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

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 mechanism¹. 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 prostanoids². 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 protection³. 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 tissue⁵. Benzylidene acetophenones or benzal acetophenones are other names for chalcones⁶. Several heterocyclic ring systems, such as isoxazoles, pyrazolines, pyrimidines, and cyano pyridines,   may be synthesized using chalcones⁷. 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 ^8–10. isoxazoles have a wide range of pharmacological and biological activities. which include antibacterial¹¹, antifungal¹², GABA antagonist¹³, anti-inflammatory¹⁴  antidiabetic¹⁵ 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.

Experimental

Materials and Method

3-methoxyacetophenon, Hydroxylamine hydrochloride and different substituted 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 1H NMR uses tetramethylsilane (TMS) as an internal standard. and ¹³CNMR spectra of the produced compounds were captured in CDCl3. 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 hours 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.

Research Scheme

Scheme 1: Synthesis of Isoxazole derivatives (4a-n). Click here to View Figure

Basic fundamentals of Isoxazole Derivative Synthesis (4a-n)

 (0.01 mole)  chalcones (1a-1n), (0.01 mole)   hydroxylamine hydrochloride were added to and Sodium acetate  ethanol (25 mL), and the mixture was mixed and then refluxed in an oil bath for six hours before being put in the cold water (50 mL). The precipitate obtained from ethanol was cleaned, filtered, and recrystallized. (Scheme-I).

{4-[4-(3-Methoxy-phenyl)-isoxazol-3-yl]-phenyl}-dimethyl-amine (4a)

Characterization Brown solid; Yield: 70%; m.p. 290-291 ºC. Elemental analysis of C₁₈H₁₈N₂O₂ calcd. (found) %: C, 73.45; H, 6.16; N, 9.52; O, 10.87. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.76-7.57 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.33 (C8), 155.35 (C13), 130.27 (C15), 112.62 (C16,C18), 130.69 (C19), 40.309 (OCH3), Mass: m/z: (M+1) 295.10.

4-(3-Methoxy-phenyl)-3-phenyl-isoxazole (4b)

Characterization White solid; Yield: 85%; m.p. 223-224 ºC. Elemental analysis of C₁₆H₁₃NO₂ calcd. (found) %: C, 76.48; H, 5.21; N, 5.57; O, 12.73. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.96 (s, 1H, CH=CH), 6.92-7.57 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.57 (C8), 130.27 (C15), 129.69 (C16,C18), 55.34 (OCH3), Mass: m/z: (M+1) 252.94.

3-(4-Chloro-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4c)

Characterization White solid; Yield: 80%; m.p. 265-267 ºC. Elemental analysis of C₁₆H₁₂ClNO₂ calcd. (found) %: C, 67.26; H, 4.23; Cl, 12.41; N, 4.90; O, 11.20. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.92-7.56 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.96 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.58 (C8), 130.91 (C15), 129.25 (C16,C18), 130.91 (C19), 55.33 (OCH3), Mass: m/z: (M+1) 286.02.

4-[4-(3-Methoxy-phenyl)-isoxazol-3-yl]-phenol (4d)

Characterization Black solid; Yield: 80%; m.p. 334-335 ºC. Elemental analysis of C₁₆H₁₃NO₃ calcd. (found) %: C, 71.90; H, 4.90; N, 5.24; O, 17.96. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617, 3432 (OH), (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.73-7.42 (m, 8H, Ar-H), 5.021 (s,1H, OH), 13C NMR (100 MHz, CDCl3) δ: 130.96 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.37 (C8), 130.69 (C15), 116.40 (C16 ,C18), 121.67 (C19), 55.34 (OCH3), Mass: m/z: (M+1) 268.07.

3-[4-(3-Methoxy-phenyl)-isoxazol-3-yl]-phenol (4e)

Characterization Black solid; Yield: 80%; m.p. 413-415 ºC. Elemental analysis of C₁₆H₁₃NO₃ calcd. (found) %: C, 71.90; H, 4.90; N, 5.24; O, 17.96. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617, 3432 (OH), (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.87 (s, 1H, CH=CH), 6.85-7.42 (m, 8H, Ar-H), 5.021 (s,1H, OH), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.96 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.36 (C8), 130.69 (C15), 118.40 (C16), 122.56 C18), 121.67 (C19), 55.34 (OCH3), Mass: m/z: (M+1) 268.04.

