Thiothymine in the synthesis of triazolotriazine and hydrazone compounds: evaluation of antimicrobial activity of some hydrazones
Hamdi M Hassaneen1, Fatma M Saleh1, Yasmin S Mohamed2, Enas M Awad3
1 Department of Chemistry, Faculty of Science, Cairo University, Dokki, Giza, Egypt
2 National Organization for Drug Control and Research, Dokki, Giza, Egypt
3 Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, Egypt
Date of Submission 05-May-2018
Date of Acceptance 05-Jul-2018
Date of Web Publication 07-Dec-2018
Prof. Hamdi M Hassaneen
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613
DOI: 10.4103/epj.epj_17_18
Background and objectives Fused triazine derivatives are known to be a very important class of compounds that have significant biological activities. Also, they showed a broad spectrum against leukemic cell lines and cancer cells. Our objective after such a study is, on one hand, to shed some light on the synthesis of triazolotriazine and hydrazone derivatives from the thiothymine compound and, on the other hand, to explore the biological activity of the isolated products from such reactions.
Materials and methods Reaction of thiothymine 3 and hydrazonoyl chloride 1 in refluxing chloroform in the presence of triethylamine yielded triazolotriazine derivatives 11. The hydrazino derivative 14 underwent condensation reactions with aldehydes, pyruvic acid, ethyl pyruvate, and ketones to give the corresponding hydrazone derivatives 15 and 16, respectively. Antimicrobial activities of some newly synthesized compounds were studied using the diffusion plate method.
Results and conclusion Both antibacterial and antifungal activities of the new synthesized compounds 14, 15h, and 16a–d were studied. Also, the minimum inhibitory concentration values for the highly efficient antibacterial compounds using the most sensitive microorganisms were determined.
Keywords: antimicrobial activities, cycloaddition reaction, hydrazone, hydrazonoyl halides, thiothymine
Hassaneen HM, Saleh FM, Mohamed YS, Awad EM. Thiothymine in the synthesis of triazolotriazine and hydrazone compounds: evaluation of antimicrobial activity of some hydrazones. Egypt Pharmaceut J 2018;17:180-9
Hassaneen HM, Saleh FM, Mohamed YS, Awad EM. Thiothymine in the synthesis of triazolotriazine and hydrazone compounds: evaluation of antimicrobial activity of some hydrazones. Egypt Pharmaceut J [serial online] 2018 [cited 2019 Jul 19];17:180-9. Available from: http://www.epj.eg.net/text.asp?2018/17/3/180/247069
Hydrazonoyl halides 1 represent a unique class of compounds which underwent 1,3-base catalyzed elimination reaction to give nitrilimines 2 (Chart 1) [1],[2],[3],[4],[5],[6],[7]. The latter are versatile synthetic intermediates especially useful for 1,3-dipolar cycloaddition reaction that have been used in the synthesis of numerous heterocycles [8],[9],[10],[11],[12],[13]. In continuation of our study on the chemistry of hydrazonoyl halides 1 [6],[7],[8],[9],[10],[11],[12],[13], we report here a synthesis of triazolotriazine and hydrazone derivatives from the thiothymine compound. Evaluation of antimicrobial activity against some microorganisms was investigated.
Melting points were determined on a Stuart melting point apparatus and are uncorrected. The IR spectra were measured as KBr pellets on an FTIR Bruker-Vector 22 spectrophotometer (Manasquan, New Jersey, United States). The 1H NMR and 13C NMR spectra were recorded in CDCl3 or [D6] dimethyl sulfoxide (DMSO) on a Varian Mercury VXR 300 spectrometer (300 MHz for 1H NMR and 75 MHz for 13C NMR) using TMS as internal standard. Mass spectra were measured on a Shimadzu GCMS-Q-1000 EX mass spectrometer at 70 eV. The elemental analyses were carried out at the Microanalytical Center, Cairo University, using Automated analyzer CHNS (Vario EL III; Elementar, Hanau, Germany). The hydrazonoyl chloride 1, 6-methyl-3-thioxo-3,4-dihydro-1, 2, 4-triazin-5(2H)-one 3, 6-methyl-3-(methylthio)-1, 2, 4-triazin-5(4H)-one 13, and hydrazine derivative 14 were prepared using the reported procedures [12],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23].
