Molecular Docking Studies, Synthesis of Novel Isoxazole Derivatives from 3-Methoxy Substituted Chalcone and Evaluation of their Anti-Inflammatory Activity

Synthesis of 3-methoxy acetophenone with substituted benzaldehydes resulted in a number of novel chalcones. The chalcones were then treated to a cyclization reaction with hydroxylamine hydrochloride in ethanol to enable the synthesis of 3-methoxy acetophenone isoxazole derivatives. After purification, the structures of the synthesized compounds were identified using TLC, FTIR, 1 H NMR, 13 C NMR and a Mass spectroscopy. The carrageenan-induced paw edema method was used to test the compounds for anti-inflammatory activity. Based on the findings, the three compounds appeared to be moderate to extremely active.


INTRODUCTION
An essential defense mechanism for the body's protection against physical or chemical injury or illness threats harm is inflammation.Often fatal illnesses including autoimmune conditions, Crohn's disease, rheumatoid arthritis, and inflammatory syndrome bowel utilize this defense mechanism 1 .The essential regulator of the inflammatory process is a prostaglandin.When prostaglandin E2 is unstable, an enzyme known as isomerase that is specialized to this intermediate transforms it into a variety of prostanoids 2 .Arachidonic acid is transformed into prostaglandin E2 by the enzyme cyclooxygenase (COX).A number of produces prostaglandins, the main mediators of inflammation, pain, and increased body temperature, important enzymes are cyclooxygenases (COX) or prostaglandin endoperoxide synthases (hyperpyrexia).Two primary COX protein isoforms that the body processes are cyclooxygenases-2 (COX-2) and cyclooxygenases-1 (COX-1).and crucial biological mediators like prostanoids, prostacyclin, thromboxane, and prostaglandins are produced by COX-1, which is also involved in the production of pain, blood clotting, and stomach protection 3 .While COX-2 has an essential function in the production of prostaglandins by inflammatory cells and the central nervous system and is engaged in pain through inflammation4.Inflammation has reduced the inhibition of COX-1, although the stomach's inner protective properties are also lost.This may cause stomach aches, ulcers, and including gastrointestinal and intestinal hemorrhage.COX-2, however, normally only affects inflammatory tissue 5 .Benzylidene acetophenones or benzal acetophenones are other names for chalcones 6 .Several heterocyclic ring systems, such as isoxazoles, pyrazolines, pyrimidines, and cyano pyridines, may be synthesized using chalcones 7 .Isoxazole is one of the five-membered heterocycles.In the molecule isoxazole, two heteroatoms, oxygen, and nitrogen, are situated next to one another.The molecular structure of the molecule is unsaturated due to two double bonds between carbon atoms [8][9][10] .Isoxazoles have a wide range of pharmacological and biological activities.which include antibacterial 11 , antifungal 12 , GABA antagonist 13 , anti-inflammatory 14 antidiabetic 15 and anticancer [16][17] .Inspired by the above facts, The current study's objective was to synthesize novel isoxazole derivatives and assess their ability to reduce inflammation.

MATERIALS AND METHOD
3-methoxyacetophenon, Hydroxylamine hy d r o c h l o r i d e a n d d i f fe r e n t s u b s t i t u t e d benzaldehydes were acquired from AB Fine Chemicals for the current study project (Nasik, India).Parecoxib, which was employed as a reference medication, was purchased from Local Market.All of the additional chemicals and solvents were provided by CDH.All of the chemicals were analytical grade and had been cleaned up before usage.
The open capillary method was employed to identify the uncorrected melting points of the produced compounds.With a Perkin Elmer 2400 analyzer, the FTIR spectra of synthetic substances were captured on potassium bromide discs.Using a Bruker Advance Neo spectrophotometer and 1 H NMR uses tetramethylsilane (TMS) as an internal standard and 13 CNMR spectra of the produced compounds were captured in CDCl 3 .In ppm, all chemical shift values are recorded.In SAIF, P. U., Punjab, India, synthetic substances underwent spectroscopic analysis.
To track the development of the reactions, silica gel G thin-layer chromatography (TLC) was employyed, and spots were seen in an iodine chamber.The carrageenan-induced paw edema method, will be used to pharmacologically evaluate the target drugs for anti-inflammatory activity.

Synthesis of substituted chalcones for (4a-4n)
Equimolar amounts substituted benzaldehyde and 3-methoxy acetophenone were mixed in pure alcohol (0.02 mol), and 10% NaOH solution (25 mL) was poured in while being stirred dropwise.The action was then permitted to continue for 3-4 h before being stopped overnight.To obtain the outcome, the reaction mixture was then submerged in cold water for a whole night.The final product was created by recrystallizing the crude product from ethanol.

3-(3-Chloro-phenyl)-4-(3-methoxy-phenyl)isoxazole (4j)
Characterization   The docking simulation was carried out using AutoDock Vina when the ligand molecules' energies were lowered.The crystal structures of the receptor protein molecules were identified using the protein data database.The intricate coordinates of the structures, which contained water molecules and other atoms, are what gave r ise to better resolution.[20]  The separated molecules (4f, 4n, 4a, 4e, 4i, 4l, 4d) 2D docked images and Parecoxib binding affinities with the protein 4COX-2 receptor in the 2D images, respectively Table 2. Cyclooxygenase-2 was used as a common medication.4f showed a significant amount of anti-inflammatory activity, whereas 4n showed the least amount.The findings indicated that the activity response was producing outcomes comparable to those of the docking research.

CONCLUSION
The synthesis of novel isoxazole derivatives was detailed in the current work.Mass Spectra, FTIR, 1 H NMR, and 13 C NMR Spectra were used to analyze the synthesized compounds.Using the carrageenan-induced paw edema method for anti-inflammatory activity, Due to the nito group's presence at the meta and para positions, the results showed that compound 4f and 4n was the most active molecule.Further research into the mechanisms of action of some of these compounds may result in the identification of novel compounds with potential anti-effects in a research investigation.