Synthesis of Novel Diclofenac Hydrazones: Molecular Docking, Anti-Inflammatory, Analgesic, and Ulcerogenic Activity

Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Department of Pharmaceutical Chemistry, Drug Exploration and Development Chair (DEDC), College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Peptide Chemistry Department, Chemical Industries Research Division, National Research Centre, Dokki, Cairo 12622, Egypt Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital and Research Center, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia


Introduction
Diclofenac is a phenyl acetic acid derivative with anti-inflammatory, analgesic, and antipyretic activity. It is a widely used NSAID for the treatment of chronic inflammatory diseases. Diclofenac is associated with serious dose-dependent gastrointestinal, cardiovascular, and renal effects. Prolonged use of diclofenac has caused gastric ulcer and bleeding. e pharmacological activity of diclofenac is the cyclooxygenase enzyme inhibition, resulting in the inhibition of prostaglandin biosynthesis from arachidonic acid [1]. Diclofenac is a nonselective COX inhibitor with complete absorption and extensive metabolism. Selective COX-2 inhibitors like celecoxib, rofecoxib, and etoricoxib are reported to have less gastrointestinal side effects [2].
Synthetic approaches based on chemical modification have been used to improve the safety profile of drugs [3,4]. Many modification methods were used, for example, hybridization and prodrugs [5][6][7]. Derivatization of carboxylic acid moiety of NSAIDs has resulted in an increase in anti-inflammatory with reduced ulcerogenic effect [8][9][10][11][12][13]. Amide modification causes greater selectivity for COX-2, resulting in gastroprotection [14,15]. e active site in COX-2 is bigger than the active site of COX-1 enzyme, so changing the size of the drug molecule may improve the binding against COX-2 [16]. erefore, the development of NSAID with an improved safety profile is still in demand. Literature survey revealed that modification of carboxylic acid moiety of diclofenac has produced significant pharmacological results [11,15,[17][18][19].
In continuation of our research in this field, herein we have synthesized novel diclofenac hydrazone derivatives and evaluated their potential anti-inflammatory, analgesic, and ulcerogenic activity [20,21].

Experiment
2.1. Chemistry. All the solvents were purchased from Merck. Compound purity was checked by thin-layer chromatography (TLC) and was carried out on silica gel 60F 254 coated plates (Merck). FT-IR was determined by Perkin Elmer FT-IR spectrophotometer. Melting points were checked by Gallenkamp melting point apparatus. 1 H and 13 C NMR of the compounds were determined by Bruker NMR 500 MHz and 125 MHz spectrophotometer, respectively. Molecular weights of compounds were determined by Agilent triple quadrupole 6410 TQ GC/MS. Elemental analysis was carried out by Elementar (Analysensysteme GmbH, Germany). e molecular docking simulations were performed using CB-Dock online server. is server employed Autodock vina for protein-ligand docking. Usually, the binding affinity of the docked ligand with the protein is estimated with vina score, which is also known as docking energies (kJ/mol). e bestdocked compound typically demonstrates low vina score or docking energies. phenyl]acetate (II).

Synthesis of Methyl
e diclofenac ester was synthesized as per the reported procedure [22].

Carrageenan-Induced Paw Edema in
Rats. An injection wad made of 0.05 mL of 1% carrageenan sodium salt (BDH) into the right hind foot of each rat under the plantar aponeurosis. e test group of rats was treated with equimolar doses orally with test drugs 1 hour before carrageenan injection. At the same time, the control group was given 5 mL/kg of normal saline and the reference group was given 10 mg/kg orally of an aqueous solution of diclofenac in 5% methyl cellulose. Percent inhibition was calculated taking the values in the control group as 0% inhibition. e measurements of foot volume were done by the displacement technique using a plethysmometer (Apelex, France) immediately after and +1, +2, and +3 hours after the injection of carrageenan. e inhibitory activity was calculated to the following formula 100 (1−a−x/b−y), where "b" is the mean paw volume of control rats after carrageenan injection and "y" before the injection whereas "x" is the mean paw volume of treated rats before injection and "a" is the mean paw volume after carrageenan injection. Student's t-test was used for the statistical calculations [27].