General procedure for the synthesis of 6-methyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazine derivatives (11A–I)
To a mixture of hydrazonoyl chloride 1 (6.0 mmol) and 6-methyl-3-thioxo-3,4-dihydro-1, 2, 4-triazin-5(2H)-one 3 (0.9 g, 6.0 mmol) in chloroform (20 ml), triethylamine (0.6 ml, 6.0 mmol) was added at room temperature. The reaction mixture was refluxed for 6 h and then cooled, the excess chloroform was removed under reduced pressure and the residue was treated with ethanol (10 ml). The solid that precipitated was collected by filteration and crystallized from a suitable solvent to give compounds 11A–I. The compounds prepared together with their physical properties are listed below:
3-Acetyl-6-methyl-1-phenyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Aa)
Yellow crystals: mp 242°C (CH3CN), yield (1.35 g, 80%). IR (KBr): ν=1709 and 1670 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.32 (s, 3H, CH3), 2.71 (s, 3H, CH3CO) and 7.46–8.06 (m, 5H). 13C NMR (75 MHz, DMSO-d6): δ=18.1, 27.5, 120.8, 128.2, 129.5, 135.6, 138.7, 148.4, 155.3, 160.4, 185.1. MS, m/z (%): 269 (M+, 5.2), 77 (100). C13H11N5O2 (269.09): C, 57.99; H, 4.12; N, 26.01. Found: C, 57.83; H, 4.15; N, 26.02.
3-Acetyl-6-methyl-1-(p-tolyl)-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Ab)
Yellow crystals: mp 232°C (DMF), yield (1.35 g, 76%). IR (KBr): ν=1712 and 1674 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.32 (s, 3H, CH3), 2.40 (s, 3H, CH3C6H4), 2.71 (s, 3H, CH3CO), 7.43 (d, 2H) and 7.90 (d, 2H). 13C NMR (75 MHz, DMSO-d6): δ=18.1, 20.6, 27.5, 109.2, 121.0, 129.8, 133.2, 137.8, 148.2, 155.2, 160.5, 185.2. MS, m/z (%): 283 (M+, 50.2), 148 (100). C14H13N5O2 (283.11): C, 59.36; H, 4.63; N, 24.72. Found: C, 59.20; H, 4.65; N, 24.76.
3-Acetyl-1-(4-chlorophenyl)-6-methyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triaz-in-5(1H)-one (11Ac)
Yellow crystals: mp 264°C (DMF), yield (1.41 g, 74%). IR (KBr): ν=1715 and 1665 (2CO) cm−1. MS, m/z (%): 303 (M+, 11.6), 247 (100). C13H10ClN5O2 (303.05): C, 51.41; H, 3.32; Cl, 11.67; N, 23.06. Found: C, 51.32; H, 3.36; Cl, 11.69; N, 23.09.
Ethyl 6-methyl-5-oxo-1-phenyl-1,5-dihydro-[1],[2],[4]triazolo [3,4-c][1],[2],[4]-triazine-3-carboxylate (11Ba)
Yellow crystals: mp 282°C (CH3CN), yield (1.35 g, 72%). IR (KBr): ν=1741 and 1672 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.37–1.41 (t, 3H, CH3CH2), 2.33 (s, 3H, CH3), 4.47–4.54 (q, 2H, CH2CH3) and 7.48–8.01 (m, 5H). 13C NMR (75 MHz, DMSO-d6): δ=13.9, 18.1, 62.8, 121.1, 128.1, 129.4, 134.5, 135.6, 148.4, 154.3, 155.2, 160.5. MS, m/z (%): 299 (M+, 7.4), 91 (100). C14H13N5O3 (299.10): C, 56.18; H, 4.38; N, 23.40. Found: C, 56.02; H, 4.36; N, 23.47.
Ethyl 6-methyl-5-oxo-1-(p-tolyl)-1,5-dihydro-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazine-3-carboxylate (11Bb)
White crystals: mp 230°C (DMF), yield (1.35 g, 69%). IR (KBr): ν=1748 (COOEt), 1672 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.38–1.40 (t, 3H, CH3CH2), 2.32 (s, 3H, CH3), 2.39 (s, 3H, CH3C6H4), 4.48–4.52 (q, 2H, CH2CH3), 7.40 (d, 2H) and 7.85 (d, 2H). 13C NMR (75 MHz, DMSO-d6): δ=13.9, 18.6, 20.6, 62.8, 110.0, 120.6, 129.6, 133.2, 134.3, 137.8, 154.3, 155.2, 160.5. MS, m/z (%): 313 (M+, 14.8), 105 (100). C15H15N5O3 (313.12): C, 57.50; H, 4.83; N, 22.35. Found: C, 57.42; H, 4.86; N, 22.38.
Ethyl 1-(4-chlorophenyl)-6-methyl-5-oxo-1,5-dihydro-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazine-3-carboxylate (11Bc)
White crystals: mp 202°C (DMF), yield (1.53 g, 73%). IR (KBr): ν=1751 and 1670 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.36–1.41 (t, 3H, CH3CH2), 2.33 (s, 3H, CH3), 4.47–4.53 (q, 2H, CH2CH3), 7.69 (d, 2H) and 8.04 (d, 2H). 13C NMR (75 MHz, DMSO-d6): δ=13.8, 18.0, 62.9, 122.4, 129.5, 132.3, 134.5, 134.6, 148.3, 154.1, 155.3, 160.3. MS, m/z (%): 333 (M+, 9.9), 334 (M+1, 2.5), 335 (M+2, 3.5), 125 (100). C14H12ClN5O3 (333.06): C, 50.39; H, 3.62; Cl, 10.62; N, 20.99. Found: C, 50.20; H, 3.65; Cl, 10.64; N, 20.95.
