Synthesis of new pyrazole-triazole hybrids by click reaction using a green solvent and evaluation of their antitubercular and antibacterial activity

Article abstract of DOI:10.1007/s11164-015-2241-9

A new series of pyrazole-based 1,2,3-triazole derivatives (6a-x) were synthesized by employing click reaction using a 2:1 mixture of PEG-400 and water as green solvent. The synthesized intermediate and final compounds were characterized by ¹H NMR, ¹³C NMR, and mass spectra and elemental analysis techniques. The structure of one of the final compounds, 6a was evidenced by single crystal X-ray diffraction study. Among the twenty-four compounds, five compounds (6a, 6b, 6d, 6f, and 6g) showed significant antitubercular activity against Mycobacterium tuberculosis H₃₇Rv with a minimum inhibitory concentration (MIC) ≤ 6.25 μg/mL. The 4-(((5-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1-cyclohexyl-1H-1,2,3-triazole (6g) was the most potent compound of the series, which showed a MIC of 3.13 μg/mL. The cytotoxicity study of active anti-TB compounds on normal Vero cells revealed that the compounds are non-toxic with a high selectivity index (>37). Most of the pyrazole-1,2,3-triazole derivatives with a 4-chlorophenyl substitution at position-5 of the pyrazole ring showed a better anti-TB activity than the corresponding 4-bromophenyl or 4-methoxyphenyl substituted derivatives. The target compounds were also evaluated for their in vitro antibacterial activity and six compounds (6a, 6c, 6d, 6e, 6l, and 6w) showed promising inhibition activity against four tested strains.

Full text of DOI:10.1007/s11164-015-2241-9

Res Chem Intermed
DOI 10.1007/s11164-015-2241-9
Synthesis of new pyrazole-triazole hybrids by click
reaction using a green solvent and evaluation of their
antitubercular and antibacterial activity
Nagabhushana Nayak1 Jurupula Ramprasad1
•
•
Udayakumar Dalimba1 Perumal Yogeeswari2
•
•
Dharmarajan Sriram2 H. S. Santosh Kumar3
•
•
S. K. Peethambar4 Rajeshwara Achur4
•
Received: 15 July 2015 / Accepted: 25 August 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A new series of pyrazole-based 1,2,3-triazole derivatives (6a–x) were syn-
thesized by employing click reaction using a 2:1 mixture of PEG-400 and water as green
1
solvent. The synthesized intermediate and final compounds were characterized by H
NMR, 13C NMR, and mass spectra and elemental analysis techniques. The structure of
one of the final compounds, 6a was evidenced by single crystal X-ray diffraction study.
Among the twenty-four compounds, five compounds (6a, 6b, 6d, 6f, and 6g) showed
significant antitubercular activity against Mycobacterium tuberculosis H37Rv with a
minimum inhibitory concentration (MIC) B 6.25 lg/mL. The 4-(((5-(4-Chlorophenyl)-
1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1-cyclohexyl-1H-1,2,3-triazole (6g) was
the most potent compound of the series, which showed a MIC of 3.13 lg/mL. The
cytotoxicity study of active anti-TB compounds on normal Vero cells revealed that the
compounds are non-toxic with a high selectivity index ([37). Most of the pyrazole-
1,2,3-triazole derivatives with a 4-chlorophenyl substitution at position-5 of the pyrazole
ring showed a better anti-TB activity than the corresponding 4-bromophenyl or
4-methoxyphenyl substituted derivatives. The target compounds were also evaluated for
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2241-9)
contains supplementary material, which is available to authorized users.
&
Udayakumar Dalimba
udayaravi80@gmail.com; udayakumar@nitk.ac.in
1
2
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Srinivasanagar, Mangalore 575025, India
Medicinal Chemistry and Drug Discovery Research Laboratory, Pharmacy Group, Birla
Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad,
Telangana 500078, India
3
4
Department of Bioinformatics, Kuvempu University, Jnanasahyadri, Shankaraghatta 577451,
India
Department of Biochemistry, Kuvempu University, Jnanasahyadri, Shankaraghatta 577451,
India
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N. Nayak et al.
their in vitro antibacterial activity and six compounds (6a, 6c, 6d, 6e, 6l, and 6w) showed
promising inhibition activity against four tested strains.
Graphical Abstract A new series of pyrazole-1,2,3-triazole derivatives were synthe-
sized by employing a multicomponent one pot click reaction. The most potent anti-TB
agent of the series showed a MIC of 3.13 lg/mL.
Keywords Pyrazole-1,2,3-triazole hybrid Á Click reaction Á Green solvent Á
Antitubercular activity Á Antibacterial activity Á Cytotoxicity
Introduction
Tuberculosis (TB) is one of the most dangerous and deadly microbial diseases. It is
caused by a bacterium called Mycobacterium tuberculosis (MTB) and is the second
greatest killer disease after HIV/AIDS among the diseases caused by a single
infectious agent. According to the World Health Organization (WHO) report
released in the year 2014, 9 million people fell ill with TB and 1.5 million died,
among which 0.3 million were HIV-positive [1]. The standard medication for the
short course treatment of tuberculosis includes a mixture of four drugs: isoniazid,
rifampicin, pyrazinamide, and ethambutol for 2 months, then isoniazid and
rifampicin alone for a further 4 months. These drugs are known as first line anti-
TB drugs [2]. However, extensively drug-resistant tuberculosis (XDR-TB) or
multidrug resistant tuberculosis (MDR-TB) even show resistance to first line
therapy. In such cases the second-line drugs are implemented for the treatment. But
these drugs are less preferred due to one of the possible three reasons: it may be less
effective than the first-line drugs (e.g., p-aminosalicylic acid); it may have toxic
side-effects (e.g., cycloserine); or it may be expensive and unavailable in many
developing countries (e.g., fluoroquinolones). All the antitubercular medicines
including the first line drugs show some side effects and also the course for the
treatment is too long. Furthermore, except for the recently introduced bedaquiline,
no new TB drugs have been introduced over the last four decades, which testifies to
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Synthesis of new pyrazole-triazole hybrids by click…
the lack of significant research in this area of pharmaceutical science. These major
issues prompted us to search for new lead molecules to act against TB.
Azole-based heterocycles, especially pyrazole and 1,2,3-triazole derivatives,
gained great significance in medicinal chemistry research due to their wide range of
biological activities. A number of pyrazole derivatives have shown significant
activities, such as antimicrobial [3, 4], analgesic [5], anti-inflammatory [6],
anticancer [7], and antitubercular [8–14]. These results gave a great momentum to
the search for potential pharmacologically active drugs carrying a pyrazole
substituent. On the other hand, with the invention of click chemistry of
cycloaddition reactions, several 1,2,3-triazole derivatives have been synthesized
and are found to possess a broad spectrum of pharmacological effects [15] such as
antimicrobial [16], anticancer [17], antifungal [18], antiviral [19], and anti-
inflammatory [20] activities. Some N-substituted 1,2,3-triazole derivatives have
shown remarkable antitubercular [21–27] properties among which derivative I-A09
[28, 29] is presently in preclinical trials. Additionally, the derivatives of 1,2,3-
triazole possess remarkable metabolic stability and are proven to be amide
surrogates in various bioactive compounds [30]. Further, recent studies on azoles
demonstrated their ability of forming hydrogen bonding and dipole interactions with
biomolecular targets, which results in improving their solubility in biological
systems [31, 32]. In recent years, the molecular hybridization concept has received
significant importance in drug design and development, which involves a
combination of pharmacophoric moieties of two bioactive elements to produce a
new hybrid lead compound with improved efficiency and efficacy, when compared
to the parent compounds. It is also found that this strategy resulted in developing
compounds with a modified selectivity profile, different and/or dual modes of
action, and reduced undesired side effects [33, 34]. With this background and
pertaining to the literature reports on promising antitubercular and antimicrobial
activities of a number of pyrazole and 1,2,3-triazole derivatives (Fig. 1), we have
planned to amalgamate these two structural units in a single molecular framework
and synthesized a library of pyrazole-1,2,3-traizole hybrid derivatives (Fig. 2).
