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

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

Accepted Manuscript In vitro antioxidant, anti-inflammatory and in silico molecular docking studies of thiosemicarbazones G.R. Subhashree, J. Haribabu...

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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

DOI:

10.1016/j.molstruc.2017.05.054

Reference:

MOLSTR 23795

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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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, 1

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R. Karvembu2,*, D. Gayathri5,* 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,

North-East Institute of Science and Technology (CSIR), Branch Laboratory, Imphal, Manipur795004, India

5

Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600025, India

$

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4

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Maduravoyal, Chennai 600095, India

Equal contribution

* [email protected], [email protected]

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Abstract

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

5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde

and

(un)substituted

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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

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(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

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 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

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Introduction

Thiosemicarbazones belong to a group of thiourea derivatives, which have been studied

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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

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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

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thiosemicarbazones against human immuno deficiency virus (HIV) was reported [9]. The presence of bulky substitution at the Ν−terminal nitrogen atom of the thiosemicarbazones

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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 3aminopyridine-2-carboxaldehyde thiosemicarbazone (triapine) [15]. Triapine has entered phase II clinical trials as a chemotherapeutic agent [16]. Another promising drug, di-2-pyridylketone 4cyclohexyl-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

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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 Shimadzu2600 spectrophotometer. NMR spectra were recorded in DMSO-d6 by using TMS as an internal

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standard on a Bruker 500 (1H) or 125 (13C) MHz spectrometer. The (Z)-2-((2hydroxynaphthalen-1-yl)methylene)-N-methylhydrazinecarbothioamide (6) was synthesized by using a reported procedure [19].

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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

crystals of 1, 2 and 7.

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and hexane, and dried in vacuum. It was recrystallized from dimethylformamide/acetone to get

(E)-2-(2-hydroxy-5-methoxybenzylidene)hydrazinecarbothioamide (1) 2-Hydroxy-5-methoxybenzaldehyde (0.152 g, 0.001 mol) and thiosemicarbazide (0.911

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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

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(DMF): λmax, nm 260, 329. FT-IR (KBr): ʋ, cm-1 3450 (OH), 3346, 3221 (N–H), 1575 (C=N), 1270 (C=S). 1H 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).

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C 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), 1584 (C=N), 1264 (C=S). 1H 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,

3H, OCH3), 3.03 (d, J = 4.4 Hz, 3H, CH3).

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aromatic), 6.84 (dd, J = 8.8, 2.9 Hz, 1H, aromatic), 6.80 (d, J = 8.1 Hz, 1H, aromatic), 3.74 (s, C 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]+.

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(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.

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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), 1569 (C=N), 1264 (C=S). 1H 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, OCH3), 3.70 (q, J = 6.9 Hz, 2H, CH2CH3), 1.18 (t, J = 7.0 Hz, 3H, CH2CH3).

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C NMR (125

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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

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(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,

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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, 3H, OCH3). 13C 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):

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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

C NMR (125 MHz, DMSO-d6): δ, ppm 179.0

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aromatic), 7.21 (d, J = 7.1 Hz, 1H, aromatic).

(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]+.

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(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), 1251 (C=S). 1H NMR (500 MHz, DMSO-d6): δ, ppm 11.68 (s, 1H, OH), 10.63 (s, 1H, NHCS),

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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, CH2CH3).

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C NMR (125 MHz, DMSO-d6): δ, ppm 180.4 (C=S), 156.3 (C=N), 144.7, 139.6,

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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)

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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, aromatic), 7.19 (d, J = 7.2 Hz, 1H, aromatic).

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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

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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

Antioxidant assay - DPPH free radical scavenging

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ellipsoidal plot, intra- and inter-molecular hydrogen bonds are represented using PLATON [22].

The antioxidant activity of the thiosemicarbazone derivatives was evaluated using the

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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

Anti-inflammatory studies

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compounds were elucidated by haemolysis assay [25].

Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids at sn-2 position of

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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

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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

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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

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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

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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 RCSBprotein 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

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site of PLA2 was represented by Ligplot using PDBSUM [33]. Surface view model was generated using PyMol [34].

Results and discussion

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Synthesis and characterization

The biologically active 5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde based (1-8)

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thiosemicarbazones

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 different spectroscopic (UV-visible, FT-IR, 1H, 13C 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,

dimethylsulfoxide. The compounds were air and light stable.

dimethylformamide

and

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Scheme 1. Synthetic route of the 5-methoxysalicylaldehyde appended thiosemicarbazone

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derivatives.

Scheme 2. Preparation of the 2-hydroxy-1-naphthaldehyde appended thiosemicarbazone derivatives.

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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

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[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]. In the 1H 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 7.477.79 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-

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1.35 ppm in the spectra of 3 and 7 attributed to the presence of CH2 and CH3 protons [25]. 13C 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 56.056.3 ppm [38]. The methyl and ethyl carbons present in complexes 2, 3 and 7 exhibited signals at

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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

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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

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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,

the

molecular

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hydrazinecarbothioamide group was almost planar with maximum deviation of −0.064(2) Å and structure

was

nearly

planar

with

dihedral

angle

between

the

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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

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one N−H...O inter-molecular hydrogen bonding interactions. Atoms N2 and N3 act as donors to S1 at 1-x, 1-y, -z and 1-x, -1/2+y, 1/2-z generating centrosymmetric dimer of R22(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

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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

centrosymmetric dimer of R22(8). Intra- and inter-molecular hydrogen bond parameters are listed

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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.

