In Vitro Testing of Salicylanilide Derivatives Against Some Fungal and Bacterial Strains

IOANA MARIA CARMEN IENASCU1,2, DIANA OBISTIOIU3, IULIANA MARIA POPESCU4, MARIANA NELA STEFANUT1, OSSER GYONGYI2, CLAUDIA JURCA5, GABRIELA CIAVOI5, EDWIN SEVER BECHIR6*, FARAH BECHIR6, ADINA CATA1 1 National Institute of Research and Development for Electrochemistry and Condensed Matter, 144 Dr. A. P. Podeanu, 300569, Timiooara, Romania 2 Vasile Goldis Western University of Arad, Faculty of Pharmacy, Department of Pharmaceutical Sciences, 86 Liviu Rebreanu, 310045, Arad, Romania 3 Banat’s Agricultural Science University, Faculty of Agriculture, Interdisciplinary Research Platform, 119 Calea Aradului, 300645, Timiooara, Romania 4 Banat’s Agricultural Science University, Faculty of Agriculture, Department of Chemistry and Biochemistry, 119 Calea Aradului, 300645, Timiooara, Romania 5 University of Oradea, Faculty of Medicine and Pharmacy, 10 P-ta 1 Decembrie, 410073 Oradea 6 Faculty of Dental Medicine, University of Medicine, Pharmacy, Sciences and Technology of Targu Mures, 38 Gheorghe Marinescu Str., 540142 Targu Mures, Romania

In the last decades, a major intensification in the prevalence of resistance to antibacterial and antifungal agents has been observed. Antibiotic resistance occurs when a drug fails the capacity to inhibit bacterial growth efficiently. Bacteria become resistant and continue to proliferate in the presence of therapeutic concentration of the antibiotics. Prescription of broad spectrum antibiotics in hospitals to treat nosocomial infections increases antibiotic resistance [1].
The implications of the appearance of resistance to current antimicrobial agents would be the following: morbidity, mortality and high health care costs both in hospitals and in the community. Therefore, the researchers have been focused on a better understanding of the mechanisms of antimicrobial resistance, developed methods to detect resistance, new antimicrobial agents for such resistant microorganisms infections treatment, and approaches to prevent the occurrence and spread of resistance in the first place [2]. So, the development of effective and non-toxic antimicrobial agents is still a vital issue due to the increase of multi-resistant bacterial strains [3][4][5][6][7]. The most affected patients are those with compromised immune systems or with other associated diseases [8][9][10][11][12][13][14].
2-Hydroxybenzamide derivatives are a class of compounds with a large spectrum of biological activity, from which, 2-hydroxybenzanilides, as well as Osubstituted 2-hydroxybenzanilides proved to be potent antimicrobial agents [15,16]. Among the currently used antibacterial agents, nitrofurazone, furazolidone and nitrofurantoin are known to contain hydrazide-hydrazone moiety [17][18][19][20]. Due to this fact, hydrazide-hydrazone * Phone: +40723396969 All authors contributed equally to the study and the publications derivatives represent a good source to develop new antibacterial agents. Development of new compounds by attaching 2hydroxybenzamides to another halogen-substituted aromatic nucleus conducted to the enhancement of the antibacterial, antiviral, anti-inflammatory and analgesic effects of these compounds. Salicylamide-O-acetic hydrazide and hydrazones displayed antiinflammatory and analgesic effect superior to salicylamide and, at the same time, inferior ulcerogenic effect [21,22]. Novel 2-hydroxy-benzamide derivatives, esters, hydrazide and hydrazones were designed [23][24][25][26][27] and investigated for their biological activity [28,29] in our laborator y. Antimicrobial activity of salicylanilide derivatives, esters, hydrazides and hydrazones, was evaluated in vitro against some bacteria and fungi. The minimum inhibitory concentrations obtained for esters were generally comparable to those for salicylanilide, but the hydrazides and hydrazones proved to possess superior antibacterial and antifungical effect compared to the control substance [29].
The aim of this work was to synthetize some novel 2hydroxy-benzamide derivatives and to evaluate their biological activity. The relationship between the antimicrobial activity and the structure of these synthesized compounds was also established.

Conventional heating synthesis
Anilide -To a mixture of salicylic acid, 2-bromoaniline and phosphorus trichloride 1:1:0.3, chlorobenzene was added. The mixture was stirred and heated to reflux for 5 h. After being cooled at room temperature, the obtained, crude product owas filtered off, washed three times successively with hot water and then three times successively with 10% sodium carbonate solution. The solid product was then filtered off, dried and recrystallized from dimethylformamide.
Ethyl/Methyl esters were added dropwise to a mixture of appropriate salicylamide and anhydrous K 2 CO 3 , refluxed in 2-butanone, ethyl/methylchloro-acetate. The Optimum molar ratio was amide:ester:K 2 CO 3 = 1:1:1. The mixture was stirred and heated to reflux for 5 h. After cooling at room temperature, the mixture was poured into water and shaken intensively. The organic phase was dried over MgSO 4 . After filtration and evaporation of solvent in vacuum, the esters were obtained in crystalline form and were recrystallized from absolute ethanol.
Hydrazide -A mixture of ethyl ester and hydrazine hydrate 1:1 was refluxed in ethanol for 3 h. The reaction mixture was cooled, the separated solid filtered off, then subjected to recrystallization from absolute ethanol.
Hydrazoneswere added to a solution of hydrazide in ethanol, the appropriate benzaldehyde was (1:1). The reaction mixture was refluxed for 5 h. The solid obtained after cooling was filtered off, washed with water and recrystallized from dimethylformamide.