Results and discussion
Chemistry
The synthetic route of intermediates 5a–c is shown in Scheme 1. The protocol
involves the Claisen condensation of 4-substituted acetophenones (1a–c) with
diethyl oxalate in the presence of sodium ethoxide in ethanol at room temperature to
yield a corresponding sodium salt of a,c-diketoesters (2a–c). Esters of 5-aryl-1-
phenyl-1H-pyrazole-3-carboxylic acids (3a–c) were synthesized in quantitatively
good yield by the conventional cyclization reaction between sodium salt of a,c-
diketoesters 2a–c and arylhydrazines in the presence of acetic acid. The ester
derivatives were then reduced to corresponding alcohols (4a–c) in good yields by
using LiAlH4 as the reducing agent. The propargylated scaffolds, 5-(4-aryl)-1-
phenyl-3-((prop-2-ynyloxy)methyl)-1H-pyrazoles (5a–c) were obtained by the
123

N. Nayak et al.
F
Cl
O
N
N
N
N
O
O
N
N
N
N
F
N
N
N
H
N
N
Cl
N
H
O
O
O
N
I (Horrocks et al, 2013)
CH3
III (Aragade et al, 2013)
II (Pandit and Dodiya, 2013)
OH
OH
Cl
O
N
N
F
N
O
N
O
N
N
N
N
OCH3
HN
N
O
O
O
NH
O2N
V (Yempala et al, 2014)
IV (Kamal et al, 2013)
N
O
I-A09 (Zhou et al, 2010)
Fig. 1 Representative pyrazole and 1,2,3-triazole based antitubercular agents (I–V and I-A09)
Various substituted aryl
and aliphatic groups
R2
O
N
N
N
N
N
R1
Cl, OCH3, Br
Potent anti-TB unit
Active antimicrobial
and anti-TB unit
Fig. 2 Design strategy for pyrazole-1,2,3-triazole hybrids
alkylation of alcohols 4a–c with propargyl bromide in the presence of sodium
hydride (NaH) (60 %) in tetrahydrofuran (THF). The targeted regioselective 1,4-
substituted 1,2,3-triazole derivatives (6a–x) were synthesized using a multicompo-
nent one pot click chemistry approach by treating the propargylated scaffolds (5a–c)
with alkyl bromides or benzyl bromides in presence of a catalytic amount of copper
sulfate pentahydrate and sodium ascorbate in a 2:1 mixture of polyethylene glycol-
400 (PEG-400) and water at 50 °C (Scheme 2). We tried using different solvent
systems for the click reaction and the optimization was carried out with the reaction
between 5a and 4-fluorobenzyl bromide (Table 1). Among the solvents used, a 2:1
mixture of PEG-400 and water was the most effective system, which yielded a
product (6a) yield of 84 %. The reaction using a mixture of t-butanol and water as
123

Synthesis of new pyrazole-triazole hybrids by click…
O
N
O
O
ONa O
a
O
c
b
N
O
R1
R1
R1
2a-c
1a-c
3a-c
O
OH
N
N
N
d
N
R1
R1
1a-5a R1 = Cl
1b-5b R1 = OCH3
1c-5c R1 = Br
5a-c
4a-c
Scheme 1 Synthesis of the pyrazole based scaffolds. Reagents and conditions: a diethyl oxalate, NaOEt,
Ethanol, 0 °C to room temperature (RT), 24 h; b phenyl hydrazine, HOAc, 105 °C, 3 h; c LiAlH4, diethyl
ether, 0 °C to RT, 1 h; d NaH (60 %), THF, propargyl bromide (80 % in toluene), 0 °C to RT, 1 h
R2
O
O
N
N
N
N
N
e
N
N
R1
R1
5a-c
6a-x
Scheme 2 Synthesis of target molecules. Reagent and conditions: e alkyl and benzyl bromides,
CuSO4Á5H2O, NaN3, sodium ascorbate, PEG-400/H2O (2:1), 50 °C, 12 h
the solvent also resulted in comparable product yield. PEG-400 is an interesting
solvent in synthetic chemistry as it is non-toxic and inexpensive. Also, it is soluble
in water but shows poor solubility in a number of organic solvents. The greener and
easy removable nature of PEG-400 prompted us to choose this solvent in the click
reaction protocol.
1
The structures of all the synthesized compounds were confirmed by H NMR,
13C NMR, and mass spectral analysis as well as by elemental analysis. Cyclization
of sodium (E)-1-(4-substituted)-4-ethoxy-3,4-dioxobut-1-en-1-olate (2a–c) to ethyl
5-(aryl)-1-phenyl-1H-pyrazole-3-carboxylate (3a–c) was evident by the presence of
1
a singlet at d 7.17 ppm due to the pyrazole ring proton, in the H NMR spectrum.
Also, the spectrum displayed an additional five aromatic protons confirming the
presence of the N-phenyl ring. The reduction of the ester group in 3a–c to give (5-
(4-substituted)-1-phenyl-1H-pyrazol-3-yl)methanol (4a–c) was confirmed by the
1
appearance of a signal corresponding to the -OH group at d 5.25 ppm in the H
NMR spectrum. The absence of ethyl carboxylate peaks in the spectrum further
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N. Nayak et al.
Table 1 Optimization of solvent for the click reaction of 5a
O
N
N
N
N
Click reaction
N
F
5a
Cl
6a
Entry
Organic solvent
Time (h)
Yielda (%)
1
2
3
4
5
6
1,4-Dioxane
Ethanol
24
24
12
12
24
24
–
54
80
84
–
t-Butanol
PEG-400
Acetonitrile
DMF
48
Reagent and conditions: 4-Fluorobenzyl bromide, CuSO4Á5H2O, NaN3, sodium ascorbate, organic sol-
vent/H2O (2:1), 50 °C
a
Isolated yield after column purification. – Required product not formed
confirms the reduction of the ester group. The alkylation of 4a–c using propargyl
bromide to yield scaffolds 5a–c was evidenced by the disappearance of –OH signal
in the 1H NMR spectrum of 5a–c. Further, appearance of two new peaks at around d
4.58 ppm for –OCH2 and d 4.25 ppm for alkyne –CH protons clearly confirmed the
formation of the required products. The formation of the triazole ring in 6a–x was
supported by the characteristic 1,2,3-triazole ring (–CH) signal in the range d
1
8.16–8.29 ppm in the H NMR spectra. For instance, in compound 6a, the triazole
ring (–CH) signal appears as a sharp singlet at d 8.22 ppm, all other aromatic –CH
signals are in the range d 7.18–7.44. The sharp singlet at d 6.69 ppm is due to
pyrazole ring –CH and the benzyl –CH2 appears at d 5.59 ppm. The –CH2 signals of
the methylenedioxy linkage appear at d 4.64 and 4.56 ppm, the higher chemical
shift value is for the –CH2 group attached to the pyrazole ring. The electrospray
ionization mass spectrum (ESI–MS) of 6a showed a molecular ion peak at m/z
474.3, which tallies with its molecular formula C26H21ClFN5O. Further 13C NMR
spectrum and elemental analysis results clearly confirms the formation of 6a. The
structural parameters, yield, and melting point data of the target compounds (6a–x)
are presented in Table 2.
