Click chemistry based regioselective one-pot synthesis of coumarin-3-yl-methyl-1,2,3-triazolyl-1,2,4-triazol-3(4H)-ones as newer potent antitubercular agents

Article abstract of DOI:10.1002/ardp.201900013

Coumarin-3-yl-methyl-1,2,3-triazolyl-1,2,4-triazol-3(4H)-ones (8k-z) were synthesized via copper(I)-catalyzed azide-alkyne cycloaddition click chemistry. The synthesized hybrid molecules were characterized by spectral studies. Compounds 8k-z were screened for their in vitro anti-TB activity by using the Microplate Alamar Blue assay and for cytotoxicity using the MTT assay. Some of the compounds were found to be most potent against the tested Mycobacterium tuberculosis H37Rv strain with a MIC of 1.60 μg/ml. Further, docking the compounds into the InhA binding pocket showed strong binding interactions and effective overall docking scores were recorded. The drug-likeness and toxicity studies were computed using Molinspiration and Protox, respectively.

Full text of DOI:10.1002/ardp.201900013

Received: 12 January 2019
Revised: 28 June 2019
Accepted: 5 July 2019
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DOI: 10.1002/ardp.201900013
F U L L P A P E R
Click chemistry based regioselective one‐pot synthesis
of coumarin‐3‐yl‐methyl‐1,2,3‐triazolyl‐1,2,4‐
triazol‐3(4H)‐ones as newer potent antitubercular agents
Shilpa M. Somagond1
Saba Kauser J. Shaikh1
Mahadevappa Y. Kariduraganavar1
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Ravindra R. Kamble1
Shrinivas D. Joshi2
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Praveen K. Bayannavar1
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Vijay M. Kumbar3
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Aravind R. Nesaragi1
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1Department of Studies in Chemistry, Karnatak
Abstract
University Dharwad, Karnataka, India
2Novel Drug Design and Discovery
Laboratory, Department of Pharmaceutical
Chemistry, S.E.T.’s College of Pharmacy,
Dharwad, Karnataka, India
Coumarin‐3‐yl‐methyl‐1,2,3‐triazolyl‐1,2,4‐triazol‐3(4H)‐ones (8k‒z) were synthe-
sized via copper(I)‐catalyzed azide‒alkyne cycloaddition click chemistry. The
synthesized hybrid molecules were characterized by spectral studies. Compounds
8k‒z were screened for their in vitro anti‐TB activity by using the Microplate Alamar
Blue assay and for cytotoxicity using the MTT assay. Some of the compounds were
found to be most potent against the tested Mycobacterium tuberculosis H37Rv strain
with a MIC of 1.60 µg/ml. Further, docking the compounds into the InhA binding
pocket showed strong binding interactions and effective overall docking scores were
recorded. The drug‐likeness and toxicity studies were computed using Molinspiration
and Protox, respectively.
3Dr. Prabhakar Kore Basic Science Research
Center, KLE University, Belagavi, Karnataka,
India
Correspondence
Dr. Ravindra R. Kamble, Department of
Studies in Chemistry, Karnatak University
Dharwad, Karnataka 580003, India.
Email: kamchem9@gmail.com
Funding information
University Grants Commission (UGC)
“Antitumor activity an integrated Approach”
a focused area of UPE FAR‐I programme,
Grant/Award Number: F. No. 14–3/2012
(NS/PE) Dated: 14–03‐2012
K E Y W O R D S
2H‐chromen‐2‐one, antitubercular activity, click reaction, cytotoxicity, drug likeness, molecular
docking, toxicity prediction
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1
INTRODUCTION
prominent successes. Therefore, to understand these mirrors of success
that proceed towards in other antibacterial drug discovery programs[4]
Tuberculosis (TB) is a lung disease generally caused by Mycobacterium
tuberculosis (Mtb), which leads to the death of almost 3 million people
every year and it is found to be a prominent bacterial infectious
agent.[1,2] The rising drug resistance and poor activity of accessible
therapies towards the concealed stage of Mtb infection have created a
clear need to develop new medicine to treat TB.[3] Thus, fast‐acting
medicines with different mechanisms of action that are not cross defiant
to the existing drugs are being keenly preferred. To overcome this
drawback two initial screening methods were being applied in TB drug
discovery‐target. First, high throughput screening and makeup minimum
inhibitory concentration (MIC) based on the screening of entire cell
bacteria. Second, the enzyme‐based high throughput screening for novel
TB drugs has been generally adopted; this methodology has not formed
Presently, there is an overwhelming call to develop new
structural categories of antitubercular agents that permit shorter
and additional effective therapies. Triazoles are shown to possess a
variety of fascinating features within the framework of medicinal
chemistry. They are stable to acidic/basic reaction and conjointly
reductive/oxidative conditions, investigative of
a high aromatic
stabilization and this scaffold is comparatively resistant to metabolic
degradation. The best‐known examples of triazole containing
structure are tazobactam, a β‐lactamase inhibitor with the broad‐
spectrum antibiotic piperacillin.[5–7] And also several 1,2,3‐triazole
analogs 1–5 (Figure 1)[8–11] have been reported with antitubercular
activity. In addition, 1,2,3‐triazoles are found in herbicides, fungi-
cides, and dyes.[12]
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Arch Pharm Chem Life Sci. 2019;e1900013
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900013

