Discovery of novel Methylsulfonyl phenyl derivatives as potent human Cyclooxygenase-2 inhibitors with effective anticonvulsant action: Design, synthesis, in-silico, in-vitro and in-vivo evaluation

Accepted Manuscript Discovery of novel Methylsulfonyl phenyl derivatives as potent human Cyclooxygenase-2 inhibitors with effective anticonvulsant action: Design, synthesis, in-silico, in-vitro and in-vivo evaluation Chandra Bhushan Mishra, Shikha Kumari, Amresh Prakash, Rajesh Yadav, Ankit Kumar Tiwari, Preeti Pandey, Manisha Tiwari PII:

S0223-5234(18)30334-9

DOI:

10.1016/j.ejmech.2018.04.007

Reference:

EJMECH 10356

To appear in:

European Journal of Medicinal Chemistry

Received Date: 23 November 2017 Revised Date:

28 February 2018

Accepted Date: 2 April 2018

Please cite this article as: C.B. Mishra, S. Kumari, A. Prakash, R. Yadav, A.K. Tiwari, P. Pandey, M. Tiwari, Discovery of novel Methylsulfonyl phenyl derivatives as potent human Cyclooxygenase-2 inhibitors with effective anticonvulsant action: Design, synthesis, in-silico, in-vitro and in-vivo evaluation, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.007. 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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Discovery of Novel Methylsulfonyl Phenyl Derivatives as Potent Human

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Cyclooxygenase-2 Inhibitors with Effective Anticonvulsant Action: Design,

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Synthesis, In-silico, In-vitro and In-vivo evaluation

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Chandra Bhushan Mishra1, Shikha Kumari1, Amresh Prakash2, Rajesh Yadav1, Ankit Kumar Tiwari1,

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Preeti Pandey2, Manisha Tiwari1*

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Delhi, Delhi-110007, India.

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India.

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Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research, University of

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School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi,

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*Corresponding Author

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[email protected] (Dr. Manisha Tiwari)

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Abstract

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A novel series of methylsulfonyl phenyl derivatives has been designed and synthesized to

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evaluate their COX-2 inhibitory activity along with anti-convulsant potential. In-vitro evaluation

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revealed that two compounds MTL-1 and MTL-2 appeared as most potent and selective COX-2

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inhibitors in the entire series. Anti-convulsant activity of both potent COX-2 inhibitors was

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assessed in sc-PTZ induced seizure test and MTL-1 excellently protected animals against PTZ

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induced seizure at the dose of 30 mg/kg. MTL-1 also indicates long duration of action in time

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course study and displayed significant seizure protection up to 6h of drug administration.

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Further, the anti-epileptogenic effect of MTL-1 has been examined in PTZ induced chronic

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model of epilepsy. The results indicated that MTL-1 had a significant anti-epileptogenic effect in

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PTZ kindled rats as compared to Etoricoxib (ETX) and PTZ alone treated group. Additionally,

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MTL-1 successfully improved cognition deficit in PTZ kindled rats, which were confirmed by

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social recognition, novel object recognition and light-dark chamber tests. Moreover, molecular

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docking and molecular simulation (MD simulation) studies were also performed to elucidate the

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interaction of MTL-1 with the active site of COX-2 and results showed that MTL-1 suitably

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binds within active site of COX-2. To investigate the safety profile of MTL-1, a sub-acute

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toxicity study was also performed and MTL-1 emerged as a new non-toxic chemical entity.

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Thus, the present investigation discovered a potent and safe COX-2 inhibitor, which is endowed

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with an effective anti-epileptic action.

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1. Introduction

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Epilepsy is a common and complex neurological disorder which affects approximately 65

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million people worldwide [1]. The disease is symptomized by recurrent and spontaneous seizures

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and caused due to uncontrolled synchronous and rhythmic firing of central nervous system (CNS)

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neurons which leads to sensory and motor dysfunctions [2]. Clinical evidences concluded that

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epilepsy is a group of more than 30 types of epileptic syndromes containing 15 different types of

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seizures [3]. The clinical hallmark of epilepsy is the continual emergence of sudden and

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unexpected seizures [4]. It is well studied that numerous neurotransmitters or neuromodulators

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have been shown to play a crucial role in the development of epilepsy [5]. Cyclooxygenase

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(COX) is also known as prostaglandin-endoperoxide synthase, exist in two isoform, COX-1 as

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well as COX-2 and is the key enzyme that converts arachidonic acid to prostaglandin, which

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impart crucial role in signaling and inflammation [6]. COX-1 is expressed in many tissues and

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prostaglandins (PGs) produced by COX-1 mediate the “housekeeping” functions like platelet

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aggregation, renal blood flow regulation and cytoprotection of gastric mucosa. On the other side,

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COX-2 is not present in most normal tissues, but its expression is induced by various stimuli

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such as lipopolysaccharides, proinflammatory cytokines (IL1b, TNFα), growth factors (fibroblast

48

growth factor, platelet-derived growth factor, epidermal growth factor), mitogens and oncogenes

49

(phorbol esters), hormones (luteinizing hormone) and disorders of water-electrolyte hemostasis,

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resulting in increased synthesis of PGs in inflamed and neoplastic tissues.

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In the brain, COX-2 isoform is predominantly expressed in glutamatergic neurons, particularly

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within cortex and hippocampus, the main area prone to onset of epileptic seizures. It was

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observed that expression of COX-2 is noticeably enhanced in the pyramidal cells of the

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hippocampus of kindled animals as compared to control [7]. Additionally, elevated expression of

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COX-2 and brain prostaglandin E2 (PGE2) levels was also noticed in lithium chloride and

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tacrine induced status epilepticus seizure models [8]. Dhir et al. studied the possible mechanism

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and suggested that COX-2 inhibitors protect PTZ induced seizure probably through the

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GABA/Benzodiazepine receptor mechanism [9]. Till date, several selective COX-2 inhibitors

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have been developed and some of them such as Celecoxib, Rofecoxib, Valdecoxib and

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Etoricoxib are being successfully used clinically [10]. These COX inhibitors also have shown

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promising anticonvulsant activity in pentylenetetrazole (PTZ) induced acute and chronic model

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of epilepsy [7]. However, some of them have low selectivity towards COX-2, poor

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pharmacokinetic properties and severe liver toxicity after long time use [11]. Therefore, it is

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mandatory to discover more potent and very selective COX-2 inhibitor with minimum toxicity.

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Methylsulfonyl (−SO2CH3) containing molecules have a successful history to discover potent

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COX-2 inhibitors and numerous clinically approved COX-2 inhibitors such as Rofecoxib,

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Etoricoxib and SC57666 (Fig.1) are examples which contain this functional group [10, 12]. By

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installing methylsulfonyl on the benzene ring numerous potent COX-2 inhibitors have been

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synthesized and tested for their COX-2 inhibitory activity [10]. Additionally, urea and thiourea

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groups appeared to be versatile, functional groups and widely used to develop various potent

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biologically active molecules, including COX-2 inhibitors as well as anti-convulsants [13, 14].

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Keeping these valuable properties of methylsulfonyl benzene, urea and thiourea functional

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groups

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(phenylcarbamothioyl)hydrazinecarboxamide derivatives where, methylsulfonyl benzene is

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linked with substituted phenyl by urea and thiourea linker. Synthesized derivatives were

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screened for their COX-2 inhibitory potential and their selectivity was assessed over COX-1

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isoform. Further, the most potent and selective COX-2 inhibitors MTL-1 and MTL-2 were

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selected for examining their anticonvulsant potential. Firstly, MTL-1 and MTL-2 were assessed

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for its anti-convulsant activity against sc-PTZ induced seizure mice model. A time course study

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was also performed to evaluate time depended anticonvulsant activity of MTL-1. Additionally,

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MTL-1 was evaluated against PTZ induced chronic model (kindled model) of epilepsy to

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confirm its anti-epileptogenic potential. Moreover, the toxicity profile of MTL-1 has been

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observed in sub-acute toxicity rat model. Furthermore, cognition enhancing ability of MTL-1 has

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been evaluated in social recognition, object recognition and light-dark test. The molecular

aimed

to

design

a

novel

series

of

N-(4-(methylsulfonyl)phenyl)-2-

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interaction of MTL-1 with COX-2 has been investigated by performing molecular docking and

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molecular dynamic simulation (MD simulation) studies. 2. Result and discussion

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2.1. Synthesis Synthesis of the designed compounds 4-14 have been synthesized according to scheme 1. Briefly, an equimolar amount of 4-(methylsulfonyl) aniline 1 reacted with an equimolar amount

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of phenylchloroformate which produced carbamate derivative 2. Further, carbamate derivative 2

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was refluxed with hydrazine hydrate to give key intermediate N-(4-(methylsulfonyl) phenyl)

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hydrazinecarboxamide 3. Finally, target compounds 4-14 were obtained by reacting key

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intermediate 3 with substituted isothiocyanates in high yields. Synthesized compounds were fully

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characterized by NMR spectroscopy, mass spectroscopy and elemental analysis.

