Synthesis, docking and ADME prediction of novel 1,2,3-triazole-tethered coumarin derivatives as potential neuroprotective agents

Article abstract of DOI:10.1007/s11164-017-3210-2

In an attempt to find potential neuroprotective agents, a series of novel 3-(1-((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl) methoxyimino) ethyl)-2H-chromen-2-one derivatives 6a–j were synthesized by using “click reaction” and evaluated for their in vit

Full text of DOI:10.1007/s11164-017-3210-2

Res Chem Intermed
https://doi.org/10.1007/s11164-017-3210-2
Synthesis, docking and ADME prediction of novel 1,2,3-
triazole-tethered coumarin derivatives as potential
neuroprotective agents
Maddineni Aruna Kumari1 Chunduri Venkata Rao1
•
•
Settypalli Triloknadh1 Nallapaneni Harikrishna1
•
•
Chintha Venkataramaiah2 Wudayagiri Rajendra2
•
•
Daggupati Trinath2 Yeguvapalli Suneetha2
•
Received: 22 June 2017 / Accepted: 17 November 2017
Ó Springer Science+Business Media B.V., part of Springer Nature 2017
Abstract In an attempt to find potential neuroprotective agents, a series of novel
3-(1-((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl) methoxyimino) ethyl)-2H-
chromen-2-one derivatives 6a–j were synthesized by using ‘‘click reaction’’ and
evaluated for their in vitro neuroprotectivity and toxicity against H2O2-induced
PC12 cell lines by using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
zolium bromide) reduction assay. Most of them exhibited moderate to good activity
in which compound 6e was found to have significant protectivity with cell viability
of 94.51 0.68% at 50 lg/ml concentration and half-maximal effective concen-
tration (EC50) value of 14.04 lg/ml against injured PC12 cell lines and low toxicity
with half-maximal cytotoxic concentration (CC50) value of 544.88 lg/ml. Fur-
thermore, molecular docking was carried out in order to understand plausible
binding modes of novel derivatives with the active site of glycogen synthase kinase-
3b enzyme, and the results were well complemented by the in vitro neuroprotective
results against H2O2-induced PC12 cell lines. Additionally, in silico ADME prop-
erties showed drug likeness with good oral absorption and moderate blood–brain
barrier permeability. The structures of all the synthesized compounds were con-
1
firmed by H NMR, 13C NMR, IR and LC–MS analyses.
Keywords Click reaction Á PC12 cell lines Á Neuroprotective activity Á Molecular
docking Á ADME prediction
&
Chunduri Venkata Rao
cvrsvu@gmail.com
1
2
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502,
India
Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
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M. A. Kumari et al.
Introduction
Neuroprotection is one of the major challenges of modern medicine for the
treatment of neurodegenerative disorders, such as Parkinson’s, Alzheimer’s and
Huntington’s diseases [1]. Increased oxidative stress has been recognized as the
main cause of many neurological disorders [2, 3]. Oxidative stress is often the result
of unregulated production of reactive oxygen species including hydrogen peroxide,
nitric oxide, super oxide and reactive hydroxyl radicals. Compared with other
organs in the body, the central nervous system is mainly susceptible to oxidative
stress because it is rich in highly oxygen-consuming polyunsaturated fatty acids and
possesses a relatively low level of anti-oxidants and low regenerative capacity.
Thus, the synthesis and development of neuroprotective agents has become an
increasingly important area of research in recent years.
Both natural and synthetic coumarins (2H-1-benzopyran-2-ones) are attractvie
oxygen-containing fused heterocycles used in drugs, dyes, perfumes, cosmetics,
optical brighteners, and fluorescent and laser dyes. Coumarins are plant flavonoids
widely distributed in nature. The potent antibiotics like novobiocin, coumermycin
and chlorobiocin are natural coumarin derivatives. The synthetic coumarin
derivatives also have a wide range of biological activities against bacteria [4],
fungi [5], tumors [6], viruses [7] and HIV protease [8]. They also act as
anticoagulants [9], free radical scavengers [10], and lipoxygenase [11] and
cyclooxygenase [12] inhibitors. In addition, coumarin derivatives exhibit good cell
permeability. Furthermore, the activity of coumarin depends on the position and
nature of the substituents present on it.
Click chemistry is a modular approach to generate 1,2,3-trazoles in a reliable and
quantitative manner. In particular, Huisgen [3 ? 2] cycloaddition between terminal
alkynes and an azide in the presence of a Cu(I) catalyst generates 1,4-disubstituted
1,2,3-triazoles. They are characterized by biological activities like antiproliferative
activity [13], antimicrobial [14], antidiabetic [15], anti-inflammatory [16], anticon-
vulsant [17], antineoplastic [18], antimalarial [19], and antiviral agents [20].
Coumarin derivatives, like 4-hydroxycoumarin, scopoletin, coumarin-3-car-
boxylicacid, fraxetin, 3,8-dimethyl-5-isopropyl-6-methoxycoumarin, etc., are natu-
rally occurring cytotoxic coumarins. Recently, it has been reported that the nitrogen
heterocycle at the 3-position of coumarin might contribute to neuroprotective
activity. Several 3-substituted coumarin derivatives have been synthesized and
proved to possess nueroprotective activity. Sun et al. [21] synthesized several
substituted piperazinyl-linked coumarin scaffolds and examined their in vitro
nueroprotective activity against OGD-induced PC12 cell lines (Fig. 1, I, II).
Fig. 1 Previously synthesized 3-substituted coumarin derivatives possessing neuroprotective activity
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Synthesis, docking and ADME prediction of novel 1,2,3…
Asadipour et al. [22] synthesized novel coumarin-3-carboxamides bearing N-benzyl
piperidine moiety and determined their inhibitory activity against AChE and BuChE
and also protectivity against H2O2 induced PC12 cell lines (Fig. 1, III). Based on the
above cited observations, we have recognized the importance of Fig. 1, nitrogen
heterocycle at the 3rd position of coumarin to develop nueroprotective agents, and
here we have reported the regioselective synthesis of novel 1,2,3-triazole derivatives
6a–j through oxime functionality at the 3rd position of coumarin and their in vitro
evaluation of neuroprotective activity against H2O2-induced PC12 cell lines using
conventional MTT assay. PC12 cells were selected due to their similarity with
dopaminergic neurons. Further, there is significant evidence that GSK-3b, a member
of the protein kinase family, plays a crucial role in neuro-degeneration [23]. Not
only that, but GSK-3b is abundantly found in the central nervous system and
granulovascular degenerated neurons [24]. Accordingly, we opted to dock our
synthesized molecules at the ATP binding pocket of the GSK-3b enzyme. In
addition, we have also used an in silico method to predict ADME properties in order
to suggest the suitability of any of the new compounds for further drug
development.
Materials and methods
Chemistry
Melting points were determined in open capillaries on a Mel Temp apparatus and
are uncorrected. All the reactions were monitored by thin layer chromatography
(TLC) on precoated silica gel 60 F254 (mesh); spots were visualized with UV light.
