Synthesis and antimicrobial evaluation of novel 1,3,4-thiadiazole derivatives of 2-(4-formyl-2-methoxyphenoxy) acetic acid

1 Introduction

The treatment of infectious disease caused by bacteria, fungi and viruses still remains an important and challenging problem because of a combination factors including newly emerging infectious diseases and increasing number of multi-drug resistant gram-positive pathogens (Tenover and McDonald, 2005; Pfeltz and Wilkinson, 2004; Roberts, 2004; Dessen et al., 2001), such as methicillin-resistant Staphylococcus aureus (MRSA), penicillin resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant Enterrococci (VRE), compounded problems in the therapeutics (Babu et al., 2008; Dalhoff, 1994). Thus it is still necessary to search for new antimicrobial agents.

Five membered aromatic systems having three hetero atoms at symmetrical position have interesting physiological properties (Hetzhein and Mockel, 1996; Sandstrom, 1968). During recent years there has been intense investigation of different classes of thiadiazole compounds, many of which known to possess interesting biological properties such as antimicrobial (Demirbas et al., 2009; Kadi et al., 2007; Bekhit and Abdel-Aziem, 2004), anti-inflammatory (Mullican et al., 1993; Song et al., 1999; Mathew et al., 2007), anticonvulsants (Chapleo et al., 1986, 1988), antioxidant (Cressier et al., 2009), anticancer (Matysiak et al., 2006; Chou et al., 2003; Radi et al., 2008) and antifungal (Swamy et al., 2006) activities. The activity of 1,3,4-thiadiazoles is possibly due to the presence of the ⚌N–C–S moiety (Bauer et al., 1966). In view of these facts, we have synthesized several new 1,3,4-thiadiazole derivatives of 2-(4-formyl-2-methoxyphenoxy) acetic acid moiety in order to study their biodynamic behavior. The present study reports the synthesis of 3-(4-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-3-methoxyphenyl)-5-(substituted)-4,5-dihydro-1H-pyrazole-1 carbothioamide (6a–s) by appropriate methods and their evaluation for antibacterial and antifungal potentials.

2 Chemistry

The synthetic route of compounds (6a–s) is shown in Scheme 1. 2-(4-Formyl-2-methoxyphenoxy) acetic acid (2) was prepared by reacting vanillin with chloroacetic acid in the presence of sodium hydroxide. Various chalcone derivatives (4a–s) were synthesized by treating (2) with different derivatives of acetophenone (3a–s). Compounds (5a–s) were obtained by refluxing (4a–k) and thiosemicarbazide in the presence of glacial acetic acid and ethanol. 1,3,4-Thiadiazole derivatives (6a–s) were obtained by cyclization of (5a–s) by treating with thiosemicarbazide and POCl₃ or PPA. The physical data of all the synthesized compounds is shown in Table 1.

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Scheme 1

Reagents: (a) chloroacetic acid, NaOH, HCl; (b) EtOH, KOH, petroleum ether; (c) thiosemicarbazide, glacial acetic acid; (d) thiosemicarbazide, PPA or POCl₃.

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3 Biological activity

All the synthesized 1,3,4-thiadiazole derivatives (6a–s) have been screened for both antibacterial and antifungal activities using cup-plate agar diffusion method by measuring zone of inhibition in mm. Eight different bacterial cultures S. aureus, Salmonella enterica, Vibrio cholera, Bacillus subtilis, Proteus mirabili, Escherichia coli V517, Mycobacterium smegmatics, Pseudomonas aeruginosa in nutrient agar medium, and one fungal culture Candida albicans in sabouraud’s dextrose agar medium (Holla et al., 2002) were used. The results were compared with positive control, the standard drug ampicillin (50 μg/ml) for bacteria and amphotericin B (50 μg/ml) for fungi and negative control, the DMSO poured disk. These sterilized agar media were poured into petri-dishes and allowed to solidify. On the surface of the media microbial suspensions were spread with the help of sterilized triangular loop. A stainless steel cylinder of 8 mm diameter (pre-sterilized) was used to bore cavities. All the synthesized compounds (50 μg/ml) were placed serially in the cavities with the help of micropipette and allowed to diffuse for 1.0 h. DMSO was used as a solvent for all the compounds, and as a control. These plates were incubated at 37 °C for 24 h and 28 °C for 48 h, for the antibacterial and antifungal activities, respectively. The zone of inhibition was observed around the cup after respective incubation and was measured and percent inhibition of the compounds was calculated.

