Abstract
Compound 2 was synthesized by reacting CS2/KOH with compound 1. The treatment of compound 2 with hydrazine hydrate produced compound 3. Then, compound 3 was converted to Schiff bases (4a–d) by the handling with several aromatic aldehydes. The treatment of triazole compounds 4a–d containing Schiff base with morpholine gave compounds 5a–d. All compounds were tested for their antioxidant and antimicrobial activities. The antioxidant test results of DPPH• radical scavenging and ferric reducing/antioxidant power methods showed good antioxidant activity. The triazole-thiol (3) was the most active, and the effect of the substituent type of the thiophene ring on the activity was same for both Schiff bases (4a–d) and Mannich bases (5a–d). Among the newly synthesized triazole derivatives, the Schiff base 4d and the Mannich base 5d carrying nitro substituent on the thiophene ring showed promising antibacterial and antifungal activity, with lower MIC values than the standard antibacterial ampicillin.
Introduction
Various 1,2,4-triazole derivatives have been reported as antimicrobial, insecticidal, fungicidal, antitumoral as well as anticonvulsant, antidepressant, hypoglycemic, antihypertensive, analgesic, and plant growth regulator anticoagulantCitation1–5. Fluconazole compounds having 1,2,4-triazole residues are powerful azole antifungal agents, and N-nucleoside ribavirin is a potent antiviral. 1,3,4-Oxadiazoles, which form an important class of heterocyclic compounds, have a broad range of biological activities such as antibacterial, antioxidant, antimalarial, antihypoglycemic, anti-inflammatory, antifungal, muscle relaxant, anticonvulsant, antimycobacterial, anticancer, genotoxic, and insecticidalCitation6–10. Hybrid 1,2,4-triazole and 1,3,4-oxadiazole compounds are new classes of azole anti-mycobacterials, which are proved to be highly active both in vitro and in vivoCitation11. Schiff bases obtained from various heterocycles have a wide range of biological activities including antifungal, antibacterial, antimalarial, antimycobacterial, antimicrobial, anti-inflammatory, antiviral, cytotoxic, anticonvulsant, antiproliferative, anticancer, and antipyretic activitiesCitation12–18. N-fuctionalized morpholines have diverse pharmacological activities. They have been reported to have antimicrobial, antiemetic, platelet aggregation inhibitory, antihyperlipo, proteinemic, and bronchodilator activity. In addition, they are also used in the treatment of pain, migraine, and asthmaCitation19–22. In recent years, the thiophene chemistry have attracted more interest because of the biological significance. Many of thiophene compounds have been widely investigated for therapeutic uses, especially as antifungal, anti-HIV, anti-inflammatory, antibacterial, germicidal, D2 dopaminergic, anticonvulsant, antiasthmatic, antiproliferative, and analgesic agentsCitation23.
We aimed to synthesize a new series of hybrid triazole molecules having Schiff base and morpholine moieties including thiophene ring in order to obtain more effective biological activities.
Experimental
Chemistry
The 1H-, and 13C-nuclear magnetic resonance spectra were recorded on a Varian-Mercury 400 MHz spectrometer, where TMS as an internal standard and DMSO-d6 as solvent were used. IR spectra were recorded on a Perkin–Elmer Spectrum one FT-IR spectrometer (resolution 4) in KBr pellets. The MS spectra were measured with a Micromass Quattro LC-MS/MS spectrometer with methanol as solvent. Elemental analyses were carried out on a C, H, N-O rapid elemental analyzer Hewlett–Packard 185 for C, H, and N, and results were within 0.4% of the therotical values. Melting points were measured on an electrothermal apparatus and were not verified.
Synthesis of 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl) methyl)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (2)
A mixture of compound 1 (0.01 mol) and CS2/KOH (0.02 mol) in ethanol/water (1:2) refluxed for about 6 h. The reaction mixture was cooled to room temperature and diluted with water. The contents were then acidified with conc. HCl (20 mL). A solid precipitate was obtained. Then it was filtered, washed with water, and recrystallized from ethanol. Yield: 90.10%, m.p(0).194–196 °C. IR (KBr, cm−1): 3305–3199 (NH2), 1667 (C=O), 1581 (C=N), 1162 (C-O); 1H NMR (400 MHz, DMSO-d6) δ: 4.09 (s, 2H, thiophene-CH2), 5.02 (s, 2H, N-CH2), 5.39 (s, 2H, NH2), thiophene H [6.94 (s, 2H), 7.36 (s, 1H), 14.00 (s, 1H, SH)]; 13C NMR (100 MHz, DMSO-d6) δ: 25.39 (thiophene-CH2), 40.51 (N-CH2), thiophene C [125.63 (CH), 127.04 (CH), 127.37 (CH), 137.38 (C)], 148.10 (C=N), 153.23 (C=O), 159.49, 178.47 (oxadiazole C); LC-MS (m/z): 311.26 (M + 1, 50%). Analysis (% Calculated/found) for C10H10N6S2O2 C:38.70/38.01, H:3.25/3.39, N:27.08/27.05.