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FIGURE 1 Tuberculostatic 1,2,3‐triazoles 1‒5
1,2,3‐Triazoles have been of interest to medicinal chemists as
Sharpless initially introduced the concept of click chemistry, by that
the Huisgen’s [2+3] cycloaddition reaction of organic azides and
terminal alkynes will be efficiently catalyzed by copper(I) ions and
may be performed at ambient temperature, which results in the
formation of single product viz., 1,4‐regioisomer of 1,2,3‐tria-
zole.[13–16] Copper(I) catalyzed chemical action of this transformation
accelerates the speed of reaction up to 107 times and hence has
placed it in a class of its own and has facilitated many novel
applications.[17–22]
pharmacophore in their elemental structures which proves the
antifungal potencies of the 1,2,4‐triazole template so that it
expresses significant antimicrobial activity.[40] Several 1,2,4‐triazole
derivatives have been screened for incising novel promising anti‐TB
candidates. Some 1,2,4‐triazole appended indole hybrids 8[41] with
substituted phenyl ring at 2nd position of indole and 1,2,4‐triazole
derivatized benzthiazole 9[42] have exhibited anti‐TB activity against
MTB H37Rv (Figure 3; MIC: 0.5 mg/ml). Hence, we were also
prompted to work on the preparation of 1,2,4‐triazole using
N‐arylsydnone which belongs to mesoionic class of compounds.
N‐Arylsydnone act as functional and novel precursor for the
synthesis of various biologically active heterocycles viz., pyrazoles,
1,3,4‐oxadiazoles, phenyl indazoles, pyrazolines, and tetrazines via
1,3‐dipolar cycloaddition and addition elimination reactions.[43]
In addition, to explore the mechanism of action of title
compounds 8k‒z and provide guidance for new drug design, SAR
analysis (Figure 4) of the reported molecules was carried out which
revealed the following information.
Coumarin (2H‐chromen‐2‐one), an oxygen heterocycle present in
the plants, which consists of various phenolic substances and is
composed of combined benzene and 2‐pyrone ring systems. Thirteen
hundred coumarins are distinctive as secondary metabolites from
plants, microorganisms, and fungi.[23,24] Coumarin and its derivatives
have diverse pharmacological activities such as antibacterial,[25]
antifungal,[26] anticoagulant,[27] antihypertensive,[28] anti‐HIV,[29,30]
antioxidant,[31] scavenging reactive oxygen species,[32] antitubercu-
losis,[33] anticonvulsant,[34] antihyperglycemic,[35] anticancer,[36] and
anti‐inflammatory.[37] Thus, the synthesis of coumarin and its analogs
has established growing attention to synthetic organic chemists as
well as biologists. 1,4‐Disubstituted bis‐chromenyl 1,2,3‐triazole
hybrids 6 and mono‐chromenyl 1,2,3‐triazole hybrids 7 (Figure 2)
have been synthesized and studied in vitro for anti‐TB activities
against M. tuberculosis H37Rv (M.tb).[38]
(i) 1,2,4‐Triazole nucleus displayed promising anti‐TB potency
against drug‐sensitive and DR‐TB including MDR‐TB, warrant
further appraisal as a novel agent to control TB in humans.[44,45]
The aromatic substitutions are preferred; the substitution at the
4th position of the aromatic substituent follow the trend
‒OCH3 > ‒H.
1,2,4‐Triazole derivatives have received great attention during
the past two decades as prospective antimicrobial agents.[39] Also,
most of the existing antifungal drugs hold 1,2,4‐triazole
(ii) 1,2,3‐Triazole as a linker to combine different anti‐TB active
skeleton[46] suggesting that the presence of 1,2,3‐triazole moiety
extremely improves the efficiency.
FIGURE 2 Chemical structures of
coumarin 1,2,3‐triazole hybrids as anti‐TB
agents

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FIGURE 3 1,2,4‐Triazole nucleus
containing potent antitubercular agents
(iii) It is evident from the SAR study that the bioactive skeleton
having the aforementioned substitution at positions 6 and 7 and
halogen (Br, Cl) at C‐6 position of the coumarin core found
responsible for the potent activity compared to methoxy,
methyl, benzo‐substitutions (Figure 4).[47]
and toxicity studies were carried out computationally to get an
insight into the structural parameters leading to biological activity.
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2
RESULTS AND DISCUSSION
Synthesis
Moreover, a recent inclination in medicinal chemistry illustrates
the recognition of molecular hybridization for drug design and
development. This has supported combining pharmacophoric units of
various bioactive substances to form a new hybrid molecule with
increased effects evaluated in comparison to the parent drug.[48–50]
Thus, in the present study new hybrid molecules are reported which
are formed by the combination of three different pharmacophoric
units such as coumarin, 1,2,3‐triazole, and 1,2,4‐triazole. Further a
library of coumarin‐3‐yl‐methyl‐1,2,3‐triazolyl‐1,2,4‐triazol‐3(4H)‐
ones 8k‒z hybrids were synthesized by click chemistry approach.
The structures of these hybrid molecules were characterized by FT‐
IR, 1H NMR, 13C NMR, mass spectral data and elemental analysis. In
vitro antitubercular activity of title compounds have been carried out
against pathogenic strain M. tuberculosis H37Rv strain and also
carried out the cytotoxicity evaluation. The computational study
(molecular docking) revealed the competent interaction of hybrid
molecules with inhibition of enzyme InhA‐D148G which assisted in
parallel to in vitro anti‐TB activity. Moreover, in silico pharmacokinetic
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2.1
Acetylenic dipolarophile 4a,b was prepared by the reaction of
substituted 2‐aryl‐2H‐1,2,4‐triazol‐3(4H)‐one 3a,b with 3‐bromo-
prop‐1‐yne employing potassium carbonate in anhydrous acetone
(Scheme 1). The ethyl‐4‐bromoacetoacetate was synthesized from
bromination of ethylacetoacetate in dry ether at 0–5°C. Under
Pechmann cyclisation, it was reacted with various substituted
phenols in presence of conc. sulfuric acid as cyclising agent.[50]
This reaction ends up with the formation of required synthon
4‐bromomethyl coumarin 6c‒j (Scheme 2). The corresponding dipolar
azide 7c–j was obtained by treating 6c‒j with sodium azide (NaN3) in
aqueous acetone at room temperature. This was followed by azide‐
alkyne cycloaddition of the acetylenic dipolarophiles 4a,b and
4‐azidomethyl coumarins 7c‒j in presence of copper ascorbate
(generated in situ) in THF/water, 1:1 at room temperature to get
exclusively one product, that is, coumarinyl‐1,2,3‐triazolyl‐1,2,4‐
triazol‐3(4H)‐ones 8k‒z in excellent yields and purity (Scheme 2).
FIGURE 4 Rational molecular hybridization design

