Synthesis, Characterization and AntibacterialActivity of Azomethine Derivatives Derived from 2-Formylphenoxyacetic Acid

Abstract

A series of eight new azomethine derivatives were synthesized by reacting 2-formylphenoxyacetic acid with aromatic amines. The chemical structures of these compounds were confirmed by means of ¹H-NMR, ¹³C-NMR, MS and elemental analysis. The compounds were assayed by the disc diffusion method for antibacterial against Staphylococcus aureus and Escherichia coli. Among the compounds tested, 2a, 2b, 2e, 2g and 2h exhibited good antibacterial activity, almost equal to that of Ciprofloxacin used as standard.

Introduction

It is evident that in azomethine derivatives the C=N linkage is an essential structural requirement for biological activity. These compounds are readily hydrolyzed under acidic conditions leading to active aldehydes which can act as alkylating agents [1]. Besides, several azomethines have been reported to possess remarkable antibacterial [2,3,4,5,6], antifungal [7,8,9], anticancer [10,11,12,13], and diuretic activities [14]. The action of aryloxyaliphatic acids on the permeability of blood vessels [15], and the antimicrobial activity against human pathogens [16] of o-substituted phenoxyacetic acid have been reported. The azomethine derivatives and their complexes derived from o-formylphenoxyacetic acid with aminothiazoles, a number of aminobenzene derivatives, some heterocyclic and aliphatic amines have revealed biological significance such as antimetabolites of pyridoxal phosphate [17], bacteriostatic activity [18], chorismate synthase inhibition [19] and antitumor activity [20]. Many attempts have been made to synthesize, characterize and to study structure-activity relationship (SAR) of Schiff bases [21,22,23,24]. In view of our ongoing preliminary investigation of the remarkable binding of azomethines derived from o‑formylphenoxyacetic acid with proteins, we decided to synthesize some new azomethines. This study was aimed at exploring the potential antibacterial activity resulting from the combination of pharmacophores in one structure. The results of this study may be useful to researchers attempting to gain more insight into the antibacterial activity of azomethine derivatives.

Results and Discussion

The azomethine derivatives 2a-h were synthesized in good yields (65 to 90%) by condensation of o-formylphenoxyacetic acid with various substituted primary aromatic amines in hot ethanol, benzene or dichloromethane (DCM) using molecular sieves as the dehydrating agent (Scheme 1).

Scheme 1. Synthesis of Azomethine derivatives

Scheme 1. Synthesis of Azomethine derivatives

It is known that condensation of amines with aldehydes is favoured by a polar medium [20]. The addition of ytterbium triflate was also found to be beneficial, as in some experiments the reaction was found not to go to completion, even after extended heating times. This may be due to the acid-base reaction occurring between the amine and phenoxyacetic acid groups in the starting materials. Addition of the Lewis acid catalyst resulted in greater conversion to product (70%) in the case of compound 2h.

The structures of the title compounds were determined by IR, ¹H- and ¹³C-NMR, and FAB mass spectrometry and the spectroscopic properties and analytical data were in accord with the proposed structures. Compounds 2a,b,d-h showed in the IR spectra an absorption band at 1630-1680 cm^-1, typical of the stretching vibrations of the C=N double bond, while the value for the hydrazone 2c was lower, at 1600 cm^-1, as expected for the electron donating amino substituent on the imine nitrogen. Two more absorption bands in the 3580-3425 cm-1 (br) and 1711-1682 range were also observed, due to -OH and C=O groups of the carboxylic acid substituent in each compound.

The ¹H-NMR spectra of 2a-h contained multiplet signals due to aromatic protons in the δ 6.39-8.31 ppm regions and singlets at δ 8.29 - 9.37 ppm from the C-H protons of the CH=N groups. In the DEPT spectra of 2a-h, the peak at δ 189.8 ppm, due to the -CHO group, disappeared and was observed shifted to δ 154.2, 157.8, 154.8, 154.9, 158.5, 160.76 164.9 and 160.9, respectively, indicating the formation of CH=N groups. The signals at δ 23.9, 20.7, and 20.6 ppm showed the presence of CH₃ groups in compounds 2a, 2b and 2d respectively. Similarly, one signal each appeared at δ 65.1, 65.5, 64.9, 65.2, 65.1 65.0 and 66.4 ppm in compounds 2a-g, while one more signal at δ 41.9, in addition to a signal at δ 65.0 in compound 2f, were due to CH₂ groups. In the ¹H-NMR spectrum of compound 2h four methylene signals were observed at δ 2.15, 2.91, 4.20 and 5.04, while the singlet of HC=N- group was observed at δ 8.37. The phenyl and imidazole protons gave rise to overlapping multiplets in the range δ 6.94-7.78. The compound exhibited three quaternary carbon signals at δ 166.0, 157.7, 124.46, four methylene signals at δ 68.9, 45.5, 37.9, 29.9, and eight CH signals in the DEPT spectrum.

