1,2,3-Triazole derivatives as antitubercular agents: synthesis, biological evaluation and molecular docking study

Article abstract of DOI:10.1039/c5md00057b

Searching for new active molecules against Mycobacterium tuberculosis (MTB) H37Ra, a small focused library of 1,2,3-triazoles has been efficiently prepared via a click chemistry approach. The newly synthesized compounds were tested against drug-sensitive MTB. Several derivatives were found to be promising inhibitors of MTB characterized by lower MIC values (5.8-29.9 μg mL^-1). Among all the synthesized 31 compounds, 15e was the most active compound against MTB. Based on the results from the anti-tubercular activity, SAR for the synthesized series has been developed. The active compounds from the anti-tubercular study were further tested for anti-proliferative activity against THP-1, A549 and PANC-1 cell lines using MTT assay and showed no significant cytotoxic activity against these three cell lines except THP-1 at the maximum concentration evaluated. Further, the synthesized compounds were found to have potential antioxidant activities with an IC₅₀ range of 10.1-37.3 μg mL^-1. The molecular docking study of the synthesized compounds was performed against the DprE1 enzyme of MTB to understand the binding interactions. Moreover, the synthesized compounds were also analysed for ADME properties and all the experimental results promote us to consider this series as a starting point for the development of novel and more potent anti-tubercular agents in the future.

Full text of DOI:10.1039/c5md00057b

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CONCISE ARTICLE
1,2,3-Triazole derivatives as antitubercular agents:
synthesis, biological evaluation and molecular
docking study†
Cite this: DOI: 10.1039/c5md00057b
Mubarak H. Shaikh,a Dnyaneshwar D. Subhedar,a Laxman Nawale,b Dhiman Sarkar,b
c
c
a
*
Firoz A. Kalam Khan, Jaiprakash N. Sangshetti and Bapurao B. Shingate
Searching for new active molecules against Mycobacterium tuberculosis (MTB) H37Ra, a small focused
library of 1,2,3-triazoles has been efficiently prepared via a click chemistry approach. The newly
synthesized compounds were tested against drug-sensitive MTB. Several derivatives were found to be
promising inhibitors of MTB characterized by lower MIC values (5.8–29.9 μg mL−1). Among all the synthe-
sized 31 compounds, 15e was the most active compound against MTB. Based on the results from the anti-
tubercular activity, SAR for the synthesized series has been developed. The active compounds from the
anti-tubercular study were further tested for anti-proliferative activity against THP-1, A549 and PANC-1 cell
lines using MTT assay and showed no significant cytotoxic activity against these three cell lines except
THP-1 at the maximum concentration evaluated. Further, the synthesized compounds were found to have
potential antioxidant activities with an IC50 range of 10.1–37.3 μg mL−1. The molecular docking study of the
synthesized compounds was performed against the DprE1 enzyme of MTB to understand the binding inter-
actions. Moreover, the synthesized compounds were also analysed for ADME properties and all the experi-
mental results promote us to consider this series as a starting point for the development of novel and more
potent anti-tubercular agents in the future.
Received 9th February 2015,
Accepted 27th April 2015
DOI: 10.1039/c5md00057b
www.rsc.org/medchemcomm
such as isoniazid and rifampicin, there is serious problem
emergence as MTB developed resistance against the first-line
1. Introduction
Tuberculosis (TB), an infection caused by Mycobacterium
tuberculosis (MTB), still remains the leading cause of world-
wide deaths among infectious diseases. The World Health
Organization (WHO) reported that more than one-third of the
world's population is infected with TB and that resulted in an
estimated 1.5 million deaths worldwide in 2013,1 out of which
0.36 million people were infected with both human immuno-
deficiency virus (HIV) and TB. The long duration of therapy
generally results in noncompliance of the treatment and
results in multidrug resistance tuberculosis (MDR-TB) and
extensively drug-resistant tuberculosis (XDR-TB), which are
highly lethal, extremely expensive and complicated to treat,
posing new challenges for the prevention, treatment and con-
trol of TB.2 Despite the availability of effective anti-TB drugs,
as well as the second-line drugs.3 Therefore, there is an
urgent need to develop new inhibitors that reduce the com-
plexity and duration of the current therapeutic treatment as
well as effectively treat MDR and XDR tuberculosis.
Oxidative stress is the major cause of tissue inflammation
in tuberculosis. During illness, due to the poor dietary intake
of micronutrients, free radicals burst from activated macro-
phages and anti-tuberculosis drugs. If these free radicals
were not neutralized by the antioxidants, this leads to pulmo-
nary inflammation.4 These antioxidants exert their effects by
scavenging or preventing the generation of reactive oxygen
species (ROS)5 which can protect the formation of free radi-
cals and retard the progress of pulmonary inflammation.
1,2,3-Triazoles have received much attention, as their
intriguing physical and biological properties as well as their
excellent stability render them promising drug core struc-
tures. The 1,3-dipolar cycloaddition reaction of a 1,3-dipole
to a dipolarophile (i.e. an acetylene or an alkyne) for the syn-
thesis of five-membered heterocycles is a well-known trans-
formation in synthetic organic chemistry.6 Recently, the
groups of Sharpless7 and Meldal8 have reported the dramatic
rate enhancement (up to 107 times) and improved regio-
selectivity of the Huisgen 1,3- dipolar cycloaddition reaction
a Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad, 431 004, India. E-mail: bapushingate@gmail.com;
Fax: +91 240 2403113; Tel: +91 240 2403313
b Combi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune 411
008, India
c Dr. Rafiq Zakaria Campus, Y. B. Chavan College of Pharmacy, Aurangabad 431
001, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5md00057b
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of an organic azide and terminal acetylene to afford, regio-
specifically, the 1,4-disubstituted-1,2,3-triazole in the pres-
ence of CuIJI) catalyst. The CuIJI)-catalyzed azide alkyne cyclo-
addition (CuAAC) reaction has successfully fulfilled the
requirement of “click chemistry” as prescribed by Sharpless
and within the past few years has become a premier compo-
nent of synthetic organic chemistry.9
derivatives and their antitubercular, antioxidant and cytotoxic
activities. In addition to this, we have also performed molecu-
lar docking study and in silico ADME prediction for the syn-
thesized compounds.
2. Results and discussion
Over the past few years, 1,2,3-triazoles have been an
important class of compounds because of their wide coverage
of biological applications including anti-tubercular,10 anti-
bacterial, anti-allergic, anti-HIV,11 anti-fungal12 and
α-glycosidase inhibitor13 activities. Moreover, 1,2,3-triazole
derivatives have been found to show a promising anti-
tubercular activity profile.14 Owing to these significant fea-
tures, a number of protocols for the synthesis of 1,2,3-triazole
compounds have been developed.15 Among them, the most
elegant and useful approach is the Huisgen 1,3-dipolar cyclo-
addition of azides and alkynes.16 In recent years, derivatives
with conjugated 1,2,3-triazoles have been synthesized and
proved to possess different bioactivities. Baltas and
coworkers17 reported 1,4-disubstituted 1,2,3-triazole deriva-
tives (A and B) (Fig. 1) exhibiting good inhibitory activities
against MTB H37Rv. Similarly, clubbed [1,2,3] triazoles with
fluorine containing benzimidazole series (C) of H37Rv
strain inhibitors have been reported.18 The 1,2,3-triazole
based compounds (D) are active against different pathogenic
and opportunistic Mycobacteria including M. avium and
MTB.19 Recently, a 1,2,3-triazole based isoniazid derivative10
(E) has been shown to possess antitubercular activity against
MTB H37Rv (Fig. 1).
2.1. Chemistry
The 1,4-disubstituted-1,2,3-triazole derivatives 13–19 have
been obtained by the fusion of substituted IJprop-2-yn-1-
yloxy)benzenes 1–7 and benzyl azides 8–12 via click chemistry
(Scheme 1). The commercially available phenols have been
alkylated with propargyl bromide in the presence of K2CO3 as
a base in N,N-dimethylformamide (DMF) affording the corre-
sponding IJprop-2-yn-1-yloxy)benzene derivatives 1–7 in good
to excellent yield (ESI†). The synthesis of benzyl azides 8–12
has been achieved from the corresponding benzaldehydes
according to a literature procedure.21 The Huisgen CuAAC
reaction has been performed on IJprop-2-yn-1-yloxy)benzene 1
with 4-nitrobenzylazide 8 in the presence of CuIJOAc)2 in
t-BuOH–H2O (3 : 1) at room temperature for 20 h affording
1,4-disubstituted-1,2,3-triazole 13a in 89% yield (Scheme 1).
