NOVEL β-LACTAMS AND THIAZOLIDINONE DERIVATIVES FROM 1,4-DIHYDROQUINOXALINE SCHIFF’S BASE: SYNTHESIS, ANTIMICROBIAL ACTIVITY AND MOLECULAR DOCKING STUDIES

A series of novel isolated β-lactams 3a-c and thiazolidinone derivatives 4a-c were successfully synthesized from reactions of new Schiff's bases 2a-c with chloroacetyl chloride and thioglycolic acid. The chemical structures of the new compounds were confirmed through different spectroscopic techniques including IR, 1 H and 13 C NMR, mass spectrometry and elemental analysis. The antimicrobial activity of the obtained compounds was assessed in vitro against gram-positive Staphylococcus aureus and gram-negative Escherichia coli bacteria and Aspergillus flavus and Candida albicans fungi. All compounds exhibited good to excellent antimicrobial activity against the tested strains. Furthermore, a molecular docking study was carried out for the synthesized compounds and the results indicated that compounds 3b and 4b display comparable binding affinity scores as that of glutamate. These two compounds are promising candidates as antibacterial and antifungal agents that would deserve further investigations.


Introduction
The β-lactam skeleton has attracted much attention from medicinal chemists for the past 60 years due to its numerous biological activities and the mechanism of action of its derivatives has been broadly reviewed [1][2][3]. As a class of organic molecules, β-lactams constitute more than 65% of the world antibiotics market in both human and veterinary medicine [4,5]. Since the discovery of penicillin, efforts have been devoted to synthesize various β-lactam derivatives bearing important biological activities [6]. Besides their antibacterial activity, β-lactam derivatives have shown antitumor [7,8], antiviral [9], antihyperglycemic [10], antitubercular [11], antileishmanial and other potent biological activities [12]. However, the evolution of antibiotic-resistant bacteria represents the main obstacle to broad the clinical application of β-lactams despite the exhaustive medicinal chemistry campaigns aiming to vary β-lactam antibiotics. Consequently, the need for new derivatives with efficient biological activities has increased.
During the past years, substituted quinoxaline derivatives have gained attention and have been progressively explored as a result of their broad spectrum of pharmacological properties [13][14][15]. Therefore, a wide variety of synthetic routes for the design of functionalized quinoxalines has been intensively studied. Quinoxaline derivatives display versatile biological activities such as antiviral [16,17], antimicrobial [18,19], anti-inflammatory [20], antitumor [21] anti-tuberculosis [22], etc. Diverse recent approaches were carried out to the existing drugs to minimize the microbial resistance. These, for the most part, necessitate structural modification of actual antimicrobial agents to improve the microbial intracellular concentration of the drug, and thereby to boost the antimicrobial activity. The throughout literature surveys indicate that some substituents, particularly chlorine atoms, of structure of the bioactive organic compounds regarded as potential drug, have a promising effect on their specific biological activity. Several previous studies have pointed out that β-lactam derivatives possessing chlorine atoms were endued with significant biological activities [23][24][25][26]. The search for novel synthetic pathways for the design of heterocycles based on the β-lactam skeleton with improved biological derivatives is being actively pursued in our laboratory [27,28].
The aim of this study was to synthesize novel β-lactams 3a-c and thiazolidinone derivatives 4a-c and to evaluate in vitro their antibacterial and antifungal activity against several strains. Molecular docking studies were carried out to estimate the binding affinities of most active synthesized compounds on a molecular level.