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SCHEME 1 Synthesis of acetylenic
dipolarophile 4a,b
3a,b
1a,b
2a,b
4a,b
:
:
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2.2
Spectral characterization
for CH2 protons attached to 1,2,4‐triazolin ring and coumarin moiety
at 5.00–5.99 ppm and 4.93–5.08 ppm, respectively. The C5‐H of
1,2,3‐triazolin and C5‐H of 1,2,4‐triazolin ring appeared as a singlet in
the range 7.70–8.46 ppm and 7.78–8.51 ppm, respectively. The C3‐H
of coumarin resonated as a singlet in the range 5.95–6.49 ppm. 13C
NMR spectral analyses showed the number of signals equivalent to
the magnetically non‐equivalent carbon atoms. Also, the mass
spectral analyses showed the molecular ion peak corresponding to
the molecular mass of title compounds.
Formation of coumarinyl‐1,2,3‐triazolyl‐1,2,4‐triazol‐3(4H)‐ones 8k‒z
is supported by spectroscopic studies viz., FT‐IR, 1H NMR, 13C NMR
and MS. FT‐IR spectral analysis of compounds 8k‒z showed a sharp
band in the range 1,710–1,730 cm−1 due to the carbonyl stretching of
coumarin moiety. Another sharp band in the range 1,690–1,715 cm−1
and
a ,
medium intense band in the range 1,640–1,610 cm−1
respectively correspond to the C=O and C=N stretching of 1,2,4‐
triazolin‐3‐one ring. 1H NMR spectral analysis showed two singlets
5c–j
6c–j
4a,b
7c–j
8k–z
5,7-di
SCHEME 2 Schematic representation for the synthesis of coumarinyl‐1,2,3‐triazolyl‐1,2,4‐triazol‐3(4H)‐on 8k–z

