In?vitro antioxidant, antiinflammatory and in silico molecular docking studies of thiosemicarbazones

Article abstract of DOI:10.1016/j.molstruc.2017.05.054

A series of 5-methoxysalicylaldehyde appended thiosemicarbazones (1–4) and 2-hydroxy-1-naphthaldehyde appended thiosemicarbazones (5–8) was obtained from the reactions between 5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde and (un)substituted thiosem

Full text of DOI:10.1016/j.molstruc.2017.05.054

Accepted Manuscript
In vitro antioxidant, anti-inflammatory and in silico molecular docking studies of
thiosemicarbazones
G.R. Subhashree, J. Haribabu, S. Saranya, P. Yuvaraj, D. Anantha Krishnan, R.
Karvembu, D. Gayathri
PII:
S0022-2860(17)30638-5
10.1016/j.molstruc.2017.05.054
MOLSTR 23795
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 December 2016
Revised Date: 23 January 2017
Accepted Date: 12 May 2017
Please cite this article as: G.R. Subhashree, J. Haribabu, S. Saranya, P. Yuvaraj, D. Anantha Krishnan,
R. Karvembu, D. Gayathri, In vitro antioxidant, anti-inflammatory and in silico molecular docking studies
of thiosemicarbazones, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.05.054.
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ACCEPTED MANUSCRIPT
In vitro antioxidant, anti-inflammatory and in silico molecular docking studies of
thiosemicarbazones
G. R. Subhashree1, $, J. Haribabu2, $, S. Saranya3, P. Yuvaraj4, D. Anantha Krishnan5,
R. Karvembu2,*, D. Gayathri5,*
1 Department of Physics, Dr. MGR Educational and Research Institute University, Maduravoyal,
Chennai 600095, India
2 Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India
3 Department of Biotechnology, Dr. MGR Educational and Research Institute University,
Maduravoyal, Chennai 600095, India
4 North-East Institute of Science and Technology (CSIR), Branch Laboratory, Imphal, Manipur-
795004, India
5 Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy
Campus, Chennai 600025, India
$ Equal contribution
* kar@nitt.edu, drdgayathri@gmail.com
Abstract
A series of 5-methoxysalicylaldehyde appended thiosemicarbazones (1-4) and 2-
hydroxy-1-naphthaldehyde appended thiosemicarbazones (5-8) was obtained from the reactions
between
5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde
and
(un)substituted
thiosemicarbazides with the view to ascertain their biological properties brought about by the
change in substitution at N-terminal position of the thiosemicarbazide derivatives. The
compounds were fully characterized by elemental analyses, and various spectroscopic techniques
(UV-Visible, FT-IR, NMR and mass). The solid-state structure of three compounds (1, 2 and 7)
was determined by single crystal X-ray diffraction method. The compounds (1, 2 and 7) have
adopted a monoclinic crystal system with P21/c (1 and 2) or C2/c (7) space group. Antioxidant
and non-haemolysis activities of the compounds (1-8) were analyzed by in vitro DPPH and
haemolysis assays, respectively. Anti-inflammatory potential was verified by in vitro PLA2
inhibition assay and in silico molecular docking study. In vitro and in silico studies revealed
promising anti-inflammtory potential of the thiosemicarbazone derivatives. Compounds 2, 4, 6, 7
and 8 showed significant anti-inflammatory activity.

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Keywords: Thiosemicarbazones, Crystals structures, Antioxidant, Anti-inflammtory, Molecular
docking.
Highlights
ꢀ Synthesis of eight bioactive thiosemicarbazone derivatives
ꢀ Thiosemicrbazones showed promising antioxidant and non-haemolysis properties
ꢀ The compounds exhibited attractive anti-inflammtory activity
ꢀ Anti-inflammtory activity was supported by molecular docking study
Introduction
Thiosemicarbazones belong to a group of thiourea derivatives, which have been studied
due to their versatile biological activities and broad application in industry, and analytical
sensitivity towards various metal ions. Biological applications of thiosemicarbazones have been
studied since 1956 when Brockman et al reported the antitumoral property of 2-formylpyridine
thiosemicarbazone derivatives [1]. Followed by this, considerable number of thiosemicarbazone
derivatives have been reported as antibacterial, antiviral, antifungal, antioxidant,
antiinflammatory and antiproliferative agents [2-5]. Further, this class of compounds has been
studied for its activity against tuberculosis, leprosy, psoriasis, rheumatism, trypanosomiasis and
coccidiosis [6,7]. The presence of nitrogen and sulfur donor atoms in the thiosemicarbazone
might be responsible for its potential biological activity [8]. Certain thiosemicarbazones showed
a selective inhibition of herpes simplex virus (HSV) infection in vitro and in vivo. The effect of
thiosemicarbazones against human immuno deficiency virus (HIV) was reported [9]. The
presence of bulky substitution at the Ν−terminal nitrogen atom of the thiosemicarbazones
derived from 2-formylpyridine and 2-acetylpyridine increases the biological activity [10-12].
The anticancer activity of thiosemicarbazones was expected to be due to their action as
inhibitors of ribonucleotide reductase [13,14], which catalyzes the rate-limiting step of DNA
synthesis. The most studied therapeutic compound among the thiosemicarbazones was 3-
aminopyridine-2-carboxaldehyde thiosemicarbazone (triapine) [15]. Triapine has entered phase
II clinical trials as a chemotherapeutic agent [16]. Another promising drug, di-2-pyridylketone 4-
cyclohexyl-4-methyl-3-thiosemicarbazone has just entered clinical trials, but RNR was not or
only in part responsible for its biological activity [17]. Based on the above facts, we intend to
report the synthesis, crystal structure, antioxidant, non-haemolysis and anti-inflammtory
properties of some thiosemicarbazone derivatives.

