In vitro inhibitory potential of two synthesized fatty amides derivatives against some microbial pathogens

In the present study, two amide derivatives with long chains; N-cyclohexyloctamide 1 and N-Phenyldodecanamide (dodecanilide) 2 were synthesized. They were tested in vitro for a wide array of antimicrobial activities against; two Gram positive bacteria ( Bacillus subtilis , Staphylococcus aureus ), four Gram negative bacteria ( Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , Salmonella enteric ), and three fungi ( Aspergillus carbonarius , Mucor ramannianus , Candida albicans ), to evaluate their potential use as antimicrobial agents. Zones of inhibition of these compounds were determined by disc diffusion assay. Results revealed high antimicrobial activities of the two amides. The new fatty N-cyclohexyloctamide 1 showed significant antimicrobial activity against S. aureus and A. carbonarius , and was more active against C. albicans than synthesized fatty anilide 2, as it caused higher zones of inhibition. On the other hand, the fatty anilide 2 demonstrated high antibacterial activity against P. aeruginosa , and was more active against S. enteric than substituted fatty amide 1. On the basis of these obtained results, it could be assumed that fatty amide derivatives may be used as components of disinfectants, and may be possible sources of new drugs effective against various infectious diseases.


Introduction
Bacteria have been reported to cause a number of life-threatening infections all over the world (Berber et al., 2003). Large number of bacteria can cause infectious diseases or contaminate food, thus affecting their quality. It is therefore necessary to control these microbes. The use of antimicrobial substances included in disinfectants for example, can control microbial growth. Zhang et al., (2010) defined the antimicrobial as a substance that reduce or completely block the growth and multiplication of microbes, and are helpful in the treatment of various infectious diseases.
Infectious diseases were the most significant causes of human death, accounting approximately for one-half of all deaths throughout the world (Gnanamani et al., 2003). Karaman et al., (2003) added that these numbers were still increasing due to the development of drug resistance in microorganisms, as a result of the indiscriminate use of antimicrobial drugs.
Thus, the search for new antimicrobial agents is an urgent need. With the advancement of Science and Technology, remarkable progress has been made in the field of medicine with the discovery of many efficient natural and synthetic drugs (Preeth et al., 2010).
Amide derivatives are important classes of organic compounds. They were associated with broad spectrum of biological activities including; antituberculosis, anticonvulsant, analgesic, antiinflammatory, insecticide, antifungal, and antitumor agents. They also showed anti-platelet activities. Pochampally et al., (2014) added that amides in conjugation with other aliphatic, aromatic and heterocyclic moieties; possessed various types of biological activities.
Anilides are important classes of amides. In the last decade, a special interest has been focused on anilides, because of their peculiar biological activities including; anti-bacterial, anti-fungicidal, anticonvulsant, anesthetic, and platelet aggregation (Narasimhan et al., 2007). Farshori et al., (2011) reported that several anilides have found wide applicability as bioactive species (i.e. antimicrobial, antioxidant and anti-atherosclerotic agents). Moreover, they were also involved as intermediate precursors in the synthesis of therapeutic agents (Malki et al., 2011;Malki et al., 2016a). For example, Cyanoacetanilides were important and versatile reagents; which have been specifically used for the synthesis of polyfunctionalized heterocycles with diverse pharmacological activities (Farrag et al., 2015). In previous studies of Malki et al., (2016b); Malki et al., (2017a), they evaluated the in vitro antioxidant activities of some synthesized amide derivatives.
Results revealed that the tested amides had variable and interesting antioxidant properties, and free radical scavenging activities; when compared to the standard antioxidants.
According to the wide variety of biological activities shown by amide derivatives, and in continuation of author's efforts in search for potent molecules exhibiting antimicrobial activities; the purpose of the current study was to evaluate the antimicrobial activities of two synthesized amides derivatives containing fatty chain alkyl, against diverse range of microorganisms including; Gram-positive, Gram-negative bacteria and fungi.

