Journal of Chemistry

Volume 2013 (2013), Article ID 638520, 8 pages

http://dx.doi.org/10.1155/2013/638520

Research Article

Synthesis, Characterization, and Biological Activity of Novel Schiff and Mannich Bases of 4-Amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione

Uzma Yunus, Moazzam H. Bhatti, Naima Rahman, Nosheen Mussarat, Shazia Asghar, and Bilal Masood

Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan

Received 23 May 2013; Revised 1 August 2013; Accepted 29 August 2013

Academic Editor: Suleyman Goksu

Copyright © 2013 Uzma Yunus et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The present work describes the syntheses and antimicrobial activity studies of a series of novel Schiff bases (4a–4i) and their Mannich bases (5a–5h) starting from 4-amino-3-(N-phthalimido-methyl)-1,2,4-triazole-5-thione (3). All the synthesized compounds were characterized by IR, ¹H-NMR, ¹³C-NMR, and MS. All the synthesized compounds were screened for four Gram-negative strains, one Gram-positive strain of bacteria, and one diploid fungal strain. In general the antimicrobial activity increased remarkably on the introduction of azomethine functionality in parent triazole (3). The antimicrobial activity further improved when morpholine group was added to them except for Enterobacter cloacae, where loss of activity was observed. The results are promising and show that the fine tuning of the structures (5a, 5b, 5e, 5f, and 5h) can lead to some new antimicrobial compounds.

1. Introduction

Many of the antibiotics presently in use are becoming ineffective due to the emergence of resistant microbial strains. It proves that the microbes are more intelligent than what is being anticipated by human beings, as they quickly develop mechanisms to intercept the antibiotic, thus making them ineffective. This situation demands the development of new antimicrobial agents which can deprive the microbes of their pathogenicity by novel multisite mechanisms of action [1–4]. The 1,2,4-triazole nucleus is the main structural unit of many medicines currently in market. Ribavirin (1), letrozole (2), fluconazole (3), itraconazole (4), and anastrozole (5) are a few to name which are currently in use as medicines (Figure 1). Many other 1,2,4-triazole derivatives are also known to possess pharmacological activities like antitubercular, anticonvulsant, anti-inflammatory, and analgesic activities [5–14]. It has been reported that triazoles are less susceptible to metabolic degradation and have higher target specificity and wider spectrum of activities as compared to imidazoles [15, 16]. Many heterocyclic systems having azomethine functionality are known to possess cytotoxic, antimicrobial, anticancer, and antifungal activities [17–21].

Figure 1: Some 1,2,4-triazole based medicine currently in use.

The literature reveals that the presence of morpholine or piperazine ring on a heterocyclic system contributes to enhanced pharmacological activities in many cases [22–24]. This could be attributed to the increased solubility of the compounds in aqueous solvents because of the formation of aminium salt [25]. These wide applications of 1,2,4-triazole Schiff and Mannich bases motivated us to synthesize new derivatives of 4-amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione and to test their potential as antibacterial and antifungal agents.

2. Experimental

General Comments. All reagents were purchased from Acros, Fluka, and Aldrich and used without further purification. All reactions were performed in standard glassware. All reactions were monitored by TLC plates precoated with silica gel Si 60 F₂₅₄ from Merck and were visualized under UV lamp at λ = 254 nm. Melting points were determined with electrothermal apparatus, Gallenkamp and are uncorrected. IR spectra were recorded on PerkinElmer spectrophotometer by ATR technique. ¹H-NMR and ¹³C-NMR spectra were recorded on Burker 300 MHz/400 MHz instruments and 75 MHz/100 MHz instruments, respectively; the solvent used is specified along with data. Mass spectra were recorded on Jeol mass spectrometer in electron ionization mode. Leco 3200CHNS analyzer was used for elemental analysis. The thiocarbohydrazide (2) and the 4-amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (3) were synthesized by the previously reported method [26, 27]. All manipulations of microbial activity were performed in laminar flow chamber (LFC) with disposable surgical gloves, all standard biosafety measures were taken, and contaminated materials after experimentation were collected in autoclave bags and autoclaved at 120°C before disposal.

2.1. 4-Amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (3)

To a clean test tube were added 0.01 mol (2.05 g) of N-phthaloylglycine and 0.01 mol (1.06 g) of thiocarbohydrazide, and the test tube was kept in a preheated oil bath. The molten mixture was kept at 145°C for 25 minutes. On cooling the mixture a solid mass was formed which was triturated with ethanol and filtered. The crude solid was recrystallized in acetonitrile and ethanol (1 : 1). Yield: 69%, white crystals, m.p. 189–192°C, IR (ATR) (v cm⁻¹), 3305.08, 3152.47 (NH), 2982.12 (Ar-H), 2952.46 (CH₂), 1767.06 (cyclic amide), 1605 (C=N), 1145 (C=S). ¹H NMR (400 MHz, DMSO-, δ ppm): 13.98 (b, 1H, NH), 7.86–7.94 (m, 4H, C₆H₄), 5.63 (s, 2H, NH₂), 4.84 (s, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-, δ ppm): 167.53 (C=S), 167.24 (C=O), 147.81 (N=C), 135.24 (N=C), (131.91, 123.86 (Ar-C)), 32.99 (CH₂). MS-E1 (M/z), Relative intensity: 275.1 (M+1, 100%), 259.1 (65%), 160.1 (80%), 130.1 (30%), 104.0 (45%), 77.1 (30%); C₁₁H₉N₅O₂S: Calculated: C, 47.99; H, 3.30; N, 25.44; S, 11.65. Found: C, 48.10; H, 3.35; N, 25.49; S, 11.85.

2.2. General Procedure for Synthesis of Schiff Bases of 1,2,4-Triazole (3) (4a–4i)

Aromatic aldehyde (15 mmol) was dissolved in 8.0 mL of glacial acetic acid, and 10 mmol of 1,2,4-triazole (3) was added. The reaction mixture was refluxed for 25 minute to one hour in a preheated oil bath. The mixture was cooled, and the solid formed was filtered and washed with cold ethanol. The solid was recrystallized from ethanol and Schiff bases (4a–4i) were obtained in good to excellent yields.

