Design and Synthesis of New Aryloxy-linked Dimeric 1,2,3-Triazoles via Click Chemistry Approach: Biological Evaluation and Molecular Docking Study

Article abstract of DOI:10.1002/jhet.3608

A quest for more potent new antitubercular agents has prompted to design and synthesize aryloxy-linked dimeric 1,2,3-triazoles (4a–j), from azides (2a-e) and bis(prop-2-yn-1-yloxy)benzene (3a–b) on 1,3-dipolar cycloaddition reaction via copper (I)-catalyzed click chemistry approach with good to better yields. The titled compounds (4a–j) were designed using molecular hybridization approach by assembling various bioactive pharmacophoric fragments in a single molecular framework. All the synthesized compounds have been screened for their in vitro antitubercular, antifungal, and antioxidant activities against their respective strains. Among them, 4h and 4i show the highest antifungal activity, whereas compounds 4h, 4i, and 4j have revealed promising antitubercular activity against their respective strains. In addition to this, most of the synthesized compounds were found as potent antifungal and antioxidant agents. A significant network of bonded and non-bonded interactions stabilized these molecules into the active site of fungal CYP51 that is realized from the obtained well-placed docking poses and the associated thermodynamic interactions with the enzyme. The synthesized compounds have also been analyzed for absorption, distribution, metabolism, and excretion properties.

Full text of DOI:10.1002/jhet.3608

Month 2019 Design and Synthesis of New Aryloxy-linked Dimeric 1,2,3-Triazoles via
Click Chemistry Approach: Biological Evaluation and Molecular Docking
Study
Tejshri R. Deshmukh,a Smita P. Khare,a Vagolu S. Krishna,b Dharmarajan Sriram,b Jaiprakash N. Sangshetti,c
d
e
a
*
Omprakash Bhusnure, Vijay M. Khedkar, and Bapurao B. Shingate
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, Maharashtra, India
bDepartment of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar,
Hyderabad 500 078, Telangana, India
cDepartment of Pharmaceutical Chemistry, Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad
431 001, Maharashtra, India
dDepartment of Pharmaceutical Chemistry, Channabasweshwar College of Pharmacy, Latur, Maharashtra, India
eDepartment of Pharmaceutical Chemistry, Shri Vile Parle Kelavani Mandal’s Institute of Pharmacy, Dhule 424 001,
Maharashtra, India
*E-mail: bapushingate@gmail.com
Received October 20, 2018
DOI 10.1002/jhet.3608
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
A quest for more potent new antitubercular agents has prompted to design and synthesize aryloxy-linked
dimeric 1,2,3-triazoles (4a–j), from azides (2a-e) and bis(prop-2-yn-1-yloxy)benzene (3a–b) on 1,3-dipolar
cycloaddition reaction via copper (I)-catalyzed click chemistry approach with good to better yields. The
titled compounds (4a–j) were designed using molecular hybridization approach by assembling various bio-
active pharmacophoric fragments in a single molecular framework. All the synthesized compounds have
been screened for their in vitro antitubercular, antifungal, and antioxidant activities against their respective
strains. Among them, 4h and 4i show the highest antifungal activity, whereas compounds 4h, 4i, and 4j have
revealed promising antitubercular activity against their respective strains. In addition to this, most of the
synthesized compounds were found as potent antifungal and antioxidant agents. A significant network of
bonded and non-bonded interactions stabilized these molecules into the active site of fungal CYP51 that
is realized from the obtained well-placed docking poses and the associated thermodynamic interactions with
the enzyme. The synthesized compounds have also been analyzed for absorption, distribution, metabolism,
and excretion properties.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
main aim of this synthetic approach is to obtain more
potent and more selective dimeric drug as compared with
monomer. It is very useful when the targeted protein
exists with two binding sites [4]. Literature survey also
reveals that dimeric compound shows better bioactivities
than monomers [5]. Keeping these facts, many
researchers focused on the synthesis and bioevaluation of
dimeric molecules as shown in Figure 1 [6].
Twin drug synthesis is one of the famous classical
approaches of a promising rational drug designing [1].
There are two types of twin drugs on the basis of
combining two similar or dissimilar moieties known as
“identical” and “non-identical” twin drugs, respectively
[2]. The identical twin drugs are obtained from
dimerization of active pharmacophoric groups and
possesses the C2 symmetry in its structural motif [3]. The
Nowadays, azole class of compounds gain more
attention to researchers, especially 1,2,3-triazoles as they
© 2019 Wiley Periodicals, Inc.

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Figure 1. Some biologically active symmetrical dimeric compounds. [Color figure can be viewed at wileyonlinelibrary.com]
have excellent biological and physical properties. They are
also easy to synthesize by copper (I)-catalyzed 1,3-dipolar
cycloaddition reaction of azide and alkyne by using
Sharpless and Meldal’s [7] approach of click chemistry.
Among the various triazoles, the 1,4-di-substituted 1,2,3-
triazoles have been found to be a promising drug core
moiety as they have proved to be bioisostere of amide
bond [8]. Literature reveals that 1,2,3-triazolyl scaffolds
have shown various bioactivities like antitubercular [9a],
antifungal [9b], antioxidant [9c], anti-inflammatory [9d],
anti-HIV [9e], antibacterial [9f], antimalarial [9g],
antiviral [9h], and anticancer [9i]. Some representative
bioactive molecules having 1,2,3-triazolyl scaffold in
their structure are shown in Figure 2 [10].
Nowadays, fungal infections are spreading worldwide at
an alarming rate [11]. This is commonly acquired in
immune-deficient patients [12]. The azole class has been
considered as the top choice in treating a wide variety of
both localized and systemic fungal infections and
particularly triazoles [13]. The triazoles generally show
high levels of efficacy across a variety of fungal pathogen
species because nitrogen atom of triazole binds to the
sixth coordination of heme iron atom of the porphyrin
and inhibits the CYP51 in the process of biosynthesis of
ergosterol [14].
Tuberculosis (TB) is a highly contagious and an
insidious disease caused by Mycobacterium tuberculosis
(Mtb) [15]. Despite TB is an ancient disease, it remains
the leading cause of morbidity and mortality worldwide
[16]. According to Global Tuberculosis Report by World
Health Organization, TB is the second leading cause of
mortality among the infectious diseases after HIV [17].