Synthesis of 4-amino-1-((4-amino-5-mercapto-4H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (3)
Hydrazine hydrate (0.03 mol) was added to a solution of compound 2 (0.01 mol) in n-butanol (50 mL), then refluxed for 4 h. At the end of this period, KOH (0.02 mol) was added to reaction medium. A solid precipitate was separated and filtered. The obtained solid was acidified with conc. HCl, filtered, washed with water, and recrystallized from ethanol. Yield: 40.00%, m.p. 216–218 °C. IR (KBr, cm−1): 3301–3260 (NH2), 1699 (C=O), 1622 (C=N); 1H NMR (400 MHz, DMSO-d6) δ: 4.07 (s, 2H, thiophene-CH2), 4.92 (s, 2H, N-CH2), 5.39 (s, 4H, 2NH2), thiophene H [6.93 (s, 2H), 7.36 (s, 1H)], 13.63 (s, 1H, SH); 13C NMR (100 MHz, DMSO-d6) δ: 25.44 (thiophene-CH2), 40.57 (N-CH2), thiophene C [125.55 (CH), 126.95 (CH), 127.34 (CH), 137.62 (C)], 147.98 (C=N), 153.42 (C=O), 147.38, 166.84 (triazole/thiol C); LC-MS (m/z): 363.04 (M + K, 100%). Analysis (% Calculated/found) for C10H12N8S2O C:37.03/37.45, H:3.73/3.80, N:34.54/34.19.
Synthesis of compounds 4(a–d)
0.01 mol compound 3 and 0.02 mol aromatic aldehydes were heated in oil bath without solvent for 2–3 h. at 160–170 °C. After cooling it to room temperature, the obtained solid was recrystallized from a mixture of DMF and water (1:5).
1-((5-mercapto-4-(thiophene-2-yl methylenamino)-4H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-4-(thiophene-2-yl methylenamino)-1H-1,2,4-triazole-5(4H)-one (4a):Yield: 86.50%, m.p.181–182 °C. IR (KBr, cm−1): 1706 (C=O), 1651 (C=N); 1H NMR (400 MHz, DMSO-d6) δ: 4.28 (s, 2H, thiophene-CH2), 5.23 (s, 2H, N-CH2), thiophene H [7.00–7.04 (m, 2H), 7.26–7.32 (m, 2H), 7.44–7.46 (m, 1H), 7.79–7.81 (m, 2H), 7.92–7.93 (m, 1H), 8.06 (s, 1H)], 9.82 (s, 1H, N=CH), 10.01 (s, 1H, N=CH), 14.17 (s, 1H, SH); 13C NMR (100 MHz, DMSO-d6) δ: 25.89 (thiophene-CH2), 40.60 (N-CH2), thiophene C [125.92 (CH), 127.21 (CH), 127.34 (CH), 128.74 (CH),128.83 (CH), 131.85 (CH), 133.62 (CH), 134.63 (CH), 136.29 (C), 136.40 (CH), 137.14 (C), 138.16 (C)], 146.25 (C=N), 149.33, 158.85 (2N=CH), 149.74 (C=O), 145.37, 162.58 (triazole/thiol C); LC-MS (m/z): 513.05 (M + 1, 50%). Analysis (% Calculated/found) for C20H16N8S4O C:46.86/46.80, H:3.15/3.05, N:21.86/21.55.