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TABLE 1 In vitro antitubercular evaluation of MIC (µg/ml) of title
compounds 8k‒z
coumarin, p‐methoxy substitution at C1 position of the 1,2,4‐
triazole), 8w (methyl at C7 position of the coumarin, p‐methoxy at
C1 position of the 1,2,4‐triazole), 8x (benzo at C5 and C6 position of
the coumarin, p‐methoxy phenyl at C1 position of the 1,2,4‐triazole)
showed excellent activity having MIC as low as 1.6 µg/ml which
proved better than the standard drug pyrazinamide having MIC of
3.12 µg/ml against M. tuberculosis H37Rv. Compounds 8k (methyl at
C7 position of the coumarin), 8m (methyoxy at C7 position of the
coumarin), 8n (t‐butyl at C7 position of the coumarin), 8u (methoxy at
C6 position of the coumarin, p‐methoxy phenyl at C1 position of the
1,2,4‐triazole) were more active as that of pyrazinamide with MIC of
3.12 µg/ml. Compounds 8l, 8r, 8q, 8y, and 8z showed good to
moderate activity with MIC of 6.25–12.5 µg/ml.
Entry Ar
R
MIC (µg/ml) IC50 (µg/ml) SI
8k
8l
Phenyl
6‐CH3
6‐Cl
3.12
6.25
3.12
3.12
1.60
1.60
6.25
477.40
291.20
127.60
111.10
769.30
072.13
228.10
292.80
1087.00
1438.00
125.20
017.81
114.70
078.54
053.21
493.00
ND
153.01
046.59
040.90
035.61
480.81
045.08
036.50
023.42
679.38
898.75
040.13
011.13
071.69
049.09
008.51
039.44
ND
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
8m
8n
8o
8p
8q
8r
6‐OCH3
6‐t‐Butyl
7‐CH3
5,6‐Benzo
7,8‐Benzo
The electron‐donating groups on coumarin and the phenyl ring
attached to 1,2,4‐triazole were generally observed to enhance
the antitubercular activity (viz., 8o, 8p, 8s, 8t, 8v, 8w, and 8x)
with MIC 1.6 µg/ml. It is well known from the literature that the
existence of these groups imparts a variety of properties including
metabolic stability, steric, electronic properties, enhanced binding
interactions, changes in physical properties and selective
reactivates.[51]
5,7‐Di‐CH3 12.5
8s
p‐Anisyl 6‐CH3
p‐Anisyl 6‐Cl
1.60
1.60
3.12
1.60
1.60
1.60
6.25
8t
8u
8v
8w
8x
8y
8z
Std
p‐Anisyl 6‐OCH3
p‐Anisyl 6‐t‐Butyl
p‐Anisyl 7‐CH3
p‐Anisyl 5,6‐Benzo
p‐Anisyl 7,8‐Benzo
The cytotoxicity results (Table 1), expressed as the IC50 for the
HEK293 (human embryonic kidney) cell line explain that all of the
tested compounds 8k‒z showed very low cytotoxicity (IC50 value
ranging from 53.21 to 898.75 µg/ml). Pavan et al. reported that
isoniazid and rifampicin, first‐line drugs in current TB treatment,
had IC50 values of 1250 and 156.3 µg/ml, respectively.[52] The
selectivity index (SI) of each compound was calculated by dividing
IC50 by the MIC (Table 1). According to Orme et al.[53] candidates
for new drugs must have a selective index equal or higher than 10,
with MIC lower than 6.25 µg/ml and low cytotoxicity. SI is used to
estimate the therapeutic window of a drug and to identify drug
candidates for further studies. Thus, in this study, it has been
identified that compounds 8k, 8o, and 8s, with SI values of 153.01,
480.81, and 679.38, respectively as very promising new TB drug
candidates. The SI of compound 8t, for example, was 898.75 and
this index is comparable to the SI of rifampicin (MIC = 0.2 µg/ml,
IC50 = 156.3 µg/ml: SI = 781[54]), which is used together with
isoniazid to treat TB.
p‐Anisyl 5,7‐Di‐CH3 12.5
3.12
–
–
Note: Std, pyrazinamide.
Abbreviation: MIC, minimum inhibitory concentration.
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2.3
Pharmacological evaluation
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2.3.1
In vitro antitubercular activity
In view of the intermolecular interactions and hydrogen binding
modes of newly synthesized coumarinyl‐1,2,3‐triazolyl‐1,2,4‐triazol‐
3(4H)‐ones 8k‒z with InhA‐D148G mutant in complex with NADH
(PDB: 4DQU) by docking simulation, the in vitro antitubercular
activity was evaluated against M. tuberculosis H37Rv strain in eight
different concentrations by using a Microplate Alamar Blue assay
(MABA) method with pyrazinamide as standard. Results for the
tested compounds are tabulated in Table 1, as MIC and the activity
ranged from 12.5 to 1.6 µg/ml. The MIC of the tested compounds
were compared with standard drugs which indicated that almost all
the newly synthesized compounds showed lower MIC values with
potent inhibitory activity against M. tuberculosis H37Rv strain.
The antitubercular activity revealed that compounds 8o (methyl
at C7 position of the coumarin), 8p (benzo at C5 and C6 position of
the coumarin), 8s (methyl at C6 position of the coumarin,
p‐methoxyphenyl at C1 position of the 1,2,4‐triazole), 8t (chloro at
C6 position of the coumarin, p‐methoxy phenyl substitution at C1
position of the 1,2,4‐triazole), 8v (t‐butyl at C6 position of the
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2.3.2
Molecular docking studies
To investigate the detailed intermolecular interactions between the
ligand and the target protein, Surflex‐Dock program was used.
Docking studies give a fair idea related to drug–receptor interactions.
Three‐dimensional structure information on the target protein was
taken from the PDB entry 4DQU. The processing of the protein
integrated the removal of the ligand and the solvent molecules as
well as the addition of hydrogen atoms. All the 16 inhibitors were
docked into the active site of enzyme as shown in Figure 5 to
compare with the in vitro antitubercular studies. The predicted
binding energies of the compounds are listed in Table 2.