Biological activity

All the synthesized compounds were screened for antibacterial activity against Staphylococcus aureus (AMJ-2005) and Escherichia coli (AMJ-2006) by the disc diffusion method. Ciprofloxacin was used as a standard. The bacterial inhibition zone values are summarized in Table 1. The MICs (minimum inhibitory concentrations) and MBCs (minimum bacterial concentrations) are presented in Table 2.

Table 1. Bacterial Inhibition Zone values.

Compound	Staphylococcus aureus (AMJ-2005)	Escherichia coli (AMJ-2006)
2a	25a	27
2b	23	22
2c	18	19
2d	13	12
2e	24	26
2f	20	21
2g	25	27
2h	26	30
Standard	25	30

^a Diameter of zone of inhibition in mm

Table 1. Bacterial Inhibition Zone values.

Compound	Staphylococcus aureus (AMJ-2005)	Escherichia coli (AMJ-2006)
2a	25a	27
2b	23	22
2c	18	19
2d	13	12
2e	24	26
2f	20	21
2g	25	27
2h	26	30
Standard	25	30

^a Diameter of zone of inhibition in mm

Table 2. MIC and MBC results of azomethine derivatives.

Compound	Staphylococcus aureus (AMJ-2005) MIC  MBC		Escherichia coli (AMJ-2006) MIC MBC
2a	6.25	12.5	6.25	12.5
2b	6.25	6.25	6.25	12.5
2c	12.5	50	12.5	50
2d	12.5	100	12.5	100
2e	6.25	25	6.5	6.5
2f	12.5	25	25	50
2g	6.25	6.25	6.25	6.25
2h	6.25	6.25	6.25	6.25
Standard	6.25	12.5	12.5	25

MIC (ug/mL) = minimum inhibitory concentration that is lowest concentration to completely inhibit bacterial growthMBC (ug/mL) = minimum bacterial concentration that is lowest concentration to completely kill bacteria.

Table 2. MIC and MBC results of azomethine derivatives.

Compound	Staphylococcus aureus (AMJ-2005) MIC  MBC		Escherichia coli (AMJ-2006) MIC MBC
2a	6.25	12.5	6.25	12.5
2b	6.25	6.25	6.25	12.5
2c	12.5	50	12.5	50
2d	12.5	100	12.5	100
2e	6.25	25	6.5	6.5
2f	12.5	25	25	50
2g	6.25	6.25	6.25	6.25
2h	6.25	6.25	6.25	6.25
Standard	6.25	12.5	12.5	25

MIC (ug/mL) = minimum inhibitory concentration that is lowest concentration to completely inhibit bacterial growthMBC (ug/mL) = minimum bacterial concentration that is lowest concentration to completely kill bacteria.

The screening data revealed that most of the tested compounds showed good bacterial inhibition. The compounds 2a, 2b, 2e, 2g and 2h exhibited good antibacterial activity, almost equal to that of the standard. The MBC of compound 2b, 2g and 2h were found to be the same as the MIC, but in most of the compounds it was two to four fold higher than the corresponding MIC result. The synthesis and bioassay of similar other azomethine derivatives and their complexes are under study.

Experimental

General

2-Formylphenoxyacetic acid, picoline, N-phenylhydrazine, p-toluidine, o-bromobenzylamine, 2,3-dichloroaniline, p-aminoacetanilide, 3-imidazol-1-yl-propylamine were obtained commercially from Lancaster Research Chemicals. 2-Amino-4-(2'-chlorophenyl)thiazole was prepared from 1-bromo-2'-chloroacetophenone and thiourea by standard methods. Solvents used were of analytical grade. ¹H- and ¹³C-NMR spectra were recorded on a Bruker DPX-400 instrument at 400 and 100 MHz, respectively. Chemical shifts are reported in ppm reference to the residual solvent signal. Mass spectra were recorded on a JEOL SX-102 instrument and IR spectra were recorded on a Perkin Elmer Paragon 1000 spectrometer. Melting points were recorded on a Stuart Scientific-SMP3 apparatus and are uncorrected.