1-((5-mercapto-4-(3-methylthiophene-2-yl) methylenamino)-4H-1,2,4-triazole-3-yl) methyl)-3-yl) methyl)-4-((3-methylthiophene-2-yl) methylenamino)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (4b): Yield: 74.60%, m.p. 160–161 °C. IR (KBr, cm−1): 1705 (C=O), 1586 (C=N); 1H NMR (400 MHz, DMSO-d6) δ: 2.30 (s, 6H, 2CH3) 4.26 (s, 2H, thiophene-CH2), 5.15 (s, 2H, N-CH2), thiophene H [6.98–7.78 (m, 7H)], 9.90 (s, 1H, N=CH), 10.09 (s, 1H, N=CH), 14.11 (s, 1H, SH); 13C NMR (100 MHz, DMSO-d6) δ: 14.25 (CH3), 25.89 (thiophene-CH2), 40.61 (N-CH2), thiophen C [125.87 (CH), 127.14 (CH), 127.27 (CH),127.34 (CH), 130.20 (C), 131.00 (CH), 131.67 (CH),131.72 (CH), 132.60 (CH), 137.17 (C), 143.92 (C), 144.01 (C), 146.19 (C)], 146.33 (C=N), 148.20, 157.82 (2N=CH), 150.00 (C=O), 145.28, 162.64 (triazole/thiol C); LC-MS (m/z): 541.17 (M + 1, 100%). Analysis (% Calculated/found) for C22H20N8S4O C:46.87/46.78, H:3.73/3.75, N:20.72/20.67.
4-((5-bromothiophene-2-yl) methylenamino)-1((4-(5-bromothiophene-2-yl) methylene amino)-5-mercapto-4H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (4c): Yield: 81.80%, m.p. 225–226 °C. IR (KBr, cm−1): 1703 (C=O), 1589 (C=N); 1H NMR (400 MHz, DMSO-d6) δ: 4.31 (s, 2H, thiophene-CH2), 5.26 (s, 2H, N-CH2), thiophene H [7.04–7.06 (m, 2H), 7.42–7.49 (m, 3H), 7.67–7.71 (m, 2H)], 9.89 (s, 1H, N=CH), 10.08 (s, 1H, N=CH), 14.23 (s, 1H, SH); 13C NMR (100 Hz, DMSO-d6) δ: 25.83 (thiophene-CH2), 40.61 (N-CH2), thiophene C [118.13 (C), 120.12 (C), 125.96 (CH),127.19 (CH), 127.30 (CH), 132.13 (CH), 132.25 (CH), 135.30 (CH), 137.04 (C), 137.07 (CH), 138.12 (C), 140.08 (C)], 146.18 (C=N), 147.84, 156.99 (2N=CH), 149.78 (C=O), 145.46, 162.54 (triazole/thiol C); LC-MS (m/z): 670.75 (M, 40%). Analysis (% Calculated/found) for C20H14Br2N8S4O C:35.83/35.78, H:2.10/2.18, N:16.71/16.82.
1-((5-mercapto-4-(5-nitrothiophene-2-yl) methylenamino)-4H-1,2,4-triazole-3-yl) methyl)-3-yl) methyl)-4-((5-nitrothiophene-2-yl) methylenamino)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (4d): Yield: 75.00%, m.p. 130–131 °C. IR (KBr, cm−1): 1713 (C=O), 1639 (C=N); 1H NMR (400 MHz, DMSO-d6) δ: 4.40 (s, 2H, thiophene-CH2), 5.35 (s, 2H, N-CH2), thiophene H [7.05–7.09 (m, 2H), 7.49 (bs, 1H), 7.96–8.35 (m, 4H)], 10.10 (s, 1H, N=CH), 10.63 (s, 1H, N=CH), 14.40 (s, 1H, SH); 13C NMR (100 MHz, DMSO-d6) δ: 25.79 (thiophene-CH2), 40.60 (N–CH2), thiophene-C [126.01 (CH), 127.22 (CH), 127.31 (CH), 130.39 (CH), 130.69 (CH), 133.28 (CH), 134.72 (CH), 136.91 (C), 143.03 (C), 145.57 (C), 153.03(C), 153.69 (C)], 146.33 (C=N), 147.25, 154.57 (2N=CH), 149.54 (C=O), 145.57, 162.69 (triazole/thiol C); LC-MS (m/z): 602.21 (M + 1, 50%). Analysis (% Calculated/found) for C20H14N10S4O5 C:39.86/39.86, H:2.34/2.28, N:23.24/23.20.
Synthesis of compounds 5(a–d)
A mixture of formaldehyde (0.02 mol) and morpholine (0.02 mol) in DMF was added to solution of compound 4 (0.001 mol) in DMF. The resulting mixture was stirred overnight at room temperature. The precipitated solids were filtered, washed with water and recrystallized from methanol.
