Antimicrobial and Anti-pathogenic Activity of New Naphtho [1,2,4] Triazol-Thiadiazin Derivatives

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Introduction
Resistance is the ability of a bacteria against the inhibitory effect of antibacterial agents on the proliferation and growth of bacteria.Through the indiscriminate use of antibiotics, resistant microorganisms have emerged during recent decades and caused numerous problems in the treatment of infections induced by these microorganisms.Therefore, an attempt at producing drugs to deal with these outstanding threats to global health is an essential need.To this end, one strategy is employing a combination of different active parts in one molecule.In this strategy, each drug moiety is designed to bind independently to different biological targets and accumulate at both target sites synchronously (1)(2)(3).It was found that 1, 2, 4-triazole and naphthalene derivatives exhibit a wide variety of pharmacological and biological activities such as antibacterial (4,5), antimycobacterial (6,7), antiinflammatory (8), antifungal (9,10), antineoplastics (10), anticancer (11), and antiviral (12) activities.Thiadiazines are also efficient antibacterial and antifungal compounds (12), and their use in the treatment of the bacteria Helicobacter pylori and their role as reverse transcriptase inhibitors of the human immunodeficiency virus have been reported (13).On the other hand, the studies conducted on a large number of p-naphthol derivatives for antibacterial and antifungal activities indicated that these compounds exhibit preferential activity against grampositive bacteria and human pathogenic fungi.In fact, in Evaluation of antimicrobial activity the present study, the synthesis of three-ring heterocycles has been designed in such a way that all three heterocycles have antimicrobial power and can exert their synergistic effect by being present in one molecule at the same time.As mentioned in the previous study, 10-substituted-5Hnaphtho [1,2-e] [1,2,4] triazolo [3,4-b][1,3,4] thiadiazin-5-ones derivatives were synthesized in the presence of different substitutions at the 5-triazole position as follows: Since the synthesis and evaluation of antimicrobial activity is an important part of our research program (14)(15)(16), in this study, we aimed to evaluate the antibacterial and antifungal activity of these derivatives against some microorganisms that cause the largest number of infections and, in some cases, have exhibited resistance to currently available antibiotics such as Methicillin-resistant Staphylococcus, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Candida tropicalis, Candida krusei, and Candida glabrata.

Antibacterial Screening
The antibacterial activity of synthesized compounds was tested against gram-positive and gram-negative bacteria, including E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, and Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 700698.These chosen strains were proceeded to screen the antibacterial activity because they are commonly isolated pathogens from hospitalized patients with intestinal ailments and blood and skin infections (17).All microorganisms were obtained from the Urmia University of Medical Sciences.The standardization of each bacterial inoculum was done following the Clinical and Laboratory Standards Institute (CLSI).Briefly, Mueller-Hinton agar (MHA) medium was used to prepare MHA agar plates which were inoculated with the bacterial suspension (10 6 CFU/ mL) uniformly onto the surface of agar plates, and wells of uniform diameter were punched on the solidifying agar media by the sterile borer.Test compounds were then dissolved in 99.5% dimethyl sulfoxide (DMSO) to obtain 1 mg/mL stock solutions.Afterward, 100 μL of each of these solutions were dispensed into the labeled wells (18)(19)(20).Ceftizoxime and ciprofloxacin (Sigma, Aldrich, US) were used with the same method as standard drugs, and DMSO was used as solvent control.Next, Petri plates were transferred to the incubator for 18 hours at 35 °C, and the final results were evaluated by measuring the average diameter of the inhibition zone around each well.Experiments were performed in triplicate.

Agar Dilution Method
The agar dilution method was performed according to the procedures described by the CLSI (21).Stock solutions of antimicrobial compounds were prepared in DMSO, added to molten MHA (Becton Dickinson, Franklin Lakes, MD, USA) at 50 °C, and then poured into Petri plates to provide two-fold dilutions ranging from 7.8 to 1000 µg ⁄mL.Bacterial inoculums were prepared from a 24-hour culture on MHA.Bacterial suspensions were applied to agar plates using a multipoint inoculator to give a final concentration of 1.5 × 10 5 CFU per spot (22).The agar plates were then incubated at 35 °C for 18 hours and were evaluated visually.The minimum inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobial agent observed in complete inhibition of visible growth or the growth of less than five colonies per spot (23).

