1,4 Benzoxazine Derivatives: Synthesis, In-Silico Studies And Antimicrobial Evaluation

Abstract

Microbial infections and rising antimicrobial resistance highlight the need for new treatments. Benzoxazine derivatives are promising candidates due to their diverse biological activities. This study synthesized and evaluated the antimicrobial properties of 2H-benzo[b][1,4] oxazin-3(4H)-one derivatives. The synthesis involved reacting 2-aminophenol with chloroacetic acid, followed by sulfonation with chlorosulfonic acid and subsequent nucleophilic substitution with aryl amines. The compounds were tested against E. coli, S. aureus, and B. subtilis, with compound 4e showing the highest activity across all strains. Molecular docking studies targeting E. coli DNA gyrase revealed that compound 4d had the strongest binding affinity, followed by compounds 4a, 4e, and 4f. Binding interactions with key amino acid residues were analyzed, providing insights for further optimization. Compound 4e’s broad-spectrum activity and high potency suggest its potential as a new antimicrobial agent, warranting further in vivo testing and toxicity evaluation.

Keywords

Introduction

Benzoxazines, characterized by their fused oxazine ring structure, exhibit a range of pharmacological effects, including anti-inflammatory, anti-ulcer, antibacterial, and antifungal properties. These compounds primarily exist in the form of aglycones in living systems, where they function as defense chemicals against various organisms such as fungi, bacteria, insects, and weeds. This defensive role is underscored by their presence as natural plant toxins in economically important species within the Poaceae family (e.g., maize, wheat, and rye) and some dicotyledonous species from the Acanthaceae, Lamiaceae, Scrophulariaceae, and Ranunculaceae families. The biological relevance of benzoxazines extends to their analogues, which include cyclic hydroxamic acids, their corresponding lactams, and ring-contracted benzoxazolinones. These naturally occurring compounds are produced as secondary metabolites in plants and have been recognized for their potential antimicrobial properties. The structural simplicity and versatility of benzoxazine derivatives make them attractive candidates for synthetic modification, aimed at enhancing their efficacy as antimicrobial agents. This study focuses on the synthesis and evaluation of 2H-benzo[b] [1,4] oxazin-3(4H)-one derivatives for their antimicrobial activity. By exploring the chemical modifications of these compounds and assessing their effectiveness against various bacterial strains, we aim to identify potent new antimicrobial agents. The findings could contribute significantly to the development of novel treatments to combat resistant microbial infections, addressing a critical need in contemporary medicine.6-10

       
           
       
    Figure 2: Synthesis pathway of Benzoxazine derivatives 4(a-g)

Table 1: Physical data for synthesized derivatives 4a to 4g:

Pharmacological Screening Procedure:

Microbial infections are an escalating threat due to rising antimicrobial resistance. Benzoxazine derivatives have emerged as promising antimicrobial agents due to their diverse biological activities. These compounds, mainly present as aglycones in plants, serve as natural defense chemicals and exhibit anti-inflammatory, antibacterial, and antifungal properties. In this study, 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized and evaluated for their antimicrobial efficacy. The synthesis involved reacting 2-aminophenol with chloroacetic acid to form 2H-benzo[b][1,4]oxazin-3(4H)-one, followed by sulfonation with chlorosulfonic acid and subsequent reaction with aryl amines, yielding potent antimicrobial derivatives 4(a-g) shown in fig. no. 3.

       
           
       
    Figure 3: Chemical structure of synthesized compounds.

The synthesized compounds were assessed for antimicrobial activity through their zones of inhibition against E. coli, S. aureus, and B. subtilis. Compound 4e emerged as the most potent, showing significant inhibition across all strains: 22 mm for E. coli, 20 mm for S. aureus, and 18 mm for B. subtilis. Compound 4a also showed notable effectiveness against E. coli (20 mm). Compounds 4b, 4c, and 4f displayed moderate activity, while 4g was largely ineffective. These findings highlight compound 4e's potential as a broad-spectrum antimicrobial agent, warranting further in vivo and toxicity studies for clinical application shown in fig no.4 and table no.2.

       
           
       
    Antimicrobial Activity of compound 4a-4f against B.subtilis.

       
           
       
    Antimicrobial Activity of compound 4a-4f against E.coli.

Figure 4: Antimicrobial Activity of compound 4a-4f against various micro organisms.

Table 2: Antibacterial activity of selected synthesized Derivatives:

Diameter in mm calculated by Vernier Caliper- means no. zone of inhibition.

