2,4-Substituted Oxazolones: Antioxidant Potential Exploration

Oxidative stress plays a role in the development of various chronic illnesses, such as cancer, atherosclerosis and neurodegenerative conditions. Lipid peroxidation that occurs during periods of oxidative stress results in the formation of lipid electrophiles and free radicals which can modify a multitude of proteins in the cell. The generation of free radicals is considered a primary factor contributing to various diseases such as heart disease and cancer. Reactive Oxygen Species (ROS), on the other hand, primarily oxidize the cell membrane due to their high concentration of unsaturated fatty acids in the lipid components. Membrane lipids cause lipid peroxidation by ROS leading to the formation of lipid hydroperoxide that can further decompose to aldehydes such as malondialdehyde, 4-hydroxynonenal, Isoprotans, cyclic endoperoxides, etc.^1–3 This lipid peroxidation results in the cross-linking of membrane proteins, altered membrane fluidity, forming lipid DNA and lipid-protein adduct, which may be disastrous to the cell function. Certain amino acid residues of protein side chains like tryptophan, cysteine and methionine are permitted for oxidation by ROS. Protein oxidation products are carbonyl compounds that have direct and indirect damage by alteration in the tertiary structure. Cross-linkages between proteins, proteolytic degradation and fragmentation. Even though DNA is a well-sheltered molecule, ROS can interact and cause various damages such as alteration of DNA base pairs, strand breaks, loss of Nitrogenous bases, damage to the sugar moiety, DNA protein cross-linkages and damage to DNA repair mechanisms. Sugar moiety is also susceptible to attack by free radicals leading to sugar peroxyl radicals and eventually resulting in strand breakage. The modification of genetic material as a cause of DNA damage results in cell death, mutagenesis and ageing.^4,5 Chronic and cumulative oxidation process results in drastic changes in DNA, proteins and lipids.

In this context, antioxidants play a crucial role in counteracting the harmful effects of free radicals by donating electrons, thereby neutralizing them and preventing further electron donation. This mechanism effectively safeguards tissues and cells from reactive oxygen species-mediated damage. Antioxidants are often described as substances that “swab off” free radicals by neutralizing them and inhibiting the pickup of electrons by nucleophiles.

Mechanochemical synthesis is one of the green chemistry techniques that involves grinding the crystals together, using different reagents in a mortar and pestle with the formation of local heat that induces a reaction between two materials in the presence of a small amount of liquid phase.^6–9 Heterocyclic Chemistry has attracted chemists to achieve the synthesis of several heterocyclic systems such as pyrazolines, pyrimidines, etc., via greener technique. This method possesses short reaction time, higher yields and products formed at normal room temperature. Moreover, it was successful and selective only for solid-state reactions rather than the liquid phase. Because the molecules are more firmly held and arranged regularly in a crystal lattice than liquid components. Based on the advantages and highlights of the MHC approach, it is envisaged that it can provide superficial access to azlactones in a more efficient, economical and greener way than conventional methods.

Azlactones are a class of small five-membered heterocycles recognized as essential pharmacophores with excellent pharmaceutical properties. They are regarded as keto derivatives of oxazolines and serve as a useful intermediate in the synthesis of alpha-amino acids, alpha-keto acids, amides, peptides, amino alcohols and polyfunctional compounds. It can also be readily prepared from N-protected amino acids by dehydration. It also possesses several biological activities like immune modulatory,¹⁰ pesticidal,¹¹ antidiabetic activity,¹² antitubercular¹³ anti-cancer,¹⁴ anti-HIV,¹⁵ anticonvulsant,¹⁶ anti-angiogenic,¹⁷ analgesic and anti-inflammatory activity.¹⁸ Recently research has explored all these biological activities and believed that most of the reported diseases would be occurring only because of a demand for antioxidants in the human system. Therefore, antioxidants have attracted considerable attention to oxidative stress, reducing free radicals and the prophylaxis of cancer and longevity. Therefore, this research paper aims to investigate the role of azlactones in antioxidant activity as well as to assess the antioxidant potential of azlactones and determine their effectiveness in scavenging free radicals and preventing oxidative damage.