X-ray crystallographic analysis of 6a
The single crystal of final compound 6a was grown by the slow evaporation of a 1:1
mixture of methanol-chloroform solution of the compound at room temperature.
The 3-dimensional structure of the molecule was established by carrying out single
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Synthesis of new pyrazole-triazole hybrids by click…
Table 2 Structural parameters, yield, melting point data, and antitubercular results of target compounds
6a–x
Compound
R1
R2
Yielda (%)
mpb (°C)
MIC (lg/mL)
6a
6b
6c
–Cl
–Cl
–Cl
4-F–C6H4–CH2–
84
86
81
114–115
80–81
6.25
6.25
12.5
4-CN–C6H4–CH2–
4-OCH3–C6H4–
CH2–
92–93
6d
6e
6f
–Cl
4-NO2–C6H4–CH2–
C6H5–CH2–
78
79
82
68
76
80
85
81
112–113
Syrup
83–84
Syrup
Syrup
96–97
70–71
Syrup
6.25
12.5
6.25
3.13
50
–Cl
–Cl
2-F–C6H4–CH2–
Cyclohexyl–
6g
6h
6i
–Cl
–Cl
Cyclopentyl–
–OCH3
–OCH3
–OCH3
4-F–C6H4–CH2–
4-CN–C6H4–CH2–
50
6j
25
6k
4-OCH3–C6H4–
CH2–
12.5
6l
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–Br
4-NO2–C6H4–CH2–
C6H5–CH2–
80
73
75
70
76
80
88
87
109–110
85–86
78–79
Syrup
Syrup
94–95
Syrup
58–59
12.5
12.5
25
6m
6n
6o
6p
6q
6r
6s
2-F–C6H4–CH2–
Cyclohexyl–
50
Cyclopentyl–
25
4-F–C6H4–CH2–
4-CN–C6H4–CH2–
50
–Br
50
–Br
4-OCH3–C6H4–
CH2–
50
6t
–Br
–Br
–Br
–Br
–Br
–
4-NO2–C6H4–CH2–
79
69
78
67
70
–
102–103
91–92
95–96
Syrup
Syrup
–
50
6u
C6H5–CH2–
25
6v
2-F–C6H4–CH2–
12.5
12.5
25
6w
Cyclohexyl–
6x
Cyclopentyl–
Isoniazid
Ethambutol
Pyrazinamide
a
–
–
–
0.1
3.13
50
–
–
–
–
–
–
Isolated yield after column purification
b
Melting points
crystal X-ray diffraction (SCXRD) analysis using a Bruker SMART APEXII DUO
CCD diffractometer. The suitable crystal required for the analysis was selected by
using a microscope and the single crystal was mounted on a goniometer tip for
X-ray exposure. The structure of the crystal was solved by the full matrix least-
squares method using the SHELXL-2013 package. All the atoms were positioned
in different Fourier maps and refined isotropically, using a riding model and all
the projections were generated using the Oak Ridge Thermal Ellipsoid Plot Program
(ORTEP). It was found that the compound 6a crystallizes in triclinic space group
´
´
˚
˚
P-1 with cell parameters;
a
(A) = 5.6750(3),
b
(A) = 10.7105(5),
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N. Nayak et al.
´
´
3
˚
˚
c (A) = 19.9112(9), Volume (A ) = 1163.87(10). The number of molecules in the
unit cell (Z) was observed to be two. The ORTEP diagram (50 % probability) of 6a
is depicted in Fig. 3. The details of bond lengths and bond angles of the compound
are given in the supplementary information.
Pharmacology
Antitubercular studies
The antimycobacterial activity of the pyrazole-1,2,3-triazole hybrids (6a–x) against
MTB H37Rv (ATCC27294) was evaluated by the agar dilution method and the
minimum inhibitory concentration (MIC) values are given in Table 2. Isoniazid,
Ethambutol, and Pyrazinamide were used as positive drug standards. MIC is defined
as the minimum concentration of compound required to completely inhibit the
bacterial growth. Among twenty-four compounds screened against MTB, 13
compounds (6a, 6b, 6c, 6d, 6e, 6f, 6g, 6k, 6l, 6m, 6p, 6v, and 6w) showed MIC in
the range 3.13–12.5 lg/mL. Out of eight 4-chlorophenyl substituted pyrazole
derivatives (6a–h), seven compounds showed significant activity against MTB,
which suggests that 4-chlorophenyl substitution at position-5 of the pyrazole (R1)
ring is a decisive factor for the activity. Compound 6g, which contain a cyclohexyl
substitution on the 1,2,3-triazole ring (R2), was found to be the most active
compound of the series with a MIC of 3.13 lg/mL. The inhibition activity of 6g is
comparable (in terms of MIC value) with that of the standard drug Ethambutol. All
the 4-chlorophenyl substituted target compounds (6a–h), except 6h, are either
Fig. 3 ORTEP diagram showing the X-ray crystal structure of 6a. (Crystal data: Chemical
formula = C26H21ClFN5O, Formula weight = 473.93, Crystal system = Triclinic, Space group = P-1,
´
´
´
´
3
˚
˚
(A) = 5.6750(3),
˚
(A) = 10.7105(5),
˚
a
b
c
(A) = 19.9112(9), Volume (A ) = 1163.87(10),
(mm-1) = 0.201,
˚
a = 79.5270(19), b = 85.8390(19), c = 78.1330(19), Z = 2, F000 = 492.0,
Temperature (T) = 296 K, Radiation wavelength (A) = 0.71073, Radiation type = Mo Ka, R-Factor
(%) = 4.3, CCDC No. = 1032622)
l
´
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Synthesis of new pyrazole-triazole hybrids by click…
equipotent or more potent when compared with their respective 4-methoxyphenyl
substituted (6i–p) and 4-bromophenyl substituted (6q–x) analogues. This structure–
activity relationship further confirms the significant contribution of the 4-chlor-
ophenyl substituent towards the inhibition activity of the molecules. The nature of
the substituent on the 1,2,3-triazole ring (R2) also affected the inhibition activity.
However, we did not observe a general trend in the variation of the activity with
respect to electron donating or withdrawing nature of these substituents. But, in case
of 4-chlorophenyl substituted derivatives (6a–h), those compounds, which carry an
electron-withdrawing functional group at R2, showed better inhibition activity than
those compounds containing an electron releasing functionality. That is, compounds
6a, 6b, 6d and 6f, which contain 4-F, 4-CN, 4-NO2 and 2-F groups (at R2)
respectively exhibited MIC of 6.25 lg/mL, whereas compounds 6c (which contain
an electron-donating 4-OCH3 group) and 6e (which has no substituent on the phenyl
ring) showed a higher MIC (12.5 lg/mL) value.