Merck silica gel (60–120 mesh) was used for column chromatography. The IR
spectra were recorded on a Perkin Elmer BX1 FTIR Spectrophotometer as KBr
1
pellets and the wave numbers are given in cm-1. H NMR (400 MHz), and 13C
NMR (100 MHz) spectra were recorded on a Bruker AMX 400 MHz NMR
spectrometer in CDCl3 solution using TMS as an internal standard. All chemical
shifts are reported in d (ppm) using TMS as an internal standard. The mass spectra
were recorded on an Agilent 1100 LC/MSD instrument with method API-ES at
70 eV. Elemental analysis was determined using a Vario Microcube Elemental
Analyzer.
General procedure for the synthesis of 3-acetyl-2H-chromen-2-one (2)
The mixture of salicylaldehyde 1 (1 mmol, 8.6 g) and ethyl aceto acetate (1 mmol,
9 ml) was stirred under cooling conditions. To this, a few drops of piperidine were
added and stirring continued a yellow solid mass was obtained which was
recrystallized from ethanol to get the pure compound 2. Yield: 95%, m.p: 110 °C;
IR (KBr, m cm-1): 1690 (C=O), 1717.5 (ester C=O), 3040 (aromatic CH); 1H NMR
(400 MHz, CDCl3) dH: 2.72 (s, 3H, CH3), 7.33–7.38 (m, 2H, Ar–H), 7.64–7.68 (m,
2H, Ar–H), 8.50 (s, 1H); 13C-NMR (100 MHz, CDCl3) d: 30.1, 116.2, 117.8, 124.1,
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M. A. Kumari et al.
124.5, 129.8, 133.9, 147.0, 154.8, 158.7, 195.0; LC–MS (positive ion mode): m/z
211 [M ? Na]? for C11H8O3.
General procedure for the synthesis of 3-(1-(hydroxyimino)ethyl)-2H-chromen-2-
one (3)
To a solution of compound 2 (1 mmol, 0.5 g) in dichloromethane, hydroxyl amine
hydrochloride (4 mmol, 0.7 g) followed by sodium acetate (6 mmol, 0.9 g) were
added and stirred at room temperature for 8 h. Then, the mixture was poured into
crushed ice and extracted with dichloromethane. The extracted organic layer was
distilled to get a solid product. It was purified by column chromatography using
hexane and ethyl acetate mixture (30%) as an eluent. Yield: 90%, m.p: 170 °C; IR
(KBr, m cm-1): 1597.9 (C=N), 1704.9 (C=O), 2890 (CH), 3043.4 (aromatic CH),
1
3227.1 (O–H); H NMR (400 MHz, CDCl3) dH: 2.28 (s, 3H), 7.26–7.35 (m, 2H),
7.55 (d, J = 6.7 Hz, 2H), 7.92 (s, 1H), 11.21 (s, 1H, O–H); 13C-NMR (100 MHz,
CDCl3) d: 15.3, 117.3, 119.6, 125.4, 125.5, 129.4, 133.0, 142.3, 154.8, 156.0, 160.3;
LC–MS (positive ion mode): m/z 204 [M ? 1]? for C11H9NO3.
General procedure for the synthesis of 3-(1-(prop-2-ynyloxyimino) ethyl)-2H-
chromen-2-one (4)
To a solution of compound 3 (1 mmol, 0.3 g) in acetone, potassium carbonate
(3 mmol, 0.7 g) was added. To this, propargyl bromide (1.5 mmol, 0.2 ml) was
added under inert atmosphere (N2 gas) and stirring continued at room temperature
overnight. After completion of the reaction, the mixture was poured into crushed
ice. The separated precipitate was filtered and dried. This solid was purified by
column chromatography using 15% hexane and ethyl acetate mixture as a mobile
phase. Yield: 85%, m.p: 130 °C; IR (KBr, m cm-1): 1605.9 (C=N), 1712.2 (C=O),
2115.7 (C:C), 3264.1 (:CH); 1H NMR (400 MHz, CDCl3) dH: 2.27 (s, 3H, CH3),
2.51 (s, 1H, acetylene CH), 4.79 (s, 2H, CH2), 7.27–7.35 (m, 3H, Ar–H), 7.55 (d,
J = 6.7 Hz, 1H, Ar–H), 7.93 (s, 1H, Ar–H); 13C-NMR (100 MHz, CDCl3) d: 14.5,
61.8, 74.7, 79.6, 116.5, 118.8, 124.6, 124.8, 128.6, 132.2, 141.5, 154.1, 155.2,
159.6; LC–MS (positive ion mode): m/z 242 [M ? H]?, 264 [M ? Na]? for
C14H11NO3.
General procedure for the synthesis of aryl azides (5)
Aniline derivative (9.1 mmol) was dissolved in concentrated HCl (91 mmol) at
room temperature and cooled to 0 °C, followed by the addition of an aqueous
solution of sodium nitrite (45.5 mmol). The reaction mixture was stirred for 30 min
at 0–5 °C. Then, an aqueous solution of sodium azide (27.3 mmol) was added and
the mixture was further stirred for 2 h at room temperature. After completion of the
reaction, the mixture was extracted with hexane. The combined organic layer was
dried over anhydrous sodium sulfate. After evaporation of the solvent the desired
azide derivative was obtained.
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Synthesis, docking and ADME prediction of novel 1,2,3…
General procedure for the synthesis of 3-(1-((1-(substituted phenyl)-1H-1,2,3-
triazol-5-yl)methoxyimino)ethyl)-2H-chromen-2-one (6a–j)
To a solution of compound 4 (0.7 mmol) in 2 ml of THF:H2O (1:1) solvent mixture,
CuSO4Á5H2O (0.17 g, 0.7 mmol) was added and the reaction mixture was stirred for
5 min. To this light blue colour mixture sodium ascorbate (0.2 g, 1.05 mmol) was
added and stirred for 15 min. The reaction mixture colour becomes dark yellow.
After that aryl azide 6 (1.05 mmol) was added and allowed to stir at room
temperature for 30 min-1 h. After that the reaction mixture was partitioned between
ethyl acetate and water. The combined organic layer was dried over anhydrous
sodium sulfate. The solvent was distilled and the crude product was purified by
silica gel column chromatography using hexane ethyl acetate mixture as an eluent.
3-(1-((1-p-tolyl-1H-1,2,3-triazol-4-yl) methoxyimino)ethyl)-2H-chromen-2-one (6a)
Yield: 70%, m.p: 120 °C; IR (KBr, m cm-1): 1609.9 (C=N), 1722.7 (C=O), 2856.2
1
(CH2), 2921.2 (=CH); H NMR (400 MHz, CDCl3) dH: 2.27 (s, 3H), 2.42 (s, 3H),
5.43 (s, 2H), 7.28–7.35 (m, 4H), 7.55 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H),
7.9 (s, 1H), 8.05 (s, 1H, triazole CH); 13C-NMR (100 MHz, CDCl3) d: 14.8, 21.3,
68.0, 116.7, 119.0, 120.7, 121.5, 124.5, 124.8, 125.1, 128.7, 130.4, 132.4, 135.0,
139.1, 141.6, 154.2, 154.6, 159.7; LC–MS (positive ion mode): m/z 375 [M ? H]?,
397 [M ? Na]? for C21H18N4O3. Anal. calcd. for C21H18N4O3: C, 67.37; H, 4.85;
N, 14.96; found: C, 67.68; H, 4.73; N, 14.69.
3-(1-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6b) Yield: 75%, m.p: 125 °C; IR (KBr, m cm-1): 1126.8 (C–F), 1603.6
(C=N), 1701.6 (C=O), 2893.5 (CH2), 3071.4 (=CH); H NMR (400 MHz, CDCl3)
1
dH: 2.27 (s, 3H, CH3), 5.43 (s, 2H, CH2), 7.29–7.36 (m, 3H), 7.50 (d, J = 5.8 Hz,
1H), 7.54 (s, 1H), 7.56–7.59 (m, 3H), 7.89 (s, 1H), 8.12 (s, 1H, triazole CH); 13C-
NMR (100 MHz, CDCl3) d: 14.3, 67.3, 116.3, 118.5, 118.7, 120.9, 123.0, 123.4,
124.4, 124.6, 128.2, 130.7, 131.5, 132.0, 137.6, 141.1, 145.3, 153.7, 154.2, 159.2;
LC–MS (positive ion mode): m/z 379 [M ? H]?, 401 [M ? Na]? for C20H15FN4-
O3. Anal. calcd. For C20H15FN4O3: C, 63.49; H, 4.00; N, 14.81; found: C, 63.88; H,
4.23; N, 14.59.
3-(1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6c) Yield: 80%, m.p: 130 °C; IR (KBr, m cm-1): 1252.2 (C–O–C), 1606.5
(C=N), 1718.2 (C=O), 2911.6 (CH2), 3070.8 (=CH); H NMR (400 MHz, CDCl3)
1
dH: 2.26 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 5.42 (s, 2H, CH2), 7.02 (d, J = 8.8 Hz,
2H), 7.27–7.35 (m, 3H), 7.54 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H, Ar–H),
7.9 (s, 1H), 8.02 (s, 1H, triazole CH); 13C-NMR (100 MHz, CDCl3) d: 14.5, 51.5,
67.8, 116.5, 118.8, 120.5, 121.2, 121.3, 123.4, 124.3, 124.6, 124.9, 128.5, 130.2,
132.2, 138.9, 154.0, 154.4, 159.5; LC–MS (positive ion mode): m/z 391 [M ? H]?,
413 [M ? Na]? for C21H18N4O4. Anal. calcd. For C21H18N4O4: C, 64.61; H, 4.65;
N, 14.35; found: C, 64.88; H, 4.73; N, 14.09.
3-(1-((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-
chromen-2-one (6d) Yield: 60%, m.p: 140 °C; IR (KBr, m cm-1): 1111.7 (C-F),
123

