Green synthesis, characterization and biological evaluation of novel chalcones as anti bacterial agents

2.1 General method for the synthesis of chalcones

To a solution of 3-acetyl-2,5-dimethylthiophene (0.38 g, 0.0025 mol) and the corresponding active aldehyde (0.0025 mol) in dry ethanol (20 ml) taken in a beaker (100 ml), a catalytic quantity of sodium hydroxide (0.05 g, 1.25 mmol) was added and the reaction mixture was heated inside a microwave oven for 30–50 s. (at 210 W, i.e. 30% microwave power) (Asiri et al., 2013). The reactions were monitored through TLC using solvent system ethyl acetate:benzene (2:8), when the reaction was found to be complete, the reaction mixture was cooled in an ice bath and the product thus formed was filtered, washed with ethanol followed by washing with water till the washings were neutral and recrystallized by distilled ethanol and chloroform.

2.2 Organism culture and in vitro screening

Antibacterial activity was examined by the disk diffusion method with minor modifications. S. aureus, S. pyogenes, S. typhimurium and E. coli were sub-cultured in BHI medium and incubated for 18 h at 37 °C, and then the bacterial cells were suspended, according to the McFarland protocol in saline solution to produce a suspension of about 10⁻⁵ CFU mL⁻¹:10 μL of this suspension was mixed with 10 mL of sterile antibiotic agar at 40 °C and poured onto an agar plate in a laminar flow cabinet. Five paper disks (6.0 mm diameter) were fixed onto nutrient agar plate. 1 mg of each test compound was dissolved in 100 μL of DMSO to prepare stock solution and from the stock solution different concentrations 10, 20, 25, 50, and 100 μg/μL of each test compound were prepared. These compounds of different concentration were poured onto disk plate. Chloramphenicol (30 μg/disk) was used as standard drug (positive control). DMSO poured disk was used as negative control. The susceptibility of the bacteria to the test compounds was determined by the formation of an inhibitory zone after 18 h of incubation at 36 °C. (Table 1) reports the inhibition zones (mm) of each compound and the controls. The minimum inhibitory concentration (MIC) was evaluated by the macro dilution test using standard inoculums of 10⁻⁵ CFL mL⁻¹. Serial dilutions of the test compounds, previously dissolved in dimethyl sulfoxide (DMSO) were prepared to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg/mL and to each tube was added 100μL of a 24 h old inoculum. The MIC, defined as the lowest concentration of the test compound, which inhibits the visible growth after 18 h, was determined visually after incubation for 18 h, at 37 °C, and the results are presented in Table 2. Tests were performed using DMSO and chloramphenicol as negative and positive controls.

Table 1 Physicochemical data of the synthesized compounds (1–8).

Compound No.		Molecular formula	Crystallization	% Yield	Reaction Time (micro wave)
1		C15H14O2S	CHCl3	88.2	30 s
2		C16H16O2S	CH2Cl2	88.5	45 s
3		C17H18O3S	CH2Cl2	82.0	43 s
4		C18H20O4S	CHCl3	88.0	50 s
5		C17H19NOS	CHCl3	86.00	35 s
6		C23H21NOS	CH2Cl2	90.0	42 s
7		C15H13NO3S	CHCl3	89.2	38 s
8		C20H20N2OS	CHCl3	89.6	45 s

3.1 Synthesis and characterization

Chalcone derivatives were synthesized by the reaction of 3-acetyl-2,5-dimethylthiophene and the corresponding active aldehyde (Scheme 1 and Table 1). The purified products were characterized by EI-MS m/z (rel. int.%): FT-IR, ¹H NMR, ¹³C NMR and elemental analysis. The IR spectra of compounds (1–8) show the characteristic band. The ν(C⚌O) peak of Act-thiophen observed at 1668 cm⁻¹ shifts to a lower frequency of 1628–1653 cm⁻¹ of chacones. This is due to the conjugation of the π-electrons on the benzene moiety with those on the ethylene moiety in the enon linkage. ¹H NMR spectra, is a proven diagnostic tool for the positional elucidation of the proton. Assignments of the signals are based on chemical shift and intensity pattern. The ¹H NMR spectra of all the compounds (1–8) measured at room temperature shows two doublets at 7.68–8.16 ppm (J = 15.6) for the CH⚌C and 6.89–7.45 ppm (J = 15.6 Hz) for the CO⚌CH indicating that the ethylene moiety in the enon linkage is in the trans-conformation which conform the formation of chalcones.

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Scheme 1 Synthetic route of the chalcones (1–8).

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¹³C NMR (CDCl₃) spectra of chalcones (1–8) were recorded in CDCl₃ and spectral signals are in good agreement with the probable structures details of ¹³C NMR spectra of all compounds and those data are given in the experimental section.

Characteristic peaks were observed in the EI mass spectra of compounds (1–8), which followed the molecular ion peak and similar fragmentation pattern.

3.2 Antimicrobial activity: disc-diffusion and micro dilution assay

The compounds (1–8) were tested for their antibacterial activities by disc-diffusion method using nutrient broth medium [contained (g/L): beef extract 3 g; peptone 5 g; pH 7.0] (Asiri and Khan 2010). The Gram-positive bacteria and Gram-negative bacteria utilized in this study consisted of S. aureus, S. pyogenes, S. typhimurium and E. coli. In the disc-diffusion method, sterile paper disks (0.5 mm) impregnated with compound dissolved in dimethylsulfoxide (DMSO) at concentration of 100 μg/mL were used. Then, the paper disks impregnated with the solution of the compound tested were placed on the surface of the media inoculated with the microorganism. The plates were incubated at 35 °C for 24 h. The growth inhibition zones after incubation are shown in Table 2. The chalcone derivative was further checked by the MIC method. The results are presented in Table 3.