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R-Group Phenyl 4-Methylphenyl 2, 6 Dimethylphenyl 4-Methoxyphenyl 4-Fluorophenyl 2, 6 Difluorophenyl

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Compound Number 4 5 6 7 8 9

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Compound Number 10 11 12 13 14

R-Group 4-Chlorophenyl 2, 3 Dichlorophenyl Benzyl Phenylethyl Ethyl

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Scheme1: Reagent & conditions: A. Phenyl chloroformate, dryTHF, pyridine, RT, 14h; B. Hydrazine hydrate, 1, 4-dioxane, reflux, 6h. C, substituted isothiocyanate, dry ACN: ethanol, reflux, 8-10h

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2.2. In-vitro COX inhibition study and Structure activity relationship (SAR) analysis

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Results of in-vitro COX inhibition assay of synthesized compounds 4-14 have been shown in

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table-1. SAR investigation revealed that all derivatives have shown selective activity against

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COX-2 isoform and these compounds appeared inactive for COX-1 isoform at the concentration

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of 10 µM. A compound containing unsubstituted phenyl ring (compound 4) showed the medium

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class of inhibitory activity towards COX-2 (IC50 = 12.80 µM). Installation of an electron

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donating group like methyl group on the phenyl ring (MTL-1) profoundly increased inhibitory

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activity towards COX-2 and shown an IC50 value 2.15µM. Further, substitution with dimethyl

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group on the phenyl ring (MTL-2) also showed similar type of inhibition. Next, the 4- methoxy

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group substitution on the phenyl ring (Compound 7) decreased the inhibitory activity against

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COX-2 as compared to methyl substitution and this derivative displayed an IC50 of 7.15 µM.

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Substitution with a strong electronegative groups like fluoro (Compound 8) provided medium

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range of inhibition (IC50 = 12.49 µM). However, di-fluoro substitution (Compound 9) yielded a

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more potent inhibitor of COX-2 (IC50 = 6.42 µM) as compared to mono fluoro substitution.

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Chloro substitution (Compound 10) on phenyl ring provided active inhibitor for COX-2 isoform

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with an IC50 of 5.32 µM. However, 2, 3 dichlorobenzene (compound 11), benzyl (compound 12),

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phenylethyl (compound 13) and ethyl derivative (compound 14) also did not show satisfactory

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inhibitory activity against COX-2.

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Thus, the SAR study clearly indicates that an electron donating group containing phenyl

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derivatives appear to be potent derivatives and especially 4-methyl substitution on the phenyl

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ring (MTL-1) appeared worthy, which displayed most potent inhibitor in the entire series.

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Table1 In vitro inhibition data of compounds 4-14 against COX-1 and COX-2

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Compounds 4 5 (MTL-1) 6 (MTL-2) 7 8 9 10 11 12 13

% inhibition (10 µM) COX-1 COX-2 b n.a 50 n.ab 75 b n.a 64 n.ab 62 b n.a 54 n.ab 62 b n.a 70 n.ab 46 b n.a 21 n.ab 22

IC50(µM)a COX-2 12.80 2.15 3.14 7.15 12.49 6.42 5.32 n.dc n.dc n.dc 5

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14 Etoricoxibd[15]

n.ab -

n.dc 1.1

32 -

128 129 130 131 132 133 134 135

2.3. Subcutaneous Pentylenetetrazole (sc-PTZ) seizure test

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Pentyleneterazole (PTZ) is a well-known chemo-convulsant and binds to the gamma-amino

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butyric acid ‘A’ subtype (GABA-A) receptor in the brain [16]. The sc-PTZ seizure test is well

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documented test to identify the anticonvulsant potential of drug candidates in pre-clinical stage

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of anti-epileptic drug discovery. Most potent COX-2 inhibitors MTL-1 and MTL-2, along with

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two standards Etoricoxib (selective COX-2 inhibitor) and Sodium Valproate (potent anti-

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epileptic drug) were evaluated in sc-PTZ induced acute epilepsy mice model (Table 2). Results

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have shown that MTL-1 prominently inhibited sc-PTZ induced convulsions at the dose of 30

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mg/kg and displayed 83% protection in both time intervals (0.5 and 4h). Additionally, MTL-1

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showed similar protection activity at a dose of 300 mg/kg. However, at the dose of 100 mg/kg

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protection percentage slightly reduced at 0.5h time interval. Another COX-2 inhibitor, MTL-2

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did not displayed promising seizure protection against sc-PTZ induced seizures and only at high

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dose (300 mg/kg) it showed 33 and 50% protection at 0.5 and 4h time intervals, respectively.

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Standard COX-2 inhibitor Etoricoxib showed satisfactory protection from seizure and displayed

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67 % protection at both time intervals. However, standard anti-epileptic drug sodium valproate at

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300 mg/kg has shown 100 and 50% protection at 0.5 and 4h time intervals, correspondingly.

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Thus, these observations point out that MTL-1 exhibited excellent protection in sc-PTZ induced

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seizure model as compared to standard drug Etoricoxib. In quantitative anticonvulsant studies,

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MTL-1 has also shown an ED50 value of 11.32 mg/kg in this model.

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IC50 values, means of three independent experiments, represent the concentration required produce 50% enzyme inhibition. b Not active, no inhibition was found at 10 µM of test compound. c Not determined. d value taken from reference 15.

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Table 2: In-vivo anticonvulsant screening of MTL-1, MTL-2, Etoricoxib and Sodium Valproate

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4hb

Compounds

161

(Dose in mg/kg)

P/Ta

% protection

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MTL-1(30)

5/6

83

5/6

163

MTL-1(100)

4/6

67

5/6

MTL-1 (300)

5/6

83

5/6

MTL-2 (30)

1/6

17

MTL-2 (100)

2/6

33

MTL-2 (300)

2/6

Etoricoxib (1)

3/6

Etoricoxib (5)

4/6

Etoricoxib (10)

3/6

P/Ta

a

6/6

83

1/6

17

2/6

33

33

3/6

50

50

3/6

50

67

4/6

67

50

3/6

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100

3/6

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P/T denotes number of protected animals verses total number of animals tested. Time intervals after drug administration.

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dose of 30mg/kg in sc-PTZ induced seizure model and seizure score was documented up to 6h

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after drug administration (Table. 3). Results indicated that MTL-1 has efficient capability to

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protect seizures with longer duration of action. MTL-1 has shown 83-67 % seizure protection up

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to 2h of drug administration. Further, the efficacy of MTL-1 was enhanced and percentage

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protection reached up to 83 % after 3-4 h of drug treatment. It was observed that MTL-1 restored

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protection up to 67 % after 6h of drug administration, which indicates its longer duration of 7

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action against seizure episodes. Hence, with this investigation MTL-1 appeared as potent anti-

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convulsant agent that endowed with longer duration action.

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Table. 3 Time course evaluation of MTL-1 in sc-PTZ seizure test with six Swiss Albino mice

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each time

Percentage seizure protection 83 67 67 83 83 67

SC

0.5 1 2 3 4 6

Protected/ Number of animals used 5/6 4/6 4/6 5/6 5/6 4/6

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2.5. Evaluation of MTL-1 in PTZ induced chronic model of epilepsy (kindling model)

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epileptogenic processes in the brain. PTZ is a potent chemo-convulsant and it increases activities

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in major epileptogenic centers of the forebrain like the amygdala and the piriform cortex [17].

