Halimi et al Tropical Journal of Pharmaceutical Research February 2017; 16 (2): 327-336 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i2.10 Original Research Article Pharmacological evaluation of novel dimers of an arylpropionic acid class of non-selective cyclooxygenase inhibitors Syed Muhammad Ashhad Halimi1*, Muhammad Saeed1, Safiullah1 and Khalid Muhammed Khan2 1Department of Pharmacy, University of Peshawar, Peshawar 25120, 2H.E.J. International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan *For correspondence: Email: [email protected]; Tel: +92 91 9216750; Fax: +92 91 9218318 Received: 4 August 2016 Revised accepted: 5 January 2017 Abstract Purpose: To explore and identify cyclooxygenase (COX) inhibitors with optimal potency and efficacy using an arylpropionic acid class of drugs as lead molecules. Methods: The selected lead molecules were dimerised through chemical processes (reflux condensation) and characterised in terms of structural properties using infrared, proton nuclear magnetic resonance, electron impact mass spectrometry, and elemental analysis techniques. The molecules were evaluated pharmacologically for acute toxicity and anti-inflammatory (carrageenan- induced paw oedema test), analgesic (acetic acid-induced writhing test in mice), and antipyretic (Brewer’s yeast-induced pyrexia test in mice) activities against control (normal saline) and relevant reference standard drugs. Docking analyses were also performed to assess possible protein–ligand interactions. Results: The test compounds were non-toxic at doses of 50, 100 and 150 mg/kg body weight, ip. Pharmacological evaluation revealed that the test compounds, TC-I through TC-IV, had significant anti- inflammatory and peripheral analgesic activities (p < 0.001). An antipyretic test showed that TC-I, -II, and -III showed highly significant antipyretic activities at all doses tested. TC-IV at 20 and 30 mg/kg body weight exhibited significant antipyretic activities (p < 0.05), while at 50 mg/kg body weight, the activity was highly significant (p < 0.001). Molecular modelling revealed strong inhibitory interactions with docking scores of 116.2, 128.8, 144.2, and 136.0 kcal/mol, respectively, in comparison with the reference ligand, flurbiprofen (94.9 kcal/mol). Conclusion: The dimerised lead drug molecules showed significant anti-inflammatory, analgesic, and antipyretic activities in animals and may further be explored as potential new drug candidates for inflammatory conditions. Keywords: Analgesic, Anti-inflammatory, Antipyretic, Arylpropionic acid, COX-2 inhibitors, Molecular docking Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION analgesic, and antipyretic activities. A diverse collection of NSAIDs, such as arylpropionic Cyclooxygenase (COX) inhibitors, non-steroidal acids, arylacetic acids, β­ketoenols, and diary anti-inflammatory drugs (NSAIDs), are commonly heterocycles, acts by inhibiting the activity of prescribed and used for their anti-inflammatory, cyclooxygenase (COX) enzymes [1]. NSAID Trop J Pharm Res, February 2017; 16(2): 327 Halimi et al selection depends on the clinical condition of the Proton nuclear magnetic resonance (1H NMR) patient under treatment, COX1 and COX2 measurements were performed using a Bruker selectivity, duration of use, and gastric ulcerative AV 300 system (400 MHz) with dimethyl and cardiovascular risks [2]. sulphoxide (DMSO) as the solvent. Electron impact mass spectroscopy (EI MS) was A prolific area of research looks at existing drug conducted with a JEOL MS Route spectrometer molecules that may be further amplified in terms (JMS 600-H). Infrared (IR) spectra were obtained of potency, efficacy, selectivity, or safety. The with a Jasco IR-A-302 spectrometer. Ultraviolet development and successful testing of molecular (UV) spectra were generated with a Thermo computations for both the receptors (proteins) Electron Vision Pro (ver. 4.10). C:H:N analyses and ligands (drugs) has further aided the efforts were performed with a Carlo of researchers, to the point of designing ErbaStrumentazione system (Mod-1106, Italy). appropriate drug structures for both known and Anti-inflammatory activity tests were conducted unknown proteins, receptors, or enzymes [3,4]. using a plethysmometer (Model LE 7500 Plan lab S.L.). Antinociceptive activity was assessed with Well-established lead NSAID molecules for a hot plate analgesiometer (Harvard Apparatus, synthesising derivatives have the advantages of USA). Antipyretic