M. A. Kumari et al.
1
1608.9 (C=N), 1703.3 (C=O), 2888.9 (CH2), 3075 (=CH); H NMR (400 MHz,
CDCl3) dH: 2.27 (s, 3H, CH3), 5.44 (s, 2H, CH2), 7.29–7.36 (m, 3H), 7.69–7.72 (m,
3H), 7.89 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 8.08 (s, 1H), 8.19 (s, 1H, triazole CH);
13C-NMR (100 MHz, CDCl3) d: 14.7, 67.4, 116.3, 117.3, 118.5, 119.6, 121.0,
123.3, 124.4, 128.2, 130.2, 131.2, 132.0, 134.3, 141.1, 143.2, 143.5, 144.3, 154.9,
156.4, 159.9; LC–MS (positive ion mode): m/z 429 [M ? H]?, 451 [M ? Na]? for
C21H15F3N4O3. Anal. calcd. For C21H15F3N4O3: C, 58.88; H, 3.53; N, 13.08; found:
C, 59.08; H, 3.73; N, 13.23.
3-(1-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-2-
one (6e) Yield: 65%, m.p: 135 °C; IR (KBr, m cm-1): 1528.5 (NO2), 1605.3
(C=N), 1706.6 (C=O), 2855.7 (CH2), 2920.0 (=CH); H NMR (400 MHz, CDCl3)
1
dH: 2.27 (s, 3H, CH3), 5.45 (s, 2H, CH2), 7.52–7.58 (m, 4H, Ar–H), 7.76 (s, 1H), 7.9
(s, 1H), 8.28–8.32 (m, 3H, Ar–H), 8.66 (s, 1H, triazole CH); 13C-NMR (100 MHz,
CDCl3) d: 13.5, 66.6, 115.5, 117.8, 118.0, 120.2, 122.2, 122.6, 123.6, 123.8, 127.5,
130.0, 130.7, 131.2, 136.9, 140.4, 144.6, 152.9, 153.5, 158.5; LC–MS (positive ion
mode): m/z 406 [M ? H]?, 428 [M ? Na]? for C20H15N5O5. Anal. calcd. For
C20H15N5O5: C, 59.26; H, 3.73; N, 17.28; found: C, 59.38; H, 3.78; N, 17.35.
3-(1-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6f) Yield: 60%, m.p:128 °C; IR (KBr, m cm-1): 1162.6 (C–F), 1602.8
(C=N), 1701.8 (C=O), 2933.1 (CH2), 3060.0 (=CH); H NMR (400 MHz, CDCl3)
1
dH: 2.19 (s, 3H, CH3), 5.36 (s, 2H, CH2), 7.19–7.27 (m, 3H), 7.43–7.48 (m, 5H),
7.82 (s, 1H), 8.03 (s, 1H, triazole CH); 13C-NMR (100 MHz, CDCl3) d: 12.7, 64.8,
114.6, 115.0, 115.9, 117.7, 118.7, 123.1, 123.7, 124.4, 126.7, 127.7, 130.0, 131.2,
133.0, 134.3, 141.1, 147.8, 152.7, 159.0; LC–MS (positive ion mode): m/z 401
[M ? Na]? for C20H15FN4O3. Anal. calcd. For C20H15FN4O3: C, 63.49; H, 4.00; N,
14.81; found: C, 63.88; H, 4.23; N, 14.96.
3-(1-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-2-one (6g)
Yield: 75%, m.p: 110 °C; IR (KBr, m cm-1): 1600.8 (C=N), 1716.3 (C=O), 2856.7
1
(CH2), 2919.5 (=CH), 3051.8 (Aromatic C–H); H NMR (400 MHz, CDCl3) dH:
2.27 (s, 3H, CH3), 5.44 (s, 2H, CH2), 7.27–7.35 (m, 3H), 7.44 (d, J = 7.5 Hz, 1H),
7.51–7.56 (m, 5H), 7.9 (s, 1H), 8.11 (s, 1H, triazole CH); 13C-NMR (100 MHz,
CDCl3) d: 14.2, 67.5, 112.9, 115.6, 117.1, 120.1, 121.6, 122.8, 128.4, 129.2, 129.8,
131.3, 131.9, 134.3, 142.7, 143.2, 149.0, 157.2; LC–MS (positive ion mode): m/z
361 [M ? H]?, 383 [M ? Na]? for C20H16N4O3. Anal. calcd. For C20H16N4O3: C,
66.66; H, 4.48; N, 15.55; found: C, 66.89; H, 4.73; N, 15.39.
3-(1-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6h) Yield: 60%, m.p: 120 °C; IR (KBr, m cm-1): 1605.4 (C=N), 1702.9
(C=O), 2888.9 (CH2), 3070.1 (=C–H); 1H NMR (400 MHz, CDCl3) dH: 2.2 (s, 3H),
5.35 (s, 2H), 7.21–7.28 (m, 4H), 7.47 (d, J = 7.47 Hz, 2H), 7.56 (d, J = 8.24 Hz,
2H), 7.83 (s, 1H), 7.98 (s, 1H, triazole CH); 13C-NMR (100 MHz, CDCl3) d: 13.5,
66.7, 115.5, 117.8, 119.5, 120.2, 122.4, 123.3, 123.6, 123.9, 127.5, 129.2, 131.2,
133.7, 137.8, 140.3, 152.9, 158.5; LC–MS (positive ion mode): m/z 395 [M ? H]?,
123