Cytotoxicity
The in vitro cytotoxicity studies of the active anti-TB compounds with MTB
MIC B 6.25 lg/mL were carried out by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay [35] against a normal Vero cell line. The results
of the cytotoxicity study are presented in the Table 3. All the active antitubercular
compounds 6a, 6b, 6d, 6f, and 6g exhibited low toxicity and the inhibition values
were between 13.22 and 29.91 % at 65 lg/mL. Further, the high selectivity index of
these derivatives ([37) clearly implies the suitability of the compounds for drug
development as new anti-TB agents.
Antibacterial studies
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella
typhi are the common pathogens, which cause harmful effects on human health.
Some pyrazole [36] and 1,2,3-triazole [24] derivatives showed promising activity
against these pathogens. Hence, we thought it was worthwhile to evaluate the
antimicrobial activity of the new pyrazole-1,2,3-triazole derivatives (6a–x) against
Table 3 Cytotoxicity of active anti-TB derivatives
Compound
Percentage inhibitiona
IC50 (lg/mL)
SIb
6a
6b
6d
6f
6g
a
29.20 2.9
29.91 3.9
20.30 1.3
13.22 6.0
17.62 5.1
[1000
231.67
986.67
656.67
646.67
[160
37.07
157.9
105.1
206.6
Percentage inhibition is at 62.5 lg/mL
b
Selectivity index (in vitro) = IC50 against Vero cells/MIC against M. tuberculosis
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N. Nayak et al.
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N. Nayak et al.
these microorganisms. The antibacterial activity of the compounds was studied
following the well plate method (zone of inhibition) by using Streptomycin, the first
class of antibiotic drug, as the standard. Compounds 6a, 6c, 6d, 6e, 6l, and 6w
showed promising activity against tested bacterial strains (Table 4). It is interesting
to note that four of these compounds (6a, 6c, 6d, and 6e) contain a 4-chlorophenyl
substitution at position-5 of the pyrazole ring. The antibacterial activity of
derivatives 6d and 6l against P. aeruginosa is very close to that of Streptomycin, the
presence of the nitro group in these molecules could be responsible for the observed
activity. It is evident from Table 4 that the target molecules show a better activity
against the P. aeruginosa strain as compared to the other three bacterial strains.
Conclusions
We described the synthesis of a new series of pyrazole-1,2,3-triazole hybrids (6a–x)
using a multistep synthetic route in which a substituted 1,2,3-triazole ring was
constructed in the final step through the click reaction protocol. The solvent system
for the click reaction was optimized in terms of the product yield and the green
solvent system, a 2:1 mixture of PEG-400 and water, was the most effective among
the solvent systems used. All the target compounds were characterized by 1H NMR,
13C NMR, mass spectral and elemental analyses. The in vitro antitubercular screening
of the molecules against MTB H37Rv strain revealed the better anti-TB activity of
4-chlorophenyl substituted derivatives as compared to their 4-methoxyphenyl and
4-bromophenyl substituted analogues. The derivative (6g) with a 4-chlorophenyl
substitution on the pyrazole ring and a cyclohexyl moiety on the 1,2,3-triazole ring is
the best antitubercular agent of the series with MIC of 3.13 lg/mL. In 4-chlorophenyl
substituted derivatives, an electron-withdrawing substituent on the 1,2,3-triazole ring
enhanced the anti-TB activity. The cytotoxicity study on active antitubercular
compounds also suggested that the compounds are nontoxic and have a high
selectivity index ([37). Further, derivatives 6a, 6c, 6d, 6e, 6l, and 6w exhibited
significant antibacterial activity against the tested bacterial strains. With regard to the
relationship between the structure of pyrazole-1,2,3-triazole hybrids and the detected
anti-TB and antimicrobial properties, it can be concluded that the chlorophenyl
substituted pyrazole scaffolds are ideally suited for further structural modifications to
obtain efficient antibacterial leads. These findings suggest that pyrazole-1,2,3-triazole
hybrids could be considered as promising molecular patterns for potential antituber-
cular and antimicrobial agents and can be used for further chemical modifications for
improving activity, which will be the main goal of our future research.
Experimental protocols
Analysis and instruments
All the reagents were purchased from leading reagent suppliers like Sigma Aldrich
(Germany), Merck (India), Loba Chemie Pvt. Ltd., and Spectrochem Chemicals Pvt.
Ltd. All the solvents were extra dried by the distillation method. Thin layer
123

Synthesis of new pyrazole-triazole hybrids by click…
chromatography (TLC) was carried out on coated aluminum sheets with 60 F254
silica gel (Merck KGaA). The intermediate and the final compounds were purified
by the column chromatography technique using silica gel 230–400 mesh size
(Merck). A Stuart SMP3 melting point apparatus was used for the determination of
the melting points of the newly synthesized compounds and are uncorrected. The
X-Ray diffraction studies were worked out by a Bruker APEX II DUO CCD
1
diffractometer. H and 13C NMR spectra of all the compounds were recorded on
400 MHz spectrometers using TMS as an internal standard. A Thermo electron
corporation EA-112 series C,H,N,S analyzer was used for the elemental analysis.
Molecular mass of the compounds was determined using a Waters Q-Tof micro
mass spectrometer with an ESI source.
General procedure for the synthesis of final compounds (6a–x)
A mixture of appropriate scaffold (5a–c) (0.1 g), bromo compound (1.1 eq), NaN3
(1.1 eq), copper sulfate pentahydrate (0.2 eq), and sodium ascorbate (0.3 eq) was
placed in a 2:1 mixture of PEG-400 and water (3 mL). The mixture was stirred at
50 °C for 12 h. Completion of the reaction was confirmed by TLC. The reaction
mixture was then filtered through Celite; the residue was washed with 25 mL of
ethyl acetate. The filtrate was extracted with ethyl acetate (25 9 3 mL). The
organic layer was separated and washed twice with ammonia solution, then with
water followed with brine solution, and dried over anhydrous sodium sulfate. The
solvent was removed under reduced pressure and the crude product was purified by
flash column chromatography (230–400 size mesh) using 60 % ethyl acetate in pet
ether to afford the pure final products (6a–x).
Spectral data of final derivatives (6a–x)
1-(4-Fluorobenzyl)-4-(((5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
methyl)-1H-1,2,3-triazole(6a) Off white solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 8.22 (s, 1H, triazole-CH), 7.38–7.44 (m, 7H, ArH), 7.18–7.29 (m, 6H, ArH),
6.69 (s, 1H, pyrazole-CH), 5.59 (s, 2H, CH2), 4.64 (s, 2H, CH2), 4.57 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) d ppm 163.55, 161.12, 150.37, 144.66, 142.65,
139.89, 133.65, 132.83, 132.80, 130.76, 130.67, 130.64, 129.65, 129.26, 129.14,
128.33, 125.70, 124.58, 116.17, 115.95, 108.18, 65.62, 63.48, 52.45; ESI–MS (m/z)
474.3 (M ? H)?; Anal. calculated for C26H21ClFN5O; C, 65.89; H, 4.47; N, 14.78.
Found: C, 65.86; H, 4.45; N, 14.81.