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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

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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

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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

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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

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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

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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

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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%

In silico molecular docking

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of PLA2 activity at 100 µM concentration.

In addition to in vitro screening of anti-inflammtory potential of thiosemicarbazone derivatives, molecular docking studies were also performed to screen the anti-inflammtory

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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

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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 elemental analysis and spectroscopic (FT-IR, UV-visible, 1H NMR,

13

C NMR and mass)

techniques. Crystal structure of representative compounds (1, 2 and 7) was determined by X-ray

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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

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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

pharmaceutical applications.

Acknowledgements

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thiosemicarbazones at the active site of PLA2, which can be further explored for their

JH thank the University Grants Commission (UGC), Government of India for financial

References

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support. DG thank DBT for Bioinformatics infrastructure facility at University of Madras.

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[34] The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger LLC. [35] K. Jeyalakshmi, N. Selvakumaran, N.S.P. Bhuvanesh, A. Sreekanth, R. Karvembu, RSC Adv. 4 (2014)17179-17195.

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[36] R.N. Prabhu, R. Ramesh, Tetrahedron Lett. 54 (2013) 1120-1124. [37] P. Kalaivani, R. Prabhakaran, E. Ramachandran, F. Dallemer, G. Paramaguru, R. Renganathan, P. Poornima, V. Vijaya Padma, K. Natarajan, Dalton Trans. 41 (2012) 2486-

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2499.

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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.

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Fig. 2a. Thermal ellipsoidal plot of 2 at 30% probability.

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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

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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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was 1,2-dithio analog of diheptanoyl phosphatidylcholine.

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Fig. 6. Percentage of PLA2 activity of the thiosemicarbazone compounds (1-8). Substrate used

Fig.

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7.

Ligplot

representation

showing the interactions of co-crystal ligand

thiosemicarbazone derivatives at the active site of PLA2.

and

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

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Table 1. Crystal data and structure refinement parameters 2 1503336 C10H13N3O2S 239.29

7 1503335 C14H15N3OS 273.35

Crystal system

Monoclinic

Monoclinic

Monoclinic

space group

P21/c

P21/c

Temperature (K)

293

293

a = 7.4847 (7)

a = 7.2023 (5)

b = 9.9713 (8)

b = 24.7874 (16)

b = 7.033 (5)

c = 14.3553 (10)

c = 6.3036 (4)

c = 18.853 (5)

C2/c

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293

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Unit cell dimensions (Å, °)

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CCDC number Chemical formula Mr

1 1503334 C9H11N3O2S 225.27

a = 26.511 (5)

β = 94.263 (4)

β = 129.707 (5)

V (Å3) Z, Dx (Mg m−3) Absorption coefficient, µ (mm-1) F(000)

1070.78 (15) 4, 1.397 0.29 472

1122.24 (13) 4, 1.416 0.28 504

2704 (2) 8, 1.343 0.24 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 θmax, θmin (°)

Fine-focus sealed tube 0.71073 BRUKER Kappa APEXII CCD 30.6, 2.5 29.1, 1.6

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β = 91.892 (3)

31.6, 2.0

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No. of measured, independent

14838, 2955,

reflections

2160

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and observed [I > 2σ(I)]

Rint h k l Absorption correction Tmin, Tmax Refinement method No. of parameters (sin θ/λ) max (Å−1) R [F2 > 2σ(F2)], wR(F2)

21885, 2929, 2165

0.026 0.035 -10 → 10 -9 → 8 -13 → 13 -33 → 33 -20 → 20 -8 → 8 multi-scan, SADABS (Bruker 2004) 0.692, 0.746 0.658, 0.746 Full matrix least-squares on F2 138 148 0.715 0.683 0.042, 0.133 0.055, 0.163

25639, 4144, 2478 0.036 -33 → 38 -10 → 10 -27 → 22 0.669, 0.746 174 0.737 0.059, 0.218

ACCEPTED MANUSCRIPT Goodness of fit on F2 (S) ∆ρmax, ∆ρmin (e Å−3)

1.07 0.32, −0.27

1.24 0.38, −0.46

1.12 0.43, −0.39

2

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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) −173.9 (3) 179.9 (3) 179.4 (3) 179.4 (3) 2.7 (4) 2.4 (4) −174.8 (3) 173.3 (2) −175.1 (3) −13.3 (4) 166.3 (2)

C8−O1 C11−N1 N1−N2 N2−C12 C12−S1 C12−N3 N3−C13 C13−C14 O1−C8−C7 C7−C11−N1 C11−N1−N2 N1−N2−C12 N2−C12−S1 N2−C12−N3 S1−C12−N3 C12−N3−C13 N3−C13−C14 O1−C8−C9−C10 C6−C7−C8−O1 C11−C7−C8−O1 C8−C7−C11−N1 C6−C7−C11−N1 C7−C11−N1−N2 C11−N1−N2−C12 N3−C12−N2−N1 S1−C12−N2−N1 N2−C12−N3−C13 S1−C12−N3−C13 C14−C13−N3−C12