3-Benzoyl-6-methyl-1-phenyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Da)
Yellow crystals: mp 224°C (DMF-Alc), yield (1.65 g, 79%). IR (KBr): ν=1715, 1663 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.32 (s, 3H, CH3) and 7.48–8.24 (m, 10H). 13C NMR (75 MHz, DMSO-d6): δ=18.0, 121.2, 128.1, 128.9, 129.5, 130.5, 134.7, 135.1, 135.7, 138.8, 148.2, 155.2, 160.7, 179.7. MS, m/z (%): 331 (M+, 37.7), 80 (100). C18H13N5O2 (331.11): C, 65.25; H, 3.95; N, 21.14. Found: C, 65.12; H, 3.96; N, 21.19.
3-Benzoyl-6-methyl-1-(p-tolyl)-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5-(1H)-one (11Db)
Yellow crystals: mp 234°C (DMF), yield (1.63 g, 75%). IR (KBr): ν=1720, 1660 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.31 (s, 3H, CH3), 2.38 (s, 3H, CH3C6H4) and 7.38–8.23 (m, 9H). MS, m/z (%): 345 (M+, 4.6), 105 (100). C19H15N5O2 (345.12): C, 66.08; H, 4.38; N, 20.28. Found: C, 65.92; H, 4.36; N, 20.29.
6-Methyl-5-oxo-N-phenyl-1-(p-tolyl)-1,5-dihydro-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazine-3-carboxamide (11Eb)
White crystals: mp 250°C (DMF), yield (1.63 g, 72%). IR (KBr): ν=3296 (NH), 1709 and 1660 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.34 (s, 3H, CH3), 2.39 (s, 3H, CH3C6H4), 7.19–7.98 (m, 9H) and 10.94 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=18.0, 20.6, 120.5, 120.9, 125.0, 128.9, 129.7, 133.4, 136.9, 137.3, 137.5, 147.9, 155.1, 157.1, 160.6. MS, m/z (%): 360 (M+, 42.7), 63 (100). C19H16N6O2 (360.13): C, 63.33; H, 4.48; N, 23.32. Found: C, 63.20; H, 4.49; N, 23.36.
6-Methyl-1-phenyl-3-(thiophene-2-carbonyl)-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Fa)
Red crystals: mp 240°C (DMF), yield (1.42 g, 67%). IR (KBr): ν=1670 and 1653 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.33 (s, 3H, CH3) and 7.39–8.40 (m, 8H). MS, m/z (%): 337 (M+, 7.5), 80 (100). C16H11N5O2S (337.06): C, 56.97; H, 3.29; N, 20.76; S, 9.50. Found: C, 56.85; H, 3.32; N, 20.78; S, 9.54.
6-Methyl-3-(thiophene-2-carbonyl)-1-(p-tolyl)-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Fb)
Yellow crystals: mp 236°C (DMF-Alc), yield (1.39 g, 63%). IR (KBr): ν=1673 and 1657 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.32 (s, 3H, CH3), 2.40 (s, 3H, CH3C6H4) and 7.39–8.38 (m, 7H). 13C NMR (75 MHz, DMSO-d6): δ=18.0, 20.6, 120.6, 121.0, 129.4, 129.8, 133.3, 137.8, 138.2, 138.9, 140.1, 146.1, 155.2, 158.6, 160.4. MS, m/z (%): 351 (M+, 68.6), 148 (100). C17H13N5O2S (351.08): C, 58.11; H, 3.73; N, 19.93; S, 9.12. Found: C, 58.02; H, 3.78; N, 19.99; S, 9.10.
3-(Furan-2-carbonyl)-6-methyl-1-phenyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]tri-azin-5(1H)-one (11Ga)
Orange crystals: mp 250°C (DMF), yield (1.23 g, 61%). IR (KBr): ν=1670 and 1661 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.32 (s, 3H, CH3) and 6.90–8.31 (m, 8H). MS, m/z (%): 321 (M+, 66.3), 168 (100). C16H11N5O3 (321.09): C, 59.81; H, 3.45; N, 21.80. Found: C, 59.73; H, 3.46; N, 21.83.
6-Methyl-1-phenyl-3-styryl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Ha)
Yellow crystals: mp 198°C (DMF), yield (1.42 g, 69%). IR (KBr): ν=1670 (CO) cm−1. MS, m/z (%): 329 (M+, 15.8), 287 (100). C19H15N5O (329.13): C, 69.29; H, 4.59; N, 21.26. Found: C, 69.18; H, 4.56; N, 21.29.