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Experimental
Materials and methods
All the chemicals were purchased from Sigma Aldrich / Merck / Alfa Aesar and used as
received. Solvents were purified according to the standard procedures [18]. The melting points
were determined on Lab India instrument and are uncorrected. The elemental analyses were
performed using a Vario EL-III CHNS analyzer. FT-IR spectra were obtained as KBr pellets
using a Nicolet-iS5 spectrophotometer. UV-visible spectra were recorded using a Shimadzu-
2600 spectrophotometer. NMR spectra were recorded in DMSO-d6 by using TMS as an internal
standard on a Bruker 500 (1H) or 125 (13C) MHz spectrometer. The (Z)-2-((2-
hydroxynaphthalen-1-yl)methylene)-N-methylhydrazinecarbothioamide (6) was synthesized by
using a reported procedure [19].
Synthesis of 5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde thiosemicarbazones
(Un)substituted thiosemicarbazide (10 mmol) was dissolved in 25 mL of ethanol and
gently heated for about 20 min. To this, ethanolic solution (15 mL) of 5-methoxy
salicylaldehyde/2-hydroxy-1-naphthaldehyde (10 mmol) and a few drops of glacial acetic acid
were added and the mixture was refluxed for 3 h. Upon cooling, a white/whitish yellow
crystalline product began to separate. This was collected by filtration, washed with cold ethanol
and hexane, and dried in vacuum. It was recrystallized from dimethylformamide/acetone to get
crystals of 1, 2 and 7.
(E)-2-(2-hydroxy-5-methoxybenzylidene)hydrazinecarbothioamide (1)
2-Hydroxy-5-methoxybenzaldehyde (0.152 g, 0.001 mol) and thiosemicarbazide (0.911
g, 0.001 mol) were used. Yield: 73%. White solid. m.p.: 201-203 °C. Anal. Calc. C9H11N3O2S
(%): C, 47.99; H, 4.92; N, 18.65; S, 14.23. Found: C, 47.83; H, 5.07; N, 18.52; S, 14.69. UV-Vis
(DMF): λmax, nm 260, 329. FT-IR (KBr): ʋ, cm-1 3450 (OH), 3346, 3221 (N–H), 1575 (C=N),
1
1270 (C=S). H NMR (500 MHz, DMSO-d6): δ, ppm 11.39 (s, 1H, OH), 9.38 (s, 1H, HNCS),
8.34 (s, 1H, CH=N), 8.25 (s, 1H, NH(NH2)), 7.79 (s, 1H, NH(NH2)), 7.51 (d, J = 7.1 Hz, 1H,
aromatic), 6.81 (d, J = 7.6, 1H, aromatic), 6.78 (d, J = 7.9 Hz, 1H, aromatic), 3.70 (s, 3H,
OCH3). 13C NMR (125 MHz, DMSO-d6): δ, ppm 177.5 (C=S), 152.6 (O–C), 151.2 (C=N),
139.0, 121.5, 118.1, 117.3, 110.4 (aromatic), 56.1 (OCH3). ESI-MS m/z = 226.61 [M + H]+.
(E)-2-(2-hydroxy-5-methoxybenzylidene)-N-methylhydrazinecarbothioamide (2)
2-Hydroxy-5-methoxybenzaldehyde (0.152 g, 0.001 mol) and 4-methylthiosemicarbazide
(0.105 g, 0.001 mol) were used. Yield: 60%. White solid. m.p.: 199-201 °C. Anal. Calc.