Antimicrobial activity evaluation
Antimicrobial activity of the 2-hydroxy-benzamide derivatives was tested against 6 bacterial strains, and 2 fungal strains, using the Disk diffusion method for susceptibility testing, according to the Standard Rules for Antimicrobial Susceptibility Testing using Impregnated Disks [30,31]. In vitro testing was performed in plates, containing microcomprimates with Nystatin for the antifungal activity and Gentamicin for the antimicrobial activity as positives controls, alongside blank filter papers impregnated with DMSO as negatives controls and filter papers impregnated with 10 µL of 20 g/L stock solution of each compound. A 10 -2 dilution of the fresh Candida cultures and a 10 -2 fresh bacteria culture was used to perform the assay, an inoculum equivalent to a 0.5 McFarland standard. The Petri plates so seeded and the respective specimens containing the tested compounds were incubated at 30 °C for Candida species and 37 °C in case of the bacterial strains, for 24-48 h. Tests were performed in triplicate. Finally, the interpretation of the result, the ratio of the antimycotic and antibacterial effect of the tested compounds, was achieved by measuring the diameter of the analyzed culture inhibition zones (including the diameter of the disc -5mm) in millimeters. The results are presented as the average of two determinations and a percentage representation of the efficacy of the compounds in relation to the effectiveness of the positive control.

Results and discussions
Twelve N-(2-bromo-phenyl)-2-hydroxy-benzamide and N-(4-bromo-phenyl)-2-hydroxy-benzamide derivatives were synthesized, except anilides, the rest of the compounds were synthesized by our team, probably for the first time, were obtained in good yields, between 72-92%. The synthesis pathway is presented in figure 1. The compounds were characterized using modern analytical methods, IR, NMR, elemental analysis, some of the data has already been published [28]. The synthesized compounds, yields and melting points are presented in table 1. Table 2 presents the diameters of inhibition in mm and the compound efficacy expressed in percentages. The results of the microbiological tests indicated that the N-(2bromo-phenyl)-2-hydroxy-benzamide derivatives were more active against the tested microbes compared to N-(4-bromo-phenyl)-2-hydroxy-benzamide derivatives, inhibition zones of 6-12 mm being obtained. The most susceptible microorganisms to the action of the N-(2-bromo-phenyl)-2-hydroxy-benzamide derivatives are Candida albicans and Streptococcus pyogenes. Anilide , ethyl ester and hydrazide inhibited both microbes, moreover, C. albicans is also inhibited by hydrazone . The most active compound against Escherichia coli and Pseudomonas aeruginosa is hydrazide which exhibited a percentage of efficacy in relation to the effectiveness of Gentamicin of 57.7% and 46.7, respectively. The highest efficacy (57.1%) against Streptococcus pyogenes was exhibited by ethyl ester , and in case of Candida albicans, the most active compounds were anilide and ethyl ester (36.9%).
Among N-(4-bromo-phenyl)-2-hydroxy-benzamide derivatives, N-(4-bromo-phenyl)-2-hydroxy-benzamide itself is the only one which exhibited antimicrobial activity. This compound is active only against Pseudomonas aeruginosa and Streptococcus pyogenes, with a percentage of efficacy in relation to the effectiveness of Gentamicin as positive control of 40 and 66.7%, respectively, being the most effective compound (14 mm inhibition zone). However, anilide presented no effect against fungal strains. So, the 4-substitution of aniline ring with bromine conducted to zero efficiency in terms of antifungal activity. Moreover, the substitution of phenolic hydroxyl, apparently, brings also no benefit.
The tested compounds presented no effect against S. aureus, S. flexneri, S. typhimurium and C. parapsilopsis at the tested concentration. C. albicans and S. pyogenes were very susceptible to the action of the N-(2-bromo-phenyl)-2-hydroxy-benzamide derivatives. The most active compound against E. coli and P. aeruginosa is hydrazide . Anilide exhibited the highest efficacy against Streptococcus pyogenes. The most potent compounds against Candida albicans were anilide and ethyl ester .The low efficiency of some of the tested compounds may be due to the low solubility of the compounds which act like a barrier against diffusion in the culture medium or due to the tested concentration. Thus, filter papers were impregnated with 10 µL of 20 g/L stock solution of compounds, meaning that the tested concentrations were 50 folds lower than gentamicin and 500 folds lower than nystatin.
Interactions between oral microbes and dental materials may also occur, although today not very numerous information are available regarding this possibility [32]. Under current circumstances, the issue of safety in terms of sterilization and disinfection in the dental office is of utmost importance, wherefore the study of substances used in disinfection and sterilization should be a constant concern for the specialists in the field.
Among N-(4-bromo-phenyl)-2-hydroxy-benzamide derivatives, only anilide exhibited antimicrobial effect, being active against Pseudomonas aeruginosa and Streptococcus pyogenes; no effect against fungal strains was obtained. In case of N-(4-bromo-phenyl)-2-hydroxybenzamide derivatives, the substitution of phenolic hydroxyl, apparently, is not favorable for antimicrobial effect, but this may also be due to the low solubility/ diffusion of the compounds in the culture medium or due to the tested concentration. These results demonstrated once more the great potential of 2-hydroxy-benzamide derivatives as antimicrobial agents.