6-Methyl-1,3-diphenyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (11Ia)
White crystals: mp 228°C (CH3CN), yield (1.39 g, 73%). IR (KBr): ν=1678 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.33 (s, 3H, CH3) and 7.39–8.22 (m, 10H). 13C NMR (75 MHz, DMSO-d6): δ=18.2, 120.3, 123.6, 127.2, 127.9, 128.9, 129.3, 131.6, 136.2, 154.4, 160.6. MS, m/z (%): 303 (M+, 47.2), 77 (100). C17H13N5O (303.11): C, 67.32; H, 4.32; N, 23.09. Found: C, 67.26; H, 4.35; N, 23.11.
General procedure for the synthesis of 3-(3-(dimethylamino)acryloyl)-6-methyl-1-aryl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (12a–c)
To compounds 11a–c (30 mmol) in dioxane (50 ml), dimethylformamide dimethyl acetal (DMF-DMA) (4 ml, 30 mmol) was added. The reaction mixture was refluxed for 6 h.The solid product was collected and crystallized from a suitable solvent to afford compounds 12a–c.
3-(3-(Dimethylamino)acryloyl)-6-methyl-1-phenyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (12a)
Orange crystals: mp 228°C (DMF), yield (7.0 g, 72%). IR (KBr): ν=1706 and 1666 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.48 (s, 3H, CH3), 3.00 (s, 3H, CH3N), 3.23 (s, 3H, CH3N), 5.8 (d, 1H, CH) and 7.26–8.15 (m, 6H, Ph and CH). 13C NMR (75 MHz, DMSO-d6): δ=18.9, 37.9, 45.8, 89.0, 121.2, 126.3, 128.1, 129.5, 136.2, 147.9, 155.8, 156.6, 161.6, 164.1. MS, m/z (%): 324 (M+, 7.5), 80 (100). C16H16N6O2 (324.13): C, 59.25; H, 4.97; N, 25.91. Found: C, 59.12; H, 4.99; N, 25.93.
3-(3-(Dimethylamino)acryloyl)-6-methyl-1-(p-tolyl)-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazin-5(1H)-one (12b)
Orange crystals: mp 240°C (DMF), yield (6.5 g, 64%). IR (KBr): ν=1700 and 1669 (2CO) cm−1. MS, m/z (%): 338 (M+, 11.8), 80 (100). C17H18N6O2 (338.15): C, 60.34; H, 5.36; N, 24.84. Found: C, 60.26; H, 5.38; N, 24.89.
1-(4-Chlorophenyl)-3-(3-(dimethylamino)acryloyl)-6-methyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]tri-azin-5(1H)-one (12c)
Orange crystals: mp 252°C (DMF), yield (6.4 g, 60%). IR (KBr): ν=1698 and 1668 (2CO) cm−1. MS (EI, 70 eV) m/z (%): 358 (M+, 12.1), 359 (M+1, 3.0), 360 (M+2, 4.2) 100 (100). C16H15ClN6O2 (358.09): C, 53.56; H, 4.21; Cl, 9.88; N, 23.42. Found: C, 53.43; H, 4.25; Cl, 9.85; N, 23.46.
General procedure for the synthesis of 3-(2-arylidenehydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15a–h)
A mixture of 3-hydrazinyl-6-methyl-1, 2, 4-triazin-5(2H)-one 14 (0.7 g, 5.0 mmol) and the appropriate aldehyde (5.0 mmol) in ethanol (30 ml) was refluxed for 6 h in the presence of few drops of acetic acid. The reaction mixture was cooled, the precipitate that got separated was collected, and crystallized from the suitable solvent to give the corresponding 3-(2-arylidenehydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15a–h).
6-Methyl-3-(2-(4-methylbenzylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (15a)
White crystals: mp 292°C (DMF), yield (0.89 g, 74%). IR (KBr): ν=3330 and 3221 (2NH) and 1678 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.10 (s, 3H, CH3), 2.33 (s, 3H, CH3C6H4), 7.2 (d, 2H), 7.80 (d, 2H), 8.04 (s, 1H, CH), 11.51 (s, 1H, NH) and 12.59 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=16.9, 21.0, 127.4, 129.2, 131.3, 139.6, 144.8, 147.9, 153.6, 162.3. MS, m/z (%): 243 (M+, 100). C12H13N5O (243.11): C, 59.25; H, 5.39; N, 28.79. Found: C, 59.16; H, 5.43; N, 28.82.
3-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15b)
Yellow crystals: mp 296−298°C (DMF), yield (1.02 g, 69%). IR (KBr): ν=3339 and 3213 (2NH) and 1665 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.09 (s, 3H, CH3), 7.38–8.44 (m, 3H, aromatic), 8.47 (s, 1H, CH), 11.81 (s, 1H, NH) and 12.72 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=16.9, 127.5, 128.9, 130.3, 133.4, 134.8, 139.5, 148.2, 153.5, 162.2, 162.8. MS, m/z (%): 297 (M+, 31.1), 298 (M+1, 7.83), 299 (M+2, 20.0), 123 (100). C11H9Cl2N5O (297.02): C, 44.32; H, 3.04; Cl, 23.78; N, 23.49. Found: C, 44.16; H, 3.08; Cl, 23.80; N, 23.53.