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C10H13N3O2S (%): C, 50.19; H, 5.48; N, 17.56; S, 13.40. Found: C, 50.04; H, 5.59; N, 17.45; S,
13.55. UV-Vis (DMF): λmax, nm 270, 337. FT-IR (KBr): ʋ, cm-1 3427 (OH), 3309, 3220 (N–H),
1
1584 (C=N), 1264 (C=S). H NMR (500 MHz, DMSO-d6): δ, ppm 11.42 (s, 1H, OH), 9.45 (s,
1H, HNCS), 8.45 (d, J = 4.3 Hz, 1H, NH-terminal), 8.34 (s, 1H, CH=N), 7.49 (d, J = 7.0 Hz, 1H,
aromatic), 6.84 (dd, J = 8.8, 2.9 Hz, 1H, aromatic), 6.80 (d, J = 8.1 Hz, 1H, aromatic), 3.74 (s,
3H, OCH3), 3.03 (d, J = 4.4 Hz, 3H, CH3). 13C NMR (125 MHz, DMSO-d6): δ, ppm 177.9
(C=S), 152.8 (O–C), 151.0 (C=N), 139.2, 121.3, 118.0, 117.3, 110.6 (aromatic), 56.0 (OCH3),
31.3 (CH3). ESI-MS m/z = 240.05 [M + H]+.
(E)-N-ethyl-2-(2-hydroxy-5-methoxybenzylidene)hydrazinecarbothioamide (3)
2-Hydroxy-5-methoxybenzaldehyde (0.152 g, 0.001 mol) and 4-ethylthiosemicarbazide
(0.119 g, 0.001 mol) were used. Yield: 77%. White solid. m.p.: 188-190 °C. Anal. Calc.
C11H15N3O2S (%): C, 52.15; H, 5.97; N, 16.59; S, 12.66. Found: C, 52.27; H, 5.83; N, 16.69; S,
12.47. UV-Vis (DMF): λmax, nm 270, 331. FT-IR (KBr): ʋ, cm-1 3440 (OH), 3321, 3204 (N–H),
1
1569 (C=N), 1264 (C=S). H NMR (500 MHz, DMSO-d6): δ, ppm 11.40 (s, 1H, OH), 9.48 (s,
1H, HNCS), 8.61 (t, J = 6.0 Hz, 1H, NH-terminal), 8.30 (s, 1H, CH=N), 7.44 (d, J = 7.1 Hz, 1H,
aromatic), 6.87 (d, J = 8.1 Hz, 1H, aromatic), 6.82 (d, J = 8.6 Hz, 1H, aromatic), 3.79 (s, 3H,
13
OCH3), 3.70 (q, J = 6.9 Hz, 2H, CH2CH3), 1.18 (t, J = 7.0 Hz, 3H, CH2CH3). C NMR (125
MHz, DMSO-d6): δ, ppm 177.8 (C=S), 152.8 (O–C), 151.4 (C=N), 139.1, 121.1, 118.3, 117.0,
110.2 (aromatic), 56.0 (OCH3), 43. 7, 14.6 (ethyl). ESI-MS m/z = 254.01 [M + H]+.
(E)-2-(2-hydroxy-5-methoxybenzylidene)-N-phenylhydrazinecarbothioamide (4)
2-Hydroxy-5-methoxybenzaldehyde (0.152 g, 0.001 mol) and 4-phenylthiosemicarbazide
(0.167 g, 0.001 mol) were used. Yield: 81%. White solid. m.p.: 212-214 °C. Anal. Calc.
C15H15N3O2S (%): C, 59.78; H, 5.02; N, 13.94; S, 10.64. Found: C, 59.62; H, 4.91; N, 13.74; S,
10.95. UV-Vis (DMF): λmax, nm 266, 337. FT-IR (KBr): ʋ, cm-1 3436 (OH), 3311, 3169 (N–H),
1580 (C=N), 1280 (C=S). 1H NMR (500 MHz, DMSO-d6): δ, ppm 11.84 (s, 1H, OH), 10.08 (s,
1H, HNCS), 9.16 (t, J = 6.0 Hz, 1H, NH-terminal), 8.47 (s, 1H, CH=N), 7.52 (d, J = 7.2 Hz, 1H,
aromatic), 7.41 - 7.45 (m, 2H, aromatic), 7.25 - 7.30 (m, 2H, aromatic), 7.00 - 7.05 (m, 1H,
aromatic), 6.91 (d, J = 7.9 Hz, 1H, aromatic), 6.85 (dd, J = 8.4, 3.9 Hz, 1H, aromatic), 3.86 (s,
13
3H, OCH3). C NMR (125 MHz, DMSO-d6): δ, ppm 178.3 (C=S), 153.0 (O–C), 151.7 (C=N),
139.4, 134.6, 128.4, 123.7, 121.9, 120.8, 118.3, 117.5, 110.4 (aromatic), 56.3 (OCH3). ESI-MS
m/z = 301.09 [M]+.

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(E)-2-((2-hydroxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (5)
2-Hydroxy-1-naphthaldehyde (0.172 g, 0.001 mol) and thiosemicarbazide (0.911 g, 0.001
mol) were used. Yield: 78%. Yellow solid. m.p.: 206-209 °C. Anal. Calc. C12H11N3OS (%): C,
58.76; H, 4.52; N, 17.13; S, 13.07. Found: C, 58.93; H, 4.47; N, 17.00; S, 13.35. UV-Vis (DMF):
λmax, nm 265, 330. FT-IR (KBr): ʋ, cm-1 3441 (OH), 3370, 3261 (N–H), 1563 (C=N), 1275
(C=S). 1H NMR (500 MHz, DMSO-d6): δ, ppm 11.70 (s, 1H, OH), 10.63 (s, 1H, NHCS), 9.10 (s,
1H, CH=N), 8.21 (s, 1H, NH(NH2)), 7.89 (d, J = 8.2 Hz, 1H, aromatic), 7.84 (d, J = 8.1 Hz, 1H,
aromatic), 7.47 (s, 1H, NH(NH2)), 7.55 (t, J = 7.4 Hz, 2H, aromatic), 7.24 (d, J = 8.4 Hz, 1H,
13
aromatic), 7.21 (d, J = 7.1 Hz, 1H, aromatic). C NMR (125 MHz, DMSO-d6): δ, ppm 179.0
(C=S), 156.6 (C=N), 144.9, 139.5, 132.2, 129.1, 128.5, 127.8, 118.2 (aromatic). ESI-MS m/z =
268.02 [M + Na]+.
(E)-N-ethyl-2-((2-hydroxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (7)
2-Hydroxy-1-naphthaldehyde (0.172 g, 0.001 mol) and 4-ethylthiosemicarbazide (0.119
g, 0.001 mol) were used. Yield: 71%. Yellow. m.p.: 222-226 °C. Anal. Calc. C14H15N3OS (%):
C, 61.51; H, 5.53; N, 15.37; S, 11.73. Found: C, 61.31; H, 5.69; N, 15.06; S, 11.91. UV-Vis
(DMF): λmax, nm 267, 334. FT-IR (KBr): ʋ, cm-1 3436 (OH), 3379, 3261 (N–H), 1568 (C=N),
1
1251 (C=S). H NMR (500 MHz, DMSO-d6): δ, ppm 11.68 (s, 1H, OH), 10.63 (s, 1H, NHCS),
10.15 (s, 1H, NH-terminal), 9.13 (s, 1H, CH=N), 7.87 (d, J = 8.0 Hz, 1H, aromatic), 7.81 (d, J =
8.2 Hz, 1H, aromatic), 7.53 (t, J = 7.2 Hz, 2H, aromatic), 7.23 (d, J = 8.8 Hz, 1H, aromatic), 7.20
(d, J = 7.4 Hz, 1H, aromatic), 3.81 (qd, J = 7.3, 5.7 Hz, 2H, CH2CH3), 1.35 (t, J = 7.1 Hz, 3H,
13
CH2CH3). C NMR (125 MHz, DMSO-d6): δ, ppm 180.4 (C=S), 156.3 (C=N), 144.7, 139.6,
132.0, 129.0, 128.7, 128.0, 118.5 (aromatic), 41.8, 14.2 (ethyl). ESI-MS m/z = 273.30 [M]+.
(E)-2-((2-hydroxynaphthalen-1-yl)methylene)-N-phenylhydrazinecarbothioamide (8)
2-Hydroxy-1-naphthaldehyde (0.172 g, 0.001 mol) and 4-phethylthiosemicarbazide
(0.167 g, 0.001 mol) were used. Yield: 81%. Yellow solid. m.p.: 213-215 °C. Anal. Calc.
C18H15N3OS (%): C, 67.27; H, 4.70; N, 13.07; S, 9.98. Found: C, 67.07; H, 4.87; N, 13.24; S,
9.90. UV-Vis (DMF): λmax, nm 265, 327. FT-IR (KBr): ʋ, cm-1 3441 (OH), 3382, 3247 (N–H),
1573 (C=N), 1279 (C=S). 1H NMR (500 MHz, DMSO-d6): δ, ppm 11.76 (s, 1H, OH), 10.60 (s,
1H, NHCS), 10.07 (s, 1H, NH-terminal), 9.17 (s, 1H, CH=N), 8.44 (s, 1H, aromatic), 8.32 (s,
1H, aromatic), 7.91 (d, J = 8.9 Hz, 1H, aromatic), 7.88 (d, J = 8.1 Hz, 1H, aromatic), 7.59 (t, J =
7.0 Hz, 2H, aromatic), 7.39 (dd, J = 14.9, 7.4 Hz, 3H, aromatic), 7.23 (d, J = 8.4 Hz, 1H,
13
aromatic), 7.19 (d, J = 7.2 Hz, 1H, aromatic). C NMR (125 MHz, DMSO-d6): δ, ppm 184.9

