Synthesis and characterization of some tetrazoles and their prospective for aerobic micro-fouling mitigation

2.2.1 Preparation of N-(1H-tetrazol-5-yl)-1-(aryl)methanimine (101–106)

A solution of 1H-tetrazol-5-amine (0.010 mol) and aromatic aldehydes (0.010 mol) (1–6) in ethanol was refluxed for 2 h in water bath Scheme 1. The resultant solution was cooled; the solid was filtered off and recrystallized from petroleum ether. Compounds 101–106 were obtained as crystalline mass (Kumar et al., 2014).

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Scheme 1

Preparation of N-(1H-tetrazol-5-yl)-1-(aryl)methanimine (101–106). 101: 1-phenyl-N-(1H-tetrazol-5-yl)methanimine. 102: N-(1H-tetrazol-5-yl)-1-(p-tolyl)methanimine. 103: 4-((1H-tetrazol-5-yl)imino)methyl)phenol. 104: 1-(4-methoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine. 105: 1-(4-chlorophenyl)-N-(1H-tetrazol-5-yl)methanimine. 106: 1-(4-nitrophenyl)-N-(1H-tetrazol-5-yl)methanimine.

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2.2.2 Preparation of 1-(4-alkoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine (107–111)

These five compounds were synthesized in two steps, Scheme 2:

1. Step 1: preparation of 4-alkoxybenzaldehyde:

2. As illustrated (Nessim et al., 2015), the five 4-alkoxybenzaldehydes were prepared via the reaction of 4-hydroxybenzaldehydes with five different alkyl bromides (C₄H₉Br, C₆H₁₃Br, C₈H₁₇Br, C₁₀H₂₁Br and C₁₂H₂₅Br).

3. Step 2: Synthesis of (107–111):

4. As discussed in Scheme 1 (Kumar et al., 2014), the five 4-alkoxybenzaldehydes reacted with 1H-tetrazol-5-amine to form compounds (107–111).

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Scheme 2

Synthesis of compounds (107–111). 107: 1-(4-butoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine. 108: 1-(4-hexylkoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine. 109: 1-(4-octylkoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine. 110: 1-(4-decylkoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine. 111: 1-(4-dodecylkoxyphenyl)-N-(1H-tetrazol-5-yl)methanimine.

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2.2.3 Preparation of stock standard solutions for biological assay

For each of the tested tetrazoles groups [(107–111) & (101–106)], stock standard solutions were freshly prepared and stored at 4 °C (Chemiasoft, 2011). Samples were dissolved in DMF that is widely known to have no antimicrobial activity against different microorganisms and is considered as negative control (Cooper, 1972).

2.2.4 Model Organisms

Identified bacterial isolates of Staphylococcus aureus, Escherichia coli and Candida albicans were provided from Cairo MIRCEN (MIRCEN).

2.2.5 Antimicrobial susceptibility test methods

1. Agar assay

Under sterilized conditions, for each of the synthesized tetrazole derivatives, agar-agar media (Balouiri et al., 2015) were freshly prepared and left till warmness and then seeded with 24 h grown bacterial suspensions (1–2 × 10⁸ CFU/mL per McFarland standard), vortexed and poured onto Petri plates (4 mm height agar layer). Agar plates were then kept till dryness and stored at 4 °C for two hours. One millimeter wells were cork borered onto agar plates, inoculated with 100 µl of each of the test samples and incubated at 37 °C ± 2 for 48 h. Each experiment was done in triplicate (Parekh and Chanda, 2007). DMF was used as a negative control. Inhibition zones were measured, and activity indices were calculated according to Dut Jasuja et al. equation (Jasuja et al., 2014) [Eq. (1)]. $$\mathit{Activity}index=Inhibitionzoneofthesample(mm)/Inhibitionzoneofthestandard(mm)$$

• Micro plate assay:

Recently, the 96-well Microtiter plate for MICs evaluation is commonly applied as time saver, a significant reducer of sample numbers experiment cost and place occupied, thus improving the repeatability of the results (Zhang et al., 2006). In a sterilized area, microbial suspensions were freshly cultured in suitable broth media (bacteria/yeast). About 0.125 μl of microbial cell suspension of 10⁷ CFU/ml count were adjusted at 550 nm absorbance. Blank broth media were inoculated in the wells. Different sample concentrations were applied from 64 to 0.5 μg/ml via 10-fold serial dilutions. A volume of approximately 180 μl of a sample concentration is then injected to each well. The next step is to inoculate about 10 μl microbial suspension into each well. Some wells are specified to solvent, blank sample (without microbes), blank broth and untreated microbial suspensions. The 96-well micro plates were then shaker- incubated for 6–8 h at incubation temperature. Coloring agent (0.4% Resazurin) was applied to each of the 96 wells and the micro plates were incubated for two hours to accomplish dye reaction (Coffey and Anderson, 2014).

2.2.6 Cell cytotoxicity assay

Cell cytotoxicity assay is designed for screening the activity of a certain compound cellular effects such as cell proliferation or in the form of direct cytotoxic effects expressed by cell death. The metabolic activity of viable cells is commonly used as the basis for absorbance assay. Cell viable count is “a measure of the number of cells that are viable (i.e. alive and capable of growth) in a given area or volume”. One use of viable cell count is to estimate cytotoxicity. Continuous UV–Vis spectroscopic measurements of a treated microbial suspension optical density in correlation to a 100% live microbial could be employed to estimate viable cell numbers and percentage (Park et al., 2012; Mageswari and Subramanian, 2012). Microbial suspensions were prepared, adjusted to 0.5 O.D. McFarland standards (1–2 × 10⁸ CFU/mL) and sterilized. For each test time interval, bacterial suspensions were centrifuged at 10,000 r.p.m. for 10 min (Sigma Centrifuge Model 3 K 300), and the supernatants were removed. A process of three subsequent washing s of the remaining pellets were carried out, suspended in sterilized deionized water and well-vortexed (Germany International Corp Vortex, Model VM-300). For each time interval, 24 mL of each test agent were injected and mixed with 6 mL of the bacterial suspension and their optical densities were measured (ATI Unicam 8625 UV/Vis Spectrophotometer). The 0-time untreated remaining bacterial suspensions were used as 100% live cells and optical density reading was measured and plate counted in CFU/mL. Eq. (2) (Park et al., 2012). Experiment was terminated after 48 hrs. of exposure. $$\mathit{Cell}ViabilityPercentage=\left\{\frac{\mathit{Absorbance}oftestsuspension}{\mathit{Absorbance}of100\%livemicrobialsuspension}\right\}\left(\%\right)$$

3.1.1 Elemental analysis

Data obtained in Table 1 showed that the calculated percentages of the elements coincides with the observed ones.

3.1.2 Infra-red spectroscopy

See Table 2.

3.1.3 Proton NMR spectroscopy of compounds (101–111)

101: C⚌CH at 9.38 singlet, 7.82 doublet, 7.55 doublet, 7.28 triplet and NH at 6.28 ppm singlet.

102: C⚌CH at 9.22 singlet, 7.68 doublet, 7.16 doublet, NH 6.32 singlet and CH₃ Protons at 2.60 ppm singlet.

103: OH at 9.98, C⚌CH at 9.18 singlet, 7.68 doublet, 7.02 doublet and NH at 6.33 ppm singlet.

104: C⚌CH at 9.36 singlet, 7.95 doublet, 7.56 doublet, NH 6.36 singlet and OCH₃ proton at 3.26 ppm singlet.

105: C⚌CH at 10.01 singlet, 7.95 doublet, 7.56 doublet and NH at 6.36 ppm singlet.

106: C⚌CH at 9.43 singlet, 8.40 doublet, 8.16 doublet and NH at 6.36 ppm singlet.

107: C⚌CH at 9.423 singlet, 7.826 doublet, 7.684 doublet, NH at 6.414 singlet, (e) CH₂ at 4.328 triplet, CH₂ Protons at 1.748 multiplet, CH₃ protons at 0.848 triplet.

108: C⚌CH at 9.246 singlet, 7.825 doublet, 7.721 doublet, NH at 6.402 singlet, CH₂ at 4.312 triplet, CH₂ 1.738 multiplet, and CH₃ at 0.842 triplet.

109: C⚌CH at 9.864 singlet, 7.855 doublet, 7.113 doublet, NH at 6.441 singlet, CH₂ at 4.063 triplet, CH₂ at 1.728 multiplet, and CH₃ at 0.857 triplet.

110: C⚌CH 9.285 singlet, 7.675 doublet, 7.482 doublet, NH at 6.421 singlet, CH₂ at 4.214 triplet, CH₂ at 1.728 multiplet and CH₃ at 0.828 triplet.

111: C⚌CH at 9.114 singlet, 7.848 doublet, 7.763 doublet, NH at 6.460 singlet, CH₂ at 4.062 triplet, CH₂ at 1.715 multiplet and CH₃ at 0.830 triplet.

3.2.1 Activity index

Screening the antibacterial activity of the synthesized tetrazoles, using diffusion techniques (Fig. 1A and B), revealed that they apparently exhibited antibacterial activities according to their main substituted groups together with the main skeleton activity. It is worth to mention that Gram-+ve bacteria showed more susceptibility towards the tested tetrazoles than Gram −ve bacteria and yeast. The composition of their microbial cell wall is considered to be a detrimental criterion influencing their response towards the introduced agents. Gram +ve bacterial cell wall main component is a thick layer of peptidoglycan, whereas peptidoglycan layer in Gram −ve bacteria are enclosed between an inner cytoplasmic layer and an outer membrane. In yeast, cell wall is made of polysaccharides, proteins, lipids and chitin that make its cell wall much thicker. So, yeast is more anti-microbially resistive than Gram −ve and Gram +ve bacteria respectively (Salton and Kim, 1996). For tetrazoles, (107–111), compound 107 revealed the highest activity index (Fig. 1A), whereas of the tetrazoles, (101–106), compound 106 was the most potent one (Fig. 1B).

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Fig. 1

Activity index of the tested groups (A) & (B) against Staphylococcus aureus, Escherichia coli & Candida albicans. (Standard deviation ± 0.02).

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3.2.2 Micro plate evaluation

Results of MIC (µg/ml) of the synthesized tetrazoles against Staphylococcus aureus were observed to be lower than Escherichia coli and Candida albicans respectively. Compound 107 seemed to be more toxic against tested microbes, where low concentration was needed to inhibit microbial growth (MIC value = 10.66666667 µg/ml) (Figs. 2 and 3A). While a minimum concentration of compound 106 (16 µg/ml) was fair enough to inhibit the growth of all the tested organisms (Figs. 2 and 3B).

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Fig. 2

Micro plate MIC evaluation of Staphylococcus aureus, Escherichia coli & Candida albicans treated with tested tetrazoles.

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Fig. 3

MICs (µg/ml) of Staphylococcus aureus, Escherichia coli & Candida albicans treated with tested tetrazoles groups (A) & (B). (Standard deviation ± 0.02).

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3.2.3 Cell viability and cytotoxicity assay

Cell Viability percentage can indirectly express sample activity and its cytotoxicity. Screening of cell viability (%) of Staphylococcus aureus, Escherichia coli and Candida albicans exposed to the two tetrazoles series, (101–106) and (107–111), are illustrated in Fig. 4A and B. Of the tetrazoles series (107–111) compound 107 showed highest activity, (average activity percentage = 93.282623%), whereas the highest average activity percentage one of series (101–106) belonged to compound 106 (90.723733%).

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Fig. 4

Cell viability % of Staphylococcus aureus, Escherichia coli & Candida albicans treated with tested tetrazoles (A) & (B) after 48 h of exposure.

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3.2.4 Antibacterial mode of action

Yet, a little is published discussing tetrazoles antimicrobial mechanism. Few suggestions were reported.

In the present work, the two evaluated tetrazoles series are suggested to have two logical modes of actions.

• Considering series (107–111), alkyl chain length seems to control their antimicrobial mechanism. Precisely, the alkyl chain length affects their trend which may be attributed to molecules lipophilicity enhancing the attacking process into microbial cell membranes leading to deterioration of its transmembrane signaling leading cytotoxic effects and death. So as the chain length decrease the efficiency increase (Gao et al., 1999).

• In series (101–106), another electrostatic mode of action is proposed. Efficacy increases with the positively charged tetrazole ring bearing electron-withdrawing group (Suzen et al., 2006). Then, electrostatic interaction with negatively charged microbial cell membranes forms reactive oxygen species. Reactive oxygen species are the by-products of an oxidative stress resulted from the electrostatic interaction.

• Generally, antimicrobial action of an agent involves inhibition of enzymes involved in cell wall biosynthesis, nucleic acid (DNA) metabolism and protein synthesis responsible for membrane structure. Microbes have their protection mechanisms against external oxidative stresses (OS) expressed in altered enzymatic activities. The generally accepted aromatic tetrazoles mode of action entails inhibition of ergosterol in the microbial membrane. Recently, proposed mechanisms of antimicrobial mode of action depend mainly on the hypothesis of electron transfer, reactive oxygen species and oxidative stress that is considered as unifying process (Kovacic and Abadjian, 2017).

Tetrazoles act as free radical scavengers. For a certain extent, their antioxidant potential depends on substituted terminal groups and structure conjugation (Chand et al., 2018). Bacterial responses to tetrazoles oxidative stress are expressed as the generation of reactive oxygen species (ROS) leading to the oxidative damage to cellular proteins. In addition, utilization of electron transfer processes (ET) could also be responsible of different physiological alterations (Kovacic and Abadjian, 2017; Kashmiri and Mankar, 2014; Kovacic and Somanathan, 2010; McDonnell and Denver Russell, 1999).