., 8f–h), 8i (e.g., 8j–m) or 8n (e.g., 8o–q)
presented a negative effect as they reduced the antitubercular
activity (Table 2). Probably, the halogen reactivity at R position
compromised the original interactions of the non-substituted com-
pounds with the bacterial target. The in silico analysis also pointed
the volume of the halogen as a feasible restrictive factor since bro-
mine, the largest halogen, is more deleterious to the antitubercular
activity than chlorine or fluorine (Fig. 4). Based on these data we
may infer that R is a steric and/or restricted position that should
be carefully considered in the future design of antitubercular
agents (Fig. 5).
Herein we also calculated the stereoelectronic properties of
these derivatives including HOMO and LUMO energy, coefficient
distribution and density, lipophilicity (c log P), molecular volume,
molecular area, polar surface area, number of hydrogen bounds do-
nors (HBD) and acceptors (HBA) groups, solubility and dipole mo-
ment (Table 2). Interestingly, we noticed an analogous behavior for
the most active compounds of each subgroup. All of them pre-
sented the highest values of HOMO and LUMO energy and dipole
moment, and the lowest volume, molecular weight and c log P of
its own subgroup (Table 2, in bold). Thus, low lipophilicity, less ste-
ric and smaller reactive groups should to be preferential for design-
ing new antitubercular molecules. More importantly, these data
suggested that more than one parameter, simultaneously and all
together, modulate the activity profile of these compounds. There-
fore the difficult of finding new antitubercular agents may be due
to the complexity of planning an active molecule that fulfills all
these structural requirements.
The analysis of the biological data of each subgroup showed
that the derivatives with the nitro group at furyl ring C-5 position
(8n–q) presented the best antibacterial profile (MIC = 2.5–12.5
lg/mL) (Table 2). According to our SAR, the active biological profile
of the 8n subgroup is due to different factors including the increase
of the number of hydrogen acceptors and dipole moment allowed

A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
5609
and polar surface area ꢁ140 Å2)35 (Table 2). This result reinforced
the promising profile of these compounds for further experimental
in vivo and in vitro biodisponibility investigation.
3. Conclusions
In summary, we reported the synthesis and the in vitro and in
silico evaluations of the N-substituted-phenylamino-5-methyl-
1H-1,2,3-triazole-4-carbohydrazides 8a–b, 8e–f, 8i–j, 8n–o as well
as of the new analogs 8c–d, 8g–h and 8p–q as antitubercular
agents. The NAH compounds 8a–d (Ar = phenyl) did not show
any antitubercular profile whereas the acylhydrazones 8n–q
(Ar = nitrofuryl) were more active than their corresponding ana-
logs in the series 8a–d (Ar = phenyl), 8e–h (Ar = bromothiophenyl)
and 8j–m (Ar = furyl). Among the nitrofuran derivatives, the non-
substituted compound 8n was the most potent molecule and
exhibited a MIC value (2.5
successful drugs such as ethambutol (MIC = 2
l
g/mL) comparable to other clinically
g/mL). The SAR
l
study pointed the presence of the furyl ring, the electronegative
group (NO2), the low lipophilicity and small volume groups at R
position as important structural features for the antitubercular
profile of these compounds. These parameters should be further
considered in future design of new and more effective compounds
and drug evaluations.
Figure 5. Comparison of the theoretical toxicity risks of the most active subgroup
(8n–q) with commercial drugs. The risks of mutagenicity, irritability and repro-
ductive negative effects are represented as numbers 1 (low), 2 (medium) or 3
(high).
4. Experimental
by the addition of the nitro group. These chemical features proba-
bly increased the receptor-ligand interactions and/or the com-
pounds binding affinity. In addition, the reduced LUMO energy
values of this subgroup points to a better electrophilic profile that
may help in orienting and/or interacting with the bacterial target.
The SAR studies involving the electrostatic potential maps
(MEP) of the compound 8n showed that the presence of a nitro
group generated a singular molecular electrostatic distribution.
The MEP revealed a strong electronic charge in the C-2 position
of the furan ring and in the nitro group at C-5 position (Fig. 4B)
for all compounds of this subgroup, which seems to be relevant
for the antitubercular activity.
Melting points were determined with a Fisher–Johns instru-
ment and are reported uncorrected. Infrared (IR) spectra were
recorded on an ABB FTLA2000–100 spectrophotometer in KBr pel-
lets. NMR spectra, unless otherwise stated, were obtained in
Me2SO-d6 or CDCl3 using a Varian Unity Plus 300 MHz spectrome-
ter. Chemical shifts (d) are reported in ppm and the coupling con-
stant (J) in Hertz. Column chromatography purification was
performed on flash silica gel from Acros Organics. Reactions were
routinely monitored by thin layer chromatography (TLC) on silica
gel pre-coated F254 using Merck plates. Microanalyses were per-
formed on
a Perkin–Elmer Model 2400 instrument, and all
reported values were within 0.4% of the calculated compositions.