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(C=S), 157.1 (C=N), 144.0, 139.6, 133.1, 132.1, 129.2, 128.7, 128.5, 128.3, 125.5, 123.9, 118.9
(aromatic). ESI-MS m/z = 321.01 [M]+.
X-ray crystallography
X-ray diffraction method was used to determine the three dimensional structure of
thiosemicarbazone derivatives. Data collection was performed with BRUKER APEX2
diffractometer using MoKα radiation [20]. Cell refinement and data reduction were carried out
using SAINT [20]. Direct methods were used for structure determination using SHELXS
program and refined with least squares refinement procedure using SHELXL [21]. Thermal
ellipsoidal plot, intra- and inter-molecular hydrogen bonds are represented using PLATON [22].
Antioxidant assay - DPPH free radical scavenging
The antioxidant activity of the thiosemicarbazone derivatives was evaluated using the
DPPH free radical scavenging assay [23,24]. Different concentrations of synthesized compounds
were used for revealing their antioxidant potential and the assay protocol is followed as reported
in [25].
Haemolysis assays
Human red blood cells (RBC) were isolated using the standard protocol [26] and diluted
with 0.1 M phosphate buffer saline (PBS), pH 7.4. Non-haemolytic potential of the synthesized
compounds were elucidated by haemolysis assay [25].
Anti-inflammatory studies
Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids at sn-2 position of
phospholipids releasing lysophospolipid and fatty acid. The released fatty acid plays a major role
in eicosanoid production in the cell [27] and in the formation of arachidonic acid followed by the
synthesis of leukotrienes and prostaglandins [28]. Over expression of PLA2 was the major
precursor of inflammation pathway and also plays an important role in carcinogenesis [29].
To analyze the anti-inflammatory potential of the thiosemicarbazone derivatives, we have
performed in vitro PLA2 assay using Cayman’s secretory PLA2 assay kit. 1,2-dithio analog of
diheptanoyl phosphatidylcholine was used as a substrate. PLA2 hydrolyzed the thio ester bond at
sn-2 position and the free thiols were detected using 5,5’-dithio-bis-(2-nitrobenzoic acid)
(DTNB) at 414 nm. Procedure of PLA2 assay was followed as mentioned in the protocol of
PLA2 assay kit of Cayman chemical. The concentration of PLA2 used in this assay caused an
absorbance increase of approximately 0.1 per min. Measured absorbance value as a function of

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time was noted and changes in the absorbance during the time interval was calculated using the
formula, ∆A414 = [A414 (time 2) - A414 (time 1)] / [time 2 - time 1].
Average change in the absorbance was calculated for the sample containing PLA2
enzyme incubated with substrate and was considered as 100% PLA2 enzyme activity. Average
change in the absorbance of the sample containing PLA2 enzyme incubated with the substrate
and thiosemicarbazones were calculated to identify whether the activity of PLA2 enzyme was
decreased in the presence of the thiosemicarbazones.
Molecular docking
To investigate the anti-inflammtory potential of the thiosemicarbazone derivatives by in
silico method, molecular docking was performed. Induced fit docking was carried out using
Schrodinger-Maestro [30] (Schrodinger LLC 2009, USA). Two dimensional coordinates of eight
thiosemicarbazone derivatives were generated using ChemSketch version 12.01[31] and
converted to 3D format followed by energy minimization using Ligprep in Schrödinger maestro.
The 3D coordinates of human non-pancreatic secretory PLA2 were downloaded from RCSB-
protein data bank (PDB) [32] and subjected to energy minimization using protein preparation
wizard. Based on the docking energy, score and active site interactions, the best protein-ligand
complex conformation was chosen. Hydrogen bonds and hydrophobic interactions at the active
site of PLA2 was represented by Ligplot using PDBSUM [33]. Surface view model was
generated using PyMol [34].
Results and discussion
Synthesis and characterization
The biologically active 5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde based
thiosemicarbazones (1-8) were synthesized from methoxysalicylaldehyde/2-hydroxy-1-
naphthaldehyde and (un)substituted thiosemicarbazide in the presence of glacial acetic acid
(Scheme 1 and 2). All the thiosemicarbazone derivatives were characterized by analytical and
1
13
different spectroscopic (UV-visible, FT-IR, H, C NMR and mass) techniques. The solid state
structure of 1, 2 and 7 was confirmed by single crystal X-ray diffraction method. The white or
yellow colored thiosemicarbazones (1-8) were insoluble in dichloromethane, chloroform,
benzene and water, but soluble in acetone, acetonitrile, dimethylformamide and
dimethylsulfoxide. The compounds were air and light stable.