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FIGURE 5 Docked view of all the title compounds at the active site of the enzyme InhA‐D148G
As depicted in Figure 6, the compound 8x makes four hydrogen‐
bonding interactions at the active site of the enzyme InhA‐D148G
(PDB ID: 4DQU), among them two bonding interactions were raised
from 1st nitrogen atom of triazole ring with hydrogens of ARG43 (–N
—H‐ARG43, 2.18 & 2.36 Å), 2nd nitrogen atom of 1,2,3‐triazole ring
makes a bonding interaction with hydrogen of ARG43 (–N—H‐
ARG43, 2.83 Å), oxygen atom of carbonyl group present on the 1,2,4‐
triazole ring makes a bonding interaction with hydrogen of ASP42
(–C = O—H‐ASP42, 2.32 Å) and remaining one interaction came from
the oxygen atom of methoxy group present on the phenyl ring
attached to 1,2,4‐triazole with hydrogen atom of GLY14 (–O—H‐
GLY14, 1.80 Å) amino acid.
As depicted in Figure 7, the compound 8t makes six hydrogen
bonding interactions at the active site of the enzyme InhA‐D148G
(PDB ID: 4DQU), among them one bonding interaction was raised
from 1st nitrogen atom of 1,2,3‐triazole ring with hydrogen of GLY96
(–N—H‐GLY96, 2.30 Å), 3rd nitrogen atom of 1,2,4‐triazole ring
makes a bonding interaction with hydrogen of SER20 (–N—H‐SER20,
TABLE 2 Surflex docking score (kcal/mol) of the title compounds 8k‒z on enzyme InhA‐D148G (PDB ID: 4DQU)
Code
8k
8l
C scorea
7.11
7.60
6.66
7.41
8.10
8.07
7.70
7.12
6.58
8.97
7.46
8.60
8.68
9.98
8.09
6.98
Crash scoreb
−2.14
−2.04
−2.05
−1.30
−1.77
−0.90
−1.58
−2.50
−1.42
−2.99
−4.95
−1.92
−0.97
−2.52
−1.12
−2.76
Polar scorec
D scored
−141.56
−137.31
−141.94
−145.63
−137.75
−129.32
−147.40
−145.62
−143.49
−172.77
−187.92
−148.45
−144.16
−161.38
−139.32
−143.09
PMF scoree
G scoref
−274.01
−248.09
−266.42
−269.18
−249.35
−207.57
−268.49
−228.70
−237.07
−308.86
−358.21
−324.49
−230.02
−341.01
−244.81
−264.18
Chem scoreg
−29.59
−33.75
−29.10
−30.69
−33.00
−36.22
−31.63
−32.89
−32.10
−38.23
−39.37
−35.50
−31.87
−41.62
−30.45
−30.24
0.95
−6.79
3.58
−12.87
−10.34
−11.70
−6.99
8m
8n
8o
8p
8q
8r
1.02
1.34
3.30
3.25
−37.31
−8.62
0.81
2.50
−6.91
8s
1.89
−23.38
−18.73
13.34
8t
3.19
8u
8v
8w
8x
8y
8z
2.10
1.85
−75.58
−13.98
−35.28
−19.94
−2.12
2.44
1.88
1.02
1.60
aC score (consensus score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and
reports the output of total score.
bCrash‐score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate penetration.
cPolar indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make no
hydrogen bonds.
dD‐score for charge and the van der Waals interactions between the protein and the ligand.
ePMF‐score indicating the Helmholtz free energies of interactions for protein–ligand atom pairs (Potential of Mean Force, PMF).
fG‐score showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies.
gChem‐score points for H‐bonding, lipophilic contact, and rotational entropy, along with an intercept term.

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FIGURE 6 Docked view of compound 8x at the active site of the enzyme InhA‐D148G
FIGURE 7 Docked view of compound 8t at the active site of the enzyme InhA‐D148G

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FIGURE 8 (a) Hydrophobic amino acids surrounding compounds 8x (green color) and 8t (cyan color). (b) Hydrophilic amino acids
surrounding compounds 8x and 8t
2.78 Å), oxygen atom of carbonyl group present on the coumarin ring
makes a bonding interaction with hydrogen of LYS118 (–C = O—H‐
LYS118, 2.50 Å), oxygen atom present in the coumarin ring makes a
bonding interaction with hydrogen of LYS118 (–O—H‐LYS118,
2.18 Å) and oxygen atom of carbonyl group present on the 1,2,4‐
triazole ring makes a bonding interaction with hydrogen of GLY96
(–C = O—H‐ASP42, 2.21 Å).
formulating the rule of five such as lipophilicity (Log P), molecular
weight (MW), number of hydrogen bond donors (donorHB) and
acceptors (accptHB), molecular volume (A)3, number of rotatable
bonds (NROTB). According to Lipinski, a compound is more likely to
be membrane permeable and easily absorbed by the body if it
matches the criteria such as (a) MW of compound is less than 500. (b)
Its lipophilicity (Log P) is less than 5. (c) The number of groups in the
molecule that can accept hydrogen atoms to form hydrogen bonds
(HBA) is less than 10. (d) The number of groups in the molecule that
can donate hydrogen atoms to hydrogen bonds (HBD) is less than 5.
Molinspiration is also used for calculation of Lipiniski’s violation. In
addition, it estimates Topological Polar Surface Area (TPSA; Å2). The
values of TPSA are used to estimate the percentage of oral
absorption (%ABS) by the formula: %ABS = 109 – 0.345TPSA.
Figure 8a,b represents the hydrophobic and hydrophilic amino
acids surrounding the compounds 8x and 8t.
All docked compounds 8k–z showed consensus score (C‐Score) in
the range 9.98–6.58 indicating the summary of all forces of
interaction between ligands and the InhA‐D148G. Charge and the
Van der Waals interactions between protein and ligands varied from
−129.32 to −187.92. The Helmholtz free energies of interactions for
protein ligands atom pairs range between −2.12 and −75.58.
However, its H‐bonding, complex (ligand‐protein), and internal
(ligand‐ligand) energies range from −207.57 to −341.01, while those
values −29.10 to −41.82 designate the ligands due to H‐bonding,
lipophilic contact, and rotational entropy, as well as intercept terms
(Table 2).
Theoretical calculation of pharmacokinetic factors (LogP, MW,
TPSA, %ABS, number of hydrogen donors and acceptors, NROTB,
volume) of all new molecules 8k‒z was carried out and entered in
Table 3.[56,57] From the data presented in Table 3, it was remarkable
that all the described title compounds possess LogP values
compatible with those required to cross membranes. TPSA, described
to be a predictive indicator of membrane penetration, was also found
to be positive. In addition, it can be observed from the table that no
violations of Lipinski’s rule (MW, LogP, number of hydrogen donors
and acceptors) were found, but only one molecule 8u is in a slight
violation regarding (accptHB < 10). This indicates a good predicted
oral bioavailability for most of the synthesized molecules.
Generally, it was observed that methoxy (OCH3) and carbonyl
(C=O) groups and more electronegative atoms present in the
molecules form the H‐bond with a substrate‐binding site of the
target enzyme and presence of electron‐donating or withdrawing and
halogen substitution on the aromatic ring attached to coumarin and
1,2,4‐triazole moiety may favor the activity, while those of coumarin,
1,2,3‐triazole, 1,2,4‐triazole and phenyl moieties help to occupy or
penetrate the molecule at the active sites.
|
2.3.4
Toxicity prediction
Toxicity prediction of a newer molecule is an important part of the
drug design and development. Computational toxicity findings are
not solely faster than the determination of toxic doses in animals,
however, they can even facilitate to reduce the number of animal
experiments. A web server Protox[58] estimates rodent oral toxicity,
which is derived from compounds of known drug candidates and
|
2.3.3
In silico pharmacokinetics evaluations
We have used Molinspiration[55] property calculation program to
evaluate whether the synthesized hybrid molecules own the correct
pharmacokinetic parameters to exhibit drug‐likeness or not. This
server predicts molecular descriptors which were used by Lipinski in