3-(2-Ethylidenehydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15c)
White crystals: mp 248–250°C (DMF), yield (0.59 g, 71%). IR (KBr): ν=3327 and 3214 (2NH) and 1666 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.89 (d, 3H, CH3), 2.13 (s, 3H, CH3), 7.42 (q, 1H, CH), 11.03 (s, 1H, NH) and 12.36 (s, 1H, NH). MS (EI, 70 eV) m/z (%): 167 (M+, 46.7), 152 (100). C6H9N5O (167.08): C, 43.11; H, 5.43; N, 41.89. Found: C, 43.02; H, 5.46; N, 41.95.
6-Methyl-3-(2-(2-methylpropylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (15d)
White crystals: mp 218–220°C (DMF), yield (0.76 g, 79%). IR (KBr): ν=3335 and 3219 (2NH) and 1670 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.05 (d, 6H, (CH3)2CH), 2.06 (s, 3H, CH3), 2.44 (m, 1H, CH(CH3)2), 7.37 (d, 1H, CH), 11.13 (s, 1H, NH) and 12.27 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=16.8, 16.9, 19.6, 30.9, 147.8, 153.7, 154.2, 162.9. MS, m/z (%): 195 (M+, 22.1), 80 (100). C8H13N5O (195.11): C, 49.22; H, 6.71; N, 35.87. Found: C, 49.13; H, 6.75; N, 35.89.
3-(2-(Benzo[d][1],[3]dioxol-5-ylmethylene)hydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15e)
White crystals: mp 292–294°C (DMF), yield (1.02 g, 75%). IR (KBr): ν=3330 and 3210 (2NH) and 1660 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.10 (s, 3H, CH3), 6.07 (s, 2H, CH2), 6.92–7.95 (m, 3H), 7.98 (s, 1H, CH), 11.50 (s, 1H, NH) and 12.63 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=16.9, 101.4, 105.5, 108.0, 123.8, 128.6, 147.9, 148.0, 148.9, 153.6, 154.0, 162.2. MS, m/z (%): 273 (M+, 67.1), 134 (100). C12H11N5O3 (273.09): C, 52.75; H, 4.06; N, 25.63. Found: C, 52.64; H, 4.10; N, 25.68.
6-Methyl-3-(2-(pyridin-4-ylmethylene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (15f)
Yellow crystals: mp 296–298°C (DMSO), yield (0.82 g, 72%). IR (KBr): ν=3329 and 3207 (2NH) and 1663 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.12 (s, 3H, CH3), 7.87–8.04 (m, 4H), 8.61 (s, 1H, CH), 11.80 (s, 1H, NH) and 12.79 (s, 1H, NH). MS, m/z (%): 230 (M+, 34.5), 120 (100). C10H10N6O (230.09): C, 52.17; H, 4.38; N, 36.50. Found: C, 52.00; H, 4.42; N, 36.57.
6-Methyl-3-(2-(thiophen-2-ylmethylene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (15g)
Yellow crystals: mp 286–288°C (DMF), yield (0.81 g, 69%). IR (KBr): ν=3339 and 3210 (2NH) and 1676 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.09 (s, 3H, CH3), 7.11–7.68 (m, 3H), 8.27 (s, 1H, CH), 11.67 (s, 1H, NH) and 12.53 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=17.9, 121.3, 129.3, 131.1, 138.9, 148.3, 153.6, 154.1, 160.4. MS, m/z (%): 235 (M+, 61.5), 96 (100). C9H9N5OS (235.05): C, 45.95; H, 3.86; N, 29.77; S, 13.63. Found: C, 45.80; H, 3.90; N, 29.81; S, 13.68.
3-(2-(1-(1H-Indol-3-yl)ethylidene)hydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (15h)
White crystals: mp 270°C (DMF), yield (1.04 g, 74%). IR (KBr): ν=3334, 3280 and 3204 (3NH) and 1664 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.13 (s, 3H, CH3), 7.11–7.85 (m, 4H), 8.37 (s, 1H, CH), 8.47 (s, 1H, CH-Indole), 11.20 (s, 1H, NH-Indole), 11.61 (s, 1H, NH) and 12.27 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=17.0, 111.2, 111.5, 120.3, 122.6, 124.1, 130.6, 137.0, 143.2, 147.7, 153.3, 154.5, 163.0. C13H12N6O (268.3): C, 58.20; H, 4.51; N, 31.33. Found: C, 58.02; H, 4.46; N, 31.27.
General procedure for the synthesis of 6-methyl-3-(2-(1-aryl-ethyl-idene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16a–h)
A mixture of 3-hydrazinyl-6-methyl-1, 2, 4-triazin-5(2H)-one 14 (0.7 g, 5.0 mmol) and the appropriate ketone or pyruvic acid or ethyl pyruvate (5.0 mmol) in absolute ethanol (30 ml) was refluxed for 6 h in the presence of few drops of acetic acid. The reaction mixture in each case was cooled; the precipitate that separated was collected and crystallized from the suitable solvent to give the corresponding hydrazone derivatives 16a–h.