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Kindling is described by repetitive administration of an initial sub-convulsive electrical or

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chemical stimulus, resulting in progressive multiplication of seizure activity, which culminates in

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generalized seizures that resembling human epileptic syndrome [18]. In the present study, PTZ

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was used to induce kindling in adult male Wistar rats to visualize anti-epileptogenic effect of

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compound MTL-1. A study conducted by Katyal J. et al. have shown that Etoricoxib (ETX)

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exerted anti-epileptogenic effect at low dose in Pentylenetetrazole-kindled rats [19]. Etoricoxib is a

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selective COX-2 inhibitor having anti-epileptiogenic potency; therefore ETX was chosen as a

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reference drug to compare the potency of test compound MTL-1. A total number of four groups

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(control; PTZ; PTZ+ ETX and PTZ+ MTL-1) were constituted. Chronic treatment for a period of

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28 days with sub-convulsive dose of PTZ (40 mg/kg) induced successful kindling in rats of all

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groups. Seizures were visualized manually by keeping the rats in individual cages after injecting

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PTZ for the period of 45 minutes. The resultant behavioral seizure activity was categorized

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according to revised Racine scale. Control group animals received only vehicle throughout the

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study and did not show any seizure like activity. The animals of PTZ group have initially

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test compound MTL-1 was administered i.p. to Swiss Albino mice (6 animals in each group).

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developed behavioral seizure patterns which were later recognized as secondary generalized

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seizures similar to the chronic epileptic disorder.

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It was clearly witnessed that animals belongs to PTZ group developed generalized tonic-clonic

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seizures gradually and significant difference (p<0.0001) was seen, when compared to control

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group animals (Fig. 2). In contrast, pretreatment with ETX in PTZ+ ETX group showed decreased

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(p<0.05) mean kindling score in comparison with PTZ group. Mortality was not observed in any

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group except PTZ group, in which two animals died due to severe tonic-clonic seizures followed

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by high pitch vocalization. However, more noticeable anti-seizure effect was shown by animals

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treated with test compound MTL-1 at the dose of 30 mg/kg. Our results have shown that MTL-1

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treated rats displayed a seizure score 1.5±0.98, 1.75±1.11, 1.79±0.93 and 2.12±1.23 at first,

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second, third and fourth week respectively as compared to ETX treated rats which were shown a

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seizure score 2.33±1.43, 2.42±1.11, 2.79±1.38 and 3.12±1.33 at first, second, third and fourth

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week, respectively.

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However, PTZ alone treated rats showed 2.70±1.27, 3.79±1.0, 4.54±0.83 and 5.08±0.58 seizure

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scores at first, second, third and fourth week respectively. Thus, these results clearly indicate that

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MTL-1 excellently reduced seizure scores as compared to ETX and PTZ alone treated rats which

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indicate strong anti-epileptic effect of MTL-1. Overall kindling results displayed that animals

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treated with compound MTL-1 pointedly (p<0.0001) attenuated seizure severity when compared to

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PTZ-kindled animals.

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2.6. Impact of MTL-1 on cognition deficit kindled rats

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Epilepsy affects the person in several aspects of life, including physical, mental and cognitive

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functions. Cognition deficit includes loss of intelligence, thinking, remembering and

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understanding [20]. According to a survey conducted by International Bureau of Epilepsy stated

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that 44% of patients with epilepsy complained of difficult learning, and 45% of slowness in

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thinking ability [21]. Therefore, anticonvulsant agents which have capability to improve

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cognition impairment along with anti-seizure activity may perform excellent management in

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epilepsy. Cognition enhancement potential of MTL-1 has been assessed in PTZ induced chronic

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model of epilepsy by performing well established social recognition, object recognition and

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light-dark test.

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2.6.1. Social recognition test

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A well recognized social recognition test has been accomplished to confirm cognition enhancing

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ability of MTL-1 in PTZ kindled rats [22]. Our results indicate that MTL-1 significantly reduced

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the T2 (second interaction trial) /T1 (first interaction time) ratio as compared to the vehicle

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which reflects the strengthening of short-term memory by MTL-1 (Fig. 3). The ratio of T2/T1 in

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MTL-1 group was 0.561± 0.139 as compared to PTZ group which has displayed T2/T1= 0.928 ±

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0.234. Thus, MTL-1 successfully ameliorated cognition deficit induced by chronic PTZ

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treatment, which evidently supports cognition enhancer potential of potent anticonvulsant agent

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MTL-1.

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2.6.2. Object recognition test

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An object recognition test is the most commonly used behavioral test and reflected as the crucial

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assessment to observe cognitive enhancer ability of pharmacological drugs [23]. In this behavioral

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test MTL-1 treated group also appeared as satisfactory cognition enhancer agents along with its

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anti-seizure effect (Fig. 4A and B). Animals treated with MTL-1 showed less percentage

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exploration preference for a similar object (3.9%) while, this percentage was increased for novel

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object (5.7%) as similar to control group animals which indicate MTL-1 significantly improved

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cognition impairment induced by PTZ.

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2.6.3. Light-dark test

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The light-dark test is widely used in pharmacology to assess cognition deficit/anxiety like behavior

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in animals and based on the native aversion of rodents to brightly illuminated areas [24]. The

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results of this experiment again advocate cognition boosting aptitude of MTL-1 and MTL-1 treated

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animals spent significantly higher time in light as compared to PTZ kindled animals (Fig. 5).

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Results showed that percentage time spent in light by MTL-1 treated group was 13.3 ± 4.6 s as

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compared to PTZ treated group which was 3.05± 1.25 s (Fig. 5A). The percentage time spent in the

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dark by MTL-1 treated group was 86.6 ± 4.6 s as compared to PTZ treated group and vehicle

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group which were shown 96.9± 1.25 and 80.6 ± 2.5 s respectively (Fig. 5B.).

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2.7. Molecular Docking study

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To determine the molecular interaction of MTL-1, a docking analysis was performed with the

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coordination of COX-2 (PDB ID: 6COX) using AutoDock 4.2. Results showed that MTL-1 is

278

preferentially accommodated at the cyclooxygenase active site of COX-2 (Fig. 6) and Arg120,

279

Ser353 and Tyr324 are involved in H-bond interaction with MTL-1. We observed that

280

methylsulfonyl benzene moiety of MTL-1 is deep merged in the lower hydrophobic channel,

281

surrounding with residues Val116, Leu117, Leu352, Tyr355, Leu359, Tyr385 and Ala527

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whereas, methyl phenyl of MTL-1 is protruded towards the upper hydrophobic domain of the

283

cavity and is engaged in hydrophobic interaction with residues: Leu352, Trp387, Ala516, Ile517

284

and Phe518.

285

2.8. Molecular dynamic simulation study

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To analyze the structural stability and molecular interaction of COX2-MTL-1 complex, a MD

287

simulation was carried out using biomolecular simulation package AMBER14 for the period of

288

250 ns at 300K. The dynamical change of all Cα backbone RMSD of COX-2 and COX-2-CX

289

complex is shown in Fig. 7A. The RMSD plot shows initial drop down in trajectory during 0-25

290

ns in water, however, change in RMSD of 1.5 Å is seen for 25-75 ns, which suggested the initial

291

perturbation in structure. The trajectory remains stable during 75-210 ns and small drift of 0.5 Å

292

is observed at 225 ns, although, it is settled quickly with the end of simulation at 250 ns, which

293

suggested the stable conformation of COX-2 in water at 300K. The RMSD trajectory of COX-2-

294

MTL-1complex shows relatively stable conformation as compared to protein in unbind state.

295

With the initial perturbation trajectory attained the equilibrium and remain stable ~85 ns of

296

simulation. We find a drift of 1.0 Å at 90 ns, which is settled after 100 ns, and slow but

297

continuous dropdown in trajectory is observed till the simulation is finished at 250 ns. The initial

298

change in confirmation suggested the spatial fitting of ligand at the active site. The stable RMSD

299

during 100-250 ns reveals the stable interaction of ligand at binding pocket of COX-2.The

300

average RMSD value 2.29±0.36 and 2.55±0.33 of COX-2 in free and bind state also indicated

301

the higher conformational stability of MTL-1 at the active site of COX-2 enzyme.