Generalities
Unless otherwise mentioned, reagents were purchased from Sigma Aldrich (Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia) with high grade of purity and used without further purification. Reaction progress was monitored using thin-layer chromatography on silica gel pre-coated F254Merck plates (Darmstadt, Germany) and spots were visualized by ultraviolet irradiation. Melting point values of the synthesized compounds were determined using a Gallenkamp electro thermal melting point apparatus. IR spectra were measured as KBr pellets on a Pye-Unicam sp 1000 spectrophotometer. 1

Antimicrobial activity
Antimicrobial activity (antibacterial and antifungal) of the tested samples was assessed following a modified Kirby-Bauer disk diffusion method [29]. Plates impregnated with filamentous fungi like Aspergillus flavus at 25°C for 48 h; gram-positive bacteria as Staphylococcus aureus; gram-negative bacteria as Escherichia coli, were incubated at 35-37°C for 24-48 h and Candida albicans and Aspergillus flavus fungi were incubated at 30°C for a period varying between 24 and 48 h. The standard disks of ampicillin (an antibacterial agent) and amphotericin B (an antifungal agent) served as positive control for antimicrobial activity and filter disks impregnated with 10 μL of solvent (distilled water, chloroform and DMSO) were used as negative controls. All experiments were repeated and carried out in triplicate in the case of a significant difference in the results and the average inhibition diameters were measured in mm/mg sample.

Molecular docking
In this study, molecular simulation and modelling were performed using Molecular Operating Environment software (MOE, Version 2010.08, Canada). Glucosamine-6-phosphate synthase enzyme (GlcN-6-P) (PDB: ID 1gdo) was downloaded from RCSB Protein Data Bank [30]. The novel synthesized compounds 3a-c and 4a-c were subjected to molecular docking via their 2D and 3D structures. Before docking, adequate steps were performed including running conformational analysis using systemic search, 2D protonation of the structures, selecting the least energetic conformer and applying the protocol.

Synthesis and characterization
The chemical synthesis of the compounds is illustrated in Scheme 1. The synthesis of new Schiff's bases derived from ethyl 3-amino-1,4dihydroquinoxaline-2-carboxylate 1 is the fundamental key in the synthesis of the conforming isolated β-lactams 3a-c and thiazolidinone 4a-c derivatives. Thus, the reaction of compound 1 with different aromatic aldehydes in ethanol (30 mL) under reflux for about 4 h [27,[31][32][33] led to the formation of the corresponding Schiff's bases 2a-c in 65-84% yield.

Scheme 1. Synthesis of the novel
The IR spectra of compounds 3a-c revealed a N-H stretching band at about 3100-3400 cm -1 and absorption bands at 1685-1690 cm -1 and 1730-1732 cm -1 corresponding to cyclic CO and CO ester, respectively. On the other hand, the IR spectrum of compounds 4a-c exhibited absorption bands at 1760-1766 cm -1 corresponding to CO related to thiazolidinones. The 1 H NMR spectrum of compounds 3a-c displayed a singlet resonate at 12.35 ppm assignable to N-H quinoxaline; the spectrum of compound 3c showed a singlet at 3.56 ppm corresponding to (3H, for OCH 3 ). Moreover, the 1 H NMR spectrum of compounds 3a-c exhibited a doublet at 5.2 ppm corresponding to -CH-N.

Antimicrobial activity
The synthesized compounds (1, 2a-c,  3a-c and 4a-c) were evaluated for their antimicrobial activity against strains of grampositive Staphylococcus aureus, gram-negative Escherichia coli, Aspergillus flavus and Candida albicans fungi. The initial screening results of in vitro antibacterial and antifungal activity are presented in Table 1. All tested compounds showed a comparatively promising activity towards gram-negative bacteria than grampositive bacteria. Moreover, the highest activity of the designed compounds was recorded against the Candia albicans fungi. Thus, the obtained results show that compound 3b exhibited the highest activity against Escherichia coli and Staphylococcus aureus bacteria. By comparing the β-lactam derivatives 3a-c to their corresponding Schiff bases 2a-c, the β-lactam derivatives exhibited better activity and higher growth inhibition for the tested strains including bacteria and fungi.
Structure-activity relationship studies were carried out to better understand the effect of benzene substitution operation on antimicrobial activity. Considering gram-negative bacteria (Escherichia coli) testing results, it is obvious that Schiff base 2b and β-lactam derivative 3b with electron-withdrawing chlorine atoms showed better antibacterial activity than all other tested compounds. Schiff base derivative 2a without substituent on the benzene ring showed good antibacterial activity against Escherichia coli. It is worth noting that all thiazolidinones derivatives exhibited low activity against Escherichia coli compared to the Schiff base parents 2a-c.
Besides, all compounds displaying an electron donating methoxy group on the benzene ring showed the lowest activity against grampositive and gram-negative bacteria. It can be initially concluded that the presence of chlorine atom as a substituent on the benzene moiety withdraw electrons by induction and give electrons by resonance convey higher antibacterial and antifungal activity to Schiff's base 2b, β-lactam 3b and thiazolidinones derivatives 4b. Whereas, the presence of the methoxy group as a substituent on the benzene ring (2c, 3c, and 4c) donates electrons by induction and reduces the antimicrobial activity of the synthesized compounds against the tested bacterial strains.