The formation of compound 13a was confirmed by 1H
NMR, 13C NMR and HRMS spectral analysis. In the H NMR
1
spectrum of compound 13a, the two methylene groups
attached to nitrogen and oxygen showed singlet at δ 5.20 and
5.67 ppm, respectively. In addition to this, the signal
observed at δ 7.64 ppm indicates the presence of the proton
on the triazole ring. In the 13C NMR spectrum of compound
13a, the signals at δ 53.20 and 62.01 ppm indicate the pres-
ence of methylene carbon attached to the nitrogen of the
triazole ring and oxygen to the phenyl ring, respectively. For
compound 13a, the calculated mass for [M+Na]+ is 333.0958,
and in HRMS, the [M+Na]+ peak is observed at 333.0967. Fur-
thermore, to expand the series, 1,4-disubstituted-1,2,3-
triazole derivatives 13b–e, 14a–e, 15a–e, 16a–e, 17a–d, 18a–e
and 19a–b with various substituents (ESI†) have been pre-
pared by the cycloaddition reaction of benzyl azides 8–12 and
In connection with the development of new active mole-
cules against MTB, a small focused library of 1,2,3-triazole
incorporated molecules has been efficiently prepared by click
chemistry. We were encouraged to design new 1,2,3-triazole
based molecules from commercially available starting mate-
rials in minimum steps with high overall yield. In continua-
tion of our earlier work20 on the synthesis and biological
properties of various heterocyclic moieties, herein, we would
like to report the synthesis of 1,4-disubstituted-1,2,3-triazole
Fig. 1 1,2,3-Triazole containing compounds possess antitubercular activity.
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Scheme 1 Synthesis of 1-benzyl-4-IJphenoxymethyl)-1H-1,2,3-triazole 13–19. Reagents and conditions: (a) propargyl bromide, K2CO3, DMF; (b)
CuIJOAc)2 (20 mol%), t-BuOH : H2O (3 : 1), rt.
alkynes 1–7 (Scheme 1 and Table 1) under similar reaction
conditions in good to excellent yields.
increase in anti-tubercular activity (MIC range = 5.8–29.2 μg
mL−1). The substitution of halogens like chloro- and bromo-
groups at R6 position (compounds 15c, MIC = 8.2 μg mL−1,
and 15e, MIC = 5.8 μg mL−1) showed a sharp increase in anti-
tubercular activity. The substitution of bromo- at R6 position
(compound 15e, MIC = 5.8 μg mL−1) was more favorable and
found to be the most active compound among all the 31 syn-
thesized compounds. The replacement of the chloro- group
from R5 (compound 15d, MIC = 25.8 μg mL−1) to R6 (com-
pound 15c, MIC = 8.2 μg mL−1) showed a 3-fold increase in
activity. All the compounds of the 16a–16e series (replace-
ment of hydrogen by the nitro- group at R2 position) were
found to be least active (MIC range = >30 to ≥ 100 μg mL−1)
among all the synthesized series. The substitution of the
methyl group at R2 position and the chloro- group at R3 posi-
tion (17a–17d series) has shown some significant activity
(MIC range = 26.9 to ≥ 30 μg mL−1). The substitution of the
chloro- group at R1, R3 and R4 positions (18a–18e series) has
not shown a significant change in activity (MIC range = 27.8
to >30 μg mL−1) except compound 18c (MIC = 7.7 μg mL−1)
bearing the chloro- group at R6 position. The replacement of
the chloro- group by the iodo- group at R1 and R4 positions
and the nitro- group at R3 position (compounds 19a–19b)
showed no significant alteration in activity (MIC = >30 μg
mL−1). None of the synthesized compounds showed activity
comparable with that of standard rifampicin (MIC = 0.04 μg
mL−1).
2.2. Biological evaluation
2.2.1. Anti-tubercular activity. All the 31 newly synthesized
compounds were screened for in vitro anti-tubercular activity,
determined by measuring inhibition of growth against aviru-
lent strain of MTB (MTB H37Ra; ATCC 25177) in liquid
medium. In a preliminary screening (ESI†, Tables S1 and S2),
the anti-mycobacterial activity of these compounds was
assessed at concentrations of 30, 10 and 3 μg mL−1 using an
established XTT Reduction Menadione assay (XRMA) anti-
tubercular screening protocol22 using first-line antitubercular
drugs rifampicin and isoniazid as reference standards, and
the data obtained are presented in Table 1. The triazole deriv-
atives 13a, 13c, 13d, 13e, 14a, 14c, 14d, 15a, 15b, 15c, 15d,
15e, 17a, 17c, 18c and 18e (MIC range = 5.8–29.9 μg mL−1)
were found to be particularly active against MTB. Compounds
13b, 13e, 14b, 14e, 16b, 17b, 17d, 18a, 18b, 18d, 19a and 19b
(MIC = >30 μg mL−1) were found to show less activity against
MTB strains. Compounds 16a, 16c, 16d and 16e (MIC = >100
μg mL−1) were found to be less active against MTB strains.
The structure–activity relationship reveals that the com-
pounds have shown significant anti-tubercular activity
(Table 1). The activity table showed that the 1,2,3-triazole ring
is important for anti-tubercular activity. The activity varies
depending on the various substituents present on the phenyl
rings. The compounds from the 13a–13e series (MIC range =
27.7 to ≥ 30 μg mL−1) do not show any significant activity
when compared with the standards. The replacement of
hydrogen with the methyl group at R3 position (14a–14e
series, MIC range = 24.2 to ≥ 30 μg mL−1) does not show any
significant change in anti-tubercular activity when compared
with compounds of the 13a–13e series. The substitution of
the chloro- group at R3 position (15a–15e series) showed an
2.2.2. Antioxidant activity. In tuberculosis, oxidative stress
may result in tissue inflammation due to anti-tubercular
drugs.4 The synthesized compounds have shown promising
anti-tubercular activity and have potential to develop as lead
compounds. Therefore, it is necessary to evaluate the synthe-
sized compounds for their antioxidant activity. Antioxidant
activities of the synthesized compounds 13–19 were mea-
sured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging assay.23 DPPH radical scavenging activity is the
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Table 1 In vitro antitubercular activity against MTB H37Ra and DPPH radical scavenging activities of compounds 13–19a
Compounds
R1
R2
R3
R4
R5
R6
Anti-TB MIC (μg mL−1
)
Anti-TB IC50 (μg mL−1
)
DPPH IC50 (μg mL−1
)
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
16a
16b
16c
16d
16e
17a
17b
17c
17d
18a
18b
18c
18d
18e
19a
19b
Rifampicin
BHT
AA
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NO2
NO2
NO2
NO2
Me
Me
Me
Me
H
H
H
H
H
H
H
—
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
I
H
NO2
H
Cl
H
H
NO2
H
Cl
H
H
NO2
H
Cl
H
H
NO2
H
Cl
H
H
NO2
H
H
H
NO2
H
Cl
28.0
>30
29.9
27.7
28.9
29.9
>30
24.2
28.9
>30
27.5
29.2
8.2
1.7
2.4
3.5
2.4
3.2
1.1
2.4
2.5
3.0
7.8
3.6
2.6
0.7
2.9
0.5
>100
8.3
53.4
26.6
43.2
0.8
2.2
0.2
2.4
2.2
2.6
0.8
2.2
0.9
2.6
25.0
0.002
NA
NA
10.1
25.3
26.1
27.3
18.3
10.9
12.1
17.3
16.3
16.3
24.2
21.3
26.1
23.3
11.0
17.1
19.1
37.3
31.2
29.4
13.4
15.6
11.4
16.5
28.9
29.2
27.2
16.4
14.0
16.8
18.5
NA
H
H
Br
NO2
H
Cl
H
Br
NO2
H
Cl
H
Br
NO2
H
Cl
H
Br
NO2
H
Me
Me
Me
Me
Me
Cl
Cl
Cl
Cl
Cl
H
25.8
5.8
>100
>30
>100
>100
>100
28.2
>30
26.9
>30
>30
>30
7.7
>30
27.8
>30
>30
0.04
NA
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NO2
NO2
—
—
—
Cl
Br
NO2
H
Cl
H
Br
NO2
Br
—
—
—
NO2
H
Cl
H
H
H
—
—
—
I
I
—
—
—
—
—
—
16.5
12.7
NA
a
DPPH: 2,2-diphenyl-1-picrylhydrazyl; BHT: butylated hydroxytoluene; AA: ascorbic acid; NA: not applicable.
most commonly used method for screening the antioxidant
activities of various natural as well as synthetic antioxidants.