M. tuberculosis has a unique ability to develop resistance
against the regularly used anti-TB drugs hence known as
drug resistant TB (DR-TB) [18]. Due to this unique
ability, not a single drug can challenge M. tuberculosis in
the arm race. A quest of the last four decades,
Bedaquiline is the first approved anti-TB drug by Food
and Drug Administration that proved as a watershed in
the battle against the MDR-TB [19].
The association of fungal coinfection is mostly observed
with immune-deficient patients [20]. The fungal
pulmonary coinfection to patients with TB is acquired at
the lungs by opportunistic fungal pathogens [21]. Mostly,
the fungal pulmonary infection does not show any
specific clinical symptoms to patients with TB because it
may be hindered by the symptoms of TB [22]. The
treatment of coinfected patients requires coadministration
of anti-TB and antifungal drugs and includes challenges
of drug–drug interactions, overlapping of toxic effects,
Figure 2. Some bioactive molecules bearing 1,2,3-triazolyl scaffold in their structures. [Color figure can be viewed at wileyonlinelibrary.com]
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Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
and pill burden on patient. This kind of lacunas may lead to
high risk at patient’s life even after the TB was cured
completely [23].
The design of aryloxy-linked dimeric 1,2,3-triazoles was
broadly divided into three segments: (i) 1,2 or 1,3-diaryloxy
moiety showing ether linkage with triazolyl units as a source
of hydrogen bond acceptor, (ii) substitutional variant unit of
respective appended cyclic/aliphatic groups that controls
In patients with TB, due to many reasons, the large
number of free radicals is released from activated
microphages [24]. The excess load of free radicals
generates oxidative stress in the body that results in cell
damage and pulmonary tissue inflammation [25]. The
excessive reactive oxygen species need to be neutralized
by antioxidants to protect the cells and tissues from
damage [26]. To fulfill the need of antioxidants and to
avoid the pill burden of patients with TB, it is necessary
to synthesize such drugs that have the properties of both
anti-TB and antioxidant [27].
By considering the need and therapeutic significance of
the above and by continuing our work on chemistry and
biology of 1,2,3-triazole-based compounds [28], here, we
have planned and synthesized aryloxy-linked dimeric
1,2,3-triazoles with hope to obtain better antitubercular,
antifungal, and antioxidant activities. In addition to this,
we have also performed in silico absorption, distribution,
metabolism, and excretion (ADME) prediction for
synthesized compounds.
lipophilicity
and
contributes
as
most
active
pharmacophoric unit, and (iii) the main backbone of the
design strategy was symmetrically situated in two 1,2,3-
triazolyl units that helps to enhance the pharmacophoric
property as they exhibit drug-like properties.
We have described here the multi-step synthetic strategy
of novel aryloxy-linked substituted dimeric 1,2,3-triazoles.
The series of derivatives of desired compounds (4a–j)
were synthesized by using copper (I)-catalyzed click
chemistry approach as shown in Scheme 3. For the
synthesis, freshly prepared various azides (2a–e) and
dialkynes (3a–b) were used as starting materials. Initially,
various azides (2a–e) were synthesized via synthesis of 2-
chloro-1-substituted ethanone derivatives (1a–e) as
intermediates as shown in Scheme 1.
The intermediates (1a–e) were obtained by reacting
chloroacetyl chloride with respective aliphatic/cyclic
secondary amines in the presence of triethylamine as a
base in chloroform at 0°C. The intermediates (1a–e) on
reaction with sodium azide in N,N-dimethylformamide
(DMF) : H2O (3:1) resulted in the desired azides (2a–r)
in good to excellent yields [30]. The 1,3-bis(prop-2-yn-1-
yloxy)benzene (3a) and 1,2-bis(prop-2-yn-1-yloxy)
benzene (3b) as a source of dialkynes were synthesized
by using commercially available resorcinol and catechol,
respectively (Scheme 2). For the synthesis of bis(prop-2-
RESULTS AND DISCUSSION
Chemistry. The designed scaffold was originated from
molecular hybridization of pharmacophoric fragments of
antitubercular [29a,b], antifungal [29c], and antioxidant
[29d] agents as shown in Figure 3.
Figure 3. Design strategy for the synthesis of aryloxy-linked dimeric 1,2,3-triazoles. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Scheme 1. Synthesis of azides (2a–e) from respective secondary amines. [Color figure can be viewed at wileyonlinelibrary.com]
Scheme 2. Synthesis of bis(prop-2-yn-1-yloxy)benzene (3a–b). [Color figure can be viewed at wileyonlinelibrary.com]
1
yn-1-yloxy)benzene (3a–b), resorcinol and catechol were
individually allowed to react with propargyl bromide in
the presence of K2CO3 as a base in DMF at room
temperature in good to excellent yields [31].
After the synthesis of all the starting materials, the
desired aryloxy-linked substituted dimeric 1,2,3-triazoles
hybrids (4a–j) were synthesized by 1,3-dipolar
cycloaddition of azides (2a–e) with bis(prop-2-yn-1-
yloxy)benzene (3a–b) as a source of dialkyne. This click
reaction was carried out in the presence of catalytic
amount of copper sulfate pentahydrate and reducing
agent sodium ascorbate in PEG-400 : H2O (3:1) for 8 h
at room temperature (Scheme 3).
of the representative H NMR spectrum of compound 4g
displays peaks at δ 5.18, 5.41, and 8.16 ppm as a singlet,
due to O─CH2, N─CH2, and amido N─H, respectively.
The presence of three characteristic carbon signals is
observed at δ 26.2, 56.1, and 164.3 ppm in 13C NMR
spectrum of 4g owing to the signals of methylene groups
of pyrrolidine and carbonyl carbon of amide, respectively.
The HRMS further strengthen the structure assigned to 4g
as it displays [M + H]+ ion peak at m/z 495.2462 for the
molecular formula C24H31N8O4 and equivalent to the
calculated [M + H]+ ion peak at 495.2469.
Biological assay. In vitro antifungal activity.
All the
synthesized compounds were screened for their in vitro
antifungal activity against five different human
pathogenic fungal strains such as Candida albicans
(NCIM 3471), Fusarium oxysporum (NCIM 1332),
All the newly synthesized compounds have been
1
characterized by using their IR, H NMR, 13C NMR, and
high-resolution mass spectra (HRMS) spectral data. One
Scheme 3. Synthesis of aryloxy-linked substituted dimeric 1,2,3-triazoles (4a–j). [Color figure can be viewed at wileyonlinelibrary.com]
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Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
Aspargillus flavus (NCIM 539), Aspargillus niger (NCIM
1196), and Cryptococcus neoformans (NCIM 576)
having minimum inhibitory concentrations (MICs) 25,
25, 12.5, 25, and 25 μg/mL, respectively. MIC values
were estimated using a standard agar dilution method
[32]. Miconazole is used as a standard drug for the
comparative study of the newly synthesized compounds.