4-(3-Methoxy-phenyl)-3-(3-nitro-phenyl)-isoxazole (4f)

Characterization Brown solid; Yield: 85%; m.p. 421-422 ºC. Elemental analysis of C₁₆H₁₂N2O₄ calcd. (found) %: C, 64.86; H, 4.08; N, 9.46; O, 21.60. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 9.03 (s, 1H, CH=CH), 6.92-8.74 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.68 (C8), 125.26 (C15), 126.80 (C17), 130.29 C18), 136.52 (C19), 55.34 (OCH3), Mass: m/z: (M+1) 297.01.

3-(3-Bromo-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4g)

Characterization Yellow solid; Yield: 85%; m.p. 294-295 ºC. Elemental analysis of C₁₆H₁₂BrNO₂ calcd. (found) %: C, 58.20; Br, 24.20; N, 4.24; H, 3.66; O, 9.69. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.96 (s, 1H, CH=CH), 6.92-7.77 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.34 (C8), 132.24 (C15), 133.80 (C17), 130.83 C18), 128.59 (C19), 55.34 (OCH3), Mass: m/z: (M+1) 330.00.

3-(4-Bromo-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4h)

Characterization Yellow solid; Yield: 85%; m.p. 294-295 ºC. Elemental analysis of C₁₆H₁₂BrNO₂ calcd. (found) %: C, 58.20; H, 3.66; Br, 24.20; N, 4.24; O, 9.69. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.92-7.64 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.58 (C8), 131.87 (C15), 130.56 (C16), 55.34 (OCH3), Mass: m/z: (M+1) 330.01.

3-(4-Fluoro-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4i)

Characterization Brown solid; Yield: 80%; m.p. 237-238 ºC. Elemental analysis of C₁₆H₁₂FNO₂ calcd. (found) %: C, 71.37; H, 4.49; F, 7.06; N, 5.20; O, 11.88. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.92-7.97 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.34 (C8), 131.07 (C15,C19), 116.87 (C16), 55.34 (OCH3), Mass: m/z: (M+1) 270.07.

3-(3-Chloro-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4j)

Characterization White solid; Yield: 80%; m.p. 266-267 ºC. Elemental analysis of C₁₆H₁₂ClNO₂ calcd. (found) %: C, 67.26; H, 4.23; Cl, 12.41; N, 4.90; O, 11.20. H, 5.34 (5.32). IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.92-7.52 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.67 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.42 (C8), 130.69 (C15), 129.16 (C16 C18), 128.91 (C19), 55.34 (OCH3), Mass: m/z: (M+1) 286.02.

3-(2-Methoxy-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4k)

Characterization Brown solid; Yield: 84%; m.p. 269-270 ºC. Elemental analysis of C₁₇H₁₅NO₃ calcd. (found) %: C, 72.58; H, 5.37; N, 4.98; O, 17.06. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.86 (s, 1H, CH=CH), 6.88-7.57 (m, 8H, Ar-H), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.86 (C4), 121.65 (C6), 155.77 (C8), 111.96 (C16), 130.92 (C17), 122.62 (C18), 131.39 (C19) 55.33 (OCH3), 56.40 (OCH3), Mass: m/z: (M+1) 282.02.

3,4-Bis-(3-methoxy-phenyl)-isoxazole (4l)

Characterization Brown solid; Yield: 84%; m.p. 269-270 ºC. Elemental analysis of C₁₇H₁₅NO₃ calcd. (found) %: C, 72.58; H, 5.37; N, 4.98; O, 17.06. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.85 (s, 1H, CH=CH), 6.85-7.42 (m, 8H, Ar-H), 3.83 (s, 6H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 129.96 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.36 (C8), 112.47 (C16), 130.69 (C17), 122.62 (C18), 125.19 (C19) 55.34 (OCH3), 55.35 (OCH3), Mass: m/z: (M+1) 286.04.

3-(4-Methoxy-phenyl)-4-(3-methoxy-phenyl)-isoxazole (4m)

Characterization Brown solid; Yield: 84%; m.p. 269-270 ºC. Elemental analysis of C₁₇H₁₅NO₃ calcd. (found) %: C, 72.58; H, 5.37; N, 4.98; O, 17.06. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=CH), 6.92-7.54 (m, 8H, Ar-H), 3.79 (s, 3H, OCH3), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.35 (C8), 114.64 (C16, C18), 130.69 (C17),   130.56 (C15, C19) 55.33 (OCH3), Mass: m/z: (M+1) 286.02.