1-((1-(morpholinomethyl)-4-thiophene-2-yl methyleneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-4-(thiophene-2-yl methylene amino)-1H-1,2,4-triazole-5(4H)-one (5a): Yield: 83.45%, m.p. 144–145 °C. IR (KBr, cm−1): 1706 (C=O), 1670 (C=N), 1112 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ: 2.71 (s, 4H, morpholine N-CH2), 3.54 (s, 4H, morpholine O-CH2), 4.15 (s, 2H, thiophene-CH2), 5.05–5.17 (s, 4H, N-CH2), thiophene H [6.91–7.93 (m, 9H)], 9.66–9.80 (m, 2H, 2N=CH); 13C NMR (100 MHz, DMSO-d6) δ: 25.88 (thiophene-CH2), 40.61 (N-CH2), 50.69 (morpholine N-CH2), 66.52 (morpholine O-CH2), 69.22 (N-CH2-N), thiophene C [125.94 (CH),127.22 (CH), 127.34 (CH), 128.76 (CH), 128.88 (CH), 131.90 (CH), 133.94 (CH), 134.68 (CH), 136.06 (C), 136.81 (CH), 137.12 (C), 138.12 (C)], 145.12, 147.12 (2C=N), 149.36, 160.29 (2N=CH), 149.78 (C=O), 163.42 (C = S); LC-MS (m/z): 612.09 (M + 1, 50%). Analysis (% Calculated/found) for C25H25N9S4O2 C:49.08/49.14, H:4.12/4.18, N:20.61/20.77.
4-((3-methylthiophene-2-yl) methylenamino)-1-((4-(3-methylthiophene-2-yl) methylene amino)-1-(morpholinomethyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (5b): Yield: 86.77%, m.p. 220–221 °C. IR (KBr, cm−1): 1701 (C=O), 1675 (C=N), 1114 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ: 2.30 (s, 6H, 2CH3), 2.67 (s, 4H, morpholine N-CH2), 3.54 (s, 4H, morpholine O-CH2), 4.16 (s, 2H, thiophene-CH2), 5.04–5.12 (s, 4H, N-CH2), thiophene H [6.89–7.00 (m, 4H), 7.32 (s, 1H), 7.72 (s, 2H)], 9.78 (d, 2H, N=CH); 13C NMR (100 MHz, DMSO-d6) δ:14.29 (CH3), 25.89 (thiophene-CH2), 40.61 (N-CH2), 50.70 (morpholine N-CH2), 66.52 (morpholine O-CH2), 69.23 (N-CH2-N), thiophene C [125.89 (CH), 127.15 (CH), 127.26 (CH), 130.00 (C), 131.05 (CH), 131.67 (C), 131.72 (CH), 132.94 (CH), 137.11 (C), 143.96 (C), 145.04 (C)], 145.39, 146.71 (2C=N), 148.21, 159.30 (2N=CH), 149.92 (C=O), 163.57 (C = S); LC-MS (m/z): 641.56 (M+, 20%). Analysis (% Calculated/found) for C27H29N9S4O2 C:50.68/50.60, H:4.57/4.45, N:19.70/19.77.
4-((5-bromothiophene-2-yl) methylenamino)-1-((4-(5-bromothiophene-2-yl)m ethylene amino)-1-(morpholinomethyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl) methyl)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (5c): Yield: 83.55%, m.p. 163–164 °C. IR (KBr, cm−1): 1704 (C=O), 1672 (C=N), 1266 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ: 2.67 (s, 4H, morpholine N-CH2), 3.53 (s, 4H, morpholine O-CH2), 4.14 (s, 2H, thiophene-CH2), 4.89–5.14 (m, 4H, N-CH2), thiophene H [6.90–7.53 (m, 7H)], 9.70 (bs, 2H, N=CH); 13C NMR (100 MHz, DMSO-d6) δ: 25.81 (thiophene-CH2), 40.61 (N-CH2), 50.69 (morpholine N-CH2), 66.51 (morpholine O-CH2), 69.22 (N-CH2-N), thiophene C [118.14 (C),120.47 (C), 125.97 (CH), 127.22 (CH), 127.36 (CH), 132.15 (CH), 132.32 (CH), 135.33 (CH), 136.97 (C), 137.46 (CH), 137.87 (C), 140.05 (C)], 145.05, 145.55 (2C=N), 147.85, 158.41 (2N=CH), 149.84 (C=O), 163.32 (C = S); LC-MS (m/z): 771.46 (M + 2, 70%). Analysis (% Calculated/found) for C25H23Br2N9S4O2 C:39.02/39.19, H:3.01/2.98, N:16.38/16.45.