The theoretical toxicity risks study (mutagenic, tumorigenic,
and irritant) of the N-substituted-phenylamino-5-methyl-1H-
1,2,3-triazole-4-carbohydrazide derivatives revealed a lower theo-
retical toxicity profile for all compounds of the most active series
(8n–q) in comparison with the commercial drug Chloramfenicol
and similar to others such as Linezolide (Fig. 5). Literature32 de-
scribes nitrofuran group as possibly involved in derivatives toxicity
profile. Since other compounds without the 5-nitrofuran group at-
tached to the acylhydrazone framework of the triazole ring were
also active in this work, this group is not essential for the antitu-
bercular activity and may be replaced to optimize these molecules.
Thus, we propose the exchange of the nitrofuran by a furan ring
substituted in the 5-position with a sulfonamide functional moiety,
which is described in the literature33 as an antimicrobial group, in
attempt to modulate the toxicity profile. In addition, since our the-
oretical study also pointed the phenylamino group at N-1 position
of the 1,2,3-triazole ring as probably involved in the detection of
the mutagenic theoretical profile (not shown), we also suggest its
substitution by benzylamino or benzyl(phenyl)amino group.
According to our theoretical evaluation, these groups decreased
the derivative mutagenic effect, leading to a safer toxicity theoret-
ical profile.
4.1. Chemistry
4.1.1. General procedure for the preparation of the N-substitut
ed-phenyltriazolyl-4-acylhydrazone derivatives 8c, 8d, 8g, 8h,
8l, 8m, 8p and 8q
To a solution of hydrazide derivatives 10c–d (0.50 mmol) in
15 mL of EtOH, an equimolar amount of the appropriate aromatic
aldehyde was added in the presence of a catalytic amount of HCl
(37% v/v). The reaction was stirred for 2 h at room temperature.
The solvent was then evaporated, and the precipitate was collected
by filtration, washed with cold water and dried under vacuum. The
NAH derivates 8c, 8d, 8g, 8h, 8l, 8m, 8p and 8q were purified by a
flash chromatography column using n-hexane/EtOAc (1:1) as the
eluent.
4.1.2. 1-(4’’-Bromophenylamino)-5-methyl-N0-(phenylmethyle
ne)-1H-[1,2,3]-triazole-4-carbohydrazide 8c
Compound 8c obtained as a white solid by condensation of 10c
with benzaldehyde. IR (KBr) mmax (cmꢀ1) 3439 and 3197 (N–H);
1646 (C@O); 1603 (C@N) 1H NMR (300.00 MHz, DMSO-d6) d 2.47
(s, 3H, CH3), 6.47 (d, 2H, J = 8.8, H-2’ and H-6’), 7.42–7.47 (m, 5H,
H-2’’, H-3’’, H-4’’, H-5’’ and H-6’’), 7.71 (d, 2H, J = 8.8, H-3’ and H-
5’), 8.58 (s, 1H, N@CH), 10.36 (br s, 1H, N–H), 12.10 (br s, 1H, NH
–N) ppm. 13C NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 112.8 (C-4’),
115.1 (C-2’ and C-6’), 127.1 (C-3’’ and C-5’’), 128.8 (C-2’’ and C-
Interestingly, our theoretical studies showed that all com-
pounds fulfilled Lipinski’s ‘Rule of five’ (c log ꢁ5, molecular weight
ꢁ500, number of hydrogen bond donors ꢁ5 and number of hydro-
gen-bond acceptors ꢁ10)34 (Table 2), also meeting at least one cri-
terion of those proposed by Veber, (number of rotation bonds ꢁ10

5610
A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
6’’), 130.1 (C-4’’), 132.1 (C-3’ and C-5’), 134.3 (C-1’’) 136.3 (C-4 or
C-5), 138.4 (C-4 or C-5), 145.4 (C-1’), 148.2 (N@CH), 156.9 (C@O)
ppm. Anal. Calcd for C17H15BrN6O: C, 51.14; H, 3.79; N, 21.05.
Found: C, 51.10; H, 4.00; N, 20.41.
