Antimicrobial drugs occupy a unique niche in the history of medicine. The increased incidence of severe opportunistic bacterial infections in immunological deficient patients together with the development of resistance among pathogenic Gram-positive and Gram-negative bacteria have revealed great need to search for new compounds that are effective against antibiotic-resistant bacteria. Literature highlights the potentials of β-phenyl acrolein as an antimicrobial [1], anticancer [2] and flavoring agent for chewing gums [3]. Many investigators have observed the importance of azomethines for their antibacterial [4,5] antifungal [6], anti-proliferative [7,8] and antipyretic properties. It is evident that azomethines with aryl substituents are more stable and readily synthesized, whereas those containing alkyl substituents are relatively unstable. Azomethines of aliphatic aldehydes are usually unstable and readily polymerizable, while those with aromatic aldehydes having effective conjugation are more stable [9].

In the present research work, a new series of azomethine derivatives of β-phenyl acrolein was synthesized (SB-1 to SB-6) and screened for antibacterial activity against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Klebsiella pneumonia and Pseudomonas aeruginosa) bacteria in order to generate potent and safer antibacterial agents.

Materials

All the solvents and chemicals used were of analytical grade and obtained from Sigma-Aldrich and Merck Pvt Ltd, India and were used without further purification.

General procedure for synthesis of azomethine derivatives of β-phenyl acrolein

Equimolar concentration of β-phenyl acrolein (0.01 M) and substituted aromatic amines (0.01 M) were dissolved in 50 mL of anhydrous ethanol separately. Solution of substituted aromatic amine was then added drop-wise into β-phenyl acrolein solution in a conical flask. The mixture was made up to 150 mL with 95 % anhydrous ethanol, and 2 to 3 drops of triethylamine (basic catalyst) was added [10].The mixture was then stirred using magnetic stirrer at 60 to 70 °C for 6 h over a water bath. The reaction was monitored by TLC. The sample mixture was evaporated under pressure at 65 °C using rotatory evaporator [11,12]. The solid obtained on concentration of filtrate was recrystallized from aqueous ethanol to yield the pure compounds SB-1 to SB-6 (physical data are given in ). The melting points of the compounds were determined on a Thoshniwal electric melting point apparatus and the values were uncorrected. The reaction was mentioned by TLC on Silica gel-GF 254 (Merck) coated plates. Spots of TLC were identified in iodine chamber.

Determination of minimum inhibitory concentration (MIC)

Test tube dilution method was used to determine minimum inhibitory concentration. One mL of sterilized media (Nutrient agar) was poured into sterile test tubes. One mL of 2000 µg/mL test solution was transferred in one tube and serially diluted to give concentrations of 1000, 500, 250 and 125 µg/mL. To all the test tubes, 0.1 mL of suspension of bacteria in saline was added and the tubes were incubated at 37 °C for 24 h. The growth in the tubes was observed visually for turbidity. MIC was determined with the lowest concentration of the sample that retarded the development of turbidity [13].

Disc-diffusion method

The disc diffusion method [14,15] was used to determine the antibacterial activity of azomethine using a 6-mm disc were prepared from Whatman’s filter paper no.1. Azomethine solutions of varying concentrations ranging from 125, 250 and 500 µg/mL were prepared. Nutrient agar was prepared, sterilized and used as the growth medium for the culture of microorganisms; 20 mL of the sterilized medium was poured into each sterilized petri dish, covered and allowed to solidify. Thereafter, the 16-h old broth cultures of the specified microorganisms were used for disc diffusion studies [13].The sample, control and standard treated discs were air-dried at room temperature, to remove any residual solvent which might interfere with the determination, sterilized and inoculated. These plates were initially placed at low temperature for 1 h to allow the maximum diffusion of compounds from the test disc into the agar plate and later incubated at 37 °C for 24 h in case bacteria [16],  after which the zone of inhibition will be easily observed.

Statistical analysis

Each experimental value is expressed as the mean ± standard error mean (n = 3). Statistical analysis was performed using GraphPad Prism 5.0 and data analyzed using one-way analysis of variance (ANOVA) for comparison between groups followed by Dunnett's multiple comparison test at a significant level of p < 0.05.

Chemistry

UV-Visible, IR, NMR and mass spectral data supported the structures of all newly synthesized compounds. The spectral data for the newly synthesized compounds are as follows.

