Synthesis , Characterization , Anti-Bacterial and Anti-Fungal Activities of New Quinoxaline 1 , 4-diN-Oxide Derivatives

A new series of quinoxaline 1,4-di-N-oxides were synthesized and evaluated for their antibacterial and antifungal activities. The best result was demonstrated by 3-amino-N-(4-methoxyphenyl)-2-quinoxalinecarboxamide 1,4-di-N-oxide 4e, MIC (0.24 μg/ml) against Aspergillus fumigatus, and (0.12 μg/ml) against Streptococcus pneumonia.


Introduction
There is a growing need to develop new antibacterial agents in order to overcome the emergence of bacterial resistance to antibiotic therapy.Quinoxaline 1,4-di-Noxide is a nucleus that displays a wide range of activities.Quinoxaline 1,4-di-N-oxides and their derivatives demonstrated excellent activities as antibacterial [1][2][3][4][5], antiplasmodial [2,6,7], antifungal [8] and as antimycobacterial [9][10][11][12][13].Encouraged by these reported activities and with the aim of searching for new, broad spectrum and more potent antimicrobial compounds which can improve the current chemotherapeutic treatments, 20 new quinoxaline 1,4-di-Noxide derivatives were synthesized and evaluated.In most of the quinoxaline 1,4-di-N-oxides reported as antibacterial and/or antimycobacterial, the carboxamide in position 2 was a common feature [11,13].Therefore, the newly synthesized compounds 3a-n possessed a carboxamide in position 2 that was substituted with various aryl moieties bearing both electron-withrawing and electron donating groups.This series of quinoxaline carboxamide was prepared from unsubstituted benzofuroxane, 3a-g to be compared to their 7-chloro counterparts 3h-n in order to study the effect of substitution of chlorine atom in position 7.

Chemistry
The synthetic route for the preparation of the quinoxaline 1,4-di-N-oxide were obtained by the Beirut reaction [14,15].This reaction, which has been referred to as the Beirut reaction, is an excellent method for preparing these heterocyclic compounds.Benzofuroxanes 1a,b were obtained by previously described methods [13,16].Thus, the appropriate BFO reacted with the corresponding β-ketoamide (2a,b) or β-cyanoamide (3a-n) in the presence anhydrous potassium carbonate as a catalyst (Scheme 1).The use of inorganic catalyst for this reaction provided better means of purification with considerably good yields, whereas the reaction of BFO with cyanoacetamide was better carried out using triethylamine as a catalyst and dimethylfomamide as the solvent.These two intermediates, β-ketoamide and β-cyanoamide, were prepared by condensation of either ethylacetoacetate or ethylcyanoacetae with different amines [11,12,17].When the target compounds were prepared from monosubstituted-BFO, the formation of isomeric quinoxaline 1,4-di-N-oxides was observed.In most cases, the 7-substituted isomer prevailed over 6-substituted isomer, and when the methoxy substituted quinoxalines were prepared, only the 7-isomer was obtained, as previously described [18,19].
Moreover, in order to investigate the antimicrobial activity of fused tricyclic and tetracyclic di-N-oxides derived from quinoxaline 1,4-di-N-oxide the phenazine derivative 5 and the indenoquinoxaline 6 were synthesized.The Beirut reaction was similarly followed using cyclic diketones such as indandione or dimidone.All the prepared compounds were characterized in light of their microanalysis and spectral data including IR, 1 H NMR and mass spectrum.

Microbiology
The newly synthesized compounds were tested for their antibacterial and antifungal activities.These assays were performed at the Regional Center for Mycology and Biotechnology, Antimicrobial unit test organisms, Cairo, Egypt.Compounds 4i and 4k were previously synthesized [17] but they were prepared again in order to perform the microbiological testing on them and compare them to their unsubstituted counterparts.The tested compounds were evaluated for their antifungal activity against (Aspergillus fumigatus (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotricum candidum (RCMB 05097) Candida albicans (RCMB 05036)).The antibacterial activity was evaluated against (Gram positive: Streptococcus pneumonia (RCMB 010010), Bacillis subtilis (RCMB 010067)) and Gram negative (Pseudomonas aeruginosa (RCMB 010046), Escherichia coli (RCMB 010052)).Amphotericin B was taken as a reference for the antifungal effect, while ampicillin was the standard used for the evaluation of anti-bacterial activity against gram positive bacteria and gentamicin was used as a standard in assessing the activity of the tested compounds against gram negative bacteria.The results expressed as the mean zone of inhibition in mm ± standard deviation beyond well diameter (6 mm) produced on the microorganisms using (10 mg/ml) concentration of tested samples, shown in Table 1.
The initial screening of the tested compounds showed promising activity of some of the compounds which encouraged the determination of their minimum inhibitory concentration (MIC) (Table 2).The best results were demonstrated by compound 4e both as antifungal and against gram positive bacteria, it possessed double the activity of the standard, amphotericin B against Aspergillus fumigatus, (0.24 and 0.49 µg/ml, respectively)and ampicillin against Streptococcus pneumonia (0.12 and 0.24 µg/ml, respectively).This compound has also displayed 4 times the activity of amphotericin B against Syncephalastrum racemosum, (7.81, 1.95 µg/ml, respectively).Other derivatives also possessed remarkable activity against Syncephalastrum racemosum as 4i which displayed double the inhibitory effect of the standard (3.9, 7.81 µg/ml, respectively), while 2a and 6 were as active as the amphotericin B. Moderate activity against gram positive bacteria Streptococcus pneumonia was also demonstrated by compounds 3b and 6.However, this  series of compounds had no effect on Candida albicans and were also ineffective against gram negative bacteria.Moreover, compounds 2b, 4g, 4k and 5 showed no activity against most of the tested strains.
A simple SAR study reveals certain features that could improve the activity such as the amino group in position 3, quinoxaline unsubstituted in position 6, the electrondonating substituents on the phenyl amino displayed better effect than electron withdrawing.As for the fused derivatives the tetracyclic indenoquinoxaline 6 was better than the tricyclic phenazine 5.