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2.7.1. Radius of gyration (Rg) determination

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Radius of gyration (Rg) is another important and valuable parameter to determine the structural

304

stability and compactness of protein. Herein, to analyze the structural integrity of protein-ligand

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complex we computed the time evolution plot of Rg backbone, as shown in Fig. 7B. As depicted

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from Fig. 6B, the structural entity of COX-2 is remained stable during the simulation of 250 ns

307

with the average Rg value 24.27±0.15 Å. We observed the initial change of 0.5 Å in Rg value of

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COX-2-MTL-1 during 0-50 ns of simulation, which is settled at 75 ns and equilibrium, is

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maintained till the simulation is finished at 250 ns. The average Rg value 24.46±0.16 Å of

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complex suggested that secondary and tertiary conformation of COX-2 is retained during the

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simulation, whereas, initial rise in Rg suggested the preferential binding of ligand at the active

312

site of COX-2.

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2.7.2. RMSF determination

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To determine the dynamic progression of COX-2 and COX-2-MTL-1, we also analyzed the

315

time–average RMSF plot of all C αatoms. As shown in Fig. 8, the average fluctuation of residues

316

having < 4-6Å, are belonging to stable conformation (α-helices and β-sheets), however, loop

317

region showed average fluctuation <6.0 Å. The structure of COX-2 consists of 40% α-helices

318

and 5% β-sheets, and loops are imbedded between the regular secondary conformations. The

319

RMSF plot of protein-ligand complex showed the least average fluctuation of residues are

320

belonging to α-helix-2 (His90), α-helix-4 (Val116, Ser119, Arg120), α-helix-7 (Asp190,

321

Gln192),α-helix-11 (His351, Leu352, Ser353),α-helix-13(Phe381, Leu384),α-helix-22 (Gly526,

322

Ala527, Ser530) and

323

suggested the coordination with ligand at the active site of COX-2.

324

2.7.3. Interaction energy (MM-PBSA) observation

325

To estimate the binding free energy of COX-2-MTL-1 complex, we used the MM-PBSA.py, a

326

compatible program with AMBER to compute various components of binding energy i.e. the

327

electrostatic, van der Walls, polar and non-polar solvation energy. Figure 9 shows that the

328

average values are converged in MMPBSA calculations. Although, we carried out MM-PBSA

329

calculations for the entire 250 ns, however, observing the convergence, results of last 50 ns of

330

simulation are shown here having binding free energy -45.02± 2.67 (Fig.9).

331

2.8. Sub-acute toxicity study

332

Toxicity of new chemical entity is considered as a major obstacle in path of drug discovery and

333

development [24]. Many of potent pre-clinically active pharmacological drugs could not approve

TE D

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305

Tyr385, Trp387, Ala516, Phe518) which

AC C

EP

residues loops (Tyr355,

12

ACCEPTED MANUSCRIPT 13

for clinical use due to their toxic nature [25]. Various reports indicate that COX-2 inhibitors

335

produce cardiovascular toxicity which might be dependent on the dose as well as on the duration

336

of treatment [26]. Cardiovascular toxicity associated with COX-2 treatment is due to an inequity

337

between COX-2 derived vasoprotective prostacyclin (PGI2) in the endothelium and COX-1

338

induced thrombotic thromboxane A2 (TXA2) in platelets [27]. It is also documented that prolong

339

use of COX-2 inhibitor showed gastrointestinal and renal toxicity [28].

340

Therefore, a sub-acute toxicity study has been performed to evaluate safety nature of novel

341

chemical compound MTL-1 in normal, healthy Wistar rats at the dose of 200 mg/kg/bwt. During

342

the whole experimental period, no sign of visible toxicity was observed. Hematological analysis

343

results have indicated that treatment with MTL-1 did not alter crucial hematological parameters

344

such as RBC, WBC and platelet counts, which indicated the nontoxic nature of MTL-1upon 14

345

days treatment (Table. 4). Further, liver function associated biomarkers were also quantified and

346

results displayed that treatment with MTL-1 did not significantly modify serum glutamate

347

oxaloacetatetransaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline

348

phosphatase (ALP), total protein, and total bilirubin as compared to non treated control animals.

349

Thus, MTL-1 did not exert any remarkable toxicity to liver upon chronic treatment (Table. 5).

350

Furthermore, renal toxicity related biomarkers such as uric acid, creatinine, and urea were also

351

deliberated after 14 days administration of MTL-1. Obtained data indicated that MTL-1 did not

352

produce significant renal toxicity upon 14 days oral administration (Table 5).

353

Overall, these preliminary toxicological evaluations have proven MTL-1 as a safe COX-2

354

inhibitor which also having valuable anticonvulsant activity and cognition boosting capability.

355 356

Table 4: Hematological parameters after oral administration of vehicle and MTL-1 for 14 days in rats.

AC C

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Hematological parameters with units Hb(g/dl) TLC (thou/mm3) Neutrophil %

Control (Vehicle, po) ± S.D 12.87±0.6

MTL-1 (200 mg/kg, po) ± S.D 14.15±0.07

7.23±1

6.25±0.64

17.33±3.21

21.5±2.1

13

ACCEPTED MANUSCRIPT 14

77.33±2.08

73±4.24

Eosinophil %

2.67±0.58

2.5±0.7

Monocyte %

2.67±0.58

2.5±0.7

Basophils %

0±0

RBC (mill/mm3)

8.19±0.64

Platelet count (thou/mm3)

1119±142.9

0.5±0.7

7.34±0.65

990±56.57

SC

357

RI PT

Lymphocyte %

Table 5: Liver and renal function test after oral administration of vehicle and MTL-1for

359

14 days in rats. Biochemical parameters (liver and kidney) with units Total Bilirubin SGOT

Alkaline phosphate

Control (Vehicle, po) ± S.D 0.27 ± 0.068

MTL-1 (200 mg/kg, po) ±S.D 0.4±0

77.7 ± 1.05

76.65±2.33

64.3±3.5

59.6±11.45

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SGPT

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358

115.9±14.5

123.5±2.12

6.7±0.26

6.55±0.21

40.83±1.16

31.45±2.62

0.63±0.05

0.9±0

3.13±0.4

1.45±0.35

Calcium

10.67±0.61

9.65±0.07

Phosphorous

7.83±0.29

7.2±0.14

144.87±0.75

145.05±0.21

5.14±0.05

4.9±0.14

107.13±0.85

104±5.66

Total protein

Creatinine

AC C

Uric acid

EP

Blood urea

Sodium

Potassium Chloride

14

ACCEPTED MANUSCRIPT 15

3. Conclusion

361

Herein, we report discovery of a novel series of methylsulfonyl benzene derivatives as potent

362

and selective COX-2 inhibitors, which endowed with admirable anti-convulsant effect. Our in-

363

vitro evaluation provided MTL-1 and MTL-2 as potent and selective COX-2 inhibitors in the

364

entire series. MTL-1 displayed excellent protection against sc-PTZ induced seizure in acute

365

models of epilepsy. Additionally, in time course study MTL-1 showed a long duration of action

366

and displayed 67% seizure protection after 6h of drug administration. Further, MTL-1 also

367

showed satisfactory protection in PTZ induced chronic model of epilepsy as compared to

368

standard COX-2 inhibitor ETX and successfully improved cognition impairment in PTZ kindled

369

rats. In sub-acute toxicity study MTL-1 did not exert any significant toxicity, as compared to

370

control group animals. Furthermore, in-silico docking and MD simulation studies revealed that

371

MTL-1 nicely fitted in active site of COX-2 with appropriate H-bond, hydrophobic and polar

372

interaction. Thus, MLT-1 became visible as a potent COX-2 inhibitor, which also showed

373

excellent anti-convulsant and cognition boosting potential without exerting any significant

374

toxicity.