The screening results were summarized in Table 1. It was
found that most of the compounds from the series
exhibited good to excellent antifungal activity.
12.5 μg/mL for strains C. albicans and F. oxysporum.
Hence, in case of these two strains, compound 4c was
proved more potent antifungal agent than miconazole,
which is used as a standard for comparative study. The 4j
hybrid shows better activity against strain C. neoformans
and for C. albicans that was equally active to miconazole.
Compounds 4d and 4g were active against any one strain
from screened strains respectively F. oxysporum and
A. niger. The remaining two compounds, 4e and 4f, do not
show any MICs near to the standard miconazole against
any screened fungal strains. Hence, both these compounds
were considered to be inactive compounds for fungal
strains. The antifungal screening results of the series show
that most of the active compounds will serve as an
important scaffold for further designing of such analogs
and development of new antifungal agents in the future.
The detailed study of Table 1 shows that compounds 4a,
4b, 4c, 4d, 4g, 4h, 4i, and 4j were found as more or
equipotent antifungal agents against some of the screened
strains as compared with miconazole. Compounds 4h and
4b were observed as most active compounds among all
the synthesized compounds because both were active
against any four strains out of five. Compound 4h is active
against all screened strains except A. niger. It was
observed that this compound was more potent than
miconazole against strain C. neoformans with MIC
6.25 μg/mL and equipotent to standard in case of
remaining strains. Similarly, compound 4b was also active
against all screened strains except F. oxysporum. It shows
better activity than miconazole in case of A. niger with
MIC 12.5 μg/mL, whereas the remaining MICs were equal
to standard. Compounds 4a and 4i were observed active
against any three strains out of five. According to the
results, 4a hybrid is equipotent to miconazole when it was
screened against strain C. albicans, A. niger, and
C. neoformans. The 4i hybrid shows better activity with
MIC 6.25 μg/mL against strain A. flavus, whereas for
C. albicans and A. niger, it was equally active to
miconazole. The activities of compounds 4c and 4j were
observed better or equal to standard against any two
strains among screened strains. Compound 4c has a MIC
Antioxidant activity.
1,1-Diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity [26] is the most
commonly used method for screening antioxidant
activities of various natural and synthetic antioxidants.
Due to intake of higher doses of antitubercular drugs over
a long period of time leads to oxidative stress in patients
with TB that may result in pulmonary tissue
inflammation. Most of the synthesized compounds
possess good antitubercular activity and have potential to
develop as a drug in the future. Therefore, it is necessary
to evaluate all the synthesized compounds for their
antioxidant activity. In this study, a lower IC50 value
indicates the greater antioxidant activity. The IC50 values,
which are the concentration required to scavenge 50% of
the radicals, were calculated to evaluate the potential
antioxidant activities. Here, the IC50 of all the newly
synthesized compounds were compared with the IC50 of
BHT (butylated hydroxyltoluene), and the results were
summarized in Table 1. It is observed that most of the
Table 1
In vitro antifungal and antioxidant activity of synthesized compounds (4a–j).
Minimum inhibitory concentration in μg/mL against fungal strains
Antioxidant activity IC50 (μg/mL)
Compound
CA
25
FO
AF
AN
CN
12.5
25
62.5
50
62.5
62.5
50
DPPH scavenging activity
4a
4b
4c
4d
4e
4f
4 g
4 h
37.5
50
12.5
25
62.5
50
37.5
25
50
50
25
NT
12.5
12.5
25
50
25
50
62.5
12.5
6.25
25
12.5
12.5
62.5
100
100
62.5
25
37.5
25
15.05 0.21
29.04 0.18
11.19 0.28
14.34 0.40
71.17 0.34
22.25 0.87
18.17 0.09
28.49 0.44
08.19 0.16
16.24 0.64
NT
25
12.5
62.5
50
100
50
25
25
25
25
NT
6.25
4i
4j
37.5
12.5
25
75
25
NT
Miconazole
BHT
12.5
NT
NT
16.47 0.18
AF, Aspergillus flavus; AN, Aspergillus niger; BHT, butylated hydroxytoluene; CA, Candida albicans; CN, Cryptococcus neoformans; DPPH, 1,1-
diphenyl-2-picrylhydrazyl; FO, Fusarium oxysporum; NT, not tested.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Table 2
In vitro antitubercular evaluation of compounds (4a–j) against Mycobacterium tuberculosis H37Rv.
Compound
4a
Structure
Log Pa
Clog Pa
MIC (μg/mL)
>25
ꢀ0.65
ꢀ0.0691996
4b
0.78
0.4008
>25
4c
1.62
1.5188
>25
4d
1.51
1.8064
>25
(Continues)
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Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
Table 2
(Continued)
Compound
4e
Structure
Log Pa
Clog Pa
MIC (μg/mL)
>25
0.15
ꢀ0.309601
4f
ꢀ0.65
ꢀ0.4192
>25
4g
0.78
0.0507999
>25
4h
1.62
1.1688
12.5
4i
1.51
1.4564
6.25
(Continues)
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T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Table 2
(Continued)
Compound
4j
Structure
Log Pa
0.15
Clog Pa
MIC (μg/mL)
ꢀ0.659601
12.5
Isoniazid
0.1
0.2
1.56
1.56
Rifampicin
Ethambutol
Ciprofloxacin
MIC, minimum inhibitory concentration.
aCalculated using ChemBioDraw Ultra 12.0.
synthesized compounds show excellent antioxidant
activities with promising lower IC50 values in comparison
with the standard used as BHT.
6.25 to 12.5 μg/mL. Among all the synthesized
compounds, compound 4i was found to have excellent
activity against Mtb with MIC 6.25 μg/mL, whereas 4h
and 4j derivatives were also found as potent compounds
among the synthesized series with MIC 12.5 μg/mL.
The activities are may be due to the presence of variant
unit in the structural framework of the compounds. All
these values of MIC are considerable by global program
for the discovery of new antitubercular drugs as threshold
for the evaluation of new therapies to cure TB. Hence,
the active compounds from the series may lead to
become able drugs to cure TB, and it seems that there is
a scope for the discovery of new therapeutic agents in the
future by generating more similar analogs of the
synthesized hybrids.