4 Results and discussion

The structures of synthesized compounds were established on the basis of their spectral data. Spectral data of compounds were in full agreement with proposed structures. The formation of 1,3,4-thiadiazoles (6a–s) was supported by the presence of N–H band in the IR spectra and absence of carbonyl stretching band of the carboxylic acid function. In general, infra red spectra (IR) revealed a bilobe of NH₂ stretch at ∼3100, 3300, and C⚌N, C–N and C⚌S peak at ∼1530, 1328, and 1126 cm⁻¹, respectively. In the nuclear magnetic resonance spectra (¹H NMR) the signals of the respective protons of the prepared titled compounds were verified on the basis of their chemical shifts, multiplicities, and coupling constants. The spectra showed a singlet at δ ∼5.2 ppm corresponding to OCH₂ group; doublet at δ ∼4.0 ppm corresponding to C4 methylene group; singlet at δ ∼3.8 ppm corresponding to methoxy group; singlet at δ ∼3.2 ppm corresponding to C5 CH group; singlet at δ ∼5.8 ppm corresponding to NH₂ and multiplet at δ ∼6.5–8.2 ppm for aromatic proton; singlet at δ ∼8.5 ppm corresponding to another NH₂ group.

The 1,3,4-thiadiazole derivative 6h showed activity against all the strains. It showed maximum activity (97%) against S. enterica (95%), against V. cholera and (87.9%) inhibition of E. coli V517 when compared with standard drug ampicillin. Compound 6e showed (93.2% and 89.5% inhibition) against S. enterica and V. cholera, respectively. Compound 6k showed maximum inhibition (87.1%) against S. aureus. Compound 6o was found to be active (96.5% inhibition) against E. coli V517 and 6p showed (90.2% inhibition) against P. aeruginosa. Rest of all the 1,3,4-thiadiazole derivatives showed moderate to good antibacterial activity. The 1,3,4-thiadiazole derivative 6h showed maximum inhibition (87.8%) whereas, compound 6e showed (83.3%) inhibition against fungal strain C. albicans. Rest of all the 1,3,4-thiadiazole derivatives showed moderate to low antifungal activity. The results are presented in Table 2.

5 Experimental

Chemicals were purchased from Merck India, Spectrochem and Sigma–Aldrich etc. Most of the solvents and chemicals used were of LR grade. The purity of the compounds was confirmed by thin layer chromatography using precoated TLC plates and solvent systems of benzene–acetone (9:1), (8:2); T–E–F (5:4:1), and chloroform–methanol (9:1), (9.5:0.5). The spots were visualized under ultraviolet lamp. Melting points were determined in one end open capillary tubes on a liquid paraffin bath and are uncorrected. Infrared (IR) and ¹H nuclear magnetic resonance (¹H NMR) spectra were recorded for the compounds on Perkin Elmer IR (ν_max in cm⁻¹) spectrophotometer in KBr pellets and Bruker model avance II (400 MHz, ¹H NMR) instrument, respectively. Chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard.

6 Conclusion

A total of 19 compounds were synthesized and screened for their antibacterial activity against S. aureus, S. enterica, V. cholera, B. subtilis, P. mirabili, E. coli V517, M. smegmatics, P. aeruginosa and antifungal activity against C. albicans. The % inhibition of all the compounds was determined by observing the zones of inhibition formed around the cup after 24 h of incubation for antibacterial and 48 h for antifungal activities. Among the tested compounds 6h, 6e, 6k, 6o, 6p and 6h, 6e possess significant antibacterial and antifungal activities, respectively while rest of all the 1,3,4-thiadiazole derivatives showed moderate antimicrobial activity as compared to standards.