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Scheme 1. Synthetic route of the 5-methoxysalicylaldehyde appended thiosemicarbazone
derivatives.
Scheme 2. Preparation of the 2-hydroxy-1-naphthaldehyde appended thiosemicarbazone
derivatives.
Spectroscopy
The formation of thiosemicarbazone derivatives (1-5, 7 and 8) was established from the
π→π* (260-270 nm) and n→π* (327-337 nm) transitions observed in their UV-visible spectra
[35]. In the FT-IR spectra of the thiosemicarbazone derivatives (1-5, 7 and 8), there was one
O−H band observed in the region 3427-3450 cm-1. The terminal N−H and NHCS stretching
bands were appeared in the region 3169-3382 cm-1. The C=N and C=S stretching bands were
observed at 1563-1584 and 1251-1280 cm-1, respectively [36].
1
In the H NMR spectra of the thiosemicarbazones (1-5, 7 and 8), the signal due to
carboxyl OH proton was found as a broad singlet in the region 11.39-11.84 ppm [37]. The
thiocarbonyl attached NH and terminal NH (2-4, 7 and 8) protons were observed at 9.38-10.63
and 8.45-10.15 ppm respectively. A singlet was appeared at 8.30-9.17 ppm for the proton present
in the azomethine group. The resonances owing to the aromatic ring protons were located at

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6.78-8.44 ppm in the spectra of all the compounds. In the spectra of 1-4, a singlet was appeared
at 3.70-3.86 ppm due to methoxy protons. Two broad singlets were found at 8.21-8.25 and 7.47-
7.79 ppm corresponding to NH2 protons in the spectra of 1 and 5. The signal due to methyl
protons was appeared at 3.03 ppm in the spectra of 2. Further, resonances at 3.70-3.81 and 1.18-
13
1.35 ppm in the spectra of 3 and 7 attributed to the presence of CH2 and CH3 protons [25]. C
NMR spectra of the compounds confirmed the presence of C=S (177.5-184.9 ppm) and C=N
(151.0-157.1 ppm). The resonance for the methoxy carbon (1-4) was observed in the region 56.0-
56.3 ppm [38]. The methyl and ethyl carbons present in complexes 2, 3 and 7 exhibited signals at
31.3, and 41.8-43.7 and 14.1-14.6 ppm, respectively. The signals of all other aromatic and
aliphatic protons / carbons were observed in the expected regions.
Three dimensional structural studies
Crystal structure of three compounds (1, 2 and 7) are reported here. Data collection and structure
refinement details are summarized in Table 1. The compounds 1, 2 and 7 crystallize in
monoclinic crystal system in P21/c (1, 2) and C2/c (7) space groups. Cell parameters for 1, 2 and
7 were a = 7.4847(7); 7.2023(5); 26.511(5) Å, b = 9.9713(8); 24.7874(16); 7.033(5) Å, c =
14.3553(10); 6.3036(4); 18.853(5) Å and β = 91.892(3); 94.263(4); 129.707(5)°, respectively,
with one molecule in the asymmetric unit. Three dimensional crystal structures of 1, 2 and 7
were determined by direct methods and refined by least squares refinement to final R value of
4.2, 5.5 and 5.9%, respectively. Thiosemicarbazone group in 1, 2 and 7 adopted E configuration
with respect to C=N moiety as evidenced from the torsion angles C6−C8−N1−N2 [−178.9 (2)]°,
C4−C8−N1−N2 [173.3(2)]° and C7−C11−N1−N2 [179.5(2)]°, respectively. In 1,
hydrazinecarbothioamide group was almost planar with maximum deviation of −0.064(2) Å and
the molecular structure was nearly planar with dihedral angle between the
hydrazinecarbothioamide N1−N2−C9(S1)−N3 moiety and rest of the molecule being 6.7 (1)°. In
2, N2 slightly deviates (0.203 (2) Å) from the planarity of N1−C9−N3−S1 and the moiety
C10−N3−C9−S1 was planar as observed from the torsion angle [0.0 (5)°]. The diheral angle
between N1−N2−C9(S1)−N3−C10 and rest of the molecule was 16.6(1)° in 2. In 7, the dihedral
angle between C1−C11/O1 and N1−N2−C12(S1)−N3-C13−C14 was 5.1(1)° indicating the near
planarity of the molecule. Bond lengths, bond angles and torsion angles were in the allowed
ranges (Table 2). Crystallographic information files have been deposited in Cambridge structure
database with CCDC numbers 1503334, 1503336 and 1503335 for 1, 2 and 7, respectively.