4-Benzylidene-2-phenyl-substituted-4(5)-oxazolone derivatives were prepared using various aromatic aldehydes and Hippuric acid using acetic anhydride and sodium acetate as catalyst. The reactant hippuric acid, acetic anhydride and sodium acetate was available at college; various aromatic aldehydes like benzaldehyde, 3,5-dimethoxy-4-hydroxy benzaldehyde, were purchased from Southern India Scientific Corporation Limited Chennai.

By using a different technique like Grind stone method, 4-Benzylidene-2-phenyl substituted-4(5)-oxazolone derivatives were synthesized. The melting points were determined using Open capillary tube method. The chemicals purity was justified using thin layer chromatographic Technique. Docking was performed for the designed compounds using Autodock version 4.2. The compounds were sketched using ChemDraw version 8.0 and geometry optimization was done by using AvogadroV1.2.0. The protein was preprocessed by UCSF Chimera, Kollmann charges were assigned, grid points were generated and it was subjected to molecular docking to analyze the active site binding affinity and its interactions which resulted in rank by energy. Visualization of the docked complexes was done by using the Biovia molecular discovery studio visualizer.¹⁹ Based on the experimental evidence and the validation report like resolution, Ramachandran outliers and side chain outliers, the pdb 2X08 was found to be selective. Moreover, the co-crystallized ligand present in the protein was found to be ascorbate which is an excellent standard that can be comparatively studied against the synthesized molecules. The compounds that ranked excellent were subjected to various in silico analysis viz., drug-likeness by Molsoft, bioactivity by Molinspiration,^20,21 ADME parameters by SWISS ADME²² and toxicity analysis by VNN-ADMET software.^23,24

A mixture of hippuric acid (1.2 mmol; 0.210 g), fused sodium acetate (1.5 mmol; 0.123 g) and appropriate aromatic aldehyde (1.0 mmol) was mixed in a porcelain mortar and pestle in the presence of a few drops of acetic anhydride (Figure 1). Grinding was carried out for different time intervals at room temperature. The completion of the reaction was determined by TLC and the reaction mixture turned to a yellow solid. To this, 15 mL of ethanol was added and the mixture was kept in the refrigerator overnight.^25–30 The resulting solid product was collected by filtration, washed with cold water and recrystallized from ethanol to give the desired azlactones. All the synthesized compounds were subjected to in vitro antioxidant activity by DPPH method and Hydroxy radical scavenging method using ascorbic acid as standard.

Figure 1:
Synthetic route for 4-benzylidene-2-phenyloxazol-5(4H)-ones (M₁-M₆).

The docking results of title compounds were presented in Table 1 with their binding energies expressed in kcal/mol. All the compounds showed excellent binding affinity towards the receptor 2X08. The physicochemical properties of the synthesized compounds were tabulated in Table 2 Drug likeness is an important criterion to justify the compound’s potential to be a drug candidate by correlating the molecular properties and structural features and its results were tabulated in Table 3. The designed derivatives were subjected to bioactivity against regular human receptors which is necessary for the development of drugs. The bioactivity scores of the compounds were calculated and reported in Table 4 for their GPCR ligand, kinase inhibitor, protease inhibitor, ion channel inhibitor and nuclear receptor ligand and enzyme inhibitor activities. The toxicity profile of the docked compounds was predicted using VNN-ADMET software and reveals results in terms of membrane transporters along with other properties like Herg blockers, MMP, AMES and MRTD values shown in Table 5.

Each compound is assigned a unique identifier for reference throughout the study (M₁-M₆), with corresponding information about its molecular weight (in grams per mole), the time taken for synthesis (in min), the melting point (in degrees Celsius) and the percentage yield of the reaction of various 4-Benzylidene-2-phenyl substituted-4(5)-oxazolone derivatives were given in Table 5.