2.3. 4-(Benzylidene)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4a)

Yield 42.8%, Yellow crystals m.p. 198–200°C. IR (ATR, v cm⁻¹): 3283.40, 3156.39 (N–H), 3012.31 (Ar-H), 2996.00 (CH₂), 1770.75 (cyclic amide), 1599.79 (C=N), 1243.82 (C–N), 1120.06 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 14.02 (s, 1H, NH), 9.92 (s, 1H, N=CH), 7.88 (m, 4H, Ph), 7.80 (d, 2H, J = 7.2 Hz, Ph), 7.59 (m, 1H, Ph), 7.50 (m, 2H, Ph), 4.99 (s, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-, δ ppm): 167.47 (C=S), 164.39 (C=O), 162.48 (N=CH), 146.52 (C=N), (135.33, 133.23, 132.29, 131.81, 129.52, 129.10, 123.90 (Ar-C)), 33.17 (CH₂). MS-E1: (m/z, Relative intensity, %): 363.3 (20%), 260.2 (100%), 242.2 (24%), 228.2 (19%), 203.2 (5%), 183.2 (15.9%), 160.2 (28.8%), 148.2 (18.2%), 130.1 (24%), 104.1 (87%), 89.1 (7.1%), 76.1 (45%). C₁₈H₁₃N₅O₂S: Calculated: C, 59.49; H, 3.61; N, 19.27; S, 8.82. Found: C, 59.48; H, 3.66; N19.30; S, 8.84.

2.4. 4-(2-Hydroxybenzylidene)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4b)

Yield 73%, white crystals, m.p. 239–241°C. IR (ATR, v cm⁻¹): 3216.31 (O–H), 3055.35 (Ar-H), 2997.4 (CH₂), 1774.02 (cyclic amide), 1601.09 (C=N), 1253.65 (C–N), 1119.56 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 13.95 (s, 1H, NH), 10.40 (s, 1H, OH), 10.13 (s, 1H, N=CH), 7.89 (m, 4H, Ph), 7.70 (dd, 1H, J = 7.9, 1.5 Hz, Ph), 7.40 (dt, 1H, J = 7.8 Hz, 1.5 Hz, Ph), 6.94 (d, 1H, J = 8.1 Hz, Ph), 6.85 (t, 1H, J = 7.8 Hz, Ph), 4.97 (s, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-, δ ppm): 167.49 (C=S), 162.37 (C=O), 160.72 (N=CH), 158.90 (C–OH), 146.56 (C=N), (135.31, 134.80, 131.82, 127.28, 123.88, 119.88, 118.62, 117.03 (Ar-C)), 33.19 (CH₂). MS-E1 (m/z, Relative intensity, %): 379.3 (M⁺, 2.7%), 347.4 (2.8%), 330.3 (5.2%), 260.2 (100%), 242.2 (8.6%), 228.2 (27.1%), 160.2 (53.4%), 148.2 (32.5%), 130.1 (57.3%), 119.1 (84.2%), 104.1 (85.3%), 91 (88%). C₁₈H₁₃N₅O₃S: Calculated: C, 56.98; H, 3.45; N, 18.46; S, 8.45. Found: C, 57.07; H, 3.54; N, 18.57; S, 8.57.

2.5. 3-(N-Phthalimidomethyl)-4-(3-pyridine)amino-1,2,4-triazole-5-thione (4c)

Yield 76.05%, off-white crystals, m.p. 219–221°C. IR (ATR, v cm⁻¹): 3041.73 (Ar-H), 2905.69 (CH₂), 1771.38 (cyclic amide), 1591.89 (C=N), 1302.07 (C–N), 1112.59 (C=S). ¹H NMR (400 MHz, DMSO-, δ ppm): 14.07 (s, 1H, NH), 10.13 (s, 1H, N=CH), 8.95 (d, 1H, J = 1.8 Hz, Ph), 8.75 (dd, 1H, J = 4.8, 1.5 Hz, Ph), 8.22 (d, 1H, J = 8.1 Hz, Ph), 7.88 (m, 4H, Ph), 7.55 (dd, 1H, J = 7.8 Hz, 4.8 Hz, Ph), 5.01 (s, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-, δ ppm): 167.0 (C=S), 162.05 (C=O), 160.88 (HC=N), 152.0 (Py-C), 151.0 (Py-C), 149.0 (C=N), (133.7, 132.2, 130.4, 123.9, 123.6, 123.0 (Ar-C)), 33.5 (CH₂). MS-E1 (m/z, Relative intensity, %): 364.0 (12.8%), 260.0 (100%), 242.0 (8.1%), 228.0 (4.6%), 183.0 (6.4%), 160.0 (16.9%), 148.0 (11.1%), 130.0 (16.1%), 104.0 (46.7%). C₁₇H₁₂N₆O₂S: Calculated: C, 56.04; H, 3.32; N, 23.06; S, 8.80. Found: C, 56.16; H, 3.45; N 23.10; S, 8.82.

2.6. 4-(3-Nitrobenzylidine)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4d)

Yield 48%, yellow powder, m.p. 258–260°C. IR (ATR, v cm⁻¹): 3200 (N–H), 3041.73 (Ar-H), 2919 (CH₂), 1770.38 (cyclic amide), 1591.89 (C=N), 1302.07 (C–N), 1112.59 (C=S). ¹H NMR (400 MHz, DMSO-, δ ppm): 14.06 (s, 1H, NH), 10.22 (s, 1H, N=CH), 8.61 (s, 1H, Ph), 8.40 (d, J = 6.8 Hz, 1H, Ph), 8.24 (d, J = 6.5 Hz, 1H, Ph), 7.85 (m, 5H, Ph), 5.01 (s, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-, δ ppm): 166.87 (C=S), 162.08 (C=O), 160.84 (N=CH), 148.21 (C=N), (146.09, 134.71, 134.61, 133.58, 131.23, 130.66, 126.65, 123.29, 122.47 (Ar-C)), 32.58 (CH₂). MS-E1: m/z, (Relative intensity, %): 407.9 (7.3%), 260.0 (100%), 242 (5.1%), 228.0 (3.5%), 160.0 (13.3%), 148.0 (17.3%), 130.0 (11.0%), 104.0 (26.2%). C₁₈H₁₂N₆O₄S: Calculated: C, 52.94; H, 2.96; N, 20.58; S, 7.85. Found: C, 53.0; H, 3.10; N, 20.78; S, 8.02.