4-((4-(((5-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-1,2,3-tri-
1
azol-1-yl)methyl)benzonitrile (6b) Off white solid; H NMR (400 MHz, DMSO-
d6) d ppm 8.28 (s, 1H, triazole-CH), 7.85 (d, 2H, ArH, j = 8.4 Hz), 7.36–7.46 (m,
7H, ArH), 7.22–7.27 (m, 4H, ArH), 6.69 (s, 1H, pyrazole-CH), 5.73 (s, 2H, CH2),
4.66 (s, 2H, CH2), 4.58 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d ppm
150.36, 144.78, 142.65, 142.02, 139.89, 133.66, 130.64, 129.65, 129.25, 129.14,
129.11, 128.34, 125.71, 125.06, 119.00, 111.38, 108.19, 65.63, 63.45, 52.62; ESI–
123

N. Nayak et al.
MS (m/z) 481.3 (M ? H)?; Anal. calculated for C27H21ClN6O; C, 67.43; H, 4.40;
N, 17.47. Found: C, 67.39; H, 4.41; N, 17.50.
1-(4-Methoxybenzyl)-4-(((5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6c) Pale yellow solid; H NMR (400 MHz, DMSO-d6)
d ppm 8.16 (s, 1H, triazole-CH), 7.38–7.22 (m, 11H, ArH), 6.91 (d, 2H, ArH,
J = 8.8 Hz), 6.69 (s, 1H, pyrazole-CH), 5.51 (s, 2H, CH2), 4.63 (s, 2H, CH2), 4.56
(s, 2H, CH2), 3.72 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d ppm 159.59,
150.39, 144.58, 142.64, 139.90, 139.79, 133.65, 130.63, 130.04, 129.92, 129.64,
129.56, 129.26, 129.14, 128.47, 128.32, 125.70, 124.32, 114.58, 114.48, 108.18,
65.61, 63.51, 55.58, 52.81; ESI–MS (m/z) 486.3 (M ? H)?; Anal. calculated for
C27H24ClN2O2; C, 66.73; H, 4.98; N, 14.41. Found: C, 66.67; H, 5.00; N, 14.43.
1-(4-Nitrobenzyl)-4-(((5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6d) Pale yellow solid; H NMR (400 MHz, DMSO-
d6) d ppm 8.29 (s, 1H, triazole-CH), 8.23 (d, 2H, ArH, j = 8.8 Hz), 7.52 (d, 2H,
ArH, J = 8.0 Hz), 7.38–7.44 (m, 5H, ArH), 7.22–7.27 (m, 4H, ArH), 6.69 (s, 1H,
pyrazole-CH), 5.79 (s, 2H, CH2), 4.66 (s, 2H, CH2), 4.57 (s, 2H, CH2); 13C NMR
(100 MHz, DMSO-d6) d ppm 150.36, 147.69, 144.81, 143.97, 142.65, 139.88,
133.66, 130.66, 129.65, 129.45, 129.14, 128.34, 125.70, 125.11, 124.39, 108.19,
65.64, 63.46, 52.35; ESI–MS (m/z) 501.3 (M ? H)?; Anal. calculated for
C26H21ClN6O3; C, 62.34; H, 4.23; N, 16.78. Found: C, 62.39; H, 4.26; N, 16.81.
1-Benzyl-4-(((5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-
1,2,3-triazole(6e) Pale yellow Syrup; 1H NMR (400 MHz, DMSO-d6) d ppm 8.20
(s, 1H, triazole-CH), 6.89–7.38 (m, 14H, ArH,), 6.69 (s, 1H, pyrazole-CH), 5.63 (s,
2H, CH2), 4.66 (s, 2H, CH2), 4.57 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d
ppm 161.12, 144.77, 144.64, 143.45 141.5, 139.89, 132.95, 132.83, 132.79, 130.76,
130.64, 130.61, 129.65, 129.26, 129.21, 128.33, 125.69, 124.57, 116.11, 115.90,
108.16, 65.62, 63.50, 52.42; ESI–MS (m/z) 456.4 (M ? H)?; Anal. calculated for
C26H22ClN5O; C, 68.49; H, 4.86; N, 15.36. Found: C, 68.55; H, 4.87; N, 15.40.
1-(2-Fluorobenzyl)-4-(((5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
methyl)-1H-1,2,3-triazole(6f) Off white solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 8.22 (s, 1H, triazole-CH), 7.18–7.40 (m, 13H, ArH), 6.69 (s, 1H, pyrazole-CH),
5.60 (s, 2H, CH2), 4.64 (s, 2H, CH2), 4.58 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d ppm 162.58, 162.12, 150.40, 143.66, 142.95, 139.91, 133.55, 132.93,
132.78, 130.66, 130.62, 130.60, 129.88, 129.64, 129.24, 128.25, 125.70, 124.68,
116.16, 115.90, 108.17, 65.63, 63.49, 52.44; ESI–MS (m/z) 474.3 (M ? H)?; Anal.
calculated for C26H21ClFN5O; C, 65.89; H, 4.47; N, 14.78. Found: C, 65.93; H,
4.48; N, 14.80.
4-(((5-(4-Chlorophenyl)-1-phenyl1H-pyrazol-3-yl)methoxy)methyl)-1-cyclohexyl-
1
1H-1,2,3-triazole(6g) Pale yellow syrup; H NMR (400 MHz, DMSO-d6) d ppm
8.20 (s, 1H, triazole-CH), 7.23–7.44 (m, 9H, ArH), 6.70 (s, 1H, pyrazole-CH), 4.63
(s, 2H, CH2), 4.58 (s, 2H, CH2); 4.47 (t, 1H, CH, J = 11.2 Hz), 2.04 (d, 2H, CH2,
J = 11.2 Hz), 1.69–1.83 (m, 8H, CH2); 13C NMR (100 MHz, DMSO-d6) d ppm
123

Synthesis of new pyrazole-triazole hybrids by click…
150.51, 144.01, 142.88, 139.94, 132.17, 130.88, 129.67, 129.60, 128.36, 125.21,
122.76, 122.32, 108.11, 65.66, 63.66, 59.42, 33.32, 25.17, 25.11; ESI–MS (m/z)
447.1 (M ? H)?; ESI–MS (m/z) 448.1 (M ? H)?; Anal. calculated for C25H26-
ClN5O; C, 67.03; H, 5.85; N, 15.63. Found: C, 67.09; H, 5.87; N, 15.65.
4-(((5-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1-cyclopentyl-
1
1H-1,2,3-triazole(6h) Pale yellow syrup; H NMR (400 MHz, DMSO-d6) d ppm
8.20 (s, 1H, triazole-CH), 7.17–7.55 (m, 9H, ArH), 6.70 (s, 1H, pyrazole-CH), 4.95
(p, 1H, CH, J = 6.8 Hz) 4.63 (s, 2H, CH2), 4.58 (s, 2H, CH2), 1.68–2.19 (m, 8H,
CH2); 13C NMR (100 MHz, DMSO-d6) d ppm 151.01, 143.98, 142.36, 139.83,
132.16, 130.01, 130.51, 129.98, 128.36, 124.67, 123.98, 122.31, 108.15, 65.69,
63.58, 59.40, 33.33, 25.17, 25.08; ESI–MS (m/z) 434.1 (M ? H)?; Anal. calculated
for C24H24ClN5O; C, 66.43; H, 5.57; N, 16.14. Found: C, 66.39; H, 5.59; N, 16.17.