375

4. Experimental section

376

4.1. Chemistry

377

Chemicals and reagents were purchased from Sigma Aldrich (USA), S.D Fine Chemicals (India),

378

Merck (Germany) and TCI (Japan). Melting points were determined with open capillaries by

379

using model KSPII, KRUSS, (Germany). The nuclear magnetic resonance (NMR) spectra were

380

obtained on high resolution Jeol-400 MHz NMR spectrophotometer (USA) DMSO-d6 using

381

tetramethylsilane (TMS) as the internal reference. Chemical shifts (δ) were expressed in parts per

382

million relative to TMS, and the following abbreviations were used to describe the peak patterns

383

when appropriate: s, (singlet); d, (doublet); t, (triplet); m, (multiplet) and brs (broad singlet). The

384

coupling constant (J) values are given in hertz (Hz). Mass spectra were recorded on an Agilent

385

6310 Ion trap LC/MS and elemental analysis (C, H and N) was performed on Elementar

386

analysensysteme.

387

4.1.1 Synthesis of phenyl (4-(methylsulfonyl) phenyl) carbamate (2)

388

An equimolar (1mM) amount of 4-(methylsulfonyl) aniline (1mM), 1 and phenyl chloroformate

389

were stirred in tetrahydrofuran (THF) with a catalytic amount of pyridine for 14h at RT.

AC C

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360

15

ACCEPTED MANUSCRIPT 16

Reaction mixture was poured in chilled water and appeared precipitate was filtered to achieve

391

carbamate derivative 2.

392

Phenyl (4-(methylsulfonyl) phenyl) carbamate (2)

393

Crystalline white solid, yield: 92%; mp: 158-1600C; 1H NMR(400 MHz, DMSO-d6): δ 3.15 (s,

394

1H, CH3), 7.23-7.28 (m, 3H, Ar-H), 7.41-7.45 (m, 2H, Ar-H), 7.71 (d, 2H, Ar-H, J= 9.1Hz), 7.87

395

(d, 2H, Ar-H, J= 9.1Hz), 10.74 (s, 1H, NH); LC–MS: m/z 291(M+1).

RI PT

390

396

4.1.2 Synthesis of N-(4-(methylsulfonyl)phenyl)hydrazinecarboxamide (3)

398

A mixture of carbamate derivative 2 and hydrazine hydrate was heated at temperature 100° C in

399

1,4-dioxane for 6h. Reaction mixture was poured in chilled water and product extracted with

400

ethyl acetate twice. Combined organic layer was treated with anhydrous sodium sulfate and

401

evaporated with reduced pressure to yield carboxamide derivative 3.

M AN U

SC

397

402

N-(4-(methylsulfonyl)phenyl)hydrazinecarboxamide (3)

404

Crystalline white solid, yield: 87%; mp: 180-1820C; 1H NMR (400 MHz, DMSO-d6): δ 3.13 (s,

405

1H, CH3), 4.42 (brs, 2H, NH2), 7.72- 7.78 (m, 5H, Ar-H+ NH), 9.13 (s, 1H, NH); LC–MS: m/z

406

229(M+1)

407

4.1.3

408

(alkyl/arylcarbamothioyl)hydrazinecarboxamide (4-14)

409

An equimolar ratio of carboxamide derivative 3 and various substituted isothiocyanates were

410

refluxed (900C) in mixture of dried acetonitrile and ethanol for 8-10h. After completion of

411

reaction solvent was evaporated and the reaction mixture was diluted with ethyl acetate. The

412

organic layer was washed with brine solution twice, dried with anhydrous sodium sulfate and

413

followed by evaporation, provided crude desired products in good yield. Crude products were

414

purified by column chromatography using chloroform: methanol (96:4) as eluent.

for

synthesis

of

N-(4-(methylsulfonyl)phenyl)-2-

EP

procedure

AC C

415

General

TE D

403

416

4.1.3.1 N-(4-(methylsulfonyl) phenyl)-2-(phenylcarbamothioyl) hydrazinecarboxamide (4)

417

White solid; yield 70%; mp: 170-1720C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13 (s, 3H, CH3),

418

7.13(t, 1H, J= 7.3Hz, Ar), 7.50(d, 2H, J= 8. 0Hz, Ar); 7.74(d, 2H, J= 8.2Hz, Ar), 7.81(d, 2H, J= 16

ACCEPTED MANUSCRIPT 17

13

8.72 Hz, Ar), 8.49(s, 1H, NH), 9.39(s,1H, NH), 9.53(s,1H, NH),9.86(s, 1H, NH);

C NMR

420

(DMSO-d6, 100 MHz): δ 43.9, 118.0, 124.9, 128.0, 128.2, 132.2, 139.1, 144.5,154.6, 181.7; LC-

421

MS: 365 (M+1); Anal.Calcd for C15H16N4O3S2: C, 49.43; H, 4.43; N, 15.37; Found: C, 49.68; H,

422

4.63; N,15.15.

423

4.1.3. 2 N-(4-(methylsulfonyl) phenyl)-2-(p-tolylcarbamothioyl) hydrazinecarboxamide (5)

424

White solid; yield 75%; mp: 182-1830C; 1H NMR (DMSO-d6, 400 MHz): δ 2.25(s, 3H, CH3),

425

3.12(s, 3H, CH3), 7.10(d, 2H, J= 8.2Hz, Ar), 7.34(d, 2H, J= 8.68Hz, Ar), 7.72(d, 2H, J= 8.6Hz,

426

Ar), 7.79(d, 2H, J= 9.1Hz), 8.4(s, 1H, NH), 9.36(s, 1H, NH), 9.44(s, 1H, NH), 9.70(s, 1H, NH)

427

; 13C NMR (DMSO-d6, 100 MHz): δ 20.5, 43.9, 117.9, 125.2, 128.1,128.5, 133.1, 134.0, 136.6,

428

144.5, 154.6, 181.8; LC-MS: 379 (M+1); Anal.Calcd. for C16H18N4O3S2: C, 50.78; H, 4.79; N,

429

14.80; Found: C, 51.01; H, 4.59; N, 15.04.

M AN U

SC

RI PT

419

430 431

4.1.3.32-((2,6-dimethylphenyl)carbamothioyl)-N-(4-(methylsulfonyl)phenyl)hydrazine

433

carboxamide (6)

434

White solid; yield 72%; mp: 185-1570C; 1H NMR (DMSO-d6, 400 MHz): δ 2.13(s, 6H, 2xCH3),

435

3.12(s, 3H, CH3), 7.01-7.05(m, 3H, Ar), 7.68-7.80(m, 4H, Ar), 8.53(s, 1H, NH), 9.25(s, 1H,

436

NH),9.40(s, 2H, 2xNH);

437

128.2, 133.1, 136.4, 136.9, 144.6, 154.4, 156.8, 181.9; LC-MS: 393 (M+1); Anal.Calcd for

438

C17H20N4O3S2: C, 52.02; H, 5.14; N, 14.27; Found: C,50.26; H,4.91; N,14.06.

439

4.1.3.4.2-((4-methoxyphenyl) carbamothioyl)-N-(4-(methylsulfonyl)phenyl)

440

hydrazinecarboxamide (7)

441

White solid; yield 68%; mp: 176-1770C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3),

442

3.68(s, 3H, OCH3), 6.83(d, 2H, J= 9.16Hz, 2H), 7.38(d, 2H, J= 9.16 Hz, Ar), 7.74-7.80(m, 4H,

443

Ar), 7.95(s, 1H, NH), 8.21(s, 1H, NH), 8.65(1H, NH), 9.31(s, 1H, NH);

444

100 MHz): δ 43.9, 55.2, 113.2, 118.0, 126.7, 128.2, 131.9, 133.1, 144.5, 154.7, 156.6, 182.2;

445

LC-MS: 395 (M+1); Anal.Calcd for C16H18N4O4S2: C, 48.72; H, 4.60; N, 14.20; Found: C,48.91;

446

H, 4.76; N,14.02.

447

4.1.3.5 2-((4-fluorophenyl) carbamothioyl)-N-(4-(methylsulfonyl)phenyl)hydrazinecarboxamide

448

(8)

TE D

432

C NMR (DMSO-d6, 100 MHz): δ 17.9, 43.9, 117.9, 126.7, 127.4,

AC C

EP

13

13

C NMR (DMSO-d6,

17

ACCEPTED MANUSCRIPT 18

449

White solid; yield 74%; mp:172-1740C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3),

450

7.37(d, 2H, J= 8.2Hz, Ar), 7.54(d, 2H, J= 8.7Hz, Ar), 7.73(d, 2H, J= 8.6Hz, Ar), 7.80(d, 2H, J=

451

9.1Hz, Ar), 8.49(s, 1H, NH), 9.40(s, 1H, NH), 9.62(s, 1H, NH), 9.92(s,1H, NH);

452

(DMSO-d6, 100 MHz): δ 43.9, 114.5, 114.8, 128.1, 133.2, 135.4, 144.4, 156.6, 132.0; LC-MS:

453

383 (M+1); Anal.Calcd for C15H15FN4O3S2: C, 47.11; H, 3.95; N, 14.65; Found: C,47.37;

454

H,3.83; N,14.41.