The quality of drug and therapeutic success depends
upon lipophilicity [34]. Each and every drug should
reach to the target site by passing through several
biomembrance for its better in vitro action [35].
Lipophilicity is one of the physicochemical property
based on Log P/Clog P values. Therefore, to study the
correlation of antitubercular activity of synthesized
compounds with lipophilicity parameters, Log P and
Clog P were calculated using ChemBiodraw ultra 12.0
software, and the results were included in Table 2. When
the value of Log P ≤ 5, then the compounds may
show better lipophilicity. It was found that all synthesized
compounds having values of Log P and Clog P are in
acceptable range that is ≤5. Hence, all the compounds
show excellent lipophilic property that helps to accelerate
the rate of therapeutic success.
Among all the synthesized compounds of the series,
the hybrids 4a, 4c, 4d, 4i, and 4j show excellent
radical scavenging activity than BHT, which is used as a
standard for comparative study. It was found that active
compounds have IC50 values in the range of 08.19–
16.24 μg/mL and found to be more potent than
BHT. Moreover, compound 4i displayed the highest
radical scavenging activity than BHT with IC50 value
08.19 μg/mL. Another most promising antioxidant activity
was shown by compounds 4a, 4c, and 4d having IC50
values 15.05, 11.19, and 14.34 μg/mL, respectively,
whereas compound 4j was observed as equipotent to BHT
with IC50 value 16.24 μg/mL. It may be due to the
presence of respective variant unit of the compound and
their position. Remaining compounds 4b, 4e, 4f, 4g,
and 4h show less antioxidant activity as compared with
BHT that may have substituent and position effect of
respective variant units present in their structures. Hence,
these compounds were considered as inactive antioxidant
compounds. However, the study of results shows that most
of the compounds from the series displayed promising
antioxidant activity.
In vitro antitubercular activity.
All the newly
synthesized aryloxy-linked dimeric 1,2,3-triazoles (4a–j)
were evaluated for their in vitro antitubercular activity
against M. tuberculosis H37Rv (ATCC 27294) in a
standard primary screening to obtain the MIC in
triplicates by using microplate Alamar Blue assay [33]. It
has been found that 4h, 4i, and 4j compounds of the
series have displayed noticeable activity against the
M. tuberculosis strain H37Rv with MICs ranging from
Molecular docking study. One of the most widely used
in silico technique for structure-based drug design is
molecular docking to model the binding mode of a ligand
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Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
into the active site of the target enzyme followed by the
prediction of its binding affinity towards the receptor
and the associated thermodynamic interactions with the
active site residues that could govern the inhibition of
the causative pathogen. These techniques have gained
significant importance in the drug discovery regimen,
especially in the case of unavailability of resources to
do the experimental enzymatic assays. The antifungal
activity demonstrated by the aryloxy-linked dimeric
1,2,3-triazoles investigated herein prompted to explore
the plausible mechanism by which these compounds
induce this activity and guide the further structure-
activity relationship. With this objective, molecular
docking was performed against the crucial fungal
enzyme-sterol 14α-demethylase (CYP51) inhibition of
which is shown to check the conversion of lanosterol to
ergosterol in the fungal cells leading to accumulation of
14α-methyl sterols in the cell and causing impaired cell
growth of fungi.
The binding affinity data revealed that all the
compounds (4a–j) could snugly fit into the active site of
CYP51, and the complex formed were stabilized by
formation of well-distributed network of several bonded
and non-bonded interactions (Table 3).
Their docking scores varied from ꢀ8.565 to ꢀ6.461
with average docking score of ꢀ7.607, while average
Glide binding energy was observed to ꢀ62.565 kcal/mol
signifying a promising binding affinity towards CYP51.
It was observed that piperidin-1-yl substitution on the
triazole rings produced the highest binding affinity (4h:
ꢀ8.565/ꢀ69.574 kcal/mol and 4c: ꢀ8.278/ꢀ67.843 kcal/
mol), which is accordance with their observed antifungal
activity. Even the other heterocycles (morpholino and
pyrrolidin-1-yl), that is, 4a, 4b, and 4f, 4g produced
moderate to good binding affinities. The acyclic
substituents (viz. N,N-diethylacetamide and N,N-
dimethylacetamide) when present on ortho di-substituted
analogs (4d and 4e) produced an excellent binding while
when present on meta di-substituted analogs (4i and 4j)
produced a lower binding score. Furthermore, a detailed
investigation was carried out for the per-residue bonded
and non-bonded interactions between these molecules and
the active site residues of the enzyme to identify the most
prominently interacting residues and also the nature of
thermodynamic interactions that can guide the lead
optimization (Table 3).
However, the analysis is discussed only for one of the
most active compounds (4h) in detail in the next section.
The most stable docking model for 4h was selected
according to the best scored conformation predicted
by the Glide extra precision (XP) scoring function
(Fig. 4). Our docking study showed that the compound is
oriented into the active site of CYP51 engaging in a
significant network of steric and electrostatic interactions.
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Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
The most prominent steric interactions were observed
through the methylene-1,2,3-triazolyl-1-piperidinyl
of 4h is also attributed to electrostatic interactions
observed with Hem moiety (ꢀ8.746 kcal/mol), Met460
(ꢀ1.996 kcal/mol), and Thr459 (ꢀ1.327 kcal/mol)
residues in the active site. Furthermore, the compound
showed a very stable hydrogen bonding interaction with
CYP51 through Met460 having a bond distance of 2.69 Å.
The hydrogen bond serves as an anchor to stabilize the
3D orientation of ligand into the active site and facilitate
the non-bonded (steric and electrostatic) interactions.
However, the interaction observed with Hem moiety (iron
metal) in the active site of CYP51 is of at most important
for antifungal activity knowing the fact that fluconazole
is also coordinated through this Heme moiety with
CYP51 indicating that the title compounds can also
exhibit an analogous mechanism of antifungal action.