M.P.165°C, yield 88%; FT-IR (KBr,cm^-1): 1506 (C=C), 1202 (C-C),1220 (C=O) (lactone), 1613 (C-N); 3014 (C-H);¹H NMR (400 MHz, DMSO): δ 7.33 and δ 7.60 (m, 5H,Ar-H), δ7.91(s, 1H,CH) δ 7.52 and δ 7.95 (m, 5H,Ar-H); MS (ESI) m/z:250 [M]+1; Anal. found C,76.8%; H, 4.0%,N, 5.61%, O, 12.82%;Calculated values: C, 77.02%, H, 4.41%, N, 5.62%, O, 12.84%.M.F:C₁₆H₁₁NO₂.

M.P.150°C, yield 90%; IR (KBr,cm^-1): 1512 (C=C), 1214 (C-C), 1750 (C=O) (lactone), 1610 (C-N), 3612 (phenolic OH), 1350 (C-O-C); 3016 (C-H); ¹H NMR (400 MHz, DMSO): δ7.33 and δ7.60(m, 5H,Ar-H),δ7.91(s, 1H,CH) δ 7.52 and δ7.95 (m, 5H,Ar-H); δ 6.72(s, 2H,Ar-H), δ 3.83(s, 6H,O-CH₃), δ 7.91(s, 1H,CH),δ 7.52 and 7.95 (m, 5H,Ar-H),δ 5.35(s, 1H, OH); MS (ESI) m/z:326 [M]+1; Anal. found C,66.39%; H, 4.62%, N, 4.30%, O, 24.56%; Calculated values: C, 66.46%, H, 4.65V N, 4.31%, O, 24.59%. M.F: C₁₈H₁₅NO₅.

M.P.135°C,yield 96%; IR (KBr,cm^-1): 1508 (C=C),1220 (C-C),1602 (C=O) (lactone), 1400 (C-N), 3010 (C-H); 3600 (OH); ¹H NMR(400 MHz, DMSO): δ 7.16(d1H,Ar-H), δ 7.53(t,1H,Ar-H) δ 6.83(s, 1H,Ar-H), δ 5.35(s, 1H,Ar-H), δ 6.70(s, 1H,Ar-H), δ 7.91(s, 1H,CH), δ 7.52 and 7.95 (m, 5H,Ar-H); MS (ESI) m/z:266 [M]+1; Anal. found C, 72.28%; H, 4.14%; N, 5.27%, O, 18.06%; Calculated values: C, 72.45%, H, 4.18%, N, 5.28%, O, 18.09%, M.F: C₁₆H₁₁NO₃.

M.P.189°C, yield 96%; IR (KBr,cm^-1) 1512(C=C),1214(C-C),1285 (C=O) (lactone),1606 (C-N), 787 (C-Cl), 3011 (C-H);¹H NMR (400 MHz, DMSO): δ 7.68 and 7.44(d,4H,Ar-H), δ7.91(s, 1H,CH),δ 7.95(d, 2H,Ar-H), δ 7.52(m, 3H,Ar-H);MS (ESI) m/z: 284 [M]+1; Anal. found C, 67.67%, H, 3.52%, N, 4.93%, O, 11.26%, Cl 12.33%; Calculated values: C, 67.74%, H, 3.55%, N, 4.94%, O, 11.28%, Cl, 12.52%. M.F C₁₆H₁₀ClNO₂.

M.P.238°C, yield 93%; IR (KBr,cm^-1):1514 (C=C), 1210(C-C),1274 (C=O) (lactone),1610 (C-N), 1562 (NO₂₎; 3015 (C-H);¹H NMR (400 MHz, DMSO): δ 7.99(d, 1H,Ar-H), δ 7.66(t, 1H,Ar-H), δ 8.14(d,1H,Ar-H), δ 8.31(s, 1H,Ar-H), δ 8.02(s, 1H,OH), δ 7.95(d, 2H,Ar-H) and δ 7.52(m, 3H,Ar-H);MS (ESI) m/z:295 [M]+1; Anal. found C, 65.24%, H, 3.39%, N, 9.51%, O, 21.72%; Calculated values: C, 65.31%, H, 3.43%, N,9.52%, O, 21.75%. M.F: C₁₆H₁₀N₂O₄.

