New Quinoxalinyl Chalcone Derivatives: Search for Potent Antimicrobial Agents

Owing to the prevalence of antimicrobial resistance and its arising threat rendered by the ineffectiveness of existing antibiotics, the present work was aimed to generate new 5/6 substituted quinoxalinyl chalcones derivatives. A total of 16 compounds were synthesized and characterized by IR, 1H NMR, 13C NMR and mass spectral studies. The antimicrobial activity against resistant pathogens was by micro-dilution assay method. The result revealed that compounds bearing 2,4-dichloro benzylidine and 4-fluoro benzylidine moiety demonstrated promising antibacterial and antifungal activities against the selected pathogenic bacteria and fungi. It was observed that chalcones of 3,4dihydroquinoxalin-2(1H)-one exhibited better activity (MIC: 2.57 μM) as compared to the chalcones of quinoxalin-2,3(1H,4H)-dione. The ClogP values of the synthesized compounds were in good agreement with antifungal activity indicated the significance of lipid solubility better cell wall penetration. Overall quinoxalinyl chalcones exhibited broad spectrum antimicrobial activity.


Introduction
Drug resistance was often observed with tuberculosis disease, but now it has percolated to other infections due to bacteria, virus, fungi, parasite etc. and evolved as "Antimicrobial resistance (AMR)". Antimicrobial resistance (AMR) is a serious threat to worldwide health that demands immediate solution from researchers because the expected mortality of infectious disease is not predictable unless newer agents are developed, especially to fight superbugs (resistant pathogens). AMR is a great challenge not only to developing counties but also to developed nations due to the huge cost and duration of antimicrobial treatment that will definitely affects the economy of people. Therefore, there is an urgent need to advance and to accelerate the antimicrobial research [1][2][3].
At this juncture, the reported antimicrobial activity for various oxoquinoxalines [4][5][6] and their diverse mechanism of action that could act as intercalating agents [7][8][9][10][11] have stimulated our team to design newer agents. In the light of literature and in continuation to our earlier reports [12][13][14][15][16], it was decided that oxoquinoxaline could be as better choice to produce new antibacterial scaffolds. Considering the present era of antimicrobial resistance and the underlying mechanism for resistance, it was our thought to design oxoquinoxaline with lipophilic side chain. As a result, aryl chalcone side chain was opted as side chain due to high lipophilic nature of benzylidine core. It is know that many existing antimicrobial molecules are ineffective for their poor cell wall permeability.
In the present work new chalcone derivatives were generated by exploiting 5 and 6 positions of quinoxaline through Friedel Crafts acylation followed by Claisen Schmidt condensation reactions. The obtained synthetic library was confirmed for its chemical structure by physical and spectral data and screened for antimicrobial activity against selected bacteria and fungi.

Materials and Methods
Purity of compounds was confirmed using precoated TLC and HPLC (Agilent LC 1200, C 18 (250 mm × 4.6 mm, 5 µ) Column) techniques. The melting point was determined by open capillary tube method and is uncorrected. Functional groups were characterized by IR spectra using a Fourier transform-Infrared spectrophotometer (Alpha E, Bruker, ATR) technique) at resolution of 4 cm-1. The protons of the synthesized structures were assigned using proton nuclear magnetic resonance spectrum ( 1 H NMR) using a Varian VXR Unity using TMS as internal standard and DMSO-d 6 /CDCl 3 as solvent). The mass spectrum from Shimadzu LCMS-2010A was used to confirm molecular weight. All the chemicals reagents used were of analytical or synthetic grade. Microorganisms used were ATCC strains and modified in laboratory as resistant species using ciprofloxacin and Griseofulvin respectively for bacteria and fungi [6].

Synthesis of 6-acetylquinoxaline-2,3(1H, 4H)-dione(2) and 5acetylquinoxaline-2 (1H)-one (5)
A mixture of compound 1 or 4 (0.015 mol), anhydrous aluminium chloride (mol) and acetic anhydride (mol) were taken in a 5 ml microwave vial and irradiated in a microwave synthesizer at 110 watt for 1 minute. The reaction mixture was then cooled and poured onto crushed ice with stirring. The precipitated product was filtered on a Buchner funnel under suction and washed with 3 × 10 ml portions of distilled water. The product was recrystallized from 95% ethanol.
In the same way antifungal activity was screened against griseofulvin resistant Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231. The resistant species were produced in laboratory as per our earlier report [6].
The obtained MIC (90% inhibition) was converted into micromolar concentration (µM). The above antifungal and antibacterial activities were performed as per the literature [12,13].