2.7. 4-(4-Chlorobenzylidine)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4e)

Yield 64.52%, white crystals, m.p. 241–244°C. IR (ATR, v cm⁻¹): 3035.73 (Ar-H), 2956 (CH₂), 1772.18 (cyclic amide), 1611.24 (C=N), 1306.75 (C–N), 1118.19 (C=S). ¹H NMR (400 MHz, DMSO-, δ ppm): 13.99 (s, 1H, NH), 9.99 (s, 1H, N=CH), 7.84 (m, 4H, Ph), 7.81 (d, 2H, J = 8.4 Hz, Ph), 7.57 (d, 2H, J = 8.4 Hz, Ph), 4.97 (s, 2H, CH₂). ¹³C NMR: (100 MHz, DMSO-, δ ppm): 166.87 (C=S), 162.05 (C=O), 162.02 (N=CH), 145.99 (C=N), (137.29, 134.74, 131.25, 130.73, 130.14, 129.12, 123.32 (Ar-C)), 32.56 (CH₂). MS-E1: (m/z, Relative intensity, %): 397.3 (14.1%), 260.2 (100%), 242.2 (23.1%), 228.2 (14.7%), 183.2 (15.7%), 160.2 (26.6%), 148.1 (15%), 137.1 (77.6%), 104.1 (61.7%), 76.1 (24.8%). C₁₈H₁₂ClN₅O₂S: Calculated: C, 54.34; H, 3.04; N, 17.60; S, 8.06. Found: C, 54.45; H, 3.16; N, 17.62; S, 8.08.

2.8. 4-(4-Bromobenzylidine)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4f)

Yield 66.13%, white crystals, m.p. 215–217°C. IR (ATR, v cm⁻¹): 3201 (N–H), 3041.73 (Ar-H), 2950.09 (CH₂), 1772.54 (cyclic amide), 1620.89 (C=N), 1302.07 (C–N), 1112.07 (C=S), 710 (C–Br). ¹H NMR (400 MHz, DMSO-, δ ppm) 14.04 (s, 1H, NH), 9.73 (s, 1H, N=CH), 7.79 (m, 4H, Ph), 7.57 (d, 2H, J = 8.4 Hz, Ph), 7.40 (d, 2H, J = 8.4 Hz, Ph), 5.0 (s, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-, δ ppm): 167.12 (C=S), 163.68 (C=O), 159.10 (N=CH), 144.94 (C=N), (134.39, 132.24, 131.85, 131.32, 130.08, 127.15, 123.75 (Ar-C)), 32.88 (CH₂). MS-E1: (m/z, Relative intensity, %): 442.9 (3.7%), 274.0 (100%), 260.0 (84.1%), 241 (16.2%), 182.9 (28.7%), 148.0 (26.3%), 127.0 (51.1%), 104 (47.4%). C₁₈H₁₂BrN₅O₂S: Calculated: C, 48.88; H, 2.73; N, 15.8; S, 7.25. Found: C, 49.00; H, 2.88; N, 15.86; S, 7.29.

2.9. 3-(N-Phthalimidomethyl)-4-(4-pyridine)amino-1,2,4-triazole-5-thione (4g)

Yield 48.02%, amorphous solid, m.p. 217–218°C. IR (ATR, v cm⁻¹): 3012.18 (Ar-H), 2896.83 (CH₂), 1771.78 (cyclic amide), 1603.02 (C=N), 1268.54 (C–N), 1119.49 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 13.90 (s, 1H, NH), 9.70 (s, 1H, N=CH), 8.66 (d, 2H, J = 7.9, Ph), 7.98 (d, 2H, J = 7.9 Hz, Ph), 7.80 (m, 4H, Ph), 4.98 (s, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-, δ ppm): 168.20 (C=S), 162.70 (C=O), 159.00 (N=CH), 152.20 (Py-C), 149.00 (Py-C), 144.30 (C=N), (132.2, 123.70, 123.10, 120.41 (Ar-C)), 37.23 (CH₂). MS-E1 (m/z, Relative intensity, %): 364.0 (10.8%), 260.0 (100%), 242.0 (6.0%), 228.0 (3.1%), 183.0 (5.1%), 160.0 (12.9%), 148.0 (6.8%), 130.0 (8.6%), 104.0 (34.3%). C₁₇H₁₂N₆O₂S: Calculated: C, 56.04; H, 3.32; N, 23.06; S, 8.80. Found: C, 56.13; H, 3.51; N, 23.10; S, 8.81.

2.10. 4-(4-Methoxybenzylidine)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4h)

Yield 36% white crystals, m.p. 239°C. IR (ATR, v cm⁻¹): 3200 (Ar-H), 2919 (sp3 C–H), 1767 (C=O of anhydride), 1602 (C=N), 1279 (C=S) and 1255 (C–O). ¹H NMR (400 MHz, DMSO-, δ ppm): 13.91 (s, 1H, NH), 9.69 (s, 1H, N=CH), 7.87 (m, 4H, Ph), 7.7 (d, 2H, J = 8.8 Hz, Ph), 7.33 (2H, J = 8.8 Hz, Ph), 4.94 (s, 2H, CH₂), 3.83 (s, 3H, O–CH₃). ¹³C NMR (100 MHz, DMSO-,δ ppm): 166.87 (C=S), 163.95 (C=O), 162.84 (N=CH), 161.99 (C–OCH₃), 145.78 (C=N), (134.72, 131.26, 130.54, 124.16, 123.30, 114.46 (Ar-C)), 55.47 (O–CH₃), 32.61 (CH₂), MS-E1: (m/z, Relative intensity, %): 393.4 (26.0%), 361.4 (4.7%), 260.2 (100%), 242.2 (8.7%), 228.3 (17.8%), 186.2 (9.8%), 160.2 (32.2%), 148.2 (10.8%), 133.1 (100%), 104 (66.4%). C₁₉H₁₅N₅O₃S: Calculated: C, 58.01; H, 3.84; N, 17.80; S, 8.15. Found: C, 58.10; H, 4.01; N, 17.85; S, 8.20.

2.11. 4-(4-Fluorobenzylidine)amino-3-(N-phthalimidomethyl)-1,2,4-triazole-5-thione (4i)

Yield 35%, light yellow crystals, m.p. 231–233°C. IR (ATR, v cm⁻¹): 3200 (Ar-H), 2912 (sp³ C–H), 1770 (C=O of anhydride), 1597 (C=N), 1279 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 14.00 (s, 1H, NH), 9.93 (s, 1H, N=CH), 7.85 (m, 6H, Ph), 7.33 (t, 2H, J = 8.4 Hz, Ph), 4.98 (s, 2H, CH₂). ¹³C NMR: (75 MHz, DMSO-, δ ppm): 166.91 (C=S), 164.62 (d,  Hz, C–F), 162.43 (C=O), 162.02 (N=CH), 145.99 (C=N), 134.76, 131.27, 131.17 ( Hz), 128.46, 123.35, 116.21 ( Hz), (Ar-C), 32.62 (CH₂). MS-E1 (m/z, Relative intensity, %): 381.0 (22.4%), 260.0 (100%), 242.0 (10.1%), 228.0 (4.5%), 183.0 (7.7%), 160.0 (18.4%), 148.0 (9.1%), 132.0 (9.3%), 104.0 (36.6%). C₁₈H₁₂FN₅O₂S: Calculated: C, 56.69; H, 3.17; N, 18.36; S, 8.41. Found: C, 56.74; H, 3.37; N, 18.39; S, 8.44.