1-(4-Fluorobenzyl)-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6i) Off white solid; H NMR (400 MHz, DMSO-d6) d
ppm 8.22 (s, 1H, triazole-CH), 7.35–7.42 (m, 5H, ArH), 7.18–7.35 (m, 6H, ArH),
6.90 (d, 2H, ArH, J = 8.8 Hz), 6.56 (s, 1H, pyrazole-CH), 5.70 (s, 2H, CH2), 4.63
(s, 1H, CH2), 4.54 (s, 2H, CH2), 3.74 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-
d6) d ppm; 163.55, 161.12, 159.68, 150.14, 144.70, 143.75, 140.25, 132.82, 132.79,
130.76, 130.67, 130.23, 129.50, 128.04, 125.62, 125.52, 124.57, 122.70, 116.16,
115.95, 114.51, 107.36, 65.73, 63.45, 55.62, 52.46; ESI–MS (m/z) 470.3 (M ? H)?;
Anal. calculated for C27H24FN5O2; C, 69.07; H, 5.15; N, 14.92. Found: C, 69.02; H,
5.17; N, 14.94.
4-((4-(((5-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)benzo nitrile(6j) Off white solid; 1H NMR (400 MHz, DMSO-
d6) d ppm 8.27 (s, 1H, triazole-CH), 7.84 (d, 2H, ArH, J = 7.6 Hz), 7.37–7.46 (m,
5H, ArH), 7.24 (d, 2H, ArH, J = 7.6 Hz), 7.14 (d, 2H, ArH, J = 8.4 Hz), 6.90 (d,
2H, ArH, J = 8.8 Hz), 6.57 (s, 1H, pyrazole-CH), 5.73 (s, 2H, CH2), 4.64 (s, 2H,
CH2), 4.55 (s, 2H, CH2); 3.74 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d
ppm 159.68, 150.13, 144.83, 143.75, 142.03, 140.25, 133.20, 130.23, 129.51,
129.11, 128.04, 125.63, 125.05, 122.70, 119.00, 114.51, 111.37, 107.38, 65.74,
63.43, 55.63, 52.63; ESI–MS (m/z) 477.3 (M ? H)?; Anal. calculated for
C28H24N6O2; C, 70.57; H, 5.08; N, 17.64. Found: C, 70.51; H, 5.10; N, 17.68.
1-(4-Methoxybenzyl)-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6k) Yellow syrup; H NMR (400 MHz, DMSO-d6) d
ppm 8.16 (s, 1H, triazole-CH), 6.90–7.43 (m, 13H, ArH,), 6.57 (s, 1H, pyrazole-
CH), 5.51 (s, 2H, CH2), 4.62 (s, 2H, CH2), 4.54 (s, 2H, CH2); 3.75 (s, 3H, OCH3),
3.73 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d ppm 159.67, 159.58,
150.15, 144.61, 143.74, 140.24, 130.23, 130.04, 129.50, 128.47, 128.04, 125.63,
124.31, 122.70, 114.58, 114.50, 107.36, 65.70, 63.46, 55.62, 55.59, 52.80; ESI–MS
(m/z) 482.1 (M ? H)?; Anal. calculated for C28H27N5O3; C, 79.84; H, 5.65; N,
14.54. Found: C, 79.86; H, 5.66; N, 14.56.
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N. Nayak et al.
1-(4-Nitrobenzyl)-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
methyl)-1H-1,2,3-triazole(6l) Pale yellow solid; 1H NMR (400 MHz, DMSO-d6)
d ppm 8.29 (s, 1H, triazole-CH), 8.23(d, 2H, ArH, J = 8.8 Hz), 7.53(d, 2H, ArH,
J = 8.0 Hz), 7.35–7.42 (m, 3H, ArH,), 7.24 (d, 2H, ArH, J = 6.9 Hz), 6.90 (d, 2H,
ArH, J = 8.8 Hz), 6.57 (s, 1H, pyrazole-CH), 5.79 (s, 2H, CH2), 4.65 (s, 2H, CH2),
4.56 (s, 2H, CH2); 3.74 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d ppm
159.68, 150.13, 147.69, 144.85, 143.98, 143.75, 140.25, 130.22, 129.50, 129.45,
128.04, 125.62, 125.09, 124.39, 122.69, 114.51, 107.38, 65.75, 63.44, 55.62, 52.35;
ESI–MS (m/z) 497.3 (M ? H)?; Anal. calculated for C27H24N6O4; C, 65.31; H,
4.87; N, 16.93. Found: C, 65.37; H, 4.89; N, 16.90.
1-Benzyl-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-
1,2,3-triazole (6m) Pale yellow solid; 1H NMR (400 MHz, DMSO-d6) d ppm 8.20
(s, 1H, triazole-CH), 7.17–7.42 (m, 11H, ArH), 6.90 (d, 2H, ArH, J = 8.8 Hz), 6.56
(s, 1H, pyrazole-CH), 5.70 (s, 2H, CH2), 4.62 (s, 1H, CH2), 4.52 (s, 2H, CH2), 3.73
(s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d ppm 159.67, 150.18, 144.95,
144.61, 143.71, 141.26, 130.25, 130.14, 129.78, 128.77, 128.34, 125.53, 124.32,
122.72, 114.58, 114.53, 107.35, 65.69, 63.46, 55.64, 52.81; ESI–MS (m/z) 452.3
(M ? H)?; Anal. calculated for C27H25N5O2; C, 71.82; H, 5.58; N, 15.51. Found:
C, 71.80; H, 5.50; N, 15.53.
1-(2-Fluorobenzyl)-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
methyl)-1H-1,2,3-triazole(6n) Off white solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 8.20 (s, 1H, triazole-CH), 6.69–7.42 (m, 13H, ArH), 6.57 (s, 1H, pyrazole-CH),
5.71 (s, 2H, CH2), 4.62 (s, 1H, CH2), 4.53 (s, 2H, CH2), 3.73 (s, 3H, OCH3); 13C
NMR (100 MHz, DMSO-d6) d ppm 163.58, 161.61, 159.72, 150.09, 144.68, 143.76,
140.25, 132.81, 132.83, 130.73, 130.70, 130.23, 129.48, 128.14, 125.63, 125.54,
124.57, 122.70, 116.19, 115.96, 114.51, 107.36, 65.72, 63.46, 55.63, 52.45; ESI–
MS (m/z) 470.3 (M ? H)?; Anal. calculated for C27H24FN5O2; C, 69.07; H, 5.15;
N, 14.92. Found: C, 69.10; H, 5.18; N, 14.91.
1-Cyclohexyl-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-
1
1H-1,2,3-triazole(6o) Pale yellow syrup; H NMR (400 MHz, DMSO-d6) d ppm
8.19 (s, 1H, triazole-CH), 6.98–7.47 (m, 13H, ArH), 6.69 (s, 1H, pyrazole-CH), 4.96
(p, 1H, CH, J = 6.8 Hz), 4.64 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 1.68–2.21 (m, 8H,
CH2); 13C NMR (100 MHz, DMSO-d6) d ppm 159.70, 150.92, 141.97, 140.86,
132.47, 131.79, 129.97, 129.83, 128.72, 125.60, 122.94, 122.33, 108.20, 65.69,
63.72, 59.42, 55.88, 33.33, 25.16, 25.09; ESI–MS (m/z) 444.1 (M ? H)?; Anal.
calculated for C26H29N5O2; C, 70.41; H, 6.59; N, 15.79. Found: C, 70.36; H, 6.57;
N, 15.83.