455

4.1.3.62-((2, 6-difluorophenyl)carbamothioyl)-N-(4-(methylsulfonyl)phenyl)

456

hydrazinecarboxamide (9)

457

White solid; yield 65%; mp: 166-1680C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3),

458

7.11(t, 2H, J= 8.26Hz, Ar), 7.32-7.39(m,1H, Ar), 7.71-7.81(m, 4H, Ar), 8.65(s, 1H, NH),

459

9.34(s,1H, NH), 9.47(s,1H,NH), 9.84(s,1H,NH); 13C NMR (DMSO-d6, 100 MHz): δ 43.8, 111.7,

460

117.8, 128.2, 128.8, 133.2, 144.3, 154.3, 157.5, 160.1,162.0,183.3; LC-MS: 401 (M+1);

461

Anal.Calcd for C15H14F2N4O3S2: C, 44.99; H, 3.52; F, 9.49; N, 13.99; Found: C,44.81; H,3.73;

462

N,14.18.

463

4.1.3.7 2-((4-chlorophenyl) carbamothioyl)-N-(4-(methylsulfonyl)phenyl)hydrazinecarboxamide

464

(10)

465

White solid; yield 67%; mp: 172-1730C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3),

466

7.29(s, 2H, Ar), 7.53(s, 2H, Ar), 7.77-7.79(m, 4H, Ar), 8.13(s, 1H, NH), 8.26(s, 1H, NH),8.99(s,

467

1H, NH), 9.34(s,1H, NH); LC-MS: 399 (M+1); Anal.Calcd for C15H15ClN4O3S2: C, 45.17; H,

468

3.79; N, 14.05; Found: C,45.43; H,4.01; N,13.81.

469

4.1.3.8 2-((2, 3-dichlorophenyl)carbamothioyl)-N-(4(methylsulfonyl)phenyl)

470

hydrazinecarboxamide (11)

471

White solid; yield 71%; mp: 288-2890C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3,

472

7.35(t,1H, J= 7.9Hz, Ar), 7.45(s, 1H, Ar), 7.52(d, J= 8.2Hz, Ar), 7.73(d, 2H, J= 8.68Hz, Ar),

473

7.80(d, 2H, J= 9.1Hz, Ar), 8.64(s, 1H, NH), 9.38(s, 1H, NH), 9.80(s, 2H, 2xNH);

474

(DMSO-d6, 100 MHz): δ 43.9, 118.0, 127.5, 128.2, 129.5, 131.6, 133.3, 144.4, 154.5, 182.4; LC-

475

MS: 432 (M+1); Anal.Calcd for C15H14Cl2N4O3S2: C, 41.58; H, 3.26; N, 12.93; Found: C,41.84;

476

H,3.09; N,12.68.

477

4.1.3.9 2-(benzylcarbamothioyl)-N-(4-(methylsulfonyl) phenyl)hydrazinecarboxamide (12)

C NMR

AC C

EP

TE D

M AN U

SC

RI PT

13

13

C NMR

18

ACCEPTED MANUSCRIPT 19

White solid; yield 75%; mp: 176-1770C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3), ,

479

4.72(d, 2H, CH2), 7.20-7.29(m, 5H, Ar), 7.72(d, 2H, J= 8.72Hz, Ar), 7.79(d, 2H, J= 8.72Hz),

480

8.40(s, 1H, NH), 8.69(s, 1H, NH), 9.27(s, 1H, NH), 9.30(s, 1H, NH); 13C NMR (DMSO-d6, 100

481

MHz): δ 43.8, 46.7, 117.9, 126.3, 126.9, 127.5, 127.8, 128.4, 133.1, 139.2, 144.5, 154.6, 182.8;

482

LC-MS: 379 (M+1); Anal.Calcd for C16H18N4O3S2: C, 50.78; H, 4.79; N, 14.80; Found: C,51.02;

483

H,4.97; N,14.54.

484

4.1.3.10 N-(4-(methylsulfonyl)phenyl)-2-(phenethylcarbamothioyl)hydrazinecarboxamide (13)

485

White solid; yield 79%; mp: 177-1790C; 1H NMR (DMSO-d6, 400 MHz): δ 3.13(s, 3H, CH3),

486

2.82(t, 2H, J= 7.8Hz, CH2), 3.64(q, 2H, J= 7.4Hz, CH2), 7.17-7.25(m, 5H, Ar), 7.73(d, 2H, J=

487

8.72Hz, Ar), 7.80(d, 2H, J= 8.7Hz, Ar), 8.22(s, 1H, NH), 8.32(s, 1H, NH), 9.18(s, 1H, NH),

488

9.28(s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): δ 34.8, 43.9, 45.2, 118.0, 126.1, 128.1, 128.3,

489

128.6, 133.2, 139.3, 144.4, 154.7, 182.1; LC-MS: 393 (M+1); Anal.Calcd for C17H20N4O3S2: C,

490

52.02; H, 5.14; N, 14.27; Found: C,52.30; H,4.99; N,14.51.

491

4.1.3.11 2-(ethylcarbamothioyl)-N-(4-(methylsulfonyl)phenyl)hydrazinecarboxamide(14)

492

White solid; yield 64%; mp: 176-1780C; 1H NMR (DMSO-d6, 400 MHz): δ 1.05(t, 3H, J=

493

7.0Hz, CH3), 3.12(s, 3H, CH3), 3.46(q, 2H, J= 6.9Hz, CH2),7.71(d, 2H, J= 9.1Hz, Ar), 7.78(d,

494

2H, J= 8.7Hz, Ar), 8.12(s, 1H, NH), 8.28(s, 1H, NH), 9.06(s, 1H, NH), 9.25(s, 1H, NH);

495

NMR (DMSO-d6, 100 MHz): δ 14.4, 38.5, 43.9, 118.0,128.1, 133.1, 144.4, 154.7, 181.9; LC-

496

MS: 317 (M+1); Anal.Calcd for C11H16N4O3S2: C, 41.76; H, 5.10; N, 17.71; Found: C,41.99;

497

H,5.29; N,17.51.

498

4.2. In-vitro COX-2/COX-1 inhibition assay

499

COX-2/ COX-1 inhibition assay has been performed according to manufacturer’s instructions

500

[29]. Briefly, stock solutions of test compounds were dissolved in a minimum volume of DMSO.

501

Provided 3ml assay buffer was diluted with 27ml of HPLC grade water and this final buffer

502

(100mM Tris HCl, PH-8.0) was used in the whole assay. Whole assay was performed in 96 well

503

plate and 150µl of assay buffer, 10 µl of heme, 10 µl of enzyme, and 10 µl of solvent were added

504

in 100% initial activity well. Further, 160 µl of assay buffer, 10 µl of heme and 10 µl of solvent

505

were added in background well. Furthermore, 150 µl of assay buffer, 10 µl of heme, 10 µl of

506

enzyme and various concentrations of inhibitor (10 µl) were added in inhibitor well. Reaction

507

mixture was incubated for five minutes at room temperature followed by addition of ADPH (10

13

C

AC C

EP

TE D

M AN U

SC

RI PT

478

19

ACCEPTED MANUSCRIPT 20

µl). Reaction was initiating by quick addition of Arachidonic acid solution (10 µl) to inhibitor

509

well and control well. After providing two minutes incubation, absorbance of plate was taken at

510

excitation wavelength 535 and an emission wavelength 590 nm. Percentage inhibition was

511

determined by subtracting each inhibitor sample value from 100% initial activity sample, divided

512

by 100% initial activity and multiply by 100. IC50 was calculated by percentage inhibition with

513

Microsoft excel.