2.12. General Procedure for the Synthesis of Mannich Bases (5a–5h)

The corresponding Schiff bases (4a–4h) (10 mmol) were dissolved in acetonitrile: dioxane (2 : 1) at RT. Then, a solution of formaldehyde (37%, 0.5 mL) and morpholine (10 mmol) in ethanol was added dropwise with vigorous stirring. The reaction mixture was stirred at RT for 2 hours and left over night. The solid product was filtered and washed with ethanol and recrystallized from acetonitrile: dioxane (2 : 1) to yield title compound.

2.13. 4-(Benzylideneamino)-1-(morpholinomethyl)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5a)

Yield 72%, m.p. 208–210°C IR (ATR, v cm⁻¹) 2958 (CH₂, asym), 2855 (CH₂, sym), 1771 (cyclic amide), 1591 (C=N), 1112 (C=S). ¹H NMR (300 MHz, CDCl₃, δ ppm): 10.50 (s, 1H, N=CH), 7.91 (dd, 2H, J = 5.7 Hz, J = 3.0 Hz, Ph), 7.80 (m, 2H, Ph), 7.77 (dd, 2H, J = 5.7 Hz, J = 3.0 Hz, Ph), 7.44–7.57 (m, 3H, Ph), 5.10 (s, 4H, 2 × CH₂, N–CH₂–N, N–CH₂–C), 3.69 (b, 4H, morpholine), 2.79 (b, 4H, CH₂, morpholine). ¹³C NMR (75 MHz, DMSO-, δ ppm): 167.20 (C=S), 163.70 (C=O), 161.06 (N=CH), 145.05 (C=N), (134.40, 132.52, 132.28, 131.87, 128.93, 128.88, 123.73 (Ar-C)), 69.08 (N–CH₂–N), (66.85, 50.68 (CH₂, morpholine)), 32.96 (CH₂). MS-E1: (m/z, Relative intensity, %): 462.1 (M+1, 20.6%), 363.0 (10.5%), 260.0 (76.6%), 242.0 (4.6%), 228.0 (5.7%), 185 (4.9%), 160.0 (27.5%), 100.1 (100%). C₂₃H₂₂N₆O₃S: Calculated: C, 59.73; H, 4.79; N, 18.17; S, 6.93. Found: C, 59.82; H, 4.85; N, 18.20; S, 6.99.

2.14. 4-((2-Hydroxybenzylidene)amino)-1-(morpholinomethyl)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5b)

Yield 74%, m.p. 214–216°C. IR (ATR, ν cm⁻¹): 2942 (CH₂, asym), 2849 (CH₂, sym), 1774 (cyclic amide), 1618 (C=N), 1111 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm) 10.43 (s, 1H, N=CH), 10.00 (s, 1H, OH), 7.88 (m, 4H, Ph), 7.66 (dd, 1H, J = 8.1 Hz, 1.5 Hz, Ph), 7.40 (t, 1H, J = 7.6 Hz, Ph), 6.94 (d, 1H, J = 8.1 Hz, Ph), 6.83 (t, 1H, J = 7.5 Hz, Ph), 5.03 (s, 2H, N–CH₂–N), 5.02 (s, 2H, N–CH₂–C), 3.53 (b, 4H, CH₂, morpholine), 2.64 (b, 4H, CH₂, morpholine). ¹³C NMR (75 MHz, DMSO-, δ ppm): 167.48 (C=S), 163.17 (C=O), 161.99 (N=CH), 159.00 (C–OH), 145.35 (C=N), (135.30, 134.98, 131.82, 127.24, 123.90, 119.86, 118.41, 117.05 (Ar-C)), 69.00 (N–CH₂–N), (66.51, 50.63 (CH₂, morpholine)), 32.27 (CH₂). MS-E1 (m/z, Relative intensity, %): 478 (M⁺, 3%), 359 (5%), 260 (31.8%), 185 (3.8%), 160 (12%), 119 (18.1%), 110.1 (100%). C₂₃H₂₂N₆O₄S: Calculated: C, 57.73; H, 4.63; N, 17.56; S, 6.70. Found: C, 57.8; H, 4.73; N, 17.57; S, 6.79.

2.15. 1-(Morpholinomethyl)-4-((pyridin-3-ylmethylene)amino)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5c)

Yield 69% m.p. 188–190°C IR (ATR, ν cm⁻¹): 2953 (CH₂, asym), 2848 (CH₂, sym), 1772 cyclic amide, 1591 (C=N), 1115 (C=S). ¹H NMR (300 MHz, CDCl₃, δ ppm): 10.13 (s, 1H, N=CH), 8.94 (s, 1H, Ph), 8.73 (dd, 1H, J = 6.2 Hz, 1.5 Hz, Ph), 7.93–7.83 (m, 5H, Ph) 5.07 (s, 2H, N–CH₂–N), 5.03 (s, 2H, N–CH₂–C), 3.64 (b, 4H, CH₂, morpholine), 2.71 (b, 4H, CH₂, morpholine), ¹³C NMR (300 MHz, CDCl₃, δ ppm): 167.13 (C=S), 163.75 (C=O), 157.17 (N=CH), 152.98 (Py-C), 150.62 (Py-C), 145.00 (C=N), (134.82, 134.45, 131.86, 128.57, 123.86, 123.73 (Ar-C)), 69.17 (N–CH₂–N), (66.87, 50.73 (CH₂, morpholine)), 32.85 (CH₂). MS-E1 (m/z, Relative intensity, %): 463 (M+1, 7.5%), 363.9 (5.0%), 260.0 (40.0%), 228.0 (5.3%), 160.0 (14.0%), 100.1 (100%). C₂₂H₂₁N₇O₃S: Calculated: C, 57.01; H, 4.57; N, 21.15; S, 6.92. Found: C, 57.10; H, 4.68; N, 21.20; S, 7.02.