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The thermal ellipsoidal plots, and intra- and inter-molecular packing are provided in Figs.
1a-3a and 1b-3b, respectively. The molecular structure was stabilized by O−H...N and N−H...N
intra-molecular hydrogen bonding interactions, generating S(6) and S(5) motifs, respectively in
all the reported crystal structures. In 1, the crystal packing was stabilized by two N−H...S and
one N−H...O inter-molecular hydrogen bonding interactions. Atoms N2 and N3 act as donors to
2
S1 at 1-x, 1-y, -z and 1-x, -1/2+y, 1/2-z generating centrosymmetric dimer of R2 (8) and chain
C(4), respectively. In N-H…O interaction, atom N3 acts as donor to O2 generating chain of
C(9). In 2, the crystal packing was stabilized by N−H…O and C−H...O inter-molecular hydrogen
bonding interactions generating chains of C(7) and C(3), respectively. In 3, the crystal packing
was stabilized by N−H...S intermolecular hydrogen bonding interaction generating
2
centrosymmetric dimer of R2 (8). Intra- and inter-molecular hydrogen bond parameters are listed
in Table 3.
DPPH assay
Free radicals-induced oxidative stress play a definite role in the development of various
metabolic disorders. Antioxidants were known to fight against free radicals and prevent the
pathological ailments caused by oxidative stress. It was important to prevent the excessive
formation of free radicals formed in human body by various external and internal stress.
Synthetic antioxidants showed stronger antioxidant activities when compared to natural
antioxidants. However the use of synthetic agents was limited because of their toxicity. Due to
this, there was a great deal of interest in finding out new antioxidant compounds which do not
show any pathological side effects [39]. In this regard, the synthesized thiosemicarbazone
derivatives were studied for their antioxidant potential using DPPH scavenging assay. DPPH
reacts with antioxidant which donate hydrogen and reduces the DPPH radical. Absorbance was
measured at 517 nm. The compounds 1-8 showed higher antioxidant activity at 25 µg/mL (Fig.
4). Further increase in concentration did not show any significant changes. Only compound 4
showed dose considerable dependent activity till 100 µg/mL. Further increase in concentration
above 100 mg/mL stabilized the activity. Compounds 1-8 have potency equal to that of ascorbic
acid at similar concentration. From the above results, it was clear that the compounds 1-8 have
high antioxidant potential.
Haemolysis assay
Interaction of the drug with the blood components particularly human RBC was an
important and inevitable phenomenon, thus assessing the haemolysis becomes crucial in

ACCEPTED MANUSCRIPT
evaluating the blood compatibility of the drugs [39]. Six different concentrations of the
thiosemicarbazone derivatives were tested for their toxicity against human RBC. Results were
compared with the control cells treated with water which produced 100% lysis. All the
compounds showed negligible percentage of hemolysis (Fig. 5). The results suggested that the
thiosemicarbazone derivatives can be used for further pharmacological actions.
In vitro anti-inflammtory PLA2 assay
In vitro PLA2 assay has been performed to screen the anti-inflammtory potential of the
thiosemicarbazones. Compounds that can inhibit the activity of PLA2 will have a greater
potential as anti-inflammtory and also as anticancer drugs. Activity of PLA2 treated with
substrate alone was considered for 100% activity of the PLA2. The activity of PLA2 enzyme
incubated with thiosemicarbazone compounds (at 100 µM concentration) along with substrate
was calculated accordingly. Fig. 6 shows the percentage activity of PLA2 incubated with
substrate alone and thiosemicarbazones along with substrate. PLA2 enzyme incubated with 2, 4,
6, 7 and 8 along with substrate showed lesser PLA2 activity suggesting the competent binding of
thiosemicarbazones against binding of substrate at the active site of PLA2. Results revealed
promising anti-inflammtory potential of 2, 4, 6, 7 and 8, and importantly 7 inhibited nearly 70%
of PLA2 activity at 100 µM concentration.
In silico molecular docking
In addition to in vitro screening of anti-inflammtory potential of thiosemicarbazone
derivatives, molecular docking studies were also performed to screen the anti-inflammtory
potential by in silico method. Molecular docking was performed with human non-pancreatic
secretory PLA2 with respective PDB code 1DCY [40]. From the induced fit docking results, it
was inferred that the thiosemicarbazone derivatives possess good anti-inflammtory property and
also showed comparable results with the co-crystal ligand. The energy, score and interactions of
co-crystal ligand (1-benzyl-5-methoxy-2-methyl-1h-indol-3-yl)-acetic acid) at the active site of
PLA2 were used to compare with the results obtained with thiosemicarbazone derivatives. The
docking energy and glide score are listed in Table 4. Ligplot representation is shown to highlight
the hydrogen bond and hydrophobic interactions at the active site of PLA2 (Fig. 7). Surface view
model of PLA2 with 7 bound at the active site is shown in Fig. 8.

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Conclusion
Eight thiosemicarbazone derivatives (1-8) have been synthesized and characterized by
1
elemental analysis and spectroscopic (FT-IR, UV-visible, H NMR, 13C NMR and mass)
techniques. Crystal structure of representative compounds (1, 2 and 7) was determined by X-ray
crystallography. Structures were refined to good R factors. Structure and crystal packing of 1, 2
and 7 were stabilized by intra- and inter-molecular hydrogen bonding interactions. Antioxidant
and non-haemolysis potential of the compounds were analyzed by in vitro DPPH and haemolysis
assay, respectively. All the thiosemicarbazone derivatives showed promising antioxidant and
non-haemolysis activity. Anti-inflammatory activity of the thiosemicarbazones was evaluated by
in vitro PLA2 assay and the results revealed that the compound 7 have the highest inhibition
potential. In silico molecular docking results revealed the binding mode of the
thiosemicarbazones at the active site of PLA2, which can be further explored for their
pharmaceutical applications.
Acknowledgements
JH thank the University Grants Commission (UGC), Government of India for financial
support. DG thank DBT for Bioinformatics infrastructure facility at University of Madras.
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Fig. 1a. Thermal ellipsoidal plot of 1 at 30% probability.
Fig. 1b. Molecular packing viewed down a axis showing intra- and inter-molecular hydrogen
bonding interactions in 1.

ACCEPTED MANUSCRIPT
Fig. 2a. Thermal ellipsoidal plot of 2 at 30% probability.
Fig. 2b. Molecular packing viewed down a axis showing intra- and inter-molecular hydrogen
bonding interactions in 2.

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Fig. 3a. Thermal ellipsoidal plot of 7 at 30% probability.
Fig. 3b. Molecular packing viewed down b axis showing intra- and inter-molecular hydrogen
bonding interactions in 7.