Synthesis, docking and ADME prediction of novel 1,2,3…
397 [M ? 3H]?, 417 [M ? Na]? for C20H15ClN4O3. Anal. calcd. For C20H15-
ClN4O3: C, 66.84; H, 3.83; N, 14.19; found: C, 66.88; H, 4.03; N, 14.09.
3-(1-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6i) Yield: 50%, m.p: 130 °C; IR (KBr, m cm-1): 1598.7 (C=N), 1704
(C=O), 2894.2 (CH2), 3068.2 (=CH); 1H NMR (400 MHz, CDCl3) dH: 2.26 (s, 3H,
CH3), 5.44 (s, 2H, CH2), 7.08 (d, J = 7.2 Hz, 2H), 7.13 (d, J = 7.5 Hz, 2H),
7.52–7.56 (m, 4H), 7.88 (s, 1H), 8.1 (s, 1H, triazole CH); 13C-NMR (100 MHz,
CDCl3) d: 13.7, 67.7, 115.5, 116.5, 119.7, 120.2, 121.5, 123.3, 123.6, 123.4, 125.5,
130.2, 131.2, 138.8, 140.3, 152.9, 153.4, 159.5; LC–MS (positive ion mode): m/z
439 [M ? H]?, 441 [M ? 3H]?, 461 [M ? Na]? for C20H15BrN4O3. Anal. calcd.
For C20H15BrN4O3: C, 54.69; H, 3.44; N, 12.75; found: C, 54.88; H, 3.73; N, 12.59.
3-(1-((1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxyimino)ethyl)-2H-chromen-
2-one (6j) Yield: 55%, m.p: 135 °C; IR (KBr, m cm-1): 1592.9 (C=N), 1704.8
(C=O), 2891.8 (CH2), 3066.2 (=CH); 1H NMR (400 MHz, CDCl3) dH: 2.27 (s, 3H,
CH3), 5.43 (s, 2H, CH2), 7.29–7.36 (m, 3H), 7.42 (d, J = 8.1 Hz, 1H), 7.54–7.58
(m, 3H), 7.89 (s, 1H), 7.98 (s, 1H), 8.1 (s, 1H, triazole CH); 13C-NMR (100 MHz,
CDCl3) dc: 14.5, 67.6, 116.5, 118.8, 119.0, 121.2, 123.3, 123.7, 124.7, 128.5, 130.2,
131.0, 131.7, 132.3, 137.9, 141.4, 145.6, 154.0, 154.5, 159.5; LC–MS (positive ion
mode): m/z 439 [M ? H]?, 441 [M ? 3H]?, 461 [M ? Na]? for C20H15BrN4O3.
Anal. calcd. For C20H15BrN4O3: C, 54.69; H, 3.44; N, 12.75; found: C, 54.88; H,
3.73; N, 12.59.
Biological activity
In vitro cytotoxic and neuroprotective activity
Cell lines The PC12 cell lines were purchased from the National Center for Cell
Sciences, Pune. The cells were maintained in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 8% horse serum, 2% FBS, 100 lg/ml penicillin and
100 lg/ml streptomycin in a water-saturated atmosphere of 5% CO2 at 37 °C. The
medium was changed every 3 days, and the confluent cells were passaged weekly
by trypsinization.
MTT assay A forty-eight-hour monolayer culture of PC12 cells at a concentration
of one lakh/well was seeded in 24-well titer plates. To the washed cell sheet with
MEM without FCS, 1 ml of medium (without FCS) was added containing defined
concentrations of the drug in respective wells. The stock drug was prepared by
adding 10 mg of drug to 10 ml of serum-free MEM to give a concentration of 1 mg/
ml. Working stock was prepared by mixing 4.5 ml of MEM and 0.5 ml of stock to
give 1 mg/ml. To the cell control wells, 1 ml MEM (w/o) FCS was added and
incubated at 37 °C in 5% CO2 environment. After incubation, 200 ll of MTT at a
concentration of 5 mg/ml was added to each well and incubated for 6–7 h in 5%
CO2 atmosphere. After incubation, 1 ml of DMSO was added to each well and left
for 45 s. Then, the quantity of metabolized MTT product dissolved in DMSO was
123