A lower IC50 value indicates greater antioxidant activity. The
IC50 (concentration required to scavenge 50% of the radicals)
values were calculated to evaluate the potential antioxidant
activities. Butylated hydroxytoluene (BHT) and ascorbic acid
(AA) have been used as standard drugs for the comparison of
antioxidant activity and the observed results are summarized
in Table 1.
According to the DPPH assay, compounds 13a, 14a, 14b,
14d, 14e, 15e, 17a, 17b, 17c, 17d, 18d, 18e and 19a exhibited
promising radical scavenging activities when compared with
synthetic antioxidant BHT (IC50 = 16.5 μg mL−1), with IC50
values of 10.1, 10.9, 12.1, 16.3, 16.3, 11.0, 13.4, 15.6, 11.4,
16.5, 16.4, 14.0 and 16.8 μg mL−1, respectively. Similarly,
compounds 13a (IC50 = 10.1 μg mL−1), 14a (IC50 = 10.9 μg
mL−1), 14b (IC50 = 12.1 μg mL−1), 15e (IC50 = 11.0 μg mL−1)
and 17c (IC50 = 11.4 μg mL−1) show excellent antioxidant
activity compared to the standard antioxidant drug ascorbic
acid (IC50 = 12.7 μg mL−1). From the synthesized compounds,
13a, 14a, 15e and 17c showed the best DPPH radical scaveng-
ing activity, while compound 16c showed the lowest activity
when compared with standards.
From the antioxidant activity data (Table 1), the synthe-
sized compounds had shown moderate to good antioxidant
activity. Compound 13a (IC50 = 10.1 μg mL−1) from the
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13a–13e series bearing R6 = NO2 group was found to be the
most active antioxidant agent among the synthesized com-
pounds than standard drugs BHT and ascorbic acid. The
replacement of R6 = NO2 group by R5 = NO2 led to a decrease
in activity by 2 fold (compound 13b, IC50 = 25.3 μg mL−1).
The replacement of the NO2 group with the chloro- group at
R6 (compound 13c, IC50 = 26.1 μg mL−1) and R5 (compound
13d, IC50 = 27.3 μg mL−1) positions did not show any signifi-
cant change in activity. The substitution of R6 = Br (com-
pound 13e, IC50 = 18.3 μg mL−1) led to an increase in activity
when compared with chloro- substituted compounds 13c and
13d. Among the 14a–14e series, all the five compounds (IC50
activity except compounds 18d (R1, R3, R4 and R5= Cl, IC50
16.4 μg mL−1) and 18e (R1, R3 and R4 = Cl and R6 = Br, IC50
=
=
14.0 μg mL−1) which showed comparable antioxidant activity
with BHT.
2.2.3. Cytotoxic activity. The synthesized 1,2,3-triazole
derivatives 13a, 13c, 13d, 13e, 14a, 14c, 14d, 15a, 15b, 15c,
15d, 15e, 17a, 17c, 18c and 18e were further assayed for their
cytotoxic activity against the three different human cancer
cell lines, THP-1 (human acute monocytic leukemia cell line),
A549 (human lung adenocarcinoma epithelial cell line)
and PANC-1 (human pancreas carcinoma cell line), using
3-IJ4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT) assay24 with 48 h exposure time of the tested com-
pounds and paclitaxel used as positive control. The observed
results are summarized in Table 2. The cytotoxic effect of
these compounds was checked on cancer cell lines using the
concentration range between 100 and 0.78 μg mL−1 to deter-
mine the growth inhibition (GI) (GI50 and GI90, Table 2). The
results indicated that, in MTT cytotoxicity studies, most
active compounds are leads as antimicrobials owing to no
significant cell toxicity against THP-1, A549 and PANC-1 cell
lines at the maximum concentration evaluated.
2.2.4. Selectivity index. According to the study of
Hartkoorn and coworkers on drug susceptibility of TB, the
anti-mycobacterial activity was considered to be specific when
the selectivity index is >10.25 The selectivity index was calcu-
lated by dividing GI50 for cell lines (THP-1, A549, PANC-1) by
the MIC for in vitro activity against active/dormant MTB.26
Growth inhibition concentration, 50% (GI50) was taken as the
lowest concentration of the compound killing 50% of the
cells as obtained from the percentage cytotoxicity curve
range
=
10.9–17.3 μg mL−1) have shown good activity
when compared with standards. There is no significant vari-
ation observed while varying the substituent such as NO2, Cl
and Br.
From the 15a–15e series, all compounds (IC50 range =
21.3–26.1 μg mL−1) except 15e (IC50 = 11.0 μg mL−1) showed
less potent activity when compared with standards. The sub-
stitution of R6 = Br (compound 15e) led to a 2-fold increase
in the activity when compared with compounds 15a, 15b, 15c
and 15d. The substitution of R2 = NO2 (series 16a–16e) led to
the least active (IC50 range = 17.1–37.3 μg mL−1) series among
the synthesized compounds. The compounds (IC50 range =
11.4–16.5 μg mL−1) with –CH3 substitution at R2 position
(series 17a–17d) showed significant better activity than stan-
dard BHT (IC50 = 16.5 μg mL−1). Compound 17c (IC50 = 11.4
μg mL−1) with R2 = CH3, R3 = Cl and R6 = Cl substitution
showed better activity than standards BHT and ascorbic acid.
The substitution of halogens like chloro- and iodo- at R1 posi-
tion (compounds 18a–18e and 19a–19b) showed moderate
Table 2 Cytotoxicity of selected compounds in 3 human cancer cell linesa
Cytotoxic profile against human cancer cell lines with SD values
THP-1
GI50
A549
GI50
PANC-1
GI50
Compound
GI90
GI90
GI90
13a
13c
13d
13e
14a
14c
14d
15a
15b
15c
15d
15e
62.9 0.9
36.4 0.9
14.6 0.5
16.2 0.9
14.2 0.8
74.1 0.5
36.7 0.4
31.1 0.1
34.9 0.5
17.4 0.9
20.9 0.5
52.9 0.3
27.9 0.8
31.2 0.2
25.1 0.3
58.7 0.1
0.14 0.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.13 0.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.72 0.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.004 0.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.07 0.6
54.3 0.6
68.3 0.3
66.4 0.3
>100
93.1 0.4
88.9 0.1
97.6 0.9
89.7 0.6
>100
>100
17a
17c
18c
84.3 0.1
88.1 0.3
>100
>100
5.82 0.1
18e
Paclitaxelb
a
GI50/GI90 in μg mL−1, after 48 h. Human cancer cell lines: THP-1 (human acute monocytic leukemia cell line), A549 from lung adenocarci-
noma and PANC-1 from pancreas carcinoma. The results show that cell viability is >80% at the highest concentration of 100 μg mL−1 against
PANC-1 and A549 cell lines. Standard anticancer drug and positive control. The GI50 values were indicated as mean calculated from three
b
independent experiments.
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plotted using Origin8. It was found that all the compounds
have selectivity index of >3 at dormant state of MTB. How-
ever, compounds 15c, 15e and 18e showed comparatively
higher selectivity index of greater than 10 against A549 and
PANC-1 cell lines (Table 3).