1-Cyclopentyl-4-(((5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-
1
1H-1,2,3-triazole(6p) Pale yellow syrup; H NMR (400 MHz, DMSO-d6) d ppm
8.20 (s, 1H, triazole-CH), 7.14–7.43 (m, 7H, ArH,), 6.91 (d, 2H, ArH, J = 8.0 Hz)
6.58 (s, 1H, pyrazole-CH), 4.90 (p, 1H, CH, J = 7.2 Hz), 4.63 (s, 2H, CH2), 4.56 (s,
2H, CH2); 3.74 (s, 3H, OCH3), 1.67–2.21 (m, 8H, CH2); 13C NMR (100 MHz,
DMSO-d6) d ppm 159.67, 150.21, 144.21, 143.72, 140.25, 130.22, 129.51, 128.04,
123

Synthesis of new pyrazole-triazole hybrids by click…
125.61, 123.14, 122.71, 114.51, 107.38, 65.73, 63.59, 61.38, 55.63, 33.30, 24.07;
ESI–MS (m/z) 430.1 (M ? H)?; Anal. calculated for C25H27N5O2; C, 69.91; H,
6.34; N, 16.31. Found: C, 70.00; H, 6.36; N, 16.33.
1-(4-Fluorobenzyl)-4-(((5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
methyl)-1H-1,2,3-triazole(6q) Off white solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 8.20 (s, 1H, triazole-CH), 7.18–7.44 (m, 13H, ArH), 6.70 (s, 1H, pyrazole-CH),
5.59 (s, 2H, CH2), 4.63 (s, 2H, CH2), 4.57 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d ppm 163.54, 161.10, 149.57, 144.68, 142.73, 141.99, 133.73, 132.91,
132.78, 130.78, 130.71, 130.54, 129.63, 129.11, 129.20, 128.30, 124.99, 124.62,
116.19, 116.06, 107.02, 65.63, 63.47, 52.45; ESI–MS (m/z) 519.1 (M ? H)?; Anal.
calculated for C26H21BrFN5O; C, 60.24; H, 4.08; N, 13.51. Found: C, 60.21; H,
4.10; N, 13.53.
4-((4-(((5-(4-Bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-1,2,3-tri-
azol-1yl)methyl)benzonitrile (6r) Yellow syrup; 1H NMR (400 MHz, DMSO-d6) d
ppm 8.27 (s, 1H, triazole-CH), 7.83 (d, 2H, ArH, j = 8.4 Hz), 7.38–7.44 (m, 7H,
ArH), 7.21–7.25 (m, 4H, ArH), 6.69 (s, 1H, pyrazole-CH), 5.73 (s, 2H, CH2), 4.65
(s, 2H, CH2), 4.58 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d ppm 150.37,
144.77, 142.58, 141.89, 139.96, 137.88, 130.59, 129.65, 129.45, 129.04, 129.13,
128.31, 127.32, 125.03, 118.99, 111.37, 108.17, 65.64, 63.46, 52.64; ESI–MS (m/z)
526.1 (M ? H)?; Anal. calculated for C27H21BrN6O; C, 61.72; H, 4.03; N, 16.00.
Found: C, 61.72; H, 4.01; N, 16.03.
1-(4-Methoxybenzyl)-4-(((5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6s) Off white solid; H NMR (400 MHz, DMSO-d6) d
ppm 8.18 (s, 1H, triazole-CH), 6.92–7.38 (m, 13H, ArH), 6.69 (s, 1H, pyrazole-CH),
5.50 (s, 2H, CH2), 4.63 (s, 2H, CH2), 4.57 (s, 2H, CH2), 3.73 (s, 3H, OCH3); 13C
NMR (100 MHz, DMSO-d6) d ppm 159.57, 150.38, 144.88, 142.64, 140.02, 139.89,
133.63, 130.62, 130.14, 129.96, 129.64, 129.55, 129.24, 129.14, 128.57, 128.30,
125.69, 124.30, 114.55, 114.38, 108.22, 65.60, 63.52, 55.57, 52.80; ESI–MS (m/z)
531.1 (M ? H)?; Anal. calculated for C27H24BrN5O2; C, 61.44; H, 4.56; N, 13.20.
Found: C, 61.49; H, 4.58; N, 13.23.
1-(4-Nitrobenzyl)-4-(((5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-
1H-1,2,3-triazole(6t) Off white solid; 1H NMR (400 MHz, DMSO-d6) d ppm 8.29
(s, 1H, triazole-CH), 8.23 (d, 2H, ArH, j = 8.8 Hz), 7.52 (d, 2H, ArH, J = 8.0 Hz),
7.38–7.44 (m, 5H, ArH), 7.22–7.27 (m, 4H, ArH), 6.69 (s, 1H, pyrazole-CH), 5.79
(s, 2H, CH2), 4.66 (s, 2H, CH2), 4.57 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6)
d ppm 150.33, 147.30, 143.98, 143.90, 142.85, 140.86, 133.88, 130.76, 128.85,
129.65, 129.12, 128.44, 125.75, 125.19, 124.45, 108.19, 65.62, 63.45, 52.34; ESI–
MS (m/z) 546.1 (M ? H)?; Anal. calculated for C26H21BrN6O3; C, 57.26; H, 3.88;
N, 15.41. Found: C, 57.21; H, 3.90; N, 14.37.
1-Benzyl-4-(((5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1H-
1,2,3-triazole(6u) Off white solid; 1H NMR (400 MHz, DMSO-d6) d ppm 8.20 (s,
1H, triazole-CH), 7.18–7.44 (m, 11H, ArH), 6.89 (d, 2H, ArH, J = 8.8 Hz), 6.57 (s,
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N. Nayak et al.
1H, pyrazole-CH), 5.69 (s, 2H, CH2), 4.62 (s, 1H, CH2), 4.52 (s, 2H, CH2), 3.73 (s,
3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d ppm 161.14, 144.73, 144.61,
143.39, 141.51, 140.39, 132.64, 132.22, 132.09, 130.89, 130.74, 130.52, 129.53,
129.26, 129.20, 128.30, 125.71, 124.54, 116.14, 115.91, 108.20, 65.63, 63.48,
52.41; ESI–MS (m/z) 501.1 (M ? H)?; Anal. calculated for C27H21BrN6O; C,
62.41; H, 4.43; N, 14.00. Found: C, 62.42; H, 4.43; N, 14.03.
1-(2-Fluorobenzyl)-4-(((5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)-
1
methyl)-1H-1,2,3-triazole(6v) Pale yellow solid; H NMR (400 MHz, DMSO-
d6) d ppm 8.20 (s, 1H, triazole-CH), 7.16–7.56 (m, 13H, ArH), 6.69 (s, 1H,
pyrazole-CH), 5.67 (s, 2H, CH2), 4.64 (s, 2H, CH2), 4.57 (s, 2H, CH2); 13C NMR
(100 MHz, DMSO-d6) d ppm 161.88, 160.90, 150.44, 143.59, 142.84, 140.01,
133.56, 132.92, 132.79, 130.68, 130.64, 130.61, 129.77, 129.69, 129.36, 128.29,
125.79, 124.67, 116.15, 115.94, 108.18, 65.62, 63.52, 52.43; ESI–MS (m/z) 519.1
(M ? H)?; Anal. calculated for C26H21BrFN5O; C, 60.24; H, 4.08; N, 13.51.