M. A. Kumari et al.
measured by determining the absorbance at 595 nm on a microplate reader by
taking DMSO as a blank.
Cell viability ð%Þ ¼ Mean OD=Control OD Â 100
Docking studies Molecular docking was used to estimate the binding free energy
and binding mode of the synthesized compounds 6a–j with GSK-3b. Molecular
docking was performed with PyRx 0.8 implementation of Auto Dock 4.2 [25] using an
empirical free energy force field and Lamarckian genetic algorithm conformational
search with the default parameters. The grid box was set around the ATP pocket in
˚
˚
˚
˚
GSK3b with a 45 A 9 42 A 9 46 A grid box having 0.375-A grid point spacing.
Target structure and ligand dataset preparation The 3D coordinates of the crystal
structure GSK-3b bounded with a native ligand, AR-A014418, in the ATP pocket
was selected from the RCSB Protein Data Bank (PDB code: 1Q5 K) as the receptor
model. The ATP pocket is present at the interface of b-strand domain (residues
25–138) at the N-terminal end and the a-helical domain (residues 139–343) is
bordered by a glycine-rich loop and hinge region [26]. Water molecules and hetero
atoms were removed from the co-crystal structure. Hydrogen atoms and Gesteiger
partial charges were added to the target protein using UCSF Chimera 1.10.2 [27].
The compounds were subjected to energy minimization using the Open Babel
module in PyRx program.
In silico ADME prediction The synthesized compounds were subjected to
prediction of ADME properties. The various ADME properties including topolog-
ical polar surface area (TPSA), molecular weight, number of rotatable bonds,
molecular volume, number of hydrogen bond donors, number of hydrogen bond
acceptors, mi Log P and violations of Lipinski rule were calculated by the
Molinspiration online property toolkit. %ABS was calculated by using the formula:
%ABS = 109 - (0.345 9 TPSA) [28]. ADME prediction properties like HIA%,
CaCO2 permeability, PPB%, and blood–brain barrier (BBB) were predicted by
using pre-ADMET online server (http://preadmet.bmdrc.org/).
Results and discussion
Chemistry
The synthesis of 1,2,3-triazole-coumarin hybrids is outlined in Scheme 1. The target
molecules synthesis consists of three parts. It involves the preparation of 3-(1-
(hydroxyimino) ethyl)-2H-chromen-2-one (3) by treating 3-acetyl coumarin with
hydroxylamine hydrochloride in sodium acetate and then treatment with propargyl
bromide under nitrogen atmosphere in the presence of potassium carbonate to form
3-(1-(prop-2-ynyloxyimino) ethyl)-2H-chromen-2-one (4) and it is further con-
verted into 3-(1-((1-(substitutedphenyl)-1H-1,2,3-triazol-5-yl)methoxyimino)ethyl)-
123

Synthesis, docking and ADME prediction of novel 1,2,3…
Scheme 1 Schematic representation for the synthesis of 3-(1-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-5-
yl)methoxyimino)ethyl)-2H-chromen-2-one (6a–j). Where R = 4-CH3: 6a, 5a; R = 2-F: 6f, 5f; R = 3-
F: 6b, 5b; R = H: 6g, 5g; R = 4-OCH3: 6c, 5c; R = 4-Cl: 6h, 5h; R = 3-CF3: 6d, 5d; R = 4-Br: 6i, 5i;
R = 3-NO2: 6e, 5e; R = 3-Br: 6j, 5j
2H-chromen-2-one derivatives 6a–j by reacting with different aryl azides 5a–
j through click reaction. Initially, the 3-acetyl coumarin (2) was synthesized by
Knovenagel condensation between salicylaldehyde and ethyl acetoacetate in the
presence of piperidine as a catalyst.
1
All the synthesized compounds were characterized by H NMR, 13C NMR, IR
and LC–MS spectra and elemental analysis. The formation of compound 2 was
confirmed by the appearance of peaks at 1717.54 and 1705.12 cm-1 due to C=O
stretching in the IR spectra. Apart from this, a signal at d 195.02 ppm in 13C NMR
due to ester carbonyl carbon indicated the formation of coumarin. The peaks at
3227.06 and 1597.88 cm-1 due to OH and C=N stretching, respectively, confirmed
the formation of oxime derivative 3. In addition to this, a signal at d 11.21 ppm due
1
to OH proton in H NMR spectra also confirmed the formation of oxime. Further,
the formation of propargyl derivative 4 was confirmed by the appearance of a peak
at 3264.07 cm-1 in its IR spectra due to :CH stretching and also by a singlet at d
2.51 ppm in 1H NMR spectra due to acetylenic CH proton. In the 1H NMR spectra,
the formation of triazole ring was confirmed by the resonance of H–C (5) of the
triazole ring in the aromatic region, i.e. from d 7.98 to 8.66 ppm.
Biology
In vitro neuroprotective activity
With all the target compounds in hand, neuroprotective activity was assayed against
oxidative stress-induced PC12 cells by using MTT assay. H2O2 was used as toxic
123