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Fig. 4. Antioxidant activity of the thiosemicarbazone compounds (1-8) at different
concentrations using DPPH assay. Each value represents a mean ± SD (n = 3).
Fig. 5. Human blood compatibility analysis of the thiosemicarbazone compounds (1-8). Each
value represents a mean ± SD (n = 3).

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Fig. 6. Percentage of PLA2 activity of the thiosemicarbazone compounds (1-8). Substrate used
was 1,2-dithio analog of diheptanoyl phosphatidylcholine.

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Fig. 7. Ligplot representation showing the interactions of co-crystal ligand and
thiosemicarbazone derivatives at the active site of PLA2.

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Fig. 8. Surface view of PLA2 with 7 (green) bound at the active site.

ACCEPTED MANUSCRIPT
Table 1. Crystal data and structure refinement parameters
1
2
7
CCDC number
Chemical formula
Mr
1503334
C9H11N3O2S
225.27
1503336
C10H13N3O2S
239.29
1503335
C14H15N3OS
273.35
Crystal system
Monoclinic
P21/c
Monoclinic
P21/c
Monoclinic
C2/c
space group
Temperature (K)
293
293
293
a = 7.4847 (7)
b = 9.9713 (8)
c = 14.3553 (10)
a = 7.2023 (5)
b = 24.7874 (16)
c = 6.3036 (4)
a = 26.511 (5)
b = 7.033 (5)
c = 18.853 (5)
Unit cell dimensions (Å, °)
V (Å3)
β
= 91.892 (3)
β
= 94.263 (4)
β = 129.707 (5)
1070.78 (15)
4, 1.397
ꢀ
(mm-1) 0.29
1122.24 (13)
4, 1.416
0.28
2704 (2)
8, 1.343
0.24
Z
, Dx (Mg m−3)
Absorption coefficient,
F(000)
472
504
1152
Crystal size (mm)
0.19 × 0.2 × 0.21
0.18 × 0.21 × 0.22
0.2 × 0.21 × 0.22
Radiation type
Wavelength (Å)
Diffractometer
Fine-focus sealed tube
0.71073
BRUKER Kappa APEXII CCD
θmax
,
θmin (°)
30.6, 2.5
29.1, 1.6
31.6, 2.0
No. of measured, independent
and observed [I > 2
reflections
σ(I)]
14838, 2955,
2160
21885, 2929, 2165
0.035
25639, 4144, 2478
0.036
Rint
h
0.026
-10
-13
-20
→
→
→
10
13
20
-9
-33
-8
→
8
-33
-10
-27
→
→
→
38
10
22
k
→ 33
l
→
8
Absorption correction
Tmin, Tmax
multi-scan, SADABS (Bruker 2004)
0.692, 0.746 0.658, 0.746
Full matrix least-squares on F2
0.669, 0.746
Refinement method
No. of parameters
138
148
174
(sin
θ
/
λ
) max (Å−1)
(F2)], wR(F2)
0.715
0.683
0.737
R [F2 > 2
σ
0.042, 0.133
0.055, 0.163
0.059, 0.218

ACCEPTED MANUSCRIPT
Goodness of fit on
F
2 (S)
1.07
1.24
1.12
ꢁρmax
,
ꢁρmin (e Å−3)
0.32, −0.27
0.38, −0.46
0.43, −0.39
Table 2. Selected geometric parameters (Å, °)
1
2
7
1.419(4)
1.373(2)
1.365(2)
1.279(2)
1.380(2)
1.339(2)
1.687(2)
1.313(2)
116.9(2)
122.4(2)
122.8(2)
115.0(1)
121.1(1)
118.8(1)
118.9(2)
122.3(1)
−0.1 (3)
179.6 (2)
179.5(2)
−179.1(2)
−179.6(2)
180.0(2)
-0.3(3)
1.414(4)
1.366(3)
1.368(3)
1.284(3)
1.382(3)
1.358(3)
1.676(3)
1.319(4)
1.447(4)
118.2(2)
122.2(2)
123.8(2)
114.6(2)
121.4(2)
118.2(2)
117.5(2)
124.3(2)
124.3(2)
6.8 (5)
1.340(2)
1.285(2)
1.373(2)
1.350(2)
1.672(2)
1.323(2)
1.449(3)
1.492(4)
123.2(2)
121.4(2)
116.4(2)
120.2(2)
119.6(2)
116.7(2)
123.7(2)
124.5(2)
110.4(2)
179.7(2)
178.6(2)
−1.4 (3)
−1.9 (3)
178.2(2)
179.5 (2)
−177.4 (2)
−6.7 (3)
173.4 (2)
175.0 (2)
−5.0 (3)
179.4 (2)
C1
−
O1
C1
−
O1
C8
−
O1
O1
−
C2
O2
O1
−
C2
O2
N1
C11
−
N1
C5
C8
N1
N2
−
C5
C8
−
N1
−
N2
−
N1
N2
−
N2
−
C12
−
N1
N2
−
N2
C12
C12
−
S1
−
C9
−
C9
−
N3
C9
C9
C1
O2
−S1
C9
C9
−S1
N3
C13
O1
−
C13
C14
C8 C7
C11 N1
N1 N2
N2 C12
−
N3
O1
C5
C8
N1
N2
C9
C9
C9
O1
O1
C2
C2
C4
C6
C6
C6
C6
C8
N1
N2
N2
−N3
−
−
−
C2
C6
N1
N3
−
C10
−
−
−
−
C1
−
O1
C5
C8
N1
N2
C9
C9
C9
−
C2
C4
N1
C7−
−
C6
C8
−
−
O2
−
−
C11
−
−
−
−N2
C4
C8
−
−
N1
N2
N2
−
−
N1
N2
N2
−
−
C9
−
−N2
−
C12
C12
−
S1
−
−S1
N1
N2
N2
−
−
C9
S1
−
−
N3
−
−N3
−
−
S1
−
C12
N3
C13
C8
−N3
S1
−
−
N3
−
−N3
C12
−
−
C13
C14
C1
C1
O1
O1
−
−
C2
C2
C3
C7
−
C7
C3
C4
C6
O2
O2
O2
N1
N1
N2
S1
C9
C1
C1
−
−
N3
N3
−
−
−
−
−
−
−
−
−
N3
O1
O1
−C10
O1
−
C9
C8
C8
C11
C11
N1
−
C10
−
−
−
−
−C2
−C2
−C7
−C3
−
C3
C7
C6
C4
O2
O2
N1
N1
N2
C9
N3
S1
C6−
C7
−
−
O1
−173.9 (3)
179.9 (3)
179.4 (3)
179.4 (3)
2.7 (4)
−
−
−
C11
−
C7
−
−O1
−N1
−N1
C3
C7
C8
C5
C7
C6
C8
N1
N1
−
−
C5
C5
C5
C8
C8
−
O1
O1
−
C2
−
C8
C6
C7
−
−
−
C7
−
−
−
−
−
−
−
−
−
C2
−
C7
−
−
−
C7
C8
C5
C3
C4
C8
−
C6
C4
C4
C4
C8
−
−
−
−
−
C5
C5
C8
C8
N1
−
C11
N1
C12
C12
C12
C12
C13
−
−
N2
C12
N1
N1
1.1 (3)
−
−
−
−
−
−
−
−
C11
−
−
−
N2
N2
−
−179.1(2)
−178.9 (2)
177.0 (2)
7.2 (3)
2.4 (4)
−
−
−
N3
−
−
−174.8 (3)
173.3 (2)
−175.1 (3)
−13.3 (4)
166.3 (2)
−
N1
N2
−
−
S1
−
−
N2
−
−
−
−
C9
N3
S1
−
N2
−
−
N3
−C13
−
−
C9
−
−
N1
N2
N2
−N2
−
S1
−
−
N3
−C13
−174.0 (1)
−
−
C9
N1
N1
−
−
C9
−
C14
−
−N3−C12
−
−
C9
−