SOMAGOND ET AL.
9 of 13
|
TABLE 3 In silico pharmacokinetics data of the title compounds 8k–z as predicted by Molinspiration
Code
8k
8l
LogPa
2.22
2.45
1.83
3.48
2.22
2.95
2.95
2.60
2.27
2.50
1.88
3.53
2.27
3.01
3.01
2.65
M.Wt (<500 amu)b
414.43
434.84
430.42
456.51
414.43
450.46
450.46
428.45
444.45
464.87
460.45
486.53
444.45
480.48
480.48
458.48
accptHB (<10)c
donorHB (<5)d
Lipinsk’s violationd
TPSAe
100.76
100.76
109.99
100.76
100.76
100.76
100.76
100.76
109.99
109.99
119.23
109.99
109.99
109.99
109.99
109.99
Volumes (A)3f
355.59
352.57
364.58
405.22
355.59
383.02
383.02
372.15
381.14
378.11
390.1
NROTBg
%ABSh
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
5
5
6
6
5
5
5
5
6
6
7
7
6
6
6
6
74.23
74.23
71.05
74.23
74.23
74.23
74.23
74.23
71.05
71.05
67.86
71.05
71.05
71.05
71.05
71.05
9
8m
8n
8o
8p
8q
8r
10
9
9
9
9
9
8s
10
10
11
10
10
10
10
10
8t
8u
8v
8w
8x
8y
8z
430.76
381.14
408.57
408.57
397.70
aLogP: logarithm of compound partition coefficient between n‐octanol and water.
bM.Wt: molecular weight.
caccptHB: number of hydrogen bond acceptors.
ddonorHB: number of hydrogen bond donors.
eTPSA: topological polar surface area.
fVolume.
g%ABS: percentage of absorption.
hNROTB: number of rotatable bonds.
their toxicity by their toxic fragments or chemical structure. The
online server Protox compares the similarity of structures of
synthesized molceules which will be loaded in a server with info of
molecules having antecedently proverbial toxicity and identifies the
toxic fragments of loaded molecules and probable toxicity targets.
Toxic doses are often known as LD50 values in mg per kg body
weight. The LD50 is the median lethal dose, meaning the dose at
which 50% of test subjects die upon exposure to a compound. The
predicted LD50 (lethal dose) for our target molecules is above
300 mg/kg and less than 1,000 mg/kg for all the tested compounds as
shown in Table 4.
analysis and screened for their anti‐TB activity. The in vitro study of
synthesized hybrids 8k–z yielded some potent entities (viz., 8o, 8p,
8s, 8t, 8v, 8w, and 8x) owning their potency in micromolar range
against M. tuberculosis H37Rv (MTB; ATCC‐27294) strain. From the
cytotoxicity study, we found that compounds 8o, 8s, and 8t, with SI
values equal or higher than 153, being more active and less toxic
than some drugs used in the treatment of TB. Molecular docking
study of all hybrid molecules 8k‒z against InhA as a targeted enzyme
for antimycobacterial activity studies was performed to learn binding
pattern of the active motifs. Compounds 8t and 8x were found to
show good binding affinity with the active binding pocket of the InhA
enzyme with the highest C score value. The obtained docking results
corroborate the experimental findings. Further, the results of
pharmacokinetic data and toxicity prediction study provide a con-
firmation to consider the active molecules as lead targets for further
progress in the drug discovery process.
The newly synthesized molecules 8k‒z come under the toxicity
category of Class IV and there are no toxic fragments present as
claimed by the developer’s limits. This toxicity prediction study
reviews that synthesized molecules can act as the lead molecules for
further analysis (Table 4).
|
|
3
CONCLUSIONS
4
EXPERIMENTAL
Chemistry
|
The regioselectivity associated with Cu(I) catalyzed azide–alkyne
cycloaddition by click reaction under environmentally benign condi-
tions has been confirmed using coumarinyl azides and acetylenic
dipolarophile (prepared from sydnone) to get the title compounds
8k–z. The synthesized compounds were characterized by spectral
4.1
|
4.1.1
General
All starting materials and reagents were of analytical grade and were
obtained from commercial suppliers and were used without further