M.P.166°C, yield 86%; IR (KBr,cm^-1): 1519 (C=C), 3014 (C-H),1204 (C-C), 1274 (C=O)(lactone), 1612 (C-N), 1242 (C-O-C); ¹H NMR (400 MHz, DMSO):δ 7.62(d, 2H,Ar-H) δ 6.94(d, 2H,Ar-H), δ 3.83(s, 3H,O-CH3),δ 7.91(s, 1H,CH),δ 7.95(d, 2H,Ar-H) and δ 7.52(m, 3H,Ar-H);MS (ESI) m/z:280 [M]+1; Anal. found C, 73.04%, H, 4.65%, N, 5.01%, O, 17.18%; Calculated values: C, 73.11%, H, 4.69%, N, 5.02%, O, 17.19%. M.F: C₁₇H₁₃NO₃.

Antioxidants plays a pivotal role in the neutralization of all free radicals generated out of different metabolic reactions. Every organism constantly produces nitrogen species and reactive oxygen species during their physiological activities. However, their level drastically increases in different pathological conditions as a result of dysfunction of pro-oxidative systems. In such cases their production and trapping should be counterbalanced only by natural anti-oxidant system. A disruption in this extends to oxidative stress causing irreversible damage to cell and their components leading to different diseased conditions. Inspite of ascorbic acid as standard, synthetic compounds are also being tested to identify the potential antioxidant capacity. Therefore, all the synthesized compounds were subjected to in vitro antioxidant activity by DPPH method and hydroxy radical scavenging method using ascorbic acid as standard.

