Drug Development of Mefenamic Acid Derivatives as Analgesic by Molecular Approach

A new compound of Mefenamic Acid derivate, 4-nitrobenzoyl-mefenamic acid has been synthesized by benzoylation reaction between mefenamic acid and 4-nitrobenzoyl chloride after prediction by in silico study/molecular approach. A derivative of mefenamic acid (4-NO2-benzoyl-mefenamic acid) has been synthesized for increase its activity as candidate of analgesic drug/inhibitor COX-2 (Cyclooxigenase-2). This compound has been purified by Column Chromatography and analyzed using TLC-Densitometry to determine purity with Rf value 0,8. The spot has good purity and then it was identified this structure using H-NMR 400 MHz and FTIR-KBr. The result showed that this compound is 4-nitrobenzoyl-mefenamic acid (4NBMA). 4NBMA gives white yellow color with melting point 198-199C. Finally, 4NBMA was tested analgetic activity by hot plate method and it showed that 4-nitrobenzoyl-mefenamic acid has been higher activity than mefenamic acid. Keyword: 4-nitrobenzoyl-mefenamic acid, analgesic, benzoylation, molecular approach. INTRODUCTION Pain is a multidimentional sensory experience. International Association for the Study of Pain (IASP) defines that pain is a sensory and unpleasant emotional experience associated with tissue damage, both actual and potential. Chronic pain becomes a serious problem if it increases rate of pain and gives chronic prevalence. Pain is one of the most frequently reported symptom occurs in one from six people in the population and it is estimated to occur in 2-40% of adult population. Some studies estimate that the prevalence of chronic pain in Europe is up to 55.2%. In Indonesia, the population of the elderly, reported that 25-50% of them experienced pain. Chronic pain causes increasing health care costs. The research in the United States showed that the cost yearly for chronic pain is estimated around 100 billion dollars. Mefenamic acid is a drug in the market as NSAIDs (Non-steroidal AntiInflammatory Drugs) which has long been used as an analgesic-inflammatory COX and widely used in the world for treatment of diseases to relieve pain/pain and inflammation such as rheumatoid arthritis, toothache, gout and peripheral muscle pain. In the effort to design and develop new drugs, the first step is modification of commercial drug that has been known its molecular structure and biological activity and was become guidance based on a systematic and rational research to reduce trial and error. Further guidance from lead compounds were developed and modified that become new compounds/derivatives and then was tested this biological activity. Because of extensive use of mefenamic acid so we effort to develop new drugs and created derivatives. Based on previous research, mefenamic acid was substituted benzenesulfonic, bromo anthranilic, paracetamol, phenoxybenzoic, cyclocarboimide the coupling reaction, cyclourea, esters and amides, hydrazine and hidramin can enhance the analgesic effect of anti-inflammatory and reduce the side effects (ulcers). In this study conducted with the benzoyl derivative substitution by topliss theory and then was predicted by molecular approach. The substitution by benzoyl based on research Jayaselli et al and Susilowati through benzoylation reaction of NSAIDs such as paracetamol and piroxicam derivatives gives greater biological activity than the lead compound. We used several substituents benzoyl derivative is based on the Topliss theory that predicted by lipophilic, electronic and steric parameters as substituents. Lipophilic parameters associated with penetration rate in biological membranes. Electronic parameters contributed in the process of drug interactions with receptor by ionization and polarization proccess thus increasing the biological effectiveness overall. Steric parameters related to the compatibility of the interaction of the compound with the receptor in the cell that it effected maximum binding orientation so increased activity. Increased lipophilic properties can be done by inserting a non-polar groups such as aromatic rings, while the increase in electronic properties can be done by inserting electronegative substituents as steric halogen. Steric properties can be done by creating a more bulky structure that serves as a shield and encourages interaction between drug and active site of