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C1−O1 O1−C2 C5−O2 C8−N1 N1−N2 N2−C9 C9−S1 C9−N3 N3−C10 C1−O1−C2 O2−C5−C4 C4−C8−N1 C8−N1−N2 N1−N2−C9 N2−C9−S1 N2−C9−N3 S1−C9−N3 C9−N3−C10 C1−O1−C2−C3 C1−O1−C2−C7 O1−C2−C7−C6 O1−C2−C3−C4 C7−C6−C5−O2 C8−C4−C5−O2 C5−C4−C8−N1 C3−C4−C8−N1 C4−C8−N1−N2 C8−N1−N2−C9 N1−N2−C9−N3 N1−N2−C9−S1

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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) 1.1 (3) −179.1(2) −178.9 (2) 177.0 (2) 7.2 (3) −174.0 (1)

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C1−O1 O1−C2 C5−O2 C8−N1 N1−N2 N2−C9 C9−S1 C9−N3 C1−O1−C2 O2−C5−C6 C6−C8−N1 C8−N1−N2 N1−N2−C9 N2−C9−S1 N2−C9−N3 S1−C9−N3 C1−O1−C2−C7 C1−O1−C2−C3 O1−C2−C3−C4 O1−C2−C7−C6 C3−C4−C5−O2 C7−C6−C5−O2 C8−C6−C5−O2 C5−C6−C8−N1 C7−C6−C8−N1 C6−C8−N1−N2 C8−N1−N2−C9 N1−N2−C9−N3 N1−N2−C9−S1

7

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1

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Table 2. Selected geometric parameters (Å, °)

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)

ACCEPTED MANUSCRIPT N2−C9−N3−C10 S1−C9−N3−C10

179.6 (3) 0.0 (5)

D−H...A 1

D−H (Å)

H...A (Å)

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Table 3. Intra- and inter-molecular hydrogen bonds

D...A (Å)

D−H...A (°)

0.82

1.96

2.680(2)

145

N3−H3A...N1

0.86

2.36

2.691(2)

103

N2−H2...S1* * 1-x,1-y,-z

0.86

2.60

3.366(2)

148

N3−H3A...S1# # 1-x,-1/2+y,1/2-z

0.86

N3−[email protected] 1-x,1/2+y,1/2-z

0.86

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O2−H2A…N1

2.66

3.268(2)

129

2.10

2.958(2)

172

2.00

2.711(3)

145

0.86

2.33

2.699(3)

106

0.86

2.34

3.038(3)

139

0.96

2.52

3.243(4)

132

0.82

1.87

2.593(3)

146

N3−H3A…N1

0.86

2.26

2.638(4)

107

N2−H2A…S1* * -x,y,-1/2-z

0.86

2.58

3.393(3)

158

@

2 O2−H2A...N1

C1−H1B...O1# # x,1/2-y,-1/2+z 7

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O1−H1…N1

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N2−H2...O2* * x, y, -1+z

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N3−H3A...N1

0.82

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Table 4. Docking energy, score and interactions of thiosemicarbazone derivatives at the active site of PLA2.

1

2

-4.655

-5.052

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-7.551

Residues

Distance (Å)

-50.522

Ca2+ His47

2.57 2.86

-35.401

Ca2+ Cys44

Tyr21, Gly22, His27, Cys28, Gly29, Tyr51, Lys62

-38.343

Ca2+ Tyr 21 Gly29 Asp48

3.12 2.62 3.28 2.75

Leu2, Phe5, His6, Gly22, Cys44, His47

-38.777

Tyr 21 His47 Asp48

2.99 2.97 2.45

4

-7.023

-37.901

His47 Asp48

2.86 2.71

5

-4.935

-35.625

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

Ca2+ Cys44

2.61 2.62

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-5.558

-5.235

-37.366

7

-5.848

-43.036

8

-6.865

-42.453

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Phe5, His6, Ala17, Ala18, Tyr21, Gly22, Gly29, Val30, Phe98

2.53, 3.11 2.70

3

6

Hydrophobic interactions

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(1-benzyl-5methoxy-2methyl-1H-indol3-yl)-acetic acid (co-crystal ligand)

Score

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Compound

Energy (kcal/mol)

Hydrogen bonding & Ca2+ interactions

Phe5, Ile9, Ala17, Ala18, Gly22, Cys28, Val30, Cys44, Tyr51, Lys62 Phe5, Ile9, Ala17, Ala18, Tyr21, Gly22, Cys28, Cys44, Tyr51, Phe98 Tyr21, Gly22, Cys28, Gly29, Val30, Lys62, Phe98 Phe5, Tyr21, Cys28, Gly29, Tyr51, Lys62

Phe5, Cys28, Gly29, Asp48, Tyr51, Lys62 Tyr21, Gly22, Cys28, Gly29, Val30, Tyr51, Glu55, Lys62, Phe98