N-(3-phenylallylidene)benzamine (SB-1): Yield: 70 %; mp: 98-100 ^ºC; Anal. Calcd. for C₁₅H₁₃N: C, 86.92; H, 6.32; N, 6.76 %. Found C, 86.89; H, 6.28; N, 6.66 %; IR (KBr, cm^-1): 3085 (=C-H stretching of aromatic ring), 3039 (=C-H stretching of alkenyl group), 1600 (-C=N stretching azomethine group), 1540-1600 (C=C- stretching of aromatic ring), 1320 (C-N, stretching of azomethine group); ¹H-NMR (500.1 MHz, CDCl₃-d, δ/ppm): 5.76 (1H, t,J = 9.5 Hz, H-2'), 6.52 (1H, d,J = 12 Hz, H-3'), 7.23 - 7.60 (10H, m, phenyl), 8.24 (1H, d,J = 7.8 Hz, H-1'); ¹³C-NMR (100 MHz, CDCl₃, δ / ppm): 120.2 (C_2'), 123.4 (C₃& C₅), 126.3 (C_5'& C_9'), 127.5 (C₁), 128.4 (C_7'), 129.2 (C_6'& C_8'), 131.1 (C₂& C₆), 135.8 (C_4'), 139.2 (C_3'), 150.4 (C₄), 164.3 (C_1'); MS (m/z, (relative abundance, %)): 207 (M⁺_, 18.9), 107, 102, 77, 53, 51, 130 (BP, 100); UV-Vis (MeOH) (l_max/nm): 353.

4-(3-Phenylallylideneamino)benzaldehyde (SB-2): Yield: 79 %; mp: 130-134 ^ºC; Anal. Calcd. for C₁₆H₁₃NO: C, 81.68; H, 5.57; N, 5.95 %. Found C, 81.56; H, 5.48; N, 5.87 %; IR (KBr, cm^-1): 3050 (=C-H stretching of aromatic ring), 3038 (=C-H stretching of alkenyl group), 2720, 2820 (C-H  stretching of aldehyde group), 1725 (C=O Stretching of aldehyde group), 1658 (-C=N stretching azomethine group), 1540-1620 (C=C- stretching of aromatic ring), 1320 (C-N, stretching of azomethine group); ¹H-NMR (500.1 MHz, CDCl₃-d, δ/ppm): 5.81 (1H, t, J = 9.5 Hz, H-2'), 6.63 (1H, d, J = 12 Hz, H-3'), 7.5 - 7.9 (10H, m, phenyl), 8.32 (1H, d, J = 7.8 Hz, H-1'), 9.94 (s, 1H, aldehyde); ¹³C-NMR (100 MHz, CDCl₃, δ / ppm): 120.5 (C_2'), 123.8 (C₃& C₅), 126.2 (C_5'& C_9'), 128.3 (C_7'), 129.4 (C_6'& C_8'), 131.8 (C₂& C₆), 135.4 (C_4'), 136.3 (C₁), 139.6 (C_3'), 155.2 (C₄), 164.6 (C_1'), 192.4 (-CHO); MS (m/z, (relative abundance, %)):  235 (M⁺_, 20.4), 158, 105, 102, 77, 53, 51 (BP, 100); UV-Vis (MeOH) (l_max/nm): 307.

4-(3-Phenylallylideneamino)phenol (SB-3): Yield: 80 %; mp: 120-130 ^ºC; Anal. Calcd. for C₁₅H₁₃NO: C, 80.69; H, 5.87; N, 6.27 %. Found C, 80.56; H, 5.78; N, 5.82 %; IR (KBr, cm^-1): 3640 (Broad, O-H Str), 3055 (=C-H stretching of aromatic ring), 3038 (=C-H stretching of alkenyl group), 1668 (-C=N stretching azomethine group), 1540-1600 (C=C- stretching of aromatic ring), 1320 (C-N, stretching of azomethine group); ¹H-NMR (500.1 MHz, CDCl₃-d, δ/ppm): 5.4 (1H, s, -OH, D₂O exchangeable), 5.72 (1H, t, J = 9.5 Hz, H-2'), 6.59 (1H, d,J = 12 Hz, H-3'), 7.4 - 7.8 (10H, m, phenyl), 8.29 (1H, d,J = 7.8 Hz, H-1'); ¹³C NMR (100 MHz, CDCl₃, δ / ppm): 118.4 (C₂& C₆), 120.3 (C_2'), 123.9 (C₃& C₅), 126.5 (C_5'& C_9'), 128.4 (C_7'), 129.2 (C_6'& C_8'), 135.8 (C_4'), 139.2 (C_3'), 141.6 (C₄), 157.2 (C₁), 164.3 (C_1'); MS (m/z, (relative abundance, %)): 223 (M⁺, 18.5), 146, 130, 102, 93, 77, 53, 51 (BP, 100); UV-Vis (MeOH) (l_max/nm): 302.

