DESIGN, SYNTHESIS AND MOLECULAR DOCKING STUDY OF NOVEL BIS-OXAZOLONE DERIVATIVES AS POTENT ANTIOXIDANT AND ANTIBACTERIAL AGENTS

. In response to the challenge of antibiotic-resistant microorganisms, oxazolone analogs are frequently used for bacterial, antiviral, and anti-inflammatory treatments. However, few studies have shown bis-oxazolone analogs possess antibacterial activities. In this study, we modified bis-oxazolone molecules with various aromatic amines to create new bis-benzamide and bis-imidazolone derivatives. These derivatives were analyzed using FT-IR, ¹H-NMR, and ¹³C-NMR spectroscopy. Molecular docking revealed favorable interactions with DNA gyrase, with compounds 3, 4a, and 5e showing higher binding affinities than penicillin G and ampicillin. These findings suggest their potential as future antimicrobial agents. The tested compounds demonstrated efficacy against bacterial strains, particularly E. coli and S. aureus , with significant activity observed in compounds 4a, 4e, 5d, and 5e. Antioxidant activity, assessed using the DPPH method, showed bis-compounds with excellent results comparable to ascorbic acid. This encourages further studies to explore their potential. Overall, the synthesized bis-oxazolone derivatives demonstrated increased medicinal activity and high potential as future antimicrobial and antioxidant agents


Chemistry
In this study, we synthesized and modified novel compounds from basic precursors to enhance their biological activity and potential as biomolecules and drug candidates.Molecular docking predicted the biological activities of ten oxazolone derivatives with different groups.Among these, compounds 3, 4a, 4e, 5d, and 5e displayed higher antimicrobial activity against E. coli compared to S. aureus at 800 µg.Additionally, DPPH measured antioxidant scavenging, showing increased activity over time across various concentrations, comparable to ascorbic acid.

Anti-bacterial activity
The compounds were tested for antimicrobial activity against E. coli and S. aureus using a dilution method [40].Results in Figure 2 showed greater effectiveness against E. coli than S. aureus for all compounds.The strong antimicrobial effect, possibly due to increased surface area interaction with the bacteria, led to significant growth inhibition.The potency was concentration-dependent, with the highest inhibition observed at 800 µg.Notably 3, 4a, 4e, 5d, and 5e exhibited the most significant activity, while others had varying efficacy against the bacterial strains.

Molecular docking
Molecular docking is one of the most common methods used in structure-based drug design to analyze the interaction between a small molecule and a protein at the atomic level.Molecular docking was performed with optimized compounds targeting DNA gyrase, an ATPase vital for bacterial growth [41,42].It controls supercoiling processes necessary for DNA replication, chromosomal condensation, and gene expression, making it an excellent drug target [14,43,44].The binding affinity of titled compounds was compared with ampicillin which was used as a positive drug for comparison purposes which was recommended for clinical therapeutic [45,46].The binding affinities of the synthesized compounds bis-oxazolone (3), 4a and 5e (-9.2, -9.3 and -9.4 kcal/mol, respectively) were higher than the binding affinity of the positive drug penicillin G and ampicillin (-7.7 and -7.9 kcal/mol, respectively) (Table 1).Thus, these three compounds could be effectively inhibiting the (DNAG) and (PBP1a), which are essential targets for the development of antibacterial medicines (Table 2, Figures 4, 5 and 6).

Materials and methods
Chemicals, sourced from reputable brands such as Scharlau, Fluka, and Riedel-de Haen, were of analytical grade. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were acquired using a Bruker spectrometer at Esfahan University, Iran, with CDCl3 and DMSO as solvents.Chemical shifts (δ) were referenced to TMS.Fourier transform infrared (FT-IR) spectra were recorded using an FT-IR-4100 (Shimadzu) spectrometer.Melting points were determined using an Electrothermal melting point device 9100.TLC, employing Merck silica gel 60 F254 plates and nhexane/ethyl acetate as a mobile phase, was utilized for purity checks with UV visualization.

Synthesis of 4-bromohippuric acid (2)
In a 250-mL round-bottom flask, a mixture of glycine (8.25 g, 0.11 mol) and potassium hydroxide (11.22 g, 0.2 mol) in 50 mL water was cooled to 0 °C.4-Bromobenzoylchlororide (21.95 g, 0.10 mol) was added in three portions with shaking.The reaction mixture was stirred for 1.0 hour.Then, the solution was acidified with conc.HCl, and the product (24 g) was collected and recrystallized from ethanol [36].

Synthesis of bis benzamide (4)
A mixture of bis-oxazolone (3) (714 mg, 1 mmol), substituted aromatic amine (3 mmol) in toluene (20 mL) in the presence trace of acetic acid was refluxed with stirring for 1.0 hour.The reaction mixture was monitored by changing of bright-yellow solution to pale yellow solution.The described product was filtered off, washed by ethanol, dried and re-washed by hot toluene then recrystallized in DMF to obtain pale yellow crystals of compound (4), Table 1, simple techniques and modified procedures were applied, resulting in good yields [38].

Synthesis of bis-imidazolone (5)
Bis imidazolone (5a-j) derivatives were synthesized in one step by mixing of bis-oxazolones (3) (1 mmol) with substituted aromatic amine (3 mmol) in acetic acid (10 mL) [39].The reaction was refluxed with stirring for (6-10) h. it was monitored by color changing to pear-green soluble solution, or in two steps by refluxing bis-benzamide (4) in glacial acetic acid for 8 hours, Scheme 5.The reaction mixtures were poured into water, filtered off, dried, and crystallized in a mixture of toluene and ethanol to obtain bright pear-green crystals of (5a-e). (

CONCLUSIONS
In conclusion, we have demonstrated the effective synthesis and characterization of eleven compounds: bis-oxazolone 1, a series of bis-benzamide 5, and a series of bis-imidazolone 5. We have assessed their antibacterial properties, revealing that the newly synthesized bis-oxazolone, bis-benzamide, and bis-imidazolone compounds showcase promising antimicrobial potential, as evidenced by antimicrobial assays.According to this study, we have noticed that our newly synthesized benzamide derivatives mostly showed significantly antibacterial activity against Gram-positives than Gram-negatives.
Molecular docking analyzed the interaction between optimized compounds and DNA gyrase, crucial for bacterial growth, revealing higher binding affinities for (3, 4a, and 5e) compared to penicillin G and ampicillin (-7.7 and -7.9 kcal/mol, respectively).These findings suggest their potential in inhibiting essential bacterial targets like DNAG and PBP1a, for antibacterial drug development, as depicted in Tables (1-4) and Figures (4)(5)(6).All investigated substances have displayed moderate to high antimicrobial activity against at least two species (E. coli and S. aureus), positioning them as excellent candidates for further biological screening tests, particularly for external wound applications.
In the DPPH antioxidant activity evaluation, bis-compounds showed excellent results, comparable to ascorbic acid.Their superior radical scavenging capabilities, due to enhanced functionality and conjugation, highlight their potential as effective antioxidant agents.These findings encourage further research to explore their therapeutic applications and develop potent antioxidant compounds for medical use.These findings highlight the multifaceted pharmacological potential of these compounds and warrant further exploration for their clinical applications in combating microbial infections and oxidative stress-related diseases.

Figure 2 .
Figure 2. Antimicrobial activity of screened compounds (3, 4a,e and 5d,e) against: a. S. aureas and b.E. coli.DPPH radical scavenging ability DPPH, a stable organic nitrogen radical, assesses antioxidant scavenging capabilities.The deep violet color of the DPPH radical signifies its electron impairment, with radical scavenging

Table 1 .
The compounds binding affinity.