activity was measured using a predicable pharmacological activities, known digital thermometer (Model CA92121, ACON doses, durations of action, and side and toxic Laboratories, USA). effects. The pharmacological evaluation of such synthesised analogues, in vivo and through Synthesis computer docking, can provide useful data for further augmentation of the existing lead Ethyl ester analogues of naproxen, flurbiprofen, molecule; for more benefits and fewer side and ibuprofen were synthesised through reflux effects, the ‘better’ structure could be screened condensation of the reactants in an equimolar out among the synthesised analogues for further (0.1 mmol:0.1 mmol) ratio in an acidic optimisation of its pharmacological activity. environment, with the addition of a few drops of Several success stories have been reported to concentrated sulphuric acid to the reaction [9]. date regarding such computer-aided drug Completion of the reaction was monitored using discoveries [5]. thin layer chromatography (TLC), with ethyl acetate and hexane as the mobile phase. The Naproxen [6], flurbiprofen [7], and ibuprofen [8], reaction duration ranged from 12–18 h. The belonging to the arylpropionic acid class of non- resulting solid products were filtered, washed selective COX inhibitors, were selected for with hot hexane (200–400 mL) and distilled water synthesis of their dimers and further exploration (400–500 mL), and then dried (Step 1). Esterified of these dimers for anti-inflammatory, analgesic, compounds thus synthesised were treated with and antipyretic activities. Molecular docking hydrazine hydrate, and again subjected to reflux analyses were also performed to assess condensation for 24–48 h, using ethanol as the probable drug-receptor interactions with this new solvent [10]. Completion of the reactions was COX inhibitor approach. ascertained using TLC. The products were then purified with different techniques (Step 2). The EXPERIMENTAL esters and hydrazides thus synthesised were refluxed overnight, in the presence of Chemicals carbonyldiimidazole (CDI) as the catalyst, using acetonitrile as the reaction medium. General Pharmaceutical-grade ibuprofen, flurbiprofen, Scheme I shows dimer formation in a three-step and naproxen were provided by Polyfine reaction. The products formed were then purified Chemipharma (Pvt.) Ltd, Hayatabad, Peshawar, similarly through the different techniques. Pakistan. Ethanol and concentrated sulphuric acid were obtained from Sigma-Aldrich, USA. Experimental animals Lambda carrageenan (Sigma, USA), glacial acetic acid (Panreac, Spain), Brewer’s yeast Mice (BALB-c) from the National Institute of (Merck, Germany), and sterile normal saline I/V Health (NIH) Islamabad, Pakistan, were used for (Otsoka Japan) were used as received. the experimental studies. Mice were fed with standard food ad libitum with free access to The purity of the products was checked on thin- drinking water, and kept at 25 ± 2°C with a 12- layer chromatographic plates of aluminium h/12-h day/night cycle. The animal studies were coated with silica gel (Kieselgel 60 F25, Merck, approved by the Ethical Committee of the Germany). Melting points were assessed with a University of Peshawar, Pakistan (ref no. 01/EC- Bicot apparatus (Bibby Scientific Ltd., UK). Trop J Pharm Res, February 2017; 16(2): 328 Halimi et al where = naproxen, flurbiprofen, and ibuprofen. Table 1: Dimers of naproxen, flurbiprofen, and ibuprofen Test Structure IUPAC name Formula compound 2-(2-fluorobiphenyl-3-yl)-N'-((S)-2-(6- C29H27FN2O3 methoxynaphthalen-2- TC-I yl)propanoyl)propanehydrazide 2-(4-isobutylphenyl)-N'-(2-(3- C26H36N2O2 isopropylphenyl)propanoyl)propanehydr TC-II azide S)-2-(6-methoxynaphthalen-2-yl)-N'- C28H28N2O4 ((S)-2-(6-methoxynaphthalen-2- TC-III yl)propanoyl)propanehydrazide 2-(2-fluoro-[1,1’-biphenyl]-4-yl)-N’-(2-(2- C30H26F2N2O fluoro-[1,1diphenyl]-4- 2 yl)propanoyl)propanehydrazide TC-IV 15/Pharm), according to international guidelines For the toxicity study, 18 mice were distributed for the handling of animals [11,12]. randomly into three groups, n = 6 each. Groups were treated with 50, 100, and 500 mg/kg body Acute toxicity test weight of the test compounds intraperitoneally (ip) through injection. The animals were observed for 6 h for any behavioural changes; Trop J Pharm Res, February 2017; 16(2): 329 Halimi et al mortality rates were determined at 24 h after hot plate test after 30- and 60-min intervals; the administration of the test