2.16. 1-(Morpholinomethyl)-4-((3-nitrobenzylidene)amino)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5d)

Yield 80% m.p. 193–195°C (decomposed), IR (ATR, ν cm⁻¹): 2940 (CH₂, asym), 2840 (CH₂, sym), 1774 (amide), 1610 (C=N), 1115 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 10.01 (s, 1H, N=CH), 8.62 (t, 1H, J = 1.5 Hz, 1.8 = Hz, Ph), 8.42 (ddd, 1H, J = 8.1, 2.1 and 0.6 Hz, Ph), 8.23 (d, 1H, J = 7.8 Hz, Ph), 7.78–7.92 (m, 4H, Ph) 7.79 (d, 1H, J = 8.2 Hz, Ph), 5.07 (2H, N–CH₂–N), 5.06 (s, 2H, N–CH₂–C), 3.54 (b, 4H, CH₂, morpholine), 2.66 (b, 4H, CH₂, morpholine). ¹³C NMR (75 MHz, DMSO-, δ ppm): 167.46 (C=S), 163.20 (C=O), 162.93 (N=CH), 148.71 (C=N), (145.42, 135.31, 133.91, 131.76, 131.25, 127.43, 123.91, 123.13 (Ar-C)), 69.12 (N–CH₂–N), (66.51, 50.62 (CH₂, morpholine)), 33.20 (CH₂). MS-E1: (m/z, Relative intensity, %): 507. (M⁺, 1.9%), 408.0 (2.5%), 260.0 (21.6%), 185.0 (2.7%), 160.0 (12.2%), 130.0 (6.1%), 100.1 (100%). C₂₃H₂₁N₇O₅S: Calculated: C, 54.43; H, 4.17; N, 19.32; S, 6.32. Found: C, 54.53; H, 4.28; N, 19.35; S, 6.33.

2.17. 4-((4-Chlorobenzylidene)amino)-1-(morpholinomethyl)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5e)

Yield 79% m.p. 178–180°C IR (ATR, ν cm⁻¹): 2958 (CH₂, asym), 2855 (CH₂, sym), 1771 (cyclic amide), 1593 (C=N), 1112 (C=S). ¹H NMR (300 MHz, DMSO-, δ ppm): 9.85 (s, 1H, N=CH) 7.88 (m, 4H, Ph), 7.80 (d, 2H, J = 8.7 Hz, Ph), 7.57 (d, 2H, J = 8.4 Hz, Ph), 5.02 (s, 4H, 2 × CH₂, N–CH₂–N, N–CH₂–C), 3.52 (b, 4H, CH₂, morpholine), 2.63 (b, 4H, CH₂, morpholine). ¹³C NMR (300 MHz, DMSO-, δ ppm): 166.95 (C=S), 163.62 (C=O), 162.73 (N=CH), 144.84 (C=N), (137.53, 134.84, 131.30, 130.60, 130.21, 129.21, 123.43 (Ar-C)), 68.60 (N–CH₂–N), (66.01, 50.14 (CH₂, morpholine)), 32.69 (CH₂). MS-E1: (m/z Relative intensity, %): (496.0, M⁺, 5.7%), 396.9 (5.1%), 260.0 (56.5%), 242.0 (2.7%), 228.0 (3.5%), 185.0 (4.1%), 160.0 (20.8%), 137.0 (29.0%), 100.1 (100%). C₂₃H₂₁ClN₆O₃S: Calculated: C, 55.59; H, 4.26; N, 16.91; S, 6.45. Found: C, 55.74; H, 4.37; N, 16.99; S, 6.47.

2.18. 1-(Morpholinomethyl)-4-((4-bromobenzylidene)amino)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5f)

Yield 39% m.p. 189−191°C IR (ATR, ν cm⁻¹): 2958 (CH₂, asym), 2854 (CH₂, sym), 1772, 1719 (cyclic amide), 1607 (C=N), 1112 (C=S). ¹H NMR (300 MHz, CDCl₃, δ ppm): 10.56 (s, 1H, N=CH), 7.86 (dd, 2H, J = 5.4 Hz, 3.0 Hz, Ph), 7.74 (dd, 2H, J = 5.4 Hz, 3.0 Hz, Ph), 7.66 (d, 2H, J = 8.4 Hz, Ph), 7.56 (d, 2H, J = 8.4 Hz, Ph), 5.05 (s, 2H, N–CH₂–N), 5.04 (s, 2H, N–CH₂–C), 3.63 (b, 4H, CH₂, morpholine), 2.73 (b, 4H, CH₂, morpholine), ¹³C NMR (75 MHz, CDCl₃,  δ ppm): 167.12 (C=S), 163.68 (C=O), 159.10 (N=CH), 144.94 (C=N), (134.39, 132.24, 131.85, 131.85, 131.32, 130.08, 127.15, 123.75, 123.68 (Ar-C)), 69.12 (N−CH₂−N), (66.84, 50.70 (CH₂, morpholine)), 32.88 (CH₂). MS-E1 (m/z, Relative intensity, %): 540 (M⁺, 3.1%), 441 (3.1%), 260.0 (84.4%), 242.0 (3.7%), 182.9 (22.6%), 160.0 (26.7%), 100.1 (100%). C₂₃H₂₁BrN₆O₃S: Calculated: C, 51.02; H, 3.91; N, 15.52; S, 5.92. Found: C, 51.00; H, 3.99; N, 15.59; S, 6.02.

2.19. 1-(Morpholinomethyl)-4-((pyridin-4-ylmethylene)amino)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5g)

Yield 69% m.p. 201–203°C IR (ATR, ν cm⁻¹): 2953 (CH₂, asym), 2848 (CH₂, sym), 1770 (cyclic amide), 1602 (C=N), 1112 (C=S). ¹H NMR (300 MHz, CDCl₃,  δ ppm): 10.10 (s, 1H, N=CH), 8.65 (d, 2H, J = 7.8 Hz, Ph), 7.97 (d, 2H, J = 7.8 Hz, Ph), 7.80 (m, 4H, Ph), 5.07 (s, 2H, N–CH₂–N), 5.03 (s, 2H, N–CH₂–C), 3.64 (b, 4H, CH₂, morpholine), 2.71 (b, 4H, CH₂, morpholine). ¹³C NMR (300 MHz, CDCl₃, δ ppm): 167.23 (C=S), 163.72 (C=O), 157.17 (N=CH), 152.97 (Py-C), 149.02 (Py-C), 145.40 (C=N), (133.82, 134.54, 131.80, 128.77, 122.96, 123.73 (Ar-C)), 70.02 (N–CH₂–N), (66.89, 50.77 (CH₂, morpholine)), 32.65 (CH₂). MS-E1 (m/z, Relative intensity, %): 463 (M+1), 363.9 (5.8%), 260.0 (72.4%), 228.0 (2.8%), 160.0 (32.2%), 104 (26.8%), 100.1 (100%). C₂₂H₂₁N₇O₃S: Calculated: C, 57.01; H, 4.57; N, 21.15; S, 6.92. Found: C, 57.09; H, 4.63; N, 21.26; S, 7.03.