Chemicals
Chemicals used were of analytical grade purchased from Sigma-Aldrich, Merck, Prolabo and Biochem.
Dodecanilide 1 was first prepared as follows; a mixture of aniline and dodecanoic acid was heated at 160-200°C for 3-4 h. Water from the reaction was continuously distilled off. Under similar operating conditions, the newly N-cyclohexyloctanamide 2 was prepared by reaction of an excess of cyclohexylamine with octanoic acid. The produced white solids were recrystallized in ethanol, and recovered as white crystals in quantitative yields. They were then characterized and their chemical structures were confirmed by spectroscopic analyses in reference to Malki et al., (2011);Malki et al., (2016b).

In vitro antimicrobial activity
The synthesized amides 1, 2 were screened for in vitro antimicrobial activities by the paper disk-diffusion assay according to Balouiri et al., (2016 (Shirling and Gottlieb, 1966).
Briefly, both amides 1 and 2 were dissolved in methanol (1 mg/mL). Sterile 6-mm diameter filter paper disks were impregnated with solution of the compound to be tested (20, 60 μg/disk), then these paper disks were placed aseptically on the surface of ISP-2 medium, seeded separately with each indicator microorganism. For positive control, Gentamicin (10 μg/disk) was used to determine the sensitivity of Gram-positive and Gram-negative bacteria, while Fluconazole and amphotericin B (25 μg/disk) were used for yeast and fungal species, respectively. Petri plates were left at 4°C for 2 h to allow diffusion of the amides in the medium, and then incubated at 37°C for 24 h for bacteria, and at 30°C 96 h for yeasts and fungi. Positive antimicrobial activity was observed as a clear zone around the disk indicating inhibition of microbial growth (Gülçin et al., 2008). The antimicrobial activity was evaluated by measuring the diameters of inhibition zones in mm. All assays were performed in triplicates, in three independent experimental sets. Data obtained was presented as mean of diameters of inhibition zones of different concentrations of each amide against each tested bacteria and fungi.

Statistical analysis
Data were analyzed with the statistical software statistica. Values were expressed as means ± standard deviations (SD). Differences were considered significant at p < 0.05.

In vitro antimicrobial potency
Both amides 1 and 2 were screened for their in vitro antibacterial and antifungal activities against different bacterial and fungal species, and results were represented in Table 1. Table 1 demonstrated that both fatty amides showed inhibitory effects against the Gram-negative bacterium S. enteric, and that fatty anilide 2 was more active than fatty amide 1. On the other hand, only anilide 2 exhibited potent inhibitory activity against Gram-negative bacterium P. aeruginosa; however, at the concentration of (60 µg/disk) none of the two amides manifested antibacterial activities against either E. coli, K. pneumonia, or against B. subtilis. Both fatty amides showed antifungal activities against C. albicans, where amide 1 showed the highest activity.

Results observed in
Only amide 1 demonstrated the ability to inhibit A. carbonarius growth, whereas the two fatty amides were inactive against M. ramannianus, which proved thus to be resistant to both amide derivatives. It should also be noted that the various inhibition assays were carried out at a charge of disk about 20 and 60 μg/disk. Maximum inhibitions were practically achieved at charge of 60 μg/disk for all the tested strains.

Conclusion
In this study, results demonstrated the potential antimicrobial properties of the tested amides. Both fatty amides showed interesting antibacterial activities against S. enteric, and antifungal activities against C. albicans.
High antifungal activity against A. carbonarius was recorded specifically for the amide 1; whereas, the fatty anilide 2 demonstrated high antibacterial activity against P. aeruginosa. The antimicrobial potency increased with increasing the corresponding concentration of the amides, and was directly related to the nature of the substituents on the amide group.
Thus both active compounds recorded in this study could be useful for developing new antimicrobial drugs, and may be used in the future for treatment of many diseases.