4-Methoxy-N-(3-phenylallylidene)benzamine (SB-4): Yield: 87 %; mp: 140-143 ^ºC; Anal. Calcd. for C₁₆H₁₅NO: C, 80.98; H, 6.37; N, 5.90 %. Found C, 80.96; H, 6.33; N, 4.89 %; 80.59; IR (KBr, cm^-1): 3052 (=C-H stretching of aromatic ring), 3037 (=C-H stretching of alkenyl group), 2934, 2876 (C-H, Str), 1658 (-C=N stretching azomethine group), 1540-1620 (C=C- stretching of aromatic ring), 1320 (C-N, stretching of azomethine group), 1255 (C-O-C, Str); ¹H-NMR (500.1 MHz, CDCl₃-d, δ/ppm): 3.73 (3H, s, CH₃), 5.74 (1H, t, J = 9.5 Hz, H-2'), 6.56 (1H, d, J = 12 Hz, H-3'), 7.3 - 7.75 (10H, m, phenyl), 8.36 (1H, d, J = 7.8 Hz, H-1'); ¹³C-NMR (100 MHz, CDCl₃, δ / ppm): 56.3 (-CH₃), 116.2 (C₃& C₅), 120.4 (C_2'), 123.7 (C₃& C₅), 126.2 (C_5'& C_9'), 126.9 (C_7'), 128.1 (C_6'& C_8'), 135.8 (C_4'), 139.3 (C_3'), 141.2 (C₄), 160.4 (C₁), 164.3; MS (m/z, (relative abundance, %)): 237 (M⁺, 19.0), 160, 130, 107, 102, 77, 53, 51 (BP, 100); UV-Vis (MeOH) (l_max/nm): 306.

4-Methoxy-2-(3-phenylallylideneamino)benzoic acid (SB-5): Yield: 80 %; mp: 150-154 ^ºC; Anal. Calcd. for C₁₇H₁₅NO₃: C, 72.58; H, 5.37; N, 4.98 %. Found C, 72.52; H, 5.39; N, 4.92 %; IR (KBr, cm^-1): 3400-2500 (OH Str of COOH), 3057 (=C-H stretching of aromatic ring), 3038 (=C-H stretching of alkenyl group), 2500-2900 (O-H of COOH, Str),  1720 (C=O of COOH Str), 1658 (-C=N stretching azomethine group), 1540-1620 (C=C- stretching of aromatic ring), 1280 (C-O stretching of COOH group), 1323 (C-N, stretching of azomethine group), 1250 (C-O-C stretching of ether group); ¹H-NMR (500.1 MHz, CDCl₃-d, δ/ppm): 3.74 (3H, s, OCH₃), 5.76 (1H, t,J = 9.5 Hz, H-2'), 6.8 (1H, d,J = 12 Hz, H-3'), 7.64 - 7.92 (10H, m, phenyl), 8.38 (1H, d,J = 7.8 Hz, H-1'), 11.00 (1H, s, COOH); ¹³C-NMR (100 MHz, CDCl₃, δ / ppm): 56.8 (-CH₃), 108.4 (C₅), 109.1 (C₃), 112.6 (C₁), 119.8 (C_2'), 126.1 (C_5'& C_9'), 127.2 (C_7'), 128.6 (C_6'& C_8'), 132.4 (C₂), 135.7 (C_4'), 138.3 (C_3'), 150.5 (C₄), 163.2 (C_1'), 167.8 (C₆) , 169.6 (-COOH); MS (m/z, (relative abundance, %)): 281 (M⁺, 20.7), 204, 151, 130, 102, 77, 53, 51 (BP, 100); UV-Vis (MeOH) (l_max/nm): 338.