2.20. 4-((4-Methoxybenzylidene)amino)-1-(morpholinomethyl)-3-(N-phthalimidomethyl)-1,4-dihydro-5H-1,2,4-triazole-5-thione (5h)

Yield 82% m.p. 218–220°C IR (ATR, ν cm⁻¹): 2939 (CH₂, asym), 2840 (CH₂, sym), 1770 (amide), 1598 (C=N), 1171 (C=S). ¹H NMR (300 MHz, CDCl₃, δ ppm): 10.21 (s, 1H, N=CH), 7.86 (dd, 2H, 5.4 Hz and 3.3 Hz, Ph), 7.72–7.74 (m, 4H, Ph), 6.93 (d, 2H, J = 8.4 Hz, Ph), 5.04 (s, 4H, 2 × CH₂, N–CH₂–N, N–CH₂–C), 3.85 (s, 3H, OCH₃), 3.65 (b, 4H, CH₂, morpholine), 2.73 (b, 4H, CH₂, morpholine), ¹³C NMR (75 MHz, CDCl₃, δ ppm): 167.15 (C=S), 163.68 (C=O), 162.64 (N=CH), 161.99 (C–OCH₃), 144.85 (C=N), (134.38, 134.31, 131.93, 130.79, 124.83, 123.74, 123.65, 114.38 (Ph-C)), (69.14, 66.87 (CH₂, morpholine)), 55.48 (O–CH₃), 50.71 (CH₂), 32.9. MS-E1: (m/z, Relative intensity, %): 492 (M⁺, 5.9%), 393.0 (9.6%), 358.0 (3.8%), 260.0 (57.2%), 228.0 (2.9%), 185.0 (3.1%), 160.0 (23%), 133.0 (71.1%), 100.1 (100%). C₂₄H₂₄N₆O₄S: Calculated: C, 58.52; H, 4.91; N, 17.06; S, 6.51. Found: C, 58.59; H, 4.92; N, 17.08; S, 6.53.

3. Microbiology

The antimicrobial activity of the compounds 3, 4a–4i, and 5a–5h was tested on one Gram-positive strain (Staphylococcus aureus) ATCC25923, four Gram-negative strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Enterobacter cloacae 13047, and Klebsiella pneumoniae ATCC 13883), and one diploid fungus (Candida albicans ATCC SC5314). Levofloxacin, amikacin, and fluconazole were used as standards. Filter paper disc method was used to evaluate the microbial activity.

3.1. Methodology

10 gm of nutrient agar broth was dissolved in 400 mL of distilled water. The media was autoclaved at 120°C for 2 hrs. The media was poured in sterilized petri plates up to 40 mL and then covered the plates with lids. The agar was allowed to set and harden. Filter papers of 5 mm diameter were cut and dipped in dilution of 100 μg/mL of each sample that is, 3, 4a–4i, 5a–5h, and standards, and were placed on seeded agar. All stock solutions of tested compounds were made in DMSO. The bacterial culture was kept at 37°C, and fungal plates were kept at 18°C for 3-4 days. After incubation, the diameter of clear zone around the discs was measured and compared against zone of inhibition formed by solutions of known concentrations of standard antibiotics. All the tests were carried out in triplicate, and the results were averaged out.

4. Results and Discussion

4.1. Chemistry

The 1,2,4-triazole (3) was synthesized by the fusion of N-phthaloylglycine and thiocarbohydrazide by the method reported earlier by our group [27]. The Schiff bases were synthesized by refluxing the triazole (3) with corresponding aldehydes in glacial acetic acid; Figure 2 describes the synthetic scheme. All the synthesized compounds were characterized by spectroscopic analysis. In the H NMR spectra of Schiff bases (4a–4i), the two protons of CH₂ group of glycine gave a singlet in the range of 4.90–5.10 ppm. The most downfield signal around 14 ppm (NH) proves that the triazole exists in thione form when in solution. The proton of azomethine linkage (N=CH) gave a singlet downfield around 9.9–10.2 ppm. The chemical shifts for the four protons of phthalimido group are in the range of 7.89–7.79 ppm as a multiplet due to AA′BB′ spin system, whereas the para-disubstituted Schiff bases (4f–4i) give a doublet for each proton characteristic of an AB spin system in the range of 8.66–7.33 ppm. In C-13, the signal around 163 ppm is of carbonyl carbon of imide (C=O), and most down field signals around 167 ppm are for thione form (C=S). All other carbons are also well justified. The molecular ion could be seen in all the Schiff bases (4a–4i). In the IR spectra, characteristic absorption bands are visible for C=S and C=N groups in the range of 1110–1120 cm⁻¹ and 1590–1600 cm⁻¹, respectively. The absorption for the C=O of cyclic amide appears around 1770 cm⁻¹ in all compounds.

Figure 2: Scheme for the synthesis of (5a–5h).

The Schiff bases (4a–4h) were reacted with formaldehyde in presence of morpholine to obtain the corresponding Mannich bases (5a–5h). The ¹H NMR spectra showed identical chemical shifts for the two methylene protons of phthalimidomethylene and the two morpholino methylene protons. The four protons either appeared as a one singlet in case of 5a, 5e, and 5h or two singlets with a chemical shift difference of only 0.01–0.03 ppm as in 5d, 5f, and 5g. The methylenes of morpholine appeared as a set of two broad signals as shown in Figure 3. The methylenes of morpholine constitute A2 M₂ spin system which gives complex second order splitting pattern and is not easy to interpret. In case of morpholine ring, the rapid ring flipping at room temperature makes axial and equatorial protons of morpholine ring almost equivalent; therefore a broad signal showing some splitting as well is observed; however, it cannot be called a triplet as neither line intensity nor coupling constant values are justified for triplet. The C-13 data IR and mass spectral data are consistent with the structure.

Figure 3: ¹H NMR signals for methylene protons of morpholine group.