1-((1-(morpholinomethyl)-4-(5-nitrothiophene-2-yl) methyleneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl) methyl)-4-((5-nitrothiophene-2-yl) methyleneamino)-3-(thiophene-2-yl methyl)-1H-1,2,4-triazole-5(4H)-one (5d): Yield: 79.80%, m.p. 140–141 °C. IR (KBr, cm−1): 1706 (C=O), 1651 (C=N),1263 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ: 2.71 (s, 4H, morpholine N-CH2), 3.54 (s, 4H, morpholine O-CH2), 4.25 (s, 2H, thiophene-CH2), 4.92–5.27 (s, 4H, N-CH2), thiophene H [6.92–8.12 (m, 7H)], 9.88 (s, 2H, N=CH): 13C NMR (100 MHz, DMSO-d6) δ: 25.85 (thiophene-CH2), 40.54 (N-CH2), 50.64 (morpholine N-CH2), 65.36 (morpholine O-CH2), 66.67 (N-CH2-N), thiophene C [126.01 (CH),127.39 (CH), 130.34 (CH), 130.67 (CH), 133.26 (CH),134.70 (CH), 136.79 (C), 143.05 (C), 153.05 (C), 153.50 (C)], 145.02, 146.33 (2C=N), 147.25, 160.10 (2N=CH), 149.56 (C=O), 162.74 (C = S); LC-MS (m/z): 701.19 (M+, 50%). Analysis (% Calculated/found) for C25H23N11S4O6 C:42.79/42.70, H:3.30/3.26, N:21.95/22.02.
Antioxidant activity tests
For antioxidant activity determinations, two or more antioxidant methods based on different strategies or chemistries are generally utilized because of inconsistencies between the results of different methods. This can be due to different reaction mechanisms and kinetics, varying solvent effects, sterical hindrance issues, and the effects of temperature, pH, and other reaction components. Two of the most widely used antioxidant methods were used in the current study to determine the antioxidant capacities of the compounds synthesized. DPPH• radical scavenging method has been used extensively for many types of samples including syntheticsCitation24. Similarly, ferric reducing/antioxidant power (FRAP) has been determined in many investigations on synthetic organics. To overcome the problem of solubility of the compounds in test reaction medium, FRAP reagent was modified to contain methanol in 3:2 ratio in water instead of using water as solvent in the original methodCitation25,Citation26.
DPPH• scavenging test
Among the most widely used spectrophotometric test methods, DPPH• radical (2,2-diphenyl-1-picrylhydrazyl) scavenging assay relies on the measurement of absorbance change as the radical is deactivated by antioxidants. The method developed by Cuendet et al.Citation24 was used with some modification. DPPH• radical was dissolved in methanol at the test concentration of 100 μM. The synthesized compounds were dissolved in DMSO at 10 mg/mL concentration. Testing solutions were serially diluted with methanol to the concentration determined by pretest experiments. Equal volumes (750 μL) of DPPH• and sample solutions were mixed, vortexed, and incubated for 60 min at room temperature. The absorbances measured at 517 nm were plotted against sample concentrations, and SC50 values (mg/mL), used to express antiradical activity, were determined from the graphs as the sample concentration reducing DPPH• concentration to half of its initial value. Reagent blank and solvent control tests were also made, and the results were used in the construction of the graphs. Lower SC50 value is an indication of higher radical scavenging potential.
The reaction kinetics of the synthesized compounds were also determined by following the absorbances at 517 nm for a period of 60-min incubation. Graph of absorbance change as a function of time was created ().