514

4.3. Pharmacology

515

Adult Swiss Albino male mice (25–30 g) and male Wistar rats (200-250 g) were used as

516

experimental animals. All animals were kept under standard animal laboratory conditions at Dr.

517

B.R Ambedkar Center for Biomedical Research, University of Delhi, India. Each animal was

518

allowed for free access to food and water, except during the experimental period. Subcutaneous

519

PTZ and PTZ-induced kindling tests were performed according to the standard protocol

520

illustrated by Antiepileptic Drug Development (ADD) program of the National Institute of

521

Health (NIH, USA). All the experimental protocols were prior approved by the Institutional

522

Animal Ethics Committee (IAEC) for animal care.

523

Chemical and drugs

524

Pentylenetetrazole was procured from Sigma Aldrich (St. Louis, USA); sodium valproate

525

(Unimed

526

pharmacological assays, the test compound MTL-1 was freshly suspended in a 1% gum acacia in

527

sterile saline solution. The vehicle represents 1% gum acacia in sterile saline. Fresh

528

pentylenetetrazole was used and dissolved in normal sterile saline to such concentrations that

529

requisite doses were administered in a volume of 10 ml/kg (mice or rats) body weight. Sodium

530

valproate and etoricoxib at desired doses were also dissolved in sterile saline before use.

531

4.3.1. sc-PTZ test

532

Pentylenetetrazole (PTZ) was administrated subcutaneously (sc) at the convulsive dose of 85

533

mg/kg (CD97). The test compound MTL-1 was administered intraperitoneally (i.p.) at the doses

534

of 30, 100 and 300 mg/kg in mice and after 0.5 h and 4 h, PTZ was injected. Standard COX-2

535

inhibitor etoricoxib was tested on three doses (1, 5 and 10 mg/kg, i.p.) while, standard AED

536

sodium valproate was tested at 300 mg/kg, i.p. at both time intervals. The mice were placed in a

TE D

Ltd,

India)

and

etoricoxib

(Themis

Medicare,

India).

For

AC C

EP

Technologies

M AN U

SC

RI PT

508

20

ACCEPTED MANUSCRIPT 21

537

clear rectangular plastic cage, permitting a full view of the animal’s seizure episodes and

538

observations were carried out for 30 min. The episode of clonic seizures, tonic seizures was

539

documented carefully according to our previous method [30-33].

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4.3.2 Time course study

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The in-vivo time course study was performed in mice to judge long duration action of MTL-1.

543

The test compound MTL-1 was administered i.p. at the fixed dose of 30 mg/kg, and the

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percentage of seizure protection was examined at varying time intervals such as 0.5, 1, 2, 3, 4,

545

and 6 h by administering convulsive dose of PTZ (85 mg/kg, sc) [30].

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4.3.3 Anticonvulsant Quantification Studies (ED50 Determination)

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The median effective dose (ED50) of MTL-1 was evaluated by sc-PTZ test using mice and was

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administered i.p. to each animal at the varied doses till three-five points were established between

550

the dose level of 0% protection and 100% protection. The ED50 and the 95% confidence interval

551

were deliberated by the Graphpad prism 5 [31].

553

4.3.3. Induction of kindling by PTZ: In-vivo Epileptiogenesis model

554

All animals were randomly divided into four groups (n=8 in each group), which are as follows:

555

1: Control group (vehicle daily, i.p. for 28 days).

556 557

2: PTZ group (PTZ at 40 mg/kg, i.p. on alternate days from 1st to 28th day + vehicle daily, i.p. for 28 days).

558 559

3: PTZ + ETX group (PTZ at 40 mg/kg, i.p. on alternate days from 1st to 28th day + etoricoxib at 5 mg/kg daily, i.p. for 28 days).

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4: PTZ + MTL-1 group (PTZ at 40 mg/kg, i.p. on alternate days from 1st to 28th day + MTL-1 at 30 mg/kg daily, i.p. for 28 days).

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Immediately, after the PTZ injection, the animals were observed for the incidence of convulsive

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behavior. The resultant behavioral seizure activity was categorized according to revised Racine

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scale: 21

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Stage 1- Sudden behavioral arrest and/or motionless staring;

567

Stage 2-Facial jerking with muzzle or muzzle and eye;

568

Stage 3- Neck jerks;

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Stage 4-Clonic seizure in a sitting position;

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Stage 5- Convulsions including clonic and/or tonic–clonic seizures while lying on the belly and/or

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pure tonic seizures;

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Stage 6- Convulsions including clonic and/or tonic–clonic seizures while lying on the side /or wild

573

jumping.

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Additionally, all tested animals were observed for next 45 min to check for lethality before

575

returning to the home cage. The animals were considered to be kindled after receiving 14th PTZ

576

injection and after having reached at least three consecutive stages 5 or 6 seizures. The collective

577

kindling score was (calculated by taking the average of the individual behavior, seizure pattern of

578

each mouse in a group and dividing them with the number of animals present in the corresponding

579

group) plotted against time duration of kindling period. On the test day, all groups except group I

580

was tested for PTZ challenge dose test consisting single injection of 35 mg/kg, i.p. This high dose

581

of PTZ shortens the threshold of seizure activity more and thus produces lethality and status

582

epileptics in kindled rats [32].

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4.3.4 Neurobehavioral Tests

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The cognition assessment of MTL-1 was performed by various types of neurobehavioral tests,

586

including social recognition test, novel object recognition test and dark/light test. These

587

experiments were performed in developed kindled rats, which consisted of three groups consist

588

of six animals in each group. Group A (vehicle only), Group B (PTZ alone) and Group C (PTZ +

589

MTL-1). All the experiments were performed in an animal experimental room where the

590

temperature (24±2˚C) was maintained. The animal experimental room was kept silent during the

591

experiments. Experiments were performed during 9.00 h to 14.00 h.

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4.3.4.1 Social Recognition Test

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The social recognition test is a widely accepted method to measure short term memory relies on

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the olfactory cues. Adult (200-250 g) and juvenile rats (60-65 g) were used and this test was 22

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performed according to the method previously reported by Timmermann et al. with minor

597

modifications [34]. Briefly, animals were acclimatized in the animal house test room for 60

598

minutes before starting social recognition test. After acclimatization, each rat was placed alone in

599

separate cages for the 30 minute habituation period. After habituation, each rat was first placed

600

with a juvenile rat in the test cage for 3 minutes and first interaction time (T1) was noticed

601

manually by stop watch. It was entitled as a first interaction trial. After the completion of the first

602

interaction trial, juvenile rats were taken back in their original cages from test cages. Second

603

interaction trial (T2) was started after 2 h of first interaction trial in which each rat was again

604

interacted with same juvenile rat for 5 minutes and second interaction time T2 was noticed. For

605

the analysis point of view the ratio of T2/T1 was calculated for each rat and averaged was taken

606

for all groups. The lower ratio of T2/T1 is indicator of improved cognition [34].

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4.3.4.2 Novel Object Recognition Test

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by Prickaerts et al., 1997 with certain modifications. This test consists of three distinct phases

612

named habituation, training and test. During the first phase, the rats were placed in the test

613

apparatus without any object for 2 days habituation, twice for 3 min in each day. Testing session

614

was commenced which consisted two trials T1 and T2, each with a duration of three minutes. In

615

T1 trial each animal was exposed with two similar objects and interaction time T1 was noted.

616

After 1 h interval period second session was started and one of the similar objects was replaced

617

with new one and interaction time T2 was noticed. Percent time exploration of both objects, i.e.

618

similar and novel by each individual animal was calculated. The exploration parameter was

619

defined by animal behavior to explore the object by directing the nose to the object at a distance

620

of no more than 2 cm and or touching the object with the nose. To avoid olfactory cues, the

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objects were meticulously cleaned with 70% ethanol after each trial. Time spent exploring each

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object was calculated by an observer blind and was expressed as a percentage of the total

623

exploration time [35].

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The Novel object recognition (NOR) test was performed in male Wistar rats as method described

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4.3.4.3 Light-dark (LD) test 23

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The light-dark test was performed to understand the role of MTL-1 in the cognitive function

627

including anxiety- like behavior. The rats which spends less time in the light region of light-dark

628

box is considered as an indication of anxiety-like behavior. The experiment was performed with

629

minor alteration in the original protocol of Chen et. al [36]. The test was performed by the light-

630

dark apparatus made up of polycarbonate comprise of two compartments (30x 30x 30cm) light

631

compartment and dark compartment. Both compartments are connected to each other by black

632

plastic tunnel (30x 30x15 cm) and separated by black lid on the junction of both compartments.