For the simultaneous detection of both active and dor-
mant stage inhibitors against tubercular bacilli, we used
XRMA protocol developed earlier.22 The assay basically fol-
lows a very similar principle of hypoxia model of dormancy.27
Under these conditions, the incubation was terminated on
the 8th day and 12th day, respectively, to identify active and
dormant stage inhibitors.
acid residues like ARG58, THR118, GLY321, GLU322 and
LYS418 and van der Waals interactions with amino acid resi-
dues like TRP16, ARG58, TYR60, PRO116, THR118, GLN120,
VAL121, GLY125, ALA126, CYS129, ILE131, ASP318, GLY321
and GLY322. The triazole ring was held in the active site
by forming various interactions with amino acid residues
TRP16, ARG58, TYR60 and THR118. Most of the hydro-
phobic interactions were formed in the presence of the
active methylene group (–CH2–). The oxygen atom (–O–)
in compound 15e had shown hydrogen bonding with
amino acid THR118 (2.46 Å). The bromo- group of com-
pound 15e had formed van der Waals interactions with
amino acid residues GLY125, ALA126 and ILE131. The
chloro- group had shown van der Waals interactions with
amino acid GLU322. Thus, halogen substituents like chloro-
and bromo- groups at R3 and R6 positions are important
for antitubercular activity.
The moderately active compounds 14e and 19b were
docked to understand the binding interactions against the
DprE1 enzyme. Compound 14e was held in the active site by
forming various hydrophobic interactions with amino acid
residues like ARG58, GLY117, THR118 and GLY321 and van
der Waals interactions with amino acids TRP16, ARG58,
TYR60, PRO116, GLY117, THR118, ILE131, ASP318, GLY321,
GLU322 and TYR415. The triazole ring was held in the active
site by forming various interactions with amino acid residues
like TRP16, TYR60, THR118 and GLY321. The amino acid
TYR60 had formed two hydrogen bonds with the 2nd (2.31 Å)
and 3rd (2.45 Å) nitrogen atoms of the triazole ring. The
bromo- group at R6 position had shown van der Waals inter-
actions with amino acids PRO116, ILE131 and TYR415. The
replacement of the chloro- group (electron withdrawing
2.3. Computational studies
2.3.1. Molecular docking study. The 1,2,3-triazole derivatives
have been reported to inhibit the DprE1 IJdecaprenylphosphoryl-
β-D-ribose-2′-epimerase) enzyme of MTB.28 DprE1 is involved
in the biosynthesis of decaprenylphosphoryl-D-arabinose
(DPA), an essential component of the mycobacterial cell
wall.29 In order to investigate the binding interactions,
the synthesized compounds 15e (most active), 14e, 19b
(moderately active) and 16a (least active) were docked against
the DprE1 enzyme (PDB ID:4FDO)30 using VLife MDS 4.3
package.31 The binding interactions of docked compounds
with the DprE1 enzyme are shown in Fig. 2. The synthesized
compounds have showed binding energy, i.e. 15e (most
active, −77.97 kcal mol−1), 14e, 19b (moderately active, −73.79
and −66.58 kcal mol−1, respectively) and 16a (least active,
−60.42 kcal mol−1). The docking study results for compound
15e revealed that it binds to the active site of the DprE1
enzyme by forming various hydrophobic bonds with amino
Table 3 Selectivity index on human cell lines against MTB H37Raa
MTB –
dormant
MIC
Selectivity index (SI)
Compound
(μg mL−1
)
THP-1 GI50 (μg mL−1
)
SI
A549 GI50 (μg mL−1
)
SI
PANC-1 GI50 (μg mL−1
)
SI
13a
13c
13d
13e
14a
14c
14d
15a
15b
15c
15d
15e
17a
17c
18c
18e
RP
28.0
29.9
27.6
28.8
29.9
24.2
28.9
27.5
29.2
8.2
25.8
5.8
28.2
26.9
7.7
62.9
36.4
14.7
16.2
14.2
74.1
36.7
31.1
34.9
17.4
20.9
52.9
27.9
31.2
25.0
58.7
100.0
2.2
1.2
0.5
0.6
0.5
3.1
1.3
1.1
1.2
2.1
0.8
9.2
1.0
1.2
3.2
2.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>3.6
>3.3
>3.6
>3.5
>3.3
>4.1
>3.5
>3.6
>3.4
>12.2
>3.9
>17.3
>3.6
>3.7
>12.9
>3.6
>2325.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>3.6
>3.3
>3.6
>3.5
>3.3
>4.1
>3.5
>3.6
>3.4
>12.2
>3.9
>17.3
>3.6
>3.7
>12.9
>3.6
>2325.6
27.8
0.043
>2325.6
a
RP: rifampicin, dormant state: a reversible state of bacterial metabolic shutdown. Selectivity index (SI) was calculated for all the titled
compounds taking into account the MIC against dormant MTB H37Ra and the IC50 on human cancer cell lines (SI = IC50/MIC) by the MTT
assay. If the SI is ≥10, then the compound is investigated further.
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group) with the –CH3 group (electron donating group) at R3
position may lead to a decrease in binding with the active
site of the enzyme compared to the most active compound
15e. Compound 19b had shown hydrophobic and van der
Waals interactions with amino acid residues like TRP16,
ARG58, TYR60, GLY117, THR118, GLN120, ASP318, GLY321,
GLU322, TYR415, ALA417 and LYS418. The triazole ring is
held in the active site by forming various interactions with
amino acids TRP16, ARG58, TYR60 and THR118. The bromo-
and iodo- groups had formed van der Waals interactions with
amino acids GLN120 and GLY117, respectively. The nitro-
group (–NO2) did not show any interaction with the active
sites of the enzyme and this may be the reason for less activ-
ity of compound 19b.
We have also docked the least active compound 16a
against the DprE1 enzyme. The compound had shown least
binding energy that is −60.42 kcal mol−1. The compound
had shown good hydrophobic and van der Waals interac-
tions, but it did not form any hydrogen bond with the
enzyme. The various amino acid residues like ARG58,
THR118, VAL121, CYS129, ILE131 and ALA417 had formed
hydrophobic interactions with compound 16a. The amino
acids like GLY55, GLY57, ARG58, SER59, TYR60, ASN63,
ALA64, THR118, VAL121, GLY125, CYS129, ILE131, GLY321,
ALA417 and LYS418 showed van der Waals interactions with
compound 16a. The triazole ring was held in the active site
by forming only weak van der Waals interactions with
amino acids ARG58, THR118, VAL121 and ALA417. The
triazole ring and nitro- groups (–NO2) had not shown any
hydrophobic interaction. Due to poor binding interactions
with the DprE1 enzyme, compound 16a may have shown
the least anti-mycobacterial activity among the synthesized
compounds. When we compare the docking pose of
compounds 15e (most active) and 16a (least active), it was
found that in compound 16a, the phenyl ring with R5 = H
and R6 = NO2 substituents was out of the active cavity of
the enzyme. In compound 15e, the phenyl ring with R5 = H
and R6 = Br was held deep into the active cavity of the
enzyme and this may be the reason why compound 15e is
more active than 16a. On the basis of activity data and
docking results, compound 15e has potential to inhibit the
DprE1 enzyme and can be processed further to develop as a
lead compound.
2.3.2. In silico ADME prediction. The success of a drug is
determined not only by good efficacy but also by an accept-
able ADME (absorption, distribution, metabolism and excre-
tion) profile. In the present study, we have calculated molecu-
lar volume (MV), molecular weight (MW), logarithm of
partition coefficient (milog P), number of hydrogen bond
acceptors (n-ON), number of hydrogen bonds donors (n-
OHNH), topological polar surface area (TPSA), number of
rotatable bonds (n-ROTB) and Lipinski's rule of five32
using the Molinspiration online property calculation
toolkit.33 Absorption (% ABS) was calculated by % ABS =
109 − (0.345 × TPSA).34 Drug-likeness model score (a collective
property of physicochemical properties, pharmacokinetics and
Fig. 2 Docking image of compounds 15e (most active), 14e, 19b
(moderately active) and 16a (most inactive). The ligands are shown in
red. The hydrophobic bonds are shown in sky blue.