2-(4-Acetamido phenyliminomethyl)phenoxyacetic acid (2a).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to 4-aminoacetanilide (0.5 mmol, 0.075 g) in dioxane (10 mL) in addition to molecular sieves and the mixture heated under reflux for 3 h under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, the yield of 2a was found to be 80%; m.p. 216 °C; HRMS (FAB, MH+) calcd. C₁₇H₁₆N₂O₄ 313.1188, found 313.1193; IR (ν_max, KBr, cm^-1) 3390, 1702, 1677, 1550; Anal. Found: C, 65.13; H, 5.17; N, 8.90. Calcd. for C₁₇H₁₆N₂O₄: C, 65.15; H, 5.15; N, 8.94; ¹H-NMR (DMSO-d₆) 2.60 (s, CH₃), 4.85 (s, CH₂), 7.09 (2H, t, J, 7.6 Hz), 7.20 (2H, d, J 8.0 Hz), 7.47 (1H, dd, J 7.6, 14.8 Hz), 7.63, (2H, d, J 8.4 Hz), 8.02 (1H, d, J 7.6 Hz), 8.93 (1H, s, CH=N); ¹³C-NMR (DMSO-d₆) 23.97 (CH₃), 65.11 (CH₂), 113.13 (CH), 119.62 (2CH), 121.27 (2CH), 121.32 (CH), 124.42 (C), 126.74 (CH), 132.69 (CH), 137.62 (C), 146.75 (C), 154.22 (C=N), 157.76 (C), 168.16 (C), 170.05 (COOH).

2-(4-Methylpyridin-2-yliminomethyl)phenoxyacetic acid (2b).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to 2-amino-4-methylpicoline (0.5 mmol, 0.054 g) in benzene (10 mL) containing molecular sieves and the mixture heated under reflux for 4 h under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, the yield of 2b was found to be 75%; m.p. 118 °C; HRMS (FAB, MH⁺) calcd. C₁₅H₁₄N₂O₃ 271.1083, found 271.1088; IR (ν_max, KBr, cm^‑1) 3390, 1706, 1683, 1598; Anal. Found: C, 66.44; H, 5.17; N, 10.35. Calcd. for C₁₅H₁₄N₂O₃: C, 66.39; H, 5.20; N, 10.32; ¹H-NMR (DMSO-d₆) 2.17 (s, 3H), 4.82 (s, 2H), 6.36 (1H, s), 6.39 (1H, d, J 1.2 Hz), 7.47 (1H, dd, J 7.6, 14.8 Hz), 7.63, (2H, d, J 8.4 Hz), 8.02 (1H, d, J 7.6 Hz), 8.93 (1H, s, CH=N); ¹³C-NMR (DMSO-d₆) 20.71 (Me), 65.52 (CH₂), 108.92 (CH), 113.38 (CH), 113.82 (CH), 121.03 (CH), 124.41 (C), 127.43 (CH), 136.18 (CH), 144.29 (CH), 148.45 (C), 158.53 (C), 157.76 (C=N), 160.47 (C), 170.48 (COOH).

2-(2-phenyl hydrazonomethyl)phenoxyacetic acid (2c).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to N-phenylhydrazine (0.5 mmol, 0.049 g) in EtOH (10 mL) in addition to molecular sieves and stirred at room temp. for 15 min. under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, the yield of 2c was found to be 90%; m.p. 102 °C; HRMS (FAB, M⁺) calcd. C₁₅H₁₄N₂O₃ 270.1004, found 270.1002; IR (ν_max, KBr, cm^-1) 3440, 1712, 1600, 1566, 1516; Anal. Found: C, 66.60; H, 5.19; N, 10.33. Calcd. for C₁₅H₁₄N₂O₃: C, 66.64; H, 5.22; N, 10.36; ¹H-NMR (DMSO-d₆) 4.78 (s, CH2), 6.74 (1H ,m), 6.95 (1H, d, J 8.0 H_Z), 7.01 (1H, t, J 7.6 Hz), 7.09, (2H, dd, J 1.6, 7.6 Hz), 7.18 - 7.27 (4H, m), 8.29 (1H, s, CH=N); ¹³C-NMR, (DMSO-d₆) 64.87 (CH₂), 111.87 (CH), 111.88 (CH), 112.39 (CH), 118.56 (CH), 121.18 (CH), 124.24 (CH), 124.61 (C), 128.91 (CH), 129.07 (CH), 129.09 (CH), 131.86 (CH), 145.37 (C), 154.87 (CH=N), 170.16 (COOH).

