Synthesis of Some Quinoxaline Sulfonamides as a Potential Antibacterial Agent

Aims: This studies aims at the synthesis of new heterocyclic systems and study its biological and pharmacological properties. Objective: This study was designed to synthesized some quinoxaline-2,3-dione with sulfonamide moiety, characterize the synthesized compounds, and study the antimicrobial properties of the synthesized compounds on some bacterial strains. Materials and Methods: Six quinoxaline-6-sulfonohydrazone derivatives were synthesized by reacting quinoxaline-6-sulfonohydrazine with some substituted benzaldehydes and ketones. The compounds were tested for their potential antibacterial properties. Results: All the test compounds possessed promising antibacterial property against a panel of bacterial strains used for this study. The MIC values exhibited by these compounds ranged between 0.0313 and 0.250 mg/mL. Among the compounds tested, compound 2 showed appreciable antibacterial activity. Discussion and Conclusion: The study concluded that all the compounds exhibited appreciable bactericidal effects towards all the bacterial strains, particularly, compound 2 This is an indication that such compounds possessing broad spectrum activities will be useful in formulating antimicrobial compounds which could be used to treat infections caused by pathogens that are now developing resistance against the available antibiotics. Original Research Article Taiwo et al.; JPRI, 33(51B): 116-132, 2021; Article no.JPRI.60636 117


INTRODUCTION
The synthesis and chemistry of quinoxalines have attracted considerable attention in the past twenty years. Quinoxaline molecules are nitrogen containing heterocycle derivatives which have broad spectrum biological and pharmaceutical applications. Quinoxaline are known for their therapeutic properties and as such attract the attention of many scientists searching for potent antimicrobial compounds. These compounds possess anti-viral [1][2][3] anti-bacterial [4][5][6][7][8][9][10] antiinflammatory [11,12] anticancer [13][14][15]. They are also used in the agricultural field as fungicides, herbicides, and insecticides [16]. This study focused more on the biological activity of quinoxaline on some bacterial strains known to cause human infections. Many of these pathogens have developed resistance against antibiotics. This has been creating a lot of headache in healthcare delivery which requires urgent solution. Thus scientists need to move faster in researches on antimicrobials in order to develop more potent antimicrobials to take care of superbugs that are now "waging wars" against the available antimicrobials. This study is one of such efforts taken to develop potent antimicrobial compounds to combat the menace of these pathogens.

General
Melting points were determined with open capillary tube on a Gallenkamp (variable heater) melting point apparatus and were uncorrected. Infrared spectra were recorded as KBr pellets on a Bruker 2000 Spectrometer. The 1 H and 13 C NMR was run on a Bruker 600 MHz spectrometer ( in ppm relative to Me4Si), Mass spectra were taken on a high-resolution (m/∆m = 30 000) Thermo Scientific LTQ-Orbitrap Discovery mass spectrometer (San Jose, CA) equipped with an electrospray ionization source at the Department of Chemistry, Portland state University, Portland U.S.A. The purity of the compounds was routinely checked by TLC on silica gel G plates using n-hexane/ethyl acetate (1:1, v/v) solvent system and the developed plates were visualized by UV light. All reagents used were obtained from Sigma-Aldrich Chemical Ltd, except Glacial acetic acid, ethanol, oxalic acid and vanillin which were obtained from BDH Chemical Limited.

Antibacterial Sensitivity Testing of Synthesized Compounds
The synthesized compounds were screened for antibacterial activity using agar-well diffusion method as described by Akinpelu et al., (2016) [18]. The test organisms were first re-activated in nutrient broth for 18 hours before use. Exactly 0.1 mL of standardized bacterial strains (10 6 cfu/mL of 0.5 McFarland standards) was transferred into Mueller-Hinton agar medium at 40 o C. With the aid of a sterile 1 mL pipette, exactly 0.2 mL of the standardized broth culture of the test organism was added to 18 mL sterile molten agar medium which had already cooled down to 40 o C and thoroughly mixed together and poured into sterile Petri dishes which were properly labeled. The medium was allowed to set and wells were bored into it using 6 mm sterile cork borer. The wells were made 5 mm to the edge of the plates and filled-up with the solution of the compounds. Care was taken not to allow the solution to spill on the surface of the medium. Streptomycin phosphate and tetracycline were used as positive controls at a concentration of 1 mg/mL respectively. The plates were allowed to stand for about one hour on the bench to allow for proper in-flow of the solution into the medium and then incubated aerobically uprightly at 37 o C for 24 hours. Care was taken not to stockpile the plates. The plates were later observed for zones of inhibition which is an indication of susceptibility of the organisms to the compounds.