4.2. Antimicrobial Activity Test

The antimicrobial activity of the 1,2,4-triazole (3) and the nine Schiff bases (4a–4i) derived from triazole (3) were tested on a Gram-positive strain (Staphylococcus aureus), four Gram-negative strains, (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella pneumoniae), and one diploid fungus (Candida albicans). Levofloxacin, amakacine, and fluconazole were used as standards. A comparison of the microbial activity of the triazole (3) and its Schiff base derivatives (4a–4i) has been made. Furthermore, the Mannich bases (5a–5h) were also screened against the above mentioned strains. The results are shown in Table 1. The triazole (3) did not show any significant activity against the strains mentioned here; however, the introduction of azomethine linkage in all cases has improved the antimicrobial activity exceptions being 4h and 4i, where the activity remained almost the same. The best activity comparable to Levofloxacin was shown by 4b and 4e against Enterobacter cloacae, whereas compounds (4a, 4c, 4d, and 4g) have displayed moderate activity against the same bacterial strain. 4a and 4b have demonstrated good activity and 4c, 4g, and 4f moderate activity against Klebsiella pneumoniae with reference to levofloxacin and amikacin. Compounds (4b, 4c, 4d, 4f, and 4g) have displayed moderate activity against fungal strain, Candida albicans, in comparison to fluconazole. It has been observed [23] that the introduction of morpholine or piprazine ring increases the antimicrobial activity in many heterocyclic systems. For instance, itraconazole, eperezolid, and linezolid antibiotics possess a morpholine or piperazine ring. These ring functions increase the solubility in aqueous solvents when transformed into iminium salts, thus increasing the bioavailability of the compound. Keeping this in mind a morpholine group was introduced in compounds (4a–4h) the resulting Mannich bases (5a–5h) were also tested against the above mentioned six strains. In general antimicrobial activity in all cases significantly increased except for Enterobacter cloacae, where loss of activity was observed. Compound (5h) showed comparable activity to levofloxacin against Escherichia coli and Klebsiella pneumoniae. 5b, 5d, 5e, and 5f showed activity very close to levofloxacin against Pseudomonas aeruginosa. 4d showed compareable activity to levofloxacin, however, its Mannich base lost all activity. 5d, 5e, and 5h showed significant increase in antimicrobial activity against S. aureus.

Table 1: In vitro antimicrobial screening of compounds (mm) conc. 100 µg/mL.

Compound no.
Microorganisms and inhibition zone (mm)

Escherichia coli
Pseudomonas aeruginosa
Enterobacter cloacae
Klebsiella pneumoniae
Staphylococcus aureus
Candida albicans

3
7.00
8.00
9.68
9.40
7.00
9.95

4a
10.25
8.60
11.65
12.35
9.10
10.00

4b
8.52
9.65
14.42
11.20
8.10
11.75

4c
6.75
8.10
8.00
10.20
7.25
11.25

4d
7.38
9.30
9.60
9.83
8.00
10.76

4e
7.30
8.37
12.20
9.42
7.67
9.72

4f
8.21
8.43
9.33
10.70
9.42
10.45

4g
7.22
10.90
7.30
7.10
7.10
12.30

4h
6.55
6.95
7.30
7.44
8.0
7.82

4i
7.1
8.11
8.63
7.50
7.43
10.28

5a
13.2
13.20
—
11.10
12.60
14.30

5b
13.0
14.10
—
11.70
11.60
15.10

5c
7.00
10.00
9.00
11.00
11.00
13.20

5d
11.60
13.80
—
12.40
14.00
12.40

5e
12.50
14.80
—
12.00
14.10
13.70

5f
11.30
14.10
—
11.10
12.20
13.20

5g
—
—
—
—
10.00
12.20

5h
14.70
11.40
—
13.20
14.20
14.60

Levofloxacin
16.50
13.85
14.80
13.95
18.32
—

Fluconazole
—
—
—
—
—
17.25

Amikacin
17.70
12.75
16.30
14.20
19.20
—

All compounds (5a–5h) showed improved activity against fungal strain Candida albicans; however, 5b demonstrated best activity, and 5a and 5h also showed good activity. The results show that the compound 5h is the most promising among the tested compounds. Our results strengthen the earlier findings by others that the presence of morpholine ring in heterocyclic molecules increases the antimicrobial activity. In conclusion the Mannich bases with electron donating substituents (–OH, –OCH₃) on phenyl ring 5b and 5h or with halogens on phenyl ring (5e and 5f) showed best activity among the tested compounds. The results are promising and show that the fine tuning of the structures can lead to some new antibacterial compounds.

5. Conclusion

A series of novel Schiff bases and their Mannich bases were synthesized to study the structure activity relationship. All the synthesized compounds were screened for their antimicrobial activities against six strains. In general the antimicrobial activity increased remarkably on the introduction of azomethine functionality in parent triazole (3). The antimicrobial activity further improved when morpholine group was added to them except for Enterobacter cloacae, where loss of activity was observed. The results show that the compound (5h) is the most promising among the tested compounds. Our results strengthen the earlier findings by others that the presence of morpholine ring in heterocyclic molecules increases the antimicrobial activity. In conclusion the Mannich bases with electron donating substituents (–OH, –OCH₃) on phenyl ring (5b and 5h) or with halogens on phenyl ring (5e and 5f) showed the best activity among the tested compounds. The results are promising and show that the fine tuning of the structures (5a, 5b, 5e, 5f, and 5h) can lead to some new antimicrobial compounds.

References

M. Koca, S. Servi, C. Kirilmis et al., “Synthesis and antimicrobial activity of some novel derivatives of benzofuran: part 1. Synthesis and antimicrobial activity of (benzofuran-2-yl)(3-phenyl-3-methylcyclobutyl) ketoxime derivatives,” European Journal of Medicinal Chemistry, vol. 40, no. 12, pp. 1351–1358, 2005.

C. G. Bonde and N. J. Gaikwad, “Synthesis and preliminary evaluation of some pyrazine containing thiazolines and thiazolidinones as antimicrobial agents,” Bioorganic & Medicinal Chemistry, vol. 12, no. 9, pp. 2151–2161, 2004.

D. Yu and G. Huiyuan, “Synthesis and antibacterial activity of linezolid analogues,” Bioorganic & Medicinal Chemistry Letters, vol. 12, no. 6, pp. 857–859, 2002.