4.1.7. 1-(4’-Fluorophenylamino)-N0-[(fur-200-yl)methylene]-5-
methyl-1H-1,2,3-triazole-4-carbohydrazide 8m
Compound 8m obtained as a white solid by condensation of 10d
with 2-furaldehyde. IR (KBr) mmax (cmꢀ1) 3297 and 3195 (N–H);
1645 (C@O); 1607 (C@N); 1H NMR (300.00 MHz, DMSO-d6) d
2.47 (s, 3H, CH3), 6.54 (d, 2H, J = 8.8; 4.4, H-2’ and H-6’), 6.64 (dd,
1H, J = 3.4; 1.7, H-3’’), 6.90 (d, 1H, J = 3.4, H-2’’), 7.10 (d, 2H,
J = 9,0; 8.8, H-3’ and H-5’), 7.85 (d, 1H, J = 1.7, H-4’’), 8.46 (s, 1H,
N@CH), 10.20 (br s, 1H, N–H), 12.14 (br s, 1H, NH–N) ppm. 13C
NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 112.1 (C-3’’), 113.2 (C-2’’),
114.6 (J = 7.7, C-2’ and C-6’), 115.9 (J = 23.2, C-3’and C-5’), 136.2
(C-4 or C-5), 137.8 (N@CH), 137.8 (C-4’’), 138.3 (C-4 or C-5),
142.5 (J = 1.7, C-1’), 149.5 (C-1’’), 156.8 (C@O), 157.2 (J = 236.9, C-
4’) ppm. Anal. Calcd for C15H13FN6O2: C, 54.88; H, 3.99; N, 25.60.
Found: C, 54.91; H, 3.99; N, 25.48.
4.1.3. 1-(4’’-Fluorophenylamino)-5-methyl-N0-(phenylmethyle
ne)-1H-[1,2,3]-triazole-4-carbohydrazide 8d
Compound 8d obtained as a white solid by condensation of 10d
with benzaldehyde. IR (KBr) mmax (cmꢀ1) 3304 and 3054 (N–H);
1676 (C@O); 1592 (C@N) 1H NMR (300.00 MHz, DMSO-d6) d 2.48
(s, 3H, CH3), 6.54 (dd, 2H, J = 8.8; 4.4, H-2’ and H-6’), 7.11 (dd,
2H, J = 8.8; 4.4, H-3’ and H-5’), 7.45–7.47 (m, 3H, H-2’’, H-4’’ and
H-6’’), 7.68–7.75 (m, 2H, H-3’’ and H-5’’), 8.58 (s, 1H, N@CH),
10.36 (br s, 1H, N–H), 12.10 (br s, 1H, NH –N) ppm. 13C NMR
(75 MHz, DMSO-d6) d 8.1 (CH3), 114.6 (J = 8.5, C-2’ e C-6’), 116.0
(J = 22.6, C-3’ and C-5’), 127.0 (C-3’’ and C-5’’), 128.8 (C-2’’ and C-
6’’), 130.0 (C-4’’), 134.3 (C-1’’), 136.2 (C-4 or C-5), 138.3 (C-4 or
C-5), 142.6 (J = 2.3, C-1’), 148.1 (N@CH), 157.0 (C@O) ppm. Anal.
Calcd for C17H15FN6O: C, 60.35; H, 4.47; N, 24.84. Found: C,
58.13; H, 4.69; N, 24.13.
4.1.8. General procedure for the preparation of the N-substitu
ted-phenyltriazolyl-4-acylhydrazone derivatives 8p–q
A solution of 5-nitro-2-furfurylidene diacetate (0.4 mmol) in a
mixture of EtOH (10.0 mL) with a 50% aqueous solution of sulfuric
acid (1.0 mL) was heated for 1–2 min on a steam bath and cooled
to room temperature. Compounds 10c–d (0.4 mmol) were then
added and the resulting mixture was stirred for two hours at room
temperature after which it was poured into crushed ice. The insol-
uble product was filtered off and then purified by column chroma-
tography using a 1:1 n-hexane/EtOAc mixture as the eluent.
4.1.4. N0-[(500-Bromothien-200-yl)methylene]-1-(4’-bromopheny
lamino)-5-methyl-1H-[1,2,3]-triazole-4-carbohydrazide 8g
Compound 8g obtained as a white solid by condensation of 10c
with 2-bromothiophene-5-carboxaldehyde. IR (KBr) mmax (cmꢀ1
)
3298 and 3165 (N–H); 1640 (C@O); 1602 (C@N) 1H NMR
(300.00 MHz, DMSO-d6) d 2.45 (s, 3H, CH3), 6.46 (d, 2H, J = 8.8, H-
2’ and H-6’), 7.27 (d, 1H, J = 0.7, H-2’’), 7.27 (d, 1H, J = 0.7, H-3’’),
7.42 (d, 2H, J = 8.8, H-3’ and H-5’), 8.66 (s, 1H, N@CH), 10.38 (br
s, 1H, N–H), 12.23 (br s, 1H, NH –N) ppm. 13C NMR (75 MHz,
DMSO-d6) d 8.4 (CH3), 113.3 (C-4’), 115.3 (C-4’’), 115.4 (C-2’ and
C-6’), 131.7 (C-2’’), 131.9 (C-3’’), 132.6 (C-3’ and C-5’), 136.4 (C-4
or C-5), 139.1 (C-4 or C-5), 141.1 (C-1’’), 142.9 (N=CH), 145.6 (C-
1’), 157.4 (C@O) ppm. Anal. Calcd for C15H12Br2N6OS: C, 37.21; H,
2.50; N, 17.36. Found: C, 37.68; H, 3.01; N, 16.81.