Molecular Docking:

Table 3: Docking score (in Kcal/mol) of the synthesized compound in active site of E. coli DNA gyrase B (PDB ID: 5L3J)

Using Maestro's ligands-interaction tool, 2D and 3D representations of ligands-protein interactions in fig. 5 and table no. 3 were generated to understand the binding mode of synthesized compounds with E. coli DNA gyrase B. Analysis revealed critical binding residues Met95, Glu50, and Ile59, with compounds 4d, 4a, and 4e forming hydrogen bonds with Asp73 at 2.17Å. These interactions highlight key molecular interactions and inform structure-based drug design. Understanding these interactions enables researchers to optimize compounds for enhanced binding affinity, aiding the development of potent, selective DNA gyrase B inhibitors for therapeutic use.

       
           
       
    
       
           
       
    Figure 5: 2D and 3D representation of Binding interaction of active compound 4a and compound 4d in the cavity of E. coli DNA gyrase B (In 2d interaction diagram Magenta arrow indicates hydrogen bonding and green line indicates hydrophobic interaction)

Table 4:Docking score (in Kcal/mol) of the synthesized compound in active site of B.subtilis DNA gyrase B (PDB ID: 6YJ7

The docking results for the synthesized compounds against S. aureus DNA gyrase are summarized in Table 4, with more negative scores indicating stronger interactions. Compound 4a had the strongest interaction with a docking score of -6.038. Compounds 4b, 4c, and 4g also showed favorable binding with low scores. Conversely, compounds 4f, 4d, and 4e exhibited higher docking scores, indicating weaker binding affinities.

Figure 6: 2D and 3D representation of Binding interaction of active compound 4e in the cavity of E. coli DNA gyrase B (In 2d interaction diagram Magenta arrow indicates hydrogen bonding and green line indicates hydrophobic interaction).

Table 5: Docking score (in Kcal/mol) of the synthesized compound in active site of S.aureas. DNA gyrase B (PDB ID: 3TTZ)

2D and 3D representations of the binding interactions of active compounds 4a and 4g in the cavity of S. aureus DNA gyrase B reveal critical interactions. In the 2D interaction diagrams, magenta arrows indicate hydrogen bonding, while green lines denote hydrophobic interactions. These visualizations help elucidate the binding modes of 4a and 4g within the enzyme's active site, highlighting key interactions that contribute to their binding affinity.

Figure 7: 2D and 3D representation of Binding interaction of active compound 4a and 4g in the cavity of S.aureas. DNA gyrase B (In 2d interaction diagram Magenta arrow indicates hydrogen bonding and green line indicates hydrophobic interaction).

CONCLUTION

In conclusion, the rising prevalence of antimicrobial resistance is a critical global health issue, demanding urgent advancements in therapeutic strategies. With 13.7 million infection-related deaths in 2019, the need for novel antimicrobial agents is evident. Benzoxazine derivatives have emerged as promising candidates due to their diverse biological activities and relative chemical simplicity, offering a versatile approach to combating microbial infections. Our study highlights the potential of these derivatives in addressing infections caused by pathogens such as Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, Klebsiella pneumonia, and Pseudomonas aeruginosa. Among the synthesized 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives, compound 4e showed the highest antimicrobial potency across all tested strains. Docking studies revealed critical interactions of compounds 4d, 4a, and 4e with the GyrB active site, identifying key amino acid residues essential for binding efficacy. These insights into the molecular mechanisms underlying their antimicrobial activity pave the way for future structure-based drug design. Continued research and development of Benzoxazine derivatives are crucial, offering potential breakthroughs in the fight against microbial infections and antimicrobial resistance. Further studies, including in vivo testing and clinical trials, are essential to validate the efficacy, safety, and therapeutic potential of these compounds. By leveraging our detailed understanding of ligand-protein interactions, researchers can refine Benzoxazine derivatives to develop more potent and selective inhibitors, ultimately improving global health outcomes.

CONFLICT OF INTEREST:

Authors don’t have any conflict of interest

ACKNOWLEDGEMENT:

I would like to express my heartfelt gratitude to ARACOP, including all teaching and non-teaching staff, and Principal Prof. Dr. R.D. Wagh, for their unwavering support throughout this research. I extend special thanks to my research guide, Prof. Dr. R.D. Wagh, for his invaluable guidance and encouragement. Their collective assistance has been instrumental in making this work possible.

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