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pharmacodynamics of a compound is represented by a numer-
ical value) was computed by MolSoft software.35
3.1. General procedure for the synthesis of 1-IJprop-2-
ynyloxy)benzene or substituted 1-IJprop-2-ynyloxy)benzene
(1–7). To the stirred solution of phenol or substituted phenol
(20 mmol) in N,N-dimethylformamide (DMF) (20 mL), K2CO3
(24 mmol) was added. The reaction mixture was stirred at
room temperature for 30 minutes, which results in the corre-
sponding oxyanion. To this mixture, propargyl bromide
(20 mmol) was added and stirred for 2 h. The progress of the
reaction was monitored by TLC using ethyl acetate : hexane as
a solvent system. The reaction was quenched with crushed
ice. In the case of a solid product, it was filtered and the
obtained crude solid product was crystallized using ethanol.
The crystallized products were obtained for the next step.
When the products are liquid, they were extracted with ethyl
acetate (20 mL × 3). The combined organic layers were dried
over MgSO4. The solvent was removed under a reduced pres-
sure and used for further reaction without purification.
A computational study of active compounds (compounds
showing IC50 values ≤2.00 μg mL−1) 13a, 14a, 15c, 15e, 17a,
17c, 18c and 18e was performed for prediction of ADME
properties and the values obtained are presented in Table 4.
It is observed that the compounds exhibited good % ABS
(% absorption) ranging from 79.40 to 95.21%. Furthermore,
only compounds 18c and 18e violated Lipinski's rule of five
(milog P ≤ 5). Compounds 13a, 14a, 15c, 15e, 17a and 17c
did not violate Lipinski's rule of five. A molecule likely to be
developed as an orally active drug candidate should show no
more than one violation of the following four criteria: milog P
(octanol–water partition coefficient) ≤5, molecular weight
≤500, number of hydrogen bond acceptors ≤10 and number
of hydrogen bond donors ≤5.36 The larger the value of the
drug likeness model score, the higher is also probability that
the particular molecule will be active. All the tested com-
pounds followed the criteria for orally active drug, and there-
fore, these compounds may have good potential for eventual
development as oral agents.
3.2. General experimental procedure for the synthesis of
substituted
1-benzyl-4-IJphenoxymethyl)-1H-1,2,3-triazole
(13–19). To the stirred solution of IJprop-2-yn-1-yloxy)benzenes
1–7 (0.5 mmol), substituted benzyl azide 8–12 (0.5 mmol)
and copper diacetate IJCuIJOAc)2) (20 mol%) in t-BuOH–H2O
(3 : 1, 8 mL) were added and the resulting mixture was stirred
at room temperature for 19–27 h. The progress of the reac-
tion was monitored by TLC using ethyl acetate : hexane as a
solvent system. The reaction mixture was quenched with
crushed ice and extracted with ethyl acetate (2 × 15 mL). The
organic extracts were washed with brine solution (2 × 15 mL)
and dried over anhydrous sodium sulphate. The solvent was
evaporated under reduced pressure to afford the correspond-
ing crude compounds. The obtained crude compounds were
recrystallized using ethanol.
3. Experimental
All the solvents and reagents were purchased from commer-
cial suppliers, Spectrochem Pvt. Ltd., Rankem India Ltd. and
Sigma Aldrich, and were used without further purification.
The progress of each reaction was monitored by ascending
thin layer chromatography (TLC) using TLC aluminum
sheets, silica gel F254 precoated (Merck, Germany), and locat-
ing the spots using UV light as the visualizing agent or iodine
vapors. Melting points were obtained by an open capillary
3.2.1. 1-IJ4-Nitrobenzyl)-4-IJphenoxymethyl)-1H-1,2,3-triazole
(13a). Compound 13a was obtained via 1,3-dipolar cycloaddi-
tion reaction between azide 8 and alkyne 1 for 20 h with 89%
1
method and are uncorrected. H NMR spectra were recorded
IJCDCl3/DMSO-d6) on a Bruker Avance 200 NMR spectrometer.
13C NMR and DEPT 135 spectra were recorded IJCDCl3/DMSO-d6)
on a Bruker Avance 200 NMR spectrometer and a JEOL ECX
400 NMR spectrometer. Chemical shifts (δ) are reported in
parts per million (ppm) using tetramethylsilane (TMS) as an
internal standard. The splitting pattern abbreviations are
designated as singlet (s), doublet (d), double doublet (dd),
triplet (t), quartet (q) and multiplet (m).The mass spectra
were recorded on a Q-TOF micromass (YA-105) spectrometer
in ESI (electrospray ionization) mode.
1
yield, mp: 96–98 °C. H NMR (200 MHz, CDCl3, δ ppm): 5.23
(s, N–CH2), 5.65 (s, O–CH2), 6.93–7.02 (m, 3H, Ar H), 7.29–
7.44 (m, 4H, Ar H), 7.61 (s, 1H, triazole H), and 8.20–8.27 (m,
2H, Ar H). 13C NMR (50 MHz, CDCl3, δ ppm): 53.2, 62, 114.8,
121.4, 124.4, 128.5, 129.6 and 141.5. HRMS calculated
[M+Na]+ for C16H14N4O3Na: 333.0958, found: 333.0967.
3.2.2. 1-IJ3-Nitrobenzyl)–4-IJphenoxymethyl)-1H-1,2,3-triazole
(13b). Compound 13b was obtained via 1,3-dipolar cycloaddi-
tion reaction between azide 9 and alkyne 1 for 20 h with 90%
Table 4 Pharmacokinetic parameters important for good oral bioavailability
Entry
% ABS
TPSA (A2)
n-ROTB
MV
MW
milog P
n-ON
n-OHNH
Lipinski's violations
Drug-likeness model score
Rule
13a
14a
15c
15e
17a
17c
18c
18e
—
—
—
6
6
5
5
6
5
5
5
—
<500
310.31
324.3
334.20
378.65
358.78
348.23
403.09
447.54
≤5
<10
7
7
4
4
7
4
4
4
<5
0
0
0
0
0
0
0
0
≤1
0
0
0
0
0
0
1
1
—
79.40
79.40
95.21
95.21
79.40
95.21
95.21
95.21
85.77
85.77
39.95
39.95
85.77
39.95
39.95
39.95
269.83
286.39
273.57
277.92
299.93
290.13
300.64
304.99
3.29
3.74
4.69
4.82
4.35
4.89
5.90
6.03
−0.72
−0.62
−0.20
−0.46
−0.01
0.27
0.21
−0.06
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yield, mp: 98 °C. 1H NMR (200 MHz, CDCl3, δ ppm): 5.23
(s, N–CH2), 5.65 (s, O–CH2), 6.94–7.01 (m, 3H), 7.24–7.35
(m, 2H), 7.57–7.63 (m, 3H), and 8.17–8.26 (m, 2H). 13C NMR
(50 MHz, CDCl3, δ ppm): 53.2, 62, 114.8, 121.4, 122.9, 123.8, 129.6,
130, 133.9. HRMS calculated [M+H]+ for C16H15N4O3: 311.1160,
found: 311.1155, [M+Na]+ for C16H14N4O3Na: 333.0980, found:
333.0980.
cycloaddition reaction between azide 12 and alkyne 2 for 21
h with 93% yield, mp: 106 °C. 1H NMR (200 MHz, CDCl3, δ
ppm): 2.17 (s, 3H), 5.18 (s, N–CH2), 5.46 (s, O–CH2), 6.93–
6.97 (m, 3H), 7.11–7.15 (m, 2H), 7.32 (m, 1H), and 7.47–7.54
(m, 3H). 13C NMR (50 MHz, CDCl3, δ ppm): 29.8, 53.5, 62.1,
114.8, 121.4, 122.7, 123.1, 129.7, 129.8, 132.4, 133.6, 145 and
158.2.
3.2.3.
1-IJ4-Chlorobenzyl)-4-IJphenoxymethyl)-1H-1,2,3-
3.2.9.