M. A. Kumari et al.
insult to introduce oxidative damage. H2O2 can generate exogenous free radicals,
which are highly reactive species which lead to lipid, protein and DNA damage.
PC12 cells are usually used for studying neurodegenerative diseases [29, 30]. In this
assay, the addition of 30 lM H2O2 to growth medium significantly reduced cell
viability to 48% compared to control. PC12 cells were incubated with coumarin
analogs at concentrations ranging from 10 to 50 lg/ml for 6–7 h, which iwass
followed by the incubation with 30 lM of H2O2 for 24 h. The protective effect of
coumarin analogs against H2O2 was determined by the cell viability through MTT
assay. MTT is named (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium
bromide. MTT is reduced by succinate dehydrogenase system of mitochondrial
living cells to produce purple formazan crystals [31, 32]. After solubilization, the
formazan can be measured spectrophotometrically and the amount of formazan
produced is directly proportional to the number of viable cells in the culture
(Table 1).
The cell viability values are shown in Table 2 and the results indicated that the
cell viability rates significantly increase in a concentration-dependent manner from
10 to 50 lg/ml. Compounds 6a, 6d and 6f also showed neuroprotectivity even at
10 lg/ml concentration, but the remaining compounds lacked protectivity against
H2O2-induced PC12 cell lines at this concentration due to toxicity. Compounds 6h,
6i and 6j did not show any neuroprotectivity even at 30 lg/ml concentration. When
the concentration reached 40 lg/ml, all compounds started to exhibit protectivity.
Highest cell viability was observed for compounds 6e, 6a, 6b, 6d and 6f at 50 lg/ml
concentrations of the test compounds. The graphical representation of these values
is shown in Fig. 2.
The EC50 values of the compounds 6a–j are in the range of 13.21 to 46.68 lg/ml
as shown in Fig. 3. Of all the compounds, 6a, 6e and 6f, possessing para methyl
phenyl, meta nitro phenyl and ortho fluoro phenyl, respectively, showed excellent
protectivity with EC50 values 13.21, 14.04 and 15.66 lg/ml, respectively. The
protectivity of compounds 6b, 6c and 6d was moderate. The remaining compounds,
6g, 6h, 6i and 6j, exhibited very poor neuroprotectivity.
The structure activity relationship of compounds 6a–j demonstrated that
substitution of the electron-withdrawing nitro group at the meta position of the
phenyl ring increased the protectivity of compound 6e against damaged PC12 cell
lines in the concentration range of 20–50 lg/ml. Moreover, it is worth noting that
the presence of the electron-releasing methyl group at the para position improved
the activity of compound 6a compared with that of compound 6e. This is possibly
due to the lower toxicity of compound 6a compared with that of compound 6e.
Compound 6a exhibited protection in the range of 10–50 lg/ml concentrations.
Looking at the halogen derivatives, the type and position of the halogen atom in the
phenyl ring have a variable influence on the activity. As the size of the halogen atom
increases, the activity decreased gradually due to stearic hindrance. This was
observed in compounds 6h and 6i. The cell viability of compound 6h which is a
para chloro-substituted derivative is more than that of compound 6i which is a para
bromo substituted derivative in the concentration range 10–50 lg/ml. Similarly, for
compound 6b (a meta fluoro derivative) protectivity is more than that of 6j (a meta
bromo derivative) from 10 to 50 lg/ml concentrations. Furthermore, the presence of
123

Synthesis, docking and ADME prediction of novel 1,2,3…
123

M. A. Kumari et al.
Table 2 Results obtained from molecular docking studies of compounds 6a–j
e
H–B length (A)
DGb (kcal/mol)a
Ki (lM)b
H–Bc
aa H–Bd
˚
Compound
6a
6b
6c
6d
6e
6f
- 7.51
- 6.86
- 6.54
- 6.21
- 8.3
3.13
9.34
3
3
0
3
4
2
1
1
0
2
Y134 (3)
2.6, 2.7, 3.1
Y134 (2), R141 (1)
2.7, 3.3, 3.3
16.06
28.05
0.82
–
–
Y134 (2), R141 (1)
2.9, 3.1, 3.4
Y134, V135, R141, R144
2.7, 3.3, 2.7, 3.0
- 6.99
- 6.69
- 6.43
- 6.97
- 6.59
7.53
R141 (2)
R141
3.1, 3.3
3.5
6g
6h
6i
12.39
19.48
7.75
Y134
2.9
–
–
6j
14.79
Y134 (2)
3.2
aBinding energy
bInhibition constant
cNumber of hydrogen bonds
dAmino acids involving hydrogen bonds
eHydrogen bond length between amino acids and compounds 6a–j
100
90
80
70
60
50
40
30
20
10
0
Control
H2O2
10µg/ml+H2O2
20µg/ml+H2O2
30µg/ml+H2O2
40µg/ml+H2O2
50µg/ml+H2O2
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
Fig. 2 Protective effects of compounds 6a–j on H2O2-induced cell death in PC12 cells. Cells were
incubated with different concentrations (10–50 lg/ml) of compounds 6a–j for 6 h and then treated with
30 lM H2O2 for 24 h. Cell viability was measured by MTT assay. Data are shown as mean SD of three
independent experiments
a fluorine atom at the ortho position improves the protectivity of compound 6f
compared with that of the meta fluorine-substituted derivative 6b in the range of
10–40 lg/ml concentrations. Moreover, compound 6f showed protectivity even
from 10 lg/ml concentrations, but for compound 6b protection starts from 20 lg/ml
123