2-(4-Methyl phenyliminomethyl)phenoxyacetic acid (2d).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to p-toluidine (0.5 mmol, 0.054 g) in EtOH (10 mL) in addition to molecular sieves and stirred at room temp. for 3 h under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, 2d was obtained in 65% yield; m.p. 148 °C; HRMS (FAB, M⁺) calcd. C₁₆H₁₅NO₃ 270.1130, found 270.1133; Anal. Found: C, 71.00; H, 5.62; N, 5.16. Calcd. for C₁₆H₁₅NO₃: C, 71.08; H, 5.59; N, 5.18; IR (ν_max, KBr, cm^-1) 3406, 1710, 1682, 1598; ¹H-NMR (DMSO-d₆) 2.32 (s, 3H), 4.84 (s, 2H), 7.06 – 7.18 (4H, m), 7.22 (2H, d, J 7.6 Hz), 7.49, (1H, t, J 7.6 Hz), 8.03 (1H, d, J 7.3 Hz), 8.94 (1H, s, CH=N); ¹³C-NMR (DMSO-d₆) 20.55 (Me-H), 65.16 (CH₂), 113.14 (CH), 120.75 (2CH), 121.25 (CH), 124.36 (C), 126.70 (CH), 129.20 (CH), 129.73 (CH), 132.75 (CH), 135.18 (C), 146.01 (C), 154.88 (CH=N), 157.82 (C), 170.05 (COOH).

2-(2,3-Dichloro phenyliminomethyl)phenoxyacetic Acid (2e).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to 2,3-dichloroaniline (0.5 mmol, 0.06 ml) in benzene (10 mL) in addition to molecular sieves and refluxed at 85 °C for 5 h under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, the yield of the viscous compound was found to be 84%; HRMS (FAB, M⁺) calcd. C₁₅H₁₁NO₃Cl₂ 324.0199, found 324.0189; Anal. Found: C, 55.53; H, 3.40; N, 4.29. Calcd. for C₁₅H₁₁NO₃Cl₂: C, 55.55; H, 3.42; N, 4.32; ¹H-NMR (DMSO-d₆) 4.83 (s, CH₂), 6.74 (1H, m), 7.01 (1H, t, J 8.0 Hz), 7.12, (2H, dd, J 1.6, 7.6 Hz), 7.39 - 7.47 (3H, m), 8.97 (1H, s, CH=N); ¹³C-NMR (DMSO-d₆) 65.11 (CH₂), 113.56 (CH), 116.69 (CH), 121.22 (CH), 123.65 (C), 127.26 (CH), 127.50 (CH), 131.52 (C), 133.84 (CH), 136.21 (CH), 146.71 (C), 146.73 (C), 158.48 (CH=N), 160.25 (C), 170.02 (COOH).

2-(2-Bromobenzyl iminomethyl)phenoxyacetic acid (2f).

2-Formylphenoxy acetic acid (0.5 mmol, 0.09 g) was added to o-bromobenzylamine (0.5 mmol, 0.112 g) in EtOH (10 mL) in addition to molecular sieves and refluxed at 85 ^oC for about 4 h under N₂ (g). After filtration, evaporation and recrystallisation from EtOH, the yield of the title compound 2f was 70%; m.p. 166 °C; HRMS (FAB, MH⁺) calcd. C₁₆H₁₄NO₃Br, 348.0235 found; 348.0237; IR (ν_max, KBr, cm^-1) 3400, 1708, 1650, 1573; Anal. Found: C, 55.12; H, 4.09; N, 4.05. Calcd. for C₁₆H₁₄NO₃Br: C, 55.16; H, 4.05; N, 4.02; ¹H-NMR (DMSO-d₆) 5.05 (s, CH₂), 5.19 (s, CH₂), 7.29 - 7.83 (7H, m), 8.31 (1H, dd, J 1.2, 8.0 Hz), 9.37 (1H, s, CH=N); ¹³C-NMR (DMSO-d₆) 41.89 (CH₂), 65.05 (CH₂), 113.80 (CH), 121.27 (CH), 123.27 (C), 124.46 (C), 127.51 (CH), 127.99 (CH), 130.39 (CH), 130.44 (CH), 132.68 (CH), 133.25 (CH), 136.22 (C), 160.76 (CH=N), 160.81 (CH), 169.76 (COOH).

(2-{[4-(2′-chlorophenyl)-thiazole-2-yl imino]methyl}phenoxy)acetic acid (2g).