These well-balanced steric and electrostatic non-bonded
interactions could snugly accommodate other molecules
(Figs. 4–12) in the series and into the active site of CYP51
with gradually decreasing affinity.
ethanone side chain of the molecule with Val461
(ꢀ3.104 kcal/mol), Leu356 (ꢀ3.811 kcal/mol), Ala291
(ꢀ2.941 kcal/mol), Phe290 (ꢀ2.458 kcal/mol), Ala287
(ꢀ1.144 kcal/mol), Tyr116 (ꢀ3.181 kcal/mol), and
Phe110 (ꢀ1.825 kcal/mol) residues, while the other
similar side chain positioned ortho to it engaged in similar
steric interactions with Met460 (ꢀ4.084 kcal/mol), Thr459
(ꢀ2.898 kcal/mol), Met360 (ꢀ1.892 kcal/mol), Pro210
(ꢀ2.313 kcal/mol), Ile105 (ꢀ2.425 kcal/mol), Tyr103
(ꢀ3.467 kcal/mol), and Val102 (ꢀ2.234 kcal/mol). The
1,2-di-substituted phenyl ring at the junction of two
substituents was engaged in a very strong van der Waals
interactions with Hem moiety Hem500 (ꢀ9.747 kcal/mol),
while Ile209 (ꢀ2.382 kcal/mol), Leu208 (ꢀ3.129 kcal/
mol), and Met106 (ꢀ3.545 kcal/mol) residues also
interacted similarly with this ring to stabilize the molecule
into the active site of CYP51. The high binding affinity
Figure 4. Binding mode of 4h into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5. Binding mode of 4b into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Figure 6. Binding mode of 4c into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Figure 7. Binding mode of 4d into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Figure 8. Binding mode of 4e into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
Figure 9. Binding mode of 4f into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Figure 10. Binding mode of 4g into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Figure 11. Binding mode of 4i into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
Figure 12. Binding mode of 4j into the active site of sterol 14α-demethylase (CYP51) (on right side: the pink lines represent the hydrogen bonding
interactions). [Color figure can be viewed at wileyonlinelibrary.com]
This information derived from the in silico binding
affinity study and more specifically from the per-residue
ligand interaction analysis could be fruitfully utilized for
the structure-based lead optimization of these aryloxy-
linked dimeric 1,2,3-triazoles to arrive at the potent
antifungal agent.
In silico absorption, distribution, metabolism, and
excretion prediction. In silico ADME profile of the newly
synthesized aryloxy-linked dimeric 1,2,3-triazoles (4a–j)
has been reported in Table 4.
The success of a drug is determined by a good efficacy
and an acceptable ADME profile. ADME properties
prediction is one of the important pharmacokinetic
parameter to predict oral bioavailability of drug. In this
study, we calculated molecular volume, molecular weight,
logarithm of partition coefficient, number of hydrogen
bond acceptors, number of hydrogen bonds donors,
topological polar surface area, number of rotatable bonds,
and Lipinski’s rule of five [36a] of all the newly
synthesized compounds using Molinspiration online
property calculation toolkit [36b]. Absorption (% ABS)
of all the derivatives of the series was calculated by [37]
%ABS ¼ 109 ꢀ ð0:345ꢁTPSAÞ:
Drug-likeness model score (a collective property
of physicochemical properties, pharmacokinetics, and
Table 4
Pharmacokinetic parameters for in silico ADME prediction.
%
TPSAb
(A2)
Lipinski
Drug-likeness
model score
Entry
ABSa
n-ROTBc
MVd
MWe
<500
miLog Pf
n-ONg
n-OHNHg
violationsh
Rule
4a
4b
4c
4d
4e
—
—
—
10
10
10
14
10
10
10
10
14
10
—
≤5
<10
14
12
12
12
12
14
12
12
12
12
<5
0
0
0
0
0
0
0
0
≤1
2
1
2
1
1
2
1
2
1
1
—
61.05
67.42
67.42
67.42
67.42
61.05
67.42
67.42
67.42
67.42
138.99
120.52
120.52
120.52
120.52
138.99
120.52
120.52
120.52
120.52
460.38
442.41
476.01
463.13
395.93
460.38
442.41
476.01
463.13
395.93
526.55
494.56
522.61
498.59
442.48
526.55
494.56
522.61
498.59
442.48
ꢀ0.08
0.25
0.33
0.44
0.28
0.13
0.28
0.30
0.42
0.30
0.15
1.03
2.04
1.73
ꢀ0.66
ꢀ0.50
0.61
1.62
1.31
ꢀ1.07
4f
4 g
4 h
4i
0
0
4j
ADME, absorption, distribution, metabolism, and excretion.
aPercentage absorption.
bTopological polar surface area.
cNumber of rotatable bonds.
dMolecular volume.
eMolecular weight.
fLipophilicity.
gNumber of hydrogen bond acceptors.
hNumber of violations.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
pharmacodynamics of a compound is represented by a
numerical value) of each and every compound was
computed by MolSoft software [38].
acetic acid to it. The obtained solid intermediates of the
respective amines were filtered, dried, and crystallized in
ethanol. The melting points of all the compounds show
A compound is considered to be an orally active
drug if there is only one violation observed out of
the following four criteria’s: miLog P (octanol–water
partition coefficient) ≤5, molecular weight ≤500, number
of hydrogen bond acceptors ≤10, and number of hydrogen
bond donors ≤5. Drug-likeness score was also calculated
in order to achieve biological activity of compound. It is
observed that all the synthesized compounds exhibited
moderate to good % ABS ranging from 61.05% to 67.42%
as depicted in Table 3. Most of the predictions were found
to be within the acceptable range. All the results show
that the compounds possess average to good potential
for the development of orally active drug molecule.
good agreement with the reported.
(b) Synthesis of azides (2a–e). The newly synthesized
intermediates (1a–e) were refluxed with aqueous
solution of sodium azide (0.1 mol) in DMF for 6 h. The
progress of the reaction was monitored by TLC using
ethyl acetate : hexane (1:9) as a solvent system. After
completion of the reaction, the reaction mixture was
poured on crushed ice. Ethyl acetate was added to the
mixture, and the organic layer was separated. The aqueous
layer was extracted with 3 × 10 mL of ethyl acetate, and
the combined organic layers were dried over MgSO4. The
solvent was removed under reduced pressure. All the
azides (2a–e) were synthesized from their respective
secondary amines and were obtained as solid compounds.
All the newly synthesized solid azides were crystallized in
ethanol and then used for further reaction. The melting
points of all freshly synthesized azides (2a–e) were in
good agreement with the reported [30].
EXPERIMENTAL
General. All the chemicals were of laboratory grade
and purchased from commercial suppliers Spectrochem,
Rankem, Alfa Aesar, and Sigma Aldrich and were used
without further purification. Purity and completion of
reaction time of all synthesized compounds were
monitored by thin layer chromatography (TLC) using
silica gel 60-F254 precoated on aluminum sheets as an
adsorbent, Merck, Germany, and visualization was accom-
plished by iodine/ultraviolet light. Melting points of all the
synthesized compounds were determined in open capillary
tubes and are uncorrected. The IR spectra (in KBr pellets)
Synthesis of bis(prop-2-yn-1-yloxy)benzene (3a–b).