1-IJ4-Nitrobenzyl)-4-IJ(4-chlorophenoxy)methyl)-1H-
triazole (13c). Compound 13c was obtained via 1,3-dipolar
cycloaddition reaction between azide 10 and alkyne 1 for 21
h with 88% yield, mp: 90–92 °C. H NMR (200 MHz, CDCl3, δ
1,2,3-triazole (15a). Compound 15a was obtained via 1,3-dipo-
lar cycloaddition reaction between azide 8 and alkyne 3 for
22 h with 90% yield, mp: 94 °C. H NMR (200 MHz, CDCl3, δ
1
1
ppm): 5.19 (s, N–CH2), 5.49 (s, O–CH2), 6.94–7.00 (m, 3H),
7.12–7.17 (d, 2H), 7.29–7.33 (m, 2H) and 7.47–7.53 (m, 2H).
13C NMR (50 MHz, CDCl3, δ ppm): 53.5, 62.1, 114.8, 121.3,
122.6, 129.4, 129.6, 133, 134.9, 145 and 158.2. HRMS calcu-
lated [M+H]+ for C16H15N3OCl: 300.0898, found: 300.0905,
[M+Na]+ for C16H14N3OClNa: 322.0718, found: 322.0725.
ppm): 5.25 (s, N–CH2), 5.53 (s, O–CH2), 7.21–7.28 (m, 2H),
7.34–7.48 (m, 4H) 7.61 (s, 1H, triazole H) and 7.81–7.85
(m, 2H). NMR (100 MHz, CDCl3, δ ppm): 53.6, 62.3, 116.1,
122.6, 123, 126.2, 129.4, 129.7, 132.4, 133.4, 144.5 and 156.7.
HRMS calculated [M+H]+ for C16H14N4O3Cl: 345.0748, found:
345.0739, [M+Na]+ for C16H13N4O3Cl Na: 367.0568, found:
367.0559.
3.2.4.
1-IJ4-Bromobenzyl)-4-IJphenoxymethyl)-1H-1,2,3-
triazole (13e). Compound 13e was obtained via 1,3-dipolar
cycloaddition reaction between azide 12 and alkyne 1 for 21
h with 89% yield, mp: 103–104 °C. 1H NMR (200 MHz, CDCl3,
δ ppm): 5.19 (s, N–CH2), 5.49 (s, O–CH2), 6.94–7.00 (m, 3H),
7.12–7.17 (m, 2H), 7.28–7.33 (m, 2H), and 7.47–7.53 (m, 3H).
13C NMR (50 MHz, CDCl3, δ ppm): 53.6, 62.1, 114.8, 121.4,
122.6, 123.1, 129.7, 129.8, 132.4, 133.6, 145.1 and 158.2.
HRMS calculated [M+Na]+ for C16H14N3OBrNa: 366.0212,
found: 366.0212.
3.2.10. 1-IJ3-Nitrobenzyl)-4-IJ(4-chlorophenoxy)methyl)-1H-
1,2,3-triazole (15b). Compound 15b was obtained via
1,3-dipolar cycloaddition reaction between azide 9 and alkyne
3 for 23 h with 89% yield, mp: 96 °C. 1H NMR (400 MHz,
CDCl3, δ ppm): 5.21 (s, N–CH2), 5.67 (s, O–CH2), 6.91–6.93
(m, 2H), 7.24–7.29 (m, 2H), 7.60–7.65 (m, 3H), 8.19 (s, 1H,
triazole H) and 8.24–8.27 (m, 1H). 13C NMR (100 MHz, CDCl3,
δ ppm): 53.2, 62.2, 116.1, 122.8, 122.9, 123.9, 126.3, 129.5,
130.4, 133.9, 136.5, 148.6 and 156.7. HRMS calculated [M+H]+
for C16H14N4O3Cl: 345.0749, found: 345.075, [M+Na]+ for
C16H13N4O3ClNa: 367.0571, found: 367.0567.
3.2.5.
1-IJ4-Nitrobenzyl)-4-IJ(p-tolyloxy)methyl)-1H-1,2,3-
triazole (14a). Compound 14a was obtained via 1,3-dipolar
cycloaddition reaction between azide 8 and alkyne 2 for 19 h
with 90% yield, mp: 90–91 °C. 1H NMR (200 MHz, CDCl3, δ
ppm): 2.17 (s, 3H), 5.22 (s, N–CH2), 5.65 (s, O–CH2), 6.94–7.01
(m, 3H), 7.29–7.42 (m, 3H), 7.63 (s, 1H, triazole H), and 8.19–
8.24 (m, 2H). 13C NMR (50 MHz, CDCl3, δ ppm): 31.1, 53.3, 62,
114.8, 121.5, 123, 124.4, 128.7, 129.7, 141.7, 145.4, 148.1, and
158.1.
3.2.11. 1-IJ4-Chlorobenzyl)-4-IJ(4-chlorophenoxy)methyl)-1H-
1,2,3-triazole (15c). Compound 15c was obtained via 1,3-dipo-
lar cycloaddition reaction between azide 10 and alkyne 3 for
1
23 h with 88% yield, mp: 80 °C. H NMR (200 MHz, CDCl3, δ
ppm): 5.18 (s, N–CH2), 5.51 (s, O–CH2), 6.91–6.93 (m, 2H),
7.16–7.18 (m, 2H), 7.24–7.29 (m, 2H) and 7.52–7.54 (m, 3H).
13C NMR (50 MHz, CDCl3, δ ppm): δ 53.6, 62.3, 116.2, 126.3,
129.5, 132.9, 135 and 156.8. HRMS calculated [M+H]+ for
C16H14N3OCl2: 334.0508, found: 334.0504, for [M+Na]+
C16H13N3OCl2Na: 356.0358, found: 356.0324.
3.2.6.
1-IJ4-Chlorobenzyl)-4-IJ(p-tolyloxy)methyl)-1H-1,2,3-
triazole (14c). Compound 14c was obtained via 1,3-dipolar
cycloaddition reaction between azide 10 and alkyne 2 for 21
1
h with 92% yield, mp: 84–85 °C. H NMR (200 MHz, CDCl3, δ
3.2.12. 1-IJ3-Chlorobenzyl)-4-IJ(4-chlorophenoxy)methyl)-1H-
1,2,3-triazole (15d). Compound 15d was obtained via
1,3-dipolar cycloaddition reaction between azide 11 and
ppm): 2.17 (s, 3H), 5.22 (s, N–CH2), 5.65 (s, O–CH2), 6.94–
7.01 (m, 3H), 7.29–7.42 (m, 3H), 7.64 (s, 1H, triazole H), and
8.19–8.24 (m, 2H). 13C NMR (50 MHz, CDCl3, δ ppm): 29.8,
53.6, 62.1, 114.8, 121.4, 122.7, 129.4, 129.5, 129.6, 133.1,
134.9, 145 and 158.2.
1
alkyne 3 for 24 h with 89% yield, mp: 104 °C. H NMR (200
MHz, CDCl3, δ ppm): 5.21 (s, N–CH2), 5.67 (s, O–CH2), 6.91–
6.93 (m, 2H), 7.24–7.29 (m, 2H), 7.60–7.65 (m, 3H), 8.19 (s,
1H, triazole H) and 8.24–8.27 (m, 1H). 13C NMR (50 MHz,
CDCl3, δ ppm): 51.8, 62.3, 116.2, 126.3, 127.8, 129.5, 129.8,
130.5, 130.6, 132.3, 133.6 and 156.8. HRMS calculated [M+H]+
for C16H14N3OCl2: 334.0508, found: 334.0504, [M+Na]+ for
C16H13N3OCl2Na: 356.0358, found: 356.0324.
3.2.7.
1-IJ3-Chlorobenzyl)-4-IJ(p-tolyloxy)methyl)-1H-1,2,3-
triazole (14d). Compound 14d was obtained via 1,3-dipolar
cycloaddition reaction between azide 11 and alkyne 2 for 22
h with 91% yield, mp: 70 °C. 1H NMR (200 MHz, CDCl3, δ
ppm): 2.17 (s, 3H), 5.20 (s, N–CH2), 5.67 (s, O–CH2), 6.95–
6.99 (m, 3H), 7.18–7.33 (m, 4H), 7.42–7.44 (d, 1H), and 7.64
(s, 1H, triazole H). 13C NMR (50 MHz, CDCl3, δ ppm): 31.1,
51.6, 62.1, 114.9, 121.4, 123.1, 127.8, 129.8, 130, 130.4, 132.4,
133.6, 144.7 and 158.3.