Determination of Minimum Inhibitory Concentrations (MICs) of the Test Compounds
Minimum inhibitory concentrations of the compounds and the standard antibioticsstreptomycin and tetracycline were carried out using a two-fold dilution method [19]. Two milliliter of different concentrations of solution of the compound was added to 18 ml of presterilized molten nutrient agar at 40 o C to give final concentrations regimes of 0.0157 and 1.0 mg/mL. The same range of concentrations was also prepared for the two positive controls. The medium was then poured into sterile Petri dishes and allowed to set. The plates were left on laboratory bench overnight to ascertain their purity The surfaces of the media were allowed to dry under a laminar flow chamber before streaking with 18 h old standardized bacterial cultures. The plates were later incubated at 37 o C for up to 72 h after which they were examined for the presence or absence of growth. The MIC was taken as the lowest concentration of the test compounds that will prevent the growth of the susceptible bacterial strains tested.

Determination of Minimum Bactericidal Concentrations (MBCs) of the Compounds and Standard Antibiotics
The minimum bactericidal concentrations of the compounds were determined as described by Oludare et al. (1992) [19] with some modifications. Samples were taken from line of streak in the plates with no visible growth in the MIC assay and sub-cultured onto freshly prepared nutrient agar medium and later incubated at 37 o C for 48 h. The MBC was taken as the lowest concentration of the compound that completely kills the susceptible test organisms.

Determination of Killing Rate of the Susceptible Bacterial Strains
The assay was carried out using each of the synthesized compounds on the viability of Enterococcus feacalis representing Grampositive organism and Pseudomonas fluorescens representing Gram-negative organism [20]. Viable counts of the test organisms were initially determined. A 0.5 mL volume of known cell density (by viable counts 10 6 cfu/mL) from each organism suspension was added to 4.5 mL of different concentrations of the synthesized compounds. The suspension was thoroughly mixed and held at room temperature (28 -30 o C) and the killing rate was determined over a period of 2 h. Exactly 0.5 mL of each suspension withdrawn at 15 Minutes time interval and transferred to 4.5 mL nutrient broth (Lab. M) recovery medium containing 3% "Tween 80" to neutralize the effect of the antimicrobial compounds carried over from the test suspensions. The suspension was shaken properly and serially diluted up to 10 -5 in sterile physiological saline. Exactly 0.5 mL of the final dilution of the test organism was transferred into pre-sterilized nutrient agar (Biolab.) at 45 o C and plated out. The plates were allowed to set and incubated in inverted position at 37 o C for 72 h. The viable counts were made in triplicates for each sample. Depression in the viable counts indicated killing by the compounds.

Chemistry
The quinoxaline-6-sulfonyl hydrazide 1 was synthesized by reacting quinoxaline-2,3-dione with excess chlorosulfonic acid to obtain the corresponding quinoxaline-6-sulfonyl chloride, which was then reacted with hydrazine hydrate in methanol to give the expected quinoxaline-6sulfonohydrazine, 1. The sequence of reactions is shown in Scheme 1.

Antimicrobial Studies
All the synthesized compounds were active against all the bacteria investigated. The zones of inhibition observed for the synthesized compounds against the test organisms ranged between 10 mm and 30 mm. On the other hand, the zones of inhibition observed for streptomycin and tetracycline against the bacteria ranged between 15 and 28 mm (Table 1). This is an indication that the synthesized compounds compared favourably with the standard antibioticsstreptomycin and tetracycline used as positive control. The MIC exhibited by the synthesized compounds against the bacterial strains ranged between 0.0625 mg/mL and 0.125 mg/mL ( Table 2). The lowest MBC against the test organism was 0.125 mg/mL while the highest MBC was 5.00 mg/mL (Table 3). On the other hand, MIC exhibited by streptomycin against the organisms ranged between 0.0078 mg/mL and 0.500 mg/mL and those exhibited by tetracycline were between 0.313 mg/mL and 0.500 mg/mL (Tables 2 and 3). The lowest MBC observed for streptomycin was 0.0313 mg/mL and the highest MBC was 0.500 mg/mL while tetracycline exhibited a range between 0.0313 mg/mL and 0.500 mg/mL. In comparison, the synthesized compounds compared favourably with the two standard antibiotics used as positive controls.
The lowest the MIC and MBC exhibited by antimicrobial compounds the better and more potent such antibiotics are. The synthesized compounds having exhibited low MIC and MBC is an indication that such compounds could be used to produce potent antimicrobial compounds that could be used to control the infections