6-Methyl-3-(2-(1-phenylethylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16a)
White crystals: mp 246°C (dioxane), yield (0.95 g, 79%). IR (KBr): ν=3328 and 3210 (2NH) and 1669 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.11 (s, 3H, CH3), 2.49 (s, 3H, CH3), 7.38–8.05 (m, 5H, Ph), 10.98 (s, 1H, NH) and 12.66 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=13.9, 17.0, 120.6, 126.7, 129.2, 137.5, 147.9, 151.8, 153.7, 160.4. MS, m/z (%): 243 (M+, 12.0), 59 (100). C12H13N5O (243.27): C, 59.25; H, 5.39; N, 28.79. Found: C, 59.20; H, 5.41; N, 28.76.
6-Methyl-3-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16b)
Yellow crystals: mp 248°C (dioxane), yield (0.98 g, 81%). IR (KBr): ν=3324 and 3224 (2NH) and 1679 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.09 (s, 3H, CH3), 2.40 (s, 3H, CH3), 7.35–8.56 (m, 4H, pyridine), 11.00 (s, 1H, NH) and 12.69 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=12.1, 16.9, 121.2, 123.8, 124.5, 136.2, 138.4, 148.2, 151.3, 153.9, 162.6. MS, m/z (%): 244 (M+, 24.9), 78 (100). C11H12N6O (244.26): C, 54.09; H, 4.95; N, 34.41. Found: C, 54.13; H, 4.90; N, 34.39.
6-Methyl-3-(2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16c)
Yellow crystals: mp 286°C (DMF), yield (0.92 g, 76%). R (KBr): ν=3329 and 32208 (2NH) and 1672 (CO) cm−1. MS, m/z (%): 244 (M+, 34.9), 243 (100). C11H12N6O (244.26): C, 54.09; H, 4.95; N, 34.41. Found: C, 54.00; H, 4.99; N, 34.35.
6-Methyl-3-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16d)
Yellow crystal; mp 268°C (DMF), yield (0.94 g, 78%). IR (KBr): ν=3341 and 3215 (2NH) and 1664 (CO) cm−1. MS, m/z (%): 244 (M+, 10.3), 64 (100). C11H12N6O (244.26): C, 54.09; H, 4.95; N, 34.41. Found: C, 53.98; H, 4.89; N, 34.44.
6-Methyl-3-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)-1, 2, 4-triazin-5(2H)-one (16e)
White crystals: mp 260°C (DMF), yield (0.90 g, 73%). IR (KBr): ν=3334 and 3214 (2NH) and 1670 (CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.10 (s, 3H, CH3), 2.33 (s, 3H, CH3), 7.07–7.60 (m, 3H, thienyl), 11.10 (s, 1H, NH) and 12.38 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=14.9, 16.8, 120.6, 127.5, 128.5, 138.2, 146.6, 152.1, 153.8, 162.3. MS, m/z (%): 249 (M+, 27.71), 59 (100). C10H11N5OS (249.29): C, 48.18; H, 4.45; N, 28.09; S, 12.86. Found: C, 48.21; H, 4.42; N, 28.07; S, 12.89.
3-(2-(1-(9H-Fluoren-2-yl)ethylidene)hydrazinyl)-6-methyl-1, 2, 4-triazin-5(2H)-one (16f)
Yellow crystals: mp 288°C (DMF), yield (1.29 g, 79%). IR (KBr): ν=3338 and 3211 (2NH) and 1677 (CO) cm−1. MS, m/z (%): 331 (M+, 24.1), 205 (100). C19H17N5O (331.38): C, 68.87; H, 5.10; N, 21.13. Found: C, 68.90; H, 5.15; N, 21.10.
2-(2-(6-Methyl-5-oxo-2,5-dihydro-1, 2, 4-triazin-3-yl)hydrazono)propan-oic acid (16g)
White crystals: mp 236–238°C (DMF), yield (0.77 g, 65%). IR (KBr): ν=3578 (OH), 3484 and 3222 (2NH), 1729 and 1660 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=2.08 (s, 3H, CH3), 2.11 (s, 3H, CH3), 11.87 (s, 1H, NH), 12.33 (s, 1H, NH) and 12.84 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ=15.9, 16.8, 146.6, 151.0, 153.4, 164.5, 165.6. MS, m/z (%): 211 (M+, 30.6), 126 (100). C7H9N5O3 (211.20): C, 39.81; H, 4.30; N, 33.16. Found: C, 39.89; H, 4.28; N, 33.13.