4.1.9. N’-[(500-Nitrofur-200-yl)methylene]-1-(4’-bromophenylam
ino)-5-methyl-1H-1,2,3-triazole-4-carbohydrazide 8p
Compound 8p obtained as a yellow by condensation of 10c with
5-nitrofuraldehyde. IR (KBr) mmax (cmꢀ1) 3435 and 3267 (N–H);
1637 (C@O); 1613 (C@N) 1H NMR (300.00 MHz, DMSO-d6) d 2.47
(s, 3H, CH3), 6.48 (d, 2H, J = 8.8, H-2’ and H-6’), 7.25 (d, 1H,
J = 4.1, H-3’’), 7.43 (d, 2H, J = 8.8, H-3’ and H-5’), 7.78 (d, 1H,
J = 3.9, H-2’’), 8.53 (s, 1H, N@CH), 10.38 (br s, 1H, N–H) ppm. 13C
NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 112.8 (C-4’), 114.6 (C-2’’),
114.9 (C-3’’), 115.1 (C-2’ and C-6’), 132.1 (C-3’ and C-5’), 135.8
(N@CH), 135.9 (C-4 or C-5), 139.0 (C-4 or C-5), 145.2 (C-1’),
145.3 (C-1’’), 151.8 (C-4’’), 157.1 (C@O) ppm. Anal. Calcd for
4.1.5. N0-[(500-Bromothien-200-yl)methylene]-1-(4’-fluoropheny
lamino)-5-methyl-1H-[1,2,3]-triazole-4-carbohydrazide 8h
Compound 8h obtained as a white solid by condensation of 10d
with 2-bromothiophene-5-carboxaldehyde. IR (KBr) mmax (cmꢀ1
)
C15H12BrN7O4: C, 41.49; H, 2.79; N, 22.58. Found: C, 41.10; H,
3439 and 3292 (N–H); 1664 (C@O); 1601 (C@N); 1H NMR
(300.00 MHz, DMSO-d6) d 2.46 (s, 3H, CH3), 6.56 (d, 2H, J = 8.8;
4.5, H-2’ and H-6’), 7.10 (dd, 2H, J = 8.8; 4.5 H-3’ and H-5’), 7.27
(m, 2H, H-2’’ and H-3’’), 8.66 (s, 1H, N@CH), 10.17 (br s, 1H, N–
H), 12.17 (br s, 1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-d6) d
8.0 (CH3), 114.6 (C-4’’), 114.7 (J = 7.7, C-2’ and C-6’), 115.9
(J = 23.2, C-3’ and C-5’), 131.1 (C-200), 131.2 (C-300), 136.1 (C-4 or
C-5), 138.3 (C-4 or C-5), 141.0 (C-100), 142.1 (N@CH), 142.4 (C-4’),
156.8 (C@O) ppm. Anal. Calcd for C15H12BrFN6OS: C, 42.56; H,
2.86; N, 19.86. Found: C, 42.13; H, 2.79; N, 18.63.
2.50; N, 18.20.
4.1.10. N’-[(500-Nitrofur-200-yl)methylene]-1-(4’-fluorophenylam
ino)-5-methyl-1H-1,2,3-triazole-4-carbohydrazide 8q
Compound 8q obtained as a yellow by condensation of 10d with
5-nitrofuraldehyde. IR (KBr) m
max (cmꢀ1) 3200 (N–H); 1664 (C@O);
1589 (C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.48 (s, 3H, CH3),
6.58 (d, 2H, J = 9.0, H-2’ and H-6’), 7.10 (d, 2H, J = 9.0, H-3’ and
H-5’), 7.25 (d, 1H, J = 3.8, H-2’’), 7.78 (d, 2H, J = 3.8, H-3’’), 8.53 (s,
1H, N@CH), 10.21, (br s, 1H, N–H), 12.59 (br s, 1H, NH–N) ppm.