Antifungal Activity Agar Dilution Assay
In vitro antifungal activity of 10-substituted-5Hnaphtho [1,2- infection in immune-compromised situations due to their great adaptability to different host niches (24).All fungi were obtained from the Shiraz University of Medical Sciences.The antifungal activity of triazole derivatives was determined using the agar-well diffusion method (25).Then, 100 µL of each fungal suspension with a cell density of 1.5 × 10 8 CFU/mL was applied on the surface of the pre-sterilized and autoclaved Sabouraud Dextrose Agar (SDA) Petri dishes and spread using a shaped glass spreader.Wells with 6 mm of diameter were made at the center of each of the SDA Petri plates with the help of a sterilized cork borer.Next, the wells were filled with triazole derivatives as prepared above with three replications for each treatment, and the zone of inhibition was calculated based on the mean values.Fluconazole (1 mg/mL) and DMSO were used as positive and negative controls, respectively.All Petri dishes, including triazoles and controls, were allowed to be diffused and incubated at 35 °C for 48 hours.The antifungal activity of derivatives was determined after incubation.
In the next step, the MICs were determined by the Agar dilution method according to the procedures described by the CLSI (26).Two-fold serial dilutions of the compounds and reference drug (fluconazole) were prepared in SDA to obtain the required concentrations of 25-1000 µg/mL.The fungal inoculates were adjusted to 0.5 McFarland standard (1.5 × 10 8 CFU/mL) (27), and the suspensions were then diluted to give 10 6 CFU/mL per spot.Petri dishes were spot inoculated with 10 µL of each prepared fungal suspension (10 6 CFU/spot) and incubated at 25 ˚C for 48 hours.At the end of the incubation period, MIC was determined, which is the lowest concentration of the test compound that resulted in no visible growth on the plate.To get the minimum fungicidal concentration (MFC), a loopful was taken from the MIC tubes and streaked on SDA plates.The growth was observed after incubation at 37 º C for 48 hours, and the lowest concentration was recorded as MFC which exhibited no growth (28).

Statistical Analysis
Statistical analysis was run using Microsoft Excel 2010 based on three independent experiments, and the results were expressed as mean.
All tested compounds (4a-4g) inhibited the growth of S. aureus and MRSA, but compounds 4a, 4b, 4c, and 4d were more active than the others.The growth of E. coli and P. aeruginosa was also inhibited by the compounds at the range of 250 µg/mL concentration.Furthermore, some of the compounds showed the same or higher antibacterial activity with ceftizoxime; however, they exhibited poor activity compared to ciprofloxacin.Additionally, the MBC of compounds was two or three-fold higher than that of the corresponding MIC results.

Antifungal Screening
The antifungal potential of tested compounds was evaluated according to their zone of inhibition and minimum inhibition concentration against tested fungal pathogens.The results were compared with the activity of fluconazole and are summarized in Table 2 and Figure 2.

Discussion
All synthesized polycyclic heterocycles were tested for their antimicrobial activities, and MICs were recorded as a minimum concentration of compounds that inhibit the growth of tested microorganisms.The obtained results revealed that our new tetracyclic heterocyclic ring system (4a-4g) has good antimicrobial activity, but the highest inhibitory activity was observed against Grampositive bacteria which can be explained by the presence of lipophilic moieties in triazolo scaffold.The widest spectrum of antibacterial activity was exerted by 4a, 4b, 4c, and 4d, suggesting that the phenyl ring, which contains an electron-withdrawing or electron-releasing substituent at the C-2 position of the [1,2,4] triazole ring exerts a good antibacterial effect against S. aureus and MRSA.These observations are similar to the results reported by Kumar et al (29).Interestingly, the most potent antibacterial compound in the present study belongs to the C-2 Hydroxyphenyl compound.As evidenced by inhibition zone sizes, the antibacterial activity of compounds against Gram-positive was somewhat stronger than that of ceftizoxime, while they displayed moderate antimicrobial activity against the Gram-negative bacterial strain.In addition, the antifungal screening results revealed that compounds are active against the fungal strains used in the study due to the presence of triazole thiadiazin moiety in 10 naphtha, which increased the antimicrobial activity.Moreover, compounds 4a, 4d, 4e, and 4f had better antifungal activity against tested fungal strains which is comparable to fluconazole, but significant activity was observed for compound 4d that was more potent than the control drug (fluconazole) against standard strains such as C. albicans, C. tropicalis, C. krusei, and C. glabrata.Additionally, the structural and antimicrobial similarity between our synthesized compounds and Fluconazole suggested that the mechanism of the action of the new compounds may be similar to that of fluconazole.Furthermore, this compound may be a good alternative for treating and controlling infections caused by MRSA, which is an extremely significant public health concern.
In case further studies establish the safety and efficiency of these compounds, they may be represented as an excellent prototype with anti-MRSA activity in the development of new drugs for medical use.

Figure 1 .
Figure 1.Inhibition Zone of Compounds against Escherichia coli and Methicillin-resistant Staphylococcus aureus in 1mg/mL Concentration

Figure 2 .
Figure 2. Inhibition Zone of Compounds against Candida tropicalis (a) and Candida albicans (b) in 1 mg/mL Concentration\