Synthesis and antimicrobial activities of some novel 1,2,4-triazole derivatives

2.1 Measurements

Melting points were determined by open capillary method and are uncorrected. The IR spectra (in KBr pellets) were recorded on a Thermo Nicolet avatar 330-FT-IR spectrophotometer. ¹H NMR and ¹³C NMR spectra were recorded (DMSO-d₆) on a Bruker (400 MHz) spectrometer using TMS as internal standard. Chemical shift values are given in δ scales. The mass spectra were recorded on LC–MS-Agilent 1100 series and API 2000 LC/MS system. Elemental analyses were performed on a Flash EA 1112 series CHNS–O analyzer. The completion of the reaction was checked by thin layer chromatography (TLC) on silica gel coated aluminium sheets (Silica Gel 60 F254). Commercial grade solvents and reagents were used without further purification. The reaction pathway has been summarized in Scheme 1.

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Scheme 1

Synthetic route for the Schiff bases 4a–f.

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2.2 Synthesis

2.3 Antimicrobial studies

All the newly synthesized compounds were evaluated for their antimicrobial activities against various microorganisms representing Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and fungus (Candida albicans), using the Minimum Inhibition Concentration (MIC) method by the serial dilution technique (Mackie and Mc Cartney, 1989). Several colonies of S. aureus, B. subtilis, E. coli and P. aeruginosa were picked off a fresh isolation plate and inoculated in corresponding tubes containing 5 mL of trypticase soya broth. The broth was incubated for 6 h at 37 °C until there was visible growth. Mc Farland No.5 standard was prepared by adding 0.05 mL of 1% w/v BaCl₂·2H₂O in Phosphate Buffered saline (PBS) to 9.95 mL of 1% v/v H₂SO₄ in PBS. The growth of all the four cultures was adjusted to Mc Farland No. 5 turbidity standard using sterile PBS. This gives a 10⁸ cfu/mL suspension. The working inoculums of above mentioned four different microorganisms containing 10⁵ cfu/mL suspension was prepared by diluting the 10⁸ cfu/mL suspension, 10³ times in trypticase soya broth.

Antimicrobial suspension was prepared by dissolving 0.5 μg of each compound in 10 mL of trypticase soya broth to get 50 μg/mL. This suspension was filter sterilized in syringe filters. To prepare the dilutions in all, for each of the eight anti-microbial compounds and standard antimicrobial i.e. Ceftriaxone, 24 tubes of 5 mL capacity were arranged in four rows with each row containing six tubes. Then 1.9 mL of trypticase soya broth was added in the first tube in each row and 1 mL in the remaining tubes. Now, 100 μl of filtered anti microbial suspension was added to the first tube in each row and after mixing the content, 1 mL was serially transferred from these tubes to the second tube in each of the rows. The contents in the second tube of each of the rows were mixed and transferred to the third tube in each of the rows. This serial dilution was repeated till the sixth tube in each of the rows. This provided anti microbial concentrations of 50, 25, 12.5, 6.25, 3.125, 1.6125 μg/mL in the first to sixth tube, respectively, in each row. Finally, 1 mL of 10⁵ cfu/mL of S. aureus, B. subtilis, E. coli, P. aeruginosa and C. albicans suspension were added to the first, second, third and fourth rows of tubes, respectively. Along with the test samples and Ceftriaxone (standard), the inoculum control (without antimicrobial compound) and broth control (without antimicrobial compound and inoculum) were maintained. All the test sample and control tubes were then incubated for 16 h at 37 °C. The results of the antimicrobial studies are summarized in Table 1.

3.1 Chemistry

Formation of N-[(4-{[(E)-substituted]amino}-5-sulfanyl-4H-1,2,4-triazol-3-yl)methyl]-4-substituted-benzamide (4) was confirmed by recording their IR, NMR, mass spectra and elemental analyses. IR spectrum of compound 4a showed absorption bands at 3322, 3046, 1642, 1596 and 1277 cm⁻¹ which is due to the N–H, C–H, C⚌O, C⚌N and C⚌S groups, respectively. The ¹H NMR spectrum of 4a showed a singlet at δ 13.99 corresponds to S–H proton. A singlet at δ 10.4 was due to N⚌CH proton. A triplet was observed at δ 9.01 was due to N–H attached to –CH₂. The appearance of doublet at δ 4.67 and multiplet at δ 7.42–8.33 was due to –CH₂ and aromatic ring protons, respectively. Similarly the mass spectrum was recorded and reported as[M+1] values. For the compound 4a, molecular weight 383 is consistent with the molecular formula C₁₇H₁₄N₆O₃S. The values for the remaining compounds have been presented under the experimental part.

3.2 Antimicrobial activity

All the newly synthesized compounds were screened for their antibacterial and antifungal activities. For antibacterial studies, microorganisms employed were S. aureus, B. subtilis, E. coli and P. aeruginosa. For antifungal studies, microorganism employed was C. albicans. Both antimicrobial studies were assessed by Minimum Inhibition Concentration (MIC) method by the serial dilution technique.

From the antimicrobial activity study, it was found that, compounds 4a–f exhibited the same activity as that of the standard drug Ceftriaxone against S. aureus and moderate activity against other bacteria B. subtilis, E. coli, P. aeruginosa and also fungus C. albicans. The observed activity may be due to presence of chloro, methoxy and nitro groups in these compounds. The antifungal activity of compounds 4a and 4f against C. albicans was found to be higher than that for the standard drug. However, remaining compounds exhibited same activity as that of standard drug. The observed activity in 4a and 4f is may be due to the presence of nitro group alone for the 4a, and in 4f, it may be due to the number and orientation of methoxy group.