Found: C, 60.29; H, 4.11; N, 13.53.
4-(((5-(4-Bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1-cyclohexyl-
1H-1,2,3-triazole(6w) Yellow syrup; 1H NMR (400 MHz, DMSO-d6) d ppm 8.19
(s, 1H, triazole-CH), 7.16–7.57 (m, 13H, ArH), 6.70 (s, 1H, pyrazole-CH), 4.62 (s,
2H, CH2), 4.57 (s, 2H, CH2), 4.35(p, 1H, CH), 2.04 (d, 2H, CH2, J = 12 Hz),
1.23–1.82 (m, 8H, CH2); 13C NMR (100 MHz, DMSO-d6) d ppm 150.47, 143.95,
142.67, 139.90, 132.07, 130.88, 129.67, 129.63, 128.35, 125.70, 122.51, 122.31,
108.18, 65.65, 63.68, 59.43, 33.32, 25.15, 25.08; ESI–MS (m/z) 493.1 (M ? H)?;
Anal. calculated for C25H26BrN5O; C, 60.98; H, 5.32; N, 14.22. Found: C, 60.94; H,
5.32; N, 14.23.
4-(((5-(4-Bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methoxy)methyl)-1-cyclopentyl-
1H-1,2,3-triazole(6x) Yellow syrup; 1H NMR (400 MHz, DMSO-d6) d ppm 8.19
(s, 1H, triazole-CH), 7.16–7.57 (m, 13H, ArH), 6.70 (s, 1H, pyrazole-CH), 4.96 (p,
1H, CH, J = 6.8 Hz), 4.63 (s, 2H, CH2), 1.67–2.19 (m, 8H, CH2); 13C NMR
(100 MHz, DMSO-d6) d ppm 150.47, 150.20, 144.17, 142.67, 139.88, 132.06,
130.87, 129.66, 129.61, 129.54, 129.06, 128.86, 128.34, 127.98, 125.69, 123.16,
122.31, 108.18, 107.89, 65.61, 63.61, 63.03, 61.96, 60.28, 33.30, 33.01, 24.53,
24.40, 24.07; ESI–MS (m/z) 479.3 (M ? H)?; Anal. calculated for C24H24BrN5O;
C, 60.26; H, 5.06; N, 14.64. Found: C, 60.24; H, 5.04; N, 14.63.
Antitubercular studies
Twofold serial dilutions of each test compound/drug were prepared and incorpo-
rated into Middlebrook 7H11 agar medium with oleic acid, albumin, dextrose, and
catalase (OADC) growth supplement to get final concentrations of 50, 25, 12.5,
6.25, 3.13, 1.56, and 0.78 lg/mL. Inoculum of MTB H37Rv ATCC 27294/XDR-TB
was prepared from fresh Middlebrook 7H11 agar slants with OADC (Difco) growth
supplement adjusted to 1 mg/mL (wet weight) in Tween 80 (0.05 %) saline diluted
to 10-2 to give a concentration of *107 cfu/mL. Five microliters of this bacterial
suspension was spotted onto 7H11 agar tubes containing different concentrations of
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Synthesis of new pyrazole-triazole hybrids by click…
the drug as discussed above. The tubes were incubated at 37 °C, and final readings
(as MIC in lg/mL) were determined after 28 days. This method is similar to that
recommended by the National Committee for Clinical Laboratory Standards for the
determination of MIC in triplicate.
Cytotoxicity
Vero (African green monkey kidney) cell line was procured from the National Centre
for Cell Sciences (NCCS), Pune, India. Stock cells were cultured in MEM supplemented
with 10 % inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin
(100 lg/mL), and amphotericin B (5 lg/mL) in an humidified atmosphere of 5 %
CO2 at 37 °C until confluent. The cells were dissociated with TPVG solution (0.2 %
trypsin, 0.02 % EDTA, 0.05 % glucose in PBS). The stock cultures were grown in
25 cm2 culture flasks and all experiments were carried out in 96 microtitre plates
(Tarsons India Pvt. Ltd., Kolkata, India). For cytotoxicity studies, each weighed test
drugs were separately dissolved in distilled DMSO and volume was made up with
MEM supplemented with 2 % inactivated FBS to obtain a stock solution of 1 mg/
mL concentration and sterilized by filtration. Serial twofold dilutions were prepared
from this for carrying out the MTT assay. The monolayer cell culture was
trypsinized, and the cell count was adjusted to 1.0 9 105 cells/mL using MEM
containing 10 % FBS. To each well of the 96 well microtitre plate, 0.1 mL of the
diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a
partial monolayer was formed, the supernatant was flicked off, the monolayer was
washed once with medium, and 100 lL of different test concentrations of test drugs
were added on to the partial monolayer in microtitre plates. The plates were then
incubated at 37 °C for 3 days in 5 % CO2 atmosphere, and microscopic
examination was carried out and observations were noted every 24 h interval.
After 72 h, the drug solutions in the wells were discarded and 50 lL of MTT in PBS
was added to each well. The plates were gently shaken and incubated for 3 h at
37 °C in a 5 % CO2 atmosphere. The supernatant was removed and 100 lL of
propanol was added, and the plates were gently shaken to solubilize the formed
formazan. The absorbance was measured using a microplate reader at a wavelength
of 540 nm. The percentage growth inhibition was calculated using the following
formula, and concentration of test drug needed to inhibit cell growth by 50 % (IC50)
values was generated from the dose–response curves for each cell line. Selectivity
index (SI = IC50 against Vero cells/MIC against M. tuberculosis) values were also
determined. The compound that exhibited selectivity index (SI) values[10 against
the Vero cell line could be considered as nontoxic [37].
ꢀ
ꢁ
Mean OD of individual test group
Mean OD of control group
% Growth inhibition ¼ 100 À
 100
Antibacterial studies
Newly synthesized organic compounds 6a–x were tested against a panel of
pathogenic microorganisms, which were maintained on nutrient agar medium at
123

N. Nayak et al.
37 °C. The cultures were inoculated into 10 mL fresh nutrient broth to yield an
initial suspension of approximately 10–100 cfu/mL. All broths were incubated
statically at the aforementioned temperature for 24 h. Susceptibility of the test
organism to the organic compound was determined by the well plate technique [38,
39]. The bacterial suspensions were serially diluted in saline and 0.1 mL from the
appropriate dilution was spread on nutrient agar. The wells were dug using a sterile
cork borer, and the organic compounds dissolved in DMSO were added (1.0 and
0.5 mg/mL). The same procedure was repeated for all microorganisms, and the petri
plates were incubated for 18 h at 37 °C. After the incubation, the inhibition zone
was measured and the values for DMSO solvent were subtracted to get the actual
values.
Acknowledgments NN and JR gratefully acknowledge the financial support provided by NITK,
Surathkal. We are thankful to MIT Manipal and Dr. Reddy’s Institute of Life Sciences, University of
Hyderabad Campus, Hyderabad for NMR and Mass spectrometer facility.
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