13C NMR (75 MHz, DMSO-d6) d 8.2 (CH3), 114.6 (C-2’’), 114.7
(J = 7.7, C-2’ and C-6’), 115.0 (C-3’’), 116.0 (J = 22,6, C-3’ and C-5’),
135.7 (N@CH), 135.8 (C-4 or C-5), 138.9 (C-4 or C-5), 142.5
(J = 1.7, C-1’), 151.8 (C-1’’), 151.8 (C-4’’), 157.2 (C@O), 157.3
(J = 237.5, C-4’) ppm. Anal. Calcd for C15H12FN7O4: C, 48.26; H,
3.24; N, 26.27. Found: C, 48.10; H, 3.10; N, 26.20.
4.1.6. 1-(4’-Bromophenylamino)-N0-[(fur-200-yl)methylene]-5-
methyl-1H-1,2,3-triazole-4-carbohydrazide 8l
Compound 8l obtained as a white solid by condensation of 10c
with 2-furaldehyde. IR (KBr) mmax (cmꢀ1) 3437 and 3220 (N–H);
1671 (C@O); 1589 (C@N); 1H NMR (300.00 MHz, DMSO-d6) d
2.46 (s, 3H, CH3), 6.47 (d, 2H, J = 8.8, H-2’ and H-6’), 6.63 (dd, 1H,
J = 2.9; 1.7, H-3’’), 6.90 (d, 1H, J = 2.9, H-2’’), 7.42 (d, 2H, J = 8.8,
H-3’ and H-5’), 7.85 (d, 1H, J = 1.7, H-4’’), 8.46 (s, 1H, N@CH),
10.39 (br s, 1H, N–H), 12.15 (br s, 1H, NH –N) ppm. 13C NMR
(75 MHz, DMSO-d6) d 8.3 (CH3), 112.4 (C-3’’), 113.1 (C-4’), 113.8
(C-2’’), 115.2 (C-2’ and C-6’), 132.3 (C-3’ and C-5’), 136.4 (C-4 or
C-5), 138.2 (N@CH), 138.2 (C-4’’), 138.7 (C-4 or C-5), 145.6 (C-1’),
149.6 (C-1’’), 157.6 (C@O) ppm. Anal. Calcd for C15H13BrN6O2: C,
46.29; H, 3.37; N, 21.59. Found: C, 46.59; H, 3.91; N, 20.69.
4.2. Pharmacology
4.2.1. In vitro evaluation of antitubercular activity
The drug potency was determined by the Microplate Alamar
Blue assay (MABA), which shows good correlation and proportion-
ality with the BACTEC MGIT 960 method. This colorimetric method
uses the Alamar Blue–resazurin-based oxidation–reduction indica-
tor to obtain drug susceptibility measurements for bacteria.

A. K. Jordão et al. / Bioorg. Med. Chem. 19 (2011) 5605–5611
5611
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lg/mL) was
defined as the lowest drug concentration that prevented a color
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butol and isoniazid were used as reference drugs.
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(Wavefunction Inc. Irvine, CA, 2000) as described elsewhere.24,27
Briefly the structures were optimized to a local minimum and
the equilibrium geometry obtained in vacuum using RM1 semi-
empirical methods. Subsequently, molecules were submitted to a
single-point energy ab initio calculation, at the 6-31G⁄ level, to cal-
culate some stereoelectronic properties and perform the SAR stud-
ies. Thus, we calculated HOMO and LUMO energies and density
isosurface, molecular weight, molecular surface area, polar surface
area, dipole moment, lipophilicity and electrostatic potential maps
for all compounds best conformation. The theoretical study of bio-
availability and toxicity properties was performed using Osiris
Property Explorer (http://www.organic-chemistry.org/) and Molin-
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bond donors ꢁ5 and number of hydrogen-bond acceptors ꢁ10;
and Veber criteria, pointing PSA values being lower than 140 Å2
and having less rotatable bounds than 10 as important features
for bioavailability.
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Acknowledgments
28. Da Silva, E. N., Jr.; Menna-Barreto, R. F. S.; Pinto, M. C. R. F.; Silva, R. S. F.;
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This work was supported by the Brazilian agencies FAPERJ
(APQ1 E-26/110.801/2009) and CNPq. Fellowships granted to
UFF, by CAPES, CNPq, CNPq-PIBIC are gratefully acknowledged.
29. Campos, V. R.; Abreu, P. A.; Castro, H. C.; Rodrigues, C. R.; Jordão, A. K.; Ferreira,
V. F.; De Souza, M. C. B. V.; Santos, F. C.; Moura, L. A.; Domingos, T. S.; Carvalho,
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