4-(3-Methoxy-phenyl)-3-(4-nitro-phenyl)-isoxazole 4n

Characterization Yellow solid; Yield: 80%; m.p. 270-271 ºC. Elemental analysis of C₁₆H₁₂N₂O₄ calcd. (found) %: C, 64.86; H, 4.08; N, 9.46; O, 21.60. IR (KBr, νmax, cm-1): 2861 (O-CH3), 1617 (Ar-H, stretching), 3551 (str, NH amide), 1636 (C=N, stretching in ring), 1026 (CO, stretching in ring), 832 (CH=CH p-disub-Aromatic). 1H NMR (500 MHz, CDCl3) δ: 8.97 (s, 1H, CH=C), 6.92-7.54 (m, 8H, Ar-H), 3.83 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 130.69 (C1), 113.76 (C2), 110.85 (C4), 121.67 (C6), 155.301 (C8), 125.54 (C16, C18), 129.67 (C15, C19) 55.33 (OCH3), Mass: m/z: (M+1) 297.01.

Figure 1: FTIR spectrum of synthesized compound 4f. Click here to View Figure

Figure 2: 1H NMR spectrum of synthesized compound 4f. Click here to View Figure

Figure 3: 13C NMR spectrum of synthesized compound 4f. Click here to View Figure

Figure 4: Mass spectrum of synthesized compound 4f Click here to View Figure

Molecular Modelling Studies

In this work, the isolated compounds (4a–4n) were docked with the target protein Cyclooxygenase-2 (PDB ID: 4COX-2) as part of studies on the molecular docking of the separated molecules. It was estimated through molecular docking studies how the test drugs would probably attach to the targeted Cyclooxygenase-2 proteins using Auto Dock Vina4.2 (MGLtools 1.5.7). 16 A graphic depicting possible docked positions and binding energy (kcal/mol) The compounds’ structures (4a–4n) were shown in the proper 2D orientations using ChemDraw 16.0. In order to lower the energy of the compounds/ligands, ChemDraw3D extreme was employed. 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 rise to better resolution. A protein preparation method with default settings was used to prepare the proteins.^18-20

Table 1: Total Binding Affinities Energy (kcal.mol-1) comparison between Parecoxib and Isoxazole derivatives (4a-4n) against the Cyclooxygenase-2 (4COX-2) receptor.

Compound Code	Involved amino acid residues within 5 Å	Interaction Hydrogen bond	Total Binding Affinity Energy (kcal.mol-1)	RMSD
4a	Cys36, Cys37, Cys41, Cys47, Cys159, Pro153, Val155, Ala156, Glu465	Cys41, Cys47	-8.9	3.395
4b	Asp 125, Ser126, Pro542, Gln543, Lys546	Ser126 Lys546	-8	34.136
4c	Asn39, Cys36, Cys37,Cys41, Cys47, Tyr136,  Pro153, Ala156,	Asn39, Cys41	-7.9	32.611
4d	Cys41, Arg44, Cys47, Leu152, Pro153, Lys468, Arg469,	Cys41	-8.5	2.003
4e	Cys41,  Arg44,  Tyr130, Leu152, Lys468, Arg469,	Cys41 Tyr130	-8.7	1.773
4f	Val 116, Arg120, Val 349, Leu352,Ser353, Leu359, Tyr 385,Phe518, Val523, Alu527, Ser530, Leu531	Arg120 Leu352	-9.1	29.013
4g	Cys41, Tyr130,  Leu152,  Pro153, Lys468		-8.4	40.552
4h	Cys36, Arg41, Cys47,  Gly135,Pro153,Ala156		-8	19.314
4i	Gln42, Asn43, Arg44, Tyr130, Pro153,  Gln461, Glu465, Lys468, Arg469,	Tyr130 Gln461	-8.6	2.573
4j	Cys47,  Tyr130,  Leu152,  Pro153, Lys468	Cys47	-8.6	40.529
4k	Asn39, Cys36, Cys41, Cys47, Tyr136, Pro153, Ala156	Asn39 Cys41	-8.3	45.408
4l	Cys41, Arg44, Glu46,Tyr130, Leu152, Lys468,Arg469	Cys41 Glu46	-8.6	2.559
4m	Pro153, Trp323,Ser548	Ser548	-8.4	29.66
4n	Pro128, Gly225,Val 228,Asn375, Arg376,  Asn537, Pro538	Gly225 Asn375	-9	21.407
Parecoxib	Cys36,Asn39,Arg44,Leu152,Pro153 Gln461	Cys36 Gln461	-9.3	3.274

 

Figure 5: 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 Click here to View Figure