receptor. While we modified mefenamic acid by functional groups that increase in lipophilic, electronic and steric parameters that influenced the DOI number: 10.25258/ijpcr.v9i1.8294 Puspaningtyasm et al. / Drug Development of... IJPCR, Volume 9, Issue 2: February 2017 Page 124 biological activity. The synthesis of mefenamic acid derivative is reacted between benzoyl derivatives and mefenamic acid via nucleophilic addition. The mechanism can be seen Figure 1. Mefenamic acid derivatives activity can be predicted before synthesized by molecular approach that used docking with Molegro Virtual Docker (MVD). The goal of docking estimates the interaction between mefenamic acid derivatives as ligands with COX-2 receptors that act as enzymes in inflammatory pain. Then from the molecular docking will be obtained compounds are the most potent predictions which will then be synthesized. The compounds synthesized were tested for purity by melting point and Thin Layer Chromatography (TLC). Further, the structure of compounds was identified by with the Infrared Spectrometry (FTIR) and Proton Nuclear Magnetic Resonance Spectrometry (1H-NMR) and then analgesic activity was tested by the hot plate method. METHODS Instrument Glass beaker (Iwaki Pyrex), bulb pipettes, pipette, stirrer, micropipette (Blaubrand® IntraEnd), chamber, magnetic stirrer, oven, analytical balance (Sartorius), Ultraviolet Lamp (UV) dryer/hair dryer, Electrothermal melting point apparatus, 1H-NMR 400 MHz JEOL Resonance, FTIR-KBr Perkin Elmer-Spectrum One, TLCDensitometry (Camag), digital cameras, ChemBioOffice 2008 trial version, Molegro Virtual Docker trial version. Material Mefenamic acid (Merck), 4-nitrobenzoylchloride (Sigma), acetone (Sigma), Silica gel 60 F 254, hexane (Merck), toluene (Merck), ethyl acetate (Merck), methanol (Merck), KBr (Merck), chloroform-d pro NMR (Sigma), tetramethylsilane (TMS) pro NMR (sigma), mice. Molecular docking Structure mefenamic acid and its derivatives as ligands drawn with Chemoffice program 2008 while 3D structure of the cyclooxygenase (COX-2) as the receptor is taken from the Protein Data Bank (PDB). Furthermore, docking ligand-protein using the program MVD 2008. Preparation Ligand and Receptor. The structure of all compounds mefenamic acid derivative according to Topliss theory (4-OCH3, 4-Cl, 4-Br, 4-F, 4-NO2, 3-NO2, 4-CF3, 3-Cl, 2Cl, H, 3.4 -Cl benzoyl) drawn using Draw Ultra 11.0 ChemBio 2008. Further optimized by using 3D Ultra 11.0 ChemBio 2008 and the minimization of the energy of the most stable ligand with MM2 to produce a ligand conformation pose consistent. Prospective pose energy minimized in the active site using a grid-based method for evaluating the protein-ligand interaction energy stored in the docking receptor Mol2. We must remove water before preparation ligand and then determine active site via Detect Cavity. Validation Docking. Validation is performed to calibrate the method of docking software. The parameter is used to assess of the validity that is RMSD value where RMSD value is less than 2 that means position of the ligand copy superimpose with native ligand so that the methods used would be more appropriate. RMSD value is also influenced by the resolution of the receptor protein and the receptor modeling methods. Validation is performed on ligand binding site pocket with 10 times replication for each receptor. Docking is operated by HP pavilion with a processor Intel (R) 2.2 GHz, 2.00 GB of RAM, and a 64 bit operating system. Docking software is done by MVD 2008 with a grid resolution of 0:30, iteration a maximum of 1500, maximum population size 50, a pose energy generation 100.00, evolution simplex used at step 300 and max distance scanning 1.00. The parameters of docking are MolDock Score, Rerank Score, RMSD (Root Mean Standard Deviation) and H Bond. MolDockScore value of ligands which has a lower energy so it is more stable in receptor binding and it can be choosen to synthesized. Synthesis of mefenamic acid derivatives The first stage mefenamic acid (2 mmol) was dissolved in 30 ml acetone and then added NaHCO3 0.19 g (2.2 mmol) and benzoyl chloride derivative which is optimum according to the prediction of molecular docking approach (4-NO2-benzoyl chloride) (2.2 mmol) in 5 ml acetone from the funnel into the flask drop by drop over 30 minutes thus stirring in ice bath. After the completion