Chemistry
The quinoxaline-2,3-dione was prepared by reacting o-phenylenediarnine with oxalic acid dihydrate thermally or by microwave irradiation in acidified water. The quinoxaline-6sulfonohydrazine was synthesize by reacting quinoxaline-6-sulfonyl chloride obtained from the reaction of quinoxaline-2,3-dione with excess of chlorosulfonic acid with hydrazine hydrate in absolute methanol(Scheme 1).

In vitro Antimicrobial Activities of the Compounds and Standard Antibiotics
The antimicrobial properties of all the synthesized compounds used in this study were investigated against panel of bacterial strains. These compounds at a concentration of 2 mg/ml were found to inhibit the growth of both Grampositive and Gram-negative organisms. This shows all the compounds to possess broad spectrum activities. These synthesized compounds showed appreciable antibacterial activity against all Gram-negative organisms used for this study. Gram negative species are reported to be more resistant to inhibition by most antibacterial compounds due to their outer membrane [21,22]. Among the Gram-negative organisms inhibited by these synthesized compounds are Pseudomonas species that are known to be more resistant to antimicrobial agents [23]. Such compounds that could inhibit the growth of Pseudomonas species could serve as a novel antimicrobial compound to manage infections caused by these opportunistic organisms. Some of the Gram-positive bacterial strains used for this study are known to cause various infections in man. For example, Staphylococcus aureus are known to cause various infections in man and animal and predominates in surgical wound infections [24]. Staphylococcus aureus are also responsible for superficial skin infection and can as well cause some life-threatening diseases such as sepsis, respiratory and septicaemia [25]. This organism has developed resistant towards many of the antibiotics used as therapy to treat infections caused by this organism.
For example, methicillin and vancomycin were adopted to treat the infections caused by Staphylococcus aureus and these drugs are no longer showing potency towards the treatment of infections caused by this pathogen [26,27]. Thus, drugs formulated from these synthesized compounds could be used to manage infections caused by Staphylococcus aureus and other organisms. Other Gram-positive organisms that were susceptible to these compounds are B. cereus known to cause food infections among other diseases, Streptococcus pneumoniae the causative agent of pneumonia. The infections caused by these organisms can be treated using drugs developed from these synthesized compounds and thus go a long way in healthcare delivery.
The assay for MIC and MBC exhibited by the synthesized compounds were also investigated. The results obtained from the assay showed that the compounds exhibited low MIC and MBC against test bacterial strains used for this study. For example, the lowest MIC observed was 0.0313 mg/mL while the lowest MBC was 0.0625 mg/mL., it has been reported that, a low MIC value of antibacterial agents indicates a better antibacterial activity [28]. This observation shows that the synthetic compounds exhibited significant antibacterial activities and thus can be used to formulate potent antibacterial compounds that could be used to manage infections caused by pathogens that are gradually developing resistant against antimicrobials.
The significant activity of the compounds could be explained on the basis of the contributions of incorporated aromatic ring which we know should increase the lipophilicity of the compounds. This Scheme 1.

Scheme 2.
increase in lipophilicity would help their permeability through the microbial cell wall and enhance the reaction of different functional groups present in the synthetic compounds to reacts with the cellular membrane of the bacterial cell and thus impaired both its functions and integrity [29,30] resulting in higher activity. The synthetic compounds may be considered as the analogue of sulfonamides (a known antimicrobial) due the presence of the R-SO2-NHR 1 group. Also the synthetic compounds contain quinoxaline and hydrazone (CH=N-NH-) group in their structure. These classes of compounds have been known to possess interesting antibacterial activities.

CONCLUSION
The synthesized quinoxaline sulfonamides exhibited appreciable antimicrobial potency against panel of bacterial strains used for this study. The compounds exhibited broad spectrum activities and thus showed a significant therapeutic action for the treatment of infections caused by pathogens. The study also confirmed the mode of action of these compounds through damage to the cytoplasm of the test bacterial strains and led to the leakage of cytoplasmic content. The bactericidal effects exhibited by these synthesized compounds thus caused the death of the test organisms. Such compounds could be used to formulate antimicrobial compounds which could be more potent than the available antibiotics used as therapy to treat infections caused by pathogens.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.