Ethyl 2-(2-(6-methyl-5-oxo-2,5-dihydro-1, 2, 4-triazin-3-yl)hydrazono)-propanoate (16h)
White crystals: mp 198–200°C (DMF), yield (0.77 g, 65%). IR (KBr): ν=3460 and 3214 (2NH), 1718 and 1672 (2CO) cm−1. 1H NMR (300 MHz, DMSO-d6): δ=1.27–1.31 (t, 3H, CH3CH2), 2.05 (s, 3H, CH3), 2.10 (s, 3H, CH3), 4.18–4.25 (q, 2H, CH2CH3), 11.25 (s, 1H, NH) and 12.45 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ=13.0, 13.9, 16.7, 61.0, 145.1, 150.1, 153.5, 161.8, 164.1. MS (EI, 70 eV) m/z (%): 239 (M+, 31.3), 166 (100). C9H13N5O3 (239.10): C, 45.19; H, 5.48; N, 29.27. Found: C, 45.02; H, 5.46; N, 29.30.
Antibacterial activity was investigated in-vitro on Gram-positive bacteria Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600) and on Gram-negative bacteria Escherichia More Details coli (ATCC 1175) and Pseudomonas aeruginosa (ATCC 10145). Moreover, antifungal activity against Aspergillus flavus (ATCC 15442) and Candida albicans (ATCC 26555) was investigated. All these microorganisms were performed at the Microanalytical Center, Cairo University.
The antibacterial and antifungal activity assays were carried out using the diffusion plate method [24],[25]. A bottomless cylinder containing a measured quantity (1 ml, mg/ml) of the sample is placed on a plate (9 cm diameter) containing a solid bacterial medium (Kirby–Bauer agar) or fungal medium (Dox's medium), which has been heavily seeded with a spore suspension of the test organism. After incubation at 35±2°C (24 h for bacteria and 5 days for fungi) the diameter of the clear zone of inhibition surrounding the sample is taken as a measure of the inhibitory power of the sample against the particular test organism. The solvent used was DMSO and the concentration of the sample used is 10 μl. The references antibiotics Ampicillin and Amphotericin were used as references to evaluate the relative potency of the tested compounds under the same conditions.
Antimicrobial activity evaluation
Antimicrobial activity of the tested compounds was determined using a modified Kirby–Bauer disk diffusion method [24]. Briefly, 100 μl of the test bacteria/fungi was grown in 10 ml of fresh media until they reached a count of ∼108 cells/ml for bacteria or 105 cells/ml for fungi [25]. A measure of 100 μl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained.
Of the many media available, NCCLS recommends Mueller–Hinton agar or fungal medium (Dox's medium) due to: it results in good batch-to-batch reproducibility. The disk diffusion method for filamentous fungi was tested by using the approved standard method (M38-A) developed by the NCCLS (2002) [26] for evaluating the susceptibilities of filamentous fungi to antifungal agents. The disc diffusion method for yeasts was developed by using the approved standard method (M44-P) by NCCLS (2009) [27].
Minimum inhibitory concentration evaluation
The minimum inhibitory concentration (MIC) values were measured by the broth dilution method [28],[29]. A measure of 500 ml of a stock solution (10.24 mg/ml) of each tested compound in DMSO was prepared and then diluted with Mueller–Hinton broth to 1024 µg/ml. The strains were grown briefly at 37°C in Mueller–Hinton media. After 5 h of bacterial growth, the bacterial culture was diluted to obtain a concentration of 5×105 cells/ml. Then, 150 µl bacterial suspensions were added to each well of the flat-bottomed 96-well tissue culture plate. Two-fold serial dilutions were carried out from the first well to the tenth well; the final concentrations of the compounds ranged from 1 to 512 µg/ml; and excess media (150 µl) were discarded from the last well. The plates were incubated at 37°C for 24 h in an electro-heating standing temperature cultivator and were read visually. The MIC of the sample showing no turbidity was recorded as the lowest concentration of compound that inhibited bacterial growth completely. Each assay was run in triplicate.
Reaction of thiothymine 3 [14] with hydrazonoyl chloride 1 in refluxing chloroform in the presence of triethylamine yielded, in each case, a single product. As expected from all our previous results with related compounds [30],[31],[32], these products did not contain sulfur. On the basis of this finding, the structures 4–9 were overlooked for the products. Spectral and microanalytical measurements agree with one of the two isomeric forms 10 or 11. The reaction pathway for obtaining 10 and 11 is believed to proceed by the first production of thiohydrazonate 5 or amidrazone 4. Since a structure like 4 is known to be stable [17],[32],[33], its formation was excluded due to failure of all attempts to its isolation. Moreover, interaction of thiouracils with halogenated reagents affords only S-substituted compounds [8],[9],[10],[11]. Upon S→N rearrangement, thiohydrazonates 5 yield the corresponding thiohydrazides 7 via intermediate 6. The latter can also be produced through 1,3-dipolar cycloaddition of nitrilimines 2 (produced by base-catalyzed dehydrochlorination of the hydrazonoyl chloride 1) to the thione group C=S. The latter intermediates 7 undergo cyclization followed by elimination of hydrogen sulphide to give the final product 11. Structural elucidations of the new compounds were confirmed by elementary and spectroscopic analyses (see Experimental). Their structures were assigned 11 rather than the isomeric structure 10 (Scheme 1). For example, the 13C NMR spectrum of 11Aa showed the signal for the carbonyl carbon resonance at δ less than 166. This indicates that N (4) is sp3-hybridized nitrogen atom as in structure 11. If this nitrogen is sp2-hybridized as in structure 10, the signal for the carbonyl carbon will appear near δ 166 [34]. Also, the IR spectrum of 11Aa showed two carbonyl bands at ν 1709 and 1670 cm−1. Its 1H NMR spectrum showed two singlet signals at δ 2.32 (3H, CH3) and 2.71 (3H, CH3CO), in addition to the multiplet signal at δ 7.46–8.06 (5H) corresponding to aromatic protons (Ph). On the basis of the above data, the structure of 11 is 6-methyl-[1],[2],[4]triazolo [3,4-c][1],[2],[4]triazine derivatives.