reagents are added, the mixture is heated at 40° C and stirer for 4 hours. When the reaction is complete, the reaction proceeds until the solvent is evaporated by the evaporator runs out then the precipitate is added with water (10 ml) and ethyl acetate (30 ml) resulting in two phases, organic and water phase. Further, 2 phase of solution was washed with HCl pH 3-4 (10 ml) and separated with a separating funnel where collected by ethyl asetat. Ethyl acetate phase was washed with NaHCO3 pH 7-8 (10 ml) and separated with a separating funnel and collected. Thus, Ethyl acetat phase was dried with sodium sulfate anhidrat and evaporated. The residue is purified by column chromatography with methanol:toluene (2:8). The crystals were collected and saved in exicator. Purity test of mefenamic acid derivatives Purity Test of mefenamic acid derivative Thin Layer Chromatography (TLC). Purification using TLC (Thin Layer Chromatography) need to mobile phase methanol: toluene (2: 8). After eluation finished and observed with 254 nm. b. Melting point range. The range of melting point requirement to determine of purity of compounds is ≤2C Identification of mefenamic acid derivatives a. Infrared spectrophotometry. A number of mixed homogeneous powder sample with KBr and made the form of pellets with hydraulic presses and the spectrum observed at wave numbers 4000-400 cm by FTIR. b. Spectro 1H-Nuclear Magnetic Resonance. Samples were prepared by dissolved in chloroform-d1 (CDCl3), with the internal standard tetramethylsilane (TMS) and irradiated with radio wave. Spectrum wave of H NMR is a graph of the amount of energy absorbed (I or intensity) against strong magnetic field. Analgesic Acivity Test Mice were divided into 6 groups, each consisting of 4 Puspaningtyasm et al. / Drug Development of... IJPCR, Volume 9, Issue 2: February 2017 Page 125 mice. Groups I was negative control (CMC Na 1%). Groups II is the positive control group that treated mefenamic acid. Groups III-VI is treated group given mefenamic acid derivative synthesized with 4 concentration. Administration of mefenamic acid derivative was given one times one day for 7 to 14 days. Method of Hot Plate Analgesic Pain stimuli used in hot plate (55-56C). Hot pain in the legs of mice led to responses raised front legs and licked. The average pet mice will provide a response to this method within 3-6 seconds. Mice weighed (25-35 g) is noted and given symbol. 30 minutes later the mice were placed on a hot plate that had been set the temperature at 50 ± 0.5C. Observe the time the mice began to put on a Table 1: Molecular Docking Scoring of mefenamic acid and its derivatives into (COX-2). No Compounds Moldockscore Rerank score RMSD H Bond 1 Native ligan -105.321 -84.4739 0.822142 0.35223 2 Asam mefenamat -107.7271 -87.27073 4.653694 -3.406527 3 4Cl-benzoil Asam mefenamat -119.3606 -53.30389 3.119501 -5.826691 4 4Br-benzoil Asam mefenamat -108.14942 -3.870076 4.080552 -0.3546422 5 4F-benzoil Asam mefenamat -118.41 -49.06499 3.218392 -5.691136 6 4CF3-benzoil Asam mefenamat -124.8416 -55.21401 3.733331 -5.601465 7 4NO2-benzoil Asam mefenamat -131.7728 -19.617856 3.013529 -5.2265942 8 2Cl-benzoil Asam mefenamat -109.707 64.167485 4.423048 2.7628988 9 4OCH3-benzoil Asam mefenamat -123.5064 -54.77227 3.061372 -5.698905 10 3Cl-benzoil Asam mefenamat -112.0296 -21.040703 2.862561 0.2114293 11 3NO2-benzoil Asam mefenamat -127.2584 -78.79086 3.738665 -5.04409 12 Benzoil Asam mefenamat -113.3884 -41.10053 3.058158 -4.8811931 13 3,4Cl-benzoil Asam mefenamat -128.4548 -54.73877 3.245705 -6.471567 Figure 1: Reaction of Mefenamic acid derivative. Figure 2: Molecular Modelling mefenamic acid by secondary sheet in COX-2 receptor. Figure 3: Molecular Modelling 4-NO2-benzoyl mefenamic acid (4NBMA) by secondary sheet in COX-2 receptor. Figure 4: Benzoylation reaction of 4-NO2-benzoyl mefenamic acid (4NBMA) between mefenamic acid and 4-NO2-benzoyl chloride. Table 2: Spectrum Characteristic Infra Red of mefenamic acid. Wavelength (cm-1) Function Groups 3311 N-H (secondary) 1511-1651 C=O Carboxyl salt 2924 C=C aromatic 893 O-substitution Puspaningtyasm et al. / Drug Development of... IJPCR, Volume 9, Issue 2: February 2017 Page 126 Table 3: Spectrum Characteristic H NMR of mefenamic acid. Chemical Shift (ppm) Integration Multiplicity Proton from groups 13 1 singlet COOH 9.5 1 singet NH 6,68-