Ferric reducing/antioxidant power test
First developed by using FeSO4 solutionsCitation25, FRAP antioxidant test method was later improved by using TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) as ferrous ion complexing agentCitation26. FRAP activities reflect the total reducing potential of samples. The method is based on the measurement of the absorbance of Fe2+–TPTZ complex at 595 nm. The expected solubility problems of the compounds when the sample solutions are mixed with FRAP reagent in the original methodCitation26 were overcome by changing the reagent solvent from water to 3:2 methanol:water mixture. Fresh FRAP reagent was prepared by combining 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3·6H2O in 10:1:1 ratio, respectively. Calibration curve was constructed with Trolox (1000-500-250-125-62.5 μM). The compound solutions of 10 mg/mL concentration and Trolox solutions (50 μL) were mixed with FRAP reagent (1.5 mL), vortexed, and incubated for 20 min at room temperature. The absorbance was read at 595 nm against water. Reagent and sample blanks were also tested, and the absorbance of these two measurements was subtracted from that of the sample mean of triplicate measurements. FRAP activities were expressed as Trolox equivalent antioxidant capacity (TEAC, μM) and calculated from the calibration graph as the corresponding Trolox concentration. Higher TEAC values mean higher FRAP and thus higher antioxidant capacity.
Antimicrobial activity
All test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam (Ankara, Turkey) and were as follows: Escherichia coli (E. coli) ATCC35218, Yersinia pseudotuberculosis (Y. pseudotuberculosis) ATCC911, Pseudomonas aeruginosa (P. aeruginosa) ATCC43288, Enterococcus faecalis (E. faecalis) ATCC29212, Staphylococcus aureus (S. aureus) ATCC25923, Bacillus cereus (B. cereus) 709 Roma, Mycobacterium smegmatis (M. smegmatis) ATCC607, Candida albicans (C. albicans) ATCC60193, and Saccharomyces cerevisiae (S. cerevisiae) RSKK 251. All the newly synthesized compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare compound stock solution of 10 μg/mL.
The antimicrobial effects of the compounds were tested quantitatively in respective broth media by using double microdilution, and the minimal inhibition concentration (MIC) values (μg/mL) were determinedCitation27. The antibacterial and antifungal assays were performed in Mueller–Hinton broth (MH) (Difco, Detroit, MI) at pH.7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI) at pH 7.0, respectively. The micro dilution test plates were incubated for 18–24 h at 35 °C. Brain Heart Infusion (BHI) broth (Difco, Detriot, MI) was used for M. smegmatis, and incubated for 48–72 h at 35 °CCitation28. The MIC was defined as the lowest concentration that showed no growth. Ampicillin (10 mg/mL), streptomycin 10 mg/mL, and fluconazole (2 mg/mL) were used as standard antibacterial and antifungal drugs, respectively. DMSO with dilution of 1:10 was used as solvent control. The results are shown in .
Table 1. Screening results for antimicrobial activity of the compounds 2–5.
Results and discussion
Synthesis
The starting compound (1) was synthesized according to published literatureCitation17. The reaction of compound 1 and carbon disulfide in presence of potassium hydroxide gave compound 2 in high yield (Scheme 1). The peaks of NH and NH2 belonging to the hydrazide (1) disappeared, and the peak of SH appeared at 14.00 ppm in the 1H-NMR spectrum of compound 2. In addition, peaks at 159.49 (oxadiazole C=N) and 178.47 (oxadiazole C=S) ppm were observed in the 13C-NMR spectrum. Compound 2 was converted to the corresponding compound 3 via the reaction with hydrazine hydrate. Triazole-thiol NH2 appeared at 5.39 ppm together with triazol-NH2 in the 1H-NMR spectrum. Triazol-thiol C = S and C=N peaks appeared at 166.84 and 147.38 ppm, respectively, in the 13C-NMR spectrum of compound 3. The reaction of triazole-thiol (3) with several aromatic aldehydes gave the Schiff bases 4a–d. The imine N=CH protons resonated as singlet at 9.82–10.63 ppm integrating for 2 protons in the 1H-NMR spectra of compounds 4a–d. Imine N=CH appeared at 147.33–158.85 ppm in the 13C-NMR spectra of compounds 4a–d.
Scheme 1. Synthetic pathway for the preparation of compounds 2–5.
The reaction of compounds 4a–d with morpholine and formaldehyde produced compounds 5a–d. In compounds 5a–d, the signal belonging to NH (SH) proton disappeared and the presence of a peak at 4.89–5.17 belonging to the N-CH2-N protons verified the formation of Mannich bases (5). The peaks of N-CH2 and O-CH2 belonging to morpholine were observed at 2.67–2.71 and 3.53–3.54 ppm, respectively, in the 1H-NMR spectra of 5a–d. In addition, the formation of Mannich base was figured out by the presence of new carbon peaks relating morpholine in the 13C spectra of compound 5.