Synthesis, docking and ADME prediction of novel 1,2,3…
50
45
40
35
30
25
20
15
10
5
0
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
Compounds 6a-j
Fig. 3 EC50 values of compounds 6a–j on H2O2-induced PC12 cells
concentration. Furthermore, the para bromo-substituted derivative 6i showed more
protectivity than that of compound 6j which is a meta bromo-substituted derivative
at 40 and 50 lg/ml concentrations. At 50 lg/ml concentration, the cell viability of
compound 6i is 75.24 0.48% where as for compound 6j it is 66.21 0.46%.
From these results, it can be understood that ortho and para positions are preferred
for halogens to exhibit good neuroprotectivity.
Neurotoxicity of compounds in PC12 cell lines
One of the major hindrances in developing effective neuroprotective drugs is their
toxicity to normal cells. In order to investigate the safety index of these potent
neuroprotective agents, all the compounds were tested for cytotoxicity by using
MTT assay in neuroblastoma cells (PC12). After incubating the cells with
compounds for different times, the CC50 values were calculated and are shown in
Fig. 4. Among all the compounds, 6a, 6e and 6f showed low toxicity with CC50
600
500
400
300
200
100
0
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
Compounds 6a-j
Fig. 4 Cytotoxicity of compounds 6a–j on PC12 cells
123

M. A. Kumari et al.
values 540, 544.88 and 548.14 lg/ml, respectively. Compounds 6b, 6c, 6d and 6g
showed moderate toxicity with CC50 values ranging from 412.36 to 482.26 lg/ml.
The remaining compounds 6h, 6i and 6j showed high toxicity.
Molecular docking studies
The docking protocol was validated using redocking experiments by removing the
native ligand Ar-A014418 from GSK-3b and docking back into the same binding
pocket using Auto Dock 4.2 in PyRx with default parameters. It showed an RMSD
˚
value of 0.689 A, obtained from all atoms and heteroatom coordinates between
experimental and redocked confirmations. Moreover, as shown in Fig. 5, the docked
native ligand (blue-colored ligand in Fig. 5) is bound tightly to GSK-3b involving
almost the same residues Ile62, Ala83, Asp133, Tyr134, Val135, and Leu188 as in
the co-crystallized structure (purple-colored ligand in Fig. 5). This indicates that
Fig. 5 Docking validation performed by re-docking native ligand into the ATP pocket of GSK-3b.
Docked pose is shown in blue and native conformation is shown in purple. (Color figure online)
123

Synthesis, docking and ADME prediction of novel 1,2,3…
these parameters are adequate in reproducing the experimental structure and can be
extended to do docking studies on our synthesized compounds.
The molecular docking results revealed that all the compounds 6a–j tend to bind
within the ATP pocket of GSK-3b and show good binding free energies ranging
from - 8.3 to - 6.21 kcal/mol. The docking results are summarized in Table 2.
Compound 6e showed the highest binding energy of - 8.3 kcal/mol and the lowest
inhibition constant of 0.82 followed by compounds 6a and 6f which showed binding
energies of - 7.51 and - 6.99 kcal/mol and inhibition constants of 3.13 and 7.53,
respectively. The more negative value of binding energy and low inhibition constant
indicate good binding affinity of the ligand towards the target enzyme. Thus, these
results correlate well with the observed in vitro neuroprotective activity.
The 3D and 2D visualization of the interactions of the most active compounds 6e,
6a and 6f within the ATP pocket of GSK-3b are shown in Fig. 6. The binding
models indicate that the compounds are held in the active site by a combination of
various hydrogen bonding and hydrophobic interactions. Four hydrogen bonds were
present in the derivative 6e, which was the highest among the series. It showed
hydrogen bonding interactions with amino acid residues Arg141, Arg144, Tyr134
˚
and Val135 at the bond distances of 2.7, 3.0, 2.7 and 3.3 A, respectively. The NH2
group of Arg141 and Arg144 residues showed hydrogen bonding with oxygen of the
nitro group on the phenyl ring (linked to the triazole) which is not seen in other
molecules. This is evidence for the requirement of hydrophilic groups to bind
strongly to the protein active site. The OH group of Tyr 134 and the NH group of
Val 135 participated in hydrogen bonding with oxygen atoms of oxime and
coumarin carbonyl oxygen, respectively. The coumarin moiety of 6e occupies the
hydrophobic pocket bounded by Leu188 and Val70 residues whereas the triazole
ring is located in a pocket defined by the IIe62 residue. Furthermore, in compound
6a, the coumarin moiety sits inside the hydrophobic pocket formed by Pro191,
Glu137 and Pro136 residues while the phenyl ring (linked to triazole) is positioned
in the pocket designed by IIe62 and Leu188 residues. In addition, it was also found
to engage in three hydrogen bonding interactions with the OH group of the Tyr134
residue using oxygen and nitrogen atoms of oxime linkage and coumarin carbonyl
˚
oxygen at the distances of 2.6, 2.7 and 3.0 A. Moreover, compound 6f participated
in hydrogen bonding with the NH2 group of Arg141 residue using oxygen atoms of
˚
oxime and the coumarin carbonyl group at the distances of 3.1 and 3.3 A. Regarding
the hydrophobic interactions of compound 6f, the coumarin moiety is engaged in the
pocket formed by Gln185 and IIe62 residues, meanwhile the phenyl ring (linked to
triazole) occupied the hydrophobic pocket formed by Tyr134, Leu188 and Ala83
residues. On the basis of activity and docking studies, it was found that compound
6e had the improved potential to protect the PC12 cell lines against H2O2 oxidative
stress.
In silico ADME prediction of compounds 6a–j
Nowadays, many potential drugs fail to reach the clinic because of ADMET
liabilities. Adsorption, distribution, metabolism, excretion and toxicity (ADMET)
processes play a pivotal role in defining the therapeutic efficacy of a drug. Drug
123