Results
The synthetic figure adopted was quite convenient and afforded pure compounds. A total of 14 compounds were obtained from Figure  1. The chalcones 3(a-f) was yellowish in nature with yield range from 50-70% whereas the chalcones 6(a-f) was cream colored crystals with yield ranging from 60-82%. All the melting point was above 300°C. The R f value of the compounds were 0.4 to 0.8.
All the compounds were relatively stable and none were found to be hygroscopic or deliquescent. The physical data of the compounds were shown in Table 1. The spectral data of the compounds illustrated in Figure 1 were satisfactory for the proposed structures. The data related to spectral interpretation is detailed in materials and methods section.
The antimicrobial activity was carried out against four bacteria and two fungi and the activities were measured by minimum inhibitory concentration (MIC in µM) using microdilution assay method. The standards used were ciprofloxacin (CIP) and griseofulvin (GSFN) for antibacterial and antifungal activity respectively. DMSO was employed as solvent blank. The results of antibacterial and antifungal activities revealed that all the chalcones exhibited considerable antibacterial and antifungal activities when compared to the intermediates (compounds 2 and 5).
The most active compounds against Staphylococcus aureus and Escherichia coli (Gram positive bacteria) were compounds 6e and 6f with MIC of 3.27 µM and 2.57 µM respectively. The compound 6e was found to be the most active compound against Proteus vulgaris and Pseudomonas aerugenosa (Gram-negative bacteria) with MIC of 3.27 µM. In the case of anti-fungal screening, compound 6c was found to be the most active against the Asperigillus niger and Candida albicans with MIC of 1.85 µM.

Discussion
The synthetic Figure was consisted of two routes respectively for 3af and 6a-f as shown in Figure 1. The product formation and the reaction progress were monitored by TLC technique. The starting materials used were quinoxaline 2, 3 (1H, 4H)-dione 1 and 3, 4dihydroquinoxalin-2(1H)-ones 4 respectively for 3a-f and 6a-f.  Further the formation of chalcones (3a-f and 6a-f) respectively from compound 2 and 5, were confirmed by two proton NMR signals accounting for unsaturated -CH=CH-system (at ~7-8 ppm). The Jvalue (~ 15 Hz) of doublets for protons of -CH=CH-system indicated the E configuration. All the compounds were obtained as stable crystals except compound 2 and 3. The yields of all chalcones were more than 60% and were colored in nature (yellowish to reddish).
The retardation factor (R f value) and ClogP for all chalcones were more than intermediate and starting materials. It indicated that the prepared chalcones are more lipophilic in nature.
The melting points were found to be more than 200°C; this could be due to considerable intermolecular forces. The physical data of the synthesized compounds are shown in Table 1 Structures of the compounds were confirmed by IR, 1 HNMR, and mass spectral data.
All above compounds were screened for antibacterial and antifungal activities and their results were tabulated in Table 2. Screening was performed using micro-dilution assay method and result was obtained as minimum inhibition concentration (MIC 90 in micro-molar concentration).
The result revealed that chalcones exhibited promising and broad spectrum activity against tested species as compared to the intermediates (>12 µM) and starting materials (>30 µM). It was noted that acetyl derivatives of quinoxalines are more active against gram positive than Gram negative bacteria. However 3,4-dihydroquinoxalin-2(1H)-one 4 and its acetyl derivatives 5 demonstrated 2 times more potent antimicrobial actvities than quinoxaline-2,3(1H,4H)-dione 1 and its acetyl derivatives 2. All chalcones were 5-10 times more potent than starting materials indicated the new scaffolds are quite acceptable for antimicrobial drug design.   Among all chalcones, 6a-f series were relatively more potent than 3a-f. It might be due to high lipophilicity. Out of all chalcones, compound 6c, 6d and 6e were found to be more potent against both bacteria (3.27-3.71 µM) and fungi (3.27-1.85 µM).  is indicates the importance of substitution on benzylidine part of chalcones where substitution on phenyl moiety increases the potency to 2-3 times as compared to unsubstituted compound 3a and 6a. 2,4,6trimethoxy substitution (compound 3e and 6e) was found to equally active against both Gram Positive and negative species. Among all, 2, 4-dichloropheyl/4 fluorophenyl substituted compounds (3c, 6c, 3f, 6f) were relatively more active against fungi than bacteria. The most potent compound of the series was 6c (dichlorophenyl substitution). Relative potency of the synthesized compounds can be observed from

Conclusions
Thus halogen substituted chalcones of quinoxaline (electron withdrawing) can be considered for development of antifungal agents whilst amino/and methoxy substitution (electron releasing) may be considered for development of antibacterial agents. Nitro substitution did not show any significant increase in antimicrobial spectrum.

Conflict of Interest
All authors expressed no conflict of interest.