Biological activities
Antioxidant activities
DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity and FRAP methods are among the most widely used methods for the determination of antioxidant activities of synthetic and natural compoundsCitation29,Citation30. The studies on the biological activities of Schiff and Mannich bases with different functional groups, including triazole and triazole-thiol, are available in the literatureCitation31. Not only triazole or triazole-thiol groups alone are effective in the antioxidant potential, but also the groups added to the structure significantly affect the activity in antioxidant activity evaluations of these bases with many antioxidant test methods, including DPPH• and ABTS radical scavenging and FRAPCitation32. Formation of Mannich bases with indole group has been demonstrated to provide weaker free radical scavengers in comparison to their Schiff base counterpartsCitation33. Schiff and Mannich bases with various functional groups have also been shown to possess anti-inflammatory and antimicrobial activitiesCitation34–36. In addition, there are a number of studies reporting good anticancer activities of Schiff and Mannich basesCitation37–39.
All of the compounds synthesized in this study showed antioxidant activity in both antioxidant tests at various extends (). The antioxidant activities were affected from the substituents of the Schiff and Mannich bases in a similar manner. The effect of the substituent type of the thiophene ring on the activity was same for both Schiff bases (4a–d) and Mannich bases (5a–d) as –CH3 > –NO2 > –Br > –H.
Figure 1. The DPPH• radical scavenging activities of the compounds expressed as SC50 values. The lower SC50 shows higher activity. Trolox was used for antioxidant activity comparison.
Figure 2. The scavenging of DPPH• radical by the compounds synthesized as a function of time, expressed by the decrease in absorbance at λmax for DPPH•.
Figure 3. TEAC (Trolox equivalent antioxidant capacity) values of the compounds synthesized and obtained from Trolox calibration graph in FRAP test. The values for 4a–d and 5a–d are the means of the series with four different substituents on the thiophene ring.
In DPPH• test, compound 3 showed the highest radical scavenging activity, expressed as the lowest SC50 value, among all the compounds synthesized (). The Schiff bases (4a–d) exhibiting slightly lower activity than compound 3 had much higher scavenging potential in comparison to Mannich bases (5a–d). The antioxidant activity was increased 13-fold in DPPH• test () and 3-fold in FRAP test () as oxadiazole-thiol compound (2) was transformed into the triazole-thiol (3) as a result of the reaction with hydrazine.
The extend of the reactions between DPPH• radical and the compounds tested were monitored for a 60-min period and appeared to reach completeness at the end of this period with most of the compounds (). This finding is in accordance with the literature reportsCitation40,Citation41.
The data obtained with FRAP assay showed compound 3 as the most active one as in the DPPH• test. Similarly, the Schiff bases (4a–d) were more active when compared with the Mannich bases (5a–d).
Antimicrobial activities
All of the synthesized compounds were observed to have antimicrobial activities. The new compounds were more active against gram-negative bacteria than gram-positive ones. All the compounds exhibited antimicrobial activities, but Schiff base 4d and Mannich base 5d including nitro substituent showed high antibacterial and antifungal activities against all test microorganisms.
Conclusion
In this study, the synthesis of new 1,2,4-triazole-1,3,4-oxadiazole, 1,2,4-triazole-triazole/thiol, 1,2,4-triazole-Schiff bases, and 1,2,4-triazole compounds possessing morpholine and Schiff base rings were reported. The structures of all the compounds were confirmed by recording their elemental analyses, IR, 1H NMR, 13C NMR, and mass spectral data. All the newly synthesized compounds were screened for their antimicrobial and antioxidant activities. Among all the synthesized triazole derivatives the nitro substituted thiophene containing compounds 4d and 5d showed high promising antimicrobial activity. The starting compound 3 (triazole-thiol) was determined to have higher antioxidant activity than all the other test compounds in both antioxidant assays; the Schiff bases showed closer antioxidant activity to compound 3. In addition, the antioxidant activities decreased as the Schiff bases were transformed into the Mannich bases, and the substituent type on the thiophene ring was determined to be affective on the antioxidant activity. The order of the effect of substituent type of the thiophene ring on the activity was –CH3 > –NO2 > –Br > –H for both Schiff and Mannich bases.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grants from Karadeniz Technical University and Scientific and Technological Research Council of Turkey (TÜBİTAK-114Z582).
Supplementary materials online only – For review only at proofing stage.
NMR spectra of the new compounds are provided as the supplementary material.
IENZ_1206088_Supplementary_Material.pdf
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