4-(3-Phenylallylideneamino)benzoic acid (SB-6): Yield: 86 %; mp: 130-140 ^ºC; Anal. Calcd. for C₁₆H₁₃NO₂: C, 76.42; H, 5.29 N, 5.52 %. Found C, 72.51; H, 5.32N, 4.88 %; IR (KBr cm^-1): 3052 (=C-H stretching of aromatic ring), 3036 (=C-H stretching of alkenyl group), 2926, 2872 (C-H stretching of methyl group), 1668 (-C=N stretching azomethine group), 1540-1600 (C=C- stretching of aromatic ring), 1320 (C-N, stretching of azomethine group); ¹H-NMR (500.1 MHz, CDCl₃-d,δ/ppm): 5.85 (1H, t, J = 9.5 Hz, H-2'), 6.76 (1H, d, J = 12 Hz, H-3'), 7.64 - 7.92 (10H, m,phenyl), 8.41 (1H, d, J = 7.8 Hz, H-1'), 11.00 (1H, s, COOH); ¹³C-NMR (100 MHz, CDCl3, δ / ppm): 120.4 (C_2'), 123.5 (C₃& C₅), 126.7 (C_5'& C_9'), 127.4 (C_7'), 128.4 (C_6'& C_8'), 129.2 (C₁), 131.2 (C₂& C₆), 135.9 (C_4'), 139.4 (C_3'), 155.4 (C₄), 164.8 (C_1'), 169.6 (-COOH); MS (m/z, (relative abundance, %)): 251 (M⁺, 32.6), 174, 130, 121, 102, 77, 53, 51 (BP, 100); UV-Vis (MeOH) (l_max/nm): 307.

Antibacterial activity

The newly synthesized β-phenyl acrolein derivatives were tested for their antibacterial potential against B. subtilis and S. aureus, K. pneumoniae, and P. aeruginosa, using gentamycin as standard. The results are given in and .

Equimolar concentration of β-phenyl acrolein and substituted aromatic amines in the presence of basic catalyst, triethylamine resulted in formation of azomethine derivatives of β-phenyl acrolein moiety shown in .

The λ_max for the newly synthesized azomethines was found to be in range from 300-440 nm. The IR stretch at around 1650-1680 cm^-1 showed the C=N bond formation. The formation of azomethines was identified by the presence of triplet between 5.7 to 5.8 ppm, in proton NMR spectra. All other aliphatic and aromatic protons were observed within the expected regions. The novel compounds were further confirmed by their characteristic mass fragment spectra. The mass fragment pattern of compound SB-4 given in , displayed parent ion peak at 237, base peak at 51, and different fragment peaks at 160, 130, 107, 102, 77, and 53.

Similarly, all the new compounds were characterized. This part confirmed the synthesis of a series of six new azomethines derivatives of β-phenyl acrolein.

The antibacterial potential of newly synthesized molecules was estimated by tube dilution and disc diffusion method; using Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram negative bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa). Tube dilution method depends upon the inhibition of growth of a microbial culture in a uniform solution of antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic [17]. In this method minimum inhibitory concentration of the test compounds was determined. Gentamycin was used as a standard drug [18].

As per the minimum inhibitory concentration study results given in and , among all synthesized compounds, SB-5 and SB-6 displayed highest MIC value of 62.5 µg/ml. The zone of inhibition experiment, revealed that at MIC of 250 µg/ml, all compounds SB-1 to SB-6 showed significant antibacterial activity (p < 0.05). SB-5 and SB-6 were active against S. aureus, P. aeruginosa and K. pneumoniae; SB-3 was active against B. subtilis and S. aureus, SB-4 was active against P. aeruginosa and S. aureus. SB-1 and SB-2 were active against S. aureus. The antimicrobial results proved that all synthesized azomethines of β-phenyl acrolein moiety possess significant antibiotic potential.

β-Phenyl acrolein derivatives have been successfully synthesized and appear to be a novel and important class of antibacterial agents against Gram-positive and Gram-negative bacteria including S. aureus, P. aeruginosa, and K. pneumonia. The synthetic route and antibacterial potential of the compounds may be useful in guiding future efforts to synthesize new compounds with improved antibacterial activity.