Treatment of compound 11A with dimethylformamide dimethyl acetal (DMF-DMA) in refluxing dioxane yielded the appropriate enamine 12. Compatible elementary and spectroscopic analyses were gained for compounds 11A and 12 (see Experimental) (Scheme 2).
Stirring of 3 with dimethylsulphate in sodium hydroxide solution yielded 6-methyl-3-(methylthio)-1, 2, 4-triazin-5(4H)-one 13 (Scheme 3) [15]. Treatment of compound 13 with hydrazine hydrate in refluxing ethanol yielded the corresponding hydrazine derivative 14 [15] (Scheme 3). Reaction of hydrazine derivative 14 with the appropriate aldehyde in the refluxing ethanol in the presence of few drops of acetic acid afforded, in each case, one isolated product hydrazone 15 (Scheme 3). The structures of isolated products were identified by their elemental and spectroscopic analyses. For example, the 1H NMR spectrum of 15a shows two singlet signals at δ 2.10 (3H, CH3) and 2.33 (3H, CH3C6H4), the pair of doublet of an aromatic protons at δ 7.20 (2H) and 7.80 (2H) in addition to two singlet signals exchangeable protons at δ 11.51 (1H, NH) and 12.59 (1H, NH). Also, its 13C NMR spectrum showed 10 signals.
Next, the treatment of hydrazine 14 with the appropriate ketone, pyruvic acid, or ethyl pyruvate in the refluxing ethanol in the presence of few drops of acetic acid afforded, in each case, one isolated product hydrazone 16 (Scheme 4). The structures of isolated products were identified by their elemental and spectroscopic (IR, 1H NMR, 13C NMR and MS) analyses (see Experimental).
Antimicrobial screening
The antibacterial and antifungal activities of new synthesized compounds 14, 15h, and 16a–d were studied by the disk diffusion method. The antibacterial activities were done on the following pathogenic organisms: the Gram-positive bacteria S. aureus and B. subtilis, the Gram-negative bacteria E. coli, and P. aeruginosa. Moreover, antifungal activities against A. flavus and C. albicans were studied. The synthesized compounds were used at a concentration of 20 mg/ml using DMSO as a solvent. Ampicillin 10 µl/disk was used as a standard antibacterial agent and amphotericin B 20 µg/ml as the standard antifungal agent. The results presented in [Table 1] suggest that two compounds 14 and 16b inhibited both bacterial and fungal species; however, the most potent compound that exhibited antibacterial and antifungal activities was 16b.
Table 1 In-vitro antibacterial and antifungal activities of compounds 14, 15h, 16a–d (inhibition zone in mm)a
Compound 16b showed high potential in growth inhibition of all tested microorganisms and exceed the activity of standard antibiotics with a relative activity of 200% in B. subtilis, 205.5% in S. aureus, 154.5% in E. coli, 211.7% in P. aeruginosa, 280% in A. flavus, and 166.6% in C. albicans. In-vitro susceptibility tests were performed to evaluate MIC by a broth dilution method [28],[35],[36]. MIC values were determined for the highly efficient antibacterial compounds using the most sensitive microorganisms. The results illustrated in [Table 2] indicated that compound 16b achieved the lowest MIC values (high efficient derivative) against the sensitive bacterial strain S. aureus and fugal strain A. flavus with MIC values of 20 and 14 μg/ml, respectively.
Table 2 Minimum inhibitory concentration of compound 16b against the tested bacteria and fungi
In summary, thiothymine 3 was proved to be a useful precursor for the convenient synthesis of a wide variety of triazolotriazine and hydrazone derivatives that have not been reported hitherto.
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Hassaneen HM
Saleh FM
Mohamed YS
Awad EM
antimicrobial activities
cycloaddition reaction
hydrazone
hydrazonoyl halides
thiothymine
Antimicrobial ac...
Minimum inhibito...
Results and disc...
[TAG2]