Analgesic Activity by Hot Plate Method.
e hot plate method is used for evaluating analgesic activity. e hot plate was maintained at 55.0 ± 5.5°C. Albino mice were used in the hot plate method. e reaction time was taken as the interval extending from the instant the animal reached the hot plate until the moment the animal licked its forefeet or jumped off. e mouse was monitored carefully for the time in seconds in which it displayed nociceptive responses, considered as the control reaction time. To avoid damage to the paws, a cutoff time of 60 s was used. e activity was evaluated after p.o. administration of diclofenac or test compounds at the equimolar dose each. e reaction time was then retested at 0 h and 30, 60, and 120 min after injection (each animal acted as its own control). e percentage changes in the reaction were then calculated. Student's t-test was used for the statistical calculations [28].

Analgesic Activity by Writhing Test.
Mice of either sex with a weight between 20 and 25 g were used. Solution of 0.1 mL of 0.6% solution of acetic acid was injected intraperitoneally. Groups of 6 animals were used for control and treated mice. Test animals were administered the test compounds or the standard drug with equimolar doses at various pretreatment times prior to acetic acid administration. e mice were placed individually into glass beakers. e mice were then observed for a period of twenty minutes and the number of writhes was recorded. e formula for computing percent inhibition is as follows: average writhes in the control group minus writhes in the drug group divided by writhes in the control group times 100% [29,30]. Student's t-test was used for the statistical calculations.

Ulcerogenic Activity.
Wistar rats were used for the ulcerogenic activity. e ulcerogenic activity was checked after peroral administration of an equimolar dose of diclofenac or test compounds. e control group was given a suspension of 1% methyl cellulose perorally. e animals were euthanized by using pentobarbitone (50 mg/kg) intraperitoneal before cervical dislocation. e stomach was opened along the greater curvature and washed with distilled water. A magnifying glass was used to examine the mucosal damage. For each stomach, the mucosal damage was assessed according to the reported scoring system [31,32]. Data expressed as ulcer index. Student's t-test was used for the statistical calculations.

Determination of LD 50 .
e Karber method was used to determine the median lethal dose (LD 50 ) of compound 8. An observation was made for 24 h after the dosing of animals. Dead rats were counted at the end of the study period.
e Karber formula was used for the calculation of LD 50 . For determination of LD 50 , an observation was made for 24 hours and symptoms of toxicity and rate of mortality were noted. Expired animals were counted at the end of the study period for the calculation of LD 50 . LD 50 � LD 100 − ×((a × b)/n), where n is the total number of animals in a group, a is the difference between two successive doses of administered extract/substance, b is the average number of dead animals in two successive doses, and LD 100 is the lethal dose causing 100% death of all test animals [33].
2.5.6. Docking Studies of Compounds. Molecular docking simulations were performed to reinforce the wet-lab analysis and get insight into the molecular mechanism of inhibition of COX-1 and COX-2 by the drugs.
ree-dimensional structure of COX-1 was downloaded from PDB (6Y3C). It is a recently resolved human COX-1 structure. Usually, in previous studies, COX-1 structures from other organisms were employed for molecular docking, which might bring some discrepancies in the results. e COX-2 three-dimensional structure was also downloaded from PDB (5F1A). e downloaded structures were visualized using PyMol and refined for the docking process by removing all of the bounded ligands and water molecules. Likewise, three-dimensional structures of all synthetic compounds were developed using Chem 3D Pro 12.0 version. e structure of a standard compound (Diclofenac) was downloaded from the PubChem database. Finally, the molecular docking simulation was performed using the CB-Dock online server. is server used Autodock vina at the backend to perform docking analysis. e docking poses of drugs with the protein structures were ranked as per decreasing energies values. e best docking pose usually has the lowest energy. e two-dimensional and three-dimensional bindings of the drugs with the proteins were visualized using LigPlot and PyMol.

Results and Discussion
To synthesize various substituted diclofenac hydrazones, the hydrazide of diclofenac (III) was used as a starting material [28]. e diclofenac hydrazide was obtained from diclofenac ester (methyl [2-(2,6-dichloroanilino)phenyl]acetate (II), which was reacted with hydrazine hydrate 99% in absolute ethanol. Hydrazones of diclofenac (1−14) were synthesized by reacting diclofenac hydrazide (III) with different substituted benzaldehydes in ethanol with few drops of glacial acetic acid, used as a catalyst in this reaction. e preparation of hydrazones of diclofenac was performed as per Scheme 1. Moreover, the structures were characterized by spectroscopic methods, elemental analysis, and mass spectrometry. e structure of the diclofenac hydrazide (III) has presented an identical NMR splitting pattern as that of diclofenac. e structures of the diclofenac acid hydrazide derivatives were confirmed on the basis of 1 H NMR analysis, which was established by the disappearance of-NH 2 protons at δ 1.97 ppm. Mass spectra confirmed the molecular masses of compounds by their molecular ion peaks.