633

Rats generally prefer to stay in the dark region due to nocturnal in nature, but more depressed

634

rats or cognition impaired rats also prefer dark region while rats having improved cognition

635

prefer light region of light-dark box. Light chamber was illuminated by a 60 W lamp. Before

636

starting experiment each animal of all groups were familiarized with light- dark box for 3

637

minutes 24h earlier and next day actual experiment was started. Animals of all groups were

638

placed in light-dark box for 3 minutes and the time spent in each compartment was noticed by

639

stop watch. The percentage time spent in each compartment was calculated for all three groups

640

[36].

641

4.3.5. Molecular Docking and MD simulation

642

The molecular docking of MTL-1was carried out with the X-ray coordinates of COX-2 (PDB ID:

643

6COX) using AutoDock 4.2. The protein and ligand files were prepared with MGL tools and

644

multiple docking was run as described earlier [37-40]. The Lamarckian genetic algorithm (LGA)

645

was applied to define the best docking conformations. As a result, 10 top-posed docking

646

conformations were obtained and the best docking pose in terms of binding free energy was

647

subjected to molecular dynamics (MD) simulation for 250 ns to determine the consistent

648

interactions using AMBER14 software. The t-leap module of AMBER was used to prepare the

649

system for protein and protein-ligand complex and protonation states of the protein residues were

650

defined with PROPKA [41]. The parameterization of protein and ligand was done with ff14SB

651

and GAFF force field, respectively [42] and, a partial atomic charge on the ligand was assigned

652

with AM1-BCC [43]. Each system was neutralized with the addition of Na+ ions. All simulations

653

were performed using explicit TIP3P water molecules padding around 10Å. Each protein-ligand

654

system was minimized in two steps using steepest descent, followed by conjugate gradient

655

minimization. In the first step, ions and solvent molecules were minimized by restraining the

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solute and in second step entire system was minimized removing the restraints. The system was

657

then gradually heated to 300 K in six steps. The simulations were equilibrated for 1ns prior to

658

production run and the production run of 250 ns was carried out for protein and protein-ligand

659

complex using NPT ensemble. The trajectory was calculated with the time step of 2fs and

660

SHAKE was used to constrain hydrogen bonds. Finally, the data was collected at every 10ps for

661

the analysis. The structural stability of the protein-ligand complex was assessed in terms of root

662

mean square deviation (RMSD) and radius of gyration (Rg) of backbone Cα atoms.

663

Statistical analysis

664

Statistical analysis was performed by one-way and two way of variance ANOVA followed by

665

post-hoc Tukey’s and bonferroni test, respectively. The p value less than 0.05 was regarded to be

666

statistically significant. Statistical analysis was done using the GraphPad Prism 51 software (La

667

Jolla, USA). Data were represented as mean ± standard deviation (Mean ± SD).

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Acknowledgment

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Chandra Bhushan Mishra is thankful to the Department of health research for financial support,

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Shikha Kumari is thankful to DBT for Post-doctoral fellowship, Amresh Prakash is thankful to

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Department of Science and Technology (DST), Delhi, India for the young scientist award.

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Rajesh Yadav is thankful to ACBR, University of Delhi for the financial support. Manisha

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Tiwari is thankful to the University of Delhi for sanctioning research funds. The University

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Science Instrumentation Center (USIC) is deeply acknowledged for providing NMR spectral

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analysis of the synthesized compounds.

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Fig. 2. The visual assessment of seizure scores in PTZ induced kindling in all groups for a period of 4 weeks. Group 1: Control group given vehicle only; Group 2: PTZ treated; Group 3: PTZ (40 mg/kg, i.p.) + Etoricoxib (ETX; 5 mg/kg, i.p.); Group 4: PTZ (40 mg/kg, i.p.) + Test compound MTL-1; 30 mg/kg, i.p.). The mean seizure scores expressed (±S.D) for each group. ***=P<0.0001 compared to control group; **= P<0.001 compared to control group; a= P<0.05 compared with PTZ group; ###=P<0.0001 compared to PTZ group. Repeated measures ANOVA followed by post hoc Tukey’s test.

848 849 850 851 852 853

Fig. 3. Graphical representation of the recognition ratio in the social recognition test performed on vehicle and PTZ-Kindled animals after administration of test compound MTL-1. The recognition ratios were calculated by dividing T2 (subsequent juvenile rat interaction time) by T1 (initial juvenile rat exploration time). Data are expressed as mean ± S.D (n=6 in each group; ** P<0.001 when group B (PTZ) was compared with group C (PTZ+ MTL-1). (One-way ANOVA followed by post hoc Tukey’s test).

854 855 856 857 858 859 860

Fig. 4. Graphical representation of the percent exploration preferences in novel object recognition test performed on vehicle and PTZ-Kindled animals after administration of test compound MTL-1. (A) denotes percentage exploration preference by animals in T1 trial with two identical objects A and A’ (B) denotes percentage exploration preference by animals in T2 trial with one similar object A and novel object B. Data are expressed as mean ± S.D for n=6 rats in each group. **P<0.01 within group A (vehicle).*P<0.05 within group C (PTZ+ MTL-1). Statistical analysis was carried out by Two-way ANOVA followed by bonferroni test.

861 862 863 864 865 866 867 868 869 870 871

Fig. 5. Effect of administration of test compound MTL-1in PTZ-kindled rats during the Light/Dark Test: (A) Denotes the percentage of time spent of test animals in light area. The significance levels of ***P<0.0001 was observed, when vehicle treated rats (Group A) were compared with PTZ-Kindled animals (Group B) and ###P<0.0001 when PTZ-Kindled animals (Group B) were compared with group C animals (PTZ+ MTL-1). * P<0.05 vehicle treated rats (Group A) versus group C (PTZ+ MTL-1). (B) Percentage time duration of test animals in dark area. Each column represents mean ± S.D (n=6 rats in each group). *** P<0.0001 Group A versus PTZ-Kindled animals (Group B) and ###P<0.0001 PTZ-Kindled animals (Group B) versus group C animals (PTZ+ MTL-1).*P<0.05 when PTZ-Kindled animals (Group B) were compared with group C animals (PTZ+ MTL-1). Statistical analysis was carried out by One-way ANOVA followed by post hoc Tukey’s test

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Figure legends Fig. 1. Marketed COX-2 inhibitors and designed novel COX-2 inhibitors

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Fig. 6. Molecular docking of MTL-1: (A) Cartoon view of docked pose with COX-2 at PyMol. (B) COX-2 active site residues involve in interactions are shown with stick and Comp. MTL-1 is represented in ball-and-stick model. Residues Arg120, Ser353 and Leu352 are involved in Hbond interaction shown with dashed line in black.

877 878 879 880

Fig. 7. (A) RMSD plots of COX (black) and complex with MTL-1 (red) in water at 300 K during 250 ns MD simulation; (B) Time evolution of radius of gyration (Rg) values, all atoms Cα backbone of COX-2 (black) and COX-2- MTL-1 (red) in water at temperature 300K for 250 ns.

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Fig. 8. RMSF plot of Cα atoms of COX-2 (black) and COX-2- MTL-1 (red) in water at temperature 300K for 250 ns.

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Fig. 9 Convergence plot of COX-2 and MTL-1 complex.

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Fig. 1

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***

Control Group

***

2

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PT Z Group PT Z + ET X Group

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PT Z + MT L-1 Group

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ACCEPTED MANUSCRIPT

1. MTL-1 has appeared as selective and potent COX-2 inhibitor in the entire series. 2. MTL-1 successfully protected animals from sc-PTZ induced seizure. 3. In chronic model of epilepsy, MTL-1 also showed excellent anti-epileptic activity.

RI PT

4. MTL-1 nicely interacted with the active site of COX-2 which was confirmed by molecular docking and MD simulation studies.

AC C

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5. It also improved cognition impairment in kindled rats and appeared non toxic in sub-acute toxicity study.