V. J. Ram and A. J. Vlietinck, “Chemotherapeutical agents. VII. Synthesis and pesticidal activities of sulphides and sulphones derived from bis[4-aryl-1, 2, 4-triazoline-5-thione-3-yl]alkane and 5-phenyl-1, 3, 4-oxadiazole-2-thione,” Journal of Heterocyclic Chemistry, vol. 25, no. 1, pp. 253–256, 1988.

R. W. Sidwell, J. H. Huffman, G. P. K. Lois, et al., “Broad-spectrum antiviral activity of virazole: 1-β-D-ribofuranosyl-1, 2,4-triazole-3-carboxamide,” Science, vol. 177, no. 4050, pp. 705–706, 1972.

J. T. Witkowski, R. K. Robins, G. P. Khare, and R. W. Sidwell, “Synthesis and antiviral activity of 1,2,4-triazole-3-thiocarboxamide and 1,2,4-triazole-3-carboxamidine ribonucleosides,” Journal of Medicinal Chemistry, vol. 16, no. 8, pp. 935–937, 1973.

M. Clemons, R. E. Coleman, and S. Verma, “Aromatase inhibitors in the adjuvant setting: bringing the gold to a standard?” Cancer Treatment Reviews, vol. 30, no. 4, pp. 325–332, 2004.

S. Budavari, The Merck Index, Merck, White House Station, NJ, USA, 12th edition, 1996.

J. Haber, “Present status and perspectives on antimycotics with systemic effects,” Journal of Czech Physicians, vol. 140, no. 19, pp. 596–604, 2001.

K. Walczak, A. Gondela, and J. Suwiński, “Synthesis and anti-tuberculosis activity of N-aryl-C-nitroazoles,” European Journal of Medicinal Chemistry, vol. 39, no. 10, pp. 849–853, 2004.

B. S. Holla, K. N. Poojary, B. S. Rao, et al., “New bis-amino-mercaptotriazoles and bis-triazolothiadiazoles as possible anticancer agents,” European Journal of Medicinal Chemistry, vol. 37, no. 6, pp. 511–517, 2002.

B. S. Holla, B. Veerendra, M. K. Shivananda, et al., “Synthesis characterization and anticancer activity studies on some Mannich bases derived from 1,2,4-triazoles,” European Journal of Medicinal Chemistry, vol. 38, no. 7-8, pp. 759–767, 2003.

M. Amir and K. Shikha, “Synthesis and anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation activities of some new 2-[(2,6-dichloroanilino) phenyl]acetic acid derivatives,” European Journal of Medicinal Chemistry, vol. 39, no. 6, pp. 535–545, 2004.

A. Almasirad, S. A. Tabatabai, M. Faizi et al., “Synthesis and anticonvulsant activity of new 2-substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles and 1,2,4-triazoles,” Bioorganic & Medicinal Chemistry Letters, vol. 14, no. 24, pp. 6057–6059, 2004.

K. El Akri, K. Bougrin, J. Balzarini, A. Faraj, and R. Benhida, “Efficient synthesis and in vitro cytostatic activity of 4-substituted triazolyl-nucleosides,” Bioorganic & Medicinal Chemistry Letters, vol. 17, no. 23, pp. 6656–6659, 2007.

M. S. Karthikeyan, B. S. Holla, and N. S. Kumari, “Synthesis and antimicrobial studies of novel dichlorofluorophenyl containing aminotriazolothiadiazines,” European Journal of Medicinal Chemistry, vol. 43, no. 2, pp. 309–314, 2008.

M. T. H. Tarafder, A. Kasbollah, N. Saravanan, et al., “S-methyldithiocarbazate and its Schiff bases: evaluation of bondings and biological properties,” The Journal of Biochemistry, Molecular Biology and Biophysics, vol. 6, no. 2, pp. 85–91, 2002.

P. Vicini, A. Geronikaki, M. Incerti et al., “Synthesis and biological evaluation of benzo[d]isothiazole, benzothiazole and thiazole Schiff bases,” Bioorganic & Medicinal Chemistry, vol. 11, no. 22, pp. 4785–4789, 2003.

O. Bekircan, B. Kahveci, and M. Küçük, “Synthesis and anticancer evaluation of some new unsymmetrical 3,5-diaryl-4H-1,2,4-triazole derivatives,” Turkish Journal of Chemistry, vol. 30, no. 1, pp. 29–40, 2006.

O. Bekircan and H. Bektas, “Synthesis of Schiff and Mannich bases of isatin derivatives with 4-amino-4,5-dihydro-1H-1,2,4-triazole-5-ones,” Molecules, vol. 13, no. 9, pp. 2126–2135, 2008.

B. Kahveci, O. Bekircan, and Ş. A. Karaoǧlu, “Synthesis and antimicrobial activity of some 3-alkyl-4-(arylmethyleneamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones,” Indian Journal of Chemistry B, vol. 44, no. 12, pp. 2614–2617, 2005.

T. Plech, M. Wujec, A. Siwek, et al., “Synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their Mannich bases bearing 3-chlorophenyl moiety,” European Journal of Medicinal Chemistry, vol. 46, no. 1, pp. 241–248, 2011.

H. Bayrak, A. Demirbas, S. A. Karaoglu, and N. Demirbas, “Synthesis of some new 1,2,4-triazoles, their Mannich and Schiff bases and evaluation of their antimicrobial activities,” European Journal of Medicinal Chemistry, vol. 44, no. 3, pp. 1057–1066, 2009.

M. Ashok, B. S. Holla, and B. Poojary, “Convenient one pot synthesis and antimicrobial evaluation of some new Mannich bases carrying 4-methylthiobenzyl moiety,” European Journal of Medicinal Chemistry, vol. 42, no. 8, pp. 1095–1101, 2007.

M. S. Karthikeyan, D. J. Prasad, B. Poojary, et al., “Synthesis and biological activity of Schiff and Mannich bases bearing 2,4-dichloro-5-fluorophenyl moiety,” Bioorganic & Medicinal Chemistry, vol. 14, no. 22, pp. 7482–7489, 2006.

L. F. Audrieth, E. S. Scott, and P. S. Kippur, “Hydrazine derivatives of the carbonic and thiocarbonic acids. I. The preparation and properties of thiocarbohydrazide,” The Journal of Organic Chemistry, vol. 19, no. 5, pp. 733–741, 1954.

U. Yunus, M. K. Tahir, M. H. Bhatti, N. Yousaf, and M. Helliwell, “2-(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-ylmethyl)isoindoline-1, 3-dione,” Acta Crystallographica E, vol. 64, no. 2, pp. o476–o477, 2008.