In a
round bottom flask, resorcinol or catechol (0.01 mol) was
allowed to react with propargyl bromide (0.02 mol) in the
presence of K2CO3 base dissolved in DMF and stirred at
room temperature for 2 h. The progress of the reaction
was monitored by TLC using ethyl acetate : hexane (1:9)
as solvent system. After completion of the reaction, the
reaction mixture was poured on crushed ice. Ethyl acetate
was added to the mixture, and the organic layer was
separated. The aqueous layer was extracted with
3 × 10 mL of ethyl acetate. Solvent was removed under
reduced pressure, and the brownish liquids of 1,3-
bis(prop-2-yn-1-yloxy)benzene (3a) and 1,2-bis(prop-2-
yn-1-yloxy)benzene (3b) were obtained from resorcinol
and catechol, respectively. The melting points were in
1
were recorded on Bruker FT-IR spectrometer. H NMR
spectra were recorded on a Bruker DRX-300 and 300-
MHz NMR spectrometer using tetramethylsilane as an
internal standard and chemical shifts are in δ (ppm). The
splitting pattern abbreviations are designed as singlet (s),
doublet (d), double doublet (dd), triplet (t), quartet (q),
and multiplet (m). 13C NMR spectra were recorded on a
Bruker DRX-75 and 125-MHz NMR in CDCl3/DMSO-
d6. HRMS were obtained using Agilent 6520 (Q-TOF)
ESI-HRMS instrument.
good agreement with the reported [31].
Synthesis of aryloxy-linked substituted dimeric 1,2,3-triazoles
(4a–j). In a round bottom flask, bis(prop-2-yn-1-yloxy)
benzene (3a–b) (0.01 mol) and the newly synthesized
azides (2a–e) (0.02 mol) were allowed to react in the
presence of aqueous solution of copper sulfate
pentahydrate as a catalyst and sodium ascorbate as a
reducing agent, stirred at room temperature in PEG-400
as a solvent for 8 h. The progress of the reaction was
monitored by TLC using ethyl acetate : hexane (2:8) as a
solvent system. After completion of the reaction, the
reaction mixture was poured on crushed ice. The
obtained solid products (4a–j) were filtered, dried, and
crystallized in ethanol-DMF.
2,2′-(4,4′-((1,3-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-morpholinoethanone) (4a).
compound (4a) was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2a) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3a) as
Synthesis of azides (2a–e).
(a) Synthesis 2-chloro-1-substituted ethanone (1a–e). In a
round bottom flask, secondary amines (0.1 mol) and
triethylamine base were dissolved in chloroform and
stirred at 0°C temperature in ice bath. The 2-chloroacetyl
chloride (0.1 mol) was added to the reaction mixture
dropwise with continuous stirring on magnetic stirrer.
After this addition, the ice bath was removed, and the
reaction mixture was stirred at room temperature for 4 h.
The progress of the reaction was monitored by TLC
using ethyl acetate : hexane (1:9) as a solvent system.
After completion of the reaction, the reaction mixture
was poured on crushed ice and neutralized by adding
The
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
white solid; yield: 92%; mp 200–202°C; IR νmax/cmꢀ1
1658.31 (C═O); 1H NMR (400 MHz, DMSO-d6, δH
ppm) 3.60 (dd, 8H, O─CH2, morpholine), 3.74 (dd, 8H,
N─CH2, morpholine), 5.26 (s, 4H, O─CH2), 5.59 (s, 4H,
N─CH2), 6.42 (s, 1H, Ar─H), 6.50 (d, 2H, Ar─H), 7.04
(t, 1H, Ar─H), 8.19 (s, 2H, triazolyl-H); 13C NMR
(100 MHz, CDCl3, δC ppm) 45.3, 51.2, 62.3, 66.5, 115.0,
121.9, 127.0, 133.8, 143.1, 148.5, 165.0; HRMS (ESI)
Calcd for C24H31N8O6 [M + H]+: 527.2367; found:
(s, 4H, N─CH2), 6.34 (d, 2H, Ar─H), 6.60 (s, 1H,
Ar─H), 7.17 (t, 1H, Ar─H), 8.07 (s, 2H, triazolyl-H);
13C NMR (100 MHz, CDCl3, δC ppm) 56.4, 78.8, 79.8,
111.9, 114.9, 122.1, 135.1, 147.4, 162.6, 178.1; HRMS
(ESI) Calcd for C20H27N8O4 [M + H]+: 443.2146; found:
443.2147.
2,2′-(4,4′-((1,2-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-morpholinoethanone)
(4f).
Compound 4f was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2a) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3b) as
white solid; yield: 91%; mp 209–211°C; IR νmax/cmꢀ1
1660.24 (C═O); 1H NMR (400 MHz, DMSO-d6, δH
ppm) 3.49 (dd, 8H, O─CH2, morpholine), 3.61 (dd, 8H,
N─CH2, morpholine), 5.15 (s, 4H, O─CH2), 5.47 (s, 4H,
N─CH2), 6.92 (dd, 2H, Ar─H), 7.18 (dd, 2H, Ar─H),
8.07 (s, 2H, triazolyl-H); 13C NMR (100 MHz,
DMSO-d6, δC ppm) 45.2, 51.1, 62.2, 66.4, 115.0, 121.8,
527.2363.
2,2′-(4,4′-((1,3-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-(pyrrolidin-1-yl)ethanone) (4b).
Compound 4b was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2b) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3a) as
white solid; yield: 90%; mp >270°C; IR νmax/cmꢀ1
1646.01 (C═O); 1H NMR (400 MHz, DMSO-d6,
δH ppm) 1.86 (q, 8H, CH2, pyrrolidine), 1.97 (q, 8H,
N─CH2, pyrrolidine), 5.18 (s, 4H, O─CH2), 5.41 (s, 4H,
N─CH2), 6.65 (s, 1H, Ar─H), 6.73 (d, 2H, Ar─H), 7.26
(t, 1H, Ar─H), 8.16 (s, 2H, triazolyl-H); 13C NMR
(100 MHz, CDCl3, δC ppm) 26.2, 56.1, 70.4, 78.8, 102.5,
108.1, 126.8, 130.6, 142.9, 159.9, 164.3; HRMS (ESI)
Calcd for C24H31N8O4 [M + H]+: 495.2469; found:
126.9, 143.0, 148.4, 165.0.