M. A. Kumari et al.
Fig. 6 Schematic diagram representing the 3D and 2D modeled binding modes of compounds 6e, 6a and
6f within the ATP domain of GSK-3b
likeness appears as a promising paradigm of a compound that optimizes their
ADME in the human body [33].
With the aim of estimating the drug-likeness of the compounds, we have
determined the compliance of the synthesized molecules to the Lipinski’s ‘rule of
five’. According to this rule, poor absorption or permeation is more likely when
there are more than five hydrogen bond donors, ten hydrogen bond acceptors, the
123

Synthesis, docking and ADME prediction of novel 1,2,3…
molecular weight is greater than 500 and the calculated log p (logarithmic ratio of
the octanol–water partitioning coefficient) is greater than 5. Molecules violating
more than one of these parameters may have problems with bioavailability and a
high probability of failure to display drug-likeness [34]. Further, the topological
polar surface area (TPSA) which is another key property in estimating drug
2
˚
bioavailability was also calculated. Generally, compounds with a TPSA [ 140 A
are thought to have low bioavailability [35]. As shown in Table 3, all the
synthesized compounds comply with these rules. Moreover, all the compounds
exhibited a greater percentage of absorption (%ABS) ranging from 64.7 to 80.5%.
Hence, theoretically, all of these compounds should have good passive oral
absorption and drug likeness.
In addition to this different ADME predictions, such as BBB penetration,
percentage of human intestinal absorption (HIA%), CaCO2 permeability and
percentage of plasma protein binding (PPB%) were predicted for all compounds.
Analyzing the ADME predictions (Table 4), it was observed that all the compounds
showed high HIA% values in the range of 94.92–99.33% and are well absorbed. The
CaCO2 cell permeability values are moderate, ranging from 6.88 to 24.79 nm/s.
Furthermore, all the compounds were strongly bound to plasma proteins with %PPB
penetration more than 91.25%. In addition, they were found to have moderate
Table 3 Physicochemical properties of the compounds 6a–j
Compound Mol.
wta
Mol. n-
volb
n-
n-ON
mi
TPSA Lipinski’s %ABSh
2 g
ROTBc
OHNH
donord
acceptore Log Pf (A )
violation
˚
Rule
6a
6b
6c
6d
6e
6f
B 500
B 5
0
B 10
B 5
3.65
3.56
3.26
4.29
3.35
3.53
3.21
3.88
4.01
4.20
B 1
0
374.4 330.3
378.3 319.0
390.4 339.6
428.3 345.3
405.3 337.4
378.3 319.0
360.3 314.0
394.8 327.6
439.2 331.9
439.2 331.9
5
5
6
6
6
5
5
5
5
5
7
7
82.53
82.53
91.76
82.53
128.35
82.53
82.53
82.53
82.53
82.53
80.5
80.5
77.3
80.5
64.7
80.5
80.5
80.5
80.5
80.5
0
0
0
8
0
0
7
0
0
10
7
0
0
0
6g
6h
6i
0
7
0
0
7
0
0
7
0
6j
0
7
0
aMolecular weight
bMolecular volume
cNumber of rotatable bonds
dNumber of hydrogen bond donors
eNumber of hydrogen bond acceptors
fLogarithmic ratio of the octanol–water partitioning coefficient
gTopological polar surface area
hPercentage of absorption. %ABS = 109 - (0.345 9 TPSA)
123

M. A. Kumari et al.
Table 4 Prediction of pharmacokinetic properties of compounds 6a–j
Compound
CaCOa2 permeability
HIAb (%)
PPBc (%)
BBBd (Cbrain/Cblood
)
6a
6b
6c
6d
6e
6f
23.43
24.79
20.74
20.93
6.88
98.81
98.93
99.33
98.78
94.92
98.93
98.93
98.22
97.78
97.78
92.27
92.73
91.25
92.87
93.60
92.14
92.79
95.95
100.00
94.88
0.117
0.479
0.355
0.443
0.193
0.374
0.319
0.345
0.325
0.548
23.39
20.84
21.82
21.99
22.14
6g
6h
6i
6j
aColon adenocarcinoma
bHuma intestinal absorption
cPlasma protein binding
dBlood–brain barrier
penetration (0.117–0.548) to the CNS through the BBB. From all these parameters,
it can be observed that, theoretically, all the compounds exhibited good absorption
and bioavailability with reasonable permeability through the BBB.
Conclusion
With the aim of synthesizing more potent neuroprotective agents, we focused our
attention on the synthesis of coumarin and 1,2,3-triazole linked moieties 6a–j by
copper(1)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) reaction to
achieve quantitative yields, and examined their neuroprotective activity against
H2O2-induced PC12 neurons and toxicity using MTT reduction assay. Of all the
compounds, 6a, 6e and 6f showed better nueroprotective activity with EC50 values
13.21, 14.04 and 15.66 lg/ml, respectively. Compounds 6f, 6e and 6a showed low
toxicity with CC50 values 548.14, 544.88 and 540 lg/ml, respectively. In addition to
that compound, 6e showed the highest cell viability (94.51 0.68%) against H2O2-
induced PC12 cell lines at 50 lg/ml concentration. From these results, it can be
concluded that compound 6e was a better neuroprotective agent with low toxicity.
Further molecular docking studies between the synthesized molecules 6a–j and
GSK-3b enzyme ATP binding pocket revealed that compound 6e showed higher
binding affinities and many more interactions when compared to other compounds.
In addition, in silico ADME prediction showed that all the compounds fulfilled
Lipinski’s rule of five with moderate potential to penetrate the BBB. These results
suggested that compound 6e can be considered as a promising candidate for further
optimization and development of potential neuroprotective drugs.
123

Synthesis, docking and ADME prediction of novel 1,2,3…
Acknowledgements One of the authors (MAK) gratefully acknowledges UGC for providing financial
assistance in the form of a BSR fellowship. Funding was provided by University Grants Commission
(Grant No. F.25-1/2013-14 (BSR)/7-187/2007 (BSR)).
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