Analgesic Activity by Hot Plate
Method. Analgesic activity was determined by the hot plate method. Based on the results of anti-inflammatory activity, compounds that showed significant anti-inflammatory activity were further tested for their analgesic activity ( Table 2). Compound 8 (R � 3,5dimethoxy-4-hydroxyphenyl) expressed highly significant analgesic activity of 204% change, compared with the reference drug diclofenac with 183.33% change. e analgesic activity of compound 3 (R � 2,4-dimethoxyphenyl) was found to be 172% change. Substitution with 2,5-dihydroxyphenyl has shown analgesic activity of 135.48% change in compound 13. Compounds 3, 8, and 13 have a significant analgesic activity as compared to the reference drug diclofenac.

Ulcerogenic Activity.
e compounds which showed highly significant anti-inflammatory and analgesic activity were screened for ulcerogenic activity (Table 4). Gastric mucosal damage was examined after oral administration of the test compounds. Ulcerogenic reduction activity between 0.00 and 0.50 ± 0.22 was observed in test compounds as compared to the diclofenac with an ulcer index of 0.66 ± 0.21. Compounds 3 and 8 showed maximum ulcerogenic reduction activities.

Toxicity of Compound 8.
To determine the median lethal dose (LD 50 ) of compound 8, the Karber method was used.
e toxicity symptoms and mortality at 24 h of observation were made. Compound 8 was found to have the median lethal dose (LD 50 ) of 168 mg/kg (Table 5). e molecular docking simulations were performed using CB-Dock online server. is server employed Auto dock vina for protein-ligand docking. Usually, the binding affinity of the docked ligand with the protein is estimated with vina score, which is also known as docking energies (kJ/mol). e best-docked compound typically demonstrates low vina score or docking energies. e accuracy of CB-Dock server is estimated to be ∼70%, which is better than its contemporary tools. Compound 8 (3,5-dimethoxy-4-hydroxyphenyl) showed better binding affinities with COX-1/ COX-2 (−9.4 kJ/mol) protein structures as compared to the standard drug, diclofenac (Table 6). Interestingly, diclofenac demonstrated approximately 20% better affinity with COX-2 (−8.1 kJ/mol) as compared to COX-1 (−6.6 kJ/mol). is data is consistent with previous experimental findings as well [34]. e visualization of the docked complexes revealed that diclofenac bounded with COX-1 and COX-2 in a small cavity at the same position (Figures 1 and 2). It seems to be very rational as both proteins share 70% sequence identity and very similar type of three-dimensional structures [35]. However, compound 8 did not bind in the same cavity where diclofenac was docked in both proteins. One of the potential reasons for this phenomenon may be the large size of compound 8 as compared to diclofenac. Compound 8 docked at the same position in both proteins as it can be visualized in Figures 3  and 4. e drug showed similar binding affinities with COX-1 and COX-2 (−9.4 kJ/mol). In COX-1, the prominent interactions are hydrophobic while, in COX-2 hydrogen, binding seems to be more critical as compared to hydrophobic interactions ( Figure 5).       Compd.

Conclusions
Novel diclofenac hydrazones (1−14) were prepared in excellent yield. e synthesized compounds were confirmed by the elemental analysis and spectral data. Chemical modification to the diclofenac hydrazide resulted in fourteen new diclofenac hydrazones. e compounds (2, 3, 7, 8, 11, and 13) were found to be the most promising compounds in anti-inflammatory activity. Compounds 3, 8, and 13 have been found to have significant analgesic activity compared to the reference drug diclofenac in analgesic activity by both hot plate and acetic acid-induced writhing method. Compound 8 containing 3,5-dimethoxy-4-hydroxyphenyl substitution was found to be a highly potent anti-inflammatory and analgesic agent. It was further evaluated for ulcerogenic activity and demonstrated significant ulcerogenic reduction activity. e median lethal dose of compound 8 was found to be 168 mg/kg. Compound 8 with 3,5-dimethoxy-4hydroxyphenyl substitution was found to be the most promising anti-inflammatory and analgesic agent with gastric sparing activity.

Highlights
Synthesis of novel diclofenac hydrazones. Spectral characterization of synthesized compounds.
In vivo anti-inflammatory, analgesic, and ulcerogenic activity of compounds. LD 50 determination of most potent compound. Molecular docking of synthesized compounds against COX−1/COX−2 binding site.

Conflicts of Interest
e authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.