3.2.13. 1-IJ4-Bromobenzyl)-4-IJ(4-chlorophenoxy)methyl)-1H-
1,2,3-triazole (15e). Compound 15e was obtained via 1,3-dipo-
lar cycloaddition reaction between azide 12 and alkyne 3 for
1
24 h with 90% yield, mp: 98 °C. H NMR (400 MHz, CDCl3, δ
3.2.8.
1-IJ4-Bromobenzyl)-4-IJ(p-tolyloxy)methyl)-1H-1,2,3-
ppm): 5.18 (s, N–CH2), 5.51 (s, O–CH2), 6.91–6.93 (m, 2H),
7.16–7.18 (m, 2H), 7.24–7.29 (m, 2H) and 7.52–7.54 (m, 3H).
triazole (14e). Compound 14e was obtained via 1,3-dipolar
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13C NMR (100 MHz, CDCl3, δ ppm): 53.6, 62.3, 116.1, 122.6,
123, 126.2, 129.4, 129.7, 132.4, 133.4, 144.5 and 156.7. HRMS
calculated [M+H]+ for C16H14N3OClBr: 379.9948, found:
379.9948, [M+Na]+ for C16H13N3OClBrNa: 401.9768, found:
401.9767.
(bs, 1H, triazole H). 13C NMR (100 MHz, CDCl3, δ ppm): 20.5,
53.7, 62.3, 113.4, 117.5, 126.6, 129.6, 129.7, 129.8, 133, 135,
137.3 and 156.7. HRMS calculated [M+H]+ for C17H16N3OCl2:
348.0664, found: 348.0664, [M+Na]+ for C17H15N3OCl2Na:
370.0484, found: 370.0486.
3.2.14. 4-IJ(3-Nitrophenoxy)methyl)-1-IJ4-chlorobenzyl)-1H-
1,2,3-triazole (16c). Compound 16c was obtained via 1,3-dipo-
lar cycloaddition reaction between azide 10 and alkyne 4 for
26 h with 89% yield, mp: 123–124 °C. 1H NMR (200 MHz,
CDCl3, δ ppm): 5.25 (s, N–CH2), 5.53 (s, O–CH2), 7.21–7.28
(m, 2H), 7.34–7.48 (m, 4H) 7.61 (s, 1H, triazole H) and 7.81–
7.85 (m, 2H). 13C NMR (50 MHz, CDCl3, δ ppm): 53.6, 62.4,
109.6, 116.3, 121.6, 122.9, 129.4, 129.5, 130.2, 132.9, 135,
143.6, 149.2 and 158.7. HRMS calculated [M+H]+ for
C16H14N4O3Cl: 345.0748, found: 345.0739, [M+Na]+ for
C16H13N4O3Cl Na: 367.0568, found: 367.0559.
3.2.19. 4-IJ(2,4,6-Trichlorophenoxy)methyl)-1-IJ3-nitrobenzyl)-1H-
1,2,3-triazole (18b). Compound 18b was obtained via
1,3-dipolar cycloaddition reaction between azide 9 and alkyne
6 for 27 h with 89% yield, mp: 170 °C. 1H NMR (200 MHz,
CDCl3, δ ppm): 5.21 (s, N–CH2), 5.70 (s, O–CH2), 7.31–7.34
(m, 2H), 7.56–7.66 (m, 2H), 7.87 (bs, 1H) and 8.19–8.27
(m, 2H). 13C NMR (50 MHz, CDCl3, δ ppm): 53.2, 66.3, 122.9,
123.8, 128.8, 130.2, 130.3, 133.9, 136.6, 148.5 and 149.4.
HRMS calculated [M+H]+ for C16H12N4O3Cl3: 404.9802, found:
404.9802, [M+Na]+ for C16H11N4O3Cl3Na: 426.9622, found:
426.9622.
3.2.15. 4-IJ(3-Nitrophenoxy)methyl)-1-IJ3-chlorobenzyl)-1H-
1,2,3-triazole (16d). Compound 16d was obtained via
1,3-dipolar cycloaddition reaction between azide 11 and
alkyne 4 for 25 h with 88% yield, mp: 86 °C. 1H NMR (200
MHz, CDCl3, δ ppm): 5.25 (s, N–CH2), 5.68 (s, O–CH2), 7.21–
7.44 (m, 6H), 7.33 (bs, 1H, triazole H) and 7.81–7.83 (m, 2H).
13C NMR (50 MHz, CDCl3, δ ppm): 51.8, 62.4, 109.6, 116.3,
121.6, 127.7, 130, 130.1, 130.5, 130.6, 132.2, 133.7, 149.2 and
158.7. HRMS calculated [M+H]+ for C16H14N4O3Cl: 345.0748,
found: 345.0739, [M+Na]+ for C16H13N4O3Cl Na: 367.0568,
found: 367.0559.
3.2.20. 4-IJ(2,4,6-Trichlorophenoxy)methyl)-1-IJ4-chlorobenzyl)-1H-
1,2,3-triazole (18c). Compound 18c was obtained via 1,3-dipolar
cycloaddition reaction between azide 10 and alkyne 6 for 27
h with 87% yield, mp: 106 °C. 1H NMR (400 MHz, CDCl3,
δ ppm): 5.20 (s, N–CH2), 5.53 (s, O–CH2), 7.20–7.37 (m, 6H)
and 7.63 (s, 1H, triazole H). 13C NMR (100 MHz, CDCl3,
δ ppm): 53.6, 66.6, 123.4, 128.9, 129.5, 130, 130.4, 133.1, 135,
143.9 and 149.6. HRMS calculated [M+H]+ for C16H12N3OCl4:
403.9628, found: 403.9704, [M+Na]+ for C16H11N3OCl4Na:
425.9526, found: 425.9526.
3.2.21.
4-IJ(2,6-Diiodo-4-nitrophenoxy)methyl)-1-IJ4-
3.2.16. 4-IJ(3-Nitrophenoxy)methyl)-1-IJ4-bromobenzyl)-1H-
1,2,3-triazole (16e). Compound 16e was obtained via 1,3-dipo-
lar cycloaddition reaction between azide 12 and alkyne 4 for
26 h with 87% yield, mp: 131–132 °C. 1H NMR (400 MHz,
CDCl3, δ ppm): 5.23 (s, N–CH2), 5.50 (s, O–CH2), 7.14–7.16
(m, 2H), 7.29–7.36 (d, 1H), 7.40–7.51 (m, 3H), 7.63 (bs, 1H,
triazole H) and 7.80–7.83 (m, 2H). 13C NMR (50 MHz, CDCl3,
δ ppm): 53.8, 62.5, 109.6, 116.3, 121.5, 123.2, 129.8, 130,
132.4, 133.3, 149.2 and 158.7. HRMS calculated [M+H]+ for
C16H14N4O3Br: 389.0250, found: 389.0257, [M+Na]+ for
C16H13N4O3Br Na: 411.0063, found: 411.0070.
nitrobenzyl)-1H-1,2,3-triazole (19a). Compound 19a was
obtained via 1,3-dipolar cycloaddition reaction between azide
8 and alkyne 7 for 26 h with 86% yield, mp: 176 °C. H NMR
(400 MHz, CDCl3, ppm): δH 5.21 (s, N–CH2), 5.70 (s, O–CH2),
7.31–7.34 (m, 2H), 7.60–7.63 (m, 2H), 7.87 (bs, 1H) and 8.19–
8.26 (m, 2H). 13C NMR (50 MHz, CDCl3, ppm): δ 53.2, 66.2,
122.8, 123.8, 128.8, 130.2, 130.3, 133.9 and 136.6.
3.2.22. 4-IJ(2,6-Diiodo-4-nitrophenoxy)methyl)-1-IJ4-bromobenzyl)-1H-
1,2,3-triazole (19b). Compound 19b was obtained via
1,3-dipolar cycloaddition reaction between azide 12 and
alkyne 7 for 24 h with 89% yield, mp: 146–148 °C. 1H NMR
(200 MHz, CDCl3, δ ppm): 5.26 (s, N–CH2), 5.54 (s, O–CH2),
7.17–7.21 (m, 3H), 7.51–7.55 (m, 2H) and 8.64 (s, 2H). 13C NMR
(50 MHz, CDCl3, δ ppm): 53.2, 66.2, 122.8, 128.8, 130.2, 130.3,
133.9, 136.6, 148.5 and 149.4. HRMS calculated [M+H]+
for C16H12N4O3BrI2: 640.8197, found: 640.8197, [M+Na]+ for
C16H11N4O3BrI2Na: 662.8017, found: 662.8017.