Biological activity

An anti-inflammatory action

Regarding the anti-inflammatory effects, the best docking score compounds were selected (Table 1). Using the carrageenan-induced paw edema method, the synthetic compounds (4f, 4n, 4a, 4e, 4i, 4l, 4d, 4g,) for anti-inflammatory activity^21-24. After two hours, the studied drugs’ anti-inflammatory efficacy is reduced by 43.95% to 62.71%. In comparison to the reference medication Parecoxib, which showed 76.24% inhibition 2 hours later and 75.93% inhibition 3 hours later, the inhibition ranged from 44.32% to 63.71% after 3 h. In comparison to the reference medicine parecoxib, the compounds (4f, 4n, and 4a) were discovered to be the series’ most powerful compounds (Table 2). Compound 4-(3-Methoxy-phenyl)-3-(3-nitro-phenyl)-isoxazole 4f showed 61.99% inhibition  2 hours later and 61.20% inhibition  3 hours later, indicating a high anti-inflammatory effect of these compounds. Whereas substance 4-(3-Methoxy-phenyl)-3-(4-nitro-phenyl)-isoxazole 4n showed inhibition at 2 and 3 hours, respectively, of 61.47% and 62.24%. After 2 and 3 hours, compound 4-[4-(3-Methoxy-phenyl)-isoxazol-3-yl]-phenyl-dimethylamine (4a) inhibited the reaction by 62.69% and 63.69%, respectively. These results served as the foundation for additional research and compound development. The most potent anti-inflammatory effect was displayed by the isoxazole derivatives (4f, 4n, 4a, 4e, 4i, 4l, 4d, 4g) including 4f, 4n, and 4a. The chemical 4g had the least amount of anti-inflammatory action. More than 50% of the compounds 4e, 4i, and 4l exhibited inhibition.

Table 2: Compounds’ anti-inflammatory activity (4f, 4n, 4a, 4e, 4i, 4l, 4d, 4g).

Compounds	Increased Paw Volume (mm)		% Inhibition		Potential
Treatments	after 2h	after 3h	after 2h	after 3h
4f	0.35 ± 0.02 ***	0.34 ± 0.02 ***	62.71	63.71	0.82
4n	0.36 ± 0.02 ***	0.36 ± 02 ***	61.47	62.24	0.81
4a	0.36 ± 0.04 ***	0.37 ±0.04 ***	61.99	61.2	0.79
4e	0.43 ± 0.01 ***	0.43 ± 0.02 ***	54.46	55	0.71
4i	0.45 ± 0.02 ***	0.43 ± 0.03 ***	52.18	55	0.69
4l	0.46 ± 0.04 ***	0.46 ± 0.05 ***	51.31	51.89	0.66
4d	0.49 ± 0.02 ***	0.48 ± 0.02 ***	47.81	50.34	0.63
4g	0.53 ± 0.04	0.53 ± 0.04 ***	43.95	44.32	0.57
Parecoxib	0.20 ± 0.02 ***	0.22 ± 0.02 ***	77.23	76.89	1
Tween 80	0.96 ± 0.02	0.97 ± 0.02	–	–	–

 

Results and discussion

Chemistry for synthesis

By following the steps in Scheme I, all novel isoxazole derivatives (4a–4n) were successfully developed. Through the use of infrared spectroscopy, FTIR, ¹H NMR, ¹³C NMR, Mass Spectra, and elemental analysis, the novel synthesized compounds were verified. substituted benzaldehyde and 3-methoxy acetophenone were combined in a Claisen-Schmidt Condensation reaction to synthesize substituted chalcones (4a-4n). Under constant stirring and substituted chalcones were cyclized with hydroxylamine hydrochloride and sodium acetate by refluxing on an oil bath for six hours to produce isoxazole derivatives (4a-4n).

Molecular model.

Cyclooxygenase-2 (PDB ID: 4COX-2) was the subject of a molecular docking investigation of target compounds (4a-4n) in relation to the reference medication Parecoxib. The target molecules (4a-4n) had lower docking scores on Cyclooxygenase-2, indicating that they could have more effective anti-inflammatory. 4f, 4n, and 4a were among the produced compounds that were most effective because they had the lowest docking scores for Cyclooxygenase-2 (4COX-2) receptor.

Discussion

Pharmacology

Albino rats of all groups were treated 0.3% sodium carboxymethyl cellulose, 100 mg per kg of the test chemicals and 10 mg per kg of parecoxib were suspended. Both the vehicle (control) and the test drugs were used. The injection is given intraperitoneally (i.p). The data showed that all tested substances significantly decreased edema caused by carrageenan, and the results are shown in 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.

Conclusions

The synthesis of novel isoxazole derivatives was detailed in the current work. Mass Spectra, FTIR, ¹H NMR, and ¹³C 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.

Acknowledgments

The management at IFTM University deserves praise for setting up the lab for the effective conduct of the intended study work’s experiments.

Conflict of interest.

There are no competing interests, according to the authors.

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