Molecular docking study
The most biologically active derivatives 3a-c and 4a-c were subjected to molecular docking study to predict their binding modes on a molecular level. Glucosamine-6-phosphate synthase enzyme (GlcN-6-P), a potential target for antibacterial and antifungal agents, was the enzyme of choice for performing the docking study. The target derivatives were re-docked into the binding site of GlcN-6-P enzyme (PDB: ID 1gdo). To do so, first, glutamate was re-docked into GlcN-6-P with a root mean standard deviation (RMSD) of 1.25, the ligand showed score binding energy (S) of -15.11 kcal/mol and hydrogen bonding with Gly99, Trp74, Cys1, His86, Arg73, Thr76, Asp123 (Figure 1). The data of this study including the energy associated with intermolecular interactions (affinity in kcal/mol) of the target compounds 3a-c and 4a-c and hydrogen bonding interactions are presented in Table 2.   Table 2). Compound 3a recorded the energy docking of -16.67 kcal/mol, and it showed four bonding as following: i) Trp74 with NH, ii) Gly99 with NH, iii) Gly99 with C=O, iv) Cys1 with C=O. Besides, compound 3b registered a docking energy value of -18.95 kcal/mole and was found to perform four hydrogen-bonding interactions: i) Trp74 with NH, ii) Gly99 with NH, iii) Gly99 with C=O, iv) Cys1 with C=O. Moreover, the candidate 3c recorded a docking energy value of -16.00 kcal/mol and four hydrogen-bonding interactions: i) Thr124 with OCH3, ii) Arg73 with OCH3, iii) His86 with OCH3, and iv) Ser176 with C=O. In addition, the target compound 4a displayed two hydrogen bonds with Thr76 and Cys1 via binding with NH and C=O groups. On the other hand, the candidate 4b showed a high docking energy value of -18.50 kcal/mol and two hydrogen-bonding interactions with Thr76 and Trp74. Finally, compound 4c demonstrated the least docking energy score of -13.22 kcal/mol with only one hydrogen bonding interaction with Cys1 amino acid.
It can be concluded that the most active compounds are 3b and 4b since they showed comparable binding affinity scores as that of glutamate. These results are considered in agreement with the experimental results.

Conclusions
Substituted β-lactams 3a-c and thiazolidinone derivatives 4a-c were successfully synthesized from the reaction of new Schiff's bases 2a-c with chloroacetyl chloride and thioglycolic acid. All reactions were prepared with satisfactory yields and spectral data (IR, 1 H and 13 C NMR) of the obtained compounds (3a-c, 4a-c) were in full agreement with the proposed structures.
All newly synthesized compounds were evaluated in vitro for their antimicrobial activity against gram-positive and gram-negative bacteria and fungi. All compounds displayed moderate to excellent antimicrobial activity against the tested strains. The biological screening proved compound 3b to be the most active compound against all tested bacteria and fungi. Furthermore, compound 4b showed good antibacterial activity when tested against Staphylococcus aureus bacteria. This behaviour was ascribed to the presence of chlorine withdrawing atom as a substituent on the benzene moiety.
It was found that all compounds carrying an electrodonating methoxy group as a substituent on the benzene ring displayed the least biological activity. Furthermore, the results from the biological testing were in agreement with the molecular docking studies. Therefore, compounds 3b and 4b represent promising candidates as antibacterial and antifungal agents that would deserve further investigations.