Chemical Methods
All the solvents used were commercially available and distilled before use.Reactions were monitored by thinlayer chromatography (TLC) on silica gel plates (60 F254), visualizing with ultraviolet.Infra red spectra (KBr) were recorded on FT-IR 5300 spectrophotometer and Perkin Elmer spectrum RXIFT-IR system (ν, cm −1 ). 1 HNMR spectra were recorded on Varian Gemini spectrophotometer (300 MHz) in DMSO-d 6 or CDCl 3 as solvent.Proton chemical shifts (d) are relative to tetramethylsilane (TMS, d = 0.00) as internal standard and expressed in ppm.Spin multiplicities are given as s (singlet), d (doublet), t (triplet), and m (multiplet) as well as b (broad).Coupling constants (J) are given in hertz.Melting points were determined by using melting point apparatus and are uncorrected.MS spectra were obtained on a GC Ms-QP 1000 EX mass spectrometer at 70 eV.Microanalyses were performed using a C H N S/O analyzer.Elemental data are within ±0.4% of the theoretical values.All yields reported areunoptimized.The chemical reagents used in synthesis were purchased from Fluka, Sigma and Aldrich.

General Procedure for Preparation of Compounds 2a,b
A warm solution of 5(6)-benzofuroxane 1a,b and the appropriate acetoacetanilide (0.01 mol) in ethanol (50 mL) was stirred at room temperature in the presence of catalytic amount of potassium carbonate.The yellow products precipitated over a period of 2 -4 h.The residual product was triturated with water, extracted with ethylacetate then the organic solvent was dried over anhydrous sodium sulphate and removed in vacuo to give yellow crystals that were recrystallized out from ethanol.6)-substituted benzofuroxane 1a,b and cyanoacetamide (0.01 mol) was stirred for 10 min at 0˚C.Over the cooled suspension, a solution of triethylamine (5 drops) in dimethylformamide was added.The mixture was allowed to stand at room temperature over 24 h and then filtered off.The red solid product was filtered and recrystallized from ethanol.

General Procedure for Preparation of
Compounds 4a-n A warm solution of 5(6)-benzofuroxane 1a,b and the appropriate cyanoacetanilide (0.01 mol) in ethanol (50 mL) was stirred at room temperature in the presence of catalytic amount of potassium carbonate.The solutions turned red and then they were stirred at room temperature for 1 -5 hr depending on the BFO and the cyanoacetanilides.The residue obtained was triturated with waterand then extracted with dichloromethane.The organic phase was dried with anhydrous sodium sulphate and filtered.The solvent was removed in vacuo and the red-brown precipitate was recrystallized out from ethanol.
Yield (67%); mp 180˚C.IR (KBr, cm   6)-substituted benzofuroxane 1a (0.01 mol) and dimidone (0.01 mol) in ethanol (50 mL) was stirred and heated at 60˚C, while ammonia gas was bubbled into the solution for 1 hr.The solution was stirred at room temperature for another 11 hr.The solvent was then removed and finally a yellow precipitate was obtained which was recrystallized out from ethanol.

Antimicrobial and Antifungal Assays
Antimicrobial activity was determined using the agar well diffusion assay method as described by [20].The tested organisms were subcultured on nutrient agar medium (Oxoid laboratories, UK) for bacteria and Saboroud dextrose agar (Oxoid laboratories, UK) for fungi.Ampicillin and Gentamycin were used as a positive control for bacterial strains.Amphotericin B was used as a positive control for fungi.The plates were done in triplicate.Bacterial cultures were incubated at 37˚C for 24 h while the other fungal cultures were incubated at (25˚C -30˚C) for 3 -7 days.Antimicrobial activity was determined by measurement zone of inhibition [21].

Determination of MIC
The minimum inhibitory concentration (MIC) of the samples was estimated for each of the tested organisms in triplicates.Varying concentrations of the samples (1000 -0.007 µg/ml), nutrient broth were added and then a loopful of the test organism previously diluted to 0.5 McFarland turbidity standard was introduced to the tubes.A tube containing broth media only was seeded with the test organisms to serve as control.Tubes containing tested organisms cultures were then incubated at 37˚C for 24 h while the other fungal cultures were incubated at (25˚C -30˚C) for 3 -7 days.The tubes were then examined for growth by observing for turbidity [22].

Table 1 . Antifungal and antibacterial activity of the newly synthesized compounds.
Data are expressed in the form of mean zone of inhibition in mm ± standard deviation beyond well diameter (6 mm) produced on a range of environmental and clinically pathogenic microorganisms using (10 mg/ml) concentration of tested samples; b NA: No activity. a

Table 2 . Antimicrobial and antifungal activity as MICS (µg/ml) of tested compounds against tested microorganisms.
a Minimum inhibitory concentration (µg/ml); b NA: No activity.