10 of 13
SOMAGOND ET AL.
|
TABLE 4 Toxicity prediction parameters of compounds 8k‒z
Code
8k
8l
Predicted LD50 (mg/kg)
Predicted toxicity class
Average similarity (%)
42.94
Prediction accuracy (%)
54.26
Toxic fragments
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
42.82
54.26
8m
8n
8o
8p
8q
8r
42.34
54.26
43.24
54.26
43.13
54.26
43.51
54.26
43.51
54.26
42.57
54.26
8s
42.76
54.26
8t
42.23
54.26
8u
8v
8w
8x
8y
8z
42.40
54.26
43.06
54.26
42.94
54.26
43.30
54.26
43.30
54.26
42.59
54.26
Class I: Fatal if swallowed (LD50 ≤ 5 mg/kg); Class II: Fatal if swallowed (5 < LD50 ≤ 50 mg/kg); Class III: toxic if swallowed (50 < LD50 ≤ 300 mg/kg); Class
IV: harmful if swallowed (300 < LD50 ≤ 2,000 mg/kg); Class V: may be harmful if swallowed (2,000 < LD50 ≤ 5,000 mg/kg); Class VI: nontoxic
(LD50 > 5,000 mg/kg).
purification (Sigma‐Aldrich, S.D. Fine, Alfa Aesar, Spectrochem). All
melting points were recorded on Coslab scientific melting point
apparatus and are uncorrected. Follow up of the reaction rates by
thin‐layer chromatography (TLC) was performed on precoated Merck
silica gel 60F‐254 plates using an appropriate solvent system and the
spots were detected under UV lamp (λ 254 nm). Infrared spectra (IR)
spectra were recorded on a Nicolet 170 SX FT‐IR spectrometer, using
potassium bromide (KBr) pellets; the frequencies are expressed in
cm−1. Nuclear magnetic resonance (1H NMR [400 MHz] and 13C
NMR [100 MHz]) spectra were recorded with a Bruker Avance FT
NMR spectrometer using tetramethylsilane as the internal reference,
with DMSO‐d6 as a solvent. The chemical shifts are accounted in
parts per million (δ ppm). Mass spectra were recorded by using mass
spectrometers Shimadzu GCMS‐QP2010S and ESI/APCI‐Hybrid
Quadrupole, Time‐of‐flight, and LCMS (Synapt G2 HDMS ACQUITY
UPLC). Elemental analyses (C, H, and N) were performed on a
Heraeus Carlo Erba 1180 CHN analyzer.
of ethyl acetate and hexane (2:8) as an eluent. After completion of
the reaction the solvent was removed under reduced pressure and
after evaporation colourless crystals of compounds 4a,b.[59,60]
|
4.1.3
6c‒j
Preparation of 4‐bromomethyl‐coumarins
Substituted‐4‐bromomethyl coumarins have been prepared via
Pechmann cyclization of substituted phenols 5c‒j with ethyl‐4‐
bromoacetoacetate.[61,62]
|
4.1.4
Preparation of 4‐(azidomethyl)‐2H‐chromen‐
2‐ones 7c‒j
4‐Bromomethyl coumarins 6c‒j (0.010 M) were taken in acetone
(20 ml) in a round‐bottom flask. To this, sodium azide (0.012 M) in
water (3 ml) was added drop wise with stirring, which was continued
for 10 hr. The reaction blend was then poured into ice‐cold water.
Separated solid was filtered and recrystallized using ethanol to afford
7c‒j.[63,64]
Please see the Supporting Information for a complete set of
spectral data. The InChI codes of the investigated compounds
together with some biological activity data are also provided as
Supporting Information.
|
4.1.5
Synthesis of coumarin‐3‐yl‐methyl‐1,2,3‐
|
4.1.2
Preparation of 2‐aryl‐4‐(prop‐2‐ynyl)‐2H‐
triazolyl‐1,2,4‐triazol‐3(4H)‐ones 8k‒z
1,2,4‐triazol‐3(4H)‐ones 4a,b
To a solution of 2‐aryl‐4‐(prop‐2‐ynyl)‐2H‐1,2,4‐triazol‐3(4H)‐ones
4a,b (1.00 M) in THF/H2O, 1:1(v/v), CuSO4·5H2O (0.15 M) and
sodium ascorbate (0.30 M) were added. The mixture was stirred at
room temperature for 15 min and 7‐methyl‐4‐azidomethyl coumarins
7c‒j (1.00 M) were added. The resulting reaction mixture was
To a solution of 2‐aryl‐2H‐1,2,4‐triazol‐3(4H)‐ones 3a,b (0.02 M) in
dry acetone, anhydrous K2CO3 (0.02 M), and 3‐bromoprop‐1‐yne
(0.02 M) were added. The reaction mixture was stirred at RT for 4 hr.
The completion of the reaction was observed by TLC using a mixture