1
3.2.17.
4-IJ(4-Chloro-3-methylphenoxy)methyl)-1-IJ3-
nitrobenzyl)-1H-1,2,3-triazole (17b). Compound 17b was
obtained via 1,3-dipolar cycloaddition reaction between azide
1
9 and alkyne 5 for 26 h with 88% yield, mp: 106–108 °C. H
NMR (200 MHz, CDCl3, δ ppm): 2.32 (s, 3H), 5.17 (s, N–CH2),
5.65 (s, O–CH2), 6.73–6.84 (m, 2H), 7.20–7.22 (m, 1H), 7.57–
7.63 (m, 3H) and 8.16–8.23 (m, 2H). 13C NMR (100 MHz,
CDCl3, δ ppm): 20.4, 53.3, 62.3, 113.4, 117.4, 123, 123.9,
126.6, 129.8, 130.5, 134, 136, 137.3, 145.1, 148.6 and 156.6.
HRMS calculated [M+H]+ for C17H16N4O3Cl: 359.0905, found:
359.0903, [M+Na]+ for C17H15N4O3ClNa: 381.0725, found:
381.0720.
3.2.18. 4-IJ(4-Chloro-3-methylphenoxy)methyl)-1-IJ4-chlorobenzyl)-1H-
1,2,3-triazole (17c). Compound 17c was obtained via 1,3-dipolar
cycloaddition reaction between azide 10 and alkyne 5 for 25
h with 90% yield, mp: 108–110 °C. 1H NMR (200 MHz, CDCl3,
δ ppm): 2.37 (s, 3H), 5.18 (s, N–CH2), 5.55 (s, O–CH2), 6.75–
6.88 (m, 2H), 7.20–7.30 (m, 3H), 7.37–7.42 (m, 2H) and 7.60
3.3. Experimental protocol for biological activity
3.3.1. Antitubercular testing using the XRMA protocol.
Compounds 13–19 were evaluated for their in vitro effects
against dormant phase M. tuberculosis H37Ra using the
XRMA protocol.22 All experiments were performed in tripli-
cate, and IC50 and MIC values were calculated from their
dose–response curves. % Inhibition = 100 − (A1 − blank)/(A2 −
blank) × 100, where A1 is the culture absorbance at 470 nm
in the presence of the compound after addition of menadi-
one, A2 is the culture absorbance at 470 nm (DMSO solvent
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control) after addition of menadione, and blank is the culture
absorbance at 470 nm of the respective data points before
addition of XTT/menadione.
obtained from the Protein Data Bank in order to prepare the
protein for the docking study. Docking procedure was
followed using the standard protocol implemented in VLife
MDS 4.3 package and the compounds were docked against
the three-dimensional structure of the DprE1 enzyme.
3.3.2. General procedure for evaluation of DPPH radical
scavenging activity. The hydrogen atom or electron donation
ability of some compounds was measured from the bleaching
of the purple colored methanol solution of 1,1-diphenyl-1-
picrylhydrazyl (DPPH). The spectrophotometric assay uses
the stable radical DPPH as a reagent. 1 mL of various concen-
trations of the test compounds (5, 10, 25, 50 and 100 μg
mL−1) in methanol was added to 4 mL of 0.004% IJw/v) metha-
nol solution of DPPH. The reaction mixture was incubated at
37 °C. The scavenging activity on DPPH was determined by
measuring the absorbance at 517 nm after 30 min. All tests
were performed in triplicate, and the mean values were
obtained. The percent of inhibition (I%) of free radical pro-
duction from DPPH was calculated by using the following
4. Conclusions
We have synthesized 1,4-disubstituted-1,2,3-triazole deriva-
tives from commercially available starting materials. Out of
the 31 screened compounds, 13a, 13c, 13d, 13e, 14a, 14c,
14d, 15a, 15b, 15c, 15d, 15e, 17a, 17c, 18c and 18e display
antitubercular activity with MIC ranging from 5.8 to 29.9 μg
mL−1 against the strain MTB H37Ra. Among the above-
mentioned active compounds, the more potent antitubercular
compound 15e having the chloro- group at R3 and the
bromo- group at R6 position of the phenyl ring shows a MIC
value of 5.8 μg mL−1. Furthermore, compounds 15c and 18c
with MIC values of 8.2 and 7.7 μg mL−1 are endowed with
better potency, being more active than the remaining com-
pounds. Further, the active antitubercular compounds were
evaluated for their cytotoxic effect against three human can-
cer cell lines THP-1, A549 and PANC-1. The cytotoxicity study
revealed that compounds 13a, 13c, 13d, 13e, 14a, 14c, 14d,
15a, 15b, 15c, 15d, 15e, 17a, 17c, 18c and 18e have shown
any cytotoxicity against A549 and PANC-1 cell lines at the
maximum concentration evaluated. Compounds 13a, 14a, 14b,
15e and 17c show potential antioxidant activity as compared
with standards BHT and ascorbic acid. In addition to this,
the molecular docking study of these synthesized triazole
derivatives has shown that these derivatives have a high affin-
ity towards the active site of the DprE1 enzyme which pro-
vides a strong platform for new structure-based design
efforts. Furthermore, analysis of the ADME parameters for
the synthesized compounds has shown that these com-
pounds have good drug-like properties and can be developed
as oral drug candidates. Thus, this suggests that the com-
pounds from the present series 13a, 14a, 14b, 15c, 15e, 17a,
17c, 18c and 18e can be further optimized and developed as
lead molecules.
equation: % of scavenging = [(Acontrol − Asample)/IJAsample
×
100)], where Acontrol is the absorbance of the control (DPPH
radical without test sample) and Asample is the absorbance of
the test sample (DPPH radical with test sample). The control
contains all reagents except the test samples.
3.4. Cytotoxic activity assay
3.4.1. Anti-proliferative activity against THP-1, A549 and
PANC-1 cell lines using the MTT assay.37 The effects of com-
pounds 13–19 were evaluated on cell growth in acute mono-
cytic leukemia cell line THP-1, lung A549 adenocarcinoma,
and pancreatic PANC-1 adenocarcinoma cell line. In vitro
cytotoxicity of all test samples was assessed on a panel of
three human cancer cell lines using the standard MTT
ij3-IJ4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay24 for measuring cellular proliferation. Each concentra-
tion was tested in triplicate in a single experiment. The via-
bility and growth in the presence of test material is calcu-
lated by using the following formula: % cytotoxicity =
(average of control − average of compound)/(average of con-
trol − average of blank) × 100), where control is the culture
medium with cells and DMSO and blank is the culture
medium without cells.
IC50 and MIC values were calculated by plotting the per-
centage survival versus concentrations, using OriginPro
software.
Acknowledgements
The authors M.H.S. and D.D.S. are very much grateful to the
Council for Scientific and Industrial Research (CSIR), New
Delhi for the award research fellowship. The authors are also
thankful to the Head, Department of Chemistry, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad
431 004, India for providing laboratory facility.
3.4.2. Selectivity index.38 The selectivity index (SI) was cal-
culated by dividing IC50 for human cancer cell lines (THP-1,
A549, PANC-1) by the MIC for in vitro activity against active/
dormant MTB H37Ra; if the SI is ≥10, then the compound is
investigated further.
Selectivity index (SI) = (IC50 − in vitro cytotoxicity on
human cancer cell lines)/(minimum inhibitory concentration
(MIC-TB) in vitro activity against MTB H37Ra).
Notes and references
3.4.3. Molecular docking study. Molecular docking study
was performed using VLife MDS 4.3 package. With this pur-
pose, the crystal structure of the DprE1 IJdecaprenylphosphoryl-
β-D-ribose-2′-epimerase) enzyme of MTB (PDB ID:4FDO) was
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