2-Formylphenoxyacetic acid (0.001 mol, 0.18 g) was added to a solution of 2-amino-4-(2′-chlorophenyl)thiazole (0.001 mol, 0.211 g) in absolute alcohol (20 mL), in addition to molecular sieves and Na₂SO₄ (anhydr.). The mixture heated under reflux for one week under N₂. The product was purified by crystallisation from EtOH and the yield of 2g was 40%; m.p. 191-193 °C. HRMS (FAB, MH⁺) calcd. for C₁₈H₁₃O₃N₂SCl 372.604 found 372.58. IR (ν_max, KBr cm^-1): 3030, 1735, 1680, 1550, 1240; Anal. Found: C, 58.0; H, 3.86; N, 7.31. Calcd. for C₁₈H₁₃O₃N₂SCl: C, 57.99; H, 3.51; N, 7.51. ¹H-NMR, (400 MHz, MeOH-d₄): δ 4.98 (2H, s, CH₂), 6.77 (1H, d, J 8.0 Hz, ArH), 6.90-7.49 (8H, m, ArH and thiazole-H), 7.99 (1H, s, CH=N); ¹³C-NMR (100 MHz, MeOH-d₄): δ 66.4 (CH₂), 164.9 (CH=N), 169.4 (2′-C), 113.3 (5′-C), 141.0 (4′-C), 122.2, 122.5, 126.2, 128.4, 129.8, 130.2, 130.8, 130.9, 135.4, 141.0, 156.3, 157.2, 172.0 (CO₂H).

2-{2-[(3-imidazol-1-yl-propylimino)methyl]phenoxy}acetic acid (2h).

2-Formylphenoxyacetic acid (0.002 mol, 0.360 g) was added to 3-imidazol-1-yl-propylamine (0.002 mol, 0.250 g) in dichloromethane (10.0 ml) in addition to 10% mmol of Yb(OTf)₃ as Lewis catalyst, and molecular sieves as dehydrating agent. The mixture was heated under reflux for 10 h under N₂. The reaction mixture was filtered through a column of silica gel, charcoal and Celite^® to remove the catalyst. The product obtained after concentration under vacuum, as viscous oil in 70% yield; HRMS: found MH⁺ 288.1344, C₁₅H₁₈N₃O₃ requires 288.1348; IR (ν_max, nujol, cm^-1): 3200-2900 (br), 1652, 1557, 1490, 1380; Anal. Found: C, 62.41; H, 6.32; N, 14.53 Calcd. for C₁₅H₁₈N₃O₃: C, 62.47; H, 6.29; N, 14.57; ¹H-NMR (MeOH-d₄): 2.15 (2H, quin, J 7 Hz, CH₂), 2.91 (2H, t, J 7 Hz, CH₂), 4.20 (2H, t, J 7 Hz, CH₂), 5.04 (2H, s, CH₂), 6.94-7.78 (7H, m, ArH and ImH), 8.37 (1H, s, CH=N); ¹³C-NMR (MeOH-d₄): 29.9 (CH₂), 37.9 (CH₂), 45.5 (CH₂), 68.9 (CH₂O), 114.8, 121.2, 121.6, 122.8, 128.4, 130.9, 137.8, 138.1 (ImC-2), 157.7, 160.9 (C=N), 166.0 (CO₂H).

Antibacterial activity

The newly synthesized compounds were screened for their antibacterial activity against locally isolated Escherichia coli (AMJ-2006) and Staphylococcus aureus (AMJ-2005) bacterial strains by the disc diffusion method [25,26]. Overnight incubated cultures of these bacteria were introduced onto the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculum. The discs measuring 6.25 mm in diameter were prepared from Whatman No. 1 filter paper and sterilized by dry heat at 140 °C for an hour. The sterile discs previously soaked in a 100 µg/ml of the test compound dissolved in DMSO were placed on the inoculated nutrient agar medium. The plates were inverted and incubated for one day at 37 °C. Ciprofloxacin was used as a standard drug. Growth inhibition zones were measured and compared with the controls. The bacterial inhibition zone values are summarized in Table 1. Minimum inhibitory concentrations (MIC) were determined by the broth dilution technique. The Nutrient Broth, which contained logarithmic serially two fold diluted amount of test compound and controls, were inoculated with approx. 5 x 10⁵ c.f.u. of actively dividing bacterial cells. The cultures were incubated for 24 h at 37 °C, and the growth was monitored visually and spectrophotometrically. To obtain the minimum bacterial concentration (MBC), 0.1 mL vol. was taken from each test and spread on agar plates. The number of c.f.u was counted after 18-24 hrs of incubation at 37 °C. The MIC and MBC are given in Table 2.