SOMAGOND ET AL.
11 of 13
|
continued to stir until the starting material was consumed (TLC). The
reaction mixture was then cooled, separated solid was filtered and
washed with water and recrystallised from ethyl acetate to get the
crystals of desired products 8k‒z.
Cat. No. 10270106, Antibiotic – Antimycotic 100× solution (Thermo
Fisher Scientific) Cat. No. 15240062, 96‐well plates.
Cytotoxicity assay
The cells were seeded at a density of approximately 5 × 103 cells/well
in a 96‐well flat‐bottom microplate and maintained at 37°C in 95%
humidity and 5% CO2 for overnight. Different concentrations (600,
300, 150, 75, 37.5, and 18.75 µM) of samples was treated. The cells
were incubated for another 48 hr. The cells in well were washed
twice with phosphate buffer solution, and 20 µl of the MTT staining
solution (5 mg/ml in phosphate buffer solution) was added to each
well and plate was incubated at 37°C. After 4 hr, 100 µl of dimethyl
sulfoxide (DMSO) was added to each well to dissolve the formazan
4‐((1‐((6‐Methyl‐2‐oxo‐2H‐chromen‐4‐yl)methyl)‐1H‐1,2,3‐triazol‐4‐
yl)methyl)‐2‐phenyl‐2H‐1,2,4‐triazol‐3(4H)‐one (8k)
Greenish yellow (yield: 95%); Mp: 102–104°C; IR (KBr, cm−1): 1723
(coumarin, C=O), 1698 (1,2,4‐triazolin, C=O), 1629 (C=N); 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.34 (s, 3H, –CH3) 4.96 (s, 2H, coumain
C4‐CH2), 5.80 (s, 2H, 1,2,4‐triazolin N4‐CH2), 5.95 (s, 1H, H3 coumarin
pyran ring), 6.96–7.70 (m, 8H, Ar‐H), 8.24 (s, 1H, 1,2,3‐triazole C5H),
8.30 (s, 1H, 1,2,4‐triazolin C5H); 13C NMR (100 MHz, DMSO‐d6) δ
(ppm): 20.32, 37.11, 48.99, 113.65, 114.09, 116.43, 119.78, 124.32,
124.80, 130.67, 133.25, 133.76, 137.45, 142.43, 149.33, 150.64,
151.19, 156.32, 159.38; MS, m/z (70 eV): 414 [M+]; CHN analysis for
C22H18N3O6 (%): Calcd: C 63.76, H 4.38, N 20.28 Found: C 63.81, H
4.40, N 20.32.
crystals, and absorbance was recorded with
microplate reader (1, 2).
a 570 nm using
Surviving cells (%) = Mean OD of test compound/Mean OD of
negative control × 100.
Using GraphPad Prism Version 5.1, we calculated the IC50 of
compounds.
|
4.2
Biological activity
Note: DMSO concentration is less 1.5% in experiments and
concentrations are in duplicates.
|
4.2.1
Antitubercular activity assay
The antitubercular activity of the compounds 8k–z was assessed
against M. tuberculosis using MABA. This methodology is nontoxic,
employs a thermally stable reagent, and shows good correlation with
proportional and BACTEC radiometric method. Sterile de‐ionzed
water (200 µl) was added to all outer perimeter wells of sterile 96
wells plate to minimize the evaporation of medium in the test wells
during incubation. The 96‐well plate received 100 µl of the Middle
Brook 7H9 broth and serial dilution of compounds was made directly
on the plate. The final drug concentrations tested were 100 to
0.20 µg/ml. Plates were enclosed and preserved with parafilm and
incubated at 37°C for 5 days. After this freshly prepared 1:1 mixture
of Almar Blue reagent (25 µl) and 10% Tween 80 was added to the
plate and incubated for 24 hr. Blue color in the well was understood
as no bacterial growth, and pink color was scored as growth. The MIC
was defined as the lowest drug concentration which prevented the
color change from blue to pink.[64]
|
4.3
Molecular docking studies
Molecular modeling was carried out using Sybyl‐X, version 2.0,[66]
running on a Intel® CoreTM i3‐2130 CPU@3.40 GHz processor using
Windows 7 professional workstation. Surflex‐Dock algorithm of
Sybyl‐X 2.0 was used to dock designed compounds. The crystal
structure of M. tuberculosis InhA‐D148G mutant in complex with
NADH was downloaded from the Protein Data Bank (PDB entry code
4DQU extracted from the Brookhaven Protein Database http://www.
rcsb.org/pdb) and used for initial docking studies. Co‐crystallized
ligand was removed from the structure, water molecules were
removed, essential H atoms were added and side chains were fixed
during protein preparation. The structure was then subjected to an
energy refinement procedure. Gasteiger‐Huckel charges[67] were
calculated for the ligand, while Amber 7FF02 was used for the
protein. The model was then subjected to energy minimization
following the gradient termination of the Powell method for 3000
iterations using the Tripos force field with nonbonding cut offset at
9.0 and the dielectric constant set at 4.0. The binding of the
substituted coumarin derivatives was also estimated using a variety
of scoring functions that have been compiled into the single
consensus score (C Score).
|
4.2.2
Cytotoxicity evaluation
Cell culture
This is a colorimetric assay that measures the reduction of yellow 3‐
(4,5‐dimethythiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) by
mitochondrial succinate dehydrogenase. The MTT enters the cells
and passes into the mitochondria where it is reduced to an insoluble,
colored (dark purple) formazan product. The cells are then solubilized
with an organic solvent (e.g., DMSO, isopropanol) and the released,
solubilized formazan reagent is measured spectrophotometrically.
Since the reduction of MTT can only occur in metabolically active
cells the level of activity is a measure of the viability of the cells.[65]
Cell line: HEK293 (human embryonic kidney); media: DMEM with
low glucose (Cat No.‐11965‐092); materials: FBS (Gibco, Invitrogen)
ACKNOWLEDGMENTS
The authors are thankful to the University Grants Commission
(UGC), New Delhi (F. No. 14‐3/2012 [NS/PE] Dated: 14–03‐2012) for
providing infrastructural and chemical facilities under “Antitumor
Activity an Integrated Approach” a focused area of UPE FAR‐I
program. We articulate our appreciation to the USIC, K. U. Dharwad,
NMR Research Centre, IISc Bengaluru and Institution of Excellence,

12 of 13
SOMAGOND ET AL.
|
Mysore University, India for carrying out the spectral analyses. We
forward our gratefulness to UGC, New Delhi for providing financial
aid to one of the author S. M. S. in the form of project fellowship
under UPE FAR‐ I program.
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