2,2′-(4,4′-((1,2-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-(pyrrolidin-1-yl)ethanone) (4g).
Compound (4g) was obtained by Cu (I)-catalyzed
1,3-dipolar cycloaddition reaction between newly
synthesized azide (2b) and 1,3-bis(prop-2-yn-1-yloxy)
benzene (3b) as white solid; yield: 92%; mp >270°C;
IR νmax/cmꢀ1 1673.60 (C═O); 1H NMR (400 MHz,
DMSO-d6, δH ppm) 1.81 (q, 8H, CH2, pyrrolidine), 1.94
(q, 8H, N─CH2, pyrrolidine), 5.14 (s, 4H, O─CH2),
5.36 (s, 4H, N─CH2), 6.90 (dd, 2H, Ar─H), 7.02 (dd,
2H, Ar─H), 8.11 (s, 2H, triazolyl-H); 13C NMR
(100 MHz, CDCl3, δC ppm) 24.0, 52.6, 56.3, 78.6,
114.8, 121.9, 122.5, 147.3, 156.4, 174.5; HRMS (ESI)
Calcd for C24H31N8O4 [M + H]+: 495.2469; found:
495.2462.
2,2′-(4,4′-((1,3-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-(piperidin-1-yl)ethanone) (4c).
Compound 4c was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2c) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3a) as
white solid; yield: 92%; mp >270°C; IR νmax/cmꢀ1
1656.42 (C═O); 1H NMR (400 MHz, DMSO-d6, δH
ppm) 1.36 (s, 4H, CH2, piperidine), 2.04 (s, 8H, CH2,
piperidine), 2.92 (s, 8H, N─CH2, piperidine), 5.14 (s,
4H, O─CH2), 5.47 (s, 4H, N─CH2), 6.92 (d, 2H,
Ar─H), 7.03 (s, 1H, Ar─H), 7.43 (t, 1H, Ar─H), 7.98 (s,
2H, triazolyl-H); 13C NMR (100 MHz, CDCl3, δC ppm)
23.2, 26.3, 55.9, 70.3, 78.7, 102.7, 108.1, 124.9, 130.4,
495.2465.
2,2′-(4,4′-((1,2-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(1-(piperidin-1-yl)ethanone) (4h).
Compound 4h was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2c) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3b) as
white solid; yield: 92%; mp >270°C; IR νmax/cmꢀ1
1679.98 (C═O); 1H NMR (400 MHz, DMSO-d6, δH
ppm) 1.39 (s, 4H, CH2, piperidine), 2.09 (s, 8H, CH2,
piperidine), 2.89 (s, 8H, N─CH2, piperidine), 5.15 (s,
4H, O─CH2), 5.51 (s, 4H, N─CH2), 6.90 (dd, 2H,
Ar─H), 7.01 (dd, 2H, Ar─H), 7.99 (s, 2H, triazolyl-H);
13C NMR (100 MHz, CDCl3, δC ppm) 24.5, 27.5, 56.5,
146.3, 158.8, 172.8.
2,2′-(4,4′-((1,3-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(N,N-diethylacetamide)
(4d).
Compound 4d was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2d) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3a) as
white solid; yield: 88%; mp >270°C; 13C NMR
(100 MHz, CDCl3, δC ppm) 21.0, 56.0, 78.8, 79.7, 108.1,
111.8, 130.5, 137.0, 146.1, 158.8, 172.5.
2,2′-(4,4′-((1,3-Phenylenebis(oxy))bis(methylene))bis(1H-1,
78.8, 79.8, 110.7, 114.9, 122.10, 147.2, 165.3, 170.7.
2,2′-(4,4′-((1,2-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(N,N-dimethylacetamide)
(4e).
2,3-triazole-4,1-diyl))bis(N,N-diethylacetamide)
(4i).
Compound 4e was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2e) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3a) as
white solid; yield: 86%; mp >270°C; IR νmax/cmꢀ1
1697.90 (C═O); 1H NMR (400 MHz, DMSO-d6, δH
ppm) 3.46 (s, 12H, N─CH3), 4.72 (s, 4H, O─CH2), 5.11
Compound 4i was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2d) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3b) as
white solid; yield: 87%; mp >270°C; 13C NMR
(100 MHz, CDCl3, δC ppm) 28.2, 56.5, 78.9, 79.9, 115.0,
116.8, 122.1, 147.5, 160.1, 177.2.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Bioevaluation of Dimeric 1,2,3-Triazoles
2,2′-(4,4′-((1,2-Phenylenebis(oxy))bis(methylene))bis(1H-1,
2,3-triazole-4,1-diyl))bis(N,N-dimethylacetamide) (4j).
of free radical production from DPPH was calculated by
the following equation:
Compound 4j was obtained by Cu (I)-catalyzed 1,3-
dipolar cycloaddition reaction between newly synthesized
azide (2e) and 1,3-bis(prop-2-yn-1-yloxy)benzene (3b) as
white solid; yield: 89%; mp >270°C; IR νmax/cmꢀ1
1679.67 (C═O).
ÂÀ
Á À
ÁÃ
%of scavenging ¼
A
control–Asample = Asampleꢁ100
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 the reagents except the test samples.
In vitro Mycobacterium tuberculosis microplate Alamar Blue
Experimental protocol for biological activity.
In vitro antifungal activity.
Antifungal activity was
determined by a standard agar dilution method as per
Clinical & Laboratory Standards Institute (formerly,
National Committee for Clinical Laboratory Standards)
guidelines [32]. These newly synthesized compounds
were screened for their in vitro antifungal activity against
five human pathogenic fungal strains such as C. albicans
(NCIM 3471), F. oxysporum (NCIM 1332), A. flavus
(NCIM 539), A. niger (NCIM 1196), and C. neoformans
(NCIM 576). The synthesized compounds and standard
miconazole were dissolved in DMSO solvent. The
medium yeast nitrogen base was dissolved in phosphate
buffer pH 7, and it was autoclaved at 110°C for 10 min.