All the six compounds were subjected to molecular docking to identify the ligand’s affinity on the receptor through different amino acid interactions. The compounds were docked through the validated target 2X08 cytochrome C oxidase and it is exhibiting different amino acid interactions proving the ligand efficiency. Compounds M₃ and M₅ showed good interaction with the amino acids by illustrating less binding energy. The 2-dimensional and 3-dimensional images with interactions of compounds M3 and M5 are shown in Figure 2. Molecular Weight (g/mol) indicates the molecular weight of each compound, providing insight into its mass and chemical composition. All compound’s molecular weights are in between the range of 249.26 to 325.32. The time required for the synthesis of each compound is documented in Time Taken (min), reflecting the duration of the chemical reactions involved in the process in between 25 to 45 min. The Melting Point is crucial for understanding the compound’s physical properties. All compound’s percentage yields are obtained in between the range of 86 to 96 which represents the efficiency of the synthesis process and higher percentage yield suggests a more efficient and successful synthesis process. All the compounds fall within the range of 2.5 to 4Å for their log P values, indicating favorable oral bioavailability according to Lipinski’s rule of five, which establishes suitable log P values. The compounds satisfy the criterion where the Total Polar Surface Area (TPSA) should be less than 140Å. With TPSA values ranging from 35-85Å, all compounds have TPSA values below 90Å, indicating enhanced penetration. Furthermore, the compounds have molecular weights between the ranges of 249.26 to 325.32 classifying them as small molecule inhibitors since their molecular weights are less than 500 Daltons. Table 3 demonstrates that the compounds adhere to the Lipinski rule, as the number of hydrogen bond donors and acceptors fall within the specified range. Additionally, the number of rotatable bonds in the compounds is less than 5, indicating favorable flexibility of the molecules. As a general rule, the larger the bioactivity score, the higher the probability that the investigated compound will be active. Therefore, a molecule having a bioactivity score of more than 0.00 is most likely to possess considerable biological activities, while values -0.50 to 0.00 are expected to be moderately active and if the score is less than -0.50 it is presumed to be inactive. The obtained results revealed that the investigated novel compounds are biologically active molecules and will produce the physiological effect by interacting with GPCR ligands, nuclear receptor ligands, protease inhibitors and other enzymes. GPCR lies between -0.95 to -0.65, showing Nuclear Receptor (-0.55 to -0.90). Protease inhibitor from -0.55 to -0.90 and enzyme inhibitor depicted from -0.25 to -0.45. For drug-induced liver toxicity, all M₁-M₅ showed toxicity except compound M₆. Furthermore, cytotoxicity was only exhibited by compound M₅, the remaining all did not indicate any toxicity. In conclusion, only compound M₆ did not depict any toxicity. All the compounds cross the blood-brain barrier, except compound M₅. Additionally, for PgP inhibitors only compounds M₄ and M₅ were not inhibited remaining all were inhibited. Similarly, with the PgP Substrate, compoundsM₁, M₄ and M₅ are not the substrate which does not aid in the elimination of the toxins from the cells, whereas M₂, M₃ and M₆ show the activity eventually acting as PgP substrates. Compound M₅ did not produce any activity whereas compound M₆ showed the activity for all the transporters. None of the drugs indicated Herg blocking activity as well as MMPS. On the other hand, the Ames test showed that only compound M₅ had mutagenicity, whereas the other four compounds were devoid of special toxicities. Likewise, MRTD was calculated least for compound M₅ and maximum for compound M₆ at 99 mg/day and 142 mg/day respectively. The structure activity relationship of 4- Benzylidene-2-Substituted oxazolones was exhibited diverse in activity. Compound M₃ substituted with hydroxyl group showed higher antioxidant activity and compound M₅ substituted with nitro group showed increase in activity whereas compounds substituted with electron withdrawing groups like 3,5-dimethoxy-4-hydroxy, 4-methoxy and 4-chloro showed substantial decrease in activity. The findings from the anti-oxidant activity reveals that the compound M₃ and M₅ showed excellent antioxidant activity against the standard ascorbic acid, whereas other compounds M₂, M₄ and M₆ exhibited moderate antioxidant activity which is closer to the values of M₃ and M₅. Therefore, compounds with excellent antioxidant activity can be further investigated for their related potential biological activities.

Figure 2:
Docking interactions of the potent compounds.

4-phenylidene-2-Substituted oxazolones were exhibited diverse in activity. Compound M₃ substituted with hydroxyl group showed higher antioxidant activity and compound M5 substituted with nitro group showed increase in activity whereas compounds substituted with electron withdrawing groups like 3,5-dimethoxy-4-hydroxy, 4-methoxy and 4-chloro showed substantial decrease in activity.

In the current research investigation, we established a simple, efficient and eco-friendly method to synthesize azlactones without solvents. This approach offers advantages such as higher yields and rapid workup compared to traditional methods. After synthesis, we conducted a comprehensive in silico evaluation, including docking studies, drug-likeness assessments, bioactivity predictions, ADME profiling and toxicity analyses, affirming the rationale and potential applicability of compounds M₁-M₆. Compounds M₃ and M₅ showed significant in vitro antioxidant activity, surpassing ascorbic acid in the DPPH assay. The binding affinity was too excellent towards the target for M₃ and M₅ exhibiting -8.9 and -10.04 kcal and the percentage of inhibition was found to be 88% and 89% respectively. The in silico findings from the docking analysis were well correlated with the in vitro antioxidant activity justifying the potency. Their substantial efficacy suggests potential therapeutic use against oxidative stress-related disorders. These findings provide a strong foundation for further exploration and development of these compounds as promising therapeutic agents, bridging efficient synthesis with detailed computational assessments for potential applications in addressing oxidative stress-related conditions.

We thank the Research Council of SRMIST and the Dean of SRM College of Pharmacy for their valuable support.