ACCEPTED MANUSCRIPT
179.6 (3)
0.0 (5)
N2
S1
−
C9
−
N3
−
C10
−C9
−
N3
−
C10
Table 3. Intra- and inter-molecular hydrogen bonds
H...A (Å)
D...A (Å)
D−H...A
D−H (Å)
D−H...A (°)
1
0.82
0.86
0.86
1.96
2.36
2.60
2.680(2)
2.691(2)
3.366(2)
145
103
148
O2
N3
N2
−
H2A…N1
H3A...N1
H2...S1*
−
−
* 1-x,1-y,-z
−
H3A...S1#
0.86
0.86
2.66
2.10
3.268(2)
2.958(2)
129
172
N3
# 1-x,-1/2+y,1/2-z
N3−
H3B...O2@
@ 1-x,1/2+y,1/2-z
2
0.82
0.86
0.86
2.00
2.33
2.34
2.711(3)
2.699(3)
3.038(3)
145
106
139
O2
N3
N2
−
H2A...N1
H3A...N1
H2...O2*
−
−
* x, y, -1+z
C1−
H1B...O1#
0.96
2.52
3.243(4)
132
# x,1/2-y,-1/2+z
7
0.82
0.86
0.86
1.87
2.26
2.58
2.593(3)
2.638(4)
3.393(3)
146
107
158
O1
N3
N2
−
H1…N1
H3A…N1
H2A…S1*
−
−
* -x,y,-1/2-z

ACCEPTED MANUSCRIPT
Table 4. Docking energy, score and interactions of thiosemicarbazone derivatives at the active
site of PLA2.
Hydrogen bonding &
Ca2+ interactions
Hydrophobic
Energy
(kcal/mol)
Compound
Score
interactions
Residues Distance (Å)
(1-benzyl-5-
methoxy-2-
methyl-1H-indol-
3-yl)-acetic acid
(co-crystal
Phe5, His6, Ala17,
Ala18, Tyr21, Gly22,
Gly29, Val30, Phe98
Ca2+
His47
2.57
2.86
-7.551
-50.522
ligand)
Tyr21, Gly22, His27,
Cys28, Gly29, Tyr51,
Lys62
Ca2+
Cys44
Ca2+
Tyr 21
Gly29
Asp48
2.53, 3.11
2.70
-4.655
-5.052
-35.401
-38.343
1
2
3.12
2.62
3.28
2.75
Leu2, Phe5, His6,
Gly22, Cys44, His47
Phe5, Ile9, Ala17,
Ala18, Gly22, Cys28,
Val30, Cys44, Tyr51,
Lys62
Tyr 21
His47
Asp48
2.99
2.97
2.45
-5.558
-38.777
3
Phe5, Ile9, Ala17,
Ala18, Tyr21, Gly22,
Cys28, Cys44, Tyr51,
Phe98
Tyr21, Gly22, Cys28,
Gly29, Val30, Lys62,
Phe98
His47
Asp48
2.86
2.71
-7.023
-4.935
-5.235
-37.901
-35.625
-37.366
4
5
6
Ca2+
Cys44
2.46, 3.05
2.95
Ca2+
Cys44
His27
Glu55
Ca2+
His27
Cys44
Glu55
2.48
2.93
3.04
3.23
2.40
3.03
2.77
3.21
Phe5, Tyr21, Cys28,
Gly29, Tyr51, Lys62
Phe5, Cys28, Gly29,
Asp48, Tyr51, Lys62
-5.848
-6.865
-43.036
-42.453
7
8
Tyr21, Gly22, Cys28,
Gly29, Val30, Tyr51,
Glu55, Lys62, Phe98
Ca2+
Cys44
2.61
2.62