With each set, a growth control without the antifungal
agent and a solvent control DMSO were included. The
fungal strains were freshly subcultured onto Sabouraud
dextrose agar and incubated at 25°C for 72 h. The fungal
cells were suspended in sterile distilled water and diluted
to get 105 cells/mL. Ten microliters of standardized
suspension were inoculated onto the control plates and the
media incorporated with the antifungal agents. The
inoculated plates were incubated at 25°C for 48 h. The
readings were taken at the end of 48 and 72 h. The MIC
was at the lowest concentration of drug preventing growth
of macroscopically visible colonies on drug containing
plates when there was visible growth on the drug-free
control plates.
assay.
The antitubercular activity of the newly
synthesized compounds (4a–j) have been screened for
their in vitro effects against Mtb H37Rv (ATCC 27294)
by using microplate Alamar Blue assay [33] for the
determination of MIC in triplicates. The MIC (in μg/mL)
was recorded as the lowest concentration/highest dilution
of the compounds/control drugs that completely
inhibited the growth of MTB cultures. The MIC values
of the compounds (4a–j) have been compared with
standard drugs (rifampicin, isoniazid, ethambutol, and
ciprofoxacin). The experimental method for antitubercular
activity is briefly described as follows.
Initially, the inoculum was prepared from fresh LJ
medium re-suspended in 7H9-S medium (7H9 broth, 0.1%
casitone, 0.5% glycerol, supplemented oleic acid, albumin,
dextrose, and catalase [OADC]), adjusted to a McFarland
tube No. 1 and diluted 1:20; 100 μL was used as
inoculum. Each drug stock solution was thawed and
diluted in 7H9-S at fourfold the final highest concentration
tested. Serial twofold dilutions of each drug were prepared
directly in a sterile 96-well microtiter plate using 100-μL
7H9-S. A growth control containing no antibiotic and a
sterile control were also prepared on each plate. Sterile
water was added to all perimeter wells to avoid
evaporation during the incubation. The plate was covered,
sealed in plastic bags, and incubated at 37°C in normal
atmosphere. After 7-day incubation, 30 μL of Alamar
Blue solution was added to each well, and the plate was
re-incubated overnight. A change in color from blue
(oxidized state) to pink (reduced) indicated the growth of
bacteria, and the MIC was defined as the lowest
concentration of drug that prevented this change in color.
General procedure for (evaluation of 1,1-diphenyl-2-
picrylhydrazyl radical scavenging) antioxidant activity.
The antioxidant activity of all the synthesized compounds
have been assessed in vitro by the DPPH radical
scavenging assay [26], and the results were compared with
the standard synthetic antioxidant BHT. The hydrogen
atom or electron donation ability of the compounds was
measured from the bleaching of the purple colored
methanol solution of DPPH. The spectrophotometric assay
uses the stable radical DPPH as a reagent; 1 mL of various
concentrations of the test compounds (5, 10, 25, 50, and
100 mg/mL) in methanol was added to 4 mL of 0.004%
(w/v) methanol 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 entered. The percent of inhibition (I %)
Molecular docking study. Molecular docking study has
been performed to investigate the binding affinity of the
new aryloxy-linked dimeric 1,2,3-triazoles (4a–4j) into
the active site of an antifungal target sterol 14α-
demethylase (CYP51) using the standard protocol
implemented in the GLIDE (Grid-based LIgand Docking
with Energetics) program integrated in the Schrödinger
Molecular modeling package (Schrödinger, LLC, New
York, NY, 2015) [39]. The X-ray crystal structure of
sterol 14α-demethylase (CYP51) enzyme co-crystallized
with its inhibitor-fluconazole with the pdb accession
code: 3KHM was retrieved from the Protein Data Bank
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

T. R. Deshmukh, S. P. Khare, V. S. Krishna, D. Sriram, J. N. Sangshetti, O. Bhusnure,
V. M. Khedkar, and B. B. Shingate
Vol 000
(http: //www.rcsb. org/pdb). The protein inhibitor complex
was pre-processed with the protein preparation wizard by
applying the OPLS-2005 force field. This involved
eliminating the crystallographic water molecules (as they
did not participate in any kind of interaction with the
enzyme), adding the missing hydrogens/side chain atoms
corresponding to pH 7.0, assignment of appropriate
charge and protonation state and finally energy
minimization of the obtained structure to relieve the steric
clashes among the amino acid residues until Root Mean
Square Deviation (RMSD) value of 0.30 Å is reached for
the heavy atoms. The 3D structures of the title
compounds in the series (4a–4j) were sketched through
the build panel in Maestro visualizer and then optimized
using LigPrep module which does the addition of
hydrogens, adjusting realistic bond angles and lengths,
and so forth. The partial charges were assigned to the
structure using the OPLS-2005 force field, and finally,
each structure was subjected to energy minimization until
its average RMSD reached 0.001 Å. Next, with Receptor
Grid Generation panel, the shape and properties of the
active site of the CYP51 enzyme was defined for which a
grid box of 10 × 10 × 10 Å dimensions centered on the
centroid of the co-crystallized ligand was generated.
Here, the co-crystallized ligand serves as the reference
coordinate to define the active site for the molecules to be
docked. With this automatic setup, molecular docking
was executed to gauze the binding affinities of the
synthesized compounds (4a–j) against CYP51 by using
the extra precision Glide scoring function. The docking
poses generated at the end of simulation were visualized
and analyzed for the mode of binding and to identify the
most significant bonded and non-bonded interactions with
the residues lining the active site using the Maestro’s
Pose Viewer utility.
Among these hybrids, 4h, 4i, and 4j have displayed
significant antitubercular activity with MIC 12.5, 6.25,
and 12.5 μg/mL, respectively. Furthermore, antifungal
activity of the newly synthesized analogs was also
evaluated against five human pathogenic fungal strains.
In addition to this, all the compounds were also
examined for antioxidant activity, and DPPH radical
scavenging assay revealed that compounds 4a, 4b, 4c, 4i,
and 4j possess more potent antioxidant activity than
BHT. So it can be concluded from the aforementioned
observations that aryloxy-linked substituted dimeric
1,2,3-triazole hybrids offer an attractive lead series for
the discovery of novel antitubercular, antifungal, and
antioxidant agents.
Acknowledgments. One of the authors T. R. D. is very much
grateful to Dr. Babasaheb Ambedkar, Marathwada University,
Aurangabad for the award of University Scholars Fellowship.
Authors are also thankful to the Head of the Department of
Chemistry, Dr. Babasaheb Ambedkar, Marathwada University,
Aurangabad 431 004, India, for providing laboratory facility.
We are also grateful to Schrodinger Inc. for GLIDE software to
perform the molecular docking studies.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet