Synthesis of derivatives azomethine compounds bonded to alkoxylated benzene and their antibacterial activity tests

Azometin compound has been synthesized from addition-elimination reaction between aromatic aldehyde compounds such as benzaldehyde, veratraldehyde, piperonal with primary amine compound from ethylenediamine. The presence of imine group (>C=N-), and alkoxy groups such as methoxy (-OCH3) and methylenedioxy (-O-CH2-O-) affected to their activity as antibacterials. In the synthesis of methoxy substituted azomethine compound from verataldehyde with ethylenediamine to produce N,N’-Bis(3,4-dimethoxybenzylidene) ethylenediamine in the form of white powder has a melting point of 164.8–166.2°C and in about 36.08% yield. In the synthesis of methylenedioxy-substituted azomethine compound from piperonal with ethylenediamine produced N,N’-Bis(3,4-methylenedioxybenzylidene) ethylenediamine in the form of greenish-white solid has a melting point of 176,3–177,4°C and in about 67,88% yield. In the synthesis of non-substituted azomethine compound from benzaldehyde with ethylenediamine produced N,N’-Bis(benzylidene)ethylenediamine in the form of orange solid has a melting point of 110.2-111.4°C and in about 34.82% yield. Antibacterial activity of compound N,N’-Bis(3,4-dimethoxybenzylidene)ethylenediamine, N,N’-Bis(3,4-methylenedioxybenzylidene)ethylenediamine, and N,N’-Bis(benzylidene) ethylenediamine with inhibitory zone diameter 0, 25; 3,08; 0.06 mm in S.aureus bacteria (G+) and 3.26; 4.61; 3.48 mm in E.coli bacteria (G−).


Introduction
Azometin compound is a compound which has an imine group (>C=N-) from the addition-elimination reaction between carbonyl compounds from aldehydes or ketones with the primary amine compound from ethylenediamine. Azometin compound has been known to play an important role in their biological activities such as antibacterials [1]. Antibacterial activity of azomethine compound is caused by the interaction of the imine group with the bacterial cell membrane resulting in bacterial cell lysis [2]. Several studies on antibacterial activity with 1 and 2 imine groups have been reported that azomethine compound with 2 imine groups has better antibacterial activity than 1 imine group [3,4].
In this study, synthesis of azomethine derivatives bound to benzene with 2 imine group (>C=N-) and substituted by alkoxy groups including methoxy (-OCH3) and methylenedioxy (-O-CH2-O-) substituent, or without substituent. First, synthesis of methoxy-substituted azomethine compound, N,N'-Bis (3,4-dimethoxybenzylidene)ethylenediamine from veratraldehyde with ethylenediamine. The second is the synthesis of methylenedioxy-substituted azomethine compound namely N,N'-Bis (3,4methylenedioxybenzylidene) ethylenediamine from piperonal with ethylenediamine. The third is the synthesis of unsubstituted azomethine as a comparison namely N,N'-Bis(benzylidene)ethylenediamine from benzaldehyde with ethylenediamine. From the three compounds that was obtained, then it was tested as antibacterials against Staphylococcus aureus (G + ) and Escherichia coli (G -) bacteria. By comparing the antibacterial activity of the three compounds, it can be seen the influence of the type of substituent bound to the azomethine compound. The presence of two methoxy groups in azomethine N,N'-Bis (3,4-dimethoxybenzylidene)ethylenediamine and methylenedioxy groups in azomethine N,N'-Bis(3,4-methylenedioxybenzylidene) ethylenediamine is expected to influence antibacterial activity compared to antibacterial activity. azomethine N,N'-Bis(benzylidene)ethylenediamine compounds without substituent.

Materials and tools
The laboratory equipments are reflux tools, glass tools, funnels, magnetic stirrers (VS 130SH) and bars, chambers, analytical balance sheets (Ohaus), petri dishes, ose needles, tweezers, micropipettes 10-100 μL, spreaders, incubators (Memmert IN55) (3,4-dimethoxybenzylidene) ethylenediamine from veratraldehyde with ethylenediamine A (0,67 mL, 20 mmol) ethylenediamine was dissolved in 10 mL ethanol in a three-neck round bottom flask. In other beaker glass, as much as (3,32 g, 20 mmol) verataldehyde was dissolved in 15 mL ethanol and mixed into ethylenediamine solution. Glacial acetic acid (5-10 drops) as an acid catalyst was added to the solution. Then the solution was stirred with a magnetic stirrer, stirring speed of 400 rpm and heated at 78 o C for 3 hours. The reaction mixture was placed on a cold water bath for 10 minutes and filtered using Whatman filter paper and funnel. The product precipitate was then recrystallized by dissolving it in hot ethanol, filtered again, washed with cold ethanol and dried. Furthermore, the azomethine compound N,N'-Bis (3,4-dimethoxybenzylidene) ethylenediamine was weighed and analyzed using melting point test, solubility test, UV-Vis spectrophotometry and IR spectrophotometry [4].

Synthesis of N,N'-Bis (3,4-methylenedioxybenzylidene) ethylenediamine from Piperonal with Ethylenediamine
A (0,67 mL, 20 mmol) ethylenediamine was dissolved in 10 mL ethanol in a three-neck round bottom flask. In other beaker glass, as much as (3,00 g, 20 mmol) was dissolved in 15 mL ethanol and mixed into ethylenediamine solution. Glacial acetic acid (5-10 drops) as an acid catalyst was added to the solution. Then the solution was stirred with a magnetic stirrer, stirring speed of 400 rpm and heated at 78 o C for 3 hours. The reaction mixture was placed on a cold water bath for 10 minutes and filtered using Whatman filter paper and funnel. The product precipitate was then recrystallized by dissolving it in hot ethanol, filtered again, washed with cold ethanol and dried. Furthermore, the azomethine  (3,4-methylenedioxybenzylidene) ethylenediamine was weighed and analyzed using melting point test, solubility test, UV-Vis spectrophotometry and IR spectrophotometry [4].

Synthesis of N,N'-Bis (benzylidene) ethylenediamine from benzaldehyde with ethylenediamine
A (0,67 mL, 20 mmol) ethylenediamine was dissolved in 10 mL ethanol in a three-neck round bottom flask. In other beaker glass, as much as (2,03 mL, 20 mmol) was dissolved in 15 mL ethanol and mixed into ethylenediamine solution. Then the solution was stirred with a magnetic stirrer, stirring speed of 400 rpm and heated at 78 o C for 3 hours. The reaction mixture was evaporated using evaporator to remove the solvents. Then, the mixture was placed on a cold water bath for 10 minutes and filtered using Whatman filter paper and funnel. The product precipitate was then recrystallized by dissolving it in hot ethanol, filtered again, washed with cold ethanol and dried. Furthermore, the azomethine compound N,N'-Bis(benzylidene)ethylenediamine was weighed and analyzed using melting point test, solubility test, UV-Vis spectrophotometry and IR spectrophotometry [5].

Antibacterial activity test
Antibacterial activity test of azomethine compound was carried out by using diffusion disc method based on measuring of minimum inhibitory concentration. The compounds tested in this research were N,N'-Bis (3,4-dimethoxybenzylidene) Table 1. The UV-Vis analysis spectra of N,N'-Bis (3,4-dimethoxybenzylidene) ethylenediamine are found at wavelengths 268,4 nm (band I) and 305,6 nm (band II). The result of UV-Vis spectra is presented in Figure 2.    Table 2. The UV-Vis analysis spectra of N,N'-Bis (3,4-methylenedioxybenzylidene) ethylenediamine is found at wavelengths 268,4 nm (band I) and 305,6 nm (band II). The result of UV-Vis spectra is presented in Figure 5.   Table 3. The UV-Vis analysis spectra of N,N'-Bis (3,4-benzylidene)ethylenediamine is found at wavelengths 243,4 nm (band I). The result of UV-Vis spectra is presented in Figure 8.

Antibacterial activity test
Antibacterial activity of azomethine compound was tested against Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria). This antibacterial testing was done by disc diffusion method. It was done by a filter paper containing a number of active compounds placed on the surface of the solid medium which had previously been inoculated by the bacteria on the surface of the medium.
Antibacterial activity of azomethine compound is caused by the presence of imine groups (>C=N-). The presence of imine groups will interfere with the normal process of bacterial cells by forming hydrogen bonds between the active centre on the bacterial cell membrane with nitrogen at the imine group of the azomethine compound. The hydrogen bond will affect the permeability of the cell wall and cytoplasmic membrane. The disturbance of permeability cell walls and cytoplasmic membranes can cause an imbalance of macromolecules and ions in cells so that the cell becomes lysis [2]. The antibacterial activity test results of azomethine compound were indicated by the measurement of compound inhibition zone diameter (mm) which is presented in table 4.    (3,4-dimethoxybenzylidene)ethylenediamine which is difficult to penetrate the bacterial cell membrane so that the inhibitory process against bacteria becomes smaller. This is supported by research conducted by Alcaraz et al. (2000) reported that the addition of methoxy groups could reduce the antibacterial activity of azomethine compounds. From this explanation, it can be concluded that the presence of methoxy groups can reduce the antibacterial activity of azomethine compound. In compound N,N'-Bis(benzylidene)ethylenediamine doesn't have a substituent in the structure of the azomethine compound so that the antibacterial activity is only affected by the imine group (>C=N-). In azomethine N,N'-Bis (3,4-methylenedioxybenzylidene)ethylenediamine has the highest antibacterial activity compared to N,N'-Bis(benzylidene)ethylenediamine and N,N'-Bis (3,4-dimethoxybenzylidene)ethylenediamine. This is because the compound N,N'-Bis (3,4-methylenedioxybenzylene)ethylenediamine has a cyclic methylenedioxy group (-O-CH2-O-) which is an electron booster. This impulse of electrons from the methylenedioxy group gives a positive mesomeric effect on the aromatic ring so that the aromatic ring is more electronegative. As a result, the N atoms in the imine group (>C=N-) are more electronegative. The free electrons of the N atom of the imine group can bond hydrogen more strongly with the active center of the bacterial cell membrane so that the inhibition of the bacteria becomes stronger.

Conclusion
From the results of this research, it can be concluded that synthesis of alkoxy-substituted azomethine compounds were carried succesfully. Synthesis of methoxy-substitued azomethine compound from veratraldehyde with ethylenediamine produce N,N'-Bis (3,4- (3,4-